Cellular Transplantation from Laboratory to Clinic
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Cellular Transplantation from Laboratory to Clinic
Editors Craig Halberstadt Dwaine Emerich
Amsterdam • Boston • Heidelberg • London • New York Oxford • Paris • San Diego • San Francisco • Singapore Sydney • Tokyo
Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK This book is printed on acid-free paper. Copyright © 2007, Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail:
[email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting “Support & Contact” then “Copyright and Permission” and then “Obtaining Permissions.” Library of Congress Cataloging-in-Publication Data APPLICATION SUBMITTED ISBN 13: 978-0-12-369415-7 ISBN 10: 0-12-369415-9 For information on all Academic Press publications visit our Web site at www.books.elsevier.com Printed in the United States of America 07 08 09 10 9 8 7 6 5 4
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Contents
Contributors .................................................... vii Preface ........................................................... xiii
6. Characterization of Islet Preparations ................... 85 Clark K. Colton, Klearchos K. Papas, Anna Pisania, Michael J. Rappel, Daryl E. Powers, Jack J. O’Neil, Abdulkadir Omer, Gordon Weir, and Susan Bonner-Weir
A. The Basic Biology of Cell Therapy 1. Immunology of Cell and Tissue Xenotransplantation ...................................... Jeffrey L. Platt
7. Encapsulated Human Islet Allografts: Providing Safety with Efficacy .......................................... 135 David W. Scharp
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8. Strategies for Cell Replacement for Kidney Failure ................................................. 155 Marc R. Hammerman
2. Current Immunosuppressive Drugs and Clinical Use .................................................... 13 Harrison S. Pollinger, Justin M. Burns, Vince P. Casingal, and Paul F. Gores
9. Strategies for Extracorporeal Devices for Kidney Failure ............................................................ 169 Khajohn Tiranathanagul and H. David Humes 10. Transplants for Hemophilia ................................ 187 Andrew Gómez-Vargas and Gonzalo Hortelano
3. Stem and Precursor Cells for Transplant Therapy ... 29 Jingli Cai and Mahendra Rao
11. Cell Therapy for Peripheral Diseases and Reconstructive Applications: Transplants for Lysosomal Storage Disease ................................ 205 Toya Ohashi and Yoshikatsu Eto
4. Creating Animals for Cell Xenotransplantation ...... 43 David White
12. Cellular Transplants for Liver Diseases ................ 215 Nicholas J. Krebs, Craig Neville, and Joseph P. Vacanti
B. Cell Therapy for Peripheral Diseases and Reconstructive Applications
13. Stem Cell Transplantation for Autoimmune Diseases ....................................... 241 Alan Tyndall and Katarina Le Blanc
5. Islet Transplants for Diabetes: The Edmonton Protocol .......................................................... 59 Peter A. Senior and A. M. James Shapiro
14. Cardiac Cell Transplantation .............................. 259 Bryce H. Davis, Harald C. Ott, and Doris A. Taylor v
Contents 15. Cylindrical Cartilage Transplantation as Tracheal Replacement .................................................... 275 Koji Kojima and Charles A. Vacanti
25. Neural Transplantation in Parkinson’s Disease ....... 439 Stephen B. Dunnett and Anne E. Rosser 26. Transplants for Chronic Pain .............................. 455 Jacqueline Sagen, Daniel A. Castellanos, and Shyam Gajavelli
16. Muscle Cell Transplants .................................... 289 Mitra Lavasani, Jonathan Pollett, and Johnny Huard
27. Cell Grafting for Spinal Cord Injury Repair: Cell Replacement and Bridging Strategies ................... 477 D. D. Pearse and D. J. Barakat
C. Cell Therapy for CNS Diseases
28. Human Embryonic Stem Cells, Dopaminergic Neurons, and Pathways for Developing a Parkinson’s Disease Therapy .............................. 523 William J. Freed, Joseph F. Sanchez, Chun-Ting Lee, Tandis Vazin, Rose Amable, Cynthia Schoen, Catherine Schwartz, Mahendra Rao, and Xianmin Zeng
17. Immunological Considerations in CNS Transplants ...................................................... 305 Kai-Christian Sonntag 18. Neurotrophic Cell Transplants for Degenerative Diseases of the Eye .......................................... 327 Chris Thanos and Dwaine F. Emerich
D. Future Avenues of Cell Therapy
19. Transplants for Glioblastoma .............................. 345 Jian Wang, A. J. A. Terzis, Karen S. Aboody, Per Oyvind Enger, and Rolf Bjerkvig
29. Concepts in Cell Therapy: From Cord Blood to Sertoli Cells .................................................... 547 Paul R. Sanberg, Denis K. English, Amal Hakki, Don F. Cameron, Svitlana Garbuzova-Davis, Alison E. Willing, Cesario V. Borlongan, Robert Henning, Stephen K. Klasko, and Nagwa S. El-Badri
20. NT2N Cell Transplantation and GDNF Treatment in Stroke: Linking Neurotrophic Factor Therapy and Neuroprotection .......................................... 353 Cesario V. Borlongan, Christine E. Stahl, Guolong Yu, Lin Xu, Takao Yasuhara, Koichi Hara, Noriyuki Matsukawa, Paul R. Sanberg, Yun Wang, and David C. Hess
30. Immunologically Privileged Environments ............ 567 Mark P. Hedger
21. NGF-Producing Cell Transplants for Alzheimer’s Disease ........................................................... 373 Ann-Charlotte Granholm
31. Converging Cell Therapy with Biomaterials .......... 591 Omar A. Ali and David J. Mooney 32. Nanotechnology’s Impact on Cell Transplantation ................................................ 611 Dwaine F. Emerich and Craig Halberstadt
22. Transplants of CNTF-producing Cells for the Treatment of Huntington’s Disease ...................... 385 Carole Escartin, Philippe Hantraye, and Nicole Déglon
33. Design and Implementation of a Cell Processing Facility ........................................................... 629 Tatsuya Kin, Mohammadreza Mirbolooki, and Jonathan R. T. Lakey
23. Choroid Plexus Epithelial Cell Transplants for Repair of the Brain ........................................... 399 Christopher G. Thanos and Dwaine F. Emerich
34. Developing Cell Therapy Products ...................... 635 Alfred V. Vasconcellos
24. Neural Transplantation in Huntington’s Disease ........................................................... 417 Anne E. Rosser and Stephen B. Dunnett
Index ................................................................... 647
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Contributors
The number in parentheses indicates the chapter to which the author contributed.
Susan Bonner-Weir (6) Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts
Karen S. Aboody (19) The Divisions of Hematology/Hematopoietic Cell Transplantation and Neurosciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California
Cesario V. Borlongan (20) Research and Affiliations Service Line, Augusta VAMC, Augusta, Georgia Department of Neurology and Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA
Omar A. Ali (31) Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts Rose Amable (28) Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
Justin M. Burns (2) Division of Transplantation Surgery, Carolinas Medical Center, Charlotte, North Carolina Jingli Cai (3) National Institute on Aging, Triad Technology Center, Baltimore, Maryland
D. J. Barakat (27) The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami School of Medicine, Miami, Florida
Don F. Cameron (29) Center for Excellence for Aging and Brain Repair, Department of Anatomy, USF Health, Tampa, Florida
Rolf Bjerkvig (19) NorLux Neuro-Oncology, Department of Biomedicine, University of Bergen, Norway and NorLux Neuro-Oncology, Centre Recherche Public Santé, Luxembourg
Vince P. Casingal (2) Division of Transplantation Surgery, Carolinas Medical Center, Charlotte, North Carolina
Katarina Le Blanc (13) Dept of Allogeneic Stem Cell Transplantation, Karolinska Institute, Stockholm, Sweden
Daniel A. Castellanos (26) Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, Florida vii
Contributors
Clark K. Colton (6) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
Svitlana Garbuzova-Davis (29) Center for Excellence for Aging and Brain Repair, Department of Neurosurgery, USF Health, Tampa, Florida
Bryce H. Davis (14) Duke University, Department of Biomedical Engineering, Durham, North Carolina
Andrew Gómez-Vargas (10) Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
Nicole Déglon (22) Atomic Energy Commission, CEA CNRS URA 2210 Unit and MiRcen ImaGene Program Stephen B. Dunnett (24, 25) School of Biosciences, Cardiff University, Cardiff, Wales, UK Nagwa S. El-Badri (29) Center for Excellence for Aging and Brain Repair, Department of Neurosurgery, USF Health, Tampa, Florida Dwaine F. Emerich (18, 23, 32) LCT BioPharma, Providence, Rhode Island Per Oyvind Enger (19) NorLux Neuro-Oncology, Department of Biomedicine, University of Bergen, Norway and NorLux Neuro-Oncology, Centre Recherche Public Santé, Luxembourg Denis K. English (29) Center for Excellence for Aging and Brain Repair, Departments of Neurosurgery, USF Health, Tampa, Florida Carole Escartin (22) Atomic Energy Commission, CEA CNRS URA 2210 Unit and MiRcen ImaGene Program Yoshikatsu Eto (11) Department of Gene Therapy, Institute of DNA Medicine, Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan William J. Freed (28) Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland Shyam Gajavelli (26) Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, Florida
Paul F. Gores (2) Division of Transplantation Surgery, Carolinas Medical Center, Charlotte, North Carolina Ann-Charlotte Granholm (21) Department of Neurosciences and the Center on Aging, Medical University of South Carolina, Charleston, South Carolina Amal Hakki (29) Center for Excellence for Aging and Brain Repair, Department of Neurosurgery, USF Health, Tampa, Florida Craig Halberstadt (32) Carolinas Medical Center, Charlotte, North Carolina Marc R. Hammerman (8) Renal Division, Departments of Medicine, and Cell Biology and Physiology, Washington, University School of Medicine, St. Louis, Missouri Philippe Hantraye (22) Atomic Energy Commission, CEA CNRS URA 2210 Unit and MiRcen ImaGene Program Isotopic Imaging, Biochemical and Pharmacological Unit, Service Hospitalier Frederic Joliot, CEA, Orsay, France Koichi Hara (20) Research and Affiliations Service Line, Augusta VAMC, Augusta, Georgia Mark P. Hedger (30) Monash Institute of Medical Research, Monash University, Melbourne, Australia Robert Henning (29) Center for Excellence for Aging and Brain Repair, Department of Internal Medicine, USF Health, Tampa, Florida viii
Contributors
David C. Hess (20) Research and Affiliations Service Line, Augusta VAMC, Augusta, Georgia Department of Neurology and Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA
Chun-Ting Lee (28) Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
Gonzalo Hortelano (10) Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada Research and Development, Canadian Blood Services
Noriyuki Matsukawa (20) Research and Affiliations Service Line, Augusta VAMC, Augusta, Georgia Mohammadreza Mirbolooki (33) Clinical Islet Transplant Program, University of Alberta and Capital Health Authority, Edmonton, Alberta, Canada
Johnny Huard (16) Department of Bioengineering, University of Pittsburgh Growth and Development Laboratory, Children’s Hospital of Pittsburgh Departments of Orthopaedic Surgery and Molecular Genetics and Biochemistry, University of Pittsburgh
David J. Mooney (31) Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts Craig Neville (12) The Center for Regenerative Medicine, Massachusetts General Hospital Department of Pediatric Surgery, Mass General Hospital for Children and Harvard Medical School
David H. Humes (9) Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Tatsuya Kin (33) Clinical Islet Transplant Program, University of Alberta and Capital Health Authority, Edmonton, Alberta, Canada
Toya Ohashi (11) Department of Gene Therapy, Institute of DNA Medicine, Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan
Stephen K. Klasko (29) Center for Excellence for Aging and Brain Repair, University of South Florida College of Medicine, USF Health, Tampa, Florida
Abdulkadir Omer (6) Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts
Koji Kojima (15) Laboratory for Tissue Engineering and Regenerative Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts Nicholas J. Krebs (12) The Center for Regenerative Medicine, Massachusetts General Hospital
Jack J. O’Neil (6) Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts Lifescan, Inc., Johnson and Johnson, Skillman, New Jersey
Jonathan R. T. Lakey (33) Clinical Islet Transplant Program, University of Alberta and Capital Health Authority, Edmonton, Alberta, Canada
Harald C. Ott (14) University of Minnesota Medical School, Center for Cardiovascular Repair, Minneapolis, Minnesota
Mitra Lavasani (16) Department of Bioengineering, University of Pittsburgh Growth and Development Laboratory, Children’s Hospital of Pittsburgh
Klearchos K. Papas (6) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts Diabetes Institute, University of Minnesota, Minneapolis, Minnesota ix
Contributors
Joseph F. Sanchez (28) Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
D. D. Pearse (27) The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami School of Medicine, Miami, Florida Anna Pisania (6) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
David W. Scharp (7) Executive Vice President and Chief Medical Officer Novocell, Inc., 31 Technology Dr., Suite 100, Irvine, CA 92618
Jeffrey L. Platt (1) Transplantation Biology and the Department of Surgery, Immunology and Pediatrics, Mayo Clinic College of Medicine, Rochester, Minnesota
Cynthia Schoen (28) Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
Jonathan Pollett (16) Growth and Development Laboratory, Children’s Hospital of Pittsburgh Harrison S. Pollinger (2) Division of Transplantation Surgery, Carolinas Medical Center, Charlotte, North Carolina
Catherine Schwartz (28) Laboratory of Neuroscience, National Institute of Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
Daryl E. Powers (6) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
Peter A. Senior (5) Assistant Professor, Department of Medicine, Clinical Islet Transplant Program, University of Alberta
Mahendra Rao (3, 28) National Institute on Aging, Triad Technology Center, Baltimore, Maryland Laboratory of Neuroscience, National Institute of Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
James A.M. Shapiro (5) Associate Professor, Department of Surgery, Clinical Islet Transplant Program, University of Alberta Kai-Christian Sonntag (17) Assistant Professor in Psychiatry/Neuroscience, Harvard Medical School, Center for Neuroregeneration Research, Udall Parkinson’s Disease Center of Excellence, McLean Hospital, Belmont, Massachusetts
Michael J. Rappel (6) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts Anne E. Rosser (24, 25) School of Biosciences, Cardiff University, Cardiff, Wales, UK
Christine E. Stahl (20) Department of Internal Medicine, Dwight D. Eisenhower Army Medical Center, Fort Gordon, Georgia
Jacqueline Sagen (26) Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, Florida
Doris A. Taylor (14) Duke University, Department of Biomedical Engineering, Durham, North Carolina University of Minnesota Medical School, Bakken Professor and Director, Center for Cardiovascular Repair, Minneapolis, Minnesota
Paul R. Sanberg (20, 29) Center of Excellence for Aging and Brain Repair, University of South Florida, College of Medicine, Tampa, Florida x
Contributors
A. J. A. Terzis (19) NorLux Neuro-Oncology, Department of Biomedicine, University of Bergen, Norway and NorLux Neuro-Oncology, Centre Recherche Public Santé, Luxembourg
Jian Wang (19) NorLux Neuro-Oncology, Department of Biomedicine, University of Bergen, Norway and NorLux Neuro-Oncology, Centre Recherche Public Santé, Luxembourg
Chris Thanos (18, 23) LCT BioPharma, Providence, Rhode Island Brown University, Dept. of Molecular Pharmacology, Physiology, and Biotechnology, Providence, Rhode Island
Yun Wang (20) Neural Protection and Regeneration, NIDA, NIH, DHHS, Baltimore, Maryland Gordon Weir (6) Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts
Khajohn Tiranathanagul (9) Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan Division of Nephrology, Department of Medicine, Faculty of Medicine, Chulalongkorn University Hospital, Bangkok, Thailand
Alison E. Willing (29) Center for Excellence for Aging and Brain Repair, Department of Neurosurgery, USF Health, Tampa, Florida
Alan Tyndall (13) Dept of Rheumatology, University Hospital, Basle, Switzerland
David White (4) Robarts Research Institute, University of Western Ontario Siebens Drake Centre, London, Ontario, Canada
Charles A. Vacanti (15) Laboratory for Tissue Engineering and Regenerative Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts
Lin Xu (20) Research and Affiliations Service Line, Augusta VAMC, Augusta, Georgia Takao Yasuhara (20) Research and Affiliations Service Line, Augusta VAMC, Augusta, Georgia
Joseph P. Vacanti (12) The Center for Regenerative Medicine, Massachusetts General Hospital Department of Pediatric Surgery, Mass General Hospital for Children and Harvard Medical School
Guolong Yu (20) Research and Affiliations Service Line, Augusta VAMC, Augusta, Georgia
Alfred V. Vasconcellos (34) LCT Biopharma
Xianmin Zeng (28) Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
Tandis Vazin (28) Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
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Preface
relieved, and then the bone was grafted back into the skull. Modern organ transplantation relied on the development of anesthetics (earliest development in 1540 by Valerius Cordus discovering the effects of ether), the development of aseptic surgical techniques in the 1800s by Joseph Lister, and the discovery of cyclosporine in the 1970s by Jean-Francois Borel with the subsequent trials in transplant patients by Roy Calne. These were only some of the discoveries that have led to today’s successful whole-organ transplantation of kidneys, lungs, livers, pancreases, heart, and other peripheral organs. Bone marrow transplantation of matched HLA donors became a successful procedure in the 1970s and 1980s. In the 1980s and into the 1990s, it was demonstrated that autologous bone marrow transplantation on irradiated patients could be used for cancer therapy. During this period, a subset population of cells (the stem cell) was identified and methods for expansion and further differentiation into the various myeloid lineages were developed. This therapy has revolutionized modern methods of treating several cancer phenotypes. Hence, the success of this cell therapy has led the impetus to develop other cell-based therapies. The complexity of whole-organ transplantation for diseases such as diabetes and neuromuscular disorders has led to the final push in cell therapy. Over the past 20 years hundreds of clinical trials have been performed to evaluate the safety and efficacy of cell transplantation ranging from pancreatic islet transplantation for diabetes to dopamine-producing cells for Parkinson’s disease.
These are exciting times for everyone involved in and interested in cell transplantation. Great progress has been made using cellular transplants for the amelioration of diseases over the last three decades. Indeed, the field has grown to its current-day accepted practice for certain diseases. As you will read, however, this scientific and clinical field continues to mature and many hurdles remain that must be overcome for cell transplantation to be routinely accepted in clinical practice. Significant advances in cell sourcing (both stem cells and/or animal cell sources), gene therapy, and immunoprotectior/novel-immunosuppressants will be focal areas for the foreseeable future. In addition, the ability to scale up these technologies and package the cells is a difficult task for the eventual delivery of viable cells to the clinician. Finally, developing reproducible transplantation strategies that will ensure high-percentage outcomes without complications also has to be developed. These obstacles are embraced by the authors of the chapters in this text as challenges that will be overcome with a greater understanding of disease mechanisms and transplantation biology. These authors provide their insights into the accomplishments and challenges of cell transplantation in specific clinical applications. Cell transplantation has passed through several revolutions in its surprisingly long history. One of the earliest recorded autograft transplants was performed to relieve cranial pressure in the Bronze age. A hole was bored in the skull, the bone removed, pressure xiii
Preface
Both successful and unsuccessful outcomes have been observed. It is the goal of this book to introduce the reader to many different potential applications of this therapy and to demonstrate not only the challenges but the clinical successes that have been achieved to date. Because the field of cell transplantation has become so broad, researchers and clinicians are finding it increasingly difficult to remain familiar with all of the different multidisciplinary aspects. Although several books exist on the topic, they tend to cover the field from a specialized perspective. A major impetus for this book was the belief that there is a need for a more inclusive approach that illuminates the rapid advances and developments that are pushing cell transplantation forward. We have organized the book into several parts to help ensure that as many aspects as possible could be covered by leading experts. In Part I, some basic biology of cell transplantation is identified, including transplant immunology, the use of immunosuppressive drugs, stem cell biology, and the development of uniquely suitable donor animals for transplantation. In Part II, recent advances in cell transplantation for peripheral and reconstructive
applications are identified, focusing on diabetes, kidney transplants, hemophilia, liver failure, autoimmune diseases, cardiac and orthopedic, and muscle transplantation. Part III describes the current state of central nervous system transplantation, describing the strategies under investigation for Parkinson’s, Alzheimer’s, and Huntington’s disease, as well as stroke and spinal cord trauma. Finally, Part IV attempts to be somewhat prescient describing future developments in the field, ranging from identifying novel transplantable cells to the combined use of biomaterials and nanotechnology with cell matrices. We find ourselves in the midst of a new era in cell transplantation. The focus of cell transplantation is shifting from an intuitively appealing but speculative concept to clinical reality. With continued time and effort our hope is that one day off-the-shelf cell-based products will exist that will be able to treat hundreds of diseases and tissue malfunctions.
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Craig R. Halberstadt Dwaine F. Emerich
A The Basic Biology of Cell Therapy
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1 Immunology of Cell and Tissue Xenotransplantation Jeffrey L. Platt Transplantation Biology and the Department of Surgery, Immunology, and Pediatrics, Mayo Clinic College of Medicine, Rochester, Minnesota
Introduction The early attempts at xenotransplantation failed, as did early attempts at human-to-human transplantation (allotransplantation). The reason for failure of xenografts and allografts was not understood until the 1940s, when the immune response of the recipient against the graft was found to be the principal biological barrier to transplantation [11]. With the advent of immunosuppressive drugs in the late 1950s, this barrier was overcome and the era of clinical allotransplantation began soon thereafter. The application of organ transplantation for the treatment of disease was among the most exciting advances in medicine and surgery. However, as in the early years of the twentieth century the availability of human donors was quite limited. Hence, efforts were made once again to use animals, such as chimpanzees and baboons, as a source of organs [31, 34]. When the recipient was treated with immunosuppressive agents, primate-tohuman xenografts functioned for weeks to months, but ultimately failed. Human-to-human transplants, however, would sometimes function indefinitely. This experience suggested that although xenotransplantation was a potential solution to organ failure xenotransplantation was impaired by an immunological barrier more severe than the barrier hindering human-to-human transplantation. Recent years have brought a better understanding of the immunological barriers to xenotransplantation and allotransplantation, and the possibility of applying genetic engineering to better address that barrier, at least in part [6]. The material following reviews the immunological barriers to xenotransplantation and discusses
Xenotransplantation refers to the transplanting of cells, tissues, or organs from individuals of one species into individuals of another species. Xenotransplantation has long been envisioned as a way of treating human diseases because animals are available in large numbers and at little expense. In contrast, when humans are used as a source of transplants for human recipients (allotransplants), the number of transplants that can be performed is limited by the availability of human donors (as low as 5% of the number needed) and the cost of these transplants can be very high [10]. The first serious efforts at xenotransplantation were made in the early years of the twentieth century. With the development of the surgical technique that could enable organ transplants to be performed (the vascular anastomosis), experimental surgeons sought a ready source of organs to treat subjects with organ failure, particularly kidney failure. Humans, living and recently deceased, were not viewed as a potential source of such organs because of such ethical questions as whether and under what conditions one could define death and whether and in what ways one could meddle with the deceased. Hence, the first attempts at clinical transplantation used animals as a source of organs [36]. The first animals used for xenotransplants were pigs and sheep (Table 1.1) [14]. The ethical hurdles to using humans as a source of organs for transplantation did not last very long. The year 1911 brought what may be the first recorded attempt to transplant an organ from one person to another [2].
Cellular Transplantation: From Laboratory to Clinic
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Copyright © 2007, Elsevier Inc. All rights of reproduction in any form reserved.
Jeffrey L. Platt
is more remote. On the other hand, cell transplantation, stem cell transplantation, and tissue engineering seem less promising for replacement of the function of structurally complex organs such as the kidney, lungs, and heart. For replacement of these organs, organogenesis (the de novo formation of organs) or xenotransplantation may be necessary. Xenotransplantation may also find application in conjunction with organogenesis. For example, one might envision “growing” human organs (perhaps derived from stem cells as a xenograft in an animal host) and then transplanting the organs to human patients [7, 24].
TABLE 1.1 Some clinical attempts at xenotransplantation. Year
Donor
Organ
Maximum Survival
1906 1964 1964 1984 1992
Pig Chimpanzee Baboon Baboon Baboon
Kidney Kidney Kidney Kidney Liver
2 days 9 months 60 days 20 days 70 days
the various approaches proposed for overcoming those barriers.
Source of Xenografts Many species have been used as sources of tissues and organs for xenotransplantation. Xenografts from sources phylogenetically closer to the recipient would be expected to provoke less immunity and to be more physiologically compatible with the recipient. Consistent with that idea, experimental cardiac xenografts from monkeys to baboons have survived greater than a year, and renal xenografts from chimpanzees to humans have survived and functioned up to nine months (Table 1.1). However, some biological barriers to xenotransplantation derive from expression of one or very few genes in the donor or recipient. These barriers do not relate directly to overall genetic difference. The genetic barrier of greatest current interest and importance is expression of a1-3 galactosyltransferase, a glycosyltransferase that catalyzes synthesis of Gala13Gal. Gala1-3Gal is a saccharide expressed by lower mammals and New World monkeys, but not by humans and old-world monkeys. Humans and old-world monkeys have “natural” antibodies specific for this saccharide, and these antibodies trigger severe reactions when organs containing Gala1-3Gal are transplanted. Indeed, many of the efforts in genetic engineering and immunosuppression for xenotransplantation are directed respectively at eradicating expression of the sugar or suppressing immunity directed against it. Today, most efforts in xenotransplantation focus on the pig as a potential source of tissues and organs. The most important reason for favoring the pig is that pigs are available in large numbers (it is estimated that more than one million would be needed for transplants each year on a worldwide basis). Another reason for favoring pigs as a source of xenografts is that pigs can be bred and genetically manipulated, as described
The Rationale for Xenotransplantation Before the barriers to xenotransplantation are discussed, one may usefully consider why with the availability of allotransplantation we should pursue xenotransplantation today. The main rationale for xenotransplantation is that animals could potentially provide a limitless source of organs and tissues for transplantation. In addition, the cost of xenotransplantation might be very much lower than the cost of allotransplantation if the latter includes the costs of additional intensive care and clinical organ harvesting. Further, under some circumstances xenotransplantation may be preferred over allotransplantation. Where organ failure is caused by a viral infection (e.g., hepatitis), xenotransplantation might be preferred because the transplant would resist reinfection by the virus that caused organ failure [21, 25, 27]. Xenotransplantation might also be preferred as a way of delivering genes of therapeutic importance [7, 27]. For example, an animal source might be genetically engineered to express a gene at a high level or under regulated conditions. An important consideration today is how to weigh xenotransplantation against other potential approaches to treating organ failure [5]. Although some new technologies (such as stem cells, tissue engineering, and cardiac assist devices) have received much attention, they have also received less scrutiny than xenotransplantation because they are so recent in their development. Most likely, these technologies will be applied in ways that fill therapeutic niches, such as repairing local defects or injury of tissues. Devices may eventually be used to replace the heart, but application for other organs 4
1.
Immunology of Cell and Tissue Xenotransplantation
by genetic engineering, is now considered routine in xenotransplantation. Another reason for this more encouraging view of the prospects for overcoming the immunological barriers to xenotransplantation stems from the fact that the barrier to successful transplantation of xenogeneic cells may be much less daunting than the barrier to successful transplantation of xenogeneic organs.
in material following. Still another reason for favoring the pig is that the organs are large enough to fulfill the needs of full-sized humans, and some strains of pigs (such as the mini-pig) may at maturity approximate human size. Finally, the microorganisms harbored by pigs and potentially infectious for humans are well known, and measures for screening for these organisms are well established. In contrast, some viruses of nonhuman primates are poorly known and potentially lethal to humans. Although the experience is limited, best present evidence would suggest that the heart, lung, and kidneys of the pig would function sufficiently to sustain the life of the human. Whether the liver would function sufficiently is a matter of controversy because of the metabolic complexity of that organ and because of the possibility that complex cascades, such as complementing coagulation, could be incompatible between pig and human.
The Type of Transplant and the Barrier to Xenotransplantation No factor has a greater bearing on the barrier to xenotransplantation than the type of transplant: isolated cells or tissues on the one hand or intact organs on the other. All transplants can arouse immune responses, and by every measure the immune response to cell and tissue grafts may be quite comparable to the immune response to organ grafts. However, the impact of the immune response on cell and tissue grafts is profoundly different than the impact on organ grafts. This difference is summarized in Figure 1.1.
The Immunological Barriers to Xenotransplantation: An Overview Xenotransplantation excites nearly every immune and inflammatory pathway known. Among the inflammatory pathways are complement, coagulation, neutrophils, and natural killer cells. The immunological pathways include both innate and elicited responses by B-cells and T-cells. So intense is the response to xenotransplantation that no approach yet used has allowed the enduring survival of a xenogeneic organ transplant in a human subject, or in an animal for that matter. One might argue cynically that this failure of success drives even more daunting regulatory hurdles (put another way, one doubts that governments would impose such severe regulatory hurdles if xenotransplantation could clearly succeed). However, despite this discouraging picture there are reasons to think that the immunological barriers to xenotransplantation can be overcome. One reason for this more encouraging view of the prospects for overcoming the immunological barriers to xenotransplantation is that in contrast to humanto-human transplantation genetic engineering might be applied to some of the barriers. For example, the author and co-workers suggested more than a decade ago that intrinsic susceptibility of a xenograft to the complement system of the recipient might be overcome by expressing human complement regulatory proteins in the graft [30]. This approach, the addressing of immunological hurdles
The Immune Response to Cell and Tissue Transplants Cell and tissue grafts provoke cell-mediated immune responses and humoral immune responses. Cellmediated immune responses give rise to cellular rejection and possibly to primary nonfunction. Humoral immune responses provide a reliable marker of immunity to the transplant, but as we shall see these responses have little or no impact on the fate of the graft. That is not to say that B-cells are unimportant in cell and tissue transplantation. As we shall discuss, B-cells have functions other than the production that may influence the fate of cell and tissue grafts. Although any antigen carried by the graft and not by the recipient can elicit an immune response, responses to major histocompatibility complex (MHC) antigens are most pronounced and have the greatest impact on the fate of the graft [28]. A complete consideration of the immune response to MHC is quite beyond the scope of this chapter. Here it is pertinent to say that transplants generate cellular and humoral responses to MHCencoded proteins. The cellular responses occur in nearly every instance and reliably and rapidly cause rejection. In fact, so reliable and rapid is rejection that the antigens 5
Jeffrey L. Platt Free tissue or cell transplant
Primary nonfunction failure of neovascularization microenvironment incompatibility
Cellular rejection
(a) Organ transplantation
Acute vascular rejection Cellular rejection
Hyperacute rejection
Chronic rejection
Accommodation
(b) Figure 1.1. The biological outcome of xenografts: impact of mechanism of vascularization. Cell and tissue xenografts derive their blood supply through the in-growth of blood vessels of the recipient. Because the blood vessels of these grafts are constructed from cells of the recipient, antibodies of the recipient do not generally bind to the blood vessels and vascular diseases of organ grafts such as hyperacute and acute vascular rejection are not observed. Rather, the grafts are mainly subject to injury by T-cells that have the ability to migrate effectively through blood vessel walls. (a) Outcome of cell and tissue xenografts. Cell and tissue xenografts are subject to primary nonfunction and cellular rejection. Primary nonfunction, the immediate failure of the newly implanted cells or tissues, may be caused by various factors, including T-cells. (b) Outcome of organ xenografts. Like cell and tissue xenografts, organ xenografts are subject to cellular rejection, and to vascular types of rejection, including hyperacute, acute vascular, and chronic rejection. Vascular rejection, particularly hyperacute and acute vascular rejection, are caused by the binding of antibodies and activation of complement of the recipient on xenogeneic blood vessels. Having blood vessels originating with the recipient, cell and tissue xenografts are not subject to this type of problem.
highly immunogenic. The factors that make an amino acid sequence more or less immunogenic are incompletely understood but include the presence of amino acids at key points that allow a peptide to be loaded onto MHC class I or class II complexes and probably the presence of consensus domains that allow the proteins to be cleaved in ways that will encourage such loading. Beyond these factors, however, is evidence that MHCencoded proteins may have evolved in ways that make them better targets for T-cell receptors. A third reason MHC-encoded proteins are such potent antigens is that when they are presented in grafts the proteins can be recognized in native form by T-cell receptors of the recipient [18]. All other antigens of the graft must be degraded and presented in association with MHC complexes of the recipient. This type of antigen presentation is known as “direct antigen presentation,” and it is thought to be quite potent. Thus, up to 10% of the T-cells in a person’s blood can recognize foreign MHC by direct antigen presentation, whereas only a very much smaller fraction (perhaps less than 0.1% of a person’s T-cells) would recognize peptides from that MHC antigen presently in association with MHC on antigen-presenting cells of the responding individual. This second type of antigen presentation is referred to as indirect antigen presentation.
were named major histocompatibility antigens [8]. The antibodies produced in response to MHC-encoded proteins were used to map the MHC locus. Other antigens (in contrast to MHC antigens) tend to trigger rejection less reliably and more slowly than MHC antigens, and hence these antigens are called minor histocompatibility antigens. However, in a given combination of donor and recipient strains some minor histocompatibility antigens cause rejection just as quickly and reliably as MHC antigens. Responses to MHC antigens are quite vigorous for several reasons. One factor accounting for the importance of MHC antigens is that they are highly polymorphic. Thus, transplants between out-bred individuals are nearly certain to contain at least one MHC antigen not present in the recipient and are thus able to generate an immune response. Most other proteins are quite similar if not identical between different individuals, and hence any other given protein is unlikely to arouse a response. However, at least some protein allotypes will distinguish the donor and recipient, and therefore polymorphism alone (although necessary) does not account for the importance of MHC-encoded proteins as antigens. A second reason MHC-encoded proteins are such potent antigens is that the polymorphic sequences are 6
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The same cannot be said for xenotransplantation. Most or all proteins are immunogenic in xenogeneic species. Therefore, one might predict that the response to xenogeneic proteins would add significantly to the immune response to a xenograft. Consistent with this concept, some have found that T-cells will proliferate to these non-MHC-encoded proteins, whereas they do not proliferate to non-MHC alloantigens. The reason T-cells do not proliferate to non-MHC alloantigens is thought to be that the number of such antigens is so small that the fraction of the T-cell repertoire that can respond is too low to be detected.
It is the usual way in which foreign antigens are recognized [33]. Following cell and tissue transplantation, both direct and indirect pathways of MHC antigen presentation can be used in stimulating responses of the recipient. If the transplant contains cells expressing MHC class II (in mice, MHC class II is expressed mainly on dendritic cells and B-cells; in humans it is expressed on these cells and also on monocytes and endothelial cells), the cell may migrate to regional lymph nodes and there stimulate T-cells of the recipient directly. However, the T-cells activated through this pathway may find relatively few cells in the transplant expressing the allogeneic MHC class II. Of course, most or all parenchymal cells will express allogeneic MHC class I. However, the T-cells of the recipient are usually found near blood vessels and not parenchymal cells — at least during the early days after transplantation — and the blood vessels derive mainly from the recipient (by in-growth). Thus, in cell and tissue grafts the main pathway of antigen presentation leading to rejection may be the indirect pathway. The pathway through which antigen presentation occurs is especially pertinent for xenografts. T-cells respond poorly to direct antigen presentation by xenogeneic antigen-presenting cells [1, 30]. The defective response is owed in part to species’ specificity of adhesion molecules that promote interaction of T-cells and antigen-presenting cells and in part to species specificity of cytokines. Thus, if rejection depended on the response of the direct pathway xenografts might be less susceptible to cellular rejection. Some have thought that these defects might allow xeonografts to be better accepted than allografts and that this susceptibility might be further improved by genetic engineering (to further dissuade interactions of recipient cells with the graft). Sadly, these hopes have not been realized. Cell and tissue xenografts are as susceptible as allografts to cellular rejection. Some differences between xenografts and allografts can still be exploited. Xenografts may depend to an even greater extent on the indirect pathway, and hence therapies that disrupt interaction of T-cells with MHC class II may be especially effective [26]. Still another consideration in the immune response to cell and tissue xenografts may be the possibility that cellular immune response will be generated to multiple proteins of the xenogeneic source. It was previously stated that most proteins of the donor will be the same (or at least not immunogenic) in an allogeneic recipient.
The Impact of Immunity on Cell and Tissue Grafts Cell and tissue grafts are subject mainly to primary nonfunction and cellular rejection. The mechanisms underlying cellular rejection have been considered elsewhere in detail [29] and will not be discussed here except as an index of the immune response.
PRIMARY NONFUNCTION If a graft fails to show evidence of engraftment or function, it is considered to have primary nonfunction. Primary nonfunction may have a variety of causes, and these causes may differ depending on the type of graft and location in which it is placed. For example, engraftment of bone marrow and hepatocytes depends very much on the microenvironment in which the graft is placed. Bone marrow appears to require healthy stromal cells to support hematopoietic stem cells (hepatocytes engraft poorly in the microenvironment of the cirrhotic liver). When cellular grafts are administered into blood vessels such as the portal vein, the grafts may be subject to injury or destruction by antibodies and complement of the recipient. All of these factors may cause failure of engraftment or failure of function. Cell and tissue grafts are also subject to allogeneic resistance caused by the action of natural killer cells of the recipient and manifest as failure of engraftment. Natural killer cells have killer inhibitory receptors that recognize self-MHC and upon doing so prevent natural killer cells from exerting cytotoxicity against cells expressing this MHC. For example, bone marrow from parental strain (homozygous for MHC) may not engraft in F1 (MHC heterozygotes) individuals because natural 7
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by CD4+ T-cells and mediated by macrophages predominates in rejection. Each of these mechanisms finds experimental support. For xenografts, the question of whether cytotoxicity or delayed-type hypersensitivity is of greater importance in causing tissue injury has not been addressed specifically. However, because xenografts usually contain a great many macrophages and seem especially to benefit from treatments aimed at CD4+ T-cells one might anticipate that delayedtype hypersensitivity might be more important than cytotoxicity as a mechanism of rejection. Consistent with this concept is the observation that CD8 binding to MHC class I may be especially impaired across species.
killer cells of the recipient lack killer inhibitory receptors that would suppress responses to the MHC class I missing in the parent. Resistance of this type would presumably be even more pronounced for xenografts because natural killer cells are stimulated by these grafts through multiple pathways [13]. Primary nonfunction may also be caused by T-cells [16]. How T-cells might cause primary nonfunction, however, is not entirely clear. Certainly T-cells respond vigorously and rapidly to allografts and xenografts. However, the kinetics of the best characterized response are seemingly too slow to account for primary nonfunction. The kinetics of the cellular immune response to allogeneic transplants have been thoroughly investigated. Typically, a cellular or tissue graft becomes vascularized over a period of three to five days and for a period of time functions normally. Only after a period of vascularization (often eight days to two weeks after engraftment) is cellular rejection and failure of function observed. This picture of function followed by failure differs from primary nonfunction, in which function is never observed. Two recent observations may help explain how T-cells could cause primary nonfunction. First, contrary to the kinetics of T-cell activation in vitro or to the kinetics of rejection, T-cell activation in lymph nodes occurs within minutes of the introduction of antigen [35]. The T-cells activated most rapidly may be memory T-cells or some other population, the role of which may be more that of helping the activation of T-cells that will contribute to the effector response. However, these firstactivated cells can produce cytokines, and it may be these cytokines that account at least in part for primary nonfunction. Second, T-cells were recently found to act on grafts nonspecifically during the period of reperfusion and doing so to contribute to ischemia-reperfusion injury. Perhaps T-cells might cause primary nonfunction through this nonspecific action.
B-cells, Antibodies, and the Barrier to Cell and Tissue Transplantation Although cell and tissue grafts stimulate a vigorous humoral immune response, antibodies appear to have a limited impact on the fate of cell and tissue grafts. For instance, studying porcine hepatocytes transplanted into rats [22], we found no evidence that humoral immune responses compromised graft function. Similar results have been observed in nonhuman primates. How does humoral immunity have such a profound impact on organ grafts and such a limited impact on cell and tissue grafts? Organ grafts are vascularized by anastomosis of graft blood vessels with the blood vessels of the recipient. Hence, the vascular supply of the organ graft is that of the organ. As the graft is perfused with the blood of the recipient, antibodies in the blood specific for antigens of the graft can bind to endothelium, activate complement, and initiate vascular diseases such as hyperacute and acute vascular rejection and possibly chronic rejection [6, 25, 27]. These diseases of organ grafts — hyperacute and acute vascular rejection and chronic rejection — are diseases of blood vessels that are not observed in cell and tissue grafts. In fact, there exists no evidence that cell and tissue xenografts would benefit from genetic manipulations such as the expression of human complement regulatory proteins or the knocking out of the enzyme that catalyzes synthesis of Galα1-3Gal (the antigen targeted by natural antiswine) that has advanced the transplantation of organ grafts [20, 37]. The reasons vascular diseases (such as hyperacute and acute vascular rejection) are not observed, and expression of human complement regulatory proteins
CELLULAR REJECTION The most important type of rejection to afflict cell and tissue grafts is acute cellular rejection. Of course, cellular rejection depends on recognition of the graft and graft antigens by T-cells. To what extent and in which way(s) tissue damage depends on the function of T-cells are less clear. For allografts, the question is whether cytotoxicity mediated by CD8+ T-cells or a reaction such as delayed-type hypersensitivity initiated 8
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stem cells success may be limited by incompatibility of xenogeneic stem cells with recipient bone marrow stroma and cytokines, and by the action of complement and of natural killer cells. Despite the difficulties in inducing tolerance between species, however, cell and tissue xenografts have achieved at least some measure of success in recipients treated with conventional types of immunosuppression. As two examples, swine hepatocytes have been transplanted into rabbits [12] and into rats [22] with enduring function and survival observed in recipients treated with only cyclosporine or with no immunosuppression. Swine substantia nigra cells have survived for months in human subjects treated only with cyclosporine [9]. Soon to be published are accounts of engrafting of pancreatic islets and hepatocytes for many months in nonhuman primates. In neither case was genetic engineering, either expression of human complement regulatory protein or knocking out of a1,3-galactosyltransferase, needed. Thus, the hurdles to the transplantation of xenogeneic cells and tissues may be far less than might be thought based on the difficulties observed with intact organs. Indeed, from the perspective that can now be suggested the greatest hurdles to successful xenotransplantation of cells and tissues may be finding appropriate clinically advantageous applications and gaining regulatory approval.
or abolished synthesis of Gala1-3Gal is not particularly beneficial in cell and tissue xenografts, are several. First, the diseases are caused by binding of antibodies of the recipient to donor blood vessels (cell and tissue grafts receive their blood supply by in-growth of blood vessels of the recipient). Second, cell and tissue grafts generally reside outside blood vessels, where the concentration of antibodies and complement are considerably lower than the concentrations in the blood. Although antibodies and complement may not contribute importantly to rejection of cell and tissue grafts, B-cells may play an important role. B-cells can specifically capture, concentrate, and present foreign antigens. B-cells also help to generate the lymphoid tissues in which antigens are presented [19, 23]. Finally, B-cells promote diversification of the T-cell repertoire [15, 17]. Given these considerations, it may not be surprising that therapies directed at B-cells may have value for promoting survival of cell and tissue grafts.
Therapeutic Approaches to Cell and Tissue Transplantation Although some important aspects of the cellular immune response to cell and tissue xenotransplantation are understood, the most important practical question — whether cell and tissue xenotransplants can be sustained by administration of available immunosuppressive drugs — is still not clear. Although in vitro studies may suggest that immune response to xenografts may be weaker than the immune response to allografts, cell and tissue xenografts are clearly subject to cellular rejection, and the kinetics of rejection of cell and tissue xenografts in untreated recipients are at least as rapid as the kinetics of rejection of allografts. Some have proposed that the cellular immune response to xenografts may be so strong that success may depend on the induction of immunological tolerance [32]. Unfortunately, the induction of an enduring state of tolerance between widely disparate species, particularly for swine transplants in primates, has not been accomplished. In part, the failure to achieve immunological tolerance between disparate species reflects the enormously difficult hurdle to achieving durable engraftment of hematopoietic stem cells [3, 4]. The difficulty in achieving tolerance between species, at least as it is pursued today, may reflect in part on mechanisms underlying primary nonfunction. Thus, if tolerance is sought by engraftment of hematopoietic
Acknowledgments Work in the author’s laboratory described in this chapter has been supported by grants from the National Institutes of Health (HL46810 and HL52297).
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35. Stefanova, I., Dorfman, J. R., Germain, R. N. Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes. Nature 420:429–434; 2002. 36. Ullman, E. Tissue and organ transplantation. Annals of Surgery 60:195–219; 1914. 37. Yamada, K., Yazawa, K., Shimizu, A., Iwanaga, T., Hisashi, Y., Nuhn, M., O’Malley, P., Nobori, S., Vagefi, P. A., Patience C.,
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2 Current Immunosuppressive Drugs and Clinical Use Harrison S. Pollinger, D.O.[1] Justin M. Burns, M.D.[2] Vince P. Casingal, M.D.[2] Paul F. Gores, M.D.[2] [1]Division of Transplantation Surgery, Mayo Clinic College of Medicine, Rochester, Minnesota. [2]Division of Transplantation Surgery, Carolinas Medical Center, Charlotte, North Carolina
The clinical application of cell transplantation is still in its infancy. However, the immunosuppressive drugs that make allotransplantation possible have been in use for over 50 years. During the latter half of the twentieth century, organ trasplantation evolved from an experimental technique performed in the animal laboratory to a mature clinical discipline affording improved health and a better quality of life for thousands of individuals. The fulcrum underpinning this successful transformation was the development of small molecule immunosuppressive agents which modulate the immune response in a non-antigen specific manner. Each of these molecules, aside from their immunosuppressive qualities, is associated with significant toxicities which precludes their use in high enough doses to provide effective prophylaxis as monotherapy against rejection. Fortunately, these drugs differ with respect to the way in which they interfere with the immune response. Thus it has been possible to implement combination therapies; minimizing drug specific side effects while taking advantage of synergies to potentiate suppression of the rejection response by the immune system. More recently biologic agents have been introduced which offer the promise of providing more specific immunomodulation. Refinement of these strategies will be essential for the continued development of cell transplantation which, in general, is a less invasive form of therapy than is solid organ transplantation.
Cellular Transplantation: From Laboratory to Clinic
The Alloimmune Response An appreciation of the host response to donor antigen is essential for understanding the mechanism of action and rationale of combination therapies. Rejection of organ and tissue allografts is dependent on the generation of cytotoxic T-lymphocytes. Effective T-lymphocyte activation requires the delivery of two signals. The first signal is provided by engagement of the T-cell receptor with donor antigen presented in context with major histocompatibility molecules on antigenpresenting cells (APC) of either host or donor origin. The second (co-stimulatory) signal is delivered by the interaction of co-stimulatory ligands of the B7 family on the APC. B7-1 (CD80) and B7-2 (CD86) bind with CD28 expressed on T-cells. When two signals are delivered, activation triggers three signal transduction pathways: (1) the calcium-calcineurin pathway, (2) the RAS-mitogen-activated protein (MAP) kinase pathway, and (3) the nuclear factor-κβ pathway. Initiation of these pathways leads to the generation of numerous cytokines that in turn leads to activation of the “target of rapamycin” and cell proliferation. Conversely, in the absence of this second signal the T-cell becomes anergic [59]. The spectrum of immunosuppressive agents currently utilized includes small-molecule drugs, glucocorticoids, depleting and nondepleting protein drugs (polyclonal
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and monoclonal antibodies), and fusion proteins [43]. These drugs deplete lymphocyte populations, divert or manipulate lymphocyte function, or block lymphocyte end products. Small-molecule immunosuppressive agents target intracellular proteins and include azathioprine, cyclosporine, tacrolimus, sirolimus, everolimus, and mycophenolate mofetil6 [43].
Azathioprine Azathioprine (Imuran) is an antimetabolite derived from the imidazole derivative 6-mercaptopurine. This agent was responsible for the initial advancement of solid organ transplantation more than 30 years ago [31]. Gertrude Elion and George Hitchings are credited with its development, for which they were awarded the Nobel Prize in 1988. However, since the introduction of cyclosporine in the early 1990s azathioprine has been relegated to the role of an adjunctive second-line drug. This purine analogue is incorporated into cellular deoxyribonucleic acid (DNA), where it functions to inhibit purine nucleotide synthesis and ultimately the synthesis of ribonucleic acid (RNA). This translates into the suppression of T-cell replication and activation. Unfortunately, azathioprine is not specific to T-cells. It is broadly myelosuppressive and is associated with a doserelated, generally reversible, depression of bone marrow function usually manifested as leukopenia and thrombocytopenia. It is an uncommon cause of anemia but can in rare cases lead to agranulocytosis, pancytopenia, and aplastic anemia. Azathioprine can be administered either orally or intravenously. Half the orally administered dose is absorbed. Serum levels are not of importance clinically because its efficacy is not concentration dependent. The kidney does not excrete the drug. Neither is the drug dialyzable. The daily oral dose is 2 to 3 mg/kg when employed as a primary immunosuppressive agent and 1 to 2 mg/kg when used as an adjunctive agent in combination with a calcineurin inhibitor. Azathioprine administration is associated with a major drug interaction. It is converted to its inactive form, 6-thiouric acid, by xanthine oxidase. Therefore, allopurinol (a powerful xanthine oxidase inhibitor) should be avoided or given with extreme care. The dose of azathioprine should be reduced by 50% and the white blood cell and platelet counts monitored more frequently when allopurinol is prescribed.
Cyclosporine Cyclosporine belongs to a family of compounds known as calcineurin inhibitors, which currently serve as the backbone of solid organ immunosuppression. Cyclosporine is a small 11-amino-acid cyclic polypeptide derived from the fungus Tolypocladium inflatum [13], which binds to cyclophilin, an intracellular protein of the immunophilin family. The resulting complex targets the protein phosphatase calcineurin, which is required for the translocation of an activation factor (NF-ATc) from the cytosol to the nucleus [17]. In the presence of a calcineurin inhibitor, the cytosolic activation factor is unable to reach the nucleus and the transcription of interleukin-2 (IL-2) and other cytokines is strongly inhibited, resulting in a significant reduction in lymphocyte activation and proliferation. The advantage of calcineurin inhibitors is the selective inhibition of the immune response. They do not hinder neutrophil function, as do corticosteroids, nor are they myelosuppressive. Patients receiving cyclosporine-based immunosuppression still possess adequate immune responsiveness to maintain host defense. This can be attributed to the fact that calcineurin inhibitors only inhibit approximately 50% of total calcineurin activity, thus enabling immune response to strong triggers of cytokine expression. The original oil-based formulation of cyclosporine (Sandimmune) has been largely replaced by a microemulsion formulation (Neoral). The new microemulsion combines a lipophilic solvent, hydrophilic solvent, and surfactant, and is available in both liquid and gel cap forms [80]. The absorption of Neoral occurs in the proximal gastrointestinal (GI) tract at a constant rate, independent of drug concentration at the absorption site. Local metabolism of cyclosporine occurs immediately in the intestinal epithelium, via the cytochrome P450-IIIa system, reducing the amount of prodrug available for uptake [52]. The remaining compound is absorbed via the portal system, with very little drug transported through lymphatics [33]. The absorption of Sandimmune from the GI tract is bile dependent and is unreliable when compared to Neoral [33]. Peak concentration in the blood is achieved in 3 to 4 hours, with only 30% of the drug reaching systemic circulation. In contrast, the absorption of Neoral is more rapid, complete, and consistent due to the more homogenous dispersion of the uniform particles of the suspension [69]. The trough concentration of Neoral does not differ significantly from Sandimmune. However, the
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microemulsion possesses a maximum absorption of 60% and has an overall bioavailability 50% greater than the oil-based formulation [53]. Patients experiencing gastroparesis, cholestasis, biliary diversion, malabsorption, and diarrhea will all have variability with respect to the absorption of cyclosporine, especially the oil-based formulation [39, 58, 73]. Regardless of the cyclosporine formulation used, 50% is bound to erythrocytes, 10% to leukocytes, and 30 to 40% to plasma proteins. Only 1 to 5% exists in the free state [57]. Up to 46% of cyclosporine is associated with high-density lipoprotein, 35% with low-density lipoproteins, and 20% with very low-density lipoproteins in plasma. The close association of cyclosporine with the low-density lipoprotein receptor may account for the hyperlipidemia often experienced with its use [84]. Cyclosporine readily accumulates in fat, liver, pancreas, heart, lung, kidney, spleen, lymph nodes, and blood. Drug concentrations remain low in the cerebrospinal fluid and central nervous system, and very small amounts can be detected in the fetal circulation [72]. The liver and bowel epithelia are the primary sites for cyclosporine metabolism. This process is under the influence of the cytochrome P450-IIIA system via demethylation [54]. More than 25 active metabolites of cyclosporine have been isolated in human blood, bile, and urine. All of these retain the functional cyclic oligopeptide structure, and all are less immunosuppressive than the parent compound [48]. Cyclosporine is eliminated by biliary excretion, with a half-life of 6 to 8 hours. Clearance is significantly decreased in patients with hepatic impairment, thus mandating longer dosing intervals and/or reduction in the dose. Any agent that alters cytochrome P450 enzyme activity will affect the metabolism and clearance of cyclosporine [103] (Table 2.1). Phenytoin and rifampin are two classic drugs that induce mixed-function oxidases of the cytochrome P450 system and cause a major fall in cyclosporine levels. This may lead to a rejection episode unless the interaction is recognized in a timely manner. Inhibitors of the cyctochrome P450 system will increase cyclosporine levels and may result in acute nephrotoxicity. Examples of cytochrome P450 inhibitors are erythromycin, ketoconazole, diltiazem, colchicine, and the fluoroquinolones [61]. The most significant and worrisome side effect of cyclosporine is nephrotoxicity. This is of critical importance with respect to renal transplant recipients because it can be difficult to distinguish cyclosporine nephrotoxicity from rejection. The toxic effects to the kidney can be
TABLE 2.1 Drug interactions with cyclosporine. Agent
Pharmacokinetic Action
Consequences
Carbamazepine Nafcillin Phenobarbital Phenytoin Rifampin
Induce cytochrome P450 enzymes
Decrease half-life blood levels, and immunosuppressive effect
Colchicine Diltiazem Fluconazole Fluoroquinolones Ketoconazole Macrolide antibiotics Oral contraceptives Verapamil
Inhibit cytochrome P450 enzymes
Increase half-life, blood levels, and toxicity or immunosuppression
Aminoglycosides Amphotericin B Cimetidine Nonsteroidals Sulfur
Interact at a glomerular or tubular level
Increase nephrotoxicity
seen early after transplantation, as the ischemic kidney is particularly susceptible to the deleterious effects of cyclosporine. There is an increased incidence of delayed renal allograft function when cyclosporine is used. After the kidney has recovered and baseline renal allograft function has been reached, high cyclosporine serum levels can lead to an increase in serum creatinine that needs to be distinguished from an acute allograft rejection episode. Often a percutaneous biopsy of the graft is required [3]. Microvascular thrombosis is another uncommon phenomenon associated with cyclosporine. These lesions mimic hemolytic-uremic syndrome and can be completely reversible if diagnosed early with the subsequent reduction in the cyclosporine level [82]. Chronic cyclosporine toxicity is associated with a slow decline of renal function and may be difficult to distinguish from chronic rejection. The major finding on biopsy is interstitial fibrosis. This chronic toxicity is not limited to renal allografts but is also seen in the native kidneys of recipients of a nonrenal organ [79]. The mechanism of cyclosporine nephrotoxicity has not been completely elucidated. It has been hypothesized that injury results from decreased renal blood
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flow with increased renal vascular resistance. The site of major insult is the afferent arteriole of the glomerular apparatus [83]. An alternative mechanism involves the decreased production of prostaglandins in the local environment of the kidney associated with cyclosporine use. This mechanism could also explain the functional and histological changes seen with the chronic use of cyclosporine [75]. Cyclosporine has been associated with a number of side effects as well. Hypertrichosis and gingival hyperplasia are cosmetically distressing to patients. These are both enhanced when cyclosporine is used in conjunction with calcium channel blockers. Cyclosporine is also linked with neurological, endocrine, and hepatic toxicity. Neurologic toxicities include fine motor tremor, headache, seizures, and a demyelinating neuropathic syndrome. Hepatic toxicities seem to correlate with elevated cyclosporine levels and are manifested by elevation of hepatic transaminase enzymes. Hypertension is a major adverse effect associated with cyclosporine-based immunosuppression. Eightyfive percent of renal transplant recipients maintained on cyclosporine require antihypertension medication [22]. The mechanism appears to be multifactorial, but can be attributed to (1) increased intracellular calcium concentrations sensitizing resistance vessels, (2) increased sodium and water retention, and (3) increased sensitivity to adrenergic stimulation.
concentrations do not need to be modified in the face of renal dysfunction. Tacrolimus resembles cyclosporine in that it is nephrotoxic and can induce hemolytic-uremic syndrome, but it is less likely to cause hyperlipidemia, hypertension, or cosmetic side effects. Tacrolimus has been associated with increased rates of posttransplantation diabetes and is more likely to induce BK-related polyomavirus nephropathy [64]. The common adverse effects of tacrolimus are listed in Table 2.2 and a side effect comparison of the calcineurin inhibitors is outlined in Table 2.3. New onset diabetes mellitus occurs significantly more often with tacrolimus than with cyclosporine, as demonstrated by both U.S. and European trials [2, 99]. These studies also demonstrated an increased incidence of neurologic sequelae (convulsions, confusion, psychosis, encephalopathy, and coma) with tacrolimus, but these events tended to be mild. TABLE 2.2 Common adverse events with tacrolimus. Gastrointestinal
Metabolic
Nephrotoxicity Neurologic
Anorexia Diarrhea Dyspepsia Nausea
Hyperglycemia Decrease in Hyperkalemia creatinine clearance
Tacrolimus Tacrolimus (FK506) is a metabolite of the fungus Streptomyces tsukubaensis. It is a macrolide antibiotic with a mechanism of action very similar to that of cyclosporine, but tacrolimus inhibits cytokine production with a potency 100 times that of cyclosporine [90]. Tacrolimus is available in an intravenous formulation and as an enteric capsule. Gastrointestinal absorption is independent of bile metabolism and occurs primarily in the small intestine. Oral bioavailability approaches 25% and is relatively consistent. Subsequently, it is rarely necessary to administer tacrolimus parenterally. Onethird of absorbed tacrolimus is bound to lipoproteins, and the remainder of the drug is bound to erythrocytes in serum. Tacrolimus differs from cyclosporine in that it is not significantly bound to lipoproteins and thus has less of an adverse effect on cholesterol metabolism [92, 93]. Metabolites are excreted in bile, with virtually no renal excretion. Therefore, drug
Confusion Encephalopathy Headache Paresthesia Psychosis Seizure Tremor
TABLE 2.3 Nonimmunologic side effect profile of tacrolimus versus cyclosporine. Parameter
Tacrolimus
Cyclosporine
Nephrotoxicity
++
++
Neurotoxicity
+
+
Diabetogenicity
++
+
Growth effects Hirsutism Gingival hyperplasia Facial brutalization Hepatotrophic effects Gynecomastia
0 0 0 + + ++ 0
+++ ++ + +++ +
Other metabolic effects Cholesterol increase Uric acid increase
0 +
++ ++
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There have been many studies over the past decade designed to examine the effects of tacrolimus in renal transplant recipients. The European Tacrolimus Multicenter Renal Study Group trial reported that the graft survival for tacrolimus versus cyclosporine was the same. However, the incidence of histologically confirmed acute rejection was lower for the tacrolimus group (25.9% versus 45.7%, p < 0.001) [63]. The complication rates encountered during the trial were similar, but each agent had a higher incidence of specific adverse events. Nephrotoxicity, tremor, diarrhea, hyperglycemia, and angina pectoris were higher in the tacrolimus group, whereas gingival hyperplasia, hirsutism, and arrhythmia were more common with cyclosporine [63]. The U.S. renal trial demonstrated a significantly higher number of patients requiring insulin therapy one year posttransplant if they were maintained on tacrolimus. However, 50% of those patients were reported as being off insulin therapy as of two years posttransplant [46]. The development of diabetes was dependent on the dose of tacrolimus and corticosteroids used to maintain graft function. Lower doses (0.18 mg/kg versus 0.3 mg/kg) were associated with a lower incidence of diabetes (10%) [30].
Sirolimus Sirolimus (rapamycin) is a macrolide triene antibiotic produced by Streptomyces hygroscopicus. It is structurally related to tacrolimus, but differs because of its unique triene ring [32]. This compound was isolated from soil found on Rapa Nui (Easter Island) in 1968. The immunosuppressive properties of sirolimus were first discovered by Suren Sehgal and co-workers in 1972 [85]. Similar to cyclosporine and tacrolimus, sirolimus is also an immunophilin binding drug. Sirolimus differs from the calcineurin inhibitors in that it does not inhibit the phosphatase calcineurin nor does it inhibit cytokine transcription. Rather, sirolimus binds FK binding proteins, which form a unique ligand complex with an enzyme termed the target of rapamycin (TOR). TOR is a protein kinase vital to the regulation of cell growth factor receptors and cell proliferation. The cumulative effect of sirolimus is inhibition of these protein kinases, which then interfere with the progression of T-cells from the G1 to S phase of the cell cycle [68]. Sirolimus is rapidly absorbed from the GI tract, reaching peak serum concentration within 2 hours. Sirolimus
has an extremely long half-life (mean 62 hours). Most of the drug is found within whole blood and tissue with less than 3% distributed within plasma [104]. It is largely metabolized by the hepatic cytochrome P450-IIIA4 system and does not require dose adjustment for renal impairment. Sirolimus and the calcineurin inhibitors are metabolized by the same enzyme systems and therefore significant drug interactions can be encountered. Cyclosporine can raise the blood level of sirolimus by as much as 80% when administered concomitantly. It is recommended that sirolimus be administered at least 4 hours after cyclosporine to limit this interaction. The side effect profile of sirolimus has a spectrum best characterized by its metabolic, hematologic, and dermatologic derangements. Most adverse effects are directly related to the inhibition of growth factors [67]. Increased low-density lipoproteins and serum triglycerides are the principal metabolic side effect. All blood elements are also affected, with anemia, neutropenia, and thrombocytopenia being the most common. In a double-blind randomized trial thirty renal transplant patients were divided into three groups to better understand the side effect profile of sirolimus [70]. All patients had stable renal function maintained on a cyclosporine/steroid regimen and were randomized to low-dose (1 to 3 mg/m2 /day), medium-dose (5 to 6 mg/m2 /day), or high-dose (7 to 13 mg/m2 /day) sirolimus therapy. The major toxic side effects were seen in the high-dose group and were predominantly hypertriglyceridemia and thrombocytopenia [70]. A deleterious effect unique to sirolimus therapy is delayed wound healing. Several reports in the literature correlate sirolimus usage with increased rates of wound complications, including fascial dehiscence [38]. Proposed mechanisms linking sirolimus to impaired wound healing include drastic reductions in the pro-inflammatory cytokines plateletderived growth factor (PDGF) and basic fibroblast growth factor (bFGF) [66]. Another postulated complication with sirolimus is an association with hepatic artery thrombosis, although this is controversial [28]. Several multicenter trials have been performed documenting the efficacy of sirolimus in combination with other agents in preventing acute rejection. Sirolimus, when used in combination with cyclosporine and prednisone, reduced rejection rates to 7% from nearly 40% when cyclosporine and prednisone were used alone [37]. The safety profile of sirolimus compared with cyclosporine was favorable, with a decreased incidence of tremor, hirsutism, and renal dysfunction but an increased incidence in aberrations of cholesterol
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metabolism [37]. There are several other trials currently underway designed to investigate the risks and benefits of sirolimus.
Mycophenolate Mofetil Mycophenolate acid (MPA) is a fermentation product of Penicillium brevicompactum and related fungi. It was found to inhibit inosine 5 -monophosphate dehydrogenase (IMPDH) and to decrease the proliferation of some cancer cells. The mofetil moiety was added later to improve its bioavailability. Interest in its mechanism of action derived from research with inborn errors of metabolism and the finding that deficiency in enzymes involved with de novo purine synthesis resulted in profound immunodeficiency [7]. Allison and colleagues discovered that lymphocytes are especially dependent on de novo purine synthesis [8]. Essentially functioning as an antimetabolite, mycophenolate mofetil (MMF) exerts its immunosuppressant effects by selectively inhibiting the formation of guanosine and adenosine, two purine nucleosides. More specifically, the conversion of inosine monophosphate into guanosine monophosphate is blocked by MPA, which depletes lymphocytes of DNA. The ability of other cells to implement the salvage pathway for purine synthesis allows MMF to target the immune system specifically. MMF has also been shown to have several secondary characteristics that contribute to its immunosuppressive properties. By depleting nucleotide pools, MMF decreases the production of several adhesion molecules (integrins and selectins), which are crucial to the inflammatory response [9]. Although the major effects of MMF are on lymphocytes, it also significantly depletes guanosine triphosphate (GTP) levels in all mononuclear cells — effectively decreasing the levels of circulating cytokines, including IL-2 and tumor necrosis factor (TNF). Allison et al. have shown that MMF can also inhibit antibody production in vitro [6]. His group concluded that MMF can inhibit primary humoral responses in vivo, but does not effectively inhibit secondary antibody responses. MMF is available for clinical use in both oral and intravenous formulations, and is generally well tolerated. The intravenous formulation is rarely needed. Oral formulations are available in both 250-mg and 500-mg capsules. The standard dose is 1 twice daily. The bioavailability of MMF is 90%, with a half-life of 12 hours. It is readily absorbed from the GI tract
and hydrolyzed to MPA in the liver. The most common side effects associated with MMF involve the GI tract. Diarrhea has been reported in up to 1/3 of patients, with nausea, dyspepsia, vomiting, and dysphagia occurring in up to 20% of patients. Most of the GI-related adverse events resolve with reduction of the daily dose. Although the action of MMF is specific to lymphocytes, the incidence of anemia, leukopenia, and thrombocytopenia is similar to that of azathioprine. The incidence of posttransplant lymphoproliferative disorder (PTLD) and opportunistic infections is similar to that of other immunosuppressive agents. Epstein-Barr virus (EBV) infection (primary or reactivated) is a major risk factor associated with development of PTLD. The European MMF trial reported a slightly increased incidence of PTLD with patients treated in the MMF group compared to patients receiving azathioprine or placebo, with an overall frequency of less than 2%. In addition, there is a slight increase in the incidence of tissue invasive cytomegalovirus (CMV) infection in patients treated with MMF, especially in patients who receive more than 3 g/day. However, this observation could be related to more frequent endoscopic surveillance in patients who received higher doses of MMF [1]. Most adverse effects of MMF are self-limited or will resolve with a reduction in dose. Only 113 of 910 (12.4%) patients enrolled in renal transplant trials examining the effects of MMF discontinued the drug because of symptoms attributable to MMF [1, 50, 88, 89]. Unlike sirolimus and the calcineurin inhibitors, MMF is not metabolized by the cytochrome P450 system, resulting in fewer significant drug interactions. MMF should be administered with caution with sirolimus and tacrolimus because both of these medications have been shown to elevate the trough level of MMF [86]. The primary goal of adding MMF to maintenance immunosuppression regimens is to decrease the incidence of acute allograft rejection. A secondary objective is to minimize the need for more toxic agents, while not precipitating increased rates of rejection. Most of the data on MMF comes from experience in renal transplantation looking at combination therapy of MMF/cyclosporine versus azathioprine/cyclosporine. The U.S. and European trials had very defined end points: biopsy-proven rejection, incidence of graft loss, and patient death [1, 88, 89]. The incidence of graft loss was significantly reduced in the MMF group when compared to the azathioprine group (1.8% versus 8.6%) [88]. Biopsy-proven rejection occurred in 79 patients (47.6%) in the azathioprine group and in
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52 patients (31.3%) in the MMF group [88]. Analysis of the data of all three renal transplant MMF trials confirmed that MMF at 2 or 3 g/day was superior to azathioprine or placebo, with an absolute reduction in the incidence of graft loss due to acute rejection of 50% [42]. This finding has profound implications, in that acute rejection episodes act as a surrogate for long-term graft survival [62]. However, despite showing significantly lower rates of acute rejection and graft loss these trials were unable to show a difference in patient or graft survival at 1 year with combination therapy of MMF/cyclosporine versus azathioprine/cyclosporine. MMF has had a major impact on solid organ transplantation. None of the long-term trials with MMF have demonstrated significant end organ toxicity, and its use has been definitively associated with decreased rates of acute rejection. Future studies will be required to determine its long-term benefits and to see if it has any effect on chronic allograft nephropathy.
Corticosteroids Corticosteroids have a profound anti-inflammatory effect and historically have formed the mainstay of immunosuppressive therapy for transplantation. However, they are associated with severe and debilitating side effects with long-term use, and with the advent of more specific immunosuppressant agents interest has grown in steroid-sparing protocols. Steroids have an effect on all mammalian tissue, which is a reflection of the fact that all cells have glucocorticoid receptors within their cytoplasm [21]. Corticosteroids have been shown to block Ca2+ ionophore-induced lymphocyte proliferation by a mechanism different from that of the calcineurin inhibitors, and have been shown to inhibit the migration of mononuclear cells from the intravascular compartment into the tissue [65]. Once within the cell membrane, steroids bind to cytoplasmic receptors and travel to the nucleus, where the complex binds to DNA sequences referred to as glucocorticoid response elements (GREs). GREs are promotor regions essential to the transcription of many cytokine genes. As a result of this interaction, all stages of T-cell activation and proliferation are inhibited. There are many adverse effects of long-term corticosteroid usage. Severe growth retardation is seen in children, and all phases of wound healing are inhibited. Steroids are associated with increased risk of avascular
necrosis (AVN) and osteoporosis. The incidence of AVN is as high as 10% in transplant patients treated with highdose protocols, and likely 1 to 2% in patients receiving low-dose regimens [101]. The AVN region most commonly seen in transplant patients involves the hips and is frequently bilateral. Eventually, most patients diagnosed with steroid-associated AVN require total hip replacement. Osteoporosis is accelerated after transplantation and can lead to pathologic fractures [45]. Diabetes is another consequence of chronic steroid use. Up to 60% of transplant patients will develop glycosuria in the first 6 months following transplantation. Of these patients, only 5 to 10% will require chronic insulin therapy [24]. Cushingoid facies and truncal obesity are common with high-dose steroid regimens. The severity of these lesions is variable and dose dependent. Peptic ulceration, colonic perforation, pancreatitis, hypertension, and psychiatric disturbances are all more frequent, especially in the early posttransplant period when steroid doses tend to be higher. Infections continue to plague transplant patients, especially those on high-dose steroid protocols. The incidence of infections in patients on “conventional” low-dose regimens is less than that found with high-dose regimens [23]. Prednisolone and prednisone (11-keto metabolite) are the most commonly used oral preparations in clinical transplantation. Methylprednisolone is the most commonly used intravenous form. Prednisone and prednisolone are rapidly absorbed from the GI tract. Peak plasma concentrations usually occur within 1 to 3 hours of ingestion [56]. Prednisone is metabolized to prednisolone by the liver. Once within the blood, prednisolone is bound to both cortisol-binding globulin and albumin. Because only the unbound fraction is available to bind to the intracytoplasmic receptors, hypoalbuminemia is associated with greater bioavailability and an increased incidence of steroid-related complications [56]. The half-life of prednisone is 60 minutes, and that of prednisolone is 200 minutes. Glucocorticoids are completely metabolized by the hepatic microsomal system. Therefore, drugs that affect hepatic metabolism will directly affect the circulating half-life of steroids [25]. One area in which steroids still have a firm indication is the treatment of acute rejection. Despite the introduction of the powerful antilymphocyte globulins and anti-T-cell monoclonal antibodies as antirejection therapy, steroids continue to be a first-line modality. For most cases, if diagnosed early acute rejection can be
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reversed with intravenous or oral prednisolone therapy [94]. The risk of serious complications increases with each course of steroid therapy, and those cases resistant to steroids should be treated with polyclonal or monoclonal anti-T-cell globulins. Regardless of how the role of steroids evolves in the management of acute rejection, they may play a more pronounced role in the prevention of chronic rejection. Support of this hypothesis stems from studies reporting an increased rate of sclerosing arteriopathy and interstitial fibrosis in patients in which steroids were withdrawn compared to the steroid-treated controls [78]. Thus, preventing chronic rejection may involve not only the degree of immunosuppression but more specifically the agent employed and the innate ability of that agent to block the inflammatory response.
Protein Drugs In 1960, polyclonal antilymphocyte sera was introduced for both the prevention and treatment of acute allograft rejection [91]. This event was novel at the time because unlike any of the chemical agents used previously these biologic agents were specific and selective for immune cells. The development of hybridoma technology in the early 1970s permitted large volumes of monoclonal antibody (MAb) to be produced against T-lymphocyte receptors and other cell membrane determinants [51]. This new field of biologic immunopharmacology has allowed for the targeting of specific cell surface markers. Moreover, newer agents are even being targeted against distinct cell protein products (cytokines). The first MAb preparation to be used clinically was OKT3, derived from a mouse source. It is reactive against the T-cell receptor (TCR) [19]. These biologic agents possess distinct characteristics from conventional immunosuppressive agents. They have been shown under certain conditions to promote a state of immunologic unresponsiveness that is long lasting despite the absence of immunosuppression [100]. Several animal model experiments over the last two decades [11, 40] have been successful in inducing immunological unresponsiveness with anti-T-cell monoclonal antibodies.
Polyclonal Antilymphocyte Sera Antithymocyte globulin is produced by immunizing either horses (Atgam) or rabbits (Thymoglobulin) with
human thymocytes. Thymocytes are either obtained from cadaveric sources or procured from the thymus gland of patients undergoing open cardiac surgery. The preparation process of the polyclonal sera involves immunizing the host animal with the selected immunogen, collecting the serum, and removing unwanted antibodies. Despite the extensive preparation process, more than 95% of the final product contains nonspecific rabbit or horse immunoglobulins. It has been estimated that less than 2% of the antibody contained within the antisera is specific for human T-lymphocytes [87]. The antibody specificity of polyclonal sera is highly variable. Most consistently there are antibodies detected against CD-2, CD-3, CD-4, CD-28, and the TCR [12]. The exact mechanism of action by which these preparations prolong graft survival is not known. Absolute lymphopenia is not a prerequisite for effective immunosuppression, although profound lymphopenia is sometimes observed after the initial injections with these agents. The lymphopenia is induced by complementmediated lysis of lymphocytes and their uptake by the reticuloendothelial system after the lymphocytes have been opsonized. The proliferation of circulating T-cells is impaired and may be due to a direct effect as well as to the induction of nonspecific suppressor T-lymphocytes [60]. Polyclonal preparations have been used as induction agents and to treat acute rejection when it occurs. In animal models, they have been employed in preconditioning protocols to induce donor-specific tolerance. Long-term renal allograft survival without long-term immunosuppression has been achieved in a nonhuman primate model with an anti-lymphocyte globulin regimen. This protocol incorporated a posttransplant course of polyclonal serum and donor bone marrow infusion to the recipient, and no further immunosuppression after posttransplant day 5 [95]. This data has provided the rationale for several human tolerance trials utilizing similar protocols. Results have been mixed, allowing steroid withdrawal in some patients in the bone marrow group. However, no attempt was made to withdraw all immunosuppression, as in the nonhuman trial [10]. The route of administration of polyclonal antibody preparations is usually intravenously through a large central vessel or arteriovenous fistula because peripheral administration can result in a painful chemical phlebitis. Polyclonal antiserum must be infused slowly over several hours. If infused too rapidly, it may result in systemic hypotension. The dose depends on the source of the preparation. The typical dose for
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the rabbit-derived preparation is 1.5 mg/kg/day, and 15 mg/kg/day for the equine-derived product. The total duration of therapy varies but typically ranges from 5 to 14 days. The degree of immunosuppression can vary widely between different preparations, as well as between different patients who receive the identical preparation. Attempts to follow the degree of immunosuppression have been made by following the absolute T-cell counts in peripheral blood. This has been helpful in predicting the likelihood of rejection resolution. Delmonico et al. reported that depression of the CD-3+ cell number to less than 10% of pretreatment values correlates with clinical recovery from rejection [26]. It must be noted that after a course of therapy with antilymphocyte serum antibodies to the preparation can be detected in many patients. If these antibodies are present in a high enough titer, they can neutralize the beneficial effects of the drug. Adverse reactions vary in incidence and severity depending on the preparation used. The most common reaction reported is fever and chills, which can occur up to 80% of the time with the first infusion. This so-called “first-dose” response is likely due to lymphocytolysis and cytokine release [16]. To help limit this phenomenon, patients can be premedicated with antihistamines, antipyretics, and steroids. Fifty percent of patients will experience thrombocytopenia and/or anemia during a course of antilymphocyte therapy, which is almost always dose related. One major complication that has been linked to over-immunosuppression with these agents is PTLD. Gaber et al. reported no clinically significant differences with adverse events between Thymoglobulin and Atgam [34]. Malignancies were reported in three patients who received Thymoglobulin and three patients who received Atgam during the one-year follow-up period. These included two cases of PTLD in each group. To date, polyclonal antilymphocyte preparations maintain a vital role in most immunosuppressive regimens both as induction agents and for the treatment of acute rejection.
Monoclonal Antibody Preparations ANTI-CD-3 Muromonab-CD3 (OKT-3) was the first monoclonal antibody approved (in 1986) for human use by the Food
and Drug Administration (FDA) [100]. Initially, their sole indication was to treat acute rejection, but monoclonal antibodies are now used to both treat and prevent acute rejection in solid organ transplantation. The biologic target of OKT-3 is the e chain of CD-3, which is closely associated with T-cell receptors on T-cells. Once bound, these antibodies prevent activation of CD-4 and CD-8 T-cells. The recommended adult dosage of OKT-3 is 5 mg/kg/day. The usual course of therapy is 7 to 10 days, but is dependent on whether the agent is being used as an induction agent or to treat steroid-resistant rejection. One advantage of OKT-3 is that it can be administered via a peripheral vein. However, it does require filtration prior to administration to remove particulate matter in the preparation. As with the polyclonal agents, antihistamines, acetaminophen, and steroids should be administered concomitantly to help reduce the incidence of the “first-dose” response. Although OKT-3 remains the prototype monoclonal antibody to which all others are compared, it does possess some innate limitations. Two of the most significant shortcomings are the cytokine release phenomenon associated with the first few infusions and the nonspecific T-cell depletion that can result in over-immunosuppression, leading to the increased risk of infection and PTLD (Table 2.4) [29]. Several multicenter trials have confirmed the efficacy of OKT-3 as an induction agent. When comparing triple therapy with cyclosporine, azathioprine, and steroids with and without OKT-3, there was a significant delay in time to the first rejection episode, decreased incidence of rejection, and fewer instances of rejection in the OKT-3 treatment group [77]. OKT-3 has also proven efficacious as a treatment alternative for acute rejection. The initial trial examining OKT-3 as a treatment for allograft rejection was conducted in Boston in the early 1980s, in the era of azathioprine and steroids [19]. This pilot trial concluded that renal allograft rejection could be reversed in the majority of recipients with a relatively small dose of OKT-3 (1 to 2 mg/kg/day). This smaller trial led to a multicenter randomized trial that demonstrated that OKT-3 could reverse 94% of initial rejection episodes compared to 74% when conventional therapy was instituted [4]. One of the initial shortcomings of the use of OKT-3 for the treatment of acute rejection was that in the azathioprine era 50% of these patients would develop a repeat episode of rejection. With the introduction of cyclosporine, this recurrent rejection rate was reduced to 20% [81].
21
Harrison S. Pollinger et al. TABLE 2.4 Toxicity profile of OKT-3 therapy. Cytokine Release Syndrome
Over Immunosuppression
Immunogenicity
Reaction Chills, fever 1st dose 2nd dose Nephrotoxicity Vomiting, diarrhea Dyspnea Pulmonary edema Aseptic meningitis Arterial thrombosis
Reaction CMV infection Fungal infection Herpes simplex PTLD
Reaction Rash Anaphylaxis
Incidence (%) 70–90 10–20 30–40 15–30 10–20 0 –4 0 –3 <1
In a multicenter trial with a cohort of 300 patients diagnosed with severe rejection refractory to highdose steroids (and in some instances anti-lymphocyte therapy), a 65% response rate was demonstrated to OKT-3, with 56% of those allografts remaining functional at 6 months [76]. OKT-3 has also been shown to be beneficial in the treatment of hepatic allograft rejection. A prospective randomized trial conducted at Massachusetts General Hospital randomized liver transplant patients with biopsy-proven rejection to 10 days of OKT-3 therapy versus high-dose steroids alone. The OKT-3 group had a significantly better allograft outcome. However, the infection rate in the OKT-3 group was higher. The recommendation was, therefore, to limit the use of OKT-3 to steroid-resistant rejection in the liver transplant population [20]. If repeated doses of OKT-3 treatment are required, there is increased risk of xenosensitization. This is due to the fact that this agent is of murine origin and can trigger the production of human anti-mouse antibodies (HAMA). The incidence of a HAMA response is 25 to 30% per exposure to OKT3 [15]. This number can be reduced to 21% if the patient is on concomitant triple therapy immunosuppression [18]. Most patients will mount a HAMA response 7 to 10 days after OKT-3 therapy has ceased. The neutralizing effect of these anti-idiotypic antibodies can often be overcome by escalating the dose of OKT-3. Many centers have adopted the philosophy of routine escalation of doses of OKT-3 without monitoring to ensure adequate serum levels and to avoid the cost associated with frequent in vitro monitoring of CD-3+ cells.
Incidence (%) 20–60 15–35 5–25 0 –8
Incidence (%) 0–5 <1
ANTI-CD-25 In the early 1990s, other potential targets of the immune response were identified that could be blocked with monoclonal antibodies. Daclizumab (Zenapax) and basiliximab (Simulect) are MAb directed against the α-chain of the IL-2-receptor (also known as CD-25). CD-25 is present in high concentrations on activated T-lymphocytes. By competitively blocking this site, IL2-dependent proliferation of T-cells is inhibited without the deleterious effects associated with T-cell depletion. Daclizumab was studied as an induction agent in two prospective randomized controlled multicenter trials of first-time recipients of cadaver-donor renal allografts. The incidence of biopsy-proven acute rejection at 6 months was reduced by 37% in the U.S. trial [97] and by 40% in the European study [74]. Daclizumab was administered on the day of transplant and at 14-day intervals thereafter for a total of five doses. Basiliximab is similarly efficacious, with pivotal trials demonstrating a reduction in the composite endpoint of death, and graft loss or acute rejection of 26% and 31%. The dosing regimen is easier to comply with because only two doses were administered: one on the day of transplantation and the other on the fourth postoperative day. Unlike the polyclonal preparation and OKT3, infusion of IL-2 receptor blockers is well tolerated by patients (the cytokine release syndrome does not occur). However, there have been reports of severe hypersensitivity reactions within 24 hours of the first dose. The most common adverse reactions of the IL-2 receptor blockers
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are nausea, fever, and headache. The incidence of PTLD and infection are similar to the other antibody preparations in clinical use.
Anti-CD-20 Anti-CD-20 (rituximab) is a humanized monoclonal antibody that has been utilized in several desensitization protocols for ABO-incompatible kidney transplants [96]. CD-20 is a common cell surface marker present on immature and mature B-cells. Historically, ABOincompatible kidney transplants have been performed after several preoperative plasmapheresis sessions with the hope of depleting the circulating anti-A or anti-B antibody levels to avoid hyperacute rejection. These desensitization protocols also employ antithymocyte globulin and other more conventional immunosuppressive agents in attempt to inhibit antibody-producing cells. Rituximab has recently been added to the desensitization armamentarium to try to deplete B-cells that are programmed to become part of the antibodyproducing population (i.e., plasma cells). Tyden et al. have recently used rituximab preemptively in combination with immunoadsorption to successfully transplant 11 ABO-incompatible kidney recipients [96]. Rituximab is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD-20 antigen found on the surface of normal and malignant B-cells. The monoclonal antibody is an IgG1 kappa immunoglobulin with light and heavy chains of murine origin and variable regions derived from human sources. The agent is well tolerated in the transplant population, but when used to treat B-cell malignancies it has been associated with life-threatening tumor lysis syndrome. As newer monoclonal antibodies are developed and a better understanding of antibody secreting cells evolves, plasma cells will be more specifically targeted and effectively destroyed. Plasma cells are protean, expressing different cell surface markers at different time points in their maturation. This selective regulation of cell surface markers makes it difficult to definitively target the plasma cell with a single agent.
Alemtuzumab Alemtuzumab (Campath-1H) is a recombinant DNAderived humanized monoclonal antibody directed against the 21–28 kD cell surface glycoprotein
CD-52 [41]. CD-52 is expressed on the surface of a variety of normal and malignant cell lines. B- and T-lymphocytes, natural killer cells, macrophages, some granulocytes, and some tissues of the male reproductive system possess the CD-52 cell surface marker at some time point in their maturation. The Campath antibody is 150 kD in size and is an IgG1 subtype. The mechanism of action of Campath-1H is antibody-dependent cell lysis [14]. The initial indication for Campath-1H was refractory chronic lymphocytic leukemia [41]. The toxicodynamic profile of Campath-1H is similar to that of the other monoclonal antibody preparations in that similar instances of infusion reactions, lymphopenia, myelosuppression, posttreatment malignancy, and infections have been reported [14]. Campath-1H has also been employed in an ex vivo fashion for the depletion of T-lymphocytes prior to bone marrow transplantation for the prevention of graftversus-host disease [41]. Within the last several years, interest in Campath-1H as an immunosuppressive agent for renal transplantation has surfaced. Preliminary data on this agent in combination with low-dose calcineurin inhibition have produced encouraging results. However, these data are limited to a small number of patients [14]. The cohort of patients involved in trials with Campath-1H consists of 31 renal allograft recipients with a mean follow-up of 21 months. The incidence of acute rejection was 20% [14]. More trials are needed to define the efficacy of Campath-H1 as an induction agent, as well as its potential benefit when used to combat acute rejection.
Co-stimulation Inhibitors Abatacept (CTLA4-Ig) is a novel fusion protein consisting of the extracellular domain of human CTLA4 (a T-cell membrane protein subserving an inhibitory function) fused to a fragment of the Fc portion of a human immunoglobulin (Ig) G1 antibody. By binding avidly to B7-1 (CD-80) and B7-2 (CD-86) on the surface of the antigen-presenting cell (APC), the interaction of the T-cell’s CD-28 molecule with the APC’s CD-80/86 is blocked and the second signal required for T-cell activation is not delivered. Abatacept has been shown to be effective in patients with psoriasis and rheumatoid arthritis [5, 35], but did not demonstrate substantial efficacy in a nonhuman primate renal transplant model [49].
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It was hypothesized that a more potent CTLA4-Ig protein could be developed by engineering a molecule with higher avidity for CD-80 and CD-86. Belatacept was derived from abatacept by a 2-amino-acid substitution, which led to a fourfold increase in avidity to CD-86 and a twofold increase in avidity to CD-80. In vitro belatacept demonstrated tenfold more potent inhibition of T-cell activation and was dramatically more effective than the parent compound in preclinical transplant models [55]. Recently, in a multicenter phase-2 study belatacept was compared to cyclosporine in patients receiving basiliximab, MMF, and steroids. Belatacept did not appear to be inferior to cyclosporine in preventing acute renal allograft rejection [98]. Importantly, belatacept was associated with an improved cardiovascular and metabolic risk profile and was not nephrotoxic. However, the incidence of infection and neoplasms as similar in the belatacept and cyclosporine groups. Thus, belatacept — despite its elegantly specific mechanism of action — still provides generalized immunosuppression and carries with it the attendant risks of such therapy.
Deoxyspergualin 15-deoxyspergualin (DSG) is a synthetic derivative of spergualin, an antibiotic produced by Bacillus laterosporous. The mechanism of action is unknown but it appears to differ from that of other available immunosuppressive agents. It binds to a member of the heat shock protein 70 family and does not compete with the immunophilins, cyclophilin, or FK binding protein [71]. DSG has been shown to inhibit several macrophage functions. It inhibits lysosomal enzyme release and superoxide production by macrophages, and blocks induction of ornithine decarboxylase activity, inhibition of which impairs macrophage function [27, 44]. This antimacrophage activity makes DSG a potentially important immunosuppressant for cell transplantation in general, and islet transplantation in particular. Macrophage proinflammatory mediators such as nitric oxide and oxygen-free radicals have been shown to be deleterious to beta cells, and macrophages have been associated with primary nonfunction of islet allografts [47]. Thus, an antimacrophage agent such as DSG (which is itself not diabetogenic) may be particularly useful as part of an immunosuppressive protocol to protect islet allografts [102]. A pilot study using
DSG in recipients of simultaneous islet/kidney allografts was successful, and further studies are underway [36]. However, the studies ongoing are of an exploratory nature and pivotal trials of this agent are still some years away.
Conclusions As a discipline, transplantation has entered an exciting time. In the last decade several novel classes of immunosuppressive agents from humanized monoclonal antibody preparations to the costimulatory inhibitors have been introduced. As always, a priority has been to maximize efficacy while simultaneously reducing toxicity. This fundamental philosophy is and will always be a daunting task for investigators striving for newer and better immunosuppressive agents. As the field of immunosuppression advances and the technologies that monitor the immune response become more complex — examining the molecular origins of immunity rather than surrogates of the immune response such as allograft function — the agents will evolve to become more specific and effective. As transplantation becomes less invasive with the advent of cellular transplantation, this will drive immunosuppression down a similar path of less toxic and more tolerable treatment regimens. Both of these disciplines will play vital roles in allowing transplantation to reach its full potential for the long-term replacement of organs and tissues.
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Harrison S. Pollinger et al. 82. Remuzzi, G., Bertani, T. Renal vascular and thrombotic effects of cyclosporine. Am J Kidney Dis 13:261–272; 1989. 83. Remuzzi, G., Perico, N. Cyclosporine-induced renal dysfunction in experimental animals and humans. Kidney Int Suppl 52:S70–74; 1995. 84. Satterthwaite, R., Aswad, S., Sunga, V., Shidban, H., Bogaard, T., Asai, P., Khetan, U., Akra, I., Mendez, R. G., Mendez, R. Incidence of new-onset hypercholesterolemia in renal transplant patients treated with FK506 or cyclosporine. Transplantation 65:446–449; 1998. 85. Sehgal, S., Baker, H., Vezina, C. Rapamycin (AY-22,989) a new antifungal antibiotic: II. Fermentation isolation, and characterization. J Antibiotic 28:727–732; 1975. 86. Shapiro, R., Jordan, M. L., Scantlebury, V. P., Vivas, C., Gritsch, H. A., Casavilla, F. A., McCauley, J., Johnston, J. R., Randhawa, P., Irish, W., Hakala, T. R., Fung, J. J., Starzl, T. E. A prospective, randomized trial to compare tacrolimus and prednisone with and without mycophenolate mofetil in patients undergoing renal transplantation: First report. J Urol 160:1982–1985; discussion 1985–1986; 1998. 87. Simpson, M., Monaco, A. Clinical uses of polyclonal and monoclonal antilymphoid sera. In: Monoclonal antibodies in transplantation, p 1. Ed by Chatenoud, L., 1995. 88. Sollinger, H. W., Belzer, F. O., Deierhoi, M. H., Diethelm, A. G., Gonwa, T. A., Kauffman, R. S., Klintmalm, G. B., McDiarmid, S. V., Roberts, J., Rosenthal, J. T., et al. RS-61443 (mycophenolate mofetil). A multicenter study for refractory kidney transplant rejection. Ann Surg 216:513–518; discussion 518–519; 1992. 89. Sollinger, H. W., Deierhoi, M. H., Belzer, F. O., Diethelm, A. G., Kauffman, R. S. RS-61443: A phase I clinical trial and pilot rescue study. Transplantation 53:428–432; 1992. 90. Spencer, C. M., Goa, K. L., Gillis, J. C. Tacrolimus. An update of its pharmacology and clinical efficacy in the management of organ transplantation. Drugs 54:925–975; 1997. 91. Starzl, T. E. Heterologous antilymphocyte globulin. N Engl J Med 279:700–703; 1968. 92. Starzl, T. E., Abu-Elmagd, K., Tzakis, A., Fung, J. J., Porter, K. A., Todo, S. Selected topics on FK 506, with special references to rescue of extrahepatic whole organ grafts, transplantation of “forbidden organs,” side effects, mechanisms, and practical pharmacokinetics. Transplant Proc 23:914–919; 1991. 93. Tanaka, H., Kuroda, A., Marusawa, H., Hashimoto, M., Hatanaka, H., Kino, T., Goto, T., Okuhara, M.
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94.
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103. 104.
Physicochemical properties of FK-506, a novel immunosuppressant isolated from Streptomyces tsukubaensis. Transplant Proc 19:11–16; 1987. Thistlethwaite, J. R. Jr., Gaber, A. O., Haag, B. W., Aronson, A. J., Broelsch, C. E., Stuart, J. K., Stuart, F. P. OKT3 treatment of steroid-resistant renal allograft rejection. Transplantation 43:176–184; 1987. Thomas, J. M., Carver, F. M., Foil, M. B., Hall, W. R., Adams, C., Fahrenbruch, G. B., Thomas, F. T. Renal allograft tolerance induced with ATG and donor bone marrow in outbred rhesus monkeys. Transplantation 36:104–106; 1983. Tyden, G., Kumlien, G., Genberg, H., Sandberg, J., Lundgren, T., Fehrman, I. ABO incompatible kidney transplantations without splenectomy, using antigenspecific immunoadsorption and rituximab. Am J Transplant 5:145–148; 2005. Vincenti, F., Kirkman, R., Light, S., Bumgardner, G., Pescovitz, M., Halloran, P., Neylan, J., Wilkinson, A., Ekberg, H., Gaston, R., Backman, L., Burdick, J. Interleukin2-receptor blockade with daclizumab to prevent acute rejection in renal transplantation. Daclizumab Triple Therapy Study Group. N Engl J Med 338:161–165; 1998. Vincenti, F., Larsen, C., Durrbach, A., Wekerle, T., Nashan, B., Blancho, G., Lang, P., Grinyo, J., Halloran, P. F., Solez, K., Hagerty, D., Levy, E., Zhou, W., Natarajan, K., Charpentier, B. Costimulation blockade with belatacept in renal transplantation. N Engl J Med 353:770–781; 2005. Williams, S. K., Wang, T. F., Castrillo, R., Jarrell, B. E. Liposuction-derived human fat used for vascular graft sodding contains endothelial cells and not mesothelial cells as the major cell type. J Vasc Surg 19:916–923; 1994. Winter, G., Milstein, C. Man-made antibodies. Nature 349:293–299; 1991. Woo, K. T., Junor, B. J., Vikraman, P., d’Apice, A. J., Kincaid-Smith, P. Serum-alkaline-phosphatase as predictor of avascular necrosis of bone in renal-transplant recipients. Lancet 1:620–621; 1979. Xenos, E. S., Casanova, D., Sutherland, D. E., Farney, A. C., Lloveras, J. J., Gores, P. F. The in vivo and in vitro effect of 15-deoxyspergualin on pancreatic islet function. Transplantation 56:144–147; 1993. Yee, G. C. Pharmacokinetic interactions between cyclosporine and other drugs. Transplant Proc 22:1203–1207; 1990. Zimmerman, J. J., Kahan, B. D. Pharmacokinetics of sirolimus in stable renal transplant patients after multiple oral dose administration. J Clin Pharmacol 37:405–415; 1997.
3 Stem and Precursor Cells for Transplant Therapy Jingli Cai and Mahendra Rao National Institute on Aging, Triad Technology Center, Baltimore, Maryland
Introduction Cell replacement therapy as a potential treatment for previously intractable diseases is now being considered a more feasible and effective treatment than even just a decade ago. Preliminary success in corneal therapy, the availability of commercial cartilage and skin replacement products, new advances in islet cell replacement, commercialization of cord blood therapy, and the potential of obtaining large numbers of cells from adult, fetal, and embryonic sources has led to the hope that stem cells may serve as a viable treatment option for treatment of a wide range of disorders, including congestive cardiac failure, lysosomal storage disorders, and metastatic tumors [15, 78]. This potential of stem cells has led to renewed commercial and regulatory interest in the field. In this chapter, we use the central nervous systems (CNS) as an example to illustrate the advances made and obstacles that must be overcome. In the first section, we discuss the current research and compare the advantages and disadvantages of different cell sources. In the second half of the chapter, we highlight the problems that must be solved for cell therapy in repair.
Some Basic Facts The CNS consists of approximately 1012 cells, of which the largest majority by far are fully differentiated postmitotic cells. Approximately 10% of these cells are neurons, whereas the remaining cells are supporting cells. For example, astrocytes provide trophic support, whereas oligodendrocytes produce myelin and ensheath
Cellular Transplantation: From Laboratory to Clinic
axons to enhance communication among neurons. Neurons communicate with each other and act in concert to regulate bodily activities. Any single neuron may make tens of thousands of connections. These connections may be made in close proximity to the soma of the cell or at distances that could be even several feet away [47]. The brain in general does not repair itself by regenerating new neurons to any large extent in most regions. Cues to direct appropriate differentiation of neural stem cells (NSCs) do not exist in all but selected areas of persistent stem cell populations. These stem cell populations are present predominantly in more cranial regions of the brain, such as the subventricular zone (SVZ) around the lateral ventricle [31, 74]. These persistent NSC populations can differentiate into glia and a small number of neurons that are directed to the olfactory bulb and the hippocampus — small but critical regions of the brain [21]. Although some controversy exists as to the extent of new neurogenesis and the number of NSCs present, it is reasonable to state that the total number of NSCs present in the adult is small, they do not participate in the repair process in most areas of the brain, and the roles of NSCs in the adult appear to be limited to very specific regions [22, 60, 68]. One more aspect of repair in the brain that must be kept in mind is that in the adult brain distances from sites of injury to stem cell niches are very large and no direct pathways for cell migration exist [45]. Radial glia that direct neuronal migration in the early developmental stages are no longer present in the adult, and fiber tracts are oriented in directions that are not useful for directing cells to cortical gray matter [77]. Thus, damage to cortical regions may not be signaled
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to the periventricular regions. Even with correct signals, there are no significant numbers of cells to migrate to that region, and even if a small number did so no cues to direct their site- and region-specific differentiation would exist [10]. As experiments have been performed, it has become clearer that different areas of the brain are structurally different and perform different functions, and that types of neurons present differ in their properties. These functional and structural differences arise very early in development and appear to include the stem cells present in those regions [47]. Thus, NSCs present in the hippocampus (or the SVZ associated with this region) differ from the NSCs present in the anterior SVZ associated with the olfactory bulb. One more critical fact to keep in mind is that the brain is relatively insulated from the rest of the body, and it is difficult to reach specific areas of the brain without causing damage as cells are being harvested and routes of administration are considered. The stem cell niches are present deep inside the brain and cannot be accessed by direct injection with any ease. Possible routes include deposition of cells in the ventricles or cerebrospinal fluid (CSF) space and delivery via the circulatory system (arterial or venous injection) [4, 19, 86]. Overall, when cell replacement therapy is considered one must take into account these basic facts while evaluating the type of cell to use, the mechanism of action of that particular cell (Table 3.1), and how one will get acceptable numbers of such cells to the appropriate location and how appropriate cues to direct differentiation will be provided. Issues that need to be considered for a successful cell therapy are summarized in Table 3.2.
TABLE 3.1 Mechanisms by which cells may have a beneficial effect. Possible Mechanisms
Examples
Replacing damaged or dead cells Supplying growth factor/gene therapy Delivering a toxic agent Mobilizing the endogenous stem cell pool Repairing damaged regions Reconstructing neural circuit
PD, AD, HD, stroke, ALS, epilepsy AD, HD, lysosomal storage diseases, brain tumor Brain tumors Stroke
HD, brain ischemia Traumatic spinal or brain injury
Cell Source Issues NEURAL STEM CELLS Stem cells are a population of cells that self-renew and are multipotent. Stem cells are classified by the tissue and the developmental stage from which they are harvested, and in general most of the tissue-specific stem cells (TSC) undergo limited but not lifelong selfrenewal. TSC have the ability to divide either symmetrically or asymmetrically. By symmetric division, stem cells give rise to two progenies whose properties remain similar. In contrast, stem cells can produce two different daughter cells by asymmetric division, with one cell being similar to itself and the other being a differentiated cell. In general, TSC derived at embryonic stages appear to self-renew for longer periods and
TABLE 3.2 Issues that need to be considered for successful therapy. Cell Issues
Host Issues
Transplantation
Choice of cell source
Diagnosis and timing of therapy Age and medical history Delivery location Dose vs. severity, brain size Surgery techniques
Monitoring cell survival
Cell quantity Directed differentiation Cell purification
Immunosuppression
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Tracing cell migration Quantifying cell proliferation Following cell differentiation Evaluating anatomical and functional integration Estimating side effects of transplantation
3.
Stem and Precursor Cells for Transplant Therapy
have shorter cycle times than TSC derived from later stages of development. In the CNS, NSCs have been isolated from different stages of development and different locations. These NSCs differ in their self-renewal capacity, position markers they express, and the proportions and phenotypes of cells they differentiate into [43, 48, 89, 90]. Both lineage-specific (positive and negative) and general stem cell markers have been identified to prospectively identify and locate stem cells in the nervous system [9, 74]. However, there is still no perfect marker (i.e., useful for all NSCs). Transplantation of NSCs has been tested in several pilot studies, and some positive and cautious conclusions can be made. For example, in Parkinson’s disease (PD) NSCs isolated from 7- to 8-weekold human embryos showed alleviation of symptoms and survival of transplanted dopaminergic neurons [17, 32]. Transplanting SVZ adult NSCs into stroke rat models significantly improved neurological function and reduced infarct size as assessed by magnetic resonance imaging (MRI) and histopathological analysis [95]. Dramatic improvement in experimental autoimmune encephalomyelitis (EAE) models has also been reported [62, 75]. However, these and other studies also identify areas of concern that need further research. Adult NSCs show a decreased capability to self-renew. In most studies, adult NSCs show shortened telomere length, longer cell cycle time, inadequate differentiation, and limited proliferation capability [6]. It also remains a problem to obtain sufficient cell numbers. In comparison, fetal cells grow better. However, it has been difficult to maintain a purified population of cells. In addition, some studies have suggested that cells change in their properties when maintained in culture, making the idea of a bank of cells difficult to implement. Delivery via arterial or venous circulation does not seem possible, and issues of homing and migration seem open to interpretation. Further, a major concern has been the inability to obtain robust differentiation after transplantation. Most reports of stem cell transplants have suggested poor survival and limited differentiation and/or limited migration [55]. Svendsen and colleagues, for example, reported that the only dopaminergic neurons they found were those that had been predifferentiated in culture [8]. It has also become clear that the brain is not an immune-privileged site, and as such nonautologous cells are likely to be rejected over time. Thus, although dramatic progress has been made in understanding the
biology of the cells, identifying methods of isolating and amplifying cells in culture, and following cells after transplantation it has been difficult to demonstrate substantial success in a clinical trial. However, current studies have identified problems and provided clues as to how such problems may be solved.
RESTRICTED PRECURSOR CELLS In contrast to the hematopoietic stem cell (HSC) model, in which the most primitive cell is the optimal cell for therapy, many researchers in the CNS field believe progenitor cells may be used for therapy as well. These restricted precursors are defined by limited self-renewal and the ability to give rise to a restricted set of mature progeny. Such restricted precursor cells (blast cells) have been identified in the CNS, and their properties have been studied carefully [57]. These include olfactory ensheathing glia, oligodendrocyte precursors, glial precursors, astrocyte precursors, and neuronal precursors [36, 50, 67, 79]. Although the restricted precursor cells do not have the same prolonged self-renewal as NSCs but have a more restricted differentiation capacity, they are nevertheless a possible cell source in many tissues and many diseases. We and others have reasoned that differentiated cells are not replaced in large numbers in many solid tissues and organs, and as such limited self-renewal may not be a limiting factor in providing long-term benefits. Furthermore, many diseases in solid tissue are diseases affecting a specific subpopulation. Given that no cues for directing differentiation of immature cells to a particular lineage exist, using selected cells may be more appropriate (i.e., glial cells for glial diseases and appropriate neuronal progenitors for neuronal diseases) [69]. In the CNS, it appears that glial proliferation after injury is robust and that cues are present in the brain to direct glial progenitors to mature, migrate, and respond after tissue damage. Thus, the possibility exists that transplanted cells can co-opt the same cues for efficient repairs. Glial progenitors have been shown to be effective in enhancing repair and remyelination in a variety of models of injury [5, 35, 42]. Neuronal progenitors have been shown to be effective in transplants in the hippocampus, and long-term reconstitution studies have shown that mature cells derived from transplanted progenitors can survive for months and years in rodent models [29, 34].
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Some investigators have reasoned that predifferentiating NSCs might be the best approach for therapy because it combines the prolonged self-renewal ability of NSCs with the ability of differentiated cells to respond to local cues. Indeed, forcing NSCs down a distinct lineage before transplantation into a rat model of spinal cord injury Hofstetter et al. [44] found more oligodendrocyte and neurons differentiated from Ngn2NSCs grafts, with better functional recovery and less neuropathic pain compared to naive NSCs. Although using progenitor cells has some undeniable advantages, several problems remain. There are no autologous sources of progenitor cells, with the exception of perhaps olfactory ensheathing glia. Amplifying sufficient numbers of glial cells from biopsy material such as that performed in temporal lobe epilepsy remains a challenge. The limited self-renewal restricts the number of cells available and shortens the time window between collection and transplantation. As with NSCs, using progenitor cells does not resolve the immune issues associated with nonautologous/ mismatched transplants. In addition, migration and integration issues that affect transplants in solid tissues remain. No data that progenitors can be delivered via arterial or venous circulation have been reported, and neuronal progenitors (unlike glial progenitors) do not migrate extensively. Although progenitor cell transplants offer some benefits in therapies for some diseases, they are not a panacea, and several issues that may be unique to progenitor cells need to be considered when evaluating these cells for therapy.
TRANSDIFFERENTIATED CELLS An alternative cell source that may resolve many of the problems associated with using NSCs or progenitor cells could be the idea of transdifferentiating autologous stem cells. Examples include HSCs and MSCs (bone marrow stromal cells) that are relatively abundant, easily collected, and may be delivered via the circulatory system [65]. One can harvest cells in sufficient numbers or obtain enough via limited amplification in culture. These cells by their very nature are migratory and survive in circulation. They have in previous studies been shown to circulate through various tissues in small but readily detectable numbers [39]. Moreover, a plethora of reports has suggested that these particular stem cells are plastic, respond to tissue damage, accumulate at sites of
injury, and can respond to local cues to differentiate in a site- and tissue-specific fashion [88]. HSCs accumulate in cardiac tissue after an infarct, and some cells transdifferentiate into cardiac muscle and others contribute to blood vessels, resulting in tissue reconstitution. Others studies have suggested similar benefits in lung, nervous system, liver, and other solid tissue [18, 38, 41]. Although controversy exists about transdifferentiation [56], we do not feel that this chapter needs to focus on the controversy. We simply want to emphasize that although the usage of transdifferentiated cells for therapy offers many potential advantages several questions and problems need to be solved. The frequency and heritability of the transdifferentiation process needs to be tested, and the persistence of the transdifferentiated cells in their new environment must be assessed over a long time period. The effect of the nontransdifferentiated population that does not respond to local cues must be considered, and more control on directing the process must be obtained. It is fair to state that it is unclear if transdifferentiated cells are better than tissuespecific stem cells or progenitor cells for all therapies and in all diseases. Rather, it will be important to weigh each of these potential cell sources carefully to determine which one will be the best cell choice for therapy. Further transdifferentiation of nonneuronal populations does not solve the problems of delivery and migration in a solid tissue organ either. Other cells that can be dedifferentiated or transdifferentiated have been described as well [84]. In the nervous system, for example, all major types of glial cells (including oligodendrocyte precursors, astrocytes, and radial glia cells) have been reported to transdifferentiate into mature neurons [13, 53, 87]. Even postmitotic neurons have been reported to reenter the cell cycle and give rise to dividing precursor cells [1, 7, 24]. The added advantage of such transdifferentiated populations compared to nonneuronal stem cell populations is that one can imagine directing surplus cells present in situ (at site of damage or close to it) to transdifferentiate into the appropriate cell type and thus circumvent the difficulties associated with delivering the right numbers of cells to the right place and at the right time. Indeed, Macklis and colleagues [14, 25] have shown that if the injury paradigm is designed correctly either stem cells present locally or transdifferentiated cells are activated to repair and regenerate damaged tissue. The results are quite spectacular, and the degree of appropriate connectivity seen is impressive. However, as with other
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3.
Stem and Precursor Cells for Transplant Therapy
transdifferentiated cells it remains to be seen whether these cells will be more stable, whether the numbers will be sufficient, and whether one will be able to induce such transdifferentiation in all injury paradigms.
ESC-DERIVED CELLS Embryonic stem cells (ESCs) are pluripotent, with an unlimited self-renewal capability, and can be induced to differentiate into any cell type of an organism. Their relatively rapid cell cycle and ability to generate all cell types and tissues make ESCs promising candidates for therapeutic applications in which large-scale expansion of cells is important. Human ESC lines, derived from embryos generated in excess for in vitro fertilization [27], can be expanded in vitro with little or no change of properties over a long time period [12, 81]. Such expanded lines can be induced by appropriate exogenous signaling molecules to generate appropriate cell types [49, 76, 91]. A brief list of various methods used for neural differentiation is summarized in Table 3.3. Similar methods have been described for mesenchymal and endodermal derivatives as well. It is quite clear that ESCs can serve a virtually unlimited source of specific cell types. New ESC lines can be obtained in a relatively high frequency [58] (up to 30% efficiency has been reported), which provides the possibility of creating a bank of
ESC lines whereby one could obtain immunologically matched cell lines. This bank would not have to be extremely large because there are only a limited number of MHC class molecules. By choosing an immunologically matched line, one could perhaps avoid the problems of long-term immunosuppressive therapy. However, issues such as obtaining sufficient numbers of cells for therapy, donor cell survival, integration, migration, and delivery still have to be resolved. ESCs in addition may have some specific problems of their own. For example, the inability of immature ESCs to respond to environmental cues and their truly impressive selfrenewal capacity permit small numbers of ESCs to form teratomas after transplantation. This imposes a far more stringent requirement of purity on ESC samples than on any other stem cell population. Cell sorting may help to overcome this problem, but it requires developing sorting and selection protocols and identifying cell-typespecific markers that may not be available in some cases. Currently, the number of ESC lines available is small (http://stemcells.nih.gov/research/registry/ ), and immunological matching is not feasible. One possible resolving strategy is nuclear transfer, which has been successfully demonstrated in some pilot studies [46] but is highly restricted in most countries. Long-term propagation is currently an absolute requirement, and although reports have suggested stability we note that growing cells in culture imposes stresses and selection pressures and changes can and do occur. Mouse ESCs
TABLE 3.3 Neural differentiation methods used with human ESCs. Progeny Phenotype
Methods
Reference
99% NCAM+, 97% nestin+, and 90.5% A2B5+ neural progenitor cells Nestin+, NCAM+ neural precursor cells, and Tuj1+ neurons Neural-tube rosettes Neural cells and glial cells 39% and 52% NF-L+ neurons MAP2+ and NF+ neurons
Prolonged culture and mechanical selection
80
Treatment of ES cell aggregates with RA
11
ES cell aggregates Noggin treatment RA and NGF treatment EB culture in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII) Co-culture with PA6 stromal cells
94 72 82 83
87% of hES colonies contained tyrosine hydroxylase (TH)-positive cells Up to 79% neurons express TH 40% of Tuj1+ neurons express TH
Co-culture with MS5, MS5-Wnt, or S2 stromal cells Co-culture with stromal cells
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93 73 71
Jingli Cai and Mahendra Rao
show a decrease in the ability to contribute to the germ line after as few as twenty passages [2, 63]. Unlike adult NSCs, which may have incorrect positional information, ESC derived cells may lack appropriate positional information. Thus, ESC biologists may face similar issues in terms of the ability of the cells to survive and integrate in a tissue- and site-specific fashion. A substitute for ESCs is embryonic carcinoma (EC) cells, which show surprisingly similar characteristics. They have the ability of extensive self-renewal and can differentiate into committed precursors and terminal differentiated cells; for example, postmitotic neurons [59]. The differentiation of EC cells is accompanied by loss of tumorigenicity [66]. NTera2 (NT2) cells originate from testicular carcinoma, and one subclone NT2/D1 is used to get mature neurons through the treatment with RA. NT2-derived neurons are commercially available as LBS neurons (Layton BioScience, Sunnyvale, CA) and have been transplanted into patients [51, 52]. Neuropathological evaluation and immunohistochemical analysis of one patient showed survival of grafted LBS neurons 27 months after transplantation and no signs of neoplasm formation [64]. However, no detailed reports on cell integration or migration are available.
We note, however, that teratocarcinoma lines do not offer any specific benefit over ESC except for the fact that they may be easier to grow. We note as well that karyotyping of EC cells and their derivatives has been reported to be abnormal [20, 61]. We believe their utility should be relegated to using them as models for potentially safer cells. The relationship of different cell types in the CNS is illustrated in Figure 3.1. It is clear from the previous discussion that each cell type offers some unique advantages or disadvantages (Table 3.4), and thus it is unlikely that one will use a single well-characterized cell type for all diseases. Indeed, it is clear that the field is moving in that direction. For some disease for which proliferation and migration are critical, more undifferentiated cells will be transplanted early in the disease course. In other diseases, in which a single cell type may be required or be sufficient such as in PD, differentiated progenitor cell populations are being evaluated. Creative studies on utilizing the propensity of cell fusion and the transient survival of transdifferentiated cells are being initiated to harness the specific positive features of different stem cell populations. Cell types that have been used in different disease models are outlined in Table 3.5.
Figure 3.1. Major differentiation pathways in the CNS. NSCs — which can be isolated from different developmental stages, derived from ESCs, or transdifferentiated from other tissue cells — can give rise to all types of mature cells in the CNS.
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TABLE 3.4 Comparison of different cell sources. Cell Type
Advantage
Disadvantage
Neural stem cells (adult and fetal)
High potential of self-renewal.
Lineage-restricted precursors
Useful for replacing a specific mature cell type. Great availability. No ethical problems. Ideal for autologous cell therapy. Great availability. Most capable of expanding.
Limited resources. Differentiation direction not totally understood yet. Fetal ones are not ethically acceptable. Limited self-renewal capability.
Transdifferentiated cells
Specialized cells
ES-derived cells
Olfactory nerve-ensheathing cells Neural crest stem cells Retinal stem cells
Remyelinating capability. Promoting axon growth. Multipotent and very plastic. Integrative plasticity and ability to differentiate into retinal neurons.
Transdifferentiation rate is very low.
Potential malignant transformation. Differentiation mechanism not clear yet. Ethical problem. Limited sources. Poor proliferation capability. Differentiation mechanisms not clear. Limited sources.
TABLE 3.5 Different cell types for therapy in different diseases. Diseases
Types of CNS Cells Affected
Most Commonly Used Cell Type in Treatment
Obstacles to Overcome
Alternative Cell Sources
Reference
PD
Dopaminergic neurons in the A9 region of the substantia nigra parsa compact Neuronal circuitry and supporting glial cells
Fetal mesencephalic neurons
Limited clinical benefits. Side effect as dyskinesias. Source and ethical problems. Potential tumorigenicity if contaminated with undifferentiated cells.
ES- or NSC-derived specific A9 dopaminergic neurons Hematopoietic stem cells
40 70 28
Low proliferation rate.
Forebrain-derived NSPCs
90
Noggin-expressing NPCs
85
Stroke
Spinal cord injury
HD AD Lysosomal storage diseases
Varied neurons and glia
Glutamate-induced neuron death Basal forebrain cholinergic neurons Abnormal morphology found in neurons
Human neuronal cells derived from NTera2/D1 teratocarcinoma cell line Spinal-cord-derived neural stem/ progenitor cells Neural precursor cells
Predominantly differentiated into astrocytes. Fetal striatum neurons Source and ethical problems. Fetal NSC-derived Source and ethical cholinergic neurons problems. Bone marrow cells with Limited delivery of the vector to the target and retrovirally transduced transient expression of lysosomal genes the gene.
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ES or adult NSC-derived 37 neurons ES or adult NSC-derived 92 neurons Gene therapy using 23 NSCs for delivery
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Difficulties Researchers Face As we have discussed before, identifying the optimum cells for therapy and obtaining sufficient numbers of cells to perform such experiments is only part of the process of translating cell therapy to the clinic. Researchers face many challenges before such therapy can be widely used in clinical practice. There are huge differences in the CNS structure and function between animal models and human beings. Most of the disease models only reflect parts of the human diseases. The mechanisms of action of many of the cells touted for therapy remain unclear. Noninvasive measures to assess cell survival, integration, migration, and differentiation after transplantation remain limited. Many of the diseases for which cell therapy is being considered are not diagnosed early, and the window of opportunity for therapy may be lost. Unlike drugs, it is also unclear how long the follow-up after such therapy should be. The FDA (Food and Drug Administration), like all of us, is uncertain and remains reluctant to allow therapy or trials to proceed in the face of such uncertainty. These difficulties (discussed in material following) need to be resolved for any cell being considered for therapy in a particular disease model.
BASIC DEVELOPMENTAL BIOLOGY BACKGROUND IS UNCLEAR Understanding the relationship between the pathology of diseases and the development of a particular system is critical in designing effective and specific therapies for any disorder. In cell therapy, one needs to also understand how cells respond to and effect changes. Perhaps the single largest problem in translating potentially exciting results to the clinic has been a difficulty in obtaining a consensus on how stem cells exert a particular effect. MSCs have been reported to have measurable effects even though they do not survive in culture. Their benefits have been variously attributed to transdifferentiation, release of growth factor in the local environment, and modulation of the immune system [16]. Likewise, we understand little of how cells fuse, how transdifferentiation actually occurs, and how stem cells (which are normally restricted to a particular niche) can sense cues, steer themselves, and respond appropriately when endogenous stem cells fail to do so. Understanding such basic issues is critical. We do not need to understand them in detail before one moves
to the clinic, but we do need to understand them well enough so that we can manipulate the system and predict the effects with some reliability and reproducibility.
ANIMAL MODELS AND TESTS ARE DIFFERENT FROM WHAT IS APPLICABLE IN HUMANS Another major problem facing translational work has been the slow but clear realization that there are species differences between stem cells as well as difficulties in extrapolating from animal models to human disease. Differences could be mundane or critical, but it is often difficult to predict a priori whether the difference will be one or the other. These differences affect experimental design and assessment of results, and raise questions about appropriateness of animal models that should be used. Examples of these differences have been reviewed elsewhere [28]. The point we wish to make is that although these issues bedevil drug evaluations they effect cell transplanters to a greater extent. The reason is that cells themselves respond to the environment and their response changes over time. This is not true for drugs, at least in the context of testing them in an animal model. This dual interaction of host and transplant adds a huge layer of complexity, which makes translating cell therapy to the clinic a lot more difficult than any other drug or small-molecule agent.
NONINVASIVE MEASURES OF CELL SURVIVAL, INTEGRATION, MIGRATION, AND DIFFERENTIATION AFTER TRANSPLANTATION REMAIN LIMITED Another critical aspect of therapy, given the persistence of stem or progenitor populations, will be developing simple noninvasive measures to follow patients subjected to therapy. This has not been as much of a problem in the hematopoietic system because accessing this system has been relatively straightforward. However, in solid tissues one will nevertheless have to estimate what cells do and whether their behaviors remain appropriate. This requires developing methods to monitor cells and to delete them should something abnormal be identified. Unfortunately, to date only a few noninvasive methods that are sensitive enough exist.
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In PD, it is possible to monitor dopamine release noninvasively [26, 54]. Lumbar puncture [3] to access CSF is often an inexpensive method of following factor levels in the CNS when cells are used as drug delivery vehicles. However, MRI and computerized axial tomographic (CT) scan — our mainstays in examining tissue — do not have sufficient resolution to look at small numbers of cells. MRI dyes such as the ferridox labeling technique enhance sensitivity but resolution is still at the level of hundreds of cells even with more powerful magnets. Likewise, no reliable method of eliminating cells has been developed. Engineering suicide genes, introducing regulatory/inducible promoters, identifying cell-type-specific toxins, and so on are all being attempted. It is fair to state that currently no off-the-shelf strategy to eliminate cells after they have been introduced exists.
PREDICTING THE BEHAVIOR OF CELLS IN A DISEASED ENVIRONMENT IS DIFFICULT An important concern related to therapy in general and stem cells in particular is that little information is available about the diseased and damaged environment and the response of stem cells or progenitors to such an environment. This is critical when translating cell therapy results in animal models to the clinic. We note that transplanting human embryonic dopamine neurons into the brains of PD patients also shows clear age-related effects, with some clinical benefit seen in younger but not in older patients [28]. Similarly, Olanow et al. showed that a significant therapy effect is observed in patients with mild PD but not in those severely affected [70]. Unfortunately, not many good models of aging and progressive damage are available. They require an extraordinarily long time to generate, often rodent models are not useful, and costs for primate models make them prohibitive. Often, the only model that seems appropriate is a human, but clinical studies prospective or retrospective are also expensive and time consuming. Further, it is difficult to imagine that anyone would consider it ethical or moral to perform experiments on humans without reasonable animal data. This potential catch-22 situation predicts that we will have failures translating promising therapy in an animal model to humans. Such failures are not unexpected, as these have been seen in medicine trials as well. The recent recalls of Tysabri and Vioxx after extensive animal studies are good examples.
We would emphasize that cell therapy supporters are concerned about the same issue and believe that the likelihood of failures remains high and may be higher in cell therapy studies when compared to drug trials. It is perhaps such valid concerns that have made the progress to the clinic slower than what patient advocates would like. Dramatic results with cell therapy have been reported in spinal cord injury models, after stroke, and in the treatment of lysosomal disorders. However, to date no clinical trial has been initiated and it is perhaps with good reason that the FDA has been slow in granting approvals. It is important to remember that the litany of problems and issues we have discussed do not amount to an insurmountable obstacle in the path of translational research. Indeed, the number of potential disease targets for therapy being considered is large, and the number of investigational new drug (INDs) applications filed has increased dramatically over the last few years. We would instead suggest to the reader that the criteria laid out in this chapter help to evaluate cells for therapy and identify problems that need to be addressed. As discussed in subsequent sections of this book, cell therapy is indeed moving toward the clinic. We would argue that, for example, based on the criteria we have outlined pancreatic islets are a good candidate for clinical therapy. The mechanism of action is known, the number of cells required is small, the cells themselves are not required to do anything other than their normal biological function, and methods of isolating, propagating (for a short time), and delivering cells have been clearly defined. Investigators have also developed adequate immunosuppressive protocols and methods of monitoring posttransplantation, and have resolved dosing, quality control, and manufacturing issues. Similarly, a reasonable case can be made for limbal stem cells for corneal injury, dopaminergic neurons for PD, MSC for mesenchymal abnormalities, and so on.
Future Prospects It is important to emphasize that since 2002 the landscape for stem cell therapy has changed. The major impetus for change has been the involvement of the FDA. The FDA has reasoned that cells need to be classified as a drug when cells are used as modified in any fashion in culture or are used in a manner that is akin to a new use or function of these cells. Because cells can change in as little as 48 hours in culture,
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the FDA has argued that even if cells were isolated from the same patient but would be implanted in the future, manipulated (minimal as this may be), exposed to animal protein, or infected with a reagent they should be considered a product. Likewise, if BMSC (bonemarrow-derived mesenchymal stem cells) were used to make neurons this would be a different use of these cells and subject to FDA purview. This has had both positive and negative effects on the industry. The process of review and time to submit an IND application need to be factored into the planning of any therapy. Likewise, scale-up, lot sizes, good manufacturing practice (GMP), and product manufacturing are all issues that need to be considered. These more rigorous criteria have prolonged the process of moving promising therapies to the clinic and have greatly increased the expense to initiate trials for studies that could previously have been performed under IRB approval. Further, the FDA itself is not clear as to what is feasible in this new and rapidly changing field, and because no cell therapy has been approved as a product under the new guidelines it is not clear to the industry and investors how to factor the risk and time involved in adhering to the new regulations. Uncertainty has led to reluctance to invest, as is evidenced by the lack of large corporation or pharmaceutical companies investing in this field. Nevertheless, we believe that the net effect of the FDA’s involvement in the field has been beneficial and has brought a much-needed rigor in a fragmented field. The GMP and manufacturing regulations now required have forced investigators to examine the compositions of their cell products in much greater detail. Comparison between samples from different investigators is now becoming possible. Results from one study can now be evaluated against those derived at another center, and improvements can now be tested in a more rational way. Greater attention is now being paid to devising surrogate measures of potency and efficacy, and dosing studies are becoming the norm. Issues of follow-up potential side effects and more quantitative measures of improvement are being devised, and more attention to appropriate animal models that better mimic the human disease target are being developed. The effect of these regulations in the long term cannot be predicted, but we think it is safe to say that overall most investigators have welcomed the oversight despite the associated regulatory burden. The FDA on its part has played a proactive role and has initiated several workshops and courses to educate academic
researchers on the new regulations, and the NIH has recognized the need to fund new GMP facilities. The NHLBI (National Heart, Lung, and Blood Institute), for example, has developed cord blood banking procedures and has provided templates for investigators who wish to obtain FDA approval. Production Assistance for Cellular Therapies (PACT) facilities is a publicly funded GMP service that will allow investigators to obtain GMPquality cells without investing huge amounts in infrastructure. The NIDDK (National Institute of Diabetes and Digestive and Kidney Diseases) has likewise worked in partnership with pancreatic islet groups to standardize protocols and studies to help the entire field move forward. The number of INDs filed has increased dramatically in the last year or so, and we feel that it is not long before a cell product will be licensed for therapy. Biotechnology investments have seen a rebound as the economic climate has changed, and a significant portion of the investment has been in stem cells, nanotechnology, and other cutting-edge therapies. Investors have recognized that although regulations and rules are a burden they do remove uncertainty, making risks and benefits easier to evaluate. We expect that once the extent of regulatory delay is known and can be factored in changes instituted in the past few years will make for a healthy investment climate. As one surveys the stem cell landscape, it becomes clear that some stem cells or progenitor cells are already in therapy, some cells are in early clinical studies, and some have great promise based on animal studies (summarized in Table 3.6). To date most of the reported benefits have come from HSC and HSC-related products. It appears to us that the next cell type that may prove of utility is the MSC where several companies and investigators have reported utility either as an adjuvant to HSC to improve homing, as a source of cells for replacing defective cells in osteogenesis imperfecta, or as a source of trophic molecules in a variety of injuries in a variety of tissues. NSCs and progenitor cells are being explored for use in lysosomal storage disorders and treatment of PD, spinal cord injury, and stroke. Stem Cell, Inc. and Geron are two companies with submitted INDs. Unfortunately, the promise of these preliminary results and the relative ease with which one can perform some of these experiments have led to a proliferation of offshore clinics that purport to use stem cells to treat any or all diseases. In our opinion, these clinics have little to recommend to the scientific community, as they remain
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Stem and Precursor Cells for Transplant Therapy TABLE 3.6 Current clinical trials of cell therapy.
Cell Type
Candidate Diseases
Clinical Trial Performed
Bone marrow stem cells
Heart failure Severe long bone nonunion fractures Acute lymphocytic leukemia Neoplasm metastasis renal cell carcinoma Metastatic or recurrent kidney cancer Recurrent testicular cancer Disorders of heart muscle Cornea disease Severe stress urinary incontinence Severe systemic sclerosis
Texas Heart Institute Aastrom Biosciences, Inc. Various places
Hematopoietic stem cells
Thigh muscle cells Cornea cells Muscle stem cell from patient arm T-cell depleted autologous peripheral stem cells Adult neural stem cells Chondrocyte cells Macrophages isolated from the patient’s own blood
Parkinson’s disease
Cedars-Sinai Medical Center, Los Angeles, CA Tigenix, Inc., Belgium Proneuron, Inc.
Damaged knee joint Spinal cord injury
unwilling to share their results, conduct appropriate double-blind trials, or subject their data to peer review. Although we do not deny that some people may obtain some benefit, we would remind all readers that purported magical remedies have been available throughout recorded history. Many never survived scientific scrutiny, and in some cases were shown to be harmful. Some that showed benefit, however, were co-opted by the scientific establishment, and those then could be improved upon, standardized, and used as a base for a further round of improvement or modification. It is this scientific method that is crucial to progress and widespread applicability, and any unwillingness to subject oneself to this scrutiny should raise a red flag of caution. Scientists themselves have not been immune to the hype, and many claims have been made about the “magical” properties of these cells — such as how they can identify sites of injury, steer themselves to that site, recognize the type of damage that exists, and begin to respond in a tissue- and site-specific manner. These claims have led to passionate counterclaims and discord within the community. We, however, believe that success for this field will come in measured paces following accepted scientific protocols and performing rigorous peer-reviewed experiments with no unseemly haste to rush to the clinic. The phrase “hasten slowly” seems appropriate here. Efficiency in experimental design and care in performing controls will reduce the number of times
Mount Sinai Hospital Osaka University Medical University of Innsbruck, Austria UPMC Cancer Centers
one rushes up a promising blind alley, and will allow one to achieve success quicker than leaping at every potentially exciting news item. There will be failures, but we will learn from them and we believe there is a high likelihood of success.
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Goldman, S. A. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat Med 9:439–447; 2003. Ogawa, Y., Sawamoto, K., Miyata, T., Miyao, S., Watanabe, M., Nakamura, M., Bregman, B. S., Koike, M., Uchiyama, Y., Toyama, Y., Okano, H. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J Neurosci Res 69:925–933; 2002. Olanow, C. W., Goetz, C. G., Kordower, J. H., Stoessl, A. J., Sossi, V., Brin, M. F., Shannon, K. M., Nauert, G. M., Perl, D. P., Godbold, J., Freeman, T. B. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson’s disease. Ann Neurol 54:403–414; 2003. Park, C. H., Minn, Y. K., Lee, J. Y., Choi, D. H., Chang, M. Y., Shim, J. W., Ko, J. Y., Koh, H. C., Kang, M. J., Kang, J. S., Rhie, D. J., Lee, Y. S., Son, H., Moon, S. Y., Kim, K. S., Lee, S. H. In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons. J Neurochem 92:1265–1276; 2005. Pera, M. F. Human pluripotent stem cells: A progress report. Curr Opin Genet Dev 11:595–599; 2001. Perrier, A. L., Tabar, V., Barberi, T., Rubio, M. E., Bruses, J., Topf, N., Harrison, N. L., Studer, L. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA. 101:12543–12548; 2004. Pevny, L., Rao, M. S. The stem-cell menagerie. Trends Neurosci 26:351–359; 2003. Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani, G., Dina, G., Galli, R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi, A. L., Martino, G. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422:688–694; 2003. Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B. E., Goldstein, R. S. Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells. Stem Cells 2005. Rakic, P. Elusive radial glial cells: Historical and evolutionary perspective. Glia 43:19–32; 2003. Rao, M. S. (ed). Neural development and stem cells, 2nd ed. Totowa, NJ, Humana Press, 2005. Rao, M. S., Noble, M., Mayer-Proschel, M. A tripotential glial precursor cell is present in the developing spinal cord. Proc Natl Acad Sci USA 95:3996–4001; 1998. Reubinoff, B. E., Itsykson, P., Turetsky, T., Pera, M. F., Reinhartz, E., Itzik, A., Ben-Hur, T. Neural progenitors from human embryonic stem cells. Nat Biotechnol 19:1134–1140; 2001. Rosler, E. S., Fisk, G. J., Ares, X., Irving, J., Miura, T., Rao, M. S., Carpenter, M. K. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 229:259–274; 2004. Schuldiner, M., Eiges, R., Eden, A., Yanuka, O., ItskovitzEldor, J., Goldstein, R. S., Benvenisty, N. Induced neuronal
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differentiation of human embryonic stem cells. Brain Res 913:201–205; 2001. Schulz, T. C., Palmarini, G. M., Noggle, S. A., Weiler, D. A., Mitalipova, M. M., Condie, B. G. Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci 4:27; 2003. Seaberg, R. M., Smukler, S. R., Kieffer, T. J., Enikolopov, G., Asghar, Z., Wheeler, M. B., Korbutt, G., van der Kooy, D. Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages. Nat Biotechnol 22:1115–1124; 2004. Setoguchi, T., Nakashima, K., Takizawa, T., Yanagisawa, M., Ochiai, W., Okabe, M., Yone, K., Komiya, S., Taga, T. Treatment of spinal cord injury by transplantation of fetal neural precursor cells engineered to express BMP inhibitor. Exp Neurol 189:33–44; 2004. Snyder, E. Y., Daley, G. Q., Goodell, M. Taking stock and planning for the next decade: Realistic prospects for stem cell therapies for the nervous system. J Neurosci Res 76:157–168; 2004. Steindler, D. A., Laywell, E. D. Astrocytes as stem cells: Nomenclature, phenotype, and translation. Glia 43:62–69; 2003. Tao, H., Ma, D. D. Evidence for transdifferentiation of human bone marrow-derived stem cells: Recent progress and controversies. Pathology 35:6–13; 2003. Walder, S., Ferretti, P. Distinct neural precursors in the developing human spinal cord. Int J Dev Biol 48:671–674; 2004. Watanabe, K., Nakamura, M., Iwanami, A., Fujita, Y., Kanemura, Y., Toyama, Y., Okano, H. Comparison between fetal spinal-cord- and forebrain-derived neural stem/progenitor cells as a source of transplantation for spinal cord injury. Dev Neurosci 26:275–287; 2004. Wobus, A. M., Boheler, K. R. Embryonic stem cells: Prospects for developmental biology and cell therapy. Physiol Rev 85:635–678; 2005. Wu, P., Tarasenko, Y. I., Gu, Y., Huang, L. Y., Coggeshall, R. E., Yu, Y. Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat. Nat Neurosci 5:1271–1278; 2002. Zeng, X., Cai, J., Chen, J., Luo, Y., You, Z. B., Fotter, E., Wang, Y., Harvey, B., Miura, T., Backman, C., Chen, G. J., Rao, M. S., Freed, W. J. Dopaminergic differentiation of human embryonic stem cells. Stem Cells 22:925–940; 2004. Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., Thomson, J. A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19:1129–1133; 2001. Zhang, Z. G., Jiang, Q., Zhang, R., Zhang, L., Wang, L., Arniego, P., Ho, K. L., Chopp, M. Magnetic resonance imaging and neurosphere therapy of stroke in rat. Ann Neurol 53:259–263; 2003.
4 Creating Animals for Cell Xenotransplantation David White Robarts Research Institute, University of Western Ontario Siebens Drake Centre, London, Ontario, Canada
The rapid advances made in molecular biology over the last two decades have opened the door to a wide range of therapeutic options that would have seemed fanciful science fiction not so very long ago. Nowhere has this advance been more spectacular or more controversial than in the genetic engineering of animals. The possibility now exists that animals can be “created” for specific therapeutic or agricultural applications. Public antagonism to genetically modified (GM) crops in certain countries has made the agricultural industry wary of pursuing use of genetically modified livestock as a component of the food chain. However, the use of genetically modified animals used for therapeutic purposes is, for the most part, considered acceptable. This chapter addresses the challenges of engineering animals — specifically the pig — as a source of cells for cell therapy in patients.
The Development of Microinjection The publication in 1982 by Palmiter and Brinster [55] of their transgenic “supermouse” and its appearance on the front cover of Nature announced the arrival of the new field of genetic engineering in the most dramatic fashion. As a matter of record, these were not the first transgenic animals (created almost by accident) produced by this group [14]. There were four different laboratories [22, 34, 83] that in 1980 demonstrated almost simultaneously that DNA injected into a mouse oocyte would become incorporated into the mouse genome. Chronologically, Gordon and colleagues were first. However, the construct used was cDNA and so was not expressed. In 1981, Costantini and Lacy and Wagner et al. [22, 83] both produced transgenic mice whose genome contained foreign globin genes, although Cellular Transplantation: From Laboratory to Clinic
neither group was able to demonstrate convincingly that the gene was indeed producing the protein in an appropriate manner. At that time, Palmiter — who was doing research into metallothioneines (MT) — had constructed a plasmid consisting of the MT promoter and the thymidine kinase gene from the herpes simplex virus [56]. This plasmid was sent to Ralph Brinster, who injected it into mouse oocytes (and fertilized eggs) and who upon inducing the promoter by exposure to cadmium was able to demonstrate an increase in thymidine kinase [14]. Brinster then took some of the injected eggs and transferred them to pseudopregnant surrogate mothers, thereby producing mice with much increased levels of thymidine kinase compared to controls. Next, the gene was incorporated into the genome in multiple copies and inherited by the offspring in a simple Mendelian fashion. The expression of thymidine kinase varied considerably between offspring [13, 56]. Thus, the technique of creating transgenic technology was firmly established. Brinster and Palmiter wanted to use this technique to cure a genetic disease. The model they settled on was the “little” mouse that lacked growth hormone and hence did not grow. Thus, a plasmid was constructed using the MT promoter and rat growth hormone. However, the experiment had to be delayed due to the time needed to build up a colony of little mice. It was during this delay that Brinster injected the growth hormone construct into the fertilized oocytes of normal mice and created the supermouse [55].
Embryonic Stem Cells and Knockouts Microinjection of DNA into fertilized oocytes provided the proof of principle that genes may be transferred successfully between species and continue to function.
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However, the technique has many limitations. The procedure itself is highly skilled, success rates are low (typically between 1 and 7% of eggs injected become transgenic animals), and results are extremely hit or miss. The location within the genome where the foreign gene becomes integrated is totally random, and for most constructs expression of the product is dependent on the site of integration. Thus, it is often the case that many transgenic animals must be produced before a founder with sufficient levels of expression can be identified. In addition, although microinjection provides a valuable methodology for adding a gene to an animal’s genome it does not provide a technology for removing a gene (termed a knockout) or replacing one gene with another (termed a knock-in). This state of affairs was at least partially rectified by the development of embryonic stem (ES) cell technology for mice. It has long been known that if the cells from the blastocysts of two different strains of mice were mixed the resultant offspring were chimeras between these two individuals. Termed allophenes or tetraparentals (because the offspring had four parents), such mice were often used to study developmental biology, tolerance, and reproductive development [6, 35]. Although most such chimeras were made between mouse strains, the technology has been applied to crossspecies chimeras [30, 62]. In 1981, Evans and Kaufman derived from mouse embryo cell lines termed ES cells, which under stringent culture conditions were immortal although remaining genetically normal. When mixed with cells of a blastocyst, these cells also produced chimeric mice. More importantly, the genetics of these ES cells could be passed on in the germ line of the chimera to produce offspring carrying the pure genetics of the ES cell line used [29]. The next challenge was to develop a technique for performing homologous recombination in these cell lines. Homologous recombination uses matching DNA sequences flanking the gene of interest to replace one gene with another. Because the new gene can be constructed to be nonfunctional, such technology allows genes to be silenced or knocked out. In 1987, two groups [28, 78] separately reported that they had successfully targeted genes in ES cells, with the latter describing new selection techniques that greatly improved the efficiency of homologous recombination. Production of transgenic mice using ES cell technology has produced many startling biological discoveries and has destroyed many cherished beliefs. However, despite several reports of
the discovery of ES cells in other species to date this technique for generating transgenics can only be applied to mice.
Dolly the Sheep and Other Animals The failure to develop ES cell technology for any species other than mice has meant that creating transgenics in larger species has been restricted to microinjection and the chance inherent in that technology. Some years ago we created pigs transgenic for a human complement regulatory protein hDAF by microinjection [24]. In total, 79 different founder transgenic animals were produced. Approximately half of these produced mRNA, and these were bred for one generation to allow a total analysis of tissue expression of the transgene. Variation in expression levels was vast both between pigs and between different cell types within the pig [23]. This inability to predict expression was both expensive and time consuming. In addition, there was no practical way of knocking out any genes from the pig. The arrival of Dolly the sheep in 1996 completely changed the situation [89]. Although the technique used for producing Dolly is generally referred to as cloning, a more accurate term would be nuclear transfer (NT). The methodology of nuclear transfer is remarkably simple in principle. The genetics desired are established in a primary cell culture (typically a fibroblast). In the case of Dolly, these were mammary cells taken from the udder of a six-year-old ewe. The mammary cells were cultured for several days and then fused with a sheep oocyte, from which the nucleus had been removed by micromanipulation. The oocyte with its new nucleus was then implanted into a foster mother. After 277 attempts, Dolly was born. Dolly’s existence challenged one of the fundamental tenets of developmental biology. Most scientists had thought cell differentiation was irreversible. After all, even over a 90-year lifespan a hepatocyte remains a hepatocyte, a nerve cell a nerve cell. The production of a live lamb from a cell taken from the udder of a 6-year-old sheep demonstrated that differentiated cells are not immutable. At the time of original publication, it was thought that the most likely explanation was that the cells used could by chance have been a mammary stem cell. It is now known that nuclear transfer can be successfully achieved from many different types of differentiated cells and in several different species. The birth of Dolly sparked a global interest
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in nuclear reprogramming of somatic cells and animal cloning. Nuclear transfer technology is now widely used to produce animals from somatic cells. In the last few years, nuclear transfer has been used in a number of different mammalian species and has resulted in live births of cattle [41], goats [4], pigs [57, 61], horses [32], cats [93], and dogs [45]. In that pluripotent ES cells in domestic animals have not yet been developed, the technology of animal cloning provides an alternative methodology for gene targeting in these animals. Genetargeted animal clones have been successfully produced in sheep [49] and pigs [59] by homologous recombination in fetal fibroblast cells followed by nuclear transfer.
Making Pigs The technology for genetically altering pigs is now in place. Although efficiencies remain low, the level of success is sufficient that lines of pigs can be created with extra genes added or genes knocked out. The question that remains unanswered is which genes could allow pigs to be designed to provide cells for therapeutic transplantation. To begin to address this question, a basic understanding of the immunology of xenotransplantation is needed. To date, all pigs have been genetically engineered with the specific aim of generating either kidneys or hearts for xenotransplantation. The value of such pigs as donors for cell therapy is slowly being addressed. Thus, it is important to focus on the rejection process involved in vascularized xenograft rejection and extrapolate only those data that are relevant to cell therapy. Hyperacute rejection causes the rapid loss of function of a vascularized graft. Complement activation, usually mediated by antibody binding, leads to platelet aggregation, vasoconstriction, and loss of vascular integrity. The cardinal histological signs of hyperacute rejection are interstitial hemorrhage, edema, focal ischemia, and thrombosis. Activation of the complement cascade is of central importance in hyperacute rejection. The complement system consists of a triggered enzyme cascade of more than twenty serum proteins and five cell surface proteins. The primary function of the system is protection against invasive microorganisms. This is achieved by complement components alone, by a lytic mechanism involving cascade to the membrane attack complex, or by the recruitment of nonspecific effector mechanisms
such as phagocyte cells. The system consists principally of two activation pathways (there is a third called the mannose binding pathway), a single terminal pathway, regulatory proteins, and complement receptors. The function of the activation pathways is to generate C3 convertases. The classical pathway consists of C1, C4, C2, and C3, and is activated by antigen-antibody complexes. C1 binds to the Fc region of an antibody, and catalyzes the proteolytic cleavage of C4 and C2, generating the classical pathway C3 convertase, C4b2a. The alternative pathway functions in the absence of antibody. There are two possible mechanisms of initiation of the alternative pathway. One is by the spontaneous hydrolysis of the intramolecular thioester bond of C3. This hydrolyzed C3 reacts with serum factors B and D and forms an initial C3 convertase (C3bBb), which is able to split multiple C3 molecules to C3b. The other mechanism is that the thioester bond of C3 is directly attacked by a hydroxyl group available at a surface, resulting in binding of a C3b-like C3 to the surface and generation of C3bBb. In solution, the C3bBb convertase is unstable and factor B is readily displaced by factor H, to form a complex that is inactivated further by factor I. Similarly, if C3b or C3b-like C3 binds to a host surface regulatory mechanisms prevent further cascade. Foreign unprotected surfaces are able to stabilize the C3bBb enzyme in its active form and protect the C3b from factor H. Because the product of the alternative pathway C3 convertase is itself a subunit of the enzyme, an amplification loop is generated. The pathways are also interactive (see Figure 4.1). The C3b generated by the classical pathway C3 convertase complexes with factor B. The complement system cascades further by the cleavage of C5. C3b achieves this by serving as an attachment site for C5. C5 attaches to C3b and assumes a conformation that renders it susceptible to cleavage by neighboring classical and alternative pathway C3 convertases. C5b generated by the cleavage of C5 may remain loosely bound to C3b. Sequential attachment of C6 and C7 to C5b forms a complex with a transient membrane binding site. The membrane-associated complex is then joined by C8, yielding C5b678. C8 directs the insertion of up to 18 molecules of C9 in the lipid bilayer, yielding the annular membrane attack complex (MAC) consisting of C5b678 and poly C9. C5b678 is able to contribute to ion and water flux across a membrane, but the cell lysis is predominantly caused by the poly-C9 complex.
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David White Classical pathway
iC4b C4 + C2
C1 + antibody MCP
C4b C3 convertase C4b2a DAF
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DAF C3 convertase C3bBb C3b MCP Alternative C3 + B pathway iC3b Figure 4.1. Activation pathways of the complement cascade, showing the sites of action of MCP and DAF.
Regulators of Complement Activation (RCA) Complement activation triggers powerful effector mechanisms against which host cells must be protected. Regulatory proteins of the complement system provide a means of preventing lysis of the organism’s own cells (autologous lysis). These regulatory proteins may be classified into two categories: • •
Inhibitors that down-regulate activation of complement in the fluid phase Regulators that control complement activation on target cells
A range of proteins fall into the second category of regulators. These include the following proteins. • • • • • •
C1 inhibitor Decay accelerating factor (DAF, CD55) Membrane cofactor protein (MCP, CD46), factor H, complement receptor 1 (CR1, CD35) Complement receptor 2 (CD21) C4 binding protein (C4bp) CD59
All of these proteins are involved in down-regulating complement action on host tissue. C1 inhibitor acts at the initial stage of the complement cascade, inhibiting C1 activation. CD59 functions by inhibiting the assembly of the MAC. The final common pathway of both the alternative and the classical complement pathway is the production of C3b, controlling both the alternative and classical pathways. DAF, MCP, and the other RCA molecules act at this critical stage of C3 activation. There is a degree of species specificity in the action of complement regulators. Indeed, complement regulatory molecules help separate self from non-self. Thus, pig RCA is not active against human complement and human regulators and is only partially active in nonhuman primates. Atkinson and colleagues have discussed these concepts in detail [2].
Inhibition of Complement Activation in Xenotransplantation There are three different approaches to the regulation of complement activation for xenografting.
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Systemic inhibition of complement activation by the administration of a soluble complement
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inhibitor (e.g., sCR1). This approach has the disadvantage of being systemic in that it blocks all complement activation. Thus, although such an approach could be acceptable in the short term, in the long term it carries with it a significant increased risk of infections, particularly in the immunosuppressed individual. Removal of recipient naturally occurring antispecies antibody or modification of the graft to remove antigenic targets (the knock-out pig). This approach does not down-regulate antibodyindependent (alternative pathway) complement activation, and is not likely therefore to completely prevent complement-mediated rejection of xenografts [40]. The specific inhibition of complement activation by expression of human regulators of complement activity on grafted tissue. This allows normal complement-dependent immunity in the host while inhibiting both classical and alternative pathways of complement activation locally on the xenograft.
It is postulated that the transgenic expression of human regulators of complement activity (RCA) on the tissues of pigs should prevent human complement activation, thus overcoming perhaps the most formidable hurdle to graft survival in pig-to-human xenotransplantation. It has been demonstrated that transfection of DAF and MCP protects animal cells from human-complementinduced lysis. DAF was more effective than MCP, and DAF/MCP transfectants were no more effective than DAF transfectants [25, 88]. DAF was therefore selected as a molecule that could potentially inhibit human complement activation on pig cells. The production of transgenic pigs expressing the human DAF molecule was undertaken with the primary purpose of establishing whether this could combat hyperacute rejection of pig organs in primate recipients. However, its value in protecting islet cells has also been evaluated.
Pigs Transgenic for Complement Regulatory Proteins It has been known for many years that inhibition of complement activation would inhibit xenograft hyperacute rejection. It was a widely held belief that porcine complement regulatory proteins were unable to regulate the activation of primate complement [2, 25]. Recent studies [15, 39] have suggested that failure of the
pig to express porcine CD55 in an appropriate fashion may be more significant than species incompatibility in complement-mediated xenograft destruction. Curiously, porcine CD55 is structurally different from other CD55 analogues and is more effective at inhibiting human complement than pig complement [58]. This is clearly an area that requires much more study. Although complement is the predominant player in hyperacute rejection, its role in vascular rejection and the relative importance of antibody and complement in the xenograft rejection process is less clear. There can be no doubt that anti-pig antibodies either elicited or “naturally occurring” are involved in the vascular rejection process [66]. However, the evidence of their ability to damage the graft in the absence of complement is based on in vitro models [7]. It is clear from recent studies with organs from Gal knockout pigs that complement in the absence of antibody binding will not cause hyperacute rejection. The role of complement in acute vascular rejection remains to be resolved. Whereas Wang and colleagues [84] were able to demonstrate that a rat heart xenograft was not rejected by a B-cell knockout mouse, Johnson and colleagues [40] demonstrated that rabbit hearts were hyperacutely rejected by neonatal pigs in the complete absence of antibody. Neither model seems of much relevance for testing pig-to-human transplants. Genetically engineered nonhuman primates with knockouts for B-cells or C3 would be needed to resolve this issue. Whatever the relative importance of antibody and complement in the xenograft rejection process, there is overwhelming evidence that expression of human complement regulatory proteins on pig cells protects them from human complement. The credit for first demonstrating this goes to a rarely quoted publication by Oglesby and colleagues [54]. Confirmation of these findings followed rapidly both in vitro [16, 25, 85] and in vivo using transgenic mice [17, 92] followed by transgenic pigs [24]. These studies used animals expressing a single complement regulatory transgene CD55, CD46, or CD59. A recent study by Loveland and colleagues [46] documented in detail the benefits of expressing complement regulatory proteins on porcine xenografts. They demonstrated that normal pig kidneys transplanted into non-immunosuppressed adult baboons were hyperacutely rejected in a median of 3.5 h, whereas CD46 transgenic grafts harvested at approximately 24-hour intervals posttransplant remained essentially undamaged for up to 68 h. The issue of how these results might apply to cell therapies has not yet
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been addressed. However, there have been some studies on islet xenotransplantation that have sought to address this issue.
Islet Xenotransplantation Since the 1970s, it has been demonstrated repeatedly in experimental animal models that islet allotransplantation could successfully be used to reverse diabetes [5, 65]. This advance has prompted research into developing methods for the isolation of human islets, which in turn has led to human islet transplantation into the liver via the portal vein of immunosuppressed patients with kidney transplants [67, 69, 74, 86]. Initial promising results of insulin independence in these patients proved short-lived, however, as it became apparent that most patients reverted back to hyperglycemia. Indeed, there is a striking anomaly when islet allotransplantation is compared to pancreas transplantation. Approximately 60% of the pancreas recipients remain insulin independent 3 years posttransplant [76], compared to only 7% at one year for islet recipients [48, 87]. The reason for such a stark difference in success rates remains obscure considering that the histocompatibility barrier, the immunosuppressive reagents employed, and the underlying autoimmune disease are the same in both types of transplant. However, it should be emphasized that islet autotransplantation for the treatment of surgically induced diabetes restores insulin independence in only 50% of cases [53, 68]. Although this demonstrates the functional capacity of islet transplantation per se, it also illustrates the problems associated with islet separation technology and the importance of islet integrity and islet cell mass to the restoration of carbohydrate control. This bleak picture of islet transplantation has, however, been transformed by the development of the Edmonton protocol [72]. The success of this protocol has been attributed to a number of factors. These include avoiding “diabetogenic” drugs, the use of freshly prepared islets, not performing transplants in kidney transplant recipients, and repeat transplantation. Although the long-term (5-year) results of the Edmonton protocol as measured by insulin independence on a relatively small number of patients have been disappointing, the overall benefit to restoration of carbohydrate control to the type 1 diabetic has been demonstrated by these allotransplants. However, as a potential cellular therapy for type 1 diabetes islet allotransplantation is never going to be a realistic option. There are approximately
8,000 type 1 diabetic patients per million people. Current organ donation rates are less than 30 per million of population. Given that the Edmonton protocol requires the islet yield from two and sometimes three pancreases, allotransplantation can at best treat 0.2% of the patient population in one year. Thus, an unlimited source of glucose-responsive cells is required as an essential component of the treatment of diabetes by cell transplantation. One such potential source could be appropriately genetically engineered pigs. Professor Carl Groth and colleagues have made a number of novel observations on the immune response elicited by adult pig islets when placed in contact with human (in vitro) or primate (in vivo) blood. Their insights have shed light on how to modify these pigs to provide islets suitable for transplantation into humans. It was believed at the outset of this study that unlike organ xenotransplants islet xenotransplants do not undergo hyperacute rejection [37]. The aim of the research was to establish if complement activation was in any way involved in the rejection of islet xenografts and if the use of islets from pigs transgenic for the human complement regulator hDAF described previously [24, 43] would benefit islet xenograft survival. In vitro analysis was performed using a loop of heparin-bonded PVC tube on a rocking device [50]. The tube contained 7 mls of whole human blood. When 5,000 porcine islets were introduced into these loops, they rapidly induced a cascade of undesirable events. These can be summarized briefly as induction of the clotting cascade with loss of platelets and WBC, activation of complement, and release of insulin (100,000 pmol/L), indicating islet cell death. This reaction has many of the pathological features of hyperacute rejection. Disappointingly, when islets from hDAF transgenic pigs whose organs were resistant to hyperacute rejection were introduced into these “loops” no difference was observed in the responses compared with that seen with nontransgenic islets. A FACS analysis of the islets from these transgenic pigs subsequently revealed that the transgene was not expressed on these islets [9]. An unexpected finding associated with this FACS analysis was that human islets (originally used as controls for the digestion process) also failed to express hDAF. Human islets expressed human CD59 well, but human MCP (CD46) was poorly expressed. In contrast, porcine islets expressed both porcine CD59 and porcine MCP at high levels (no reagent to detect porcine DAF was available). The expression levels of the major
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xenogeneic epitope Gal were low or undetectable on API, whereas significant levels of Gal were present on fetal islet cells. Porcine and human endothelial cells subjected to the same digestion protocol as the islets expressed appropriate levels of DAF, MCP, and CD59. The observation that human islets do not express hDAF stimulated Groth and colleagues [10, 11] to introduce human islets into the loop with ABO-compatible human blood taken from healthy (i.e., not diabetic) volunteers. These human islets induced responses similar to those seen with adult pig islets. The clotting cascade was activated, platelets and white cells were lost, complement was activated, and insulin was released. In these experiments, however, the amount of insulin released was significantly less (33,600 pmols/l) than that seen with the pig islets. The majority of this insulin release occurred during the first 10 minutes of perifusion and could not be accounted for by the levels of glucose in the blood. In all of these in vitro experiments, both with human and porcine islets, the relationship between the observed events remains unclear. For example, does the complement activation initiate the clotting cascade or vice versa? Is complement activation directly responsible for the destruction of islets or are the released proinflammatory products (C3a and C5a) activating granulocytes and monocytes that subsequently damage the islets. In vivo studies were undertaken by transplanting adult pig islets intraportally into cynomolgus monkeys. Two groups of monkeys (n = 4 per group) were transplanted with 20,000 IE/kg API. One group had been pretreated with sCR1 to inhibit complement activation. Levels of
insulin in the blood were then monitored over the next hour. The results (see Figure 4.2) show that there is a significant insulin release (interpreted as a surrogate for islet destruction) in the untreated group. This insulin release is greatly reduced when complement activation is inhibited [50]. All of these data taken together strongly support the contention that adult pig islets exposed to human or primate blood suffer significant immediate posttransplant damage. This conclusion is supported by the studies of Contreras and colleagues [20, 21] using rhesus monkey serum and by Rayat et al. [64] using human serum, both of whom reported similar porcine islet cell destruction. In contrast, Mirenda and colleagues [51, 52] failed to achieve islet cell lysis when exposing pig islets to human serum that was diluted 50%. This suggests that destruction of porcine islets is highly concentration dependent (though of what is not known). It is also unclear as to what percentage of islets is rendered nonfunctional by this humorally mediated damage. Furthermore, if pig islets are damaged by humoral factors in primate and human serum why do they survive when transplanted into nude diabetic mice [26]? There are at least three possible explanations for this: (1) nude mice have inadequate complement activity [71], (2) antibody critical to the destruction of the islets is T-cell dependent [82], or (3) the site of transplantation (in this case, under the kidney capsule) confers some protection to the pig islets. In support of this last hypothesis, Brandhorst and colleagues [12] demonstrated that pig islet transplanted under the kidney capsule of nude diabetic rats could maintain reasonable carbohydrate control while
Insulin (pmol/L)
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Figure 4.2. Intraportal transplantation of porcine islets into primates. Graph shows the effect of complement inhibition (TP 10 = sCR1) on posttransplant insulin levels [50].
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Days posttransplant Figure 4.3. Adult porcine islet function in nude rats (intraportal versus subcapsular transplants). Adapted from [12].
transplantation via the portal vein to the liver resulted in failure (see Figure 4.3). These experiments give important pointers as to the ways in which pigs might be genetically engineered to reduce the immune responses described previously. The failure of the human transgene to be expressed on the islet b cells also highlights the importance of selecting the appropriate promoter when constructing animals specifically designed to provide therapeutic cells. At present, the only animals that have been genetically engineered with the specific aim of providing cells for xenotransplantation are tilapia (a fish) into which the human insulin gene has been introduced [60].
Clinical Islet Xenotransplantation Dr. Rafael Valdes at Hopital de Infantil de Mexico in Mexico City has performed a small controversial study of porcine to human islet transplantation in type 1 diabetic patients. Twelve patients with C-peptide negative type 1 diabetes were transplanted with neonatal pig islets (5,000/kg) and Sertoli cells (35 to 100/islet) derived from an SPF herd of pigs. The cells were inserted into neovascularized collagen tubes created by implanting a closed stainless steel mesh containing an inner Teflon stent subcutaneously 8 weeks prior to transplantation [80]. No immunosuppression was given. Of the 12 transplanted patients, 6 have functioning grafts as defined by reduction of exogenous insulin requirement HbA1c < 8% and production of porcine insulin
in response to IV glucose challenge. Biopsies of the grafts revealed the presence of insulin- and glucagonpositive cells, although the overall architecture of the islets was destroyed. Follow-up extends to more than 4 years, with two patients currently insulin independent. The presumption is that these cells survive in this subcutaneous environment because the Sertoli cells induce a T-cell deficit via Fas-Fas ligand interactions [8]. The ability of Sertoli cells to provide tropic and nutritive factors that stimulate germ cells and support islets in vitro [47, 75] must also be taken into account when interpreting the overall results of this study. However, it remains unclear how these islets can survive in the presence of both preexisting and elicited anti-pig antibody. It seems possible that the artificially created collagen chamber may provide a protective environment comparable to that of the kidney capsule in nude mice and rats. This chamber cannot, however, be considered an immunologically privileged site, nor can it be compared with encapsulation technology. Although the Sertoli cells may prevent T-cell-mediated rejection, there are clearly other factors that protect the islets from destruction by antibody and complement. There is a temptation to speculate that graft accommodation [1, 3] may be taking place. However, there is currently no evidence to support (or refute) this hypothesis. Curiously, in all of the reported studies in the literature involving the transplantation of porcine islets under the kidney capsule of nude mice not a single investigator has published an analysis of the antibody responses produced by these animals. Given the recent elegant studies of
50
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de Vriens and colleagues [81, 82] on accommodation of hamster hearts in nude rats, this is clearly an area of research that needs to be further explored. Simeonovic and colleagues [73] published a detailed analysis of such antibody responses in immunocompetent mice and reported that the capacity of such hyperimmune sera to induce rejection of pig proislets (recently transplanted into CD4 T-cell-depleted recipients) after adoptive transfer was highly dependent on levels of IgG3. In contrast, Gourlay and colleagues [36] reported that adoptive transfer of hyperimmune anti-pig antibodies into nude mice bearing subcapsular porcine islets for more than 70 days failed to induce islet destruction. Transfer of immune T-cells did induce rejection, whereas naïve T-cells had no effect. In neither of these studies did the authors measure the complement competence in the recipients. These studies suggest that once revascularized islet xenotransplants become relatively insensitive to antibody destruction, particularly T-independent antibody. A detailed analysis of the elements involved in providing protection to these islets could well provide evidence for genetically engineering pigs that would give better survival in this interesting new approach to cell therapy.
Clinical Neural Xenotransplantation Another potential area for developing genetically engineered pigs is the supply of fetal neurons for the treatment of Parkinson’s disease and Huntington’s disease. The observation that human fetal neurons are able to survive and function when allografted into the adult brain without the use of immunosuppressive drugs suggests that cellular therapy is a promising approach to achieve neuronal replacement for treatment of diseases of the adult central nervous system (CNS). The use of human fetal tissue is limited by ethical, infectious, regulatory, and practical concerns. Pigs are a reasonable alternative source of fetal neuronal tissue because of their brain size, large litters, and the extensive experience in rearing them in captivity under controlled conditions. Fink and colleagues [31] have reported preliminary clinical studies of porcine fetal neural cells grafted unilaterally into Parkinson’s disease patients. Clinical improvement of 19% was reported in 10 patients assessed 12 months after unilateral striatal transplantation of 12 million fetal porcine ventral mesencephalic cells. In a single autopsied patient, some porcine cells were detected 7 months after transplantation [27]. Twelve Huntington’s disease
patients showed no change in total functional capacity score 1 year after unilateral striatal placement of up to 24 million fetal porcine striatal cells. Xenotransplantation of fetal porcine neurons is a promising approach to delivery of healthy neurons to the CNS. However, minimizing rejection remains a significant challenge in which genetic engineering of the donor species could play an important part.
Other Transgenic Pigs Generated for Xenotransplantation There have been a number of other genetically engineered pig lines developed for xenotransplantation. Although these have been produced primarily with organ transplantation as the goal, their potential in cell therapy should not be neglected. A brief summary of the current status of these pigs is included here.
KNOCKOUT PIGS The antibody response induced by pigs in humans is predominantly against the Gal epitope. For this reason several groups [38, 42, 44, 59, 63, 77] have knocked out the galactosyl transferase gene (Gal-KO), which controls its expression. The presumption that this would prevent vascular rejection was by no means certain because aggressive vascular rejection will occur between Gal-positive donors and recipients such as hamster to rat and pig to dog. There is a unanimous consensus that this manipulation prevents hyperacute rejection [19, 43, 91] even in porcine lungs [70]. This observation eliminates the concept that in pigto-primate transplantation hyperacute rejection could be induced by activation of the alternative pathway of complement. The most complete analysis of the effect of this knockout on graft survival has come from the Sachs group. Hearts from Gal-KO pigs transplanted heterotopically into baboons using an antiCD154 monoclonal antibody-based immunosuppressive regime regimen survived between 2 and 6 months, with a median of 78 days [43]. The predominant lesion associated with graft failure was a thrombotic microangiopathy, with resulting ischemic injury. Four baboons receiving either xeno-thymokidneys or vascularized thymic lobe plus kidney from Gal-KO pigs all survived longer than 2 months. Furthermore, none
51
David White
of these kidneys was lost to rejection [90, 91]. Studies by Chen et al. using Gal-KO kidneys have been less encouraging [19]. Recipients survived between 8 and 16 days, with 4 of 6 grafts failing due to severe acute humoral xenograft rejection. Circulating induced anti-non-Gal antibodies, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells, were markedly elevated at rejection. The immunosuppression used in these studies was different from that of the Sachs group in that it was not based on an anti-CD154 monoclonal antibody regime, nor was a thymokidney technique used. With several groups just starting to test Gal-KO organs in vivo, and with the development of Gal-KO hCD55 transgenic pigs [77], there is obvious potential for the advancement of xenotransplantation toward the clinic.
CONTROL OF THROMBOSIS Microthrombi formation and fibrin deposition are universal pathological findings in the acute vascular rejection of a xenograft. Several of the studies reviewed here and elsewhere have also reported systemic coagulopathys in recipients of xenografts. These observations have started to focus attention on whether it is possible to genetically engineer pigs to be phenotypically less pro-coagulant. Although these studies are still in their infancy, two deserve special mention here. Ghanekar and colleagues [33] cloned the porcine equivalent of fibrinogen-like protein 2 (fgl2), which is an inducible prothrombinase that plays an important role in the pathogenesis of fibrin deposition during viral hepatitis. Studies of pig-to-baboon kidney xenografts undergoing AVR in vivo revealed induction of pfgl2 expression on graft vascular endothelial cells that could account for the thrombosis characteristic of this form of rejection. In support of this, hearts transplanted heterotopically from fgl2 knockout mice into Lewis rats were devoid of thrombosis upon rejection, whereas transplants from wild-type mice developed AVR with intravascular thrombosis. One awaits the arrival of a pfgl2 knockout pig with interest. Chen and colleagues [18] have produced transgenic mice that express membrane-tethered fusion proteins based on human tissue factor pathway inhibitor or hirudin. Heterotopic hearts transplanted into non-immunosuppressed Lewis rats survived 6.7 and 7 days, respectively, whereas wildtype controls survived only 2.8 days. When recipient rats were immunosuppressed with Cyclosporin A wild-type
hearts, the hearts were rejected in 3 days, whereas hearts from both transgenic mice survived for more than 100 days showing normal histology. It can only be a matter of time before the production of pigs bearing these transgenes.
Conclusions and Future Directions The development of nuclear transfer techniques in the pig has given an impetus to genetically engineering of this species for the purpose of providing organs and tissues for clinical transplantation. It would seem that the extent of modifications should be limited only by imagination. Indeed, the number of research teams producing transgenic pigs has increased, as has the variety of transgenes and knockout options that can be applied. These include complement regulatory strategies, epitope elimination by knockouts, control of thrombosis, and even somewhat curiously the addition of HLA-D molecules [79]. Xenograft survival times continue to improve in the exacting and expensive pig-to-baboon model. However, these have yet to consistently achieve results comparable to current allograft survival. This limitation poses a serious dilemma for the future development of xenografting. Short- to medium-term allograft results are so successful that until they can at least be approximated by xenografts the application of xenotransplantation in the clinic will be problematic. Perhaps the most promising avenue for moving xenotransplantation to the clinic is in the deployment of cell therapies. Both islet and neural xenotransplants from pigs to patients are in current clinical practice. Both offer the advantage that there is no appropriate supply of human cells as an alternative source either because of donor shortage or ethical inhibitions. Both techniques avoid the use of immunosuppressive drugs, and perhaps using cells from specifically engineered porcine donors could in both cases enhance the results achieved.
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Rowe, J. D. Production of sheep-goat chimeras by inner cell mass transplantation. J Anim Sci 65:325–330; 1987. Ramsoondar, J. J., Machaty, Z., Costa, C., Williams, B. L., Fodor, W. L., Bondioli, K. R. Production of alpha 1,3galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase. Biol Reprod 69:437–445; 2003. Rayat, G. R., Rajotte, R. V., Elliott, J. F., Korbutt, G. S. Expression of Gal alpha(1,3)gal on neonatal porcine islet beta-cells and susceptibility to human antibody/complement lysis. Diabetes 47:1406–1411; 1998. Reckard, C. R., Barker, C. F. Transplantation of isolated pancreatic islets across strong and weak histocompatibility barriers. Transplant Proc 5:761–763; 1973. Richards, A. C., Davies, H. F., McLaughlin, M. L., Copeman, L. S., Holmes, B. J., Dos Santos Cruz, G., Bycroft, S., Chavez, G., White, D. J., Schuurman, H. J., Cozzi, E. Serum anti-pig antibodies as potential indicators of acute humoral xenograft rejection in pig-to-cynomolgus monkey kidney transplantation. Transplantation 73:881–889; 2002. Ricordi, C., Tzakis, A. G., Carroll, P. B., Zeng, Y. J., Rilo, H. L., Alejandro, R., Shapiro, A., Fung, J. J., Demetris, A. J., Mintz, D. H., et al. Human islet isolation and allotransplantation in 22 consecutive cases. Transplantation 53:407–414; 1992. Robertson, G. S., Dennison, A. R., Johnson, P. R., London, N. J. A review of pancreatic islet autotransplantation. Hepatogastroenterology 45:226–235; 1998. Scharp, D. W., Lacy, P. E., Santiago, J. V., McCullough, C. S., Weide, L. G., Boyle, P. J., Falqui, L., Marchetti, P., Ricordi, C., Gingerich, R. L., et al. Results of our first nine intraportal islet allografts in type 1, insulin-dependent diabetic patients. Transplantation 51:76–85; 1991. Schroeder, C., Allan, J. S., Nguyen, B. N., Wu, G., Zhang, T., Azimzadeh, A. M., Madsen, J. C., Schuurman, H. J., Sachs, D. H., Pierson, R. N. III. Hyperacute rejection is attenuated in GalT knockout swine lungs perfused ex vivo with human blood. Transplant Proc 37:512–513; 2005. Schulz, G., Staffileno, L. K., Reisfeld, R. A., Dennert, G. Eradication of established human melanoma tumors in nude mice by antibody-directed effector cells. J Exp Med 161: 1315–1325; 1985. Shapiro, A. M., Lakey, J. R., Ryan, E. A., Korbutt, G. S., Toth, E., Warnock, G. L., Kneteman, N. M., Rajotte, R. V. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238; 2000 Jul 27. Simeonovic, C. J., Townsend, M. J., Wilson, J. D., McKenzie, K. U., Ramsay, A. J., Matthaei, K. I., Mann, D. A., Young, I. G. Eosinophils are not required for the rejection of neovascularized fetal pig proislet xenografts in mice. J Immunol 158: 2490–2499; 1997. Socci, C., Falqui, L., Davalli, A. M., Ricordi, C., Braghi, S., Bertuzzi, F., Maffi, P., Secchi, A., Gavazzi, F., Freschi, M., et al. Fresh human islet transplantation to replace pancreatic
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endocrine function in type 1 diabetic patients: Report of six cases. Acta Diabetol 28:151–157; 1991. Suarez-Pinzon, W., Korbutt, G. S., Power, R., Hooton, J., Rajotte, R. V., Rabinovitch, A. Testicular sertoli cells protect islet beta-cells from autoimmune destruction in NOD mice by a transforming growth factor-beta1-dependent mechanism. Diabetes 49:1810–1818; 2000. Sutherland, D. E. International Pancreas Registry. Minneapolis, University of Minnesota, 1997. Takahagi, Y., Fujimura, T., Miyagawa, S., Nagashima, H., Shigehisa, T., Shirakura, R., Murakami, H. Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III. Mol Reprod Dev 71: 331–338; 2005. Thomas, K. R., Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512; 1987. Tu, C. F., Hsieh, S. L., Lee, J. M., Yang, L. L., Sato, T., Lee, K. H., Weng, C. N., Mao, S. J., Tsuji, K., Lee, C. J. Successful generation of transgenic pigs for human decay-accelerating factor and human leucocyte antigen DQ. Transplant Proc 32:913–915; 2000. Valdes, R., Martin, S., Cravioto, A., Tenopala, J. Biological encapsulation as a new model for preservation of islets of Langerhans. Transplant Proc 30:481; 1998. Vriens, P. W., Pollard, J. D., Hoyt, G., Morris, R. E., Scheringa, M., Bouwman, E., Robbins, R. C. Hamster cardiac xenografts are protected against antibody mediated damage, early after transplantation to Lewis rats. Xenotransplantation 8:239–246; 2001. Vriens, P. W., Stoot, J. H., Hoyt, G., Scheringa, M., Bouwman, E., Robbins, R. C. Pre-transplant blood transfusion induces tolerance to hamster cardiac xenografts in athymic nude rats. Xenotransplantation 8:247–257; 2001. Wagner, T. E., Hoppe, P. C., Jollick, J. D., Scholl, D. R., Hodinka, R. L., Gault, J. B. Microinjection of a rabbit betaglobin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc Natl Acad Sci USA 78:6376–6380; 1981. Wang, H., DeVries, M. E., Deng, S., Khandaker, M. H., Pickering, J. G., Chow, L. H., Garcia, B., Kelvin, D. J., Zhong, R. The axis of interleukin 12 and gamma interferon
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regulates acute vascular xenogeneic rejection. Nat Med 6: 549–555; 2000. Wang, M. W., Wright, L. J., Sims, M. J., White, D. J. Presence of human chromosome 1 with expression of human decay-accelerating factor (DAF) prevents lysis of mouse/human hybrid cells by human complement. Scand J Immunol 34: 771–778; 1991. Warnock, G. L., Kneteman, N. M., Ryan, E. A., Rabinovitch, A., Rajotte, R. V. Long-term follow-up after transplantation of insulin-producing pancreatic islets into patients with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 35:89–95; 1992. Weir, G. C., Bonner-Weir, S. Scientific and political impediments to successful islet transplantation. Diabetes 46: 1247–1256; 1997. White, D. J., Oglesby, T., Liszewski, M. K., Tedja, I., Hourcade, D., Wang, M. W., Wright, L., Wallwork, J., Atkinson, J. P. Expression of human decay accelerating factor or membrane cofactor protein genes on mouse cells inhibits lysis by human complement. Transpl Int 5 Suppl 1:S648–650; 1992. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., Campbell, K. H. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813; 1997. Yamada, K. Life-supporting pig-to-baboon renal transplantation using GalT knockout donors: Benefit of cotransplanting a vascularized donor thymic graft. Kidney Int 68:1971; 2005. Yamada, K., Yazawa, K., Shimizu, A., Iwanaga, T., Hisashi, Y., Nuhn, M., O’Malley, P., Nobori, S., Vagefi, P. A., Patience, C., Fishman, J., Cooper, D. K., Hawley, R. J., Greenstein, J., Schuurman, H. J., Awwad, M., Sykes, M., Sachs, D. H. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med 11:32–34; 2005. Yannoutsos, N., Ijzermans, J. N., Harkes, C., Bonthuis, F., Zhou, C. Y., White, D., Marquet, R. L., Grosveld, F. A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection. Genes Cells 1:409–419; 1996. Yin, X. J., Lee, H. S., Lee, Y. H., Seo, Y. I., Jeon, S. J., Choi, E. G., Cho, S. J., Cho, S. G., Min, W., Kang, S. K., Hwang, W. S., Kong, I. K. Cats cloned from fetal and adult somatic cells by nuclear transfer. Reproduction 129: 245–249; 2005.
B Cell Therapy for Peripheral Diseases and Reconstructive Applications
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5 Islet Transplants for Diabetes: The Edmonton Protocol Peter A. Senior, BMedSci, MBBS, Ph.D., MRCP (UK) Assistant Professor, Department of Medicine, Clinical Islet Transplant Program, University of Alberta
A. M. James Shapiro, MBBS, Ph.D., FRCS, FRCPC Associate Professor, Department of Surgery, Clinical Islet Transplant Program, University of Alberta
Introduction Diabetes is a common condition whose prevalence is increasing rapidly worldwide [63]. Diabetes is characterized by hyperglycemia due to an absolute or relative deficiency of insulin. Chronic hyperglycemia is associated with a number of chronic complications that place a huge burden on affected individuals, their families, and health care systems. Chronic complications include microvascular disease; diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy; and macrovascular disease. Diabetic retinopathy is the most common cause of blindness in working-age adults. End-stage renal disease due to diabetic nephropathy is the leading indication for renal replacement therapy (renal dialysis) [5]. Macrovascular disease, predominantly coronary artery and cerebrovascular disease, is responsible for the majority of deaths in people with diabetes [69, 107]. Diabetes is associated with a twofold to fourfold increased risk of death [68] and a significant reduction in life expectancy. Insulin is a peptide hormone secreted by beta cells in response to blood glucose levels. Beta cells make up approximately 40% of cells in the islets of Langerhans. Islets are found in the pancreas, which is predominantly an exocrine gland. The islets make up the endocrine component that represents a tiny proportion of the pancreas. Insulin plays a key role in glucose homeostasis. Its major action is to lower blood glucose levels by promoting glucose uptake into fat, liver, and muscle, Cellular Transplantation: From Laboratory to Clinic
and by inhibiting glycogenolysis and gluconeogenesis. Insulin also has important effects on fat and protein metabolism. In clinical practice, insulin is administered subcutaneously by injections several times per day or continuously using a pump.
PATHOGENESIS OF DIABETES Type 1 diabetes results from an autoimmune process characterized by lymphocyte infiltration of the islets and “insulitis” [45, 86]. This leads to an absolute deficiency of insulin over a relatively short period. Males and females are affected equally, and there is a peak incidence during early teenage years and the disease usually presents before the age of 40. Insulin therapy is required in all cases in order to prevent metabolic decompensation. Formerly, the terms juvenile onset and insulin-dependent diabetes were used for this condition. The majority (> 85%) of diabetes worldwide is due to type 2 diabetes, formerly known as maturity onset or noninsulin-dependent diabetes. Hyperglycemia in type 2 diabetes is due to a relative deficiency of insulin. Defects in insulin action (insulin resistance) and of insulin action have both been described [34]. Type 2 diabetes is associated with obesity and physical inactivity, which are both associated with insulin resistance. Hyperinsulinemia as a compensatory response to insulin resistance, in a vain attempt to maintain euglycemia, is commonly seen in pre-diabetes and in the early stages of type 2 diabetes. Nevertheless, the natural history of type 2 diabetes is
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generally for insulin secretion to progressively decline over a number of years. Thus, although type 2 diabetes is usually treated by lifestyle changes and oral medication insulin therapy may be required with longer duration of diabetes.
run their blood sugars high deliberately in order to avoid hypoglycemia.
LIMITATIONS OF INSULIN THERAPY FOR DIABETES
A cure for type 1 diabetes is a highly attractive proposition and is the ultimate goal for many researchers and lay organizations (e.g., the Juvenile Diabetes Research Foundation is “dedicated to finding a cure”), as well as for affected individuals and their families. A number of strategies for restoration of endogenous insulin secretion have been considered and tested. Prevention of type 1 diabetes has thus far been unsuccessful [4, 44], although much has been learned about identifying individuals at risk. It is now possible to predict, with reasonable accuracy, the likelihood of developing diabetes in families at risk for type 1 diabetes [86]. Similarly, efforts to reverse type 1 diabetes in its early phases by manipulating the immune environment have shown little benefit [1, 21, 125]. Prevention of type 2 diabetes has been more successful, with lifestyle modification as well as pharmacological interventions proving effective [64, 65, 133]. Transplantation has been another strategy to restore functional beta cell mass. Whole-pancreas transplantation was pioneered in the late 1960s and is now well established as a treatment for selected cases of type 1 diabetes. Most commonly, pancreas transplantation is performed simultaneously with a kidney transplant [51]. Solitary pancreas-alone or pancreas after kidney transplants are, however, being performed more commonly than before [51]. Whole-pancreas transplantation as a treatment for type 1 diabetes is subject to a number of important limitations. The transplant involves a major and complex surgical procedure, with a substantial risk to the patient’s life. At one year, graft survival is > 80%, with patient survival being approximately 95% [51]. In addition, lifelong immunosuppression is required, further adding to the risks of pancreas transplantation. The very limited supply of suitable organs is a major obstacle limiting the availability of this treatment. Despite these limitations, successful pancreas transplantation can result in a robust and sustained restoration of euglycemia [105] superior to that achievable with insulin therapy and without the risk of hypoglycemia. Indeed, this success has been associated with the reversal of chronic diabetic complications [38].
The discovery of insulin and its introduction to clinical practice by Banting and Best, assisted by MacLeod and Collip, in 1921 was a major advance in diabetes care [17]. Prior to this, type 1 diabetes was associated with a dire prognosis and was universally fatal, usually within weeks or months. Although preventing these early deaths, the improved survival with insulin therapy was followed by a huge burden of complications and premature deaths from coronary artery disease and renal failure. Progress has been made over the years. Advances in insulin preparations, delivery devices, and technology for self-monitoring of blood glucose level have been helpful. Intensive insulin therapy can delay the development or progression of microvascular complications of diabetes [3], and the proportion of people developing microvascular complications appears to be declining [57]. However, these complications are not completely prevented, most likely because of an inability of insulin therapy to achieve euglycemia or normoglycemia. In contrast, the restoration of normoglycemia by successful whole-pancreas transplantation can lead to a resolution of the histological changes of diabetic nephropathy [38]. Thus, despite progress in insulin therapy diabetes continues to have a major impact on the lives of people with type 1 diabetes. Multiple daily injections, frequent selfmonitoring of blood glucose levels, careful attention to dietary carbohydrate intake, and exercise are required to achieve good glycemic control. This is associated with a reduced risk of, but is not a guarantee of freedom from, complications. Hypoglycemia is a major factor limiting the ability of insulin therapy to achieve normoglycemia. The intensive insulin therapy employed in the diabetes control and complications trial was associated with a threefold increase in hypoglycemia [3]. Hypoglycemia is a serious complication that can be associated with coma, seizure, or even death. Fear of hypoglycemia is a further difficulty, leading many individuals with type 1 diabetes to
ALTERNATIVE APPROACHES
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Islet Transplants for Diabetes: The Edmonton Protocol
Islet transplantation seeks to achieve the benefits of endogenous insulin secretion from the restoration of functional beta cells but with reduced risk. Major surgery is not required, and potential complications from multiple anastamoses and transplantation of the exocrine pancreas are avoided. The smaller amount of transplanted tissue may also represent a reduced alloantigen load.
Historical Perspectives on Islet Transplantation Islet transplantation is not a new idea. An attempt to transplant fragments of ovine pancreas into a child with diabetic ketoacidosis was reported in the nineteenth
century [137]. Interest waned following the discovery of insulin, but was rekindled as a result of the poor outcomes of early whole-pancreas transplants [129]. (See Figure 5.1.) The successful isolation of rat islets from the pancreas was pioneered at Washington University, St. Louis, under the direction of Dr. Paul Lacy in the 1960s [67]. Subsequently, transplantation of these islets was shown to reverse chemically induced diabetes in animal models [9]. Despite much enthusiasm and the initial success of islet transplantation in animal models, the transition to clinical practice proved difficult and frustrating. To further emphasize the slow pace of clinical islet transplantation, it was 1989 before insulin independence was achieved, albeit briefly, following islet transplantation in a type 1 diabetic subject with a previous kidney transplant [114].
Figure 5.1. Islet milestones.
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Improved outcomes in clinical islet transplantation were reported not long afterward. Insulin independence rates of more than 50% at one year were achieved in 10 patients receiving combined liver and islet allotransplants following upper abdominal exenteration (including pancreatectomy) for cancer [103]. However, insulin independence in this series of islet allotransplants was not achieved in any patients with preexisting diabetes. Interestingly, the immunosuppression received by the subjects receiving combined liver and islet transplants did not include steroids, in contrast to the regimen employed in diabetic subjects. Successful islet transplantation, predominantly in association with kidney transplantation, in type 1 diabetes was achieved by the European GRAGIL consortium (based in Geneva and Giessen) [12]. This improved success was attributed to advances both in peritransplant management and in immunosuppressive therapy. However, insulin independence rates at one year were only about 20%. Early experience in auto islet transplantation (which did not require immunosuppression) highlighted a
number of problems in using the islet preparations available at that stage. These preparations were impure, containing a large amount of exocrine tissue and other contaminants. Consequently, several serious complications were encountered: portal vein thrombosis, portal hypertension, and ultimately liver failure [134]. At this point, islet allotransplants were generally combined with another solid organ transplant that required and justified immunosuppression. Despite earlier optimism, serious complications and relatively disappointing insulin independence rates suggested that the benefits of islet-alone transplantation were outweighed by the risks. The future of islet-alone transplants seemed in doubt. The report in 2000 of insulin independence in all of seven type 1 diabetic subjects receiving islet-alone transplants at the University of Alberta in Edmonton [121] was followed by much interest in the medical and scientific communities as well as among the general public. Subsequently, there has been a huge expansion in clinical islet transplantation as well as basic and clinical research. (See Figure 5.2.)
Figure 5.2. Islet transplant activity (1999–2005).
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Islet Transplants for Diabetes: The Edmonton Protocol
Current Clinical Islet Transplantation PANCREAS SELECTION AND HARVESTING Human islets are isolated from pancreata harvested from brain dead donors. Donor selection is an important factor contributing to the likelihood of isolating a sufficient quantity of high-quality islets. The pancreas is a sensitive organ. Activation of the exocrine pancreas digestive enzymes leading to autodigestion is associated with damage to islets. Furthermore, islets are highly susceptible to ischemia because they are metabolically active and require excellent blood flow to maintain their health [20, 74]. Careful procurement of pancreata from stable donors with minimal periods of warm or cold ischemia is a key element for successful islet transplantation. The advent of the two-layer method using perfluorocarbon (thought to improve oxygen delivery to the islets) has been associated with improved islet yields [79, 100, 132]. It can be difficult to separate islets from exocrine tissue in younger donors, and islet mass also seems to be less in lean donors. Generally, pancreata from slightly older and heavier donors (> 45 years) are preferred because of improved islet yield [71, 84]. Care is taken to avoid potential donors who have known or undiagnosed diabetes. The optimal donor for islet transplantation is relatively distinct from that for whole-pancreas transplantation, where a young lean donor is optimal. This is fortunate because it avoids competition for organs between islet and whole-pancreas programs [104].
ISLET ISOLATION AND CULTURE Even though the pancreas is a sensitive organ, the process of islet isolation [102] requires the pancreas to be subjected to a hostile environment. The initial stage involves the cannulation of the pancreatic duct and controlled distension of the pancreas with collagenase [72]. Subsequently, the pancreas is cut into small pieces and placed in the Ricordi chamber, a stainless steel container, along with stainless steel marbles. The chamber is perfused with a blend of enzymes at 37◦ C and agitated. With this combination of chemical and mechanical dissociation, islets are freed from exocrine tissue. Digestion of the pancreas is generally complete within less than 30 minutes. Islets are separated from exocrine tissue over a series of density gradients using a COBE cell
separator. Generally, the final islet preparation consists of < 5 cc of tissue. The use of liberase as the digestive enzyme for islet isolation represented an advance over the commercially available collagenases used previously, which exhibited substantial variability in enzyme activity between batches [75]. Liberase is a purified blend of collagenase isoforms 1 and 11. Earlier reports suggested that there was good reproducibility between Liberase lots [70]. Nevertheless, substantial variation in islet yields persisted. More recently, evaluation of collagenase blended with a neutral protease at the time of use has yielded promising results [23]. Prior to release, islets are subject to a number of quality control tests, including a gram stain to rule out bacterial contamination and an assay to exclude significant amounts of endotoxin. Further assays of biological efficacy can also be employed, including measurement of insulin content, insulin secretion in response to low and high glucose (stimulation index), or bioassays such as transplantation into diabetic mice. The release criteria are outlined in Table 5.1. Previously, freshly isolated islets were suspended in transplant medium and infused immediately [121]. Long-term culture of islets is associated with a loss of endocrine cells [115]. However, it is clear that islets can be successfully cultured for several days prior to transplantation without significant loss of potency [56]. Indeed, the ability to culture islets prior to transplantation has a number of advantages. Safety is improved because transplants can be scheduled when the entire transplant team can be present. Time is also available to administer conditioning or other immunosuppressive therapy, avoiding exposure of islets to the cytokine release associated with many cell-depleting induction therapies. Logistically, patients no longer need to live close to the transplant center for indeterminate
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TABLE 5.1 Release criteria for clinical islet transplantation. Parameter
Cut Point
Islet number Packed cell volume Islet viability Endotoxin content Gram stain
> 4,000 ie/kg < 10 cc > 70% < 5 EU/kg No bacteria seen
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periods of time. Indeed, islets can be successfully shipped from the isolation facility to distant transplant centers [48]. This opens up the potential for a small number of core islet isolation facilities, which would likely have economies of scale because islet isolation must be performed in a cGMP facility. Preliminary data from a multicenter trial of islet transplantation indicate superior outcomes from experienced centers and confirm the steep learning curve associated with islet isolation [120, 123]. Thus, the concentration of expertise in a small number of facilities seems sensible. Although there is generally some loss of islets during the culture period, there is an increase in islet purity because of more rapid loss of exocrine tissue. This increased purity, along with the associated reduction in the volume of tissue to be infused, has further safety advantages. Smaller and more pure preparations are associated with a reduced risk of portal hypertension and portal thrombosis. Thus, the ability to culture islets prior to transplant has a number of benefits both in terms of practicality and safety.
QUANTITY OF ISLETS NEEDED TO CURE DIABETES There is substantial heterogeneity between isolated islets, particularly in terms of their size but also in their shape. The ability to quantify islet number is important in order to compare clinical outcomes. The concept of an islet equivalent (IE) as the unit of enumeration of islets is a result of the variability in islet size. An IE attempts to combine islet size and number into one parameter. The reference value for an IE is a spherical islet with a diameter of 150 µm. Counts of islets in a small aliquot are performed in duplicate. After staining with dithizone, islets are counted manually under the microscope and a tally of islets of different sizes kept [10, 101]. From this an estimate of the number of IEs can be made. Counting islets is time consuming and there is substantial variation between observers in the estimates. This is exacerbated by the irregular, nonspherical nature of most islets. A number of strategies to automate counting islets and improve both the accuracy and precision of the count are being explored [47, 73]. Precise estimates of the number of islets in a human pancreas are difficult to find. It seems that under certain physiological conditions beta cell mass can expand;
for example, during pregnancy (which is a temporary state of insulin resistance) [14]. There is still debate as to whether this is the result of islet hyperplasia or islet hypertrophy (i.e., an increased number of islets or an increase in islet size), although animal data would favor the latter [18]. The most reliable technique for estimating islet number is using stereological principles [19]. This technique has been used to reliably estimate islet number and volume in animal models. Despite this uncertainty, it is clear that glucose tolerance can be maintained even despite significant reductions in total beta cell mass. Rates of diabetes following partial and subtotal pancreatectomy, particularly for neoplasia, provide the best data to support this. Diabetes is a universal complication after total or subtotal pancreatectomy, where more than 80% of the pancreas is removed. Removal of smaller proportions of the pancreas (40 to 80%) is associated with the development of diabetes in less than 50% of subjects [126]. There seems to be a relationship between glucose tolerance and the amount of pancreas removed [124]. The same phenomenon seems to apply in diabetes. Although difficult to study directly, it is estimated that despite the progressive loss of beta cells in type 1 diabetes euglycemia can be maintained until approximately 90% of the beta cell mass is lost [135]. Thus, it would be expected that replacement of only a small proportion of a normal total complement of islets would be required to restore insulin independence following islet transplantation. Insulin independence in the original series of seven patients from the University of Alberta was achieved after infusion of approximately 11,000 IE/kg in two (or more) islet infusions [121]. In a follow-up report, infusion of > 9,000 IE/kg (approximately 857,000 islets per recipient) was necessary for insulin independence to be achieved [108]. Although this total represented approximately 85% of the approximate one million islets in a normal pancreas, and although insulin independence was achieved, the ability of the transplanted islets to secrete insulin fell well short of that in normal control subjects. During the intravenous glucose tolerance test, the acute insulin response to glucose was 19% and the area under the curve for insulin was 36% compared with controls [108]. These data support suggestions that a significant proportion of islets are lost during the transplant procedure or fail to engraft [16]. More recent data indicate, however, that insulin independence can be achieved with a smaller number of
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islets (7,271 IE/kg), and indeed from a single donor [55]. In the 5-year follow-up data from 69 patients transplanted at the University of Alberta there is no longer any clear relationship between insulin independence and number of islets infused [109]. A further area of uncertainty in relationship to the number of islets infused is whether a greater islet mass will give more robust, sustained insulin independence. The natural history of type 2 diabetes seems to suggest that after a period of initially successful compensation for insulin resistance there is a steady decline in beta cell mass with time [136]. The concept of islet stress and islet rest (to preserve and sustain beta cells) would support the hypothesis that a larger islet mass would be more likely to provide longer-term insulin independence. Although the notional number of islets infused in islet transplantation approaches the number in a normal pancreas, the number of islets successfully engrafting is almost certainly a fairly marginal islet mass. Sustained (> 5 years) insulin independence is unusual after islet transplantation [109] but much more common after successful whole-pancreas transplantation, where a full complement of islets is transplanted within a well-vascularized graft with an intact microvascular structure.
program have often led to the acceptance of organs from marginal donors that previously would have been declined. Consequently, the proportion of isolation procedures yielding preparations suitable for clinical use has remained static, or perhaps even declined in some well-established centers. The inefficiency of the isolation procedure contributes to the need for more than one islet infusion to attain insulin independence. Thus, currently the ratio of pancreata to insulin independence is probably greater than 4. Even if this ratio were improved to 1 or even 0.5 (one pancreas treating two patients), the current supply of pancreata means that islet transplantation would remain a very specialized treatment for a highly selected minority of type 1 diabetic patients. Single-donor success is an important goal if islet transplantation is to be used more widely. It is achievable but probably relies, in part at least, on very strict selection of donor and recipient selection, and on strict criteria for islet preparations (in terms of islet number and purity) [122]. Insulin independence is much more likely in small insulin-sensitive individuals.
Clinical Islet Transplantation RECIPIENT SELECTION
Inefficiency of Islet Isolation and Transplantation High-quality pure islet preparations have been a key development contributing to recent advances and success in clinical islet transplantation. Unfortunately, it is not possible to reliably achieve an islet preparation suitable for transplantation from every pancreas that undergoes processing. Generally, about half of attempted isolation procedures yield an islet preparation of sufficient quantity or quality to be transplanted. The remainder are utilized for research — if consent for such use has been given. This inefficiency is a major hurdle. Limited organ supply is the rate-limiting step facing most solid organ transplant programs. Islet transplantation faces the same issue. This limited supply further highlights the inefficiency of the islet isolation procedure. Recently, the expansion in the number of islet transplant centers has been associated with a decline in the number of organs being offered to any individual center. A reduced number of donor organs coinciding with a desire to expand the clinical
Clinical islet allotransplantation is associated with a number of short-term procedural risks, particularly from bleeding or portal vein thrombosis. In the longer term, the major risk is from the lifelong immunosuppression required to prevent rejection. Consequently, much effort is made in assessing potential islet transplant recipients. The aim of the assessment process is first to identify individuals in whom the benefits of islet transplantation outweigh the risks, and second to ensure that the risks are fully understood by the applicant. Thus, islet transplantation has tended to be restricted to those individuals most severely affected by diabetes. In particular, individuals at high short-term risk from their diabetes — often because of frequent, severe, and recurrent hypoglycemia — are often suitable candidates for islet transplantation. Unstable blood glucose control (despite an optimized insulin regimen) and progressive secondary complications of diabetes have also been identified as potential indications for islet transplantation. In our center, advanced microvascular complications are a caution because approximately 10% have required laser photocoagulation for diabetic retinopathy
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following transplant [109], whereas progression of albuminuria has been noted in 40% of subjects [118]. Early after transplant, there was a clear decline in renal function in a small number of subjects with preexisting renal impairment [108]. Heavy proteinuria, apparently associated with sirolimus, has also been noted in three cases [116]. Other functions of assessment are to exclude any alternative approach that could be used to address the difficulties for which islet transplantation may be indicated or the presence of other co-morbidities. Such co-morbidities could increase the risk of the procedure (e.g., underlying coronary artery disease, hepatic hemangiomata, and thrombophilia) or the risk of side effects of immunosuppression (e.g., gastroparesis and anemia). The wait for islet transplantation can be lengthy. Currently, the wait in Edmonton is approximately 2 years. Consequently, our practice is to review listed candidates on an annual basis to ensure that they still have indications for islet transplantation and have not developed any contraindications, and to provide an update on the latest clinical outcomes.
CROSS-MATCHING, TISSUE TYPING, AND IMMUNOSUPPRESSION Similar to liver transplantation, islet grafts have been assigned to ABO-compatible recipients and have not been matched for HLA type. Earlier, a cross match between donor spleen cells and recipient blood was routinely performed retrospectively for first transplants. Prospective cross matches are performed for individuals with high PRA (panel reactive antibody) or prior to second, or subsequent, islet infusions. There has been a transition from the earlier hemagglutination-based techniques for measuring PRA and performing the cross match to a flow-cytometrybased technique. It has become apparent that the new flow method is much more sensitive than the older assays. Preliminary retrospective analysis by the flowbased method of the “negative” (by the old method) cross matches in cases with early graft loss has suggested that alloimmunity and HLA mismatch may have played a significant role in the loss. Previously, the loss of these grafts had been postulated to be mediated mainly by recurrent autoimmunity.
SELECTING A RECIPIENT FOR AN ISLET PREPARATION
TRANSPLANTATION IN INDIVIDUALS WITH HIGH PRA
When an islet preparation of sufficient quality becomes available, a suitable recipient must be identified and called to the transplant center. Because islet transplants can be successfully scheduled, listed recipients no longer have to reside in Edmonton. Nevertheless, they must be prepared to travel to Edmonton at short notice. Key factors influencing recipient selection include ABO blood type, body weight, and the number of islets in the preparation. Preparations containing larger numbers of islets will preferentially be given to a larger individual. If small numbers of islets are given to a large recipient, it is likely that multiple procedures would be required to achieve insulin independence. This should be avoided because there are operative risks with each procedure. In addition, multiple procedures are associated with a sequential rise in portal pressure [27] and with an increased risk of bleeding (Villiger, in preparation). Patients who require a further transplant because they have not yet attained insulin independence are transplanted in preference to those who have not yet received any islet infusion.
Because the current flow-based assay for PRA is more sensitive, there has been an increase in the number of cases with positive PRA. This has necessitated the development of procedures to facilitate the identification of suitable islet preparations for recipients with high PRA. One aim has been to avoid the need to call a patient from a distant location to the transplant center only to find that the prospective cross matching is positive. This involves several steps.
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The specificities of the antibodies causing the positive PRA (i.e., the HLA types the recipient has antibodies against) are identified. When the tissue typing of the donor is known, this can be used to rule out recipients with specific antibodies to that donor’s tissue type. Blood from recipients with high PRA is sent to the HLA lab each month and stored. If a recipient does not have specific antibodies against a donor, these samples can be used to perform a preliminary cross match against donor spleen cells. Where this is
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negative, the patient can be called to the transplant center. A final cross match is performed at the time of transplant.
The process for identifying a suitable candidate is thus significantly more complex and involved than when the current clinical islet transplant program was initiated.
IMMUNOSUPPRESSION Successful islet transplantation for type 1 diabetes requires immunosuppression that can effectively control alloimmunity but can also control the autoimmune process that caused diabetes in the first place. A further challenge is to avoid the use of agents that are known to be toxic to islets — particularly corticosteroids, which have been the mainstay of immunosuppression since successful allotransplantation was first developed. The avoidance of steroids in islet transplantation seems to have been a key step in the recent advances in successful islet transplantation. In place of steroids, daclizumab (a humanized antibody to the interleukin 2 receptor) is administered intravenously immediately prior to transplantation at a dose of 1 mg/kg. This was repeated every 2 weeks for 5 doses in our initial transplants. Currently, our standard immunosuppressive regimen repeats the dose of daclizumab only once. Maintenance immunosuppression was achieved using sirolimus and a low dose of tacrolimus. Sirolimus is a novel agent that appears to be associated with less nephrotoxicity and diabetogenicity than calcineurin inhibitors (such as cyclosporine and tacrolimus). A loading dose of sirolimus (0.2 mg/kg) is given prior to transplant, followed by 0.15 mg/kg. The dose is then adjusted subsequently to achieve trough levels between 12 and 15 ng/ml for the first 3 months and between 7 and 10 ng/ml subsequently. A low dose of tacrolimus was used and adjusted to maintain trough levels between 3 and 6 ng/ml. The success of this regimen described initially at the University of Alberta has been replicated at other centers as part of a multicenter trial [120, 123]. Success has been reported with other regimens using antithymocyte globulin combined with etanercept (a monoclonal antibody that binds to tumor necrosis factor alpha) for induction and a combination of sirolimus and mycophenolate mofetil ± very-low-dose tacrolimus for maintenance [55].
Other immunosuppressive agents have been used in some cases because of drug intolerance or other side effects. Most of our experience is with a combination of tacrolimus, using higher doses with target trough levels between 8 and 10 ng/ml, and mycophenolate mofetil (up to 2 g/day as tolerated). This combination is better tolerated in individuals who are sirolimus intolerant and seems to be reasonably efficacious in terms of preserving islet function. In a small number of cases, the use of these higher doses of tacrolimus was accompanied by predictable nephrotoxicity. We are less confident in the ability of sirolimus combined with mycophenolate mofetil to provide adequate immunosuppression. The use of this combination is further limited by the side-effect profile of these drugs, which both affect the GI tract. A number of new immunosuppressive protocols are being developed and evaluated. Some of the strategies being tested include the use of antibodies to deplete the lymphocyte pool [120]. One hope is that as the immune system is reconstituted this approach may facilitate the development of tolerance. Tolerance is a major focus of much basic research because this would permit the minimization or even the avoidance of long-term immunosuppression.
THE TRANSPLANT PROCEDURE Islets are removed from culture media, recounted, and resuspended in approximately 100 ml of transplant media in a closed infusion bag system. Islets are then infused, aseptically, into the main portal vein under gravity, with regular monitoring of portal venous pressure before, during, and after the infusion. Access to the portal vein is achieved by a transcutaneous transhepatic approach using a combination of ultrasound and fluoroscopy to guide the radiologist. The procedure is performed using local anesthesia, combined with opiate analgesia and hypnotics given as premedication. Initially, a branch of the right portal vein is cannulated using a 22-gauge Chiba needle and a guide wire advanced into the main portal vein. A catheter is advanced over the guide wire and positioned proximal to the confluence of the portal vein. A portal venogram is performed routinely to confirm the position of the catheter. A description of the procedure has been published [89]. Portal venous pressure is measured by an indirect pressure transducer. Islet infusion did not proceed if
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initial pressure was > 20 mmHg or if portal pressure rose to > 22 mmHg or more than twice the baseline value [89]. Unfractionated heparin (35 units per kg per 5 cc of islet tissue) is mixed with the islet preparation immediately prior to infusion to reduce the risk of portal vein thrombosis. Some centers have advocated the use of intravenous heparin to provide full anticoagulation. The disadvantage of such anticoagulation in islet allotransplantation where a transhepatic puncture provides access to the portal vein is the increased risk of bleeding. Full heparinization is required in islet autotransplantation where large volumes of unpurified islet tissue (with an even greater potential to promote thrombosis) are infused. In autotransplantation, islets are infused into the portal vein under direct vision during the laparotomy and any bleeding can be controlled directly. Upon completion of the islet infusion, the catheter is withdrawn. A number of modifications have been made to this part of the procedure, largely to reduce the frequency of bleeding from the hepatic puncture. Earlier, gelatin sponge (gelfoam) was used to embolize the catheter tract. Subsequently, metal coils in combination with a fibrin “tissue glue” (Tisseel, Baxter) were used. Currently, a microfibrillary collagen (Avitene, Davol) is being employed without the use of metal coils. This compound can be mixed with contrast to permit visualization of its placement, reducing the risk of embolization into the portal vein. The avoidance of coils is advantageous for future imaging because the metal coils produce artifacts on MR images and cast acoustic shadows on ultrasound studies of the liver. Patients are instructed to lie on their right-hand side for 4 hours following the procedure. An ultrasound examination is performed the following day to rule out any free fluid in the abdomen and to confirm that the portal vein is patent and has normal flow. The ultrasound is repeated at one week to exclude any late portal vein thrombosis.
INTRAHEPATIC ISLET ENGRAFTMENT Although islets represent a tiny proportion of the total pancreas (1%), they receive a disproportionate share of the pancreas’ blood supply (15%) [20, 58]. This reflects the fact that islets are metabolically active. Islets are accustomed to an abundant oxygen supply. Hypoxia is therefore a major metabolic challenge to isolated islets whose microvasculature has been disrupted. Isolated
islets are now reliant on passive diffusion of oxygen. Larger islets may be even more affected because of the greater distance over which oxygen has to diffuse. The situation may not be improved following islet infusion. Islets embolize into distal branches of the portal vein, where they lodge in presinusoidal capillaries [134]. The oxygen tension within islets (pO2 8 to 10 mmHg) is lower than in the portal vein (10 to 15 mmHg), both of which are significantly lower than the oxygen tension seen in the intact pancreas (40 mmHg) [25, 96, 119]. Engraftment refers to the process whereby islets become revascularized. Although infused into the portal vein, it would seem that engrafted islets derive their blood supply from the arterial circulation [8]. Generally, revascularization is believed to take about two weeks. Nevertheless, the vascular density observed in transplanted rodent islets at one month is only half that seen in the native pancreas, suggesting that the blood supply of transplanted islets may always be suboptimal [26]. Hypoxia is likely a key factor responsible for the very marked early loss of transplanted islets. The process leading to cell death has already begun even before transplantation. Some 30% of cells within islets can be seen to be undergoing apoptosis, with beta cells particularly affected, immediately following isolation [90]. After transplantation there is further loss of cells mediated by nonimmune factors. Some studies in immunodeficient mice have demonstrated that 70% of beta cells in transplanted islets are lost in the first month [32]. This loss does not seem to be related to the transplant site (islet grafts in rodents are often placed under the kidney capsule or in the spleen as well as intraportally) [25]. Experience in clinical islet transplantation would tend to be consistent with perhaps only one-third of islets engrafting successfully [99]. Islets that successfully survive this hypoxic stress face further challenges. One of these is an inflammatory response due to activation of the clotting cascade and platelets immediately after islets come into contact with blood [13]. This response has been termed the instant blood-mediated inflammatory reaction (IBMIR). Islets constitutively express tissue factor, a potent activator of the clotting cascade, causing the IBMIR [81]. The thrombosis resulting from IBMIR is associated with the formation of a fibrin capsule around newly implanted islets and disrupted islet morphology [13, 81], and may be associated with a negative outcome in clinical islet transplantation [59]. This early inflammatory process may represent a key initiating event leading to
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macrophage infiltration and perhaps ultimately to activation of alloimmune responses. Conversely, platelet activation may be an important precursor to the establishment of a vascular supply to these newly engrafted islets [22].
Why the Liver? Insulin secretion from beta cells within native islets is relatively complex. Although it is clear that insulin is secreted in response to increases in blood glucose levels, other secretagogues (e.g., arginine) also stimulate insulin secretion. The arrangement of alpha, beta, and delta cells within islets is not random [88]. Somatostatin secreted from delta cells has paracrine actions modulating insulin secretion [50]. The autonomic nervous system represents a further influence on insulin secretion and may influence pancreas regeneration [62]. Variations in pulse frequency and amplitude are seen to occur as insulin secretion changes in different physiologic states [61]. In the native pancreas, islets receive their blood supply from the systemic circulation while insulin secreted by islets drains into the portal vein and thence to the liver. The liver extracts much of the insulin during the first pass. Consequently, in the postprandial state the concentration of insulin in the systemic circulation is significantly lower than in the portal vein. Circulating insulin levels are significantly lower in whole-pancreas transplant recipients where the venous drainage of the graft is into the portal rather than the systemic circulation [95]. First-pass metabolism in the liver affects other substances relevant to intraportal islet transplantation, including drugs and nutrients. Intraportal infusion of islets may permit delivery of pancreatic hormones, particularly insulin, directly to the liver. The ability to deliver insulin and glucagon directly to the liver is attractive from a physiological perspective. The liver is a key site of insulin action. Insulin promotes glycogen synthesis and turns off both glycogenolysis and gluconeogenesis. Glucagon has the opposite effects to insulin on glycogenolysis and gluconeogenesis, raising blood glucose levels. Glucagon plays an important role in preventing hypoglycemia. (In addition to the loss of insulin secretion, normal glucagon secretion is lost in type 1 diabetes and contributes to the susceptibility to hypoglycemia seen in some patients [46].) The secretion of insulin from intrahepatic islets may not be exactly equivalent to portal delivery of insulin
from the native pancreas. Normally, insulin secretion will result in uniform concentration of insulin being delivered throughout the liver. With intrahepatic islet transplants, there may be areas of the liver surrounding engrafted islets that have relative hyperinsulinemia. This localized hyperinsulinemia may explain the patchy focal steatosis observed in some patients after clinical islet transplantation [15]. The intrahepatic location of transplanted islets has a number of potential advantages and disadvantages. The concentration of orally administered immunosuppressant drugs in the portal circulation is significantly higher than in the systemic circulation because of hepatic first-pass metabolism [35]. Potentially this could be advantageous, in that islets and the liver could be exposed to effective concentrations of drugs using lower doses with reduced potential for systemic toxicity. In fact, monitoring venous trough levels assesses dosing of most immunosuppressive drugs. The current recommended levels are relatively high in comparison to solid organ transplants. Peak drug levels in the portal system are likely very high and potentially may be toxic to islets in the longer term [11]. Another potential disadvantage is the exposure of islets to high concentrations of nutrients in the portal vein, not yet subject to the first-pass metabolism in the liver. Potentially, this may be associated with supramaximal stimulation of islets in the postabsorptive state, which may have deleterious effects on long-term islet function.
Peritransplant Glucose Management Careful blood glucose control around the time of transplant is important. Immunosuppressive induction regimens are often accompanied by a number of side effects. Nausea and vomiting are particularly common. This together with the need to fast prior to the procedure increases the risk of hypoglycemia, which after all is the leading indication for islet transplantation. Frequent measurements of capillary blood glucose are recommended and appropriate adjustments made to insulin doses in order to avoid hypoglycemia. Generally, intravenous infusions of insulin and dextrose are started prior to the transplant procedure in order to maintain euglycemia during the transplant. Some animal data imply superior outcomes if islets are transplanted into a euglycemic rather than a hyperglycemic environment [16], although the levels of hyperglycemia
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were far higher than would be expected in clinical practice. Immediately following the islet infusion, the insulin and dextrose infusions can be stopped. The blood glucose should continue to be monitored. Hypoglycemia is not uncommon in the first 12 hours after the infusion. This likely represents unregulated insulin release from transplanted beta cells, perhaps because of injury or even death of islets. In some cases, moderate doses of intravenous dextrose (5 to 10 g per hour) are required to maintain euglycemia. In some experimental protocols intravenous insulin (1 unit per hour) has been continued after the islet infusion, along with a variable-rate glucose infusion adjusted to maintain euglycemia. This approach has been advocated by centers reporting single-donor success [55]. As well as maintaining strict euglycemia, the insulin infusion is postulated to have an antiinflammatory action. Although maintaining euglycemia in order to avoid an additional metabolic stress to newly transplanted islets seems intuitively sensible, there is no clear human data indicating the superiority of intravenous insulin over the initial approach in Edmonton of recommencing subcutaneous insulin if capillary blood glucose levels rose above 8 mmol/l (144 mg/dl) premeal or above 10 mmol/l (180 mg/dl) 2 hours following a meal. In terms of diet, patients are advised to continue to follow a healthy diet as recommended by national diabetes associations. Three regular meals per day are preferred. Additional snacks, required prior to transplant to avoid hypoglycemia, are generally no longer required even if the individual has not attained complete insulin independence. Patients are encouraged to take regular exercise, as this will facilitate good blood glucose control.
Posttransplant Monitoring After transplant, regular and careful monitoring of immunosuppressant levels is required, both to ensure that effective levels are rapidly achieved and maintained and to ensure that levels do not overshoot the target, leading to toxicity. Meticulous self-monitoring of blood glucose level is recommended to guide dosing of supplementary insulin in those who are not yet insulin independent. Insulin requirements tend to increase over the first two weeks, usually reflecting improved appetite that is generally impaired after starting immunosuppressants.
Over the subsequent few weeks, insulin requirements are reduced, presumably reflecting improvements in islet function accompanying engraftment. A decision regarding the need for a further islet infusion is generally made after one month. By this point, further improvements in islet function are unlikely to be significant and the very common elevations of hepatic enzymes will usually have resolved [98].
Assessment of Islet Function Graft function can be assessed in a number of ways; for example, using a range of metabolic tests. The interpretation of the results is not always clear. It is unusual for these data to influence clinical care significantly, unless the test indicates complete graft loss, when a decision to discontinue immunosuppression is clarified. It is likely that in this situation the test will likely be merely to confirm and document the clinical impression. Frequent severe hypoglycemia, erratic blood glucose levels, and increased insulin requirements to pretransplant levels all suggest graft dysfunction or failure. The simplest estimates of graft function can be derived from records of self-monitoring of blood glucose and daily insulin requirements. These are not completely precise because they can be influenced by factors other than graft function, such as diet and exercise. Some patients’ dietary restrictions and excessive exercise, resulting from a desire to maintain euglycemia, have resulted in an unhealthy amount of weight loss. Fasting and postprandial estimates of blood glucose and C-peptide levels can give some impression of graft function. The interpretation of C-peptide levels can be difficult, particularly in individuals who are using insulin. C-peptide levels will vary depending on blood glucose levels that are variable. Fasting C-peptide levels, for example, could be low, even below the level of detection by the assay, but would not necessarily reflect graft dysfunction if the fasting glucose were also low. In contrast, undetectable C-peptide levels in the face of hyperglycemia would indicate significant graft dysfunction. The slow turnover of glycated hemoglobin A1c , used to monitor long-term glycemic control in diabetes, means it is not useful as an early marker of graft dysfunction. Other standardized tests include a mixed-meal test or a standard 75-g oral glucose tolerance test. A disadvantage of these tests is that they cause hyperglycemia, which indeed is the stimulus for islets in these tests.
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Intravenous arginine will also stimulate insulin secretion from islets but by a glucose-independent mechanism, thus avoiding hyperglycemia. Again, the interpretation of these data is not always straightforward because within an individual there can be discordance between the results of oral mixed-meal and intravenous arginine tests. More specialized tests seek to tease out more detailed information about the ability of transplanted islets to secrete insulin. The intravenous glucose tolerance test permits the estimation of a number of parameters, including the acute insulin response to glucose as well as the area under the insulin curve and calculation of a disposition index. The glucose-potentiated arginine test relies on the fact that insulin secretion in response to arginine is markedly augmented by prior infusion of glucose. This is thought to indicate the maximum capacity of the islets to secrete insulin and has been proposed as a marker of functional beta cell mass [131]. Data from islet transplant recipients show a weak relationship between glucose-potentiated arginine insulin secretion and the number of transplanted islets [92]. Better tools to assess functional engrafted beta cell mass are still required. Other aspects of islet function have been assessed following islet transplantation, including glucagon secretion. Human studies of islet transplantation have demonstrated that the glucagon response to hypoglycemia is not restored to normal [94]. This absence of glucagon secretion is in contrast to studies in dogs receiving intraperitoneal islet transplants, which did secrete glucagon in response to hypoglycemia [53]. It also contrasts with whole-pancreas transplantation where alpha cells appear to function normally [93].
Outcomes Recent advances in islet transplantation using highly purified preparations with steroid-free immunosuppression have been associated with substantially improved outcomes. Definitions of success in islet transplantation have, of recent times at least, focused on insulin independence. Previously, when islet transplantation was less successful the presence of detectible c-peptide was the mark of success. Although insulin independence would seem to be a clearly defined, objective, and categorical variable it can contain a spectrum of success that may only be partial. Insulin independence, for example, might
rely on excessively strict dietary or exercise regimens. Similarly, individuals can be insulin independent and yet continue to have blood glucose values in the diabetic range in response to an oral glucose tolerance test — a situation not dissimilar to people with diet-controlled type 2 diabetes. Thus, insulin independence can be a relatively arbitrary state because there are no agreed levels at which insulin should either be discontinued after transplant or reinitiated with a failing graft. Insulin independence is nonetheless a very visible mark of success. The appetite for a cure for diabetes, with freedom from insulin injections as a badge of success, is huge in the medical and surgical communities as well as among patients and the general population. Some steps toward developing tools quantifying success in islet transplantation as more than just a dichotomous phenomenon have been made. The potential benefits of such a tool could be the ability to decipher incremental improvements in outcomes of different islet transplant protocols. To view anything short of insulin independence as a failure would be to underestimate the significant benefits of endogenous insulin secretion in terms of hypoglycemia avoidance and stable glycemia. There are also suggestions that such “partial success” may have benefits in terms of diabetes complications [39, 40]. The beta score is such a tool developed in Edmonton [110]. This tool gives a score for each of four parameters: fasting glucose, HbA1c , insulin independence, and stimulated c-peptide (with a maximum score of two for each parameter). The score correlates well with blood glucose levels 90 minutes after a mixed-meal test.
Insulin Independence Following initial reports of insulin independence from Edmonton there has been a rapid expansion in islet transplantation. It is estimated that approaching 500 individuals with type 1 diabetes have now received islet transplants at more than 40 institutions worldwide [120]. Edmonton’s initial success has been replicated at other centers. Insulin independence rates at one year continue to be substantial. Combined data from Edmonton, Miami, and Minneapolis indicated a one-year insulin independence rate of 82% [120, 122]. Nevertheless, the preliminary findings of the Immune Tolerance Network sponsored multicenter trial found that success rates at the three most experienced sites (> 80%) were much higher and less variable than in newly established sites (0 to 63%) [123].
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Longer-term insulin independence rates are perhaps less impressive. Of the first 69 patients transplanted in Edmonton, survival analysis indicates insulin independence rates of only 10% at 5 years [109]. Nevertheless, graft survival at 5 years as indicated by ongoing c-peptide production was 80%. Insulin independence was achieved following a single islet infusion in six subjects, after two infusions in 34, and after three infusions in 8. Three subjects did not achieve insulin independence despite receiving more than 18,000 IE/kg. Thus, insulin independence for more than one month was achieved in 48 completed transplants. The median follow-up in completed transplants was 34.9 months (range 4.1 to 67.8). Insulin independence was maintained for a median of 17 months (interquartile range 6.2 to 26.2). C-peptide persisted longer: median 27 months (range 17.9 to 36.9) [109]. (See Figure 5.3.)
Insulin Secretion and Blood Glucose Control C-peptide secretion and glucose tolerance in response to a mixed-meal test were superior in insulin-independent individuals compared with those with ongoing c-peptide production but who had resumed insulin therapy [109]. A decline in the c-peptide increment following the mixed-meal test seemed to precede the resumption of insulin therapy [109]. There was no evidence to suggest that insulin resistance contributed to the need to resume insulin therapy [109]. Blood glucose control was best in insulin-independent subjects (HbA1c 6.2%; IQ range 5.9 to 6.3), although not
Figure 5.3. Five year islet function–University of Alberta.
strictly normal (< 6.1%); intermediate in those resuming insulin therapy (6.8%; IQ range 6.5 to 7.0); and worst in subjects who had lost graft function (8.1%; IQ range 7.0 to 8.8) [109]. (See Figure 5.4.)
Hypoglycemia and Glycemic Lability Although insulin independence following islet transplantation is almost synonymous with success, there are other worthwhile outcomes that indicate clinical success. Prevention of severe hypoglycemia and stable, predictable blood glucose controls are important clinical outcomes because these are the main indications for islet
Figure 5.4. HbA1C insulin independent.
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transplantation. These improvements are highly valued by patients, their families, and their doctors. Scores to objectively quantify both hypoglycemia (HYPO score) and glycemic lability (lability index) have been developed [112]. Both scores correlate well with the clinical impression of experienced diabetologists. Islet transplant candidates have scores that are significantly higher than typical type 1 diabetic controls. Successful islet transplantation has led to normalization of both the HYPO score and lability index [112]. These objective improvements would confirm the clinical impression. Furthermore, the reductions in hypoglycemia and increased stability are attributable to graft function, rather than improved self-care, in that the loss of graft function has been associated with a deterioration in both of these scores to pretransplant levels [112]. The improvements in HYPO and LI seem to be maintained with ongoing graft function, even if insulin therapy is resumed. This partly reflects the smaller insulin doses required in those with persistent graft function who require supplemental exogenous insulin. It also reflects the buffering role played by functional islets because they secrete insulin in a glucose-dependent manner. Thus, on occasions when insulin requirements are lower than usual (perhaps because of increased physical activity or lower carbohydrate intake) even if the supplemental insulin dose was not reduced insulin secretion from engrafted islets would decrease as glucose levels fell, reducing the risk of hypoglycemia.
Microvascular Complications Amputations, blindness, and kidney failure are complications of diabetes that are greatly feared by patients. Although the risks of complications can be reduced by tight glycemic control, they are not abolished. Furthermore, most patients with type 1 diabetes are unable to achieve the strict targets for glycemic control recommended by their doctors. As indicated earlier, hypoglycemia is a major factor that limits the ability to achieve glycemic targets with insulin therapy. It is often hoped that islet transplantation will enable people with diabetes to achieve superior glycemic control and thus prevent microvascular complications. While this seems a reasonable premise, it will likely take some time to accumulate sufficient data to confirm this unambiguously. Furthermore, there are some data that would suggest this view may be overly optimistic.
There are data examining the ability of islet or pancreas transplants to prevent complications in animal models of diabetes. A consistent finding in these studies is that reversal or prevention of complications is much more likely when transplantation (and presumably normalization of glycemia) takes place soon after the onset of diabetes [80, 97]. Another theme that emerges is the importance of achieving euglycemia. In a direct comparison between islet and pancreas transplantation in diabetic rats, superior glycemic control was achieved after pancreas transplantation, and only pancreas transplantation was able to prevent glomerulopathy (the early stage of diabetic nephropathy) [127]. In human studies of whole-pancreas transplantation, there is data to suggest a beneficial effect on microvascular complications. For example, reduced proteinuria at one year has been observed [31]. However, sustained normoglycemia over 10 years is required before regression of the histological changes of diabetic nephropathy can be observed [38]. Simultaneous pancreas and kidney (SPK) transplantation is associated with a reduced need for laser therapy for diabetic retinopathy compared with kidney transplantation alone [66]. However, more than 30% of SPK recipients required laser within 3 years following transplantation [66]. This raises the possibility that there may be perpetuating factors other than hyperglycemia in operation in the context of established complications. Advanced glycated end (AGE) products, for example, turn over slowly and are prosclerotic. Thus, AGEs could promote ongoing fibrosis that is typically seen in diabetic nephropathy. The strongest evidence for an effective means of preventing microvascular complications in humans is from the diabetes control and complications trial [3]. This trial examined the ability of intensive insulin therapy to prevent (primary prevention) and delay the progression (secondary prevention) of microvascular complications (predominantly retinopathy). Two points are worth noting before this data is extrapolated to the islet transplant setting. First, complications in the secondary prevention arm were at a relatively early stage. Second, rates of nephropathy in the intensively treated secondary prevention arm were the same as in the conventionally treated primary prevention arm, suggesting that it was more difficult to prevent nephropathy in those with established microvascular complications. Currently, most patients receiving clinical islet transplants have a long duration of diabetes and many have established complications. The animal and human
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data would suggest that although islet transplantation could reasonably be assumed to slow the progression of microvascular complications in many people it will be difficult to prevent them completely. The ability to reliably regress long-established complications will likely require sustained, robust euglycemia. Whether islet transplantation can reliably deliver the same degree of euglycemia as whole-pancreas transplantation remains to be demonstrated. In the last 5 years in Edmonton, laser photocoagulation has been required in 14% of patients receiving islet transplants [109]. In many cases this was after a relatively short period following islet transplantation. This is probably to be expected because although ultimately beneficial improved glycemic control is associated with a short-term worsening of diabetic retinopathy [2]. Concern regarding the impact of islet transplantation on renal function was raised in early reports from Edmonton [108, 121]. A decline in glomerular function rate was observed in two patients with preexisting renal dysfunction [108]. However, it is most likely that this decline was due to the hemodynamic effects of tacrolimus rather than the progression of diabetic nephropathy. Nevertheless, subsequent data from Edmonton indicate that development or progression of albuminuria (a typical feature of diabetic nephropathy) is seen in a significant proportion (40%) of islet transplant recipients [118]. The relative contributions of the transplant itself, and the direct and indirect effects of the immunosuppressant drugs, remain to be clarified. Immunosuppressants could have a direct impact on renal function because they are nephrotoxic. These drugs also increase blood pressure that accelerates diabetic nephropathy. Sirolimus is associated with hypertension, but has been thought to be free of direct nephrotoxic side effects. This assumption has been questioned recently with several reports of heavy proteinuria developing after renal transplantation [36] and now after islet transplantation [116]. Other data suggest that ongoing islet graft function, albeit short of insulin independence, may be associated with superior renal function in the context of kidney plus islet transplantation [41]. Fiorina et al. demonstrated reduced albuminuria and improved renal graft survival in those with continued c-peptide production compared with those who had lost islet graft function. There was no difference in GFR, however, and the differences in albuminuria could be attributed to superior blood pressure control. The improved survival of kidney grafts in those with surviving islet grafts arguably has more to do
with immunological matching of the grafts than with a protective effect of endogenous insulin secretion. Although earlier reports in pancreas transplantation have suggested a beneficial effect on diabetic neuropathy, we have not seen any clear positive or negative effect of islet transplantation at this stage.
Cardiovascular Disease Cardiovascular disease is the leading cause of death in people with diabetes. It is not surprising that there has been interest in the impact of islet transplantation on cardiovascular disease and cardiovascular risk factors. It is clear that hyperglycemia is a risk factor for cardiovascular disease, even into the so-called normal range. Changes to the diagnostic criteria for diabetes have reflected this [6]. The new condition of prediabetes (impaired fasting glucose and impaired glucose tolerance) is associated with increased risk not only for diabetes but for cardiovascular disease [6]. Despite the clear associations between hyperglycemia and cardiovascular disease, to date there are no clinical trials that have demonstrated the ability for improved glycemic control to reduce cardiovascular events. Nevertheless, preliminary data from long-term follow-up of the DCCT cohort are promising. Six and a half years of tight glycemic control was associated, some 10 years later, with a 57% reduction in risk of cardiovascular end points (heart attack and stroke) (Nathan et al. ADA, 2005). Although relatively young, and with low cholesterol levels and little hypertension, coronary artery disease is common (43% of asymptomatic applicants) in prospective islet transplant recipients [117]. Following transplantation, there is an increase in the proportion of individuals receiving antihypertensive and lipid-lowering therapies [109]. Although hypertension and dyslipidemia (recognized adverse effects of sirolimus) are more common, we have not seen as yet any evidence of progression of cardiovascular disease either clinically or on annual myocardial perfusion imaging following islet transplantation. Other groups have demonstrated improvements in surrogate markers of cardiovascular disease and endothelial function following islet transplantation. Again, in the context of islet plus kidney transplant individuals with persistent c-peptide production had improved endothelial function, reduced progression in intima-medial
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thickness (a marker of atherosclerosis), and the suggestion of fewer cardiovascular deaths [40].
Risks of Islet Transplantation The risks of islet transplantation can be divided broadly into four categories. • • • •
Procedural risks Side effects of immunosuppressant drugs Risks of long-term immunosuppression As yet “unknown” risks of this novel treatment.
These have been described in detail previously [111], but a brief summary here is helpful. The major procedural risks of islet transplantation via a transcutaneous approach to the portal vein are bleeding from the hepatic puncture. One case of upper GI bleeding immediately following islet transplantation suggested the formation of a fistula between a hepatic vessel and the biliary tree. In a small number of cases, bleeding has been associated with cardiovascular instability and necessitated blood transfusion. Bleeding and biliary punctures have been associated with abdominal pain, which although requiring opiate analgesia in some have been self-limiting and have not required specific therapy. Portal venous thrombosis has, fortunately, been an uncommon complication. There have been a small number of branch portal vein thromboses (< 4% of procedures), but none affecting the main portal vein. These have resolved after a period of anticoagulation. There is a rise in liver enzymes in almost all subjects following clinical islet transplantation. In some cases, these have been to levels greater than ten times the upper limit of normal [98]. The very marked elevations noted in a few patients are thought to be due to a specific batch of enzymes used in the preparation of the islets. The more modest elevations seen routinely are thought to reflect the inflammatory response to the graft and the islet transplant procedure itself. Generally, these elevations are asymptomatic and resolve spontaneously within 4 to 6 weeks [98]. There have been no procedural deaths. There was one postoperative myocardial infarct following severe vomiting and inadequate beta blockade (due to omission of regular betablockers because of immunosuppressant related nausea) in a patient with preexisting coronary disease.
The immunosuppressants used in islet transplantation have a number of side effects. Side effects of sirolimus and tacrolimus include nausea, oral ulceration, GI upset (either diarrhea or constipation), fatigue, anemia, neutropenia, edema, tremor, acne, hypertension, and dyslipidemia. Of the two, tacrolimus is better tolerated, but tacrolimus is used at lower doses when combined with sirolimus in islet transplantation. More side effects of tacrolimus (nephrotoxicity, tremor, and anxiety) are seen when higher doses of tacrolimus are used. Increased doses of tacrolimus are used when sirolimus is not tolerated and mycophenolate mofetil is substituted for sirolimus. Mycophenolate is associated with gastrointestinal side effects; mainly diarrhea, which usually improves with dose reductions. Long-term immunosuppression is associated with risks of infection, particularly with atypical organisms. Cytomegalovirus (CMV) can be transmitted from donor to recipient. CMV disease can pose a risk to the graft and to the recipient. Rates of CMV transmission seem to be lower in islet transplantation than in solid organ transplantation, perhaps reflecting the small amount of highly purified and lymphocyte free tissue that is transplanted. In Edmonton, two cases of possible seroconversion have been observed following transplantation but no cases of CMV disease. One case of late CMV seroconversion, perhaps indicating subclinical CMV disease, has been reported in another islet transplant center [43]. Neoplasia is another risk, with posttransplant lymphoproliferative disease (PTLD) a serious malignancy associated with immunosuppression in solid organ as well as bone marrow transplantation [130]. Currently, no cases of PTLD have been reported after clinical islet transplantation. The long-term consequences of intraportal islet transplantation and intrahepatic insulin secretion remain unknown. The secretion of insulin is not entirely physiological and glucagon secretion appears abnormal [94]. Hepatic steatosis, observed on ultrasound and MRI after islet transplantation, is likely associated with intrahepatic insulin secretion [15, 77]. Frequently, the steatosis is patchy, presumably reflecting the fact that islets are scattered throughout the liver. There is the suggestion that steatosis may indicate graft stress, rather than health [15]. A uniform homogeneous pattern of hepatic steatosis is generally seen in association with the hyperinsulinemia seen in insulin resistant states, such as type 2 diabetes. Hyperinsulinemia seems to be a key underlying feature in nonalcoholic steatohepatitis (NASH), which is improved by interventions that reduce insulin
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resistance [33]. A proportion of subjects with NASH seem to be at risk for liver cirrhosis and liver cancer. The steatosis seen after islet transplantation thus warrants careful observation. Other, as yet unknown, risks may become apparent with long-term follow-up of islet transplant recipients.
Summary Over time, as the number of islet transplant procedures and the duration of follow-up increase the effectiveness and benefits, as well as the limitations, of islet transplantation become clearer. Recent developments in islet transplantation undoubtedly represent a very significant step forward in terms of insulin independence and graft survival rates compared with historical data. Furthermore, the procedure seems to be safe, particularly in contrast to whole-pancreas transplantation that continues to have finite perioperative mortality. Nevertheless, questions remain about the degree of glycemic control achieved by islet transplantation, its longevity, and the ability of islet transplantation to have a clear beneficial impact on micro- and macrovascular complications. It does seem clear, however, that islet transplantation is effective for reducing hypoglycemia and stabilizing glycemic control [113]. Successful islet transplantation also reduces the fear of hypoglycemia [60] and anecdotally has a hugely positive impact on the quality of life of selected patients with type 1 diabetes who have the most difficult problems with hypoglycemia and lability. Although encouraging, it is clear that there are a number of challenges that must be overcome. One of the challenges is the apparent decline in graft function over time. The reasons for declining graft function over time are unclear. Consequently, it is difficult to know in which way efforts to intervene should be directed. There are currently no markers for rejection. Nor is it possible to reliably biopsy transplanted islets. (A small number of liver biopsies, performed for various reasons, have incidentally noted the presence of functional islets on some occasions, but not in all cases.) Such graft dysfunction could be due to rejection caused by alloimmunity, which in other transplant settings would be treated by increased immunosuppression. However, there is the suspicion that some late graft dysfunction may reflect immunosuppressant toxicity. For example, the antiproliferative effects of sirolimus may prevent islet proliferation or neogenesis. Additionally, autoimmunity is an additional
immune challenge in islet transplantation that could lead to graft dysfunction. Thus, currently islet transplantation is a specialized treatment that can be a life-altering treatment for a highly selected group of type 1 diabetic patients. It is clear that there is scope for significant improvements that hopefully will permit the full potential of this treatment to be realized and the availability of this treatment can be expanded to a broader audience.
Other Transplant Settings AUTOISLET TRANSPLANTATION So far the discussion of islet transplantation has been confined to islet-alone allotransplantation for type 1 diabetes. Autotransplantation of islets following pancreatectomy has been used for many years, albeit in relatively small numbers of cases. Many of the concerns and cautions surrounding clinical islet allotransplantation do not apply to autotransplantation. There is neither allo- nor autoimmunity to be concerned about, nor is there any need for long-term immunosuppression. Nevertheless, the risks of portal vein thrombosis have been considerable, particularly because large impure preparations of islets have tended to be used. The expertise of the islet isolation lab in Edmonton has facilitated islet autotransplantation locally. Isolating islets from these diseased pancreata, which are often fibrotic due to chronic pancreatitis, is not always easy. Nevertheless, the avoidance of the cytokine storm seen in brain-dead donors and minimized cold ischemia time mitigates this difficulty [29].
ALLOTRANSPLANTATION FOR SECONDARY DIABETES Islet allotransplantation has been restricted to patients with type 1 diabetes, not least because they are insulin deficient and insulin sensitive. As such, the likelihood that islet transplantation could have a beneficial effect is maximized. In particular, the ability to achieve insulin independence is enhanced. Individuals with secondary diabetes, due to pancreatic disease or following pancreatic surgery, could, however, be considered for islet allotransplantation because such patients may experience the same difficulties with hypoglycemia and glycemic lability as the type 1 patients currently considered for
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clinical islet transplantation. One important exclusion would be those with diabetes secondary to pancreatectomy for cancer.
ISLET ALLOTRANSPLANTATION FOR TYPE 2 DIABETES Some have raised the question of islet allotransplantation for type 2 diabetes. Generally, type 2 diabetes is associated with insulin resistance rather than with insulin deficiency. Most affected patients are heavier than type 1 subjects, and many are overweight or obese. Although in long-standing type 2 diabetes insulin deficiency can develop [135], the majority of subjects continue to have ongoing endogenous islet function. The increased demand placed on transplanted islets because of insulin resistance and greater body weight means that insulin independence would be difficult to achieve. Whether a reduction of insulin requirements by 40 or 50 units of insulin per day is worthwhile in an individual taking 140 units per day is unclear. It is therefore uncertain how beneficial islet transplantation would be in type 2 diabetes and whether these benefits would justify the risks. Particularly in that currently the organ supply is so limited the consensus seems to be to exclude type 2 diabetic patients from islet transplant programs.
OPTIMIZING ENGRAFTMENT The loss of islets during and immediately after transplantation is a major challenge. Developments in the use of agents to prevent the IBMIR [49, 82], the overexpression of antiapoptotic genes [37], and the avoidance of immunosuppressive agents that prevent islet regeneration will hopefully enhance islet engraftment. The ability to expand the number of beta cells either prior or subsequent to islet transplantation would be hugely beneficial. The ability of a number of growth factors to perform in this manner is being explored, including GLP-1, exendin-4, EGF, gastrin, INGAP, and hepatocyte growth factor [24, 76, 85, 87, 106, 128].
SINGLE-DONOR TRANSPLANTATION Optimization of islet isolation and engraftment, and developments in the ability to expand the beta cell mass, will play a role in facilitating reliable single-donor transplant success. Currently, single-donor success has been reported by the Minnesota group [55]. It is not clear whether developments in islet isolation or transplantation procedures, immunosuppression, or careful selection of donors and recipients are the most important factors leading to this success [122]. Nevertheless, single-donor success is an important milestone in the progress of islet transplantation.
Future Directions Despite rapid progress, much remains to be done before the dream of a cure for diabetes will be realized. Research focused in a number of areas holds promise for the future — some in the nearer term, others further into the future.
OPTIMIZATION OF ISLET ISOLATION Currently, islet isolation remains inefficient and unpredictable. Ultimately, the aim would be to develop the ability to reliably isolate sufficient islets from a single-donor pancreas to treat one or more diabetic recipients. The complexity of the islet isolation process means that there are a large number of variables that must be considered in order to optimize the yield of transplantable preparations. Improvements in organ procurement, transportation, and islet culture (as well as a reliable supply of enzymes with predictable performance) will all be helpful.
LIVING DONOR ISLET TRANSPLANTATION The first successful living donor islet transplant was reported recently by a group from Japan [78]. Achieving insulin independence following the infusion of islets isolated from a distal pancreatectomy (i.e., half a pancreas) underscores the major insults (cold ischemia and the proinflammatory cytokines associated with brain injury [29]) to which islets are exposed when isolated from cadaveric donors. Living donor transplantation could help overcome some of the difficulties of organ supply and may improve tissue matching. Making islet transplantation an elective procedure has benefits in terms of the conditioning treatments that can be given to the recipient even prior to islet isolation, let alone prior to islet infusion. Nevertheless, there are finite risks for the donor, from the procedure itself in the short term and of developing diabetes in the longer term [52, 129]. The use of a laparoscopic approach to distal pancreatectomy in
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the donor and careful selection of lean donors with good beta cell reserve should minimize these risks. Expectations of the isolation lab’s performance will be heightened because processing the pancreas from a living donor raises the stakes compared to a cadaveric donor pancreas. Living donation may be an attractive option for children with diabetes. A smaller number of islets may be required for insulin independence in view of their smaller size. Current immunosuppression may make this scenario less attractive because of the risks of long-term immunosuppression and the requirement for beta cell mass to expand to meet the increased insulin secretory demands associated with growth and puberty.
MINIMAL IMMUNOSUPPRESSION AND TOLERANCE Although sirolimus and tacrolimus have played a key role in the success of the “Edmonton protocol,” these drugs have side effects (some serious) that limit their tolerability. Further developments of novel immunosuppressant therapies that are more “islet friendly” with fewer nonimmune side effects would likely be associated with improved outcomes [120]. Together, these improvements would have a synergistic effect improving the risk/benefit ratio for islet transplantation. Some of these “islet friendly” approaches may have the potential to induce tolerance in animal models [120]. Tolerance refers to the concept of retraining the immune system such that it can accept an allotransplant as self. This would obviate the need for immunosuppression. Although full tolerance may be a distant possibility, interim steps might permit minimization of the immunosuppression required following transplantation. Clinical trials of such strategies are being designed.
ALTERNATIVE SOURCES OF ISLETS The limited organ supply is a major barrier to more widespread application of islet transplantation. Even with successful single donor protocols, the number of available organs is far outstripped by the number of people with diabetes who potentially could benefit from islet transplantation. A great deal of research is focused on the identification of alternative sources of islets or insulin-secreting cells. Xenotransplantation of porcine islets is being actively explored. Pork insulin can effectively control diabetes
in humans and was a standard therapy until the advent of recombinant human insulin. Porcine pancreata represent a potentially unlimited supply of organs from which islets can be isolated. Nevertheless, xenotransplantation poses a number of substantial challenges, not least the hyperacute rejection mediated by alpha Gal and other xenoantigens [30]. Ensuring the safety of xenotransplantation, particularly because of the potential for zoonoses, and the risks of porcine endogenous retrovirus (PERV) infections are further challenges [42]. Stem cell research and attempts to promote transdifferentiation of progenitor and ductal cells into insulinsecreting cells are another focus [54, 91]. Other groups are attempting to enable differentiated cells, including hepatocytes and myocytes, to secrete insulin in a glucose-dependent manner by a number of different approaches [7, 28, 83, 138].
Conclusions Some of the initial enthusiasm surrounding the initial reports of insulin independence after islet transplantation from Edmonton has been tempered by the realization that we are still a long way from the cure for diabetes we continue to pursue. Nevertheless, the Edmonton protocol has been an important step in this ongoing journey. It has helped raise the profile of islet transplantation among the general public as well as people with diabetes. Furthermore, it has provided a major impetus for further research as well as attracting increased commitment to research funding. The decline in insulin independence rates despite ongoing graft function raises important questions about the biology of transplanted islets and represents a challenge to be overcome. Islet transplantation as a clinical treatment for people with type 1 diabetes is now a reality, which is making a positive impact on the lives of individuals previously blighted by diabetes. The challenges that lie ahead are undoubtedly substantial, but the goal of a cure for diabetes remains incredibly worthwhile.
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6 Characterization of Islet Preparations Clark K. Colton (1), Klearchos K. Papas (1 and 3), Anna Pisania (1), Michael J. Rappel (1), Daryl E. Powers (1), John J. O’Neil (2 and 4), Abdulkadir Omer (2), Gordon Weir (2), and Susan Bonner-Weir (2) (1) Department of Chemical Engineering, Massachusetts Institute of Technology Cambridge, Massachusetts (2) Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts (3) Diabetes Institute, University of Minnesota, Minneapolis, Minnesota (4) Lifescan, Inc., Johnson and Johnson, Skillman, New Jersey
Introduction
•
Following the initial report in 2000 from Edmonton of insulin independence in the first seven patients treated with a new protocol [171], nearly 500 patients with type 1 diabetes received islet transplants at 43 institutions worldwide as of mid 2005, and high rates of insulin independence were observed at 1 year in the leading transplant centers [170]. Despite these very promising results, various problems currently remain to be solved, including the following. •
•
•
Initial insulin independence usually requires an average of 12,000 islets/kg body weight, or about 850,000 islets for a 70-kg human, which corresponds to the islet yield from two or more human cadavers [150, 160]. For islet transplantation to be a widely accepted treatment modality, it is essential that the donor/recipient ratio be brought down to 1:1 [90]. Although several centers report insulin independence with some single donors [90, 152, 160], success with single donors is limited by the quality of the islet preparation, which is related in part to the quality of the donor pancreata [70, 152]. Even experienced centers can obtain clinical-grade preparations in no more than about 50% of islet isolations [175]. Patients typically have glucose intolerance. It has been suggested that functional capacity for insulin secretion is only about 20 to 40% of that in nondiabetic patients [158, 159].
Cellular Transplantation: From Laboratory to Clinic
There is a progressive loss of insulin independence over time, leaving only about 50% of patients still insulin free at 2 years and 10% at 5 years [160].
The entire process (from brain death of the donor and organ removal to the posttransplant period) can last several days, during which a variety of factors and sequential insults can lead to a substantial loss in islet mass and viability [152]. The quality of the original islets of the donor, itself an important factor, can only decrease. Donor brain death could exert a negative influence on islets by evoking release of inflammatory cytokines [31, 34, 44, 95], but this postulate is based only on experiments in rodents with extreme brain destruction. Hypoxia, or even anoxia, occurs to varying degrees during pancreas retrieval, storage, and islet isolation. Mechanical and enzymatic disruption adds stresses during islet isolation. Hypoxia likely occurs during most conventional islet culture conditions and may be severe during islet shipment. These stresses, in turn, lead to mitochondrial dysfunction—including excessive levels of reactive oxygen species (among other possible sources of ROS), intracellular redox imbalance, accumulation of reduced pyridine nucleotides, mitochondrial membrane depolarization, cytochrome c release, and change in energy state resulting from insufficient ATP generation. All of these are associated with activation (as cause and/or effect) of apoptotic and necrotic pathways [23, 113]. Last, following transplantation islets are exposed to hypoxia until vascularization occurs and to presumed stress
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Colton et al.
from the immune system. It has been hypothesized that islets are further damaged by instant blood-mediated inflammatory reaction, which may be induced by inflammatory mediators such as tissue factor and MCP-1 following contact of the islets with ABO-compatible blood [90]. To move forward in development of improved methods, it is essential to have assays to assess meaningful characteristics of islet preparations. Unfortunately, the tools in clinical use today are rather blunt instruments. Currently used parameters are not capable of quantifying the “dose” of islets transplanted and are not predictive of transplantation outcome [42, 46, 156]. Development of improved methods will have a number of benefits in terms of facilitating the ability of practitioners to (1) predict transplantation outcome, (2) improve islet isolation procedures, (3) make more efficient use of islets, (4) standardize test conditions in research, and (5) facilitate FDA licensing [47, 191] and insurance coverage. In this chapter we examine methods for in vitro characterization of islet preparations. After a description of islet isolation processes, and consideration of the information we need, we divide methods into three groups: (1) islet purity and composition, (2) quantity of islets, and (3) viability of islets. We also review assays currently in use, as well as new methods under study at various laboratories with special attention to our own efforts. In examining these methods, we give attention to utility in terms of adequacy for the intended purpose,
accuracy and precision, time required to perform the assay and obtain a result, and cost in terms of materials and equipment.
Islet Isolation HUMAN ISLETS The pancreas is surgically removed from the donor by a surgical team, stored in University of Wisconsin (UW) preservation solution [73] on ice and shipped to the processing facility. The pancreas may be stored by the so-called two-layer method, in which the pancreas floats at the interface between UW solution and oxygenated perfluorocarbon (PFC)—an oxygen carrier— in order to reduce oxygen limitations and ischemia. Extensive studies have been performed on the purported advantages provided by the two-layer method [53, 71, 93, 96, 109, 185], but recent measurements suggest that the current technique provides little, if any, amelioration of oxygen supply limitations in the tissue [120]. In the islet isolation process (Figure 6.1), the pancreas is digested enzymatically with mechanical agitation by automatic means or hand shaking. It was first described by the laboratory of Lacy [155] in 1988, and since then several improvements have been reported. Briefly, the pancreas is dissected from the duodenum, and the
Distension with Collagenase/Protease Solution Ricordi Chamber (Shaker) Density Gradient Centrifugation
Ischemic Conditions
Exocrine Tissue
Islet Preparation
150 µm
1-2% original pancreas volume
Figure 6.1. Schematic diagram of human islet isolation. The pancreas is distended with an enzyme blend and placed in the Ricordi chamber. Through enzymatic digestion and mechanical disruption, islets are liberated from the pancreas and purified with a density gradient centrifugation.
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6.
Characterization of Islet Preparations
main and accessory ducts are clamped and divided. An enzyme blend that includes collagenase is injected through a cannula placed at the pancreatic duct to allow the organ to distend. Collagenase was originally used as the dissociation enzyme but has been substituted in most centers by Liberase, a standardized mixture of purified enzymes (primarily two types of collagenase) from Roche Applied Sciences, because of improved islet yields from human pancreata [98–100]. After distension, the pancreas is cut into a number (typically 8 to 10) of small pieces that are placed in the dissociation chamber, which is called a Ricordi chamber. The chamber consists of an upper conical part that is separated by a mesh from the lower cylindrical part, in which stainless steel spheres and the pancreatic pieces are placed. The lower part has two inlets at its base, whereas the conical part has an outlet at its top. The pancreatic tissue is digested by a combination of enzymatic digestion at 37◦ C and mechanical agitation of the spheres provided by manual or motorized shaking of the chamber. The enzyme is circulated by a peristaltic pump through the chamber and heating system, which allows the enzyme to be activated in the chamber. Continuous flow allows the islets to be collected, when desired, from the top of the conical part of the chamber after they are released. Cooling and dilution prior to collection of the islets protect them from further enzymatic action. The outlet of the chamber where islets are collected is periodically sampled to monitor the dissociation process and to decide when to stop the enzymatic digestion. At the end of the dissociation process, the cell aggregates are collected in separate containers, and the pancreatic ducts and vessels are retained in the cylindrical part of the chamber by the mesh. In the final purification step, density gradient centrifugation is used to separate the small islet fraction from the non-islet fraction (exocrine: duct, acinar), usually using a specialized cell processor (COBE 2991 Cell Processor). The gradient medium used for human islet isolations is Ficoll, and the density gradients are continuous (between 1 and 1.11 g/ml). At the end of the purification step, the upper layers usually contain highly purified islets at a density of about 1.08 g/ml, whereas less pure and embedded islets are found within more dense layers. The tissue is emptied into about 14 tubes or flasks, and tubes 4 through 9 (in order of elution) contain most of the islet population. The content of individual tubes is accessible for assays and experiments. Usually, all fractions containing significant
numbers of islets are combined in clinical preparations for transplantation.
RAT AND PORCINE ISLETS Isolation of rat pancreatic islets was first successfully performed and described by Lacy and Kostianovsky, and current procedures are based on their method—a two-step process involving digestion with collagenase followed by sedimentation [94]. This method was later modified to include discontinuous Ficoll gradient purification [167]. Some centers use different gradient media, such as Histopaque. The pancreas is distended with chilled Hanks’ Balanced Salt Solution (HBSS) supplemented with glucose and antibiotics or UW solution. The pancreas is dissected from the surrounding tissue and stored in supplemented HBSS or UW solution. The pancreas is distended in a similar way as for the human islet isolation, placed in a 50-ml tube, and then digested with crude collagenase or the more well-defined collagenase preparation known as LiberaseRI in a static incubation for about 20 minutes in a water bath at 37◦ C. After the digestion, cold medium supplemented with serum is added to stop the digestion. The tissue is washed in fresh medium and passed through a mesh (about 400 mm) to remove large fibrous pieces. The collected tissue is centrifuged, washed, and resuspended in the density gradient medium with culture medium on top. Islets are collected from the interface at a density about 1.077 g/ml. Islets are washed in fresh medium two to three times and purified further with sedimentation. Porcine islets are isolated in a manner similar to that of human islets [19, 115, 157, 197]. Pancreata are distended with a collagenase solution and digested using a modified semiautomated method. The digestion takes place also at 37◦ C and lasts 40 to 60 minutes. The purification step also takes place with a Ficoll gradient in a COBE 2991 Cell Processor using layers with densities of 1.11, 1.096, and 1.06 g/ml, and most of the islets are collected between 1.11 and 1.096 g/ml. Some centers follow different protocols for porcine islets, with a static incubation in a chamber similar to the Ricordi chamber. Islets isolated from rat and porcine pancreata usually contain very little contamination by other pancreatic cells. By contrast, human islet preparations that are transplanted typically consist of about 50% non-islet components on average, largely acinar and duct cells [60, 86, 116, 169, 177].
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What Do We Want to Know? The general areas used to characterize islet preparations prior to transplantation include (1) safety, (2) identity, (3) quantity, (4) viability, (5) potency, and (6) other. The information needed reflects the answers to the following questions.
transplantation outcome and for testing in vitro islet quality assays. Development of meaningful assays of islet preparations has proven to be a challenge and is under study in several laboratories. Why are islet preparations so difficult to characterize? There are several reasons. •
•
•
•
•
•
•
Safety: Is the preparation sterile? Is it free from microbial cells, mycoplasma, endotoxins, and other adventitious agents? Purity and composition: What fraction of the preparations is islet (endocrine) and non-islet (exocrine) tissue? What is the cell composition (i.e., what fraction is b cells, non-b islet (a, d, PP) cells, duct, and acinar cells)? Quantity: How much total tissue is present? What is the tissue volume? What is the number of cells? What is the volume and number of cells of islet and non-islet tissue? What is the volume and number fraction of the individual component cells, especially b cells? Viability: For the total tissue and for the islet tissue, how much is viable? What fraction of the tissue is viable? How, indeed, do we define viability? How do we determine what is alive now? How do we deal with cells that are alive now but destined to be dead later because of irreversible commitment to the cell death process? Potency and function: What is the insulin secretory capacity in terms of glucose-stimulated insulin secretion? Do the islets cure diabetes when transplanted in a laboratory animal? Other: Can anything be learned from gene expression profiling? Are there other parameters of interest?
In this chapter, we focus on the second through fourth of these areas. The fifth area, potency and function, is also of interest but is not covered here. Glucosestimulated insulin secretion measured with freshly isolated islet preparations does not appear to correlate with transplantation outcome [177]. Transplantation of varying quantities of islets into the renal capsule of an immunodeficient mouse has arguably been the standard for more than 15 years [150, 155] for predicting clinical success, but the results are apparent only after a number of days and are therefore of retrospective value. Nonetheless, it is a potentially useful assay for correlating potency or dose to
•
•
•
Islets are cellular aggregates with a variety of nonsymmetric shapes and wide size distributions that are difficult to quantify accurately. Visual size estimation is prone to error, is operator dependent, and has large intrinsic uncertainty. Human preparations have varying amounts of impurities. Distinguishing between the properties of islet and exocrine tissue is difficult. The islet is a moving target. Islet volume decreases with time after isolation. Stress repeatedly occurs during pancreas retrieval, storage, islet isolation, culture, and shipment. Some cells are lost to apoptosis and necrosis. Recovered cells are likely not representative of the original islet. Many techniques suitable for cells are inapplicable to islets because of their 3D structure. Unfortunately, the islets cannot be usefully dissociated into dispersed cells by agitation and incubation with serine proteases such as trypsin because from 30 to 50% of the cells are lost during dissociation [140, 141, 193]. Cell damage may result from initiation of apoptosis arising from separation of the cell membrane from the extracellular matrix [55, 186], as has been demonstrated in trypsin dissociation of epithelial cells such as hepatocytes [172]. Thus, cells recovered from the dissociation process may not be representative of cells in the original islet.
Islet Purity and Cell Composition Light microscopic observation following staining with dithizone (diphenylthiocarbazone, DTZ) is the commonly accepted technique employed currently to assess the purity of islet preparations. Because of its inadequacies, a variety of new techniques have been examined over the past few years, as summarized in Table 6.1. In this section, each method is briefly summarized, including discussion of its strengths and weaknesses. There follows an in-depth discussion of recent studies examining morphological analysis by electron and light
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TABLE 6.1 Measurements of islet purity and cell composition. Tissue Assayed
Parameter Measured
Method
Instrumentation
Intact Islet Preparation
Volume Fraction of Islets
DTZ Staining
Light Microscope
Tissue Morphology
Light Microscope
Volume Fraction of Cell Types
Immunohistochemistry
Laser Scanning Confocal Microscope
Number Fraction of Cell Types
Ultrastructural Analysis
Electron Microscope
Number Fraction of Cell Types
Immunohistochemistry
Fluorescence Microscope
Dispersed Single Cells
Laser Scanning Cytometer
microscopy and a comparison of cell composition data from these and other studies in the literature.
DTZ STAINING DTZ, a zinc-specific binding dye, is used to obtain an estimate of the volume fraction of islets in a sample [30]. Zinc is specific to islets and therefore islets stained with DTZ have a red color and can be visually differentiated from exocrine tissue. DTZ is usually dissolved in dimethylsulfoxide (DMSO) and diluted with HBSS to form a working solution. Aliquots from the islet preparation are incubated with DTZ, and the fraction of islets is estimated visually under a microscope as the volume fraction of cells stained red. Purity assessments by DTZ are rough visual estimates, and they usually grossly overestimate islet purity, as has been previously reported [116, 177].
MORPHOLOGICAL ANALYSIS Morphological analysis is usually carried out by electron or light microscopy. Aliquots from the islet preparation are processed using a standard procedure [116], during which they are fixed, dehydrated, cleared, embedded, cured, and trimmed to produce 1-mm sections in epoxy resin. This process takes about 3 days for light and 4 for electron microscopy. Frozen sections can be prepared in less than an hour.
ELECTRON MICROSCOPY (EM) ANALYSIS For ultrastructural determinations, ultrathin sections of islets are cut and stained with lead citrate and uranyl acetate and viewed on an electron microscope. Images from each block are collected with systematic sampling and examined on photographic prints. For the studies done by our group that are described in this chapter, between 500 and 800 cells are classified into cell types by their ultrastructure and granule morphology. Secretory granules of islet endocrine cells are too small to be seen with light microscopy, but zymogen granules of acinar cells are evident, which allows acinar contamination to be easily identified on the 1-mm thin plastic sections used as a preliminary assessment for sections to be taken for EM. At the EM level, cell boundaries are identified. Then each cell is assigned to a category of islet [I] cells, including b and non-b endocrine cells (a, d, PP), and non-islet (NI) cells including acinar and ductal cells. b cells have distinctive granules with an electron-dense core containing insulin crystals surrounded by a lighter halo. In addition, human b cells have characteristic lipid inclusions. Non-b cells (a, d, and PP) have more homogeneous granules without the crystalline structure and without the halos of b cells. Although these three non-b islet cells can be distinguished from one another by granule morphology, for quantitation purposes they can be classified together as non-b islet cells. Acinar cells have easily identifiable large electron-dense zymogen granules and stacks of endoplasmic reticulum. Duct cells can be identified by their size, shape, little endoplasmic
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reticulum, and lack of granules. Occasional dead cells or endothelial cells are categorized as “other.” The resulting cell composition is based on number, not volume, of cells and yields the number fraction fi of each cell type defined as the number ni of cells of type i divided by the total number of cells, nTC . LIGHT MICROSCOPY (LM) ANALYSIS For LM determinations, 1-mm sections are analyzed at 420x by stereological point counting [192] in which islet and non-islet tissue are distinguished (as described in the caption to Figure 6.2) at positions defined by a grid of points covering adjacent, non-overlapping fields. The points that lie in the islet and non-islet tissue are counted to obtain the volume fraction of islet tissue (not cell number). An example is shown in Figure 6.2. Islets are highly vascularized tissue, and freshly isolated islets have large dilated vascular spaces, the size of which decreases with time. Although the vascular space can be included within the domain of the islet, we have chosen to keep track of it separately. Assuming that the photomicrograph represents a plane through the tissue, the probability of a random point falling into a certain type of tissue is proportional to the volume fraction of that tissue [192]. The number of points falling on islet tissue (PI ), non-islet tissue (PNI ), and vascular space (PV ) is determined, and the volume fraction of islets I is
calculated from I =
PI + PV , PI + PV + PNI
(6.1)
where the numerator is the number of points falling within the domain of islets and the denominator is the total number of points. An entirely equivalent procedure is to first ignore the vascular space and calculate IXV =
PI , PI + PNI
(6.2)
where IXV is the islet volume fraction exclusive of islet vascular spaces. The tissue is reanalyzed at higher magnification (830x) to obtain the vascular void fraction VI in the islets from VI =
PV . PV + PI
(6.3)
By rearranging Equation 6.1 and substituting Equations 6.2 and 6.3, the islet volume fraction is then calculated from
I =
IXV 1 − VI IXV + (1 − IXV ) 1 − VI
.
(6.4)
Islet
Vascular Space
NonIslet
Figure 6.2. Sample field of view of a slide analyzed by light microscopy using an 830x magnification in an Olympus BH2 microscope. Intercepts of tissue with a 90-point grid are counted as islet, non-islet, or vascular space. Islet tissue is distinguished from non-islet tissue (exocrine or ganglia) by its cordlike pattern of cells (often around vascular spaces), lack of visible granulation, and occasional visible small lipid droplets, whereas the exocrine tissue is in the form of either large duct sheets or folded sheets of polarized columnar epithelia or clumps of small duct and acinar tissue. The acinar tissue is initially granulated with large stained granules, although after several days of culture these are less pronounced, and the small ducts are only faintly stained and surrounded by acinar tissue.
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The vascular volume correction in Equation 6.4 puts the entire expression on a basis of total volume (including vascular volume). Equation 6.4 is equivalent to Equation 6.1, as can be verified by substituting Equations 6.2 and 6.3 into 6.4. In the analysis of samples reported in this chapter, we use the procedure that employs Equations 6.2 to 6.4 because separate estimation of vascular void fraction VI at higher magnification provides a more accurate and precise result. With islet preparations that average about 50% islet volume fraction, use of a total of 500 to 800 counted points yields a standard error equal to 3 to 5% of the mean value [192]. EM offers capability for identifying individual cell composition, whereas LM provides only islet volume fraction. The LM assessment method described is less time consuming than EM but still suffers from the time required to process the tissue. An appealing prospect is to use frozen sections, which can use the same LM approach. The time for tissue preparation and analysis should only take a few hours, which is fast enough to be valuable for evaluation of clinical samples prior to transplantation.
OTHER APPROACHES USING IMMUNOHISTOCHEMICAL ANALYSIS Immunohistochemistry can be applied to blocks of tissue or single cells from dissociated islets. Blocks of pancreatic tissue or islets are processed with a standard procedure of fixation, dehydration, and embedding [89, 138, 140, 177] to produce sections (paraffin 5 to 7 mm thick, frozen 8 to 15 mm). The sections are then incubated overnight with the primary antibodies (to insulin, glucagon, somatostatin, and pancreatic polypeptide), washed, and then incubated with the secondary antibody to which a fluorochrome is bound. The sections can be analyzed either by fluorescence or laser scanning confocal microscopy. Preparations of paraffin sections also require several days, making these measurements useful retrospectively. Single cells can be analyzed by a laser scanning cytometer, which is slide based and is a cross between a flow cytometer and a static image cytometer. STANDARD FLUORESCENCE MICROSCOPY The fraction of cells specific to each antibody is determined by examination under a fluorescence microscope, and results can reflect number fraction or volume fraction, depending on how the analysis is carried out.
Problems with immunohistochemical staining include the possibility of overlapping cells on paraffin sections and nonspecific staining. LASER SCANNING MICROSCOPY Cell composition estimation with immunohistochemistry has recently been performed with laser scanning confocal microscopy [22, 25] or laser scanning cytometry [75, 76] to analyze cell composition. Unlike conventional fluorescence microcopy, light is collected from a single focal plane in laser scanning confocal microscopy. The specimen (islet or pancreatic tissue) is scanned point by point and line by line, and a single image is created. Evenly spaced optical sections obtained by moving the focal plane in the axial direction can assemble a 3D image. These images can be analyzed by image analysis software to determine the number or volume fraction of different cell types. Samples analyzed to date include pancreatic blocks [25], wholemount islets [22], and dispersed cells [75]. The procedure employed with dispersed cells is summarized later in this chapter in Figure 6.18 in connection with cell viability measurements. Dispersed cells do not require the confocal feature and can be prepared in a few hours, but their use entails the problems identified with islet dissociation. If cell composition is based on counting the number of cells stained with a specific antibody, the number fraction is estimated [75]. If the normalization is based on a volume basis using a 3D reconstruction or other technique, the volume fraction is estimated [23]. A problem with current laser scanning microscopes is their very high cost, which makes them available in only a limited number of centers.
COMPARISON OF EM AND LM MORPHOLOGICAL ANALYSIS In a recent study [116], we carried out morphological analysis of 33 individual freshly isolated clinical islet preparations by EM and found it to be a valuable tool for assessing cellular composition. In a subsequent study [142], we examined the 1-mm sections from 27 of these blocks by LM to assess the purity of the preparations. Results of these two studies are summarized and compared between themselves in this section and with results using other techniques in the next section. Because results of these various studies are expressed on the basis of either number (in our case EM) or volume (LM) fraction, and the distinction is often blurred,
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Colton et al. TABLE 6.2 Estimated volume (mm3 ) of pancreatic cells. Symbol
Native Pancreas
Freshly Isolated
νb νNb
1,000 400
950 400
νA νD νO
1,550 200 200
1,200 200 200
Islet Beta Non-Beta (Mainly Non-Islet Acinar Duct Other
a)
we also address the mathematical equations needed to convert between number and volume bases. This conversion is also required for calculating various quantities of interest. The cell volume estimates used in carrying out calculations are summarized in Table 6.2. Values for b and non-b cells (assumed to have properties of a cells) are taken from measurements with freshly isolated and purified rat islets [137, 141]. The b-cell volume for freshly isolated islets reflects an estimated 5% loss from the commonly accepted value of about 1,000 mm3 for the native pancreas [18, 32], which is consistent with a partial degranulation during isolation and dispersion. Acinar volume in the mature pancreas on average is within the 1,300- to 1,800-mm3 range reported for the adult rat [4, 5, 164], and it is assumed that about 20 to 25% of the volume in freshly isolated preparations is lost from degranulation. Values for duct and other cells are from our observations [17]. After several days in culture, surviving b cells regranulate. Acinar cells are more prone to death when cultured and not as well studied. Data from 27 freshly isolated clinical islet preparations are summarized in Table 6.3. Columns 2, 3, and 5 through 7 contain the number fraction of individual islet and non-islet cell types from EM ultrastructural analysis. Column 4 contains the number fraction of all islet cells (fIC ). The number fraction of non-islet cells (fNIC ) is simply fNIC = 1 − fIC . Columns 8, 9, and 10 contain the volume fraction quantities defined by Equations 6.2 through 6.4, respectively, from LM morphological analysis. The latter quantity, (I )LM , is the purity (i.e., the volume fraction of islets by LM morphology analysis). The average number fraction of b cells in the entire
preparation is 35.8%, and the average number fraction of islet cells in the preparation is 48.4%. The latter value is about 8% smaller than the average volume fraction of islets by LM, which is 52.3%. Although the precision of the mean values as expressed by SD or SEM (Table 6.3) is respectable because of the large number of preparations examined, values for individual samples vary widely, largely because of differences in purity between preparations. An objective of further analysis is to use the EM number fraction data (Table 6.3) together with the volume of each cell (Table 6.2) to estimate the volume fraction of islets so as to enable proper comparison with the value from LM analysis and to provide the basis for comparison with values in the literature. The results of these calculations, summarized in Table 6.4, make use of the basic number-volume relationships for cells summarized in Table 6.5. Equations 6.5 through 6.9 (Table 6.5) show how the numbers of individual cell types can be summed to determine the number fractions fIC and fNIC . Recognizing that the volume of each cell type is the product of the number of cells and the volume per cell of that type, Equations 6.10 through 6.14 (Table 6.5) lead to definitions of volume fractions fIC and fNIC for islet and non-islet cells, respectively, as the total volume of each cell type divided by the total volume of all cells. Next, the average cell volume for islet (ν IC ) and non-islet (ν NIC ) cells and for the total of all cells (ν TC ) is evaluated as the volume of cells of a particular type divided by the number of cells of that type. These relations reduce to Equations 6.15 through 6.17 (Table 6.5), respectively, in terms of number fractions and volume per cell of individual cell types. Equation 6.18 (Table 6.5) is especially important because it relates the volume fraction of islet cells (fIC ) to the number fraction (fIC ) by the ratio ν IC ν TC . If that ratio is unity, meaning all islet cells have the same average volume as all non-islet cells, then fIC = fIC . Last, the number fraction fbIC and volume fraction fbIC of b cells among all islet cells are given by Equations 6.19 and 6.20 (Table 6.5). Columns 12, 13, and 14 of Table 6.4 contain the estimated average cell volume for different cell combinations. The average for islet cells over all preparations 3 is 806 ± 9 mm . The ratio ν IC ν TC in column 15 averages 1.09 ± 0.02, and 22 of 27 samples fall between 1.0 and 1.15. This means that the volume correction associated with different cell sizes is usually small, and to a rough approximation the volume fraction of all cells
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TABLE 6.3 Data from EM and LM morphological analysis and DTZ staining. Number Fraction Cells by EM
Volume Fraction by LM
Non-islet
DTZ
Beta
Islet NonBeta
Total
Acinar
Duct
Other
Islet Tissue w/o Vascular Void
Sample
fb
fNb
fIC
fA
fD
fO
(IXV )LM
VI
(I )LM
(I )DTZ
1
2
3
4
5
6
7
8
9
10
11
1–16 2–06 2–08 2–17 2–19 2–20 2–21 2–22 2–24 2–25 3–06 3–10 3–13 3–16 3–19 3–21 3–23 3–25 3–28 4–01 4–06 4–07 4–10 4–11 4–15 4–23 4–24
0.220 0.186 0.322 0.291 0.308 0.490 0.176 0.529 0.267 0.398 0.287 0.354 0.330 0.525 0.343 0.507 0.637 0.532 0.162 0.362 0.378 0.489 0.323 0.452 0.358 0.131 0.303
0.126 0.036 0.131 0.136 0.060 0.160 0.154 0.179 0.120 0.114 0.149 0.092 0.102 0.128 0.077 0.056 0.216 0.162 0.088 0.149 0.079 0.191 0.170 0.163 0.269 0.036 0.058
0.346 0.222 0.453 0.427 0.368 0.650 0.330 0.708 0.387 0.512 0.436 0.446 0.432 0.653 0.420 0.563 0.853 0.694 0.250 0.511 0.457 0.680 0.493 0.615 0.627 0.167 0.361
0.338 0.416 0.301 0.288 0.387 0.194 0.355 0.161 0.305 0.200 0.281 0.130 0.326 0.157 0.224 0.287 0.054 0.115 0.414 0.191 0.340 0.099 0.199 0.170 0.089 0.407 0.349
0.307 0.330 0.228 0.270 0.201 0.129 0.285 0.092 0.300 0.284 0.242 0.383 0.231 0.185 0.321 0.109 0.016 0.150 0.303 0.262 0.172 0.149 0.190 0.179 0.243 0.384 0.247
0.009 0.032 0.018 0.015 0.044 0.027 0.030 0.039 0.008 0.004 0.041 0.041 0.011 0.005 0.035 0.041 0.077 0.041 0.033 0.036 0.031 0.072 0.118 0.036 0.041 0.042 0.043
0.332 0.087 0.469 0.435 0.356 0.653 0.285 0.690 0.432 0.535 0.467 0.540 0.407 0.692 0.441 0.624 0.857 0.716 0.179 0.546 0.456 0.702 0.509 0.674 0.695 0.105 0.385
0.142 0.045 0.146 0.147 0.139 0.117 0.131 0.115 0.137 0.162 0.178 0.059 0.197 0.077 0.125 0.140 0.203 0.177 0.135 0.162 0.083 0.173 0.096 0.114 0.214 0.170 0.148
0.367 0.091 0.508 0.474 0.391 0.681 0.314 0.716 0.468 0.579 0.516 0.555 0.461 0.709 0.474 0.659 0.883 0.754 0.201 0.589 0.478 0.740 0.534 0.700 0.744 0.124 0.424
0.58 0.30 0.55 0.70 0.60 0.80 0.50 0.75 0.35 0.50 0.75 0.80 0.70 0.70 0.70 0.85 0.90 0.95 0.30 0.75 0.60 0.80 0.90 0.90 0.90 0.37 0.77
Mean SD COV SEM
0.358 0.129 0.36 0.025
0.126 0.056 0.44 0.011
0.484 0.163 0.34 0.031
0.251 0.108 0.43 0.021
0.229 0.089 0.39 0.017
0.036 0.024 0.67 0.005
0.491 0.192 0.39 0.037
0.138 0.042 0.30 0.008
0.523 0.195 0.37 0.038
0.68 0.19 0.28 0.04
that are islet cells is approximately 10% larger than the number fraction that are islet cells. Next, we must account for the extracellular volumes VIEC and VNIEC within the islet and non-islet domains, retrospectively. Volume definitions and relationships are shown in Figure 6.3. In each tissue, the
Vascular Void
Whole Islet
Volume Fraction Islet
total volumes VI and VNI in the islet and non-islet domains, respectively, are the corresponding sums of the extracellular volume and the cell volume, Equations 6.21 and 6.24 (Figure 6.3). The definitions of corresponding volume fractions for islet cells IC and islet extracellular space IEC , Equations 6.27
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Colton et al. TABLE 6.4 Calculated quantities from EM and LM morphological analysis. Average Cell Volume (mm3 )
b Cells in Islets Number Fraction
Volume Fraction by EM
Islet Equivalent
Cell Volume Fraction
Islet Volume Fraction
Whole Islet Cells Islet
Number of cells Total b
Islet
Non-islet
Total
Size Ratio
Sample
ν IC
ν NIC
ν TC
ν IC /ν TC fbIC
fbIC
bI
(fIC )EM (I )EM
nIE
1
12
13
14
15
16
17
18
19
20
21
22
1–16 2–06 2–08 2–17 2–19 2–20 2–21 2–22 2–24 2–25 3–06 3–10 3–13 3–16 3–19 3–21 3–23 3–25 3–28 4–01 4–06 4–07 4–10 4–11 4–15 4–23 4–24
750 861 791 775 860 815 693 811 779 828 762 837 820 842 849 895 811 822 756 790 855 796 760 804 714 831 862
717 735 750 703 812 754 730 751 698 610 698 435 774 652 586 857 567 576 752 591 826 509 593 642 439 689 746
728 763 769 733 830 794 718 794 729 721 726 614 794 776 697 878 775 746 753 692 839 704 675 742 611 712 788
1.03 1.13 1.03 1.06 1.04 1.03 0.97 1.02 1.07 1.15 1.05 1.36 1.03 1.08 1.22 1.02 1.05 1.10 1.00 1.14 1.02 1.13 1.13 1.08 1.17 1.17 1.09
0.636 0.838 0.711 0.681 0.837 0.754 0.533 0.747 0.690 0.777 0.658 0.794 0.764 0.804 0.817 0.901 0.747 0.767 0.648 0.708 0.827 0.719 0.655 0.735 0.571 0.784 0.839
0.806 0.925 0.854 0.874 0.886 0.635 0.888 0.836 0.924 0.879 0.731 0.875 0.841 0.892 0.821 0.901 0.885 0.907 0.914 0.936 0.956 0.875 0.886 0.814 0.852 0.893 0.919
0.570 0.654 0.604 0.618 0.627 0.449 0.628 0.591 0.654 0.622 0.517 0.619 0.595 0.631 0.581 0.638 0.626 0.642 0.646 0.663 0.676 0.619 0.627 0.576 0.603 0.632 0.650
0.356 0.251 0.466 0.451 0.381 0.667 0.319 0.724 0.414 0.587 0.458 0.608 0.446 0.708 0.512 0.574 0.892 0.764 0.251 0.583 0.466 0.768 0.555 0.667 0.732 0.195 0.395
0.386 0.276 0.498 0.483 0.412 0.695 0.347 0.749 0.445 0.618 0.490 0.638 0.478 0.734 0.544 0.605 0.904 0.786 0.276 0.614 0.498 0.791 0.587 0.695 0.757 0.216 0.426
1670 1455 1583 1616 1456 1537 1806 1544 1607 1513 1643 1497 1527 1487 1475 1399 1545 1524 1656 1586 1465 1574 1647 1557 1754 1506 1453
1062 1219 1125 1101 1218 1159 963 1154 1108 1176 1082 1188 1166 1195 1204 1260 1153 1168 1073 1123 1212 1132 1079 1144 1001 1181 1220
Mean SD COV SEM
806 47 0.06 9
674 110 0.16 21
745 61 0.08 12
1.09 0.08 0.08 0.02
0.739 0.086 0.12 0.017
0.867 0.066 0.08 0.013
0.613 0.047 0.08 0.009
0.526 0.179 0.34 0.034
0.554 0.177 0.32 0.034
1560 95 0.06 18
1140 69 0.06 13
and 6.26 (Figure 6.3), respectively, are based on total islet volume. The volume fractions of non-islet cells NIC and non-islet extracellular space NIEC , Equations 6.23 and 6.22 (Figure 6.3), respectively, are based on the total non-islet volume. Capital letters are used to distinguish the volume fractions in Figure 6.3, which are based on total islet or non-islet
nbIE
tissue volume (including extracellular volume), from the volume fractions in Table 6.5 designated by lowercase letters, which are based on total cell volume (Equations 6.13 and 6.14) or total islet cell volume (Equation 6.20). Available data for estimating IEC and NIEC are limited. The extracellular spaces in islet and non-islet tissue
94
6.
Characterization of Islet Preparations
TABLE 6.5 Basic number-volume relationships for cells in islet preparations. All Cells in Preparation Number
nIC = nb + nNb nNIC = nA + nD + nO nTC = nIC + nNIC
(6.5) (6.6) (6.7)
Number Fraction
Volume
Cells
VIC = Vb + VNb = nb νb + nNb νNb (6.10) VNIC = VA + VD + VO = nA νA + nD νD + nO νO (6.11) VTC = VIC + VNIC (6.12)
Islet Non-islet Total
Volume Fraction
nIC nTC nNIC = = 1 − fIC nTC
VIC VTC VNIC = = 1 − fIC VTC
fIC =
(6.8)
fIC =
(6.13)
Islet
fNIC
(6.9)
fNIC
(6.14)
Non-islet
(6.15)
Islet
(6.16)
Non-islet
(6.17)
Total
(6.18)
Islet
Average Cell Volume
nb νb + nNb νNb 1 VIC nTC nb νb + nNb νNb = = = fb νb + fNb νNb nIC nIC nIC nTC fIC VNIC 1 ν NIC = = (fA νA + fD νD + fO νO ) nNIC fIC VTC ν TC = = fb νb + fNb νNb + fA νA + fD νD + fO νO nTC ν IC =
Cell Number Fraction/Volume Fraction Conversion
fIC =
VIC nIC ν IC ν IC = = fIC VTC nTC ν TC ν TC Islet Cells
β Cell Number Fraction fbIC =
nb nIC
=
nTC nb nIC nTC
=
fb
(6.19)
fIC
β Cell Volume Fraction fbIC =
Vb VIC
=
nb νb nIC ν IC
=
fb νb
(6.20)
fIC ν IC
of the mouse in vivo are 39 and 26% of tissue water, respectively, based on an estimated tissue water content of 75% [107]. Thus, IEC = 0.29 and NIEC = 0.19 when based on total tissue volume. This in vivo value for islets is comparable to an in vitro measurement in toad fish islets [31]. For islets, we keep separate track of the vascular volume fraction VI , defined by Equation 6.25 (Table 6.3), the mean value (Table 6.3, column 9) of
which is about 14% from LM data. Thus, the interstitial volume fraction is about 0.29 − 0.14 = 0.15. If the same value applies to non-islet tissue, its vascular volume fraction is about 0.19 − 0.15 = 0.04. The vascular volume fraction is similar for freshly isolated rat islets [15]. After 24 hours in culture, the vascular volume collapses completely, leading to a substantial reduction in rat islet volume [16], whereas the collapse is substantial
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Colton et al.
Islet
Non Islet Vascular + Interstitial
VNIEC
Extracellular
Vascular VVI
VIEC
Interstitial VINT
VIXV
Acinar Cells
VNI
VNIC
Cellular
β Cells
VIC
Duct Cells Non β Cells
Other Cells
VNI = VNIEC + VNIC
Volume
VI = VVI + VIXV = VIEC + VIC
(6.21)
VVI VI V ΦIEC = IEC VI ΦVI =
Volume Fraction
ΦNIEC =
VNIEC VNI
ΦNIC = 1−ΦNIEC = Overall Purity
ΦI =
VI VI + VNI
VI
(6.22)
VNIC VNI
ΦIC = 1−ΦIEC =
(6.23)
(6.24) (6.25) (6.26)
VIC VI
VIC VIC VTC φIC 1−ΦIEC 1−ΦIEC 1−ΦIEC = = = φIC VIC VNIC VIC VTC VNIC VTC 1−ΦIC + + + 1−ΦIEC 1−ΦNIEC 1−ΦNIEC 1−ΦIEC 1−ΦIEC 1−ΦNIEC
(6.27)
(6.28)
Figure 6.3. Volume definitions and relationships in islet preparations.
but not complete after several days in culture of human islets. Returning to Figure 6.3, Equation 6.28 (Figure 6.3) for the volume fraction islets I is the key relationship, and its derivation is given in sequential detail. The first step involves use of Equations 6.23 and 6.27 (Figure 6.3) for expressing VNI and VI , respectively. All terms are then divided by the total cell volume VTC so that use can be made of volume fraction fIC from Equation 6.13 (Table 6.5), which in turn is related to number fraction fIC from Equation 6.18 (Table 6.5). Equation 6.28 (Figure 6.3) thus provides the bridge-relating number fraction to volume fraction data. Using Equation 6.28 (Figure 6.3), the average value of islet volume fraction (I )EM computed from EM cell number fraction data is 0.554 (Table 6.4, column 20), which compares well with the value measured directly by LM, (I )LM = 0.523 (Table 6.3, column 10). There
is exceptional agreement for volume fractions above 0.3. This agreement validates the use of LM point counting for determining islet purity. Several other quantities in Table 6.4 are of interest. An islet equivalent (IE), a sphere of diameter 150 mm, has a volume VIE = 1.77 × 106 mm3 . The volume of cells in an IE is given by the product of (1 − IEC ) and VIE , and thus the number of cells nIE in an IE is nIE =
(1 − IEC ) VIE ν IC
(6.29)
and the number of beta cells in an IE is nbIE = fb nIE .
(6.30)
The averages for all preparations (from columns 21 and 22 of Table 6.4, respectively) are nIE = 1,560 islet cells and nbIE = 1,140 b cells per IE.
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6.
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Other b cell quantities calculated from EM data are shown in columns 16 through 18 of Table 6.4. The number fraction of b cells among all islet cells fbIC = 0.739 becomes fbIC = 0.867 on a cell volume basis. The volume fraction of the entire islet that is b cells bI is calculated from bI = (1 − IEC ) fbIC .
(6.31)
The average volume fraction of b cells in whole islets is bI = 0.613. Coefficients of variation for these last two quantities are 8%, thereby demonstrating that the volume fraction of b cells relative to all islet cells is highly reproducible from sample to sample. Whereas the number fraction-volume fraction conversion is insensitive to the value for islet extracellular volume fraction IEC , the quantities calculated from Equations 6.28 through 6.31 are much more sensitive. For the record, the mean values in Tables 6.3 and 6.4 are slightly different from those originally reported [116] because only 27 of 33 preparations, for which we had both LM and EM data, are included here.
COMPARISON OF MORPHOLOGICAL ANALYSIS WITH DTZ STAINING Purity measurements with DTZ staining (Table 6.3, column 11) give an average volume fraction islets (I )DTZ = 0.68, about 30% higher than the value of 0.523 from direct LM measurements and 23% higher than the value of 0.554 calculated from EM data. In many individual cases, the measurement from DTZ staining is much higher than that from LM point counting; thus, DTZ staining may provide a gross overestimation of islet purity. A similar discrepancy occurs with individual samples when DTZ staining is compared with volume fraction estimated from EM number fraction data [116].
COMPARISON AMONG VARIOUS STUDIES b-cell composition data from a variety of studies are compared in Table 6.6. The data is tabulated either as volume fraction of the islet that is b cells bI or
TABLE 6.6 Comparison of literature values for b-cell content in islets. Tissue Examined
Freshly Isolated Islets Cultured Islets1 Cultured Islets2 Dissociated cells Dissociated cells Pancreatic block Pancreatic block Pancreatic block Pancreatic block Pancreatic block Pancreatic block
b Cells in Islet Cells
Method
EM Morphology IHC-LSC Microscopy IHC IHC-LS Cytometry IHC IHC-LSC Microscopy IHC IHC IHC IHC IHC
Volume Fraction
Number Fraction
bI
fbIC
0.61 0.54 0.67 0.43 0.47 0.46 0.523 0.594 0.68 0.755 0.635
0.74 0.65 0.80 0.51 0.57 0.55 0.62 0.71 0.80 0.90 0.76
IHC = Immunohistochemistry with standard fluorescence microscopy unless otherwise noted. Bold face number is data as reported. Companion number is calculated using equations in this chapter. 1 Islets shipped and cultured for 48 hr total. 2 Islets cultured 7-14 days under conditions favoring b-cell enrichment. 3 Lean, nondiabetic subjects. 4 Korean subjects. 5 Volume-weighted calculated value for 90% glucagons-rich, PP-poor lobes, 10% PP-rich lobes.
97
Reference
O’Neill et al., 2006 [116] Brissova et al., 2005 [22] Keymeulen et al., 1998 [86] Ichii et al., 2005 [77] Street et al., 2004 [177] Cabrera et al., 2006 [25] Butler et al., 2003 [24] Yoon et al., 2003 [198] Kloppel et al., 1985 [88] Stefan et al., 1982 [176] Malaisse-Lagac et al., 1979 [104]
Colton et al.
number fraction of islet cells that are b cells fbIC . All studies make use of immunohistochemical methods in one form or another except for our recent EM morphological analysis [116]. That study is also the only one with freshly isolated whole islets. The b-cell volume fraction of the islet averages about 61% for the three studies with islets. Islet volume fraction in cultured islets that were shipped is lower [22], possibly reflecting preferential b-cell death under stress. Conversely, in one study the islet culture method may have favored b (but not a) cell survival and accounted for an increased volume fraction of b cells [86]. The two studies that used dispersed islets [75, 177] yield uniformly lower values for b-cell content, which may result from selective b cell loss incurred with islet dissociation, as described previously. Data from islets embedded in the pancreas span a wide range [24, 25, 88, 104, 176, 197]. All studies except Cabrera [26] report data as volume fraction of b cells in islets, and these values average 63%, which is comparable to the value obtained from preparations of isolated islets.
SAMPLING ISSUES The weak link in characterizing islet preparations by all of the methods described in this section, and in the other sections as well, is the initial aliquot sampling in which a sample with a volume typically about 100 ml is removed from a vessel containing the entire islet preparation in 250 ml of suspension. The extent of systematic error incurred in this initial sample is unknown. The best current practice is to keep the suspension evenly distributed during sampling by repetitive inversion of the tube and to sample with a consistent procedure every time. Starting with an initially well-mixed uniform suspension, sedimentation will begin immediately if it is not eliminated or sufficiently minimized. Differences in size and density of the aggregates will lead to significant differences in settling velocity, further aggravating the situation by causing additional gradients in size and composition so that a sample from a small volume may no longer be representative of the entire volume. A second issue is local variations in the sample (independent of sedimentation) that reflect intrinsic differences in tissue properties. Because these differences should be random, precision in estimating mean values improves as more tissue is analyzed and follows standard statistics.
TABLE 6.7 Methods for measuring the quantity of islets in an islet preparation. Type of Quantity Parameter Measured
Method
Volume
Islet Volume
DTZ Staining Large Particle Flow Cytometry
Total Tissue Volume
Tissue Pellet Packed Cell Volume Ultrasound Scattering
Number of Cells Total DNA DNA Content Total Intact Cell Nuclei Nuclei Counting
Quantity of Tissue Methods for measuring the quantity of islets in an islet preparation are summarized in Table 6.7. It was recognized around 1990 that counting the number of islets was a poor way to quantify the amount of islet tissue because of the intrinsic variability in islet size and shape [44]. It was recommended that the total volume of islets in a DTZ-stained preparation be estimated and be reported in terms of the number of islet equivalents [21, 151, 156, 166] in an effort to standardize islet quantification and improve accuracy and precision, and it remains the standard method. An alternative is to measure total tissue volume and determine islet volume fraction independently, as described previously. A different and potentially more accurate approach is to measure the total number of cells and to combine it with an estimate of the number fraction of islet cells. There is increasing interest in quantifying the b cells in an islet preparation. This can be estimated from the number or volume fraction of b cells in islets, as described in the previous section, combined with the total number or volume of cells, respectively.
VISUAL COUNTING OF DTZ-STAINED ISLETS The conventional method is based on microscopic visualization of size and manual counting of samples stained with DTZ [97]. Islet samples are stained with DTZ and are visually examined under the microscope.
98
6.
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Islets are divided in different groups of 50-mm increments based on their diameter and an average conversion factor is used for all islets within each 50-mm range. The volume of islets in the preparation is calculated assuming that the islet is a perfect sphere. This method is operator dependent and discrepancies as much as 40% have been observed between different operators analyzing exactly the same photomicrographs of an islet preparation [3]. The method is also prone to error because the actual shape of the islet is not spherical [50, 187], which introduces an upward bias leading to overestimation of volume no matter how the measurement is made. With few exceptions [61], the potentially large magnitude of this overestimation is generally not appreciated. Dionne [36] and later Avgoustiniatos [6] carefully examined rat islets hand picked to be among the most spheroidal and rotated them over three axes in a microscope with a calibrated reticule. Assuming that the islets could be represented as ellipsoids of revolution with half axes a, b, and c, Avgoustiniatos found on average b/a = 0.82 and c/a = 0.60. The volume of an ellipsoid is given by 4 V = pabc. 3
(6.32)
Setting a = 1, the quantity 3V/4p = abc is 0.492. If the ellipsoid is lying on a surface with its smallest axis c perpendicular to the surface, the most likely situation, it appears as an ellipse with half-axes a and b. Because the half axis c is usually not measured with DTZ staining, estimates of the radius of an equivalent sphere must be made from a and b. Table 6.8 summarizes the value of 3V/4p = r 3 for an equivalent sphere having various possible radii: (1) the longest half-axis, (2) arithmetic average of two half-axes, and (3) geometric average of two half-axes. This latter case would arise if the area of the ellipse, which is pab, were made equivalent to that of a circle, pr 2 . The ratio of the computed sphere volume to the true volume of the ellipsoid is about 1.5 for the two most reasonable estimates of r, an overestimation of 50%. This simple calculation should serve as a red flag for the possibility of gross overestimates of IE number with current techniques.
LARGE PARTICLE FLOW CYTOMETRY Flow cytometry is well established for characterization of single-cell preparations, but size limitations in
TABLE 6.8 Errors incurred by en-face sizing of ellipsoids as equivalent spheres. Selected Radius r
r3
Volume of Sphere Volume of Ellipsoid
a a+b 2 (ab)1/2 4 3
Vsphere = pr3
1.0
2.03
0.754
1.53
0.743
1.51 4 3
Vellipsoid = p abc
a = 1, b = 0.82, c = 0.6, abc = 0.492. Ellipsoid is assumed to lie on surface with smallest axis perpendicular to surface.
traditional flow cytometers render their use inapplicable for islet preparations. Preliminary experiments with islet preparations using a large particle flow cytometer have recently been reported [49]. The new instrument has the ability to analyze large objects, such as islets, with minimal shear forces and to sort these objects (using object size, optical density, and intensity of fluorescence markers as the analytical criteria) with a gentle pneumatic sorting mechanism that minimizes cell damage. In experiments performed with islet preparations [49], a standard calibration curve was created with microspheres of known diameter to correlate time-of-flight measurements to particle diameter. Newport Green staining allowed discrimination between islet and nonislet tissue and yielded an estimate of purity. In five of six islet preparations, islet volume averaged 14% higher than that measured with conventional DTZ staining (about 100% higher in the sixth preparation), but purity measured with the flow cytometer was lower. Islet enumeration by the large particle flow cytometer has advantages over manual counting. The diameter of each islet is measured rather than being grouped in 50mm ranges. The method is automated, which decreases the operator-dependent error intrinsic to manual counting. A large number of islets can be analyzed nondestructively, thereby eliminating the possibility of sampling error. However, the method gives estimates with slightly greater volume than manual counting, which itself is thought to overestimate volume, suggesting that
99
Colton et al.
improved calibration methods are needed. Furthermore, the equipment represents a large capital cost.
PACKED CELL VOLUME (PCV) MEASUREMENTS Measurement of the packed cell volume (PCV) of a pellet is among the standard parameters used to quantify islet preparations [116]. The islet preparation is centrifuged to create a packed pellet, the supernatant is removed to the pellet surface, and a known volume of medium is added. The tissue is resuspended in fresh medium and carefully aspirated in a serological pipette to avoid forming bubbles because they interfere with measurements. The final volume of the tissue suspension is measured, and the volume of the packed cell pellet VPC is calculated as the final volume of the tissue suspension minus the volume of the fresh medium added.
The packed cell volume of a human islet preparation can be combined with data on composition, such as from the EM or LM morphological analyses described previously, to calculate various properties of interest [116], as summarized in Figure 6.4. The packed cell pellet (which consists of islet tissue, non-islet tissue, and aggregates of the two) also contains microscopic void spaces between the components with a void volume VVPC , which is distinct from the extracellular space within the domain of the tissues, and a corresponding void fraction VPC , Equation 6.33 (Figure 6.4), based on the entire packed cell volume. If this void fraction parameter is known, the total tissue volume VT (compose of islet VI and non-islet VNI volumes) is calculated from Equation 6.34 (Figure 6.4). The volume of islets and islet cells and the number of IE and islet cells are calculated successively from Equations 6.35, 6.36, 6.38, and 6.39 (Figure 6.4), respectively. Volume and number of b cells are calculated from Equations 6.37 and 6.40 (Figure 6.4) respectively, by using cell composition data. Equation 6.37
Packed Cell Pellet Void
Non-islet
VVPC
VNI
Islet
Volume Fraction Voids
ΦVPC =
VVPC VPC
(6.33)
VI VT
VPC Number
Volume Total Tissue
VT = (1 − ΦVPC) VPC
(6.34)
Islets
VI = ΦI VT
(6.35)
NIE =
VI VIE
(6.38) Islet Equivalents
Islet Cells
VIC = (1 − ΦIEC) VI
(6.36)
nIC =
VIC νIC
(6.39) Islet Cells
Beta Cells
Vβ = φβIC VIC
Vβ nβ = ν = fβIC nIC (6.40) Beta Cells β
= φβIC (1 − ΦIEC) VI = φβI VI
(6.37)
Figure 6.4. Volume and number definitions and relationships in a packed cell pellet.
100
6.
Characterization of Islet Preparations
is expressed several different ways. It is reasonable to assume that IEC is approximately constant in all freshly isolated islet preparations, and fbIC is remarkably consistent between preparations (Table 6.4). Therefore, Equation 6.37 indicates that the volume (and the number) of b cells in a preparation is directly proportional to the volume of islets in that preparation [116]. We have combined PCV measurements made with centrifugation at 248xg for 1 minute with the LM volume fraction and EM cell composition data for the 27 clinical islet preparations summarized in Tables 6.3 and 6.4. We used a preliminary estimate of VPC = 0.3 for the void volume fraction of the packed cell pellet, which was determined from mannitol distribution space measurements in packed rat islet preparations. This value falls at the low end of the range of 0.26 to 0.48 estimated for packed spheres [106]. Nonspherical shape [40] and wide size distribution [35] would tend to decrease VPC slightly, whereas red cell packing measurements under comparable conditions suggest it could be substantially higher [72]. In view of the uncertainty in VPC , these calculations (summarized in Table 6.9) may be considered preliminary. Packed cell, islet, and b-cell volumes averaged 1.9, 0.58, and 0.35 ml, respectively. IE number, obtained by combining PCV and LM data, averaged 327,000 (corresponding to 370 million b cells). IE by DTZ staining averaged 428,000, about 31% higher than by PCV/LM, and the DTZ estimate was higher in 24 of 27 preparations. PCV measurements are attractive because they are simple. When combined with purity estimation from morphological analysis, they provide an estimate of islet volume thought to be better than visual counting with DTZ staining. That estimate can be useful as a check on the occurrence of aberrant estimates by DTZ staining. However, further work to better define the packed cell void volume in human preparations is warranted.
ULTRASOUND PULSED DOPPLER MEASUREMENTS A new ultrasonic pulsed Doppler technique (USPD) was recently described that measures the ultrasonic power backscattered from suspended particles and hence has the potential to provide rapid automated measurement of total tissue volume using only a few hundred islets [1]. The system uses a single small probe transducer (diameter 4 mm), small sample volumes (about 1 ml or less)
in a stirred cell, data acquisition in about 30 minutes, and capability of measurements with a few hundred islets. Measurements with this system have been made with b cell lines, rat islets, human islets, and exocrine tissue. In one study, a 1.2-ml volume sample was placed in a temperature-controlled acrylic test chamber stirred to suspend the tissue. The transducer, seated flush with the wall, injected a series of tone bursts at 16 MHz into the sample that was focused by a lens into a small volume situated 1.6 mm from the chamber wall. The islets were set in motion by the stirring and by acoustic streaming away from the transducer generated by the high-intensity ultrasound. Backscattered Dopplershifted energy reflected from the moving particles and returned to the transducer was conditioned and passed to a digital oscilloscope for processing. A high-resolution power spectrum was computed from which the total backscattered power was determined. The backscattered power was plotted against the total cell concentration as measured by nuclei counting (described in material following), yielding a linear correlation for high and low purity islets as well as exocrine tissue, as shown in Figure 6.5. With further study, this method may be very attractive for measuring the total tissue volume in an islet preparation.
NUCLEI COUNTING Nuclei counting is a standard method for enumeration of individual cells [54, 163]. The release of nuclei from cells disrupted with citric acid occurs without nuclei breakdown or cell losses. With addition of a surfactant, nuclei counts can be used to quantitatively assess cell number in suspensions of cell aggregates [125]. We investigated nuclei counting for determining the number of cells in an islet preparation [143, 144]. We developed a cell and islet disruption protocol, compared three different counting methods, and evaluated the accuracy and precision of the methods. We also measured the DNA content of islet cells and compared it with values reported in the literature. Finally, we combined nuclei counting with islet purity measurement by LM morphology analysis to quantify islets in impure preparations. A schematic diagram of the method and the counting options is shown in Figure 6.6. An aliquot of cells or islets in suspension is incubated for 5 minutes with a lysis solution consisting of citric acid and surfactant,
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Colton et al. TABLE 6.9 Data and calculated quantities from packed cell volume measurementsa . Volume (ml)
Number
Packed Cell Pellet
Islets
Beta Cells
Sample
VPC
VI
1
2
1–16 2–06 2–08 2–17 2–19 2–20 2–21 2–22 2–24 2–25 3–06 3–10 3–13 3–16 3–19 3–21 3–23 3–25 3–28 4–01 4–06 4–07 4–10 4–11 4–15 4–23 4–24 Mean SD COV SEM
IE by DTZ
Beta Cells by
Vb
PCV/LM Staining (NIE )PCV/LM (NIE )DTZ
PCV/EM nb ×106
3
4
5
6
7
3.50 5.00 3.50 1.70 2.20 1.20 2.00 2.00 4.00 1.50 1.10 0.80 1.35 0.70 1.70 0.90 1.20 1.20 3.70 1.00 1.20 0.80 0.60 1.20 1.50 2.80 2.20
0.90 0.32 1.25 0.56 0.60 0.57 0.44 1.00 1.31 0.61 0.40 0.31 0.44 0.35 0.56 0.41 0.74 0.63 0.52 0.41 0.40 0.41 0.22 0.59 0.78 0.24 0.65
0.51 0.21 0.75 0.35 0.38 0.26 0.28 0.59 0.86 0.38 0.21 0.19 0.26 0.22 0.33 0.26 0.46 0.41 0.34 0.27 0.27 0.26 0.14 0.34 0.47 0.15 0.42
508,000 179,000 704,000 319,000 340,000 323,000 249,000 566,000 741,000 343,000 224,000 176,000 246,000 196,000 319,000 234,000 419,000 358,000 295,000 233,000 227,000 234,000 127,000 332,000 441,000 137,000 369,000
544,000 400,000 451,000 460,000 370,000 382,000 326,000 499,000 524,000 266,000 341,000 298,000 269,000 445,000 469,000 289,000 319,000 600,000 814,000 243,000 516,000 532,000 290,000 497,000 447,000 387,000 570,000
539 219 792 367 397 270 291 623 903 398 216 203 273 231 345 279 489 428 355 288 285 270 148 356 496 161 446
1.87 1.15 0.62 0.22
0.58 0.27 0.47 0.05
0.35 0.17 0.48 0.03
327,000 155,000 0.47 30,000
428,000 129,000 0.30 25,000
373 180 0.48 35
IE by
a Quantities calculated as follows: Column 2 from Equation (6.34); Column 3 from Equation (6.35) with
I from Equation (6.20) and with fb and fIC from EM data; Column 5 from Equations (6.38); Column 7 from Equation (6.40) with fbIC from Equation (6.19) and nIC from Equation (6.5) with parameters from EM data.
from LM data; Column 4 from Equations (6.36) and (6.37) with
and this suspension is then sheared by vortex mixing of cells or flow of islets through a hypodermic needle to liberate nuclei. Three different methods can be used for counting the liberated nuclei: (1) a sample is stained with crystal violet to darken the nuclear envelope and
fbIC
the nuclei are counted visually using a hemacytometer, (2) a sample is analyzed using aperture electrical resistance measurements with the Coulter Multisizer II, or (3) a sample is stained with the fluorescent dye 7-AAD that binds to cellular nucleic acids with very
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6.
Characterization of Islet Preparations
8 High purity human islets 30% human islets
7 Backscattered Power (µW)
Exocrine tissue
6 5 4 3 2 1 0
0
1
2 3 4 5 6 Cell Concentration (106 cells/ml) by Nuclei Counting
7
Figure 6.5. The backscattered power measured by the USPD technique is plotted against the total cell concentration as measured by nuclei counting, yielding a linear correlation for high and low purity islets as well as exocrine tissue.
high affinity. This sample is analyzed in a Guava PCA flow cytometer, a small unit that is especially convenient for this assay. Discrimination of nuclei by the Guava PCA system relies on the 7-AAD intensity and
the forward scatter, which in part reflects the relative size of the particles passing through the flow cell. The forward scatter threshold is set so that only particles large enough to be nuclei are counted, and debris is ignored. Only the stained nuclei display high 7-AAD intensity so that the system provides an accurate count of the nuclei population. Analysis of triplicate samples containing about 1,000 nuclei requires about 1 hour for visual counting, 11 minutes for the aperture resistance method, and only 6 minutes for the Guava PCA flow cytometer. The accuracy of the nuclei counting method was verified with a stock nuclei suspension prepared from INS-1 cells diluted to produce samples covering a 104 -fold range of concentration that were counted by the three different methods. With each method, the measured nuclei concentration agreed with the calculated nuclei concentration until the point where the relationship between the two departed from linearity (7 × 104 nuclei/ml for aperture resistance, 6 × 105 nuclei/ml for flow cytometry) as a result of interference effects at higher concentrations. The precision of nuclei counting measurements approximately followed Poisson statistics. With INS-1 cells, the precision improved as the number of nuclei counted increased with a coefficient of variation about 3% for about 1,000 nuclei counted. With islets,
Citric Acid Surfactant
lls
Ce
Vortex Mixing (Cells) Shearing Through Needle (Islets)
Isle
ts
103
nuclei x 3 Time (min) Visual Counting Hemacytometer
70
Aperture Resistance Coulter Multisizer II
16
Flow Cytometer Guava PCA
11
Liberated Nuclei
Crystal Violet
40 % (v/v) Glycerol in Isoton II
7-AAD
100 µm
Figure 6.6. Schematic diagram of protocol developed for nuclei counting of cell and islet samples. Islet or cell aliquots are incubated with a lysis solution (citric acid and surfactant). Nuclei are liberated by either vortex mixing for cells or shearing through a needle for islets. The liberated nuclei can be analyzed with three different methods: visual counting in the hemacytometer, aperture resistance measurements in the Coulter Multisizer II, and flow cytometry in the Guava PCA.
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the coefficient of variation decreased as the number be expressed in terms of the number of islet equivaof islets sampled increased with a value of about 6% lents NIE by use of Equation (6.41). Two quantities for 125 IE or more sampled. Additional validation of from the EM cell composition results (Table 6.4) are the technique was obtained by counting nuclei and the used in this calculation: the average value of the ratio cell suspension from which they were prepared. Fur- ν IC ν TC = 1.09 and the average number of cells in thermore, in a comparison of nuclei counts obtained an islet equivalent, nIE = 1,560. Had the analysis made with flow cytometry to DNA measurements from serial use of EM morphological analysis, it would have started dilutions of stock suspensions of cells and islets, DNA directly with the number fraction islet cells, fIC . concentration was linearly proportional to nuclei conThe analysis outlined in Figure 6.7 has been applied centration. to samples of freshly isolated human islet preparations These studies have demonstrated that nuclei counting for research in an ongoing study. For each preparation, can rapidly provide accurate and precise measurements the total number of IE was estimated by visual countof the number of cells with an intact nucleus in islet ing of DTZ-stained islets for the entire preparation. preparations. Nuclei counting to obtain the total number A known fraction of that preparation was processed of cells can be combined with morphological analysis for nuclei counts within 4 to 6 hours after isolation. (EM or LM) to quantify the number of islet cells and Samples of the same preparation were saved to be proislet equivalents in an impure human islet preparation. cessed for LM morphology analysis. Visual counting We compared the number of IE obtained by two substantially overestimated the number of IE compared methods: visual counting of DTZ-stained islets and to LM/nuclei counting. On a graph on which the number LM/nuclei counting for six research human islet of IE from visual counting was plotted against the numpreparations. The calculation procedure is summarized ber of IE from LM/nuclei counting, a straight-line fit in Figure 6.7. LM morphology analysis provides a mea- to the data yielded a slope of 1.90, which corresponds surement of the islet volume fraction, I , which is to 90% overestimation by visual counting with DTZsuccessively converted to islet cell volume fraction fIC stained islets. The large overestimation associated with and then to the islet cell number fraction fIC , which visual counting may reflect two factors: (1) errors assoin turn is combined with the total cell number nTC to ciated with the volume calculation and (2) regions of yield the number of islet cells, nIC . This latter param- dead cells within the tissue that do not possess intact eter suffices to characterize the preparation. It can also cell nuclei.
LM Morphology Volume Fraction Islets
Nuclei Counting Total Number Nuclei
nTC
(ΦI)LM Eqn (6.28) Figure 6.3
Islet Cell Volume Fraction
φIC
Eqn (6.18), Table 6.5
νIC νTC
Number Islet Equivalents
NIE =
Islet Cell Number Fraction
fIC
= 1.09
nIC (6.41) nIE
Eqn (6.8) Table 6.5
Table 6.4
nIE = 1560
nIC
Number Islet Cells
Figure 6.7. Calculation of number of islet cells and islet equivalents in impure human islet preparations from nuclei counting and LM morphological analysis.
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DNA CONTENT DNA measurements are used to quantify the total amount of tissue in an islet preparation, in that each cell with a single nucleus should carry the same amount of DNA. DNA is quantified by fluorospectrophotometry using a commercially available dye that undergoes strong fluorescence enhancement when bound to cellular nucleic acids. The fluorescence intensity is linearly related to the concentration of nucleic acids in the sample, and the DNA concentration is determined from a calibration curve created with a DNA standard. DNA measurements are common and are advantageous because they are not operator dependent, as is visual counting [11, 44, 133]. The limitations with DNA measurements are the time requirements and the stability and variability of the DNA standard. Therefore, comparisons between different laboratories may be difficult to perform on the same basis and consequently difficult to compare. To explore these problems, we performed DNA measurements with two different commonly used DNA-binding fluorescent dyes (Cyquant and Picogreen) following the same protocol and using the same lambda DNA standard for bTC3 cells and rat islets. Values measured with the CyQUANT dye (6.2 ± 0.6 and 6.4 ± 0.8 pg/cell for bTC3 cells and rat islets, respectively) were significantly higher (p < 0.05) than corresponding values measured with PicoGreen dye: 5.6 ± 0.8 and 4.9 ± 0.9 pg/cell for bTC3 cell and rat islets, respectively. Such differences may be in part responsible for the problems with inconsistency in DNA comparisons between different laboratories. Another concern with the DNA measurements is the degradation
A. Rat
of the standard with time. We found that the slope of the standard calibration curve measured with the same lambda DNA standard decreased about 35% over a 5-month period. This decreased slope was mirrored by a decrease in the amount of DNA measured with control tissue samples run parallel to the standard. Thus, frequent change to a new standard and recalibration is essential to achieving reproducible results. We find it useful to combine nuclei number and DNA content assays. Measurements made with CyQUANT dye over a 3-year period with 37 different batches of rat islets yielded 6.5 ± 1.9 pg/cell (Figure 6.8a). Measurements performed with 26 preparations of freshly isolated human islets yielded 6.9 ± 2.3 pg/cell (Figure 6.8b). These values are comparable to the expected value of about 6.5 pg/cell for human DNA [40, 139]. Similar measurements with human islets received from various centers after being shipped (21 preparations) yielded 8.5 ± 2.3 pg/cell (Figure 6.8c). These data suggest that some islet cells may die during shipment and release DNA that is not completely degraded and contributes to the higher measured value. This is another reason why assay of DNA alone may not provide an accurate measurement of the number of cells in an islet preparation and why nuclei may provide better accuracy.
ESTIMATION OF ISLET EQUIVALENTS IN THE PANCREAS The number of IE in a normal pancreas was recently estimated to average about 500,000 IE [90] on the basis of pancreatic islet volume estimates of 0.5 to 1.3 cm3 made
B. Human (Freshly Isolated)
16
Frequency
14
5
8
12
4
10
6 3
8 4
6
2
4
2
1
2 0
C. Human (Shipped) 6
10
0
2
4
6
8
10
12
0
0
2
4
6
8
10
12
0
0
2
4
6
8
10
12
DNA Concentration (pg DNA/cell) Figure 6.8. Distributions of DNA content per cell (pg DNA/cell) from (a) 37 rat islet preparations, (b) 26 freshly isolated human islet preparations, and (c) 22 human islet preparations received from other centers after shipment.
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with pancreata from Japanese cadavers and reported in 1978 [161]. By comparison to the commonly reported average value of about 500,000 IE isolated from a cadaver pancreas, it was concluded that a major fraction of the native islets is being recovered by current isolation methods. We have made a calculation based on more recent estimates that the volume fraction of b cells in the pancreas averages about 1.9% [24, 197]. Assuming a pancreas tissue density of 1.1 g/ml, a volume fraction b cells in islets of 0.61 (Table 6.6), and an average pancreas weight of 84 g [116], we estimate that the islet volume is 2.4 ml, which corresponds to 1.3 million IE per pancreas. Furthermore, our data presented earlier in this section suggest that the average number of IE in an islet preparation is substantially less than 500,000 IE. Taken together, these estimates suggest that only a small fraction of islets in the native pancreas are actually recovered during islet isolation.
Viability Membrane integrity measurement by visual assessment following staining with fluorescein diacetate and
propidium iodide (FDA/PI) is the most commonly employed method of assessing fractional viability of islet preparations. This assay usually gives erroneously high values that are not predictive of the transplantation outcome. Membrane integrity tests differentiate only between dead (membrane permeable) and non-dead (membrane impermeable) cells but fail to distinguish healthy cells from cells that have entered the cell death process but have not reached the membrane permeabilization step. Consequently, there is no standard assay currently employed that gives a meaningful measure of islet viability. A variety of alternative techniques are currently under investigation, as summarized in Table 6.10. These alternatives include new dyes for membrane integrity tests and new ways of quantifying membrane integrity tests with islets, assays of events associated with apoptosis, and measures of mitochondrial function. The latter category includes measurements related to cell redox state, rate of oxidative phosphorylation assessed by ATP content or oxygen consumption rate, and mitochondrial membrane potential. In this section, methods for each type of assay are briefly summarized, with more in-depth examination of assays developed in our laboratory. We conclude with a comparative study
TABLE 6.10 Methods for measuring the viability of islets in an islet preparation. Parameter Measured
Tissue Assayed
Method
Cell Membrane Integrity
Intact Islet Preparation
Live/Dead (Membrane Permeable) Fluorescein Diacetate/Propidium Iodide Alternative Dyes Dead Quantitative Nuclei Counting/7-AminoActinomycin D
Apoptotic Events
Intact Islet Preparation
Magic Angle Spinning 1 H-NMR Spectroscopy
Intact Islets (Disrupted)
Early: Signaling Pathway-Caspase Activation Late: Nucleosome DNA Fragmentation
Fixed Tissue or Cells
Phosphatidyl Serine Translocation-Annexin V DNA Fragmentation-TUNEL
Intact Islet Preparation
Redox State of the Cell: Tetrazolium Salts MTT, MTS Oxidative Phosphorylation Energetic State (ATP, ATP/ADP) Oxygen Consumption Rate
Dispersed Single Cells
Mitochondrial Membrane Potential Fluorescent Dyes JC-1, TMRE
Mitochondrial Function
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of the transient response of cells and islets to various stresses as reflected by assays of each major type, followed by a summary of data suggesting that oxygen consumption rate data combined with cell number or DNA data are predictive of transplantation outcome in the immunodeficient mouse.
CELL MEMBRANE INTEGRITY LIVE/DEAD STAINS Membrane integrity stains have been used to assess islet viability since the late 1980s [9, 10, 151, 156]. These stains assess whether cell membranes have become permeable to molecules that normally cannot permeate through the membrane. In most cases, permeabilization of the cell membrane signifies one of the final stages in the cell death process. Molecules that normally pass easily through the phospholipid bilayers of the cell membrane include hydrophobic, nonpolar, or small uncharged polar molecules, whereas those that do not pass easily include large uncharged, polar, or charged molecules. Some dyes commonly used in live/dead staining are shown in Table 6.11 [79]. It is generally thought that nonpolar dye molecules can easily permeate across cell membranes and that polar dye molecules cannot [10, 65, 66, 85, 92, 156], although the structures in Table 6.11 suggest that charge may also play a role. A normally membraneimpermeable dye enters a cell only when its membrane has been compromised. Thus, by staining cells with two different colored dyes (one that permeates and one that does not) it is possible to determine the relative quantity of cells that have intact cell membranes and those that have compromised membranes. Acridine orange (AO), which binds to all nucleic acids and is membrane permeable, together with propidium iodide (PI)—a membrane-impermeable dye that also binds to all nucleic acids—is one of the original combinations of membrane integrity dyes applied to islets [7, 8]. PI fluoresces red when bound to nucleic acids, whereas AO fluoresces green when bound to double-stranded DNA and red when bound to singlestranded DNA or RNA. In some studies, AO is replaced with fluorescein diacetate (FDA), a colorless ester until cleaved by nonspecific cellular esterases, causing it to fluoresce green [101, 102]. The cleaved product accumulates in cells that have intact membranes, resulting in a strong green fluorescence. Cells with compromised membranes do not accumulate the dye and therefore do
not give a strong green fluorescence. The membrane integrity stains are added to islet samples and incubated for a specified period of time. After incubation, the islets are observed under a fluorescence microscope to visually assess the approximate volume fraction of cells stained red compared to those stained green. After looking at many islets, an estimate is made of the overall average volume fraction stained red. In one study, viability was determined in a different fashion. A total of 50 islets were sampled and each islet was categorized as viable or nonviable, and then the overall fractional viability was calculated. Islets having more than 50% red staining on the surface or periphery or those with more than 25% centrally stained red were considered nonviable [10]. In that study, other dye combinations were also examined with islets: (1) SYTO-13 (Molecular Probes Eugene, OR), a nucleic acid stain that fluoresces green when bound to DNA, (2) ethidium bromide (EB), which fluoresces a red-orange color that intensifies when binding to DNA, and (3) Calcein AM (C), a green membrane-permeable fluorescent dye that is hydrolyzed by esterases, causing it to become negatively charged and be retained in the cytoplasm, along with ethidium homodimer (EH), a positively charged fluorescent nucleic acid stain that cannot cross intact cell membranes. C/EH and SYTO/EB provided substantial differential staining capability, whereas FDA/PI had high background fluorescence and obscured signal within the central regions of the islets, resulting in an overestimate of viability. Calcein AM was detrimental to the islets and caused significant fragmentation. SYTO/EB was suggested as a more accurate replacement for FDA/PI. Membrane integrity measurements are very quick and easy to perform, making them attractive for use immediately prior to transplantation. However, there are several problems that render them of limited value. Several studies have shown that the working stain solution must be diluted to a specific target concentration before being added to a sample [7, 29, 62, 85, 101, 102, 110, 111, 135, 174]. Too concentrated a solution results in significant background fluorescence, whereas too dilute a solution does not provide enough fluorescence. Timing is also critical, in that enough time needs to be allowed for the dyes to diffuse into the islet, but not so much that artifacts such as islet fragmentation occur. Perhaps the biggest problem with membrane integrity assays is that there is a lack of correlation between membrane integrity staining and other viability assays, including mitochondrial function assays such as MTT
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Colton et al. TABLE 6.11 Dyes commonly used in live/dead stains [79].
Membrane Impermeable
Membrane Permeable
Propidium Iodide (PI)
Acridine Orange (AO)
Fluorescein diacetate (FDA) Ethidium Bromide (EB)
Calcein AM (C) Ethidium Homodimer (EH)
7-aminoactinomycin D (7-AAD)
and ATP [29, 85, 111, 174, 196]. This may reflect the fact that mitochondrial dysfunction occurs early, whereas compromise of cell membranes occurs later in the cell death process [168]. QUANTITATIVE MEMBRANE INTEGRITY MEASUREMENT A major problem with visual estimation in membrane integrity tests for islets is that there are limitations in
viewing a 3D structure in a 2D plane, and it is necessary to guess how observations in a 2D field apply in three dimensions to the islet as a whole. This limitation can be improved to some degree by focusing through the islet and looking at multiple planes, but this in itself is difficult because each viewed plane may be obscured or affected by the previous or subsequent plane. In either case, this method is subjective, operator dependent, and not quantitative, and thus prone to error.
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One way around the problems of estimating the volume fraction of membrane-compromised cells in the 3D islet structure is to dissociate the islets into single cells and apply the membrane integrity test to the dispersed single cells after dissociation. However, dissociation of islets with trypsin (followed by dye exclusion measurements using the liberated cells) is not a suitable option because a substantial fraction of cells is lost during dissociation [139, 140, 193] and/or damaged, as described previously. We have developed a new method for making quantitative membrane integrity measurements that makes use of repeated nuclei staining in conjunction with flow cytometry to quantify the fraction of cells with intact membranes in islets, thereby eliminating entirely the need for observation of a 3D pattern in a microscope
Cells
Islet
Nuclei of membranepermeable cells are stained
7-AAD
Wash, disrupt tissue Citric Acid and Surfactant
Cells: Vortex mixing Islets: Shearing through needle
Flow Cytometer
All nuclei are stained
7-AAD
Flow Cytometer
Fraction (f) of cells permeable to 7-AAD
N1
=
N2
N1 N2
Figure 6.9. Diagram of the protocol for quantitative membrane integrity measurements with sequential 7-AAD staining. In the first step, cells or islets are stained with 7-AAD. Cells are disrupted with a lysis solution and shear, and nuclei are counted to give the number of membrane-permeable cells in the original suspension (N1 ). In the second step, the nuclei are stained with 7-AAD to label all cells, and the total number of cells is counted (N2 ). The fraction f of permeable cells is f = N1 /N2 .
[145, 146]. The procedure is summarized in Figure 6.9. Islets are stained with 7-aminoactinomycin D (7-AAD), a membrane-impermeable fluorescent dye with high affinity for cellular nucleic acids, to label nuclei in all cells with compromised membranes. Unbound 7-AAD is removed by washing. Islets are disrupted by mixing with a lysis solution (0.1 M citric acid and 1% Triton X-100) combined with high shear. An aliquot of the nuclei suspension is immediately analyzed with a flow cytometer, and labeled nuclei are immediately counted. Another equal aliquot of the stained nuclei suspension is stained again with 7-AAD, thereby labeling all previously unlabeled nuclei, and the total number of nuclei is counted. The fraction of cells with compromised membranes is estimated as the ratio of the initially stained nuclei from cells with compromised membranes (first measurement) to the total number of nuclei (second measurement). In contradistinction to the situation of islet dissociation to produce single cells, in which many cells are lost, all nuclei are recovered quantitatively following islet disruption, thereby making this procedure accurate. To validate the assay [146], islets were damaged by heat-induced killing (which compromised all cell membranes) and then mixed in various proportions with islets that had not been heat treated. The fraction of cells with compromised membranes, as measured by the 7-AAD sequential staining procedure, was equal to the fraction of heat-treated cells in the mixtures over the entire range studied. Furthermore, fractional membrane integrity assessed with repeated nuclei staining by 7-AAD correlated linearly with fractional viability measured using the MTT assay (described in material following). We concluded that sequential staining of nuclei with 7-AAD before and after cell disruption provides an objective, rapid, accurate, and precise quantitative estimate of the fraction of cells in islet preparations that has compromised membrane integrity. This assay is most useful with preparations of high purity because it does not distinguish between islet and non-islet cells. The comparison between 7-AAD membrane integrity measurement and MTT cell viability assay was carried out with islets that had undergone heat-induced cell killing, which leads to permeabilization of the cell membrane. In many other situations where stress is applied to intact islets, loss of cell membrane integrity occurs late in the cell death process, whereas the MTT assay responds to earlier steps that affect the redox potential of the cell. In our own hands, we have found
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that the two assays usually agree only after extended periods (24 to 48 hours) of culture under stable conditions [147, 148], as described in a subsequent section, during which cells that have entered the cell death process are able to reach the stage of membrane damage. Thus, caution must be exercised in interpreting the meaning of quantitative membrane integrity measurements with islets, especially within one day after isolation.
APOPTOSIS Apoptotic cell death is characterized by specific physical and molecular changes in the cell that start when the cell receives a pro-apoptotic stimuli and end when the cell nucleus breaks up into apoptotic bodies of various size. Once a cell passes a certain point in the apoptotic process it is destined to die. Although the exact point at which a cell is unable to recover is not known, insight can be gained into the overall health of a population of cells by measuring various characteristics of the apoptotic process known to occur before cell death. We focus here on some of the more widely studied apoptotic events that have been examined with islet tissue. These events include DNA fragmentation as measured by TUNEL and the nucleosome assay, phosphatidylserine externalization as measured by Annexin V staining, and caspase activation, all of which are characteristic events in the apoptotic process. In addition, we briefly discuss use of NMR spectroscopy to measure changes in the structure of intracellular lipids that correlate with apoptosis. As shown in Table 6.10, some of these assays (caspase activation, nucleosome, DNA fragmentation) can be used with intact islets, whereas others require dispersed single cells or islet histological sections. MAGIC ANGLE SPINNING 1 H NMR Nuclear magnetic resonance spectroscopy is widely used as a tool for medical diagnosis and research. A variant, high-resolution magic angle spinning proton NMR (HRMAS 1 H NMR), has recently been implemented as a method of assessing properties of cells. Cheng et al. [27, 28] used HRMAS 1 H NMR to evaluate human breast ductal carcinomas and spermine in human prostate tissue samples. Blankenberg et al. [14] used the same method to quantify apoptotic cell death in Jurkat T-cells in studies of acute
lymphoblastic leukemia. Recently, HRMAS 1 H NMR was used to examine porcine islet preparations that had been exposed to hypoxic conditions typical of culture and shipment [121]. Changes in NMR spectra of cellular lipids, in particular the ratio of methylene (CH2 ) resonance to the methyl (CH3 ) resonance, have been associated with apoptosis. The data of Papas et al. [121] exhibited changes in the methylene-to-methyl resonance ratio that correlated to the degree of hypoxia. These data suggest that HRMAS 1 H NMR is a promising technique in assessing islet quality, and in particular apoptosis, especially because it can be applied to small quantities of intact islet preparations. Further studies are needed to correlate this method with other measures of apoptosis. CASPASE Caspase activity has been suggested as a reliable marker of early apoptosis that detects apoptotic tissue earlier than DNA fragmentation assays such as TUNEL [26]. When cells are exposed to pro-apoptotic stimuli, a cascade of events begins in which one or more caspases are activated. By measuring the amount of activated caspase(s), one can detect whether or not apoptosis is occurring [182]. It is necessary to choose in advance the caspase(s) to be assayed. For example, caspase 8 and 9 both initiate the cascade of events, but caspase 8 is usually activated by external signals via membrane death receptors whereas caspase 9 is usually activated by cytotoxic agents that damage mitochondria, allowing cytochrome c leakage into the cytosol [182]. Caspase 3 and 7 are common downstream effector caspases associated with some forms of b-cell apoptosis [26, 45, 105, 149, 183, 195]. Two methods can be used to assess caspase activity in islet tissue. Each can be adapted for any one of the caspases that may be active. In one method, samples for histology are sectioned and immunostained for caspase activity. This method allows observation of an elevated level of caspase in a sample and identification of the cells that contain the activated caspases. It has been used in several studies involving islets [45, 82, 149]. A second method makes use of a hydrolysis reaction with a reporter molecule attached to a peptide by aspartic acid. Caspases have a unique specificity because they contain a critical cysteine residue that allows them to cleave other proteins at sites next to an aspartic acid residue [182]. In this assay, intact islet tissue is washed and lysed to free the activated caspase, which then
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cleaves the reporter molecule-peptide complex, releasing the reporter molecule and resulting in a color change or fluorescence. This type of assay can be completed in several hours and has been applied to several islet studies in order to quantify caspase activity [26, 105, 183, 195]. NUCLEOSOME DETECTION Detection of cytoplasmic histone-associated DNA fragments (mono- and oligo-nucleosomes) is performed with a photometric enzyme immunoassay (e.g., Cell Death Detection ElisaPLUS from Roche Applied Sciences or Nucleosome ELISA assay from Oncogene research products). The cytoplasm of apoptotic cells is enriched with nucleosomes from DNA degradation, which can occur several hours before the plasma membrane breaks down. Intact islets can be analyzed because the assay requires the tissue to be lysed in order to free up fragmented DNA. The assay is based on a quantitative sandwich enzyme immunoassay principle using monoclonal antibodies directed against DNA and histones. Aliquots of a cell or islet lysate are transferred to a multi-well plate. An immunoreagent, prepared with anti-DNAperoxidase (POD) and antihistone-biotin, is added in each well, and the samples are incubated. During incubation, the antihistone antibody binds to the histone component of nucleosomes and also attaches the immunocomplex to the streptavidin-precoated plate via its biotinylation. The anti-DNA-POD antibody reacts with the DNA component of the nucleosomes. At the end of the incubation, unbound antibodies are removed by a washing step. Finally, the amount of nucleosomes retained in the POD immunocomplex is quantified photometrically with ammonium 2, 2 -azino-bis(3)ethylbenzthiazoline-6-sulfonate as a substrate and the sample absorbance is reported relative to that of a control. Detection of nucleosomes has been used as an indicator of apoptosis for a variety of cells, including intact islets [78, 136] that have been stressed. Both apoptotic and necrotic cells can strain positive for DNA fragmentation using the TUNEL assay [41, 51, 56, 64, 184], as described in material following, and cells in both states are likely to be positive for DNA fragmentation as measured by the nucleosome assay. ANNEXIN V Translocation of phosphatidylserine (PS) from the inner to the outer side of the cell membrane occurs during the
early stages of apoptosis. Although PS is not externalized in necrosis, it would appear as if it were because cell membranes are disrupted during necrosis. Thus, staining for PS results in identification of both necrotic and apoptotic cells. Using Annexin V (a binding protein with a high affinity for PS) and PI (the dye commonly used for membrane integrity measurements), Vermes et al. [188] developed a flow cytometric assay to differentiate between apoptotic and necrotic cells and detect early stages of apoptosis when PS is externalized. Cells were stained with both Annexin V and PI, incubated in the dark, and then assessed using bivariate flow cytometry to identify healthy intact cells, apoptotic cells, and necrotic or late apoptotic cells. In addition to distinguishing among apoptotic and necrotic or late apoptotic cells, Annexin V staining consistently indicates cell damage earlier than the TUNEL DNA fragmentation assay [26, 134], demonstrating that PS externalization is an early indicator of apoptosis. Annexin V staining has been used in many studies with islet cells [26, 48, 87, 108, 114]. However, Annexin V staining is quantified through flow cytometry and thus requires single cells for analysis, which suffers from the problems identified previously.
TUNEL DNA fragmentation is a characteristic event when a cell dies. A commonly used method of detecting DNA strand breaks is the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay [57]. The technique is applied to histological sections that allow the user to not only ascertain whether DNA fragmentation occurred, which could also be determined through gel electrophoresis, but to observe the specific cells that contained fragmented DNA. DNA fragmentation as measured by the TUNEL assay is traditionally thought of as an indicator of apoptosis. However, both apoptotic and necrotic cells can stain positive using TUNEL. Therefore, TUNEL is a marker of cell death, but not necessarily of apoptosis specifically [41, 51, 56, 61, 184]. Terminal deoxynucleotidyl transferase (TdT) is used to incorporate biotinylated dUTP to the 3 -OH ends of DNA that are exposed during DNA cleavage [57]. Once labeled, a second incubation amplifies the signal, resulting in a color change or fluorescence. The labeled cells may be visualized by light or fluorescent microscopy,
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depending on which molecule is used in the final incubation. Several studies have used TUNEL to assess islet quality in response to a stress and/or treatment [26, 82, 132, 149]. It is a valuable research tool for observing the specific cells going through the cell death process. Because TUNEL requires that histological samples be taken, prepared, and stained, it is advantageously used retrospectively, rather than for prospective assessment of an islet preparation prior to transplantation. Although TUNEL is usually used on histological sections in islet research, it can also be quantified by using fixed dispersed single cells with flow cytometry [118], but with the disadvantages associated with using single cells obtained from islet dissociation (as discussed previously). Measurements of the specific changes that occur during apoptosis may provide important insight into viability of islet preparations, and different techniques can be employed with histological sections, single cells from dissociated islets, or intact islets. Assays requiring histological sections, such as the TUNEL assay and one type of caspase assay, require significant time and are thus inappropriate for fast analysis, but may be beneficial for posttransplantation analysis because the cells in which apoptosis is occurring can be identified. Assays using dissociated islet cells are questionable because a significant fraction of cells is lost or damaged during islet dissociation [139, 140, 193], making Annexin V and TUNEL by flow cytometry prone to artifact. Assays starting with intact islets are useful for rapid measurement because no significant preparation is required for analysis. As a result, the nucelosome assay (a second form of caspase assay) and HRMAS 1 H NMR are attractive for islet quality assessment. Of these three assays, the caspase assay may be the most useful indication of apoptosis, per se. The nucleosome assay detects DNA fragmentation that occurs in both necrotic and apoptotic cells. As such, it is a better indicator of all cells dying by both apoptosis and necrosis. HRMAS 1 H NMR has the potential to be useful, but requires specialized equipment and therefore may be unattractive to small institutions that do not have the resources.
MITOCHONDRIAL FUNCTION The function of mitochondria is to convert ADP to ATP, the energy currency of the cell, according to the following approximate reaction (which also
includes cytosolic glycolysis). Glucose + 36 ADP + 36 Pi + 6O2 → 6CO2 + 42H2 O + 36ATP
(6.42)
If mitochondrial function ceases, oxidative phosphorylation ceases, and the cell will die unless it is able to produce ATP by anaerobic metabolism. Furthermore, the ATP/ADP ratio is important for insulin secretion in b cells. Thus, the process of oxidative phosphorylation should be useful in assessing viability. ATP concentration and ATP/ADP ratio provide an index of the energetic state of the cell. Because the ATP production rate is six times the oxygen consumption rate (OCR), as shown in Equation 6.42, OCR measurements provide a direct assessment of the rate of oxidative phosphorylation assuming that all other O2 -consuming reactions in the cell are negligible, which is reasonable for b cells. ATP generation is driven by the gradient in hydrogen ions developed across the inner mitochondrial membrane, and thus the mitochondrial membrane potential is another quantity that can be used to assess mitochondrial function. Last, because the mitochondria are partially responsible for the maintenance of the redox state of the cell, assays of the redox state are often classified as mitochondrial function assays. We begin with the assays of the redox state that make use of tetrazolium salts. REDOX STATE OF THE CELL: TETRAZOLIUM SALTS Mosmann et al. [112] first described the use of an assay that distinguished viable from nonviable cells by their ability to cleave tetrazolium salts. This technique was later applied to the viability assessment of islets [81]. Currently, there are at least three different tetrazolium compounds in widespread use for viability assays for islets and islet cells: MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide), XTT (2,3bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino) carbonyl)-2H-tetrazolium hydroxide), and MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium, inner salt). Each of these compounds (MTT, MTS, and XTT) is reduced by viable cells to form a dye molecule with an absorbance spectrum that is different from the parent dye, which allows for easy colorimetric measurement using a multiwell plate reader. The product of MTT reduction is not water soluble, and it accumulates within cells that must be lysed and the reduced MTT solubilized as the last
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step in the assay. The products of XTT and MTS reduction are soluble, no subsequent lysis step is required with them, and it is possible to follow the concentration in solution, which increases with time. The process by which the tetrazolium salts are reduced is complex and not completely understood. Therefore, the assay results are prone to artifacts [13]. Reduction of these dyes is dependent on NADH and NADPH concentrations within the cells, which is a measure of the redox state of the cell. However, this reduction is dependent on intermediate electron acceptors, such as phenazine methosulfate (MTS and XTT), or on microsomal enzymes in the case of MTT. Superoxide is involved with the reduction of XTT and MTS, and the process is sensitive to changes in environmental oxygen. Reduction of these dyes also occurs by medium components, such as l-cysteine and l-ascorbic acid, independently of processes within cells. MTT and XTT assays (and presumably MTS) are very sensitive to the glucose concentration in the medium [189]. Finally, unexpected reduction of MTT and XTT has been observed in islets treated with the cytokines IL-1 and IFN-g [9] and has been attributed to the diaphorase activity of inducible nitric oxide synthase, which is unaffected. Viability assays using tetrazolium salts are widely used despite the drawbacks mentioned previously because they are very easy to perform, inexpensive, and can be used with intact islets. No additional lab equipment beyond a plate reader is required, and the tetrazolium dyes are affordable. Cells or islets are added to the assay reagent (medium with tetrazolium salt) and incubated for a period of time. Because MTT is insoluble, the accumulation of the reduced product within the cells is lethal, and all cells are usually killed within about 4 hours [58]. After this time, the reaction ceases, and there is no further accumulation of the reduced MTT. This product is solubilized with DMSO or SDS, and the absorbance is measured using a plate reader. Because the XTT and MTS products are soluble, they are relatively nontoxic, and it is possible to fit the early data for product concentration to a linear function of time, the slope of which is the rate of product formation and therefore a measure of the redox state of the cell [13]. Because the assays using tetrazolium salts are sensitive to a wide variety of variables that are incompletely known, it is not possible to develop a standard calibration curve relating a particular assay reading to a specific viability or to accurately relate the absorbance
reading from one islet preparation to another. However, the method is very useful for comparing the effects of a particular variable on a single islet preparation when the measurements can be made with a control, so that the fractional viability relative to the control can be measured. Nonetheless, we have found that these assays are not universally applicable and are capable of providing inexplicable results similar in nature to those described in the literature. The most striking example we have observed is a large variation in the extent of MTT reduction at 37◦ C in islets and cells that have been cultured at reduced temperatures (4 to 24◦ C), allowed to recover to 37◦ C, and then retested (unpublished data). Thus, assays using tetrazolium salts are useful for the assessment of islet viability in experiments involving a control, if used with a precisely followed standard protocol, and in combination with other viability assays if warranted by the results. OXIDATIVE PHOSPHORYLATION ATP Concentration Viable cells produce ATP as an energy intermediate. A viable cell therefore has a pool of ATP available for many cellular energy-requiring processes, whereas a dead cell does not, and this property can be exploited for measurement of cell viability [84]. In principle, the number of viable cells can be determined by lysing them to release their ATP into solution, quantifying the ATP concentration with a luceriferasebased luminescence assay, and converting the total amount of ATP into a viable cell number if accurate values for the cell-specific ATP content of viable cells are known. The ATP assay itself is inexpensive and easy to perform with a microplate reader. Furthermore, measured changes in ATP concentration (and associated changes in ATP concentration) within b cells play an important role in secretion of insulin [34]. For all of these reasons, ATP quantification is an attractive candidate as a marker for assessment of islet viability. It can be quite difficult to use an ATP assay to get an accurate result for this purpose because the cellular ATP pool size is very sensitive to changes in environmental conditions. In a single islet equivalent, ATP is produced at an estimated rate of approximately 60 pmol per minute in nutrient-free media at a high glucose concentration [44]. The ATP content of b cells in a single islet is approximately 14 pmol [59]. Based on these estimates, the half-life of ATP within an islet is only
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about 7 seconds. Given this very short half-life, a small change in environmental conditions that affects either ATP production or utilization rates can cause significant variation in the total ATP isolated from tissue. For example, a two-step wash of islets in ice-cold buffer, as was done by Noguchi et al. [114], will almost certainly produce very different results from those that would be obtained if cultured islets were immediately lysed. Islets are especially difficult to assay, in that they are large aggregates, and cells in the outer and inner regions may experience very different microenvironments depending on the assay conditions. For the reasons mentioned previously, the application of the ATP assay to islet quality assessment is quite difficult. When Brandhorst et al. [20] attempted to use ATP content measurements for quality assessment of human islets, they found a coefficient of variation (COV) of 130%, compared to a COV of 45% for glucose stimulation index with the same islets. Others have reported better results [80] but have not performed a thorough evaluation of their method. At the current time, no work has been published that validates a precise protocol for the use of ATP quantification for islet quality assessment. Such work needs to be done before the ATP assay can find widespread use for islet quality assessment. Similar limitations apply to measurement of the ATP/ADP ratio.
Respirometry, the measurement of oxygen consumption rate (OCR) of living cells and tissues, was originally used with islets to study carbohydrate metabolism. Because quantities of islets available were in the ml range, a very sensitive method using Cartesian-divers was originally developed [67–69, 194]. Subsequent studies to measure OCR have made use of perifusion systems [74, 127, 128, 179] and continuously stirred chambers [119, 126], together with a Clark-type electrode. The electrochemical sensor in microelectrode form was unstable and required frequent calibration, which rendered the procedure tedious. Large polarographic sensors were stable but too large for measurements with islets. The development of optical fiber sensors that rely on the effect of oxygen in altering the decay of phosphorescent or fluorescent intensity following irradiation made possible rapid, continuous, stable measurements of pO2 in solution. Dionne [36] was apparently the first to use optical-fiber-based sensors using this principle in a perifusion system [38] designed to examine the effects, Oxygen Consumption Rate
both transient and steady-state, of reduced oxygen on insulin secretion rate by islets [37]. Since then, systems using these or equivalent sensors for measurement of the partial pressure of dissolved oxygen pO2 have developed in three directions: (1) perifusion bioreactors, (2) continuously stirred chambers, and (3) static culture devices. Perifusion Bioreactor Sweet et al. [180] described a system referred to as flow culture that enabled continuous measurement of OCR by pancreatic islets over extended periods of time. The system featured aseptic conditions, temperature control, and collection of outflow fractions for measurement of insulin and other products. Measurement of OCR was based on the difference between inflow (pO2,in ) and outflow (pO2,out ) oxygen partial pressures according to OCR = Q a (pO2,in − pO2,out ),
(6.43)
where pO2 is in mmHg, a is the Bunsen solubility coefficient of oxygen in medium (1.27 nmole O2 /cm3 mmHg at 37◦ C), Q is medium flow rate (cm3 /min), and OCR has units of mole O2 /min. The system was used to demonstrate effects of hypoxia on insulin secretion, membrane integrity, and redox state of cytochromes in islets. The latter measurements made use of a separate continuous spectrophotometric analyzer. A subsequent study [178] investigated continuous noninvasive measurements of cytochrome c redox state and OCR as indices of electron transport, from which ATP/ADP/Pi ratios were estimated. The flow culture system is an elegant tool for studies involving long-term culture, simultaneous OCR and insulin secretion measurements, or studies involving multiple changes in environmental parameters where the transient dynamics of the response are to be followed. It is inherently more complex and time consuming than use of alternative instrumentation, such as the stirred chamber, and therefore less efficient when multiple samples or replicates need to be examined in a short period of time. Stirred Chamber Together with Instech Laboratories, we developed [131] a very small stirred chamber system (Micro Oxygen Uptake System, Model FO/SYS210T, Instech Laboratories, Plymouth Meeting, PA) specifically designed for measurements with islets that has been described in more detail recently [130]. The chamber, shown schematically in Figure 6.10,
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Sealing cap Medium initially equilibrated with air Stirred Chamber (200 µl) Islets or Cells
O2 Sensor Stirring bar
has a volume of about 200 ml (measured gravimetrically) and is stirred with a tiny, glass-coated magnetic stirring bar. The chamber is water-jacketed for temperature control and contains a fiber-optic oxygen sensor that has a fluorophor gel overlain by silicone rubber at the tip of the fiber. The sensor is calibrated at 0 and 160 mmHg. Titanium is used for the chamber body and for the oxygen probe jacket because the metal is oxygen impermeable and inert, has high thermal conductivity, and will not introduce artifactual drift arising from surface oxidation. After cells or islets are loaded through the top of the chamber, a beveled glass plug is inserted, any excess fluid is expelled from a port, and the plug is rotated to seal the chamber. The stirrer speed is controlled by a potentiometer, normally set at position 3 (out of 10), which corresponds to a rotation rate of 53 rpm that is just enough to suspend the islets off the bottom surface. The oxygen leakage rate ranges from 0 to 0.2 mmHg/min mmHg and depends on the sealing plug used. Virtually 100% of the tissue added can be recovered from the chamber for use with further measurements. Figure 6.11 is a photograph of the entire system. All OCR measurements are carried out at a defined set of conditions: Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 mM HEPES buffer and no added serum (so as to minimize bubble formation) at 37◦ C. After islets are added and the chamber is sealed, thermal equilibration at 37◦ C occurs in about 15 seconds. After a transient increase in pO2 resulting from the decreased oxygen solubility at higher temperature, pO2 decreases
Figure 6.11. Photograph of the Micro Oxygen Uptake system. The sensors at the back of the two titanium chambers are connected to the spectrophotometer. The box on top of the spectrophotometer is the stirring speed control. (Photograph courtesy of Instech Laboratories.)
with time. If the viability, as reflected by OCR, does not change during the course of the experiment (and the minimum pO2 in the islet remains far above the Michaelis constant for oxygen consumption), the slope pO2 t is constant (as depicted in Figure 6.12) for various values of the estimated number of viable islet
Islets added 160 Slope = 140
pO2 (mm Hg)
Figure 6.10. Schematic diagram of water jacketed titanium chamber with fluorescence-quenched O2 sensor for OCR measurement. A magnetic stirring bar at the bottom of the chamber ensures the cells or islets are in suspension during the measurements. After the cells or islets are loaded, the chamber is sealed, and the partial oxygen pressure is measured through the fiber-optic O2 sensor.
∆pO2 ∆time
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Time from Islet Addition (min) Figure 6.12. Oxygen partial pressure pO2 versus time after islet addition for different numbers of viable islet equivalents (VIE). The data are fitted to a straight line. Data from preparations with different numbers of viable IE are superimposed beginning at the same starting point of 160 mmHg. Measurements were conducted in the range of 160 to 60 mmHg, where islet OCR is not oxygen limited. Note the linearity of the plots even up to 50 minutes of continuous measurements.
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Oxygen Consumption Rate (nmol/min) Figure 6.13. Coefficient of variation (COV) versus OCR or number of viable IE for measurements conducted with rat, porcine, and human islets in the OCR measurement apparatus. Data for single and triplicate measurements are shown. For plotting purposes, number of viable IE was calculated assuming (OCR/DNA)viable = 500 nmol/min mg DNA, 6.5 pg DNA/cell, and 1,560 cells/IE.
equivalents (VIE) added to the chamber. With 250 VIE, a measurement takes about 20 minutes. The OCR is calculated from pO2 , (6.44) OCR = Vch a t where Vch is the chamber volume. Dividing both sides by the number of cells nC in the chamber and rearranging yields pO2 a OCR t , (6.45) = n C cell Vch where the quantity nC /Vch is the cell concentration measured, for example, by nuclei counting. The quantity OCR/DNA can be calculated from Equation 6.45 if the denominator is replaced by DNA concentration in the chamber. Because the predominant source of scatter is the oxygen sensor, the uncertainty in the estimate of the slope from a single measurement is excellent with a coefficient of variation (COV) of about 2% with 250 VIE and 1% with 500 VIE (Figure 6.13). With triplicate samples from the same preparation, the COV is higher
because of sampling errors and possible heterogeneity of OCR/cell in different islets. COV is about 10% for 250 VIE and 6% for 500 VIE. Figure 6.14 shows an example of triplicate measurements of a fresh sample of INS-1 cells. Stirring in the chamber for 15 minutes at the low speed employed has essentially no effect on membrane integrity of human or rat islets measured by 7-AAD sequential staining. At much higher stirring speeds, an increase in the fraction of cells with compromised membranes is observed with rat but not with human islets. Healthy islets that produce a constant slope on the first measurement, if left in the chamber, provide the same slope on a second measurement. When islets that have been stressed by exposure to varying degrees of hypoxia are analyzed by both OCR and MTT measurements, the OCR is linearly proportional to the MTT absorbance reading. Curvature in the plot of pO2 versus time, which leads to a decrease in slope with time, is an indicator that the rate of oxidative phosphorylation is decreasing. This reduction in mitochondrial function suggests that some cells in the islets are dying. We have observed this behavior on a number of occasions when we have had an opportunity to initiate OCR measurements with rat islets immediately (within 10 minutes) after isolation. An example of the raw pO2 versus time plots from these measurements is given in Figure 6.15. The ratio of the slope at pO2 = 120 mmHg to the initial value at 160 mmHg was about 0.5 at the two higher stirring speeds and about 0.75 at the lowest speed. When measured again at 4 hours after isolation, the rat islet samples invariably produced a straight line with constant slope. This finding suggests that a substantial fraction of islet cells may be in the process of dying immediately after isolation is completed. The rapid decrease in mitochondrial function disappears by 4 hours after isolation, suggesting that this aspect of the cell death process has stopped. Whether this initial decrease in mitochondrial function is reversible is unknown. On rare occasions, we have observed continued decrease in mitochondrial function associated with curvature of the pO2 versus time plot after 4 or more hours. These preparations are always associated with a large fraction of cells having compromised membranes. We do not have similar data with human islet preparations immediately after isolation, but we have no reason to expect that the behavior of human islets immediately after isolation is any different from that of rat islets.
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200 6 x 106 INS-1 cells/ml
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Time (min) Figure 6.14. Actual trace of measured oxygen partial pressure versus time from representative triplicate OCR measurements with INS-1 cells over a 70-minute period. Each measurement was performed with a fresh cell sample and at a setting of 3 on the potentiometer, corresponding to 53 rpm. The cell concentration, measured by nuclei counting, was 6 × 106 nuclei/ml. The data were fitted to a straight line in the steepest portion of the trace. The slopes for each measurement are listed in the figure. The mean of the three slopes is 13.8 ± 0.3 (mean ± SD) mmHg/min. The mean value of OCR/cell for these measurements was 2.94 ± 0.24 fmol/min nucleus, with the standard deviation obtained from propagation of error analysis.
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Figure 6.15. Triplicate OCR measurements performed with rat islets immediately (10 minutes) after the isolation at stirring speed settings of 9, 6, and 3, corresponding to rotational rates of 321, 187, and 53 rpm, respectively. The slopes were not straight, which is an indication of islets dying in the chamber during the measurements. The ratio of the final slope to the initial slope for each setting was 0.49 for setting 9, 0.46 for setting 6, and 0.74 for setting 3, suggesting that stressed fragile islets are more likely to die when stirred more vigorously. Similar observations were made with other rat islet preparations examined right after isolation, indicating that the islets did not have time to recover from the stress of isolation. When OCR measurements were performed with islets from the same preparation 4 hours after isolation at a setting of 3, a straight line with constant slope was obtained.
Figure 6.16 summarizes pooled data collected from rat, porcine, and human islet preparations at Joslin Diabetes Center, Harvard Medical School, and at the Diabetes Institute, University of Minnesota. The left-hand column shows the frequency distribution of the specific oxygen consumption rate, OCR/DNA. If it is assumed that there is a unique maximum
value (OCR/DNA)viable that characterizes viable cells, the ratio of (OCR/DNA) of an islet preparation to (OCR/DNA)viable represents the fractional viability of the islet preparation. For the data plotted in Figure 6.16, this ratio was calculated using (OCR/DNA)viable = 500 nmol/min mg DNA, a value comparable to the highest value of OCR/DNA we have measured with islets of
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Figure 6.16. Comparison of OCR/DNA measurements and FDA/PI membrane integrity measurements as indicators of fractional viability of rat, porcine, and human islet preparations. Membrane integrity and OCR measurements were made immediately after isolation and 4 hours later, respectively. Left-hand column is the frequency distribution of OCR/DNA. Right-hand column is fractional viability based on OCR/DNA (calculated as described in text) plotted versus fractional viability by FDA/PI. These data indicate that OCR/DNA is a more sensitive measure of fractional viability than live/dead staining.
any species. Membrane integrity was also measured by staining with FDA/PI for many (but not all) of the same preparations and expressed as fractional viability. The resulting data points are plotted in the right-hand column. Whereas the fractional viability calculated from the OCR/DNA ratio ranges from nearly 0 to 100%, the fractional viability data from FDA/PI staining are almost entirely confined to values greater than 80%, thereby demonstrating the insensitivity of membrane integrity measurements as an indicator of fractional viability as currently practiced with islet preparations. The main technical problem we have observed with the stirred chamber is occasional bubble formation. This can be minimized by careful handling of caps to prevent damage by chipping, use of a slight excess tissue suspension volume, and ensuring that fluid thermally
equilibrates before sealing. We also found that bubble problems are most frequent with new chambers and disappear with use, suggesting that passivation of nucleation sites occurs with time. It is desirable to periodically check that the stirring bar is rotating properly and to recoat the sensor every 6 months. In our experience with the Instech Micro Oxygen System Uptake, it has provided rapid, accurate, and precise measurements of the OCR of islet preparations, and it has been used reliably in our laboratories by more than 15 technical staff for over 1,500 measurements (not including replicates) from more than 300 islet preparations of various species. Static Culture A novel device for the measurement of oxygen consumption rate in tissue samples
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Idealized Well (assumed) (1) Steady State
Actual Well Round bottomed well
(2) Perfect cylinder Oxygen permeable walls
(3) Impermeable walls (4) Perfectly still medium
Medium mixed by plate movement
(5) Uniform cell distribution
Cells collect in middle of well
(6) Thin uniform sensor material
Varying thickness of silicone rubber and sensor
Figure 6.17. Schematic representation of an idealized and real well in the oxygen biosensor system.
was recently introduced [63] and used for measurement of OCR of islet preparations [52, 190]. The oxygen biosensor system (OBS) consists of an oxygen sensitive fluorophor embedded in silicone rubber at the bottom of wells in a 96-well plate. Cells or tissue are placed in the wells and consume oxygen, thereby causing the oxygen partial pressure (pO2 ) at the bottom of the well to decrease. This decrease is detected by the oxygen sensor in the well bottom, as shown in Figure 6.17. The OBS is easier to use than the stirred chamber described in the preceding section, and is therefore attractive for islet quality assessment. Unfortunately, the ease of use of the OBS is offset by difficulties in obtaining accurate and meaningful OCR estimates. Unlike the flow culture system and the stirred chamber, for which OCR can be calculated from measured parameters using straightforward material balances, the OBS uses a measurement of pO2 and relies on a theoretical model of a complex physical arrangement. To estimate OCR using the OBS, one has to convert the pO2 at the bottom of the well to the OCR of the cells or tissues. This conversion requires that the oxygen species conservation equation be applied to the entire system and solved using appropriate initial and boundary conditions, a complex endeavor. To simplify analysis of the OBS data, several assumptions have been invoked that allow computation of OCR from pO2 measurements in the OBS [63]. These assumptions include (1) the system is at steady state, (2) the well geometry is perfectly cylindrical, (3) the wells are fabricated from oxygen impermeable material, (4) the medium is completely stagnant, (5) there is
a uniform and infinitely thin cell layer, at the bottom, overlaying (6) a uniform infinitely thin oxygen sensor material. These assumptions lead to the integrated form of Fick’s first law of diffusion, OCR =
ADa pO2a − pO2b , h
(6.46)
where A is the surface area of a well, h is the medium depth, pO2a is the ambient oxygen partial pressure, and pO2b is the oxygen partial pressure at the well bottom measured by the OBS. When using Equation 6.46 for OCR estimation, the accuracy of the result is dependent on the validity of the assumptions stated. Many of the assumptions are not strictly applicable to the OBS system, as illustrated in Figure 6.17. To test the validity of these assumptions and estimate the extent of error incurred by their use, we developed a complete finite element mass transfer model of the OBS well using COMSOL Multiphysics commercial software. If the system were ideal in every other aspect, it should attain about 90% approach to steady state within approximately 40 min. However, because the walls are constructed of oxygen-permeable materials, the estimated 90% approach to steady state is actually not reached until after 260 minutes have passed. This is longer than desirable, although it does not preclude use of the system for OCR measurement. Inaccuracies in assumptions regarding the system geometry, cell layer thickness, and oxygen sensor thickness (assumptions 2, 5, and 6) have only a minimal effect on the OCR estimate and the combined error
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introduced by all of these assumptions is 10% or less. Assuming that the data for polystyrene oxygen permeability in polystyrene microplates reported by Arain et al. [3] are accurate, leakage of oxygen through the bottom of the well (assumption 3) produces a somewhat larger error. If the tissue at the bottom of the well is too closely packed, it becomes oxygen starved in the center. It is therefore critical that well bottom pO2 not get much below 75 mmHg if the OBS is being used with islets. The assumption of stagnant medium (assumption 4) is more problematic. The plate is never truly stationary as most incubators are subject to minute vibrations and thermal variations that can potentially introduce some flow within the wells. Furthermore, the transport of the plate from the incubator to the plate reader and movement of the plate in the plate reader between readings can induce significant disturbances in the liquid within the wells. The substantial adverse effect of motion in the plate reader has been demonstrated convincingly by comparing the transient reequilibration with oxygen at the bottom of wells initially purged with nitrogen and filled with either water or an agarose gel that prevents fluid motion (unpublished data). Reported OCR values measured with the OBS do not agree with measurements made with other methods. The reported value of approximately 800 fmol/min IE [190] together with an estimate of 1,560 cells per IE, corresponds to an OCR of about 0.5 fmol/min cell, roughly a factor of 5 lower than average values for islets measured in our laboratory using the stirred chamber system. Similarly, values for CHO cells of 0.5 fmol/min cell measured in the OBS system are an order of magnitude lower than what is found using a commercial respirometer [83], which is similar to what we find with other mammalian cell lines. An additional problem is large well-to-well variability that results when small numbers of islets (less than 100) are used in each well, which is essential if the islets are to be kept from being oxygen starved. To circumvent this problem in the current system, many wells need to be used, which increases the amount of labor. The published data and our own experience suggest that obtaining accurate results is difficult or impossible in the system as it is currently constructed and used. Future use will require a significant redesign of the OBS and/or the protocols for using it. Even in the absence of accuracy, the OBS could still be a useful tool if it can reproducibly provide results that are related to the OCR of islet preparations in a consistent way or
that are otherwise useful. Demonstration of this will require extensive research and a better understanding of the OBS and why it gives erroneous OCR readings. Development of standardized protocols that give identical results in different laboratories will almost certainly be very challenging, but may nonetheless be possible. Whether the OBS or a similar system can ultimately be used for OCR measurement to assess islet quality is a question that has not yet been answered. Because of the simplicity of use of a static culture system, further effort in this direction is definitely warranted. MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) Viable mitochondria are polarized and have a membrane potential of about 140 mV (negative on the inner leaflet) due to the proton pumping to the cytoplasm that is a central feature of mitochondrial function [12]. Loss of mitochondrial function is a feature that characterizes dead cells. A decrease in mitochondrial membrane potential (MMP) is also usually (but not always) one of the earliest events that occurs during apoptosis [103]. This potentially makes MMP assessment useful for identifying damaged cells that are committed to cellular death. Two dyes are currently widely used for the measurement of MMP, JC-1 (5,5 ,6,6 -tetrachloro1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide) and mitochondrial-selective rhodamines including rhodamine 123 and tetramethylrhodamine ester (TMRE). These dyes are lipophilic cations that can freely diffuse across cellular membranes, and they are preferentially accumulated in mitochondria due to the membrane polarization [103]. JC-1 forms J-aggregates (linear chains of dye molecules, the spectroscopic properties of which differ markedly from those of the dye monomer) at high concentrations in polarized mitochondria. These aggregates are accompanied by a spectral shift in the emission wavelength [173]. This spectral shift can then be used to estimate membrane potential. In contrast, the rhodamine derivatives do not show a shift in fluorescence due to an accumulation in the mitochondria (relative to the cytosol), and thus membrane potential is estimated from a change in the absolute fluorescence of the dye [165]. The spectral shift of the JC-1 dye makes it preferable for MMP measurement to determine viability, in that a ratio of fluorescence intensities (J-aggregate:monomer) can be taken that will normalize the fluorescence signal to account for changes in the absolute amount
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of tissue and number of mitochondria. JC-1 is also more specific for detecting mitochondrial depolarization because rhodamine 123 has several energy independent binding sites within some cell lines that can cause high fluorescence readings even in the absence of an MMP [162]. Application of MMP assays to islets is constrained by the same problem described for membrane integrity dyes (i.e., the difficulty in using standard fluorescence microscopy to assess the staining with MMP sensitive dyes within the 3D structure of an islet). Optical sectioning with two-photon microscopy has been used for islets stained with the MMP sensitive dyes [181]. Mitochondria throughout islets were stained with TMRE, but diffusion of the dye to the center of the islets was slower than with other dyes studied, which suggests that the issue of dye transport within the islet needs to be carefully studied if MMP sensitive dyes are to be used with whole islets. In an attempt to circumvent the problem posed by the structure of islets, Ichii et al. [75, 77] dissociated islets, then stained the dispersed cells with JC-1 or TMRE, and acquired data using a flow cytometer. They also combined TMRE staining with (1) a zinc binding dye, Newport Green (NG), so that two-color flow cytometry could be performed to estimate the fraction
of b cells (NG positive) that also had high levels of TMRE staining, and (2) 7-AAD, so that cells with compromised membrane integrity were excluded. b-cell fractional viability was defined as the fraction of cells within the NG bright cells that were TMRE positive (after 7-AAD positive cells were excluded). Thus, this index did not include either necrotic cells or late-stage apoptotic cells with compromised membranes or positive 7-AAD signal and therefore included only early apoptotic cells. Another significant problem associated with this method is that it requires dissociation of the islets, and the dispersed single cells are likely not representative of the original islet, as described previously in relation to cell composition analysis. Ichii et al. [75] defined a b-cell viability index as the product of the b-cell number fraction in islets (fbIC ) measured with LS cytometry and the b-cell fractional viability determined by TMRE/Newport Green staining measured with flow cytometry, as is summarized in Figure 6.18. They correlated the b-cell viability index of human islet preparations with the outcome of transplantation into immunodeficient mice. There was considerable overlap of the b-cell indices of successes and failures, but they were able to identify a value for the b-cell viability index below which no islet preparations cured.
Islets Antibodies to Pancreatic Polypeptide Insulin Somostatin Glucagon Fluorescent Tag
Dissociate (Accutase) Single-Cell Suspension
CompuCyte Laser Scanning Cytometer
BD FACScan Flow Cytometer 7AAD− NGbright TMRE+
Count Stained Cells
β-cell fraction =
Incubation with Newport Green (Zn) TMRE (Mitochondrial Membrane Potential) 7AAD (Membrane Integrity)
β β+α+δ+pp = 0.51
β-cell Fractional Viability (FV)
β-cell Viability Index = β-cell fraction • β-cell FV Figure 6.18. Schematic representation of the experimental procedure described by Ichii et al. [77]. Islets are dissociated enzymatically and mechanically into single cells. An aliquot is processed for LSC after being stained with specific antibodies to obtain the b-cell number fraction. The average value reported was 0.51. A second aliquot is processed through a flow cytometer after being stained with dyes sensitive to mitochondrial membrane potential to obtain the b-cell fractional viability. The b-cell viability index is the product of the b-cell fraction and the b-cell fractional viability.
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Development of technologies that enable quantitative imaging of MMP dyes within intact islets would be an important advance. Large diameter flow cytometry and two-photon microscopy are two options that might be explored for future use of MMP sensitive dyes for islet quality assessment. Unfortunately, both of these methods require very expensive equipment operated by highly trained individuals, which would limit their applicability to the field, at least in the near future.
COMPARISON OF VIABILITY ASSAY RESULTS WITH STRESSED CELLS AND ISLETS On the basis of the review presented here, we hypothesized that (1) membrane integrity tests with islets often give erroneously high values that are not predictive of transplantation outcome because they fail to distinguish healthy cells from cells that have entered the cell death pathway but have not yet reached the membrane permeabilization stage, which often occurs later in the cell death process, and (2) mitochondrial function tests provide the earliest indication of damage to cells that eventually leads to cell death. To test this hypothesis, we studied the transient response of rat and human islets and Jurkat and INS-1 cells to stresses causing extensive cell death [147, 148]. Anoxia was used to induce islet cell damage, in that this is a condition that is routinely encountered during pancreas storage and islet isolation, high-density culture, and shipping. The cell lines tested were resistant to anoxic insult, and thus alternative stresses were chosen for these cell lines (1 mM camptothecin and 5 mM streptozotocin for Jurkat and INS-1 cells, respectively). Mitochondrial function of the islets was assessed using OCR and ATP concentration measurements, and membrane integrity was assessed with FDA/PI and 7-AAD sequential staining. In addition to these aforementioned assays, the mitochondrial function of the cell lines was also assessed with MTT, MTS, and the MMP-sensitive dye JC-1. Two additional dyes, trypan blue and sytox orange, were used to probe membrane integrity. Furthermore, the use of cell lines allowed for two flow cytometer-based apoptotic event-indicating assays to be tested (a multicaspase assay and an annexin V assay) that could not be done with whole islets. The results of these experiments are summarized in Figure 6.19. The mitochondrial function and membrane
integrity data were all normalized by their value at time zero, when stress was first applied. The results of different mitochondrial function assays (OCR measurements, MTS, MTT, ATP concentration) were closely correlated and followed a similar trend. The same was true for membrane integrity assays, but the behavior of the two assay types diverged sharply. After a very short lag period, fractional viability by mitochondrial function decreased continuously and relatively rapidly, whereas the fraction of intact cells by membrane integrity varied slowly at first and began to decrease only at longer times. Early apoptosis events, a cell death measure, in Jurkat cells followed the time course of mitochondrial dysfunction, as did phosphatidyl serine externalization (Annexin V) in INS-1 cells, but caspase activation was slower. The time scale over which the cells or islets underwent the cell death process varied with the combination of cell types and stresses, but the relative trends were the same. Mitochondrial function assays likely provide a more accurate assessment of the health of an islet preparation because they are able to identify cells that are damaged but do not yet have compromised membranes. In some cases, the damage may be reversible, and removal of the stress might be sufficient to rescue some of the cells. This has not been explored and warrants further study. Nonetheless, in most cases it seems likely that islet cells that have been damaged to such an extent that their mitochondrial function has been abolished or greatly reduced are not likely to recover. Therefore, mitochondrial function assays provide the most meaningful measurement of fractional viability of islet preparations and should be included in any rigorous viability assessment protocol.
OXYGEN CONSUMPTION RATE AND CELL QUANTITY MEASUREMENTS PREDICT TRANSPLANTATION OUTCOME IN MICE We have explored the ability of OCR measurement to predict transplantation outcome. The results of these studies [91, 122–124, 129, 131] demonstrated that oxygen consumption rate and cell quantity measurements can predict transplantation outcome in immunosuppressed or immunodeficient mice. In the first series of experiments [91, 123, 124, 131], rat islets were transplanted under the kidney capsule of diabetic Balb/C
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6.
Fraction Apoptotic Cells (■)
1
Characterization of Islet Preparations
Jurkat Cells
INS-1 Cells
Suspension Culture 1µm Camptothecin
Surface-attached culture 5 mM Streptozotocin
Mitochondrial Function OCR, ATP, MTT, MTS Apoptosis Events Annexin V, Multi-caspase activation Membrane integrity Trypan blue, FDA/PI, 7-AAD, LDS 751/Sytox Orange
0.8 0.6
Apoptosis Events
0.4
Assays performed:
0.2
Rat, Human Islets
0
Anoxia, 37 °C
Relative Fraction of Viable Cells (●)
1.2
Membrane Integrity
1
1
0.8
0.8
0.6
0.6
Mitochondrial function
0.4
0.4 0.2
0.2 0 0
0 10
20
30
40
50
0
2
4
6
8
10
12
0
10
20
30
40
Relative Fraction of Cells with Intact Membranes (▲)
1.2
50
Time of Stress Exposure (hr) Figure 6.19. Cells and islets were subjected to the indicated stress for time periods up to 48 hours. Measurements made with membrane integrity, apoptotic event, and mitochondrial function assays are plotted versus time of exposure. For sake of clarity, the results from assays of the same class were pooled, and the average at each time point plotted.
mice treated with anit-CD4 antibody. Random, nonfasting blood glucose was monitored daily in the late morning or early afternoon. Blood glucose less than 200 mg/dl for at least 7 days was considered a cure. Analysis of initial experiments demonstrated that OCR of the transplanted islets did not always predict success or failure and that prediction improved by use of a second parameter, OCR/DNA. This finding led to a systematic plan to cover a range of parameters in the experiments. The idealized approach for makeup of the islet transplant experiment is shown in Table 6.12 and is expressed in terms of the number of viable and dead islet equivalents in a representative target transplant preparation. The parameter values chosen were based on early estimates for (OCR/DNA)viable = 463 nmol/min mg DNA and OCR = 7.6 pmol/min for a viable islet equivalent. We do not know if these values are correct, or even if there are single unique values to characterize fully viable islets. These values represent our best estimate at the time of the experiment and do help to provide a framework for one possible interpretation of the measured parameters. OCR/cell could have been used in place of OCR/DNA. Use of the latter quantity reflects
TABLE 6.12 Conceptual strategy for rat islet transplantation experiments.a OCR (nmol/min)
Fractional Viability (%) 10
50
90
3.75
500 Viable IE 4500 Dead IE
500 Viable IE 500 Dead IE
500 Viable IE 50 Dead IE
0.75
100 Viable IE 900 Dead IE
100 Viable IE 100 Dead IE
100 Viable IE 11 Dead IE
46
232
417
OCR/DNA (nmol/min mg DNA) a Each entry represents the properties of a target preparation in order to
cover a wide range of OCR and OCR/DNA values.
the fact that the nuclei counting assay was not available at the time that the experiments began. In actual practice, the maximum OCR/DNA was fixed by the properties of the isolated islets, and lower values were obtained by exposure to hypoxia through
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high-density culture. The results of the transplantations can be summarized in tabular or graphical form [91, 123, 124], in which each entry represents the cure/no cure results for 1 to 6 preparations having a defined set of OCR and OCR/DNA values. The data show that cure rates increased with increasing OCR. At constant OCR, cure rates increased with increasing OCR/DNA. Furthermore, the data mostly segregate into two regions representing all cured or all not cured, separated by a third small region of mixed results. This pattern allows prediction of transplantation outcome in the immunodeficient mouse for a rat islet preparation having any arbitrary combination of OCR and OCR/DNA values. The second study was carried out with high-purity (>85% by DTZ staining) human islet preparations in diabetic nude mice. The strategy for the makeup of the islet preparations was the same but the available range of OCR/DNA was smaller. In this study, success was defined as diabetes reversal (blood glucose < 200 mg/dl) for at least two consecutive readings taken daily up to 10 days posttransplant and every 3 days thereafter. The results have been summarized elsewhere [122, 129]. Although the regions of cure and no cure are not as crisply defined as in the rat, the overall results were similar in that data segregated into regions of mainly cure and mainly noncure. Statistical analysis yielded sensitivity (inclusion of all true positives) and specificity (avoidance of false positives) values of 91% and 74%, respectively. It was concluded from these data that prospective OCR and OCR/DNA measurements could effectively replace the retrospective nude mouse bioassay in assessing human islet quality prior to transplantation.
the JDRF Center for Islet Transplantation at Harvard Medical School, the Diabetes Research and Wellness Foundation, and an important group of private donors. Technical assistance was also provided by the Diabetes and Endocrinology Research Center (DERC) of the Joslin Diabetes Center supported by the NIH (P30 DK36836-16).
List of Symbols
Postscript Although this chapter has focused on assessing the properties of islet preparations, many of the same issues apply to other forms of cell therapy, and many of the techniques discussed here are likely to have wider applications if they do not already.
A a b c D EM f fA fD fi fIC fO fNb fNIC fb fbIC h LM nA nC nD ni (NIE )PCV/LM (NIE )DTZ
Acknowledgments This study was supported by grants from the NIH (NCRR ICR U4Z RR 16606 and R01-DK063108-01A1),
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nNIC nNb nO
Surface area of a well Half-axis of a symmetrical ellipsoid Half-axis of a symmetrical ellipsoid Half-axis of a symmetrical ellipsoid Diffusion coefficient of oxygen in medium Electron microscopy Fraction of cells permeable to dye Number fraction of acinar cells among all cells Number fraction of duct cells among all cells Number fraction of cell type i Number fraction of all islet cells Number fraction of all “other” cell types among all cells Number fraction of non-b cells among all cells Number fraction of all non-islet cells Number fraction of b cells among all cells Number fraction of b cells among all islet cells Medium depth Light microscopy Number of acinar cells Number of cells in the OCR chamber Number of duct cells Number of cells of type i Number of IE calculated from packed cell volume and light microscopy data Number of IE calculated from DTZ staining data Number of non-islet cells Number of non-b-cells Number of other cells
6.
nTC nb nIC nIE NIE nbIE N1
N2
OCR PI PNI pO2,a pO2,b pO2,in pO2,out PV Q r t V VA Vch VD Vellipsoid VI VIC VIE VIEC VINT VIXV VNI VNIC VNIEC
Characterization of Islet Preparations
VNb VPC VO Vsphere VIE VT VTC
Total number of cells Number of b-cells Number of islet cells Number of cells in an islet equivalent volume Number of islet equivalents Number of b cells in an islet equivalent volume Number of nuclei initially labeled with dye (cells with compromised membranes) Total number of nuclei labeled with dye after cell lysis (total cell number) Oxygen consumption rate Number of points falling within the domain of islets Number of points falling within the domain of non-islets tissue Ambient partial pressure of oxygen Partial pressure of oxygen at the well bottom Partial pressure of oxygen into the system Partial pressure of oxygen out of the system Number of points falling within the domain of islet vascular space Medium flow rate Radius of a sphere Time Volume Volume of acinar cells Volume of OCR chamber Volume of duct cells Volume of an ellipsoid Total volume of the islet domain Volume of islet cells Volume of an islet equivalent (1.77 × 106 mm3 ) Extracellular volume within islet domain Volume of interstitial space in islet Volume of islet cells and interstitial space within the islet domain Total volume of the non-islet domain Volume of non-islet cells Extracellular volume within the non-islet domain
VV VVI VVPC Vb a fIC fNIC fbIC I IC IEC IXV NIC NIEC
VI VPC bI (fIC )EM (I )DTZ (I )EM (I )LM (IXV )LM νA νD νNb
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Volume of non-b-cells Volume of packed cell pellet Volume of other cells Volume of a sphere Viable Islet Equivalents Total tissue volume Total volume of all islet and non-islet cells Vascular volume within the islet domain Volume of vascular voids in islet Void volume of packed cell pellet Volume of b-cells Solubility of oxygen in medium Volume fraction of islet cells among all cells Volume fraction of non-islet cells among all cells Volume fraction of b cells among all islet cells Volume fraction of islets Volume fraction of islet cells within the islets Volume fraction of islet extracellular space within the islets Volume fraction of islets exclusive of islet vascular spaces Volume fraction of non-islet cells based on the total non-islet volume Volume fraction of non-islet extracellular space based on the total non-islet volume Volume fraction of vascular voids within islets Void fraction of packed cell pellet Volume fraction of b cells within whole islet volume fIC calculated from electron microscopy ultrastructural analysis I calculated from DTZ staining data I calculated from electron microscopy ultrastructural analysis I calculated from light microscopy morphology analysis IT calculated from light microscopy morphology analysis Cell volume of acinar cells Cell volume of duct cells Cell volume of non-b-cells
Colton et al.
νO νb ν IC ν NIC ν TC
Cell volume of other cells Cell volume of b-cells Average cell volume for islet cells Average cell volume for non-islet cells Average cell volume for all of the cells
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Colton et al. 167. Scharp, D. W., Kemp, C. B., Knight, M., Ballinger, W. F., Lacy, P. E. The use of ficoll in the preparation of viable islets of Langerhans from the rat pancreas. Transplantation 16; 1973. 168. Schwartzman, R. A., Cidlowski, J. A. Apoptosis: The biochemistry and molecular biology of programmed cell death. Endocr Rev 14:133–151; 1993. 169. Sever, C. E., Demetris, A. J., Zeng, J., Caroll, P., Tzakis, A., Fung, J. J., Starzl, T. E., Ricordi, C. Composition of human islet cell preparations for transplantation. Acta Diabetol 28:233–238; 1992. 170. Shapiro, A. M. J., Lakey, J. R. T., Paty, B. W., Senior, P. A., Bigam, D. L., Ryan, E. A. Strategic opportunities in clinical islet transplantation. Transplantation 79:1304–1307; 2005. 171. Shapiro, A. M. J., Lakey, J. R. T., Ryan, E. A., Korbutt, G., Toth, E., Warnock, G., Kneteman, N., Rajotte, R. V. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238; 2000. 172. Smets, F. N., Chen, Y., Wang, L. J., Soriano, E. Loss of anchorage triggers apoptosis (anoikis) in primary mouse hepatocytes. Mol Gen Metab 75:344–352; 2002. 173. Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., Steele, G. D., Chen, L. B. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a j-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 88:3671–3675; 1991. 174. Smith, A. L., Smith, H. V. A comparison of fluorescein diacetate and propidium iodide staining and in vitro excystation for determining Giardia-intestinals cyst viability. Parasitology 99:329–331; 1989. 175. Smith, R. M., Gale, E. A. M. Survival of the fittest? Natural selection in islet transplantation. Transplantation 79:1301–1303; 2005. 176. Stefan, Y., Orci, L., Malaisse-Lagae, F., Perrelet, A., Patel, Y., Unger, R. H. Quantitation of endocrine cell content in the pancreas of nondiabetic and diabetic humans. Diabetes 31:694–700; 1982. 177. Street, C. N., Lakey, J. R. T., Shapiro, A. M. J., Imes, S., Rajotte, R. V., Ryan, E. A., Lyon, J. G., Kin, T., Avila, J., Tsujimura, T., Korbutt, G. S. Islet graft assessment in the Edmonton protocol: Implications for predicting long-term clinical outcome. Diabetes 53:3107–3114; 2004. 178. Sweet, I. R., Cook, D. L., DeJulio, E., Wallen, A. R., Khalil, G., Callis, J. B., Reems, J. A. Regulation of ATP/ADP in pancreatic islets. Diabetes 53:401–409; 2004. 179. Sweet, I. R., Cook, D. L., Wiseman, R. W., Greenbaum, C. J., Lernmark, A., Matsumoto, S., Teague, J. C., Kronh, K. A. Dynamic perifusion to maintain and assess isolated pancreatic islets. Diabetes Technol Ther 4:67–76; 2002. 180. Sweet, I. R., Khalil, G., Wallen, A. R., Steedman, M., Schenkman, A., Reems, J. A., Kahn, S. E., Callis, J. B. Continuous measurement of oxygen consumption by pancreatic islets. Diabetes Technol Ther 4:661–672; 2002.
181. Takahashi, N., Nemoto, T., Kimura, R., Tachikawa, A., Miwa, A., Okado, H., Miyashita, Y., Iino, M., Kadawaki, T., Kasai, H. Two-photon excitation imaging of pancreatic islets with various fluorescent probes. Diabetes 51 S1:S25–S28; 2002. 182. Thornberry, N. A., Lazebnik, Y. Caspases: Enemies within. Science 281:1312–1316; 1998. 183. Todaro, M., Di Gaudio, F., Lavitrano, M., Stassi, G., Papaccio, G. Islet beta-cell apoptosis triggered in vivo by interleukin-1 beta is not related to the inducible nitric oxidensynthase pathway: Evidence for mitochondrial function impairment and lipoperoxidation. Endocrinology 144:4264–4271; 2003. 184. Torres, D., Munell, F., Ferrer, J., Reventos, J., Macaya, A. Identification of necrotic cell death by the TUNEL assay in the hypoxic-ischemic neonatal rat brain. Neurosci Lett 230: 1–4; 1997. 185. Tsujimura, T., Kuroda, Y., Kin, T., Avila, J. G., Rajotte, R. V., Korbutt, G. S., Ryan, E. A., Shapiro, A. M. J., Lakey, J. R. T. Human islet transplantation from pancreases with prolonged cold ischemia using additional preservation by the twolayer (UW solution/perfluorochemical) cold storage method. Transplantation 74:1687–1691; 2002. 186. Valentijn, A. J., Zouq, N., Gilmore, A. P. Anoikis. Biochem Soc Trans 32; 2004. 187. Van der Burg, M. P., Scheringa, M., Basir, I., Bouwman, E. Assessment of isolated islet equivalents. Transplant Proc 29:1971–1973; 1997. 188. Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. A novel assay for apoptosis flow cytometric detection of phosphatidyl serine expression on early apoptotic cells using fluorescein labeled Annexin V. J Immunol Methods 184:39–51; 1995. 189. Vistica, D. T., Skehan, P., Scudiero, D., Monks, A., Pittman, A., Boyd, M. R. Tetrazolium-based assays for cellular viability: A critical examination of selected parameters affecting formazan production. Cancer Res 51:2515–2520; 1991. 190. Wang, W., Upshaw, L., Strong, D. M., Robertson, R. P., Reems, J. A. Increased oxygen consumption rates in response to high glucose detected by a novel oxygen biosensor system in non-human primate and human islets. J Endocrinol 185:445–455; 2005. 191. Weber, D. J., McFarland, R. D., Irony, I. Selected Food and Drug Administration review issues for regulation of allogeneic islets of Langerhans as somatic cell therapy. Transplantation 74:1816–1820; 2002. 192. Weibel, E. R: Practical Methods for Biological Morphometry. London, Academic Press, 1979. 193. Weir, G. C., Halban, P. A., Meda, P., Wollheim, C. B., Orci, L., Renold, A. E. Dispersed adult rat pancreatic islet cells in culture: A, B, and D cell function. Metabolism 33:447–453; 1984.
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197. Yonelawa, Y., Matsumoto, S., Okitsu, T., Arata, T., Iwanaga, Y., Noguchi, H., Nagata, H., O’Neil, J. J., Tanaka, K. Effective islet isolation method with extremely high islet yields from adult pigs. Cell Transplant 14:757–762; 2005. 198. Yoon, K. H., Ko, S. H., Cho, J. H., Lee, J. M., Ahn, Y. B., Song, K. H., Yoo, S. J., Kang, M. I., Cha, B. Y., Lee, K. W., Son, H. Y., Kang, S. K., Kim, H. S., Lee, I. K., Bonner-Weir, S. Selective beta-cell loss and alpha-cell expansion in patients with type 2 diabetes mellitus in Korea. J Clin Endocrinol Metab 88:2300–2308; 2003.
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7 Encapsulated Human Islet Allografts: Providing Safety with Efficacy David W. Scharp, M.D. Executive Vice President and Chief Medical Officer, Novocell, Inc., 31 Technology Dr., Suite 100 Irvine, CA 92618
Restrictions Preventing Wide Acceptance of Clinical Islet Transplantation with Immunosuppression Recent publications of the long-term results of clinical islet transplantation in hypoglycemic unaware type 1 diabetic recipients suggest excellent insulin independence rates at 12 months between 75 to 85% in the best centers [46, 52, 97]. These results follow intrahepatic injections of partially purified islets into the portal vein of the liver. However, the duration of insulin independence declines yearly to 8% by 5 years at the best centers. Lower success rates are seen from many of the other islet transplant centers, as reported in the Collaborative Islet Transplant Registry report from 2004, providing an average insulin independence rate of 55% at 12 months postimplant. In that this average result included the best centers, many islet transplant center individual results must be significantly below this average. Unfortunately, the first restriction limiting the expansion of clinical islet transplantation continues to be this wide variance of results between centers, suggesting the lack of standardized techniques involved in donor selection, pancreas procurement and shipment, islet processing and preservation, and islet transplantation techniques [46, 50, 82, 83]. In terms of the second restriction, immunosuppression, the majority of patients transplanted with primary human islets received a variation of the Edmonton protocol of immunosuppression — initially thought to reduce complications related to cyclosporine and other agents used
Cellular Transplantation: From Laboratory to Clinic
prior to 2000 [105]. However, the recipients’ physical costs required for them to obtain significant reduction in insulin requirements, including temporary insulin independence, following islet transplantation are proving very high in terms of suffering major adverse events related to their required immunosuppression [51, 104]. In fact, several recipients have elected to stop their immunosuppression while attaining insulin independence due to its side effects [96]. Additional side effects have been documented directly related to the injection of the islets into the liver by percutaneous catheterization of the portal vein leading to portal vein thrombosis or hemorrhage [119], as well as focal hepatic steatosis [60, 79]. Even if these complications and restrictions present in today’s clinical islet transplant recipients could be overcome, the limited numbers of organ donors available relative to the greater than one million potential type 1 recipients in the USA that could theoretically benefit from the procedure, severely restrict the opportunity to transplant islets to no more than a few thousand patients each year. However, the very high charges related to pancreas procurement from the not-for-profit organ procurement organizations (per pancreas at $30,000) and the high pancreas processing costs (at $10,000 each) have limited the potential number of islet transplants in the USA to less than 100 implants per year on average since 2000. The sum of all of these restrictions currently limits islet allograft transplantation under immunosuppression to the category of an academic center demonstration product, regulated by the FDA under Investigator INDs [123]. To expect wide acceptance of
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Following the lead of the Edmonton islet transplantation centers, the selection of potential islet transplant recipients without kidney transplants was limited to a subgroup of diabetics with hypoglycemic unawareness. Yet, this group of potential islet transplant recipients reduces in number each year at major centers providing diabetes care and education due to improved insulin Complications Related to delivery by new drug combinations, such as long-acting insulins and improved ultrashort-acting insulins and by a Immunosuppression greater use of insulin pumps [94]. However, the majority Recipients of whole organ grafts have little choice of type 1 diabetics is not taken care of in these optibut to accept full immunosuppression as a requirement mal diabetic treatment centers, leaving them at higher for transplant success, as confirmed by the thousands risk for developing diabetic complications. These same of potential recipients who die every year from their patients are also at higher risk for developing severe organ failure related to organ shortages in the USA hypoglycemic unawareness when trying to improve and Europe. Patients in this position of needing a their glycemic control through more aggressive insulin heart, lung, liver, kidney, or intestinal transplant for life treatment without adequate training and education. readily accept the complications of immunosuppression Now that longer-term results are available for clinical because the alternatives for them are not compatible islet transplant recipients, there is a greater appreciation with life. Diabetic patients who suffer renal failure for the risks of complications related to immunosupsecondary to their diabetic complications do have a lim- pression in these patients. Shown in Figure 7.1, are the ited choice in renal dialysis versus a pancreas or islet major complications after intra-hepatic, free islet allotransplant with immunosuppression. Although the dura- grafts under full immunosuppression, summarized from tion of life on chronic dialysis is significantly less for recent publications. It is estimated that approximately 50% of diabetic the diabetic patient than the non-diabetic patient, the comparative results for pancreas transplantation remain recipients of islet allografts under immunosuppression controversial. In consideration of transplanting diabetic suffer serious adverse events related to these complipatients, there is a different risk/benefit ratio involved cations. Developing a way to implant islet allografts in their choices compared to the choice left for patients without immunosuppression would be a major step with diseases that result in immediately life-threatening forward in advancing islet transplantation without these organ failure. The patients with diabetes suffer from complications. One choice for eliminating immunosupboth acute and chronic complications of their disease pression is the development of a safe and effective that reduce not only their quality of life but their dura- encapsulation technology for islets. tion of life in those adversely affected. So many type 1 diabetics make the decision to seek either a whole-organ pancreas transplant or an islet transplant, both under Cartoon Descriptions of Encapsulated Cell full immunosuppression, to avoid the progression of Therapy Immune Protection their diabetic complications over time. In the patients with renal failure secondary to diabetic complications, Before discussing the development of encapsulated islet the election of a renal transplant under immunosuppres- technology, the following three figures present in a carsion becomes a reasonable choice for many. Because toon fashion how islet encapsulation protects the cells immunosuppression is already required for the kidney from immune destruction. Figure 7.2 shows how unprotransplant, adding a pancreas or islet transplant results in tected islet allografts are destroyed by the host immune additional morbidity and/or mortality related especially system. Following transplantation into the host, antigens to adding the pancreas but also to the islet transplant. from the islet allograft are identified and engulfed by However, in those patients without renal failure the elec- antigen-presenting cells or dendritic cells. These antition to undergo an islet transplant requiring immunosup- gens are processed by these cells into the appropriate pression is one that cannot be taken lightly when one small peptides that are then presented upon the surconsiders the risks related to immunosuppression that face of the APCs in a specific HLA-linked process. are involved. When a T-lymphocyte locates the APC and its projected clinical islet transplantation to achieve its potential as a new therapy for diabetes as a commercial product, these limiting restrictions must be significantly reduced or eliminated.
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Figure 7.1. Potential complications following free islet allografts with immunosuppression.
Figure 7.2. Pathways leading to free islet allograft rejection.
islet antigen, it attaches to the same HLA linked structure. However, no information is transferred to the T-lymphocyte until two adjacent connections are made between the cells. The CD40 structure from the APC has to chemically bind to the CD154 ligand from the
T-lymphocyte. In a similar manner, the CD80 or CD86 structure from the APC must also bind to the CD28 ligand of the T-lym cell. Once both are connected, the antigen information is then passed to the T-lym helper cell that begins to
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process and convert it to a T-lym killer cell that is specifically able to recognize this antigen. Following the APC making all of its connections to the T-lym helper cell, the APC releases the cytokine IL-1 that diffuses into the T-lym cell to produce IL-2. This cytokine stimulates expansion of the T-lym killer cells that contain the islet allograft marker. These cells circulate in the host until they come in contact with the islet allograft cells containing the correct antigen and attack the islets directly and kill them through cell-mediated killing. If, however, under different conditions the CD80/CD86 structure of the APC attaches instead to the CD152 ligand of the T-lym helper cell, a T-lym regulatory cell is produced that limits the production and expansion of T-lym killer cells and can reduce or stop the destruction of islet allografts. Another function of the T-lym helper cell is the passage of the donor antigen information to B-lym cells for the production of specific antibodies to the islet allograft cells. These donor specific antibodies can also migrate throughout the host and kill the allograft cells in combination with complement. In a similar way, donor APCs contained within the islet graft can migrate out of the islet and process the donor antigens with the recipient T-lym helper cells that may make even more potent killer cells than those processed by host APCs. A number of immunosuppressive drugs have been developed that attack different portions of this process
of immune destruction of allograft cells, as shown in Figure 7.3. Prednisone, azathioprine, and CelCept all reduce the ability of the APC to release IL-1, which reduces the ability to expand T-lym killer cells, and thus reduces the immune destruction of the islet allograft. Cyclosporin and Prograf each attack the T-lym helper cell’s ability to produce IL-2, which also reduces the ability to expand T-lym killer cells. Rapamune also reduces the ability to release IL-2 from the T-lym helper cell, but under a different mechanism. Zenapax binds the IL-2 receptor on the T-lym killer cell, which also reduces the ability to expand the T-lym killer cells. Anti-CD3 antibody treatment nonselectively kills all T-lymphocytes, regardless of type, and protects islet allografts by reducing all T-lym cells. Thyroglobulin similarly nonspecifically kills all T-lym cells, which results in similar graft protection. Two very specific and interesting monoclonal antibodies have been produced by Biogen and IDEC that specifically bind the CD154 binding site on the T-lym killer cell. The Biogen antibody is very specific in preventing islet allograft rejection, even as a single agent, but coagulation crises and a death of a recipient have prevented further drug development. There are always new immunosuppressants being developed with the hope that one will hit the mark with minimal complications. As noted in Figure 7.1, that is not the experience with today’s drugs. Therefore, encapsulation is an attractive approach
Figure 7.3. Targets for immunosuppressive agents to reduce free islet allograft rejection.
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Figure 7.4. Immune protection offered by cell encapsulation without immunosuppression.
to prevent today’s significant immunosuppressive drug side effects. While protecting cells by encapsulation initially seems very easy, a major impediment to the development of encapsulated cell therapy has been the inability to find encapsulating substances that cause no macrophage or APC activation on their own. As shown in Figure 7.4, if the coating has no reactivity many of the antigens crossing from the encapsulated cells are simply ignored. If the coating nonspecifically activates the responding host cells, an inflammatory response begins that attracts cells that release many cytokines around the coated cells that readily penetrate the coating and kill them. In addition, host cells accumulate on the surface of the capsule and prevent adequate oxygen diffusion to the encapsulated cells, leading to their death. With excellent capsule biocompatibility, some of the antigens are processed by the host APCs and create functional T-lym killer cells. However, the cell coating prohibits these T-lym killer cells from contacting their targets so that no damage is done. The cell coating also prevents the donor APCs from escaping the coating, which are thus prevented from interacting with the host immune cells. Even with antibodies produced against the donor antigens, they are not effective if the coating prohibits their crossing to the encapsulated cells. In addition, even if the porosity is sufficiently large to permit antibodies to cross the membrane, the much larger complement is
prevented from crossing the membrane — which then prevents encapsulated cell destruction.
Development of Encapsulated Islet Transplantation Although there were a few early primitive ways attempted to protect cells, the concept of protecting islets from immune attack by placing them behind a permselective barrier originated in current time with the pioneering 1965 work by Thomas Chang [18], who encapsulated islets within microcapsules. As investigations have grown over the years, the ability to encapsulate islets has focused in three main directions (as shown in Figure 7.5): (1) the use of macrocapsules such as planar devices using flat sheet membranes and the use of hollow fibers, (2) the use of microcapsules using hydrogel encapsulation, and more recently (3) the production of direct conformal polymer coatings placed on the surface of the islets as a thin protective coating.
MACROCAPSULES Turning first to review macrocapsule development, it began with the use of available renal dialysis fibers. Chick was one of the first to use this technology to
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Figure 7.5. Islet encapsulation factors required to achieve both clinical and commercial relevance.
perfuse nutrient fluid through the hollow fibers and place the islets around these fibers [21] and then implant them into diabetic rodents [22]. Others continued these types of studies with demonstration of normalization of blood glucose levels [1, 5, 112]. Enlarging the diameter of these fibers led to the development of intravascular hollow fibers for islet implants with one device functioning as an ultrafiltration chamber [92]. Others (Biohybrid and W. R. Grace) developed a single coiled tube surrounded by islets as a diffusion device (hockey puck) inserted into an artery and containing functional islet xenograft cells from dogs or pigs [23, 111]. This device has given some of the longest xenograft results with normal glycemia for several months in diabetic large animals. However, practical problems of hemorrhage and thrombosis kept this device from being developed further. Instead, this group placed the islets within the larger tubes of acrylic polymer hollow fibers and implanted them into the peritoneal cavity of diabetic rodents and dogs with demonstration of the ability to reverse diabetes in many of these recipients [71–74], including discordant islet xenograft islets into rats [70]. However, problems of fiber fragility and limited survival of islets only on the inside surface of these fibers stopped this development. An additional vascular device was developed by the Gore companies that was patented [12, 13] but was never published, with all development on this device currently stopped.
Cytotherapeutics, Inc., used a different polymer to produce a variety of constructs of hollow fibers (Figure 7.5) of several diameters with good diffusion characteristics [34, 35, 47]. This development led to studies with islets that demonstrated the feasibility of these implants in diabetic animals [69] and to a clinical trial as a phase 1 study in nondiabetics, type 1 diabetics, and type 2 diabetics [100, 106]. The lack of host reactivity to the fiber implanted in the subcutaneous site led to survival of the human islet allografts within the fibers that were protected from autoimmune recurrence as well as allograft rejection. However, the packing density was too low to develop this device into a product for diabetes because the length of the fiber would not be clinically relevant to implant a curative dose of human islets. Another company in Europe, Inserm, investigated the potential of their dialysis hollow fibers made of AN69 acrylonitrile into an encapsulation device for islets [32, 64, 65] with demonstration of effectiveness in rodent studies [89] as well with using encapsulated porcine islet xenografts in rodents [33]. Rodent studies continue with these fibers [63, 85]. A planar device containing flat sheet membranes (Figure 7.5) was developed by Baxter as part of their gene therapy program. It also contained outer support structures, a Teflon-based membrane to induce angiogenesis, and a hydrogel membrane for permselectivity [11, 86]. A number of studies of this device
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examined neovascularization, local host responsiveness to xenografts, and release of various products from encapsulated cells [8–10]. Attempts to develop this device for a diabetes product were made in collaboration with Neocrin Co. However, Neocrin stopped the development in favor of developing PEG coating of alginate microspheres containing islets, a technology discussed later in this section. The Baxter device was obtained by Theracyte for exploring several potential cell therapy applications [62, 80, 116], but was recently sold to LCT. Another planar device was being developed by Islet Sheet Medical (now called Cerco Medical) made of supported alginate sheets being implanted into the peritoneal cavity of dogs [110] as an all-in/all-out type of device [2, 3]. There are many other types of membrane materials (polyurethanes, biopolymers, amino-silicate, cellulose, polysulphones, nucleopore, and polyvinyl alcohols) and configurations, and efforts are underway to continue to develop a clinically and commercially relevant macrodevice for the treatment of diabetes. Yet, limiting factors for these macrodevices remain to be solved, such as their relatively large size, choices as to whether they grow into the host that requires an effective method to flush and reload them, and whether they are to be an all-in/all-out device that requires absolutely perfect biocompatibility and suitable strength.
MICROCAPSULES The second major area of cell encapsulation development is the microcapsule, usually defined as a hydrogel matrix formed into a sphere (with a usual diameter of 300 to 800 microns) in which islets or other cells are randomly distributed (Figure 7.5). Whereas Chang actually initiated this technology in 1965 [17–19], it was the publication of the Lim and Sun work in 1980 [49, 75, 76] that popularized it and enabled the many studies that continue today. There are a multitude of publications and patents around the hydrogel microcapsule technology. The majority of the work centers on the original alginate microcapsule, with a permselective component of polylysine (PLL) or poly-ornithine. The original production of these alginate microcapsules consisted of suspending the cells in alginate and flowing them through a double needle apparatus to form microdroplets. The inner needle contained the cells/alginate mixture, and the outer needle used forced air to reduce the surface tension of the forming alginate droplets and hence reduce the
size of the beads. These alginate microdroplets, ranging in size from 300 to 800 microns, were caught in a high-calcium (80 mM) solution that immediately cross-linked the alginate (retaining its spherical shape). Alginate microspheres have been extensively studied because of the material’s ability to rapidly encapsulate cells by dropping into a cyto-compatible solution and forming a spherical shape. There are very few other hydrogel materials that have these properties, and those that do are typically cytotoxic when forming solid spheres. Following the early discoveries of the potential merits of using alginate for coating cells, there have been many improvements in this technology to increase the efficiency and to reduce the size of the alginate microcapsules [18, 24, 36–38, 54, 109, 122, 127]. In terms of rodent animal implants, a large number of feasibility studies have been published using rodent isograft and allograft sources of islets that have demonstrated the ability to reverse diabetes for many weeks and months using the alginate microcapsule with a PLL coating [4, 20, 29, 45, 66]. Protection against autoimmune diabetes of the NOD mouse and the BB rat (as well as successful xenografts) has also been demonstrated with these capsules in diabetic rodent recipients [39, 42, 59, 67, 77, 78, 84, 124]. Efforts to improve encapsulated islet viability postimplant have recently included the use of cross-linked hemoglobin within the alginate capsule [16] to increase oxygen carrying capacity and implanting into a prevascularized subcutaneous site [120]. Another group has added human serum albumin into highly purified alginate microcapsules and achieved long-term success of rat and human islet xenografts implanted into the peritoneal cavity without requiring immunosuppression [102]. The ability to demonstrate efficacy in large animals has been far more difficult and so far not readily reproduced by other investigators. Soon-Shiong published reversal of spontaneous diabetes in dogs using alginate PLL microcapsules by intraperitoneal implantation that may have also utilized cyclosporine as a single low-dose immunosuppressant [107]. A report by Sun in 1996 demonstrated reversal of several cynomolgus primates receiving intraperitoneal implants of alginate PLL microcapsules without any immunosuppression for a few months to two years with minimal capsule overgrowth and partial function in other recipients [113]. This work has not been successfully repeated at this time. A recent preliminary publication is suggesting that alginate-based encapsulation of porcine islets
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into the peritoneal cavity of strep-induced diabetes in cynomolgus monkeys ameliorates their diabetes without the use of immunosuppression [41]. All of the recipients received a second implant 3 months after the first implant. Insulin requirements were reduced to 43% in these recipients, with 1 out of 8 becoming insulin independent at 36 weeks. There is another microcapsule approach to the alginate-based capsules that cross-links the alginate by a different method than calcium alone. This is the work of Wang and Hunkeler that uses multiple components added to the alginate that forms the capsule by polyelectrolyte complexation of sodium alginate and cellulose sulfate with poly(methylene-co-guanidine). They have demonstrated the ability to alter the protein diffusivity profiles across a wide range of molecular weight and alter the strength of the microcapsule as well [6, 87, 93, 125]. Whether these changes will result in major improvements in their in vivo studies remains to be determined.
MICROCAPSULE LIMITATIONS A major limitation of the alginate microcapsule system is the variable biocompatibility of this material that has been the subject of a number of studies and potential improvements. Many investigators have struggled with the problem of biocompatibility of the alginate capsule that may be due to the encapsulating alginate itself, to poorly encapsulated islets that are close to the surface of the capsule, to nonuniform alginate coverage of the highly reactive PLL, and to responding macrophages that are activated by the alginate, which subsequently develops a reaction to the antigens from the encapsulated cells [27, 30, 31, 68, 126]. Yet, a recent report from the Joslin group has used a purified alginate cross-linked with barium without the use of PLL and found diabetic NOD recipients implanted with alginate microcapsules had their diabetes reversed without autoimmune recurrence [40]. Another set of problems arises from the inability to precisely control the protein diffusivity profile in alginate microcapsules that may be necessary to protect the cells from the host immune system reacting to the implant. This lack of control of permeability permits different immune components to cross from the host into the encapsulated cells and for different antigens to cross from the encapsulated cells out to the host. The results of unlimited diffusion in both directions across the alginate
membrane can be damaging to the encapsulated cells and can lead to a buildup of protein and cells on the outside of the capsule. Such protein accumulation and layers of cells on the surface of encapsulating materials leads to reduction of the diffusion of oxygen and nutrients to the cells and waste products from the cells [28, 98, 103, 121]. Attempts to control protein diffusivity characteristics of the alginate microcapsule have not been sufficiently successful to date, with effort now focused on making the microcapsules as small as possible. Some have made small alginate microcapsules and have demonstrated some success in animal models [14]. Yet, another shortcoming of the alginate microcapsule is the fact that there is a finite limit as to how small one can make them in terms of adequately coating the islets. Because the location of the islets within the microcapsule is completely random at the time of the rapid cross-linking of the droplet falling into the calcium solution, as one reduces the droplet in size to < 300 microns in diameter portions of islets within the droplet begin to stick out of the coating and cannot be adequately coated. The only way to cover these exposed islets is by repeating the droplet process, which then results in the production of large capsules. This is a primary shortcoming of this technology of coating islets with alginate. Working with other hydrogels such as agarose or chitosan has not gotten around these basic microcapsule problems [57, 58, 81, 95, 117]. The clinical problem is trying to find a site that will accept the 150 mls of encapsulated islets whose unencapsulated volume is only 2 to 3 mls.
CONFORMAL COATINGS Perhaps the ideal type of encapsulation technology is the development of hydrogel conformal coatings placed directly on the surface of the islets to provide immune protection. Several methods have been tried, but have proven to be quite difficult to develop into clinically relevant approaches [7, 61]. Hubbell has invented a method of interfacially photopolymerizing poly(ethylene glycol) diacrylates directly on the surface of islets to form a cross-linked hydrogel membrane that conformally covers each islet as a separate entity [25, 26, 55, 56]. These PEG conformal coatings have successfully eliminated diabetes in rats using PEG-coated porcine islets for more than 100 days after intraperitoneal implants. This study demonstrated that a 30-day course of low-dose cyclosporine decreased the reaction
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to the capsules immediately after implantation, with a concomitant increase in the number of surviving islets, thus demonstrating a normal glucose tolerance test as compared to those animals that received the same number of capsules without cyclosporine [53]. The use of low-dose cyclosporine had shown to be safe but not effective in a series of studies using the drug to prevent the clinical onset prior to or at time of the diagnosis of type 1 diabetes [15, 44, 88, 90, 101]. This is the technology that Neocrin chose to develop for encapsulated porcine islet xenografts transplanted into diabetic cynomolgus primates, as referred to earlier. Yet, when these capsules that worked so well in the rodent were placed in the primate little success was achieved because the primates reacted totally differently to the PEG coatings. This emphasizes a basic problem in cell encapsulation in the reliance on rodent results because they simply have a high risk of not being predictable for large animal results. This risk using rodents to predict large animal results is especially notable with microcapsule studies. Only a few investigators have tried to cross into large animal studies with microcapsules and conformal coatings. To date, none of the few reported successes in large animals has been confirmed.
Clinical Trials of Encapsulated Human Islets Very few of these encapsulated islet methods have resulted in human clinical trials. One trial was sponsored by Cytotherapeutics, Inc., as an Investigator IND approved by the FDA from Washington University in St. Louis, Missouri — with David Scharp, M.D., the principal investigator of the study. Cryopreserved human islets were encapsulated within the hollow fiber that was inserted in the subcutaneous tissue of the anterior thigh in three nondiabetic, three type 1 diabetic, and three type 2 diabetic patients as a subtherapeutic study of encapsulated islet survival. The encapsulated human islets were protected from both autoimmune destruction from the type 1 diabetes as well as from allograft rejection during the two-week implant exposure in these non-immunosuppressed recipients. Viable islets were removed from the hollow fibers postimplant and demonstrated stimulated insulin release in vivo [100]. Another encapsulated human islet study was conducted as an Investigator IND by Patrick Soon-Shiong from VivoRx (now Amcyte) using alginate microcapsules to contain the human islets. Three patients
were implanted in the intraperitoneal cavity with large volumes of alginate capsules that were approximately 600 microns in diameter. One patient had partial insulin reduction to a significant level for a few weeks before returning to the diabetic state. These results were also published [108]. Another human islet encapsulation product (W. R. Grace and Circe Biomedical) was approved by the FDA for clinical trials, but the phase I/II trial was never initiated due to significant adverse reactions in the last preclinical animals studied. This was an intravascular device that was anastamosed to a major limb artery. Many months of function with elimination of the exogenous insulin requirement were demonstrated as a xenograft product in preclinical canine recipients of porcine and bovine encapsulated islets [111]. Problems with hemorrhage and thrombosis of this device in the preclinical animals prevented clinical trials from being initiated and the product was dropped from further development. Although there have been additional patients with diabetes implanted with encapsulated human islets, these small studies were conducted outside of the USA and were performed without FDA approval. Some of these results have been published, but essentially all have been premature and poorly documented studies that would have great difficulty in passing FDA scrutiny. One example of this type of study is the one performed in Mexico using a large bore device to induce a vascularized scar in which to implant porcine islet xenografts associated with Sertoli cells without immunosuppression [118].
Novocell PEG Encapsulation Technology The PEG conformal coatings have been developed based on J. A. Hubbell’s invention [55, 56] and applied to islet allografts. As shown in Figure 7.6, there are four major reagents required to produce these interfacial conformal coatings on the surface of islets. Poly(ethylene glycol)-triacrylate (PEG-3A) is a threearmed PEG with an acrylate group conjugated to the end of each of the arms of the monomer. The photoinitiator is eosin Y (EY) conjugated to another polymer that binds the cell aggregate more effectively than eosin Y itself. The radical generator is triethanolamine (TEOA), and the co-monomer accelerant is n-vinylpyrrolidone (n-VP). As shown in the figure, the PEG-3A preassembles itself in water into a 3D network because the PEG portion is hydrophilic and the acrylate portion
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Figure 7.6. Novocell’s PEG-based islet encapsulation technology required to form islet conformal coatings.
is hydrophobic, forming foci of acrylates linked by the PEG arms. Although it preassembles in water, the material is not cross-linked. The TEOA and n-VP are added to the PEG-TA in the aqueous bulk phase. The uniqueness of this approach is to specifically bind the EY to the surface of the cell cluster or islet converting the bulk phase polymerization into an interfacial polymerization. This means that when the laser light is applied the EY assumes a high-energy state that is passed to the TEOA, forming very high-energy radicals. These radicals initiate the polymer cross-linking from the cell surface outwardly by covalently bonding the acrylates. This product has been designed to have a range of cell aggregate or islet coating that is approximately 25 to 50 microns in thickness. The radicals binding the acrylates essentially spot weld the preassembled network of PEG-3A into a polymer with the nVP acting as the accelerant. Because the EY photoinitiator is on the surface of the cell cluster, the PEG coatings form conformally on the surface of each islet, making a protective coating by interfacial polymerization. This PEG conformal coating exhibits sufficient porosity to permit nutrients, glucose, and oxygen to pass into the encapsulated islets and allows insulin to pass freely out while keeping the host immune cells from contacting the encapsulated islets. The alginate/acrylate bond is an ester bond that is biodegradable in the body. Additional compounds can
be added at this junction to vary the duration of the coatings in the body. Novocell has the exclusive license to these Hubbell patents for the treatment of any human disease or disorder.
BIOCOMPATIBILITY Referring back to Figure 7.2, regarding clinical and commercial efficacy requirements, the first is the requirement for biocompatibility. Both macrocapsules and microcapsules have had a great deal of difficulty achieving adequate biocompatibility results in animal studies. There has been relative ease of demonstrating biocompatibility in rodents, with far more difficulty in primates. This permselective capsule or coating containing living and functioning cells has to uniquely have no inherent ability to stimulate the host immune system or the effectiveness will be lost, destroying the contained cells. Biomaterials that by themselves stimulate the immune system are in a sense placing the cells inside an immune reactive coating that can be considered a Freud’s adjuvant that results in an inflammatory and an immune response covering the device and destroying the contained cells by cytokine storm. One of the challenges facing the PEG conformal coating was to develop a polymer coating that is not reactive within
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Figure 7.7. Multi-species biocompatibility with PEG polymer coatings.
the host. After a significant period of time, this has been accomplished, as shown in Figure 7.7.
SMALL ANIMAL STUDIES There have been a large number of mouse implants used to develop the PEG conformal coatings at Novocell. The only result discussed in this chapter
is the experience of implanting encapsulated mouse islet allografts into spontaneously diabetic NOD mice. Mouse islets from C57 mouse donors have been encapsulated with PEG conformal coatings and implanted into the peritoneal cavity of diabetic NOD mice without using any immunosuppression. The results are shown in Figure 7.8. These results clearly show that encapsulated mouse islet allografts can be readily implanted into diabetic
Figure 7.8. Novocell results of PEG encapsulated islet allografts implanted into NOD diabetic mice without immunosuppression.
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NOD recipients without the use of any immunosuppression and achieve > 100 days of relatively normal mouse blood glucose levels. This means that both allograft immune reactivity and autoimmune reactivity are prevented from destroying the encapsulated islets by use of the PEG coating alone.
NONHUMAN PRIMATE SAFETY AND EFFICACY STUDIES There have been several studies completed with PEG conformally coated islets in the nonhuman primate and an abstract was recently presented on these data [99]. These studies are in the process of being published and cannot be duplicated in this chapter. However, Figure 7.9 shows the long-term results of one of the pilot animals and demonstrates the ability to achieve long-term insulin independence following subcutaneous implants with only 30 days of low-dose cyclosporine. After nine months, additional low-dose insulin was given for several months. Then a second subcutaneous implant of encapsulated islet allografts was given and the recipient again achieved insulin independence with improved glycemic control. Histology at time of sacrifice confirmed a well-vascularized encapsulated islet allograft implant site with surviving islets and very little host responsiveness. The results of these studies were submitted to the FDA in a phase I/II IND application that has been recently approved for the start of a clinical
trial in type 1 diabetic recipients. Figure 7.10 demonstrates purified primary human islets prior to and after PEG conformally coated human islet allografts.
HUMAN USE OF PEG COATINGS Although the PEG conformally coated human islet allografts have not had any previous human testing, some similar PEG polymers have been used in patients as tissue sealants. The Focal Company utilized a similar process to place a coating on the surface of the lung that had just undergone a partial resection in an FDAapproved product called FocalSeal-L. The PEG polymer coating was used as a sealant to prevent air leakage from the staple holes that closed the partially resected lung remnant, avoiding the use of a chest tube after the operation. The surface of the lung was “painted” with the eosin Y photoinitiator. Then the PEG monomer, the radical generator, and the accelerant co-monomer were layered on top of the eosin Y, coating the operated site of the lung surface. A light source was used to cross-link the PEG into a polymer coating the surface during the operative procedure. This product’s in-life function was designed to be in terms of a few weeks because several molecules connected with ester bonds were inserted between the PEG portion and the acrylate terminations of the arcylated PEG. The PEG coating was successful in preventing air leaks in animals undergoing pulmonary surgery [91].
Figure 7.9. Novocell results of PEG encapsulated islet allografts implanted subcutaneously into a diabetic baboon with only 30 days of low dose cyclosporine after implants and followed for 86 weeks.
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Figure 7.10. PEG encapsulated conformal coatings on human islets of different sizes.
It was approved by the FDA and used successfully to seal air leaks after pulmonary and cardiac surgery in humans [43, 48]. In addition, it has been used to seal against anastamoses after intestinal surgery in animals [114]. Genzyme purchased Focal and continued selling this product for a time, but has since stopped the
sales of the product. A similar product, AdvaSeal, is a polyethylene glycol-based synthetic hydrogel with two components (primer and sealant) polymerized by bluegreen light and designed to biodegrade by hydrolysis within a few weeks [115]. It is sold in Europe by Johnson & Johnson’s Ethicon division.
Figure 7.11. Potential opportunities for encapsulated cell therapy products.
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ENCAPSULATED CELL THERAPY FOR THE TREATMENT OF MANY DISEASES When encapsulated islet transplantation shows success in its clinical trials, there will be opportunities to explore many other disease areas that could be treated using this approach. Figure 7.11 shows many of these potential opportunities. Several therapies could be easier to bring to successful completion than the treatment of diabetes. With the conformal coating technology proven, the critical factor for pursuing these other diagnoses is the availability of a clinically relevant cell source to use as the encapsulated cells. Today, the number of potentially treated diseases far exceeds the available cell sources for encapsulation. However, there have been a number of recent advances in the development of differentiated cells from both adult stem cells and embryonic stem cells. There will surely be a time in the near future when the limitation of an adequate cell source will no longer be a problem for the treatment of many diseases. As this occurs, encapsulated cell therapy will provide the vehicle to deliver these new functional cells to the disease host and will eliminate the risks of immunosuppression.
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8 Strategies for Cell Replacement for Kidney Failure Marc R. Hammerman Renal Division, Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, Email:
[email protected]
Introduction There was a time not long ago when there was no treatment for kidney failure. Reflecting in 1994 on the first year of his nephrology training at Washington University in 1958, Peter A. F. Morrin notes, “The common problems of nephrology were essentially the same as they are today, but our knowledge of the underlying pathophysiology and our ability to deal with them were significantly less. Dialysis for acute renal failure was considered an experimental therapy and limited to a few centers with special interests in the area. There was no effective treatment for chronic renal failure [31].” At present, dialysis and renal allotransplantation are standard medical treatments. Although life-preserving, the former therapy replaces only a small fraction of normal kidney function and has considerable morbidity [2]. The number of human organs available limits the latter therapy [16]. Although it is certain that dialysis and renal transplantation will continue to be employed for the foreseeable future, it is likely that these modalities will eventually be supplanted by one or more alternatives currently under development that employ cell transplantation or the growing of a new kidney altogether (renal organogenesis). In that humans and pigs are of comparable size and share a similar renal physiology, and because pigs are plentiful and can be bred to be pathogen free, pigs represent an ideal substitute donor [8, 16]. Unfortunately, the transplantation of whole vascularized organs such as the kidney originating from pigs into the group of primates that includes humans, the great apes, and old-world monkeys is rendered problematic because of Cellular Transplantation: From Laboratory to Clinic
the processes of humoral rejection (hyperacute and acute vascular rejection) that occur across this xenogeneic barrier [8]. Hyperacute rejection occurs as a result of the binding of preformed or natural xenoreactive antibodies present in the circulation of hosts to cells of the donor species followed by activation of the hosts’ complement system. Approximately 85% of the natural antibodies in humans that bind to pig cells are directed against galactosealpha-1, 3-galactose (alpha-gal), a sugar expressed on the vascular endothelium of cells in most mammals but not in humans, great apes, and old-world monkeys [8]. The etiology of acute vascular rejection is multifactorial and incompletely understood. Several of the processes implicated as causative of acute vascular rejection, reflect a fundamental incompatibility between host-proteins protein-systems and the vascular endothelium of the donor. Factors thought to contribute include circulating xenoreactive antibodies that trigger adverse reactions in endothelial cells of the transplant, the failure of primate natural killer cells to recognize the MHC I molecules of pigs, and molecular incompatibilities between porcine-proteins/receptors and circulating primate/human-protein systems such as clotting factors [8]. Humoral rejection following the transplantation of pig kidneys into nonhuman primates can be ameliorated or overcome through the use of genetically altered organs originating from pigs transgenic for the human complement activator, decay-accelerating factor (hDAF) [9], or the use of organs from transgenics that do not express alpha-gal [58]. Unfortunately, neither the immunosuppressive regimens used for pig to primate
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Marc R. Hammerman
kidney transplantation nor the outcomes [9, 58] would be acceptable in humans. Transplantation of kidneys from pigs transgenic for hDAF in combination with host immunosuppression and splenectomy enabled survival for up to 78 days in cynomolgus monkeys with no kidneys of their own. However, there was a high incidence of adverse events such as the development of edema (or ascites), vomiting, diarrhea, and the development of lymphoproliferative disorders. All recipients had to be euthanized because of renal failure, gastrointestinal hemorrhage, or pancreatitis [9]. Survival for as long as 83 days was achieved following xenotransplantation of kidneys from alpha-gal-deficient donors into immunosuppressed thymectomized and splenectomized baboons, provided as vascularized pig thymic tissue was cotransplanted. Recipients of xenografts without thymic tissue rejected their grafts after approximately one month [58]. Baboons died from serum sickness, infection, or myocardial infarction. In contrast to xenotransplantation of whole vascularized organs from pig to primates, nonvascularized cell and tissue grafts might be feasible, in that they are less susceptible to humoral rejection [8, 16]. Cellular transplants such as pancreatic islets from pigs can be transplanted into humans without triggering hyperacute or acute vascular rejection [16]. Early in its development, the renal metanephric anlage is avascular [3, 23] and is therefore a “candidate” for cell transplantation. The metanephric kidneys originate during the fifth week of gestation in humans, during day 12 of embryonic rat development (E12), during day 11 of embryonic mouse development (E11), and during day 24 to 28 of embryonic pig development (E24-28) when outgrowths of the mesonephric ducts (so-called ureteric buds) collect about their distal ends — intermediate mesoderm (metanephric blastema) located caudal to the mesonephros. Numerous outgrowths arise from the distal end of the ureteric bud that push radially into the surrounding mass of metanephric blastema and give rise to the collecting ducts of the kidneys. The proximal ends of the ureteric bud give rise to the ureter and renal pelvis. The metanephric blastema differentiates into all tubular structures of the adult nephron with the exception of the collecting system [23]. The mature kidney is a remarkably complex structure, the function of which is dependent on the growth and differentiation of its precursor cells within the intermediate mesoderm into a mature organ consisting of many different cell types. Al-Awqati and Oliver have
estimated that there are at least 26 terminally differentiated cell types in the kidney of a newborn mouse that arise from at least four cell types present in the undifferentiated metanephric blastema when renal development begins [3]. Delineation of 26 terminally differentiated nephron cell types takes into account cell morphology, location, and function [3]. In order for glomerular filtration, reabsorption, and secretion of fluid and electrolytes to take place in a manner that will sustain life the nephrons must be integrated in three dimensions with one another and with a collecting system, the origin of which is the ureteric bud [23]. Vascularization must occur in a unique organ-specific manner from endothelial precursors that may originate from both inside and outside the developing renal anlage [1, 24, 42, 49]. In addition to its filtration, reabsorptive, and secretory functions, the kidney is an endocrine/metabolic organ. For example, it is a major site of erythropoietin [15], renin [14] synthesis, 1-a hydroxylation of 25(OH)D3 [6], and 5’ deiodination of thyroid hormone [46]. Cell transplantation has limitations. For example, it is conceivable that endocrine functions of the kidney (such as erythropoietin production) could be recapitulated by transplanting one particular type of renal cell. Similarly, it is possible that replacement of one or another type of injured renal cell could enhance the function of damaged tubules. However, it is difficult to imagine how glomerular filtration and reabsorption in kidneys could be reconstituted de novo by infusion of individual cells. One potential approach to replacing such functions is through organogenesis or the growing of kidneys in situ post transplantation of stem cells or renal anlagen [8]. During the past decades, a number of different cell and organogenesis approaches have been applied to develop new therapies for acute and chronic renal failure. These include (1) integration of new nephrons into the kidney, (2) growing new kidneys in situ/renal organogenesis, (3) use of stem cells, and (4) generation of histocompatible tissues using nuclear transplantation/therapeutic cloning. These approaches circumvent humoral rejection and can thus be used as part of a xenotransplantation strategy employing pig tissues.
Integration of New Nephrons into the Kidney The methodology for studies directed toward integrating new nephrons into the kidney derives from a literature
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describing the transplantation of renal metanephric anlagen derived from embryos. Renal anlagen have been transplanted successfully to a number of sites, including the chorioallantoic membrane of developing birds [49], the anterior chamber of the eye [24], beneath the renal capsule [10, 45], into a tunnel fashioned within the renal cortex of recipients [55, 57], and intraperitoneally [10, 22, 40–45, 47]. Most studies that employed renal subcapsular renal anlage transplantation, placement into the anterior chamber of the eye, and onto the chorioallantoic membrane were conducted to define the immune response to fetal kidney transplants or to delineate the means by which renal anlagen are vascularized. However, information that emerged from these studies led to approaches designed to employ renal anlage transplantation as a means to enhance renal function. It had been speculated that developing nephrons implanted beneath the renal capsule [45] or into tunnels fashioned in the cortices of host kidneys [55, 57] might become incorporated into the collecting system of the host, and thereby increase host renal function. Woolf et al. implanted pieces of sectioned renal anlagen originating from embryonic day (E)13-E16 mice into tunnels fashioned in the cortex of kidneys of newborn outbred mice. Differentiation and growth of donor nephrons occurred in the host kidney. Glomeruli were vascularized, mature proximal tubules were formed, and extension of metanephric tubules into the renal medulla was observed. However, incorporation of donor nephrons into the collecting system of hosts was not demonstrated [55, 57]. We performed experiments similar to those of Woolf et al. in which E15 Sprague Dawley rat metanephroi were implanted beneath the renal capsule of adult Sprague Dawley hosts. Hosts received no immunosuppression. E15 metanephroi contained segments of ureteric bud and condensing metanephric blastema, but no glomeruli [45]. To determine whether subcapsularly transplanted rat metanephroi became integrated into host kidneys, we examined kidneys of host rats 6 weeks post-subcapsular transplantation. To clear blood from the organ, kidneys were back-perfused. This results in a blanching of the kidney as blood is replaced by perfusate. Normally, the entire kidney blanches. However, following perfusion of kidneys that contained a transplanted metanephros blood remained in the transplanted structure relative to the host kidney (Figure 8.1a, cortex arrows). Most likely, this reflects a reduced perfusion in chimeric
Figure 8.1. (a) Photograph of a mid-sagittal section obtained following perfusion of a kidney originating from a rat, 6 weeks posttransplantation. Arrows show portions of unperfused transplanted kidney. Photomicrographs of hematoxylin and eosin-stained kidneys: (b) glomerulus (g) within transplanted kidney and (c) glomerulus (G) within host kidney. (Reprinted with permission [45].)
blood vessels (derived from transplant and host kidneys) that have been shown to supply subrenal capsularly transplanted metanephroi relative to perfusion in those supplying only the host kidney. Blood could be traced into the papilla of the host kidney (Figure 8.1a, medulla, arrows). Histological examination of kidneys showed that glomeruli (g) in the transplanted kidney (Figure 8.1b) had been poorly perfused relative to glomeruli present in the host kidney (Figure 8.1c), in that they contained more red blood cells. Also, glomeruli in the transplanted kidneys (g) were smaller than those in the host kidney (G). Collecting ducts from transplanted kidneys migrated toward the papilla of host kidneys in parallel with the vasculature [45]. However, like Woolf et al. [55, 57] we were unable to determine that any connection between the collecting systems of donor and host kidneys was made [45]. In addition, we found that the growth of transplanted metanephroi was constrained by their placement beneath the host kidney capsule. The studies previously summarized show that it is possible to integrate new filtering nephrons into kidneys. If integration were accompanied by incorporation into the collecting system of hosts, metanephros subrenal capsular transplantation would represent a strategy for increasing renal function. However, to be applicable to humans with end-stage renal disease metanephroi must incorporate into host nephrons in end-stage kidneys that are small and fibrotic. Such conditions are not duplicated by subcapsular transplantations performed thus far.
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Organogenesis Perusal of the literature provides four theoretical reasons why the use of transplantation of developing metanephroi might be advantageous relative to developed kidneys. First, if developing renal anlagen are obtained at sufficiently early stage antigen-presenting cells (APCs) that mediate “direct” host recognition of alloantigen or xenoantigen would be expected to be absent from the renal anlagen because they would not have yet developed in the donor and migrated into the renal anlage [17, 32]. Second, donor antigens such as MHC class I and II may not be expressed on developing renal anlagen to the extent they are expressed in adult kidney [12, 51]. Third, the immune response to transplanted fetal renal tissue differs from that to adult tissue [11, 13]. Fourth, one might expect a transplanted renal anlage to be supplied by blood vessels of host origin and as such render irrelevant the processes of hyperacute and acute vascular rejection [42].
ANTIGEN PRESENTATION The first studies to address the question of APC depletion in renal anlagen were performed by Foglia et al., who transplanted renal anlagen, obtained from outbred Sprague Dawley rat embryos aged E15 to E21, beneath the renal capsule of adult Sprague Dawley hosts. Fetal renal allograft growth and survival was age dependent in that the growth and differentiation in situ over a 15- to 30-day period was best for renal anlagen obtained from E15 embryos and worsened progressively for renal anlagen obtained on E16 to E21. The developed E15 renal anlagen showed maturation of renal elements when examined 10 days posttransplantation and no sign of rejection, whereas E20 renal anlagen had a poor renal architecture and a dense lymphocytic infiltrate after a comparable period [17]. In contrast to renal anlagen obtained on E15, liver tissue harvested on E15 and transplanted beneath the renal capsule of hosts underwent little growth and prompt rejection [17]. Velasco and Hegre transplanted renal anlagen or liver tissue from E15, E17, E18, and E19 Fisher rat embryos (RT1lv1 ) beneath the renal capsule of MHC (RT1) incompatible Wistar Furth adult rats (RT1u ). All fetal hepatic grafts were rejected by 10 days posttransplantation. In contrast, the degree of rejection of the renal anlagen was age dependent — those from E15 embryos showing minimal or moderate rejection
and those from older embryos showing more intense rejection. If liver and renal anlagen from E15 embryos were co-transplanted at different sites into Wistar Furth rats, renal anlagen underwent a more severe rejection than if they were implanted without the liver [54]. APCs populate liver well before E15 in rats, but are not present in the circulation until several days later [32]. It was speculated that the absence of APCs in renal anlagen obtained from E15 embryos together with their presence in liver tissue obtained concurrently could explain the differential fate of renal anlagen transplanted with or without liver. Under the former conditions, but not the latter, direct presentation of donor antigens to host T-cells could take place [54].
EXPRESSION OF CLASS I AND II ANTIGENS Statter et al. transplanted renal tissue originating from E14-adult C57Bl/6 mice (H-2b ) beneath the renal capsule of adult congenic B10.A hosts (H-2a ). Expression of donor and host-specific class I (H2Kb ) and class II (Abb ) transcripts in donor tissue were low at E14 and increased progressively in renal tissue from older mice. After transplantation, surviving kidney grafts showed enhanced expression of class I and class II transcripts. However, neither class I nor class II protein could be detected in transplanted renal anlagen in contrast to its presence in transplanted adult renal tissue [51]. Dekel and co-workers have carried out a series of investigations in which human adult or embryonic kidney tissue is transplanted beneath the kidney capsule of immunodeficient rats [severe combined immunodeficiency (SCID/Lewis and SCID/nude chimeric rats)] [12, 13]. Human adult kidney fragments transplanted beneath the renal capsule of such rats survive for as long as 2 months posttransplantation. The overall architecture of the transplanted kidney tissue and the normal structure of individual cells in glomeruli is preserved. The intraperitoneal infusion posttransplantation of allogeneic human peripheral blood mononuclear cells (PBMCs) results in rejection of adult human grafts. Human fetal kidney fragments transplanted beneath the renal capsule of immunodeficient rats display rapid growth and development. Glomeruli and tubular structures are maintained for as long as 4 months posttransplantation. In contrast to the case for transplanted adult human kidney fragments, infusion of allogeneic human
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PMBC into hosts results in either minimal human T-cell infiltration or T-cell infiltrates that do not result in rejection, and do not interfere with the continued growth of the human fetal renal tissue [12, 13]. Fetal human renal grafts have reduced expression of tissue HLA classes I and II relative to the adult human renal grafts, consistent with a reduced effectiveness in inducing an alloantigen-primed T-cell response [13].
IMMUNE RESPONSE AND IMMUNE RECOGNITION Dekel et al. have shown that an allogeneic immune system appears to mount a T-helper 2-biased response when the target organ is of fetal origin resulting in enhanced survival of transplanted fetal renal tissue relative to adult kidneys, against which a T-helper 1-biased response is mounted [13]. Subsequently, this group established that development of immunological maturity in the human kidney is a relatively late event during gestation. Thus, developing kidneys had restricted expression of multiple factors that determine immune recognition. As would be expected [13, 51], 13 of 57 genes that were significantly up-regulated in adult versus fetal kidney tissue belonged to the HLA class I and class II systems. In addition, molecules that mediate trafficking of leukocytes into the graft such as the chemokines RANTES and MCP-1, the adhesion molecule E-selectin, proinflammatory cytokines such as osteopontin, and complement genes known to be associated with innate immunity had reduced expressions in embryonic relative to adult kidneys [11]. Reduced immunogenicity of human or pig kidneys isolated early during renal organogenesis transplanted into immunodeficient mice was confirmed by demonstrating the absence of cellular rejection following infusion of human PBMCs in hosts [11].
chimeric. Its ability to attract its own vasculature in situ establishes the potential for a transplanted renal anlage to attract a vasculature from a suitable vascular bed such as the peritoneal membrane [11, 42].
Isotransplantation/Allotransplantation of Embryonic Kidneys to Enhance Renal Function Renal anlagen transplanted into a host rat’s fold of omentum undergo differentiation and growth in hosts that are not confined by a tight organ capsule [45]. A renal anlage in a retroperitoneal dissection from an E15 rat embryo is shown in Figure 8.2a. The ureteric bud is delineated by an arrowhead. If transplanted with its ureteric bud attached, the renal anlage enlarges and becomes kidney-shaped within 3 weeks (Figure 8.2b). The ureteric bud differentiates into a ureter (Figure 8.2b, arrowhead). In contrast to transplanted developed kidneys that undergo acute rejection [45], renal anlagen transplanted into non-immunosuppressed hosts have a normal kidney structure and ultrastructure postdevelopment in situ and become vascularized via arteries that originate from the superior mesenteric artery of hosts and veins that originate from the host omentum (Figure 8.3) [22, 40, 45]. Figures 8.4a and b show hematoxylin- and eosin (H&E)-stained sections of an E15 rat renal anlage consisting of branched utereric bud (ub) and undifferentiated metanephric blastema (mb). H&E-stained sections of a renal anlage 6 weeks posttransplantation into the omentum of a host rat show developed cortex with a labeled glomerulus (g), proximal tubule (p) with
VASCULARIZATION OF TRANSPLANTED RENAL ANLAGEN The major vessels supplying the kidney originate from lateral branches of the abdominal aorta that terminates in a plexus of arteries in close proximity to the renal pelvis, the renal artery rete [34]. During its development, the renal anlage attracts the major portion of its vasculature from the developing aorta [56]. In that its blood supply originates outside the developing organ, the kidney is
Figure 8.2. (a) Photograph of retro-peritoneal dissection from an E15 rat embryo showing renal anlage or metanephros (m) and ureteric bud (arrowhead). (b) Photograph of a developed renal anlage (m) in the omentum of an adult host rat 3 weeks posttransplantation. Arrowhead shows developed ureter. Magnification is shown. (Reproduced with permission [22].)
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Figure 8.3. (a) Radiocontrast image of kidney (K) and developed renal anlage (M), 6 weeks posttransplantation into the peritoneum of a host rat. SMA (superior mesenteric artery); (b) artery (a) and vein (v) supplying the developed renal anlage or metanephros (m) originating from the host’s peritoneum. Magnification is shown for image B. (Reproduced with permission [22, 40].)
Figure 8.4. Photomicrographs: (a and b) H&E-stained mid-sagittal sections of a renal anlage originating from an E15 rat embryo; (c and d) H&E-stained sections of a developed renal anlage 6 weeks posttransplantation into the omentum of an adult host rat. Shown are ureteric bud (ub), metanephric blastema (mb), glomerulus (g), proximal tubule (p), brush border (arrowhead), distal tubule (d), and collecting duct (cd). (Reproduced with permission [41, 45].)
a brush border membrane (arrowhead), and distal tubule (d) (Figure 8.4c). A developed medulla with a labeled collecting duct (cd) is shown in Figure 8.4d. Electron microscopy of a developed renal anlage [21] reveals normal renal structures (Figure 8.5). Developed renal anlagen transplanted onto the omentum produce urine that is excreted in the normal manner following
Figure 8.5. Electron micrographs of transplanted rat renal anlagen. Labeled glomerular capillary loops show: (a) mesangial cell (m), (b) endothelial cell (en), and (c) epithelial cell (ep), endothelial cell (en), podocytes (pd), and a basement membrane (arrows). (d) A proximal tubule (pt) with a brush border membrane (arrowhead). (e) Proximal tubule (pt), distal tubule (dt), and collecting duct (cd). Magnifications shown for images c and e. (Reproduced with permission [21]).
ureteroureterostomy between transplant and host, a procedure that can be readily carried out if renal anlagen are implanted in close proximity to the host ureter [45]. Levels of renal function in transplanted renal anlagen [glomerular filtration rate (GFR)] were determined by measuring inulin clearance in otherwise anephric rats. In initial experiments, GFRs were very low [45]. However, as shown in Table 8.1 incubation of renal anlagen with growth factors prior to implantation increased GFRs more than a hundredfold compared to those in rats with non-growth-factor-incubated renal anlagen implanted concurrently [22]. GFRs in growth-factor-treated renal TABLE 8.1 Urine volumes and inulin clearances.
Urine volume (UV) (ul/hour) Inulin clearance (GFR) (ul/min/100g) (ul/hour) UV/GFR
Vehicle
Gr Factors
31 ± 9.1
621 ± 62
0.24 ± 0.06 38 0.8
27 ± 8.2 5313 0.12
Some data are expressed as mean ± SEM. UV/GFR is calculated using values expressed as ul/hour. (Reproduced with permission [22].)
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% Survival
anlagen are about 6% of normal. This represents a 250fold increase relative to the values obtained in our original studies employing non-growth-factor-treated renal anlagen (0.11 ± 0.02 ul/min/100 g rat weight) [45]. Renal plasma flow, another parameter of renal function, was measured in transplanted renal anlagen by calculating P-aminohippurate (PAH) clearances. The ratio of GFR/PAH clearance (filtration fraction) was 0.6, higher than normal for the rat (0.3 to 0.4) [22]. However, it is comparable to filtration fractions measured in rats with reduced renal function (0.4 to 0.5), and demonstrates that tubules in transplanted renal anlagen transport PAH [22]. Urine flow rates in transplanted rats were calculated by measuring urine volumes (UV) as a function of time. UVs are about 12% of the inulin clearance (GFR) measured in growth-factor-treated renal anlagen (Table 8.1). This value is comparable to the upper range of normal for a mammal (humans). The UV/GFR of 0.12 demonstrates that transplanted renal anlagen can concentrate urine [22]. Hemodialysis provides renal failure patients with GFRs that are about 10% of normal. Therefore, 6% of normal approximates a level of renal function that would be expected to preserve life. Indeed, life can be prolonged in otherwise anephric rat hosts by prior transplantation and ureteroureterostomy of a single renal primordium [40]. Survival as a function of time postremoval of all native renal mass (all renal function from the implant) is shown in Figure 8.6. Control rats (no transplanted renal primordia) lived 67 ± 2.7 hours (range 48 to 78 hours) post-removal of all native host renal mass. Rats in the TX group (transplanted primordium, but with the ureteroureterostomy severed such
100 90 80 70 60 50 40 30 20 10 0
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Days Figure 8.6. Survival of rats as a function of time post removal of both kidneys. Control rats (n = 13) had no transplanted renal primordia. Rats in the TX group (n = 4) had a transplanted renal primordium, but with the ureteroureterostomy severed such that urine was discharged into the peritoneal cavity. Rats in the TX-EXCR group (n = 5) had a transplanted renal primordium with an intact uteretoureterostomy that permitted excretion of urine. (Reproduced with permission [40].)
that urine was discharged into the peritoneal cavity) lived 65 ± 6.0 hours (range 55 to 76 hours), no longer than controls. Rats in the TX-EXCR group (transplanted primordium with intact uteretoureterostomy that permitted excretion of urine) lived 125 ± 12 hours (range 108 to 170 hours), significantly greater than control or TX rats [40]. Using inbred congenic rats (PVG-RT1C and PVGRT1avl ), we have shown that renal anlagen can be transplanted across the RT1 locus into non-immunesuppressed hosts. A state of peripheral immune tolerance secondary to T-cell “ignorance” permits the survival of transplanted renal anlagen. Most likely the “ignorance” results from the absence of APCs originating from the donor in the embryonic renal tissue and from the consequent absence of direct presentation of transplant antigen to host T-cells (presentation by donor dendritic cells to host T-cells) [44], as was shown previously for subrenal capsular transplants [54]. Transplantation of renal anlagen from E28 pigs to adult outbred pigs can be carried out without host immunosuppression [48].
Availability of Source Material for Renal Anlage Transplants In the case of human renal allotransplantation, there is an unavoidable delay between the time of harvest from donors and the time of implantation into recipients. Prior to removal from the donor, human renal allografts are flushed with a preservation solution (often University of Wisconsin, UW, solution) and stored subsequently in ice-cold UW solution. Theoretically, renal anlagen could be harvested immediately prior to implantation into humans. However, practically it would be best if like most human renal allografts renal anlagen could be stored in vitro for a period of time prior to transplantation. The ability to store renal anlagen would permit their distribution to sites for transplantation distant from the site of harvesting and would allow time to plan the transplant procedure. To determine whether renal anlagen can be stored in vitro prior to transplantation, we transplanted renal anlagen from E15 rat embryos into the omentum of non-immunosuppressed uni-nephrectomized (host) rats either directly or suspended in ice-cold UW preservation solution for 3 days prior to implantation. The size and extent of tissue differentiation preimplantation of E15 renal anlagen implanted directly are not
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distinguishable from the size and differentiation of renal anlagen preserved for 3 days. By 4 weeks posttransplantation, renal anlagen that had been preserved for 3 days had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts with normal structure had developed. At 12 weeks posttransplantation, inulin clearances of preserved renal anlagen are comparable to those of renal anlagen that had been implanted directly, consistent with the viability of preserved renal anlagen [41].
We transplanted renal anlagen from an E15 Lewis rat embryo across a concordant xenogeneic barrier into the peritoneum of 10-week-old C57Bl/6J mice [42]. Two weeks later, either no trace of the renal anlage could be found in mice or a yellowish piece of tissue, too small to embed, was observed in the omentum. In contrast, in mice that receive co-stimulation blocking agents hCTLA4Ig, anti-CD45RB, and anti CD154, the transplanted rat renal anlage undergoes differentiation and growth in situ (Figure 8.7) [42].
To gain insight into the origin of the vasculature (donor versus host) of renal anlagen transplanted in the omentum, using our rat/mouse model, we stained developing rat renal anlagen using mouse specific antibodies directed against the endothelial antigen CD31. The vasculature of the transplanted developed rat kidney transplanted into the mouse is largely of mouse origin, including glomerular capillary loops. In contrast, glomerular capillary loops in rat renal anlagen transplanted into rats do not stain for mouse CD31 (Figure 8.8) [42]. Using a highly disparate model (pig to rodent) we transplanted E28 pig renal anlagen (Figure 8.9a) consisting of undifferentiated stroma (s) branched ureteric bud (ub) and primitive developing nephrons (arrows) into the peritoneum of Lewis rats [43] or C57Bl/6J mice [48]. Five to seven weeks posttransplantation, no trace of the renal anlagen could be found in hosts that received no immunosuppression. In contrast, Figures 8.9b through i illustrate that by 7 weeks after transplantation in anti-CD45/tacrolimus-treated rats the E28 PRA have undergone differentiation and growth [43]. Shown in Figure 8.9b is a developed anlage in a rat’s omentum. Shown in Figure 8.9c is its developed ureter (u). Figure 8.9d is a mid-sagittal section that
Figure 8.7. (a) Photograph and (b through d) photomicrographs of H&E-stained sections of rat renal anlagen 2 weeks posttransplantation into a mouse omentum showing: (a) the developed renal anlage or metanephros (m); (b) a nephrogenic zone (NZ), cortex (c), and medulla (M); (c) developing nephron (arrowhead) in the NZ; and (d) developed glomerulus (g) deeper within the cortex. Magnifications shown for images a, b, and d (for c and d). (Reproduced with permission [42].)
Figure 8.8. Photomicrographs of stained sections of rat renal anlagen 2 weeks posttransplantation into a mouse omentum (a and b) or a rat omentum (c and d) stained using anti-mouse CD31. Arrowheads show positively staining glomeruli (a) and negatively staining glomeruli (c). Arrow shows positively staining glomerular capillary loops (b). Glomeruli (g) are labeled. Magnifications shown in a (a and c) and b (b and d). (Reproduced with permission [42].)
Xenotransplantation of Embryonic Kidneys
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Figure 8.9. Photomicrographs (a and e through i) and photographs (b through d) of pig renal anlagen. (a) E28 anlage (s, stroma; arrow, ureteric bud; arrowhead, developing nephron); (b through i) Pig renal anlagen 7 weeks posttransplantation in a rat omentum: (b) developed anlage in situ, (c) anlage after removal from the omentum (u, ureter), (d) mid-sagittal section of an anlage (arrowheads delineate lobules), (e) cortex with the nephrogenic zone delineated (arrows), and (f) Cortex with a glomerulus (g) proximal tubule (pt) and distal tubule (dt) labeled. Arrow delineates glomerular capillary loop. (g) A large artery (a) and vein (v). (h) Medulla with collecting ducts (cd) and thin loop of Henle (tlh) labeled. (i) Ureter. Magnifications are shown for a; b through d (in c); e, g, and i (in e); f (in f); and h (in h). (Reproduced with permission [43].)
delineates the lobular structure of developed pig renal anlage. Figure 8.9e shows a section of the cortex with the nephrogenic zone delineated (arrows). Figure 8.9f shows cortex with a glomerulus (g) proximal tubule (pt) and distal tubule (dt) labeled and a glomerular capillary loop (arrow). Figure 8.9g shows a large artery (a) and vein (v). Figure 8.9h shows medulla with collecting ducts (cd) and a thin loop of Henle (tlh), and Figure 8.9i, the ureter in cross section. The developed pig renal anlage is slightly larger in volume (diameter and weight) than a normal rat kidney.
Dekel et al. have successfully transplanted renal anlagen originating from pig embryos (of ages ranging from E20-21 to E27-28) beneath the renal capsule of immunodeficient mice. They found that most transplants from the E20-21 donors fail to develop or evolve into growths containing few glomeruli and tubules, but did find other differentiated derivatives such as blood vessels, cartilage, and bone. Furthermore, renal anlagen from E24-25 donors also contained nonrenal cell types and disorganized cell clusters. In contrast, the transplants originating from E27-28 pig embryos all exhibited significant growth and full differentiation into mature glomeruli and tubules [11]. Exactly as was the case in our studies of rat renal anlagen transplanted into mice [42], Dekel et al. found mouse CD31 expression in external vessels as well as developing glomeruli and small capillaries of pig renal anlage xenografts, consistent with a host origin for the vasculature of the developed renal anlage cellular transplants [11]. In addition, Dekel et al. transplanted adult pig kidney tissue or E27-28 pig renal anlagen beneath the renal capsule or onto the testicular fat of immunocompetent Balb/c mice. Some hosts were treated with CTLA4-Ig. Evaluation of adult or E27-28 embryonic tissues 2 weeks postimplantation into non-CTLA4-Igtreated hosts showed rejection of tissues. In CTLA4Ig-treated hosts, most E27-28 renal anlagen underwent growth and differentiation. In contrast, all adult kidney grafts had a disturbed morphology, necrotic tissue, and a high degree of lymphocyte infiltration. The authors interpreted these data as being consistent with an immune advantage of the developing precursor transplants over developed adult kidney transplants in fully immunocompetent hosts [11]. Finally, Dekel and co-workers implanted metanephroi from E70 human embryos intraperitoneally into NOD/SCID mice. Transplanted kidneys survived for more than 2 months posttransplantation, and became vascularized and differentiated into normal-appearing tubules and glomeruli [10]. Hybridization to cDNA arrays of RNA derived from normal human renal anlage at 8, 12, 16, or 20 weeks of gestation demonstrated a subset of 240 genes, the expressions of which changed substantially with time. The induced genes were classified as cell cycle regulators, transcription and growth factors, and signaling, transport, adhesion, and extracellular matrix molecules. Clustering analysis of global gene expression in transplants at 2, 6, and 10 weeks posttransplantation revealed a temporal profile of gene expression similar to that observed in the normal human
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kidneys during development, consistent with recapitulation of a renal developmental program [10]. Comparison of the expression profiles of developing metanephric transplants to a Wilms’ tumor specimen revealed no similarity. It was concluded that there is no threat of malignant transformation after transplantation of human kidney precursors [10].
Stem Cells For some clinical applications of tissue engineering, embryonic stem (ES) cells may represent a useful starting material [18]. A possible example is provided by studies of neuron repopulation. Mouse ES cells exposed to retinoic acid are induced to express neural associated genes, and they exhibit neurite outgrowth and can generate action potentials [5]. Neural-differentiated mouse ES cells transplanted into a rat spinal cord 9 days after injury survive; differentiate into astrocytes, oligodendrocytes, and neurons; and migrate from the edge of the lesion. Hind limbs of transplanted rats are able to support weight and exhibit partial coordination not found in hind limbs of control animals [30]. Human ES cells transplanted into immune-deficient mice develop into teratomas containing structures from all three germ layers, including glomeruli and renal tubules [35, 53]. However, growing kidneys from ES cells may be more problematic than their use for neuron repopulation. With the exception of its nerve supply, the kidney is a mesodermal derivative. Therefore, it is conceivable that each of its original cell types, plus the ureteric bud, could originate from a renal-specific descendant of an ES cell. Al-Awqati and Oliver have speculated that if it is present at all such a precursor might be located somewhere within the intermediate mesoderm at a given stage of embryogenesis [36]. Oliver et al. have shown that cultures of metanephric blastema can generate not only renal epithelial cells but cells that are positive for a-smooth muscle actin, indicating that they are myofibroblast precursors. They can also generate cells that express receptors for vascular endothelial growth factor, consistent with an endothelial lineage. Their observations suggest that the metanephric blastema may contain embryonic renal stem cells [36]. If renal-precursor cells exist and can be identified, it might be possible to induce the precursor cells to divide, and have the progenitors organize themselves into a kidney. However, to accomplish such a feat an orchestration
of gene expression would need to be externally imposed that almost certainly would be far more complex than that required to induce ES cells to develop into a single cell type or even multiple related cell types such as neurons and glia [5, 30]. The discovery of bone-marrow-derived stem cells that possess the ability to differentiate into multiple cell lineages has led to the hypothesis that such cells become incorporated into a number of organs as part of normal processes of cell turnover or organ repair. In support of bone marrow participating in kidney cell turnover, it was demonstrated that Y-chromosome-containing cells can be detected among renal interstitial and tubular epithelial cells of female mice into which male-derived bone marrow was transplanted [38, 39], and that green fluorescent protein (GFP)-labeled cells can be found among the mesangial cell population of kidneys in mice with GFP-tagged bone marrow [25]. Apropos of bone-marrow-derived cell participation in renal repair, Lin et al. [28] and Kale et al. [26] showed that in mice the cells from adult bone marrow are mobilized into the circulation by transient renal ischemia and home specifically to injured regions of the renal tubule. Loss of stem cells following bone marrow ablation resulted in more renal dysfunction postischemia, whereas stem cell infusion reversed this effect [28]. It was shown in male human recipients of kidneys from females that following an episode of acute tubular necrosis (ATN) 1% of renal tubules contained cells with Y chromosomes. In contrast, no Y-chromosomecontaining cells were seen in transplanted kidneys of similarly sex-mismatched males who did not develop ATN. This finding suggests that recipient derived cells do not routinely repopulate the transplanted kidney but may participate in repair processes [20]. The studies previously cited are consistent with a bone marrow source for renal stem cells. However, the results of these studies must be interpreted with caution. As noted by Poulsom, partial repopulation of the kidney with cells that have come to resemble their neighbors is not the same as showing that the organ has acquired new resident stem cells with functional competence as diverse and broad as that expected of an indigenous population [39]. Maeshima et al. have shown recently that mouse kidneys contain cells that retain a bromodeoxyuridine (BrdU) label for extended periods of time as a result of slow turnover. Such cells, termed label-retaining cells (LRCs), are believed to represent the stem cell compartment in a number of tissues but had not previously
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been identified in the kidney. During recovery after renal ischemic injury, LRCs in mouse kidneys underwent cell division and became positive for proliferating cell nuclear antigen (PCNA). In contrast, PCNA positive (but BrdU negative) tubular cells were rarely observed, suggesting that the cells proliferating during the process of tubular regeneration are derived from LRCs. At an early stage of regeneration, descendants of LRCs expressed a mesenchymal marker, but became positive for an epithelial marker after several cell divisions, consistent with proliferation and differentiation into epithelial cells during the process of repair postischemic injury [29]. Oliver et al. found that low-cycling BrdU-retaining cells were sparse in adult rodent kidneys except in the renal papilla [37]. During the repair phase following induction of transient renal ischemia in rats, the BrdU-retaining cells entered the cell cycle and quickly disappeared from the papilla. Single-cell clones of the papillary cells coexpressed mesenchymal and epithelial proteins and gave rise to myofibroblasts and cells that expressed neuronal markers consistent with a stem cell identity. Following isolation of the “papillary stem cells” and injection into the renal cortex, the cells incorporated into renal parenchyma [37]. If LRCs are renal stem cells and do participate in the process of recovery following renal ischemic injury, identification of agents that enhance differentiation of LRCs and their use as therapeutic modalities could provide a new approach to the treatment of renal disease [29, 37]. Brodsky et al. demonstrated that injection via the aorta of adult human endothelial cells into athymic nude rats subjected to renal ischemia resulted in a dramatic protection of the kidney against injury and dysfunction [7]. Morphological studies demonstrated the engraftment of injected human cells into the rat renal vasculature, and suggested that the infusion/engraftment ameliorated the endothelial dysfunction that occurred postischemia. Infusion of skeletal-muscle-derived stem cells from adult mice that had been differentiated in vitro along the endothelial lineage had a similar salutary effect on the course of acute ischemic injury in mice [4]. Advances in understanding the molecular biology of rodent renal development have led to the ability to culture the components of the developing rat kidney (the ureteric bud and metanephric blastema) in isolation from one another. Steer et al. have described a method for subculturing and propagating either component in
isolation [52]. Ureteric buds can be combined with freshly isolated metanephric blastema to form a large number of rat “neokidneys” derived from a single progenitor in vitro. It remains to be determined whether or not “neokidneys” will engraft posttransplantation. Nonetheless, the authors provide interesting speculations regarding potential advantages to “growing neokidneys” in preparation for subsequent transplantation into animal or human hosts. First, neokidneys could be grown from component parts. A colony of neokidneys derived from a single renal component could lead to a large supply of genetically identical renal tissue. Second, isolated mesenchyme or blastema could be transfected with constructs designed to regulate the growth characteristics of the neokidney or enhance the function of one or more cellular components as needed. Finally, it might be possible to create a chimeric structure using the ureteric bud as a scaffold that could be recombined with alloor xeno-derived nonrenal mesenchymal cells. Such cells could be engineered to differentiate into a renal phenotype when exposed to kidney-specific signals emanating from the scaffold [52].
Generation of Histocompatible Tissues Using Nuclear Transplantation/Therapeutic Cloning Lanza et al. have created bioengineered tissues from cardiac, skeletal, and renal cells cloned from adult bovine fibroblasts. They transplanted subcutaneously into an adult bovine host cultured dispersed kidney cells from the host’s E56 cloned embryo, seeded on collagencoated cylindrical polycarbonate membranes. Cloned and passaged kidney cells expressed renal-specific proteins in vitro, including synaptopodin, aquaporin-1, aquaporin 2, and Tamm-Horsfall protein. After expansion, the cells also produced both 1, 25(OH)D3 , and erythropoietin [27]. Straw-colored fluid was produced by “renal units” that had differentiated in situ into glomerulus-like and tubule-like structures and that had undergone vascularization. Chemical analysis of collected fluid suggested unidirectional secretion and concentration of urea nitrogen and creatinine. Cells within “renal units” produced synaptopodin, aquaporins-1 and 2, and Tamm-Horsfall protein. No rejection response was detected in hosts to the cloned renal cells [27].
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organogenesis delineated herein will assume a place as part of mainstream clinical practice.
The use of exogenous renal cells or groups of renal cells for transplantation and organogenesis confers an array of therapeutic advantages relative to the transplantation of whole kidneys [19, 33, 50]. Thus, (1) renal cells can be infused [7, 37] or implanted at optimal therapeutic locations, including natural (renal) sites [1, 10–13, 17, 24, 45, 51, 54–57], immunoprivileged sites [1, 12], or ectopic (nonrenal) sites [1, 10, 11, 22, 40–45, 47, 49]; (2) renal cells can be manipulated prior to transplantation to enhance their function or reduce their immunogenicity [4, 7, 52]; (3) renal cells can be banked and cryopreserved [41]; and (4) renal cells can be combined with different cell types in the same graft [29, 37] or with noncellular biomaterials [27]. Sources for cell replacement can be autologous [7, 10, 55, 57], allogeneic [4, 17, 24, 40, 41, 44, 45, 47, 51, 54], or xenogeneic [1, 10–12, 42, 43, 48, 49, 52]. Issues relating to the prospective removal of an organ from one individual and its placement in a second individual evoked a good deal of controversy among scientists, physicians, and ethicists when transplantation was in its infancy. Questions about how and when organs could be retrieved from humans who were in the process of dying and whether or not a living donor should be allowed to assume the risks associated with organ donation were actively debated. Today, we discuss how the very limited number of organs should be allocated [8]. The use of cells for transplantation and organogenesis raises questions at least as vexing as those that first arose in the context of organ transplantation. They include whether therapeutic cloning of animals might result in transplantable tissues [27], whether the use of tissues derived from human embryos [10–13] should be permitted, whether it is justifiable to use animals as a source of cells or tissues for transplantation into humans [8, 16], whether potential complications and risks that accompany the use of cells and tissues from various sources can ever be adequately conveyed to patients as part of informed consent, whether the public health is placed at risk by the use of cells or tissues derived from animals [8], and whether health care dollars are wisely spent to develop and implement these technologies. Today, the work described is at the cutting edge of biomedicine, and many therapeutic concepts it embraces are novel and controversial. No doubt, during the coming decades the tenets of renal cellular therapy and
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9 Strategies for Extracorporeal Devices for Kidney Failure Khajohn Tiranathanagul, M.D. (1 and 2) and H. David Humes, M.D. (1) (1) Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan (2) Division of Nephrology, Department of Medicine, Faculty of Medicine, Chulalongkorn University Hospital, Bangkok, Thailand
Introduction The numbers of patients who suffer from renal failure, either acute or chronic, are high. Acute renal failure (ARF) affects up to 30% of patients in intensive care units and approximately 5% of hospitalized patients overall [4]. Furthermore, this condition has a high mortality rate [68]. Chronic renal failure (CRF), or chronic kidney disease (CKD), is quite common as well. In the United States, over 8.3 million Americans have CKD of at least stage 3 (glomerular filtration rate less than 60 ml/min) [9]. Moreover, a significant number of them will develop end-stage renal disease (ESRD), which has a fatal natural history [9]. Fortunately, the kidney is unique among body organs in that it is the first organ for which substitutive therapy has been available for both acute and chronic organ failure. Human hemodialysis (HD), an extracorporeal renal replacement therapy, was first performed in 1926 [49]. The first successful human allograft kidney transplantation was performed in 1954 [71]. Finally, chronic peritoneal dialysis using the permanent indwelling catheter was initiated in 1968 [72]. Of these various treatments, HD is the therapy most frequently administered to ESRD patients in the majority of countries, including the United States [1]. However, expected survival times for transplant patients are two to three times higher than those of patients on dialysis [1], suggesting that the current dialytic approaches that use a purely artificial device to replace only the excretory function of the kidney result in continuing clinical deterioration. The problem is expected to increase in magnitude, because the population of patients with
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ESRD has been increasing faster than the rate of kidney transplants. The statistics for ARF are similarly grim, with no notable change in overall survival rates from the predialysis era. Moreover, ARF as a component of multiorgan dysfunction syndrome (MODS) has an especially poor prognosis, with survival rates ranging from only 10 to 30%. However, recent advances in cell therapy and tissue engineering have led to the development of bioartificial constructs that can approximate, and may eventually replicate completely, the functionality of the healthy kidney. The early development of these bioartificial kidney constructs has focused on their use in extracorporeal circuits, but it is expected that once the therapeutic benefits have been established and the fundamental technical difficulties addressed the natural evolution of the technology will lead to the development of an implantable bioartificial kidney.
Normal Kidney Function Understanding of kidney physiology and functionality is a prerequisite for the development of a successful bioartificial kidney, whether extracorporeal or implantable. The kidney has a number of essential roles in the body. Its major role is to maintain the constant extracellular environment that is essential for the appropriate functioning of cells. This is accomplished by excretion of metabolic waste products such as urea, creatinine, and uric acid, and by exclusively adjusting the excretion of water and electrolytes to balance with their intake and endogenous production. The ability of the kidney
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to individually regulate these excretions is largely a function of tubular reabsorption and secretion after glomerular filtration. In addition to its excretory role, the kidney is also responsible for the secretion of hormones that are critical in maintaining hemodynamics (renin, angiotensin II, prostaglandins, nitric oxide, endothelin, and bradykinin), red blood cell production (erythropoietin), and bone metabolism (1,25-dihydroxyvitamin D3 or calcitriol). The basic functional element of the kidney is the nephron. There are approximately one million nephrons in each human kidney. Each nephron comprises three major parts: the glomerulus, the tubule, and the collecting duct (Figure 9.1). The glomerulus consists of a tuft of small, highly permeable capillaries interposed between the afferent and efferent arterioles and located inside a hollow structure called the Bowman’s capsule. The Bowman’s capsule opens up into a series of tubules (proximal tubule, loop of Henle, and distal tubule) and then into the collecting duct, all of which are lined by a continuous layer of epithelial cells. The first step in the excretory process of the nephron is the generation of an ultrafiltrate (UF) of plasma across the glomerulus into the Bowman’s space. The glomerulus is designed to provide efficient ultrafiltration of as much as one-third of the plasma entering it, removing waste products of metabolism from the circulation but retaining important circulating components such as albumin. This high rate of ultrafiltration across the glomerular capillary results from hydraulic pressure generated by the heart and vascular tone of the preglomerular and postglomerular arterioles and from the high hydraulic permeability of the glomerular capillary wall. To achieve the required semipermeability
Bowman’s capsule
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Figure 9.1. Schematic of a nephron, the functional element of the kidney.
characteristics and extremely high ultrafiltration rates, the glomerular membrane evolved into a complex structure comprising a specialized fenestrated endothelium, a charged basement membrane, and highly differentiated epithelial cells called podocytes. Together, these components impart to the glomerular membrane its extraordinary characteristics, clearly unmatched by purely artificial membranes. The UF is modified in two ways: by reabsorption and by secretion while passing through the series of tubules and the collecting duct. Reabsorption refers to the removal of a substance or water from the UF, whereas secretion refers to the addition of a substance to the UF. The different tubular segments and the collecting duct make various contributions to these processes. Ultrafiltration by the glomerulus in normal human kidneys produces approximately 130 liters per day; daily urinary volume is roughly 2 liters. Thus, more than 98% of the glomerular UF is reabsorbed by the renal tubules. The bulk of reabsorption, 50 to 65%, occurs along the proximal tubule. Similar to glomerular filtration, fluid movement across the renal proximal tubule cell is governed by physical forces. Unlike the fluid transfer across the glomerular capillary wall, however, tubular fluid flux is principally driven by osmotic and oncotic pressures rather than hydraulic pressure. Renal proximal tubule fluid reabsorption is based on active Na+ transport, requiring the energy-dependent Na+ /K+ -ATPase located along the basolateral membrane of the renal tubule cell to promote a small degree of osmotic difference (2 to 3 mOsm/kg H2 O) across the renal tubule sufficient to drive isotonic fluid reabsorption due to the very high diffusive water permeability of the renal tubule cell membrane. Once across the renal proximal tubule cell, the transported fluid is taken up by the peritubular capillary bed because of the high oncotic pressure gradient that results from the high rate of protein-free filtrate formed in the proximate glomerular capillary bed. As can be appreciated, an elegant system has evolved in the nephron to filter and reabsorb large amounts of fluid in bulk to attain high rates of metabolic product excretion while maintaining regulatory salt and water balance. When functioning properly, all kidney components work together to ensure the removal of toxic compounds from plasma while retaining useful components (e.g., glucose and proteins), maintaining appropriate fluid volumes, controlling the levels of ions, and regulating the function of other organs in the body. It is also important to note that evolution ensured that the kidney fulfills
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its role even under stress, during conditions of high toxin load. As a result, only a small portion of the overall excretory capacity of the kidneys is required under normal conditions and for sustaining life. Accordingly, the approach to a tissue-engineered renal replacement construct becomes easier to entertain.
Current Status of Renal Support Even though healthy kidneys have reserve capacity, they still cannot maintain functionality when faced with significant injury. Renal failure can be classified by onset and reversibility into ARF (which is potentially reversible) and CRF (or CKD), which is caused by permanent damage to the kidney. ARF and CKD are different disease processes. Current therapies for these disorders are suboptimal. Most ARF is caused by ischemic damage resulting from shock during infection or major operative procedures and by toxins, including antibiotics and chemotherapeutic agents. The development of ARF in a hospitalized patient results in a five- to eightfold higher risk of death [68]. Current treatment with either intermittent or continuous extracorporeal renal replacement therapy (HD, hemofiltration, and hemodiafiltration) — with its small- and middle-molecule solute and fluid clearance function — reduces death from hyperkalemia, volume overload, and uremic complications. Although increasing doses of treatment from alternate-day to daily intermittent HD [66] and from standard to high-volume continuous venovenous hemofiltration (CVVH) [62] may improve survival, patients with ARF still have high mortality rates. The cause of death in these patients is usually the development of systemic inflammatory response syndrome (SIRS) — often secondary to bacterial infection or sepsis — with resulting cardiovascular collapse, ischemic damage to vital organs, and multi-organ failure. However, if the patient survives the episode of ARF the regenerative repair processes within the kidney can result in a return of kidney function in almost all patients. CKD resulting in ESRD, unlike ARF, is not a reversible process. Systemic diseases, most commonly diabetes and hypertension, result in progressive kidney damage and scar formation. Once the patient loses 90% of kidney function, chronic renal replacement therapy is required. Current treatment options include transplantation and dialysis (HD and peritoneal dialysis). Kidney transplantation is a remarkable clinical
achievement providing full kidney function to patients with ESRD. This approach, however, has risks of opportunistic infection and development of malignancies due to the requirement of long-term immunosuppressive medication. Unfortunately, transplantation is recognized as an impractical solution in most cases of ESRD due to the shortage of available organs for transplantation. In the United States, less than 10,000 kidney transplants are performed each year, with the current number of patients awaiting transplantation approaching 60,000. Presently, HD remains the primary treatment modality for the management of patients with ESRD. It is used for greater than 90% of dialysis-dependent ESRD patients in the United States, with the remaining patients being treated with peritoneal dialysis [1]. Although HD has dramatically changed the prognosis for ESRD, the procedure does not offer complete renal replacement therapy. Unlike transplantation, HD provides only small uremic toxin and excess fluid removal, and usually only on an intermittent basis. Indeed, HD does not replace the homeostatic, regulatory, metabolic, and endocrine functions of the kidney. Current data from the Hemodialysis (HEMO) study in HD [14] and the ADEMEX (ADEquacy of PD in MEXico) study in peritoneal dialysis [55] fail to reveal a survival benefit from increased clearance of small solutes. These studies suggest that conventional diffusion-based therapies may be limited in their ability to influence outcome. Convective-based therapies that remove not only low but also middle molecular weight solutes (particularly in the range of 1,000 to 12,000 daltons) are currently used worldwide [70]. The simplest procedure is high-flux hemodialysis (HFHD), which uses the biocompatible high-flux hemodialyzer and is characterized by high permeability for water and low and middle molecular weight molecules. It removes more middle molecular weight solutes, especially b2 -microglobulin (b2 M, 11,818 daltons), than conventional HD [56]. Although several short-term studies have demonstrated improved patient outcomes [42], the recently concluded HEMO study showed no overall benefit in reduced mortality from the use of high-flux membranes during a 5-year follow-up when compared with conventional HD using low-flux biocompatible membranes [14]. Convective therapies include hemofiltration (HF) — a purely convective technique — and hemodiafiltration (HDF), a hybrid technique incorporating elements of both diffusion and convection. HDF seems to be more popular than HF and is employed via several methodologies to achieve the effect of combining diffusion and
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convection within a single treatment module comprising on-line HDF [26], convective-controlled double highflux HDF (CC-DHF) [58], and internal HDF [63]. Clearance of small molecular weight solutes is comparable between HFHD and HDF, but clearance of middle molecules in HDF — including b2 M, which can increase at higher substitution volumes — far exceeds that in HFHD [41]. Pre-dialysis b2 M levels in HDF were significantly reduced compared to those in low-flux dialysis after 3 months of treatment, and this difference was maintained in a prospective study over a 2-year period without definite short-term clinical benefit [82]. Indeed, a historical prospective study of 6,440 patients showed clinical benefit from HDF or HF over HD in terms of significantly lower relative risk of surgery for carpal tunnel syndrome, the most common presentation of b2 M amyloidosis [43]. However, improvement in mortality could not be documented in either short-term prospective randomized trials or large historical prospective studies [43, 70, 82]. Thus, these convective therapies are still suboptimal for ESRD patients.
Cell Therapy and Tissue Engineering Approach to Renal Replacement Therapy Cell therapy is a new and exciting therapeutic approach to acute and chronic diseases, including those of the kidney. Interest in this approach stems in part from the growing appreciation that most disease processes are not due to the lack of a single protein but result from alterations in the complex interactions of a variety of cell products. Cell therapy and tissue engineering depend on the isolation and growth in vitro of specific cells and on the capacity of these cells to replace in vivo important differentiated processes or organ functions deranged or lost in various disease states. One of the most challenging tasks in cell therapy is replacement of the functions of a complex organ such as the kidney. However, there are several promising cell-based approaches to renal failure. One approach is based on the concept of direct administration of cells into areas of prior tissue injury. In an animal study, transplanted renal subcapsular nephrons (metanephroi) became integrated into host kidneys, but no evidence of a successful connection between collecting systems was found [61, 83]. This technique has been applied to other organs. For example, skeletal myocytes have been injected into post-myocardial infarction scar tissue [23], and neuronal cells have been injected into the brain of patients
with Parkinson’s disease [21]. However, safety concerns have been raised because these studies resulted in high rates of cardiac arrhythmias [48] and severe uncontrollable dyskinetic activity [13], respectively. Another possible cell therapeutic alternative, ongoing in animal study, is the transplantation of metanephroi into adult hosts to grow new kidneys in situ [24], including the prospect of xenotransplantation of metanephroi into humans. A third promising approach, still in its infancy, is the tissue engineering of new kidney grown from either embryonic [39] or adult stem cells [54]. Stem cells are characterized by a high capacity for selfrenewal and the ability to differentiate under defined conditions into specialized cells that develop into the structure and functional components of a physiological organ system. For example, neural differentiated mouse embryonic stem (ES) cells have been successfully transplanted into rat spinal cord [47]. Unlike spinal cord, however, the kidney is a remarkably complex organ that orchestrates the performance of a variety of processes by many different types of cells. The development of a therapy to replace these many renal processes is clearly a formidable task. An approach that integrates cell-based therapy with dialysis technology has resulted in the engineering of an extracorporeal bioartificial kidney [35] that is currently undergoing clinical trial. This approach presently may be the best strategy for attaining comprehensive renal replacement therapy and could eventually lead to the development of an implantable bioartificial kidney.
Engineering a Bioartificial Kidney REQUIREMENTS A bioartificial kidney designed for full renal replacement would replace all essential kidney functions, including excretory (filtration but also reabsorption and secretion), metabolic, and endocrinologic functions. Filtration is the task of the glomerulus, but most of the kidney’s work is accomplished by the tubule and its interstitium — especially the renal proximal tubule — which receives the filtrate from the glomerulus and performs the bulk of the reclamation of salt, water, glucose, small proteins, amino acids, glutathione, and other substances. Furthermore, the renal proximal tubule also has other critical metabolic, endocrine, and immunologic roles, including excretion of acid as ammonia and hydroxylation of 25-OH-Vit D3 .
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All current dialysis techniques mainly substitute for only the filtration function of the failed kidney but do not address the reabsorptive, metabolic, endocrine, and immune roles that may contribute to the survival rates of renal failure patients. At present, ESRD is recognized as a disease associated with chronic inflammation [2, 38, 86] that predisposes patients to cardiovascular diseases and acute infectious complications, the two most common causes of death despite adequate HD (Figure 9.2). Clinically, the chronic inflammatory state in ESRD patients — which is independent of dialyzer membrane activation and clearance — is represented by elevated levels of C-reactive protein (CRP)
RPT cell loss
Chronic proinflammation
Accelerated atherosclerosis, malnutrition
Early mortality
(a)
RPT cell necrosis
Diminished host defense
Peripheral vasodilation
Excessive proinflammation
Sepsis
Hypotension
Multiorgan failure
Death
Susceptibility to infection
(b) Figure 9.2. (a) Possible intervention with RPT cell therapy to interrupt the chronic pro-inflammatory state of patients with end-stage renal disease (ESRD). The pro-inflammatory state of ESRD is a prominent cause of the accelerated atherosclerosis process, which leads to early death in this patient population. (b) Schematic representation of the progression of events in acute renal failure (ARF). The intervention of renal proximal tubule (RPT) cell therapy may interrupt this pathophysiologic spiral that leads to death.
and proinflammatory cytokines, equivalent to a chronic proinflammatory state [27]. All of these parameters are associated with enhanced mortality in ESRD patients and might have many causes, including the clearance of glutathione (a key antioxidant), negative nitrogen balance and energy loss in the clearance of peptides and amino acids, loss of tubular cell function in oxidative deamination, gluconeogenesis, and loss of cytokine and hormone metabolic activity in the kidney [19]. Likewise, the propensity of patients with ARF to develop SIRS and sepsis suggests that renal function, specifically renal tubular cell function secondary to acute tubular necrosis (ATN), plays a critical immunomodulatory role under stress states (Figure 9.2). The roles of the renal tubular cells in glutathione reclamation, glutathione peroxidase synthesis, other middle molecule metabolism, and activation of vitamin D3 (with its important immunoregulatory functions) are well-recognized pathways to maintain important tissue integrity and host defense under stress conditions [28]. A less recognized role of mammalian renal proximal tubule cells is their potential immunoregulatory function. They are antigen-presenting cells [65, 78, 84] that have co-stimulatory molecules [80] and that synthesize and process a variety of inflammatory cytokines [40, 60, 77, 85]. Accordingly, a bioartificial kidney ideally would have not only a filtration component but also components that perform the functions of the tubules (proximal tubule, loop of Henle, distal tubule, and collecting duct). In comparison with current treatments based on artificial membranes, substantial clinical benefits may be achieved even without incorporating the functionality of the loop of Henle and the distal nephron. This notion is supported by clinical experience with patients who suffer moderate renal insufficiency but are able to maintain adequate fluid and electrolyte homeostasis even though their kidneys cannot provide the fine control of water and salt homeostasis associated with the healthy kidney. Thus, a bioartificial kidney requires at least a filtration component and a proximal tubule component to replace kidney function and improve the prognosis of patients with renal failure.
HEMOFILTER Filtration by the glomerulus is the first step in the excretory process in normal kidney. The glomerulus removes solutes from the blood by convective transport. This
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function is practically replaced in conventional HD, where solute removal is driven by a concentration gradient of the solute across the membranes and is therefore predominantly a diffusive process. However, solute transfer also occurs convectively by a process of ultrafiltration of water and solutes across the membrane. This convective transport is independent of the concentration gradient and depends predominantly on the hydraulic pressure gradient across the membrane [8]. To successfully replace the filtration function of the nephron, the artificial hemofilter must be able to replicate the ability of the kidney glomerulus to efficiently remove blood toxins. This requirement can be satisfied if the artificial membrane permeability is high enough to permit pressure-driven ultrafiltration fluxes instead of relying on diffusion alone. Ultrafiltration removes water and effectively clears solutes that are within the molecular weight cutoff of the membrane. In contrast, clearance by diffusion depends on concentration gradients and solute mobility. As a result, diffusion is ineffective for the removal of large and/or highly dilute solutes. Both diffusive and convective processes occur during traditional HD, but most solute movement is achieved via diffusion. Recognition of the inherent limitations in diffusive transport has led to the development of membranes with high hydraulic permeability and therapeutic extracorporeal modalities for renal failure, including HDF and HF in which blood cleansing is achieved via convective HF rather than diffusive HD. Removal of uremic toxins, predominantly by the convective process, has several distinct advantages because it initiates the glomerular process of toxin removal with increased clearance of higher molecular weight solutes and removal of all solutes (up to its molecular weight cutoff) at the same rate. Distinct differences are apparent between diffusive and convective transport across a semipermeable membrane. The clearance of a molecule by diffusion is negatively correlated with the size of the molecule. In contrast, clearance of a substance by convection is dependent on size up to a certain molecular weight. The bulk movement of water carries passable solutes along with it in approximately the same concentration as in the fluid. Although HF can be achieved successfully with purely artificial membranes, new research in biomaterials and tissue engineering holds promise for significant future improvements. Modern membranes are used reliably all over the world in countless HD, HF, and HDF procedures. Typically, membranes are used in the form
of hollow-fiber modules and are based on cellulose or synthetic polymers (including polysulfone, polyacrylonitrile, and polyamide) used as such or modified with a variety of agents [10, 25, 79]. Current technology introduces considerable advances in a number of areas, including biocompatibility and consistency of pore size. However, there is much room for improvement in the performance of current membranes. Improvements in membrane biocompatibility have reduced inflammatory reactions, but off-the-shelf polymeric materials still induce a cascade of biochemical events leading to blood coagulation, thrombus formation, and frequently complement activation and inflammation. Thus, anticoagulation (most commonly the systemic administration of heparin) is required to solve these problems. The use of anticoagulants, however, may lead to additional complications such as hemorrhage, and thus researchers are trying to improve anticoagulation therapy by finding alternative antithrombotic agents (such as hirudin [16]) and methods of administration, such as direct incorporation onto the polymeric membrane [69]. The incorporation of a confluent endothelial monolayer on a porous membrane or other promising materials, such as silicon nanoengineered ultrafiltration membranes [18], may provide a solution to the membrane/blood compatibility problems and form the basis for the development of a bioartificial hemofilter (BAHF). In addition to improved biocompatibility, a bioartificial membrane is inherently better suited to replicate glomerular filtration and thus provide a complete functional replacement for the glomerulus. Owing to the relative clinical success of HD treatments and the complexity inherent in tissue engineering for kidney replacement, the literature is devoid of promising efforts to develop a BAHF. Nevertheless, the feasibility of the BAHF concept is supported by many existing studies. For example, the endothelial seeding of synthetic vascular grafts may inhibit long-term deposition of platelets and formation of thrombus [67] and improve long-term patency after implantation [36]. In addition, the antithrombotic properties of endothelialized grafts may be further improved by endothelial cells (ECs) engineered to secrete anticoagulation factors [7]. Accordingly, ECs engineered to express an anticoagulant factor could be utilized to coat vascular grafts and stents [11, 81] and semipermeable membranes and thus improve long-term performance in vivo. The same approaches can be extended to improve the patency of endothelialized HF membranes.
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Although a hemofilter would ideally contain ECs isolated from the glomerulus [22], financial and technical considerations limit the potential application of this autologous cell source for the endothelialization of the hemofilter membrane. Alternatively, autologous ECs can be isolated from peripheral blood vessels [3] or derived from progenitor ECs isolated from bone marrow [46] and then induced toward specific morphogenic changes, including the formation of fenestrae [15]. It is thus evident that all fundamentals are in place for the development of a hybrid hemofilter comprising a semipermeable inanimate membrane that provides the desired filtration characteristics and a confluent endothelial monolayer that imparts superior blood compatibility. It may also be possible to develop a BAHF in which living cells and their extracellular matrix (ECM) provide filtration functionality mimicking the glomerulus. Even though the protocols required for this step are not yet fully established, continuous advances in our understanding of the in vitro and in vivo interactions between different glomerular cell types — as well as between cells and ECM or growth factors — suggest that such a development is forthcoming. Pore size is another critical issue in hemofilter design. Conventional polymeric membranes are typically cast or spun from polymers in solution, resulting in a dispersion of pore sizes and geometries in a single fiber or sheet that causes incomplete molecular weight cutoff in convective transport. New techniques using a nanocontrol spinning procedure could help solve this problem by producing high-flux membranes in which the porosity of the inner layer is finely controlled at the nanoscale level, resulting in an increased number of pores while the spectrum of pore diameters is narrowed and concentrated near the desired values [64]. However, polymeric membranes are basically thick and require high hydraulic pressure gradients to produce UF when compared to the glomerulus. Several materials and hemofilters fabricated with the aid of modern nanotechnology, including a silicon nanoengineered ultrafiltration membrane [18] and a human nephron filter [51], attempt to approximate glomerular function in terms of constant pore size in a thin membrane. Solutions to these problems could further the development of a wearable or implantable bioartificial kidney. Conventional membranes remove solutes only on the basis of molecular size, whereas the glomerulus also employs charge selectivity by the basement membrane. This nonselective nature of conventional membranes is another limitation to overcome. One possible solution to
this problem is the growth of basement membrane cells on the hemofilter membrane. Another approach is the design of “smart” membranes that have selective transport characteristics and that contain a predetermined number and size of nonpassive nanoengineered pores that have specific interactions with solutes and solvents at the atomic level [51]. We recently successfully fabricated an implantable BAHF to initiate new tissue formation with a capillary bed surrounding synthetic hollow fibers without EC lining. UF draining into an artificial bladder in a rat with protein permselective properties was produced from this implant device [75]. Purely artificial hollow fiber high-flux membranes have been successfully employed in the hemofiltration component in both continuous and intermittent extracorporeal renal replacement therapies (convective therapies), as described previously. Although research into the ideal BAHF is ongoing, commercially available high-flux hemofilters have been used in conjunction with a bioartificial renal tubule assist device (RAD) to create an extracorporeal bioartificial kidney.
BIOARTIFICIAL RENAL TUBULE The efficiency of reabsorption, which depends on natural physical forces governing fluid movement across biologic as well as synthetic membranes, requires specialized epithelial cells to perform vectorial solute transport. A population of cells residing in the adult mammalian kidney has retained the capacity to proliferate and morphogenically differentiate into tubule structures in vitro [33] and can be used as the key cellular element of a tissue-engineered renal tubule device. Implementation of any device based on cell therapy, including a tissue-engineered renal tubular device, requires a steady and predictable supply of tissues from which cells may be isolated and cultured. Currently, these cells must be procured through the harvest of animal or human tissue. Until stem cells can be isolated and induced to differentiate into organ-specific cell types, the supply of cells available for cell therapy will be constrained. The bioartificial renal tubule can be readily conceived as a combination of living cells supported by polymeric substrata, using epithelial progenitor cells cultured on water- and solute-permeable membranes
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the majority of the filtrate — may be sufficient to replace required tubular function and sustain fluid electrolyte balance in a patient with ESRD.
Implantable Bioartificial Kidney
Figure 9.3. Schematic of a tissue-engineered renal tubule. Renal epithelial cells from a confluent monolayer along the inner surface of a polysulfone hollow fiber with pre-adhered matrix molecules.
seeded with various biometric materials so that expression of differentiated vectorial transport as well as metabolic, endocrine, and immunologic function is attained (Figure 9.3). With appropriate membranes and biomatrices, immunoprotection of cultured progenitor cells has been achieved concurrent with long-term functional performance as long as conditions support tubule cell viability. This bioartificial tubule has been shown to transport salt and water effectively along osmotic and oncotic gradients [44]. The bioartificial proximal tubule satisfies the major requirement of reabsorbing a large volume of filtrate to maintain salt and water balance within the body. The need for additional tubule segments to replace other nephronal functions, such as the loop of Henle to perform more refined homeostatic elements of the kidney (including urine concentration or dilution), may not be necessary. Patients with moderate renal insufficiency lose the ability to finely regulate salt and water homeostasis because they are unable to concentrate or dilute, yet they are able to maintain reasonable fluid and electrolyte homeostasis due to redundant physiologic compensation via other mechanisms. Thus, a bioartificial proximal tubule — which reabsorbs isoosmotically
The development of a bioartificial filtration device and a bioartificial tubule processing unit leads to the possibility of an implantable bioartificial kidney consisting of the filtration device followed in series by the tubule unit. The filtrate formed by the device would flow directly into a tubule unit lined with a confluent monolayer of renal tubule cells for regulated transport function. The tubule unit should maintain viability, because metabolic substrates and low-molecular-weight growth factors are delivered to the tubule cells from the ultrafiltration unit [50]. Furthermore, immunoprotection of cells grown within the filter membrane — which might be hollow fibers or a new material such as silicon nanoengineered ultrafiltration membrane — is achievable due to the impenetrance of immunologically competent cells through the filter [52]. Therefore, rejection of transplanted cells will not occur. This device could be used either extracorporeally or implanted within a patient. The specific implant site for a bioartificial kidney will depend on the final configuration of both filtration and tubule units. As presently conceived, the BAHF can be formulated by one of two approaches: initiation of new tissue formation with a capillary bed surrounding synthetic hollow fibers or an EC-lined bioartificial filter membrane. Either construct is connected to an arteriovenous circuit using the common iliac artery and vein, similar to the surgical connection in renal transplantation. The filtrate is connected in series to the proximal tubule unit, with the reabsorbate transported back into the systematic circulation. The processed filtrate exiting the tubule unit drains to a proximate ureter and is excreted as urine via the recipient’s own urinary collecting system. Although the ultimate goal of this approach is to construct a fully implantable bioartificial kidney, the pathway to achieve this goal will likely consist of a series of intermediate devices providing both clinical therapeutic benefit and substantial experience in the use of more elementary tissue-engineered devices [28]. The initial stage in this developmental process, currently in clinical evaluation, is an extracorporeal RAD designed to optimize current HF approaches.
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Extracorporeal Bioartificial Kidney BIOARTIFICIAL RENAL TUBULE ASSIST DEVICE (RAD) Replacement of the multivariate tubule functions of the kidney cannot be achieved with inanimate membrane devices, as has been accomplished with the renal ultrafiltration process, but requires the use of the naturally evolved biologic membranes of the renal tubular epithelium. In this regard, the tissue engineering of a bioartificial renal tubule as a cell therapy device to replace this missing component can be conceived as a combination of living cells supported on appropriate synthetic scaffolds [50]. Because the scaffolding material provides the required physical support for the tubule cells, it should not degrade considerably over the life of the bioartificial construct. Furthermore, it should permit the unhindered transport of solutes reclaimed by the tubule cells on one side to the filtered blood circulating at the other side, provide a large surface area to facilitate this mass transport, be compatible with blood, and not leach cytotoxic products into the circulation. Therefore, a bioartificial tubule can be constructed utilizing renal tubule progenitor cells [31, 33] cultured on semipermeable polymeric hollow-fiber membranes on which an extracellular matrix has been layered to enhance the attachment and growth of the epithelial cells [74]. These hollow-fiber synthetic membranes not only provide the architectural scaffold for these cells but also allow for differentiated vectorial transport and metabolic and endocrine activity and provide immunoprotection, as has been observed in the long-term implantation of the bioartificial pancreas in a xenogeneic host [52]. With appropriate membranes and biomatrices, immunoprotection of cultured progenitor cells can be achieved concurrent with long-term functional performance as long as conditions support tubule cell viability [50]. The technical feasibility of an epithelial cell system derived from cells grown as a monolayer along the luminal surface of a single polymeric hollow fiber, called a bioreactor, has been achieved [44] (Figure 9.4). Critical to the success of this cell therapeutic strategy is the isolation and growth in vitro of proximal tubular cells. These cells, obtained from adult tissue, have stemcell-like characteristics [59]. Alternatively, embryonic stem cells exposed to the appropriate environmental cues in vitro could provide a reliable cell source (Figure 9.5). Whereas studies aimed at reproducibly
Cells
Extracellular matrix
Hollow fiber
Figure 9.4. Electron micrograph of a tissue-engineered bioartificial renal tubule. The nucleus (N) is indicated. Black arrows delineate apical microvilli (a differentiated morphologic characteristic of proximal tubule cells), and the white arrow identifies the tight junctional complex of a transporting epithelium.
guiding the differentiation of adult or embryonic stem cells into renal tubule cells are ongoing, a method to isolate and grow renal proximal tubule progenitor cells from adult mammalian kidneys has been shown [31, 33]. This research was prompted by clinical and experimental observations suggesting that renal proximal tubule progenitor cells must exist, as tubule cells have the ability to regenerate after severe nephrotoxic or ischemic injury to form a fully functional and differentiated epithelium.
CHARACTERIZATION OF A SINGLE HOLLOW-FIBER BIOREACTOR Cellular attachment, stability, and confluence on the interior lumen of the bioreactor are of paramount importance. To promote attachment of the cells, the luminal surface of the polysulfone membrane was coated with ProNectin-L [53]. Laminin and collagen type IV, key components of the tubular basement membrane, also provide an effective biomatrix for cell attachment
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(a)
Primary culture and expansion of differentiated somatic cells Dissection and purification
(b)
Adult/embryonic stem cells
Committed progenitors
1. Growth factors 2. Extracellular matrix molecules
Differentiated somatic cells
1. Growth factors 2. Extracellular matrix molecules
Figure 9.5. (a) A methodology for the purification and expansion of primary cells from adult kidney exists. Primary cells are retrieved from postmortem kidneys through a regimen that includes enzymatic digestion, sieving, and centrifugation. Cells are expanded in vitro prior to seeding into a bioartificial renal tubule assist device (RAD). Progenitor and stem cells have been purified from other tissue sources, including muscle and skin. (b) Alternative methodologies for generation of fully functional differentiated somatic cells are currently being investigated by stem cell researchers. The working hypothesis is that adult or embryonic stem cells can differentiate into somatic cells if placed into the appropriate chemical and physical environment. A major challenge of this effort is to reproduce the complex temporal, spatial, and signaling events that occur during development.
and growth. After seeding of the hollow fiber with tubule cells, the hollow fiber was perfused with culture media. Because newly seeded cells need time to attach, perfusion was initially performed via diffusion from the exterior through the polysulfone membrane — and after cell attachment convective flow through the interior of the fiber was initiated. A graduated increase in flow (and, thus, shear forces) was used to condition the cells and minimize cellular detachment. Studies demonstrated that confluence was reached in 7 to 10 days. After 14 days in culture the hollow fiber bioreactor was assessed for cellular confluency and viability. Light microscopy of fixed sections showed evidence of a confluent monolayer formed on the inside of the hollow fiber [34]. Experiments were undertaken to verify the confluency of the monolayer of cultured cells. C14 -labeled inulin, a compound freely filtered by the glomerulus and by dialysis membranes but not absorbed nor secreted by the tubule, was perfused through the lumen of the
bioreactor. The amount of radioactivity in the luminal fluid and in the extraluminal fluid was compared to verify minimal inulin transport across the monolayer, with less than 2% leak across the monolayer [44].
CHARACTERIZATION OF THE RAD The single hollow fiber system has subsequently been scaled up into an extracorporeal RAD utilizing a standard high-flux HF cartridge containing renal tubule cells grown along the inner surface of the fibers. In vitro experiments have tested transport and metabolic functions of the RAD utilizing porcine renal proximal tubule progenitor cells grown intraluminally within these cartridges with membrane surface areas of 97 cm2 to 0.7 m2 , resulting in a device containing up to 108 to 109 cells [34]. Porcine cells were used because the pig is currently considered the best source of organs for both
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human xenotransplantation and immunoisolation cell therapy due to its anatomic and physiologic similarities to human tissue and the relative ease of breeding pigs in large numbers. The non-biodegradability and pore size of the hollow fibers allow the membranes to act as both scaffolds for the cells and as an immunoprotective barrier. Studies of the RAD have demonstrated that the confluent monolayer cells exhibited morphological characteristics (e.g., apical microvilli, endocytic vesicles, and tight junctional complexes) typical for differentiated tubule epithelia, as well as differentiated active transport properties, differentiated metabolic activities, and important endocrine processes [34]. In particular, these in vitro studies demonstrated the ability of the RAD to actively transport sodium, glucose, bicarbonate, and organic anions, such as PAH. The RAD also reabsorbed significant amounts of water in response to oncotic and osmotic pressure gradients. Vectorial transport of sodium and water across the monolayers was assessed by perfusion of the lumen and the extracapillary space (ECS) with calibrated flows of culture media and measurement of timed collections of media from each flow. The RAD was tested to see if transport could be increased by creating an oncotic driving force across the monolayer, and if that increase could be abrogated by ouabain (a specific inhibitor of Na+ /K+ -ATPase), the enzyme responsible for the bulk of sodium transport across the luminal surface of the cell. Results suggested that the transport was an active process, rather than passive flow governed by Starling forces across a semipermeable membrane. To increase the oncotic pressure, bovine serum albumin (BSA) was added to the ECS perfusate, and then ouabain was also added to the ECS perfusate. The data are summarized in Table 9.1. As expected, the transport was active and mediated by Na+ /K+ -ATPase [34]. The metabolic characteristics of the cultured proximal tubule cells — the transport of glucose, bicarbonate, and
glutathione — were explored and the data expressed in terms of fractional reabsorption accomplished by the RAD. For each molecule, fractional excretion was measured in the absence or presence of a known inhibitor of an enzyme essential for the reabsorption. In the case of glucose, the compound phlorizin was used; for bicarbonate, a carbonic anhydrase inhibitor (acetazolamide) was used; and for glutathione, an inhibitor of g -glutamyltranspeptidase (acivicin) was used. The data are summarized in Table 9.2. In each case, there was evidence of active transport and specific inhibition [34]. In addition, renal proximal tubule cells cultured in the RAD or in culture plates maintained their ability to synthesize and excrete ammonia (a function critical for the maintenance of acid-base homeostasis in the body), produce 1,25-(OH)2 -Vit D3 (the active form of vitamin D), and through metabolic degradation and transport of its constituent amino acids remove glutathione from the perfusate. It is thus evident that the RAD has the ability to replicate the major differentiated transport, metabolic, and secretory functions performed by the healthy renal proximal tubule. Success in preclinical studies of the RAD using porcine cells led to human clinical trials using human cells. Accordingly, biocomparability studies have been completed of the metabolic, transport, and endocrine functionality of RADs containing either human proximal tubule cells (HPTCs) [32] or porcine proximal tubule cells (PPTCs). Human cells were obtained from transplant organs determined to be unsuitable for transplantation. Metabolic assessment included ammonia and glutathione metabolism. HPTCs produced 48.4 ± 10.4 mg ammonia/106 cells/24 hours. Ammonia production rates were pH responsive in both cell types. Glutathione metabolism in HPTCs and PPTCs was 1.11 ± 0.07 and 0.86 ± 0.21 ng/cm2 /hour, respectively.
TABLE 9.2 Solute transport.a
TABLE 9.1 Fluid transport.a Ouabain Experiments
Absolute Reabsorption (ml/30 min)
Inulin Leak (percent)
Baseline Albumin Albumin and ouabain
1.05 ± 0.40 4.87 ± 0.66 1.73 ± 0.43
3.25 ± 0.21 3.10 ± 0.21 2.98 ± 0.19
Molecule
Condition
Fractional Reabsorption (%)
Glucose
Baseline Phlorizin Baseline Acetazolamide Baseline Acivicin
25.0 ± 1.2 9.2 ± 0.7 21.9 ± 1.8 4.1 ± 0.5 44 ± 2 25 ± 4
Bicarbonate Glutathione
aAdapted from reference [31].
aAdapted from reference [34].
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Glutathione metabolism was inhibited in both cell types by the addition of acivicin, a specific inhibitor of glutathione metabolism. Reabsorption was evaluated with glucose transport. HPTCs and PPTCs reabsorbed 3.6 ± 0.4 and 2.6 ± 0.5 mg glucose/106 cells/24 hours, respectively. Glucose transport in both cell types was inhibited by the addition of phlorizin, a specific inhibitor of glucose reabsorption. Endocrine function was assessed by 1,25-(OH)2 -Vit D3 production. With the addition of parathyroid hormone, HPTCs and PPTCs converted 4.9 ± 0.6 and 7.8 ± 0.5 pg/106 cells/24 hours, respectively. Cytokine production was also measured. IL-8 secretion was 8.54 ± 2.91 and 4.77 ± 1.69 ng/106 cells/ 24 hours for HPTCs and PPTCs, respectively, when cells were stimulated with endotoxin. These data suggest that HPTCs and PPTCs are biocomparable with respect to transport, metabolic, and endocrine functions in cell culture [32].
viability and functionality, with fewer than 105 cells lost during a 24-hour perfusion period. Treatment with the cell RADs maintained BUN and plasma creatinine levels similar to those of acellular control RADs. Plasma + HCO− 3 , Pi , and K levels were more similar to normal values in cell RAD-exposed animals than in animals receiving sham treatment. RADs with cells reabsorbed 40 to 50% of UF volume. Furthermore, active reabsorption of K+ , HCO− 3 , and glucose and excretion of ammonia were accomplished only in cell RADs. Glutathione processing by RADs with cells was also demonstrated, with greater than 50% glutathione removal from UF presented to the device. Finally, uremic animals receiving cell therapy attained normal 1,25-(OH)2 -Vit D3 levels, whereas sham treatment resulted in a further decline from the already low plasma levels. Thus, these experiments clearly showed that the bioartificial kidney successfully replaces many kidney functions in acutely uremic dogs [30].
EXTRACORPOREAL BIOARTIFICIAL KIDNEY CIRCUIT
BIOARTIFICIAL KIDNEY IN ARF
The extracorporeal bioartificial kidney consists of a filtration device (a conventional high-flux hemofilter) followed in series by the tubule RAD unit [17, 30, 32]. Specifically, blood that is pumped out of a patient enters the fibers of the hemofilter, where UF is formed and delivered into the fibers of the tubule lumens within the RAD downstream to the hemofilter. Processed UF exiting the RAD is collected and discarded as “urine.” The filtered blood exiting the hemofilter enters the RAD through the extracapillary space port and disperses among the fibers of the device. Upon exiting the RAD, the processed blood is returned to the patient’s body via a third pump (Figure 9.6). The RAD is oriented horizontally and kept in a 37◦ C temperature-controlled environment to ensure optimal functionality of the cells. Studies have shown that the bioartificial kidney using a RAD consisting of either porcine or human cells replaces filtration, transport, metabolic, and endocrine functions of the kidney in acutely uremic dogs [30, 32]. Twenty-four hours after bilateral nephrectomies, the dogs developed symptoms of ARF with elevated blood urea nitrogen (BUN) and plasma creatinine levels. The dogs were treated with HF and either a RAD cartridge containing tubule cells or a sham control cartridge containing no cells. Treatment was administered daily for either 7 or 9 hours for three successive days or for 24 hours continuously. The cell RADs maintained
Current therapy for ischemic or toxic ARF or ATN is predominantly supportive in nature. Uremia is treated with either intermittent HD or continuous HF, treatments that have had substantial impact on this disease process over the last 25 years. However, patients with ATN still have an exceedingly high mortality rate of greater than 50%, especially in sepsis [68], despite maintenance of normal electrolyte balance and improvement in the uremic state. The high mortality is due to the propensity of these patients to develop SIRS followed by MODS [5]. The sequential failure of organ systems apparently unrelated to the site of the initial insult has been correlated with altered plasma cytokine levels observed in sepsis [12, 45, 57]. Because ARF secondary to ischemic and/or nephrotoxic insults arises from ATN, predominantly to renal proximal tubule cells, replacement of the functions of these cells during the episode of ATN and in conjunction with HF would provide almost full renal replacement therapy. The addition of metabolic activity such as ammoniagenesis and glutathione reclamation, endocrine activity such as activation of vitamin D3 (low levels of which appear to correlate with high mortality rates in hospitalized patients) [73], immunoregulatory support, and cytokine homeostasis may provide additional physiologic replacement activities to change the current natural history of this disease process [28].
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Strategies for Extracorporeal Devices for Kidney Failure Extracapillary space Fiber wall Proximal tubule cells RAD cartridge Heat exchanger
Pump 2
Ultrafiltrate reservoir
Luminal space
Pressure monitor
Processed ultrafiltrate (urine)
Heat exchanger Post RAD blood
Post hemofilter blood (into RAD ECS) Ultrafiltrate (into RAD luminal space)
Hemofilter
Pump 1
Replacement fluid
Pump 3
Venous blood
Figure 9.6. Schema of the extracorporeal circuit for the bioartificial kidney, consisting of a synthetic hemofilter and the RAD cartridge.
Recent experiments have investigated whether treatment with the bioartificial kidney would alter the course of ARF with sepsis in animal models. Mongrel dogs that underwent surgical nephrectomy-induced ARF were treated with CVVH and either a RAD containing cells or an identically prepared sham cartridge. After 4 hours of therapy, 2 mg/kg intravenous endotoxin was infused over 1 hour to simulate gram-negative septic shock. Mean peak levels of an anti-inflammatory cytokine, IL-10, and mean arterial pressures were found to be significantly higher in cell-treated animals [17]. To further assess the effect of the bioartificial kidney in ARF with bacterial sepsis, dogs were nephrectomized
and 48 hours later administered intraperitoneally with 3 × 1011 E. coli cells/kg body weight [20]. Immediately after bacteria administration, animals were placed in a CVVH circuit with either a RAD with cells or a sham RAD without cells. Cell RAD treatment maintained better cardiovascular performance, as determined by mean arterial blood pressure and cardiac output, for longer periods than sham RAD therapy. All sham RAD animals expired within 2 to 10 hours after bacteria administration, whereas all cell RAD-treated animals survived greater than 10 hours. Plasma cytokine levels in the bacteremic animals were assessed. IL-10 levels were significantly elevated in the cell RAD group compared
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with the control group. A significant correlation between the rise in plasma IL-10 levels and the decline in mean arterial pressure was observed. The cell RAD maintained renal metabolic activity throughout the septic period [20]. In another study, pigs with normal kidney function were administered intraperitoneally with 3.0 × 1011 E. coli cells/kg body weight. One hour later, animals were placed in a CVVH circuit containing either a RAD with cells or a sham RAD without cells. All animals developed ARF with anuria within 2 to 4 h after bacteria administration. Cell RAD treatment maintained better cardiovascular performance, as determined by cardiac output and renal blood flow, for longer periods than sham RAD therapy. Consistently, the cell RAD group survived longer than the controls (10 ± 2 h versus 5 ± 1 hours, respectively). RAD treatment was associated with significantly lower plasma circulating levels of IL-6 and interferon (INF)-g . These data demonstrate that septic shock results in early ARF and that RAD treatment in a bioartificial kidney circuit improves cardiovascular performance associated with changes in cytokine profiles and confers a significant survival advantage [29].
PHASE I/II CLINICAL EXPERIENCE WITH A HUMAN RENAL TUBULE ASSIST DEVICE
and endocrinologic conversion of 25-(OH)-Vit D3 to 1,25-(OH)2 -Vit D3 by the RAD tubule cells were demonstrated. All 10 patients were critically ill with ARF and MODS, with predicted hospital mortality rates between 80 and 95%. One patient expired within 12 hours after RAD treatment due to his family’s request to withdraw ventilatory life support. Another patient expired after a surgical catastrophe (toxic megacolon) required discontinuation of RAD treatment after only 12 hours. Of the remaining eight patients, six survived past 28 days with renal function recovery. The other two patients died from nonrecoverable complications unrelated to RAD therapy and ARF, including fungal pericarditis and vancomycin-resistant enterococcus septicemia in one patient and ischemic colitis with bowel perforations in the other patient. Plasma cytokine levels suggest that RAD therapy produces dynamic and individualized responses in patients depending on their unique pathophysiologic conditions. For the subset of patients who had excessive pro-inflammatory levels, RAD treatment resulted in significant declines in granulocyte-colony stimulating factor (G-CSF), IL-6 (a proinflammatory cytokine), IL-10 (an anti-inflammatory cytokine), and especially IL-6/IL-10 ratio, suggesting a greater decline in IL-6 relative to IL-10 level and the less proinflammatory state. The phase I/II results were encouraging and led to an FDA-approved, randomized, controlled, openlabel phase II investigation at 10 clinical sites to assess the safety and early efficacy of this cell therapy approach. This study involved 58 ICU patients with dialysis-dependent ARF. Forty patients were randomized to receive both CVVH and RAD therapy, and 18 received only CVVH. Interim analysis found that the 28-day all-cause mortality in patients receiving RAD therapy was 34%, significantly lower than the 56% rate in CVVH-only patients, suggesting a treatment advantage with RAD therapy. A large, multicenter clinical trial is planned to confirm the efficacy of this therapy [76].
With these encouraging preclinical data, the FDA approved an Investigational New Drug (IND) application to study the RAD containing human cells in patients with ATN receiving CVVH. Human kidney cells were isolated from kidneys donated for cadaveric transplantation but found unsuitable for such purpose because of anatomic or fibrotic defects. The initial results in the first 10 treated patients demonstrated that this experimental treatment can be delivered safely under study protocol guidelines in this critically ill patient population for up to 24 hours when used in conjunction with CVVH [35]. These data also indicated that the RAD maintains and exhibits viability, durability, and functionality in this ex vivo clinical setting. Cardiovascular stability of the patients was maintained, and increased native renal function as determined by eleBIOARTIFICIAL KIDNEY IN ESRD vated urine outputs temporally correlated with RAD treatment, a finding that requires additional study. The A bioartificial kidney for long-term use in ESRD, similar isolated and expanded human cells also demonstrated to short-term use in ARF, would integrate tubular cell differentiated metabolic and endocrinologic activity therapy and the filtration function of a hemofilter. As in this ex vivo treatment. Glutathione degradation noted previously, ESRD patients on conventional renal 182
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replacement therapy are at high risk for cardiovascu- Summary lar and infectious diseases. A recent clinical trial failed to show survival benefit from increased doses of HD The kidney contributes in many important ways to body above what is now standard care [14]. This suggests homeostasis. Even though its main role is excretion, the that there are important metabolic derangements not kidney’s metabolic, endocrine, and immune functions adequately treated with conventional dialytic treatment. are also important. Advances in current renal replaceData from the survival of renal transplant recipients — ment therapies have improved the prognosis of both which far exceed those from the survival of age-, sex-, acute and chronic renal failure patients. However, these and risk-matched controls awaiting transplant — also therapies still do not confer the same survival advansuggest that there is some metabolic function provided tage as a functional kidney. Renal failure continues to by the kidney that transcends this organ’s filtration func- cause a heavy burden of morbidity and mortality owing tion. Patients with ESRD display elevated levels of CRP to the fact that current treatment modalities replace only (an emerging clinical marker) and pro-inflammatory part of the multiple functions of the kidney. Cell therapy cytokines, including interleukin (IL)-1, IL-6, and tumor and tissue engineering are two exciting fields in translanecrosis factor (TNF) [2, 38, 86]. All of these param- tional medicine that offer hope of better reproducing a eters are associated with enhanced mortality in ESRD functional kidney in renal failure patients. Accordingly, patients. Specifically, IL-6 has been identified as a sin- a bioartificial kidney consisting of a RAD containing gle predictive factor closely correlated with mortality in cultured renal proximal tubule cells has been successHD patients [2]. Although all ESRD patients could con- fully engineered. The device has demonstrated renal ceivably benefit from a bioartificial kidney, patients in differentiated activities in vitro and ex vivo in several the inflammatory stage who display elevated levels of large animal studies and has been shown to confer a certain markers of chronic inflammation (most notably survival advantage in models of bacterial sepsis. This IL-6 and CRP) would likely benefit most and will bioartificial kidney is currently undergoing a phase II be the target population for clinical study in the near clinical trial in ARF. The therapeutic modality employed future. in this device could represent a significant step in efforts For the ESRD patient population, however, there are to bridge the gap in functionality between wholly artifiobvious limitations in using an extracorporeal RAD con- cial renal replacement therapy and normal native kidney nected to an HF circuit. Ideally, a bioartificial kidney functions. suitable for long-term use in ESRD patients would be capable of performing continuously, like the native kidney, to reduce risks from fluctuations in volume status, Disclosures electrolytes, and solute concentrations and to maintain acid-base and uremic toxin regulation. As in ARF, cell HDH is a shareholder of RenaMed Biologics, Inc., and therapy currently can be administered only intermit- Innovative BioTherapies, Inc., biotechnology spin-off tently. These additional functions require the eventual companies of the University of Michigan. design and manufacture of a compact implantable or wearable bioartificial kidney. The advent of silicon bulk and surface micromachining offers hope for the Acknowledgements development of such a device [18]. These technologies allow the fabrication of pores, beams, gears, and This work was made possible by a fellowship awarded pressure sensors and the patterned deposition of cells to KT by the International Society of Nephrology. in engineered micro- and nanoenvironments. Surface micromachining has been used to provide capillaryREFERENCES like conduits for blood flow in hepatocyte tissue cultures, and micropatterning has been used to control 1. USRDS 2004 annual data report. Am J Kidney Dis 45:8–280; cell growth and differentiation [6, 37]. We look for2005. ward to the possible use of these emerging technologies 2. Bologa, R. M., Levine, D. M., Parker, T. S., Cheigh, J. S., in the manufacture of a bioartificial kidney for uniSerur, D., Stenzel, K. H., Rubin, A. L. Interleukin-6 preversal optimal renal replacement therapy for ESRD dicts hypoalbuminemia, hypocholesterolemia, and mortality in patients. hemodialysis patients. Am J Kidney Dis 32:107–114; 1998. 183
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10 Transplants for Hemophilia Andrew Gómez-Vargas (1) and Gonzalo Hortelano (1 and 2) (1) Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada (2) Research and Development, Canadian Blood Services
Hemophilia Congenital hemophilia is a bleeding disorder characterized by a defective clotting factor VIII (hemophilia A) or factor IX (hemophilia B). The genes for FVIII and FIX are both located on the X chromosome. Men carry one copy of the X chromosome, whereas women carry 2 copies — both of which must bear a defective mutation for severe hemophilia to manifest clinically. As a result, hemophilia affects mostly males: 1 in 5,000 males in the case of hemophilia A [42], and 1 in 30,000 males for hemophilia B [74]. A reduced functional activity of these clotting factors can result in life-threatening bleeding episodes, particularly in the event of intracraneal hemorrhages [133]. Continuous bleeding episodes lead to arthopathy at a young age, often requiring joint replacement. Current treatment of hemophilia is based on regular infusion of plasma derived from recombinant factor VIII or IX protein [38]. The safety of plasma-derived products came into question during the tainted blood scandal of the 1980s, when a large proportion of severe hemophiliacs in North America and Western Europe were infected with HIV and hepatitis through infusions prepared from tainted blood. Although there is now a minimal risk of viral transmission by protein replacement due to the viral inactivation processes currently used, the risk of transmission of novel blood-borne pathogens cannot be completely ruled out. Furthermore, there are potential side effects, such as thrombotic complications, associated with contaminants [48]. Recombinant proteins are attractive alternatives to plasma-derived products, although their high cost is a significant hurdle limiting widespread use. Therefore, an improved therapy for
Cellular Transplantation: From Laboratory to Clinic
hemophilia is desirable. Gene therapy may represent such an alternative. Hemophilia was recognized very early as an ideal candidate for gene therapy strategies. Its clinical manifestations are the consequence of the lack of functional activity of a single protein that circulates in the blood. Furthermore, years of clinical practice indicate that increasing the circulating levels of the deficient clotting factor to over 1% of the normal level can significantly improve the phenotype of the disease from severe to moderate [111]. Importantly, a number of different cells are able to secrete functional FVIII and FIX. Finally, there are excellent murine and canine models for both hemophilia A and B that mimic the human condition and are very suitable for testing new therapies. Viral vectors are being developed as promising gene delivery vehicles to treat genetic diseases such as hemophilia. Several in vivo gene therapy approaches based on the direct administration of adenovirus, retrovirus, and adeno-associated (AAV) viral vectors have been evaluated in preclinical and clinical trials of both hemophilia A and B. Although the results are encouraging, there are concerns related to possible inadvertent germ-line transfer. In addition, recent unanticipated immune responses to AAV vectors in a hemophilia gene therapy trial highlight the complexity of gene delivery [134]. Transplantation of cells producing clotting factors would obviate the need for repeated infusions, as well as avoid the risk of transfusion-related viral infections such as HIV [33]. This prompted the rapid development of ex vivo gene therapy approaches based on the transplantation of recombinant cells [120], a strategy that has been pursued since the dawn of gene therapy as a discipline
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15 years ago. Given the unique capabilities of many cells to graft in different tissues, cell transplantation might be a suitable source of therapeutic products for patients suffering from a metabolic disease. Cell transplants can be engineered by introducing genes of clinical relevance. Once transplanted, grafted cells might provide a sustained supply of therapeutic product to the host, becoming a less expensive treatment than the current therapy based on repeated protein replacement (Figure 10.1). The main limitation of cell therapy is the risk of rejection. The high cost associated with the engineering of autologous cells makes the more cost-effective strategies of allogeneic and even xenogeneic transplantation of immunoprotected cells more attractive. A number of transplantation protocols based on the use of genetically Virus receptors
engineered cells for the treatment of hemophilia have been reported. Recombinant transplanted cells have some important advantages over in vivo gene therapy approaches, such as vector persistence in grafted cells [36] and long-term survival of transplanted myoblasts [97]. Although experimental results have generated more questions than they have answered, the process has been very helpful in focusing attention on the key prerequisite of identifying suitable cell types for transplantation, establishing efficient gene transfer and transgene expression technologies, and understanding the risks associated with the procedure of cell transplantation. Although hFVIII is thought to be produced primarily in the liver, and not to be synthesized by many cells [15], FVIII mRNA is found in many tissues [126]. Indeed, a number of engineered cell types have produced FVIII.
Engineering of target cell Receptor-mediated endocytosis nucleus mRNA
cytoplasm
genome endosome DNA-polymer conjugate +/− ligand
Transplantation of cells
Naked DNA DNA liposomes
Transplanted cell cytoplasm
nucleus
secretion
mRNA genome
transport
protein FVIII
FIX
blood Figure 10.1. Transplantation of genetically engineered cells.
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Likewise, different cells can produce hFIX. Functionally active FIX must undergo substantial posttranslational modifications. Thus, as long as a cell is able to perform the required posttranslational modifications functional FIX can be transported to the bloodstream. The cells that have demonstrated the capability of synthesizing hFIX with biological activity are hepatocytes [131], skin fibroblasts [104], myoblasts [56, 57], embryonic stem cells [71], and endothelial cells [130]. The various cell types employed in transplants for hemophilia are discussed in material following.
Stem Cell Transplantation in Hemophilia Cellular transplantation, in the form of bone marrow transplants, is an established clinical procedure and the gold standard against which other cell transplantation therapies should be measured. Currently there are approximately 45,000 patients worldwide treated every year by HSC transplantation, a number that has been increasing during the past decade [108]. Regenerative medicine and cell therapy have become a major focus of modern therapeutic approaches for the treatment of a wide variety of inborn, acquired, and degenerative diseases. Data collected from scientific evidence have demonstrated the therapeutic potential of stem cells in tissue repair and maintenance of a missing organ, tissue, or cellular function [58]. However, the limited number of hematopoietic stem cells (HSCs) that can be collected narrows this practice, given that engraftment success is closely related to the number of cells transplanted [123]. Current research focuses on the task of replacing the function of a deficient gene due to inborn defects such as hemophilia. The ability of HSCs to permanently reconstitute the hematopoietic and the immune system after transplantation makes these cells a desirable tool for gene therapy approaches. Therefore, HSCs are considered a suitable target cell population in gene therapy for genetic disorders susceptible to bone marrow transplantation. However, because most HSCs are quiescent it is clear that effective strategies for expansion of HSCs are needed to improve the therapeutic potential of this approach. HSCs are particularly attractive in the gene therapy field because they can be easily harvested from donors, can be effectively genetically engineered in vitro, and are transplanted into patients by wellestablished transplantation methods currently used in the clinic [3]. In addition, hematopoietic stem cells have
an extended lifespan and a huge repopulation potential [18]. However, stem cells are undifferentiated, have not become a mature cell type, and do not express the repertoire of proteins produced by a differentiated cell [114]. Therefore, successful stem cell transplants for the treatment of hemophilia have to rely on the ability of stem cells to survive for a long time in the appropriate conditions while expressing the therapeutic product. Stem cell transplantation is scientifically and technically a difficult challenge. Manipulation of fetal and adult stem cells in vitro can lead to the development of abnormal karyotypes [21], raising concerns about their tumorogenic potential. Together with the mixed pattern of gene expression, HSCs may contribute to latent deleterious effects seen in the host [121]. Another obstacle for successful gene delivery into HSCs is that these cells are very susceptible to de novo methylation in immature cells such as embryonic stem (ES) cells and embryonal carcinoma (EC) cells, resulting in inhibition and/or silencing of the transgene expression in vivo [69]. In addition, recent experiments have shown the importance of the immune response in mediating stem cell graft rejection, demonstrating that cell transplantation for hemophilia is not a simple task [49]. Adult bone marrow cells can be further divided into undifferentiated parenchymal cells, generally referred to as stem cells, and partially differentiated mesenchymal stromal cells (which play a supportive role for the former).
ADULT PARENCHYMAL BONE MARROW CELLS In 1945, a civilian population was exposed to lethal doses of irradiation. It soon became apparent that as a consequence of total body irradiation blood components can be regenerated by bone marrow cells [61]. This finding led to the treatment of patients afflicted with cancer using bone marrow transplants (BMTs) [68]. The same approach can be applied to inherited diseases such as hemophilia. A hypothetical successful treatment for hemophilia using cell transplants is based on the successful engraftment of cells in the recipient tissues. The first study involving murine hematopoietic stem cells expressing factor VIII was published in 1992 [53]. The authors evaluated the potential of bone marrow cells to express human factor VIII (hFVIII) and the therapeutic potential of these cells through their transplantation in lethally irradiated mice. Richard Morgan used a similar approach in 1998 [24]. In this study, the authors
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reported induction of immune tolerance against hFVIII in 50% of the hemophilia A mice previously immunized with hFVIII. Even though it is a very important advance in the field of gene therapy to achieve immune tolerance against the transgene product, hFVIII protein could not be detected in the plasma of the animals that received the graft in vivo. Furthermore, myeloablative conditions were required to achieve long-term survival of the engineered engraftment, and this is not an acceptable treatment modality for hemophilic patients.
ADULT MESENCHYMAL BONE MARROW CELLS To overcome the limitations previously described as a potential treatment for hemophilia, bone-marrowderived mesenchymal stem cells have been evaluated as an alternative. These spindle-shaped cells are part of the bone-marrow stroma forming a reticular matrix that supports hematopoiesis and contributes to bone formation [119]. Mesenchymal cells have basically the same advantages of nonstromal bone marrow cells, are easily obtainable, and can be expanded, engineered in vitro, and reinfused into the patient without evidence of side effects [94]. The use of these cells obviates the myeloablative treatment of the host, thus reducing the risks associated with this treatment [16]. Chuah et al. [16] showed hFVIII therapeutic levels in the plasma of recipient mice of 13 ± 4 ng/ml (or 13% of physiological levels) infused with transfected bone-marrow-derived stromal cells, but these levels subsequently declined to basal levels by 3 weeks postinjection due to promoter inactivation. The study also concluded that 10% of the stromal cells were engrafted in the spleen and persisted for at least 4 months after transplantation in the absence of myeloablative conditioning.
EMBRYONIC STEM CELLS Tissue-specific stem cells have also been proposed for this therapy. However, there are problems associated with this approach, including difficulties in isolation and limited quantities of these cells within the tissue. Embryonic stem cells (ESCs) may solve these issues [41]. ESCs have the potential to be differentiated into specific lineage pathways and therefore be used as cell replacement therapy for many innate and acquired diseases. Despite these advantages, the field of transplantation of ESCs is still full of hurdles such as maintaining
transgene expression in vivo after transplantation and differentiation [59, 78], allograft rejection [22], and the increased risk of tumurogenesis [112]. However, the therapeutic potential of ESCs is substantial if the cells would successfully engraft. For instance, in the hepatic parenchyma ESCs can be effective as gene replacement vectors in correcting liver-based genetic disorders such as hemophilia A and B. Cells with hepatocyte phenotypes have been obtained from ESCs in vitro [45]. However, achieving complete functional activity has proven very difficult [25, 113]. A study conducted by Frelinger demonstrated the engraftment, persistence, and functional activity of allogeneic ESCs transplanted in the liver of hemophilia B mice [26]. This proof-ofconcept study highlights the principle that ESCs are likely to have clinical relevance for replacement of hepatic function, in that up to 500 ng of FIX (or 10% of physiological levels) levels of circulating hFIX were detected — which in some animals lasted at least 115 days after transplantation.
Human Keratinocytes Keratinocytes are epithelial cells that form the superficial layer of the skin. Keratinocytes are particularly suited for transplantation given their great accessibility throughout the body surface [9]. Transplantation of skin keratinocytes has been very successful and is a well-established procedure in the clinical field for the regeneration of the epidermis in burn patients [23]. The grafting process of keratinocytes can be monitored noninvasively, and the graft can be removed should it become necessary. Some of these cells retain the ability of self-renewal throughout the adult life. The potential of the process for transgene expression has been evaluated with various therapeutic products, such as human growth hormone [5]. However, the capacity of keratinocytes to transport an active transgene product to the bloodstream is limited. In 1993, Watt et al. demonstrated that keratinocytes transduced with a retrovirus vector containing the hFIX gene under the control of the retroviral LTR promoter were able to induce the production of functional clotting factor [37]. Moreover, nude hemophilia B mice transplanted with these cells achieved a modest (∼ 0.1% of normal physiological levels) amount of hFIX in the systemic circulation for a week after skin transplantation, suggesting the potential of keratinocyte transplantation as an attractive therapy for hemophilia. On the other
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hand, if this type of graft strategy is to become a reality in the treatment of hemophilia long-term expression of FIX is clearly essential [31]. To achieve sustained transgene delivery, expression vectors that do not use retroviral promoters (such as housekeeping genes) should be considered. Nonetheless, an important hurdle in the transplantation of recombinant keratinocytes is the generation of an immune response specific against the transgene product [109]. An attempt to overcome these aforementioned difficulties was explored using transplantation of recombinant keratinocytes enclosed in a silicone transplantation chamber to improve the survival of the genetically modified cells. Furthermore, hFIX expression was under the control of the cytomegalovirus (CMV) promoter and the human papilloma virus-16 keratinocyte-specific enhancers [91, 125]. Graft survival and expression of hFIX persisted for a year after transplantation, suggesting that long-term expression may be achieved with this methodology. Even though hFIX levels were sustained, transgene expression was not therapeutically representative. However, it is important to highlight that the strategy to increase hFIX expression [90] and the use of silicone chambers to avoid graft rejection [93] were successful. Devices such as immunoisolation chambers that form a physical barrier between the graft and the host immune system have been used to improve xenoimplantation [87]. In this context, graft rejection is mediated by independent histocompatibility complex mechanisms such as inflammatory cytokines and natural killer (NK) cells [17]. The silicone chambers partially protect the implanted graft against NK activity, but do not prevent cytokine diffusion inside the device [99]. Although silicone is a biocompatible material, it still attracts immunoglobulins and complement fractions with lytic activity [122]. However, it must be noted that skin grafts are acutely rejected despite intensive and long-term immunosuppressive therapy. Skin Langerhans (dendritic) cells play a crucial role in the process of graft rejection because they are involved in the processing and initiation of this reaction. Therefore, subcutaneous implantation is a particularly antigenic location, given the natural environment of the skin immune system. Transplantation of keratinocytes transduced by the vector containing hFIX gene was successful in nude mice, and human factor IX was detected in plasma at very low levels for 4 to 5 weeks, the duration of graft survival [65]. However, it is recognized that the main obstacles facing this type of application are (1) that
transgene expression by keratinocytes is very limited and (2) that the transport of the transgene product from the skin to circulation is not very efficient [28]. Nonetheless, therapeutic levels of FVIII (up to 20% of normal) were achieved in double mutant mice (FVIII-deficient and immunodeficient) that received a graft from a transgenic mouse expressing high levels of hFVIII under the control of a skin promoter [27], suggesting the feasibility of this approach.
Fibroblasts Fibroblasts are cells derived from connective tissue that secrete an extracellular matrix rich in collagen among other macromolecules. The observation that recombinant retroviruses can stably integrate a transgene into the genome of transduced fibroblasts enhanced interest in using these cells as a vector for gene therapy [66]. Previously it has been shown that recombinant fibroblasts can serve as effective biological pumps for the supply of missing enzymes and growth factors in vivo [85]. Different studies have disclosed the necessary conditions for using genetically modified fibroblasts as a strategy for protein replacement therapy in vivo. These include the presence of extracellular matrix that fibroblasts require for in vivo survival. For better engraftment results, this extracellular matrix should be matured in vitro before transplantation. The viability of transplanted fibroblasts is enhanced if the matrix is partially sequestered behind a coating of host fibroblasts, and if immortalized cells are used [76]. The previous observations support the notion that prolonged expression of transgenes by transplanted fibroblasts can be achieved in vivo, indicating their therapeutic potential in gene therapy applications [34]. In 1993, Hoeben et al. demonstrated that transfected fibroblasts can produce biologically active hFVIII; and after transplantation in immunodeficient animals the implanted fibroblasts survived in vivo for 2 months [54]. Circulating antigen in animals was not detectable, but recovered cells continued to produce hFVIII in vitro. In agreement with these results, other studies have shown the feasibility of genetically modified fibroblasts in producing hFIX [68, 75]. However, the delivery of hFVIII is hampered by low expression levels from the cells due to the instability of FVIII mRNA [64]. To avoid this obstacle, the DNA coding for the B domain not required for the biological function of the FVIII protein was removed from the transgene.
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Allotransplants of recombinant fibroblasts producing B-domain-deleted hFVIII (BDDFVIII) were implanted into the spleen [135]. Intraspleenic transplantation avoided graft immune rejection and achieved hFVIII secretion into the bloodstream, obtaining encouraging results suggesting the feasibility of this approach [137]. A clinical trial was conducted in hemophilia A patients using genetically modified autologous fibroblasts to evaluate the production level and duration of circulating therapeutic hFVIII [102]. A total of either 1 × 106 or 4 × 106 autologous recombinant fibroblasts was enclosed in a collagen matrix and transplanted intraperitoneally with laparoscopic guidance. The results showed that the implantation procedure was safe, with no evidence of development of an inhibitory immune response against the transgene product. The investigators reported an increase of the circulating levels of hFVIII beyond background in four out of six patients, with a partial and transient correction of the disease phenotype — which highlights the potential clinical efficacy of this strategy. However, this system raises concerns regarding the fate of recombinant transplanted fibroblasts in hemophilic patients and the fact that the technology has to be tailored for each patient individually. Although significant and functional hFVIII expression can be difficult to achieve in many cells, reports using genetically modified fibroblasts with hFIX are many. Transduction of skin fibroblasts using a retroviral vector in order to express hFIX, and their posterior transplantation in vivo, reached significant levels of circulating transgene product for 8 weeks in experimental rodents in 1993 [136]. Similar studies were made in rabbits achieving continuous expression of hFIX for 10 months after implantation of autologous fibroblasts engineered with a retroviral vector, and a different preclinical trial showed transgene persistence for almost 2 years [14]. Brauker et al. conducted a study in athymic rodents implanted with recombinant human fibroblasts secreting hFIX encapsulated in biocompatible chambers [8]. The transgene product persisted for 140 days, and the cell viability was high. In contrast, in animals implanted with nonencapsulated cells the graft did not survive for more than 4 days [8]. This experiment demonstrated that using an immunoisolation device can avoid graft rejection, improve implant survival, and protect the transgene expression against a specific immune response. We shall discuss this topic later. An important finding was the importance of maintaining long-term cell viability. This study raised the issue of overproducing transgene expression. A significant increase in
FIX production by recombinant fibroblasts was met with a decrease in the biological activity of the cells, and hence therapeutic potential. Thus, efforts to maximize FIX production must ensure that the cellular machinery is not saturated in order to adequately perform the required posttranslational modifications of the produced FIX. A clinical trial performed in China suggested the benefits of fibroblast transplantation in two hemophilic B patients that received genetically modified autologous skin fibroblasts transduced with a retroviral vector expressing hFIX. Both patients had a small increase in the circulating levels of the transgene product that lasted for 1 year. At the same time, they decreased the amount of recombinant hFIX infused over the same period of time [77, 95]. This result highlights the potential benefits of this approach in offering a simple, safe, and effective strategy for the treatment of hemophilia B.
Hepatocytes Over the last 30 years, research in experimental animals and several clinical trials have brought us to the threshold of hepatocyte transplantation for the treatment of acquired liver diseases and many inherited metabolic disorders [70]. Very small and uncontrolled series of human hepatocyte transplantation have been conducted in the treatment of individual cases. Although the data are sparse and inconclusive, the feasibility and safety of this procedure have been demonstrated [55]. Liver transplantation can cure both forms of hemophilia, demonstrating that the liver is a major site of hFVIII and hFIX synthesis [63, 126]. In preclinical animal studies, hepatocytes injected intraperitoneally, intraportally, or into the spleen settle down in the diseased liver. Therefore, genetic modification of hepatocytes that can be transplanted becomes a potential treatment for many inborn metabolic deficiencies, including hemophilia [98]. Among the prerequisites for this cellbased therapeutic strategy, there must be a sufficient amount of highly differentiated hepatocytes. Hence, a well-established in vitro cell-culture technique is necessary to yield a reproducible number of proliferating hepatocytes, and to preserve the physiological cell function and transgene expression in cells that have been genetically modified [19]. Transplantation of isolated hepatocytes into the liver via the portal vein of patients with Criglar-Najjar syndrome and glycogen storage disease has produced
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encouraging results [32, 83]. However, this approach is highly limited by the total number of cells that can be transplanted and the serious complications generated as a consequence of the procedure [105]. This strategy might be successful for the treatment of hemophilia because these cells are very susceptible to be modified genetically and some studies have shown engraftment survival for at least 1 week in different disease models, such as acute liver failure and glycogen storage disease type 1a. Hence, to be efficient a greater number of hepatocytes have to engraft and the cells need to survive for a longer period of time. Recently, Ohashi et al. reported the survival of syngeneic hepatocytes for 140 days posttransplantation by embedding the hepatocytes in an extracellular matrix extracted from Engelbreth-Holm-Swarm cells and implanted under the kidney capsule [86]. Hemophilic A mice transplanted with murine hepatocytes presented a sustained increase of FVIII activity up to 13% of the physiologic levels that lasted 5 weeks after implantation with concomitant hepatocyte survival. This proof-of-concept study should encourage further experimentation into using genetically modified hepatocytes for transplantation for the treatment of hemophilia and other different diseases affecting the liver.
Myocytes The implantation of cultured myogenic cells (myoblast transplantation) is an experimental strategy that is being explored for the potential treatment of myopathies. Myoblast transplantation may have two main advantages: recombinant myocytes can act as a vehicle of delivery for molecules with potential treatment for particular diseases such as hemophilia or genetic myopathies, and because they can maintain stable cell proliferation they can establish a steady pool of transgene expression [117]. Important conditions for successful myoblast transplantation include an appropriate immunosuppression regimen, and a careful distribution of sufficient myoblast cells into the recipient tissues [106]. Different reports have shown the importance of the immune response to mediate myoblast graft rejection in a very short period of time after implantation [96]. To determine the efficacy of myoblast transplantation it is necessary to evaluate many critical parameters such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression [97].
The outcome of many studies of myoblast transplantation made in experimental animals has not been ideal because they have involved transplantation of established myogenic cell lines or primary muscle cultures. These cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells that will not fuse to host fibers [6, 43]. Furthermore, for both cell populations immune suppression of the host has been necessary for long-term retention of transplanted cells [96] because these cells undergo rapid and massive death after injection into the host [52]. The speed with which the transplanted myoblasts die after implantation suggests the involvement of nonspecific mechanisms used by the host immune system [11]. This phenomenon even occurs when myoblasts are injected into a fully histocompatible host [4], in contrast to the long-term survival of transplanted myoblasts derived from histocompatible whole or sliced muscle grafts [29]. Some observations have concluded that even the conditions of myoblast isolation and cell culture procedures are relevant for the survival of transplanted cells [7]. Factors such as media components, serum added to the culture media, and proteolytic enzymes alter donor myoblasts antigenicity [60]. Recent observations have demonstrated that exposing whole muscle to culture conditions have detrimental consequences for the survival of this tissue after transplantation [107]. This unexpected and lethal effect of tissue culture reagents on cells that have been cultured in vitro and subsequently transplanted has major implications for the grafting of many cell types. Engineering myoblasts for transgene expression of recombinant products is a powerful tool in cell biology, but post-mitotic cells (such as myoblasts) are difficult to transfect by conventional methods [84]. In 1993, Kurachi demonstrated that myoblasts could survive after transplantation as muscle precursor cells, in addition to persistent expression of transgenes delivered by myoblast-mediated gene transfer [128]. Later, this group achieved transgene expression of hFIX in immunodeficient mice, reaching < 1% of the physiologic levels, by transplanting recombinant myoblasts that had been transfected with a retroviral vector [127, 129]. These results demonstrated that myoblast-mediated gene therapy for hemophilia is feasible and safe. However, it also highlighted the necessity of developing different strategies to augment myoblast survival and fusion with myofibers.
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growth factor (VEGF), known to be a potent proangiogenic factor. This study has shown that such an innovative strategy facilitates vascular growth, thus improving the survival of the transplanted myoblasts [115]. Therefore, the use of a lentivirus vector (rather than retrovirus) appears to have contributed to the successful outcome of this study.
Endothelial Cells
Figure 10.2. Alginate microcapsules (∼ 300mm φ) containing genetically engineered myoblasts secreting hFIX.
To overcome the problems associated with graft rejection and differentiation of recombinant myoblasts, a physical barrier was developed that protects the cells inside biocompatible microcapsules that avoids direct contact between the transplanted cell and the effector phase of the immune system (Figure 10.2). Immunoisolation improved the survival of the implanted allogeneic myoblasts, with sustained transgene expression for at least 7 months [56]. However, after 2 weeks of continuous hFIX delivery recipient mice developed a specific immune response against the transgene, at which point hFIX was no longer detectable in the circulation. These results suggest that encapsulation of recombinant myoblasts has therapeutic potential for the treatment of hemophilia, as an allogeneic alternative to autologous transplantation, but once again highlights the importance and difficulty of regulating the host immune system. The same strategy was tested in animals for the treatment of hemophilia A using recombinant encapsulated myoblasts as a continuous source of factor VIII replacement. However, immunocompetent and immunodeficient animals showed transient levels of circulating antigen, suggesting a nonspecific immune mechanism as the primary factor for the decline of transgene expression, such as inhibition of the LTR viral promoter driving transgene expression in vivo [35]. The group of Thierry Vanden Driessche and Marinee Chuah in Leuven, Belgium, have shown that the survival of implanted allogeneic myoblasts may be considerably increased when the transplanted cells are engineered to express (in addition to FVIII) vascular endothelial
Traditionally, endothelial cells have not attracted a lot of interest as a treatment for hemophilia. However, human umbilical endothelial cells (HUVECs) were shown to deliver therapeutic levels of hFVIII in mice [10]. Complementing the evidence in favor of transplanted endothelial cells as a source for clotting factors, Kumaran et al. have shown recently that intraperitoneal transplantation of liver cells from a normal mouse partially corrected the bleeding phenotype of hemophilia A mice [67]. Interestingly, it was not hepatocytes (as one might have expected) that contributed to the production of FVIII. Instead, the authors presented data suggesting that liver endothelial cells were providing most of the functional FVIII. Further, the intraperitoneal transplantation of MS1 pancreatic mouse endothelial cells also achieved phenotypic correction in FVIII-deficient mice [73]. Thus, endothelial cell transplantation may be a suitable approach for the treatment of hemophilia A.
Vector Choice and Design of Gene Expression The choice of vector is critical in determining the final outcome of any gene therapy strategy. Furthermore, the vector should be selected in parallel with the choice of cell line to be encapsulated, not independent from it. There are several important considerations that highlight this requirement. First, each vector has advantages as well as limitations. It is thus wise to select vectors with certain characteristics that are well suited to express high and sustained levels of the transgene in the chosen cell line. Second, certain promoters are known to be downregulated in vivo under certain circumstances, such as the type of cell line used, the backbone of the expression vector, or the nature of the transgene being expressed. In the particular case of hemophilia, there is a clear distinction between the expression of FIX and FVIII. FIX can be expressed relatively easily, which is in fact one of the
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reasons for the rapid advance of research on hemophilia B gene therapy. In contrast, due to the distinct characteristics of its gene FVIII expression entails a much higher degree of difficulty. Vectors that can deliver sustained levels of FIX in vivo can suffer promoter inhibition and thus result in transient transgene delivery when FVIII is expressed. This underscores the importance of coordinating the choice of vector and cell line for each given transgene. Gene transfer of hematopoietic stem cells has been demonstrated using many viral and nonviral vectors with varying degrees of efficiency [20]. Perhaps oncoretroviral vectors have been the most widely studied vectors to date. They are based on the type C murine oncoretrovirus such as the murine leukemia virus. An appealing property of retroviral vectors is that upon transduction of cells the viral genome integrates into the host cell’s genome in apparently random fashion [47]. As a result of this property, all of the progeny derived from a single cell transduced with retrovirus will contain the provirus, and therefore can potentially express the therapeutic product. Therefore, an integrating vector is advantageous for the treatment of chronic diseases such as hemophilia. The oncoretroviral genome is very simple, requiring the expression of only three genes. The gag, pol, and env genes code for structural, replicative, and envelope proteins, respectively. Each end of the integrated proviral genome contains long-term repeat (LTR) sequences with promoter activity for driving the expression of the viral genes [46]. Most of the current strategies that use transplantation of engineered cells modify the retroviral vectors in different ways in order to improve their safety. First, the vectors only contain the genetic information necessary for the reverse transcription of the viral RNA into DNA, retaining the signal for nuclear transport and integration into the host genome. Second, the vectors are adapted to accept the therapeutic gene, in this particular case hFVIII or hFIX. Third, the vectors are modified to optimize transgene expression in the target cell and its progeny for many cell generations lasting for months or years after transplantation [72]. Hence, a retroviral-based gene therapy vector to engineer transgene expression by HSC could potentially provide a cure for hemophilia by achieving longterm transgene expression. However, two prior studies demonstrated the inactivation of the LTRs from the retrovirus, precluding long-term expression [12, 103]. To overcome this limitation, the LTRs have been modified to prevent the binding of transcriptional repressors
that prevents sustained expression [13]. The vectors can be further improved by introducing putative transcriptional sites for transcription activators [100]. Clearly, the final goal of all of the changes introduced to retroviral vectors is the increase and duration of transgene expression in cells after transplantation [44]. However, despite all of the modifications introduced to maintain hFVIII expression in vivo after xenotransplantation of a murine model with human HSC transgene expression declined concurrently with a decline in the transcriptional level of hFVIII mRNA [118]. This finding suggests different causes for this decline, such as inactivation of the LTR promoter activity decreasing transgene expression, inability of different cells to produce FVIII, or inactivation of FVIII transcription in the xenograft. It is very important to point out two important limitations in the last two studies. First, researchers used human HSC. Unfortunately, it has proven to be quite difficult to transduce human HSCs, because of their nondividing nature. Oncoretroviral vectors can only access the nucleus during cell division, when the nuclear membrane is disrupted [116]. Further, the immune response that mediates rejection against allograft versus xenograft is very different. Whereas the first one is dependent on a specific immune response, xenograft destruction is mediated by an innate mechanism that not only destroys the graft but generates cytokines’ microenvironments that might preclude or inactivate transgene expression. Even though published studies have shown persistence of the transplanted cells for at least 3 months [101], it is not possible to discard the innate immune response as a mechanism that mediates inactivation of the therapeutic expression of the transgene. Given the difficulties shown in different experiments, other retroviral vectors have been explored — such as lentivirus vectors, which are based on human immunodeficiency virus (HIV). Lentiviral vectors have been gaining momentum because they do not require cell proliferation for transduction, a huge breakthrough for engineering HSC [110]. This vector is also being modified to comply with the safety concerns that arose from the use of retroviral vectors in clinical trials [1, 125]. Because these vectors have shown an improved efficacy in gene delivery in preclinical models, they were tested to evaluate the ability of human HSC transduced with this type of virus to sustain hFVIII transgene expression in vivo [118]. In this study, in agreement with previous results [53, 132], transplantation of transduced human HSC did not result in detectable levels of hFVIII
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expression in the plasma. However, 6 weeks after transplantation the graft was recovered, showing that human monocytic cells derived from the transplanted cells secreted functional hFVIII ex vivo, suggesting the therapeutic potential of this strategy for long-term delivery of recombinant clotting factors in vivo. Recently, Moayeri et al. [82] bioengineered a murine stem cell virus (MSCV), another retroviral vector to produce B-domain deleted hFVIII that enhances the secretion of the transgene product, transducing murine stem cells [51]. Previously, it has been reported that this type of construct has more efficient expression, secretion, and persistence in vivo [51], in that it stabilizes the transcriptional process and the hFVIII mRNA [81]. In agreement with other approaches in gene therapy for hemophilia A, this investigation reported the engraftment of recombinant bone marrow cells in immunodeficient hemophilia A mice, resulting in sustained therapeutic plasma levels of B-domain deleted hFVIII and long-term correction of the hemophilic phenotype for at least 22 weeks after transplantation [92]. These findings open a new avenue for stem cell transplantation as a therapeutic approach for hemophilia. Kikuchi et al., using a similar strategy but transducing murine bone marrow cells with a simian immunodeficiency virus carrying the full gene for hFVIII, reported similar results. Circulating antigen was detected at lower levels than those obtained by Moayeri, persisting for 60 days after transplantation into immunodeficient SCID mice [62].
Mechanisms to Prevent Immune-Mediated Rejection of Cell Transplant for the Treatment of Hemophilias The discovery of the histocompatibility system (HLA) in 1967 launched the field of transplantation. More than 800,000 such transplants have been performed during this time [79]. Although matching of donor and recipient for MHC antigens is of great importance, the development of pharmacologic agents and antilymphocyte antibodies that interfere with the process of graft rejection has played a crucial role in the success of transplantation during the past 2 decades. New levels of understanding of the mechanisms involved in graft rejection are leading to the development of newer immunosuppressive agents or methods that inhibit various components of the rejection process. It is also important to point out that the same mechanisms the
immune system uses for battling viral infections or to fight what is not recognized as self can be activated against transplanted cells and/or new transgene products that are established as foreign. The goal of the therapeutic transplantation is to create a state of antigen-specific tolerance in the recipient, whereby the graft is not rejected and the patient will not need a lifetime of medical therapy. Therefore, new strategies need to be developed and tested in order to overcome the hurdles this field faces. Immunosuppressive medications prescribed to prevent cell graft rejection are effective in lowering the incidence of rejection, but they produce significant side effects and do not induce a state of transplantation tolerance. Because of this, most of the studies of cell transplantation for hemophilia treatment use immunodeficient animals or long-lasting immunosuppressive treatments in order to avoid graft rejection or a specific immune response directed against the transgene product. Ironically, though, in the particular case of factor VIII replacement therapy some nonspecific mechanisms also mediate the inhibition of transgene expression, abrogating the continuous infusion of the therapeutic product. Most of the studies using hematopoietic stem cell transplantation for the treatment of hemophilia have not resulted in detectable FVIII expression. However, human monocytic cells transplanted in mice did secrete functional hFVIII ex vivo and were able to induce immune tolerance against factor VIII observed after transplantation of transduced bone marrow cells [24]. Monocytes are professional antigen-presenting cells (APCs) that can be differentiated in different types of dendritic cells. Presentation of antigens on these cells will induce specific T-cell tolerance by mechanisms of clonal deletion and/or development of anergy [80]. Therefore, central or peripheral tolerance may be achieved. Indeed, transplantation of genetically modified HSCs secreting hFVIII in nonhuman primates has led not to therapeutic levels of hFVIII but rather unexpectedly to a state of immune tolerance to hFVIII infusions [50]. Implantation of embryonic stem cells in the liver of complete mismatched hemophilia B recipient animals resulted not only in long-term survival of the transplanted cells but in a sustained correction of the defective phenotype with induction of immune tolerance against hFIX [26]. It is unclear whether this tolerance induction is the consequence of long-term hepatic chimerism between the host liver cells and
196
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the embryonic stem cells or the graft per se inducing immune tolerance, but it has been reported in other models of stem cell transplantation [30, 50].
Encapsulation A different strategy is to protect transplanted living cells from the cytotoxic activity of the host immune system by mechanically blocking immune attacks using synthetic membranes around the grafted cells, obviating the need for immunosuppression [88]. There are many polymeric biocompatible devices that have been developed for immunoisolation of transplanted cells secreting hormones, neurotransmitters, growth factors, or other bioactive therapeutic products. These devices entrap allogeneic or xenogeneic cells in semipermeable membranes that provide the ability to transport important molecules for cell survival such as nutrients, oxygen, proteins, and toxic products of cell metabolism [89]. The membrane used for encapsulation of living cells must be biocompatible in the recipient and suitable to protect the cells it encloses (Figure 10.3). Ideally, the material for encapsulation should not evoke fibrous tissue reaction, macrophage activation, and cytokines or cytotoxic release [88].
Ahead Looking The use of transplanted cells for the treatment of chronic disorders such as hemophilia is particularly attractive, and the feasibility of this approach has been
shown (Table 10.1). This strategy allows unique advantages over the direct administration of viral as well as nonviral vectors. Viral vectors typically elicit immune responses in the host, thus posing a challenge for the long-term delivery of therapeutics needed for hemophilia. Furthermore, due to the immune responses generated readministration of viral vectors is currently not feasible. Nonviral vectors generally do not elicit as strong an immune response, but the level of delivery currently achieved is often modest [39]. A notable exception is the extremely efficient hydrodynamic shock technique [2], based on the very fast (5 to 7 seconds) intravascular administration of DNA in a large volume (equivalent in the mouse to the total volume of blood of the animal). Unfortunately, this harsh technique appears an unlikely candidate for use in humans. Immunoprotected cells may offer a continuous source of therapeutics for the treatment of hemophilia. Arguably, the single biggest hurdle facing gene therapy is gene delivery. Therefore, the advantage of immunoprotected cells for the continuous and systemic delivery of FVIII and FIX into the circulation becomes selfevident. The versatility of multiple potential cells that may be used, as well as a variety of possible implantation sites, is a further attractive feature of this strategy. Nevertheless, immunoprotected cells may also elicit unwanted immune responses that could reduce the duration and levels of therapeutic delivery. Key to the success of this strategy is the judicious choice of cell type. The role of the cell cannot be underestimated, in particular for the treatment of chronic diseases such as hemophilia. Our laboratory has already described the importance of the cell used in the generation of an
Immune responses
Triggering factors
Novel transgene (FIX / FVIII)
Humoral
Allo/xenogeneic Cellular products and debris
Cellular
Complement
Polymer
Figure 10.3. Immune responses to implanted encapsulated cells.
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Andrew Gómez-Vargas and Gonzalo Hortelano TABLE 10.1 Cell lines used in transplantation for hemophilia. Type of Cell
Vector
Gene
Transgene Expression
Peak Levels
Reference
Murine BM (autograft) Murine BM (allograft) Mesenchymal cells (xenograft) Murine BM (autograft) Murine stem cells (allograft) Human stem cells (Xenograft) Hepatocytes (allograft) Keratinocytes (xenograft) Keratinocytes (xenograft) Human fibroblast (xenograft) Mouse fibroblast (allograft) Fibroblast (autograft) Rat fibroblast (allograft) Rabbit fibroblast (allograft) Rabbit fibroblast (autograft) Human fibroblast (xenograft) Human fibroblast (xenograft) Human fibroblast (autograft) Human fibroblast (autograft) Murine hepatocytes Murine myoblast (allograft) Murine myoblast (allograft) Murine myoblast (allograft) Murine myoblast (allograft) Endothelial cell (allograft)
Retroviral Retroviral Retroviral Lentiviral Murine SC virus SIV virus None Retroviral Retroviral Retroviral Adenovirus Plasmid Retroviral Retroviral Retroviral Retroviral Retroviral Retroviral Retroviral None Retroviral Retroviral Plasmid Plasmid NA
hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFIX hFIX hFIX hFVIII hFVIII hFVIII hFIX hFIX hFIX hFIX hFIX hFIX hFIX HFVIII HFIX HFIX HFIX HFVIII HFVIII
None None ∼ 3 months None ∼ 5.5 months ∼ 2 months ∼ 3.3 months ∼ 1 week ∼ 12 months None ∼ 2 days ∼ 1.5 months ∼ 2 months ∼ 3 months ∼ 18 months ∼ 5 months ∼ 12 days ∼ 5.5 months ∼ 14 months ∼ 5 weeks ∼ 5 months ∼ 1 month ∼ 2 weeks ∼ 2 weeks ∼ 7 days
None None 12 ng/ml None 505 mUI/ml 3.6 ng/ml 500 ng/ml 2.8 ng/ml 2.75 ng/ml None 17 ng/ml 19 ng/ml None 480 ng/ml Stable 20 ng/ml 203 ng/ml 105 ng/ml Stable 220 ng/ml ∼ 100 ng/ml ∼ 13% activity 10–30 ng/ml 1,000 ng/ml 65 ng/ml 20 ng/ml 20 ng/ml
68 53 94 101 1 44 25 23 5 28 34 54 102 135 136 14 8 77 137 105 129 84 128 127 35
immune response [40, 124]. It is ultimately the choice of protective polymer and cells that will determine the outcome of this strategy for the treatment of hemophilia. Safety is always of paramount importance when considering new therapies. However, safety is perhaps even more critical in the treatment of hemophilia, given the safe and efficacious treatments currently available for hemophiliacs. Thus, any new therapy must match at least the excellent record of recombinant- and plasmaderived coagulation factors. As a result, a promising clinical trial of hemophilia B based on direct administration of AAV was halted in 2004 when 2 patients developed an immune response against the vector and presented with transiently elevated liver enzymes, even though both patients had therapeutic circulating levels of FIX in the blood. This highlights the fact that although hemophilia is an excellent model for gene therapy research in preclinical studies it may not be as good a model for clinical trials. Although hemophilia
is currently leading the transition from bench to bedside for the treatment of monogenic diseases, it is possible that approved gene and cell therapeutic products may become available first for other more serious diseases without currently available treatment.
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11 Cell Therapy for Peripheral Diseases and Reconstructive Applications: Transplants for Lysosomal Storage Disease Toya Ohashi and Yoshikatsu Eto Department of Gene Therapy, Institute of DNA Medicine, Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan
Background Lysosomal storage disease (LSD) is characterized by genetic deficiency of lysosomal enzymes and subsequent accumulation of undegraded substrates in cells, resulting in cell dysfunction. More than 40 diseases are known as LSDs and each disease is associated with a deficiency of a different enzyme. Each disease is rare, but as a group the incidence is one per 7,000 to 8,000 live births [36, 45]. The accumulated compounds are lipid, carbohydrate, glycosaminoglycans, and others. All LSDs are inherited as autosomal recessive except for three X-linked diseases: Fabry disease, mucopolysaccharidosis (MPS) II, and Danon disease [39, 51]. Most, but not all, of the lysosomal enzymes are localized in the lysosome via mannose 6-phosphate receptor (M6PR) mediated transport from the trans-Golgi network. Part of the lysosomal enzyme is excreted outside the cell and taken up by other cells via M6PR on the cell surface and subsequently localized in the lysosome. This phenomenon is called cross collection, which provides a rationale for developing clinical strategies to treat these diseases using bone marrow transplantation (BMT) and enzyme replacement therapy (ERT) [10]. The clinical symptoms of LSD vary among the diseases, are usually multisystemic, and can include localization in the brain, heart, kidney, spleen, lung, and bone. It is not clearly understood as to why substrate accumulation leads to cell dysfunction. However, in some cases accumulated substrate is highly cytotoxic, Cellular Transplantation: From Laboratory to Clinic
as in the accumulation of galactosylpsycosine in Krabbe disease [21]. Currently, two therapies for LSD are available: ERT and BMT. ERT is available for Gaucher disease, Fabry disease, and MPS I. ERT is currently being developed for Pompe, mucopolysaccharidosis (MPS) type II, Niemann-Pick B, and MPS VI. In this chapter, the detail of enzyme replacement therapy will not be discussed, but progress of this therapy is very remarkable and the detail of enzyme replacement therapy is nicely reviewed elsewhere [3, 6]. BMT has had various degrees of success in diseases such as Gaucher disease, MPS, globoid-cell leukodystrophy, and metachromatic leukodystrophy. However, BMT and ERT both have inherent problems. BMT has a high morbidity and mortality rate. The mortality rate of BMT is 10% if the transplant were performed from HLA-identical siblings, and 25% if BMT was performed from mismatched donors [18]. Another limitation of BMT is donor shortage. Because LSD is a genetic disease, the HLAidentical siblings may also have the same disease or be heterozygote of the disease. On the other hand, ERT also has obstacles to overcome. The price of an enzyme is very expensive and it is difficult for the enzyme to cross the blood/brain barrier. Moreover, due to the few patients for some of these diseases pharmaceutical companies have not dedicated the resources for developing replacement enzymes. To overcome the obstacle of current therapies, other approaches (such as using another stem cell other than hematopoietic stem cells) in combination with gene therapy have been tried for the
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treatment of several of these human diseases. Recent development in stem cell biology has provided scientists with the ability to perform transplantation studies for various LSD using animal models. In this chapter, the current status of BMT is summarized, and recent research in the development of stem cell transplantation for LSD (including our data) is presented.
BMT The rationale of using BMT for the treatment of LSD is that the transplanted hematopoietic stem cells can differentiate into their progeny and then be distributed throughout the whole body. The new hematopoietic cells can then secrete the missing enzymes, which are then subsequently taken up by the neighboring cells. The identification of hematopoietic cells in the brain as microglia provides further evidence that the BMT can be effective for neurological involvement. In fact, neurological involvement of LSD has been halted by BMT. The first successful use of BMT for LSD was published in 1981 [17]. In this report, Hobbs et al. described one Hurler patient (MPS type I) who underwent BMT. The MPS I patient, who was one year old, received bone marrow from his mother. The result was very encouraging. His hepatomegaly disappeared and urinary excretion of glycosaminoglycans was reduced. Moreover, his neurological deterioration was halted. From this very impressive result, it was expected that most of the MPS patients could be treated by BMT. From this initial study, many BMTs have been tried for several types of LSDs. Variants of MPS also showed encouraging results using BMT. These included MPS I [13, 15, 20, 42, 43, 49], MPS VI (Maroteaux-Lamy syndrome) [1, 14, 16, 28, 29, 33, 35, 32], and MPS VII (Sly syndrome) [28, 54]. In LSDs other than MPS — such as Krabbe disease [4, 28, 31], Metachromatic leukodystrophy [28, 30, 34], alpha-mannosidosis [12, 28, 53], and Wolman disease — encouraging results were also obtained. However, long-term effects of BMT for LSD are very difficult to evaluate, because each disease is rare and has a wide spectrum of clinical phenotypes. Moreover, the natural history of these diseases is not fully understood. Thus, it is very difficult to conclude that BMT really has changed the clinical course of the disease using very sophisticated clinical studies. For these reasons, in combination with the high morbidity, the clinician has had a difficult decision whether to perform BMT for LSD patients. In this regard, Peters et al.
at the University of Minnesota published practice guidelines on when to use BMT therapy [44]. Their opinion on when to use BMT for each LSD case is as follows. In MPS IH (severe form of MPS I), BMT should be performed before 18 months old. After 18 months, a profound neural involvement has occurred and it is very difficult to restore this level of severe brain damage. In MPS IS (milder form of MPS I), it is a difficult decision whether to perform BMT because of the development of an ERT. In this type of MPS I, usually brain involvement is very minor and therefore the effect on the brain can be disregarded. As mentioned previously, the shortcoming of ERT is that an enzyme cannot cross over the blood/brain barrier. Because MPS IS does not have severe neurological involvement, ERT may be recommended. However, ERT for MPS I is not available in all countries and thus for those countries without this option BMT may be the first choice of these disorders. MPS II has a similar clinical condition to that of MPS I, with some positive and negative results associated with BMT. However, the data indicate that MPS II is not a good candidate for BMT. BMT has also been used for the treatment of Gaucher disease. Because of the development of ERT for Gaucher disease and many positive results of ERT, BMT is not the first line of therapy. However, ERT does not arrest neural involvement of Gaucher disease. Hence, BMT is only indicated for type III Gaucher disease for a potential ability to arrest or prevent the progression of brain involvement. Globoid-cell leukodystrophy is characterized by a deficient activity of galactocerebrosidase, which results in massive accumulation of galactosyl psycosine. The accumulation of this very toxic substance causes severe demyelination [21]. There are two types of globoid-cell leukodystrophies. One is an early onset type, which is called Krabbe disease. The other is a late onset type. There are some positive reports for BMT for Krabbe disease, especially for late onset type [4, 28, 31]. Although BMT for early onset Krabbe disease is not usually recommended, BMT in the neonatal period can halt the progression of neurological symptoms and prevent demyelination. Metachromatic leukodystrophy (MLD) is characterized by a deficient activity of arylsulfatase A, which degrades a glycolipid, known as sulfatide. Progressive accumulation of sulfatide in myelin forming cells, socalled oligodendrocytes, leads to massive demyelination. MLD is divided into a late infantile type, a juvenile type, and an adult type according to age of onset.
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The most prevalent type is late infantile type and typically clinical symptoms start between 4 months to 4 years of age. In this type of MLD, BMT may be recommended only for presymptomatic patients or patients with very mild neurological dysfunction. Usually, BMT is not recommended for patients with late infantile MLD because severe neurological symptoms already exist when the diagnosis is made [28, 30, 34]. In these patients, BMT does not improve the clinical course, but could even further worsen the disease state. The LSDs cited previously have the greatest history of BMT. The other existing LSDs have had limited experience with BMT, and hence it is not possible to make any conclusions on the benefit of this therapy at this time. As was mentioned previously, the main limitation of BMT for LSD is donor shortage. To overcome this limitation, unrelated adult donors are recruited. However, it usually takes too long to find an adult donor for a rapidly progressive disorder such as LSD. Banked umbilical cord blood is another source of hematopoietic stem cells, and has certain benefits over BMT such as a rapid availability. Umbilical cord blood transplantation has been performed for several LSDs, including Krabbe disease [9] and Hurler disease [50]. In Krabbe disease, cord blood transplantation was performed in the neonatal period, before clinical symptoms appeared. The results were much more impressive than those of BMT in children after symptoms had developed. In Hunter syndrome, cord blood transplantation was performed in 20 patients. The majority of the patients who received cord blood cell transplantation had improved neurocognitive function and decreased somatic symptoms.
Other Cell-based Therapies for LSD Recently, stem cell biology techniques have developed considerably. We and other laboratories have developed stem cells, other than hematopoietic stem cells, as a vehicle for the delivery of the missing enzyme to enzyme-incompetent cells located throughout the whole body. The stem cells we have examined include neural, mesenchymal, and embryonic. Stem cells have obvious advantages when compared to other committed cells, such as self-renewal and differentiating capability into a variety of cell types. These abilities suggest that transplanted cells can make a variety of progeny and renew themselves and continuously stay within the patients for long periods of time. If these cells can express and
excrete the missing enzyme, the targeted cells can then take up the enzyme. The first published approach using stem cells for the treatment of LSD was using neural stem cell transplantation in an MPS VII mouse model. Snyder et al. isolated and cultured neural stem cells and transplanted these cells into the lateral ventricle of an MPS VII mouse during the neonatal period [48]. These cells migrated extensively throughout the brain and remained viable in the brain for almost the entire life of the mouse. The therapeutic effect of the cell transplantation looked very promising. Substrate storage material in various cells was reduced to almost normal levels. This observation raised the question whether human neural stem cells could behave in a similar fashion as murine stem cells. We established a human neural stem cell line and introduced the beta-glucuronidase gene, which is nonfunctional in MPS VII disease [37]. After introduction of the normal gene, the cells expressed 87-fold more enzyme than in nontransduced human neural stem cells. Overexpression of beta-glucuronidase did not alter the morphology or growth of the neural stem cells. We transplanted these cells into the lateral ventricle of a neonatal MPS VII mouse. At 10 days posttransplantation, these cells were identified in the olfactory bulb, septum, cerebral cortex, hippocampus, mesencephalon surrounding the cerebral aqueduct, and ependyma of the cerebral ventricles. The enzymatic activity in the various brain areas was increased greater than the wild-type control (Figure 11.1). The accumulation of Chondroitin sulfate, which is one of the glycosaminoglycans that increases in MPS VII, was significantly reduced (Figure 11.2a and b). Pathological improvement was also significant (Figure 11.2c). The storage material in the cells disappeared both in the neuron and the glia. The observations were almost compatible with those that were observed in the murine neural stem cell study. However, the extent of cell migration was limited and the fate of the transplanted cells was short. Ten days after transplantation, betaglucuronidase activity was reduced to a mutant level (Figure 11.3). Even though we used immunosuppressive agents to prevent immunologic reaction of the host mouse, xenotransplantation may be the cause of this limited fate of the cells. To further investigate whether the reduction in number of transplanted cells resulted from apoptotic cell death, we performed TUNEL staining on the brain section. This assay indicated that the transplanted cells were dead through the process of apoptosis.
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Figure 11.2. The contents of total chondroitin sulfate (T-CS) (a) and hyaluronic acid (HA) (b) were significantly reduced in whole brain extracts of the MPS VII mouse brain that received cell transplantation compared with wild type controls. Glycosaminoglycan burden neurons were found in the cortex in non-transplanted mice, but were not identified in the cortex of transplanted mice (c).
Usually neural stem cells are created from fetal tissues. This might be an ethical problem for use in a clinical setting. To avoid these ethical issues, other stem cells have been evaluated. Recently, it was reported that some stem cells isolated from a specific germ layer
differentiate into cells of a different germ layer. For example, mesenchymal stem cells usually differentiate into mesodermal tissue — such as bone, cartridge, and fat tissue — but can also differentiate into neural tissues that originally develop from ectodermal tissue
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[2, 23, 24]. This suggests that mesenchymal stem cells can behave like a neural stem cell located in the brain. We transplanted mesenchymal stem cells that were isolated from mouse bone marrow into the lateral ventricle of neonatal MPS VII mice [47]. Mesenchymal cells that were used in this experiment had the capacity to differentiate into neural cells. After transplantation, the cells migrated from the transplanted site to brain parenchyma in a similar fashion as neural cell transplantation. The enzymatic activities in various brain regions were increased at 2 weeks after transplantation but declined at 8 weeks posttransplantation (Figure 11.4). The pathological improvement was significant, and the amount of glycosaminoglycan accumulation was significantly reduced (Figure 11.5). Brain sections were stained with antibodies against neural and glial cell markers. These stained sections indicated that the transplanted cells do not express any neural cell markers. This observation suggests that mesenchymal stem cells have a capacity to migrate in the brain but lose their neural differentiating capacity in vivo, or that the brain environment is not suitable for neural differentiation. With the exception of treatment of LSD in neural tissue, mesenchymal cells do not necessarily have to differentiate into neural cells. The only requirement for the transplantation of mesenchymal cells into the brain is that they engraft for an extended period of time, with a concomitant excretion of the missing enzyme with a potential integration with the recipient cells. In this regard, the mesenchymal stem cell is promising for treatment of LSD.
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Figure 11.4. Beta-glucuronidase activity of coronally divided (part a–e) brain sections from MPS VII recipient and untreated wild-type control mice at 2 weeks and 8 weeks after transplantation.
Jin et al. transplanted mesenchymal stem cells directly into the brains of Niemann-Pick disease mice [19]. They transduced the mesenchymal stem cells with a retroviral vector to overexpress acid shingomyelinase, and injected these cells into the hippocampus and cerebellum of these mice. The transplanted cells migrated away from the injected site and remained viable for more than 6 months. The overall enzymatic activity was low, but the life span of the transplanted mice was significantly longer than the control mice. Moreover, the Purkinje cell loss was significantly delayed. These observations taken together suggest that mesenchymal cell transplantation into the brain is a promising treatment of neurological involvement of LSD in animal models. Mesenchymal cell transplantation has an obvious advantage over other therapies. Mesenchymal cells can be cultivated from the patients’ own bone marrow. This eliminates immunological concerns and ethical issues. Moreover, mesenchymal stem cell transplantation has already been studied in a clinical setting. Koc et al. reported the intravenous allogeneic mesenchymal stem cell infusion for MLD (n = 6) and MPS-I syndrome (n = 5) in patients with the expected goal that mesenchymal stem cells could migrate and differentiate into bone, cartilage, and peripheral and central nerves and
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(b) 8 wk
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Total Chondroitin sulfate (TCS) Hyaluronic acid (HA) Figure 11.5. The contents of total chondroitin sulfate (T-CS) and hyaluronic acid (HA) were significantly reduced in whole brain extracts of the MPS VII mouse brain that received cell transplantation compared with wild type controls at least 16 weeks after transplantation.
clean up the storage material in these tissues [22]. All of these patients had received a successful BMT. Usually, after a successful BMT bone marrow stroma cells (from which mesenchymal stem cells originate) are recipient type. That may be one of the reasons BMT for bone, cartilage, and brain is generally not satisfactory. Two out of 6 patients were infused with mesenchymal stem cells into their bone marrow 60 days after a BMT infusion. In 4 MLD patients, nerve conduction velocities were improved significantly, and in all of the patients bone mineral density was improved. Unfortunately, there was no improvement in brain function. In this study, mesenchymal stem cells were transplanted intravenously. Thus, if mesenchymal stem cells were transplanted directly into the brain the improvement of brain function may be expected. In addition to mesenchymal stem cells, other cells have also been examined for the treatment of LSD. Amniotic cells have a capacity to differentiate to neural cells, and the transplantation of genetically engineered human amniotic epithelial cells in the brain can cure the lysosomal storage disease of MPS VII mice [25, 26, 27]. ES (embryonic stem) cells are currently being investigated in regenerative medicine, and Lau et al. are trying
to use ES cells to treat MPS III [32]. This study is still in its early stage, but there are huge possibilities. The main obstacle to be overcome in the use of ES cells is to prevent teratoma formation. Macrophage transplantation has also been tested as a possible treatment of LSD. It was thought that the macrophage was the primary cell responsible for the therapeutic role in BMT for LSD. In fact, we and others have demonstrated that macrophage transplantation exhibits almost equal effects with BMT [11, 40].
Organ Transplantation Cell transplantation has aimed at providing a continuous supply of the enzyme from the transplanted cells and hence reducing the storage material in the diseased cells. Unfortunately, in some instances organs have irreversible damage. Once irreversible organ damage has occurred, organ transplantation is required. In this regard, kidney transplantation for end-stage renal failure of Fabry disease has become a life-saving procedure. Renal transplantation for Fabry disease has a
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clear benefit. If the transplantation results in a success, the kidney function is restored and the tissue remains free of accumulation of storage material [8, 38, 41, 46]. Some cells from the recipient have accumulated glycolipid, but these are very rare and only identified by electron microscopy. Cardiac transplantation was also performed for Fabry [5] and Danon diseases [7], which are associated with a severe cardiac dysfunction.
Conclusions Recent advances in transplantation-based therapy for LSD is amazing. Stem cell technology especially opens up a new avenue for regenerative medicine. In future prospects, the ability to provide stem-cell-based, protein, small-molecule, and gene therapies will improve the quality of life for LSD patients.
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31. Krivit, W., Shapiro, E. G., Peters, C., Wagner, J. E., Cornu, G., Kurtzberg, J., Wenger, D. A., Kolodny, E. H., Vanier, M. T., Loes, D. J., Dusenbery, K., Lockman, L. A. Hematopoietic stem-cell transplantation in globoid-cell leukodystrophy. N Engl J Med 338:1119–1126; 1998. 32. Lau, A. A., Hemsley, K. M., Meedeniya, A., Hopwood, J. J. In vitro characterization of genetically modified embryonic stem cells as a therapy for murine mucopolysaccharidosis type IIIA. Mol Genet Metab 81:86–95; 2004. 33. Lee, V., Li, C. K., Shing, M. M., Chik, K. W., Lam, C. W., Tsang, K. S., Pong, H., Huen, K. F., Yuen, P. M. Umbilical cord blood transplantation for Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI). Bone Marrow Transplant 26:455–458; 2000. 34. Malm, G., Ringden, O., Winiarski, J., Grondahl, E., Uyebrant, P., Eriksson, U., Hakansson, H., Skjeldal, O., Mansson, J. E. Clinical outcome in four children with metachromatic leukodystrophy treated by bone marrow transplantation. Bone Marrow Transplant 17:1003–1008; 1996. 35. McGovern, M. M., Ludman, M. D., Short, M. P., Steinfeld, L., Kattan, M., Raab, E. L., Krivit, W., Desnick, R. J. Bone marrow transplantation in Maroteaux-Lamy syndrome (MPS type 6): Status 40 months after BMT. Birth Defects Orig Artic Ser 22:41–53; 1986. 36. Meikle, P. J., Hopwood, J. J., Clague, A. E., Carey, W. F. Prevalence of lysosomal storage disorders. Jama 281:249–254; 1999. 37. Meng, X. L., Shen, J. S., Ohashi, T., Maeda, H., Kim, S. U., Eto, Y. Brain transplantation of genetically engineered human neural stem cells globally corrects brain lesions in the mucopolysaccharidosis type VII mouse. J Neurosci Res 74:266–277; 2003. 38. Mosnier, J. F., Degott, C., Bedrossian, J., Molas, G., Degos, F., Pruna, A., Potet, F. Recurrence of Fabry’s disease in a renal allograft eleven years after successful renal transplantation. Transplantation 51:759–762; 1991. 39. Nishino, I., Fu, J., Tanji, K., Yamada, T., Shimojo, S., Koori, T., Mora, M., Riggs, J. E., Oh, S. J., Koga, Y., Sue, C. M., Yamamoto, A., Murakami, N., Shanske, S., Byrne, E., Bonilla, E., Nonaka, I., DiMauro, S., Hirano, M. Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature 406:906–910; 2000. 40. Ohashi, T., Yokoo, T., Iizuka, S., Kobayashi, H., Sly, W. S., Eto, Y. Reduction of lysosomal storage in murine mucopolysaccharidosis type VII by transplantation of normal and genetically modified macrophages. Blood 95:3631–3633; 2000. 41. Peces, R. Is there true recurrence of Fabry’s disease in the transplanted kidney? Nephrol Dial Transplant 11:561; 1996. 42. Peters, C., Balthazor, M., Shapiro, E. G., King, R. J., Kollman, C., Hegland, J. D., Henslee-Downey, J., Trigg, M. E., Cowan, M. J., Sanders, J., Bunin, N., Weinstein, H., Lenarsky, C., Falk, P., Harris, R., Bowen, T., Williams, T. E., Grayson, G. H., Warkentin, P., Sender, L., Cool, V. A., Crittenden, M., Packman, S., Kaplan, P., Lockman, L. A.,
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12 Cellular Transplants for Liver Diseases Nicholas J. Krebs (1), Craig Neville (1 and 2), and Joseph P. Vacanti (1 and 2) (1) The Center for Regenerative Medicine, Massachusetts General Hospital (2) Department of Pediatric Surgery, Mass General Hospital for Children and Harvard Medical School
Introduction Liver failure is caused by a number of factors such as viral infection, a reaction to drugs, exposure to toxins, genetic abnormalities, or vascular complications. Consequently, most diseases of the liver — including primary biliary cirrhosis, nonalcoholic fatty liver disease, cancer, and various metabolic disorders — are capable of compromising the functioning of the liver. Currently, the only successful therapy for the treatment of end-stage liver disease is orthotropic liver transplantation (OLT), which includes cadaveric whole-organ transplantation, reduced-size liver transplantation, splitliver transplantation, and living donor transplantation. Indeed, thousands of lives are saved each year through this method of treatment. However, thousands continue to wait for this life-saving procedure with no certainty of an organ becoming available. Despite the success of OLT, there are many limitations imposed by this approach, including (1) a severe shortage of donor organs, (2) extremely high costs associated with the procedure and concurrent care, and (3) requirement for life-long immunosuppressive drugs in defense against rejection. According to the Scientific Registry of Transplant Recipients, at the start of 2005 there were 17,563 patients waiting for a liver transplant with 2,424 mortalities occurring the previous year. In addition, the costs associated with treating liver disease through OLT in the United States are well over $9 billion annually. Consequently, there has been an increased interest in developing alternative approaches for treating liver disease. Alternatives have focused on various cellular transplantation methodologies, including
Cellular Transplantation: From Laboratory to Clinic
xenotransplantation, direct hepatocyte transplantation, and implantable hepatic tissue-engineered constructs. To create a living cell-based replacement system for patients experiencing liver failure scientists must formulate a solution that recapitulates vital liver functions in addition to the correct environmental conditions and cues necessary for cellular growth, proliferation, and differentiation. Major functions of the liver that must be recapitulated include (1) filtration and storage of blood, (2) metabolism of carbohydrates, proteins, fats, and hormones, (3) formation of bile, (4) storage of glycogen, vitamins, and iron, and (5) formation of coagulation factors. Assist devices like the bio-artificial liver are intended only as a temporary bridging mechanism for survival until a liver becomes available for transplant. Transplanted cells from the liver would allow for a more permanent treatment of liver failure because they can perform most liver-specific functions. Several animal models have been developed to transplant hepatocytes for therapeutic purposes, including direct injection into the spleen or liver, encapsulation within biocompatible membranes, attachment to microcarrier beads, and injection into the abdominal cavity [31, 84, 116]. Compared to OLT, direct hepatocyte transplantation has several distinct advantages. First, hepatocytes from a single donor can be used for more than one recipient. Second, morbidity rates are much lower for hepatocyte transplantation than OLT because cellular transplantation does not involve a surgical procedure. Finally, hepatocytes can be used temporarily until the patient’s liver can regenerate. However, to this point hepatic cell transplantation has met only limited success because only a small number of cells can be transplanted, providing limited long-term function.
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Nicholas J. Krebs, Craig Neville, and Joseph P. Vacanti
As a result of this limitation, scientists have focused on creating solutions that improve cell viability and mass while offering long-term functionality, including the use of transplanted stem cells to encourage liver regeneration. Using stem cells as therapeutic agents for tissue regeneration in vivo also has the potential to mitigate abnormalities common to some liver diseases while addressing the need for continual renewal. The unique pluripotent and multipotent capabilities of stem cells, along with their potential for long-term self-renewal, make them ideal sources for tissue regeneration. As a result, there has been much interest in using stem cells to repair or regenerate tissues and organs that have been damaged by disease. However, it is not yet clear which stem cell types are most appropriate and through what mechanism tissue can be regenerated using these cell sources — by differentiation, transdifferentiation, or fusion. Embryonic, adult (hematopoietic and mesenchymal), fetal, and hepatic stem/progenitor cells are all being carefully considered and debated as to their utility and practicality for regenerating liver tissue in vivo. In addition to transplanting cells directly, the transplantation of liver cells as a component of tissueengineered constructs has become the model for fully functional liver replacement. Tissue engineering of the liver has the added advantage of giving cells a matrix upon which they can organize, grow, and proliferate. Such scaffolds offer an increased surface area for cell attachment to accommodate a large number of hepatocytes. Furthermore, many of these scaffolds are made of biodegradable materials, allowing new tissue to form during resorption of the polymer. An added benefit to a tissue-engineering approach is the ability to harness the potency of stem cells in the context of a complete microenvironment or cellular niche. Understanding and utilizing the potential of stem cells will allow scientists to form tissue that is morphologically and physiologically more similar to native tissue than those constructs that only use differentiated parenchymal cells. Ultimately, the goal of a tissue-engineered approach is to create an implantable liver replacement to provide augmented function to those patients with acute and chronic liver disease. In this review we will examine cellular-based therapies for treating liver disease. First, we will define essential functions of the liver that must be recapitulated for successful treatment, examine the components of such therapies, and finally look at current cutting-edge cellular-based approaches for treatment that are being developed.
Liver Organization, Function, and Disease LIVER ANATOMY The liver is the largest organ of the body, accounting for nearly 1/50 its entire mass or approximately 1.5 kg in the adult human. Specialized metabolic, secretory, and synthetic functions occur through interactions between blood and hepatocytes (parenchymal liver cells) in units referred to as liver lobules. Nearly 100,000 lobules comprise an adult liver. They are cylindrical structures between 0.8 to 2 mm in diameter. Each lobule, as depicted in Figure 12.1, has three to seven sets of portal triads — defined by a portal venule, hepatic arteriole, and bile duct on the periphery and a central vein at the center [49]. The smallest functional unit of the liver is the acinus, which is defined by two central veins at the ends of the long axis and a portal triad at the center (Figure 12.2). An acinus is smaller than a lobule, being a diamondshaped mass of liver parenchyma that is supplied by a terminal branch of the portal vein and of the hepatic artery and drained by a terminal branch of the bile duct. Each lobule and acinus is composed of hepatocytes arranged in sheets called hepatic cellular plates, which radiate outward away from the central vein. Each hepatic plate has a thickness of two hepatocytes, with a space between adjacent cells where small bile canaliculi are located. Each bile canaliculus, comprised of epithelial cells called cholangiocytes, empties into
Hepatic sinusoids
Central vein
Liver cells Kupffer cell Hepatic cellular plate
Hepatic portal vein Hepatic artery Bile duct Figure 12.1. Liver lobule. Depiction of liver lobule showing essential components, including portal triad (hepatic artery, hepatic portal vein, and bile duct), hepatic sinusoids, hepatic plates, and parenchymal cells (hepatocytes) [49]. (Copyright Elsevier, Inc. Used with permission.)
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Portal triad Central vein
Central vein
Figure 12.2. Liver acinus. Photograph of a liver acinus comprised of two central veins at the ends of the long axis and a portal triad at the center. An acinus is the functional unit of the liver, smaller than a liver lobule, being a diamond-shaped mass of liver parenchyma that is supplied by a terminal branch of the portal vein and of the hepatic artery and drained by a terminal branch of the bile duct.
a bile duct in the fibrous tissue separating the adjacent liver lobules. As bile is formed, it empties into these ducts then leaves the liver to be stored in the gall bladder before use during digestion. Adjacent to each bile duct in the fibrous septa are small portal venules that receive blood from the venous outflow of the gastrointestinal tract by way of the portal vein. Consequently, blood from the GI tract enters the liver through the portal system, where it then empties into flat, branching hepatic sinusoids (or spaces) found between neighboring hepatic plates. As blood enters the sinusoids it interacts with the hepatocytes before entering the central vein. Hepatocytes are in continuous contact with portal blood from the GI tract. In addition to the portal venules found in the interlobular septa, there are hepatic arterioles that supply the septal tissues between adjacent lobules with oxygenated blood. Many of these small arterioles also empty directly into the sinusoids. Hepatocytes are not the only cells of the hepatic plates. Kupffer cells are interspersed in the endothelium lining the sinusoid. The endothelial lining of the sinusoids has pores or fenestrations 50 to 300 nanometers in diameter that allow free passage of solutes into the pericapillary region between the endothelial cells and hepatocytes called the space of Disse. This is in turn connected with lymphatic vessels, allowing excess fluid entering the spaces to be removed by the lymphatics. An abundance of basement membrane proteins and proteoglycans (such as collagen type IV and fibronectin) are located here.
As a result of organization into hepatic plates, hepatocytes have two distinct domains that cause these cells to be polarized. First, the basal domain is defined by the cellular regions facing the sinusoid. Here, microvilli project into the space of Disse and form nearly threequarters of the total cell surface area. This domain is continuous with blood and is responsible for the absorption and secretion of plasma components. The apical domain is the cellular region of contact between adjacent hepatocytes where bile canaliculi are formed and is responsible for the secretion of bile salts. In addition to the baso-lateral to apical polarization, hepatocytes along the hepatic plate differ in their function due to the unidirectional flow of blood through the liver. Perfusion studies demonstrated a unique zonal distribution of hepatocytes along the hepatic plate. Hepatocytes near the portal venules and hepatic arterioles are perfused first with blood and so receive higher concentrations of oxygen. Those cells nearest the central vein are perfused last, with less oxygenated blood. Consequently, periportal hepatocytes undergo mitosis and regenerate, whereas pericentral hepatocytes are responsible for processing toxins by the cytochrome oxidase P450 system.
LIVER FUNCTION As seen from the complex anatomy and interdependency of cell types observed in the liver, total replacement of liver function will be difficult to recapitulate in an engineered system. However, there are critical liver-specific functions that must be replaced to mitigate the degenerative effects of liver disease. These functions include metabolism of macromolecules and metabolic homeostasis, protein synthesis, detoxification of drugs, and the conversion of heme into bilirubin and biliverdin. The liver is a large group of highly metabolic cells that shares and distributes substrates and energy from one metabolic system to another in the body. Moreover, it processes and synthesizes multiple compounds that are delivered to the entire body. Because the liver is vital for metabolic homeostasis, it plays a central role in the degradation of carbohydrates, fats, and proteins. Of the metabolic functions the liver is responsible for, protein metabolism is probably the most important. Without these functions, death would ensue within a few days. Those functions that are most critical include (1) deamination of amino acids, (2) urea formation
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for the removal of ammonia from bodily fluids, (3) synthesis of plasma proteins, and (4) the interconversion of amino acids and the synthesis of other compounds from amino acids. Deamination of amino acids is necessary before they can be used for energy or converted into other macromolecules. Ammonia is a toxic by-product of many reactions that must be removed by conversion to urea. If urea production ceases, ammonia builds up in the blood causing hepatic coma and death. The liver produces most plasma proteins, between 15 and 50 grams per day. In individuals with chronic liver disease, plasma proteins fall to dangerously low levels causing edema and ascites. An essential liver function is synthesis of specific amino acids and compounds through transamination. Catastrophic failure in other organs results if critical amino acids are not available. The liver controls certain aspects of fat metabolism and catabolism that other tissues cannot, including (1) oxidation of fatty acids to supply energy for other bodily functions, (2) synthesis of large quantities of cholesterol, phospholipids, and lipoproteins, and (3) the synthesis of fats from proteins and carbohydrates. Most cholesterol synthesized in the liver is converted into bile salts, which are secreted into the bile for fat emulsification in the small intestine. Phospholipids and the remaining cholesterol are packaged into lipoproteins and transported to other tissues through the blood. Finally, almost all fat synthesis that occurs from carbohydrates and proteins in the body is done in the liver. After fat is synthesized in the liver, it is transported to adipose tissue in lipoproteins where it is stored. Carbohydrate metabolism in the liver can be broken down into several discrete functions, including (1) the storage of glycogen, (2) the conversion of galactose and fructose to glucose, (3) the production of glucose via gluconeogenesis, and (4) the formation of many compounds from intermediate metabolites during carbohydrate metabolism. The liver is essential for the balance of glucose in the blood. Individuals with poor liver function often experience severe increases in blood glucose concentrations after eating meals rich in sugars and carbohydrates. In addition, there are several other functions that the liver performs, including hemoglobin destruction through bilirubin formation, iron storage as ferritin, vitamin storage, synthesis of coagulation factors, and drug detoxification and hormone removal. Kupffer cells associated with the endothelium are responsible for phagocytosis of the hemoglobin resulting from lysed red blood cells. Vitamins stored in relatively large quantities
in the liver are vitamins A, D, and B12 . Finally, the liver is the primary means by which the body removes drugs and excess hormones secreted by the endocrine glands, such as thyroxine, estrogen, cortisol, and aldosterone.
Overview: Requirements for Cell-based Therapies for Liver Disease When examining the requirements for cell-based therapies to treat liver disease, three essential and interlocking factors must be considered within the context of developing a functional replacement strategy: (1) maturational lineage mechanisms when deciding cell type, (2) soluble signals, such as hormones, growth factors, nutrients, and gas exchange, and (3) insoluble signals from cellular interactions with extracellular matrices. Each of these factors operates both independently and interdependently in guiding growth and differentiation of cells, and directs the remarkable regenerative properties of the liver.
LIVER DEVELOPMENT Liver development begins when a diverticulum of the caudal foregut and the septum transversum, a part of the splanchnic mesenchyme unit. Shortly after, immature hepatic cells (hepatoblasts) begin to form as the liver bud following interactions between the epithelial cells of the endoderm with cardiogenic cells of the mesoderm [32]. Critical to the early development of the liver is the epithelial-mesenchymal interactions, which determine what progenitor cells differentiate into hepatocytes, cholangiocytes, and fenestrated endothelia. During the early stages of development, the liver consists of bunches of primitive hepatocytes bound by a continuous layer of endothelium without a basement membrane. Hematopoietic cells are also present. The continuous endothelium becomes fenestrated and the portal vasculature signals the production of basement membrane. As the portal interstitium forms, it is believed to trigger the development of bile ducts, in addition to the formation of the portal triads as the interstitium surrounds the portal venules, hepatic arterioles, and bile ducts. While the portal vasculature develops, the hepatoblasts proliferate and differentiate to form hepatic plates [147]. Just prior to birth, the fetal liver begins its process of metabolic zonation and physical organization.
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LIVER REGENERATION The liver has a much greater capacity for self-renewal and regeneration than most organs. Studies in rats have shown that following a 70% resection of the liver a remarkable compensatory response by the remaining liver restores the total mass and hepatocyte cell number within a couple of weeks. For many years, scientists believed that hepatocytes are quiescent cells with a very limited capacity for regeneration. Recent findings suggest that hepatocytes can replicate 70 times or more [180], a result contrary to the long-held thought that hepatocytes could only replicate once or twice before undergoing apoptosis. As our understanding of the mechanisms and necessary cellular interrelationships needed for liver regeneration is limited, it is a major field of investigation.
Cell Types The type and source of cells to be used in a cellbased therapy for the recapitulation of complex vital organ function must be carefully considered. The liver is an intricate organ composed of endothelial cells and a multitude of parenchymal and nonparenchymal cells, including hepatocytes, cholangiocytes, stellate cells (Ito cells), oval cells, fibroblasts, and Kupffer cells. It is difficult to precisely define exact liver-specific functions to a single specific cell type without considering the interactions between cells, extracellular matrices, and chemical mediators. For this discussion, we will focus on the cell types that currently have the most significant roles in cell-based therapies, including differentiated hepatocytes, hepatic progenitor cells, and stem cells (embryonic and determined), together with environmental factors such as the extracellular matrix, soluble signaling molecules, and co-culturing conditions.
STEM CELLS Stem cells are undifferentiated or unspecialized cells, having the ability for continuous self-renewal through symmetric mitosis while maintaining the potential to produce multiple lineages. Stem cells can be isolated both from early-stage embryos and adult tissue [102]. Further divisions of adult-derived stem cells can be applied when examining the potential for differentiation into more specialized cells. Progeny of stem cells
with a more restricted lineage are often referred to as progenitor cells, and classified as either early or late depending on their state of differentiation. Early progenitors (or determined multipotent stem cells) can possess very similar characteristics as their pluripotent precursors, whereas late progenitors (or committed progenitors) may have differentiated to a point that only allows them to produce one or two adult cell types. Totipotent embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst [124, 156]. As a result of their toti/pluripotency, ESCs may prove to be an important cell type for creating cellular transplants for liver disease. Determined stem cells are derived directly from embryonic stem/germ cells, and have a restricted genetic potential due to the mechanism of cellular commitment that occurs between the 8-cell and 16-cell stages of mitotic division. Consequently, determined stem cells are multipotent, meaning that they have the capacity to differentiate into a specified cell type depending on their location. Undifferentiated determined adult or somatic stem cells are located throughout the body in differentiated tissues and organs. Determined adult stem cells have the ability to regenerate and differentiate into several cell types that are specific to the resident tissue. The two major populations of adult stem cells in the body are hematopoietic and mesenchymal stem cells. Hematopoietic stem cells are precursors for the lymphoid and myeloid cells of the blood, which help to maintain blood lineages [107]. These cells have been isolated in human cells having a CD34+/Thy1+ phenotype (a known hematopoietic marker), along with the C1q complement receptor marker, for hepatic lineages [22]. Hematopoietic stem cells have demonstrated selfrenewal through replication while being able to differentiate into a variety of multipotent progenitors and lineage-specific progenitors, such as hepatoblasts (oval progenitor cells or intrahepatic stem cells). Mesenchymal stem cells are multipotent progenitor cells found in the bone marrow and peripheral blood that differentiate into osteoblasts, chondrocytes, adipocytes, and fibroblasts. Currently, they have no unique combination of cell surface markers (allowing them to be easily defined), but they can be isolated as a growing adherent population. Moreover, a subpopulation of mesenchymal stem cells can differentiate into hepatocyte-like cells [132]. Therefore, because they can differentiate and possibly transdifferentiate into multiple lineages (including connective tissue and hepatic precursors)
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there is a potential to use these cells for some regenerative capacity in the liver [115]. Three competing models describe the presence and location of determined hepatic stem cells, called oval cells or hepatoblasts. Model one argues no such cells exist and that all liver cells are coequal and fully capable of clonal growth [63, 88]. Model two differs somewhat, suggesting the presence of small residual populations of hepatic stem cells that remain silent, indicating that all liver regeneration in the adult is derived from adult liver cells that are coequal and capable of clonal growth [44, 61, 138]. Finally, model three describes the presence of determined hepatic stem cells within the liver that reside in specified niches or microenvironments. This niche is comprised of cells, extracellular matrix, and soluble factors released by cells in the niche to help maintain the phenotype of the liver stem cells. The location of these niches has been specified at two possible locations: in the Canals of Hering [109, 155] or in the periductular/intraportal zone of the liver [20, 137]. Regardless of their location, the purpose of hepatic stem cells is thought to be for the maintenance or repair of a specific tissue. As a result, hepatic stem cells could potentially be an optimal source for in vivo tissue repair of the liver. During development, hepatoblasts (or intrahepatic oval progenitor cells) give rise to specific cells of the liver, including hepatocytes and cholangiocytes [44]. Recent studies suggest that these stem cells have tremendous potential for replication and the repopulation of hepatocytes in liver failure models. However, few oval cell-specific markers exist to aid in their identification and isolation.
COMMITTED PROGENITOR CELLS Committed progenitors are cells derived directly from determined stem cells (e.g., oval progenitor cells), with a very restricted ability to differentiate into multiple cell types. These cells are often the precursors to a single fully differentiated adult cell type. For example, in the liver there are committed hepatic and biliary progenitors that differentiate into hepatocytes and bile duct epithelial cells. The former are called primary hepatocytes (or small hepatocytes) and differ from mature hepatocytes in that they are direct progenitors of the mature cells with full capacity to regenerate. Because these cells are phenotypically closest to the functional cells of the liver, the development and subsequent use of committed progenitor cells for cell-based liver therapies would
translate into an improved therapeutic opportunity to recapitulate acinar heterogeneity.
FULLY DIFFERENTIATED HEPATOCYTES Hepatocytes are the most important and best studied of the cell types comprising the liver. Hepatocytes can be isolated and used in many forms, including freshly isolated mature and primary cells, cultured cells, and transformed cells from hepatocarcinomas. As a result of their essential role to liver function, mature hepatocytes are the most common cellular component in cell-based therapies today. These cells require specific environmental cues to maintain the necessary hepatic phenotype in vitro. Creating a stable extracellular environment is essential for the stabilization of any mature hepatocyte culture. Mature cells have been used in extracorporeal devices as well as in hepatocyte transplantation studies. Mature hepatocytes can be obtained by perfusing with a solution of collagenase in intact or resected liver [71]. In addition to utilizing mature primary hepatocytes, the development of hepatic cell lines for use in cellular therapy is a strategy aimed at overcoming the limited growth potential of mature cells. Transformed hepatocytes have been altered genetically such that they have a near limitless capacity to maintain growth in vitro. Transformed hepatocytes have been extensively utilized in an attempt to model and recapitulate liver function in vitro. For instance, the human hepatoblastoma HepG2 cell line is widely used in the deployment of hollow fiber cartridge-based BALs [62]. Transfer of specific viral oncogenes that can divide while expressing an intermediate stage of differentiation is a process called immortalization. For example, the SV40 T antigen protein — which binds to the cell cycle regulator proteins Rb and p53 — has been utilized to reinitiate proliferation in quiescent hepatocytes [67, 81, 162]. Unlike totally transformed hepatocytes, these immortalized cells can maintain hepatic function without expressing tumorogenicity. Despite this advantage, immortalized cells still have their shortcomings. For example, over time immortalized cell lines such as the SV40T hepatocyte lines senesce and enter crisis phase. Furthermore, immortalized hepatic cell lines do not perform all of the necessary liver-specific functions that mature and primary cells do, and they do not respond to many important physiological cues necessary for proper tissue formation.
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CELL SOURCES
TABLE 12.1 Cell sources for liver therapies.
When studying cell-based therapies, a challenging problem is obtaining a tissue or cell source that is in suitable condition while balancing functionality with viability and safety. Consequently, it is imperative to choose a cell source that is easy to obtain while having a predictable phenotype and a robust expression of desired functional traits. The supply of allogeneic or autologous human tissue is unpredictable at best. In general, primary human tissues are difficult to preserve and culture for long periods of time, and there is always a concern that a disease from harvested cells may be transmitted to an ill or immunocompromised recipient. From totipotent stem cells to fully differentiated hepatocytes, primary allogeneic or autologous tissues are, however, the most ideal sources for cells when considering the development of cell-based therapies because they are the most physiologically and functionally relevant strategies for replacement. Cell lines are a popular source for cells because they are easy to handle, expand, and store. Unfortunately, many cell lines are tumor-derived immortalized lines, which cause concern for metastasis or for the transmission of infectious agents from the tumor cells. Consequently, cell lines can be studied and used as a model in vitro but their potential for use in humans is very limited and probably unlikely. Even the use of partially transformed non-tumor-derived or normal cell lines is unlikely because culturing these cells inherently causes partial or complete transformation of cellular function. Thus, these “normal” cell lines are not able to mature through their usual lineage to yield cells that are capable of intermediate or advanced functioning [118, 119, 138]. Finally, the use of xenogeneic tissues, especially porcine tissues, has garnered much attention in the field of cellular transplantation due to the unlimited source and controlled environment in which the animals are raised [4, 5, 18]. Porcine hepatocytes exhibit functional capacities similar to that of human hepatocytes with respect to urea synthesis, albumin production, and P450 cytochrome activity [35, 154]. However, there is a concern that such tissues would propagate the transfer of zoonoses that are nonpathogenic in animals into humans, with the potential for pathogenesis to occur. Therefore, due to the concerns regarding the use of cell lines and xenogeneic tissues for cellular transplant therapies the use of human-derived cells is currently the
Cell Source
Critical Issues
Sourcing, ability to expand, Primary cells: safety (PERV), phenotype Human (autologous, allogeneic) stability, immunogenicity Xenogeneic (porcine) Immortalized/transformed: Simian virus 40 (SV 40), telomerase, tumor-derived, spontaneously immortalized
Safety (suicide genes), tumorgenicity, efficacy, genotype stability
Sourcing, differentiation, Stem cells: phenotype instability, Embryonic, hematopoietic, safety (tumorgenicity), mesenchymal, liver progenitors immunogenicity Used by permission of Mary Ann Liebert, Inc.: Tissue Engineering, (2002, tbl. 1, p. 727).
most appropriate choice because they present much less risk to the recipient. However, much work must be done to ensure that allogeneic or autologous cells are readily available; easy to use, manipulate, and store; and functionally viable in an engineered system for therapeutic benefit. Potential cell sources for cell-based therapies are detailed in Table 12.1.
CELLULAR NICHES OF THE LIVER The microenvironments that cells reside within play an essential role in determining their gene expression and growth potential, which have been recognized as lineage-dependent factors. Consequently, the position of a cell within its lineage is due to at least a combination of three environmental factors: (1) autonomous intracellular mechanisms that are dependent on the time of cellular division or division number, such as controls on mitosis and cytokinesis, (2) signal transduction mechanisms activated by cell-cell interactions, gradients of soluble factors, nutrients, and gases, and gradients of extracellular matrix components, and (3) mechanisms affecting the mass transfer of soluble factors, nutrients, and gases to the cells. Despite all of the known factors that induce and promote cell growth and differentiation, it is imperative to choose a type and source of cells that have predictable phenotypes. In this regard, more than any other factor, microenvironmental cues regulate and stabilize the phenotype of hepatocytes and other hepatic cells as they
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grow and proliferate. If hepatic cells do not have the proper environmental cues, they can rapidly lose mature morphology and potentially dedifferentiate — causing them to lose necessary functional capabilities. Some of the most important environmental cues that should be recapitulated when designing cell-based therapeutics for liver disease, especially with tissue-engineered constructs, include an appropriate extracellular matrix as found in the space of Disse, physiomechanical stimuli as a result of the sinusoidal blood flow, and cell-cell interactions present in the hepatic cord.
EXTRACELLULAR MATRIX Extracellular matrix (ECM) is an insoluble group of molecules produced by most cells and found between homotypic cells at their lateral borders and between heterotypic cells at their basal borders. The lateral ECM in the liver consists primarily of cell adhesion molecules and proteoglycans but not collagens, fibronectins, or laminins, whereas the basal ECM consists primarily of cell adhesion molecules, proteoglycans, basal adhesion molecules (fibronectins or laminins), collagens, tentactin, and tenascin. A major function of the ECM is to provide an architecture upon which cells can grow in three dimensions and provide appropriate signals that are transmitted by specific cell surface receptors. The ECM can also serve to stabilize or immobilize soluble signals.
(EGF), fibroblast growth factor (FGF), interleukin6 (IL-6), transforming growth factor (TGF-a and TGF-b), and insulin-like growth factor (IGF) control the growth and differentiation of hepatic progenitor cells and hepatocytes. Corticosteroids, amino acids, nicotinamide, and dimethyl sulfoxide (DMSO) are used to stimulate hepatic function and differentiation.
CO-CULTURING INTERACTIONS Hepatocytes exhibit increased functional activity when co-cultured with other hepatic cells [87, 93]. For instance, rat primary hepatocytes cultured with fibroblasts [9] or endothelial cells [42] are better able to maintain their viability and function. In addition, co-culture with bone marrow stromal cells [95] or non-parenchymal liver cells [87] facilitates proliferation and differentiation. Cells from the islets of Langerhans in the pancreas encourage hepatic growth by supplying hepatic cells with essential hormones, mainly insulin and glycogen [59]. Lower cell densities promote proliferation, whereas higher cell densities drive hepatic differentiation [149]. The restoration of hepatic cell interactions within spheroid aggregates promotes the formation of bile canaliculi, gap junctions, and tight junctions [17, 73, 130]. The environment of the hepatocyte as shaped by both the surrounding cell population and ECM plays an integral role in shaping tissue morphology and function.
SOLUBLE SIGNALS
Discussion: Current Cell-based Transplants There is a range of soluble factors that are required for Liver Disease for hepatic growth, proliferation, and differentiation, including autocrine factors (those factors produced by a cell that act on the same cell), paracrine factors (factors produced by one cell that act on a neighboring cell or cells), and endocrine factors (factors produced by one cell that act on a target cell population of a different tissue type a distance away, carried by the blood or lymph). Culturing media can be supplemented with such factors as hormones, corticosteroids, cytokines, and amino acids to help stabilize cell morphology, survival, and function. Defined media containing growth factors, cytokines, and other molecules have been developed to support hepatocyte proliferation or to induce differentiation [12, 28, 54, 70]. Hepatic growth factor (HGF) regulates hepatocyte growth, proliferation, and motility. Epidermal growth factor
HEPATOCYTE TRANSPLANTATION An early study demonstrated the transplantation of hepatocytes into the red pulp of the spleens of rats [94]. Further studies showed that these ectopic hepatocytes were functional, exhibited extensive proliferation while surviving for at least 21 months in vivo, and formed hepatic-like structures [23, 134]. Inclusion of non-parenchymal liver cells with implanted hepatocytes increased cell survival and function [135]. The development of therapeutic cellular transplants for liver disease requires three essential elements: (1) adequate cell mass, (2) satisfactory engraftment of transplanted cells, and (3) subsequent proliferation to repopulate the native liver [136]. In addition, the approach will depend on
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the nature of the disease being treated. Certain genetic disorders affecting the function of a single enzyme will require a different approach than treating chronic liver failure caused by cirrhosis. The hepatic cell mass required to correct a single gene defect affecting a single enzyme is significantly less than that required to treat chronic or acute liver failure. In specific instances, 1 to 5% of liver mass was sufficient to replace lost function resulting from a single enzyme defect [36, 100, 135]. However, chronic or acute liver failure would presumably require significantly more than 10% of the total liver mass to be transplanted to provide adequate liver function. Transplanting a sufficient number of hepatocytes to replace liver function during total organ failure remains a significant challenge. In the near term, hepatocyte transplantation is likely to be restricted to the treatment of specific diseases and will not be a therapeutic option for treating fulminant hepatic failure. Engraftment of implanted hepatocytes occurs through a mechanism that requires initial damage to the native liver and prevention of native hepatic regeneration. The initial site of hepatocyte engraftment occurs within the sinusoids, causing both acute portal hypertension and transient ischemia-reperfusion injury [136]. Portal hypertension usually resides after 2 to 3 hours, but the reperfusion injury can last for several months as a result of the interference to normal blood flow caused by opening of additional vascular channels and infusion [48]. Consequences of ischemia reperfusion events include enzyme induction, disruption of gap junctions, activation of apoptotic pathways, and activation of Kupffer cells. TNF-a released by Kupffer cells increases endothelial cell permeability, improving access of transplanted cells to the hepatic plates. However, the TNF-a can also induce inflammation resulting in clearance of the transplanted cells.
models for the direct transplantation of hepatocytes in vivo demonstrated that a single hepatocyte has the potential to undergo 34 population doublings, giving rise to approximately 1.7 × 1010 cells [125]. Initial seeding mechanisms used intravenous or peritoneal administration of a hepatocyte suspension, primarily due to the ease of accessibility. However, later studies showed that recapitulation of liver-specific function requires a microenvironment resembling that of the liver, including a basement membrane substrate and a venous blood supply to promote active cell attachment and induce needed biophysical forces [37]. Due to this fact, the splenic pulp and host liver itself have become the primary sites of implantation [47]. Initial studies of direct transplantation used the stromal tissue of the host in addition to the hepatocytes to provide a matrix for the hepatocytes to grow and differentiate on [11, 83, 96]. Results demonstrated that there was some level of correction for metabolic defects and induced liver failure during periods ranging from 2 weeks to 1 year [82, 106, 159]. However, these same studies revealed significant necrosis and granuloma formation in the transplanted hepatocytes throughout this implantation period [50]. Moreover, further studies examined the implantation of hepatic aggregates intrasplenically for up to 2 years. These aggregates revealed improved long-term survival and more fully differentiated hepatic function relative to individual hepatocytes, despite the decreased rate of proliferation seen with the aggregates [129, 130]. In addition, early studies were complicated by discrepancies in distinguishing transplanted cells from native host cells, and difficulties quantifying the mass of liver tissue needed to partially or fully replace liver function [105, 151]. Methods have been established to overcome the former, such as labeling transplanted cells with the fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl-ester.
DIRECT INJECTION
MICROCARRIER TRANSPLANTS
Perhaps the simplest form of adjunct internal liver support is the direct transplantation of hepatocytes into a host. In general, this form of treatment is most beneficial to patients with non-life-threatening inherited metabolic diseases such as Crigler-Najjar syndrome type I because the efficiency of hepatocyte engraftment is quite low [10, 36]. Mature adult hepatocytes directly injected into a recipient have the potential to engraft, proliferate extensively, and sustain liver function. In fact, early
As an alternative to directly injecting hepatocytes into a host, hepatocytes have been injected as a component of microcarrier systems. Microcarriers have the distinct advantage of giving cells important ECM features that critically affect cell survival and function [11]. Furthermore, microcarriers offer a large surface area for hepatocyte attachment (allowing for the transplantation of a higher cell density), and offer sites for bioactive molecule attachment, such as proteins, to enhance the
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delivery of localized factors. One major disadvantage of microcarriers is their inability to reproduce a 3D structure like that of native tissue. Thus far, the most encouraging microcarrier studies have involved the attachment of hepatocytes to type I collagen-coated dextran beads [25–27, 99]. The functionality of the attached cells was examined by monitoring how certain metabolic defects were corrected in Gunn rats that are UDP-glucuronyltransferase deficient [34] and that demonstrate high accumulation of unconjugated serum bilirubin [46]. In addition, Nagase analbuminemic rats were studied, which model an inherited defect in albumin synthesis, resulting in the production of only trace amounts of plasma albumin [101]. Both species of rat displayed improvements in metabolic function over 1 month. However, the data did not suggest any improvement during long-term studies [27]. Additional work using hepatocytes attached to various microcarrier systems in these species of rat subjected to a lethal liver resection in controls demonstrated similar capabilities of providing sustained metabolic support for up to 1 month [26]. Secondary to sustaining metabolic function, these studies showed that certain microcarrier systems could promote intraperitoneal engraftment of hepatocytes without signs of significant inflammation or toxicity while inducing cell organization and neovascularization within the microcarrier-hepatocyte aggregates [26, 27]. Furthermore, more advanced materials were used to fabricate microcarrier beads, such as modified poly(2-hydroxyethyl methacrylate) (pHEMA), to achieve better attachment of hepatocytes for culture in bioreactors before injection [29–31]. In addition, more recent studies using porous poly(lactic-co-glycolic acid) (PLGA) as a polymer for microcarrier beads have shown that biodegradable biocompatible materials coated with permissive ECM (e.g., heavily cross-linked dextran coated type I collagen) are the most ideal materials for attachment and expansion of hepatocytes ex vivo. However, further studies will be needed to prove that PLGA is an appropriate microcarrier material for long-term hepatocyte survival in vivo.
hepatocytes within a semipermeable membrane is that the therapeutic cells are isolated from the host to prevent undesired immune response as a result of antibodyantigen interactions between host and transplant. In addition, this barrier allows for the reproduction of essential environmental cues and features of the liver while allowing for the identification of transplanted cells to quantify survival and viability. Moreover, encapsulation also promotes essential transport of nutrients and oxygen into the capsule due to the small size of such molecules. However, despite this advantage mass transport of these molecules into the center of the aggregate becomes severely limited as the volume of the aggregate increases (i.e., volume is proportional to the radius cubed), causing cells at the center to lose function and undergo necrosis [113]. Consequently, the size of the aggregate is limited, which directly reduces the therapeutic potential of this approach. Collagen and alginate are the most popular encapsulating materials because they form a thin layer around the cells in the core region, as shown in Figure 12.3 [17, 38, 39, 52, 104, 165]. Encapsulation of hepatocytes within an alginate-polylysine membrane has been investigated due to successful encapsulation and delivery of therapeutic allogeneic and xenogeneic islets of Langerhans [80, 150]. In vitro studies over 5 weeks with this membrane material have demonstrated continuous, although decreasing, protein and metabolite
ENCAPSULATED TRANSPLANTS In addition to attaching hepatocytes to microcarrier systems to improve the engraftment of cells into the host, hepatocytes have been encapsulated within semipermeable membranes to improve cell survival and viability. The primary advantage of encapsulating
Figure 12.3. Encapsulated hepatocytes. Photograph of spheroids composed of alginate, a natural biodegradable polymer, encapsulating mature hepatocytes. Spheroid diameter is approximately 300 mm. Scale bar is equivalent to 60 mm.
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production [13]. In vivo studies in Gunn rats showed that alginate-polylysine encapsulated hepatocytes decreased serum bilirubin levels for up to 4 weeks while increasing the survivability of these animals by up to 80% when subjected to a model for fulminant hepatic failure [31, 165]. However, no such function has been demonstrated over longer periods of time. Because autologous and allogeneic sources of hepatocytes are difficult to obtain, and because no immunosuppressive therapy currently exists to mediate xenogeneic rejection, scientists have examined the feasibility of encapsulating xenogeneic hepatocytes into small microspheres to deliver therapeutic benefit. Theoretically, encapsulating xenogeneic or allogeneic hepatocytes within a semipermeable membrane should provide a necessary barrier to protect transplanted cells from the immune system of the recipient. This would be accomplished by preventing high-molecular-weight antibodies and complement in addition to leukocytes from reaching the hepatocytes [16]. One study demonstrated that encapsulated porcine hepatocytes were able to remove ammonia during liver failure and amino acids, such as phenylalanine, during phenylketonuria [75]. However, results such as these have not been universal and long-term recapitulation of hepatic function using encapsulated hepatocytes has not been demonstrated. In summary, the transplantation of hepatocytes directly or within the auspices of a microcarrier or encapsulation system has provided evidence of sufficient short-term hepatic support for many non-lifethreatening metabolic diseases of the liver. However, it is apparent that these therapeutic modalities are very limited with respect to offering fully functional liver replacement therapies for patients with long-term and/or end-stage liver disease. Thus, other options must be developed to accommodate this need, including the use of stem cell therapies and implantable tissue-engineered constructs.
STEM CELL THERAPIES Research in recent years has suggested that there are several different candidate stem cells that have the potential to be sources of functional liver cells for transplantation. The list of proposed cells includes ESCs, intrahepatic oval progenitors, and determined non-hepatic adult stem cells. What is not clear at this time, however, is how to isolate these cells efficiently, how to regulate differentiation effectively, and by what
mechanisms these cells can be converted into functional liver cells (whether it is through differentiation, transdifferentiation, or possibly fusion with other cells). Nonetheless, a number of criteria must be met before stem cells can be therapeutically beneficial, including (1) extensive expansion in vitro, (2) differentiation into mature hepatic cells, (3) the introduction of minimal immunogenicity, and (4) reconstitution of functional liver tissue when transplanted in vivo. Totipotent or pluripotent ESCs that give rise to every cell type in the body are an appealing choice for a cell-based transplant therapy to fight liver disease. Differentiation of ESCs in vitro is challenging, but it does to some degree replicate normal embryonic development. Furthermore, until recently it has been very difficult to unequivocally exhibit the differentiation of ESCs into hepatocytes due to the lack of hepatic markers, until markers such as OV-6 (monoclonal antibody against cells isolated from carcinogen-treated liver) were discovered. To date, one of the most convincing arguments for ESC differentiation into hepatocytes was demonstrated by Jones and colleagues [58]. They used mouse embryos generated with a gene trap insertion (I.114) into an ankyrin repeat-containing gene (Gtar). Results of the gene trap showed exclusive expression of b-galactosidase in early liver development, suggesting that this was indeed differentiation into hepatocytes — later confirmed by coexpression of other functional hepatic markers including albumin and transferrin [58, 161]. Other groups have demonstrated in vitro differentiation of rat and human ESCs into hepatocytes with expression of hepatic markers such as a-fetoprotein and albumin [19, 76, 167]. However, despite these promising results data also suggest that ESC use in cell transplantation for patients suffering from liver disease may be precluded by their tumorigenic potential [43, 86, 98, 103, 112, 131]. Currently, the tumorogenicity of embryonic stem cells is being investigated to establish means by which this property can be controlled [6, 86, 112]. In addition, it is still unclear whether or not ESC differentiation into hepatocytes yields functional cells and a sufficient number of cells to be useful in cell-based therapies. Consequently, major questions must be answered with further experimentation to prove that ESCs are a viable option for cell-based therapies for liver disease. Currently, resident hepatic stem cell progenitors are being investigated for their utility in replacing lost liver function in vivo. Recent findings have shown that following liver injury with agents known to induce
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carcinogenesis there is significant proliferation of cells at the canal of Hering that have oval nuclei and a high degree of proliferation [155]. In addition, identical cells are found in the pancreas of animals that have been subjected to a diet lacking copper. Following oval cell production and the reintroduction of copper into the diet, there is a subsequent appearance of hepatocytes in the pancreas derived from these oval cells that exhibited specific hepatic markers. Upon implantation into rats, these pancreatic oval cells started to express proteins characteristic of hepatocytes [21]. Recently, there have been widespread debates about the potential of certain determined adult stem cell types to differentiate into mature cells of a different cell type through a process called metaplasia. There have also been numerous reports and a growing body of evidence suggesting that differentiated cells of one specific tissue type are converted to another differentiated cell type through adult stem cell intermediates in a process called transdifferentiation. Extrahepatic stem cells thought to undergo metaplasia and transdifferentiation are bone-marrow-derived stem cells (e.g., hematopoietic and mesenchymal stem cells) and pancreatic stem cells. Although determined adult stem cells have a lower degree of potency, they have not been found to be tumorigenic like ESCs, making them a more popular choice for research and clinical programs devoted to cellular transplantation or bioartificial organ development [6, 51, 117]. Although bone-marrow-derived stem cells are inherently derived from the embryonic mesoderm, the developmental potential of these cells is not limited to mesodermal fate because they can populate endodermal and ectodermal tissues. In fact, numerous studies document the ability of hematopoietic and mesenchymal stem cells to differentiate into hepatocytes and cholangiocytes and serve as intermediates during the transdifferentiation of pancreatic cells [24, 41, 55, 79, 85, 115, 123, 142]. Moreover, this evidence also shows that cells of macrophage-monocyte lineage are involved in this metaplastic conversion. Despite the evidence for this conversion, the frequency of differentiation has been very low (i.e., 10−4 to 10−6 ), many results have been difficult to replicate, and there has been limited repair of liver damage. As a result, these stem cells are not the primary vehicle for liver repair under physiological and most pathological conditions [45]. This evidence suggests, however, that exogenous bonemarrow-derived stem cells have a more pronounced role if extensive liver regeneration has depleted the normal
hepatic pool of progenitors. Ultimately, the capacity for marrow-derived stem cells to replace endogenous hepatocytes lost to trauma and disease depends on their ability to differentiate and expand into functional hepatocytes in vivo. Consequently, the differentiation of bone-marrow-derived stem cells must be more efficient and better understood before therapeutic applications can be developed. Transdifferentiation is thought to involve a change or shift in the transcription of a subset of specialized genes responsible for differentiation, clonally changing the phenotype of marrow-derived stem cells or pancreatic stem cells into that of a hepatocyte. Thus far, direct transdifferentiation of cell populations through hematopoietic cell intermediates into hepatocytes has also not been conclusively shown to be a mechanism for liver regeneration in vivo. The only evidence for the direct transdifferentiation of marrow-derived stem cells into hepatocytes is the lack of a demonstrated alternative mechanism [157]. Perhaps simpler mechanisms can explain the presence of hepatocytes in hematopoietic cultures [including metaplasia] or the reprogramming of hematopoietic nuclei during prolonged culture, which would effectually increase their plasticity to generate hepatocytes [57, 127, 143]. In addition, the transformation of mesenchymal epithelial cells could potentially explain the acquisition of hepatocyte phenotypes and function by some hematopoietic stem cells in culture [57, 120]. Despite the lack of direct evidence, it has not been ruled out that hematopoietic stem cells — if cultured extensively — could increase their plasticity and differentiate into hepatocytes [57, 127, 143]. One mode of transdifferentiation that has been extensively studied and characterized is the conversion of pancreatic cells into hepatocytes. Both tissue types arise from the endoderm during development. The appearance of hepatocytes in the pancreas has been demonstrated in rodent models in response to various experimental conditions including copper depletion of the diet followed by copper repletion after transplantation of epithelial cells and in transgenic mice overexpressing keratinocyte growth factor in the pancreas [21, 69, 121, 122]. Thus far, two distinct in vitro models have been developed to demonstrate the transdifferentiation potential of pancreatic cells. The first utilizes the pancreatic cell line AR42J and the second uses embryonic pancreatic tissue from mice [72, 139, 140]. In each case, the transdifferentiated hepatocytes express proteins normally found in native liver — including albumin, transferrin, and glucose-6-phosphatase [140].
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Some studies have discredited the process of metaplasia or transdifferentiation in the liver, in favor of a new mechanism for hepatic regeneration referred to as cell fusion [160, 169]. Fusion in the liver occurs when a hepatocyte fuses with another cell to form a heterokaryotic hybrid cell that initially contains the cellular organelles of both cell types. To express the hepatocyte phenotype of such a hybrid cell, the nucleus of the non-hepatic cell must be extruded or its genes must be reprogrammed to allow for the translation of hepatocyte-specific proteins [157]. The fusion of hepatocytes with hematopoietic cells in Fah−/− mice has been shown in three independent studies that use three independent methods for cell detection [14, 158, 160]. Moreover, the hematopoietic nuclei of the heterokaryotic hybrid cells demonstrated downregulation of hematopoietic genes and the up-regulation of hepatocyte genes to correct the metabolic deficiencies characterized by the Fah−/− hepatocytes [158]. Perhaps the best characterized evidence for fusion has been seen between hepatocytes and cells of the myeloid
lineage (e.g., macrophages and granulocyte-macrophage precursors) in both healthy animals [1, 146] and in Fah−/− animals [14, 146], as depicted in Figure 12.4. These later studies also showed that hematopoietic cells do not undergo a secondary fusion with hepatocytes in the form of hepatocyte-hepatocyte fusion, which would imply that hematopoietic differentiation into hepatocytes occurs first. In addition to the differentiation and/or fusion of determined stem cells into mature functional hepatocytes, there is extensive evidence that resident intrahepatic hematopoietic stem cells help maintain the replacement of hepatocytes through indirect mechanisms. These mechanisms include the production of cytokines and growth factors that support hepatocyte formation during embryogenesis [53, 66]. In fact, numerous studies modeling liver failure have demonstrated that infused hematopoietic cells do indeed support liver function and repair in adult animals through the production of critical cytokines and growth factors without the formation of new hepatocytes [2, 168].
Polymorphonuclear granulocyte
MEP CLP HSC CMP
GMP
Macrophage
Transdifferentiation?
Hepatocyte
Fusion
Fusion
Fusion
Figure 12.4. Fusion as mechanism for liver regeneration. Both Willenbring et al. [164] and Camargo et al. [14] give strong evidence that the liver regenerates following bone marrow transplantation through the fusion of hepatocytes and hematopoietic stem cell progenitors, most likely macrophages. Shown are the differentiation of hematopoietic cells to their myeloid lineages and the proposed mechanism of fusion. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; MEP, megakaryocyte-erythroid progenitor. (Reprinted by permission from Macmillan Publishers Ltd: Nature Medicine, 2004.)
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These results are proof that determined stem cells can have alternative benefits to in vivo tissue repair, separate from serving as a potential cell source. Some of the major problems associated with using stem cells for liver transplant therapies include the limited availability of normal tissue, the lack of an effective purification scheme to isolate these cells, the insufficient identification of proper preservation techniques and conditions, our inability to define proper ex vivo expansion and differentiation conditions before implantation, and our lack of understanding of how tissue-specific stem cell niches can be vital determinants of the specificity of differentiation of stem cells. As a result, these issues must be resolved before stem cells and their mechanisms of differentiation can be fully understood as potential methodologies for hepatocyte regeneration.
IMPLANTABLE TISSUE-ENGINEERED LIVER Tissue engineering is truly an interdisciplinary field that applies principles and methodologies from engineering, cell and developmental biology, and materials science (among others) to help create relevant biological alternatives for tissue and/or organ transplantation following disease or traumatic injury. The field of tissue engineering has emerged over the last two decades to address the serious problems associated with transplantation, including the shortage of available donor organs, the necessity for life-long immunosuppression, and the potential for serious complications during surgery. To date, there has been much progress in the field of tissue engineering — especially with respect to the development of dermal and connective tissue replacements — but further work must be done to develop more complex and highly vascularized vital organs such as the liver and kidneys. Currently, however, rapid advances are being made in areas such as cell typing and sourcing, coculture optimization, scaffold design and manipulation, systems modeling, and microfabrication technology to help design complex tissue-engineered systems for vital organs. In addition, the search for and use of an appropriate stem cell type in tissue engineering is an emerging concept that will play a major role in the fabrication of tissue-engineered vital organs [64]. Now, as a result of these advances, tissue engineering has become a major thrust area in the rapidly growing field of regenerative medicine.
Strategies for tissue engineering can be categorized into one of two categories: acellular matrices and cellularized constructs. The approach of implanting acellular matrices is dependent on the body’s ability to repopulate the matrix with proper orientation and population of functional cells. Acellular matrices are generally prepared by enzymatic digestion of cellular components to produce collagen-rich matrices that slowly degrade after implantation, allowing ECM and native cells to repopulate the matrix. However, this approach is not as widely used in tissue engineering of the liver as in other organs such as the bladder. The paradigm of cell-based tissue engineering, as seen in Figure 12.5 combines living cells with a natural or synthetic biodegradable polymer scaffold and appropriate growth factors to create a 3D tissue construct that is functionally and structurally similar to native tissue either upon or after implantation. Consequently, the development of a living construct for implantation requires several important features, including (1) appropriate cell type(s) and source(s), (2) custom-designed polymeric scaffolds to serve as an ECM for cells, (3) hepatotrophic stimulation with soluble factors essential for cell growth, proliferation, and/or differentiation, and (4) an in vitro bioreactor configuration to cultivate the construct in an in vivo-like environment before implantation. Thus, when cellular constructs are formed for tissue engineering purposes a small piece of donor tissue is dissociated into individual cells, which are sorted then expanded in culture, attached to a scaffold, and reimplanted into the host following expansion within a bioreactor. Tissue engineering of the liver is extremely complex, involving numerous cell types, a high degree of vascularization, and microenvironments that must work in concert to form a fully functional organ. Hepatocytes are highly metabolic and require immediate access to oxygen and nutrients to sustain function. Despite the challenges involved, this approach to treating liver disease is very promising because it would offer patients a potential treatment alternative for end-stage liver disease that is permanent and functions more like native tissue.
CELL TYPING AND SOURCING The source of donor tissue for tissue engineering can be heterologous, allogeneic, or autologous. The preferred cell source for liver constructs is autologous hepatocytes
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Tissue-engineered organ
In vitro bioreactor: Tissue expansion through: • Growth • Proliferation • Differentiation
Cell type(s)/source(s):
Scaffold:
Soluble factors:
Requirements: • Recapitulate vital organ functions • Limit immune responses • Easy to isolate, purify, and cultivate • Expansion in vitro to appropriate mass • Preserve and store when needed
Requirements: • Provide 3D architecture • Provide or direct vascularization • Mimic native ECM • Deliver appropriate nutrients, O2, and growth factors • Remove metabolic wastes
Requirements: • Stimulate growth, proliferation, and differentiation of transplanted cells • Regulate other cell cycle activities • Not affect native environment once implanted
Figure 12.5. Tissue engineering paradigm. Fundamentally, tissue engineering is an interdisciplinary field that utilizes a combination of cell types and sources, biomaterials as scaffolds, and suitable biochemical factors to improve or replace biological functions through the creation of new tissues and organs [74].
as they do not initiate an autoimmune rejection in the recipient. Differentiated hepatocytes are often difficult to culture in vitro due to their low proliferative ability. Consequently, freshly isolated hepatocytes and other parenchymal cells of the liver with high viability are often used in tissue engineering. Techniques for harvesting parenchymal cells from the liver have been described extensively in the literature, primarily through a two-step collagenase digestion [8, 133]. In addition to mature hepatocytes, highly proliferative hepatocytes have been discovered in non-parenchymal cell fractions of the liver [91, 92, 153]. These cells, known as small hepatocytes, are direct progenitors of mature hepatocytes, and because they have a high capacity for proliferation they may be a better source for initial seeding of scaffolds. Co-cultures of hepatocytes with other parenchymal and/or nonparenchymal cell fractions have demonstrated improvements in the morphology of tissue
regeneration in vitro, in vivo, and as a part of tissue engineered constructs. For instance, co-cultures of hepatocytes and cholangiocytes have demonstrated ductal formation both in vitro and in vivo [144], with histological evidence of their formation in polymer scaffolds. In addition, hepatocytes co-cultured with nonparenchymal endothelial-like cells revealed an increase in the level of ECM production, whereas co-culture with pancreatic islet cells aided hepatocyte survival, growth, and maintenance [56, 148]. From these studies it is known that hepatocytes should be co-cultured with other hepatic cells for maximum viability and function.
SCAFFOLDING: THREE-DIMENSIONAL SPATIAL ARCHITECTURE Following the in vitro expansion of donor cells, they must be seeded statically or dynamically onto a support vehicle for implantation. As a result, 3D scaffolds for use
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in tissue engineering must perform a variety of essential functions needed to stimulate the development of an appropriate functional cell mass for implantation. Therefore, an ideal scaffold should mimic the native ECM to help sustain and guide the growth, proliferation, differentiation, and gene expression of cells by regulating the appropriate mass transfer of nutrients, oxygen, and soluble factors to the developing tissue. In addition, the scaffold should be biodegradable so that as the growing tissue expands into the native environment it will not be restricted or constrained by the scaffold. Furthermore, scaffolds must provide adequate surface area and void volume for cell attachment and migration. Finally, scaffolding materials should be biocompatible, meaning that they invoke minimum immune responses upon implantation and degradation. As a result of these considerations, some of the most critical design features of scaffolds include its physical and chemical properties, 3D architecture, surface chemistry, and mechanical and degradation properties. When designing cell-based constructs for tissue engineering applications, cells are first seeded onto scaffolds synthesized or derived from an appropriate biomaterial and then cultured in an appropriate organ-specific environment. Because most mammalian cell types are anchorage dependent, the biomaterial of choice should provide a cell-adhesion substrate to stimulate cell attachment and cell-cell communication. In addition, biomaterials for tissue engineering must provide necessary mechanical support against in vivo forces to ensure that the predefined 3D structure is maintained during tissue development [68]. Moreover, specific biomolecules, such as growth factors and soluble peptides, can be loaded into the construct with cells to help regulate cell function and morphology. Consequently, the biomaterial must not contain functionalities that will interfere with the delivery of important biomolecules. Biocompatibility is a major consideration when choosing a scaffolding material and is characterized by fibrosis, vascularization near the implant site, and local and systemic inflammation [3, 89]. An ideal material would be biocompatible, as it can degrade and be resorbed in vivo with minimal tissue response. If a chosen material is incompatible, it will elicit a major inflammatory and/or foreign body response that will lead to rejection and/or necrosis. Once the scaffold begins degrading, the breakdown products are metabolized and removed at a rate that prevents the buildup of these by-products in tissues of the body [7]. Recent attention to developing biocompatible materials as scaffolding
materials for tissue engineering has inspired the creation of many new synthetic or naturally derived polymers that have a wide range of properties and applications.
CLASSES OF POLYMERS Generally speaking, there have been three classes of polymers used to develop cell-based constructs for tissue engineering: (1) naturally derived materials (e.g., collagen and alginate), (2) acellular tissue matrices (e.g., bladder submucosa and small intestinal submucosa), and (3) synthetic polymers [e.g., polyglycolic acid (PGA), polylactic acid (PLA), and poly(lacticco-glycolic) acid (PLGA)]. Each of these classes of materials has been tested with respect to their biocompatibility [110, 111], and each demonstrates a different degree of compatibility depending on the breakdown products and the level of the immune response generated upon implantation. Naturally derived materials and acellular matrices are advantageous because they can induce less of an immune response in vivo as a result of better biological recognition. However, these materials are not as readily available and their physical properties are not reproducible from batch to batch. Conversely, synthetic polymers can be reproduced on a large scale with controlled physical properties of strength, degradation rate, and microstructure, but they often induce more of an immune and/or inflammatory response than their natural and acellular counterparts. The types of scaffolding materials used in tissue engineering thus far are outlined in Table 12.2. The most widely used natural materials for tissue engineering scaffolds are collagen and hydrogels such as alginate. Collagen is the most abundant structural protein in the body and is a major component of many ECMs from connective tissue to the liver. It can be extracted from tissue through treatment with enzymes and a salt/acid extraction [78]. Although collagen is degraded in vivo by lysosomal enzymes, varying the density of the implant and the extent of cross-linking of the collagen chains can control its rate of degradation. In addition, collagen contains many cell-adhesion domain sequences (e.g., RGD) that may promote retention of native phenotype. Collagen has been used in many forms for hepatic applications, including as a coating on plastic surfaces to provide attachment for hepatocytes [141], for encapsulation of hepatocytes to form spheroids [52, 77, 166], and to entrap hepatocytes in porous synthetic sponges [60, 152]. Although it
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TABLE 12.2 Classification of scaffolding materials for tissue engineering. Material Class
Material Type
Naturally derived
Collagen, hydrogels (e.g., alginate and Matrigel), chitosan Decellularized matrices: Bladder submucosa Small intestinal submucosa
Biologic acellular
Synthetic polymers
Biodegradable: Polyglycolic acid (PGA) Polylactic acid (PLA) Poly(lactide-co-glycolic) acid (PLGA) Poly-L-lactic acid (PLLA) Polycaprolactone (PCL) Polyhydroxybutyrates (PHBs) Polyethyleneglycol (PEG) Poly(glycerol-sebacate) (PGS) Non-biodegradable: Polyurethanes (PU) Polyethylenes (PE) Polysulfones (PS) (e.g., polyethersulfone) Polycarbonate (PC) Polydimethylsiloxane (PDMS)
Copyright 2004 from Tissue Engineering of the Liver by Sery et al. Reproduced by permission of Routhledge/Taylor & Francis Group, LLC.
is important for hepatocyte attachment, collagen is difficult to use as a scaffolding material due to its limited chemical and physical properties. Alginate, an FDA-approved polysaccharide isolated from seaweed, is composed of copolymers of D-mannuronate and L-guluronate at different concentrations depending on the alginate type. It has been used as both a cell immobilization matrix [80] and as an injectable cell delivery material [145] due to its gentle gelling properties in the presence of divalent cations. Moreover, alginate offers better physical characteristics for hepatocyte entrapment and function (i.e., enhanced albumin secretion and P450 function) when compared to using collagen. Consequently, it has been used to entrap hepatocytes and form spheroids for loading into synthetic scaffolds. Acellular biologic matrices are formed by decellularizing native matrices of the body, leaving a collagen-rich network upon which cells of interest can be seeded. They are often prepared by chemically and/or mechanically manipulating tissue to strip the cellular component
off its ECM. These scaffolds slowly degrade in vivo and are remodeled by transplanted cells as they grow into the scaffold. Unfortunately, despite their promise these scaffolds are not utilized for hepatic tissue engineering because of availability, quality of tissue, and concerns over immune rejection. Perhaps the most widely studied and utilized scaffolding materials for hepatic tissue engineering are those of the synthetic class — especially polymers composed of polyesters of naturally occurring a-hydroxy acids, including PGA, PLA, and PLGA [113]. These materials, like collagen and alginate, have gained FDA approval for use in humans as a result of their biocompatibility and degradation mechanism through nonenzymatic hydrolysis — a mechanism of degradation seen throughout the body. Moreover, the byproducts of their degradation are nontoxic natural metabolites that are eventually eliminated from the body as carbon dioxide and water [40]. Another advantage of using these polyesters is that they can easily be formed into any 3D shape with a highly defined microstructure and porosity that can degrade at a rate ranging from weeks to years depending on the molecular weights of the reactants, monomer mass distribution in copolymers, chain structure, and surface and internal pore structures [40]. Because designing scaffolds for hepatic tissue engineering requires a consistently defined 3D architecture to achieve proper tissue morphology, these synthetically derived polymers are an ideal choice in this regard. One drawback to these polymers is that they tend to lose mechanical strength after implantation, especially if the degradation rate is tailored to only a few months for complete resorption. Other synthetic degradable polymers used in tissue engineering include polycaprolactone (PCL), poly-bhydroxybutyrate (PHB), and poly(glycerol-sebacate) (PGS). PGS is a novel biodegradable elastomer formed from the esterification-type polycondensation reaction between glycerol and sebacic acid, which have both been previously approved for human use by the FDA. Many of the physical properties of PGS can be tailored to meet the needs of specific applications, including its degradation rate, elasticity, and surface chemistry for cell attachment. In addition, PGS is more biocompatible than most of its synthetic counterparts, is inexpensive to produce in large quantities, and is highly reproducible from batch to batch [71]. Consequently, PGS is potentially a more ideal polymer for hepatic scaffolds than most other synthetic polymers.
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POLYMER CONFIGURATIONS AND MODIFICATIONS
including portacaval shunting, partial hepatectomies, and chemical injury.
One of the primary goals when designing scaffolds for tissue engineering is to recreate, as accurately as possible, the native ECM that cells encounter in vivo. Due to the high metabolic demand of hepatocytes, scaffolds must be at least 90 to 95% porous to allow sufficient surface area for the exchange of nutrients and wastes [90]. Attempts at recreating the ECM of the liver within a scaffold have led to the modification of the polymer surfaces to enhance cell adhesion, spreading, and function. Modification types include micropatterning and integration of adhesion factors such as glycosaminoglycans (GAGs) and short peptide sequences comprising ECM proteins (e.g., collagen, fibronectin, elastin, and laminin). Two of the most common short peptide sequences added to the surfaces of scaffolds are Arg-Gly-Asp (RGD) — an adhesion domain found on collagen, fibronectin, and vitronectin — and Tyr-IleGly-Ser-Arg (YIGSR), an adhesion domain on laminin [15]. Intermediate molecules such as polyethyleneglycol (PEG) are often used to link adhesion factors to the surface of the polymer. In addition, certain hepatic genes can also be expressed during seeding to encourage attachment. Because hepatocyte integrins on the membrane bind to adhesion domains on the ECM (i.e., RGD and YIGSR), genes coding for integrins can be expressed for prolonged periods to allow for efficient attachment.
BIOREACTORS AND IMPLANTATION
SOLUBLE FACTORS Soluble factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor, insulin, transferrin, prolactin, triiodothyronine, and growth hormone [97] are critical components of tissue-engineered constructs because they are soluble peptides capable of binding to cellular receptors — causing either permissive or preventive signals to initiate differentiation and/or proliferation [163]. For hepatic constructs, other soluble factors such as hepatocyte growth factor (HGF), hepatic stimulatory substance (HSS), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) are important for hepatotrophic stimulation in vivo due to the competition transplanted cells have with native cells. To counteract the effect of native tissue, other specialized techniques for hepatotrophic stimulation are used,
One of the most significant factors involved with developing tissue-engineered constructs for implantation occurs during the expansion phase of the tissue when cells require a microenvironment that subjects them to vital physiological stresses, parameters, and constraints such as appropriate levels of pressure, sheer, flow, compliance, and resistance. To achieve these parameters, cell-growth systems (bioreactors) can generally provide more efficient delivery of O2 and removal of CO2 , and a more physiological pH. Consequently, during the cultivation of the construct in a bioreactor these environmental stresses must be considered and implemented into its design. A typical closed-circuit design for a bioreactor has several components, including a chamber with the scaffold and seeded hepatocytes, a pump and tubing to recirculate medium, a reservoir for excess media storage, an air trap to catch air bubbles, and an oxygenator to promote exchange of oxygen and carbon dioxide. Importantly, hepatocytes cultured on a scaffold within a bioreactor exhibit better function with a more stable phenotype compared to static 2D culture [65]. Following tissue development in a dynamic environment, tissue-engineered constructs are ready for implantation. The site of implantation is crucial because the construct needs exposure to an environment that will stimulate proper growth and function of cells in vivo. Consequently, the in vivo animal model must be chosen carefully.
CRITICAL FACTORS AND ADVANCES IN LIVER TISSUE ENGINEERING One of the primary limitations of developing cellbased tissue-engineered constructs for implantation is the inherent difficulties associated with growing specific cell types in large enough quantities needed for minimal replacement of liver-specific function. Even when considering the liver’s high capacity for regeneration in vivo, cell expansion in vitro is challenging. Certainly, the development of tissue-engineered cellular constructs that can recapitulate vital liver functions has been hindered by the inability to expand donor cells in vitro. However, recent advances have been made
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through the examination of tissue-specific stem cells and committed precursor cells and how they respond to natural environmental cues for growth, proliferation, and differentiation. As a result, problems that arise with in vitro expansion of cells in a tissue-engineered construct may be overcome as scientists continue to harness the potential of stem cells and committed progenitor cells. All cells in a tissue-engineered construct require access to essential molecules to survive, grow, differentiate, and function, including oxygen, glucose, amino acids, hormones, and growth factors. Thus, a welldeveloped vasculature is needed to achieve sufficient delivery and removal of these essential factors. In addition, there needs to be a delicate balance between the delivery and consumption rates of these molecules with the removal rate of toxic byproducts and wastes, and thus scaffolds must be designed to help facilitate this balance. This is most commonly achieved by altering the porosity of the polymer, inducing angiogenesis into the scaffold, and/or incorporating preexisting microfabricated vascular networks as a component of the scaffold. Other than recognizing the importance of having an established vasculature, one of the most critical problems associated with engineering more complex tissues and organs such as the liver is the lack of an intrinsic vascular supply to the entire developing construct. When cells are not within several hundred microns of a vascular supply, they will undergo necrosis and die. Early work focused on implanting poly(vinyl alcohol) (PVA) prevascularized sponges seeded with hepatocytes. Results demonstrated that cells on the periphery had high rates of survival, growth, and function, whereas cells at the center did not survive. Thus, these studies demonstrated that total implantable mass is limited by the size of the construct, which can only be on the order of 1 to 2 mm thick. To overcome these limitations, several novel technologies have been developed to prevascularize tissue-engineered constructs, including the induction of angiogenesis, 3D printing, and microfabrication processes utilizing Micro-Electro-Mechanical Systems (MEMS) technologies. Initial attempts at vascularizing tissue-engineered constructs were aimed at inducing angiogenesis into the bulk of the tissue. Studies showed that after introducing endothelial cells and vascular endothelial growth factor (VEGF) into constructs the amount of neovascularization that occurred tripled compared to negative controls without VEGF [114, 126]. Indeed, further studies demonstrated an increase in viability and function
of hepatocytes with the addition of angiogenic factors and the subsequent neovascularization of the construct. However, as the size of the constructs was increased to achieve the necessary mass of functional tissue angiogenesis could only be induced into a certain percentage of the construct. As a result, angiogenesis has not been an effective strategy for vascularizing tissue-engineered constructs. Advances in the 3D printing of biodegradable polymers have allowed scientists to create complex custom scaffolds with intricate networks replicated from existing networks using computed tomography (CT) scans [65]. For instance, PLGA has been used successfully to create porous scaffolds for bone-tissue-engineering applications. Despite these advances, current limitations with the resolution of this technology do still exist, inhibiting the replication of capillary networks into scaffolds for organs such as the liver. With the goal of augmenting liver function, our lab has designed and fabricated a multilayer microfluidic device that approximates native vasculature and parenchymal environments of the liver. These scalable devices have alternating vascular and parenchymal layers. An adjacent vascular network supports seeded hepatocytes in each parenchymal layer. A polyethersulfone membrane separating the vascular and parenchymal layers permits the mass transport of gases and nutrients, much like the sinusoidal fenestration of hepatic endothelium. These devices sustained longterm hepatocyte viability and function in vitro through continuous perfusion of the vascular channels. Serum proteins such as albumin and transferrin produced by hepatocytes readily transverse the membrane to the vasculature network. In addition to maintaining synthetic function, liver cells also maintained P450-based drug metabolism. The devices are currently being tested in vivo in rat and porcine liver failure models.
Immune Response Modulation and Tolerance As with current forms of liver transplantation, the immune response to antigens will need to be carefully considered when developing cell-based transplant modalities for the treatment of liver disease. This is primarily a problem when both xenogeneic, allogeneic, and nonautogenic stem cells such as embryonic stem cells. Currently, the predominant antirejection therapy involves immunosuppression where the immune system
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of the host is suppressed to prevent cross reactivity between host T-cells and histocompatibility antigens presented by donor cells, mainly those of leukocytes and dendritic cells. Ideally, however, to counteract rejection without suppressing the immune system of the host novel strategies should be employed to manipulate immune cell interactions with better efficacy and less toxicity. For example, one strategy could use more selective techniques for isolating only those cell populations that are therapeutically necessary and hence eliminating unnecessary populations containing cross-reactive antigens. Another method to reduce the potential for rejection could be to develop parallel cell types with the therapeutic cells, producing a state of mixed chimerism for tolerance induction [33]. Furthermore, treatments that involve autologous cell sources will not induce immune hypersensitivity responses. However, they may likely induce localized inflammatory responses due to some levels of necrosis and apoptosis. These responses could potentially lead to impairment in the uptake of cells or constructs, or the differentiation of transplanted cells into undesired cell types. Thus, for autologous transplants understanding the effects of local inflammation on cellular physiology may lead to ideal therapeutic modalities for autologous cell-based transplants [102]. Overall, much effort is needed with respect to modulating immune responses to transplanted cells and/or tissue-engineered constructs to ensure that these therapies can be effective alternatives for patients in end-stage liver failure who do not receive a liver transplant.
Conclusions and Future Directions In order to achieve long-term recapitulation of liver function, a number of factors must be satisfied when developing cellular therapies, including (1) repopulation of damaged tissue with an adequate cell mass, (2) differentiation of cells into appropriate phenotypes to achieve replacement of hepatic function, (3) production of ECM and adoption of cells to an appropriate 3D shape as guided by the support scaffold, (4) integration of implanted cells into existing diseased tissue with little or no immunological rejection, and (5) minimal associated biological risks to the recipient. Without one of these components the therapy will not be successful. At the experimental level, isolated cell transplantation has provided very valuable insight into the cell biology of hepatocytes in vivo, but this approach to
treating advanced liver disease does not offer longterm therapeutic benefits in its current state for several reasons [136]. First, the inefficient engraftment of hepatocytes (∼ 10%) and low cell viability/survival remain major limitations of this approach. Methods for improving cell adhesion and translocation of hepatocytes into the existing parenchyma are crucial factors that will need to be improved in the future. Second, transplanted hepatocytes require significant time to engraft into the host and grow, which further limits this option for treatment. For effective treatment, a cell-based therapy needs to replenish between 10 and 30% of the acutely or chronically diseased liver. Because the normal adult liver contains some 1011 hepatocytes a minimum of 10 billion cells would be required for engraftment, which is far beyond present capabilities of this technique. Third, studies have shown that the limited life span of mature adult hepatocytes can contribute to the development of cirrhosis because of their limited viability and subsequent conversion to fibrotic tissue [128]. In conclusion, hepatocyte transplantation does offer some promise as a therapeutic alternative for treating a subset of hepatic syndromes but faces several obstacles before it can be an effective option for treating chronic liver disease. Over the past several years there have been rapid advances made in stem cell research to better understand how stem cells’ tremendous potential for differentiation and proliferation can be utilized for regenerative medicine and cell-based therapies for disease. Evidence exists that there are bipotential hepatic progenitor cells in the liver capable of producing hepatocytes and cholangiocytes. Whether or not these cells will be able to be routinely isolated, expanded, and differentiated efficiently for therapeutic purposes has yet to be determined. The field of tissue engineering has made significant advances during the past 20 years toward the goal of developing functional liver replacement constructs. Yet, despite these advances there is still no functional solution to the organ shortage problem. Many issues must be addressed before the use of stem cells and tissueengineered constructs can become a reality in the clinic. First, we must better understand the complexities of tissue and organ development; specifically, the biology of cellular differentiation, proliferation, regeneration, and signaling between cells and their environment. Without a complete understanding of these factors, creating viable cell-based therapies for diseases such as end-stage liver failure may never happen.
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Appropriate animal models must also be developed to optimally translate experimental protocols and results from animal studies into human studies, especially pertaining to issues of immune rejection and the need for lifelong immunosuppression. Furthermore, the controversies associated with the ethical, legal, and social issues of certain areas of stem cell research and stem cells’ potential clinical applications must be carefully examined to ensure that science remains as the vehicle for hope and not harm. Finally, because many technical questions remain unanswered the development of cell-based hepatic transplant therapies requires close multidisciplinary collaborations among physicians, engineers, chemists, and biologists. Without this synergy, the future of regenerative medicine will be in serious question. With this synergy, however, the aim of creating functional cell-based transplant therapies — including the development of an implantable tissueengineered liver — can and will be realized. After all, advancing treatment and care for patients to save lives is and must be our ultimate goal.
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13 Stem Cell Transplantation for Autoimmune Diseases Alan Tyndall (1) and Katarina Le Blanc (2) (1) Dept of Rheumatology, University Hospital, Basle, Switzerland (2) Dept of Allogeneic Stem Cell Transplantation, Karolinska Institute, Stockholm, Sweden
Introduction Over the past 10 years a coordinated program has been running internationally under the auspices of the European Group for Blood and Marrow Transplantation (EBMT), the European League Against Rheumatism (EULAR), the International Bone and Marrow Transplantation Registry (IBMTR), and the National Institute for Health (NIH) to explore the role of profound immunoablation followed by hematopoietic stem cell transplantation (HSCT) in the treatment of severe autoimmune disease (AD). The concept arose from observations in patients undergoing HSCT for conventional indications such as malignancy or aplastic anemia, and in whom coincidentally an AD was present, that the AD may also respond [66]. In addition, extensive animal AD model studies were supportive [109], including autologous “cure” and tolerance induction in the buffalo rat arthritis model [47]. This model closely resembles human AD in that a genetic predisposition and an environmental trigger are required to initiate a chronic self-perpetuating disease. However, the limitations of animal models in guiding treatment of AD are emphasized by the fact that although about 200 methods have been described in preventing or delaying the onset of type 1 diabetes in the non obese diabetic (NOD) mouse [88], very few have been shown to reverse established AD, one of which is HSCT [38]. The first consensus statement concerning the use of HSCT in the treatment of severe AD was published in April of 1995 [65], and the first case report in October of
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1996 [104]. Results of the autologous HSCT programs have suggested that in favorable outcomes a resetting of a dysregulated autoaggressive immune system may be occurring, rather than total ablation of autoimmuneinducing cells [23]. Results of allogeneic HSCT are as yet unclear due to small numbers and heterogeneous patient groups and treatment regimens. This chapter summarizes the data of the phase I and II trials, describes the phase III trials resulting from this experience, and summarizes the data concerning immune reconstitution and possible future directions. In addition, the emerging role of mesenchymal stem cells in the treatment of graft versus host disease (GvHD) and possibly autoimmune disease is summarized.
Autoimmune Disease Autoimmune disease is thought to result from the aberrant activation of the immune system through a combination of polygenetic and environmental factors together with the failure of immunoregulatory mechanisms that normally maintain immune tolerance [51]. Most patients are treated with drugs that suppress immune-mediated inflammation, which although being nonspecific are successful in the acute phase in most cases. However, less success has been achieved with chronic AD and chronic organ transplant rejection, despite ongoing immunosuppression [27]. Despite the plethora of involved immunological and inflammatory mechanisms,
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including multiple redundancy pathways, some key molecules represent “tipping points” (meaning a point at which intervention may tip the balance between auto and protective immunity). These molecules are tumor necrosis factor (TNF) and the lymphocyte homing molecule a4b1 integrin. Blockade of both of these molecules has resulted in major clinical benefit in various ADs, but oversuppression leads to infection and long-term tolerance has not been achieved [27]. Most ADs in humans share several or all of the following features, despite the varied clinical expression. They are polyclonal, with rarely a defined inciting single antigenic epitope, and by the time of clinical disease expression there has been extensive epitope spreading and effector cell recruitment [14]. Attempts to induce tolerance in AD through specific peptide immunization although effective in animal models [83] have also not yet translated to clinical usefulness, though some early promising results have been seen in RA [84]. The innate immune system and tissue environment probably play a vital role in determining whether an antigen will evoke an immune reaction or anergy/tolerance [68, 72], and a genetic component is present but not sufficient. Some genes encode for major histocompatibility complex (MHC) molecules, but multiple other genes on different chromosomes play a role. The recent reevaluation of a subset of CD4+CD25+ T-cells with suppressor activity [94, 114] supports this concept of dysregulation rather than “all or nothing” events, as in malignant clonal disease. Presentation of self-antigens probably occurs continuously, but under normal circumstances produces apoptosis, anergy, or tolerance if presented without co-stimulatory molecules. Typically, such peripheral tolerance occurs when nonprofessional APC lacking B7 (CD80) [44] are involved. Which T-cells are needed for this autoaggressive reaction? It is known that autoreactive T-cells may escape thymic deletion and remain in the periphery, but with low affinity. Under the circumstances described previously, these lymphocytes may be activated and induce an autoimmune process. This reaction is in turn controlled by regulatory T-cell subsets, especially early in the process. Breakdown of this regulatory network over time allows clinical expression and the development of chronic AD. Reversal of this vicious circle and reinstitution of the normal regulatory network, but not eradication of the last single autoreactive cell, is one of the postulated mechanisms behind the concept of HSCT for treatment of AD.
COINCIDENTAL AD IN PATIENTS RECEIVING HSCT FOR ANOTHER INDICATION A number of case reports have been published over the past 20 years describing patients receiving allogeneic and autologous HSCT for a conventional indication (e.g., aplastic anemia or malignancy) in which a coincidental AD improved or even fully remitted [66, 110]. In the majority of initial reports, allogeneic HSCT was employed. Many of these patients remained free of both diseases, the hematological and the autoimmune disease. In some patients relapse occurred, and in one such patient full engraftment with donor-type lymphocytes [69] was observed. More recent reports have included durable responses following autologous HSCT, emphasizing the fact that genetic predisposition alone is not sufficient for AD expression [14]. Transfer of AD through allogeneic HSCT — including Crohn’s disease [99], myasthenia gravis, thyroid disease, insulin-dependent diabetes mellitus, celiac disease, and psoriasis with arthritis [74] — has also been described. In one patient with leukemia, production of autoantibodies (anti Clq) was detected following HSCT from a donor with known systemic lupus erythematosus (SLE), but clinical disease did not develop [102]. These autoantibodies later disappeared. The interpretation of these cases is influenced by the fact that there is selection bias and details of AD severity or extent that are often lacking. However, there is sufficient evidence in these reports to assume some modification of the AD process following HSCT, justifying further clinical trials.
TREATMENT OF HUMAN AUTOIMMUNE DISEASE WITH HEMATOPOIETIC STEM CELL TRANSPLANTATION Currently about 800 patients worldwide have received an HSCT as treatment of an AD alone, 624 (596 autologous) of whom are registered in the European Group for Blood and Marrow Transplants (EBMT) and the European League Against Rheumatism (EULAR) database (Table 13.1) and the rest registered in the International Bone Marrow Transplantation Registry (IBMTR) in the United States. In the EBMT/EULAR database the majority of patients has had either severe MS or scleroderma, more correctly called systemic
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function to ensure a decent quality of life if the immunological/inflammatory process were arrested or reversed and have sufficient capacity to withstand the HSCT procedure. In general, these guidelines were followed. As the program proceeded, certain clinical parameters and treatment-related factors emerged as being associated with an unacceptable risk, such as a mean pulmonary artery pressure > 50 mm Hg in SSc, high disability scores in MS, and total body irradiation (TBI) without lung shielding in SSc. This experience was then exploited in the design of the phase III randomized studies. Following the initial consensus meetings, single case reports and small series of patients transplanted for the treatment of severe AD have been published. These reports demonstrate heterogeneity of patient selection, target AD, and outcome, and have in part formed the basis for further prospective trials. In the EBMT/EULAR database, the most commonly transplanted diseases were MS, SSc, RA, JIA, and SLE, and the data came from more than 100 transplant centers in more than 20 countries. There were longlasting responses in all disease categories, but they were achieved at a price, the overall actuarially adjusted transplant-related mortality (TRM) being 7% [31, 32]. This was higher than the predicted 3% for autologous HSCT overall and reflects the general overall level of illness and multiorgan involvement of many AD patients compared with, for instance, breast cancer patients undergoing high-dose chemotherapy and HSCT. In fact, there is a marked difference between AD groups with a TRM of 11% in SLE and only one patient (1.4%) with RA. There are also different rates and degrees of responses.
TABLE 13.1 EBMT/EULAR Autoimmune Disease Autologous HSCT Database. Disease and Disease Category* Neurological disorders Multiple sclerosis Myasthenia gravis Polyneuropathy Amyotrophic lateral sclerosis Guillain-Barré syndrome Rheumatological disorders Systemic sclerosis Rheumatoid arthritis Juvenile idiopathic arthritis Systemic lupus erythematosus Dermatomyositis Mixed connective tissue disease Behçet’s disease Psoriatic arthritis Ankylosing spondylitis Sjogren’s syndrome Vasculitides Wegener’s Cryoglobulinemia Not classified Hematological immuncytopenias Immune thrombopenia Pure red cell aplasia Autoimmune hemolytic anemia Thrombotic thrombocytopenic anemia Evans syndrome Gastrointestinal Enteropathy Inflammatory bowel disease Other Total
No 183 2 3 2 1 88 72 54 66 7 4 5 2 2 1 4 4 2 12 4 5 3 2 2 3 3 536
*Status as of October of 2004.
Phase I/II Studies sclerosis (SSc). There are no reliably effective alternative treatment options for these disorders. However, as experience grew other ADs were transplanted, largely in the context of combined phase I and II trials and following the consensus guidelines developed at international meetings [70, 108] early in the program. The essential components of these were that the HSCT regimens were limited to a small number of protocols only and that patients should have had failed conventional therapy and have a poor prognosis concerning life or vital organ function. There should also have been enough reversible or maintainable vital organ
MULTIPLE SCLEROSIS The analysis of the first 85 patients in the EBMT database showed a three-year progression free survival of 78% in the patients with secondary progressive disease, and 66% in those patients with primary progressive disease (Figure 13.1). There were five deaths from treatment and two from progressive disease [26]. Gadolinium-enhancing lesions (considered a surrogate marker of activity) were largely eliminated, but functional deterioration may continue in advanced disease
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5% improved then progressed sustained improvement 33%
TRM not transplanted progressed, no response stable too early
20%
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5% 15%
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Figure 13.1. Clinical outcome in 85 multiple sclerosis patients following HSCT. About 2/3 of the patients examined sustained benefit from the HSCT despite some degree of relapse. The transplant-related mortality of just under 9% is to be compared with a predicted 50% five-year survival of this subgroup of scleroderma patients.
due to inflammation-triggered progressive apoptosis and brain atrophy. A more recent analysis was performed on 183 registrations, of which 99 were the secondary progressive form, 19 primary progressive, and 41 relapsing forms of the disease (courtesy of R. Saccardi and the EBMT autoimmune disease working party). Median age was 34 and median disease duration 6.7 years. Of the 178 evaluable transplanted patients, 163 received a peripheral blood stem cell transplant, mobilized with Cy and G-CSF in 126 and followed by conditioning with the quadruple therapy BEAM (BCNU, VP-16, ara-C, and melphalan) and ATG in 74 patients. BEAM alone was used in 30 patients. Purging, largely using CD34+ selection, was used in 97 and not in 77 patients. The absolute TRM was 5.5%, with no deaths occurring in the past three years, probably due to more careful patient selection such as excluding patients with a very high EDSS score or severe co-morbidity such as active infection. The phase I/II data were exploited in developing the Autologous Stem Cell Transplantation International Multiple Sclerosis (ASTIMS) trial, with patients being randomized to transplant with BEAM plus ATG and no purging, or to mitoxantrone. In the retrospective analysis, no patient receiving this regimen died from treatment-related causes, and using the ASTIMS
selection criteria TRM was only 1.8%. Further information is available on the web site www.astims.org. This study is still recruiting, and no interim analysis has been yet performed. In the United States, a multicenter NIH-sponsored study is in the advanced planning phase (C. Bredeson, personal communication).
SYSTEMIC SCLEROSIS (SSC) Several transplant protocols were used in SSC, largely either Cy-based protocols (4g/m2 Cy mobilization and Cy 200 mg/kg body weight conditioning) or radiation (8 Gy/Cy 120 mg/kg body weight). A French study showed a positive outcome [24], and an analysis of all phase I/II pilot studies showed in the first 65 patients an improvement of 25% or more in the skin score (measured by the modified Rodnan method) in 70% of the patients, with a TRM of 12.5% [8]. With further patient recruitment and longer-term follow-up, the TRM of the EBMT registered patients fell, considered to be related to more careful patient selection. Lung function tended to stabilize, and some factors were identified as potentially hazardous for HSCT (e.g., pulmonary hypertension > 50 mmHg), mean pulmonary arterial pressure, severe cardiac involvement, severe
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10% 25% 2% improved then progressed sustained improvement
7%
TRM not transplanted progressed, no response stable
10%
too early unknown
9% 34% Figure 13.2. Clinical outcome in 57 patients with systemic sclerosis followed up to 6 years after HSCT. None of the patients receiving the ASTIMS protocol experienced transplant-related mortality in this retrospective analysis, and there has been no further TRM in the past three years.
pulmonary fibrosis, and uncontrolled systemic hypertension. A long-term follow-up of this cohort showed an overall TRM of 8.7%, no further transplant-related deaths, and durable remissions in more than one-third of patients [25] (Figure 13.2). The encouraging results from the phase I/II studies led to a prospective randomized comparative trial of HSCT (Cy, ATG, and CD34 selected graft) versus monthly intravenous pulse Cy 750 mg/m2 for 12 months, designated the Autologous Stem Cell Transplantation International Scleroderma (ASTIS) trial (www.astistrial.com) that has so far randomized 52 patients (24 HSCT), with no treatment-related deaths in either arm. A multicenter U.S. study of 19 SSc patients utilizing a regimen of Cy 120 mg/kg, TBI 8 Gy, and equine ATG 90 mg/kg body weight and a CD34 selected graft product showed a sustained benefit in 12 patients at median follow-up of 14.7 months [71]. Four patients died, three from treatment-related causes and one from disease progression. In two cases a fatal regimen-related pulmonary toxicity occurred, which was not seen in the subsequent 11 cases when lung shielding was employed. Twelve patients had a sustained and significant improvement of skin score and functional status to a degree not previously seen with other treatment modalities. A prospective randomized study using similar selection criteria, control arm, and end points as the ASTIS trial is planned in the United States under the auspices of the NIH. The treatment arm will, however, be different, being Cy and radiation based and allowing a comparison
between the various regimens (K. Sullivan, personal communication).
RHEUMATOID ARTHRITIS A retrospective analysis of the first 78 registered patients showed significant improvement, with 67% achieving an ACR-50 response at some time posttransplantation [98]. Most of the patients had failed a median of 5 (range 2 to 9) conventional disease-modifying antirheumatic drugs (DMARDs) before the transplant. Some degree of relapse was seen in 73% of patients posttransplantation, but this was in most cases relatively easy to control with drugs that had proven ineffective pretransplantation. At 12 months posttransplantation, more than half the patients had achieved an ACR-50 or more, and of these just over 50% had not restarted DMARDs. The median follow-up was 18 (6 to 40) months, and at this time the majority of patients received a conditioning regimen of Cy 200 mg/m2 alone and received peripherally harvested stem cells after either granulocyte colony stimulating factor (GCSF) or Cy/G-CSF (equal numbers) mobilization. Only one TRM was reported: a patient who five months posttransplantation (busulphan/Cy), died from sepsis, with a coincidental non small-cell lung carcinoma being discovered at autopsy. In the opinion of the investigators, this was not considered to be a transplant-induced tumor. A multicenter trial in Australia failed to show
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any advantage of CD34+ selection of the graft after non myeloablative conditioning with Cy [75].
PHASE I/II JUVENILE IDIOPATHIC ARTHRITIS A total of 54 children with idiopathic juvenile arthritis, largely the systemic form called Still’s disease, have been registered. Most of these cases were treated in two Dutch centers using stem cells obtained from the bone marrow and a conditioning protocol of Cy 200 mg/kg body weight, TBI 4 Gy, and ATG [112]. In the follow-up report of 34 patients there were 18 complete drug-free remissions and 6 partial remissions [111]. In those attaining remission, the corticosteroid dose could be reduced and some patients experienced puberty and catch-up growth. Three patients died from hemophagocytic syndrome, also called the macrophage activation syndrome, thought to be related to intercurrent infection or uncontrolled systemic activity of the disease at the time of transplantation. Protocols were modified accordingly, such that systemic activity is controlled before the transplant with methyl-prednisolone intravenously. Since this modification, no further such deaths have occurred. The results of phase I/II trials in JIA using Cy alone versus Cy and TBI suggested no advantage of the TBI (N. Wulffraat, personal communication). Further phase II studies will be performed to assess the optimal regimen for a phase III study.
SYSTEMIC LUPUS ERYTHEMATOSUS Of the 55 registrations in the EBMT/EULAR database, most had either renal or CNS involvement, and 21 had failed conventional Cy treatment. A peripheral stem cell source after mobilization with Cy and G-CSF was used in the majority. Twenty-three patients received a conditioning with Cy and ATG and 11 with Cy plus TBI, and four other regimens were employed. An unselected graft was used in 29, with CD34 selection in 19. There were five deaths due to treatment and one from progressive disease, resulting in an actuarially adjusted TRM of 10% [1–19]. In those 53 patients with sufficient data for analysis, 66% achieved a “remission,” defined as an SLEDAI (SLE Disease Activity Index) of ≤ 3 and steroid reduction to < 10 mg/day. Of those achieving remission, 32% subsequently relapsed to some degree and were mostly
easily controlled on standard agents that had previously been ineffective. There were 12 deaths after 1.5 (0 to 48) months, of which seven (12%) were related to the procedure [40, 41] in these severe SLE patients. Traynor and colleagues reported on nine patients with severe SLE who were mobilized in a transplant protocol [105]. One died as a result of infection following mobilization, and another three months later from active CNS lupus, having not proceeded to transplantation. The seven remaining were free of signs of active lupus at a median follow-up of 25 months posttransplantation. The high-dose chemotherapy consisted of cyclophosphamide 200 mg/kg, methylprednisolone 1 gm, and equine ATG 90 mg/kg. This cohort has since been extended, with no further TRM and improvement of pulmonary function tests [106] and antiphospholipid syndrome [100] being reported. A prospective phase II b study is being prepared together with the NIH in the United States (R. Burt, personal communication).
CROHN’S DISEASE Autologous HSCT has been performed in about 16 patients for treatment of refractory Crohn’s disease. The largest series from Chicago showed sustained clinical improvement in 11/12 patients [79], with a median follow-up of 18.5 months (range 7 to 37). A case of a 53-year-old male was reported from Italy with sustained clinical benefit at 5 months follow-up. Initially he responded clinically but not endoscopically to the mobilization with Cy 2g/kg and G-CSF, and therefore HSCT was performed with Cy 200 mg/kg, equine ATG, and a CD34 selected graft [93]. Protocols have generally followed published guidelines [35] and a multicenter trial is about to be launched in Europe under the auspices of the EBMT and the European Crohns Colitis Organization (ECCO) (C. Hawkey, personal communication). This will compare patients who after mobilization with Cy and G-CSF are randomized to immediate HSCT (Cy 200 mg/kg and rabbit ATG 7.5 mg/kg body weight) or delayed by 12 months. The study is called the Autologous Stem Cell Transplantation International Crohns (ASTIC) trial, with details available at www.ebmt.org. The numbers of cases with vasculitis, Behçet’s disease, relapsing polychondritis, and other ADs are too small to draw meaningful conclusions, with further phase I and II standardized protocol pilot studies proceeding.
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Other Issues ALLOGENEIC HSCT Several factors have been limiting for autologous transplants. First, regimens have been less than maximally intensive and thus have not achieved eradication of immunologic memory. Second, reinfusion of potentially pathogenic T-cells and B-cells is a potential cause of relapse. The latter has been thought to justify allogeneic transplantation, with its attendant higher risks (10 to 30% mortality), mainly relating to graft versus host disease (GvHD). Several case reports and series support this notion. A child with autoimmune hemolytic anemia that was refractory to immunosuppression and splenectomy had only a seven-week remission after autologous transplantation, but was still in remission 18 months after an HLA-identical unrelated donor transplant [15]. A similar refractory case of Evans syndrome was reported from Geneva [67]. A graft-versusautoimmunity effect has been proposed in a patient given an allogeneic stem cell transplant for chronic myeloid leukemia, who also had severe psoriasis [96]. This is also compatible with long-term control in RA in a small number of allografted patients. These patients had received conditioning regimens very similar to those given in trials of autografting in RA (i.e., Cy 200 mg/kg), and the longer remission in the allografted patients suggests that the type of graft rather than the conditioning regimen determined the outcome. However, both longterm remissions with autologous HSCT and relapses following allogeneic HSCT (with full donor chimerism) have been observed in AD posttransplantation, as well as newly occurring AD [4, 89, 90, 97]. Newer techniques with non-myeloablative conditioning regimens may reduce early transplant-related mortality to less than 10%, making allogeneic HSCT for AD more acceptable. The theoretical concept of tolerance induction in the thymus due to stable mixed chimerism has been recently elegantly reviewed [103]. There have been several early reports, including one with an SLE/scleroderma overlap syndrome [45] and one with RA [9]. Still, the risk of GvHD remains, and it is unclear whether the target for HSCT in AD can be defined as clearly as in malignant and inherited disorders. Carefully selected cases with early but high-risk disease and low risk for transplant-related mortality (young age, HLA identical siblings) should provide an answer. For these reasons, allogeneic HSCT for the treatment of severe AD must await further refinement of
the transplant procedure, and in particular the prevention of GvHD. The international guidelines stipulate that autologous HSCT should be the preferred approach [33]. So far, this has been largely adhered to with allogeneic HSCT for AD alone having been performed mainly in refractory cytopenias [32]. Despite new developments, arguments not to use allogeneic HSCT in the first instance remain the same. Treatment-related toxicity is high, and GvHD cannot yet be avoided and might interfere with the preexisting disease without the potential additional benefit of “graft-versus-autoimmunity.” Unlike malignancy, there is no definable clone of autoaggressive cells to be eradicated. Furthermore, incomplete or slowed immune reconstitution after allogeneic HSCT might lead to late development of a donor-type AD, even more so in predisposed patients. It remains open whether reduced intensity conditioning regimens might alter these perspectives, in that they have been shown to reduce early mortality. So far, they have not reduced risk of GvHD and long-term followup is required. Still, there is a consensus that it might be appropriate under carefully selected conditions to begin the planning of phase I/II studies to evaluate the role of allogeneic HSCT. Conditioning with Cy ± ATG as used for aplastic anemia for many years might be the most appropriate choice.
IMMUNE RECONSTITUTION In general, immune reconstitution must be considered separately for autologous and allogeneic transplantation. According to the many variations of each procedure, the impact of individual regimens on immune reconstitution may differ, even before considering the effect of different diseases and age groups. Among the possible measures for immune reconstitution are cell surface markers to determine the appearance and development of different cell lines, the response to infectious agents and vaccination, and the repertoire of adaptive immunity to nonself and self-antigens. In the autologous setting, the method used for mobilization, the extent of the immunoablative regimen, and the negative and positive selection of the autograft may influence reconstitution of the hemopoietic and immune systems. In children transplanted for nonautoimmune diseases, CD4+CD45RO+ peripheral T-cell expansion occurred within 16 days posttransplantation. Natural killer (NK) cell (CD16+/−CD56+) counts normalized
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rapidly. After day 30, an inverted CD4/CD8 ratio was still present, and T-cell (CD3+, CD3+CD4+, CD4+CD45RA+) recovery was delayed up to 24 months after the autograft [43]. In adults, lymphocyte subset recovery and T-cell receptor (TCR) beta-chain variable region did not differ between patients receiving unmanipulated or CD34+-selected autologous HSCT [23]. The reconstitution of the immune system after allogeneic HSCT is complex, and may be significantly impaired by chronic GvHD. After allogeneic HCST with full donor chimerism, recovery of TCR rearrangement diversity is more rapid in younger individuals. Recovery in older patients was slower, but little difference remained at 9 months. In this series, low levels of TCR rearrangement correlated with severe opportunistic infections and with GvHD [59]. The reconstitution of recipient NK cell repertoire after HLA-matched HSCT followed the killer immunoglobulin-like receptor (KIR) pattern of the donor in the majority of cases, whereas in the others no uniform pattern was evident and severe clinical complications occurred [95]. The finding of T-cell receptor excision circles (TRECs) in T-cells recently exiting the thymus [20] has allowed a more detailed analysis of normal and autoaggressive T-cell reactions following HSCT for AD. Following HSCT for AD, some adult patients have shown an increase in the number of lymphocytes bearing TRECs, indicating that the thymus may become reactivated and theoretically capable of inducing central tolerance. Patients treated with allogeneic HSCT have a higher rate of infections compared with those receiving autologous HSCT [21]. EBV activation after allogeneic HSCT was associated with low CD8+ levels, and a high cellular viral load preceded reactivation [12]. The rate of severe infections after allogeneic HSCT appears to depend on the source of the graft, in that it was significantly higher in marrow recipients than in patients receiving filgrastim-mobilized peripheral blood stem cell grafts [101]. In one study following autologous HSCT for SSc, the patients who had longer responses had a tendency to more sustained impairment of B-cell and CD4+ T-cell reconstitution [23].
ABLATIVE THERAPY WITHOUT HSCT Hematopoietic stem cells resist the cytotoxic effects of cyclophosphamide, and therefore theoretically an HSCT
is not needed following aplasia induction and G-CSFsupported reconstitution. Such a strategy has been successfully employed in aplastic anemia and applied to SLE [80]. Early results are encouraging, but a significant number of patients had not had conventional pulse cyclophosphamide therapy and the reconstitution times (especially for platelets) were prolonged compared to rescue with HSCT. Both procedures remain research-based rather than standard therapy.
Mesenchymal Stem Cell Immunomodulation of Autoimmune Disease During the past several years the collaboration between transplanters and specialists in AD has raised the possibility of synergy between the emerging use of mesenchymal stem cells (MSCs) in acute GvHD and AD. Mesenchymal stem cells (MSC) are pluripotent cells capable of differentiating in vitro and in vivo to different MSC lineages, including adipose, bone, cartilage, and myelosupportive stroma [29, 36, 81, 85]. MSCs were initially identified in bone marrow and later in muscle, adipose tissue, synovial membranes, and other connective tissues of human adults [28, 42, 77, 92]. They may be isolated from other cells in the bone marrow by adherence to plastic and consecutive passaging, with subsequent proliferation to form spindle-shaped cells in confluent cultures. Because of our current inability to isolate MSCs prospectively, due to their rarity in vivo and lack of characteristic cell surface markers, current data are based on studies performed on cells expanded in vitro. MSCs have been defined by using a combination of phenotypic markers and functional properties. Controversy still exists over the in vivo phenotype of MSCs (Table 13.2). However, ex-vivo-expanded MSCs do not express the hematopoietic markers CD34, CD45, and CD14 [16, 81]. In addition to their pluripotentiality, they can be identified as cells that stain positive for CD73, CD105, CD166, CD90, and CD29 by flow cytometry [5, 6, 16, 81, 85]. MSCs also express various integrin a- and b-subunits and their noncovalent associations that constitute receptors for extracellular matrix components, including collagen, laminin, fibronectin, and vitronectin [13, 16, 64, 81]. In vitro, MSCs have vast proliferative potential, can clonally regenerate, and can give rise to differentiated progeny. When MSC are injected into newborn mice or are infused in utero, they engraft in multiple organs and
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TABLE 13.2 Phenotypes and characteristics of multipotent mesenchymal stromal cells (MSCs). Surface antigen
Other name(s)
Location/function
Positive/negative
CD29
none
positive
CD73*
SH-3, SH-4 epitopes Ecto- 5‘-nucleotidase Thy-1
Leukocytes Integrin b1 sub unit T and B cell subsets
CD90 CD105*
SH-2 epitope Endoglin (TGF b receptor)
CD166
Activated Leukocyte Cell Adhesion Molecule (ALCAM)
CD14 CD34
Myelomonocytes none
CD45*
Leukocyte Common Antigen ( LCA)
CD34+ prothymocytes Functn : unknown Endothelial cells Activated mono/macro Bone marrow cell subsets Functn : binds TGF-b Activated T - cells Thymic epithelium Fibroblasts/neurons Functn : ligand for CD 6 LPS receptor Hematopoietic precursors Capilliary endothelium Functn : unknown All hematopoietic cells Functn : augments T/B cell receptor signalling
positive positive positive
positive
negative negative
negative
* the most important
demonstrate site-specific differentiation [60, 82]. However, MSCs have not yet been shown to have indefinite self-replicatory ability, have not demonstrated an ability to give rise to more than one type of mature daughter cells, and have not been proven capable of regenerating or maintaining a whole tissue compartment, thus fulfilling all expectations of a true stem cell. Regardless of whether or not MSC are true stem cells, clinical benefit from MSC may not require sustained engraftment of large numbers of cells. It is possible that a therapeutic benefit can be obtained by local production of growth factors and a provision of temporary immunosuppression. MSCs rapidly expand more than a billionfold when cultured in vitro. They secrete cytokines important for hematopoiesis and have the capacity to maintain and expand lineage-specific colony-forming units from CD34+ marrow cells in long-term bone marrow culture [2, 10, 63]. In experimental animals, MSCs promote engraftment of unrelated and umbilical-cord-derived
HSCs particularly when the HSC dose is limiting [2, 3, 39, 62]. MSCs are not immunostimulatory in vitro. They do not induce lymphocyte proliferation when co-cultured with allogeneic lymphocytes, nor are they targets for CD8+ cytotoxic lymphocytes or KIR-ligand mismatched NK-cells [17, 46, 57, 107]. After uterine transplantation of human MSCs into sheep, the cells persist long term and demonstrate site-specific differentiation [60]. Long-term persistence of fully mismatched allogeneic fetal-liver-derived MSCs was reported after transplantation into an immunocompetent human fetus with osteogenesis imperfecta in the third trimester of gestation [54]. No immunoreactivity was observed when patient lymphocytes where reexposed to the graft in vitro at different time points posttransplantation, indicating that MSCs can be tolerated when transplanted across MHC barriers in humans. Immunologic tolerance to mouse MSCs was reported by Saito et al. in immunocompetent rats allowing engraftment in the injured
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myocardium [91]. In spite of the cells’ inability to induce alloreactivity, human MSCs were rejected in a discordant xenogeneic model after infusion into immunocompetent Sprague-Dawley rats after myocardial infarction [34]. Both CD4+ and CD8+ lymphocytes bind to MSCs, and the affinity is increased for activated T-cells [64]. Several adhesion molecules expressed by MSCs are essential for the interaction with T-cells. Vascular cell adhesion molecule 1, ICAM-2, and lymphocyte function-associated antigen 3 (LFA 3) are present on unstimulated MSCs, whereas the expression of ICAM-1 is inducible [16, 64, 81, 107]. In vitro results indicate that MSCs possess immunosuppressive properties. Rodent, baboon, and human MSCs suppress lymphocyte proliferation in mixed lymphocyte cultures (MLCs) or induced by mitogens in a dose-dependent fashion [7, 17, 46, 50, 58, 107]. The suppression is MHC independent, and most marked if MSCs are added on the first day of a six-day culture (Figure 13.3). In human cell cultures, the magnitude of suppression is not reduced when the MSCs are separated from the lymphocytes in transwells, indicating that cell-to-cell contact is not required [17, 87, 107]. The T-cells do not become apoptotic or anergic because they can be restimulated if the MSCs are removed [7, 61, 62]. MSCs reduce the formation of cytotoxic lymphocytes and NK-cells in MLC and favor the
differentiation of CD4+ T-cells with presumed regulatory activity, coexpressing either CD25 or CTLA4 [61, 87]. Co-culture of purified subpopulations of immune cells with human MSCs showed that the cytokine secretion profile of dendritic cells, naïve and activated T-cells, and NK-cells was altered to a more anti-inflammatory or tolerant phenotype [1]. MSCs caused Th1 cells to decrease INFg secretion, whereas Th2 cells increase their expression of IL4. Cell cycle analyses showed that T- and B-cells stimulated in the presence of MSCs arrest at the G1 phase, as in division arrest anergy [30]. The mechanisms underlying the immunosuppressive effect remain to be clarified. Di Nicola et al. proposed that two soluble factors, hepatocyte growth factor (HGF) and transforming growth factor-b1 (TGF-b1), were involved. The addition of anti-HGF and anti-TGF-b1 partially restored the proliferation of CD2+ cells in the presence of MHC [17]. These results could not be reproduced using unfractionated PBL [55]. Aggarwal and Pittenger suggested that MSC-produced prostaglandin E2 accounted for reduced lymphocyte proliferation [1]. Another study suggests that indoleamine 2,3deoxygenase-mediated tryptophan depletion by MSCs can act as a T-cell inhibitory effector mechanism [73]. Indoleamine 2,3-deoxygenase, which is induced by INFg , catalyzes the conversion from tryptophan to kynurenine and inhibits T-cell responses. However, in the hands of Tse et al. neither MSC production of
Percentage proliferative response
120 100 80 60
*
*
40 20 0 PBL+PMA
+10% MSC
+10% MSC Transwell
+0.1% MSC
Figure 13.3. Immunomodulatory effect of allogeneic MSC on PMA-induced T-cell proliferation. Stimulating peripheral blood lymphocyte (PBL) cells with 1 mg/ml phorbol myristate acetate (PMA) gave a proliferation of 76,432 +/− 13,556 counts per minute (CPM), which in the figure has been set to 100% to reduce the variation in response between different PBLs. Addition of 10% irradiated MSCs inhibited the proliferation. This was observed when the MSC were in contact (n = 5) and when separated from the PBLs by a transwell insert (n = 5). (Reprinted from Experimental Cell Research, Vol. 305, Rasmusson I., Ringdén O., Sundberg B., Le Blanc K., Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. (Figure 1, p 35) pp 33–41, 2005.)
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IL-10, TGF-b1, and prostaglandin E2 nor tryptophan depletion in the culture medium was responsible for the immunosuppressive effect [107]. In addition, MSCs produce bone morphogenetic protein 2 (BMP-2), which may mediate immunosuppression via the generation of CD8+ regulatory T-cells [19]. The controversial data may be due to the use of MSCs generated by different techniques, including the use of different stimuli, culture conditions, doses, and kinetics as well as different lymphocyte populations tested. Such differences may in turn affect cytokine and chemokine secretion, with seemingly contradictory results. In addition, species-specific differences (particularly between murine and human MSCs) add to the confusion [50]. Suppression may also be mediated by different mechanisms depending on the T-cells’ stimulus. For instance, MSCs increased the transcription and translation of IL-2 and soluble IL-2 receptors in MLC, whereas the levels decrease if MSCs are present among mitogen-stimulated lymphocytes [86]. Similarly, depending on the kinetics MSCs can enhance or suppress IL10 levels in MLC. An immunosuppressive effect of MSC in vivo was first suggested in a baboon model, where infusion of ex-vivo-expanded donor or third-party MSC delayed the time to rejection of histoincompatible skin grafts [7]. MSCs also down-regulate bleomycin-induced lung inflammation and fibrosis in murine models if given early (but not late) after the induction [78]. MSCs adopt an epithelial-like morphology. Of notice is the fact that the epithelial crosstalk with endothelium via integrin av b6 controls alveolar flooding [76]. A similar effect was seen in a murine hepatic fibrosis model (carbon, tetrachloride induced) using an MSC line bearing the fetal liver kinase-1 (FLK1) marker [22]. This is a transmembrane tyrosine kinase found on endothelial cells and hematopoietic progenitors. Its ligand has been characterized as the vascular endothelium growth factor (VEGF) [11]. As with the mouse lung fibrosis model, MSCs assumed an epithelium-like morphology and expressed low levels of albumin as a hepatocyte marker. Treatment was only effective early (less than a week) after the damage onset. Two reports of autoimmune animal model responses have recently appeared. In the experimental allergic encephalomyelolithis (EAE) murine model of Uccelli et al., both clinical and histological improvement occurred. The responses were dependent on time of MSC treatment (the earlier the better), and were reversed with IL-2-treatment, indicating that anergy rather than apoptosis had occurred [113]. However,
in a murine model of arthritis collagen-induced arthritis (CIA) was not improved by the addition of MSCs and the in vitro immunosuppressive effects were reversed by the addition of TNFa. MSCs were not found in the joints [18]. Ex-vivo-expanded MSCs have been infused in several phase I studies [37, 48, 49, 52, 53]. No adverse events during or after MSC infusion have been observed, and no ectopic tissue formation has been noted. After infusion, MSCs remain in the circulation for no more than an hour [49]. Although consistent stromal cell chimerism has been difficult to establish, low levels of engrafted MSCs have been detected in several tissues. It is possible that sufficient therapeutic benefit is obtained by local production of growth factors and the provision of temporary immunosuppression by MSC infusion. Infusion of haploidentical MSCs to a patient with steroid-resistant severe acute GvHD of the gut and liver promptly improved liver values and intestinal function [56]. Upon discontinuation of cyclosporine, the patient’s acute GvHD recurred but was still responsive to a second MSC infusion. Lymphocytes from the patient, when investigated on multiple occasions after MSC infusion, continued to proliferate against lymphocytes derived from the haploidentical MSC donor in co-culture experiments. This suggests an immunosuppressive effect of MSC in vivo, rather than a development of tolerance. The EBMT is currently planning protocols for prevention and treatment of acute GvHD through the Stem Cell Subcommittee (W. Fibbe, K. Le Blanc, and F. Frassoni, personal communication).
Conclusions In carefully selected cases of severe refractory AD, immunoablation followed by autologous HSCT has shown to have an acceptable benefit/risk ratio so that phase III randomized comparative studies are being planned or are running internationally in SSc, MS, SLE, and RA. Only through these trials will the true place of such treatment be established. Although relapses have occurred in significant numbers, many of these patients have been rendered sensitive to standard treatment that were ineffective pretransplant. A concept of “resetting” the autoimmune process is emerging. The place of allogeneic HSCT, with its attendant theoretical advantage of tolerance induction, is being considered — taking into account the unresolved problem of GvHD. Such an HSCT program has also opened
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an opportunity to study immune reconstitution during remission and relapse, hopefully shedding light on the early phases of autoimmune disease induction. The “holy grail” of tolerance induction without immune suppression in the treatment of severe AD remains the aim, but at the moment is still elusive. With this aim in mind, the basic science programs integrated in the running of prospective trials will shed new light on AD mechanisms and the logical manipulation of them. Regulatory cells such as CD4+CD25+ T-cells and possibly mesenchymal stem and progenitor cells as immunomodulators will play an increasing role in this “cellular therapy” era.
Acknowledgments We would like to thank the Horton Foundation (Switzerland), AMGEN, and EULAR for unrestricted supporting research grants, the Swiss National Research Foundation (grant number 31-45938.95), the Swedish Cancer Society, the Children’s Cancer Foundation, the Swedish Research Council, the Tobias Foundation, the Stockholm Cancer Society, the Swedish Society of Medicine, the Sven and Ebba-Christina Hagbergs Foundation, and the Karolinska Institute. In addition, our gratitude to Dr. Chiara Bocelli-Tyndall for data retrieval and analysis and Dr. R. Saccardi, Chairman, EBMT Autoimmune Disease Working Party.
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14 Cardiac Cell Transplantation Bryce H. Davis (1), Harald C. Ott (2), and Doris A. Taylor (1 and 3) (1) Duke University, Department of Biomedical Engineering, Durham, North Carolina (2) University of Minnesota Medical School, Center for Cardiovascular Repair, Minneapolis, Minnesota (3) University of Minnesota Medical School, Bakken Professor and Director, Center for Cardiovascular Repair, Minneapolis, Minnesota
Background Ischemic heart disease led to over 900,000 deaths in the year 2002 alone [2], primarily due to consequences of acute myocardial infarction (MI). As individuals survive MI due to better medical management, they often develop heart failure. Heart failure is an increasing problem in the world, with over 500,000 new cases reported each year in the United States. Mortality of patients who reach New York Heart Association class IV heart failure can reach 60% within one year and 80% within five years. The American Heart Association reports that over $390 billion a year is spent on treatment and missed work from cardiovascular disease, and estimates are that $28 billion of that is spent on treatment options for heart failure [2]. Current treatment options for heart failure can be loosely grouped into four categories: therapy with drugs, implantation of devices, surgical therapies (including organ transplantation), and new experimental therapies. Current drug therapies typically revolve around the use of ACE inhibitors to reduce blood pressure by blocking conversion of angiotensin I to angiotensin II and enhancing the action of kinins; beta blockers to inhibit deleterious effects of the sympathetic nervous system; and diuretics to reduce pulmonary and peripheral edema [34]. Current devices used include biventricular pacemakers that reduce the incidence of dangerous electrical abnormalities that can cause sudden death and left ventricular assist devices (LVADs), which assist in the pumping of blood, allowing the left ventricle to face a decreased load and hopefully to remodel and heal. Surgical therapies can include reshaping of the dilated left
Cellular Transplantation: From Laboratory to Clinic
ventricles (Dor procedure) and cardiac transplantation. However, only 2,500 transplants are done each year because donor tissue is severely limited [34], leaving approximately 50,000 patients per year with few or no options. Thus, new therapeutic options in clinical evaluation are novel, experimental therapies, including angiogenesis [38, 49, 90], gene therapies [32], and cell therapy. This chapter focuses on the use of cell therapy to treat cardiovascular disease. The goal of cell therapy to treat cardiovascular disease is twofold. Immediately after MI, the goal is to prevent cardiomyocyte death, restore blood flow, and eliminate negative remodeling. In HF, the goal is to replace the akinetic dead tissue of scarred areas of the heart with functioning contractile cells. Such an objective can be achieved in one of four ways. The first option is to cause surviving cardiomyocytes to divide and repopulate the damaged area. This technique has shown little promise to date, primarily because the level of proliferation shown has been insufficient to compensate for the loss of cardiomyocytes in the infarct area [13, 25, 72]. Further, there is the concern that cells induced to reenter the cell cycle could continue to proliferate in an uncontrolled manner, leading to further structural deterioration or even tumor formation. This limits enthusiasm for this procedure until it is more controllable. A second option for restoration of cellular contractility within the infarct area is the use of genetic manipulation to turn fibroblasts or other cell types present in the infarct into contractile cells. This approach has yielded some successful preclinical results [56, 91], but the clinical applicability of this technique remains in question due both to the limited clinical success of gene
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Bryce H. Davis, Harald C. Ott, and Doris A. Taylor
therapies and the further theoretical limitation imposed by the small number of live target cells within the infarct capable of taking up and expressing the required genes. A third option, which has been focused on as of late, is inducing progenitor cells from other tissues — including the bone marrow and blood — to migrate into the infarcted tissue and differentiate into contractile tissue. Examples of this approach include administration of growth factors such as granulocyte colony stimulating factor (G-CSF) and stem cell factor (SCF) to induce existing stem cells to divide and migrate to the tissue of interest [44, 63, 66]. These approaches show some promise, but the limited vascularity and reduced perfusion of large infarct areas may limit the number of stem cells that can migrate into the damaged regions and thus limit this technique to peripheral repair of the damaged region. A further potential limitation is the lack of control over where the cells are induced to migrate, potentially leading to unintended consequences. One such consequence could be angiogenesis in unintended areas such as in retina or in nascent, previously undetected tumors. Thus, this technique, while promising, will likely not move into large-scale use without coincident targeting of cells to the regions of interest. The final method currently in use, and the technique on which this chapter focuses, is direct delivery of cells to the damaged areas of the heart. This transplantation technique is the most clinically controllable, and as a result has been studied extensively in the laboratory over the past 15 years and clinically since 2001 [53]. The cells delivered directly into the failing heart can be classified into two major groups: contractile cells and noncontractile cells.
Contractile Cells The main function of the heart is to pump blood throughout the body. From this functional view, it appears obvious that when considering a cell type to replace damaged myocardium we should use contractile cells. The major contractile cell types currently being studied for their ability to repair ischemic heart are cardiomyocytes and skeletal myoblasts.
CARDIOMYOCYTES Replacing cardiomyocytes is a major goal of cardiac cell therapy. Thus, mature cardiomyocytes themselves
would seem the ideal choice to use for cardiac repair. They are contractile and have ideal mechanical and electrical properties to integrate with the surrounding myocardium. The problems associated with using adult cardiomyocytes, however, are profound. Primary among these is finding a renewable source of these cells, as adult cardiomyocytes do not readily divide in vitro and thus cannot be expanded to the numbers required for repair. Deriving cells from donor hearts adds the problem of potential immune rejection, requiring immunosuppressive therapies. Even if a source of adult cardiomyocytes could be found, another major problem exists: adult cardiomyocytes have a very limited ability to survive in an ischemic environment (hence the original infarct). Taken together, these limitations suggest cardiomyocytes may have a very limited role in cell repair unless these cells undergo some manipulation to render them capable of division and survival in vivo. To overcome this, some groups have begun to use fetal or neonatal cardiomyocytes for preclinical cardiac repair. These studies show that in infarcted hearts, fetal cardiomyocyte transplantation can improve function, including LV dimensions and developed LV pressures [55, 58, 71, 77, 79, 83]. In addition, transplanted cells could be found in injected hearts up to 6 months after delivery — a marked improvement over adult cardiocytes. Cardiomyocytes injected into uninjured hearts demonstrated evidence of cell-cell coupling with host cardiomyocytes. Unfortunately, the hostile environment of scar present in infarct has so far prevented any evidence of coupling between transplanted and native cardiomyocytes [71] in injured myocardium. Nonetheless, fetal or neonatal cardiocytes are an alternative to adult cells. However, finding a pool of fetal or neonatal cardiomyocytes itself raises a number of ethical questions that currently limit their potential as well. A new type of contractile cell that must now be considered is the cardiac stem cell (CSC). Although the evidence for cardiac repair with these cells is limited, their potential to mature into cardiomyocytes makes them an attractive candidate. Again, these cells have primarily been isolated from neonatal heart [47], and to a limited extent from adult myocardium [4, 12, 62]. Their use preclinically is intriguing and suggests that the future of cardiac repair may involve endogenous stem cells. CSCs from postnatal rat hearts can be isolated using LIM-homeodomain transcription factor islet-1 (isl1). It is possible to expand these cells in vitro when coupled with a cardiac mesenchymal
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feeder layer. Further, when these cells are co-cultured with neonatal cardiomyocytes, they are able to electrically integrate with myocardial cells in vitro by formation of gap junctions [47]. CSCs isolated from adult hearts — including those from acutely infarcted, failing, and uninjured transplant hearts — have been identified by their expression of c-kit, MRD1, and Sca-1 and their lack of expression of hematopoietic lineage markers [101]. These cells show the ability to differentiate down myocyte, smooth muscle cell, and endothelial cell pathways, but their ability to form mature cells of these types (or cardiomyocytes) is as yet unknown. Oh et al. suggest that endogenous Sca-1 positive CSCs may be able to differentiate into functional cardiomyocytes, but their differentiation potential within infarct scar is as yet unknown [61]. To date, methods for the harvest, expansion, and in vitro growth of these precursors are limited. This, combined with their unknown differentiation potential, makes their clinical use at this time highly unlikely. Nonetheless, their biology is interesting and bears watching for future developments.
SKELETAL MYOBLASTS The contractile cell type currently undergoing the most preclinical and clinical study is the skeletal muscle precursor cell, or skeletal myoblast. Myoblasts, also called satellite cells, are typically quiescent cells lining the interior basal membrane of skeletal muscle fibers. Myoblasts have several distinct advantages over cardiomyocytes in their potential use as cell therapy agents for heart failure. Most distinctive among these are myoblasts’ replication, or growth potential. A single several-gram autologous skeletal muscle biopsy from a patient has the potential to expand into hundreds of millions of cells, even in patients up to and exceeding 90 years of age [10]. This seemingly unlimited pool of autologous cells overcomes the greatest hurdles facing cardiomyocytes: number of cells and immune rejection. Another advantage myoblasts have over cardiomyocytes as a cell source is that myoblasts are much more resistant to ischemic damage than cardiomyocytes. Because cardiomyocytes are some of the most metabolically active cells in the body, they require direct contact with capillaries to supply their high demand for nutrients and oxygen (hence their inability to survive once a region becomes ischemic or hypoxic). Myoblasts, in contrast, are found lining muscle fibers’
basal membrane and are often far from capillaries. In vitro studies have shown that myoblasts are able to survive under 0.2% oxygen and 100 mg/l glucose (10× lower than typical growth media for cardiocytes or skeletal muscle) [18]. This ability of myoblasts to survive under ischemic stress is of vital importance when considering a cell for repair of infarcted myocardium. In preclinical studies, myoblasts have the ability to proliferate and differentiate into mature muscle in infarcted myocardium. The resulting cells not only possess characteristics common to skeletal muscle but express proteins such as slow-myosin [27, 29], cardiacactin, and cardiac troponin, typically found in cardiomyocytes. This pattern of expression suggests that the transplanted myoblasts may have formed a contractile cell type better suited to the cardiac workload. Most importantly, myoblast transplantation improves both global and local indicators of heart function following myocardial infarction [1, 5, 7, 8, 58, 59, 93, 95], and prevents or ameliorates negative remodeling of the left ventricle (LV) [48, 61, 93]. By both pressure-volume loops and echocardiography, myoblast transplantation has been shown to improve global LV function [6, 7, 9, 59, 64, 93–95]. In fact, myoblast therapy has been shown to turn dyskinetic regions of damaged heart into areas with synchronous contraction, as measured by tissue Doppler imaging [6, 67–69], direct visualization, or MRI. It is not completely understood how myoblasts are able to synchronously contract with the uninjured portion of the heart, as there is no evidence of cell-cell coupling between transplanted myoblasts and native cardiomyocytes. It has been suggested that the synchronous contraction is due to stretch or electromechanical-activated receptors [6], and the timing of the initiation of regional contraction of the myoblasts — a few milliseconds after the initiation of systole — suggests that this is the case. It is also possible that by promoting angiogenesis, myoblast therapy allows hibernating regions of muscle to recontribute to the cardiac workload [15]. There are, however, negative aspects to using skeletal muscle precursor cells to treat cardiac injury. Skeletal myoblasts and mature skeletal muscle cells behave electrically different than cardiac cells. Their action potential magnitude is similar, but the action potential duration differs by an order of magnitude [26, 36]. Further, myoblasts — even when injected into uninjured myocardium — are unable to form electrical connectivity to native cardiomyocytes [73].
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Unfortunately, the difference in electrical activity of the implanted myoblasts can lead to arrhythmic activity in the heart, including but not limited to ectopic beats and tachychardia [54]. Fortunately, once this potential risk was understood electrical abnormalities have been controllable in the clinical setting and have caused relatively few life-threatening events (see the “Clinical Studies” section following). More recently, with the use of low-dose antiarrhythmic therapy and more careful screening of the electrical stability of patients, this concern has decreased significantly.
EMBRYONIC STEM CELLS
When discussing “stem” (or progenitor) cells, the cell type that comes to most minds is embryonic stem cells. Embryonic stem cells (ESCs) possess a number of properties that could make them attractive candidates to treat ischemic heart disease. ESCs have active telomerase, which makes them able to self-renew and provide a seemingly unlimited number of cells to be used for cell therapy. Further, ESCs have the ability to differentiate into true cardiomyocytes, with the ideal electrical and mechanical properties to replace damaged heart tissue. In addition, prior to differentiation ESCs do not have the extremely high metabolic demands of mature cardiomyocytes. Aside from the obvious ethical and moral Noncontractile Cells debate raised by the use of embryonic stem cells, these benefits may make embryonic stem cells seem the perAlthough contractile cells would seem the ideal choice fect candidate to use for transplantation into infarcted to repair infarcted myocardium, restoring blood sup- myocardium. ply to the infarct is also a goal. Furthermore, given Despite the benefits, however, a number of hurdles the limitations of both contractile cell types discussed, exist. First, the transplantation of ESCs into host hearts other noncontractile cells have been proposed as agents would require immunosuppression to prevent immune for cardiac repair. These noncontractile cells encom- rejection and/or graft versus host disease. In addition, pass both cells that will remain noncontractile, such as transplantation of embryonic stem cells into infarcted fibroblasts, and cells that may hold the potential to dif- heart tissue, which is essentially scar, could potentially ferentiate down vascular and muscle lineages, such as lead the embryonic cells into differentiation down a nonvarious types of stem or progenitor cells. cardiac lineage as they respond to the local milieu. Thus, ESCs could easily become adipocytes, fibroblasts, or even other myogenic cells. For this reason, the most successful work with embryonic stem cells as a cell type for FIBROBLASTS transplantation into infarcted myocardium has thus far Fibroblasts are connective tissue cells that excrete extra- focused on differentiating the ESCs into cardiomyocytes cellular matrix proteins and form underlying tissue or pre-cardiomyocytes prior to implantation [31, 84]. stroma. In fact, fibroblasts, not cardiomyocytes, are In this state, embryonic cells show the potential to the most abundant cell type in the human heart. Their improve LV ejection fraction (EF) and regional wall use as preclinical therapeutic agents has primarily been motion in Sprague-Dawley rats. Some groups, in order to determine whether stable noncontractile cells can to overcome potential immunologic hurdles, propose to improve cardiac function. In our hands, transplanted use cardiomyocytes derived from embryonic stem cells dermal fibroblasts were able to improve material and to precondition autologous adult stem cells toward a cardiastolic properties of infarcted heart but were not diac phenotype by use of co-culture or cell fusion [28]. able to improve systolic function [35]. This failure The differentiated autologous stem cells would then be to improve contractility is likely due to fibroblasts’ purified from the immunogenic ES cells and used for inability to contract, but cell-related improvements in cell therapy. Although ESCs offer great future potential as a source diastolic dysfunction suggest that these cells may have a role in diastolic heart failure. Nonetheless, because of cardiocytes or pre-cardiocytes for cardiac repair, they fibroblast therapy was unable to improve systolic func- remain clinically unusable to date. This is primarily tion these cells have not moved into clinical studies. due to their pluripotentiality and their corresponding Instead, noncontractile cells moved forward are those capacity to form teratomas in vivo if the cells are not that have shown potential to differentiate into contrac- extensively purified prior to delivery. This inability to control cell fate and the potential deleterious outcome tile cells. 262
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limits current enthusiasm for these cells except as an experimental tool.
BONE-MARROW-DERIVED STEM CELLS To move toward progenitor cells that have the potential to become cardiomyocytes but overcome the ethical and immunological hurdles of embryonic stem cells, researchers have begun to examine autologous sources of stem cells, such as bone marrow and peripheral blood. Adult human bone marrow and blood contain a number of cell populations that have recently been shown capable of differentiation into cells other than blood. They include hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and subsets of each of these, including CD34+ progenitors, multipotent adult progenitor cells (MAPCs), and CD14+ blood outgrowth cells. Similar progenitor cell populations have also been isolated from umbilical cord blood. HEMATOPOIETIC STEM CELLS Historically, hematopoietic stem cells (HSCs) have been thought of as cells that differentiate only into various blood and immune cells (i.e., red and white blood cells). These cells are typically identified by labeling with CD34 and/or AC133 for human cells, and in mice are examined to be negative for a cocktail of mature hematopoietic cell markers (lineage-negative) and sometimes positive for Sca-1 and c-kit (CD117). Over the past few years, it has been shown that HSCs have the ability under the right conditions to differentiate into various cell types, including cardiomyocytes [105]. Although HSCs may be able to become cardiomyocytes, there is as yet no evidence that they can transdifferentiate into cardiomyocytes when transplanted into infarcted myocardium [20, 57]. Perhaps because of this lack of differentiation in infarct, recent studies suggest that HSCs may not have the potential of some other cell types discussed here to improve LV function following transplantation into infarcted myocardium [20]. The studies on treatment of myocardial infarction using AC133+ cells in human patients are discussed in the “Clinical Studies” section following. MESENCHYMAL STEM CELLS Mesenchymal stem cells (MSCs) are rare pluripotent progenitor cells also known as bone marrow
stromal cells. In the past, MSCs were shown to differentiate into a number of mesenchymal cell types — including adipocytes, fat, bone, cartilage, and skeletal muscle [40, 102] — both in vitro and in infarcted rat myocardium [33]. There is also some evidence that upon injection into myocardium these MSCs can differentiate into cardiomyocytes [42]. In a number of studies, transplanted MSCs have been shown to engraft at high numbers in infarcted heart [78] and lead to a number of functional benefits. In fact, a number of studies suggest that MSCs can home to sites of injury following injection into the coronary or even peripheral vasculature [14, 88]. When in infarcted hearts, MSCs have been shown to increase neovascularization by promoting angiogenesis [46, 78] and to improve regional contractility and global diastolic function [100]. In fact, one recent study from our group suggests that MSCs and skeletal myoblasts improve function after ischemia-induced cardiac injury to a similar degree when compared in a side-by-side fashion [98]. Despite reports that MSCs can differentiate into cardiomyocytes in vivo, current studies suggest that this can only happen when MSCs are in contact with native cardiomyocytes [88] and thus cannot happen in interior infarct tissue. These data imply that the optimal time course of therapy using MSCs may be early in the disease state, when surviving cardiomyocytes are present in the infarct. More recently, MSCs have been touted as immune privileged cells capable of allogeneic administration in vivo with few or no negative consequences [39]. This is possibly the most tantalizing aspect of these cells for cardiac repair: the potential to develop an off-the-shelf product for use in many patients. Although this is a relatively recent finding, clinical studies with intravenous administration of allogeneic MSCs have begun recently. ENDOTHELIAL PROGENITOR CELLS Recent research has shown that the number of endothelial progenitor cells (EPCs) present in vascular circulation is increased in patients following acute myocardial infarction [81]. As EPCs are thought to be partially responsible for neoangeogenesis [41], researchers assume these EPCs respond to the ischemic damage in the heart and migrate to damaged areas to induce formation of neovasculature. In support of this, Aicher et al. showed that when EPCs were injected into rats in either the tail vein or left ventricular cavity animals who had ischemic myocardial injury showed a greater than twofold increase in the accumulation
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of infused EPCs when compared to animals undergoing sham surgery [3]. Further evidence showed that rats undergoing intravenous injection of EPCs following ischemic myocardial injury showed improvements in ventricular dimensions, fractional shortening, and regional wall motion when compared to animals injected with culture media control [43, 52]. It is likely that the improvements seen in this study may be secondary to the improved vasculature and myocardial perfusion and subsequent rescue of hibernating myocardium. Human EPCs are typically thought to originate from cells expressing CD133 (AC133), CD34, VEGF-R2, and occasionally CD14. The number of EPCs present in human patients decreases with age, and mirrors a rapid increase in cardiovascular deaths. It has been suggested that this correlation is due to EPCs role in maintaining vascular health [45]. Recent data have shown that the number of circulating EPCs and their ability to migrate decreases in patients with increased risk of coronary artery disease, including myocardial infarction [76]. These data suggest that a reduction in the number and/or functional capacity of EPCs may be a critical factor in ischemic cardiovascular events. Along this line, our group recently published data showing that a reduction in vascular progenitor cells (VPCs), defined by expressing CD31 but not CD45 and thought to be related to EPCs, is associated with aging and disease state in a genetic model of atherosclerosis. We showed that delivery of functionally viable cells could even prevent the progression of atherosclerosis in a genetic mouse model of atherosclerosis [70]. Further studies using EPCs to treat myocardial disease in humans are discussed in the “Clinical Studies” section following. CORD BLOOD CELLS A relatively new source for stem or progenitor cells is umbilical cord blood, which contains most if not all of the bone-marrow-derived cell types discussed previously. Cord blood cells are easily obtained, have the potential to develop into multiple lineages, do not pose the ethical questions raised by embryonic stem cells, and are less immunogenic than their bone marrow counterparts — which means that a larger percentage of the population could receive cells from matched donors. Further, if cord blood cells are isolated and stored at birth these cells could provide an autologous source of stem cells to treat later myocardial damage. Current studies in animal models show that mixed cord blood
cells injected directly into infarct sites show the potential to improve LV ejection fraction, anteroseptal wall thickening, and dP/dt(max), while decreasing infarct size [30]. In addition, injection of cord blood cells intravenously in mice following ligation-induced ischemic cardiac injury showed approximately 20% higher capillary density in infarct borderzones when compared to untreated animals [50]. Recent data from our group suggest that human-cord-blood-derived CD34+ cells may be capable both of preventing injury progression in nude rats if administered shortly after MI, and of partially reversing systolic and diastolic dysfunction in the failing heart. Unfortunately, no evidence yet suggests that cord blood cells injected into infarcted portions of the myocardium have the potential to differentiate into mature cardiomyocytes. Overall, however, it appears that cord blood cells may provide an interesting cell of choice for further study in treatment of myocardial injury.
Cell Delivery Timing and Techniques Aside from the choice of cell type, perhaps the most important aspect of cell therapy is the choice of the technique for and timing of cell delivery. It may seem obvious, but injuries at different states of repair require different approaches to treat (Figure 14.1). That is to say, in the same manner that a recent infarct may require different cell choices than a healing scar, it is likely that similar injuries at differing times will require alternative cell delivery modalities. Figure 14.1 shows some of the cell types that have been proposed for cardiac repair and conditions when each may be appropriate. Importantly, the figure also shows the critical relationship between neovascularization and generation of newly contracting muscle; that is, the need for both blood supply and muscle for optimal outcome. Careful choice of cell type could lead to better functional outcomes of cell therapy.
TIMING OF CELL THERAPY Some researchers hypothesize that the timing of cell therapy will greatly determine the cell type necessary to achieve maximum benefit, suggesting that whole bone marrow or various subsets of bone-marrow-derived and blood-derived progenitor cells will be most effective at treating myocardial infarction in the acute stage of
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Injury determines choice of cell type
Acute MI
Heart Failure
Angiogenesis (Vascular Cells)
Myogenesis (Contractile Cells)
ESCs HSCs MSCs EPCs CD34+ BM Cells Cord blood PCs
Cardiomyocytes Cardiac Stem Cells Myoblasts Differentiated Stem Cells
Figure 14.1. Choosing the ideal cell type(s) for cardiac repair depends on the type of injury and the desired outcome. In cases of refractory angina, acute MI, or other settings where restoring blood flow and rescuing injured myocardium is the desired outcome, cells with vascular potential are the likely choice. Conversely when restoration of contractile function is required, the likely therapeutic option is contractile cells. In both cases, cross-talk between angiogenesis and myogenesis may be the most important aspect of treatment. If newly formed vessels do not target the appropriate region, they are less useful. Likewise, if newly formed contractile cells do not receive adequate perfusion or blood supply they will not survive. Combined therapies may be the treatments of the future.
injury, where angiogenesis is likely sufficient before significant negative remodeling has begun to occur [46, 63, 88]. These cell types have shown an increased ability over other cells studied to improve angiogenesis and to rescue hibernating myocardium [43, 46, 78]. Further, as mentioned previously a number of multipotent progenitor cells require the presence of native cardiomyocytes to allow proper differentiation into cardiomyocyte-like cells [57]. On the opposite end of the spectrum, myoblasts, cardiomyocytes, or progenitor cells with a pre-initiated tendency toward a muscle phenotype would likely provide more benefit when treating patients with end-stage heart failure (where myogenesis is required). These patients require a therapy that will provide new cells capable of aiding the contractile ability of the heart wall. Figure 14.2 shows a relative timeline outlining at which stage of disease different cell therapy options might best improve cardiac function. Although each patient’s disease may progress at a different rate, using these guidelines may help to standardize treatment options to improve patient outcome.
CELL DELIVERY TECHNIQUES Choice of cell delivery technique can also be crucial to the success of cell therapy. Cells can be delivered
into the peripheral or coronary circulation, into the myocardium via a catheter, or by direct surgical injection via an open approach, a minimally invasive procedure, or robotically. They can be released into the intravenous or intra-arterial coronary circulation or they can be injected directly into the ventricular wall. Further, injections can be performed in a single bolus or in a series of smaller injections [65]. These varied techniques will result in a wide range of possible distribution patterns for the transplanted cells. Unfortunately, little research has been done to quantitatively compare differing cell delivery techniques for each cell type. It is likely that comparisons of cell delivery will be primarily preclinical because clinical studies will have to take the target patient population into account. For example, patients shortly after acute MI may be unable to undergo an open chest procedure necessitated by direct surgical injection, whereas patients with end-stage heart failure may not have patent vessels to allow intracoronary delivery. To combat this limitation, studies have recently been done to examine a method to use a video-assisted thorascopic technique to visualize the infarct and directly inject myoblasts [99]. We have also begun to explore the use of roboticassisted cell delivery. Data suggest that robot-assisted injection into the beating heart is feasible and allows minimally invasive targeted cell delivery to a severely
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Cardiac Damage
Inflammation Angiogenesis (Vascular Cells)
Cardiocyte Apoptosis
Remodeling/compensation
Scar Expansion Myogenesis (Contractile Cells)
Decompensation
Progression to HF Figure 14.2. Heart failure is a progressive disease resulting from acute or chronic cardiac injury. This figure shows a progression from acute myocardial injury to heart failure with indications of when therapy should focus on cells with vascular vs. contractile potential and indicating that paracrine effects are likely the therapeutic effects of both types of cells.
injured failing heart. In addition, a number of studies have been done to examine endoventricular injection of cells into the center of the infarct scar via a catheter [17, 19, 21]. For this approach, a NOGA catheter (which allows electromechanical mapping) can be used to allow the surgeon to ensure cell delivery to the desired scar location. Another option that has begun to be studied is to use stereotactic magnetic navigation (which has been used previously for RF ablation) for targeted catheter delivery of cells [23]. Finally, in an effort to combine the benefits of catheter and direct surgical injection methods some researchers have begun to study a technique called transvascular cellular cardiomyoplasty, where catheter needles are guided through the coronary arteries or coronary veins and positioned directly into the injured heart [82, 96, 97].
Clinical Studies MYOBLASTS The first clinical trial using cell therapy to treat heart disease was initiated by Philippe Menasche and colleagues in 2000 [54]. In this trial, an average of 870 × 106
cells (at least 85% were identified as myoblasts by a positive staining for CD56) were injected into the scarred LV concomitantly with coronary artery bypass grafting (CABG). Over several years following transplantation, significant improvements in LV function were seen, including EF and regional wall thickening. Unfortunately, no definitive conclusions can be drawn from the functional improvements seen, as no control group was included in the study and the functional improvements could theoretically be in response to CABG. One other interesting piece of data coming from the study is that 4 of 10 patients experienced some form of ventricular tachycardia. Fortunately, in none of the patients were these arrhythmia fatal, and data suggested that appropriate prophylaxis by amiodarone could minimize electrical events [54]. The data generated in this study provided the impetus to perform a new trial begun in 2003. The MAGIC (Myoblast Autologous Graft in Ischemic Cardiomyopathy) trial is a phase 2 clinical trial that will examine up to 300 patients in North America and Europe and be completed by the end of 2006. Results of this extensive study are eagerly anticipated. In a separate trial, where myoblasts were injected concurrently with CABG (12 patients) or LVAD as a
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bridge to heart transplant (6 patients), patients showed improved myocardial perfusion and EF. Further, 4 of the 5 patients who underwent heart transplant showed areas of engrafted myoblasts within infarcted myocardium [22]. Another myoblast trial, where myoblasts were injected as sole therapy (no CABG), treated patients who were in symptomatic heart failure with 196 +/− 105 × 106 myoblasts injected directly into the infarct area via an NOGA-guided catheter system [85]. These patients showed improved wall motion and a trend toward increased EF over 3 to 6 months. Taken together, these data suggest that myoblasts can be delivered late in the disease process (even in heart failure), implant and survive within infarcted myocardium, and improve both diastolic and systolic function.
SAFETY Early reports of electrical instability in patients after receipt of autologous skeletal myoblasts have led to doubts about the safety of these cells as a treatment in the injured heart. However, conflicting data and the early versus later results suggest that several considerations should be made of these data. First, patients who received myoblasts in the earliest clinical studies [54, 85] were extremely ill patients with an expected high potential for negative electrical events. In fact, many of the patients who were included in the early trials met the MADIT-II criteria, which were presented after those trials began and suggested that all patients who meet those criteria be treated with AICDs (automatic implantable cardio-defibrillator). As a result, in more recent clinical studies where myoblasts are being used to treat heart failure many investigators have only enrolled patients who receive AICDs or low-dose antiarrhythmic agents prophylactically. This has significantly reduced the reported incidence of adverse events. For example, in the MAGIC trial as reported at the American Heart Association meetings in November of 2004, the incidence of electrical instability in patients post myoblast delivery was approximately 10%, lower than the initial 40% reported by the same group. Whether this is due to a better selection of patients in the second study, the co-administration of antiarrhythmic agents, or an improved safety profile of the cells remains to be determined. Furthermore, in clinical studies in the United States, Dib and colleagues have not reported an increased incidence of electrical instability after myoblast administration, nor have Chachques and
colleagues in preclinical studies [16]. Again, whether this represents a difference in patient selection or cell product remains unclear. Nonetheless, this combination of data suggests that autologous myoblast delivery for patients with heart failure has potential as a relatively safe and efficacious product. Randomized controlled trials underway at present are the only way to fully address these issues, and their results should be eagerly anticipated.
BONE MARROW STEM CELL In a trial similar to that performed on myoblasts by Menasche and colleagues, 1 to 5 × 106 AC133+ bone marrow stem cells (see “Hematopoietic Stem Cells” section previously) were injected into the infarct borderzone of 6 patients, with concurrent CABG. In contrast to myoblast studies, this study examined treatment of acute myocardial infarction. A total of 12 patients were treated, and they showed increased perfusion in treated areas and improved LV dimensions and EF compared to controls [86, 87]. Further, unlike patients in myoblast trials the improvements in this trial occurred without any incidence of electrical abnormalities. Another clinical study examining stem cells of bone marrow origin (the TOPCARE-AMI study) injected 16 +/− 12 × 106 circulating progenitor cells (CPCs) or 213 +/− 75 × 106 bone marrow progenitor cells (BMCs) into the infarct artery of patients (n = 30 for CPCs and n = 29 for BMCs) 4.9 +/− 1.5 days after acute myocardial infarction. At 4 months post therapy, LV end-diastolic volumes and EF were improved in both cell groups compared to controls, but no significant difference between groups was observed. By one year, EF was still significantly improved, infarct size was decreased, and there was no evidence of reactive hypertrophy. These results suggest favorable modeling in response to cell therapy [76]. What is very interesting to note is that similar results were seen between the cell-treated groups, despite the fact that an order of magnitude more BMCs were injected than CPCs. These data, when combined with the data by Stamm et al. mentioned previously, suggest very favorable response to bone-marrow-derived stem cell therapy following acute myocardial infarction, with improved myocardial performance secondary to improved cardiac perfusion. What remains unsure is the response of latestage injuries to bone marrow stem cell therapy. To combat this, an addition to the TOPCARE-AMI study
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(the TOPCARE-HF study) has been planned. With evidence on the reduced number and migratory capacity of EPCs shown in preclinical data [76], it will be interesting to see if cells from these patients are as viable in improving cardiac function. In Germany, a randomized bone marrow transfer to enhance ST-elevation infarct regeneration (BOOST trial) compared 30 patients receiving optimal standard care following infarction to 30 patients receiving optimal standard care plus 24.6 × 108 ± 9.4 × 108 (mean ± SD) BMCs 4.8 ± 1.8 days after infarct. Six months after therapy, patients receiving BMCs showed significantly enhanced LVEF when compared to control patients. Further, systolic function was improved without any incidence of arrhythmic events or restenosis of stents [103]. Finally, in Belgium, a recent clinically controlled trial compared the ability of autologous bone-marrowderived cells (average of 172 × 106 mononuclear cells) to a placebo for ability to improve function after acute MI. In this study, therapy with bone-marrowderived cells was able to reduce infarct size, but no improvement in LV function was seen when compared to placebo treatment. In fact, over 4 months the LVEF of the placebo group tended to improve more than patients receiving BMCs, though results were not statistically significant [37]. These results show that there is still work to be done to understand how cell therapy can improve function following ischemic myocardial injury.
state of the differences in the use of myoblasts versus bone-marrow-derived stem cells for myocardial repair.
OPTIMIZING CELL TYPE AND DELIVERY TECHNIQUE FOR DIFFERENT CV DISEASE STATES As discussed previously, one of the major problems in comparing individual studies — both preclinically and clinically — is the lack of similar cell type (e.g., EPCs in one study may be defined differently than EPCs in another), lack of similar delivery method, or even lack of similar disease state in patients at various stages of ischemic heart disease. Even with a similar cell type, functional outcome could be greatly affected by varying distribution of injected cells from intravascular to intramyocardial, from the infarct periphery to the infarct center, and from a single bolus to multiple microinjections. Undoubtedly, cell delivery matters. Cells injected into the coronary circulation are much more likely to remain in the infarct periphery or borderzone than cells directly injected within the center of the scar. Furthermore, the numbers of cells typically injected into intravenous or coronary circulation vary by an order of magnitude from direct surgical injections (Table 14.1). Although it will require significant resources, it is necessary to take a thorough approach to optimize (and standardize) cell type, treatment time, and cell delivery techniques for a range of ischemic cardiac injuries.
Challenges for Future Improvement
SURVIVAL OF TRANSPLANTED CELLS
Cardiovascular cell therapy is here! Yet, despite the preclinical and clinical successes achieved so far a number of hurdles still exist before cell transplantation will be a routine accepted procedure for the treatment of cardiac disease. The timing, the method of delivery, and even which cell type to use remain to be optimized for various types of cardiac injury. Survival of the transplanted cells needs to be improved, and we need to understand how to program transplanted cells to reproducibly and robustly differentiate into various cell types, including endothelial cells, smooth muscle cells, and cardiomyocytes. Finally, we must learn how to best mechanically and electrically integrate the transplanted cells into the existing cardiac environment to maximize functional improvement while minimizing any electrical abnormalities. Table 14.1 outlines the current
Currently, one of the largest hurdles of cell transplantation is the limited survival of transplanted cells. Most reports suggest that 70 to 90% of all transplanted cells die within the first few days of transplantation into infarct scar [51, 80]. Studies have shown that a subset of the transplanted cells survive and multiply [11], but it is unlikely that this multiplication can make up for the massive early cell death. Preclinical data suggest a dose response for several cell types, indicating that improving the number of surviving cells is critical to maximize functional improvement after cell therapy [24]. The incidence of cell death could be associated to one of several factors. First is immune rejection, which can be ruled out because of the use of autologous cells in the majority of treatments performed to date. Another possibility
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TABLE 14.1 Comparison of clinical studies using myoblasts and bone-marrow-derived cells for cardiovascular repair. Currently, myoblasts show greater potential to treat late-stage disease due to their myogenic potential and contractility. Bone-marrow-derived stem cells have shown little potential to differentiate into myogenic cells when in scar tissue, but they have shown the ability to improve angiogenesis and perfusion in acute MI, leading to improved LV protection. Cell Type
Patients
Dose
Delivery
Positives
Negatives
Myoblasts
• Post MI
25–1,200 ×106
• Direct injection
• Reverse
• Electrical
• Heart
remodeling • Intraventricular
failure
• Increase wall
motion
abnormalities • Low survival
• Increase
contractility Bone marrow stem cells
• Acute MI • Angina
2–250 ×106
• Intravascular
• Angiogenesis
• Intracoronary
• Increased
• Low myogenic
differentiation perfusion
• Low survival
• Increased
wall motion
is the inflammatory response secondary to needle puncture during delivery. The inflammatory response of cell injection has proven to be mild in most cases, but this possibility should be explored further. The problem faced is determining whether inflammation is an initiator of the death of transplanted cells or a response secondary to the death of the transplanted cells. The most likely hypothesis is cell apoptosis brought on by exposure to the ischemic environment of the infarct scar. This hypothesis is strengthened by data showing that survival of neonatal cardiomyocytes more than doubled when injected into 2-week-old cardiac granulation tissue or normal myocardium versus myocardial scar tissue in rats [74]. Further, preconditioning of cells before transplantation via heat shock or transfecting cells with the survival factor Akt provides protection of cells from damage by hypoxia or glucose deprivation in vitro and helps to increase their survival rate in vivo [74, 89, 105]. Ideally, more work will be focused on this area of improved cell survival in the future to better define the relationship between the microenvironment of the infarct scar and outcome of the transplanted cells. Only by doing so can we better design methods to maximize cell survival and thus to increase the benefits versus the risks of cardiovascular cell therapy.
DRIVING STEM CELLS TOWARD A CARDIAC PHENOTYPE The ideal cell for myocardial repair will be able to withstand the harsh scar environment at the time of implantation but become a fully functioning cardiomyocyte or vascular cell over time. As discussed previously, none of the progenitor cells currently used satisfies both of these criteria in high proportions. Therefore, it is important to keep working toward understanding the differentiation of progenitor cells into a cardiomyocyte phenotype. The goal, then, might be to initiate a pathway of differentiation prior to implantation that would occur slowly enough to allow neovascularization to provide nutrients for the emerging nascent myocardium.
ELECTRICAL INTEGRATION As discussed previously, myoblasts have significantly different electrical properties from cardiomyocytes. These differences can lead to electrical abnormalities, including ventricular tachycardia. For cardiovascular cell therapy to reach its potential, it will be critical
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to electrically integrate transplanted cells into the surviving myocardium. This problem may be approached by genetically altering transplanted cells, by developing new adjunctive safety measures, or preferably by somehow conditioning the transplanted cells to become true cardiomyocytes that can survive in an injury milieu.
4. 5.
Conclusions Cell therapy for myocardial repair is emerging from preclinical studies and clinical trials as a potentially viable option in the treatment of heart disease. The results so far have been exciting, but caution must be maintained. For cell therapy to reach its potential, we must use preclinical research to overcome the four major hurdles that still exist in cell therapy for myocardial repair: picking the proper cell type and delivery options for various forms of myocardial damage, maximizing survival of transplanted cells, properly differentiating progenitor cells down cardiomyocyte and/or endothelial cell pathways, and electrically integrating the transplanted cells. To achieve these goals will require the use of cuttingedge technologies, such as tissue engineering, new imaging modalities, and molecular biology. Clinical trials must be developed to better test the safety and efficacy of cell therapy in side-by-side comparisons in a variety of myocardial injuries, from acute myocardial infarction to end-stage heart failure. We have the opportunity to create a new era in the treatment of heart disease. Doing so will require continued bench-tobedside and back-to-bench evaluations as we learn from early clinical studies, develop new preclinical models, and ultimately design trials and tools to maximize the potential of cell therapy.
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Bryce H. Davis, Harald C. Ott, and Doris A. Taylor 84. Smits, A. M., van Vliet, P., Hassink, R. J., Goumans, M. J., Doevendans, P. A. The role of stem cells in cardiac regeneration. J Cell Mol Med 9:25–36; 2005. 85. Smits, P. C., van Geuns, R. J., Poldermans, D., Bountioukos, M., Onderwater, E. E., Lee, C. H., Maat, A. P., Serruys, P. W. Catheter-based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischemic heart failure: Clinical experience with six-month follow-up. J Am Coll Cardiol 42:2063–2069; 2003. 86. Stamm, C., Kleine, H. D., Westphal, B., Petzsch, M., Kittner, C., Nienaber, C. A., Freund, M., Steinhoff, G. CABG and bone marrow stem cell transplantation after myocardial infarction. Thorac Cardiovasc Surg 52:152–158; 2004. 87. Stamm, C., Westphal, B., Kleine, H. D., Petzsch, M., Kittner, C., Klinge, H., Schumichen, C., Nienaber, C. A., Freund, M., Steinhoff, G. Autologous bone–marrow stemcell transplantation for myocardial regeneration. Lancet 361:45–46; 2003. 88. Strauer, B. E., Brehm, M., Zeus, T., Kostering, M., Hernandez, A., Sorg, R. V., Kogler, G., Wernet, P. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106:1913–1918; 2002. 89. Suzuki, K., Smolenski, R. T., Jayakumar, J., Murtuza, B., Brand, N. J., Yacoub, M. H. Heat shock treatment enhances graft cell survival in skeletal myoblast transplantation to the heart. Circulation 102:III216–221; 2000. 90. Syed, I. S., Sanborn, T. A., Rosengart, T. K. Therapeutic angiogenesis: A biologic bypass. Cardiology 101:131–143; 2004. 91. Tam, S. K., Gu, W., Nadal-Ginard, B. Molecular cardiomyoplasty: Potential cardiac gene therapy for chronic heart failure. J Thorac Cardiovasc Surg 109:918–923; discussion 923–914; 1995. 92. Taylor, D. A. Cellular cardiomyoplasty with autologous skeletal myoblasts for ischemic heart disease and heart failure. Curr Control Trials Cardiovasc Med 2:208–210; 2001. 93. Taylor, D. A., Atkins, B. Z., Hungspreugs, P., Jones, T. R., Reedy, M. C., Hutcheson, K. A., Glower, D. D., Kraus, W. E. Regenerating functional myocardium: Improved performance after skeletal myoblast transplantation. Nature Medicine 4:929–933; 1998. 94. Taylor, D. A., Silvestry, S. C., Annex, B. H., Glower, D. D., Kraus, W. E. Skeletal myoblast therapy in cardiovascular disease. In: Gene Transfer in Cardiovascular Biology: Experimental Approaches and Therapeutic Implications, pp 355–375. Ed by Marsh, K. L. Norwell, MA, Kluwer Academics, 1996. 95. Taylor, D. A., Silvestry, S. C., Bishop, S. P., Annex, B. H., Lilly, R. E., Glower, D. D., Kraus, W. E. Delivery of primary autologous skeletal myoblasts into rabbit heart by
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15 Cylindrical Cartilage Transplantation for Tracheal Replacement Koji Kojima, M.D., Ph.D, and Charles A. Vacanti, M.D. Laboratory for Tissue Engineering and Regenerative Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts
Introduction This chapter provides a review of the advances over the past 10 years in the development of a tracheal replacement within the field of tissue engineering and organ regeneration. In the last decade, numerous attempts have been made to generate tracheal replacements [14]. It is well known that direct anastomosis is currently the most effective way to treat patients with tracheal defects. However, such an approach has some limitations. For example, in adults the conventional limit for successful tracheal resection and anastomosis is half the tracheal length, and in small children the limit is only about one-third. This limitation becomes problematic for clinicians because many illnesses, such as stenosis resulting from either post-intubation tracheal granulation or tumors, create defects larger than those that can be treated effectively. Currently, these patients are treated with laser therapy and stents. Some of these treatments include the fashioning of a tracheal conduit from various tissues and materials, using autografts [9, 24, 30], allografts [22, 25, 27], prosthetic materials [28, 29], or a combination of the three [2, 31]. However, to date none of these techniques has proven to be clinically practical. For this reason, the application of tissue-engineering techniques using a patient’s own cells to generate new structural tissues is an ideal solution to overcoming the shortage of reliable tissue for transplants [41, 43]. The field of tissue engineering combines biology, engineering, and polymer chemistry to produce new tissues in vitro for subsequent in vivo placement [6] for subsequent growth of the tissues. Such an approach has been
Cellular Transplantation: From Laboratory to Clinic
used to produce islet cells of the pancreas and liver hepatocytes and to reconstruct cartilaginous structures [8, 17, 35], bone [44], and skin [3, 16, 26]. However, few studies have focused on the reconstruction of the trachea [13, 15, 20, 21, 23, 34, 42]. Recently, Lee and colleagues demonstrated that a 5-mm × 5-mm full-thickness rabbit tracheal wall excision could be replaced with an implanted cell-seeded polymer scaffold [23]. Although the authors detected epithelialization, they were unable to detect any viable chondrocytes in the implanted specimens. Other investigators have also reported limitations involving tissue-engineered replacements. Fuchs and colleagues reported that prenatal tracheoplasty using engineered cartilage is effective for severe congenital tracheal malformations but that it is limited to use in adults [13]. It is our view that the cartilage component of the trachea is essential. The structure of the trachea is somewhat unusual in that its cartilage element extends about two-thirds of the way around the tracheal circumference. In addition, the flexibility and stiffness of the tracheal cartilage are an important factor in determining the airway cross-sectional area during dynamic collapse of the organ in a forced expiration. Another characteristic of the trachea is its constant exposure to the outside environment, which increases the chances of infection, vascular erosion, and extrusion. Previous attempts at engineering tracheal equivalents have found these obstacles difficult to overcome. Nevertheless, it is widely believed that by developing a functional tracheal replacement clinicians will be able to delay and possibly prevent the death of the patient, which current treatments unfortunately cannot always prevent.
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We believe that the combination of tissue engineering and cell-culture techniques, along with proper methods for scaffold seeding and preparation, could provide a solution to these serious problems by generating an ideal cartilaginous tracheal component. It is presently still unknown which source of cartilage is the most effective and which requires the least invasive procedure for harvesting the cellular sample needed for constructing a tissue-engineered trachea. We have observed that the nasal septum cartilage is a potential site for obtaining an adequate sample of tissue for further tissue-engineering studies [19]. The advantage of using hyaline cartilage from the nasal septum is that it not only has properties similar to those of tracheal cartilage but is a source of epithelial cells and connective tissues. The obvious benefit is that one is able to obtain from the same small nasal septum biopsy the three cell types necessary for in vitro 3D tissue construction. However, cartilage harvested from nasal tissue should not be considered the universal or unique source for all cases of tracheal injury in which tissue engineering could be beneficial. For instance, the availability of nasal cartilage may be limited in children or victims of smoke inhalation, and its use as a source of cartilage could be counterproductive. Other cellular sources that have the potential for use in tissue-engineering studies need to be investigated. Recently, adult stem cells were shown to be a possible source that could be exploited by tissue engineers. Our laboratory believes that stromal cells hold the greatest therapeutic potential for engineering bone and cartilage. An additional advantage of using stromal stem cells is that obtaining them is less invasive than removing a sample of nasal cartilage.
Cylindrical Tube: Comparison of Tracheal and Nasal Chondrocytes One of the main issues in cartilage tissue engineering is determining which tissue harvesting technique is the simplest, safest, and least invasive. An optimal source of cartilage must also be determined. Currently, five body sites are being investigated as the main supply of cartilage for the purposes of tissue engineering: trachea, nasal septum, knee, and costal cartilage (all of which are hyaline), and ear cartilage, which is elastic. Ear cartilage is the simplest to harvest for tissue engineering because it fulfills the necessary criteria for a source site, such as simplicity of collection, safety, and minimal surgical
invasiveness. However, its use as a source of cells for generating hyaline cartilage is still debated. In contrast, the nasal septum cartilage possesses properties similar to those of tracheal cartilage. For instance, chondrocytes, epithelial cells, and connective tissue can be harvested from the same small nasal septum biopsy. Therefore, we have studied both sheep tracheal and nasal septum chondrocytes to determine the harvesting cell yield and the quality of the engineered cartilage. The study was also designed to evaluate the feasibility of making tissue-engineered cartilage in a cylindrical shape to mimic the structure of the native trachea. For this study, 5-mm × 5-mm samples of sheep nasal septum cartilage (N = 6) and two to three tracheal rings (N = 6) were obtained from 2-month-old sheep. The chondrocytes were isolated from each cartilage sample by digestion in 0.3% collagenase type II (Worthington Biochemical, Freehold, NJ) at 37◦ C in a shaker for 5 to 8 h with gentle shaking. The resulting cell suspensions were passed through a 100-mm cell strainer (Becton Dickinson and Company, Franklin Lakes). Both chondrocyte preparations were cultured for 2 weeks in Ham’s F-12 media (Gibco, Grand Island, NJ) containing 10% fetal calf serum (Gibco) with 292 mg/ml L-glutamine, 10,000 U/ml penicillin G, 10,000 U/ml streptomycin sulfate, 25 mg/ml amphotericin B, and 50 mg/ml ascorbic acid. The culture medium was changed every 3 days. After 2 weeks, a confluent monolayer was observed. The chondrocytes were then harvested via digestion with 0.05% trypsin-EDTA (Gibco). Each preparation of isolated cells was counted in a hemocytometer, and the viability was determined using the trypan-blue (Sigma-Aldrich, Irvine, CA) exclusion method. Both chondrocyte suspensions were concentrated at 50 × 106 cell/ml and seeded onto 30-mm × 40-mm nonwoven meshes of polyglycolic acid (PGA) fibers (Davis & Geck, Danbury, CT). Both cell-polymer constructs were then incubated in vitro for 1 week and then wrapped around a silicone tube 7 mm in diameter × 30 mm long, which was subsequently implanted into a subcutaneous pocket on nude mice (tracheal cartilage, N = 6; nasal septum cartilage, N = 6) (Figure 15.1). The implants were recovered at 8 weeks and analyzed histologically and biochemically. The tissue was assayed for cartilage-specific extracellular matrix components, including proteoglycans and collagen. Throughout the experiment, all animals received humane care in compliance with the Principles of Laboratory Animals Care formulated by the National Society for Medical Research and the Guide for the
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Figure 15.1. Schematic diagram of methods for isolation, culture, and implantation of cells for tissue engineering of the trachea.
Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health (NIH publication 85-23, revised 1985). In the study, the cell-polymer constructs formed cartilage de novo in vivo in the shape of cylinders after 8 weeks. The gross appearance of both the tracheal-chondrocyte-derived cartilage and the nasal septum-chondrocyte-derived cartilage tissue-engineered tracheas (tracheal TET, nasal TET) was very similar to that of native tracheal cartilage (Figure 15.2). Each exhibited a translucent white appearance reminiscent of the hyaline cartilage of native trachea. The consistency and elasticity were also comparable. Histological examination of both tracheal TET and nasal TET using hematoxylin and eosin (H&E) staining showed the presence of mature cartilage. Safranin-O staining showed that the cells in both tissue-engineered cartilage samples were organized into lobules with large round and angular lacunae, each containing a single chondrocyte (Figure 15.3). In summary, both samples had a cartilaginous histology similar to that of native tracheal cartilage. In addition, six samples of each engineered trachea and six samples of native trachea were analyzed for proteoglycan and collagen content. Samples were digested by the addition of 1.0 ml of 100 mM sodium phosphate, 10 mM sodium EDTA, and 10 mM cysteine hydrochloride (Sigma). The specimens were incubated in a 60◦ C water bath for 24 hours [18].
The glycosaminoglycan (GAG) content of the tissue digested was quantified according to a previously described method [11]. Briefly, 50 ml of papain digest was added to 2 ml of 1,9-dimethylmethylene blue dye at pH 3.0 with absorbencies detected at 490 nm with a spectrophotometer immediately after the addition of the dye. GAG contents of the specimens were determined using chondroitin 6-sulfate from shark cartilage (Sigma) as a standard. All samples and standards were analyzed in duplicate. The chloramine T method was used for hydroxyproline quantification [38]. Briefly, the papain digests were hydrolyzed with equal volumes of 6N HCl at 115◦ C for 16–24 hours in screw cap glass tubes. Contents of each tube were washed out and transferred into borosilicate 12-mm × 75-mm glass tubes and dried for 5 hours. Chloramine T hydrate (98%, Sigma) and p-dimethylaminobenzaldehyde (Ehrlich’s reagent, Sigma) were added to hydrolyzed specimens and absorbencies were detected at 560 nm with a spectrophotometer immediately after the addition of the dye. The collagen content in experimental samples was determined using a hydroxyproline standard from Sigma. The proteoglycan contents of the tracheal TET, nasal TET, and native tracheal cartilage were 84.3 ± 7.5 mg/mg, 97.1 ± 3.2 mg/mg, and 120.0 ± 9.2 mg/mg, respectively. The collagen contents of the tracheal TET, nasal TET, and native tracheal cartilage were 1.25 ± 0.21 mg/mg, 1.28 ± 0.20 mg/mg, and 1.36 ± 0.13 mg/mg, respectively.
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(a)
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Figure 15.2. Appearance of tracheal TET (a) and cross-section (b), and nasal TET (c) and cross-section (d) at 8 weeks. Each of the TET specimens had an appearance similar to native tissue. (See color plate.)
Previously frozen samples of native and both tissueengineered tracheas were thawed at room temperature while immersed in PBS (Gibco) for approximately 30 minutes. Circumferential strips of tissue were carefully cut from all samples with a razor blade to ensure that the specimens consisted primarily of cartilage. Rectangular strips were cut to approximately 20 mm × 5 mm, and the precise length, width, and thickness were determined to within 0.1 mm with calipers. The samples were tested within 30 minutes of being cut, and all samples remained hydrated until the time of testing. The specimens were placed in stainless-steel serrated tensile grips (Harvard Apparatus, Holliston, MA) mounted in a Dynastat mechanical spectrometer (IMASS, Hingham, MA). Specimens were mounted with an initial grip-to-grip distance of 10 mm and were
subjected to ramp displacements at a rate of 0.020 mm/s for 150 s up to a maximum total displacement of 3 mm. The resultant loads were recorded to within 10 mN at a frequency of 2.5 Hz for the duration of testing. Applied displacements were normalized to the initial grip-to-grip distance to yield values for tissue strain. Sample width and thickness were used to calculate the cross-sectional area, which was used to convert measured loads to stresses. The tensile modulus was determined from the stress-strain data by calculating the slope of the linear region of the curve (generally 15 to 25% strain). Moduli for native and both groups of TET were compared by single-factor ANOVA, using a t-test with Bonferroni correction for pairwise comparison and with a p = 0.05 as the minimum level of significance. The statistical power of these studies was 0.84 with a minimum detectable difference of 0.5 MPa.
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(c) Figure 15.3. Safranin-O staining of native tracheal cartilage (a), tracheal TET (b), and nasal TET (c). Histologically, the tracheal TET and nasal TET were indistinguishable from native tracheal cartilage. Similar appearances were noted with H&E staining (not shown). See color insert.
The stress-strain response for both normal and TETs was nonlinear, with an initial soft “toe” region at low strains followed by a stiffer, more-linear region at higher strains. The modulus of native trachea was 10.6 ± 1.8 MPa, significantly higher than that of tracheal tissue engineered from tracheal (1.4 ± 0.4 MPa) or nasal (1.4 ± 0.5 MPa) chondrocytes. There was not a significant difference between the moduli of tracheal tissue engineered from tracheal or nasal chondrocytes (Figure 15.4). In these experiments, a single 5-mm biopsy of nasal septum yielded enough chondrocytes to generate one cylindrical cartilage tube.
Cylindrical Tube: Composite Chondrocyte and Epithelial Cells In this study, we evaluated methods for adding an epithelial lining to an engineered trachea. Nasal chondrocytes
Figure 15.4. (a) Representative stress-strain curves for native trachea, and tracheal tissue engineered from nasal and tracheal chondrocytes. (b) Tensile modulus of native trachea, and tracheal tissue engineered from nasal and tracheal chondrocytes.
were combined with epithelial cells to create a composite engineered trachea from the same nasal specimens. As in the previous study, 5-mm × 5-mm samples of sheep nasal septum cartilage were obtained from 2-month-old sheep (N = 6). Chondrocytes were isolated and harvested as described previously. Chondrocyte cells were counted and resuspended to a final concentration of 50 × 106 /ml. The suspensions were seeded onto 30-mm × 40-mm × 2-mm nonwoven meshes of PGA fibers. Cell-polymer constructs were incubated in vitro for 1 week and then wrapped around a silicon tube 7 mm in diameter × 30 mm in length. These composite tracheas were implanted subcutaneously in athymic mice and harvested after 6 weeks. Nasal epithelial tissues were separated from the underlying nasal septum cartilage. Epithelial cells were also obtained from the mucosal lining of the nasal septum tissue by culturing 2-mm × 2-mm explants in Ham’s F-12 media (Gibco), including 10% FCS (Gibco) with L-glutamine and an antibioticantimycotic solution, 5 mg/ml human apo-transferrin
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(Sigma, St Louis, MO), 5 mg/ml human recombinant insulin (Sigma), and 10 mg/ml epithelial growth factor (EGF) (Peprotech, Rocky Hill, NJ). The culture medium was changed every 2 days, and the explants were removed at 2 weeks. The resulting cells were trypsinized, counted, and replated at a density of 2,500 cells/cm2 in T175 flasks. Cells were passaged upon confluence for up to 4 to 6 weeks. Pluronic F-127 NF (BASF, Mount Olive, NJ) was dissolved at 4◦ C in culture medium to a final concentration of 23% (w/w) and filtered through a cold 0.22-mm filter (Figure 15.2b). Nasal epithelial cells at a concentration of 50 × 106 cells/ml were suspended in 23% pluronic F-127 and injected into the implanted cylindrical tube of cartilage that was generated in the nude mouse around the silicone tube template. Four weeks after the injection of epithelial cells, the animals were sacrificed and the implants were harvested. It is important to note that the silicone tube had been removed before the pluronic and epithelial cell mixture was injected and that a cylindrical tube of cartilage remained. Cell-polymer constructs formed de novo cartilage in the shape of cylinders lined with a pseudo-stratified columnar epithelium after 10 weeks. The gross appearance of the TET looked very similar to that of native tracheal cartilage. The lumen was lined with epithelial structures (Figure 15.5). The consistency and elasticity were also comparable to that of native trachea. Histological examination of engineered trachea using H&E stains showed the presence of mature cartilage and the formation of an epithelial layer, with a distinct interface between them. Safranin-O staining
(a)
showed that the TET was organized into lobules with round, angular lacunae, each containing a single chondrocyte. The GAG and collagen content of the TET were similar to that of native trachea [20].
Helical and Y-Shape (Carina) The previous experiment produced a cartilaginous tissue that had epithelialization. This is a significant improvement over current therapies but was not without limitations. For example, the tube was not flexible, which if used as a tracheal replacement might be uncomfortable if not stressful for the patient. In addition, the diameter of the trachea TET was not large enough to be clinically useful for treating patients. Therefore, the next logical step was to try to engineer a replacement with a clinically relevant shape and the physical characteristics of native trachea. Again, 5-mm × 5-mm samples of sheep nasal septum cartilage were obtained from 2-month-old sheep. Using these small samples of sheep nasal septum cartilage, we separated out the connective tissue from the underlying cartilage. Chondrocytes were isolated from cartilage by the same procedure. Epithelial cells were not used because we attempted more complicated geometries, such as a Y-shaped structure, to simulate the carina. Fibroblasts were obtained from the connective tissue by culturing 2-mm × 2-mm explants in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, L-glutamine, and an antibioticantimycotic solution.
(b)
Figure 15.5. Appearance of tissue engineered trachea (a). Section shows a distinct structure containing both cartilage (arrowhead) and epithelial lining (arrow) (b).
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After 2 weeks, once a confluent monolayer was detected chondrocytes and fibroblasts were harvested via digestion with 0.05% trypsin-EDTA. Chondrocyte suspensions were concentrated at 50 × 106 cells/ml and placed on 100-mm × 10-mm × 2-mm nonwoven meshes of PGA fibers. Similarly, fibroblasts were concentrated at 25 × 106 cells/ml and seeded onto 50-mm × 10-mm PGA meshes. The chondrocyteseeded mesh was placed in the grooves of a helical template 20 mm in diameter × 50 mm in length made from a Silastic ERTV mold-making kit (Dow Corning,
(a)
Midland, MI) (N = 4) or a silicone Y-shape prosthesis (Dumon silicon stent) (Kapitex Healthcare Limited, West Yorkshire, England) (N = 4) (Figure 15.6). Both the helical and the Y-shaped templates were then covered completely with the fibroblast-seeded meshes. All templates were then implanted into nude rats for 8 weeks. When the specimens were recovered, they had generated cartilage (as observed in the previous studies) and the fibroblasts had generated connective tissue that linked the cartilage rings (Figure 15.7).
(b)
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Figure 15.6. Helical template fabricated using a silicone mold-making kit (a). The chondrocyte-seeded matrix was placed in the grooves of the template and before the entire template was wrapped with the fibroblast-seeded mesh (b). Y-shaped prosthesis (Dumon silicon stent) (c). Placement of the chondrocyte-seeded matrix (d).
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(a)
(b)
Figure 15.7. Helical tissue-engineered cartilage (a) and Y-shaped tissue-engineered cartilage (b) were harvested from a nude rat at 8 weeks. In both views, similarity to native tracheal tissue is evident. There is a distinct structure containing both cartilage and connective tissue, just as in normal trachea or carina. (See color plate.)
Autologous Engineered Trachea The final step was to use the same approach in an autologous model rather than in a nude model. This time we attempted to show that the seeded templates could be implanted into the neck of a sheep (adjacent to the trachea) instead of into the back of a nude rat and with similar findings. Our ultimate future goal is to attach the engineered segment to a sheep’s trachea. We obtained two separate 5-mm × 5-mm samples of nasal septum from each 2-month-old sheep to generate two identical templates. The chondrocytes were harvested from cartilage and fibroblasts from connective tissue and the cells cultured as previously described. After 2 weeks, the chondrocytes (50 × 106 cells/ml) and fibroblasts (25 × 106 cells/ml) were seeded onto PGA fibers and cultured. The chondrocyte-seeded fibers were placed in the grooves of a helical template 20 mm in diameter × 50 mm in length made from a Silastic ERTV mold-making kit (as previously described), forming a whole template that was then wrapped with the fibroblast-seeded fiber. Two identical templates were generated from each sheep. We intended to develop two experimental models of TET. In one model,
one of the whole templates would be implanted into a subcutaneous pocket in the dorsum of an athymic rat and harvested at 8 weeks. In the second model, the other whole template would be implanted into a sheep through a longitudinal incision made in the neck of the sheep just above the sternocleidomastoid muscle. During implantation, a pocket was created under the sternocleidomastoid muscle, and the implants derived from cells from each sheep were inserted into the pocket of the same sheep from which they were obtained. After 8 weeks, the TET was harvested from the neck and a 5-cm-long resection was done on the sheeps’ cervical trachea. The lungs were ventilated via a second endotracheal tube placed during the surgical procedure. The TET was implanted by an end-to-end anastomosis using a 3-0 absorbable running suture (Figure 15.8). Postsurgery, the sheep that had received the TETs breathed spontaneously with no subcutaneous emphysema and were voluntarily ambulatory. The animals’ survival time ranged from 2 days to 7 days. The sheep with the longest survival times were sacrificed because of extensive tracheomalacia by day 7. The animals with the shortest survival times were sacrificed because of stenosis that occurred by day 2. The gross appearance of
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(a)
(b)
(c)
(d)
Figure 15.8. Implantation of tissue-engineered trachea in sheep. Pocket was created under the sternocleidomastoid muscle and implant was placed in this pocket (a). The sheep TET was harvested from the sheeps’ neck at 8 weeks (b). A 5-cm defect in the cervical trachea was created and the lungs were ventilated via a second endotracheal tube intubated through the operative field (c). The sheep tissue-engineered trachea was implanted by an end-to-end anastomosis (d). (See color plate.)
N = 6 ± S.D.
80
p<.001
p<.001
70 60 GAG (mg/mg)
the TET in both TET from a nude rat and TET from the corresponding sheep looked very similar to native trachea. There was a distinct structure containing both cartilage and connective tissue, similar to native trachea. Upon histological examination, the engineered tracheal surfaces of both groups were smooth and cartilage formation was evident. H&E staining revealed the presence of mature cartilage surrounded by connective tissue, as observed in native trachea. GAG content of the native tracheal cartilage, TET from sheep, and TET from rats were 62.8 ± 5.1, 23.8 ± 5.6, and 48.6 ± 9.7 mg/mg, respectively (Figure 15.9). These data indicate that the failure in the sheep TET is probably due to the low levels of collagen type II and GAG.
50 40 30 20 10 0 native
Sheep TET
Rat TET
Figure 15.9. Glycosaminoglycan (GAG) content of native and tissue engineered trachea (TET) (N = 6 ± SEM).
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Marrow Stromal Cells with TGF-b2 Released from Biodegradable Microspheres This study was designed to evaluate the feasibility of using autologous sheep marrow stromal cells cultured onto a PGA mesh to engineer a helix-shaped cartilage equivalent of a functional trachea. Transforming growth factor-b (TGF-b) has been shown to play a major role in cartilage development, and studies have demonstrated that TGF-b2 helps support chondrogenesis in developing 3D tissue constructs. Therefore, we explored the potential benefit of local delivery of TGF-b2 to cells using biodegradable microspheres. We were able to deliver TGF-b2 locally by coupling its release to the degradation of a biodegradable hydrogel prepared by cross-linking acidic gelatin with glutaraldehyde. This gelatin hydrogel incorporating TGF-b2 effectively promoted cartilage regeneration in vivo. The gelatin with an isoelectric point (IEP) of 5.0, prepared through alkaline processing of bovine bone, was kindly supplied by Nitta Gekatin (Osaka, Japan). It is called “acid gelatin” on the basis of its low isoelectric point. Gelatin microspheres were prepared by chemical cross-linking of gelatin in a water-in-oil emulsion state, as described previously [39]. Briefly, an aqueous solution of 10 wt% gelatin (10 ml) was preheated at 40◦ C and then added drop wise into 375 ml of olive oil preheated at 40◦ C, while an impeller stirring at 420 rpm was run for 10 min to yield a water-in-oil emulsion. The emulsion temperature was decreased to 4◦ C, followed by further stirring for 30 min for the natural gelation of the gelatin aqueous solution. Cold acetone (100 ml) was added to the emulsion, and stirring was continued for 10 min. The resulting microspheres were washed three times with cold acetone, collected by centrifugation (5,000 rpm, 4◦ C, 5 min), fractionated by size by sieves with apertures of 70 and 100 µm, and air dried at 4◦ C. The average diameter of the microspheres used was 75 µm. The non-cross-linked and dried gelatin microspheres (20 mg) were placed in 20 ml of 0.1 wt% Tween 80 aqueous solution containing 100 µl of 25 wt% glutaraldehyde solution and stirred at 4◦ C for 24 h to allow the gelatin to cross-link. After the microspheres were washed by centrifugation (5,000 rpm, 4◦ C, 5 min) with double distilled water (DDW), they were agitated in 20 ml of 100 mM aqueous glycine solution at room temperature for 1 h to block the residual aldehyde groups of unreacted glutaraldehyde. The resulting microspheres were washed three times with DDW by centrifugation and freeze dried.
Gelatin microspheres were impregnanted with TGF-b2 by pouring 20 µl of 100 mM phosphate-buffered saline solution (PBS, pH 7.4) containing 20 µg of TGF-b2 into 2 mg of the freeze-dried gelatin microspheres, which were stored overnight at 4◦ C. The TGF-b2 was completely incorporated into the gelatin microspheres by this impregnation procedure, because the volume of the TGF-b2 solution was small as compared to the total volume that can theoretically be incorporated into the microspheres. Sheep bone marrow was obtained by iliac crest aspiration from 6-month-old sheep. The aspirates were cultured in DMEM containing 10% fetal calf serum with 292 mg/ml L-glutamine, 10,000 U/ml penicillin G, 10,000 U/ml streptomycin sulfate, and 25 mg/ml amphotericin B. Culture medium was changed every 2 days. A confluent monolayer was obtained after 2 weeks. Cells were harvested via digestion with 0.05% trypsin-EDTA. The isolated cells were counted with a hemocytometer, and their viability was determined by the trypan-blue exclusion method. Cell suspensions were concentrated at 50 × 106 cell/ml and seeded on 100-mm × 10-mm × 2-mm nonwoven meshes of PGA fibers. The serum-containing medium was then replaced with DMEM with ITS+Premix (Becton Dickinson), L-ascorbic acid 2-phosphate (Sigma), 100 nM dexamethasone, TGF-b2 (10 ng/ml) (Peprotech), and IGF-1 (25 ng/ml) (Peprotech). The cell-seeded mesh samples were cultured continuously for 1 week in vitro. The structures were then placed in the grooves of a helical template 20 mm in diameter × 50 mm in length made with a Silastic ERTV mold-making kit. Cell-polymer samples were coated with gelatin microspheres with (N = 6) or without (N = 6) TGF-b2. The composite structures were implanted subcutaneously in athymic rats and harvested after 6 weeks. The implants were analyzed histologically and biochemically. Tissues were assayed for cartilage-specific extracellular matrix components, including proteoglycans and collagen. Sections of the samples were stained with hematoxylin, eosin, Safranin-O, and a mouse monoclonal antibody to type II collagen. The tissue appearance of the specimens prepared using gelatin microspheres without TGF-b2 showed fibrous remnants of PGA. However, the tissue appearance of specimens prepared with gelatin microspheres incorporating TGF-b2 appeared to be cartilage and to be neovascularized on the surface of the engineered cartilage.
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The gross appearance of the TET was solid and shiny white with a cartilaginous circular helix formed. The TETs showed great similarity to native trachea, and like native cartilage were stiff to the touch yet flexible. Upon histological examination, the TET surface was smooth and cartilage formation was evident. H&E and Safranin-O staining demonstrated hyaline cartilage tissue that closely resembled that of native tracheal cartilage, with a similar overall morphology. Type II collagen staining was strongly positive in both native and engineered cartilage. Six samples of TET and native trachea were analyzed for proteoglycan and collagen content. Proteoglycan content of the TET was 49.57 ± 6.81 mg/mg and that of native tracheal cartilage was 63.05 ± 7.3 mg/mg. The collagen content of the TET was 0.35 ± 0.37 mg/mg and of native tracheal cartilage was 0.49 ± 2.43 mg/mg. Glycosaminoglycan and hydroxyproline contents were also similar to native cartilage levels.
Discussion The trachea is a unique and complex organ because of its structural properties and specific function. We believe that the requirements for developing an ideal TET are (1) simple and minimally invasive harvest procedures that yield enough cells in a relatively short time, (2) a biocompatible and biodegradable scaffolding that avoids inflammatory responses, (3) proper flexibility, stiffness, and compliance of the engineered tissue, (4) an airtight structure, (5) successful neovascularization, (6) epithelial regeneration, and (7) growth of the insert with the recipient, which is especially important in the treatment in children. If we postulate that living cells are required to fabricate tracheal tissue by tissue-engineering techniques, much still needs to be learned about cell morphogenesis and biomaterials, especially about biodegradable materials used as scaffolds. Moreover, it is necessary to learn about cell- or tissue-biomaterial interactions and their wound-healing responses. As we increase our understanding of normal tissue development, tissuebiomaterial interactions, and wound healing, we may be able to use this knowledge to our advantage in the rational design of living tracheal tissue. A major challenge in this endeavor is the acquisition of living cells for the treatment of tracheal defects. The ultimate goal in this regard is to discover how to replace critical tracheal defects with a large-scale tissue-engineered implant.
Eventually, we may be able to obtain a large number of cells from a universal cell source from the patient’s own body, thus avoiding any immunologic difficulties. These universal cells could come from multiple cell types — such as autologous cartilage tissue, adult stem cells, embryonic stem cells, and bone marrow — all of which may be suitable for generating engineered tissues. However, the cell source should be readily available and easily obtainable. Because of the tremendous potential of stromal stem cells derived from bone marrow for generating tissues, they are being studied aggressively as a source of cells for bioengineering [32, 33]. Moreover, to our advantage there is evidence suggesting that these cells have the potential for exploitation in the development of a TET. Meanwhile, patients could potentially be treated with nasal septum or costochondral cartilage, an option that presently is better understood. The ear is probably the most convenient source of cartilage to harvest for use in tissue engineering. Such harvesting is safe and minimally invasive. However, its use as a source of cells for generating tracheal hyaline cartilage is not optimal because ear cartilage is elastic and more flexible than the rigid hyaline cartilage of native trachea. A TET made from elastic cartilage would lack the rigidity of hyaline cartilage and would collapse easily. In contrast, nasal septum cartilage is very similar to tracheal cartilage. Furthermore, chondrocytes, epithelial cells, and connective tissue can be harvested from the same small nasal septum sample. The single procedure of harvesting a 5-mm portion of nasal septum tissue yields enough cells to make a cylinder of cartilage in 2 weeks. However, it may not be possible to harvest nasal septum from some patients, such as burn victims or children. Therefore, alternative cell sources must be found. For this reason, we are studying whether stromal cells derived from bone marrow are a viable alternative to the nasal biopsy. Marrow stromal cells can be obtained through a less invasive aspiration technique, which renders it more advantageous. As described previously, we have successfully demonstrated the feasibility of engineering tracheas using sheep marrow stromal cells as a cellular source. Moreover, engineering the tracheal equivalents with supplemental TGF-b2 seemed to have a positive effect, allowing the newly formed tissue to retain a cartilaginous phenotype in the nude rat model. Therefore, we hope that in the future we will be able to use marrow stromal cells for the production of TETs in a human autologous model.
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Thousands of patients each year require at least a partial tracheal replacement. However, there are some illnesses that affect the trachea but do not require a full tracheal replacement. Smoke inhalation injury and Kartagener’s syndrome are examples of illnesses that require only the replacement of tracheal epithelium. For this reason, our laboratory designed a rigid tube structure that had a cartilaginous framework and that was lined with stable epithelial mucosa, a technique that can potentially be used to treat these patients. A murine study presented here demonstrated that it is possible to generate a tissue-engineered cartilage cylinder with an epithelial lining with a single procedure that could be used for repairing potentially fatal tracheal defects created by long circumferential resections. Because of the mouse’s small size, however, we were unable to generate a large enough tracheal replacement and therefore switched to larger animals. The use of nude rats allowed us to bioengineer larger tracheas and more complicated forms, such as the Y shape of the carina. These additional implant shapes allowed us to test other tissue parameters and variables, such as the rigidity of the cartilaginous rings and the flexibility of the fibrous portion. In fact, we showed in the autologous model section of our studies that TET samples obtained from rats were comparable to native trachea with respect to rigidity and flexibility. However, TET from sheep collapsed easily. The difference in mechanical characteristics between the TET from rats and the TET from sheep became apparent in the biochemical analyses of the tissues. TET from sheep had a much lower GAG content than did the TET from rats, as determined by proteoglycan levels. The presence of GAG in the neomatrix provides quantitative evidence of the production of extracellular matrix by the chondrocytes seeded onto the PGA. Because proteoglycans are the major structural component of the cartilage extracellular matrix and are responsible for tissue rigidity [12], it is logical that in addition to collapsing easily the TET from sheep should also be more compliant. Implanting the sheep marrow engineered tracheas into sheep represents an autologous transplantation model, which renders the study more clinically relevant than studies involving experimentally immunodeficient animals. However, because of this change in animal immunogenicity we suspect that the mechanism behind the decreased proteoglycan content in the sheep TET implanted into sheep probably involves an inflammatory response to the implant by the animal’s immune system. It is well known that PGA elicits a strong inflammatory
response that can inhibit the formation of cartilage [7]. This response is likely to involve interleukin-1, which has been reported to induce the degradation of cartilage, resulting in significant loss of tissue and subsequently of mechanical function [5]. Therefore, we investigated the molecular mechanism of inflammation in vitro and in vivo. Our data suggested that the hydrolysis of PGA to glycolide activates the classical complement pathway. Furthermore, the complement cascade is amplified via the alternative pathway and inflammation is induced by the generation of C5a. Thus, we believe that the inhibition of C5a may provide a potential therapeutic approach to limiting the inflammation associated with PGA-derived materials following implantation [10]. To protect implanted cells and/or tissues from damage by the host innate immune response, it will be important to control the acute-inflammation process. This can be done either by blocking proinflammatory signals, such as complement activation [4], or by promoting the anti-inflammatory/pro-resolution signals of acute inflammation [37]. Indeed, most of the current research and clinical efforts concentrate around the initiation and progression of the acute inflammatory response, and attempt to block these processes by developing antagonists. On the other hand, little is known about the mechanism of resolution of inflammation and return to homeostasis, which occurs spontaneously in healthy tissues. The resolution of inflammation is an active process controlled by endogenous agonists. For this reason, the identification and effective use of these pro-resolution agonists, such as lipoxins [36] and resolvins [1], would be a novel approach to improve the functional outcome of implanted tissues by preventing acute damage and/or by promoting assimilation. Furthermore, the use of alternative biomaterials as scaffolds for cartilage growth, such as pluronic and polycaprolactone, needs to be examined as well. Finally, there is also a need to address how to maintain the vascular supply to the implant, possibly through the release of b-FGF from biodegradable microspheres [40] or by some other delivery system.
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16 Muscle Cell Transplants Mitra Lavasani (1 and 2), Jonathan Pollett (2), and Johnny Huard (1–3) (1) Department of Bioengineering, University of Pittsburgh (2) Growth and Development Laboratory, Children’s Hospital of Pittsburgh (3) Departments of Orthopaedic Surgery and Molecular Genetics and Biochemistry, University of Pittsburgh
Introduction Usually, the term translational medicine refers to the progression of research from laboratory investigation to clinical application. This chapter discusses the “reverse” flow of scientific discovery from “the bedside” to “the bench.” For many years, clinicians have studied the use of muscle cell transplantation to treat muscular dystrophy. Unfortunately, this approach has not led to significant improvements in patients’ survival or quality of life. Here we describe how researchers examining the failure of muscle cell transplantation discovered a novel postnatal stem cell that has significant therapeutic value for muscle transplantation and other tissue regeneration applications. These findings all stem from the translation of research from clinical studies back into the laboratory setting (Figure 16.1).
Duchenne’s Muscular Dystrophy Duchenne’s muscular dystrophy (DMD) is a member of a family of muscular dystrophies that constitute a group of genetic degenerative diseases that primarily affect the skeletal muscles. In addition to being a fatal disease that warrants intense scientific investigation, DMD is an archetypal disease for the study of therapies based on muscle cell transplantation. DMD, a devastating degenerative muscle disease that affects approximately 1 in 3,500 boys in all populations, is an X-linked recessive disorder [29] caused by a mutation in the dystrophin gene that prevents the production of a functional protein [49, 50, 73, 130, 137]. Located in the sarcolemma of the myofiber [4, 14], dystrophin
Cellular Transplantation: From Laboratory to Clinic
plays a role in maintaining plasma membrane integrity and stability [62, 89, 105]. Specifically, dystrophin and dystrophin-associated protein complex (DAPC) form a link between the intracellular actin-based cytoskeleton and the extracellular matrix (ECM). The amino terminus of dystrophin binds to F-actin and the carboxyl terminus binds to the DAPC at the sarcolemma [13, 19]. Whereas mutations in the dystrophin gene cause DMD, mutations in any of the other components of the DAPC cause other inherited forms of muscular dystrophies [18, 24, 122]. Destabilization of the DAPC due to the absence of dystrophin results in diminished levels of the membrane proteins, and the muscle cell becomes compromised [123]. Disruption of this complex also increases the muscles’ susceptibility to contraction-induced injury and sarcolemmal damage and often results in membrane leakage. Furthermore, the DAPC plays a signaling role, the loss of which also contributes to pathogenesis [13, 18]. The skeletal muscles of DMD patients undergo repeated cycles of degeneration and regeneration that exhaust the regenerating potential of myogenic precursor cells and lead to massive connective tissue formation, which results in muscle necrosis and fibrosis [29]. The result of this process is progressive weakness and wasting of the muscles [36]. Despite extensive efforts to develop an effective approach to deliver dystrophin in dystrophic muscle (including cell and gene therapy), there is no treatment that substantially slows the course of DMD or enables regeneration of the diseased muscle tissue. DMD patients are usually wheelchair bound by 12 years of age, have an abnormal electrocardiogram by the age of 18, and die in their late teens or early twenties due to congestive
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Mitra Lavasani, Jonathan Pollett, and Johnny Huard Treatment of DMD with myoblast cell transplantation
Myoblast transplantation yields poor engraftment
Characterization of engrafting cells that display increased regenerative capacity Identification of MDSCs
MDSCs used for skeletal muscle regeneration
MDSCs used for bone and articular cartilage repair
MDSCs used for non-DMD muscle applications (cardiac/urinary tract)
MDSCs used for peripheral nerve regeneration
Figure 16.1. From bedside to bench and back. A stepwise pathway of the progression from myoblast transplantation to the discovery of MDSCs and the subsequent relevance of MDSCs to current and future clinical trials.
cardiac and respiratory failure. This disease progression indicates that any therapeutic agent must target not only the skeletal muscle (including the diaphragm) but the cardiac muscle. Although recent advances in clinical management have significantly improved the life expectancy of DMD patients, their functional ability, and their quality of life [8, 45, 91, 119], researchers have not yet identified a highly effective treatment.
ANIMAL MODEL OF DMD Mdx mice are biochemically and genetically homologous to humans with DMD. These mice exhibit dystrophin deficiency due to a spontaneously occurring point mutation in the dystrophin gene that generates a premature stop codon [118]. With less than 10% of the normal amount of dystrophin and less than 0.1 to 0.01% of muscle fibers that stain positive for dystrophin, mdx mice are considered good genetic homologues of DMD [5]. Although mdx mice appear normal at birth, their skeletal muscles show signs of extensive muscle degeneration by 3 to 5 weeks of age [23, 101, 124]. In this early phase, this acute muscle degeneration is accompanied by effective muscle regeneration that leads to transient muscle hypertrophy [23, 101]. After the mice reach
52 weeks of age, the degeneration and regeneration cycle continues at lower but relatively constant levels throughout the life of the animal. This milder histopathological condition results in slower progression of the disease in mice. Indeed, the muscle of mdx mice differs from that of persons with DMD in that the mdx muscle exhibits a greater degree of compensatory muscle regeneration [23]. However, the phenotype of the mdx diaphragm of all ages and the postural muscles (such as the soleus) of old mdx (65 to 104 weeks of age) corresponds closely with many pathological effects seen in DMD patients [101, 121], which makes this animal model a convenient choice for use in efforts to develop therapies to improve muscle regeneration and functional recovery.
CURRENT TREATMENTS AND THEIR LIMITATIONS There is no effective treatment for DMD. At this point in time, the National Institutes of Health (USA) are recruiting for only six studies focused on the treatment of muscular dystrophy, and only four of the six involve DMD. Historically, researchers have used two different therapeutic approaches to try to deliver
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normal dystrophin to murine and human dystrophic muscle: gene therapy and cell therapy based on myoblast transplantation (MT). GENE THERAPY Although this chapter focuses on muscle cell transplantation, it is crucial to mention gene therapy in any discussion of currently available therapies for DMD. Gene therapy, an experimental procedure aimed at supplementing or manipulating the genetic information responsible for genetic anomalies or deficiencies, has been a major focus of DMD research over the last few years. To effectively treat DMD, the therapy must avoid several key obstacles, including an immune reaction to the dystrophin gene, inefficient gene transfer in adult skeletal muscles, and minimal persistence of gene expression. The first potential problem, the onset of an immunologic reaction, may not be of as high a concern as previously believed. Advances in viral delivery — such as the use of adeno-associated virus (AAV), functional mini- and micro-dystrophin, and gutted vectors with large insertion capacities and minimized immunogenicity — have been tested with good results in mdx mice [30, 38, 44, 109, 116]. Recent research by Ferrer and colleagues [33] has shown that the presence of other dystrophin isoforms and revertant fibers — muscle fibers that express a smaller but functional dystrophin protein due to exon skipping — likely prevent a serious immune response. If true, this would greatly benefit patients undergoing dystrophin gene therapy. Effective gene therapy does, however, require overcoming the problems of inefficient gene transfer and persistence of gene expression, because gene correction has to occur in the vast majority of muscle fibers of a DMD patient (approximately 40% of the total body mass). On average, gene transfer into skeletal muscle will transduce cells residing only within a few millimeters of the injection site [46, 96]. Thus, the treatment of an entire muscle would require multiple injections (perhaps hundreds of injections for the heart or diaphragm) and is thus not clinically feasible. To overcome this difficulty, researchers have focused considerable attention on optimizing the delivery mechanisms for the dystrophin gene. Demonstrating resultant improvements in vectorology, a recent study reported 52% of fibers expressing dystrophin after 1 year [28], and another study showed that a single injection of viral vectors resulted in a nearly 40% improvement in muscle
function and dystrophin expression in 25 to 30% of fibers [26]. The administration of adeno-associated virus type 6 (AAV-6) serotype through the tail vein has also resulted in very efficient gene delivery to many muscles [37]. Systemic delivery of nonviral vectors has led to dystrophin expression in 40% of fibers [81], with widespread transgene expression in approximately 10% of the fibers in all leg muscles after injection into the tail artery or vein [80]. Phase I human trials using plasmid DNA delivery of dystrophin have begun [111]. Although gene therapy holds promise as a way of restoring dystrophin expression to the muscle of mdx mice, the therapeutic potential of just restoring the damaged gene is limited. As stated previously, the disease progression of DMD includes not only muscle degeneration but muscle fibrosis and poor innervation and vascularization of the damaged muscle. Thus, although gene therapy may be an effective means of delivering dystrophin and slowing or halting disease progression it cannot repair the damage already afflicted upon the tissue and therefore must be applied as soon as possible after the damage occurs. A cell-based therapy or combination therapy (cell therapy and gene therapy) would likely be more effective for the treatment of this debilitating disease because such an approach would enable delivery of both the missing protein and cells that can repair the damaged muscle and possibly even prevent future damage. MYOBLAST TRANSPLANTATION The most common experimental therapy for DMD involves transplantation of primary myoblasts into damaged muscle. The transplanted myoblasts then contribute to the formation of new muscle fibers during repair and regeneration and help to deliver dystrophin [52, 56, 65, 66, 71, 92, 98, 100, 127, 129]. Early animal experiments and clinical trials, however, revealed that although myoblast transplantation is feasible and donor cells can fuse with host myofibers at the site of injection and produce normal dystrophin [93, 99] the quantity of muscle fibers expressing dystrophin is not therapeutically significant and the approach is rather inefficient [56, 65, 87, 90, 127]. The results of those studies may also have been influenced by the poor dissemination of the donor cells after injection, their low survival rate, and the immune rejection of the cells by the host system [9, 31, 41, 47, 54, 120]. When performing animal experiments, researchers have used immunedeficient animals or immune-suppressive regimens
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[56, 57, 70, 102], pre-irradiation of the injected muscle [93], or myonecrotic agents [58, 129] to improve the success of MT. Although these approaches can improve the restoration of dystrophin in mdx mice, the success of MT remains rather limited and for the most part the technique is clinically impractical. Because the efficiency of MT is hampered by many limitations (for example, immune rejection, poor donor cell migration, and rapid cell death after transplantation) [128], many researchers have focused on improving this technique. Recent reports indicate some progress on this front. Tremblay and associates have shown that blocking myostatin, a growth factor that negatively regulates the size of muscles, increases the size of muscle fibers after MT in mdx mice [11]. Furthermore, the mdx muscle fibers’ resistance to damage induced by exercise increased and the success of MT improved in general [11]. Because myoblast allografts are particularly immunogenic and researchers have not yet found a way to induce transplant tolerance in mdx mice, Tremblay’s group tried to induce “central tolerance” [17]. They reported that the addition of specific antibodies to myoblast transplants uniformly resulted in long-term (> 100 days) engraftment of allogeneic myoblasts and tolerance of follow-up MTs [17]. Although many researchers have focused on ways to improve the outcome of MT [11, 17, 71], few groups
Skeletal
have investigated why only some transplanted cells engraft well or have attempted to determine which specific population of cells regenerates dystrophic muscle. However, two key findings inspired researchers to pursue this line of research: only a small population of cells survived MT [9, 10, 31, 47, 48, 70], and the few cells that remained viable after irradiation were sufficient to regenerate the damaged muscle [9, 10, 39, 93]. Researchers hypothesized that the cells able to survive irradiation and those able to regenerate dystrophic muscle were perhaps the same cells and constituted a special subpopulation of muscle-derived cells. Using a pre-plating enrichment technique, researchers separated and isolated three distinct populations of cells: early pre-plate (EP), late pre-plate (LP), and long-term proliferating (LTP) cells [106, 107] (Figure 16.2). These cell fractions were distinguishable on the basis of their adhesion to collagen-coated flasks, survival after transplantation, and regeneration capabilities in dystrophic muscle. EP cells, the fraction of more committed myogenic cells, divide only a limited number of times before fusing into myotubes. LP cells grow more slowly and exhibit delayed fusion. Both types of cells ultimately fuse and are no longer able to be cultured, and display only a limited regeneration capacity in dystrophic muscle [63]. The third population of isolated cells (LTP cells) are highly proliferative, adhere late in
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Figure 16.2. Schematic of the pre-plate technique used to isolate muscle progenitor cells from a muscle biopsy specimen. (See color plate.)
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Figure 16.3. Comparison of EP (myoblast) and LTP (MDSC) transplantation into dystrophic muscle as visualized through dystrophin (red) immunofluorescence staining. (See color plate.)
the pre-plate process, and show a superior ability to regenerate muscle [107] (Figure 16.3), characteristics that led researchers to believe that these cells are a population of early myogenic progenitor cells that reside within skeletal muscle. Further investigation of these LTP cells revealed that they have a high potential to undergo myogenic differentiation in vitro [107] and generate significantly more dystrophin-positive myofibers in vivo than do satellite cells [63, 107], and thus have great therapeutic potential. After clonal selection, these LTP cells displayed the ability to repopulate the bone marrow [20] and undergo differentiation toward various mesodermal lineages, including myogenic, osteogenic, chondrogenic, adipogenic, neural, and endothelial lineages [1, 16, 75, 79, 104, 107]. These cells are believed to be the cells that survive (rather than undergo rapid cell death) after MT and can repopulate and regenerate the skeletal muscle. Because of their ability to differentiate into multiple lineages and undergo self-renewal, LTP cells are considered a type of stem cell. They have since been termed muscle-derived stem cells (MDSCs) [16, 20, 25, 53, 63, 75, 79, 104, 107].
as a new area of focus in regenerative medicine. The utility of stem cell therapy is based on the cells’ unique characteristics and behaviors, including (1) selfrenewal that results in a large number of progeny, (2) long-term proliferation, (3) multilineage differentiation that increases the efficiency of stem cells’ incorporation into injured or diseased tissues, and (4) the ability of transplanted stem cells to differentiate into tissue-specific cell types and participate in the regenerative process through cell fusion, cell-to-cell contact, or release of cellular mediators, including cytokines and growth factors. Recent studies have revealed multipotent stem cells in various adult tissues. Adult stem cells isolated from the bone marrow [12, 32, 40, 42, 76], the adult musculature [6, 42, 63, 107, 126], the neuronal compartment [22, 35], and various mesenchymal tissues [135, 136] can all differentiate toward the myogenic lineage (both in vitro and in vivo) if exposed to the appropriate environmental cues. Although these different types of cells appear able to differentiate toward the myogenic lineage, most have a limited ability to regenerate skeletal muscle.
STEM CELL THERAPY FOR DMD
Postnatal (Adult-derived) Stem Cell Therapy Stem cell therapy holds tremendous potential for the treatment of pathologic conditions and thus has emerged
MDSCs appear to be very good candidates for treatment of muscular disorders, particularly because these cells can efficiently regenerate skeletal muscle, are easy to obtain by superficial muscle biopsy
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(that is, in a relatively noninvasive manner), can be expanded to generate the required number of cells, and, most importantly, might enhance restoration of the neural and vascular supply during muscle regeneration by undergoing multipotent differentiation into endothelial and neural lineages [107]. The impressive outcome after MDSC transplantation also might be tied to MDSCs’ immune-privileged behavior [107, 128], a trait they share with other types of stem cells [51, 64, 84, 110]. MDSCs also can differentiate into muscle, nerve, and vascular tissue — the three primary types of tissue required for proper muscle healing [107]. In addition, a recent study has shown that MDSCs can engraft in the hearts of mdx mice [103]. Thus, MDSCs appear able to regenerate not only skeletal muscle [79, 107] but cardiac muscle [97] — two key targets of researchers working to develop a novel therapeutic approach for DMD.
COMBINATION THERAPY As stated previously, cell therapy alone may not be sufficient for successful treatment of DMD. A combinational approach comprising both gene therapy and muscle cell transplantation may be more effective. Various growth factors are thought to play a role in different stages of muscle regeneration [3, 34] by stimulating quiescent myogenic stem cells to undergo activation, proliferation, and terminal differentiation [2, 60, 114, 115]. To restore normal tissue architecture, these factors must act to maintain a balance between growth and differentiation. In a mouse model, direct injection of insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and to a lesser extent nerve growth factor (NGF) have enhanced muscle regeneration in lacerated, contused, and strained muscle [55, 67, 68, 88]. However, these growth factors have relatively short biologic half-lives in vivo. Therefore, the safety and ease of direct injection of human recombinant growth factors for treatment of muscle injuries are practically hindered by the high concentrations of growth factors typically required to produce a beneficial effect. For this reason, researchers often pretreat implanted stem cells with growth factors to improve engraftment or use genetically engineered stem cells that act as growth factor delivery systems. By combining gene therapy and muscle cell transplantation, researchers can implant cells that both better respond to stimuli within injured tissue and more effectively participate in the healing process by delivering growth factors. Early experiments using this approach
have shown that the pretreatment of myogenic cells in culture with bFGF promotes cell proliferation and results in as much as a fourfold increase in myofiber regeneration in vivo [72]. Although extensive research has been performed on the use of IGF-1 and bFGF, a recent study that used MDSCs to deliver NGF also yielded promising results. This study demonstrated that NGF stimulation enhances engraftment efficiency threefold and improves the regeneration capacity of MDSCs in mdx skeletal muscle [77]. The significant improvement in the transplantation efficiency of NGF-stimulated MDSCs suggests that the release of local environmental cues (1) can trigger the differentiation of muscle stem cells toward nonmyogenic lineages after transplantation of the cells into dystrophic muscle and (2) may contribute to the regeneration of functional muscle tissue by enhancing vascular and neuronal supplies. The development of such a novel therapeutic strategy holds tremendous potential for the treatment of pathologic conditions associated with poor muscle regenerative capacity, such as those observed after serious muscle injury or resulting from muscular dystrophies.
Other Therapeutic Applications of MDSCs The ability of MDSCs to proliferate in vivo for an extended period of time, their capacities for self-renewal and multipotent differentiation, and their immuneprivileged behavior suggest that MDSCs are a unique cell population. Although MDSCs have primarily been used for muscle regeneration, they also have been used for experimental treatment of a variety of diseases and disorders, including urologic dysfunction and repair applications that require bone, articular cartilage, cardiac, or peripheral nerve healing.
UROLOGIC DYSFUNCTION Uncontrolled or involuntary leakage of urine defines a condition called urinary incontinence. Urinary incontinence can occur due to intrinsic sphincter deficiency (ISD), which results from a malfunction of the external urethral sphincter. Unfortunately, the most common cause of ISD is idiopathic, and it appears to be a product of aging [82]. ISD can also result from incontinence surgery or radical prostatectomy. Regardless of how ISD develops in most patients, it results in significant myogenic damage. This damage is often due to
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direct injury of the sphincter muscle or the pudendal nerve (or both). Cellular uromyoplasty, the use of autologous myogenic progenitor cells to treat urologic dysfunction, is a novel strategy designed to augment urethral sphincter contraction by regenerating new and functional muscle. Thus far, experimental data obtained from mouse and rat models confirm the feasibility and potential benefit of injecting cells into both the urethral wall and the bladder [4, 21, 132, 133]. The injection of muscle precursor cells after induction of sphincteric damage by a myotoxin agent appears to accelerate the recovery of striated muscle mass with respect to both myofiber number and diameter [131]. ISD was one of the earliest models used to study MDSC transplantation. Yokoyama and associates [134] attempted to use autologous MDSCs to treat stress urinary incontinence. Histologic examination showed that the MDSCs had integrated with cells in the striated muscle layer of the mid-urethra and that this layer was contiguous. The researchers observed a significant amount of reporter gene expression by MDSCs for up to 30 days after implantation, no negative effects on bladder function, and no histologic evidence of inflammation at the site of injection [134]. After using a sciatic nerve transection model that involves denervation of the striated muscle layer of the sphincter, researchers injected MDSCs and observed significant and clinically relevant improvements in leak point pressure [78]. MDSC injection into the cryo-injured bladders of rats led to the formation of innervated donorderived myofibers as early as 2 weeks after implantation, and strip force measurements revealed improved tissue contractility [59].
BONE AND ARTICULAR CARTILAGE HEALING Significant progress has been made in the use of stem cells (particularly MDSCs) to improve bone healing. Members of our laboratory have previously shown that MDSCs can undergo chondrogenic and osteogenic differentiation and therefore are prime candidates for use in therapies for a wide variety of injuries and diseases, including fracture nonunion, skeletal reconstruction, and osteoarthritis. Researchers studying orthopedics often use tissue engineering to replace structural tissue. Therefore, the effort to enhance bone regeneration has focused on identifying the best scaffold material and the most efficient cellular delivery system. Although
scientists are finding ways to engineer scaffold material that releases osteoinductive factors within the damaged environment, cellular delivery appears to be the most controllable aspect involved in the promotion of bone healing. Proper bone regeneration comprises four different overlapping processes: (1) acute response to injury, (2) intramembraneous bone formation, (3) chondrogenesis and endochondral bone formation, and (4) bone remodeling. Bone fractures generally heal well. However, delayed union, nonunion, and incomplete repair can result when healing fails. The precise cause of healing failure is unknown, but two of the most widely cited reasons are insufficient blood supply and poor or deficient bone stock, both of which could result from an insufficient amount of osteogenic cells caused by a poor supply of osteogenic stem cells or the inhibited differentiation of progenitor cells at the bone healing site. Members of our laboratory have used MDSCs engineered to express bone morphogenetic proteins (BMPs) to induce bone formation and to heal critical-sized calvarial defects in mice and long-bone defects in rats [117]. Observations 4 weeks after treatment have revealed that MDSC-mediated gene therapy induces significantly better bone formation than does direct delivery of BMP-2 [95]. To enhance the efficacy of MDSC-based gene therapy for bone healing of criticalsized bone defects, we have used angiogenic factors to improve and accelerate the bone-healing process. Experiments with MDSCs transduced with both BMP-4 and vascular endothelial growth factor (VEGF) have demonstrated synergistic effects resulting in the promotion of both angiogenesis and osteogenesis [104]. The co-implantation of stem cells expressing VEGF can enhance the bone formation and bone healing elicited by BMP4-expressing stem cells [104]. However, the synergistic effect of these two growth factors depends critically on their ratio, with an improper ratio leading to detrimental effects on bone healing. Further studies revealed that the synergistic interactions between BMP-4 and VEGF during bone formation involve multiple mechanisms in the endochondral pathways, including an increase in stem cell recruitment to the bone formation site to enhance cartilage formation, the acceleration of cartilage resorption, and improved cell survival at the site of bone formation [104]. Human muscle-derived cells transduced with a human recombinant BMP-2 also can promote osteogenesis in vivo, as indicated by osteocalcin synthesis and calcium phosphate deposition [94].
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Although bone formation is a complicated and multistep process, articular cartilage formation may be the most challenging type of orthopedic regeneration to promote. Articular cartilage damage has many causes, including genetic defects, progressive osteoarthritis, or physical destruction due to injury. In contrast to bone, articular cartilage has a very limited regenerative capacity and rarely heals when damaged. We have explored the use of MDSCs to repair articular defects. Muscle-derived cells transplanted into articular defects in rabbits improved the healing of the defects with an efficiency equivalent to chondrocyte transplantation [1]. MDSCs also can undergo chondrogenic differentiation in vitro after BMP-4 stimulation and can assist in the repair of articular cartilage in vivo. We transduced the MDSCs used in that study to express BMP-4, and when implanted into articular cartilage defects the transduced cells improved the repair of the cartilage [75]. This repaired cartilage survived for the duration of the study (6 months) and showed no signs of calcification [75]. These results demonstrate the robustness of MDSCs used as therapeutic tools and could lead to the development of novel strategies for improving articular cartilage healing.
CARDIAC REPAIR Exogenous cell transplantation (such as skeletal myoblast transplantation) is believed to hold promise as a possible treatment of ischemic myocardial diseases and a potential alternative to heart transplantation. Researchers have used both intramyocardial injection and intra-arterial methods to successfully deliver skeletal myoblasts to the heart [69, 108, 125]. Results of myoblast or satellite cell-mediated therapy demonstrate that myogenic cells obtained by skeletal muscle biopsy can engraft successfully in cardiac muscle [15, 61]. These findings led to the first human clinical trial involving autologous skeletal myoblast transplantation in injured hearts, which produced some promising results [86]. However, various limitations similar to those observed after myoblast implantation in skeletal muscle make the use of stem cells (rather than myoblasts) more attractive. In a preliminary proof-of-concept study, Sakai and associates have shown that retrovirally transduced MDSCs delivered to cardiac muscle exhibit a high level of b-galactosidase expression [112]. Our group has also examined the ability of MDSCs to engraft in the heart. MDSCs implanted into dystrophic mdx hearts
generated large grafts [103]. These grafts contained numerous dystrophin-expressing myocytes and consisted primarily of skeletal muscle myofibers with a small number of donor-derived hybrid cardiac-skeletal cells, the existence of which we attribute to the fusion of donor cells with host cardiomyocytes. Members of our laboratory also have demonstrated that the implantation of MDSCs in infarcted hearts yields better results than does MT. The MDSCs exhibit improved and more persistent engraftment, and, more importantly, induce more neoangiogenesis, prevent fibrotic remodeling of the damaged area, and improve cardiac function [97]. These findings further substantiate the therapeutic value of MDSCs used for muscle-mediated therapies.
PERIPHERAL NERVE REGENERATION Peripheral nerve injuries are commonly encountered clinical problems that often lead to long-term functional deficits and expensive health care, lost employment, and social disruption [83]. These injuries frequently result from trauma, sports-related accidents, military activities, and degenerative muscle diseases (such as dystrophies). The repair of peripheral nerve damage — primarily aimed at restoring nerve continuity to facilitate axonal regrowth — is often incomplete despite vast improvements in microsurgery techniques based on nerve autografts [43, 85], the standard approach to repairing severe nerve injuries that requires grafting a healthy nerve or vein from another part of the body to the damaged nerve [113]. Although the use of nerve grafts to bridge large gaps is the “gold standard,” this approach does not always result in complete functional recovery. Reinnervation may be unsatisfactory, and neuroma and scar tissue formation are common. Muscle denervation (resulting in atrophy) is an adverse effect of injury to the sensory or motor nerve. Improvements in nerve repair techniques could facilitate better restoration of normal sensation, reduce denervation-induced atrophy of muscles, and minimize pain. Schwann cells express all the neurotrophic factors, neural adhesion molecules, and extracellular matrix proteins and are integral to peripheral nerve regeneration [27]. Therefore, researchers continue to pursue the development of cellular therapies based on populations of stem cells capable of Schwann cell differentiation. In previous studies [7, 27, 74], bone marrow stromal cells have displayed the ability to at least partially repair
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peripheral nerves. However, MDSCs appear to be particularly good candidates for treatment of neuromuscular disorders because MDSCs can efficiently regenerate skeletal muscle and, most importantly, might enhance restoration of the neural and vascular supply during muscle regeneration by undergoing multipotent differentiation into endothelial and neural lineages [107]. MDSCs also can up-regulate the regenerative process and thereby facilitate more active and directed axonal growth. Our group has reported that MDSCs isolated from normal mice can differentiate into several cell types, including skeletal muscle cells, Schwann cells, vascular endothelial cells, and hematopoietic cells [20, 107]. However, the potential natural involvement of MDSCs in nerve regeneration remains undetermined. Preliminary results generated by our laboratory indicate that MDSCs isolated from normal neonatal mouse skeletal muscle express both neuronal and glial cell markers in vitro, which at least partially explains the ability of MDSCs to adopt neuronal phenotypes. The results of preliminary in vivo studies performed in our laboratory have shown a significant number of b-galactosidaselabeled MDSCs in fully regenerated sciatic nerves 6 weeks after MDSC implantation. The regenerated sciatic nerve exhibited both neurofilament (postmitotic neuronal marker) and CNPase (oligodendrocyte marker) immunoreactivity. These studies indicate that MDSCs can differentiate into Schwann cells in response to nerve injury, which suggests that neurotrophic factors found within nerve tissue stimulate the growth and differentiation of MDSCs toward the neurogenic lineage. This study both strongly supports the existence of a population of MDSCs that have the ability to repair peripheral nerve and suggests that these cells might be useful as the basis for cell-based therapies to treat neuromuscular diseases.
alleviate numerous devastating diseases. In particular, MDSC-based therapy holds vast therapeutic potential due to MDSCs’ survival ability, unique immuneprivileged behavior, long-term proliferative potential, and pluripotency. Such characteristics strongly indicate that these cells could be useful for the treatment of DMD and other conditions of the musculoskeletal system. These cells provide a good example of what can happen when clinicians and researchers work hand in hand to appropriately interpret the outcome of clinical applications. Much work remains to be done before applications based on these cells progress from bench back to the bedside, but the preliminary data reviewed here strongly suggest that the future application of these techniques is promising.
Acknowledgments We wish to thank Ryan Sauder for his excellent editorial assistance with the manuscript, Dr. Burhan Gharaibeh for his assistance with the artwork in Figure 16.2, and Ken Urish for providing the EP image shown in Figure 16.3. This work was supported in part by grants to Dr. Huard from the National Institutes of Health (R01 AR049684, R01 DE13420, R01 DK55387, R01 AR049398), the Department of Defense, the Muscular Dystrophy Association (USA), the Pittsburgh Tissue Engineering Initiative (PTEI), the William F. and Jean W. Donaldson Chair at Children’s Hospital of Pittsburgh, and the Henry J. Mankin Endowed Chair at the University of Pittsburgh.
Conclusions This chapter provides an example of a significant discovery that resulted from what researchers initially perceived as a negative clinical finding, which they then explored further in the laboratory. The main stumbling block impeding many therapeutic approaches, including the treatment of DMD, is the difficulty associated with delivering therapeutic agents at levels sufficient to provide benefit. Stem cells offer many exciting possibilities for the development of novel treatments to 297
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study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplant 2:99–112; 1993. Urish, K., Kanda, Y., Huard, J. Initial failure in myoblast transplantation therapy has led the way toward the isolation of muscle stem cells: Potential for tissue regeneration. Curr Top Dev Biol 68:263–280; 2005. Vilquin, J. T., Wagner, E., Kinoshita, I., Roy, R., Tremblay, J. P. Successful histocompatible myoblast transplantation in dystrophin-deficient mdx mouse despite the production of antibodies against dystrophin. J Cell Biol 131:975–988; 1995. Watkins, S. C., Hoffman, E. P., Slayter, H. S., Kunkel, L. M. Immunoelectron microscopic localization of dystrophin in myofibres. Nature 333:863–866; 1988. Yiou, R., Dreyfus, P., Chopin, D. K., Abbou, C. C., Lefaucheur, J. P. Muscle precursor cell autografting in a murine model of urethral sphincter injury. BJU Int 89:298–302; 2002. Yokoyama, T., Huard, J., Chancellor, M. B. Myoblast therapy for stress urinary incontinence and bladder dysfunction. World J Urol 18:56–61; 2000. Yokoyama, T., Pruchnic, R., Lee, J. Y., Chuang, Y. C., Jumon, H., Yoshimura, N., de Groat, W. C., Huard, J., Chancellor, M. B. Autologous primary muscle-derived cells transfer into the lower urinary tract. Tissue Eng 7:395–404; 2001. Yokoyama, T., Yoshimura, N., Dhir, R., Qu, Z., Fraser, M. O., Kumon, H., de Groat, W. C., Huard, J., Chancellor, M. B. Persistence and survival of autologous muscle derived cells versus bovine collagen as potential treatment of stress urinary incontinence. J Urol 165:271–276; 2001. Young, H. E., Duplaa, C., Young, T. M., Floyd, J. A., Reeves, M. L., Davis, K. H., Mancini, G. J., Eaton, M. E., Hill, J. D., Thomas, K., Austin, T., Edwards, C., Cuzzourt, J., Parikh, A., Groom, J., Hudson, J., Black, A. C. Jr. Clonogenic analysis reveals reserve stem cells in postnatal mammals: I. Pluripotent mesenchymal stem cells. Anat Rec 263:350–360; 2001. Young, H. E., Mancini, M. L., Wright, R. P., Smith, J. C., Black, A. C. Jr. Reagan, C. R., Lucas, P. A. Mesenchymal stem cells reside within the connective tissues of many organs. Dev Dyn 202:137–144; 1995. Zubrzycka-Gaarn, E. E., Bulman, D. E., Karpati, G., Burghes, A. H., Belfall, B., Klamut, H. J., Talbot, J., Hodges, R. S., Ray, P. N., Worton, R. G. The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 333:466–469; 1988.
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17 Immunological Considerations in CNS Transplants Kai-Christian Sonntag, M.D., Ph.D. Assistant Professor in Psychiatry/Neuroscience, Harvard Medical School, Center for Neuroregeneration Research, Udall Parkinson’s Disease Center of Excellence, McLean Hospital, Belmont, Massachusetts
Introduction For a long time, the CNS has been thought to be an immune-privileged site based on several factors — including the blood-brain barrier (BBB), lack of lymphatic vessels, absence of peripheral professional antigen-presenting cells (APCs), the presence of transforming growth factor b (TGF-b), and high expression of FasL, a member of the tumor necrosis factor (TNF) family [47, 81, 153]. However, this view has now been revised based on recent observations that allogeneic and xenogeneic grafts can be rejected in the CNS [169], as well as on a better understanding of the mechanisms involved in CNS immunity — which reveal that immunologically activated cells can cross the BBB. We are also made aware of the existence of CNS cells with antigen-presenting capacity and a lymphatic drainage system to deep cervical lymph nodes. These observations have led to the notion that “The privileged status of the brain is now regarded as the result of a balance of regulated events that produce either immune privilege or effective responses” [12]. A revised view of CNS immunology can have major impact on the understanding of the immunological processes involved in the survival of implanted material to the CNS and might open new avenues in the development of immunemodulatory approaches to induce transplantation tolerance. This chapter aims to summarize the factors involved in CNS responses to grafted material, with an attempt to focus on the concept that all of the immune reactions observed in the periphery can potentially also take place in the CNS once its integrity is compromised by disrupting the BBB (which allows
Cellular Transplantation: From Laboratory to Clinic
a direct interaction between the peripheral and the CNS immunity). In addition, current approaches to overcome graft rejection in allo- and xenogeneic transplantation settings in the CNS are discussed, including some new concepts about the induction of graft-specific tolerance in organ transplantation and the inhibition of inflammatory processes in CNS diseases (which might also have implications in alleviating inflammatory responses in CNS transplantation).
CNS Immunology and Its Immune Privilege THE FACTORS INVOLVED IN CNS IMMUNITY There are two barriers that separate the CNS from the peripheral system: the BBB (comprising 99% of the capillary endothelium) and the blood-cerebrospinal fluid (CSF) barrier located at the choroid plexus epithelium of the four ventricles. The BBB and the blood-CSF barriers consist of a vast network of brain capillaries, with unique anatomical structures [125]. Tight junctions play a major role by providing a continuous seal around the lateral membranes of the brain microvascular endothelial cells (BMECs). The integrity of these tight junctions determines the permeability of the BBB, and therefore, the normal physiological state of the CNS. Each type of these cellular junctions consists of a network of specific intracellular and transmembrane proteins, which include occludins, claudins, junctional adhesion molecules (JAMs), platelet endothelial cell adhesion molecules (PECAM-1, CD31), G-proteins,
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protein kinase C, GTPases, and others [125]. This complex system is the guardian of CNS homeostasis which can be dramatically compromised under injury or inflammatory conditions and is vulnerable to proinflammatory molecules (see material following). There are no lymphatic vessels in the brain. However, the perivascular spaces surrounding the larger brain vessels are thought to serve as a lymphatic drainage system [130]. Thus, lymphatic drainage can occur and antigens can be transported from the brain to deep cervical lymph nodes leading to T-cell activation, B-cell proliferation, and antibody production [34, 129]. Although there are no peripheral APCs — such as dendritic cells (DCs) — entering the CNS [61], the perivascular spaces contain macrophage-like cells with antigen-presenting capacity [56]. These APCs reside locally and are continuously replaced by cells from the monocyte/macrophage lineage [87, 154]. The perivascular cells exhibit all features of APCs, such as inflammation-activated expression of major histocompatibility complex (MHC) class II and costimulatory molecules. However, it is not fully understood how they participate in the stimulation and/or reactivation of CNS-migrating T-cells [2]. Another type of APCs in the CNS is microglia, which reside in the CNS parenchyma and are derived from bone marrow precursors in early CNS development [35]. In response to inflammatory stimuli, the microglia acquire a macrophage phenotype — including phagocytic activity and the up-regulation of MHC class II adhesion molecules such as leukocyte functionassociated molecule 1 (LFA-1, CD11a) and intercellular adhesion molecule 1 (ICAM-1, CD54) — and the costimulatory molecules CD40, CD80 (B7-1), and CD86 (B7-2) for the promotion of T-cell activation [71, 80]. In addition, activated microglia release mediators — including chemokines (IL-10, IL-12, transforming growth factor (TGF)-b, and prostaglandin E2 (PGE2)) — that regulate the function of T helper cells [2]. In general, resting parenchymal and perivascular microglia are weak APCs and require activation through pre-activated T-cells and their release of proinflammatory molecules such as interferon (IFN)-g , tumor necrosis factor (TNF)-a, and IL-1b; free radicals such as nitric oxide (NO); and fatty acid metabolites such as eicosanoids and quinolinic acid to become functional APCs [2]. Astrocytes are also involved in inflammatory CNS processes by producing immune-regulatory cytokines (chemokines and PGE2). They can also inhibit IL-12
production of microglia, but do not seem to function as strong APCs [2]. Astrocytes can be stimulated in vitro by IFN-g , TNF-a, or IL-1 to express adhesion and costimulatory molecules — MHC class II, ICAM-1, and vascular cell adhesion molecule 1 (VCAM-1) [145] — although this has not been confirmed in vivo [2]. In some circumstances (such as multiple sclerosis), astrocytes express CD1 molecules for the presentation of lipid antigens to T-cells, indicating that they might be involved in nonpeptide antigen presentation [15]. Astrocytes secrete trophic factors [e.g., macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF)] and thus play a role in regulating microglia function (see material following). Another molecule involved in regulating CNS immunity is the Fas ligand (FasL, CD95L, Apo1L), which binds to its receptor Fas (CD95 or APO-1) and induces apoptosis of Fas-expressing cells [113]. FasL is a type II membrane protein and is expressed by activated cytotoxic T-cells (CTLs), NK cells, and cells in immuneprivileged sites — such as Sertoli cells of the testis, stroma cells of the eye, astrocytes, glia, and neurons [7, 16, 32, 47, 52, 58, 106, 128, 161]. Fas is a type I membrane protein and a member of the TNF receptor family [72]. It is widely expressed in many different cell types, including activated T-cells and B-cells, nonlymphoid hematopoietic cells such as immature myeloid cells, monocytes, granulocytes, hepatocytes, and some tumor cells [54, 91, 113]. FasL works as a death factor, and the Fas/FasL system is involved in maintenance of cellular homeostasis (e.g., cell differentiation and regulation of hematopoiesis programmed cell death and regulation of T-cells) [113, 132, 158, 164]. In the CNS, expression of FasL is thought to contribute to the protection of neural cells against Fas-expressing immune-reactive T effector cells [57, 106, 110, 173]. It is of interest to note that Fas does not exclusively function as a mediator of apoptosis, but can also stimulate proliferation, regeneration, and cytokine production [1, 40, 144, 162]. In neurons, Fas-mediated activation can lead to neurite outgrowth in vitro and in vivo [39], suggesting that FasL might have functions in the CNS other than inducing apoptosis. Finally, the CNS (like other immune-privileged sites, such as the eye and testis) contains locally immunosuppressive factors, which include TGF-b and related cytokines. TGF-b is the ligand for immunophilins, and together with other cytokines has anti-inflammatory and antimitotic effects [152].
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THE MECHANISMS OF CNS IMMUNITY CNS immunity involves cells and factors, which are similar or comparable to that of the peripheral immune system and follow similar rules of activation and regulation. An important mechanism for the activation of an immune response is antigen presentation, which requires specific cells and molecules. Key molecules are MHC classes I and II, which are also known in humans as human leukocyte antigens (HLAs) (Figure 17.1). Their main function is to scavenge peptides, present them on the cell surface, and signal T-cells or B-cells. MHCs serve as the regulator for the immune system to distinguish between foreign and self. The MHC genes are highly polymorphic, and each individual gene has its own set of multiple class I and class II alleles, which determines its allogeneic profile. Whereas the MHC class I receptor is expressed on all nucleated cells, MHC class II is expressed on B-cells, activated T-cells, monocytes, and typical APCs such as macrophages
and dendritic and Langerhans cells. Peptides bound to MHC class I or II complexes are recognized by T-cells expressing the T-cell co-receptor CD8 (cytotoxic T-cells) or CD4 (inflammatory and helper T-cells), respectively (Figure 17.1). This highly complex and polymorphic system is the basis for cellular, and partly humoral, immunity and specifies the immune responses. The activation of an immune response requires two major steps: antigen presentation and immune response. In general, antigen presentation occurs on all cells that are able to process, transport, and exhibit antigenic peptides on MHC class I and/or II molecules, whereby antigens can be “self” or “non-self.” In normal physiology, presentation of self-antigens does not mount an immune response — in contrast to non-self antigens, which activate either B-cells or T-cells. Selfantigens are constantly loaded on class I molecules and serve as maintenance of the innate immune system. These antigens derive from endogenous cellular pathways, which involve the ubiquitin-proteasome-system
(a)
(b)
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CD3 CD28 CTLA4 CD8 T cell
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Figure 17.1. Schematic representation of antigen presentation on APCs to CD4 or CD8 T-cells (modified from [73]). MHC class I receptors contain three a chains and an additional molecule b2-microglobulin (b2m) — which is located outside the HLA region (a) — whereas the class II receptors consist of two chains (a and b) (b). The molecular structures of the MHC class I and II molecules resemble alpha helices and beta pleated sheets that form an antigen-binding cleft for the exhibition of peptides (9 amino acids in case of MHC class I and 12 to 24 in case of class II). The floor and the wall of this cleft are highly polymorphic and affect the binding of the individual peptides on each HLA molecule. In addition, polymorphism exists for the external loops and the alpha helices of the MHC surface antigens, which are important for binding the T-cell receptor (TCR):CD3 complex. The peptides bound to MHC class I or class II complexes are recognized by T-cells expressing the T-cell coreceptor CD8 (cytotoxic T-cells) (a) or CD4 (inflammatory and helper T-cells), respectively (b). For the activation of a T-cell, two signals are necessary: the interaction of the TCR:CD3 complex plus the CD4 or CD8 coreceptor with the MHC:peptide complex and the amplification of the initial signal through co-stimulation mediated by receptor-ligand interactions, such as CD28 or CTLA4 on T-cells with B7.1 (CD80) or B7.2 (CD86) on APCs and CD40 (on B-cells) with CD40L on T lymphocytes. Provision of the first signal is unique to the APCs and determines the specificity of the T-cell response, whereas the signals through co-stimulation determine activation or anergy of the T-cells independent of the first signal.
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(UPS) to degrade intracellular proteins into peptides — are not entirely understood, but are similar to those which are then assembled with newly synthesized in the periphery and involve a cascade of molecules HLA class I molecules in the endoplasmatic reticu- such as selectin and carbohydrate adhesion molecules, lum and transported to the cell surface through the leukocytes and chemokines, G-protein linked recepGolgi apparatus. This mechanism, usually referred to tors, integrin/immunoglobulin adhesion molecule interas the endogenous pathway, occurs in most T-cells. actions, and others [125]. Monocytes can also migrate The HLA class II molecules are also synthesized in to the CNS parenchyma, possibly by using the b2 intethe endoplasmatic reticulum. However, their loading grin complement receptor-3 (CR-3 or Mac-1) [66, 142] with peptides follows different rules. In the exogenous and other molecule interactions such as VLA-4 with pathway, class II receptors express self- or foreign VCAM-1 [49, 98], the integrin-associated molecule antigens, which are derived from proteins that have been CD47 with thrombospondin, or the monocyte ligand taken up by phagocytosis and endocytosis and were signal regulatory protein-a (SIRP-a) — which is excluproteolytically degraded in the endo- and lysosomal sively expressed on myeloid cells and neurons [36] or compartments. In contrast to class I, loading of the class junctional proteins like JAM [101]. Activated monoII molecules with peptides occurs in endosomal and cytes in the CNS differentiate into macrophages and lysosomal vesicles and involves the chaperone HLA- take part in the inflammatory processes by releasDM to ensure stabilization of the complex and export ing cytokines and serving as APCs [87, 154]. Finally, to the cell surface. The exogenous pathway is gener- neutrophils can pass the BBB possibly by vascular ally used by APCs, which are highly endocytically and endothelial growth factor (VEGF) induction, which has phagocytically active. Both pathways contribute to nor- been shown to stimulate neutrophil transendothelial mal immunological homeostasis and play major roles in migration across BMECs in vitro [88]. MHC class I expression in the normal brain is inflammation and immune reactions to grafted material. An immune response is triggered by APCs (dendritic restricted to endothelial cells, but can be induced on cells, macrophages, B-cells, endothelial cells) by CNS cells (including neurons) during inflammation MHC:peptide complex interaction with the T-cell recep- and/or by IFN-g or TNF-a [26, 116]. There is controtor (TCR):CD3 complex and the CD4 or CD8 co- versy about the ability of the BMECs to serve as APCs. receptor (Figure 17.1). This determines the specificity Depending on the experimental context, BMECs have of the interaction and requires autoimmune, allogeneic, been shown to express MHC class II and co-stimulatory or xenogeneic peptides to stimulate the T-cells. In addi- molecules in vitro [99, 122]. However, their ability to tion, activation of T-cells requires co-stimulation by present antigens to T-cells seems to vary [22, 103, 131]. receptor-ligand interactions with CD28, or CTLA4 with Recently, it has been demonstrated that BMECs prefer B7.1 (CD80), or B7.2 (CD86) and CD40 with CD40L to stimulate IL-4 - producing Th2 helper cells, which in (Figure 17.1). Thus, activated T-cells are primed to spe- turn down-regulate cytotoxic Th1 helper cells (suggestcific antigenic peptides, and after stimulation activate a ing a differential activation of CD4-positive T-cells to cascade of signal transduction pathways that eventually maintain immune-privilege of the CNS) [46]. Activated T-cells in the CNS can cause immune leads to the production and release of many cytokines and up-regulation of other molecules to boost their pro- responses to the target antigen and/or are restimulated liferation and cytotoxic (CD8 T-cells) or helper (CD4 upon encounter of the antigen presented by the local APCs (mainly perivascular macrophages, parenchymal T-cells) potential (see materials following). In normal physiology, the BBB prevents the entry microglia, and possibly astrocytes) [145]. Thus, primof immunoglobulins and immune cells into the CNS, ing of immune responses most likely does not occur in but allows the entry of activated cells such as T-cells, the CNS. However, the connection between the CNS monocytes, NK cells, and possibly neutrophils [125]. and the peripheral system by exit of antigens through Cell migration through the BBB is tightly regu- slow lymphatic drainage allows for activation of T-cells lated by adhesion molecules, chemokines, cytokines, and B-cells in lymph nodes and spleen and entry of the and matrix metalloproteinases, which normally prevent activated lymphocytes into the CNS [34, 129]. The lymthe circulation of immune cells through the CNS phocyte functions are then controlled by microglia cells, parenchyma. In general, activated T-cells can migrate which either maintain T-cell function and propagation to the CNS, but only those with specificity for CNS and/or sustain their function, but not expansion and surantigens are retained [62]. The migration processes vival [2]. In turn, microglia activity is regulated by 308
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the immune-suppressive CNS environment (e.g., neurotrophins, adenosine, PGE2, norepinephrine, and/or factors released by astrocytes such as TGF-b), which inhibits IFN-g -inducible MHC class II and ICAM-1 expression on microglia and macrophages and downregulates their antigen-presenting function [117].
Transplantation to the CNS and the Mechanisms of Graft Rejection Cell replacement paradigms for the treatment of neurodegenerative, inflammatory, or ischemic injury have been developed in animal models and have reached clinical application in the cases of Parkinson’s disease (PD) and Huntington’s disease (HD) (see material following). Although much attention has been paid to in vivo development (or integration and function of the grafts in the host), potential immunological problems have received very little attention. In animal models, immune reactions toward transplants in the CNS have been studied. However, in clinical CNS transplantation settings no systematic studies that address graft tolerance or rejection have been conducted. A common observation in all studies is that the amount of transplanted cells into the CNS dramatically decreases in time. Although this cell loss can be attributed to nonimmunological factors (e.g., apoptosis or necrosis [174]), it is now becoming evident that biologically relevant immune responses also play a role in grafting to the CNS. The mechanisms of these immune responses are complex, strongly depend on the cell type implanted, and involve active regulation of immune reactivity by the CNS microenvironment [26, 112, 159, 167].
THE ROLE OF THE GRAFT The type of the graft determines the immune response in the CNS. Grafts can be of autologous (self), syngeneic (same species, same MHC haplotype), allogeneic (same species, different MHC haplotype), or xenogeneic (cross species) origin. Cells and molecules from these origins have their own characteristics, and dependent on the transplantation situation cause strong (xenograft), less strong (allograft), or very little to no (syngeneic, autologous) immune reactions. In addition, the cell types of the grafted material play an important role in initiation of immune responses. For example, fetal ventralmesencephalon- or substantia nigra-derived
cell implants consist of heterogeneous cell populations that include neurons, microglia, astroglia, precursors, endothelial, and blood cells — all of which have different effects on the host immune system and provide major and/or minor MHC antigens presented by host APCs when they are degraded in the CNS. An interesting new population for cell repair in the CNS is stem cells (SCs), which have the potential to develop into cell populations that can be used in CNS transplantation settings. There are several types of SCs, which are currently being investigated as sources for cell replacement in the CNS [160]. Embryonic stem (ES) cells are the most immature cell type and have been used, among others, to produce dopaminergic (DA) neurons for PD therapy paradigms [93, 147] (see contributions in this book). Based on their xenogeneic or allogeneic nature, ES cells are a potential target for rejection. ES cell and ES-cell-derived graft rejection in the CNS has been observed in xenogeneic transplantation settings in animal models (mouse, primate, and human to the rat brain), indicating that immature in vitro or in vivo differentiated cells from ES cells are to some extent immunogenic. Determined by RT-PCR, mouse ES cells and embryoid bodies (EB) express a variety of factors in vitro — such as stem cell factor (SCF); erythropoietin (EPO); EPO receptor; the growth factors GM-CSF, M-CSF, and G-CSF plus receptor; the interleukins IL-6 and their IL-2, -3, -4, and -11 receptors; and leukemia inhibitory factor (LIF) receptor [96, 104, 139, 170]. In addition, an ES cell subpopulation of 33% expressed the T-cell coreceptor B7.1, whereas B7.2, CTLA4, and CD28 could not be detected [95]. In these studies, B7.1, B7.2, and CD28 were up-regulated and a low expression of CTLA4 in a CD45-positive subpopulation of embryoid body cells was expressed after 12 days in culture. In addition, functional assays demonstrated that B7.1 could bind its ligand CTLA4. However, it is not clear if this also correlated with concordant expression of the HLA and other molecules necessary to mount an immune response. In one study, no HLA class II expression was observed in immature and differentiated ES cells. However, low levels of MHC class I could be detected, which were dramatically increased when IFN-g was added to the cultures [42]. In vivo, however, undifferentiated and partially differentiated human ES cells did not stimulate proliferation of alloreactive primary human T-cells when transplanted in immune-competent mice [92]. Because the alloreactive T-cells were still responsive to ES cell-specific alloantigens but failed to proliferate after reexposure to the
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ES cells, it was suggested that ES cells possess unique immune-reactive characteristics. How these data have an impact on the immunological aspects of ES cell transplantation in the CNS will be the challenge of future studies using this cell source for therapeutic paradigms. It should be noted that possible solutions to overcome rejection processes could be therapeutic cloning using nuclei from recipient somatic cells, which could result in less immunogenic transplants [68, 69, 83]. However, this method is still in its infancy and it needs to be demonstrated that this methodology reveals functional cells when differentiated and grafted to the CNS. In contrast to ES cells, adult SCs have the advantage that they are, theoretically, not immunogenic when derived from the recipient (autologous) and reimplanted after differentiation and/or as immature cell populations. Clonogenic human CNS stem cells (hCNS-SC) can be isolated from fresh human fetal brain tissue and show engraftment, proliferation, migration, and neural differentiation when transplanted into brains of immunodeficient mice [163]. However, there is currently no data indicating that this form of cell isolation and implantation has a potential to be adapted in clinical cell replacement paradigms in the CNS of adults. There have been several other SC-like sources tested in CNS transplantation paradigms. These include an immortalized murine neuroepithelial SC (MHP36) in a rat stroke model [112], xenogeneic embryonic porcine expanded neural precursors (EPNP) in a rat model of PD [3], and the rat neural hippocampal progenitor cell line HiB5 and the striatum-derived cell line ST14A in an allogeneic transplantation model for tumor inhibition [150]. The outcome of graft survival and function strongly depended on the cell source, the model used, modes of transplantation, and the CNS environment. No strong immune responses toward the MHP36 cells were observed, and this was independent of the use of immunosuppression (CsA), the local (intact versus ischemically lesioned hemisphere), and the global (lesioned versus sham) brain environment [112]. It should, however, be noted that the MHP36 cells could down-regulate MHC class I and II upon differentiation in vitro, which might have contributed to their survival in vivo [111]. No immune response was evoked after transplantation of the rat neural cell lines in the allogeneic anti-glioma model [150], which is consistent with a recent report demonstrating that adult hippocampal progenitor cells exhibit low MHC class I and II and increased anti-inflammatory TGF-b1 expression and thus do not mount a peripheral blood mononuclear cell
(PBMC) response in vitro [76]. Similar observations of PBMC unresponsiveness have been made with in vitro expanded neural precursors that either expressed high levels of MHC class I and II molecules — but not the costimulatory proteins CD40, CD80, and CD86 [118] — or had no class I expression at all [100]. In addition, some of the transplanted EPNP survived under the protection of CsA and could develop into mature neurons [3]. In general, there is little data with SC and/or SCderived CNS grafts in terms of immunogenicity. From the studies discussed previously, there is evidence that immature cells are less immunogenic than mature transplants. However, observed graft rejection without immune suppression (especially ES cell implantation in xenogeneic transplantation settings) points to the necessity of recognizing and further investigating the immune responses involved in this field of CNS grafting. Another factor involved in graft survival in the CNS is how the grafted material is implanted. In general, solid tissue grafts consist of highly antigenic microvessels, which connect with the host vascular system and usually cause strong rejection processes. In contrast, cellular (suspension) grafts do not have their own microvessels and are secondarily vascularized by host endothelial cells — which makes them less immunogenic [10, 28]. In addition, nonneuronal tissue does not develop BBB-type microvessels, in that graft-specific vessels in solid tissue (skin) grafts in the CNS survive and anastomose with the host vessels of the BBB [151].
THE ROLE OF THE HOST The reaction of the CNS to the graft depends on the type of the transplanted material and follows the regular immunological rules as observed in peripheral organ transplantation. The graft-induced activation of T-cells and the mounting of an immune response can be initiated by two different pathways: the direct or the indirect pathway of antigen presentation [55]. The direct pathway requires the presence of donor-type APCs, which have been co-transplanted to the host. This is usually the case in large organ transplants or tissue material, which contains remaining cells from the immune system (mainly in the organ parenchyma). The donor APCs come into effect in the recipient and connect with the immune system by presenting donor antigens processed in the endogenous pathway and loaded on their MHC class I molecules, which are then recognized by the host-type CD8-positive T-cells — triggering a cytotoxic
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immune reaction. In addition, donor-type peptides are also presented on the MHC class II molecules, which initiate the CD4 T-cell-mediated immune responses. The direct pathway of antigen presentation can cause a vigorous immune reaction for two major reasons. First, due to the mismatch of the donor-type HLA molecules (which can be allo- or xenogeneic) there is usually a strong reaction between donor APCs and the host T-cells (direct recognition). In this pathway, the role of the presented antigen is not as important. Second, the donor APCs activate a heterogeneous T-cell population with a high precursor frequency and thus trigger a fast and strong reaction to the transplanted tissue. The indirect pathway of antigen presentation usually does not involve donor APCs. In this pathway, donortype allogeneic or xenogeneic peptides are presented by HLA:antigen complexes from recipient APCs [24]. The antigens are processed by the APCs using the exogenous pathway of antigen presentation, and the proteins are derived from polymorphic donor molecules shed by the graft. Thus, the peptides presented in the indirect pathway are derived from donor-type HLA and minor histocompatibility molecules or tissue-specific antigens. The immune responses initiated by the indirect pathway are usually not as vigorous when compared to those triggered by the direct pathway. Indeed, it has been estimated that the frequency of T-cells that respond to allo-peptide presentation on host-type HLA molecules is about 100-fold lower than the frequency of T-cells that recognize allo-antigens in combination with donor-type class I or II [64]. The direct and indirect pathways of antigen presentation play a role in both allo- and xenograft rejection, in that both MHC class I and II can present donortype allo- or xenopeptides via the indirect pathway and thus activate CD4- and CD8-positive T-cells. However, there is a preference for triggering T helper cell function by antigen presentation through the indirect pathway, whereas triggering cytotoxic T-cells preferentially occurs by mechanisms involved in the direct pathway [50]. Because passenger donor-type APCs are immediately active when placing a graft, direct recognition of allo- or xenogeneic HLA:antigen complexes initiates early and acute rejection. In contrast, indirect allo- or xenopeptide recognition is more important in chronic rejection processes, when allogeneic or xenogeneic immune cells are no longer present in the host. Humoral immune responses are mediated by B lymphocytes, which are able to recognize proteins, carbohydrates, glycoproteins, and glycolipids that are identical
to the antibodies they produce. Once B lymphocytes have been primed to a specific antigen, they become plasma cells and mature to antibody-secreting B-cells. The antibodies detect their specific antigen on the cell surfaces and bind and connect with the factors of the complement cascade — leading to cell destruction. Antibodies can be induced by the grafted tissue itself or can be natural. These natural or preformed antibodies are present at the time of transplantation and crossreact with the graft-specific antigens, which is a major complication in xenotransplantation to humans and nonhuman old-world primates. In contrast to all other mammals, humans and old-world primates have a mutation in the a-1,3 galactosyltransferase (a-1,3-Gal) and do not express the a-1,3-Gal polysaccharide epitope on lipids and proteins. However, probably due to expression of the a-1,3-Gal epitope on bacteria in the intestine humans and nonhuman primates raise high titers of anti-Gal “natural antibodies” during the first years of life. These antibodies can bind to transplanted a-1,3-Gal-positive xenogeneic tissue, activate the complement cascade, and cause hyperacute rejection [53].
THE MECHANISMS OF GRAFT REJECTION IN THE CNS In transplantation immunology, foreign antigens are introduced into the host via the transplanted material (i.e., solid organ or tissue grafts and cell suspensions). Professional APCs, including macrophages and other inflammatory cells that release inflammatory factors such as TNF-a and IL-1, play a major role in the initiation of immune responses. These cells present antigens to the T-cells, triggering a cascade of signal transductions and secretion of cyto- and lymphokines [i.e., IL-2 and the upregulation of its receptor (IL-2R) on cytotoxic T-cells] which proliferate, infiltrate, and attack the graft and destroy the foreign cells by release of granzyme B, perforin, and other cytotoxic molecules. Activated B-cells can also be involved in the rejection processes by the production of antigen-specific antibodies and the induction of complement-induced cytotoxicity. In addition, T helper cells are activated, which release different sets of cyto- and lymphokines and regulate T effector and B-cell functions. A chain reaction of multiple immunological events is initiated, whose focus is to specifically destroy the transplanted foreign material.
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Basically, all immune reactions observed in the periphery can potentially also take place in the CNS. However, due to its unique anatomical and cellular structures the CNS is usually protected against these influences. Thus, the healthy CNS shows very little or no immune responses to antigens, which are only poorly processed (Figure 17.2). To induce immune responses and even graft rejection, the protective structures need to be disturbed and an interplay between cells from the CNS and the periphery needs to be initiated (Figure 17.2). As a rule of thumb, the immune system communicates with the CNS through glial and endothelial cells — which can up-regulate MHC class I and II complexes and present antigens — whereas
neurons and oligodendrocytes are usually not directly involved in this interaction [115, 166]. Thus, the CNS-type APCs activate lymphocytes, which have entered the CNS and trigger an antigen-specific immune response. These mechanisms are similar to inflammatory scenarios following traumatic injury, post-infectious encephalomyelitis, or autoimmune diseases — in which the effective lymphocytes react to CNS antigens they are usually not exposed to. Part of these responses is due to the effect of proinflammatory molecules such as TNFa, IL-1b, and INF-g or chemokines, which are secreted by the migrating lymphocytes and the CNS cells [114]. In addition, “homing factors” such as ICAM-1 and vascular cell
To peripheral and central lymphoid systems
(a)
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Figure 17.2. Schematic representation of the normal CNS immunology and the sequential events involved during and after graft implantation (a to d). An explanation of the depicted cells and molecules is given in (e). The anatomical structures of the CNS separate it from the periphery by forming the blood-CSF barrier and the BBB, which consist of a vast capillary system [125]. Cells and molecules of the immune system enter the CNS through the arterial system and can migrate through the blood-CSF barrier or the BBB into the CSF or CNS parenchyma, respectively, where they reside or reenter the periphery by the venous system. In normal physiology, the BBB prevents the entry of immunoglobulins, immune cells, and other molecules to the CNS, but activated cells such as lymphocytes can pass [125] (a). If the activated lymphocytes are not further stimulated in the CNS by APCs such as perivascular macrophages, parenchymal microglia, or astrocytes they do not render an immunological response and leave the CNS [62]. In addition, antigens can be drained from the CNS to deep cervical lymphnodes through the perivascular spaces, which serve as a lymphatic system [23, 119, 130, 165]. This well-balanced and regulated homeostasis is severely compromised in inflammation and injury, two major factors associated with graft implantation (b). Damage to the blood-CSF barrier and the BBB causes their disruption and allows the entry of immune cells and molecules to the CSF and CNS parenchyma, where they initiate a cascade of events aiming to destroy the foreign material (see text for details). Some of these events take place immediately and involve the activity of cytotoxic T lymphocytes (CTLs), NK cells, and possibly anti-Gal in xenotransplantation [11, 25, 86]. In addition, there is massive cell death of the grafted material providing allo- and xenoantigens, which are taken up and processed by the migrating host APCs (c). In general, all mechanisms and events, which are involved in the cellular and humoral immune responses to grafts in the periphery, potentially take place during this vulnerable period in CNS implantation. Once the blood-CSF barrier and the BBB are sealed, the CNS regains its normal homeostasis (d). However, now a graft is in place, which connects with the host system by developing its own structure (i.e., further differentiation of implanted immature progenitors into mature cellular phenotypes), integrating into the CNS parenchyma, and secondary vascularization by the host vascular system. In addition, peripheral and CNS APCs have taken up graft-specific antigens, which they present to T-cells triggering and maintaining an active immune response using the indirect pathway of antigen presentation (see text for details). These immune responses are part of the “slow” function of the normal CNS immunity and can therefore cause slow and long-lasting rejection processes with poor susceptibility to immunesuppressive treatments.
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To peripheral and central lymphoid systems
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To peripheral and central lymphoid systems
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Graft Figure 17.2. Continued.
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(d)
To peripheral and central lymphoid systems
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(e) Dendritic cells Lymphoctes (T, B, NK cells) Monocytes/macrophages Endothelial cells Microglia Oligodendrocytes Astroglia Neurons
Antibodies Host antigen Graft antigen
APCs presenting Graft antigen
Graft cell Dead graft cell Figure 17.2. Continued.
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adhesion molecules expressed on the CNS endothelial cells are important for the migration of immune cells to the CNS [13, 107]. In the CNS parenchyma, chemokines such as IL-8, inducible protein-10, RANTES, macrophage inflammatory protein-1, and monocyte chemoattractant protein-1 play a role in cell migration [107]. Neuropeptides — such as vasoactive intestinal polypeptide (VIP), somatostatin substance P, catecholamines, norepinephrine, and others — are released from neurons or glia and can modulate the function of the inflammatory lymphocytes by acting on specific receptors [18, 74, 105, 123]. A critical factor for initiating an immune response to foreign material in the CNS is the disruption of the BBB, which enables the communication between the “sealed” environment of the CNS with the immune system in the periphery (Figure 17.2). Once the BBB is open, APCs, lymphocytes, and preformed antibodies migrate to the damaged site and the immune processes are started. Because the transplanted material into the CNS usually consists of cell suspensions or small tissue chunks, usually no passaging donor-type APCs are present. In addition, due to very little expression of HLA molecules grafts such as stem cells and fetal cell suspensions do not function as strong APCs. Moreover, a high percentage of the transplanted cells die during or immediately after grafting and provide a good source for allo- or xenoantigens, which are taken up and processed by the host APCs. Thus, the immune reactions at the site of transplantation are triggered by the recipient’s immune-competent cells and mainly involve the indirect pathway of antigen presentation. These processes are slow and long lasting, and their susceptibility to immune-suppressive treatments are poor. It should be noted that some of the grafted cells further differentiate in the host CNS and can potentially up-regulate MHC class I and II molecules — and thus might gain immunological capacities. However, it is not clear if this is a major factor contributing to delayed graft rejection. Some of the immunological mechanisms involved in CNS grafting have been studied in experimental transplantation settings using fully MHC mismatched allografts or xenografts. Both rejection and spontaneous long-term graft survival have been observed without immunosuppression (see material following). There is no clear rule when graft acceptance occurs. However, the site of transplantation (e.g., away from the ventricles or sites, where microglia and astroglia have been already activated [44]), or an atraumatic stereotactic technique can positively influence graft survival. Interestingly,
immunization can also occur in the brain. When a second graft is implanted long term after a first graft, the previous graft is not affected and the second implant is not necessarily rejected, although an inflammatory response is observed around both grafts [43]. In addition, strongly preimmunized hosts immediately reject subsequent tissue grafts to the brain. This is also the case when a CNS graft is challenged with a strong stimulus (e.g., a skin graft) short term after transplantation [43]. In contrast, if the skin graft is placed long term after the CNS implant the first graft is not promptly rejected, although a slow chronic rejection might occur. These data indicate that immunization in the CNS is regulated by the effector system and is relatively weak when compared to the effects in the peripheral system. As mentioned previously, the immune responses in the host depend on the type of graft implanted. Organ transplantation of xenografts in humans or oldworld primates is associated with a strong reaction of preformed natural anti-Gal antibodies toward the a-galactosyl antigen on the endothelial cells of the transplanted tissue. This causes hyperacute rejection within minutes by rapid activation of the complement cascade, platelet aggregation, microthrombi formation, and ischemia [5]. The presence of the a-Gal epitope has been described on endothelial cells and microglia in the ventro-mesencephalon (VM) of pigs, but not on neurons and astrocytes [156]. In addition, there are three other known non-a-1,3-Gal epitopes on embryonic porcine dopaminergic cells. These react with IgM present in human serum [155]. Although an involvement of complement has been implicated in the rejection processes of porcine VM grafts in the brain of rats [11], a direct role of hyperacute rejection in xenogeneic CNS transplantation has not been described. In general, it cannot be ruled out that damage of the BBB during implantation leads to leakage of immune reactive complement-activating antibodies, which can cause immediate rejection of part of the graft. Another factor in allo- or xenograft rejection is NK cells, which belong to the group of bone-marrowderived lymphocytes. NK cells resemble many functions of cytotoxic T-cells, such as cytokine production and cytotoxicity to target cells, which they recognize via the MHC class I receptor. However, NK cells do not rearrange the genes of the T-cell receptor, and thus react to target T-cells independently of specific antigen recognition. The exact role of NK cells in neural (xeno)transplantation to the CNS is not entirely clear. It has been found that NK cells isolated from human
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blood do not lyse porcine VM cells in vitro [156]. However, these studies did not delineate the reactivity of NK cells to the individual cell populations in the VM suspension. It is known that human NK cells can recognize the a-Gal epitope expressed on porcine microglia and endothelial cells but not on neurons and astrocytes [70]. In addition, NK cells can lyse target cells via antibody-dependent T-cellular cytotoxicity (ADCC), which is mediated by subclasses of immunoglobulin G that cross-link the NK with its target cell. ADCC has been shown to be a mechanism in lysis of porcine VM cells by natural antibodies in vitro [155], and might be involved in NK-cell-mediated lysis of VM grafts in vivo. As discussed previously, heterogeneous cell populations (including neurons, microglia, astroglia, precursors, endothelial, and blood cells) have different effects on the host immune system. All these cells can provide minor MHC antigens presented by host APCs and induce an immune response based on the indirect pathway of antigen presentation. Some of the transplanted cells, such as endothelial cells and microglia, can express MHC class I molecules — and the latter and astrocytes also express MHC class II [82]. However, there is little evidence that these cells function as APCs. Although neurons do not express MHC class II, some neuronal subtypes can up-regulate class I molecules in response to inflammatory stimuli in vitro [116] but cannot mount a T-cell response in the host. Thus, in xenograft rejection endothelial cells and microglia are highly immunogenic based on their recognition by both the indirect and direct pathways, whereas neurons and astrocytes are recognized only by the indirect pathway and are thus less immunogenic [25].
Strategies for Overcoming Graft Rejection in the CNS Transplantation to the CNS is a relatively new field when compared to organ or bone marrow transplantation, and many of the strategies for inducing tolerance in the CNS have been adapted from the experiences gained in peripheral transplantation settings. There are several ways of overcoming host immune responses, which attack the various steps in the cascades of the rejection processes [21]. These include nonspecific immunosuppressive strategies using immune-modulatory drugs, antibodies against T-cells, and depletion of immunereactive antibodies or T-cells or the induction of specific tolerance by T-cell anergy, graft-specific immune
protection, or “educating” the host immune system to specifically being nonreactive to the transplanted foreign material (see material following).
THE INDUCTION OF NONSPECIFIC IMMUNE TOLERANCE The “classical” way of inducing hyporesponsiveness of the host immune system is the use of immunesuppressive drugs such as corticosteroids, cyclosporin A (CsA), FK506, and others, which mainly aim to inhibit T-cell alloreactivity. They are also widely used in experimental neural xenografting such as in rats and primates, but their protective actions are only partial [126, 127]. In clinical CNS transplantation, the most available data are from trials in PD patients, which received allogeneic or xenogeneic fetal tissue [6, 9, 19, 94]. These studies did not systematically consider or address the effects of immune-suppressive drugs on the survival of the transplanted neural cells, and altogether the data are patchy and difficult to interpret due to variations in the experimental settings. For example, multiple donors have been used to achieve sufficient graft effects, with the likelihood of a multiplication of donor-type antigens [121]. In addition, the transplanted tissue has been stored in hibernation media [138, 148] or even cultured for 4 weeks prior to transplantation [51], and it is unknown how this might have affected the antigeneity of the neural transplants. In some studies no immunosuppression was used [59, 78, 94]. T-cell infiltrates have been observed postmortem in brains from two patients 12 and 18 months after grafting, and although CsA was discontinued there was functional graft survival in both patients [78, 79]. Taken together, there is evidence in clinical neural transplantation of fetal tissue into the brain that chronic inflammatory responses occur that do not necessarily reject the graft but can lead to microglia activation and the production of cytokines and neurotrophic factors [12]. In addition, there seems to be agreement that the use of immune-suppressive treatment is beneficial for long-term graft survival and that a combination of immune suppressive drugs before, during, and after transplantation of the embryonic neural tissue protects the graft from rejection and even from metabolic insult during the preparation phase [127, 168]. There have also been trials with embryonic neural porcine tissue in PD patients. In one study, six patients received pig fetal VM cells and either CsA or immunomasking of anti-SLA I antigens (see material
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following) [140]. The patients were followed for 12 months. Two patients were reported to have a 15% improvement in the clinical rating scales, but there were no differences in improvement in the group data (20% for the CsA group and 19% for the immunomasking group). One patient, who died from pulmonary embolism 7 months after transplantation, was autopsied and had only 638 pig DA cells plus lymphocyte infiltration in the graft despite immunosuppression with CsA [37]. In a second multicenter study, 10 patients were grafted with the porcine tissue and 8 patients were randomized as controls undergoing sham surgery and sham immunosuppression [48]. Both groups (experimental and controls) showed a modest improvement (20 to 25%) in the PD rating scale and the reasons for this improvement are unknown. Taken together, these data indicated that the immunosuppressive regimen used were most likely insufficient to prevent the xenogeneic immune responses, implying more aggressive strategies for future xenografting to the CNS. Although immunosuppressive drugs usually induce nonspecific hyporesponsiveness, the induction of tolerance — which is the nonresponsiveness of the T-cells to graft-specific antigens — would be preferable because this conserves immune competence to all other antigens. There are two types of such T-cell tolerance, which include central and peripheral mechanisms of T-cell activation. The central form requires the deletion of graft-reactive thymocytes (discussed in material following). In contrast, the induction of peripheral T-cell tolerance is not necessarily graft specific and refers to the depletion of T-cells at time of transplantation and/or interfering with their activation. There have been several approaches in experimental neural grafting to induce peripheral tolerance such as depletion of T-cells in rodent recipients prior to transplantation, which led to indefinite survival of xenogeneic rodent neural, but not human cells [120, 172]. As mentioned previously, xenotransplants (such as porcine embryonic neuroblasts) in the brain of mice and rats are rapidly rejected over a period of days and weeks by cellular and humoral immune responses [25]. This involves infiltration with CD8 T-cells, IgM, and complement deposition [11]. Based on the observation that porcine VM cells survive better in immunoglobulindeficient mice [86], attempts to overcome the humoral immune responses have been made by transient application of complement-depleting cobra venom factor — leading to prolonged graft survival short term without preventing graft rejection long term [11].
In addition, transgenic porcine VM tissue expressing human complement inhibitor CD59 or human a-1,2-fucosyltransferase in combination with anti-C5 (H transferase) antibody treatment has been used [33]. Although the expression levels of these transgenes are low in the embryonic pig brains [60], the transgenic cells survived up to 12 weeks posttransplantation to the striatum of parkinsonian primates [33]. Cellular immune responses also play a critical role in xenograft transplantation to the CNS, as evidenced by the observation that in athymic (nude) mice CNS xenografts indefinitely survive [67]. In vitro, porcine-cell-induced proliferation of human CD4 and CD8 T-cells can be blocked by pretreatment of the pig cell cultures with human serum and complement [27]. Interestingly, when these mixed glia cultures were pretreated with purified anti-Gal and complement the content of microglia was reduced and the induction of CD4 T-cell proliferation was abolished in vitro [25]. Donor tissue treatment with anti-Gal and complement might also remove highly immunogenic endothelial cells and might reduce antibodies and NK cell responses, which in part also react to the a-1,3-Gal epitope [53, 70]. However, currently there is no in vivo data available indicating that these approaches could have clinical relevance. In mice, it has been shown that blocking the T-cell costimulation by short courses of treatment with CD40L, LFA1, or CTLA4Ig can improve xenograft survival [84, 85]. In addition, the use of anti-IL-2 receptors can increase the survival of grafted tissue in the ventricular system [171]. Other approaches to overcoming the cellular immune responses in xenotransplantation include the masking of MHC class I molecules on the donor cells with F(ab)2 antibody fragments. In vitro, masking of swine lymphocyte antigen I (SLA I) on porcine embryonic cells could reduce the proliferative response of human CD8 T-cells up to 52% [38]. In addition, pretreatment of transplanted fetal cells prior to implantation with the F(ab)2 antibody increased the survival in the host striatum (52%) when compared with untreated controls (7%) [41, 126]. Another approach to bypassing cellular immune responses was attempted in an allogeneic mouse neural transplantation setting by culturing embryonic VM cells in the presence of IFN-g to induce MHC class I expression [14]. Depletion of the MHC-class-I-expressing cells prior to transplantation by fluorescence activated cell sorting (FACS), improved graft survival, and demonstrated that CNS immunogenicity could be reduced. However, when translated to a rat model of PD depletion of IFN-g -induced MHC
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class-I-positive VM cells did not reveal behavioral recovery after implanting in the striatum of lesioned animals [25]. As discussed previously, microglia cells play a critical role in T-cell activation in the CNS and are involved in CNS cellular immune responses such as infection and autoimmune and neurodegenerative diseases [97]. Activated microglia cause cytotoxicity by antigen presentation to stimulate T-cells and by the release of proinflammatory cytokines such as IL-1b and TNF-a, and the latter can also be in the absence of leukocytes [17, 109]. Therefore, microglia have been thought to be a target for alleviating inflammatory processes involved in CNS disease [8, 109]. One example, among others, is the inhibition of cyclooxigenase (COX) — which is part of the synthesis of prostanoids such as prostaglandins, prostacyclins, and thromboxanes [108]. There are two isoforms of COX (the constitutively expressed COX-1 and the inducible COX-2 isoform), and both are targets of nonsteroidal anti-inflammatory drugs (NSAIDs). In the CNS, the COX isoforms are expressed in normal physiology and contribute to synaptic activity, memory, and functional hyperemia [108]. In addition, there has been increasing evidence that COX-2 is also linked to neurodegenerative processes, although it is not clear whether it plays a direct role in disease [108]. It is known that growth factors, cytokines, and proinflammatory molecules can trigger and sustain the expression of COX-2 in the CNS — and microglia seem to have regulatory functions in these processes [108]. Moreover, COX-positive activated microglia in the striatum have been associated with inflammatory and neurodegenerative processes in PD [77]. Part of these activities have been attributed to microglia-mediated toxicity through TNF-a, to which particularly dopaminergic (DA) neurons are susceptible [63, 149]. Therefore, the inhibition of COX-2 by the selective COX-2 inhibitor celecoxib has been hypothesized to protect 6-hydroxydopamine (6-OHDA) toxicity to DA neurons in a rat model of PD — and application of celecoxib 1 hour prior to 6-OHDA administration led to an inhibitory effect on microglia function (but not on those of astrocytes) and a protective effect on DA cell degeneration [135]. Another molecule that interferes in microglia activation is naloxone, which preferentially inhibits the production of superoxide free radicals (ROS) [97]. ROS is an upstream event for the regulation of the production of other proinflammatory factors (TNF-a and IL-b) in activated macrophages [65, 136]. Similar to the experiments with celecoxib, naloxone
had an inhibitory effect on microglia activation in vitro or in vivo and predominantly down-regulated the production of TNF-a, IL-b, nitric oxide, and ROS [97]. Taken together, these results might also have implications in cell transplantation to the CNS, and especially to prolonged microglia activation in chronic graft rejection.
THE INDUCTION OF GRAFT-SPECIFIC TOLERANCE The use of immunosuppressive drugs in clinical transplantation has been proven to greatly improve the survival and function of the transplants, but such nonspecific immunosuppression also causes complications involving infection, cancer, and autoimmune diseases [143]. This is also potentially true for the induction of peripheral tolerance by immune-modulatory molecules, which interfere in specific functions of immune-reactive cells. The complications associated with nonspecific immune tolerance indicate a need for the development of strategies for overcoming graft rejection by inducing specific tolerance to the transplanted donor cells without abrogating the functional immune system. Graft-specific or central tolerance requires the deletion of graft-reactive thymocytes, which are immature T-cells primed to react against the graft antigens. The induction of central tolerance can only be achieved by total eradication of immune-reactive T-cells in the mature state, whereas graft antigens must be present in the thymus to render the maturing new thymocytes nonresponsive to the grafted tissue. Initial studies to achieve central tolerance have used total body irradiation followed by allogeneic bone marrow transplantation (BMT) to create a chimerism in the recipient consisting of its own and the donor-type MHC class I and II molecules [157]. The so-conditioned recipients are tolerant of allogeneic organ grafts, which are fully MHC mismatched to the host but MHC matched to the donor bone marrow. Although these experiments have proven the principle of central tolerance, their clinical applicability is limited due to the requirement of adequate donor bone marrow and complications related to the allogeneic BMT. To overcome some of these obstacles, a new approach has been tested to create a “molecular chimerism” by introducing allogeneic MHC genes into the recipient BM cells prior to organ
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transplantation [45, 90, 146]. In a large miniature swine animal model, retrovirus-mediated transfer and expression of allogeneic class II transgenes in the recipient BM in combination with a short course of CsA treatment allowed for prolonged survival (transfer of MHC class II DR) or even tolerance (transfer of class II DQ) to transgene class-II-matched kidney allografts [146]. Although the exact mechanism of this tolerance induction was not known, class II transgene expression in the lymphoid lineage and the graft itself were sequentially implicated in the induction of tolerance to the organ grafts. The results from the studies on MHC class II have now led to a new concept of the role of this molecule in the induction of tolerance. It is suggested that class-II-derived peptides are loaded and expressed on self-class II in a complex named T-Lo, which controls regulatory T-cells (T-reg) [89]. Self- or allogeneic class II peptides in the context of transplantation are then presented on BM derived class-II-positive APCs (possibly dendritic cells (DCs)), which are involved in T-reg differentiation in the thymus and in the activation of Tregs in the periphery to mediate suppression of cytotoxic T-cells. This mechanism of regulating T-cell immunity by self-presentation of class II peptides has also been extended to explain susceptibility to several autoimmune diseases, such as systemic lupus erythematosus, insulin-dependent diabetes, rheumatoid arthritis, and possibly neurodegenerative diseases such as multiple sclerosis — which have been associated with particular MHC class II haplotypes [89]. It is of interest to note that T-regs have also been implicated in regulating T-cell responses to neurodegeneration and that their activity can be controlled by dopamine [74, 75]. How these mechanisms, and especially the regulatory function of class II molecules and/or neurotransmitters in context of T-reg activities, are related to CNS transplantation is speculative. However, recent findings in CD4 T-cell-mediated neuroprotection to injured mouse facial motoneurons imply that the T-cell regulation requires both resident microglia in the central compartment and a BM-derived APC in the peripheral compartment [29]. It is therefore feasible to suggest that induction of central and/or peripheral tolerance would also be beneficial for the survival of intracerebral grafts. It is interesting to note that human hematopoietic CD34-positive SCs derived from cord or peripheral blood can migrate to the brain and develop into perivascular and ramified microglia after infusion into severe combined immunodeficient mice [4].
In addition, microglia can be derived from mouse ES cells, which migrate exclusively to the brain after intravenously injected into recipient animals [163]. These avenues of generating CNS-type APCs in combination with genetic engineering technologies might offer new opportunities to develop future approaches for the induction and maintenance of specific tolerance in the CNS. The finding that FasL is an important factor in immune-privileged sites and can induce apoptosis in immune-reactive T-cells and NK-cells has prompted interest in using FasL as an immunomodulatory tool to protect allogeneic or xenogeneic cells against the cellular immune responses. A graft-protecting function of FasL was shown in several models of allotransplantation, including genetically engineered FasL expressing DCs — which promoted allograft survival and induction of antigen-specific immunosuppression [20]. However, FasL expression also induced self-apoptosis and/or massive infiltration of polymorphic mononucleocytes (PMN), leading to an accelerated rate of allograft destruction [20]. Although the exact mechanisms of these processes are still unknown, there is some evidence that FasL can up-regulate chemokines such as IL-8 [30] or act directly as a chemoattractant for PMN [124, 141]. In addition, it has been shown that the different immune-modulatory functions of FasL depend on the microenvironment of its actions. For example, a protective role of FasL in a non-immune-privileged site such as the skin was only observed in the presence of TGF-b, which prevented granulocyte infiltration [31]. FasL has also been considered to protect endothelial cells in a xenotransplantation setting. In vitro, expression of human FasL on porcine endothelial cells could indeed induce apoptosis in human T-cells and NKcells and had a positive effect on adhesion of PMN and transendothelial migration of NK-cells, but was not sufficient to protect the porcine cells against cytotoxicity mediated by the NK-cells [102]. Although the immunological functions of FasL are attributed to graft rejection in the periphery, they also might play a role in transplantation of cells into the CNS — especially when one considers that FasL expression on CNS cells contributes to its natural immunoprivileged state [47]. For example, it has been shown that the CNS provides a microenvironment that allows the survival of transplanted FasL-expressing Sertoli and astrocytoma cells in allogeneic and xenogeneic CNS transplantation models [133, 134, 137]. Because the effect of FasL on Fas-expressing lymphocytes is immediate and does not
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require other means of cell activation, it could be a potent factor in lysing the first wave of immigrating immune-reactive cells when the BBB is compromised during implantation. Thus, the expression of FasL on transplanted cells as an immune-protective molecule might abrogate T-cells and NK-cell reactivity in neural allo- and xenotransplantation settings to the CNS.
Acknowledgments I want to thank Dr. Susan L. Andersen for both critical discussion and reading of the manuscript.
Summary It has now become evident that the immune privilege of the CNS is not absolute and contains many elements similar to that seen in peripheral immunity. However, the CNS has its own immunological integrity — including its own mechanisms for interacting with the peripheral system. In normal physiology, CNS immunity is well organized — rendering a balanced homeostasis and protecting the CNS from potentially dangerous immune reactions. The BBB plays a major role in this system and compromising its integrity ultimately leads to dysfunction through invasion of peripheral immune cells and molecules and an activation of the CNS defense mechanisms. Transplantation to the CNS plays into these scenarios by disrupting the BBB, causing injury and immune invasion. During this temporary phase of CNS vulnerability, the transplanted material locally interacts with both the peripheral and the CNS immune system — triggering a cascade of events aiming to destroy the implanted cells. This includes the exposure of graft-specific antigens on both peripheral and CNS APCs. Once the CNS has sealed the BBB and regained its normal homeostasis, the initiated immune responses (which can lead to long-lasting rejection processes) to the graft are controlled by the CNS immunity. Drug-induced or other peripheral immunemodulatory approaches have been used to achieve immune unresponsiveness. However, there is evidence that these nonspecific regimens might be insufficient to prevent long-lasting chronic rejection. In addition, they are associated with side effects due to compromising the immune system itself. Possible solutions could be the induction of graft-specific tolerance by modulating either peripheral and/or CNS immunity. This involves, for example, the induction of regulatory immune cells (e.g., T-regs), inhibition of graft-specific APCs (e.g., microglia), and the expression of immunemodulatory molecules on the grafted cells themselves (e.g., FasL). 320
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18 Neurotrophic Cell Transplants for Degenerative Diseases of the Eye Chris Thanos (1 and 2) and Dwaine F. Emerich (1) (1) LCT BioPharma, Providence, Rhode Island (2) Brown University, Dept. of Molecular Pharmacology, Physiology, and Biotechnology, Providence, Rhode Island
Introduction Ophthalmic diseases involving degeneration of the retina, particularly those associated with aging, are increasingly prevalent. Annually, 200,000 cases of inherited retinal degeneration are reported, whereas the total number of people inflicted with macular degeneration is 8 to 10 million [43]. The insecurity and fear associated with the loss of a sense such as vision is devastating, and most forms of blindness are currently untreatable and palliative at best. Although disease progression varies in duration and rate of pathogenesis, the impact of retinal disorders creates a uniform urgency among patients and clinicians to develop novel effective treatments. Lately, a potent class of growth factors has given patients with retinal degenerative disorders hope for attenuation or potentially reversal of disease pathology. Neurotrophic (NT) factors have long been recognized for their roles in biologic regulation of the central nervous system (CNS), and more recently for their potential as therapeutic agents for a variety of neurodegenerative disorders. Advances in biotechnology, including gene therapy and drug delivery, are making it increasingly possible to safely and effectively deliver these compounds in a stable and continuous fashion directly to CNS sites (including the eye). Ophthalmic diseases involving neurodegeneration of the retina as a primary pathologic effect — such as retinitis pigmentosa (RP) — or resulting from a cascade of related events (as in glaucoma) are all potential candidates for neuroprotective therapy with neurotrophic
Cellular Transplantation: From Laboratory to Clinic
factors. Degenerative disorders causing photoreceptor cell dysfunction and apoptosis can be ameliorated with neurotrophin therapy. Because the eye is an immunoisolated organ relying on neuronal impulse from the sensory retina for function, it is an ideal candidate for successful cell-based therapy or traditional protein delivery. This chapter describes the current methods of neurotrophic factor delivery to the eye, with emphasis on recent clinical trials for RP.
Anatomy of the Eye The human eye is spherical, roughly 2.5 cm in diameter, weighs approximately 7.5 grams [68], and is divided into three fluid-filled chambers (Figure 18.1). The anterior chamber contains a fluid called aqueous humor and consists of the space occupied between the iris and cornea. The cornea is a transparent dome-shaped membrane that refracts light and aids in focusing the eye. It consists of five layers, including an epithelial layer five to six layers thick that is capable of regeneration. Aqueous humor, the fluid between the cornea and iris, is produced by the ciliary body. The iris consists of muscles that contract and relax to maintain the appropriate level of light that enters the eye through the pupil. The posterior chamber, between the iris and lens, also contains aqueous humor and is separated from the vitreous chamber by the posterior lens capsule. The lens is composed of layers of dense connective tissue fibers and is lined with epithelium. The lens is held in place
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Chris Thanos and Dwaine F. Emerich Cornea Iris Conjunctiva Aqueous humor
Ciliary body
Lens Zonule fibers
Rectus tendon
Ora serrata Optic axis
Visual axis
Vitreous humor Pigment epithelium
Retina
Sclera Choroid Lamina cribosa Optic nerve
Disc
Fovea
Macula lutea Sheath
Figure 18.1. General eye anatomy. (Courtesy of Helga Kolb, Webvision, University of Utah, http://webvision.med.utah.edu.)
by zonule fibers that extend from the ciliary body and is enveloped in a thick fibrous sheath called the lens capsule. The vitreous chamber contains several milliliters of a highly viscous gel (the vitreous body). The vitreous consists of a collagenous network of supporting structures (Cloquet’s canal) and a fluid rich in hyaluronic acid and glycosaminoglycans that maintains the geometry and appropriate pressure in the eye. Fluid exchange with the anterior chamber provides pressure regulation between the two compartments. The vitreous lies next to the retina and associated structures. Figure 18.2b depicts an illustration of the histologic appearance of the various retina components. The anterior aspect of the retina begins just posterior to the ora serrata, which itself is just posterior to the pars plana (the region free of photoreceptors). Posteriorly, the retina thickens until is reaches the macula. Choroidal vasculature makes up the retinal blood supply, and the optic nerve provides neuronal connection with the brain via ganglion cell axons. The retina, approximately 500 microns thick, contains several layers of rods and cone photoreceptors. The focal point of the retina, the macula, is the region densest in cones and is the most linear projection of the optical pathway. The eye is surrounded by a thick fibrous layer called the sclera, which is encapsulated by the conjunctiva (the outermost loose fibrous membrane). The organization of the retina includes vertically and horizontally integrated neurons (Figure 18.2). As light enters the eye and passes through the lens it is projected
onto the retina, where it passes through the inner limiting membrane, ganglion cells, and other neurons into the rods and cones. These photoreceptors are supported on their periphery by retinal-pigmented epithelium (RPE) that maintain the health of the photoreceptors by providing growth factors as well as phagocytosing rod outer segments during turnover. Following light absorption into photoreceptors, a biochemical impulse subsequently propagates into an electrical impulse back through the retina into the ganglion cells and to the brain. As discussed in material following, a substantial amount of experimental end points center on changes in retinal layers such as the appearance of the ganglion cell layer or the number of nuclei in the outer nuclei layer (ONL). The vasculature of the retina is derived from the choroid as well as the central retinal artery. Specifically, the choroids supply the outer retinal layers, whereas the inner layers and optic nerve head receive additional supply from the central retinal artery. Figure 18.3 shows a fundus photograph of a fluorescein-labeled adult human retina with apparent vessel architecture.
Cell Encapsulation for Immunoisolation Given the immunoisolatory nature of the eye, the potential for graft rejection is reduced compared to other potential implant sites in the body. Regardless, there is an inherent safety to using encapsulated cells over unencapsulated material in that the graft is protected from the immune system and is easily retrieved. In the eye, this is important in case there are hyperinflammatory responses to the graft or secreted product. There are generally two categories for cell immunoisolation by encapsulation: micro and macro. Each of these has benefits and limitations, as outlined in Table 18.1 [18, 40, 77, 90]. Macroencapsulation involves filling a hollow, usually cylindrical, selectively permeable membrane with cells — generally suspended in a matrix — and then sealing the ends to form a capsule. Polymers used for macroencapsulation are biodurable, with a thicker wall than that found in microencapsulation. Although thicker wall and larger implant diameters can enhance long-term implant stability, these features may also impair diffusion, compromise the viability of the tissue, and slow the release kinetics of desired factors. Macrocapsules can be retrieved from the recipient and replaced if necessary.
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Neurotrophic Cell Transplants for Degenerative Diseases of the Eye Pigment epithelium Rods Cones Outer limiting membrane Muller cells Horizontal cells Bipolar cells Amacrine cells Ganglion cells Nerve fiber layer
(a)
Inner limiting membrane
Pigment epithelium Rods and cones Photoreceptors Outer nuclear layer Outer plexiform layer
Horizontal cells Bipolar cells
Inner nuclear layer
Amacrine cells
Inner plexiform layer
Ganglion cells Ganglion cell axons
Ganglion cell layer
(b) Figure 18.2. Retinal anatomy. (a) Retinal layer schematic, (b) 3D cutaway view of retina with histologic architecture. (Courtesy of Helga Kolb, Webvision, University of Utah, http://webvision.med.utah.edu.)
Figure 18.3. Funduscopy of a normal human retina. (Courtesy of Helga Kolb, Webvision, University of Utah, http://webvision.med. utah.edu.)
Macroencapsulation is generally achieved by filling preformed thermoplastic hollow fibers with a cell suspension. Pumping a solution of polymer in a watermiscible solvent through an outer annular region of a nozzle while an aqueous solution is concurrently pumped through a central bore forms the hollow fiber. The polymer precipitates upon contact with the water and forms a cylindrical hollow fiber with a permselective inner membrane (or “skin”). Further precipitation of the polymer occurs as the water moves through the polymer wall, forcing the organic solvent out and forming a trabecular wall structure. The hollow fiber is collected in a large aqueous water bath, where complete precipitation of the polymer and dissolution of the
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Chris Thanos and Dwaine F. Emerich TABLE 18.1 Advantages and disadvantages of unencapsulated, micro- and macroencapsulated implants.
Unencapsulated cells or tissue
Microencapsulation
Advantages
Disadvantages
• Permits anatomical integration between the
• Likely requires immunosuppresion
host and transplanted tissue • Good cell viability and neurochemical diffusion
• Limited tissue availability
• Permits use of allo- and xenografts without
immunosuppression • Thin wall and spherical shape are optimal for cell viability and neurochemical diffusion Macroencapsulation
• Difficult retrieval • Societal and ethical issues • Mechanically and chemically
fragile • Multiple implant sites • Limited retrievability
• Permits use of allo- and xenografts without
• Dimensions may limit
immunosuppression • Reasonable mechanical stability • Adequate cell viability and neurochemical diffusion • Retrievable
neurochemical diffusion and cell viability • Multiple implant sites • May produce significant tissue damage or displacement during implantation
organic solvent occurs. The ends of the hollow fiber are then sealed to form a macrocapsule. This final step, which provides the barrier paramount for successful immunoisolation, is not trivial because reliably sealing the ends of capsules can be difficult. A second method of macroencapsulation, called coextrusion, avoids the sealing problem by entrapping cells within the lumen of a hollow fiber during the fabrication process [3]. Pinching the fiber before complete precipitation of the polymer causes fusion of the walls, providing closure of the extremities while the cells are inside. The advantages of coextrusion over loading preformed capsules include distributing the cells more uniformly along the entire length of the fiber, reducing the shear stresses on the cells during the loading process, and the potential for mass production. Although polymer capsules are relatively sturdy, it is critical to ensure that the encapsulating membrane is compliant enough to meet the dynamics of the surrounding tissue while remaining mechanically resilient to resist failure during device implantation/retrieval. To accomplish this, the macrocapsule can be designed to provide added strength and device integrity. One approach includes the addition of a cross-linked hydrogel (e.g., a 2% alginate solution) within the device. This modification was observed to enhance structural
support during the implantation procedure [78]. However, with time the hydrogel loses structural integrity and does not provide added strength — especially with regard to tensile strength, an important consideration for device retrieval. Mechanical supports can also be used to provide added strength to the membrane/device. Such supports are better served from within the device to not impede diffusion between the encapsulating membrane and the surrounding tissue. Titanium wires and braided materials are examples of approaches that may be used to provide added tensile strength [82].
Cell Encapsulation Engineering Cells placed within encapsulation devices generally fall into one of three categories. The first category is represented by primary post-mitotic cells such as islets of Langerhans for diabetes, adrenal chromaffin cells for chronic pain, or hepatocytes for liver diseases. Second, immortalized (or dividing) cells such as PC12 cells have been utilized to deliver dopamine for Parkinson’s disease. The third category is typically cell lines that have been genetically engineered to secrete a bioactive substance such as baby hamster kidney (BHK) cells engineered to secrete factors such as nerve growth
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factor (NGF) for a potential therapy in AD. Dividing tissue has advantages over post-mitotic tissue. It can be expanded and banked, and thus more easily tested for sterility and contaminants. However, dividing tissue is also constrained by the potential for overgrowth within the capsule environment, resulting in an accumulation of necrotic tissue that could potentially diminish the membrane’s permeability characteristics — further reducing cell viability and neurochemical output. In vivo, extracellular matrices (ECMs) provide control of cell function through the regulation of morphology, proliferation, differentiation, migration, and metastasis [24, 27]. Within a capsule, ECMs were originally employed simply to prevent aggregation of cells (immobilization) and resultant central necrosis, but have since been found to be beneficial to the viability and function of cells that require immobilization. They also serve as a scaffolding for anchorage-dependent cell lines. For example, adrenal chromaffin cells have been immobilized in alginate to prevent aggregation that in turn prevents central necrotic cores from forming [2]. The chromaffin cells appear to thrive in alginate, whereas mitotically active fibroblasts do not. In this case, the use of alginate is essential to the optimal functioning of this device because some anchoragedependent cells such as fibroblasts or endothelial cells are present with the adrenal chromaffin cells. In the absence of alginate or similar immobilizing matrices, the fibroblasts can expand and overgrow the encapsulated milieu, resulting in a device deficient in bioactive factors produced from the chromaffin cells [85]. In contrast, BHK cells — a fibroblastic cell line — prefer collagen, whereas PC12 cells exhibit a preference for distribution within precipitated chitosan (which provides a scaffolding structure on which the cells anchor) [26]. In addition, chitosan has been shown to be an effective substrate for the attachment and spreading of fibroblasts [30]. To provide a substratum designed for more specific functions, the matrix material can be manipulated chemically or mechanically — which in turn may influence cell attachment, differentiation, and/or proliferation. For example, peptides such as arginine-glycine-aspartic acid (RGD) have been immobilized on a variety of surfaces to promote cell adhesion [59]. Integrin receptors on the cell surface membrane interact with the RGD sequence, a known ligand for fibronectin receptors. Glass microbeads have been modified by attaching RGD or tyrosine-isoleucine-glycine-serine-arginine (YIGSR) motifs to provide sites for cell adhesion [59]. Spherical ferromagnetic beads have been coated with
specific receptor ligands to mediate cell attachment [87]. With competitive binding assays and a mechanical stress testing apparatus, the endothelial cell’s interaction with the ECM receptor (integrin beta-1) supported a force-dependent stiffening response — whereas nonadhesion receptors did not. A laminin-derived oligopeptide sequence, CDPG-YIGSR, also has been derivitized within an agarose hydrogel and permitted a dosedependent increase in neurite outgrowth using neuronal cell bioassays [10]. Similarly, YIGSR and isoleucinelysine-valine-alanine-valine (IKVAV) — both of which are found in laminin — have been immobilized on surfaces to promote neuronal cell adhesion and neurite outgrowth [84]. Poly(ethylene oxide) (PEO)-star co-polymers have been fabricated as a potential synthetic extracellular matrix [16]. The star co-polymers provide many hydroxyl groups where various synthetic oligopeptides can be attached to desired specifications. The survival and differentiation of encapsulated cells can be influenced by the matrix interactions. A variety of matrices for use in immunoisolatory devices — such as alginate, rat tail collagen extracts, gelatin shards, porous gelatin or collagen microcarriers, carrageenan, chondroitin sulfate, fibrin, hyaluronic acid, the positively charged substrate chitosan, and an acrylamide-based thermoresponsive gel — are available and were recently reviewed [31]. All in all, successful cell encapsulation involves the choice of the cells to be encapsulated, the type of intracapsular matrix used, and the ability to control membrane properties such as geometry, morphology, and transport. The interactions between the encapsulating membrane characteristics and the capsule core, or matrix within, should be rigorously characterized to determine the optimal configuration for each cell type.
Neurotrophic Factors in the Eye Neurotrophic factors are capable of attenuating or reversing neuronal degeneration in the CNS as well as the eye. They are secreted by tissues throughout the body but are most abundant in the CNS. Although they are present throughout life in varying amounts, they are most abundant in early development and embryogenesis of the CNS. The retina contains CNSassociated neurons, making it an excellent potential site for neurotrophin therapy. Several endogenous NT factors are secreted by cells in the eye (i.e., lens epithelium and retinal pigmented epithelium), some of which
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Chris Thanos and Dwaine F. Emerich TABLE 18.2 Therapeutic neurotrophic factors in the eye. Neurotrophic Site of Action Factor
Delivery Vehicle
Reference
CNTF
Retinal ganglion cells Retinal degeneration
Intravitreal injection Intravitreal injection Adenoviral delivery Encapsulated cell technology
19, 62 15, 51, 52 53 82, 83
BDNF
Retinal ganglion cell cultures Optic nerve crush Retinal pigmented epithelium Retinal ganglion cells
Media exchange mRNA detection Media exchange Muller cell delivery of adenoviral vector Intravitreal injection Intravitreal injection Doxycycline-induced secretion
49 38 44 21
Retinal hypoxia Retinal degeneration
47 51 67
GDNF
Retinal ganglion cells Retinal degeneration
Intravitreal injection Subretinal injection Intravitreal injection Adenoviral delivery
50 34 51 61, 91
LEDGF
Retinal degeneration
Intravitreal injection
56
PEDF
Retinal degeneration
Lentivirus-mediated subretinal transfer Mini-osmotic pumps Intravitreal injection
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have a paracrine-like mode of action. These growth factors include, but are not limited to, vascular endothelial growth factor (VEGF) [11], insulin-like growth factor (IGF) [89], lens epithelium-derived growth factor (LEDGF) [56], bFGF, and CNTF [88].
Ciliary Neurotrophic Factor (CNTF) One of the best-characterized and most potent neurotrophic factors studied in retinal disorders is CNTF. CNTF is a 23-kDa protein containing 200 amino acid residues and is a member of the IL-6 superfamily. Neurotrophic activity is mediated by binding to the CNTFα receptor in addition to gp130 and leukemia inhibitory factor receptor (LIFR) through the Janus kinase pathway (JAK/STAT) [51]. Cytokines are bound to their receptor, where kinases are activated to modulate phosphorylation of specific tyrosine residues on receptors, leading to activation of STATS (signal
5 23, 69
transducer and activation of transcription proteins). STATS translocate to the nucleus, where they bind to a specific enhancer protein, causing a subsequent biologic response [74]. CNTF was initially purified from chick embryonic eye tissues by Varon et al. in the late 1970s, and was shown to have neurotrophic activities similar to NGF [57]. Subsequent work with motor neurons extended these findings, demonstrating CNTF’s broad potential as a therapeutic neurotrophic factor. In 1990, Sendtner et al. [76] showed that the remodeling of early postnatal rat sciatic nerve was accelerated in the presence of CNTF. This quickly led to experimentation in animal models, showing that following transection mRNA for CNTF was increased in local tissues [60] and that retrograde neuronal death could be attenuated in the presence of CNTF [17]. Applications of CNTF to ophthalmology were quickly realized when Mey et al. demonstrated neuroprotection of axotomized rat retinal ganglion cells [62].
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Following optic nerve (ON) transection, purified CNTF with GFP, was correlated with photoreceptor rescue as or brain-derived neurotrophic factor (BDNF) was assessed histologically by ONL count. Cells of the inner administered and animals were allowed to survive for nuclear layer, photoreceptors, and retinal pigmented up to 7 weeks. Retrograde staining with carbocyanine epithelium showed varying levels of gene transfer based dye revealed twofold to threefold more viable ganglion on GFP localization. CNTF treatment resulted in a fourcells in both treatment groups compared to the controls. fold to fivefold increase in the thickness of the ONL This initial work was replicated and extended when compared to controls. A CNTF analogue (Axokine, Regeneron PharmaceuCui et al. [19] showed in the axotomized hamster that intravitreal delivery of CNTF every 5 days for 4 weeks ticals) has been tested in the feline model of autosomal enhanced healing of a peripheral nerve graft apposed to dominant rod-cone dystrophy [15]. Unilateral intravitthe lesion. These benefits were specific, as NGF, BDNF, real injections were delivered 10 days after birth and neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) were repeated monthly. Clinical and histologic characterization, using TUNEL staining, was carried out at were ineffective. The neuroprotective effects of CNTF have been con- 9.5, 13.5, and 17.5 weeks. Assessed by counting nuclei firmed in models of retinal degeneration [51]. Certain of the rod outer segment, these injections prolonged factors — including BDNF, CNTF, IL-1 beta, and acidic photoreceptor survival but there was a minimally invafibroblast growth factor — are highly neuroprotective sive side effect of subcapsular cataract and mild retinal following light-induced damage to the retina. Intravit- folds. Sham injections, as well as injections of BDNF, real injections of these neurotrophic factors (1 mg/ml) were ineffective. Importantly, this study demonstrated were made 2 days prior to placing the animals in a fluo- that repeated administration maintained a pharmacorescent light chamber for 1 week. The contralateral eye logic effect over several months after the discontinuation was injected with vehicle as a control. Photoreceptor of treatment. protection, as determined by quantifying the thickness of the rod ONL, was confirmed for each agent tested. These results were elaborated in a series of experiments Brain-derived Neurotrophic Factor using focal mechanical injury in the rat retina [89]. Following injury by creating a 1-mm incision through BDNF is a 27-kDa protein from the NGF family. BDNF the outer retina, mRNA was extracted and analyzed is secreted in various regions of the CNS — including using Northern blot analysis. Increases in the mRNA cortical neurons in response to stress and in the striatum, content for CNTF and bFGF were associated with injury where it normally acts as a survival factor for striatal and persisted for the full 10-day study period. Peak con- neurons. In the 1980s, Barde et al. characterized BDNF centrations of CNTF were detected at 4 days after injury, in the developing embryo and studied its effect on senwhereas bFGF levels peaked earlier. sory neurons. Neuron-enriched cultures were incubated In addition to its effects in acute lesion models, with either purified BDNF or radiolabelled NGF. The CNTF is also neuroprotective in more chronic spon- neural crest and placode in the developing chick embryo taneous mutant mouse models of retinal degeneration both responded to BDNF [54, 55]. NGF, in contrast, only [52]. In one study, three types of rhodopsin-deficient showed surface receptor activity in neurons of the neural mutant mice — Q344ter, nr/nr, and opsin(−/−) — as placode. Later, the same group demonstrated activity in well as light-damaged albino mice retinas were exposed chick sensory neurons [20]. Day 3-to-14 neurons were to intravitreal injections of BDNF, NT-3, NT-4, CNTF, grown in a collagen gel and supplemented with BDNF. leukemia inhibitory factor, bFGF, NGF, and IGF-2 either Again, cells from the neural crest and placode showed alone or in combination. In one set of the mutants extensive neurite outgrowth that peaked at day 10 to (Q344ter), CNTF was neuroprotective — whereas nr/nr 12 in response to BDNF. Seeking similar results in the retinal degeneration was slowed by BDNF. The other retina, Johnson et al. looked to retinal ganglion cells as NT factors tested were ineffective. CNTF was also pro- potential targets [49]. Day 17 retinas from Wistar rats tective in the opsin(−/−) mouse when delivered with were isolated and ganglion cultures were seeded. In the an adenoviral construct including GFP [53]. Subreti- presence of BDNF, cultures maintained cell number in nal injections were performed at 2 to 5 days of age, Thy-1 positive neurons — whereas these same neurons and gene transfer and efficacy was assessed after 4 were reduced nearly tenfold in the BDNF(−) controls and 12 weeks. Transfection of host tissue, localized and NGF(+) conditions. 333
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Following this elaborate work in neuronal cultures, it was quickly realized that BDNF might play a role in endogenous repair processes following injury. Following a crush of the optic nerve (ON), BDNF mRNA is increased within the ganglion cell layer [38]. BDNF also leads to a dose-dependent increase in bFGF in retinal pigmented epithelial cell (RPE) systems [44], illustrating the often interrelated and synergistic complex profile of many of these neurotrophic factors. An adenoviral delivery system was engineered in Muller cells to deliver BDNF to injured ganglion cells of rats [21]. Transfected Muller cells were assessed in vitro for activity in co-cultures containing chicken embryo lumbar dorsal root ganglia in addition to transfected Muller cell conditioned media. After confirming that the activity of the adenoviral-delivered BDNF was comparable to that of the purified protein, the cell line was intravitreally injected into rats with both axotomized and intact retinas. Systemic immunosuppresion, used to control for an immune-mediated decrease in efficacy due to the adenovirus, extended transgene expression from 14 to at least 30 days. In both cases, efficacy peaked at 10 days but declined by 16 and continued to decline thereafter. It was suggested that an immune-mediated down-regulation of NT receptors and intermediates occurred. In a chemical-induced model of retinal hypoxia, Nakayama et al. [47] tested the effects of intravitreally injected BDNF on cell survival. Two days following injection of 0.1 to 10 mg BDNF, potassium cyanide (an oculotoxic agent) was administered. An initial tracing study showed that the intravitreal dose was localized primarily to the retina, although measurable quantities existed in the ciliary body, lens, sclera, iris, and cornea. Based on morphometric analysis as well as TUNELstaining for cell death, all doses significantly increased cell survival compared to naive cohorts. LaVail’s work, mentioned previously [51], provided support for the NT activity of BDNF in the light-damage model of the rat retina. In addition, Campochiaro and colleagues [67] showed that BDNF slowed retinal degeneration in a double transgenic mouse model of inherited retinal degeneration. Doxycycline-inducible secretion of BDNF from photoreceptors was engineered. In a hypoxia-induced cell death model, animals stimulated with doxycycline showed a roughly twofold increase in the ONL. This trend remained constant in parallel experiments using the Q344ter rhodopsin mutation mouse strain after 3 weeks, although the magnitude of the effect was somewhat reduced.
Electroretinography showed supportive evidence for neuroprotection and photoreceptor rescue due to BDNF activation.
Glial-cell-line-derived Neurotrophic Factor (GDNF) GDNF is a member of the TGF-b superfamily and is secreted as a 60- to 70-kDa protein. It was originally purified from the conditioned medium of a rat glioma cell line, has potent trophic activity for embryonic midbrain dopamine neurons [4], and continues to be evaluated in Parkinson’s disease. Binding of GDNF by its homodimeric receptor tyrosine kinase (RET) complex leads to activation of the TrkA pathway, which in the case of GDNF is highly complex and interdependent. In the early 1990s, Stromberg et al. [48, 80] used various animal models to show in vivo protection of developing dopamine neurons by GDNF. A creative choice for transplant site in some of their experiments was the eye, where (for example) fetal ventral mesencephalon was inserted into the anterior chamber and exposed to injections of GDNF (0.1 or 1.0 mg/eye). Transplant volume doubled and quadrupled, respectively, compared to controls — and the amount and relative proportion of tyrosine hydroxylase-positive cells increased in response to GDNF exposure. For the immediate purpose of the study, the eye provided an immunoisolated environment — similar in nutrient abundance and oxygen tension as the brain, where transplanted tissue could be localized, monitored, and characterized following neurotrophic factor treatment. For the present discussion, these studies confirmed that GDNF was well tolerated in the eye. Axotomized ganglion cells of the retina benefit from GDNF [50]. Di-I labeled retina flat mounts were assessed for cell survival after 14 days following ON transection and intravitreal injections of 200, 500, or 2,000 ng GDNF at days 4, 7, and 10. At day 14, histologic assessment revealed that only 15% of untreated retinal ganglion cells survived, whereas 34% survived in the two higher GDNF doses. Although GDNF showed somewhat lower efficacy than BDNF in the same model, it affords greater protection than other NT factors (such as NT-4) and probably mediates cell death by hindering apoptosis. Retinal protection was assessed in the rd/rd mouse model of retinal degeneration [34]. The rd/rd mouse,
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an extremely rapid degenerative model, was injected subretinally with GDNF (330 ng in 1 mL) or PBS vehicle at 13 and 17 days of age. Electroretinograms (ERG) were taken at day 22, followed by sacrifice and histological processing at day 23. ONL thickness was characterized, and the treated group was compared to animals receiving sham surgery. ERG showed measurable a- and b-waves in the GDNF-treated group, whereas the sham and control groups showed no activity. Stereologic cell assessment and whole mount cell counts showed that the group exposed to GDNF had roughly 1.5 times as many nuclei as the sham surgery group and twice that of the control. The photoreceptor rescue demonstrated by this study shows the potential for GDNF as a treatment for hereditary disorders of retinal degeneration such as retinitis pigmentosa. The S334ter-4 rhodopsin transgenic rat, a disease model having a deficiency of the C-terminus of proteins localized to the outer rod segments, has pathology similar to that of human RP. Using recombinant adenoviral vector-mediated delivery, AAV-GDNF was injected subretinally in conjunction with AAV-GFP for localization and characterization of gene transfer efficiency [61]. For at least 45 days, GDNF slowed photoreceptor degeneration in this model as assessed histologically (ONL thickness of 23.8 mm compared with controls at 15.4 mm) as well as by ERG. Another AAV-GDNF construct was used to study the NT potential against retinal ischemia in normal rats [91]. Three weeks following intravitreal injections of AAV-GDNF, retinal ganglion cells were damaged by elevating intraocular pressure to 110 mmHg with an anterior chamber injection of balanced salt solution. Ischemia and reperfusion were confirmed by ophthalmoscopy. As a function of total protein in the vitreous, relative intraocular GDNF concentrations over 3 weeks were roughly 38 pg/mg. Morphometric analysis to quantify retinal thickness as well as cell number of retinal ganglion cells showed that GDNF-treated animals had healthier retinas than the controls. By ERG, it was evident that b-wave amplitude recovered in the treated group.
Other Neurotrophic Factors As mentioned previously in the context of other experiments relating to the CNTF, BDNF, and GDNF, other
NT factors have significant ophthalmic potential — whereas still other NT factors such as NT3, NT4, NGF, and IGF-2 [51, 52, 89] have produced more modest levels of efficacy in both light-damaged and genetic models of retinal degeneration. Another growth factor produced in the eye, lens epithelium-derived growth factor (LEDGF), is a survival factor for lightdamaged retinal cells [56]. Sprague-Dawley rats, Royal College of Surgeons (RCS) rats, and P23H rats (having a rhodopsin mutation) received intravitreal injections of LEDGF separated by 2 days. Retinal damage was then induced by exposure to light of 2,000 lux for 48 hours. Ganzfeld ERG was carried out after 10 days (normal rats), 10 weeks (RCS), or 1 month (P23H), depending on the relative progression of the disease in the various models. Histology to characterize the ONL thickness was performed following ERG. Preservation of retinal thickness was observed in RCS and light-damaged rat retinas with roughly twofold increases in thickness compared to controls. Both light-damaged and RCS rats had improved ERG profiles with significantly improved b-waves, whereas the P23H showed no effect. Pigment epithelium-derived growth factor (PEDF) was also studied in the RCS rat [64]. This group used a lentivirus-mediated subretinal transfer of PEDF around the perimeter of the retina and followed retinal function with ERG, morphology, and TUNEL staining. Gene transfer was confirmed in the retina at 4 and 24 weeks following injection and histology, and ERG confirmed protection of photoreceptor cells. PEDF has also been linked to choroidal vascularization in wet age-related macular degeneration (AMD). Holekamp et al. [46] showed that in a prospective case-controlled study that there was less PEDF in the vitreous of patients’ eyes affected with AMD compared to patients having other eye disorders. Subsequent examination of PEDF in a model of laser-induced choroidal neovascularization in the mouse confirmed its potential [5]. PEDF was loaded into subcutaneously implanted mini-osmotic pumps. Seven days after laser ablation of the retina, eyes were enucleated and analyzed for choroidal neovascularization using FITC perfusion and confocal microscopy. This analysis revealed a dose-independent effect on neovascularization and endothelial cell migration, as lower doses were slightly inhibitory but higher doses led to an unpredictable outcome. Duh et al. recently showed that PEDF suppressed ischemia-induced choroidal neovascularization and
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VEGF-mediated angiogenesis [23, 69]. In C57BL/6 mice exposed to 75% oxygen from postnatal day 7 to 12 to induce angiogenesis, 2 mg PEDF was injected unilaterally at days 12 and 14. On postnatal day 17, eyes were enucleated and neovascularization was compared to the contralateral untreated control eye. Intravitreal injections of PEDF reduced neovascularization by as much as 83% over control eyes, and reduced VEGF-induced cell proliferation by 48%. Although the mechanism of this inhibition is unclear, the authors speculate that there was a direct inhibition of VEGF by PEDF.
Neurotrophic Encapsulated Cell Therapy: Proof of Principle in the CNS Extensive background work on the NT effects of various encapsulated factors has been carried out in the CNS, with no more elaborate proof of principle than in animal models of Huntington’s disease (HD). HD is an inherited progressive neurological disorder characterized by a severe degeneration of basal ganglia neurons, particularly the intrinsic neurons of the striatum. Accompanying the pathological changes is a progressive dementia coupled with uncontrollable movements and abnormal postures. From the time of onset, an intractable course of mental deterioration and progressive motor abnormalities begins — with death usually occurring within 15 to 17 years. Overall, the prevalence rate of HD in the United States is approximately 50 per 1,000,000 [29]. At present there is no treatment that effectively addresses the behavioral symptoms or slows the inexorable neural degeneration in HD. Intrastriatal injections of excitotoxins such as quinolinic acid (QA) have become a useful model of HD and can serve to evaluate novel therapeutic strategies aimed at preventing, attenuating, or reversing neuroanatomical and behavioral changes associated with HD [7–9, 73]. The use of trophic factors in a neural protection strategy may be particularly relevant for the treatment of HD. Unlike other neurodegenerative diseases, genetic screening can identify individuals at risk — providing a unique opportunity to design treatment strategies to intervene prior to the onset of striatal degeneration.
RODENT STUDIES Infusions of trophic factors such as NGF or implants of cells genetically modified to secrete NGF have proven
effective in preventing the neuropathological sequelae resulting from intrastriatal injections of excitotoxins, including QA [35–37, 75]. We [28, 29] examined the ability of encapsulated trophic-factor-secreting cells to affect central striatal neurons in a series of defined animal models of HD. Rats received implants of NGFor CNTF-producing cells into the lateral ventricles. One week later, the same animals received unilateral injections of QA (225 nmol) or the saline vehicle into the ipsilateral striatum. An analysis of Nissl-stained sections demonstrated that the size of the lesion was significantly reduced in those animals receiving NGF and CNTF cells compared with those animals receiving control implants. Moreover, CNTF cells attenuated the extent of host neural damage produced by QA as assessed by a sparing of cholinergic, diaphorase-positive, and GABAergic neurons. Neurochemical analyses have confirmed the protection of multiple striatal cell populations using this strategy. Importantly, behavioral studies offer additional and compelling evidence of neuronal protection that can be produced in animal models of HD. These same animals show improved performance on motor as well as learning and memory tasks, indicating that the anatomical protection afforded by trophic factors in this model is paralleled by a robust and relevant behavioral protection [25].
PRIMATE STUDIES The ability of cellularly delivered trophic factors to preserve neurons within the striatum in a rodent model of HD led to similar studies in nonhuman primates [44]. Polymer capsules containing CNTF-producing cells were grafted into the striatum of rhesus monkeys. One week later, a QA injection was placed into the putamen and caudate proximal to the capsule implants. As seen in the rodent studies, the volume of striatal damage was decreased and both GABAergic and cholinergic neurons destined to degenerate were spared in CNTF-grafted animals. Although all animals had significant lesions, there were threefold and sevenfold increases in GABAergic neurons in the caudate and putamen, respectively, in CNTF-grafted animals relative to controls. Similarly, there were a 2.5-fold and fourfold increases in cholinergic neurons in the caudate and putamen, respectively, in CNTF-grafted animals. Using quantitative morphological assessment of DARPP-32 optical density, a marker of GABAergic terminals, monkeys receiving QA lesions had significant
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reductions in DARPP-32 immunoreactivity within the globus pallidus and substantia nigra. The lesion-induced decrease in GABAergic innervation for both of these regions was prevented in CNTF-grafted monkeys, demonstrating that this treatment strategy protected GABAergic neurons and sustained their normal projection systems [32]. The intrinsic striatal cytoarchitecture can be preserved in monkeys by CNTF grafts, and once exposed to these grafts the cells apparently maintain their projections. But are the afferents to the striatum, specifically from the cerebral cortex, also influenced by these grafts? This may be particularly critical if some of the more devastating nonmotor symptoms seen in HD result from cortical changes secondary to striatal degeneration. Because layer V neurons from motor cortex send a dense projection to the post-commissural putamen — a region that was severely impacted by the QA lesion — the effects of QA lesions and CNTF implants on the number and size of cortical neurons in this region were examined. Although the QA lesion did not impact the number of neurons in this cortical area, layer V neurons were significantly reduced in a cross-sectional area on the side ipsilateral to the lesion in control-grafted monkeys. This atrophy of cortical neurons was virtually completely reversed by CNTF grafts [28]. A recent set of studies using CNTF-producing cells in 3NP-treated monkeys have replicated and extended these results [63]. Following 10 weeks of 3NP treatment, monkeys displayed pronounced chorea and severe deficits in frontal lobe cognitive performance as assessed by the object retrieval detour test. Following implantation of CNTF-producing cells, a progressive and significant recovery of motor and cognitive recovery occurred. Histological analysis demonstrated that CNTF was neuroprotective and spared NeuN and calbindin-positive cells in the caudate and putamen. Recently, clinical trials were initiated to determine the safety and tolerability of CNTF-producing cells implanted into the lateral ventricle of HD patients [6, 12]. Recently, clinical trials were completed to determine the safety and tolerability of CNTF-producing cells (0.15 to 0.50 g CNTF/day) implanted into the lateral ventricle of HD patients [6, 12]. HD symptoms were also analyzed using neuropsychological, motor, neurological, and neurophysiological tests, and the striatal pathology was monitored using MRI and PET-scan imaging. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle. The capsule was retrieved and exchanged for a new one
every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed. However, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous cell survival, however, stresses the need for improving the technique.
Clinical Trials: Cell Therapy in the Eye RETINITIS PIGMENTOSA RP is an inherited degenerative disease of the retina that affects roughly 1 out of 4,000 individuals worldwide [13]. With a complex genetic profile leading to a heterogeneous molecular etiology, RP is a challenging therapeutic target. Although more than 100 RP-inducing mutations have been identified, gene therapy is still in the early stages of development [33]. Work in the field, discussed previously, has developed a variety of relevant animal models — leading to the clear demonstration that molecules such as CNTF have therapeutic potential. However, because of its relatively short halflife in vivo [22, 81] — as well as the associated costs of protein purification — intravitreal injections of CNTF are both prohibitively expensive and inefficient. CNTF has an elimination half-life of 120 to 400 minutes following a subcutaneous bolus injection [14], establishing the need for a sustained mode of delivery. Repeated local bolus injections would also introduce protein concentrations outside the optimal therapeutic range, and they are unrealistic due to patient compliance. In the case of RP, the disease course is a slow chronic degenerative progression, and the optimal profile of a therapeutic should target similar kinetics. There has been extensive research into delivery of therapeutic agents to the posterior segment of the eye using a range of delivery systems. Biodegradable microspheres composed of poly(lactic acid) or poly(glycolic acid) have been studied in the rabbit eye [45], Vitrasert and inward-releasing scleral plugs have been used to deliver ganciclovir [58, 92], and several groups have
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attempted to deliver therapeutics across the sclera [41]. These and other delivery systems are well suited for delivery of small molecules, but there are currently few viable approaches for long-term protein delivery to the vitreous. Neurotech has developed a macroencapsulated solution for chronic delivery of CNTF to the posterior cavity of the eye [82]. Using encapsulated cell technology (ECT), this group has created a CNTF-secreting cell line from human retinal pigmented epithelium that is attached to a thermoplastic support within a semipermeable membrane. The membrane, roughly 1 cm in length and 1 mm in width, allows diffusion of nutrients into the cells and secreted proteins out of the device but rejects cells of the immune system. Although the eye carries a certain level of immunoprivilege, the immunoisolatory barrier certainly adds protection to the fragile cells contained within. The cells are introduced in a biologically inert medium into the internal volume of the membrane and sealed off with a biocompatible adhesive. In the end of the device is a titanium anchor that allows surgical placement into the pars plana such that the distal tip is freely floating in the vitreous but is somewhat restricted from motion due to the anchor. Based on the geometry of the eye and the device, physical contact with various structures of the eye is limited — reducing the likelihood of long-term complications. The CNTF-secreting cell line used in the ECT device is neuroprotective in various models of retinal degeneration [82]. The cell line, derived from the retinal pigmented epithelium of a human donor, was transfected with a CNTF gene — and stable cell lines were screened and selected. S334ter-3 rats with rapid retinal degeneration due to a rhodopsin mutation were injected with roughly 100,000 CNTF-secreting cells (called NT-201 cells) into one eye, and control injection of non-CNTF-secreting parental cells (called NT-200) was administered to the contralateral eye. A bolus injection of 1 mg recombinant CNTF was given to a cohort of rats for comparison. Eyes collected at postnatal day 20 were assessed histologically for ONL thickness. The amount of CNTF delivered from the cell injection (as measured by ELISA) was roughly 10 times less than the purified protein injection, but cell-based delivery was continuous as cell viability was confirmed using a GFP-transfected NT-200 cell line. Control eyes showed a rapid degenerative process, with only one row of nuclei remaining — whereas eyes injected with NT-201 preserved 5 to 6 layers. In contrast, the recombinant CNTF injection left only 2 to 3 layers, clearly
showing the advantage of continuous cell-based protein delivery. Subsequently, NT-201 cells encapsulated in the ECT device were characterized in the rcd1 canine model of retinal degeneration. These animals, carrying a mutation in the PDE6B gene, were implanted with devices at 7 weeks of age — a time in development when the eye could accommodate the geometry of the device. After 7 weeks in vivo, devices were explanted and assayed for CNTF secretion. Eyes were processed for histology and morphometrically characterized based on ONL thickness. Control eyes having roughly 2 to 3 layers of nuclei confirmed the progression of the disease from postnatal day 1 to the 7-week age at implant. In comparison, ECT-implanted eyes contained 5 to 6 layers of nuclei. Neuroprotection was dose dependent, as doses ranging from 0.2 ng/device/24 hours to 15 ng/device/24 hours produced increasing and statistically significant levels of protection and associated thickness of the ONL. Cell viability was completely maintained within the device at 7 weeks. Pharmacokinetic analysis in rabbits was carried out on two sets of the NT-201 cell line that were engineered to produce two different doses [83]. ECT devices secreting approximately 20 or 60 ng CNTF per day were implanted in normal rabbits and were explanted along with the vitreous between 1 and 365 days for CNTF analysis and determinations of cell viability within the device. Over the course of one year, cells within both device types secreted CNTF along linear pharmacokinetic profiles. Devices with the lower dose had an associated vitreous half-life of 88 days, whereas high-dose devices had a vitreous half-life of 126 days. High-output devices secreted roughly tenfold higher levels of CNTF to the vitreous. Histology confirmed stable cell viability throughout the study. Based on these robust preclinical results, Neurotech has tested its device in a phase I clinical trial in patients with RP [79]. Ten patients were implanted with either 6-mm or 11-mm devices and were followed for 6 months. There were no significant adverse events reported, and it was reported that some patients could read more lines in the visual examination chart [1].
AGE-RELATED MACULAR DEGENERATION (AMD) AMD is a disease of the choroidal vascular system of the retina and is the number one cause of blindness
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in people over 60 years of age [43]. The macula, a portion of the central retina richest in photoreceptors, undergoes a degenerative process — leading to the formation of avascular and nonfunctional regions. Factors leading to the disease include hereditary factors, age, exposure to UV light, and high blood pressure. The most common form of AMD — avascular (or “dry”) AMD — accounts for 90% of cases, whereas the neovascular form (or “wet” AMD) is considered the most treatable and is limited to 10% of the affected population. Dry AMD is characterized by small yellowish deposits, or drusen, on the surface of the retina resulting from macular destruction over a period of years. In neovascular AMD, a small front of developing microvasculature typically penetrates through Bruch’s membrane (a supportive layer of the retina) and grows beneath the macula. The localized vascularization can lead to swelling, bleeding, and formation of scar tissue, which in turn reduces the area of functional retina and decreases the effective visual field of the patient. Typically, AMD visual fields are still present around the periphery but central vision suffers significantly. To date, the primary factor implicated in the angiogenesis of choroidal vasculature is VEGF [86]. Cytokine feedback, hypoxia, and other environmental stimuli can lead to the synthesis of VEGF mRNA and secretion by retinal pigmented epithelium. This in turn modulates the cascade of subsequent events, leading to angiogenesis and choroidal neovascularization. Several therapies are under investigation, including inhibition with antibodies or antibody constructs [42, 69], but the half-life of these molecules is relatively short and repeated instillation is necessary to provide effective chronic treatment. In addition, although the eye is somewhat isolated from the rest of the body by the blood-retina border there exists potential for systemic exposure potentially leading to complications. Adenoviral vector-mediated PEDF gene transfer, a localized and potentially a long-term therapeutic solution, has been explored in detail by Camopchiaro et al. [23, 39, 65, 66, 72]. Through gene transfer to the retina, PEDF secretion is increased and in turn inhibition of VEGF-related neovascularization is modulated. This neurotrophic strategy is being commercialized by GenVec, who is currently in clinical trials with AdPEDF [70, 71]. The phase I clinical trial — a multicenter openlabel dose escalation trial of intravitreally administered AdPEDF to patients with severe exudative AMD — revealed safety (as there were no apparent dose-related toxicity or serious adverse events reported). At the
higher dose levels, an increase in visual acuity and an increase in gross morphologic appearance of the retina were noted.
Conclusions NT factors have obvious potential for therapy of the retina. In numerous animal models, repeated intravitreal injections of purified NT factors significantly impact the course of rapid retinal degeneration. Currently, the most widely studied factors (CNTF, BDNF, GDNF, PEDF, and others) have been targeted as neuroprotective therapies for the degeneration of retinal ganglion cells following injury, for the rescue of photoreceptors in chronic degenerative states, and for the inhibition of vasculogenesis in the exudative form of AMD. Clearly there is a widespread and growing appeal and acceptance of the therapeutic potential of these potent molecules in the ophthalmologic community. The clinical trials cited here, as well as pending trials, suggest that as a general treatment, NT factors have an acceptable risk relative to the therapeutic potential in the clinic. Interestingly, NT products that meet this apparent requirement and that are in the clinic are built into a controlled delivery platform that circumvents the need for repeated intravitreal or subretinal injections. The Neurotech ECT device and GenVec’s adenoviral mode of delivery both provide long-term therapy with a single administration, although they differ in their longevity of efficacy. Advances in tissue engineering and gene therapy have allowed the pioneering work of Varon, Stromberg, LaVail, Steinberg, Campochiaro, Wen, and others on purified NT proteins to begin to realize potential success on a much broader scale. CNTF, as previously discussed, has shown and continues to show extremely high levels of efficacy throughout NT factor research. However, only recently has such success (even in human patients) been as obvious as with the tissue engineering strategies discussed within. Adenoviral vectors and gene transfer can provide longer-term delivery and enhance efficacy compared to repeated protein injections. Genetically modified cells delivering continuous therapeutic doses of NT factors protected by the immune system and implanted in close proximity to the target site are the state of the art. It will be with great anticipation that the next generation of NT therapies for the retina involving these concepts for multiple disease indications are developed and tested in the clinic.
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19 Transplants for Glioblastoma Jian Wang (1), A. J. A Terzis (1), Karen S. Aboody (2), Per Oyvind Enger (1), and Rolf Bjerkvig (1) (1) NorLux Neuro-Oncology, Department of Biomedicine, University of Bergen, Norway and NorLux Neuro-Oncology, Centre Recherche Public Santé, Luxembourg (2) The Divisions of Hematology/Hematopoietic Cell Transplantation and Neurosciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California
Introduction Of the primary brain tumors, intracranial neoplasms that originate from neuroglial cells are the most frequent and are collectively known as gliomas [1]. Gliomas are classified according to their line of differentiation, displaying features of astroglial, oligodendroglial, or ependymal cells. Thereafter, they are graded according to their histological appearance and degree of malignancy. The most malignant form is the glioblastoma multiforme (GBM), which is composed of poorly differentiated neoplastic cells with areas of vascular proliferation and/or necrosis [25]. A hallmark of gliomas is their diffuse infiltrative nature that renders them impossible to cure by surgical resection. Hence, these patients have a dismal prognosis with a median survival of 9 to 12 months [25]. These tumors appear most frequently in adults, with a peak incidence between 45 and 70 years of age. The incidence of malignant gliomas is in the range of five to seven new cases per 100,000 [25]. At present, there is an urgent need for new treatment alternatives for this devastating disease. Surgery and radiotherapy and/or chemotherapy are the main treatment modalities currently available to patients with primary brain tumors. However, due to limited successes of these therapeutic strategies alternative therapies such as immunotherapy, genetherapy, antiangiogenic therapy, and cell-based therapies are currently under preclinical and clinical evaluation. This chapter focuses on the current use and limitations of various animal models to study glioma growth and progression, and the potential use of cell-based therapies toward the disease. It highlights some important areas of research
Cellular Transplantation: From Laboratory to Clinic
related to cell therapies of GBMs. However, it is beyond the scope of this chapter to provide a complete overview of the vast number of therapeutic strategies currently directed toward malignant brain tumors.
Animal Models The establishment of animal models for gliomas has followed three research lines: the chemical induction of brain tumors, the development of transgenic models, and the xenotransplantation of human tumors in immunocompromised animals.
CHEMICALLY-INDUCED MODELS Chemically-induced brain tumors (mainly established in rodents) have been initiated by their exposure to carcinogenic hydrocarbons and other miscellaneous chemical agents, inducing DNA mutations. In general, three different approaches of chemical induction have been used: induction by (1) polycyclic hydrocarbons (PHC) in mice, (2) intravenous injection of methylnitrosourea (MNU) in adult rats and rabbits, and (3) ethylnitrosourea (ENU) given transplacentally to rats [24]. From these models, a number of tumorigenic cell lines have been established — many of which are routinely used to assess therapeutic efficacy such as the 9L and C6 models established in Fischer and Wistar rats, respectively. However, in most cases these cell lines show limited single-cell infiltration into the surrounding tissue.
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GENETIC MODIFICATION STRATEGIES In general, genetic modification strategies have followed two directions: germ line and somatic gene modification in mice. Germ line modification involves the introduction of specific genes into mammalian germ lines, which include transgenic approaches and knockout strategies. Transgenic mice express a particular gene driven by cellspecific promoters, whereas knockout mice lose expression of the targeted gene in all cells that would normally express it [3, 4]. For example, transgenic mouse models for glioblastoma have been initiated by expressing v-src kinase under the control of the glial gibrillary acidic protein (GFAP) gene regulatory elements in astrocytes. It was shown that the tumor progressed from lowgrade astrocytoma to glioblastoma. However, the incidence of manifest tumors was relatively low (< 20%) [59]. Others have developed experimental gliomas by expressing oncogenic V12Ha-ras specifically in astrocytes, and showed that tumor progression was dependent on p21 activation [12]. Alternatively, K. M. Reilly and colleagues have shown in knockout animals that the loss of tumor suppressor function by the mutation of Nf1 and Trp53 induced low-grade astrocytomas at a young age that eventually progress to glioblastoma [45]. Somatic modification, as an alternative method for genetic alteration, has been widely developed to initiate gliomas. This strategy has mainly been based on retrovirus-mediated gene transfer of specific oncogenes. For example, it has been shown that mouse gliomas can be induced by a recombinant Moloney murine leukemia virus encoding platelet-derived growth factor (PDGF) B-chain [56]. Furthermore, Eric Holland and co-workers have shown that combined Ink4a-Arf loss will increase tumor incidence in both glial progenitor cells and astrocytes during mouse gliomagenesis [20]. However, Ink4a and Arf have been shown to have independent and differential tumor suppressor functions in the glial cell compartment [57]. As known from previous studies [11], the receptor tyrosine kinases (RTKs) such as EGFR or PDGFR lead to activation of multiple downstream signal transduction pathways in human glioblastoma (e.g., the Ras-MAP and the PI3K-AKT pathways). However, it is still not clear which specific pathways orchestrate the initiation of human gliomas. Somatic cell gene transfer models have both advantages and disadvantages. On one hand, somatic modification models may be more efficient than germline models if a combination of more than two mutations is to be studied. In addition, only a few numbers of cells can
be targeted, which reduce the opportunity to introduce secondary genetic alterations. Moreover, the tumors that are established are genetically and histologically similar to human glioblastomas, and the time for their development is relatively short compared to tumors derived from germline modification models. On the other hand, the tumor incidence is less than 100% in somatic cell gene transfer models, and there is a variation in latency time — probably due to the fact that each animal must be injected with virus. Consequently, there is also a variation in location and size of the tumors.
XENOTRANSPLANTATION MODELS The first xenotransplantations of human brain tumors dates to 1945, when Green and Arnold successfully transplanted human glioblastoma into the anterior chamber of the eye of guinea pigs [16]. However, subsequent experiments showed an unreliable tumor take — often difficult to reproduce [48]. In fact, heterologous transplantation of human glioblastoma had little success until the advent of the nude mouse and rat in the 1970s [14, 19, 38]. Since then, a plethora of experiments of human brain tumor xenografts in nude mice and rats has been reported — mainly using human cell lines. However, most of these tumor cell lines have been passaged numerous times in vitro, which is accompanied by a genetic drift leading to less invasive tumors [29]. By using spheroids derived directly from human glioblastoma biopsies, Engebraaten et al. showed that highly invasive tumors developed when implanted in the nude rat brain [13]. The infiltrative growth pattern was characterized by tumor cell migration along white matter tracts, occupying both hemispheres in the terminal stage. Furthermore, it was found that the disease course was highly reproducible when the experimental conditions were standardized. Recently, tumors have also been initiated in immunodeficient animals by the implantation of cancer stem cells isolated from human GBMs. Interestingly, the cancer stem cell tumors show an infiltrative phenotype similar to that observed in human GBMs [50].
New Treatment Principles for Gliomas The continued dismal prognosis of glioblastomas has led to an increased focus on new treatment principles for the disease, including but not limited to gene therapy
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(thymidine-kinase suicide therapy, antisense inhibition of tumor growth factor receptors, and conditionally lethal viral vectors), immunotherapy (antibody, tumor cell vaccines, and adoptive transfer of activated lymphocytes), and antiangiogenesis approaches [4, 7, 15, 28, 37, 46, 60].
GENE THERAPY Gene therapy means the transfer of a gene into cells to treat a disease. This transfer can be mediated by viral or nonviral vectors. The gene used may restore signaling pathways by replacing defective genes, induce an immune response, or encode an enzyme that converts a pro-drug into a toxic metabolite. Most clinical trials using gene therapy approaches [30, 41, 42] have involved viral vectors, as they provide a higher transduction efficacy than nonviral vectors [8]. In this context, clinical trials have been conducted both for newly diagnosed and recurrent gliomas utilizing retroviral, adenoviral, and herpes simplex vectors [9]. The majority of these studies have involved transfer of a gene encoding the enzyme thymidine kinase (tk) from herpes simplex virus [32]. This enzyme converts the pro-drug ganciclovir into a toxic metabolite that triggers apoptosis in the target cell. Retroviral vectors encoding tk have been delivered by direct stereotactic injection [42], by free-hand injection at the time of craniotomy, and by Ommaya reservoir installation [39]. In a phase III trial of 248 patients, injection with retrovirus producer cells encoding tk did not prolong the time to tumor progression or overall survival [41]. Subsequent clinical studies concluded that these findings resulted mainly from a limited gene delivery [17, 40]. The low transduction efficacy observed in cancer patients in vivo may be due to the fact that the target cell population often constitutes a solid heterogeneous tumor mass. At present, gene therapy has not improved the outcome for glioma patients. Furthermore, gene therapy has in some instances showed toxicity and side effects [53], emphasizing the need to better address safety issues.
IMMUNOTHERAPY Malignant gliomas are well suited for immunotherapeutic intervention because most patients, following resection and radiation therapy, achieve a state of
minimal disease burden and the tumors remain confined to the CNS. This makes local administration of effector cells feasible, and numerous trials have involved the administration of ex-vivo-activated lymphocytes to glioblastoma resection cavities [18, 21, 22, 26, 31, 33]. However, despite a variety of effector cell types used (lymphokine-activated killer cells, tumor-infiltrating lymphocytes, and cytotoxic T lymphocytes), significant therapeutic efficacy has not been documented. Still, anecdotal long-term survivors have been reported. At present, new and better strategies are under development to improve the antitumor potency of the lymphocyte effector cells. For example, the interleukin (IL) 13receptor alpha2 (IL13Ralpha2) has been shown to be abundantly expressed by glioma cells and is not otherwise detected within the CNS. Glioblastoma cells have been targeted with IL13Ralpha2-specific cytolytic T-cells. This has been done by an ex-vivo genetic modification to express a membrane-tethered IL13 cytokine chimeric T-cell antigen receptor, or zetakine. These modified T-cells have shown an impressive antitumor effector function, including tumor cell cytolysis. In vivo, the adoptive transfer of IL13-zetakine clones results in the regression of established human glioblastoma orthotopic xenografts. Pilot clinical trials have now been initiated to evaluate the feasibility and safety of local-regional delivery of autologous IL13zetakine redirected CTL clones in patients with recurrent GBM. The IL13-zetakine is a prototype of a new class of chimeric immunoreceptors that signal through an engineered immune synapse composed of membranetethered cytokine muteins bound to cell-surface cytokine receptors on tumors [23].
Stem Cell Therapy Both neural stem cells (NSCs) and mesenchymal stem cells (MSCs) show an extensive inherent migratory behavior toward areas of pathology. When implanted into a diseased or injured CNS, the stem cells migrate extensively over long distances and engraft within areas of discrete as well as diffuse abnormalities. The engraftment may be followed by an integration and differentiation of the stem cells, due to their pluripotency, endowing them with the capability to replace diseased tissues. Recently, it has been shown that engrafted NSCs may affect the cellular microenvironment, promoting protection and/or regeneration of host neural cells [36]. Thus, stem cells represent attractive candidates for
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the treatment of various CNS pathologies, including brain tumors. Brain tumors are, as before mentioned, generally difficult to treat due to the extensive infiltrative nature of the tumor cells and the distortion of normal brain structures. The unique ability of stem cells to “home in” to the invading tumor cells and to deliver a desired therapeutic gene product makes NSCs a very promising cellular delivery vehicle for brain tumor therapies. Engraftable NSCs can be engineered to express cytolytic viruses and genes coding for antitumor cytokines, pro-drug-converting enzymes, antiangiogenic agents, and various neurotrophic factors for targeted delivery to tumors. When tagged with reporter genes, injected NSCs can be visualized with the use of novel imaging techniques. The tropisms toward experimental brain tumors have been demonstrated for both NSCs and MSCs [1, 35] (Figure 19.1). In this context, it has been shown that NSCs and MSCs genetically engineered to express a therapeutic substance or pro-drug-activating enzyme show significant therapeutic efficacy toward experimental gliomas [1, 6, 34]. However, recent findings also suggest the possibility that stem cells may themselves be tumorigenic [47]. Although mesenchymal stem cells can be managed safely during a standard ex vivo expansion period of 6 to 8 weeks, the cells can undergo spontaneous transformation following long-term in vitro culture. Thus, spontaneous transformation of human adult stem cells may occur, supporting the hypothesis of a cancer stem cell origin. This study also underlines the importance of biosafety studies of stem cell biology to effectively exploit their full therapeutic potential.
Cell-based Delivery Systems The use of producer cells secreting therapeutic substances has been limited due to their rejection by the host’s immune system. Cell encapsulation in alginate has been one strategy to circumvent the problems of graft-host incompatibility [51]. Alginates are natural polysaccharides that are usually isolated from brown seaweed, where they encompass the extracellular matrix of the organism. The alginate polymers are composed of two linked anionic monomers (G blocks and M blocks) interdispersed with regions of mixed monomers (MG blocks). The alginate form a gel in the presence of divalent cations such as Ca2+ , which binds via free carboxyl groups to the G blocks. The pore size of the gel is in the range of 5 to 200 nm [10]. The alginates can be made more or less dense, stable, and biocompatible depending on cation ions used and the composition of the gelling solution [58]. In general, specific producer cells can be engineered to secrete therapeutic proteins that are then encapsulated in alginate. In this way, proteins may be released from the encapsulated producer cells and pass freely through the pores, whereas the producer cells are sheltered from the cells of the immune system. For example, encapsulation of insulin-producing islets of Langerhan’s subsequently implanted in diabetic patients demonstrated the potential of cell encapsulation for human diseases [51]. Several studies have described how producer cells behave in the microenvironment within alginate beads [44, 54]. After an initial adaptation to the new environment accompanied by a modest proliferation, the cells
Figure 19.1. Low (A) and high (B) power field images of invasive edge of a murine glioma as indicated by black arrows. b-galactosidase positive neural stem cells (blue) distribute throughout the main tumor mass and migrate to the surrounding invading tumor islands (red, spindle shape), as well as next to infiltrating tumor cells (1). See color insert.
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undergo a period of increased proliferative activity and spheroid formation lasting 2 to 3 weeks. These spheroids can then establish a steady state where the formation of new cells equals the cell loss. In addition, other studies have reported the delivery of recombinant gene products to the CNS [46], such as human growth hormone (hGH) released from encapsulated mouse myoblasts. In vitro analyses demonstrated a stable expression of hGH for up to 75 days. After implantation into the lateral ventricles of mice, quantification of hGH using ELISA confirmed detectable levels up to 16 weeks after implantation, with a peak at 8 weeks. The distribution of hGH was assessed by immunohistochemistry, which showed that it was present within a radius of 1.5 to 2 mm from the implantation site at a decreasing gradient. Similar experiments measuring the release of antibodies from encapsulated hybridoma cells showed a steady increase for 12 days, after which it stabilized [54]. After implantation of the beads in the rat brain, immunoglobulins could be detected at a distance of 1 mm from the implantation site. Thus, encapsulated producer cells may successfully adapt to the CNS microenvironment and maintain a transgene expression for several weeks following implantation. Furthermore, therapeutic studies have demonstrated the efficacy of encapsulated 293 cells releasing the angiogenesis inhibitor endostatin against experimental gliomas implanted in rats [43]. In vivo release of endostatin was confirmed by immunohistochemistry of the tumor tissue as well as by Western blot of the cerebrospinal fluid. This therapeutic approach was associated with suppressed tumor growth and significantly prolonged survival. In humans, Aebischer et al. reported the intrathecal delivery of CNTF into the lumbar space of patients with amyotrophic lateral sclerosis [2]. Nanogram levels of CNTF were measured in the patients’ CSF, whereas it was undetectable prior to implantation. In glioma patients, treatment with encapsulated cells has not yet been conducted but may offer unique opportunities.
Future Prospects Modern techniques of biology have provided us with tools that offer molecular insight into the mechanisms that drive tumor progression. We are now able to better study the complex and abundant regulatory mechanisms and signaling pathways of malignant cells on
a molecular level. Recent studies demonstrate that the inhibition of one particular pathway often triggers compensatory changes in others, which counteract changes in the tumor environment [27]. However, some signaling molecules integrate several pathways and are critical for cellular function [49]. Drugs can now be designed that act specifically on these key players, thereby attacking the whole cell. A main objective is therefore to identify vital components within the cancer-cell-signaling network that can be targeted. The presence of cancer stem cell pools may have important therapeutic implications, and whether these cells respond to cytotoxic drugs and radiation as do those of the remaining glioma cell population is not clear. If they are less sensitive, other regimens may be needed to target such cell populations. Furthermore, it is important to establish whether these cancer stem cells (like normal neural stem cells) have the capacity to differentiate while simultaneously loosing their proliferative potential. If so, tumor growth may be suppressed by drugs that induce differentiation. Interactions between the cancer cells and tumor stroma may also be targeted therapeutically. The formation of new host-derived vessels to meet the tumors’ increasing demands for nutrients is triggered by tumorderived growth factors and is a target for antiangiogenic therapy. However, blocking tumor angiogenesis will not target the invasive cell population. This cell population alone can mediate a lethal disease course in the absence of angiogenesis, which implies that antiangiogenic therapy will have to be supplemented with other therapies targeting the invasive compartment within tumors. The efficacy of any drug relies on sufficient delivery. Several reports suggest that insufficient biodistribution may limit the efficacy of both established and experimental therapies [5, 55]. Alternative modes of delivery have therefore been extensively studied to optimize the biodistribution of therapeutic substances [47, 54]. In gene therapy, the lack of effect observed in clinical studies has been attributed to a low transduction efficacy [17], as well as to lack of delivery to distant tumor cells [17]. Vectors have now been developed with the ability to replicate within transformed cells. These cells are destroyed by oncolysis, whereas nearby untransformed cells are spared [8]. Further refinements may provide clinicians with potent vectors that have the capacity to penetrate the tumor tissue while only replicating in neoplastic cells. Cell-based therapy with encapsulated producer cells may offer sustained release for long periods, and can
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maintain high local tissue concentrations for substances with a short biological half-life. Producer cells can be genetically engineered to secrete numerous substances, and can subsequently be cryopreserved in freeze libraries [52]. On the basis of biopsies from the patients, treatments may then be tailored individually using genetically engineered cells that release active substances fitted to the biological profile of the tumor. When these factors are brought into play with a force and synchronicity exceeding that of the brain tumor itself, progress will be made.
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20 NT2N Cell Transplantation and GDNF Treatment in Stroke: Linking Neurotrophic Factor Therapy and Neuroprotection Cesario V. Borlongan (1 and 2), Christine E. Stahl (3), Guolong Yu (1), Lin Xu (1), Takao Yasuhara (1), Koichi Hara (1), Noriyuki Matsukawa (1), Paul R. Sanberg (4), Yun Wang (5), and David C. Hess (1 and 2) (1) Research and Affiliations Service Line, Augusta VAMC, Augusta, Georgia (2) Department of Neurology and Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA (3) Department of Internal Medicine, Dwight D. Eisenhower Army Medical Center, Fort Gordon, Georgia (4) Center of Excellence for Aging and Brain Repair, University of South Florida, College of Medicine University of South Florida College of Medicine, Tampa, Florida (5) Neural Protection and Regeneration, NIDA, NIH, DHHS, Baltimore, Maryland
Acknowledgments CVB is supported by the VA VISN7 Career Development Award, the VA Merit Review, and the American Heart Association Grant-in-Aid Award.
The Regenerative Stroke Brain The “nonregenerative central nervous system” dogma is arguably a historical past. Accumulating scientific evidence demonstrates that diseased or aging brain cells can potentially be rescued and have their functions restored. Cell replacement therapy has emerged as the current translational research trend and in the future could provide a promising treatment intervention for various neurological disorders. Laboratory investigations have shown that cell replacement therapy is effective in animal models of neurological disorders. Moreover, cell transplantation regimens have begun their transition from the laboratory to a clinical setting. Limited clinical trials of neural transplantation have already been initiated in chronically ill patients. The use of neuroteratocarcinoma cells for transplantation therapy in stroke is one such approach that has reached the clinic. Procurement logistics and novel
Cellular Transplantation: From Laboratory to Clinic
neuroprotective functions associated with this cell line allow neuroteratocarcinoma cells to serve as efficacious alternatives to using fetal cells as donor cell grafts for stroke therapy, although the optimal transplantation regimen must still be determined. Stroke is the third leading cause of death, which affects more than half a million people each year in the United States alone. Despite scientific progress in our understanding of stroke, there are currently about 3 million stroke survivors, many of which suffer from significant cognitive and functional disabilities. Rehabilitation therapy has helped some stroke survivors recover, but many patients still experience permanent loss of independent function. The cost for rehabilitation and lost wages is estimated at $30 billion each year, and thus represents a significant financial impact on society. Current stroke treatments are typically limited to supportive care and secondary stroke prevention, resulting in only limited improvement in cognitive and motor function. Intravenous tissue plasminogen activator administration has been effective in ameliorating the neurological deficits arising from acute stroke, although this treatment strategy is also problematic because of its extremely limited window of efficacy in that the drug needs to be administered within 3 hours of stroke onset. Current treatments remain directed
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toward acute stroke and are rather limited in their application and efficacy. No therapy has been proven effective for treating chronic stroke, which is associated with significant morbidity and mortality. This unfortunate scenario exemplifies an urgent need to examine the efficacy of novel experimental treatments, such as neural transplantation, especially for chronic stroke.
Initial Cell Transplantation Studies Using Fetal Tissues Researchers interested in delivery of exogenous proteins into the central nervous system (CNS) have used approaches combining cellular and gene therapy strategies. Finding a transplantable and transfectable cellular platform has been the key step in generating a local delivery system for gene products of therapeutic value [7, 78, 102]. In addition, a recognized problem is continuous secretion of the gene product in order to achieve a sustained therapeutic effect from application of a gene product to affected regions of the CNS. To this end, we and others have demonstrated sustained protein delivery via transplantation of cells genetically engineered to express the therapeutic protein of interest [47]. The foundation of neural transplantation is based on promising laboratory findings in animal models of Parkinson’s disease (PD) and Huntington’s disease (HD) [3, 7]. Clinical trials of neural transplantation have been performed in more than 350 PD, HD, and stroke patients. Although not a setback, a clinical concern in neural transplantation is that patients have demonstrated variable degrees of clinical improvement — owing in part to the low viability of the grafts [2, 4, 29, 54, 64, 76, 77]. Because graft survival is greatly altered by the host immune response, cells that can avoid immunosurveillance — particularly autologous cells such as the transplant recipient’s own adrenal cells or stem cells — may limit graft rejection [21, 28, 48, 79, 92, 97, 98]. Fetal cells persist as the most widely studied graft source for transplantation. However, due to logistical and ethical issues inherent with primary fetal cell use, establishing a nonprimary fetal graft source appears more advantageous. Nonprimary fetal cells include cultured neuronal stem and progenitor cells; cells engineered to secrete neurotransmitters or neurotrophic factors such as immortalized cell lines, fibroblasts, and astrocytes; paraneuronal cells (which naturally synthesize neuronal substances and/or have neuron-like properties);
and bridge-inducing cells that create grafts that assist in the physical reconstruction of lost axonal pathways. When intracerebrally transplanted in animal models of CNS disorders, these nonfetal cells have been generally demonstrated to partially reconstruct the neuronal circuitry and form functional synapses [25, 30, 39, 45, 63]. The use of cell lines or neural stem cells, compared with primary fetal cells, is associated with less controversy. These cells are readily available, can be maintained in culture indefinitely, and can be sorted in homogeneous population with their phenotypic features fully characterized. Accordingly, establishing human neuronal cell lines or neural stem cells as vehicles for cellular and gene therapy in the CNS disorders circumvents the many logistical and ethical concerns surrounding the use of fetal cells.
NT2N Cells: Cancer Cells Turned Neuronal Cells One of the many nonfetal cell lines is the embryonal carcinoma cell line (NT2 cells) derived from a human teratocarcinoma. These NT2 cells can be induced to differentiate into post-mitotic neuron-like cells referred to as NT2N neurons [51, 70, 71, 102]. The neural differentiation process consists of a six-week retinoic acid (RA) treatment period. Following this RA exposure, the NT2 cells — which share many characteristics of neuroepithelial precursor cells — cease expressing neuroepithelial markers and instead develop neuronal markers [83, 84]. Subsequent exposure to mitotic inhibitors produces > 99% pure populations of terminally differentiated NT2N neurons [56]. Furthermore, NT2N neurons exhibit outgrowth processes and establish functional synapses. Mature NT2N neurons are virtually indistinguishable from terminally differentiated postmitotic embryonic neurons. Of note, these neurons do not divide, and they maintain a neuronal phenotype over a long-term period [83]. NT2 cells, unlike other germ-cell tumor lines, do not give rise to progeny committed to other well-defined neural or nonneural lineages in response to RA or any other differentiating agent [83, 84]. Based on this unique property of NT2 cells, they are considered in vitro equivalents of CNS neuronal progenitor cells [57, 102]. Interestingly, in addition to their use as alternative graft sources for transplantation therapy in CNS disorders both NT2 cells and NT2N neurons can be genetically
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engineered, allowing for the expression of a gene product of interest in vitro and possibly in vivo. In addition, these applications can be exploited to reveal the cellular and molecular biology of neurons [57, 102].
Normal Host Brain Microenvironment and NT2N Cell Grafts Both in vitro and in vivo laboratory studies reveal interplay between the transplant host and the NT2N cell grafts. In normal rodent brains, transplantation of NT2N neurons showed that purified NT2N neurons survive, mature, and integrate well with the host nervous system [51, 70, 71]. From a developmental cell biology perspective, such transplantation setup allows for the direct examination of the growth and maturation of human neuronal cells in an in vivo CNS environment that otherwise could not be fully investigated in an in vitro setting. The use of NT2N neurons offers many advantages over the use of human fetal neurons. For example, NT2N neurons appear to have a better graft survival by 15%, excellent in vitro and in vivo grafted cell homogeneity, and a high degree of host reinnervation [70, 71, 102]. In support of the post-mitotic feature of NT2N cells, histological examinations have revealed no observable tumorigencity — as well as neoplasticity in NT2N cell grafts over prolonged transplantation periods of more than one year [51, 70, 71]. Immunosuppression was performed in these studies because rodents were used as transplant recipients of human-derived NT2N cells. However, we have observed that some non-immunosuppressed transplant recipients tolerated the grafts (depending on the transplant target brain area), suggesting that specific brain sites may be more conducive than others for NT2N cell transplantation. The observation that some brain areas may tolerate the cross-species grafts even without immunosuppression also suggests that the host microenvironment may play an important role in proliferation and survival of grafted cells. Despite solid evidence of NT2N cells achieving features of fully differentiated neurons following treatment with RA and mitotic inhibitors (and these cells do not revert to a neoplastic state after transplantation) concerns abound on the possibility of quiescent mitotic capacity in grafted NT2N neurons that may be stimulated by the host microenvironment. The literature on studies examining transplantation of NT2N neurons reveals no indication that these grafted cells
reacquire mitotic features, at least not when they are transplanted into the striatum. In contrast, evidence exists that proliferation and survival of parent NT2 cells are affected by the host microenvironment [51, 70, 71]. The neoplastic potential of grafted NT2 cells has been explored by grafting these cells into different regions of the brains of subacute combined immunodeficient (SCID) mice and nude mice [51, 70, 71]. It was observed that the anatomical site into which the NT2 cells were implanted significantly influenced the survival, proliferation, and differentiation of NT2 cells. Histological results revealed that the NT2 cells continued to proliferate and undergo an apoptotic-like cell death with minimal capacity to differentiate into neurons following implantation into the subarachnoid space and superficial neocortex. However, when NT2 cells were transplanted in the lateral ventricles, liver, and muscle the grafted cells rapidly progressed into bulky lethal tumors within 10 weeks after transplantation. The observed tumorigenic and neoplastic state of grafted NT2 cells was in sharp contrast to the phenotypic features displayed by NT2 cells transplanted into the caudoputamen of SCID mice. Caudoputaminal grafted NT2 cells stopped proliferating, showed no evidence of necrosis or apoptosis, and did not form tumors after more than 20 weeks posttransplantation. Furthermore, neuronal phenotypic markers demonstrate that the majority of NT2 cell grafts in the caudoputamen differentiate into post-mitotic immature neuron-like cells. These marked differential histopathological effects produced by the caudoputamen and other brain transplant target sites suggest that the choice of host microenvironment for transplantation of NT2 cells critically influences the eventual survival, proliferation, and differentiation fate of grafted cells. These observations support the notion that the host microenvironment, in this case the caudoputamen, may promote signaling molecules or other cues such as cell-cell contacts — which are capable of regulating the fate of grafted NT2 cells. Of note, RA has been shown to be present in both developing and adult rodent striatum tissues [111] and is believed to potentially influence cell fate [23, 24]. Because RA and mitotic inhibitors are primarily used as factors in the differentiation process of NT2 cells into NT2N in vitro, it is possible that the presence of RA in the striatum influences the nontumorigenic fate of NT2 cells following transplantation into this brain area. Such differentiation effects of the host microenvironment to NT2 cells warrant further examinations into the influence of specific brain transplant
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target sites on survival, proliferation, and differentiation of NT2N neurons.
Stroke Host Brain Microenvironment and NT2N Cell Grafts Secondary cell death ensues following the onset of many neurological disorders, as exemplified in stroke progression characterized by increasing size of the ischemic core over time. For successful neural transplantation therapy, identifying a conducive host microenvironment as a transplant target site would likely enhance the therapeutic outcome. In stroke animal models, the reported NT2N neuronal graft survival rate of 15% [86] is a bit higher compared to fetal cell grafts, but this rate is still low considering that ischemic stroke is not limited to a specific cell population. Moreover, stratified ischemic zones exist; namely, the predominantly necrotic core and the apoptotic penumbra. To produce therapeutic effects following an ischemic stroke, either via pharmacologic treatment or cell replacement therapy, the consensus is to target the ischemic penumbra rather than the core. Such preferential penumbral rescue is logical because apoptotic cell death accompanying the penumbra may be potentially reversed as opposed to the necrosis inherent in the core. Targeting secondary cell death mechanisms, as in the case of ischemiainduced apoptosis, suggests that the ischemic penumbra seems a more conducive host microenvironment than the ischemic core. Nonetheless, the brain damage that accompanies stroke, regardless of location in the penumbra or core, is characterized by the degeneration of many cell populations and brain structures. Accordingly, a higher number of cells with high viability and increased survival ability must be transplanted into the ischemic regions. Despite the moderately conducive nature of the ischemic penumbra, the extensive brain area encompassing this region may require multiple brain targets to repair the damaged neuroanatomical circuitry. Multiple intracerebral transplantations may not be feasible, however, because of the trauma associated with such an invasive surgical procedure. In addition, different types of donor cells may need to be transplanted considering that many cell populations are destroyed in stroke. NT2N neurons have been shown to differentiate into dopaminergic and GABAergic neuron-like cells [35, 36, 66, 114]. Alternatively, RA-naive NT2 cells may possess some
multipotent properties, such as those attributed to neural stem cells, and these features may be potentially advantageous for generating different cell populations. Indeed, it has been demonstrated that transplanted human neural stem cells can mature into the phenotype of cells that are undergoing cell death in brains of animals that were introduced to neuronal injury [26]. Of note, NT2 cells can differentiate into neurons when transplanted into the caudoputamen. Accordingly, when contemplating the transplantation regimen NT2N and NT2 cell grafts may both be beneficial for focal caudoputaminal stroke. We speculate that the pluripotent features of NT2 cells and the highly differentiated neuronal-like characteristics of NT2N cells can be designed to target specific stroke types in order to enhance their therapeutic effects. The proliferative capacity of the multipotent NT2 cells may be exploited by transplanting these cells in a patient with a large striatal stroke, whereas the fully differentiated NT2N cells may be more appropriate when transplanted in a patient with a highly localized striatal stroke. These speculations need to be tested in the laboratory.
Preclinical Studies of NT2N Cell Grafts in Stroke The availability of animal models of stroke allows preclinical validation of safety and efficacy of experimental therapies. The rodent model of middle cerebral artery (MCA) occlusion replicates many pathophysiological changes seen in clinical cerebral ischemia, allowing for investigations of treatment strategies for stroke. The potential benefits of neural transplantation of NT2N neurons to correct the neurobehavioral deficits associated with cerebral ischemia have been examined in this MCA occlusion model. We have demonstrated that NT2N neuronal grafts ameliorate ischemia-induced behavioral dysfunctions as early as 1 month posttransplantation [8–10]. Compared with the transplantation of fetal striatal cells, which were previously shown to reverse motor abnormalities in stroke rats, NT2N neuronal grafts induced a significantly greater robust recovery. Our efficacy studies provide the basis for proceeding with safety as well as mechanistic investigations on NT2N cell grafts. A basic study to demonstrate that the observed therapeutic benefits were derived from the grafted NT2N cells is to show that cell viability or cell graft survival
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influences the behavioral recovery of the transplanted stroke animals. We observed that pretransplantation viability and posttransplantation survival of NT2N neurons are highly correlated with the functional recovery of transplanted stroke animals [9]. These observations suggest that the positive behavioral effects seen in transplanted stroke animals can be attributed to viable and functional NT2N neuronal grafts. During the pretransplantation period, NT2N neuron viability counts revealed a variable range of 52 to 95%. Within-subject comparisons of pretransplantation cell viability and subsequent behavioral changes in transplanted stroke animals revealed that a high cell viability just prior to transplantation surgery correlated highly with a robust and sustained functional improvement in transplant recipients. In addition, histological analysis of grafted brains revealed a positive correlation between the number of surviving NT2N neurons and the degree of functional recovery. Such correlations between pretransplantation viability or posttransplantation survival of grafted cells and behavioral outcome have also been reported in fetal tissue transplantation. Another simple study to demonstrate graft-derived behavioral effects is to use a dose-response paradigm, in that manipulating the cell dosage should influence the behavioral recovery of the transplanted stroke animals. We observed correlations between motor recovery and neuronal regeneration in stroke animals, and dosedependent functional effects of NT2N neuronal grafts have also been noted [86]. Stroke animals that received 40, 80, or 160 × 103 NT2N neurons dose-dependently exhibited performance improvements in both the passive avoidance and elevated body swing tests. Moreover, dose-dependent survival of NT2N neuronal grafts was observed in that grafts of 80 or 160 × 103 NT2N neurons demonstrated a 12 to 15% survival of NT2N neurons in the graft whereas grafts of 40 × 103 NT2N neurons demonstrated only a 5% survival. It is possible that NT2N neuronal grafts were affected by progressive stroke, suggesting again the influence of the host microenvironment. Correlational analyses revealed that ischemic animals that received 80 or 160 × 103 NT2N neurons produced a significantly better amelioration of behavioral dysfunctions as compared to those that received lower dosages of NT2N neurons. In concert with the earlier speculation that varying extent of stroke brain damage would require manipulation of the number of donor cells, this study demonstrated that transplantation of more viable NT2N neurons is required to rescue
larger stroke-induced brain damage [86]. Accordingly, NT2N cell viability at pre- and posttransplantation — as well as the extent of stroke brain areas — should be considered when determining the optimal dosage of NT2N neurons for transplantation. Now that we have demonstrated graft-derived therapeutic benefits, the next step is to address whether there is enough NT2N cell repository that can accommodate a clinical trial. This concern of NT2N neuron procurement feasibility becomes more critical when considering diseases, such as stroke, with a very narrow therapeutic window. In this disease scenario, there is an immediate need for availability of an ample supply of NT2N cells. Because NT2N cells are generated from the highly proliferative NT2 cells, there will be sufficient donor cells available within the stroke therapeutic window. Moreover, our study shows that NT2N cells remain highly viable even after longterm cryopreservation, allowing storage of the cells in stroke clinical centers. The open-label clinical trial of NT2N cells revealed that transplanted patients who had a stroke at least six months prior to NT2N neuronal transplantation displayed some trends of clinical improvement. Although such transplantation therapy could potentially reverse chronic stroke, better functional outcomes may be achieved if treatment is initiated acutely (< 3 hours) post-stroke when brain damage would presumably still be limited. A strategy incorporating immediate availability and transplantation of NT2N neurons appears to be an appropriate regimen for stroke therapy. Although an immediate transplantation procedure within the 3-hour stroke therapeutic window is indicated, one must also consider the severe inflammatory glial response that accompanies the early stages of the disease. Controversy exists as to whether such inflammatory response works for or against graft survival [6, 15]. For example, during the early post-stroke period, cytokines and inflammatory signals are highly elevated [14, 38] — which that could be harmful to grafted cells. Thus, transplantation in acute stroke could be detrimental to grafted cells. However, there is also evidence that glial cells or macrophages following stroke may produce chemoattractants, and these cues may guide grafted cells to the site of injury [16, 58, 65]. These data suggest that an early transplantation at a stage of high glial response may aid in cell graft trafficking toward appropriate stroke target sites. Accordingly, the glial response/inflammation may produce both inhibitory and
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facilitatory effects during the early periods following stroke, particularly on the migration of grafted cells — and these effects warrant further investigations. In previous studies using the chronic stroke model, however, NT2N cell grafts have been shown to migrate away from the original transplant site [8–10]. Stimulating NT2N cells to migrate in acute and chronic stroke may pose a challenge considering the large extent of brain damage inherent in the disease. Additional laboratory studies are warranted to clearly examine how to modulate the glial response/inflammation following stroke and transplantation to enhance NT2N cell graft survival and migration, as well as functional benefits. As mentioned previously, the timing of transplantation after stroke is critical. Because handling freshly cultured NT2N neurons would not be altogether feasible in the clinic, cryopreserved NT2N neurons are recommended. Compared to freshly harvested fetal cells, these cultured NT2N cells allow examination of the cells for possible infectious diseases. Such determinations can be performed well ahead of the scheduled transplant surgery, and therefore a more efficient transplantation protocol can be achieved with the use of these cells as compared to using fetal cells. In the laboratory, cryopreservation of NT2N neurons did not produce any significant deleterious effects on the viability of the cells prior to or after transplantation in stroke animals [9]. This sustained viability of NT2N neurons following cryopreservation fares much better than fetal cells, which after cryopreservation display a significant decline in viability (rendering them nontransplantable). Cryopreservation of NT2N neurons thus facilitates immediate transplantation of the cells in acute stroke. Transplantation of these clonal cells’ NT2N neurons also allows a logistical advantage of conducting neural transplantation in a wider therapeutic window following stroke. Because the success of treating cerebral ischemia depends highly on the timing of intervention, the ready availability of clone cells as a graft source would significantly reduce the time between the ischemic event and the therapeutic intervention. Nonetheless, the robust recovery of animals with a stable stroke following transplantation of NT2N neurons suggests the possibility of treating stroke patients even with a long delay after a stroke episode. Whether enhanced functional outcomes can be achieved with an acute stroke transplantation regimen versus a delay in transplantation after stroke (i.e., allowing disease-triggered host inflammation to subside) will require further laboratory investigations.
Immunosuppression and NT2N Cell Grafts Despite its immunoprivileged property, the brain still mounts an immune response leading to neuronal graft rejection, especially in the case of cross-species transplantation. Such graft rejection is accompanied by deterioration of graft-mediated therapeutic benefits. Indeed, the sustained motor and cognitive improvements noted in NT2N neuronal transplanted animals were only observed with systemic administration of cyclosporin A (CsA) immunosuppression [8–10]. In contrast, the behavioral recovery in the non-immunosuppressed animals transplanted with NT2N neurons began to diminish by about 2 months posttransplantation. Moreover, histological analysis revealed surviving NT2N cells in the brains of immunosuppressed transplanted animals but not in non-immunosuppressed transplanted animals. The near absence of visible grafts in nonimmunosuppressed animals transplanted with NT2N neurons suggests host immunological rejection of the grafts, as observed previously [103]. Nonetheless, the magnitude of the behavioral recovery produced by NT2N cell grafts in rats that did not receive CsA was greater than that seen in animals with ischemia-induced brain injury followed by injections of rat fetal cerebellar cells or medium alone, suggesting that NT2N cell grafts promote behavioral effects at early time periods posttransplantation even in the absence of immunosuppression. However, these non-immunosuppressed animals, despite displaying significant improvements as compared to control animals at 6 months posttransplantation, still remained impaired as compared to immunosuppressed animals that received NT2N cells — suggesting that CsA immunosuppression enhanced the survival of grafted NT2N cells as previously reported [103]. Moreover, histological examinations revealed many surviving NT2N cells in immunosuppressed transplanted animals exhibited a robust functional recovery for more than 6 months posttransplantation. Based on these results, the need for chronic immunosuppressive therapy as an adjunct to the transplantation of human NT2N cells in rats appears necessary in order to obtain optimal and sustained functional improvement as well as prolonged graft survival. Nonetheless, we found no evidence from these studies or any other previous reports indicating that transplanted NT2N cells (with or without immunosuppression) produce any deleterious effects on the host brain [51, 70, 71]. Chronic immunosuppression accompanying NT2N transplantation in the clinic may not be necessary
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because NT2N neurons are human-derived cells. Some clinical trials of human fetal cell transplantation for PD have in fact found that the absence of immunosuppression does not deleteriously affect the survival of fetal cell grafts and their ability to produce clinical improvement [27, 37]. In addition, preliminary data suggest that NT2N neurons may have immunosuppressive properties [89]. Recent studies have suggested that stem cells only minimally elicit an immune response and may even secrete their own immunosuppressant factors following intracerebral transplantation [26]. Similar immunosuppressant factors may also be secreted by NT2N cells, which may exert localized immunosuppression within the transplant site and allow them to circumvent host immunosurveillance. Thus, long-term systemic immunosuppression may not be necessary in humans. However, recent studies have indicated that immunosuppressants and their analogs exert neuroprotective effects in CNS disorders, including stroke [6, 7]. Perhaps adjunct limited immunosuppression with NT2N cell transplantation should be considered for enhanced graft survival and functional effects.
Neurotrophic Factors Influencing Therapeutic Benefits of NT2N Cell Grafts Because of robust behavioral effects immediately following NT2N transplantation in stroke animals, we postulated that neurotrophic factors secreted by the transplanted NT2N cells might have mediated functional recovery, at least for the early posttransplantation period. This finding supports neuronal rescue via neurotrophic factor therapy. In many preclinical and clinical studies of neural transplantation, the use of neurotrophic factors has been shown to significantly enhance the survival rate of grafted cells [3, 19, 41]. Direct infusion of neurotrophic factors alone or their use as a transplant facilitator by either pretreating donor cells or coadministration during and postneural transplantation therapy has been proven efficacious in CNS animal models. Thus, administration of neurotrophic factors may serve as another adjunct to neural transplantation. One of the most potent neurotrophic factors is glialcell-line-derived neurotrophic factor (GDNF; see latter sections of this chapter). Encouraging laboratory results have been reported in neural transplantation of GDNFsecreting fetal kidney cells for stroke [17, 99]. Interestingly, an in vitro study has demonstrated that NT2N cells respond positively to putative neurotrophic factors
secreted by an immortalized human fetal astrocyte cell line [101]. Although our initial study ascribed the therapeutic benefits of NT2N cells to neurotrophic factors secreted by these cells [10], we did not demonstrate any direct evidence that neuroprotection was indeed a function of NT2N neuronal grafts. The first suggestion that neuroprotection by NT2N neurons could be mediated by a neurotrophic factor mechanism was reported recently in a study showing that NT2N neurons are positive for GDNF mRNA [59]. Because GDNF has been found to be neuroprotective for stroke animals, the indication that NT2N neurons can exert GDNF expression offers a mechanistic explanation for the observed neuroprotection by NT2N neuronal grafts in stroke. Indeed, although a similar pattern of behavioral recovery was observed in animals that received NT2N neurons and those that received fetal striatal transplants the NT2N-transplanted animals showed a more robust recovery at 1 month posttransplantation. This effect of NT2N neurons also was evident in transplanted non-immunosuppressed animals. Because no evidence has been reported of neural transplants replacing lost host brain tissue at this early posttransplantation period, the observed functional effects may be due to the release of trophic factors from the grafted NT2N neurons. Another indication of trophic factors mediating NT2N’s action is that the effective dose of transplanted NT2N neurons needed to produce functional recovery was 10 times less than transplanted striatal cells. Based on these observations, we postulated that at the early posttransplantation period trophic factors secreted by NT2N neurons enable stroke animals to display functional improvement, whereas at later posttransplantation periods connectivity with or repair of the stroke brain by NT2N neurons may be needed for stable functional recovery. Similarly, at later posttransplantation periods NT2N cells might have replaced the degenerated host brain cells. It has been shown that after transplantation into nude mice NT2N cells can integrate and change phenotype into neurons similar to the target neurons, such as striatal neurons [51, 70, 71, 83]. NT2N neurons can potentially become striatallike neurons and may also be capable of secreting neurochemicals or even performing functions of lost striatal cells of the host brain. Indeed, NT2N cells can be stimulated through application of neurotrophic factors such as acidic fibroblast growth factor and activating factors such as catecholamines or forskolin to express the rate-limiting enzyme in catecholamine
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biosynthesis — tyrosine hydroxylase (TH) [43, 90]. Of note, in vitro studies have shown that the percentage of TH-positive neuron-like cells in the NT2N cells treated with RA co-cultured with striatal extracts exceeded by greater than tenfold the percentage of TH-positive cells induced in sister cultures exposed to retinoic acid alone [51, 70, 71]. Of note, many of the behavioral dysfunctions seen in a stroke model are dopamine-mediated behaviors [8–10]. Thus, the possibility that NT2N cell grafts can be induced by the host microenvironment — particularly the remaining host striatal neurons or the whole host striatum itself — to secrete dopamine would greatly contribute to the amelioration of ischemiainduced behavioral deficits. It has also been shown that the host microenvironment of the adult mouse striatum appears to have the potential ability to induce grafted NT2 cells to differentiate progressively into fully mature adult CNS neurons [51, 70, 71]. The striatum may exert similar neuronal differentiation effects on grafted NT2N neurons. Such further neuronal differentiation of NT2N following transplantation is important, especially if it is necessary to target specific disease types that entail degeneration of different cell populations. For example, subsets of NT2N cells have been shown to express neuronal markers for dopaminergic and GABAergic neurons [42, 115] that would be an appropriate cell graft source for PD and HD, respectively. Because multiple cell populations are affected by stroke, the ability of NT2N cells to differentiate into many cell types will be advantageous. An alternative strategy that could potentially extend the efficacy of NT2N cell transplantation to a variety of stroke types and other neurological disorders is to manipulate the microenvironment. For example, because the striatum appears as the most conducive site for NT2N cell grafts making neurotrophic factors found in the striatum available to other brain areas — such as the cortex or hippocampus, which are also damaged in stroke — may enhance NT2N cell graft survival and functional effects in these brain regions. Neurotrophic factors are only one of many possible mediators of therapeutic benefits of NT2N cell grafts, and it is likely that other mechanisms are involved.
Clinical Trials of NT2N Cells in Stroke Patients The preceding preclinical studies demonstrating successful implantation of human-derived NT2N neurons into rat brains were used as bases for pursuing limited
clinical trials of cell transplantation in stroke patients. The target stroke patients chosen were at a chronic stage because laboratory data indicated the possibility of reversing motor symptoms associated with a stable stroke. The Food and Drug Administration approved phase I clinical trials of transplantation of NT2N neurons to evaluate this therapy in the treatment of patients with stable stroke. NT2N cells were transplanted into patients with basal ganglia stroke and fixed motor deficits, including 12 patients aged 44 to 75 years with an infarct of 6 months to 6 years who were stable for at least 2 months [53]. Serial evaluations at 12 to 18 months showed no adverse cell-related serologic or imaging-defined effects. Because this was an open-label trial, we caution to the interpretations of the results. At best, the data suggest that transplantation of NT2N cells is feasible in patients with motor infarction. Long-term monitoring of NT2N cell grafts in stroke patients reveals safety of the cells. Although the intracranial transplantation of certain stem cell lines has been shown to induce tumor formation when cells were implanted into the cortex [102], no evidence was found of neoplastic formation from NT2N transplants into the striatum [51]. The presence of RA in the striatum may have aided in suppressing tumor formation following NT2 cell grafts and may have facilitated further differentiation of NT2N cell grafts into neuronal lineages. Consistent with this evidence, the previously cited report demonstrated no serious adverse events in transplanted stroke patients at one year posttransplantation. Thus, it appears that grafted NT2N cells do not exhibit neoplasticity, thereby preventing any tumor formation. However, because the intrastriatal transplantation of NT2N cells has been the preclinical and clinical approach that has demonstrated consistent nontumorigenic outcome following NT2N cell grafts, extending the transplant target sites to other brain areas outside the striatum must be approached with caution. At this time, future transplantation trials should be limited to targeting the striatum, thus focusing only on striatal stroke patients. Transplanting NT2N cells into other brain areas should incorporate a strategy to ensure that the graft material is nonresponsive to tumor formation cues from the host microenvironment or to suppress the host microenvironment from releasing such tumorigenic signals. A noninvasive detection of graft viability remains a challenge in the field of neural transplantation. A subsequent clinical report evaluated the function of NT2N transplanted cells using PET scanning [67].
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Uptake of fluorodeoxyglucose (FDG) was measured at baseline and at 6 and 12 months after transplantation of NT2N neurons. At 6 months posttransplantation, 7 of 11 patients showed > 10% uptake increase in FDG in the area of cell implantation, and this increase correlated with clinical improvement measured by stroke scale values. In a recent study that reported the first postmortem brain in an NT2N transplanted patient at 2 years posttransplantation [74], histological examination revealed neurofilament immunoreactive neurons resembling those seen in NT2N neurons in vitro. The observed NT2N cell graft survival in this patient suggests that these transplanted cells mediated functional outcome. The PET and histological data from transplanted stroke patients allow some comparisons with long-term graft survival of fetal ventral mesencephalic neurons in PD patients who also died from causes unrelated to the transplants. In both stroke and PD patients, robust graft survival was seen using PET scans and was accompanied by expression of neuronal phenotypes in grafted cells postmortem. No overt side effects of the transplants in these patients were observed, indicating that the grafts did not exacerbate disease progression. These parallel clinical outcomes seen in stroke and PD patients support the use of NT2N cells as an efficacious alternative to fetal cells. Moreover, the use of PET and possibly in vivo functional magnetic resonance imaging for noninvasive monitoring of NT2N cell grafts should allow better visualization and tracking of transplanted cells. This clinical trial also allowed us to partially examine the therapeutic cell dosage required to detect clinically relevant improvements. In this clinical study, there was no observed difference in outcome between transplantation of 2 million or 6 million cells. Interestingly, increased uptake of FDG persisted in only 3 patients at the 12-month posttransplant evaluation [67]. However, clinical improvement was maintained in 6 of the 7 patients who showed an initial increase in uptake of FDG. This finding suggests that improvement in the clinical exam may be mediated by a factor that only requires functional effects of transplanted cells during a critical time period, or even a limited number of NT2N cells may promote some degree of functional recovery. This notion of minimally required viable cell grafts for functional effects is also true for fetal cell transplantation — at least in PD, wherein it has been suggested that as few as 300 dopaminergic neurons could exert behavioral recovery [11]. However, because recurrent stroke episodes may likely ensue following
the initial stroke transplanting more viable NT2N cells may be required for long-term improvement. The notion is that if grafted cells are affected by ongoing neurodegeneration transplantation higher number of viable cells may be needed for stable therapeutic outcome. However, recent reports on PD clinical trials have suggested that too many grafted cells may be deleterious. Accordingly, additional studies are needed to determine an optimal cell dosage for NT2N cells that produce a therapeutic outcome but are not accompanied by side effects. Reservations have been raised about this clinical trial of transplantation of NT2N neurons [116]. The preceding clinical trial is an open-label study and was thus not designed to prove efficacy. At best, the results from this phase I study revealed that NT2N neuronal transplantation is feasible and safe. The follow-up studies suggest no malignant tumor formation over moderate posttransplantation periods of 2 years. Continuous monitoring of the transplanted patients at longer periods of time is being performed and should reveal further safety issues associated with the transplantation therapy. To optimize the protocol to achieve effective and consistent improved clinical outcomes, carefully designed laboratory studies and limited clinical trials need to be considered. Subsequent clinical trials should determine the optimal number of cells needed to achieve significant clinical improvements and should develop imaging techniques that would allow characterization of grafted cells to assess viability, migration, differentiation, and graft-host integration over the NT2N graft maturation period.
Recent Advances on the Use of NT2N Cells for Transplantation Therapy Recent laboratory studies have made further progress in demonstrating the potential for therapeutic efficacy of NT2N cell transplants and for elucidating the mechanisms underlying the effects of these grafts. One recent study has demonstrated that defined populations of genetically modified human NT2N neurons are a practical and effective platform for stable ex vivo gene delivery into the CNS. This study successfully displayed stable, efficient, and nontoxic gene transfer into undifferentiated NT2 cells using a pseudotyped lentiviral vector. NT2 cells were differentiated into NT2N neurons via treatment with RA and then transplanted into the
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striatum of adult nude mice. Transduced NT2N neurons survived and continued to express the reporter gene for long-term time points in vivo. Transplantation of NT2N neurons genetically modified to express nerve growth factor also significantly attenuated cognitive dysfunction following traumatic brain injury in mice [106]. Transplantation of NT2N cells has also been extended to PD animal research. Lithium (Li+) treatment of NT2N neurons was found to increase tyrosine hydroxylase (TH) expression when cells are transplanted into the striatum of hemiparkinsonian rats. Histological analysis in this study demonstrated that there was a significantly better survival of cells in the group treated briefly with lithium, thus providing an option for enhancing NT2N graft survival in future transplantation studies [107]. Another PD-related study has found that NT2N neurons induced by retinoic acid express the Nurr1 receptor, which has been shown to be essential for the development, differentiation, and survival of midbrain dopamine neurons. The study also confirmed the coexpression of Nurr1 and tyrosine hydroxylase in NT2N neurons. These findings suggest that Nurr1 may be important during the development of NT2N neurons and could also be involved in their differentiation into the dopaminergic phenotype [68]. Transplantation of NT2N cells has also been studied recently in an animal model of spinal cord injury. The histological data from one such study revealed that graft survival in rats that received transplants was displayed in 66.7% of the surviving grafted animals. Fiber outgrowth was also observed in both rostral and caudal directions bridging the lesion. The results of this study suggest that NT2N grafts have the potential to structurally reconnect the proximal and distal spinal cord across the region of injury, thus presenting the future possibility to extending the clinical use of these transplants to spinal cord injury as well [18]. Moreover, transplantation of NT2N neurons could potentially be an effective means of reestablishing electrical connectivity in the injured spinal cord. In another spinal cord study, rats were given a complete spinal cord contusion injury — producing a complete loss of motor evoked potentials — and then selected rats underwent transplantation with NT2N cells within the contusion site either immediately after injury or at a delayed point 2 weeks following injury. Rats receiving delayed transplants displayed a significant functional recovery as demonstrated by the return of motor evoked potentials as well as a modest improvement of motor function, again
suggesting the potential for NT2N cell transplantation to restore function in spinal cord injury [87]. Although the previously cited studies provide support to the therapeutic efficacy of NT2N cells, a recent study shows that embryonic cortical neurons and NT2N have different network properties [33]. Neurons derived from the human NT2 cell line were found to form networks with a clustered neuritic architecture in vitro, whereas primary dissociated embryonic rat cortical neurons were found to display a more homogeneous cell assembly. In addition, NT2N neurons showed a mostly uncorrelated firing pattern in contrast to the primary dissociated embryonic rat cortical neurons that displayed highly synchronized bursting. These findings indicate the need for more in-depth characterization of NT2N cell grafts.
NT2N Cells and Gene Therapy NT2 cells are transfectable and capable of differentiating into postmitotic neuron-like cells (NT2N cells) following treatment with retinoic acid, thereby allowing this human neuronal cell line to serve as a “platform” for gene therapy applications for treating CNS disorders [102]. The phenotypic characteristics of both NT2 and NT2N cells suggest the likelihood that they are an excellent platform for ex vivo gene therapy in the CNS. However, stable gene expression in NT2N cells has not been optimal. To circumvent the problem undifferentiated NT2 cells were initially targeted with a pseudotyped lentiviral vector encoding the human elongation factor 1-alpha promoter and the reporter gene eGFP [106]. Expression of eGFP was maintained even after the cells were differentiated into NT2N neurons and following transplantation of the cells into the striatum of adult nude mice. Furthermore, using the same approach of lentiviral transfection in NT2 cells permits the transplantation of the genetically modified NGF-expressing NT2N cells to attenuate the cognitive decline in traumatically brain injured mice [62]. Another study demonstrated that a liposome-mediated gene transfer of beta-galactosidase (beta-GAL) reporter gene obtained a high transfection rate in NT2 cells [52]. Other reports have shown the efficacy of NT2 cells, as well as the NT2N cells, for other viral vector systems such as the human immunodeficiency virus type 1 (HIV-1) infection strategy [73], cationic liposomes complexed with plasmid DNA-mediated transfection vectors with the constitutive cytomegalovirus promoter or the
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hypoxia-inducible VEGF promoter [13], Tet-regulated herpes simplex virus vectors [91], recombinant SV40 vectors [20], and adenovirus-mediated gene delivery [40]. These recent gene therapy-related studies provide evidence that the undifferentiated NT2 cells and the postmitotic NT2N cells are an efficacious vehicle for delivery of exogenous proteins into the brain.
Glial-cell-line-derived Neurotrophic Factor: A Dopaminergic Cell-survival-enhancing Agent and Potent Stroke Neuroprotective Drug Exogenous treatment with glial-cell-line-derived neurotrophic factor (GDNF) has provided symptomatic relief in animal models of neurological disorders. Two neurological disorders that have been the target of GDNF therapy include stroke and PD. Both stroke and PD lead to long-term and debilitating abnormalities in victims. Initial evidence of GDNF neuroprotective and regenerative effects was described in midbrain DA neurons, but subsequently extended to other CNS regions. Because of overlapping etiologies between stroke and PD, therapeutic outcomes with GDNF in either one or both diseases have contributed to the increased interest in our understanding of neuronal cell death — as well as to the development of neurotrophic-factor-based treatment strategies. GDNF is a member of the transforming growthfactor-beta (TGF-B) superfamily. The GDNF gene is found in all major regions of the brain [75, 3a]. The GDNF protein has garnered special attention for its neuroprotective capacity associated with DA neurons and spinal cord motor neurons. The therapeutic effect that GDNF has on ischemic brain injury is correlated with antiapoptotic signaling. In rat models, GDNF up-regulates antiapoptotic factors such as Bcl-2 and Bcl-X — thereby reducing caspase activity and the occurrence of apoptosis [49]. The GDNF family ligands (GFL) are comprised of GDNF, neurturin, artemin, and persephin. Each GFL has its own glycosylphosphatidlyinositol (GPI)-linked receptor. The GFL protein attaches to a GPI-linked GFR alpha factor, and this binding event activates GDNF. The formation of GDNF ligand receptor complex initiates downstream signaling through the proto-oncogene, c-Ret, and tyrosine kinase. GDNF binding to GFR alpha also activates a mitogen-activated protein kinase
(MAPK), which results in an enduring change in gene expression [75, 3a]. Downstream signaling also occurs through activation of cytoplasmic Src family tyrosine kinase when c-Ret is not available [85]. This signaling in the absence of c-Ret occurs through contact with the neural cell adhesion molecule (NCAM), a possible alternative signaling receptor for GDNF. Following ischemia and reperfusion, c-Ret mRNA is expressed over a slower and more gradual timescale, whereas GFR-alpha mRNA is expressed immediately after ischemia and sustains a high level through reperfusion. Considering the difference in each receptor’s uptake, it is feasible to assume that GFR and c-Ret are up-regulated in different areas of the brain following ischemic insult. The up-regulation of both receptors during the times following immediate ischemic insult and reperfusion allows for the quick response of the GDNF ligand to the area of damage. Excitatory amino acids and their receptors are released in an increased amount during ischemic neuronal insult. GDNF mRNA in the rat brain has been shown to be up-regulated to combat excitotoxicity caused by glutamate, kainate, and ischemia [49]. Cortical neurons in culture show that GDNF limits excitotoxicity via activation of the MAPK pathway. GDNF, therefore, appears to affect neurons in the area of ischemic injury by attenuation of excitotoxicity. Nitric oxide (NO) levels are also increased by ischemic insult. NO release following MCA occlusion in rat brain is virtually blocked by the presence of GDNF [105]. GDNF limits the negative effects of NO release following ischemic insult [60]. These observations suggest that GDNF is primed to respond to ischemic brain insult. Following an episode of MCA occlusion, the receptor GFR alpha is noticeably increased in the areas of ischemic insult [88]. Before reperfusion, the area of ischemic injury is presumably up-regulated by GFR alpha. After reperfusion in the same study, a second increment in up-regulation of GFR alpha is observed—which likely coincides with reperfusionassociated injury. GDNF can be found in high concentrations within the fetal kidney (FK) [100], but not in adult kidney and brain tissue [96, 100]. GFR alpha is similarly found at high concentrations within the developing metanephric kidney [110], and both GDNF and GFR alpha are found to play an important role in the maturation of the renal cortex when observed in animal models [72, 82]. Animals with a GFR alpha-1 deficiency
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displayed complete bilateral renal agenesis and ureteral deficits [12]. The elevated levels of GDNF found in FK provide tissue for transplantation to areas of ischemic injury. Cerebral damage caused by MCA occlusion in rats [17] and mice [32, 4a] has shown that transplantation of FK tissue to the cortical areas surrounding the area of injury reduced infarction volume and led to significant behavioral improvement. The beneficial effect of FK tissue was not observed when adult kidney tissue was transplanted to the area of cerebral damage, further indicating that FK tissue (but not adult kidney tissue) is a rich source of GDNF.
GDNF Protein Delivery into the Brain Gene therapy techniques that incorporate GDNF are becoming increasingly assessed for therapeutic effectiveness. Successful GDNF delivery to a targeted area of injury within the brain, however, is made difficult by the blood-brain barrier (BBB). The blood-brain barrier (BBB) consists of tight junctions and forbids the transport of large proteins (less than 600 to 1,000 Da) into the brain parenchyma. Although transport of GDNF into the parenchyma may be possible during times of cerebral ischemia, when the tight junctions of the BBB are compromised the tight junctions are compromised several hours after the stroke onset [49] — voiding any therapeutic effects. Thus, successful delivery of GDNF to ischemic brain injury requires a transport-facilitated strategy across the BBB. The delivery route of GDNF into the brain creates a safety issue due to the risk of GDNF interfering with other receptors and other neurons. In vivo studies have demonstrated that pretreatment with GDNF protein can be administered via intracerebroventricularly or intraparenchymally [105] to the cortical surface or hippocampus [69] and such direct brain delivery results in reduced cerebral infarction. An intracerebral administration of GDNF to the neonatal rats reduced the damage caused by hypoxic and ischemic brain damage [44]. In animal studies, intracerebral introduction of GDNF protein has protected injured DA neurons, promoted regenerative sprouting from axotomized DA axons, and stimulated DA turnover and release in rescued neurons [32, 4a]. Intracerebral introduction of GDNF protein, however, poses the risk of GDNF interaction with other receptors as well as presenting a surgical risk with the chance for brain hemorrhage during or following the procedure.
To avoid the risks associated with intracerebral GDNF delivery, recent studies have also explored peripheral delivery of GDNF. For example, GDNF can effectively bypass the BBB in the form of TAT-GDNF. The application of TAT-GDNF in adult stroke animals resulted in reduced caspase-3 and apoptotic activity, reduced DNA fragmentation, and increased neuronal survival [49]. Although GDNF delivery to areas of cerebral injury has increased DA levels through DA neurons, as well as benefiting motoneurons (equally important in the case of PD), GDNF protein application in clinical trials has produced mixed results. A phase II study with PD patients showed that continuous administration of GDNF directly into the putamen did not alter brain functioning. In addition, monthly injections of GDNF intracerebroventricularly to PD patients resulted in considerable side effects [22, 3b]. There were also no clear detections of GDNF even reaching the targeted areas within the brain. Taken together, these studies emphasize the need for a more reliable delivery of GDNF protein to the brain. In vivo and in vitro studies verify the regenerative effect GDNF has on DA neurons and motoneurons. A safe and efficient method of delivery must be identified to maximize these effects of GDNF for the treatment of neurodegenerative disorders.
Viral Vectors for GDNF Gene Therapy In contemplating about a technique for GDNF delivery into the brain, the approach must include a method that will allow for a stable release of GDNF over time. The need for prolonged GDNF bioavailability in the brain is critical, especially in the case of neurodegenerative disorders such as PD — when a specific cell population progressively deteriorates. Notwithstanding, such GDNF secretion in the ischemic brain appears to be equally important for optimal benefits. Although stroke has been traditionally considered an acute neurological disorder, accumulating evidence suggests an ongoing cell death cascades even at long delay periods following the initial ischemic insult. These observations categorize stroke, at the very least its secondary pathological symptoms, as a progressive disorder that would likely require chronic therapy. Rather than direct insertion of GDNF protein, a longer-lasting GDNF gene can be administered (and targeted at the area of ischemic insult) in order to create continuous expression and release of GDNF. Gene therapy provides a technique that allows for the longevity of GDNF gene expression.
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Studies have utilized viral vectors to supply GDNF. Viral vectors with GDNF gene are considered for ability to cross the BBB, and also because of the longevity of GDNF expression afforded by the viral vector. Viral vectors coupled with gene therapy present a unique option for the delivery of GDNF to an area of recent ischemic injury. Commonly used viral vectors include adenoassociated viral (AAV), herpes simplex viral (HSV), adenoviral (Ad), and Sendai virus vector. Viral vector usage has the benefit of GDNF expression for longer amounts of time compared to other methods of GDNF protein delivery. Sustained GDNF expression has successfully been observed in vivo via application of viral vector/GDNF to the nigrostriatal region of the brain [32, 4a]. Studies with viral gene transfer of GDNF show a decrease in ischemic tissue loss, a reduction in caspase activity, decreased terminal deoxynucleotidyl transferasemediated dUTP biotin in situ, nick end labeling (TUNEL), a reduction in brain infarction, and a restoration of motor behaviors in animals with cerebral ischemia [104, 109, 113]. Furthermore, the use of the Sendai virus [95], AAV [104], and adenoviral [109, 113] vectors have resulted in the successful penetration of GDNF gene to ischemic brain areas. Treatment with HSV amplicon/GDNF for one month following an incident of stroke resulted in a decrease in animal tissue loss [36]. Importantly, viral vector transmission confers the protective benefits of GDNF protein to areas with cerebral ischemia. In the same study with HSV amplicon/GDNF, pretreatment injections of the vector into the cerebral cortex lessened the damage to motor movement. In the case of stroke, for viral vector/GDNF to be beneficial the viral vector/GDNF must be initiated two to three days before MCA occlusion [36, 94, 95, 101]. Vector administration one hour to three days following the reperfusion of the MCA did not result in the same level of protection as the more immediate treatment. GDNF gene therapy has been observed and successful in other neuronal tissues. Application of recombinant AAV (rAAV) with GDNF gene to the retina [108] resulted in a decrease of ischemic injury. In gerbils with transient cochlear ischemia, a pretreatment of Ad/GDNF decreased progressive cell and hearing losses [35]. Currently, there are no studies that have examined viral vectors that are capable of being “switched on” and “shut off” to control GDNF secretion. The creation of a viral vector/GDNF that allows a switch to turn on and off GDNF secretion would be favorable
to manipulate expression of GDNF within the brain. The threat of perpetual GDNF expression bears special importance, as there is a critical time constraint on viral vector/GDNF administration following ischemic injury. Unregulated expression of GDNF during a time when its therapeutic effects are minimal has the potential to react with other receptors and alter the action of other neurons. In addition, a decision must be made concerning how much GDNF gene to incorporate into the viral vector, to provide benefit, and to avoid adversely affecting surrounding components.
Limitations of the Viral Vector Strategy for GDNF Therapy Gene therapy has been used as a potential cure for disease, from cancer to hearing loss. The main protocol is to insert the therapeutic gene of interest into a virus and administer the recombinant virus to the subject. GDNF has also been used in this manner, as mentioned previously in this chapter. However, there are drawbacks to utilizing this method as a potential vehicle for transport of GDNF into the brain. The problems with the process itself are all related to the immune system. The shortlived nature of the virus, which is due to its inability to reproduce at an efficient level, reduces the efficacy of its gene product. There is also the host immune system, which actively destroys any foreign material present in the body. In relation to host defenses, the threat of toxicity and associated inflammatory response generated pose a vital concern to the health of the subject. Aside from these general caveats with gene therapy, there are also concerns over the GDNF protein. As noted previously, GDNF can provide neuroprotection if given before an ischemic episode and up until an hour after reperfusion. After 1 hour [113] to 3 days [36] following reperfusion, the activity of GDNF delivered via viral vectors was decreased. This gives credence to the suggestion that GDNF activity may be inhibited during the later stages of reperfusion [112]. However, in a study involving 6-OHDA-lesioned rats [31, 32], in which neuroprotection was achieved by GDNF, the authors suggest that carefully manipulating the dose of the viral vector may overcome the issue of decreased activity. Therefore, decreased GDNF activity may be alleviated by determination of an optimal viral load required for stroke subjects. Even if the optimal viral concentration of GDNF is identified, the expression of GDNF at high levels can
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be toxic. This is evidenced by the discontinuation of Amgen GDNF/PD clinical trials due to concerns over cerebral toxicity in a past rhesus monkey experiment and the development of antibodies to GDNF among the patient pool [34]. Low-level expression of GDNF has been demonstrated, though, as having beneficial neurological effects in the same rhesus monkeys [34]. Due to the sudden onset of stroke, a low-level release of GDNF can act as a therapeutic molecule to protect against neuronal damage. In addition to gene therapy, other methods of introducing GDNF protein into the brain have been investigated. Further evaluation of these approaches to deliver the protein and gene into the brain may reveal the optimal dose, timing, and route for GDNF therapy.
Stroke Pathology as Guide for Optimizing GDNF Therapy Stroke leads to a hypoxic environment that triggers an ischemic cascade resulting in irreversible neuronal damage. GDNF induces the inhibition of NMDAinduced calcium influx and stimulates the MAPK pathway in vitro [75]. Experiments utilizing hippocampal brain slice [5] and cortical cultures [75] have indicated that pretreatment with GDNF decreases the level of NMDA-induced neuronal toxicity. In addition to NMDA-mediated neurotoxicity, ischemia cell death cascades also include the production of reactive oxygen species, as well as apoptosis-like processes [36, 46, 50]. Of note, exposure of primary cortical cultures with the HSV amplicon-based vector containing the gene for GDNF, alleviated hydrogen-peroxide-induced neuronal damage [36]. In vivo experiments revealed GDNF reduced caspase immunoreactivity and TUNEL labeling [116, 117], indicating the protein’s antiapoptotic property of GDNF. Similarly, decreased caspase-3 expression and DNA fragmentation was achieved by the TAT-GNDF protein in stroke adult mice [49]. As noted previously, the initial cell death inherent in stroke is abrupt and devastating — thus requiring immediate therapeutic intervention. However, equally destructive to the CNS is the secondary ischemia-associated cell death that progresses over time and that without subsequent treatment is likely to exacerbate the stroke pathology. For example, edema formation becomes apparent after stroke and needs to be addressed. Accordingly, when contemplating GDNF therapy the timing of administration after stroke is critical. Interestingly,
GDNF has been shown to relieve brain edema during ischemia [2]. Despite numerous in vitro and in vivo studies identifying the effects of GDNF in response to stroke, no clinical trials have been carried out to date. As mentioned previously, GDNF has been used in clinical trials for PD [34, 80, 81]. In designing clinical trials of GDNF in stroke patients, one needs to consider an in-depth understanding of the stroke pathology in addition to optimizing the viral vector delivery system if pursuing gene therapy.
Combined NT2N Cell Transplantation and GDNF Therapy Transplantation of cells, which secrete a high level of GDNF, has been utilized as a method of neuroprotection [47]. Stem cells, as well as NT2N cells, have been demonstrated as viable sources of GDNF due to their plastic state of gene expression and totipotent nature. Neural stem cells have been identified with an ability to secrete neurotrophic factors [61], such as mesenchymal stem cells, which have also been shown to reduce stroke deficits in transplanted rats [55]. Alternative GDNFsecreting cells, such as pineal gland and carotid body, have also been transplanted and demonstrated to exert benefits in stroke models. NT2N cells, stem cells, and other novel tissues that secrete neurotrophic factors (especially GDNF) should be further explored as efficacious graft sources for transplantation therapy in stroke. Investigations into transplantation of NT2N cells, combined with gene therapy, should allow unique opportunities on the regulation of GDNF expression (e.g., the use of hypoxia response element) [93] in the brain. Manipulation of NT2N cells for gene therapy would aid in stable and efficient GDNF expression during and after an ischemic episode. Safety and efficacy data of such genetically engineered NT2N cells are warranted to facilitate the entry of such therapy in the clinic.
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21 NGF-producing Cell Transplants for Alzheimer’s Disease Ann-Charlotte Granholm Department of Neurosciences and the Center on Aging, Medical University of South Carolina, Charleston, South Carolina
The Septo-hippocampal, NGF-dependent Cholinergic System in Alzheimer’s Disease and Animal Models A wealth of information from the last few decades demonstrates unequivocally that basal forebrain cholinergic neurons depend on NGF for trophic support [40, 41, 83, 84]. When NGF is withdrawn from these cells, they respond by decreasing firing rate, acetylcholine release, and cholineacetyltransferase (ChAT) activity [6, 7, 15, 39]. With continued loss of the growth factor, the neurons withdraw dendritic processes, atrophy, lose their phenotype, and eventually die [11, 12, 16, 32]. A natural loss of NGF support for these neurons occurs in the aged rat [16] — as well as in specific models for Alzheimer’s disease (AD), such as the Ts65Dn mouse with a segmental trisomy of chromosome 16 [17, 43–45]. A model of this NGF dependency is shown in Figure 21.1 The model is built upon data from many different research groups [5, 25, 38, 39, 47, 51, 52, 55, 77, 81, 86, 95]. Despite the formation of the hypothesis of lost NGF transport in AD (formulated by Mufson and collaborators [65] more than a decade ago) we still have not been able to completely sort out if this loss of trophic support to basal forebrain cholinergic neurons depends on decreased release of NGF from hippocampal neurons, altered signaling or response of the high-affinity NGF receptor TrkA, or altered transport of the NGF-TrkA complex from hippocampus to the basal forebrain (see Figure 21.1). A recent study from our group suggests that it is not the loss of NGF release from hippocampal neurons that is the culprit (at least not during aging in Cellular Transplantation: From Laboratory to Clinic
animal models), in that we found that NGF release from in vitro slices of aged hippocampus was not impaired either in terms of baseline release or pharmacologically induced release by glutamate or high potassium administration [91]. Our current working hypothesis is therefore that it is rather signaling or transport of the growth factor, at least in the aged rat model, that leads to impaired NGF levels in the basal forebrain. Previous work has shown that NGF binds both to the low-affinity neurotrophin receptor (p75) and the highaffinity TrkA receptor [4, 41, 55] at the terminal level, and that the TrkA-NGF complex is transported to the basal forebrain neurons via retrograde axonal transport [16, 24, 65]. Although the TrkA receptor seems to be fairly selective to the NGF ligand and transmits positive signals (cell survival and growth), the p75 receptor binds all of the neurotrophins and transmits both positive and negative (such as apoptosis signaling) signals [55]. Contrary to the classical transmitter receptors, TrkA receptors respond to absence of ligand with reduced synthesis and expression and increased ligand with increased synthesis and expression, respectively [10–12, 37, 55]. In the aged rat, it has been shown that low TrkA expression in individual basal forebrain cholinergic neurons is correlated with propensity to degenerate [16]. Consequently, studies have shown that a comparatively early change in the basal forebrain of AD patients includes a reduced TrkA labeling in nucleus basalis cholinergic neurons [18, 19, 66, 67]. However, it has not yet been explored whether the loss of TrkA expression in AD patients is simply due to lack of NGF in the basal forebrain or perhaps altered second-messenger
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Copyright © 2007, Elsevier Inc. All rights of reproduction in any form reserved.
Ann-Charlotte Granholm
Young, healthy system: Basal forebrain cholinergic neuron
Hippocampal neuron
Ach release
NGF transport = NGF = trkA -Loss of trophic support -Reduced trkA -Disturbed signaling?
Alzheimer or aging: Reduced Ach release
? ↓
Cholinergic neuron degeneration
NGF trkA ↓
Reduced NGF transport
Figure 21.1. Schematic drawing demonstrating the proposed relationship between NGF transport from the hippocampus to basal forebrain and maintenance of cholinergic neuronal function and neurotransmission. There is a reciprocal relationship between NGF and cholinergic neurons, in that NGF stimulates the firing rate and acetylcholine (ACh) release from cholinergic terminals. This, in turn, stimulates release of more NGF from the hippocampal neuron. In addition, the presence of NGF solicits more production of the high-affinity receptor TrkA from cholinergic neurons, and the presence of NGF also sequesters the high-affinity receptor to the surface. During aging and/or Alzheimer’s disease, the levels of NGF are found to be either unaltered or elevated in the hippocampus but reduced in the basal forebrain. An early change that is seen in both animal models and humans with AD is the loss of TrkA receptors in basal forebrain neurons. It is not known yet whether the TrkA loss is primary or secondary to the disease process.
* 20
**
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systems that preclude normal functioning of the receptor, even though many investigators have shown that NGF treatment ameliorates cognitive loss and cholinergic atrophy in animal models [11–12, 32, 43, 62, 63, 73, 85, 87]. It is possible that it is altered protein synthesis or metabolism, or altered axonal transport mechanisms in the AD brain, that give rise to secondary alterations in trophic factor receptor functioning. In fact, in vitro data have shown that b-amyloid alters the affinity of NGF to its TrkA receptor — and NGF in turn has been shown to affect amyloid processing, suggesting a direct link between amyloid accumulation and NGF function [38, 46, 78, 80]. However, to our knowledge this hypothesis has not been proven in intact animal models or human tissues yet. A few years ago, we examined if exogenous NGF could alter the existing imbalance between basal forebrain (low) and hippocampal (high) endogenous NGF levels in memory-impaired aged rats (see Figure 21.2) [2]. Because NGF does not readily cross the bloodbrain barrier [36], we administered NGF conjugated to a transferrin receptor antibody (OX-26-NGF), a drug delivery method that was developed by Alkermes, Inc.
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12 8 4 0 Hippocampus IMP
Non-IMP
Basal forebrain IMP-NGF
Figure 21.2. NGF levels in the basal forebrain and the hippocampus in aged rats with (impaired = IMP) or without (non-IMP) memory loss. Note that exogenous NGF administration, using the OX-26-NGF conjugate, leads to a normalization of NGF levels—both in the basal forebrain and in the hippocampus. (Adapted from Albeck et al., 2003.)
[1, 2, 11, 12, 34, 53]. By using this systemic administration of conjugated growth factor, we hoped to restore NGF levels in the areas in need of extra growth factor (basal forebrain), without increasing the levels further in those areas that already had elevated or unchanged
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levels (hippocampus and cortex). Surprisingly, the conjugated NGF administration not only restored memory but normalized NGF levels in both basal forebrain and hippocampus of the aged rat (see Figure 21.2). Although the NGF injections elicited an increase of endogenous NGF levels in the basal forebrain, they also decreased the endogenous levels in the hippocampus — presumably because cholinergic neurons now received sufficient amounts of the growth factor (see Figure 21.2) or because the TrkA signaling system or the axonal transport now worked more efficiently. The effects on memory and on the growth factor levels remained for at least two months following cessation of NGF injections, suggesting that the endogenous system was functioning more normally in NGF treated rats. These data clearly suggest that it is possible to “jumpstart” the endogenous NGF system to function normally when NGF is administered to the appropriate brain region, even in aged animals. These studies demonstrate that NGF levels are abnormally low in the basal forebrain of memory-impaired animal models and individuals with severe AD (see Figure 21.2), and the primary goal of this chapter is therefore to review the current and past attempts to replace NGF to these neurons. This is done via transplantation of brain regions, peripheral tissues, cell lines, or biomaterials that secrete NGF and therefore restore physiological levels of NGF in the vicinity of cholinergic cell bodies — with the ultimate goal of restoring memory function.
Brain Tissue Grafts Lending Trophic Support to the Host Brain or to Adjacent Grafts It is well known that transplants can benefit in terms of growth, vascularization, and neuronal survival from placement next to a brain region that normally supports the grafted neuronal population. For more than two decades now, we and others have demonstrated powerful growth stimulation in double grafts of different brain regions [8, 20, 22, 70, 92]. In our studies, we have largely utilized the intraocular grafting method for assessment of interregional growth stimulation of transplanted tissue, in that this method allows one to explore growth and vascularization of the transplants by actually visualizing them through the translucent cornea (for method, see Figures 21.3a and 21.3b). Using this transplantation parameter, we found that fetal brain
(a) Intraocular transplant
pupil
septal
hippocampal
(b) Intraocular grafting method Measure graft growth
Anterior eye chamber
Inject growth factors Figure 21.3. The intraocular transplantation method. (a) Microphotograph of a double transplant in oculo, consisting of septal forebrain region (septal) and hippocampal region (hippocampal) from fetal rat. The tissues had grown in the eye chamber for 8 weeks when the photograph was taken. See color insert. (b) Grafting method. The fetal tissue is inserted in the eye through a razor blade slit in the cornea and is placed on the anterior surface of the iris. The growth of grafted tissue can be measured through the translucent cornea on lightly anesthetized host rats. Drugs can be injected via the sclera using a guided needle between the posterior surface of the iris and the lens, to protrude through the pupil into the anterior chamber of the eye.
stem, hypothalamic, and forebrain tissue stimulated the growth of hippocampal grafts significantly. These tissues also increased growth factor levels in the hippocampal graft [20, 92]. Figure 21.4 demonstrates that single intraocular septal and hippocampal grafts have a similar imbalance in NGF levels as previously shown in the septum and hippocampus of aged cognitively impaired rats [2] (Figure 21.2). Thus, single septal grafts had low levels of NGF and hippocampal grafts had high levels of NGF compared to the same areas in noncognitively impaired aged rats or young control rats. These data suggest that the intraocular transplant scenario mimics the situation in these two brain regions in situ during aging or disease, making this a useful model for examining biological effects of NGF without
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Ann-Charlotte Granholm (a) Hippocampus
(b) Basal forebrain
NGF levels pg/mg
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(c) Double grafting enhances growth factor levels
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Colchicine Figure 21.4. ELISA measurements of NGF (a and b) and BDNF (c) levels in intraocular grafts. (a) NGF levels are elevated in aged impaired hippocampus in situ, but also in single intraocular hippocampal grafts. (b) NGF levels are, on the other hand, reduced in both septal tissue in situ from aged memory impaired animals and in single septal grafts, suggesting that a similar regulatory mechanism exists for NGF in situ and in the transplanted isolated tissues. (c) BDNF levels are increased significantly in intraocular grafts when these are grafted with adjacent brainstem grafts (Hip d) compared to levels seen in single hippocampal grafts (Hip s). Colchicine treatment significantly reduced levels in double grafts to those seen in single grafts (Hip d colchicine). These studies suggest that axonal transport may regulate growth factor levels also in intraocular double grafts of brainstem and hippocampus. (c is a modified bar graph from Willis et al., 2005.)
the complex environment of the entire brain in situ. In other recent studies, we found that BDNF levels, on the other hand, were low in single hippocampal grafts and were elevated by grafting adjacent to a brain stem graft containing the locus caeruleus [92] (Figure 21.4c). Administration of the axonal blocker colchicine reduced BDNF levels in intraocular hippocampal grafts to levels seen in single hippocampal grafts, suggesting that BDNF had been transported from the brain stem to the hippocampal graft [92]. Thus, intraocular grafts can produce appropriate levels of growth factors, and adjacent grafting elicits stimulation of growth factor
production and increased graft survival and growth. Further, the data support the notion that normal axonal transport processes are in place in grafted tissues using our paradigm — making it possible to examine growth factor transport properties between two brain regions when isolated in the anterior chamber of the eye. The intraocular grafting system is also useful for assessing direct dose response relationships of grafted tissues for different pharmacological agents. Hence, chronic administration of NGF can be achieved in the intraocular graft system by injecting the growth factor through the sclera — bringing the needle between the lens and iris through the pupil into the anterior chamber of the eye (Figure 21.3b). It was found that NGF administration for the first 6 weeks post-grafting elicited stimulatory effects on intraocular septal grafts, both when NGF was injected directly into the eye chamber [29] and when injected systemically using our OX-26-NGF conjugate described previously [10, 34]. The number of surviving cholinergic neurons was elevated in the septal transplants, and the density of both low- and high-affinity NGF receptors was also increased with the chronic NGF administration. Interestingly, work from other groups has shown that pre-treatment in vitro of fetal septal neurons with NGF prior to grafting does not increase graft survival [42] — suggesting that grafted cholinergic cells have to have access to the growth factor during their post-grafting development in order to thrive. NGF has also been injected acutely into intraocular septal grafts to assess rapid electrophysiological effects of this neurotrophic factor [74]. NGF infusion elicited a fairly slow onset (20 minutes) increase in firing rate of septal cholinergic neurons, which was blocked by p75 (low-affinity neurotrophin receptor) antibodies [74]. The same effect was not seen in intraocular hippocampal or spinal cord grafts, suggesting that these rapid NGF effects may be limited to cholinergic neurons. We have recently seen similar rapid effects of NGF on TrkA expression in septal cholinergic neurons in situ after an acute injection of NGF into the hippocampus [91] (Figure 21.5). This rapid NGF effect did not appear to be transported to the basal forebrain via classical axonal transport (colchicine did not block the effect), and was blunted in aged animals, perhaps providing a cellular mechanism for the loss of NGF signaling to the basal forebrain in the aged animal [91]. We are currently exploring the biological significance and the specific intracellular signaling properties of this rapid NGF transport in the central nervous system.
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extension of injured adult cholinergic neurons. When the peripheral cells were grafted in a tract toward the hippocampus, cholinergic neurites were guided toward the lesioned hippocampus and finally had completely reinnervated the deafferented target region. It is interesting that peripheral and central tissues can support each other by simply providing a source of growth factors during the right moment, whether it is during development or after an injury in the adult nervous system. These natural sources for growth factors (Schwann cells) have henceforth been replaced with other peripheral cells such as fibroblasts, which have been genetically modified to produce the needed growth factors (discussed further in material following).
TrkA optical density
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Figure 21.5. NGF administration into the hippocampal formation (50 microgram, stereotaxic injection) gave rise to a rapid increase (within 15 min) of TrkA immunoreactivity in the basal forebrain. This response of the high-affinity NGF receptor to NGF injections was blunted in aged rats compared to young rats, suggesting that there is an altered response to NGF with aging. Studies are now ongoing to explore the mechanism for this phenomenon, both in the aged and young rat brain. (Adapted from Williams et al., 2005.)
Peripheral Tissue Grafts Lending Trophic Support to Host Brain or Adjacent Grafts Some of the earliest transplantation work demonstrated that central neurons survive poorly when grafted. Numbers of between 5 and 15% survival have been reported, depending on the transplantation site, the age of donor tissue, and the support rendered to the grafted cells. It was shown that peripheral nerve grafts could be utilized as a growth factor source, and some studies successfully included peripheral nerve as a growth and survival factor in intracranial grafting experiments [21, 33, 59, 79]. It was demonstrated that peripheral nerves secrete excessive amounts of NGF also when grafted to the CNS [21, 71, 72], but to our knowledge the peripheral grafts have only been used for transplantation experiments in Parkinson’s disease or Parkinson’s disease animal models and not in AD patients or models. Experiments were performed early regarding transplantation of peripheral ganglion cells to the basal forebrain of rats, but to our knowledge these studies were not continued — presumably due to lack of long-term survival of the grafted ganglion cells [96]. However, cultured Schwann cells and their associated extracellular matrix were grafted intracranially to rats with fimbria fornix lesions by Kromer and collaborators [68]. The investigators found that this cell source provided a significant stimulation, particularly for fiber
Engineered Cells or Biomaterials That Produce NGF When polymer biomaterials were developed further in the 1980s and 1990s, scientists started using these biomaterials to produce spheres or rods that could deliver growth factors for a number of weeks or months when grafted to the brain or spinal cord of injured individuals [13, 60]. One of the most common forms of these polymer materials is biodegradable microspheres of poly–(DL-lactide–co-glycolide containing the growth factor of choice, which may be delivered for as long as 8 to 10 months after implantation [27]. Other investigators have demonstrated that polymer pellets secreting NGF have to be placed at least within 1 to 2 mm of the cells in order to have effect. In addition, the basal forebrain is a difficult target to hit, making these grafts cumbersome to perform in a reproducible manner [60]. Mahoney and Saltzman [60] found that effective treatment with the polymer implants required millimeter-scale positioning of the NGF source and efficacy correlated with the spatial distribution of NGF concentration in the tissue. Because the human forebrain is divided into functional regions that are several centimeters apart and of irregular shape, these investigators suggested that using polymer microspheres may be a technically challenging way of replacing NGF in the brain of patients with AD [60]. Neurotrophic factors can be delivered via gene therapy using either ex vivo (in vitro modification of cells) or in vivo (modification of cells in brain parenchyma) gene therapy [89]. For NGF secretion, by far the most common method has been the ex vivo transfection of cells for later implantation into the brain, even though
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some investigators have opted for in vivo transfection — using, for example, adenoassociated viral vectors (AAV) [50]. Many different cell lines have been genetically modified to produce NGF for transplantation either encapsulated into polymer capsules or directly into the brain parenchyma. These include skeletal muscle cells [26], schwannoma cell lines [82], primary fibroblasts [9, 35, 49, 88, 89], hamster kidney cells [54], testicular Sertoli cells [23], and other cell types and cell lines such as NT2N [57] and HEK-293 cells [64]. The basic concept of gene therapy with NGFproducing cells is depicted in Figure 21.6 [15, 50, 89]. Even though different cell lines and primary cells are used by different research groups, essentially the cells are genetically modified to produce NGF, are tested for release properties in vitro, and are then grafted to the brain parenchyma — usually in the medial septal or nucleus basalis region in close proximity to the basal forebrain cholinergic neurons (see Figure 21.6). The number of transplanted cells depends, of course, on the amount of NGF released by each cell in vitro, and on the specific release properties of the cells to be grafted. Instead of injecting unprotected transfected cells into the brain parenchyma, other investigators have opted to utilize encapsulated cells that have been modified
2. Retroviral NGF transfection
to secrete NGF [56, 82]. Schinstine et al. [82] found that an encapsulated schwannoma cell line (SCT-1 cells) transfected with human NGF cDNA protected adult rat cholinergic neurons from lesion-induced degeneration, using the fimbria fornix model. One definite advantage with the encapsulation is that it is possible to retrieve the grafted cells if the brain reacts abnormally or there are severe side effects. This is, of course, not possible to do with intraparenchymal grafts. Both of these methods have been brought to clinical trials and are currently being tested for safety and efficacy by different clinical research groups (see material following). Collectively, transplantation of genetically modified cells into the brain represents a promising strategy for the delivery and expression of specific neurotrophic factors for intervention in neurodegenerative diseases. The use of specific regulatable promoters may provide control of gene expression required for dose-specific or time-specific therapeutic treatment schedules. One such promoter is the zinc-inducible metallothioneine promoter for regulated expression of NGF that has recently been described [93]. It is likely that built-in safety measures such as these regulatable promoters will increase the community’s trust in gene therapy for future clinical use.
1. Skin biopsy from patient
3. Transplant cells to nucl. basalis Ach release
5. Normalized NGF production in hippocampus
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= NGF = trkA Ex.NGF fibroblast
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Figure 21.6. Schematic drawing demonstrating the principle for ex vivo gene transfer in human AD brains. Fibroblasts are obtained via a skin biopsy and are genetically modified to produce NGF in vitro. The cells are then transplanted to the basal forebrain (nucleus basalis region) in the patient. The thought is that the exogenous NGF may stimulate production of TrkA and also stimulate cholinergic neurons so that the endogenous NGF transport system may be enhanced. Preliminary studies indicate that fibroblasts can produce NGF for at least 18 months when implanted in the human brain [90].
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Clinical Applications Even though the animal models for Alzheimer’s disease described herein exhibit some of the neuropathological features of the disease, none of them can reproduce the complex scenario in the brain of an individual with this neurological disease. The absence of a suitable animal model for Alzheimer’s disease therefore leaves therapeutic trials in human subjects a necessity. Thus, although rodent and primate studies performed for several decades now on NGF delivery and effects in the adult or developing nervous system have taught us a lot about clinical possibility of this powerful peptide (see previous material) it is only through clinical trials that its true effects and safety for the human being can be monitored and assessed. To begin with, it is a challenge to deliver this peptide into the human brain in a safe and efficient manner [9, 25, 35, 40]. NGF does not readily cross the blood-brain barrier (BBB) due to its size and polarity [34, 38, 75]. However, because choline acetyltransferase inhibitors (which represent the most common group of pharmacological compounds used for treatment of AD today) are not targeted toward restoring dying or degenerating neurons (merely replacing the lost transmitter), NGF lends promise as a therapeutic agent with the hope that we will be able to not only quench further death of cholinergic neurons but restore function of borderline degenerating cells that are currently undergoing phenotypic loss and/or cell death processes. Therefore, it is an important quest to find ways in which NGF can be safely and efficiently administered directly where it is needed, in the vicinity of the cholinergic cell bodies in the basal forebrain.
ADMINISTRATION OF NGF VIA INTRAVENTRICULAR INFUSION A recent clinical trial with a small number of patients in Sweden [30, 47] has revealed that administration of NGF via a cannula implanted into the lateral ventricle leads to intolerable side effects, most prominently to back pain associated with the NGF infusion, despite positive effects on cognitive performance and nicotine receptor uptake in the brain [30, 47, 69]. The pain associated with NGF infusion was dose dependent and disappeared after cessation of drug treatment [30, 47]. The same research group in Sweden is now getting ready to apply a novel strategy for NGF delivery in
humans, utilizing polymer-encapsulated cells similar to the technology described previously [27]. The refined encapsulated cell biodelivery (ECB) technology has been developed by NsGene A/S, a Danish biotechnology company, and contains a human clonal cell line expressing therapeutic levels of NGF in a device suitable for intraparenchymal implantation. Preclinical experiments in animal models reveal that this mode of administration of the growth factor is quite promising. The devices function and secrete NGF for at least 6 months in vivo in the non-immunosuppressed pig brain and thereby fulfill the criteria set for the initiation of clinical trials (Lars Wahlberg, NsGene A/S, personal communication). More preclinical and clinical studies have to be performed to confirm that this will be a viable therapeutic option for AD patients in the future.
ADMINISTRATION OF NGF VIA TRANSPLANTATION OF TRANSDUCED CELLS Recently, a phase I trial was performed on eight individuals with mild AD where the patients received autologous fibroblast transplants that were genetically modified to produce NGF (Figure 21.6) [90]. The cells were implanted in the nucleus basalis region using stereotaxic surgery. The first two subjects received unilateral injections, whereas the other six received bilateral grafts. The fibroblasts were obtained from skin biopsies and were modified to produce and secrete human NGF via retroviral vectors, as previously described by this group for rodents and primates [15, 88, 89]. The preliminary data from this phase I trial suggested an improvement in the rate of cognitive decline (tested with the Mini-Mental State Exam, MMSE, and an Alzheimer Disease Assessment Scale, ADAS-Cog) and did not reveal any specific toxic effects of the transplanted cells, at least not up to 24 months following the surgery. In addition, postmortem examination of one of the subjects revealed that the implant location was appropriate, with many NGF-producing grafted cells (5 weeks post-grafting) and improved cholinergic neuron morphology in the vicinity of the grafted cells [90]. Further, cholinergic neurites from the host brain innervated the graft and appeared to be attracted by the grafted cells, suggesting, indeed, that biological amounts of NGF were released from the grafts for at least 5 weeks post-grafting. These data are promising and suggest
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continued studies for NGF-producing autologous cell grafts in AD patients.
Past Concerns and Future Promise for NGF Therapy in the Alzheimer Patient At the end of the 1980s (1988–1989), there were major discussions related to the safety of NGF for clinical trials in AD patients [see especially the discussion in Neurobiology of Aging, Volume 10(5), 1989]. Although some believed that NGF may actually be damaging and accelerate the neuropathology associated with plaques and tangles in AD, others were strong proponents for the therapeutic drug. Now, almost two decades later, there are ongoing clinical safety trials with NGF-producing human fibroblasts. These phase I trials (see previous material) do not reveal increased amyloid formation, as feared by some in the earlier discussions, although it may be too early to tell what the long-term consequences of NGF treatment are in the human brain [90]. Before these human studies were initiated, neuropathology studies were performed in the primate by Tuszynski and Kordower — both of whom found that amyloid deposits in the aged primate brain were unaltered by long-term NGF administration [54, 88]. Thus, it seems that this earlier fear of severe side effects for NGF on amyloid plaques may have been put to rest. The selectivity of NGF for cholinergic neurons compared to the more generalized degeneration found in the brain of individuals with Alzheimer’s disease was also brought up as a potential limitation for the NGF treatment approach (see Neurobiology of Aging, Volume 10(5), 1989). It is well known that both serotonergic [3, 28] and noradrenergic [61] CNS neurons as well as cortical and hippocampal neurons undergo a significant degeneration in AD patients in addition to the basal forebrain cholinergic neurons. It was suggested that using trophic factors supporting a broader variety of neuron classes (such as FGF) might be more appropriate for this generalized neurodegenerative disease. Acidic FGF is produced by basal forebrain cholinergic neurons [48, 94] and has recently been shown to act in an autocrine fashion to support these neurons [48] and is present in noradrenergic and serotonergic neurons in the brain [14]. Thus, it is possible that this growth factor may also be a plausible target for growth factor replacement therapy in the future, although we are not aware of clinical trials for AD involving this interesting growth factor. It may be risky utilizing such a general growth
factor from the perspective of side effects along with the beneficial effects of the factor. An interesting line of work has recently raised the attention of the growth factor community. Mature NGF protein is processed from a larger precursor protein, proNGF [31]. ProNGF is actually the dominant form of NGF in mouse, rat, and human brain tissue, and Fahnestock and collaborators have shown that it is this immature form of NGF that accumulates in target tissues (cortex and hippocampus) in the AD patient [31, 76]. This increase may reflect a biologically active proNGF, or a decrease of processing to mature NGF in AD. Further work is needed to determine whether proNGF and NGF interact in the AD brain, as well as if processing of proNGF can be accelerated to promote mature NGF — in that proNGF has been implicated in more deleterious effects of NGF, such as the p75-mediated degenerative signaling [58]. Other developments of novel pharmacological intervention for AD may include the feasibility of developing small molecules that mimic only specific domains of neurotrophins and modulate signaling without the damaging side effects on other systems. Developing smaller peptides may help in getting the drug across the BBB, but may also lead to peptides that only bind to TrkA receptors and therefore limit the biological effects to beneficial ones — perhaps even development of peptides that do not bind to spinal cord or DRG neurons. With the protein chemistry entering into a new phase of “peptide design,” one should be able to limit biological activity to the wanted effects in the future. This may be a worthwhile endeavor. It is, of course, possible that the cholinergic neurons in aging and AD do not die simply because of trophic factor deprivation (i.e., axotomy, target loss, and so on). It is possible that it is due to the activation of an endogenous apoptotic program requiring mRNA and protein synthesis. The possible mechanistic relationship of neuronal death to the death of other cell types both in the developing and adult organism is not known yet. If this hypothesis of active death is true, loss of transcriptional control of this program may be involved in neuronal attrition in aging or neurodegenerative disease. Thus, it is possible that individuals susceptible to AD and other degenerative diseases simply have a programming defect leading to fatal malfunctioning of certain central neurons. If this is the case, neuroprotective therapies could merely delay the process and we will never find a “cure” for AD. Regardless of the cause of the disease, it has been outlined in this chapter that NGF levels are diminished in the basal forebrain of AD patients, and many studies
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have suggested that cognitive performance, neuronal health, and cholinergic activity are improved with NGF treatment in animal models and humans with the disease. Therefore, even though we are not closer to determining the cause for this devastating disease scientists have developed a therapeutic intervention that may have significant beneficial effects for the patient. The next few years will definitely determine whether local intraparenchymal transplantation of NGF-producing cells holds the key for successful treatment of AD.
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Acknowledgments Sincere thanks are due to Dave Albeck, Heather Bimonte, Cristina Bäckman, Maria Curtis, Chris Hunter, Sonya Juthberg, Alfred Moore, Matt Nelson, Linda Sanders, Brice Williams, and Lauren Willis. Without these individuals, the work described herein would not have been possible. Thanks are also due to Maria Eriksdotter Jönhagen and to Barry Hoffer for valuable discussions throughout the years. The work was supported by grants from the National Institutes on Aging (AG04418, AG12122, and AG10755).
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47. Jönhagen, M. E. Nerve growth factor treatment in dementia. Alzheimer Disease & Associated Disorders 14 Suppl 1: S31–388; 2000. 48. Kanda, T., Iwasaki, T., Nakamura, S., Kurokawa, T., Ikeda, K., Mizusawa, H. Self-secretion of fibroblast growth factor-9 supports basal forebrain cholinergic neurons in an autocrine/paracrine manner. Brain Research 876(1–2):22–30; 2000. 49. Kawaja, M. D., Rosenberg, M. B., Yoshida, K., Gage, F. H. Somatic gene transfer of nerve growth factor promotes the survival of axotomized septal neurons and the regeneration of their axons in adult rats. Journal of Neuroscience 12(7):2849–2864; 1992. 50. Klein, R. L., Hirko, A. C., Meyers, C. A., Grimes, J. R., Muzyczka, N., Meyer, E. M. NGF gene transfer to intrinsic basal forebrain neurons increases cholinergic cell size and protects from age-related, spatial memory deficits in middle-aged rats. Brain Research 875(1–2):144–151; 2000. 51. Knusel, B., Gao, H. Neurotrophins and Alzheimer’s disease: Beyond the cholinergic neurons. Life Sciences 58(22):2019–2027; 1996. 52. Koliatsos, V. E., Price, D. L., Clatterbuck, R. E., Markowska, A. L., Olton, D. S., Wilcox, B. J. Neurotrophic strategies for treating Alzheimer’s disease: Lessons from basic neurobiology and animal models. Annals of the New York Academy of Sciences 695:292–299; 1993. 53. Kordower, J. H., Mufson, E. J., Granholm, A.-Ch., Hoffer, B. J., Friden, P. Delivery of trophic factors to the primate brain. Exp Neurol 124:21–230; 1993. 54. Kordower, J. H., Mufson, E. J., Fox, N., Martel, L., Emerich, D. F. Cellular delivery of NGF does not alter the expression of beta-amyloid immunoreactivity in young or aged nonhuman primates. Experimental Neurology 145 (2 Pt 1):586–591; 1997. 55. Lad, S. P., Neet, K. E., Mufson, E. J. Nerve growth factor: Structure, function and therapeutic implications for Alzheimer’s disease. Current Drug Targets: Cns & Neurological Disorders 2(5):315–334; 2003. 56. Lindner, M. D., Kearns, C. E., Winn, S. R., Frydel, B., Emerich, D. F. Effects of intraventricular encapsulated hNGF-secreting fibroblasts in aged rats. Cell Transplantation 5(2):205–223; 1996. 57. Longhi, L., Watson, D. J., Saatman, K. E., Thompson, H. J., Zhang, C., Fujimoto, S., Royo, N., Castelbuono, D., Raghupathi, R., Trojanowski, J. Q., Lee, V. M., Wolfe, J. H., Stocchetti, N., McIntosh, T. K. Ex vivo gene therapy using targeted engraftment of NGF-expressing human NT2N neurons attenuates cognitive deficits following traumatic brain injury in mice. J Neurotrauma 21(12): 1723–1736; 2004. 58. Longo, F. M., Massa, S. M. Neurotrophin-based strategies for neuroprotection. J Alzheimer’s Disease 6 Suppl 6:S13–17; 2004. 59. Lopez-Lozano, J. J., Bravo, G., Brera, B., Millan, I., Dargallo, J., Salmean, J., Uria, J., Insausti, J. Long-term
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Ann-Charlotte Granholm 72. Olson, L. Grafts and growth factors in CNS. Basic science with clinical promise. Stereotactic & Functional Neurosurgery 54–55:250–267; 1990b. 73. Pallage, V., Toniolo, G., Will, B., Hefti, F. Long-term effects of nerve growth factor and neural transplants on behavior of rats with medial septal lesions. Brain Research 386(1–2):197–208; 1986. 74. Palmer, M. R., Eriksdotter-Nilsson, M., Henschen, A., Ebendal, T., Olson, L. Nerve growth factor-induced excitation of selected neurons in the brain which is blocked by a low-affinity receptor antibody. Experimental Brain Research 93(2):226–230; 1993. 75. Pardridge, W. M. Blood-brain barrier drug targeting: The future of brain drug development. Molecular Interventions 3(2):90–105; 2003. 76. Peng, S., Wuu, J., Mufson, E. J., Fahnestock, M. Increased proNGF levels in subjects with mild cognitive impairment and mild Alzheimer disease. Journal of Neuropathology & Experimental Neurology 63(6):641–649; 2004. 77. Perry, E. K. Nerve growth factor and the basal forebrain cholinergic system: A link in the etiopathology of neurodegenerative dementias? Alzheimer Disease & Associated Disorders 4(1):1–13; 1990. 78. Price, D. L., Koliatsos, V. E., Sisodia, S. S., Koo, E. H., Martin, L. J., Walker, L. C., Appelgate, M. D., Cork, L. C. Amyloid-related proteins and nerve growth factor in Alzheimer’s disease and animal models. Clinical Neuropharmacology 14 Suppl 1:S9–14; 1991. 79. Rosenfeld, J. V. Current issues in neural transplantation. Annals of the Academy of Medicine, Singapore 22 Suppl 3:464–469; 1993. 80. Rossner, S., Ueberham, U., Schliebs, R., Perez-Polo, J. R., Bigl, V. Neurotrophin binding to the p75 neurotrophin receptor necessary but not sufficient to mediate NGF-effects on APP secretion in PC12 cells. J Neural Transm Suppl 54:279–285; 1998. 81. Salehi, A., Delcroix, J. D., Swaab, D. F. Alzheimer’s disease and NGF signaling. Journal of Neural Transmission 111(3):323–345; 2004. 82. Schinstine, M., Fiore, D. M., Winn, S. R., Emerich, D. F. Polymer-encapsulated schwannoma cells expressing human nerve growth factor promote the survival of cholinergic neurons after a fimbria-fornix transection. Cell Transplantation 4(1):93–102; 1995. 83. Scott, S. A., Crutcher, K. A. Nerve growth factor and Alzheimer’s disease. Reviews in the Neurosciences 5(3):179–211; 1994. 84. Siegel, G. J., Chauhan, N. B. Neurotrophic factors in Alzheimer’s and Parkinson’s disease brain. Brain Research, Brain Research Reviews 33(2–3):199–227; 2000. 85. Sinson, G., Voddi, M., McIntosh, T. K. Combined fetal neural transplantation and nerve growth factor infusion: Effects on neurological outcome following fluid-percussion brain
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22 Transplants of CNTF-producing Cells for the Treatment of Huntington’s Disease Carole Escartin (1), Philippe Hantraye (1 and 2), and Nicole Déglon (1) (1) Atomic Energy Commission, CEA CNRS URA 2210 Unit and MIRcen Program (2) Isotopic Imaging, Biochemical and Pharmacological Unit, Service Hospitalier Frederic Joliot, CEA, Orsay, France
Introduction
neurotrophic factor (CNTF), a trophic factor for striatal neurons.
Cell grafting provides an efficient and valuable therapeutic solution for several pathologies caused by the dysfunction and/or degeneration of cells or whole organs such as the heart, liver, and kidney (see other chapters). Cell grafting also shows great promise for treating several brain disorders, in particular neurodegenerative diseases in which specific neuronal populations die. Among these diseases, Huntington’s disease (HD) is a fatal neurological disorder that has no efficient treatment. HD is a genetic disease caused by the progressive degeneration of striatal neurons and is accompanied by severe motor and cognitive symptoms [49]. In the absence of a specific agent to block disease progression, an alternative therapeutic approach may be to graft embryonic neurons in the striatum to restore the functional integrity of the cortico-striatopallidal circuitry. Cell grafting may also provide a way of delivering therapeutic molecules locally to vulnerable neurons. The idea here is to help the host neurons fight against disease progression rather than simply grafting new neurons that will be subjected to a deleterious environment. The grafted cells are engineered to release a neurotrophic factor that promotes neuronal function and survival. These cells are implanted in the vicinity of the cells suffering from the disease. This chapter illustrates this original therapeutic strategy, which has recently been tested in HD patients. The treatment uses encapsulated genetically engineered cells overexpressing ciliary
Cellular Transplantation: From Laboratory to Clinic
Huntington’s Disease CLINICAL FEATURES Huntington’s disease is an autosomal dominant genetic neurodegenerative disorder that affects one person in 10,000 in Europe and North America [49]. Symptoms first appear around the age of 40 years and slowly worsen, leading to death 20 years later. The principal clinical features of Huntington’s disease are choreiform movements, dyskinesia, and dystonia. These movement disorders are associated with frontal-type cognitive deficits and psychiatric disturbances. In addition, patients often suffer mood disorders, depression, and personality changes. As the disease progresses, the cognitive alterations aggravate and evolve toward dementia [49].
NEUROPATHOLOGICAL FEATURES Postmortem studies of HD brains have indicated that this pathology is associated with a severe atrophy of the striatum (caudate and putamen), accompanied by an enlargement of the cerebral ventricles. Within the caudate-putamen complex, the prominent neuronal
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striatal loss mainly affects GABAergic spiny projection neurons, whereas the striatal interneurons are largely unaffected [60]. To a lesser extent, neuronal loss is also visible in some cortical areas, pallidal segments, several thalamic nuclei, and the substantia nigra. Positron emission tomography (PET) studies have demonstrated striatal hypometabolism measured with 2-[18 F]-fluoro-2-deoxy-D-glucose as a tracer even before strong clinical symptoms and massive neuronal loss occur [63].
Despite the well-defined molecular alteration of a single protein (i.e., polyglutamine repeats), the etiology of the disease remains complex and multifactorial [51]. Excitotoxicity, metabolic and mitochondrial dysfunction, transcriptional dysregulation, protein aggregation, and apoptosis are all factors that may be involved in neuronal death in HD.
Models of Huntington’s Disease PHENOTYPIC HD MODELS
ETIOLOGY OF HUNTINGTON’S DISEASE In 1993, a key step toward the understanding of the disease etiology was made when the gene and the mutation involved in HD were characterized [93]. The mutation responsible for HD consists of an extension of CAG triplets (which encode the amino acid glutamine) in the N-terminal part of a protein called huntingtin (Htt). Htt is a ubiquitously expressed high-molecular-weight protein. The normal function of Htt is still not fully known, but the protein is involved in transcriptional control, intracellular trafficking, and metabolic regulations [19, 22, 44, 48]. The elongated polyglutamine tract in the mutated form of Htt (muHtt) seems to confer a toxic gain of function to the protein by altering the strength of the interaction between Htt and its numerous proteinic partners [48, 65]. For example, the transcription factor CREB binding protein (CBP) is sequestered by muHtt, which leads to transcriptional alterations [70]. However, neuronal dysfunction and death may also be due to a loss of function of normal Htt. Wild-type Htt has been shown to exert antiapoptotic effects on cultured cells submitted to a wide range of challenges [79]. Wild-type Htt also increases the level of expression of the neurotrophic factor BDNF [106]. Htt is a substrate for several proteases, such as caspases and calpain [102]. Proteolysis of muHtt produces N-terminal fragments that tend to aggregate within neurons. These aggregates, which in most cases are ubiquitinated, are found either in the nucleus or in the dystrophic dendrites of the neurons [29]. The physiological/pathological role of these aggregates is still a matter of debate. The aggregates may either trigger the pathological accumulation of proteins inside the cell (thus participating in cellular dysfunction) or help sequester muHtt, preventing its abnormal interactions with key proteins inside neurons [12, 80].
Many valuable animal models of Huntington’s disease have been developed. The first models of HD were based on intracerebral injections of excitotoxins, such as quinolinic acid, which trigger striatal degeneration through an NMDA receptor-dependent mechanism. Excitotoxic striatal lesions in rats [14] and primates [39, 47, 52] reproduce many of the neuropathological and behavioral deficits typical of HD, including preferential degeneration of GABAergic medium-size spiny striatal neurons and dopamine agonist-induced dyskinesia and chorea. In addition, mitochondrial inhibitor lesion models have been developed. In particular, systemic administration of the succinate dehydrogenase inhibitor 3-nitropropionic acid (3NP) induces progressive bilateral lesions in the caudate-putamen complex associated with motor and cognitive symptoms reminiscent of HD [18, 75]. These phenotypic models have been extensively used to test therapeutic compounds or to study the mechanisms involved in the selective degeneration of striatal neurons [17].
GENETIC HD MODELS With the discovery of the mutation responsible for HD, genetic models have become available. Different strategies (knock-in, YAC, viral gene transfer) have been used to induce the expression of Htt fragments having varying numbers of polyglutamine repeats. Genetic in vitro models have been developed using either nonneuronal (HEK, HN33 cells) or neuronal (PC12, neuroblastoma, ST14A) cell lines. These in vitro models have all shown that mutation in the Htt protein triggers several cellular dysfunctions and increases the vulnerability of the cells to apoptotic stimuli [90]. Aside from cell models, several transgenic mice models of HD have also been developed. Depending on the genetic
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background, the length of the huntingtin fragment, and the regulatory sequences, the phenotypes are slightly different but they reproduce some of the key features of Huntington’s disease, such as striatal atrophy, gliosis, aggregate formation, and motor deficits. However, transgenic mice models of HD usually present limited neuronal degeneration in the striatum and are therefore not well suited to the study of neuroprotective treatments. Other useful genetic models of HD have been created in invertebrates. A fragment of mutated huntingtin has been expressed in the photoreceptors of Drosophila [101] and in the sensory neurons of the nematode C. Elegans [36]. These invertebrate models of HD allow fast screening of genetic factors that may alter the disease process [55]. Genetic models are valuable tools for reproducing and understanding the pathogenic mechanisms linked to Htt mutations and for identifying new potential therapeutic targets [13].
Therapeutic Strategies for Huntington’s Disease As the cellular and molecular mechanisms involved in neuronal death in HD are still not fully characterized, developing efficient therapies remains a challenge. Until now, symptomatic treatments to relieve depression and mood disorders have been mostly offered to patients suffering from HD. Neuroleptics are also given in an attempt to control dyskinesia. However, none of these treatments is fully satisfactory and several alternative therapeutic strategies are being actively pursued. These strategies can be divided into pharmacological, restorative, and neuroprotective approaches. The pharmacological approach is rather selective in that it targets specific cellular dysfunctions and death pathways. The other two approaches are more global and aim at promoting the general function and survival of striatal neurons.
PHARMACOLOGICAL APPROACH Many different pharmacological agents with known effects against excitotoxicity, apoptotic cascades, metabolic dysfunction, or protein aggregation have been developed and tested in various animal models of HD [13]. Riluzole and remacemide are two antiexcitotoxic agents that have been successfully tested in phenotypic and genetic models of HD [37, 45, 85]. Antiapoptotic
compounds such as caspase 1, 3, and 8 inhibitors have shown beneficial effects on disease progression in transgenic models of HD [24, 72]. Creatine and coenzyme Q10 have been tested as alternative energetic substrates to improve brain metabolic status [38]. Other strategies aiming at reducing aggregate formation through transglutaminase inhibition reported an increased survival and a reduction of movements disorders in transgenic HD mice [54]. However, aggregate formation may well be a cellular defense mechanism against toxic huntingtin fragments. Therefore, interfering with such a cellular response may accelerate disease progression [8]. Despite these encouraging results, all clinical trials performed to date with these compounds have proven unsuccessful, with only minor or insignificant therapeutic effects [61, 89, 94, 95, 97, 100].
CELL GRAFTING A very different therapeutic approach consists of replacing dead striatal neurons with grafted embryonic neurons. Many grafting experiments have shown this restorative strategy to be valuable in both rodents and primates [46, 57, 74]. Based on these data, clinical trials have been initiated in France [10], the USA [41], and the UK [81]. In this volume, Dunnett and Rosser present a detailed description of this approach which improves motor and cognitive symptoms as well as striatal and cortical metabolic deficits [10, 42].
NEUROTROPHIC FACTORS The third approach tries to promote the survival of striatal neurons using neurotrophic factors. Rita Levi-Montalcini et al. isolated the first neurotrophic factor, nerve growth factor (NGF), in 1968. Since then, many other neurotrophic factors — such as brainderived neurotrophic factor (BDNF), glial-cell-linederived neurotrophic factor (GDNF), and CNTF — have been identified and characterized. These proteins act through different transduction pathways to inhibit developmental cell death, improve neuronal survival, or induce neuronal differentiation [40, 96]. Although their mechanisms of action are not always well understood, neurotrophic factors offer new opportunities in the treatment of neuronal death in HD. Several neurotrophic factors — such as NGF, BDNF, GDNF, and CNTF — have all shown significant neuroprotective effects in several HD models both in vitro and in vivo [5].
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Among these factors, CNTF seems to be particularly interesting as it protects striatal neurons against several toxic challenges such as excitotoxicity, metabolic impairment, and muHtt toxicity both in vitro and in vivo.
Ciliary Neurotrophic Factor (CNTF) DISCOVERY OF CNTF CNTF was first described by Adler et al. [1] as a factor promoting the survival of neurons of the chick ciliary ganglion in vitro. CNTF is a 23kDa protein that belongs to the alpha-helical cytokine family. It is primarily expressed in the nervous system and is most abundant in the peripheral nerves, olfactory bulb, and optic nerve [86]. Astrocytes in the central nervous system, and Schwann cells in the peripheral nervous system, express CNTF. CNTF lacks a classic signal peptide, and is therefore considered a “lesion factor” that is only released in the extracellular space when astrocytic plasmatic membranes are disrupted [86]. CNTF binds to a tripartite receptor comprising gp130, the LIF receptor b, and the CNTF receptor a [91]. CNTF binding to its receptor triggers the activation of the Jak-Stat pathway that activates the phosphorylation cascade of Jak2 and Stat3/Stat1. Phosphorylated dimers of Stat enter the nucleus and act as transcription factors for a subset of genes that contain promoter consensus sequences composed of TTN[5]AA [16]. Among them, CNTF increases the level of expression of glial fibrillary acidic protein (GFAP) [26], neuropeptides VIP and somatostatin [78], and immediate early genes such as tis-11 and c-fos [56]. CNTF can also signal through the mitogen-activated protein (MAP) kinase pathway and the PI3 kinase pathway [6, 30, 62] that mediate broad-range effects inside responsive cells. CNTF is a pleiotropic molecule that acts on numerous cell types. CNTF induces cholinergic differentiation [83], promotes oligodendrocytes maturation and survival, and improves the myelinization process [11, 92]. CNTF also induces the differentiation of oligodendrocyte-type-2-astrocyte (O-2A) precursor cells into astrocytes [50]. In addition, CNTF exerts strong neuroprotective and neurotrophic effects on several classes of neurons. CNTF improves photoreceptor survival in several models of retinopathy [23]. CNTF also has neuroprotective effects in the pmn and wobbler mice models of motoneuronal death [67, 88], and protects from the retrograde neuronal death induced by
axotomy [25, 87]. Above all, CNTF protects striatal neurons in several animal models of HD.
NEUROPROTECTIVE EFFECTS OF CNTF IN HD MODELS NEUROPROTECTIVE EFFECTS OF CNTF IN PHENOTYPIC MODELS OF HD The neuroprotective effect of CNTF in HD were first demonstrated in the quinolinate phenotypic rat model [7]. Rats that received axokine, a CNTF analogue, in the striatum through an osmotic pump had smaller quinolinate-induced lesions than control groups. The neuroprotective effects of CNTF were later demonstrated in nonhuman primates using the quinolinateinduced striatal lesion model [35]. In this study, CNTF not only protected striatal neurons from the excitotoxic challenge but protected the cortical afferent neurons and striatal axonal projections to the pallidum and the substantia nigra pars reticulata. A subsequent study in a primate model of progressive striatal degeneration — the chronic 3NP model — also demonstrated that CNTF had strong neuroprotective and neurorestorative properties on striatal neurons. In this study, CNTF administration was started only after the appearance of the first motor and cognitive symptoms and was found to protect striatal and cortical neurons from degeneration and to reverse ongoing motor and cognitive deficits [68]. Many experimental results have since confirmed the neuroprotective properties of CNTF in phenotypic HD models using other delivery systems, such as viral vectors [28, 69, 82]. NEUROPROTECTIVE EFFECTS OF CNTF IN GENETIC MODELS OF HD As genetic models of HD became available, the neuroprotective effects of CNTF against the pathogenic mechanisms triggered by muHtt were also studied. Saudou et al. were the first to demonstrate neuroprotective effects of CNTF against muHtt toxicity in primary striatal cultures transfected with the N-terminal part of muHtt. In this in vitro model, neurons accumulate intracellular aggregates of muHtt fragments and undergo a progressive degeneration through apoptosis. Recombinant CNTF significantly improved cell survival without affecting aggregate formation [84]. Recently, the neuroprotective effects of CNTF were also demonstrated
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by lentiviral gene transfer in primary cultures of striatal neurons transfected with a fragment of muHtt. In this progressive in vitro model of HD, CNTF overexpression prevented neuronal death [104]. Finally, the effects of CNTF were studied in a transgenic mouse model of HD by lentiviral gene transfer [105]. These mice express the full-length muHtt with 72 CAG repeats in a yeast artificial chromosome (YAC). Mice that were six to seven months old had motor hyperactivity that was significantly reduced by treatment with CNTF. Histological examinations showed a significant decrease in the number of dark cells in the striatum. However, due to the lack of significant neuronal death in this transgenic HD model, clear-cut neuroprotective effects of CNTF were difficult to demonstrate. Nevertheless, these data show that CNTF is a valuable agent for developing a neuroprotective/neurorestorative therapeutic treatment for HD.
STRATEGIES FOR CNTF DELIVERY TO THE BRAIN Although CNTF is a promising trophic factor for HD treatment, delivering it to the brain in a clinical application is a challenge. CNTF cannot cross the blood-brain barrier that isolates the brain from the rest of the body. Moreover, due to its pleiotropic effects CNTF injection in the periphery causes major side effects such as weight loss, dry cough, asthenia, and fever in patients [20, 21]. Consequently, CNTF needs to be delivered directly into the brain. Several strategies have therefore been developed to deliver the protein (see Figure 22.1). OSMOTIC PUMPS One strategy has been to use a minipump filled with recombinant protein that is connected to a cannula
(3A)
(3B)
(2)
(3C) (3D)
(1)
Figure 22.1. Strategies for peptide delivery to the brain. To deliver a therapeutic molecule such as CNTF to brain cells that are naturally isolated from the rest of the body by blood-brain barrier, several strategies can be envisioned: (1) Osmotic minipumps filled with recombinant peptide provide a direct way of delivering the protein of interest to the lateral ventricle or directly into the brain parenchyma. (2) Viruses that encode the gene of interest are used as vectors to infect targeted cells in the brain. Classical viral vectors used for gene therapy in the central nervous system come from adenoassociated viruses, adenoviruses, and lentiviruses. (3) Cultured cells can be genetically modified after transfection with plasmids (3A) or infection with viral vectors (3B) to express the gene of interest. They can then be grafted in the vicinity of vulnerable neurons. These cells may be encapsulated to protect them from host responses following implantation (3C). Alternatively, transgenic cells can be directly grafted into the affected structure (3D).
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implanted inside the brain parenchyma, close to the targeted neuronal population. This system allows a regular delivery of protein inside the brain. Alternatively, the cannula can be implanted into the lateral cerebral ventricle (intracerebroventricular infusion), leading to a broader diffusion of the protein inside the brain. Trophic factor delivery through a minipump has already been tested in patients suffering from amyotrophic lateral sclerosis and Parkinson’s disease [43, 71, 76]. However, there are some limitations to this approach. First, the therapeutic agent must be stable at body temperature and should tolerate long-term storage inside the pump. Second, the pump must be regularly refilled, which may require repeated surgical interventions. Third, it is necessary to have a high quantity of peptide inside the pump to reach an optimal concentration of the therapeutic protein inside the brain away from the cannula, which makes this therapeutic strategy very costly. IN VIVO GENE THERAPY Alternatively, the brain cells themselves could be used to directly produce the therapeutic molecule through genetic manipulation. Such an in vivo gene therapy approach relies on viral gene transfer to allow stable expression of the transgenic trophic factor in the immediate vicinity of the targeted cells. Gene transfer can be achieved with several types of viral vectors (such as those derived from adeno-associated viruses, from adenoviruses, or from lentiviruses) that can infect nondividing cells and transfer the transgenic sequence into the host genome. Gene therapy for the nervous system has been largely tested in animals [27]. Over the last few years, the efficacy and safety of viral vectors have been improving and significant therapeutic effects of several transgenic proteins have been demonstrated. However, the transfer of in vivo gene therapy from the bench to the clinic is still a challenge. Only a few therapeutic trials for brain diseases have been launched to date [31]. The major problems of gene therapy are the large size of the human brain and its relative heterogeneity. Above all, safety concerns still limit this approach and additional experiments regarding the immune response, vector recombination with wild-type viruses, mutational insertion, and the possibility of shutting down transgene expression are still needed. CELLULAR DELIVERY The third approach consists of grafting genetically modified cells expressing the protein of interest within
the affected brain region. This strategy can thus be regarded as ex vivo gene therapy. Genetically engineered cells can either be implanted directly into the brain parenchyma [99] or be placed in a capsule of biocompatible polymers — to protect them from host immune response — and then implanted [3] (Figure 22.2). The delivery of therapeutic proteins by means of encapsulated cells has several advantages. It allows a high level of transgene expression by transplanted cells without the need of costly recombinant proteins while still being totally reversible, as encapsulated cells can be retrieved if any side effect is observed.
Cellular Vectors for CNTF Delivery in HD PRODUCTION AND TESTING OF ENGINEERED CELLS Cell encapsulation is based on the immunoisolation of exogenous cells by specific permeable polymer membranes [33]. The membrane is semipermeable: it allows the bidirectional transport of small molecules — such as oxygen, nutrients, and trophic factors — but stops higher-molecular-weight molecules (such as antibodies) and prevents contact between encapsulated cells and immune system cells (Figure 22.2). Therefore, xenogeneic cells can be used and immunosuppressant treatment after implantation is not needed. The biocompatibility of polymer capsules has been extensively tested in vitro and in vivo [2]. The capsules induce a very limited glial/immune response and are well tolerated inside the brain irrespective of the encapsulated cells. The encapsulated cells are selected according to their availability (expansion, growth capacity), their ability to stably express the gene of interest at a high level, and their potential to survive in the environment of the brain parenchyma. Several cell lines have been tested: PC12 cells [59, 103], adrenal chromaffin cells [4], and baby hamster kidney (BHK) fibroblasts [3]. In the present study, BHK cells were genetically modified and used for the production of CNTF-releasing capsules. The detailed procedure for BHK-hCNTF production and evaluation has been described by Aebischer et al. [3]. Briefly, BHK cells were transfected with an expression plasmid containing the human CNTF gene fused to the murine immunoglobulin signal peptide
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Extracellular matrix
BHK cells
Glue
Titanium connector
Semi-permeable membrane
Silicone catheter
Figure 22.2. The capsule. The capsule is composed of a polymer membrane filled with engineered cells, closed with glue at one end, and connected to a silicone catheter at the other end to facilitate manipulation and retrieval. A matrix to promote their anchorage and survival surrounds the cells. The capsule contains a small radio-opaque titanium connector that allows its detection in vivo by magnetic resonance or X-ray imaging.
under the mouse metallothioneine I promoter. The geneexpressing thymidine kinase was inserted into this plasmid, allowing transfected cells to be destroyed with ganciclovir in case of capsule disruption. A BHK-hCNTF clone was selected and a cell bank was produced [2]. These encapsulated cells have been extensively tested for tumorigenicity in rodents, sheep, and primates and for sensitivity to ganciclovir [2]. The capsule was also tested to evaluate the retention capacity for several classes of viruses and was found to be an effective barrier against viral diffusion [9]. Finally, the toxicity of secreted CNTF was also evaluated and was shown to be minimal. The genetically modified cells were loaded into a capsule made from polyethersulfone polymer and connected to a silicone tether to allow easy manipulation of the capsule. The production of CNTF can be evaluated before implantation using bioassays and ELISA. Typical CNTF production was about 500 ng/day/105 cells.
PRE-CLINICAL STUDIES IN HD ANIMAL MODELS Genetically modified encapsulated cells that secrete human CNTF were first tested in rodent models of
HD [32]. Capsules of BHK cells were implanted in the lateral ventricle of rats before ipsilateral striatal injection of the excitotoxin quinolinate. A significant neuroprotective effect of CNTF was demonstrated. The protection was greater for glutamic acid decarboxylase (GAD) or choline acteyltransferase (ChAT) immunopositive neurons than for NADPH diaphorase-positive neurons. A reduction in quinolinate-induced lesion size was associated with a beneficial effect on apomorphine-induced rotations. This therapeutic approach was then tested using the same encapsulation technique in a primate model of the disease. CNTF-secreting cells were implanted prior to quinolinic acid injections in the striatum of cynomolgus monkeys [35]. The quinolinate-induced lesion in the striatum was significantly reduced by CNTF production from encapsulated cells, with a net preservation of GABAergic, cholinergic, and NADPH diaphorase neurons. DARPP32 immunostaining also showed a preservation of GABAergic innervation of the pallidum and substantia nigra pars reticulata. More interestingly, histological studies also showed a significant reduction of neuronal atrophy in the layer V of the motor cortex. These neurons send axons to striatal neurons and are affected (although to a lesser extent) in HD. These results strongly suggested that the implantation of a
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capsule of CNTF-expressing cells in the striatum may protect the entire basal ganglia circuitry. This therapeutic strategy has also been tested in the 3NP primate model [68]. In addition to neuropathological alterations, this model presents the subtle motor and cognitive dysfunctions seen in HD patients [18, 75]. Monkeys chronically treated with 3NP (during five months) showed neuronal loss in the caudate-putamen and prefrontal cortex associated with cognitive deficits and hypokinesia. After two months of 3NP treatment, the implantation of two CNTF-releasing capsules in the caudate and putamen led to a progressive recovery of motor and cognitive symptoms. Moreover, there was a greatly reduced loss of NeuN and calbindin immunopositive neurons in the striatum and neurofilament protein (NFP)-positive neurons of cortical layer V cortex. In addition to the strong neuroprotective potential of CNTF for vulnerable neuronal populations in HD, these experiments demonstrated the feasibility and safety of the encapsulation technique. Encapsulated cells were viable and expressed significant levels of CNTF even after several months of implantation. Moreover, capsule implantation in either the ventricle or striatal parenchyma induced no detectable inflammation or immune response, confirming the biocompatibility of the device.
Clinical Phase I Trial for Encapsulated Cells in HD RESULTS OF THE TRIAL The strong neuroprotective effects of encapsulated CNTF-producing cells in several animal models of HD and the demonstrated safety of this technique have led to a phase I clinical trial. The clinical protocol has been extensively described by Bachoud-Levi et al. [9]. The CNTF-secreting BHK cells have been previously used in a phase I clinical trial with patients suffering from amyotrophic lateral sclerosis (ALS) [3]. In this trial, there was a sustained production of CNTF for at least 17 weeks by the capsules and a detectable increase in CNTF levels in the cerebrospinal fluid. No immune response or tissue rejection toward the capsule was observed. Six HD patients (three men, three women, stages 1 or 2) were enrolled in the HD trial. A capsule of CNTFsecreting BHK cells was stereotaxically implanted in the right ventricle of each patient. Bloch et al. recently
reported the outcome of this trial [15]. The trial was originally designed to last one year, with a capsule being exchanged every six months, but was extended to two years on the request of the enrolled patients to continue the trial. The study originally aimed to evaluate the feasibility and tolerability of intraventricular implantation of capsules of CNTF-secreting cells in patients. Similar to ALS trial [3], no side effects related to CNTF or the surgical procedure were observed during the two-year follow-up. This demonstrates that a local delivery of CNTF inside the brain is a suitable method for avoiding CNTF-mediated peripheral effects (weight loss, dry cough, fever). There was no detected immune response or inflammation and the capsules could be retrieved easily every six months without any cell adhesion. The surgical procedures, capsule implantation, and retrieval were well tolerated and validated. However, there were some limitations in the use of BHK cells. Before capsule implantation, the level of released CNTF was highly variable (from 47 to 619 ng CNTF/ml/24 h) despite a standardized procedure for capsule production. Above all, the level of CNTF production after capsule retrieval was drastically reduced (approximately 10% of CNTF initial production). Of the 24 capsules implanted, only 11 released CNTF at a rate greater than 10 ng/ml/24 h. CNTF levels were detectable in cerebrospinal fluid with only four capsules. A few surviving cells were observed in explanted capsules and the level of CNTF production was directly related to the rate of BHK cell survival. Therefore, the CNTF levels inside the striatum may have been far below a physiological concentration. This may explain the lack of clinical improvement seen during the trial as assessed by motor, neuropsychological, neurological, and neurophysiological tests. A potential benefit of CNTF treatment was only detected using electrophysiological recordings in two patients who had received the most active capsules. Patient 3 recovered a detectable median nerve somatosensory-evoked potential for at least nine months after implantation. Patient 6 had normalization of the electromyographic silent period to transcranial magnetic stimulation. Although the exact cellular and electrophysiological basis for these recordings is unknown, alterations of these electrophysiological parameters may be a consequence of dysfunction of neuronal cells and synaptic communications. Accordingly, the loss of somatosensory-evoked potential is correlated to a decrease in glucose consumption [64], and the duration
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of the silent period is correlated to the severity of chorea in HD patients [77]. A recovery of both parameters has been observed after successful cell transplants in HD patients [10]. The recovery of electrophysiological recordings may be related to a neurotrophic effect of CNTF on neurons, improving their survival and function, or to a direct effect of CNTF on neuronal synaptic properties. Importantly, some patients presented psychiatric disturbances (such as depression and mood disorders) after the final capsule retrieval. This may be due to the trial ending and the loss of hope for clinical improvement. These emotions need to be taken into account in the design of future trials. In conclusion, this clinical phase I trial demonstrated the feasibility and tolerability of repeated intraventricular capsule implantation, with no damaging side effects seen after two years. However, several improvements to the technique are needed to increase the probability of seeing a beneficial effect related to CNTF delivery.
PERSPECTIVES IMPROVING CNTF DELIVERY TO PATIENTS The main limitations of this trial were the poor delivery of CNTF to striatal neurons, which no doubt prevented the detection of significant therapeutic effects. This can be attributed to several factors: the low survival of BHK cells, the limited number of implanted capsules (i.e., only one), and the location of the device, which was far from the vulnerable neurons. There are several explanations for the low survival rate of grafted cells inside brain parenchyma. BHK cells are mitotic cells that lack contact inhibition. Therefore, encapsulated BHK cells tend to divide and proliferate inside the capsule, limiting space and nutrient resources. Dying cells may also form cellular debris that accumulates inside the capsule that may be toxic to resident cells. Therefore, it is necessary to find resistant cells that survive better inside the capsule and express a high level of the trophic factor over a long period of time. The use of postmitotic cells may help the survival and function of the cells inside the capsule. An improved survival rate of the cells will also avoid the need of a surgical procedure every six months to replace the capsule. In addition, implanting a higher number of capsules and/or implanting them directly into striatal parenchyma, as in the primate experiment [68], may improve the outcome of the approach. This will certainly
lead to a higher CNTF concentration around affected striatal neurons and prevent dilution of CNTF inside cerebrospinal fluid. Recent experiments have shown that the neuroprotective effects of CNTF were highly dependent on the distance between encapsulated cells and target neurons [34, 58].
OTHER DELIVERY STRATEGIES: TOWARD IN VIVO GENE THERAPY In vivo gene therapy could also be used to obtain high levels of CNTF expression that would be appropriate for a significant physiological effect (both on neuronal survival and clinical symptoms). Viral vectors — such as adenoassociated viruses, lentiviruses, or adenoviruses — can transduce high numbers of cells and lead to stable transgene expression. CNTF-encoding lentiviruses have already shown strong neuroprotective effects in rodent models of HD [28, 82, 105], with no inflammatory response or major side effects being observed. The same encouraging results were obtained with CNTF-encoding adenoviruses in the 3NP-rat model of HD [69]. A large number of cells are transduced and the level of expression of human CNTF is high (from 2.8 to 15 ng/mg protein depending on the promoter) and stable over time [82]. Moreover, as CNTF is secreted the volume of the region in the brain where CNTF effects are visible is high and greatly exceeds the volume of transduced cells [66]. The use of viruses may prove to be an answer to some of the problems encountered with encapsulated cells, such as low levels of CNTF expression, limited spatial diffusion of CNTF through the human striatum, and a decrease in CNTF delivery over time. However, additional experiments still need to be carried out, in particular in primate models, to determine the feasibility and safety of this approach. Concerns such as long-term inflammation or immune response and the risk of insertional mutagenesis or recombination of vectors with wild-type viruses need to be addressed. A few clinical trials involving gene therapy have already been carried out [31]. Some used a direct gene transfer by adeno-associated viruses [53, 73], whereas another used ex vivo gene therapy with implantation of autologous fibroblasts infected with an NGF-encoding retroviral vector [98]. This clinical trial performed on eight Alzheimer’s disease patients has already reported beneficial effects in terms of clinical symptoms and brain metabolism [99]. However, gene therapy is still in its early days and extensive studies
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in animal models are required to confirm the feasibility, biosafety, and therapeutic efficacy of such treatments.
Conclusions The transplantation of engineered encapsulated cells has opened up a new field in therapeutic applications. For Huntington’s disease, which still lacks a treatment, the possibility of transplanting engineered cells overexpressing CNTF to prevent striatal cell death and alleviate clinical symptoms may lead to new therapies. The transplantation of encapsulated cells has many advantages compared to standard cell grafting: the method is reversible, does not involve immunosuppressive treatments, and provides trophic support for resident neurons through CNTF delivery. However, this technology needs to be improved further in terms of capsule location, choice of encapsulated cells, and level of CNTF production. Fundamental and clinical research needs to be continued to better evaluate this original therapeutic strategy with regard to other existing strategies, such as in vivo gene therapy and fetal cell transplants. The ultimate goal is to offer the best therapeutic solution to HD patients and families.
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23 Choroid Plexus Epithelial Cell Transplants for Repair of the Brain Christopher G. Thanos and Dwaine F. Emerich LCT BioPharma, Inc., Providence, Rhode Island
Introduction Within the central portions of the brain there are four interconnected channels referred to as the ventricles that are filled with cerebro-spinal fluid (CSF). The choroid plexus (CP) lies within these ventricles, where it produces the CSF and forms a unique interface between the peripheral blood and the CSF. The CP was first identified in the third century BC, but its functions have gone largely unexplored. Even contemporary neuroscience texts only briefly mention the role of the CD in CSF manufacture and formation of the blood-CSF barrier. Although studies looking at the physiology of the CP are in their infancy, several converging lines of evidence suggest that the CP plays pivotal roles in an extraordinary range of processes that establish, survey, and maintain the biochemical and cellular status of the central nervous system (CNS) under both normal and pathological conditions. As we learn more about the expansive participation of the CP in brain function across the life span of the individual, it becomes more apparent that this small and complex organ is subject to subtle anatomical and physiological changes that can have wide-ranging consequences. As outlined in Table 23.1, there is a long list of medical conditions with distinct choroidal pathology ranging from normal aging to Alzheimer’s disease (AD) and stroke [24, 76, 83]. Causal relationships between choroidal changes and these medical conditions are poorly understood in many cases. It is also likely that some changes in the CP are actually downstream sequelae from the primary clinical condition. These relationships will undoubtedly become clearer with further investigation. A basic tenet of this chapter
Cellular Transplantation: From Laboratory to Clinic
is that independent of whether the pathology of the CP is directly or indirectly linked to a given disease the brain is subjected to an additional stress that over time may exacerbate the primary medical condition or lead to new ones. At the same time, the CP appears to assist in recovery processes by secreting neuroprotective compounds [10] and acting as a site of neurogenesis [11, 49] — suggesting both an ongoing and “as-needed” role in cellular repair and replacement in the CNS. Here we discuss the role of the CP in development, normal brain functions, neuronal surveillance, aging, and selected CNS disorders. We also present the notion of using transplantable CP cells to repair the damaged brain and spinal cord.
Basic Structure of the Choroid Plexus The CP is located throughout the ventricles of the brain. Within the lateral ventricles, it projects from the choroidal fissure and extends from the interventricular foramen to the end of the temporal horn. It projects into the third and fourth ventricles from the ventricular roof. Grossly, the CP is lobulated with a single continuous layer of cells derived from the ependymal lining of the ventricles. Despite being derived from the ependymal lining, these cells possess epithelial cell characteristics and are often referred to as choroidal epithelial cells. The choroidal epithelial cells rest on a basal lamina, contain a large central spherical nucleus with abundant cytoplasm, and possess numerous villi on their luminal surface [14]. Adjacent epithelial cells are connected by tight junctions to physically restrict the movement
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Christopher G. Thanos and Dwaine F. Emerich TABLE 23.1 Medical conditions with choroid plexus pathology. Disease
Pathology
Aicardi syndrome Aging
Choroid plexus tumors/cysts common. Atrophy of choroid plexus, decreased CSF production, diminished polypeptide synthesis, and metabolic activity. Up-regulation of lysyl oxidase in choroid plexus of SAO mice and in ALS patients. Choroid plexus of ALS patients has elevated SOD activity/enhanced Cu/Zn-SOD staining. Atrophy of choroid plexus, decreased CSF production, diminished polypeptide synthesis, and metabolic activity beyond that seen in age-matched controls. Amyloid deposits seen in choroid plexus of humans. Enhanced expression of mRNA encoding the Toll-like receptor 2 in choroid plexus of mice. Choroidal lesions in subset of patients. Calcification of choroid plexus and increased CSF protein.
ALS
Alzheimer’s disease Amyloidotic polyneuropathy Autoimmune encephalomyelitis Chester-Erdheim disease Choroidal-cerebral calcification syndrome Dandy-Walker syndrome Depression Diabetes Folate deficiency Goodpasture syndrome Hendra virus encephalitis Huntington’s disease Leigh disease Listeriosis Lupus Menkes disease Minamata disease Mitochondrial encephalomyopathy Myotonic dystrophy Neu-Laxova syndrome Schizophrenia Sturge-Weber syndrome Trypanosomiasis Tuberculosis meningitis Unknown syndrome Von Hippel-Lindau disease
Hypoplasia of choroid plexus in rats following injection of 6-aminonicotinamide. CSF levels of TTR decreased in depressed patients. Intraperitoneal streptozotocin in rats disrupts ion transport in choroid plexus. Cerebellar syndrome, spinal muscular atrophy, and hearing loss associated with defect in choroid plexus folate binding. Depostion of host IgG, IGM, and Beta 1c in choroid plexus in autoimmune nephritis. Choroid invasion of virus in symptomatic guinea pigs. Enhanced immunostaining of 3-nitrotyrosine in transgenic R6/2 mice. Ubiquitous increase in mitochondria in epithelial cells of choroid plexus. Up-regulation of ICAM-1 in choroid plexus associated with leptomeningitis in infected mice. MRL-lpr mice show infiltration of lymphoid cells in choroid plexus. Mottled gene expression highest in choroid plexus of macular mutant mice. Mercury deposition on choroid plexus. Increased mitochondria, loss of microvilli, attenuated apical processes, and electron dense bodies in epithelial cells of choroid plexus. Accumulation of myotonic dystrophy protein in choroid plexus of rats and humans. Choroid plexus cysts. Calcification of choroid plexus and associated hallucinations. Angiomatous enlargement of choroid plexus in humans. Trypanosomes and inflammatory cells in choroid plexus of rats infected with Trypanosoma brucei brucei. Fibronectin and its receptor (VLA-4) also increased. Granulomatous lesions of choroid plexus. Calcification of choroid plexus associated with moderate facial irregularities in a mother and twin daughters. No impact on intelligence. Choroid plexus papilloma with chromosome 3 allele loss.
of substances to and from the CSF (i.e., the bloodCSF barrier). Ultrastructurally, the epithelial cells of the CP contain numerous mitochondria needed to maintain their high respiratory metabolism and energy requirements [12]. Their golgi apparatus contains columns of cisternae and smooth endoplasmic reticulum, and clear
vesicles are distributed throughout the apical cytoplasm. Underlying the epithelial cells and basal lamina is a dense vascular bed that provides a blood flow four to seven times greater than the rest of the brain [22]. The capillaries are large, with thin fenestrated endothelial walls and bridging diaphragms overlying
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the fenestrations. An extensive array of adrenergic, cholinergic, peptidergic, and serotoninergic nerve fibers innervate the blood vessels and the epithelium [71].
Traditional Roles of the Choroid Plexus The most recognized function of the CP is CSF production [90]. In humans, CSF volume is 80 to 150 mls, and new CSF is formed at a rate of approximately 500 ml/day. CSF is produced mainly by active secretion with water entering the CSF from the blood along an osmotic gradient or by specific water channels such as aquaporin [72]. The epithelial cells replenish the CSF by moving Na+ , Cl− , and HCO− 3 from the blood to the ventricles to create the osmotic gradient that drives the secretion of H2 O. The CSF is a clear and slightly viscous liquid with few cells and little protein [77]. The CSF has a lower pH and concentrations of glucose, potassium, calcium, bicarbonate, and amino acids than blood plasma. In contrast, sodium, chloride, and magnesium content are greater in CSF than in plasma. Folate levels are two to three times higher in CSF than in plasma, and the tetrameric human plasma protein transthyretin (TTR) accounts for 25% of all CSF proteins [91, 103]. Within the CP, the barrier function is shifted from the vasculature to the epithelium, where tight junctions form between the epithelial cells to confer the permeability properties of the individual cells [101]. On the external surface of the brain, the epithelial cells fold over onto themselves — forming a double-layered structure between the dura and pia known as the arachnoid membrane. As in the CP, tight junctions between adjacent cells restrict the passage of substances from the blood across the arachnoid membrane. The arachnoid membrane is generally (but not completely) impermeable to hydrophilic substances, and its role in forming the blood-CSF barrier is largely passive [33].
Choroid Plexus as a First Line of Defense for the Brain Lying within the central ventricular system, the CP is in an ideal position to monitor the CSF for the presence of noxious compounds or potentially damaging cellular invasion. The CP protects the brain against acute neurotoxic insults by using a complex, multilayered detoxification system [29, 31]. The CP aids or impedes the overall biodistribution of drugs and toxic
compounds by being the source of a full complement of metabolizing enzymes, including phases I through III enzymes for functionalization, conjugation, and transport of drugs. CP contains (1) high concentrations of glutathione, cysteine, and metallothioneine that potently sequester toxic agents circulating in the CSF, (2) protective enzymes such as superoxide dismutase, glutathiones-transferase, and glutathione peroxidase and reductase to provide a barrier protecting against free-radical oxidative stress, and (3) organic ion transport systems and multidrug resistance proteins for exportation of noxious compounds from the CSF. A second example of the monitoring and modulating role of the CP comes from a recent understanding of its function within the neuroimmune system [18]. Traditionally, the CNS has been considered an immunologically privileged site with no inherent need for immunosurveillance. The first indication that the CP mediates interactions and/or signaling between the peripheral immune system and the brain came from demonstrations that the CP contains inducible lymphoid cells. Following systemic administration of lipopolysaccharide or IL-6 there is a rapid and transient induction of interleukin IL-1b and TNF-a. This activation initiates a process that ultimately spreads throughout the brain, suggesting that the CP relays information between the immune system and the brain through the coordinated local induction of proinflammatory cytokines [54]. Choroidal epithelial cells also constitutively express MHC class I molecules, and in some species (such as mice) they express MHC class II molecules that are inducible with infectious agents such as rabies virus [44, 84]. In vitro, epithelial cells present foreign antigen and stimulate T lymphocyte proliferation through an MHC class II restricted mechanism [69]. Accessory molecules important for leukocyte adhesion — such as L-selectin, ICAM-1, and VCAM-1 — are found at low levels on CP epithelial cells but can also be up-regulated during inflammation [95, 106]. Other cells (including the Kolmer cells of the CP) that normally act as phagocytic scavenger cells also display inducible MHC class I and II antigens and proliferate when challenged with endotoxins [59]. This antigen presentation capacity implies that the CP is part of an intrinsic surveillance system defending against blood-borne pathogens and CSF-localized antigens. The pathogen-induced inflammation within the CP is actually not surprising given the tropism that bacteria, parasites, and viruses such as Neisseria meningitidis and Trypanosoma brucei, Sendai virus, mumps virus,
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and perhaps even HIV-1 and human T-cell leukemia virus-1 (HTLV-1) have for the CP [57, 93]. HIV-infected T lymphocytes and monocytes are often observed in the stroma and supraepithelial portions of the CP, suggesting that the CP is a pathway of entry for infected cells into the brain [19, 34]. HIV-1-positive cells are initially found in the subarachnoid and perivascular spaces [3] but extend into the CSF of HIV-1 and HTLV-1 infected patients during the subsequent stages of infection [60, 66]. The means by which infected leukocytes or activated T lymphocytes cross the CP is unknown, but it is intuitively obvious that their crossing can be disastrous. The CP might be involved in the entry of activated, myelin-directed autoreactive T lymphocytes during multiple sclerosis (MS) [93, 107]. Activated T lymphocyte infiltration into the brain results in the formation of demyelination plaques that underly the clinical symptoms of MS. Because these plaques are frequently located in the periventricular area, the CP may constitute a preferential way for T lymphocytes to reach these structures. It should be noted that T lymphocytes and T lymphocyte chemoattractants are found in the CSF drawn from MS patients.
TABLE 23.2 Receptors in the choroid plexus. Angiotensin II Anionic pesticides Antipyrine Apolipoprotein E Apolipoprotein J (clusterin) Atrial natriuretic peptide Atropine Barbitol Bradykinin Brain-derived neurotrophic factor Cefodizime Cimetidine Corticotropin releasing factor Digotoxin Diphenhydramine Endothelin Gentimicine Insulin Insulin-like growth factor Interleukin-1 Leptin Lidocaine Methadone Methotrexate Morphine Nerve growth factor Neurotrophin-4 Penicillin Prolactin Proline Salicylic acid Tetrahydrocannabinol Transforming growth factor-b Vascular endothelial growth factor Vasoactin intestinal polypeptide Vasopressin
Central Secretory Role of the Choroid Plexus in Brain Function The CP is in the perfect location for distributing molecules both locally and globally to the brain. The CP possesses numerous specific transport systems, contains a broad array of receptors, and serves as a major source of biologically active compounds (see Tables 23.2 and 23.3). These capabilities allow the CP to monitor and respond to the biochemistry of the brain by manipulating and maintaining baseline levels of the extracellular milieu throughout the CNS [10, 92]. The molecules secreted by the CP gain proximal and distal access to the brain parenchyma via volume transmission, convective distribution, and intraparenchymal diffusion/receptor-mediated retrograde transport to neurons whose endings are located near the ependyma or the pia-astroglial membrane [2, 23, 67]. In this broad view, the CP plays an integral role in autocrine/paracrine and distal/endocrine-like effects on target cells in the brain by producing CSF and polypeptides and distributing them from the ventricles to cells within the brain and spinal cord. Together with its secretory capacity, the CP possesses an extensive array of polypeptide receptors,
Table modified from [10].
suggesting currently unknown roles in receptor-ligand mediated regulation of choroidal hemodynamics, transport, secretion, and CSF clearance.
The Choroid Plexus in CNS Early Development CPs develop early during embryogenesis (seventh week of gestation in humans), where they help control the
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neurons from the ventricular and subventricular zone to the cortical plate is governed by gradients of soluble factors — such as Slit proteins secreted by the CP [40, 70]. Hu [40] demonstrated in vitro that a soluble factor related to Slit2 is secreted by the CP to diffuse through the CSF and aid in establishing a gradient of a repulsive cue guiding cortical neurons away from the ventricular surface. Along these same lines, Yamamoto and colleagues showed that the CP modulates neurite outgrowth in the developing cerebellum [109]. Using co-cultures of explanted cerebellum and fourth ventricle CP from fetal and infant rat, it was confirmed that CP secretes a soluble neurite-growth factor that is biphasic and correlates with the major milestones of cerebellar morphogenesis. The importance of the CSF distribution of soluble factors during development is also highlighted by the eloquent work of Miyan and colleagues [47, 61, 65, 73], who showed that hydrocephalus in rats significantly impairs cortical development — leading to the suggestion that the circulating CSF and its associated factors are vital for development along the entire length of the neural tube.
TABLE 23.3 Polypeptides in the choroid plexus. Adrenomedullin Apolipoprotein J b-Amyloid precursor protein Basic fibroblast growth factor 1 & 2 Brain-derived neurotrophic factor Prostaglandin D synthase Cystatin C Endothelin-1 GD-15 GDNF Hepatocyte growth factor Insulin-like growth factor-II Insulin-like growth factor binding protein 2–6 Interleukin-1b Interleukin-6 Nerve growth factor Neuroglobin Neurotrophin-3 and 4 Transferrin Transforming growth factor-alpha Transforming growth factor-b1-3 Transthyretin Tumor necrosis factor-alpha Vascular endothelial growth factor Vasopressin
The Choroid Plexus in Aging
Table modified from [10].
extracellular environment [15]. Within the immature brain, the blood-CSF barrier is functional and limits passage of substances from the CSF to the brain parenchyma. However, whereas the CP is functional early in development its permeability is tailored for the embryonic brain — allowing low-molecular-weight compounds to enter the brain more easily than in adulthood. Among the compounds secreted by the developing CP are morphogens, mitogens, and trophic factors that guide and pattern both the general and specific growth of the CNS [5, 27, 35, 40, 55, 70, 99, 109]. For instance, the embryonic CP contains high levels of IGF-II. Based on the localized and high expression of IGF receptors in the floor plate of the hindbrain, it has been hypothesized that CP-derived IGF-II diffuses to and binds to IGF receptors on the floor plate cells and activates their role in guiding spinal axon growth [5]. As pointed out by Straziele and colleagues [93], additional support for the role of CP in morphogenesis comes from demonstrations that the radial migration of cerebral cortical
The majority of our knowledge about the morphology and function of human CP during aging comes from control tissues in studies investigating changes in Alzheimer’s disease (AD). In humans, the height of CP epithelial cells decreases by about 10 to 11% during life [85]. The aged epithelial cell cytoplasm becomes rich with Biondi ring tangles and lipofuscin deposits [104], and the nuclei appear irregular and flattened as the basement membrane thickens [85]. The stroma also thickens (and contains collagen fibers, hyaline bodies, calcifications, and psammoma bodies), and the infiltrating arteries become thicker and fragmented [83, 89]. These changes are not species specific, as similar changes have been seen in the aged mouse and rat choroid epithelial cells [88, 94]. The functions of the CP are energy dependent, and the aging CP cannot maintain its normal energy output. Synthesis of enzymes needed for anaerobic respiration and oxidative phosphorylation decline in aging rats with lactate dehydrogenase and succinatedehydrogenase decreasing 9 and 26%, respectively [26]. There are age-dependent increases in the number of epithelial cells deficient in cytochrome C oxidase, altering the respiratory mitochondrial chain and
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decreasing cellular production of ATP [64]. Reductions in Na+ K+ -ATPase and the Na+ K+ -2Cl− co-transporter also occur [13]. These anatomical and enzymatic deteriorations could lead to diminution of CSF secretion, which is decreased by as much as 45% in animal models [76]. Due to the decreased secretion and the simultaneously increased CSF volume caused by brain atrophy, CSF turnover takes significantly longer in elderly rats (7.9 hours) than in young rats (2.2 hours). In man, CSF production has been reported to diminish with age, from 0.41 ml/minute at 28 years to 0.19 ml/minute at 77 years. Coupled with age-related cerebral atrophy, the turnover of CSF decreases to less than two times daily in elderly subjects versus three to four times per day in young adults. Elevated CSF/plasma ratios have been reported for several proteins during aging [76]. The effects of these cumulative changes on brain functioning have not been directly tested, but it seems reasonable to assume that such dramatic alterations in the CP and CSF would trend toward inadequate distribution of nutritive substances, additional cellular stress, and reduced clearance of toxic compounds — all of which could play a part in age-related cognitive and motor decline or the development of specific neurological disorders.
Choroid Plexus and Neurodegeneration: Alzheimer’s Disease and Ischemia as Examples ALZHEIMER’S DISEASE As the impairments in CP function increase, the associated medical consequences become more devastating. The age-related deficiencies of the CP are exacerbated in AD. Epithelial cell atrophy is greater, with cell height decreasing up to 22% relative to age-matched controls [85]. Greater intracellular distribution of lipofuscin vacuoles and Biondi ring tangles occurs [64, 104]. The epithelial basement membrane becomes very irregular and thickens an additional 28% beyond that seen in agematched controls [85]. The stroma of the villi becomes fibrotic with extensive vascular thickening [45, 85], and numerous hyaline bodies and calcifications are found along with deposits of IgG, IgM, and C1q along the epithelial basement membrane. Further decreases in CSF secretion occur, with turnover requiring up to 36 hours in AD patients [83].
The further atrophy of the choroidal epithelial cells in AD is associated with pronounced decreases in secretory activity and transport functions. Levels of TTR, a CP synthesized molecule that associates with b-amyloid peptide to form complexes, are > 10% lower in AD [87]. Ascorbic acid and a-tocopherol levels, the two major scavengers of free radicals of CSF, are decreased in AD — likely adding to oxidative stress [80, 96]. CSF folate and vitamin B12 (important for methylation of numerous molecules) are significantly lower [42, 82, 86], whereas homocysteine — which mediates lipid peroxidation and increases the production of toxic (E)-4-hydroxy-2-nonenal — is increased in AD CSF. The impaired ability of the CP to clear molecules from the CSF of AD patients has potentially profound implications [83]. In rats, clearance of intraventricularly injected b-amyloid peptide decreases from 10.4 µl/minute at 3 months of age to 0.71 µl/minute at 30 months. Consequently, the brain content of amyloid peptide increases from 7% at the end of CSF perfusion in young rats to 49% in old animals [76]. The increase of b1-40 and b1-42 amyloid peptide levels in elderly humans could be related to decreased clearance from the CNS. Decreased CSF production could also enhance protein glycation and the formation of b-amyloid oligomers [83]. AD brain contains elevated levels of glycation products and deposits of amyloid peptide. Senile plaques and fibrillary tangles contain advanced glycation products [83]. Glycation promotes protein aggregation, the polymerization of tau microtubule associated proteins, and protein b-amyloid peptide aggregation. The decrease of CSF turnover, the increase of protein glycation, and the diminution of b-amyloid clearance could also induce oligomer formation and retention. As pointed out by Serot and colleagues [83], these changes could lead to an even greater impoverishment of the brain — leading to methylation problems, increased oxidative stress and lipid peroxidation, decreased amyloid clearance, augmented tau protein polymerization, and amyloid peptide oligomer and fibrillo formation.
ISCHEMIA Numerous anatomical and secretory changes occur in CP following cerebral ischemia [24]. Middle cerebral artery (MCA) occlusion in rats induces apoptotic cell death, with nuclear DNA breaks occurring after 6 hours of ischemia [32]. The DNA fragmentation and cell death
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in the CP can affect neighboring neuronal systems. Following ischemia, the CA1 pyramidal neurons in the hippocampus undergo delayed neuronal death, evident around 48 hours after the insult. It is unclear why this delayed cell death occurs, but an analysis of the time course of cell loss in the CP versus the hippocampus supports the possibility that ischemia initiates a cycle of cell loss in which the death of CP cells at least precedes and perhaps adversely affects neighboring neurons [25]. DNA fragmentation occurs in the CP 24 hours after ischemia but diminishes by 48 hours. In contrast, at 48 hours DNA breaks are widespread in the medial CA1 region and at 72 hours DNA fragmentation spreads laterally within the CA1 region [24]. Subsequent studies confirmed and extended these data, showing a dramatic increase in TUNEL-positive cells in the CP at 18 to 36 hours post ischemia but not at 48 hours. No TUNEL-positive cells were seen at 24 hours in the CA1 hippocampal region, clearly demonstrating that the cell death in the CP preceded cell death in the CA1 region. Whether CP cell death impacts distant neurons through disruption of the blood-CSF barrier or changes in the production of specific secreted factors remains unknown. The CP may produce or fail to produce factors capable of minimizing neuronal damage. Indeed, there is evidence of time-dependent increased production of factors capable of mitigating neuronal loss. These changes include (1) elevations in hippocampal cystatin C, a cysteine protease inhibitor produced by the CP, following ischemia that is temporally similar to the delayed neuronal death within the hippocampus [74], (2) enhanced growth hormone reactivity following ischemia in rats [79] in injured neurons, axons, glial cells proximal to the infarction, and on the CP epithelial cells, and (3) increased expression of TGFb (isoforms b1, b2, and b3) in CP following ischemia in rats [10, 50, 51]. In each case, augmented synthesis of these choroidal molecules may play a role in neuronal survival and the recovery of function. Indeed, intraventricular administration both of TGFb1 and growth hormone reduces the size of the infarct following ischemia [36, 37, 79]. These data also highlight the complexity of the response of the CP to an ischemic event and imply that CP cell loss can adversely impact the death of neighboring neurons even though at the same time surviving CP cells may produce factors that can beneficially affect the outcome of neurons. Extensive cell death in the CP will lead to a leaky blood-CSF barrier, allowing substances released into the CSF to gain greater access to parenchymal tissues. Whether this altered CSF
composition is toxic or beneficial to neurons has never been directly tested. One important experiment would be to sample the CSF from animals at different times postischemia and inject that CSF into normal and ischemic animals [24]. If the CSF contains harmful or beneficial substances, the extent of the lesion should be impacted accordingly. A careful analysis of the composition of the CSF together with detailed histological studies of the impact of the agents within the CSF on different neuronal populations would shed considerable light on the role of the CP following ischemic damage. Such studies should also not rely on a single experimental model of inducing ischemia.
Choroid Plexus and Neurogenesis In the last 10 years it has become apparent that the brain is not static and immutable but possesses a considerable ability to generate new cells from locally discrete populations of stem cells. Although the ependymal and subependymal layers of the lateral ventricles have received greater attention as stem cell sources, the CP is an extension of these structures and also possesses remarkable neurogenic capabilities. First, proliferative and immunocytochemical markers clearly show stem cells in the CP. CP epithelial cells actively generate mitogens such as amphiregulin for regulating stem cell proliferation and neurogenesis [4, 20, 43, 53]. Second, choroidal stem cells proliferate in response to trauma. Li and colleagues [58] demonstrated that CP cells proliferate and differentiate after stroke in adult rats. MCA occlusion in rats increased bromodeoxyuridine immunoreactivity colocalized with neuronal nuclear antigen and glial fibrillary acidic protein (GFAP) immunoreactivity in the CP of the ischemiaaffected hemisphere. Third, transplant studies demonstrate that when grafted into damaged regions of the CNS CP cells have the ability to differentiate. Kitada et al. [49] isolated CP from green fluorescent protein (GFP)-transgenic mice and grafted it into the damaged spinal cord of syngeneic mice. One week after injection, GFP-positive transplanted cells became immunohistochemically positive for GFAP but negative for neuronal markers. Two weeks after grafting, immunoelectron microscopy showed that the GFP-positive transplanted cells that had gained GFAP immunoreactivity contained numerous bundles of intermediate filaments, a morphological characteristic of astrocytes, and were in close contact with adjacent host tissue. Future studies should
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focus on isolating these cells, evaluating their multilineage potential and exploring pharmacological means of facilitating the native neurogenesis response that occurs following stroke to further optimize the endogenous repair mechanisms of the CP.
Harnessing the Choroid Plexus for Transplantation Therapy: Preliminary Studies The profound age- and disease-related changes in CP have clear implications for understanding disease processes but also raise the unexplored possibilities that replacing damaged CP or transplanting healthy CP into local areas of cell degeneration can be therapeutic [16]. The diminished function of the aged CP may be much like any number of diseases characterized by secretory cell dysfunction. In principle, transplanting or replacing a failing organ (such as CP) or specific cell type is a logical and direct means of restoring lost function. There is also the potential of harnessing the polypeptide synthesis of the CP through focused local delivery after transplantation into an ectopic brain region. The endogenous role of the CP in growth factor and nutrient production makes these cells a novel and viable source of stable dose-controlled polypeptide delivery. Although the notion of using transplanted CP cells to the damaged brain regions is a new idea, the studies conducted to date support the concept and warrant further investigation. CP isolated and maintained in vitro exerts potent neuroprotective effects [6, 7]. Conditioned media from alginate encapsulated and cultured CP protected embryonic cortical neurons against serum deprivation-induced cell death. This effect was dose dependent and nearly complete when neurons were cultured with 10 to 30% conditioned media. Conditioned media from CP also enhanced neurite outgrowth and dopamine uptake on cultured neuroblastoma cells and on embryonic mesencephalic tissue, respectively. These data dovetail nicely with a study where mouse CP epithelial cells were cultured with dorsal root ganglion (DRG) neurons [9]. After 4 to 5 hours of co-culture, the DRG neurons developed elongated neuronal processes with elaborate branching patterns over the surface of the epithelial cells. The ability of CP cells to provide a scaffold for the extension of neurites is consistent with its known production of extracellular matrices, including laminin and fibronectin [75, 110]. The trophic and tropic effects of CP establish
potentially excellent circumstances for the protection and repair of damaged CNS architecture. The delivery of neurotrophic factors via CP transplants to the site of injury also offers theoretical promise for treating spinal cord trauma. Recently, Ide and colleagues [41] grafted syngeneic fragments of CP into the dorsal funiculus (C2 level) in a damaged adult rat spinal cord. At various times posttransplant, subsets of animals were evaluated histologically to confirm cell survival and determine any regenerative effect on the damaged spinal cord. Electron microscopy and fluorescence histochemistry showed that epithelial cells of the grafted CP survived well and induced a robust regeneration of the damaged axons of the spinal cord. Injections of horseradish peroxidase into the sciatic nerve labeled regenerating fibers extending from the fasciculus gracilis into the graft within 7 days posttransplant. This effect was evident for at least 10 months. Some axons elongated rostrally into the dorsal funiculus, and longduration-evoked potentials were recorded 5 mm rostral to the lesion 8 to 10 months after grafting. These findings indicate that CP epithelial cells have the ability to facilitate axonal growth, suggesting that they are capable of exerting both trophic and tropic effects in vivo.
Immunoisolation within Alginate Microcapsules Enables the Use of Xenogeneic Choroid Plexus Transplants Immunoisolation is based on the observation that xenogeneic cells can be protected from host rejection by encapsulating (surrounding them within) an immunoisolatory semipermeable membrane. Single cells or small clusters of cells can be enclosed within a selective semipermeable membrane barrier that admits oxygen and required nutrients and releases bioactive cell secretions but restricts passage of larger cytotoxic agents from the host immune defense system. The selective membrane eliminates the need for chronic immunosuppression of the host and allows the implanted cells to be obtained from nonhuman sources, thus avoiding the constraints associated with cell sourcing that have limited the clinical application of unencapsulated xenogeneic cell transplantation. There are generally two categories for cell immunoisolation by encapsulation; namely micro- and macro-, each with some benefits and limitations [17]. To date, all of the studies using encapsulated CP for CNS transplant
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studies have employed microcapsules formed using alginate. Alginate is one of the most frequently investigated biomaterials for cell encapsulation and is a polysaccharide composed of guluronic (G) and mannuronic (M) acid linked by (1,4)-b- and -b-glycoside bonds. The ratio of these monomers contributes directly to certain physical characteristics of the polysaccharide. Once cationically cross-linked, materials high in G due to a more networked structure resulting from b(1-4) bonds are more brittle whereas those high in M — with more linear b(1-4) linkages — exhibit decreased 3D cross-linking and greater elasticity. Alginate has long been studied as a biomaterial in a wide range of physiologic and therapeutic applications. Its potential as a biocompatible implant material was first explored in 1964 in the surgical role of artificially expanding plasma volume [68]. More than a decade later, the matrix capability of alginate for cell support was realized in vitro in a series of experiments that demonstrated microbial cell survival for 23 days [48]. Over the last 20 years, there has been remarkable progress in alginate cell microencapsulation for the treatment of diabetes [21, 28, 38, 52, 56, 81, 98, 102], chronic pain [46], hemophilia [30, 39], CNS disorders [1, 62, 63, 78, 97, 100, 105, 108], and others.
Characterization of Alginate and Encapsulated Choroidal Epithelial Cells Prior to cell encapsulation, alginate powder is typically reconstituted in a suitable buffer, and a variety of purification techniques are employed to rid the solution of proteins, endotoxin, and polyphenols. These include solvent extraction, sequential filtration, charcoal extraction, dialysis, and others. Contaminant removal is essential to maintaining the optimal balance of hydrophilicity and to preventing inflammation related to endotoxin. It is the purification process that will ultimately determine the final physical and chemical characteristics of the encapsulated cell product, as fine variations in copolymer ratio, molecular weight, and purity can all be controlled at this step. Following purification and reconstitution of the alginate solution at a suitable pH, quality control analysis is carried out to maintain optimal operating specifications for encapsulation and subsequent in vivo longevity. The final purified alginate can be characterized both as a raw material and as a formed capsule using the analytical techniques outlined in Table 23.4. Prior to encapsulation, neonatal porcine CP (7 to 14 days of age) is isolated from the lateral ventricles and dissociated using conventional collagenase
TABLE 23.4 Analytical techniques for alginate characterization. Analytical Method
Purpose Bulk Material Analysis
1 H-NMR
SEC-MALLS Dynamic viscosity Protein concentration FTIR Endotoxin
Uronic acid ratio (M:G). Critical for physical and chemical stability of formed capsules. Weight-average molecular weight (Mw ) calculations for overall chain length and sample homogeneity (polydispersity). Used to calculate intrinsic viscosity and molecular weight. Useful for controlling droplet formation and encapsulation procedure. Bulk quantification of alginate purity and potential for degradation and host response. Alginate purity and comparison against a standard. Useful for characterizing stability based on ratio of alginate to polycation peaks. Quantification of endotoxin impurities per FDA guidelines and to minimize tissue response. Alginate-polycation Microcapsules
Viability Diffusion Burst Post-encapsulation phenotype Microbiology/virus-screening morphometry
Vital dye for determination of dosing, vitality, and biomass of encapsulated product. Predictor of isolation capability and pharmacokinetic potential. Bulk modulus of material and ultimate physical strength of microcapsule. Confirmation of potential cell functionality and morphology. Screen for product acceptance and lot release. Batch polydispersity and average size of capsules and their respective wall thickness.
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digestion procedures. The resulting cell clusters are groupings of epithelial cells ranging from 50 to 200 µm in diameter. Prior to encapsulation, cell cluster viability is confirmed by staining with a representative sample of the harvested cells with a vital dye. As shown in Figure 23.1, the viability of the isolated CP is excellent (typically > 95%). The encapsulation process does not impact cell viability, and these cells can be maintained in culture for months if needed or desired. The cultured CP clusters also appear to maintain the typical genotypic and phenotypic characteristics of the native undigested tissue. Because the epithelium of the choroid plexus is rich in tight junctions and lined with microvilli, it is possible to use immunocytochemical techniques to identify tight junctional complexes (ZO-1 [zonnula occludens]: a tight junction-associated polypeptide) and the tubulin associated with the cytoskeleton of the microvilli. Figure 23.2 shows a representative sample of both undigested CP and several freshly digested CP clusters.
Figure 23.1. Viability of unencapsulated and encapsulated CP cell clusters. Cells were stained with a vital dye (live/dead, molecular probes) and analyzed with confocal laser-scanning microscopy. Using simultaneous two-wavelength capture, green (live) and red (dead) cells are visualized. Cell clusters in (a) are approximately 100 µm in length, and capsules in (b) are approximately 500 µm in diameter. See color insert.
Following confirmation of cell viability and phenotype, the CP cells are encapsulated in alginate microcapsules by extruding a mixture of cells dispersed in 1.7% sodium alginate through a droplet-generating apparatus into a bath of cations. This process is typically performed at an encapsulation density of 10,000 to 50,000 clusters or 200,000 to 5 million cells/ml alginate. The cells, entrapped in the calcium-alginate gel are coated twice with polycations, followed by an outer coat of alginate. The central core of alginate is liquefied by the addition of a suitable chelator. The resulting microcapsules have a diameter of between 500 and 750 µm (Figure 23.1).
Encapsulated Xenogeneic Choroid Plexus Transplants in Animal Models of Stroke Stroke is the third leading cause of death and a leading health care burden in developed countries. Although the incidence of stroke has declined over the past decades, there are no effective treatments for mitigating the neuronal loss following stroke. Neural transplantation may be one means of repairing the stroke-ravaged brain, as validated in numerous laboratory studies. Initial clinical trials of neural transplantation therapy for stroke have also been initiated with generally encouraging results. The majority of these laboratory and clinical studies is attempting to integrate transplanted cells into the brain after the initial trauma for reconstructive purposes. Delivery of therapeutic molecules via cell transplantation soon after stroke can also be used to reduce or prevent the disease pathology. Based on these considerations, isolated CP obtained from rodents was tested for its neuroprotective effects in a conventional rodent model of stroke [6, 7]. Rats received a one-hour MCA occlusion immediately followed by transplantation of alginate-encapsulated CP on the cortex overlying the striatal region that would be normally infarcted following MCA occlusion. Behavioral testing on days 1 to 3 following surgery using the elevated body swing test and Bederson neurological examination revealed profound motor and neurological impairments in control animals that were significantly improved in the study group who received subdural alginate-encapsulated CP transplants. Histological analysis revealed that the behavioral improvements were accompanied by a significant decrease (approximately 35 to 40%) in the volume of striatal infarction as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. This paradigm might have actually
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Figure 23.2. Choroid plexus immunohistochemistry captured with confocal laser-scanning microscopy. ZO-1 is shown in green and a-tubulin is red. (a) intact CP, 40X; (b) CP cluster, 40X; (c) CP cluster, 60X. See color insert.
underestimated the therapeutic potential of CP grafts because the effecter molecules were required to diffuse out of the capsules and through several millimeters of cortical tissue. Accordingly, the concentrations of therapeutic molecules reaching the infarcted region were modest compared to those that might be achieved locally. Nonetheless, even under these less than ideal conditions a significant structural and functional benefit was produced by the CP transplants. Future studies should, however, carefully consider alternative transplant sites as well as the possibility of using single-cell suspensions of choroidal epithelial cells to potentially augment the benefits obtained to date.
Encapsulated Xenogeneic Choroid Plexus Transplants in a Rat Model of Huntington’s Disease Huntington’s disease (HD) is a devastating autosomal dominant neurodegenerative disorder characterized by an intractable course of mental deterioration and
progressive motor abnormalities that invariably results in death. There are no effective treatments. Unlike many other neurodegenerative diseases, the polyglutamine expression in HD permits an unequivocal diagnosis of HD early in life, even in utero. The ability to identify presymptomatic individuals provides the opportunity to design interventions that could intercede before the development of substantial neurodegeneration and the expression of the behavioral changes. Accordingly, the preservation of the neuronal cytoarchitecture and physiology of the striatum could be maintained while forestalling the debilitating consequences of the disease. Recent preclinical studies have demonstrated that encapsulated cell-based intracerebral delivery of neurotrophic factors preserves multiple populations of striatal neurons, including GABAergic neurons in animal models of HD. To determine if CP transplants have therapeutic potential in HD neonatal porcine CP was encapsulated within alginate microcapsules and tested for its neuroprotective potential in a rat model of Huntington’s disease [8]. In these studies, the animals received stereotaxic transplants of either empty capsules or
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CP-loaded capsules directly into the striatum. Three days later, the same animals received unilateral injections of the excitotoxin quinolinic acid (QA; 225 nmol) into the ipsilateral striatum. Transplanted animals gained body weight post surgery more rapidly than controls (Table 23.5) and demonstrated significant improvements on the placing test. After surgery, animals were also behaviorally tested for function of their forepaws using the placement test. As expected, the QA lesion produced a profile of anatomical and behavioral effects that resembles those seen in the clinical condition. When given 10 trials on the behavioral test, the control rats were only able to make one or two directed motor responses. In stark contrast, the rats receiving CP transplants were virtually indistinguishable from normal animals on this task, as they made greater than 9 out of 10 correct responses. Nissl-stained sections further demonstrated that CP transplants significantly reduced the volume of the striatal lesion produced by QA by approximately 80%.
Encapsulated Xenogeneic Choroid Plexus Transplants in a Monkey Model of Huntington’s Disease Based on the positive neuroprotective effects observed with CP transplants in the QA rodent model of HD, we recently conducted a similar experiment in monkeys. Seven young adult cynomolgus primates were
employed in this study. Using stereotaxic techniques, 20 cell-loaded capsules were loaded into a cannula and implanted into the head of the caudate and the right putamen. A total of four monkeys received cell-loaded implants, whereas three monkeys served as controls and received implants of empty capsules. Seven days following capsule implantation, each monkey received an injection of QA (5 µl for a total of 900 nmol of QA) approximately 2 mm posterior to the previous implant site. All monkeys were sacrificed four weeks after the QA lesion. The brains were removed and frozen sections (40 um) were cut on a sliding microtome. A mouse antineuronal nuclei (NeuN) monoclonal antibody was used to label striatal neurons for subsequent quantitative determinations of striatal cell counts and lesion volumes. The number of NeuN immunoreactive (NeuN-ir) neurons within the caudate and putamen nuclei was estimated stereologically using an optical fractionator unbiased sampling design. Using stereological principles, NeuN-ir neurons in each case were sampled by a uniform, systematic, and random design procedure. The total number of NeuN-ir neurons within the caudate and putamen was calculated using the following formula: N = Q−•1/ssf•1/asf•1/tsf. Q was the total number of raw counts. The volume of intact striatum was also estimated on a series of equispaced NeuN-ir sections along the striatum. The histological results paralleled those observed in the previously described rodent study (Table 23.6). In controls (animals receiving QA and empty capsule implants), QA administration produced a large lesion
TABLE 23.5 Weight loss in QA-lesioned rats receiving choroid plexus transplants. Days Post QA
QA /QA + empty capsule implant QA + choroid plexus implant
1
3
5
7
24
105.13 106.54
97.59 110.18∗
95.9 115.91∗
97.94 123.97∗
104.29 131.39∗
∗ p < 0.05 versus control. Data are presented as percentage of pre-surgery body weight.
TABLE 23.6 Neuronal counts in QA-lesioned monkeys receiving choroid plexus transplants. Treatment
Intact Striatum
Lesion/Implanted Striatum
Cell Loss (%)
QA + empty capsule implant QA + choroid plexus implant
41959437 ± 1309554 42031113 ± 409306
23965075 ± 1557936∗ 38615375 ± 6012797+
43 8
∗ p < 0.0001 versus intact striatum, +p < 0.001 versus QA lesioned striatum.
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Figure 23.3. Low- (a, b) and high-power (c, d) photomicrographs through the striatum of NeuN immunostained sections from (a, c) QA lesion plus empty capsules and (b, d) QA plus CP containing capsules in monkeys. Note a large degenerative area seen in both caudate and putamen nuclei in QA lesion side (a, arrows). In contrast, the lesion size was significantly diminished in the CP transplanted striatum (b, arrows). At higher magnification, there were almost no NeuN immunoreactive neurons observed in the QA lesion area (c). (d) Numerous healthy-appearing NeuN immunostained neurons were seen in CP transplanted striatum. Scale bar in a represents 0.5 cm in b.
in both the caudate and putamen nuclei — as shown in NeuN stained sections (Figure 23.3). The lesion site encompassed much of the caudate and putamen nuclei before the anterior commissure. With the exception of some occasional NeuN-positive debris and shrunken neurons, the lesion core was virtually devoid of NeuN positive neurons. In the same cases, a mild lesion was observed along the needle tract in the overlying cortex but no lesion was seen in the nucleus accumbens. In contrast, the size of lesion was notably reduced in animals receiving QA and implants of encapsulated CP. In these animals, the core of lesion was minimal and limited to a small defined area at the tip of the injection site. Immediately outside of this central core, but still adjacent to the needle tract, numerous healthy NeuN-ir neurons with dendritic NeuN immunoreactivity were observed within both the caudate and putamen nuclei. Stereological counts of NeuN-ir neurons were performed to quantify the degree of neuronal loss produced by QA and the protection mediated by implants of encapsulated CP. This analysis confirmed the gross histological assessment, revealing that relative to the intact striatum QA produced a marked loss of NeuN-ir striatal neurons (43%) that was significantly prevented by
prior implants of encapsulated choroid plexus (only an 8% loss of neurons; Table 23.6, Figure 23.3). Results from the quantitative analysis of the volume of intact striatum paralleled the stereological cell counts described previously. Relative to the intact striatum, animals receiving QA and empty capsules exhibited large lesions characterized by a 40% decrease in striatal volume (745.508 mm3 vs 446.825 mm3 ). Conversely, the striatum volume was 672.228 mm3 in animals previously implanted with encapsulated CP, which did not differ significantly from the volume of the intact striatum. Together, these studies are the first demonstration that implants of CP can provide trophic influences to degenerating striatal neurons in the rodent and primate brain and suggest that this strategy may ultimately prove relevant for the treatment of HD.
Conclusions Sometimes history repeats itself in unexpected ways. The earliest proposed function of the CP was that it provided a source of the pneuma or animal spirits that gave energy and motion to the entire body. This idea
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certainly seems odd today, but it might turn out to effectively capture the essential and far-reaching role of the CP in enabling neural function and the spirit of its central role in the CNS. As we now know, the production and distribution of CSF by the CP provides mechanical protective buoyancy for the brain and spinal cord, serves as a nutritive milieu for neurons and glial cells by secreting numerous polypeptides and growth factors, and acts as a vehicle for removing waste products of cellular metabolism. We suspect that the functions and potential of choroidal cells will become more apparent and appreciated over time. There are three interrelated reasons for this opinion. First, several convergent data sets are documenting an increasingly detailed role of CP secretory products in normal brain development and functioning. Within the CNS, the CP is a robust producer of compounds that are vital for growth and function. The role of the CP in maintaining extracellular concentrations of an array of proteins puts this tiny collection of cells at the heart of understanding the complex intertwining of nutritive and protective factors in developmental biology. Second, profound changes in CP secretion and function occur together with many pathological conditions, including aging and degeneration. Because the polypeptides carried by the CSF are distributed throughout the neuroaxis, changes in the basic secretory and transport functions of CP lead to consequences that are both local and comprehensive. A greater understanding of the role of the CP in CNS diseases will lead to a concomitantly greater elucidation of the biological underpinnings of disease processes and endogenous repair processes. Third, refining our understanding of the role of CP in CNS diseases will suggest new strategies to prevent or minimize those disease processes. Even within this brief discussion, numerous gaps are apparent in our knowledge. Although there is no doubt that the CP is important for brain development and maturation, its precise function is vaguely understood. The developing CP provides the surrounding tissue with morphogens, mitogens, and trophic factors important for axonal guidance and cell migration. However little is known about how specific barrier functions and secretory profiles are changed with time and how these changes contribute to development, aging, normal function, and pathology. With regard to CNS pathology, changes in CP secretion, transport, and metabolism are associated with both acute and chronic trauma. However, no direct causal relationship has been established between a particular change in secreted products from
CP and neuronal degeneration. Finally, a greater understanding of the role of the CP in CNS diseases may continue to bolster the emerging concept of using transplantable CP cells for CNS diseases. The few transplant studies conducted to date have been generally encouraging but have not yet focused on determining the means by which transplanted CPs exert their beneficial effects. Future efforts will need to systematically approach each potential clinical indication with emphasis on optimizing the donor source and age of the transplanted cells; determining whether specific cell types within the CP (i.e., purified epithelial cells) are most beneficial; identifying the optimal post injury timing, transplant location, and dosage of cells to be grafted; whether CP functions within parenchymal tissue in the same manner as within the CSF; the mechanism by which transplanted CP affords beneficial effects; and whether these benefits are greater because of the native ability of CP to secrete a physiologically balanced and temporally adjusted cocktail of bioactive compounds versus delivery of single agents.
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24 Neural Transplantation in Huntington’s Disease Anne E. Rosser and Stephen B. Dunnett School of Biosciences, Cardiff University, Cardiff, Wales, UK
Huntington’s Disease Huntington’s disease (HD) is the commonest dominant genetic disease of the nervous system. First described by George Huntington in 1872 [99], HD is characterized by a complex of motor, cognitive, and psychiatric symptoms, typically with onset in middle age and progressing to death within 15 to 20 years. HD runs in families with an autosomal dominant mode of inheritance, with linkage studies suggesting a single gene mutation located to the distal arm of chromosome 4 [88]. The affected gene, huntingtin, was finally isolated and sequenced in 1993 [3]. The normal huntingtin protein is involved in important aspects of intracellular signaling, trafficking, and metabolism, the precise details of which are still to be enumerated [106]. The normal form of the gene contains a short CAG trinucleotide repeat, encoding a polyglutamine repeat in the huntingtin protein. In the mutation, this is expanded beyond a critical 37 to 39 repeat length. Whereas disease onset is most commonly around the middle years, 30 to 50 years, a juvenile form of the disease occurs in 5 to 10% of patients with the longest (> 50 to 60) repeat expansions [114]. The core pathology of Huntington’s disease involves a progressive degeneration of the basal ganglia involving loss of striatal neurons, moderate gliosis associated with cell death, atrophy of the caudate and putamen nuclei, and associated enlargement of the lateral ventricles [89]. The cellular degeneration commences in the caudate nucleus, progressing in a rostral, lateral, and ventral direction first to encompass the putamen and later to involve degeneration outside the basal ganglia in areas to which the striatum is connected — in particular the neocortical mantle on the afferent side and downstream motor centers such as globus pallidus, thalamus, and substantia nigra on the efferent side. Within the
Cellular Transplantation: From Laboratory to Clinic
striatum the major population of medium spiny projection neurons are more affected than the medium and large striatal interneurons, although these two are also affected as the disease becomes more advanced. More recently, the cellular pathology of the disease has been associated with the appearance of intracellular aggregates of truncated fragments of the mutant huntingtin protein in the cytoplasm and accumulating in the nuclei of affected cells [42, 51], which suggest a developing cellular dysfunction leading to eventual cell death. Optimism that targeting this process may yield novel effective therapies has been promoted by the report of Yamamoto and colleagues that blocking the expression of huntingtin in a conditional transgenic mouse (in which the mutant gene is expressed under Tetoff control) after inclusions and behavioral dyskinesias have already developed results in the gradual disappearance of the pathology and a parallel remission of symptoms [188]. As a consequence, the abnormal cleavage, folding, and aggregation of mutant huntingtin has in recent years become a major focus of study, seeking both to understand the pathological processes that lead to targeted striatal dysfunction and cell death at the molecular level [13], and to evaluate the huntingtin protein as a surrogate marker for therapeutic targets that can inhibit inclusion formation in high throughput screens [98]. An alternative approach to genetic and pharmacological therapies is to seek to repair the damage caused by the disease process by replacement of lost or damaged cells, which is where the transplantation approach comes into play. Encouraged by the preliminary successes in animal models and open label trials in Parkinson’s disease (see the parallel chapter in this volume), applications of embryonic neural transplantation have been considered in a variety of other diseases, including
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stroke, multiple sclerosis, and spinal cord injury — of which studies in Huntington’s disease have perhaps developed the furthest. Although the ultimate goal may be to suppress or inhibit expression of the mutant gene or to manipulate the downstream molecular interactions of its protein products so as to block the pathogenic process itself, there are likely to remain for the foreseeable future many seriously damaged patients with a presently untreatable progressive condition, for whom the option of repair by cellular replacement remains the only hope. Recent advances of cell transplantation in animal models of HD and initial pilot trials in patients are the topic for review in the present chapter.
Functional Animal Models The basal ganglia have long been associated with motor disorders, and early neurologists knew that striatal damage in animals can result in motor disinhibition such as Magendie’s description of a syndrome of “obstinate progression” [117]. Throughout the first half of the twentieth century, experimental studies in animals were confounded by the difficulty of making focal lesions in the caudate nucleus and putamen without damaging the internal capsule fibers. This is particularly acute in rodents in which the caudate and putamen merge into a single neostriatal nucleus punctuated by multiple small internal capsule fiber bundles, although it remained possible to study the effects of nonspecific lesions in primates in which the large brain allowed targeting of the caudate nucleus or putamen with electrodes for making electrolytic lesions [53, 161].
STRIATAL EXCITOTOXINS This problem was essentially solved with the introduction of kainic acid, the first of the excitotoxic amino acids, which targets neurones while sparing glial cells and axon fibers [125]. Thus, injection of kainic acid into the striatum causes cell loss, atrophy, and neurochemical changes associated with damage of intrinsic striatal neurons while sparing the reciprocal corticothalamic fibers passing through the nucleus [41, 124]. From the very first, the cellular and neurochemical changes were associated with the similar pathology observed in Huntington’s disease in man [41, 124]. This was closely followed by behavioral studies, indicating that such lesions caused motor [121, 150, 167],
regulatory [63, 164, 166] and cognitive [52, 62, 164, 166] deficits in rats akin to the major classes of symptoms in the human disease. Nevertheless, kainic acid is not an ideal toxin. The animals can be quite sick after lesion surgery, they are prone to epileptic seizures, and additional damage is often induced in remote structures of the brain, such as the hippocampus [15]. Consequently, we have seen a range of alternative models developed over the last two decades targeting striatal neurons with different mechanisms of cell toxicity — of progressively greater relevance to the pathogenesis of Huntington’s disease, summarized in Table 24.1. The first resolution to this issue was the identification that other excitotoxic amino acids could match kainic acid for neuronal targeting while exhibiting lesser side effects and eliminating the problems of remote damage. Ibotenic acid was the first widely used alternative [87, 170], and much of our present knowledge about good behavioral tests for characterizing the motor and cognitive deficits of striatal damage was acquired in the 1980s using this toxin [163]. Meanwhile, an alternative excitatory amino acid, quinolinic acid, has gained increasingly wide use [171]. Quinolinic acid is an endogenous metabolite of tryptophan implicated in the kynurenine pathways [94]. It produces a more selective profile of toxicity, targeting the medium spiny projection neurons with relative sparing of the large and medium aspiny interneurons of the striatum — which mimics the specific profile of cell loss observed in Huntington’s disease [9, 70]. Together, these observations suggest that quinolinic acid may be an intrinsic component of the disease process and as a consequence this toxin is nowadays probably the most widely used excitotoxin for inducing striatal lesions in animal studies of striatal dysfunction and repair.
METABOLIC TOXINS Excitotoxicity is not the unique process by which cells die, but is one component in a “cycle of neurodegeneration” in which glutamate-mediated excitotoxicity, calcium influx, impaired energy production, and impaired handling of reactive oxygen species (oxidative stress) augment each other and converge on both apoptotic and necrotic processes of cell death [38, 69, 139]. Diverse sources of information from positron emission tomography, postmortem studies, and magnetic resonance spectroscopy all suggest impaired energy
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Approach
Effects
Method or Tool
Excitotoxic amino acids
Glutamate receptor agonists inducing excitotoxic lesions in target neurons, sparing fibers of passage
Kainic acid
Toxins that disrupt mitochondrial energy production and cell death
Malonate
11
3-Nitropropionic acid
12
Exon 1 fragments
Transgenic mice
119
Full-length htt Expanded CAG Tet-off regulation AV or LV vectors
YAC mice Knock-in mice Conditional knock-in Viral vectors
97 181 188 43
Metabolic toxins
Genetic manipulations
metabolism in the striatum of Huntington’s patients — even early in the disease before extensive tissue loss — that may account for the targeting of the disease to the basal ganglia as well as the mechanism for targeting of excitotoxicity there [10, 14]. In detailed spectrophotometric analyses, Browne and colleagues [31] showed that key components of the mitochondrial complex II and III chain for oxygen phosphorylation and energy metabolism were reduced in postmortem Huntington’s disease striatum. Similar patterns of disturbed energy production can be mimicked both in cell culture and in animal models using a variety of “metabolic toxins” that target key components of the mitochondrial electron transport complexes, including aminooxyacetic acid, the methylpiridinium ion, 3-nitroproprionic acid (3-NP), and malonate [14]. 3-NP is of particular interest because peripheral administration leads to selective targeting and focal bilateral degeneration in the neostriatum [12]. Chronic drug delivery by prolonged repeated low-dose administration typically produces more stable and consistent focal lesions, which have been shown to be associated with both motor and cognitive deficits in both rodents [24, 25] and primates [145]. Thus, a distinct advantage of 3-NP as an animal model is its efficiency and ease of administration, but this is offset by its relative variability from animal to animal and the fact that effective lesions tend to involve gross necrosis and holes in the striatum rather than the selective neuronal loss that is typically sought.
Exemplar References 41
Ibotenic acid
170
Quisqualic acid
171
GENETIC MODELS Following discovery of the HD gene in 1993, a major advance in recent years has come with the development of genetic models of the disease, involving alternative strategies for insertion and expression of the gene mutation both in cells and in experimental animals. Mangiarini and colleagues [119] introduced the first effective HD transgenic strains of mice, of which the R6/2 line still remains the most widely studied in the field. The R6/2 mice carry four or five copies of exon 1 of the human htt gene with the polyglutamine stretch expanded to approximately 145 repeats [119]. The mice exhibit onset of a neurological syndrome involving ataxia, abnormal locomotion, and posture at about 6 weeks of age, with subsequent weight loss and death by 13 to 17 weeks of age. Mice in advanced disease exhibit striatal atrophy but little striatal cell loss, and it was in this strain that the early appearance of neuronal intranuclear inclusions was first identified [42]. The development of motor, cognitive, and physiological symptoms has been characterized in detail [37, 115, 132], and this strain has also been widely used to characterize the effects of a number of potential pharmaceutics [5, 49, 130, 157]. However, both pathology and associated functional symptoms appear to extend well beyond the striatum [131, 132], and thus this model may be less suitable for the explicit analysis of cell-based repair strategies such as transplantation.
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In the subsequent years, a variety of different transgenic strains have been developed using both full-length huntingtin and the exon 1 fragment with different repeat lengths in the normal and mutant range, as well as knock-in of expanded repeats into the normal murine gene location and inducible models in which expression is regulatable using the Tet-off system (see Table 24.1). The age of onset and profile of pathology and behavioral syndromes can vary dramatically between strains and have been the subject of several recent detailed reviews [13, 126]. For evaluating the efficacy of cell transplantation, we need to identify a model with selective striatal pathology and a slow progressive time course that mimics the profile observed in the human disease pathologically and behaviorally.
Functional Experimental Transplants EXPERIMENTAL TRANSPLANTATION OF STRIATAL NEURONS The essential conditions for good functional survival and integration of embryonic donor tissues in the adult brain were first worked out for dopamine cells, and are described in the accompanying chapter on Parkinson’s disease. Similar methods and techniques have been found to be effective for transplanting striatal neurons as dissociated cell suspensions into the lesioned striatum of adult rats and monkeys, where the grafted cells can survive and integrate with a high degree of reliability [102, 105, 109, 169]. However, a number of key differences emerge in comparison with dopaminergic nigral grafts. First, whereas studies of nigral grafts have focused almost exclusively on the dopaminergic neurons, the striatum contains multiple cell types, characterized both morphologically and neurochemically. All main types of striatal neurones are seen to develop also within grafted striatal tissues [40, 93]. Nevertheless, striatal grafts exhibit a distinctive patchy appearance when staining with acetylcholinesterase or other markers of striatal neuropil [103]. This can be explained by our knowledge of normal striatal development. The striatum, which develops from the lateral and medial ridges of the ganglionic eminence (LGE and MGE, respectively) in the floor of the embryonic lateral ventricle [173], is the source of precursors for multiple forebrain structures — including globus pallidus and ventral neocortex as well as striatum. The patchiness represents the
internal organization of the grafts into separate striatallike and non-striatal-like compartments (designated the P and NP zones, respectively) [86]. Second, striatal grafts are typically placed into a homeotopic location (i.e., back into the lesioned striatum), from which intrinsic neurons are lost rather than into the denervated terminal areas typical of the ectopic location in which nigral grafts exert their functional effect. In this location, not only do grafted striatal neurons retain the capacity to extend axons to distant targets in the globus pallidus and substantia nigra; they also attract afferents from host neurons that have lost their intrinsic targets in the course of the striatal lesions [39, 182–184]. As such, striatal grafts appear to integrate fully into the host neuronal circuitry and reconstruct the key elements of a connected cortico-striato-pallidal network that may underlie the functional recovery reported with such grafts [39, 56]. The connections provided by striatal grafts with the host brain appear to be functional in physiological studies. This issue was first evaluated in oblique corticostriatal tissue slices studied up to 6 months posttransplantation, in which it was shown that stimulation of corticostriatal afferents yielded physiologically appropriate extracellular responses from neurons within striatal grafts, indicative of reformation of corticostriatal terminals on grafted neurons [162]. Monosynaptic inputs were confirmed in a subsequent intracellular recording study, but suggested some differences from the normal striatum involving higher input resistance values of the grafted cells and inhibitory as well as the normal excitatory responses to stimulation of adjacent neurons. This suggested that the full density and richness of internal connections were not fully restored and that the grafted cells expressed distinctly immature characteristics, at least by 2 to 6 weeks after transplantation as studied in this second report [178]. A subsequent series of studies by Wilson and colleagues in in vivo preparations confirmed monosynaptic cortical and thalamic inputs to anatomically identified medium spiny-like neurons within striatal grafts, which again involved both excitatory and inhibitory postsynaptic potentials and changes in input resistance and latency that suggested absence of strong inward rectification that may be attributable to complex intraneuronal regulation in the normal striatum [187]. Pharmacological analysis has confirmed that the afferent synapses are glutameteric (as in the normal corticostriatal input) but the target neurons may develop hyperexcitable levels of response to inputs [172], which are typically of less
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than normal density. Nevertheless, several key aspects of the reformed corticostriatal connections can restore key aspects of the normal circuitry. Thus, studies by Calabresi and colleagues indicate that the normal corticostriatal synapse exhibits bidirectional plasticity, with long-term depression (LTD) of the extracellular field recordings in response to a tetanus of high-frequency stimulation under normal tissue bath conditions but long-term potentiation (LTP) to a similar tetanus when magnesium is absent from the perfusate [35]. This is reproduced in striatal grafts. Thus, in a recent study we have replicated the monosynaptic extracellular field responses to corticostriatal stimulation in striatal grafts in the oblique corticostriatal slice preparation, and sustained depression in the response following tetanic stimulation with magnesium present in the medium but potentiation in its absence [123]. This suggests not just an ability of the grafted cells to relay and transduce host inputs but an ability of the grafted cells to sustain plasticity appropriate to a role in motor learning, characteristic of normal striatal function (see material following).
PATTERNS OF FUNCTIONAL RECOVERY There are now numerous reports that striatal grafts can alleviate a range of behavioral impairments when transplanted into animals sustaining striatal lesions as models of the pathology of Huntington’s disease (see Table 24.2). These include recovery not only of motor impairments in tests of locomotor activity [48, 102, 165], rotation [61, 136], postural bias [22], and skilled reaching and paw use [61, 78, 129] but in cognitive tests such as T maze alternation [47, 104], operant delayed alternation [67], or active and passive avoidance [149]. Although the bulk of functional studies has been conducted in animals in which the striatal lesions have been sustained using excitotoxic lesions, recovery has also been seen when the striatal damage has been caused by electrolytic lesions [149], ischemic middle cerebral artery occlusion [111, 135], the cholinergic toxin AF64A [82] and metabolic toxins [23], and in monkeys [9, 109, 144] as well as rats. A detailed review of the patterns of behavioral recovery is beyond the scope of the present chapter but is available elsewhere [56, 65].
TABLE 24.2 Functional recovery after striatal transplants in rats with striatal lesions. Function
Test
First Reports of Recovery with Striatal Grafts (reference)
Locomotor activity
Digiscan activity monitor Photocell open field Photocell cages
48 102 104
Coordination and balance
Omnitech rotarod Paw-reaching tray test Paw-reaching staircase test
81 61 129
Rotation and turning
Rotometer bowls Elevated body swing test
Motor habit learning
9-hole box operant RT tasks Transfer of training probes Skinner box operant RT task
122 26 55
Memory
Step through passive avoidance Step down passive avoidance Radial maze
149 111 111
Cognition
T maze delayed alternation Operant delayed alternation Morris water maze Operant DRL schedule
104 67 4 156
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Although transgenic mice may appear to offer better models of the human disease within which to evaluate graft-derived repair, only two such studies have been reported. The first indicated significant alleviation of deficits in body weight loss, locomotor hypoactivity, and excessive grooming in R6/2 mice [60], but the effects were small and transient and only provide a brief delay in the profound decline in the mice from 13 weeks of age, compatible with the interpretation that grafts targeted at the striatum have little effect on the much more widespread pathology exhibited by this strain. In the other study, HD transgenic mice received cortical grafts in the anterior cingulate cortex, which delayed the onset of abnormal paw clasping but did not affect other measures of motor coordination [177]. This study concluded that graft studies needed to target cortical as well as striatal components of the pathology in HD. However, both studies highlight the need to develop better genetic models of the disease process, and not least that models appropriate for probing the progress of cellular pathology pharmacologically [13, 126] may not be the most appropriate for evaluating cell replacement therapies. For this second purpose we need strains that show focal striatal pathology that is characterized by rank cell loss in addition to the more typical inclusion formation, astrocytosis or global atrophy. The present focus in our lab is to provide a detailed behavioral characterization of different strains that may provide a more suitable framework for future graft studies [16, 28, 29, 37, 57, 115, 130].
MECHANISMS OF FUNCTIONAL RECOVERY Grafts can exert their effects on host function by a variety of more-or-less specific mechanisms, from entirely nonspecific effects of graft surgery through trophic stimulation of host plasticity and diffuse release of deficient neurochemicals that can restore host function by essentially pharmacological means to actual replacement of lost neurons that become integrated into the host neuronal circuitry so as to provide true “brain repair” [18, 59]. Although it may be natural to conclude true repair when the grafted cells are seen to establish anatomical connections with the host brain, in fact in most circumstances less specific mechanisms can account for the recovery observed. Nevertheless, striatal grafts may provide one of the clearest models within
which graft-derived recovery is not only dependent upon but actually mediated by circuit reconstruction. Some of the evidence for this strong hypothesis is indirect. Thus, whereas deficits in a disease such as Parkinson’s can be significantly alleviated pharmacologically — witness the dramatic benefits brought since the advent of l-dopa — it has not proved possible similarly to alleviate any of the specific symptoms of Huntington’s, in particular in the cognitive domain. Present evidence suggests that recovery of the wide range of functions alleviated by the grafts by a simple pharmacological mechanism is implausible. As outlined in the introduction, the key cognitive symptoms of Huntington’s disease are of the frontal type and believed to represent a disruption of the frontostriatal circuitry. When performance is restored in grafted rats tested on behavioral tasks (such as delayed alternation) dependent on the integrity of the striatal circuitry, and both corticostriatal and striatopallidal connections are seen to be restored through the grafts, it is parsimonious to at least hypothesize that the recovery is dependent on the reformation of those connections. In a first series of disconnection studies, we have confirmed that performance of operant delayed alternation is critically dependent on an intact corticostriatal circuitry [64], and we are currently in the process of using a similar disconnection paradigm to determine whether recovery on the same task provided by striatal grafts [67] is similarly dependent on the integrity of the anatomically reconstructed graft circuits. A third angle on the issue has come through the analysis of the involvement of the normal and grafted striatum in motor learning. The striatum is not simply an “enabling” structure but is critically involved both in the selection and initiation of action, and in the learning of motor skills and habits through experience [79, 85, 127]. We have studied habit learning using a choice reaction time task originally described by Carli and colleagues [36], in which rats learn to make rapid responses to brief light stimuli in an array of holes in the curved wall of a nine-hole box operant chamber. Accurate performance requires the animals to learn specific stimulus-response (S-R) associations that need not be collocated. Performance is disrupted both by nigrostriatal lesions [36] and striatal lesions [30, 128], both of which specifically disrupt response on the contralateral side, irrespective of the location of the imperative stimulus. Striatal grafts alleviate the deficits in responding on the contralateral side [26, 122], an effect that has been replicated in a classical Skinner box with retractable
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levers [55]. Critically, when the grafted rats are first returned to the operant chambers 3 to 4 months after the lesion (by which time long-distance axon growth to and from the grafts is approaching asymptote), the grafted rats are as impaired as lesioned rats. However, with further training the grafted rats relearn the task contingencies, whereas the lesion rats remain severely impaired [26, 122]. These results suggest that the grafts are not simply reactivating the striatum to enable accurate skills and habits to be expressed but rather that the striatum is a substrate for habit learning (i.e., the lost former skills and habits must be relearned through specific training) [54]. Moreover, both transfer of training and delayed implantation experiments [26, 27] suggest that the grafts actually provide the neural substrate for the relearning to take place, a hypothesis that receives further support from the demonstration (described previously) of physiological plasticity at the cortico-striatal synapse itself. Together, these observations provide strong support for the hypothesis that under appropriate conditions striatal grafts can reconstruct damaged circuits in the basal ganglia as the basis for recovery of function in the host animals. Because the striatal lesion models reflect many aspects of the focal striatal pathology of Huntington’s disease, it has therefore been a natural extension of the clinical trials of cell transplantation in Parkinson’s disease to consider whether a similar transplantation strategy may be relevant to Huntington’s disease.
Clinical Trials Although repair of the complex connected circuits of the striatum may appear likely to prove difficult, Huntington’s disease exhibits a number of features that make it a suitable target for cell transplantation [147]. First, it is a strongly genetic disease and the advent of precise genetic testing a decade ago now allows accurate diagnosis, even early in the course of the disease. Second, the disease is progressive (and thus once expressed the patient can envisage a relentless decline leading eventually to death), and presently untreatable. Third, although in its advanced stage Huntington’s disease involves extensive degeneration throughout the brain the earliest and most severe degeneration is in an identified population of cells (the medium spiny neurons of the striatum) that can be targeted for protection or replacement. The bulk of symptoms in early and middlestage disease can be attributed to loss of the striatal
projection neurons, and it remains unknown whether maintaining their survival would modify the course of more widespread aspects of the disease. Finally, of course, positive results in many animal studies have demonstrated that long-distance circuit reconstruction in this system is not only possible but reasonably reliable and associated with significant functional recovery.
PRECLINICAL STUDIES The animal studies described previously provide evidence of efficacy and a biological rationale for proceeding to clinical trials, but prior to launching the first systematic clinical studies a number of issues pertaining to aspects of cell preparation, implantation, and assessment protocols required considerable development. In terms of donor tissue handling, it was necessary to demonstrate the accuracy of dissection protocols, assess the optimum gestational age of fetal tissue harvest, and optimize methods for tissue preparation prior to implantation. Both cell culture and xenotransplantation studies have provided validation of the anatomical landmarks used to dissect fetal striatal tissue by demonstrating that the dissected tissues do indeed contain striatal neurons and not significant numbers of contamination cells [83, 100, 153]. As discussed previously, regardless of how careful the dissection striatal grafts typically have a patchy appearance due to the presence of both striatallike and non-striatal-like compartments. Isacson and colleagues showed that if donor tissue was restricted to that derived from the LGE, rather than whole ganglionic eminence (WGE, i.e. LGE plus MGE), a higher proportion of striatal-like cells were seen in the grafts — as determined by concentrations of acetylcholinesterasepositive neuropil [46, 143]. This confirmed using other measures such as DARPP-32 staining of medium spiny projection neurons [46, 133, 134, 141]. This suggested that an LGE dissection would be optimal for maximizing specificity and functional recovery. However, many striatal interneurons originate from the MGE [120], and one study by Watts et al. [180] demonstrated that a dissection that combines both LGE and MGE yielded a higher total number of DARPP-32 cells and more convincing evidence for functional recovery — suggesting that cells from both compartments contribute to the mature striatal phenotype. Most clinical studies have selected a whole ganglionic eminence
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dissection, and the final resolution of this issue will await the results of these trials along with further laboratory data. To date, the optimum gestational age of the donated fetal striatal tissue has been estimated largely from studies using donor tissue from rodents. Fricker and colleagues [77, 78] have shown that striatal transplants derived from younger E14 rodent donors (corresponding to the stage of peak neurogenesis in the developing ganglionic eminence) and implanted into adult rat striatum yielded larger grafts, better differentiation of the DARPP-32 phenotype, and improved recovery on tests of skilled paw use than did tissue from older fetuses. This gestational age corresponds to Carnegie stages 19 to 23 through, that is, 7 to 9 weeks of gestation for dissection of striatal tissues from human donors [34]. The estimate has been validated by xenotransplantation of human fetal striatum into immunosuppressed rat hosts [83, 153]. These grafts survived well, containing neurons that differentiated into a variety of appropriate striatal transmitter phenotypes [153, 154], and were able to alleviate some behavioral deficits in the host rats [152]. However, it should be noted that the human-to-rat xenografts typically exhibit a rather low proportion of acetylcholinesterase-rich and DARPP-32-rich P zones [32, 84], probably because human tissue takes much longer than the rat to develop. Thus, a firmer estimate of optimal gestational age will require longer host survival times to allow full differentiation of the implanted tissue. Donor fetal striatum can be prepared for surgical implantation either as tissue pieces or as a dissociated cell suspension, and there are both theoretical and practical issues that may influence the choice of preparation. To date there has only been one study that directly compared the two methods [179], and in this case there was no clear difference between the two approaches for rodent allografted fetal striatum. Tissue pieces may have the theoretical advantage of retaining a degree of organization, but the disadvantage of also containing cells (such as endothelial cells) that could provoke an immunological response. In addition, tissue pieces do not come under the recently published EU tissue directive, and thus can be handled within a normal laboratory and hospital environment, rather than requiring the “Good Manufacturing Practice” (GMP) facilities needed for dissociated cell suspensions [76]. Nevertheless, dissociation of the cells has the benefit of allowing greater standardization, has a reduced risk of eliciting an immune response, and has the theoretical advantage
of generating cells that will be better able to integrate within the host. Again, the final resolution of this issue will require further work and will benefit from the fact that the centers involved in the current clinical studies (using one of these techniques) also use common assessment protocols (based on CAPIT-HD, see material following), which will allow a direct comparison of outcomes to be made. Moreover, the precise methods of preparation will need to be validated for each protocol and source of tissue. For example, Fricker and colleagues [76] found that trypsinization of rat striatal tissue prior to dissociation resulted in larger surviving grafts that also showed improvements in rotational behavior. Moreover, these grafts contained more striatal tissue and more DARPP32-positive medium spiny neurons than grafts implanted as tissue fragments. One technical advance that has facilitated the coordination of tissue donation and neurosurgery is the development of hibernation procedures that allow donated cells to be stored for a limited period of time [100, 101, 168]. Essentially, this entails storing the tissue at 4 to 8 ◦ C in a nonphysiological medium that slows cell metabolism. Viability of human fetal striatal tissue has been demonstrated for tissue stored for up to 7 days in this way [100, 101], and this method of storage was used in the NEST-UK safety study [160]. More recently, the EU tissue directive [1] has come into being and stipulates that all cells used in human trials or therapy should be donated, procured, tested, processed, stored, and distributed according to the quality control standards of GMP. In practice, this means a more stringent level of environmental control, based on rather different principles to those that govern the design and operation of regular category II tissue facilities found in most laboratories; a more detailed specification and validation of standard operating procedures; and regular inspection for accreditation by a government inspectorate (in the UK, the Medicines and Healthcare products Regulatory Agency MHRA). These regulations do not apply to tissue pieces for transplantation that are not processed further post-collection and are not placed into any storage, but do apply to tissue suspensions and to any storage or distribution beyond the center where the tissue is collected. In the UK, the implementation of GMP standards has required major adaptation to existing laboratory facilities, resourcing of materials to GMP grade, and retraining of staff in order to comply with the new regulations — all of which have placed a delay on the process of the ongoing UK trial.
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Finally, there has been a need to develop and refine assessment protocols to be able to determine the longitudinal impact of grafts in small numbers of patients in a slowly progressive neurological disease. Although the Unified Huntington’s Disease Rating Scale (UHDRS) has provided a useful index of the stage of the disease [110], there was a requirement for careful analysis of the consistency of disease progression and determination of the most reliable tests in cognitive, motor, or psychiatric domains that can be used repeatedly to characterize this. Thus, a standardized core assessment protocol for intracerebral transplantation (CAPIT-HD) was developed to allow comparison of small numbers of patients in multiple centers under standardized conditions [155]. This protocol has also proved useful for assessment of natural history [7, 95]. An important aspect of assessment is imaging of the graft, to demonstrate graft survival and function. Although MRI can be used to image the gross atrophy of the striatum in HD and can demonstrate the site of graft placements, it cannot be used to detect graft tissues with certainty or to determine whether the grafts comprised healthy or dying or striatal or nonstriatal neurones [137, 138]. Guzman and colleagues [90] suggested that the survival and development of the grafts could at least be detected, in particular with T1 weighting, even if not studied in detail. By contrast, the enlarged scale provided by MRI imaging of primate brain has yielded more clear-cut resolution of striatal grafts in T1-weighted MRI [50, 108]. Although it has lower spatial and temporal resolution than MRI, greater definition of striatal grafts has been achieved using PET. Using a small animal scanner for rats, Torres et al. [175] found that striatal grafts could be clearly demarcated using the D2 receptor ligand raclopride, which provides greater discrimination of the grafts from background than either the D1 ligand SCH-23390 or the metabolic marker 2-fluorodeoxyglucose. Moreover, the technique clearly distinguished striatal graft tissues from control grafts of cortical tissue, and the raclopride signal correlated highly with postmortem survival of striatal-like neurons within the grafts [175]. In a subsequent study comparing different ages of donor tissue there was again a close relationship between the degree of restoration of the PET raclopride binding constant in the grafted striatum and recovery of skilled paw reaching [78]. Preliminary data have recently been accumulated to replicate this method in primates [17, 108]. As a consequence of the
animal studies, the use of longitudinal PET scans with raclopride is now recommended as a core component of the CAPIT longitudinal assessment protocol for clinical trials in HD (see material following).
CLINICAL TRIALS IN HUNTINGTON’S DISEASE The first pilot clinical trials of neural transplantation in patients commenced in 1990 (see Table 24.3). The early studies (from Cuba, Czechoslovakia, and Mexico City) each provided brief clinical accounts of implantation protocols and reported no major side effects. Each of these studies involved tissue implantation within 1 to 2 hours of spontaneous abortion of the donated human fetal tissue, whereas most subsequent studies have been based on tissue donation from elective abortion. Oleg Kopyov and colleagues at the Good Samaritan hospital in Los Angeles undertook the first extensive series of implants. This group reported on safety [113], benefit in motor and neuropsychological tests on small numbers of patients, and graft survival and neuronal differentiation by MRI in a total of 14 patients [96, 112, 148, 158]. Although paving the way for subsequent trials, and providing sophisticated imaging of graft survival and differentiation, this series has provided a rather limited descriptive account of clinical outcomes, and has raised concerns about the lack of systematic assessment of the patients within a clear experimental trial design. Marc Peschanski and Anne-Catherine Bachoud-Lévi and colleagues have initiated a second major study in Créteil, France. This study is the first to be undertaken using CAPIT-HD for patient assessment, and detailed reports of the surgical methods, safety, functional efficacy, and imaging in the first five patients have now been published [6, 8, 80, 146]. In this series, three of the five patients showed a good clinical response in reduction of motor signs and improvement in UHDRS scores, which were associated with a positive graft signal in FDG-PET and with restoration of lost sensoryevoked potentials in the electrophysiology tests. A fourth patient never showed any positive response and exhibited no sign of surviving grafts in the physiological and imaging assessments, whereas the fifth patient began to show signs of recovery until suffering a complete relapse immediately after an acute fever followed by change of MRI signal in the grafted region and loss
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TABLE 24.3 Clinical trials of cell transplants in Huntington’s disease∗ Study
n
Donor Tissue/Side
Implant Tracks
Immune Treatment
n
Weeks
Dissect
Cuba & Czech 4
2–3
?
VM or 2–3 ? [B] WGE
Mexico City
2
1
E12–13 WGE
CN cavity
Los Angeles
≥ 14 5–8
E8–10
Boston
12
35–38 Porcine TGE
Tampa
7
2–8
E8–9
1 CN + 4 Pu [B] 2CN + 4 Pu [U] pcPu [B]
Créteil
5
2–4
E7.5–9 WGE
2CN + 3 Pu [B]
CyA 1 year
London
2
NEST-UK
4
2–3
E8–12
2CN + 4 Pu [U]
Triple
LGE
LLGE
WGE
Safety
Efficacy
Not yet possible to No pathological determine immunological response [174] CyA+Pred No surgical incidents or Slow progression of disease subsequent SEs [116] Not Safe — no serious SEs [113]Benefit motor [112], limited reported neuropsych tests [148] CyA or Safe — no serious SEs No change over 12 months anti-MHC [71] [71] CyA 6mo 1 death, 3 subdural Modest (ns) changes in hematomas motor tests at 12 mo [92] CyA
Procedure safe [6] Motor & electrophysiol Mild psychiatric SEs improvements [8] over 4 yr Possible psychiatric SE in Improvement in chorea in 1 one patient of 2 patients Only SEs related to immune No change in first 6 months suppression [160]
Anatomy and Imaging MRI guided stereotaxy, no reported follow-up Not reported MRI MRS [158] and FDG PET [112] Not reported MRI and PET [92] 2 post mortem cases with good survival [74] MRI and FDG PET — graft survival in 3 functional cases [80] MRI and D2 R PET — survival in PET MRI and D2 R PET — grafts in MRI? PET
∗ Based on Dunnett and Rosser [66].
Abbreviations: [B], bilateral implants; CN, caudate nucleus; CyA, cyclosporin A; En, weeks of embryonic age; LGE, lateral ganglionic eminence; LLGE lateral aspect of the lateral ganglionic eminence; Pred, prednisolone; pcPu, postcommissural putamen; Pu, putamen; SE, side effect(s); Triple, combined cyclosporin A, prednisolone, and azothiaprine; WGE, whole ganglionic eminence; [U], unilateral implants; VM, ventral mesencephalon.
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of physiological markers of graft survival, raising the possibility of graft rejection. On the basis of these preliminary results, the French team embarked on a multicenter “French-speaking” trial with more recent extension to Switzerland, Belgium, the Netherlands, Germany, and Italy to evaluate the ease of translating complex tissue handling and surgical protocols among nonspecialist neurosurgical centers. This study is ongoing and efficacy reports are not available at the time of writing. An open-label trial from Tampa, Florida, reported on outcome in seven patients, of which six showed moderate improvement whereas one showed significant deterioration resulting in an overall lack of significant change [92]. However, three of the patients developed subdural hematomas after the surgery, which may be due to the fact that the stage of disease appears to have been more advanced in this series in comparison to other studies, suggesting that implanting tissue into the already heavily atrophied basal ganglia may involve significantly higher risk than operations in patients at an earlier stage of the disease. One patient in this series died at 18 months posttransplantation due to causes unrelated to the surgery, and a detailed anatomical analysis of the postmortem brain has indicated healthy surviving grafts and good differentiation of the grafts into mature striatal-like tissue containing all striatal cell phenotypes examined [74]. Another designed trial involves a multicenter collaboration among six UK centers (NEST-UK). We have so far reported on neurosurgical safety in the first four patients over 6 months after unilateral transplantation, with the only complications being mild and reversible disturbance of routine hematological and biochemical indices associated with triple immunosuppression treatment, which has in all cases been controlled by titration of drug dose [160]. On the basis of this pilot safety study, we have now proceeded to the preliminary efficacy stage of the trial according to the CAPIT-HD protocol in 10 patients after bilateral implantations. One other study is worthy of note, involving implantation of striatal xenografts derived from porcine donors [71]. Of the 12 patients in this study, half were treated with cyclosporin immunosuppression, whereas in the other half the fetal porcine tissues were treated with a monoclonal antibody directed against surface MHC I molecules [142]. However, surviving grafts were not detectable on MRI, and the treatments had no functional benefit for the patients, which may not be surprising as fully effective immunosuppression strategies for
xenografts have still not been resolved and similar approaches to those used here have been seen to yield rather poor graft survival in experimental monkeys and in a postmortem from a patient with Parkinson’s disease [45].
Side Effects and Outstanding Issues ETHICS For trials of human neural transplantation the ethical debate focuses on two areas: the collection and use of human fetal tissues for research and clinical use and the issues associated with experimental surgery in HD patients. The legality of performing elective surgical termination of pregnancy varies worldwide. However, where it is legal the debate focuses on the details of collection and use of the fetal tissues. The main thrust of recent debate has centered on the possibility that the wish of researchers and clinicians to use fetal tissues could influence the decision to proceed with an abortion and the methods by which this may take place [19, 20, 33, 44, 176]. Concerns have centered around the possibilities that a woman’s choice on whether to seek or proceed with a termination might be influenced by the perception that it could aid others and that the medical decisions on the treatment of the woman and whether, when, and how to undertake the termination might be influenced by the potential subsequent use of the tissue. To address these concerns, a consortium of NECTAR (the Network of European CNS Transplantation and Restoration) generated a detailed set of guidelines for ethical tissue collection [19] that were adopted for self-regulation by all participating European neural transplantation centers. Subsequent discussions at national and international levels have led to most countries now formally adopting similar regulations or guidelines. For example, in the UK guidelines were proposed in a Royal Commission chaired by the Reverend Polkinghorne [151], and subsequently clarified by guidance notes from the Department of Health [2]. In essence, these guidelines set out to ensure separation of the medical process of elective termination of pregnancy from the process of collecting and using the fetal tissues, with the aim that neither a woman’s decision nor the medical practice to terminate a pregnancy should in any way be influenced by the decision to donate the tissue for purposes of research or treatment.
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The ethics of experimental surgery are concerned with a number of issues directed at protection and management of the patients in experimental trials of neural transplantation in HD. Two concerns have dominated this discussion: the nature of informed consent and the use of sham surgery as a control procedure. As with any experimental procedure, it is essential that the patient is fully informed and that he or she is able to consider and understand properly all of the issues surrounding the operation before giving consent. In the case of a highly experimental intervention, such as neural transplantation at this stage of its development, most authorities would agree that the patient must give full informed consent at the time of surgery, and that consent in advance of the procedure or consent by proxy is not sufficient. The consenting procedure requires special attention in conditions such as HD in which cognitive decline is part of the disease. Thus, it is important that the patients’ cognitive and psychiatric statuses are carefully assessed and that the physician in charge is fully convinced that the patient is capable of making an informed decision. For this reason, the European groups contributing to the multicenter development of a core assessment protocol for transplantation in Huntington’s disease (CAPIT-HD) elected to restrict experimental trials of neural transplantation in HD to early to moderate stages of the disease [155]. A second group of ethical issues relate to study design, and specifically how to produce meaningful results with the minimum number of volunteers and avoiding the bias of placebo effects. These design issues require application of meticulous and adequate data collection, and the selection of sensitive and valid end points. An issue peculiar to surgical interventions is whether or not to include sham surgery (i.e., a double blind placebo group in which surgery is performed but the potentially modifying intervention is not delivered). The proponents of sham surgery argue that meaningful results cannot be obtained without proper blinding of both the patient and the researcher so that surgical studies conform to the principles that govern the execution of large drug trials [75]. The counterarguments are that sham surgery, even with general anesthetic alone, involves an unacceptable risk; that sham operations are not the most efficient way forward, particularly during relatively early clinical studies where protocols are still being worked out by a series of careful case-by-case pilot studies; and that the double blind placebo-controlled trials performed to date [73, 140] are so drastically underpowered (due to the small numbers of patients that can
be included using current techniques) as to be relatively uninformative in addressing the key experimental issue [21]. Although placebo trials may be appropriate in the future, once the optimal protocols are established we consider that they are at present premature while suitable neural transplantation techniques are still being ascertained [58, 186].
LIMITATIONS OF TISSUE SUPPLY As with PD (see accompanying chapter), the most pressing problem is the scarcity of human fetal donor tissue of sufficient quality and reliability. However, this problem is of a different order of magnitude in HD because it appears that developing medium spiny neurons survive the transplantation process better than do developing dopamine cells. Moreover, the developing striatum is a larger structure than the ventral mesencephalon and contains many more cells of the required phenotype. Thus, it is likely that patients with HD will need maximally 2 to 3 fetal donors per side, rather than the larger number required for PD transplants. Indeed, a rather different anxiety surrounded the transplantation of striatal tissue as clinical trials were getting underway in the late 1990s, which was the fear of potential uncontrolled overgrowth of transplanted human fetal striatal tissue [84]. Such concerns have waned with reports of appropriate morphological development and functional recovery in primate allografts [108, 109], and it appears that such fears were due simply to the small size of the rat brain compared to that of the human from which the grafted tissue was derived. To date, there have been no reports of overgrowth in any of the transplanted HD patients. Ultimately, a source of tissue other than human fetal striatal tissue will be required to allow transplantation to be a widespread therapeutic intervention in HD, and it seems at the moment that this is most likely to come from some stem cell source. Stem cells have the capacity for self-renewal, thus producing expansion of numbers (which circumvents the supply problem). Use of stem cells would also allow a much greater degree of standardization, screening, and quality control than is possible with fetal striatal tissue. However, there are a number of serious safety and biological problems (largely pertaining to the differentiation of specific neuronal phenotypes from stem cell sources) that need to be solved before such cells have
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SURGICAL AND POSTOPERATIVE ISSUES
a realistic prospect of providing a useful alternative in patients [107].
TISSUE COLLECTION AND PREPARATION Details of collection will vary from place to place and the collection procedures described here are largely with reference to our own experience [66, 159]. Human fetal CNS tissue must be collected and processed under sterile conditions and dissected in a tissue culture hood using a dissecting microscope. Fetal tissue is always to some extent fragmented at collection, but provided this is not extreme striatal tissue can be identified in approximately 30 to 40% of cases. The correct anatomical identification of striatal tissue is critical both for reasons of safety and for the transplant to be effective. The inclusion of nonstriatal tissue could potentially lead to abnormalities of tissue growth, as has already been reported for inappropriate dissection of the mesencephalon for transplants in Parkinson’s disease [72, 118]. The second potential risk is the transfer of donor infections, although this risk is smaller for neural transplantation than in whole organ transplants from cadavers because the placental barrier protects the fetus. In our own study, fetal tissue assessment was undertaken primarily by screening maternal blood a week prior to tissue collection [68]. Screening can only be performed, of course, for a defined list of pathogens for which effective tests are available and are not undertaken for other pathogens for which this does not apply (e.g., prion diseases). In addition, for some tests there is a small risk that the patient has become infected too recently to have yet mounted a detectable antibody response. Thus, although the risk of passing on an infection through fetal tissue implantation is believed to be low it does exist and must be explained as part of the consenting procedure to potential tissue recipients. Following dissection, the tissue is prepared for implantation either as a suspension or as minced tissue pieces. Sterility of the technique and purity of the reagents used to digest the tissue (in the case of a suspension) must be considered for tissue safety, an issue that is now largely addressed through the GMP applications of the EU tissue directive. Once implanted, the cells must survive, grow, and make connections. Cell death or rejection of the graft tissue does not appear to cause any adverse effects as the debris appears to be rapidly and efficiently cleared without signs of accompanying inflammation or significant scarring.
Stereotaxic implantation of the injection needle itself carries a defined but small neurosurgical risk of hemorrhage that is generally assessed, from extensive experience with similar methods in biopsy surgery, as less than 1% per needle pass. It is clear that out of the several dozen patients worldwide who have received transplants for Huntington’s disease serious perioperative complications are rare. Indeed, for the great majority of patients that have received intrastriatal human fetal tissue there has been few reports of any adverse effects, and none in our own series. However, the consequences of an adverse surgical outcome may be devastating and the potential risks include intracranial hemorrhage and stroke. These should be small risks in centers in which there is established expertise in stereotaxic surgery. As described previously, clinical studies to date suggest that intracerebral hemorrhage is uncommon overall and is related to more advanced disease with associated cerebral atrophy [92]. The need for immunosuppression has not been proven, although most centers have elected to give immunosuppression for a period of months to cover the likely period in which the blood-brain barrier will be open, during which the graft is theoretically most vulnerable to immune attack [185]. The most common approach is to treat with the “triple” therapy of cyclosporin, azathioprine, and prednisolone, usually at lower levels and for a shorter period of time than those received by whole organ transplant patients. The effects of immunosuppression are now well known and if serious side effects occur the immunosuppression can be discontinued in the knowledge that this does not necessarily condemn the graft to rejection. To date the adverse effects of immunosuppression in these studies have been reversible.
Summary and Future Prospects Studies in experimental animals have demonstrated the efficacy of transplanting fetal neural tissues into the adult brain to repair the damage and alleviate motor and cognitive symptoms associated with focal striatal lesions. These studies have provided the basis for initial clinical trials in patients. Due in part to the relatively long time (commonly 18 to 24 months) required to start seeing the positive effects of grafts, it will be some years yet before the impact of such surgery in HD can be
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assessed. Even then, the human fetal tissue grafts will provide proof of principle only due to limited availability of human fetal tissue. If positive, it will be necessary to identify a reliable, standardizable, and quality controlled source of tissue for this approach to be widely applied. Consequently, there is active investigation of alternative sources of cells for transplantation, of which the two most promising at present appear to be to use expanded neural stem cells or xenografts of fetal striatal tissues. However, significant problems with each remain to be solved, and clinical trials with these alternative sources are not yet warranted.
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Acknowledgments Our own studies are supported by the Medical Research Council, the Hereditary Disease Foundation, and the Lister Foundation.
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25 Neural Transplantation in Parkinson’s Disease Stephen B. Dunnett and Anne E. Rosser School of Biosciences, Cardiff University, Cardiff, Wales, UK
Parkinson’s Disease Parkinson’s disease is the most common neurodegenerative disease of middle to old age, involving primary degeneration of the dopaminergic cells of the substantia nigra — leading to loss of dopaminergic regulation of the forebrain centers of the basal ganglia and a consequent impairment in a wide range of voluntary motor function. There are rare cases of identified gene mutations causing familial parkinsonism. The symptoms can also be caused by a number of industrial, agricultural, and other chemical toxins, and a parkinsonian movement disorder is a common side effect of many neuroleptic drugs. However, etiology remains unknown in the common idiopathic form of the disease. Degeneration of the dopamine neurons of the substantia nigra pars compacta is associated with a cellular pathology of characteristic inclusions known as Lewy bodies, which comprise aggregates of a-synuclein and other proteins in the cell cytoplasm, although there is disagreement about whether this should be taken as the critical feature for final diagnosis of true Parkinson’s disease in any of its forms. The primary motor symptoms of Parkinson’s disease involve a classic triad of rigidity, bradykinesia, and tremor, and a variety of other more variable impairments — including stooped posture, lack of facial expression, and disturbance of balance, gait, and handwriting. In addition, many patients exhibit cognitive symptoms (typically of the frontal type) involving impairments in planning and executive function, reflecting the fact that the basal ganglia are a major target of outputs of association (and in particular prefrontal) as well as motor cortex. Although the cognitive symptoms can occur early in the course of Parkinson’s disease, their neuropathological basis is less clear — whether due
Cellular Transplantation: From Laboratory to Clinic
to impairments in dopamine projections to the cortex, to impairments in relay of cortical information through the dopamine denervated basal ganglia, or to some other non-dopaminergic aspects of the disease. Thus, Lewy first described the typical inclusion bodies that bear his name in the cholinergic nucleus basalis of Meynert of Parkinson’s disease patients (not in the substantia nigra itself), and there is increasing recognition of the difficulty in drawing a firm distinction among the cognitive symptoms of Parkinson’s disease, cortical Lewy body disease, and frank dementia. Dopaminergic replacement with the dopamine precursor l-dopa (when used in combination with the peripheral DOPA-decarboxylase inhibitor carbidopa to block cardiovascular side effects) has provided the mainstay of pharmacological therapy for Parkinson’s disease over the last 35 years, and it remains powerful and effective for controlling symptoms in the early and middle stages of the disease. However, l-dopa does not alter the progression of the underlying degeneration, and in late disease the effectiveness of the drug declines, including a shortening of effective action, a narrowing of the window of effective dose, on/off fluctuations, and often disabling dyskinetic side effects. Attempts have been made to extend the duration of effective therapy and modify the course of side effects using a variety of other direct and indirect agonist drugs and preparations for more sustained delivery, but with only rather limited success. In parallel with developing novel pharmaceuticals, alternative surgical approaches have been adopted in recent years, initially using lesions in various structures that can modify, and hopefully compensate for, functional activity in the basal ganglia outputs already disturbed by the disease — most notably in the motor nuclei of the thalamus, globus pallidus, and subthalamic
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nucleus [55]. More recently, significant advances in surgical technique have been achieved replacing overt lesions with deep brain stimulation via adjustable electrodes positioned in these same areas, with the advantages that the precise electrode placement and stimulation parameters can be titrated to achieve optimal effect and that the procedure is reversible [7]. However, deep brain stimulation is a treatment primarily involving symptom suppression. It does not halt the progression of the disease, and evidence for stable long-term benefit awaits the results of ongoing studies. Consequently, there remains a significant rationale for developing alternative therapies that might actually repair the neuronal damage caused by the disease, either by promoting intrinsic regenerative processes or by extrinsic cell replacement. The development of cell transplantation as a novel therapy to repair the damage of Parkinson’s disease must be assessed not just in terms of whether it is effective in its own right but how it compares for reliability, efficiency, and efficacy in advanced disease with symptomatic pharmaceutical and surgical treatments. In addition, as we can expect the molecular and genetic basis of Parkinson’s disease to be unravelled over the coming decade transplantation will also need to be compared with other yet-to-be-identified strategies that manipulate intrinsic processes of degeneration and regeneration to alter the course of the disease itself.
Functional Animal Models 6-HYDROXYDOPAMINE The starting point for all new therapies is a valid animal model of the disease. One major reason Parkinson’s disease has been at the forefront of experimental neurology for developing new cell-based therapies is that powerful pharmacological agents have been available since the 1950s for the selective manipulation of catecholamine systems in the brain, and effective toxins for inducing selective degeneration in these neurons have been available since the 1960s. Foremost among these is 6-hydroxydopamine (6-OHDA), an analogue of dopamine which is taken up and concentrated into catecholamine neurones by an active uptake channel and then metabolized intraneuronally to yield toxic free radicals that kill the neurones [92]. Although 6-OHDA does not cross the blood-brain barrier, when injected into the neuropil of the brain it is concentrated by
and selectively kills all catecholamine neurones in the vicinity. If injected into the lateral ventricles, the toxin will induce extensive bilateral forebrain depletions of both dopamine and noradrenaline. Without further treatment, forebrain noradrenaline projections are also disrupted, but selective dopamine denervation can be achieved by pretreating animals with the noradrenaline uptake inhibitor des-methyl imipramine. Alternatively, the toxin can be injected into terminal areas, such as the neostriatum, resulting in a rapid loss of dopaminergic terminals and a progressive but typically partial retrograde degeneration in the substantia nigra. For more complete selective lesions, the most effective site for destruction of the dopaminergic neurons of the substantia nigra is to inject the toxin stereotaxically into the medial forebrain bundle — where the axon fibers are at their most concentrated as they pass through the posterior hypothalamus, leading to effective degeneration of all dopamine projections from the ventral mesencephalon to the dorsal and ventral striatum, amygdala, septum, and frontal cortex on the side of the injection.
BILATERAL LESION SYNDROME Bilateral dopamine depletions by intraventricular or nigrostriatal injection produce a profound impairment for the engagement of any voluntary movement in animals. They cease feeding, drinking, and grooming, and are profoundly akinetic, exhibiting a catalepsy in which they actively resist all movement [28, 66, 99, 108]. If the lesions are complete, the bilateral lesion syndrome is extremely debilitating. The animals will die unless they are tube fed and actively nursed for a protracted period. Conversely, if the lesions are only partial there can be considerable spontaneous behavioral recovery, as spared neurons compensate for those that are lost. In view of the difficulty of assessing behavioral recovery associated with specific treatments such as cell transplantation against either type of baseline, the bilateral lesion model has not been widely used to assess experimental reparative treatments.
UNILATERAL LESION SYNDROME By contrast, the unilateral lesion model has been widely utilized [28]. The animals exhibit predominantly unilateral motor symptoms on the side of the body contralateral to the dopamine-denervated hemisphere, leaving the
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ipsilateral side to maintain the full range of regulatory activities necessary for full health. Rats with unilateral 6-OHDA lesions show marked postural asymmetries and a bias for turning away from the intact side (i.e., ipsilateral to the lesion). This side bias is exacerbated by activating stimuli or drugs, such as amphetamine, which will induce strong turning (“rotation”) at a rate of 10 to 15 turns per minute for the several hours the drug is active [101]. Amphetamine is an indirect agonist that enhances dopamine activation in the intact striatum — an effect that is eliminated from lesioned terminals, resulting in the ipsilateral motor asymmetry. Conversely, direct dopamine receptor agonists such as apomorphine induce rotation in the opposite direction, contralateral to the lesion. This is due to preferential activation of supersensitive receptors on postsynaptic neurons that have been denervated of their intrinsic dopaminergic inputs by the lesion [100]. Although at face value drug-induced rotation has little similarity to the precise symptoms of Parkinson’s disease, it is very widely used as an experimental measure because the test is highly reliable and the rate of turning correlates closely with the extent of underlying denervation [49, 91] — providing a sensitive, noninvasive behavioral assay of denervation and recovery of dopamine innervation on an animal-by-animal basis. In addition to the motor asymmetry measured by the rotation test, rats with experimental dopamine denervation exhibit deficits in a range of motor coordination, sensorimotor, and motivational functions [28, 66] — and some of the tests that have been used to assess transplant function in nondrugged animals (such as forelimb placing, stepping, and reaching) will be described in due course. There is also some evidence that striatal dopamine depletions will induce deficits on cognitive tests (in particular ones related to prefrontal function, such as T-maze alternation), but this issue has proved technically difficult to explore because of the confounding motivational and regulatory impairments induced by the bilateral lesions necessary to disrupt bilaterally represented cognitive processing. At present the closest approach to cognitive deficits in animals with dopaminergic lesions may be provided by loss of reward signaling on just one side after unilateral lesions [21, 26].
MPTP Whereas 6-OHDA has provided the toxin of choice for studies in experimental rats, the discovery of the
dopaminergic neurotoxicity of the heroin analogue 1-methyl-4-phenyl-tetrahydropyridine (MPTP) first in drug addicts and subsequently in experimental monkeys has provided a powerful alternative model system for development of experimental therapeutics for Parkinson’s disease [53]. MPTP induces selective nigrostriatal DA degeneration, even after peripheral administration, leading to a movement disorder very similar to the symptoms of idiopathic Parkinson’s disease — other than its rapid onset and occurrence in typically younger subjects. MPTP-induced Parkinson’s disease is equally responsive to l-dopa treatment, including the fact that the affected individuals rapidly develop the side effects typical of advanced disease. The advantage of MPTP as an experimental model is that it can be administered peripherally, and the dose regime can be titrated to achieve a required degree of degeneration and disability. In large animals such as primates, unilateral lesions can be readily achieved by intracarotid administration to target just one hemisphere without the need for stereotaxic surgery [5]. MPTP is not an effective dopamine neurotoxin in rats because of species differences of metabolism — MPTP is not itself toxic but is converted by monoamine oxidase (MAO) in its A isoform to the active toxin, whereas rats almost exclusively use the B isoform of MAO — but it is effective and widely used in mouse models [93]. Consequently, MPTP has been widely used in mice and primates over the last two decades both for theoretical studies of the fundamental mechanisms involved in dopamine neuron cell death [86] and in the development of novel therapeutics of all classes [53].
TRANSGENICS Although mutations in a-synuclein and in several Park genes can cause an inherited form of Parkinson’s disease in rare pedigrees, transgenic models based on these mutations have as yet to yield useful models of human Parkinson’s disease for experimental therapeutics.
Functional Experimental Transplants Transplantation of dopamine neurons in rats and monkeys with experimental dopamine depletions has been the most widely studied system of functional cell transplantation in the nervous system, and has provided the experimental basis for the pioneering clinical trials
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of neural transplantation in Parkinson’s disease patients. The first studies were undertaken in this system because of the particularly favorable features of the 6-OHDA model system: the availability of a selective neurotoxin for a single class of neurons with clear and simply measurable behavioral effects, the possibility of selective pharmacological manipulation, and a histochemical method for selective visualization of the target neurons in the period before the widespread availability of immunohistochemical methods. Nevertheless, even in the first experimental studies the potential clinical application to Parkinson’s disease was a matter for speculation [82].
EXPERIMENTAL TRANSPLANTATION OF DOPAMINE NEURONS The essential donor conditions for catecholamine neurons to survive transplantation into the adult nervous system were first determined by Olson and colleagues using an in oculo model [80]. Whereas catecholamine neurons of the peripheral nervous system (such as the chromaffin cells of the adrenal medulla) survive transplantation into the anterior eye chamber of adult rats at any donor age, central neurons are only viable if taken from the developing embryonic brain [78, 79]. Empirical studies revealed that optimal donor ages differ for each population of neurons, typically being around the time when the particular population is undergoing final division — at which stage specific neuronal fate is determined [80]. The optimal age for dopamine neurons of the substantia nigra is embryonic day E11-13 in the rat, when crown-rump length is in the 10- to 13-mm range such that pieces of tissue dissected from the ventral mesencephalon containing the developing substantia nigra survive and mature, contain many dopamine neurones, and will give rise to a dopaminergic innervation of co-grafted striatal tissues. Similarly in the brain, pieces of embryonic ventral mesencephalon survive transplantation into appropriate spaces in the brain — such as the lateral ventricle or a pre-prepared cavity through the adjacent cortex [14, 82, 96]. When implanted into the brains of rats with unilateral 6-OHDA lesions, the dopamine neurons in the grafts were seen to survive and give rise to a new dopaminergic innervation of adjacent dopamine-denervated targets in the host striatum [9, 41]. Moreover, from the very first studies simple functional screens were undertaken using the amphetamine and
apomorphine rotation tests, with the demonstration of good recovery of grafted rats from the lesion-induced turning syndrome [9, 14, 82]. Although drug-induced rotation was clearly sensitive for detecting clear functional effects of the grafted cells, early studies found rather more limited recovery on other tests of motor and sensorimotor disability [9, 33]. Although the first studies (whether in the brain or in oculo involved transplanting pieces of embryonic brain stem, the experimental development of graft-derived repair has been greatly enhanced by the development of the cell suspension method in which the tissues are dissociated using protocols first developed for cell culture and then prepared as a concentrated suspension of cells that can be injected stereotaxically into the host brain [13]. This has several distinct advantages: grafts are placed with stereotaxic accuracy, they provide good survival in the depths of the brain neuropil, they allow a combination of different tissues or multiple grafts in the same animal, surgery avoids the trauma involved in creating and implanting into cavities, and experimental groups may be matched to receive a standard graft preparation.
PATTERNS OF FUNCTIONAL RECOVERY One of the first principles to be determined using the cell suspension methodology was the importance of graft placement. The striatum is topographically organized in terms both of cortical inputs and functions subserved. Correspondingly, where a graft is placed in an animal with extensive denervation will determine which symptoms recover. In terms of the early standard tests, rotation is dependent on lesions and reinnervation in dorsal striatum, sensorimotor deficits in the lateral striatum, and locomotor activity in the ventralstriatum/nucleus-accumbens [31, 33, 34]. Moreover, the delivery of multiple graft placements provides a broader profile of recovery involving an additive effect of each placement in turn [31]. However, there have remained other tests that have been more resistant to recovery by grafts (see Table 25.1). Thus, lateral striatal lesions disrupt skilled paw reaching as well as performance on sensorimotor tests of contralateral neglect. However, whereas the grafts alleviate the latter deficit, the former has been resistant to recovery in many different experiments by several laboratories [36, 71, 81] — although one series of studies suggests that multiple micrografts
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TABLE 25.1 Functional recovery after ventral mesencephalic grafts in parkinsonian rats. Test
Comment
Reference
Tests That Reveal Recovery Rotation, amphetamine Rotation, apomorphine Rotation, spontaneous Sensorimotor neglect Side bias ICSS via electrodes implanted in grafts Lateralized visual discrimination Coordination and balance Stepping Akinesia
Dependent on graft-derived innervation responsive to drug Dependent on chronic dopamine release to normalize receptor sensitivity Reduced in unilateral lesion rats, contralateral to graft in bilateral lesion rats Dependent on lateral striatum Spontaneous choices in maze or runway Grafts sustain self-stimulation; suggests recovery in reward signaling Operant test in 9-hole box; suggests extinction rather than motor deficit Tested on variable-speed rotarod Variable recovery only in some studies; best with combined CP and SN grafts Reinnervation of ventral striatum in bilateral lesioned rats
14, 31 31, 82 10 31, 33 10, 27 37 26 89 4, 26, 81 32, 54
Tests That Do Not Recover Disengage test Hoarding Skilled paw reaching Cylinder placing Feeding/drinking
Ipsilateral stimuli override contralateral attention Dependent on ventral striatum but not restored by ventral grafts No recovery in most studies Typically does not recover Profound aphagia/adipsia after bilateral lesions
reaching all areas of the striatum may be effective [75]. Although lateral grafts restore attention to salient contralateral stimuli [31, 34, 36], they continue to neglect contralateral stimuli when already attending to an ipsilateral one — as revealed in the “disengage test” [65], a phenomenon similar to extinction in the human neuropsychological literature on sensory neglect. Similarly, in grafts in the ventral striatum that alleviate the locomotor hypoactivity of mesolimbic dopamine depletions the grafts are less effective in reversing the food hoarding deficits associated with the same ventral system [50, 51]. Several different but overlapping hypotheses have been put forward to account for the incomplete profiles of recovery typically reported [12, 107]. On the one hand, it may be that the grafts fail to reinnervate a critical terminal area for the particular function — or that normal performance is dependent on several different systems, only some of which are restored. On the other hand, grafts are typically placed in an ectopic location
65 51 36, 71, 81 26 10, 32
to provide a reinnervation of denervated terminals in the striatum. As such, they may provide a reactivation of striatal targets sufficient to alleviate deficits in performance that are attributable to loss of dopaminergic activation or arousal. Even in an ectopic site, the grafted neurones do appear to be under some local regulation, both by sprouting of reciprocal connections from host striatal neurones into the grafts and by collateral regulation at the level of presynaptic terminals within the host striatum. Nevertheless, a graft in the striatum does not reconstruct the nigrostriatal circuitry and will not restore performance on any task dependent on the normal dopamine system transducing afferents to the substantia nigra and relay of patterned information to the remote striatal target. It is likely that both additivity of placements and reformation of an appropriate circuitry will play important roles in optimizing the profile of recovery. When allografts are placed in the substantia nigra they survive but give rise to only very limited fiber outgrowth in
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the adult brain, and no reinnervation of distant targets [11]. Combined nigral and striatal grafts have in some cases provided better recovery than striatal grafts alone [67, 70], but this may represent additivity of placements rather than any restoration of nigrostriatal communication. There is some evidence that VM grafts derived from human donors do show more extensive nigrostriatal regrowth [103], but it remains unclear whether this is due to the significantly greater growth potential of developing human neurones or to the failure to recognize species-specific factors inhibiting longdistance regeneration in the adult brain. The alternative approach is to manipulate the adult brain environment to provide the stimulation and guidance cues to allow developing axons from VM grafts placed in the brain stem to grow to distant targets in the forebrain. This has been achieved both by manipulating the host glial environment through the medial forebrain bundle [73] and by laying down a track of glia to provide a bridge across which developing and/or regenerating axons can grow [35, 106]. Although such bridges have been seen to provide a functional reinnervation of the striatum sufficient to alleviate performance on the most sensitive rotation tests, the density of reinnervation over such distances remains sparse and has to date been insufficient to affect tests such as paw reaching.
Clinical Trials On the basis of the observed recovery in simple motor deficits (in particular, rotation) associated with dopamine lesions in rats, a neural transplantation strategy was rapidly tried in Parkinson’s patients in the early 1980s. Although the functionally beneficial effects of VM grafts in rats have been confirmed subsequently in other species, including primates [1, 3, 6, 29, 94], it is noteworthy that the primate studies did not precede embarkation on the human trials.
ADRENAL GRAFTS The first issue to be addressed in any clinical transplantation program is the source of donor tissue. To circumvent the social and ethical sensitivities that would be involved using human fetal donor tissues, the first trials sought an alternative source of suitable catecholamine neurons. The chromaffin cells of the adrenal medulla synthesize and secrete dopamine as well as adrenaline
and noradrenaline in vitro, survive transplantation well in the anterior eye chamber transplantation model, and were found to alleviate apomorphine-induced rotation in the rat lesion model [40]. In addition, it is possible to use one of the patient’s own adrenal glands in an autotransplantation strategy to obtain perfect tissue matching and thereby circumvent any potential immunological problems. Although the first study from Sweden of adrenal transplantation to the caudate nucleus in two parkinsonian patients found no detectable benefit [2, 59], a subsequent report from Mexico City suggested quite dramatic recovery in two further patients [63]. With the perceived simplicity of the procedure, this report stimulated a series of small trials in many neurosurgical centers during the late 1980s, resulting in several hundred patients being transplanted worldwide using a variety of surgical approaches [44, 87]. Although at the time the situation was very confusing, a consensus has emerged [47, 87]. First, some functional change was detectable in some patients, significant in statistical terms but minor in terms of clinical benefit. Second, adult adrenal tissues do not survive well and any functional benefit is not long lasting. Third, autografts may not be an ideal donor source because all catecholamines are significantly depleted in the adrenal as well as the brain in Parkinson’s disease. Fourth, the surgical procedure carries significant morbidity as well as unacceptable mortality, not only due to the open ventricular neurosurgery employed by the original Madrazo technique but to the major abdominal operation involved in harvesting the donor tissue in patients that are already elderly and frail [47, 102]. Although several of these concerns could be addressed by refined procedures, the development of fetal grafting strategies largely replaced the earlier adrenal transplantation approach in the 1990s.
ETHICS OF FETAL TISSUE DONATION With the realization that adrenal grafts did not provide a significant broad or sustained alleviation of deficits in either experimental animals or man, the development of an effective clinical therapy required addressing the social and ethical issues involved for developing protocols that did work well experimentally; namely, use of fetal tissues. In societies where elective abortion is not itself permitted, there have been some attempts to collect and harvest tissues for transplantation from spontaneous abortions [64]. These, however, have not met
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great success both because of the practical difficulties of tissue collection and because of higher likelihood of fetal abnormalities in such tissues. Where elective abortion is allowed, detailed professional and political discussions (initiated in Sweden but undertaken in several countries) have led to the conclusion that use of human fetal tissues for research and therapy is not unethical, provided appropriate steps are undertaken to ensure that consent and decision to donate the tissues for research or therapy should have no influence on the decision, timing, and procedures for termination of pregnancy itself [15, 85]. In practice, this has led to ethical guidelines that impose different levels of separation between the gynecological teams consenting and undertaking the terminations of pregnancy and collecting the tissue, from the neurological team working with the recipient patients and the neurosurgeons undertaking the transplantation [85]. Once an ethical sourcing of human fetal donor tissues has been established, extensive studies have been undertaken to validate the handling, dissection, preparation, storage, and implantation of human VM tissues for transplantation, in particular using xenografts to the immunosuppressed parkinsonian rat model to validate according to principles of dopamine cell survival, fiber growth, and functional recovery in rotation tests [19, 23, 42, 88, 98]. Of particular importance in these studies has been not only the determination of the optimal developmental stages in human fetal development for survival of dopamine cells within the grafts [19, 42] but more recently the development of storage protocols that allow the period that cells remain viable for transplantation from a few hours with standard preparations to up to 5 to 7 days cool storage in hibernation media [52, 90], considerably enhancing the logistics of scheduling of the neurosurgical operations.
80% and the patient showed a dramatic improvement in neurological movement test scores during defined “off” to close to normal speeds, a dramatic reduction in rigidity, an improvement in the duration of “on” state following a single dose of l-dopa, and a clear and significant improvement of [18 F]-dopa uptake in the grafted putamen on PET scan. Although this study involved only a single patient and a relatively short follow-up period (five months after surgery), subsequent studies have shown similar improvements of the majority of 17 patients grafted in the Lund series [61] (see Table 25.2), with the beneficial effects in some patients lasting in excess of ten years [83]. Moreover, the clinical effects of the graft are typically associated by a reduction in the need for l-dopa (see Table 25.2) and at least one patient has remained in 100% “on” for more than six years following complete cessation of l-dopa [58, 83]. The Lund series provides probably the clearest evidence of sustained beneficial effects in a small number of patients studied in great detail, but similar clear benefits have also been reported from several other centers (see Table 25.2) [8, 30, 77]. Nevertheless, if the alleviation can be apparently so dramatic we must consider why so few patients have been grafted. Approximately 300 worldwide have been reported over the 15 years since the first reports of success. The problems are of several types: practicalities of tissue availability, relatively poor graft survival, disputes about trial design; incomplete recovery and how well transplants work in comparison with the best available alternatives; and the emergence of disturbing side effects in a proportion of patients.
FETAL VM TRANSPLANTATION
The first and foremost problem encountered is the difficulty of obtaining human fetal donor tissue of sufficient quality and reliability. Embryonic dopamine cells are relatively fragile, and are highly sensitive to oxidative stress and other stressors — such that typically only 5 to 10% survive the transplantation process. Although considerable advances have been made in promoting cell survival using a variety of trophic, antioxidative, antiapoptotic, and other neuroprotective strategies [18], it remains the case that multiple donors are required for each transplant. A second practical factor limiting availability is the worldwide trend to increasing the
The first clear evidence of viable functional transplantation of cells in Parkinson’s disease patients was reported by Lindvall and colleagues at the University of Lund in 1990 [60]. In this single case study, a patient who was exhibiting marked dyskinetic complications and up to 50% of each day in the “off” state prior to surgery received implants of dissociated VM suspensions derived from four human embryos in three tracks into the left putamen. Following surgery, the proportion of each day spent in “on” increased to
Side Effects and Problems LIMITED TISSUE AVAILABILITY
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Stephen B. Dunnett and Anne E. Rosser TABLE 25.2 Functional trials of bilateral ventral mesencephalic grafts in Parkinson’s disease.1 n3
# VM4
Target5
PET F-DOPA Uptake6
OL
4 2 5
4.9 2.5 2.8+L
Pu C+Pu C+Pu
60/52 87/68 55/48
−30 −50 − 40
−59 −50 −43
−37 −35 −45
46 105 20
OL
6
3–4
PPu
61/55
−30
−43
−16
48
1 4
PPu
++ + + ++
−10 −17
+7 −2
−20 −20
76
Pu
30/42
−6 −33
15 −66
—
24 74
—
68 69
Design2
Lund
UPDRS Motor7
Time in Off7
l-dopa Dose7
Reference
Tampa PDBPC
11 12
Créteil
OL
3 6
1–1.5 3
Halifax
OL
2 3
3.25+G
PPu Pu+SN
107/62
−32
−50
Denver
DBPC
2
Pu
40/−
−18
—
19
0
39
1. Expanded from previous review article [30]. 2. OL, open-label; DBPC, double-blind placebo control. 3. Number of subjects in trial. 4. Number of embryonic ventral mesencephali per graft. 5. C, caudate nucleus; SN, substantia nigra; Pu, putamen; PPu, posterior putamen. 6. % change in Ki uptake constants from pre-graft level and as % of normal; ++, + + ++, levels of improvement in one study providing absolute change but not baseline values. 7. % changes posttransplantation of each measure (negative scores indicate improvement).
proportion of medical versus surgical procedures for terminations of pregnancy, in particular for fetuses at the earlier stage of gestation (6 to 8 weeks) optimal for transplantation in Parkinson’s disease. Third, until recently the short duration for viability of donor fetal tissues required that the transplantation had to be undertaken on the day of collection, imposing marked logistic problems on coordinating neurosurgery with the timing of tissue availability, especially when dependent also on multiple donations. As outlined previously, this problem is beginning to be overcome with the introduction both of more viable hibernation methods [52, 90] and the use of factors in clinical trials to promote survival [20, 69]. However, although each of these practical issues is potentially soluble, no procedure dependent on separate donations of fetal tissues, especially when collected via the intrinsically variable protocols involved in termination of pregnancy, can ever achieve the levels of standardization, demonstrated pathogen-free status, and quality control that would meet optimal
medical standards of good manufacturing and clinical practice.
UNRELIABLE BENEFIT AND INCOMPLETE RECOVERY Whereas transplants can work demonstrably and dramatically well in some patients and for some symptoms, there is considerable variability in response for reasons that are not well understood. As with VM grafts in experimental animals, so also in patients some parkinsonian symptoms (such as bradykinesia and rigidity, as measured in simple neurological tests of speed of repetitive limb movements) are more responsive to the implants than others (such as axial impairments, balance, and tremor). Moreover, there can be considerable variability from patient to patient. One factor is variability in graft survival. Indeed, a significant increase of the PET fluorodopa uptake constant Ki to near-normal
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values — which indicates extensive replacement of dopaminergic terminals in the striatum — appears to be a necessary although not sufficient criterion for significant clinical recovery. Accurate diagnosis of idiopathic Parkinson’s disease remains a critical issue for all intervention trials, and the failure to gain significant benefit with a good surviving graft in at least one transplant patient is most likely related to a misdiagnosis of PD in a patient with multiple-system atrophy. All present trials involve attempts to standardize preparation and implantation protocols, including use of a common set of target sites. As we come to understand more about striatal topography, it is likely that improved benefit could be achieved by selection of target sites for each individual patient based both on a rationale selection to match the profile of symptoms and on improved resolution of diagnostic imaging [30].
TRIAL DESIGN A recurring area of contention remains the issue of how best to design the most informative trials for novel surgical therapies such as neural transplantation in Parkinson’s disease. The gold standard of trial design developed for assessing the effects of pharmaceutical drugs and medicines is the double-blind placebo controlled trial. In particular, there is significant concern that placebo effects might provide spurious benefits when working with highly motivated clinician scientists and desperate patients, which has led to strong arguments that the only way to obtain unbiased informative results is through a double-blind procedure in which patients are randomly allocated to experimental or control groups and both the patient and the clinical assessor are ignorant of each individual’s allocation until trial closure. As a corollary, in order for the patients and their physicians to remain ignorant of status it has been argued that all subjects should go through a sham surgical procedure [43] — which is likely to include surgical preparation, scanning, anesthetization, and bone surgery whether or not the individual is allocated to the experimental treatment arm. Two trials have now been undertaken using such double-blind protocols [39, 76]. Whereas the grafts were seen to survive well in both studies, as evidenced by PET imaging, neither demonstrated significant benefit of the surgical intervention on the primary outcome variables, which has led a widespread perception that transplants “don’t work” and that further trials should be halted.
We believe that this conclusion is not only premature but inappropriate [30]. First, there is an inconsistency in the standard applied to drug and surgical trails. Standard drug trials typically require several hundred patients per group to achieve the appropriate statistical power, whereas the two recent transplant trials used 11 to 19 patients per group. Nevertheless, even though not significant in the primary outcome variables, secondary variables and longer analyses beyond the point of un-blinding clearly suggested greater improvements in the transplant groups, suggesting that the formal trials were simply underpowered. Second, the design of the first study — with selection of subjective ratings of perceived improvement (rather than the more objective neurological scores) as the primary outcome and with an early closure only 13 months after surgery — almost seems to be maximally sensitive to placebo over biological effects of the experimental surgery. It is therefore noteworthy that neither study found evidence of strong or significant placebo improvement on the various neurological measures of performance. Of more fundamental concern is whether full-scale double-blind controlled trials are appropriate at this stage in the development of neural transplantation. Surgical trials have a number of distinct features when compared with standard drug trial design. First, experimental surgical treatments are permanent and not reversible, in contrast to drug trials in which drug administration can be discontinued. Second, because all surgical operations involve some risk, sham surgery is itself considered unethical by many [16]. Third, transplantation in a progressive disease such as Parkinson’s involves assessment against a chronic but variably progressing baseline, and transplanted cells require long periods to grow and connect and are likely to require several years to achieve optimal function. Finally, it could be argued that protocols are still at an early stage of development appropriate for pilot studies but are not yet optimized to the level required for a formal trial of efficacy [104]. It has therefore been argued that the more effective strategy is to proceed in a step-by-step manner studying individual patients longitudinally and in detail, using standardized and well-validated assessment protocols [56] in order to achieve a progressive refinement of surgical protocols [16].
DYSKINESIAS The controlled trials have served one important function, and that has been to identify a particular aspect of side
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effects of transplantation that had been overlooked in the open-label trials. Five of the 33 patients eventually receiving grafts in the Denver series [39] exhibited severe “runaway” dyskinesias even when the patients were withdrawn from l-dopa. Similarly, in the Tampa series up to half of the grafted patients exhibited dyskinesias, although these were largely resolved by l-dopa withdrawal. These side effects have caused widespread concern and calls for a stop to further transplantation studies. Although Freed and colleagues attributed the effect to dopamine overdose [39], this seems unlikely in view of the fact that the level of dopamine replacement indicated by the PET scans was lower than in other studies that had not found similar effects [17]. It is possible to model levodopa dyskinesias in experimental animals [22, 72], and whereas early studies reported that VM grafts reduced levodopa-induced dyskinesias [57] more detailed analysis has revealed a more complex interaction of reducing many drug-induced side effects while introducing novel limb hyperkinesias [95]. From consideration of both clinical and animal studies, present attention is now focusing on the possibility that some implantation protocols are more likely to produce “hot spots” of innervation surrounded by zones in which extensive denervation is accompanied by marked changes in receptor sensitivity, leading to abnormal spatial and temporal patterns of target activation [25, 62]. Retrospective reanalysis of the data from both the Lund and Denver series suggests that the major factor in the development of dyskinesias may be the magnitude of preexisting dyskinesias prior to transplantation surgery [38, 45] rather than a problem caused by the grafts de novo.
COMPARISON WITH DBS
confronted in the transplantation field — although the control issue of comparing subjects with the stimulator switched on versus off clearly aids experimental design. Both types of intervention exhibit distinctive complications. There is, however, one major difference in that DBS provides essentially symptomatic relief and does not alter the fundamental deficit of dopamine denervation nor the progressive nature of the underlying disease. By contrast, when working optimally VM transplantation offers the prospect of achieving structural repair that may alter the progression of the disease itself. Thus, with their different mechanisms of action and time course of effect nigral transplantation and DBS in principle offer complementary approaches to treatment. In particular, in the same way as we argued for the need in the transplantation field for refined methods of imaging and diagnosis to tailor the transplant to the individual patient so also we need to consider whether the two treatment approaches have different profiles of efficacy on the range of parkinsonian symptoms. To date there have been no systematic comparisons of the two approaches. If it turns out that some symptoms are more resistant to amelioration than others, irrespective of treatment modality, the choice between DBS and transplantation will be based on other practical, economic, and clinical factors. However, because the two approaches do involve fundamentally different mechanisms of action it is likely that they have distinct profiles of benefit. With comparable practicality and safety, the choice should then be based on the specific acute and long-term status and needs of each individual patient.
Summary and Future Prospects
A final factor influencing the current perception of transplantation therapies for Parkinson’s disease is the growing availability of deep brain stimulation as a viable surgical alternative [7]. In spite of the now many published reports of the benefits of deep brain stimulation, there has been criticism following systematic review that there is a paucity of clear evidence for longterm benefit in properly controlled trials [97]. However, exactly the same argument has been leveled against the neural transplantation literature [84]. DBS also involves experimental surgery in progressive disease, and is subject to the same difficulties of conducting properly controlled clinical trials against a slow progressive baseline
Both animal studies and clinical trials of neural transplantation suggest that embryonic VM cells can survive transplantation, replace lost dopamine innervation in the striatum caused by toxic damage or disease, and have a functional impact on the host sufficient to alleviate many of the symptoms associated with the lesion. On the basis of the animal studies, there are now a series of trials — both open-label and with controlled designs — in Parkinson’s disease patients that support the proof of principle that grafts can partially repair the cell loss associated with this disease and offer clinically significant benefit in alleviating some of the patients’ symptoms. However, the clinical trials have highlighted
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first that not all symptoms are equally responsive to the treatment and the response can vary significantly from patient to patient and second that the graft procedure can have distinct adverse effects in some patients, in particular in the form of uncontrolled dyskinesias. The problems as well as the benefits are manifested equally in the animal models, which offer the tools of addressing and resolving the complications now that they have been clearly identified. The causes of Parkinson’s disease remain unknown, and at present we have no prospect of halting the disease. Although other pharmacological and surgical therapies are available, cell transplantation remains the only one that offers true repair — even if only partial and incomplete — of the neurodegeneration caused by the disease. Experimental studies of striatal topography, enhanced cell survival, and understanding and circumventing the causes of the dyskinetic side effects of VM grafts are all essentially soluble through refinements in present methods. A more fundamental issue is the need to identify alternative sources of suitable cells for transplantation that are both practical and ethical, in that primary human fetal cells donated from elective abortion can never provide the consistent supply of standardized and fully validated cells required for widespread application. Consequently, there is an active search worldwide for a suitable quality-controlled alternative, of which (based on present knowledge) the front-runner is a source of stem cells, whether of embryonic (ES), fetal, or adult origin. An effective source will have to meet a number of criteria, including that the cells (1) remain stable under extensive (even if not indefinite) expansion, (2) can be reliably differentiated into an appropriate phenotype with at least the same capacity for growth, connection, integration, regulated dopaminergic synthesis, and synaptic release as primary developing dopamine neurones, (3) can be produced and distributed under validated conditions of GMP quality control, and (4) are safe, including there being no capacity for uncontrolled tumor formation. It must be recognized that the availability of suitable cells that meet these criteria is not imminent, due to the complexity of the regulatory as well as scientific issues that need to be resolved, and contrary to some overoptimistic claims to the contrary. Nevertheless, these goals are achievable, and given the levels of investment committed to this area we should expect suitable cells to become available within the foreseeable future. Then, the translation of stem cells
for transplantation into the clinic will need to resolve exactly the same issues related to patient selection, implantation parameters and placement, study design, patient assessment, and identification and control of side effects as already confronted in the trials using primary fetal cells. Consequently, we believe that the present widespread disappointment with primary VM tissue transplantation is misplaced. The present use of human fetal cells provides a proof of principle of the feasibility of cell-based surgical therapy in Parkinson’s disease and identifies the key issues that have to be resolved for taking any source of cells into the clinic. The conventional approach has provided both the animal models and clinical framework that will allow the rapid and effective translation of alternative cell sources, such as stem cells, to clinical applications when those alternatives are ready.
Acknowledgments Our own studies are supported by the Medical Research Council, the Parkinson’s Disease Society of Great Britain, the Lister Foundation, and the Wellcome Trust.
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79. Olson, L., Seiger, Å. Brain tissue transplanted to the anterior chamber of the eye. 1. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons reinnervating the rat iris. Z Zellforsch Mikrosk Anat 135:175–194; 1972. 80. Olson, L., Seiger, A., Strömberg, I. Intraocular transplantation in rodents: A detailed account of the procedure and examples of its use in neurobiology with special reference to brain tissue grafting. Adv Cell Neurobiol 4:407–442; 1983. 81. Olsson, M., Nikkhah, G., Bentlage, C., Björklund, A. Forelimb akinesia in the rat Parkinson model: Differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test. J Neurosci 15:3863–3875; 1995. 82. Perlow, M. J., Freed, W. J., Hoffer, B. J., Seiger, Å., Olson, L., Wyatt, R. J. Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 204:643–647; 1979. 83. Piccini, P., Brooks, D. J., Björklund, A., Gunn, R. N., Grasby, P. M., Rimoldi, O., Brundin, P., Hagell, P., Rehncrona, S., Widner, H., Lindvall, O. Dopamine release from nigral transplants visualized in vivo in a Parkinson’s patient. Nat Neurosci 2:1137–1140; 1999. 84. Polgar, S., Morris, M. E., Reilly, S., Bilney, B., Sanberg, P. R. Reconstructive neurosurgery for Parkinson’s disease: A systematic review and preliminary meta-analysis. Brain Res Bull 60:1–24; 2003. 85. Polkinghorne, J., Hoffenberg, R., Kennedy, I., Macintyre, S. Review of the guidance on the research use of fetuses and fetal material. London, HMSO, 1989. 86. Przedborski, S., Vila, M. MPTP: A review of its mechanisms of neurotoxicity. Clin Neurosci Res 1:407–418; 2001. 87. Quinn, N. P. The clinical application of cell grafting techniques in patients with Parkinson’s disease. Prog Brain Res 82:619–625; 1990. 88. Redmond, D. E. Jr., Naftolin, F., Collier, T. J., Leranth, C., Robbins, R. J., Sladek, C. D., Roth, R. H., Sladek, J. R. Jr. Cryopreservation, culture, and transplantation of human fetal mesencephalic tissue into monkeys. Science 242:768–771; 1988. 89. Rozas, G., Labandeira Garcia, J. L. Drug-free evaluation of rat models of parkinsonism and nigral grafts using a new automated rotarod test. Brain Res 749:188–199; 1997. 90. Sauer, H., Brundin, P. Effects of cool storage on survival and function of intrastriatal ventral mesencephalic grafts. Rest Neurol Neurosci 2:123–135; 1991. 91. Schmidt, R. H., Björklund, A., Stenevi, U., Dunnett, S. B., Gage, F. H. Intracerebral grafting of neuronal cell suspensions. III. Activity of intrastriatal nigral suspension implants as assessed by measurements of dopamine synthesis and metabolism. Acta Physiol Scand Suppl 522:19–28; 1983. 92. Schwarting, R. K., Huston, J. P. The unilateral 6-hydroxydopamine lesion model in behavioral brain research: Analysis of functional deficits, recovery and treatments. Prog Neurobiol 50:275–331; 1996.
93. Sedelis, M., Schwarting, R. K., Huston, J. P. Behavioral phenotyping of the MPTP mouse model of Parkinson’s disease. Behav Brain Res 125:109–125; 2001. 94. Sladek, J. R. Jr., Collier, T. J., Haber, S. N., Deutch, A. Y., Elsworth, J. D., Roth, R. H., Redmond, D. E. Jr. Reversal of parkinsonism by fetal nerve cell transplants in primate brain. Ann N Y Acad Sci 495:641–657; 1987. 95. Steece-Collier, K., Collier, T. J., Danielson, P. D., Kurlan, R., Yurek, D. M., Sladek, J. R. Jr. Embryonic mesencephalic grafts increase levodopa-induced forelimb hyperkinesia in parkinsonian rats. Mov Disord 18:1442–1454; 2003. 96. Stenevi, U., Björklund, A., Svendgaard, N. A. Transplantation of central and peripheral monoamine neurons to the adult rat brain: Techniques and conditions for survival. Brain Res 114:1–20; 1976. 97. Stowe, R. L., Wheatley, K., Clarke, C. E., Ives, N. J., Hills, R. K., Williams, A. C., Daniels, J. P., Gray, R. Surgery for Parkinson’s disease: Lack of reliable clinical trial evidence. J Neurol Neurosurg Psychiatry 74:519–521; 2003. 98. Stromberg, I., Bygdeman, M., Goldstein, M., Seiger, Å., Olson, L. Human fetal substantia nigra grafted to the dopamine-denervated striatum of immunosuppressed rats: Evidence for functional reinnervation. Neurosci Lett 71:271–276; 1986. 99. Ungerstedt, U. Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta Physiol Scand Suppl 367:95–122; 1971. 100. Ungerstedt, U. Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta Physiol Scand Suppl 367:69–93; 1971. 101. Ungerstedt, U. Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behaviour. Acta Physiol Scand Suppl 367:49–68; 1971. 102. Waxman, M. J., Morantz, R. A., Koller, W. C., Paone, D. B., Nelson, P. W. High incidence of cardiopulmonary complications associated with implantation of adrenal medullary tissue into the caudate nucleus in patients with advanced neurologic disease. Crit Care Med 19:181–186; 1991. 103. Wictorin, K., Brundin, P., Sauer, H., Lindvall, O., Björklund, A. Long distance directed axonal growth from human dopaminergic mesencephalic neuroblasts implanted along the nigrostriatal pathway in 6-hydroxydopamine lesioned adult rats. J Comp Neurol 323:475–494; 1992. 104. Widner, H. NIH neural transplantation funding. Science 263:737; 1994. 105. Widner, H., Tetrud, J., Rehncrona, S., Snow, B., Brundin, P., Gustavii, B., Björklund, A., Lindvall, O., Langston, J. W. Bilateral fetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP). N Engl J Med 327:1556–1563; 1992.
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107. Winkler, C., Kirik, D., Björklund, A., Dunnett, S. B. Transplantation in the rat model of Parkinson’s disease: Ectopic versus homotopic graft placement. Prog Brain Res 127:233–265; 2000. 108. Zigmond, M. J., Stricker, E. M., Deficits in feeding behavior after intraventricular injection of 6-hydroxydopamine in rats. Science 177:1211–1214; 1972.
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26 Transplants for Chronic Pain Jacqueline Sagen, Daniel A. Castellanos, and Shyam Gajavelli Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, Florida
Introduction Despite recent advances in the understanding of mechanisms underlying persistent pain development and maintenance, the therapeutic management of chronic pain remains a significant clinical challenge. Although potential novel molecular and pharmacologic targets are being actively pursued in preclinical studies and early clinical trials, the majority of interventive pain management therapies still relies on two major classes of compounds and their derivatives: nonsteroidal antiinflammatory drugs (NSAIDs) and the opioid family, both of which have been in use for thousands of years. In addition, pharmacologic approaches for chronic conditions can be limited by untoward side effects and by the development of tolerance and reduced effectiveness over the prolonged treatment periods required. Thus, alternative strategies as well as identification of new pharmacologic targets are active areas of research aimed at improving the therapeutic management of chronic pain. This chapter focuses on the potential for cellular transplantation approaches to address both of these aims. Cell transplantation strategies can be explored for a number of goals in the treatment of CNS disorders, ranging from replacement of lost or damaged neural circuitry to supplementation or provision of therapeutic pharmacologic molecules. Because pain management traditionally relies to a large extent on pharmacotherapy, the latter approach (and perhaps the simplest) — that of utilizing cellular transplants for provision of therapeutic molecules — has been the emphasis in the majority of preclinical studies in this field. The treatment of pain may be particularly amenable to this approach because cells can be placed noninvasively into the CSF, where they can act as pharmacologic “mini-pumps” to deliver
Cellular Transplantation: From Laboratory to Clinic
therapeutic molecules to their target sites in the spinal cord. However, an alternative strategy may be replacement or supplementation of cells thought to become damaged or dysfunctional in pain-processing regions of the spinal cord, particularly for the treatment of chronic pain following injury to the CNS. These strategies are addressed in the following sections.
Cellular Replacement Strategies in the Management of Chronic Pain Although the vast majority of cellular transplantation approaches for chronic pain management has utilized the cellular mini-pump approach, it is possible to envision a cellular replacement strategy for more severe cases of chronic pain where overt loss or damage to neural circuitries has been postulated as an underlying mechanism. As an example, a likely candidate could be the particularly vulnerable inhibitory interneurons in the spinal cord dorsal horn, which are thought to restrict ongoing pain under normal circumstances. It has been hypothesized that excessive excitatory amino acid release following injury to the peripheral or central nervous system results in excitotoxic insult to these small inhibitory interneurons in the spinal cord [25]. Following peripheral nerve injury, an increased incidence of hyperchromatic “dark neurons” in the superficial spinal or medullary dorsal horn is found, and this can be further exacerbated by a pharmacologic blockade of inhibitory neurotransmission [51, 127]. Dark neurons may be indicative of transsynaptic degeneration or atrophy, most likely including functionally impaired inhibitory interneurons [127, 128]. Coinciding with the rise of dark neurons in the dorsal horn are spontaneously active neurons and neurons with expanded
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injections. Once grooming behavior is initiated, the targeted area is photographed and drawings made on a standardized leg drawing. Preliminary studies examined the fate and behavioral outcomes following transplantation of neural progenitors containing enriched GABAergic populations in QUIS lesioned rats. After QUIS lesions, following the onset of excessive grooming BrdU-labeled rat cortical progenitor cells were transplanted into the lesion sites at T12-L1. Grooming areas were measured before transplantation and at perfusion 3 weeks after transplantation. Grooming was reduced or stabilized in a majority of animals (> 80%) that received neural progenitor grafts, in contrast to progressively increased grooming area in controls (Figure 26.1). When excessive grooming was reversed or substantially attenuated, numerous GABA-IR cells were observed in the transplant area (Figure 26.2). Fewer GABAergic cells were associated with modest or no changes in grooming behavior. These results suggest that neural progenitor transplants may be useful in restoring lost dorsal horn neurons and alleviating central pain following SCI. In another preliminary study, Hendricks et al. [57] reported reduced grooming and allodynia in QUIS lesioned mice receiving transplants of predifferentiated
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receptive fields that respond to non-noxious stimulation of adjacent dermatomes [3, 130]. g -aminobutyric acid (GABA) is a major inhibitory neurotransmitter concentrated in the superficial laminae of the dorsal horn [80, 131], where nociceptive processing predominates. An important role for GABA in sensory processing is suggested by physiological and behavioral studies, which indicate primarily an inhibitory function in the transmission of noxious stimulation [63, 73]. Thus, a loss of GABAergic inhibitory mechanisms in the spinal dorsal horn can lead to sustained hyperexcitability and consequent persistent pain. In support for this, GABAimmunoreactivity (GABA-IR) is reduced in laminae I–III after sciatic nerve transaction [16], and reductions in dorsal horn GABA-IR and GAD are found following chronic constriction injury (CCI) of the sciatic nerve [34, 61, 86, 98]. Cell death using TUNEL labeling and stereological estimates from EM sections of the superficial dorsal horn has also been observed following CCI and sciatic neurectomy [5, 18, 143]. A dramatic loss in spinal GABAergic neurons also occurs after ischemic spinal injury, and GABA agonists can reverse mechanical allodynia in the early post-injury phase [56]. Findings in our laboratory have also indicated a selective loss in GABA-IR in the superficial dorsal horn following excitotoxic spinal cord injury using the quisqualic acid lesion model SCI. Thus, a cellular replacement approach for chronic pain management — particularly following injury to the nervous system — may aim at replacing lost or damaged GABAergic interneurons in the spinal cord dorsal horn. In preliminary studies in our laboratory [70], GABAergic neural progenitor cells were transplanted into the spinal dorsal horn of animals that had undergone spinal cord excitotoxic injury 10 to 14 days earlier. The excitotoxic model takes advantage of the potential involvement of glutamate toxicity in the pathophysiology of SCI pain by utilizing direct intraspinal injections of glutamate agonist quisqualic acid (QUIS). This results in a central pain syndrome that includes mechanical and cold allodynia, thermal hyperalgesia, and spontaneous excessive grooming behaviors, and has been utilized extensively by colleagues at The Miami Project to characterize SCI pain and potential therapeutic interventions [7, 8, 40, 153, 154]. Excessive grooming behavior is thought to reflect the condition of compulsive, selfdirected behavior in patients with neuropathic pain and painful dysesthesia [81]. The excessive grooming is progressive and typically targets dermatomes associated with spinal segments adjacent to the site of QUIS
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Figure 26.1. Progression of overgrooming, an indicator of dysesthetic pain, in rats with excitotoxic spinal cord injuries. Rats received excitotoxin lesions using quisqualic acid (QUIS) injections into the spinal dorsal horn. Following the onset of overgrooming behaviors, animals received either grafts of GABAergic neural progenitors or sham injections at the spinal cord injury site. The progression of grooming was monitored for 3 weeks posttransplantation.
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Figure 26.2. Appearance of GABAergic progenitor cells (green cells) grafted near QUIS injury site in the spinal dorsal horn 4 weeks following transplantation. See color insert.
mouse embryonic stem cells. The phenotypic fate of the transplanted cells has not yet been determined. Another group recently reported recovery of both motor and sensory function, including reduced hypersensitivity to noxious heat stimuli, by grafts of mixed neural and glial restricted precursors (NRP/GRP) following a moderate spinal contusion injury [89].
including catecholamines and proteins), and (4) cells may be combined or genetically manipulated to deliver a therapeutic “cocktail” of multiple desired agents for synergistic effects or for treatment of chronic pains of different etiologies. It should, however, be kept in mind that there are also some drawbacks and limitations associated with this approach for chronic pain management: (1) because cellular delivery is a biological process, it is limited to agents that can be manufactured and secreted by cells (i.e., natural or naturally derived agents), (2) achievable levels of a given agent that can be manufactured and delivered by the cells may be limited and insufficient for reaching therapeutic efficacy, (3) success of cell-based therapy is dependent on the survival of implanted cells, which may be limited by immunologic factors, nutrient and oxygen supply, and by diffusion of sufficient amounts of therapeutic molecules to sufficient target distances in the host, and (4) since cells produce a multitude of substances in addition to those of therapeutic interest, and can probably never be completely defined, safety issues are complex and extra care must be taken in order to ensure that the cells do not release potentially detrimental agents when implanted in the host nervous system.
Adrenal Medullary Chromaffin Cells for Analgesic Delivery
Cellular Pump Strategies in the Management of Chronic Pain The strategy taken in the majority of transplantation studies for pain has been the cellular mini-pump approach. This is conceptually fairly straightforward, simply requiring the grafted cells to act in a local secretory fashion to provide a constant and replenishable source of analgesic substances. Potential benefits of this approach are several: (1) therapeutic agents can theoretically be delivered on a long-term basis, during the life span of the transplanted cells (this could reduce or completely eliminate the need for repeated administration of pharmaceutical analgesics, along with their attendant side effects), (2) therapeutic agents with biological half-lives too short to be delivered by any other means, such as labile neuropeptides, can potentially be used), (3) biologically active agents can be delivered at focal sites, such as regions in the CNS, avoiding complications associated with systemic delivery and allowing delivery of agents that would otherwise have limited penetration past the blood-brain barrier,
Although numerous donor cell types have been explored (see material following), the earliest studies and consequently the majority of work using this approach has focused on the utility of chromaffin cells from the adrenal medulla as a graft cell source. Chromaffin cells have the advantage of functioning in an endocrinelike secretory fashion, and thus possess the inherent capability and cellular machinery to act as a living mini-pump. Thus, they may be an ideal cell source for achieving continual delivery of therapeutic molecules to the spinal intrathecal space. Chromaffin cells of the adrenal medulla produce and secrete a variety of neuroactive substances with analgesic or antinociceptive activity, notably catecholamines and opioid peptides. Numerous studies have demonstrated that chromaffin cells co-release opioid peptides and catecholamines [76, 91, 138]. Because antinociceptive synergism has been widely reported using a combination of opioid and adrenergic agonists and the coadministration of subeffective levels of these agents may produce potent analgesia
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while reducing the development of tolerance [23, 115, 144, 151], another potential advantage of chromaffin cells as transplant donors for pain relief is the natural combination and secretion of these particular classes of agents. Further, adrenal medullary chromaffin cells produce numerous other agents, such as endogenous potential NMDA antagonists such as agmatine and peptide histogranin [72, 139] and a “cocktail” of neurotrophic factors and neuropeptides that may provide neurotrophic support, including basic fibroblast growth factor (b-FGF), transforming growth factors b (TGF-b), interleukin-1 (IL-1), and neurotrophin (NT)-4/5 [133, 135]. Chromaffin cells have also been reported to produce antioxidants ascorbate and NO scavenging heme proteins [13], which could potentially act downstream in spinal hyperexcitability cascade implicated in pathological pain processes.
Preclinical Studies Using Adrenal Medullary Transplants in Pain Models Adrenal medullary tissue or isolated chromaffin cell transplants have shown efficacy in various preclinical pain models, including acute pain models [12, 79, 87, 92, 101, 105, 106, 108], the formalin test [93, 119, 120, 125, 141, 150], chronic inflammation [107, 140], neuropathic pain models [21, 43, 52, 54, 77, 78], central pain models [8, 47, 157], and wind-up [58]. Although the majority of reported studies has shown positive analgesic or antinociceptive effects of the transplants, analgesic effects of chromaffin cell transplants are not always observed, as described in recent reports using acute pain models and the formalin test [74, 75]. There is also variability in the relative potency of transplants to reduce pain symptoms in various models and/or outcome measures, suggesting differential sensitivity to therapeutic molecules secreted by the cells. Variations in cell preparations or transplantation procedures may also account for some of these differences.
Adrenal Medullary Transplants in Acute Pain In general, using acute antinociceptive assays such as tail flick or paw pressure tests, baseline nociceptive thresholds are not altered appreciably by chromaffin cell or adrenal medullary transplants but rather require stimulation of cell surface nicotinic receptors [105, 106,
108, 141]. This is most likely due to the necessity of achieving relatively high levels of secretion of analgesic substances in order to reduce sensitivity to intense acute noxious stimuli. This hypothesis is supported by spinal superfusion studies that have indicated a correlation between antinociception and spinal CSF levels of metenkephalin and catecholamines following nicotine injections in animals with adrenal medullary transplants [102, 103]. A notable exception was achieved using superpurification of bovine chromaffin cells by ferromagnetic beads coated with isolectin B4 from Griffonia simplicifolia [87]. In this case, antinociception to both A- and C-fiber-mediated noxious stimuli was achieved even in the absence of nicotinic stimulation. In other cases, modest antinociception can be markedly enhanced using nicotinic agonists. This ability to enhance analgesic efficacy pharmacologically could prove a useful clinical feature, for example, during episodes of breakthrough pain. Thus, studies in our laboratory have explored the possibility of enhancing analgesic efficacy of adrenal medullary transplants using novel nicotinic agonists. As a proof of concept, recent studies have examined the use of potent nicotinic agonist epibatidine in transplanted animals [12]. After adrenal medullary (but not control) transplantation, nociceptive thresholds to acute noxious stimuli were modestly but consistently elevated (Figure 26.3). Following intrathecal injection of epibatidine, acute nociceptive response latencies were only marginally elevated in control animals at the highest dose test, with some attendant motor side effects. In contrast, in adrenal medullary transplanted animals epibatidine markedly elevated responses to acute noxious stimuli in a dose-related fashion (Figure 26.3). Similar potent and enhanced antinociceptive effects of epibatidine in adrenal (but not control) transplanted animals were observed using the formalin test [12].
Adrenal Medullary Transplants in Chronic Pain: Potential Mechanisms With regard to tonic or chronic (non-acute) pain models, numerous distinctive rodent models have been explored using adrenal medullary or chromaffin cell transplants, and there are several hypotheses regarding their underlying mechanisms in reducing pain. In particular, the contribution of NMDA antagonist activity in these effects has been hypothesized. The formalin test offers a valuable paradigm in this regard, as its two components (acute and tonic) are
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Figure 26.3. Effect of i.t. epibatidine in rats with spinal cord subarachnoid transplants on the tail flick and paw pressure tests. Following baseline evaluation (“before TP”), rats were transplanted with either control (“muscle TP”) or adrenal medullary (“adrenal TP”) tissue in the spinal subarachnoid space. Rats were tested 1 and 2 weeks following transplantation. Two weeks after transplantation, rats were i.t. injected with either epibatidine (0.01, 0.03, 0.1 mg) or vehicle (“saline”) and tested 5 minutes following injection. *p < 0.05 versus control transplant; #p < 0.05 versus saline. (Taken from Caban et al. [12].)
likely mediated by pharmacologically distinguishable mechanisms. The first phase (which occurs 1 to 5 minutes after formalin injection into the hindpaw) resembles other responses to acute noxious stimuli, whereas the second phase (occurring approximately between 20 and 50 minutes after a period of quiescence) is thought to be initiated by activation of NMDA receptors [48, 60, 136, 152]. Although both phases are suppressed by adrenal medullary allografts in the spinal subarachnoid space, block of the first but not of the second
phase is attenuated by pretreatment with the opiate antagonist naloxone or the a-adrenergic antagonist phentolamine [120]. However, suppression of the second phase may be mediated by interference in NMDA-initiated events. This possibility is supported by findings that adrenal medullary transplants diminish the exaggerated responses to noxious and innocuous stimuli that are usually seen following direct intrathecal injection of NMDA [121]. An NMDA-dependent mechanism is additionally supported by the blockade of NO synthase and cGMP increase following peripheral nerve injury [53, 78, 117], and by reduction in the activation of the immediate early gene c-fos following formalin [119] in animals with adrenal medullary (but not control) transplants. Chromaffin cells produce peptides with NMDA antagonist-like activity, including histogranin [72], and the direct intrathecal administration of this peptide or derivatives attenuates the second phase of the formalin test [122] but not the first. Histogranin also reduces NMDA-mediated nociceptive responses [55], and reduces pain-related behavior in peripheral nerve injury [118]. In further exploration of this hypothesis, neurophysiological differences between rats with adrenal medulla and control (striated muscle) spinal subarachnoid grafts were compared using the phenomenon called wind-up, in which rapid electrical stimulation of peripheral C-fibers potentiates neuronal responses in lamina V neurons [58]. Like the late formalin response and neuropathic hyperalgesia, windup is mediated by nitric oxide and NMDA-subtype glutamate receptors [22]. Under halothane anesthesia, lamina V cells were tested for wind-up by stimulating the skin electrically at C-fiber intensities with the baseline 0.1-Hz stimulation rate raised to the 1-Hz test rate for 20 seconds. Rats with adrenal transplants showed suppressed or markedly attenuated wind-up, whereas control transplanted animals showed potentiation resembling the normal intact responses. These findings also support the hypothesis that NMDA receptors are involved in chromaffin cell transplant analgesia. Neuroprotective actions may also contribute to the analgesia produced by adrenal medullary transplants. Thus, some of the neurotrophic factors secreted by chromaffin cells are neuroprotective [66, 133, 134]. In support for this, adrenal medullary transplants have been found to reduce the increased incidence of hyperchromatic “dark neurons” in the dorsal horn following peripheral nerve injury [51], which are thought to be indicative of damaged or dying cells [127]. In addition,
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these transplants have been found to reduce the loss or possibly rescue compromised GABAergic interneurons in the spinal dorsal horn [34, 61], which have been hypothesized to limit spinal hyperexcitability (and hence pain) in the normal intact spinal cord [5, 16, 18, 34, 56, 61, 86, 98, 143]. Neuroprotection may be related to mechanisms that lower NMDA-mediated facilitation, in that overstimulation of NMDA receptors leads to oxidative damage.
Adrenal Medullary Transplants in Chronic Pain of Differing Etiologies As discussed previously, adrenal medullary and chromaffin cell transplants have been shown to reduce chronic pain symptoms in a fairly wide variety of animal models, including neurogenic inflammation (formalin test), arthritic inflammation (adjuvant models), peripheral neuropathic pain (constriction nerve injuries), and spinal cord injury pain (excitotoxic injuries, hemisection, spinal ischemia models) [8, 21, 43, 47, 52, 54, 77, 78, 93, 107, 119, 120, 125, 140, 141, 150, 156, 157]. Although suppression of pain behaviors using most if not all of these models has been reported to be robust, some variability does appear to exist. In particular, significant but incomplete suppression of inflammatory pain symptoms is often obtained using the formalin test, particularly for phase I [12, 90, 119, 150], with some reports of ineffectiveness using this model [74, 75]. Similarly, adrenal medullary transplants appear to be less robust in reducing hindpaw inflammatory pain (unpublished data) than peripheral neuropathic pain using the same behavioral end points [21, 43, 52, 54, 77, 78]. One possible explanation for this is a modest but incomplete responsiveness to NMDA antagonists (including histogranin [49, 50]), in contrast with effects on chronic constriction nerve injury pain [55]. Of note, adrenal medullary transplants appear to be particularly robust and consistent in reducing central neurogenic pain in three distinct spinal cord injury pain models [8, 47, 156, 157]. Thus, certain types of pain may be more amenable to intervention using adrenal medullary transplants at least in an unmodified form, whereas others may require additional manipulations to be fully effective in alleviating pain symptoms (see future approaches following). As an example of the efficacy of unmodified adrenal medullary transplants in reducing neurogenic pain, a model of brachial plexus avulsion was recently explored in our laboratory [69]. Brachial plexus avulsion is
among the most common of the clinical neuropathic pain syndromes, and the resultant pain is often severe and permanent and poorly treated by current therapeutic interventions. Following brachial plexus avulsion, the incidence of early pain in humans is reported to be as high as 90%, and becomes intractable in 20 to 30% of patients [9, 149]. Pain following brachial plexus avulsion is very rarely controlled by pharmacologic intervention [24], and the only surgical procedure known to be effective is the DREZotomy, an ablative and irreversible procedure [123, 160]. To evaluate the potential for adrenal medullary transplants to alleviate brachial plexus avulsion pain the cervical posterior rhizotomy model was used, as it mimics the pathologic situation following traumatic brachial plexus avulsion in humans [2]. Because the origin and localization of neurogenic pain are in the upper segments, animals received adrenal medullary (or control) transplants placed in the cervical subarachnoid space, in contrast to the more traditionally used lumbar spinal graft placement site. Animals underwent a right posterior cervical rhizotomy extending from C5 to Th1, and were clinically evaluated daily for self-directed behaviors indicative of ongoing pain, including onset, dermatomal extent, and severity. Figure 26.4 shows a comparison in the appearance of self-directed behaviors indicative of neurogenic pain between animals receiving adrenal medullary versus control striated muscle transplants. In animals with muscle control transplants, self-directed behaviors appeared in 83.3% of the group, with a mean delay between rhizotomy and onset of self-directed behaviors of 8 days. In contrast, only 30.8% of the animals implanted with chromaffin cells exhibited any signs of self-directed behaviors, and these had a mean onset delay of 14 days. This extent of suppression of self-directed behaviors by adrenal medullary transplants is similar to that observed following DREZ (which results in approximately 60 to 70% suppression [45]). These findings suggest that a transplantation approach may offer a nonablative alternative in the management of more severe intractable neurogenic pains such as that resulting from dorsal root avulsion.
Clinical Trials Using Adrenal Medullary or Chromaffin Cell Transplants for Chronic Pain Nearly all of the clinical data using cell transplantation for pain management has been obtained from patients
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with severe pain secondary to inoperable cancer. Both human adrenal medullary tissue allografts and encapsulated bovine chromaffin cell have been studied, under the relevant regulatory guidelines for human subjects. An early study was performed by a team at the University of Illinois [145], who implanted adrenal medullary tissue dissected from adult human adrenal glands obtained through the regional organ bank. The glands were screened for pathogens, dissected and cultured for 7 to 10 days, and assessed for viability and identity using catecholamine assays and histology. The tissue was implanted in the subarachnoid space of patients by lumbar puncture. In four of five treated patients, marked reductions were observed in Visual Analog Scale (VAS) pain ratings over 2 to 8 weeks following transplantation. There was also a concomitant reduction or complete cessation in required additional pain medication. Three of these patients were pain-free until death (ranging between 4 and 13 months after transplantation). A group at the University of Toulouse [67] carried out a similar study, initially on seven cancer patients with intractable pain, and more recently including a total of 15 patients [68]. Patients were selected for the study based on the failure of systemic opioids
due to the persistence of undesirable side effects. All patients were implanted with intrathecal morphine delivery pumps, and by this means their pain scores (VAS) were maintained at low levels (0 to 2) via adjustment of the intrathecal morphine dosage. This allowed for evaluation of analgesic efficacy based on the ability of the patients to reduce or stabilize requirements for intrathecal morphine for maintenance of pain control following transplantation. This mode of evaluation was selected because it can be readily compared with other cancer pain patients who overall require progressive dose escalation as the disease progresses [113]. Of the 14 evaluable patients, 5 were able to discontinue intrathecal morphine, 2 had a long-lasting reduction of intrathecal morphine dosage, 5 were stabilized and kept on the same morphine doses through the remainder of their lives, and 2 required increased morphine doses to control their pain. In most cases, CSF metenkephalin levels, which were also monitored, appeared to be related to the degree of pain control. Two other centers have reported on human adrenal medullary allografts: the Universidad Nacional Autónoma de México (Dr. R. Drucker-Colin, personal communication) and the Universidad Autónoma de Madrid [137]. Both involved just one cancer pain
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patient, the former showing “striking results” and the second a “disappointing outcome” with only transient pain reduction following the implantation. The use of xenogeneic sources of chromaffin cells has also been explored in preclinical studies (see previous material), attempted in clinical trials using encapsulation approaches with semipermeable membranes to prevent graft rejection. Phase I clinical trials were conducted to assess safety and preliminary efficacy using macroencapsulated bovine chromaffin cells [1, 10, 11, 104]. At the University of Lausanne, seven patients with severe pain received encapsulated bovine chromaffin cells (approximately 2 × 106 cells in double-membrane PAN/PVC macrocapsules). The implants were placed in the lumbar cistern via lumbar puncture, and were fastened by a silicone tether sutured to the lumbodorsal fascia, which was completely covered after closure of the incision. Aside from post-puncture headaches that were occasionally noted, adverse effects from the procedure were otherwise absent. Six of the patients in the study suffered from pain secondary to malignancy, and one had unrelieved neurogenic pain secondary to thoracotomy and scoliosis. Four of the patients were receiving epidural morphine at the time of the implant. Those patients reported decreased opioid need postimplantation, but only a modest improvement or no worsening in pain ratings. The others had not been receiving oral or epidural morphine treatment prior to implant, and those patients reported improved pain ratings. Histologic examination of the spinal cords was possible in some cases and revealed no obvious pathology. In addition, capsules that were retrieved confirmed chromaffin cell viability by catecholamine release and immunostaining. A similar phase I safety trial using macroencapsulated bovine chromaffin cells was conducted in the United States [11, 104]. It included 19 patients with intractable cancer pain whose life expectancies were under 5 months. In 15 of the patients, pain relief using aggressive trials of opioids had previously proved inadequate, whereas 4 patients were limited in their opioid intake by major side effects or individual resistance. Capsules containing bovine chromaffin cells were provided by CytoTherapeutics, Providence, Rhode Island. Fifteen patients received capsules containing 1 × 106 cells (5.0 cm in length), and 4 patients received capsules containing 3 × 106 cells (7.0 cm in length) — in both cases implanted, as in the preceding study. In this study, mildly adverse events were similar to other lumbar puncture procedures and were
readily resolved (post-lumbar puncture headaches, subcutaneous fluid collections). Analgesic efficacy was suggested by reduced VAS pain scores in 9 and by reduced opiate requirements in 8 of the 15 patients. It appeared as though improved pain control was observed in patients with pain localized to body regions innervated by lumbar and sacral nerves, suggesting that the lumbar placement of encapsulated cells may not allow sufficient diffusion of neuroactive agents to higher segments for upper-level pain. Based on these initial findings suggesting safety and potential efficacy of this approach, a large multicenter placebo study (controlled with encapsulated bovine chromaffin cells) was sponsored by CytoTherapeutics and Astra AB Sweden. Patients with terminal-cancerrelated pain were accrued for the trial in Switzerland, Czech Republic, and Poland. Although findings from these studies have not been published, reports indicated that there was no difference in pain reduction between encapsulated bovine cell implants and placebo control capsules containing matrix only. Surviving chromaffin cells with evidence of catecholamine secretion were found in retrieved capsules. This approach has thus far not been further pursued. A possible explanation for the failure is the relatively low numbers of cells (hence achieving subtherapeutic doses of released analgesic agents) that can be accommodated by the capsules when scaled up for human use. It may be that the human spinal CSF space, compared with the other species studied, allows lower maximal doses to be achieved by the small numbers of encapsulated cells, or that it permits less production of natural analgesic agents. Thus, future modifications may include bioengineered cells or cell lines producing higher levels or more synergistic analgesic agents to overcome these limitations. Another clinical trial utilizing encapsulated bovine chromaffin cells has taken place at PLA General Hospital in Beijing, China [155]. This trial has involved 100 terminal cancer patients with moderate or severe intractable pain. Bovine chromaffin cells were microencapsulated in alginate-polylysine-alginate (APA-BCC). A potential advantage of this approach is the ability to transplant greater numbers of chromaffin cells, because the microcapsules are not limited by the physical dimensions of macrocapsules. Doses of APA-BCC used in this trial ranged from 0.5 × 107 to 1.5 × 107 cells per implant. Results reported a response rate of 84 to 96% within 5 days of implantation, and this was long lasting and dose related. There were
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also reported concomitant discontinuation or decreased intake of oral morphine, and reported improvements in quality-of-life measurements. Although these findings are promising, it should be noted that no placebo controls were included in the study and further study details are awaiting publication as a full report.
Cell Lines for Analgesic Delivery TUMOR CELL LINES Although chromaffin cells have been most widely explored as a platform cell vehicle for delivery of analgesic agents, several cell lines have also been employed toward this end. An advantage of tumor cell lines is the ability to obtain large quantities of cells that can be more homogeneous than primary cell cultures. In addition, tumor cells are generally more readily transfected with genes of interest to produce additional therapeutic molecules (i.e., analgesic peptides) than less rapidly dividing or post-mitotic cells. However, caution must be taken to ensure safety and inability to produce unregulated growth or tumors in the host, as well as chromosomal stability over multiple generations. Spontaneous tumor cell lines — such as the adrenal pheocytochromal PC12 cell line, which would seem an obvious choice for analgesic delivery — have been studied extensively as a potential source for grafts in a variety of neurodegenerative diseases. However, pheochromocytoma cell lines may be inappropriate for therapeutic use due to the great risk of unrestricted cell division and tumor formation. Moreover, pheochromocytoma cell lines such as the rat PC12 line may not behave as normal mature chromaffin cells in that they appear to maintain or dedifferentiate to a less mature phenotype, synthesizing primarily norepinephrine precursors and reduced levels of opioid peptides. One of the earliest examples using tumor cell grafts for pain is the AtT-20 cell line (originally derived from a mouse anterior pituitary tumor) which synthesizes and secretes opioid peptide b-endorphin. These cells have been implanted in both mouse and rat spinal intrathecal space and tested in analgesiometric assays, particularly for alterations in acute pain sensitivity [110, 147, 148]. When implanted at lumbar levels, baseline pain responses to acute noxious stimuli were not altered. However, when b-adrenergic agonist isoproterenol was administered intrathecally with a phosphodiesterase
inhibitor (to increase b-endorphin release from the cells), antinociception (using tail flick and hot plate tests) was observed [147, 148]. The antinociceptive effects of isoproterenol were dose related, naloxone reversible, and peaked at 5 to 10 minutes postinjection, returning toward baseline by 30 minutes. In another study, AtT-20 cells were immunologically isolated in polymer capsules and implanted in the spinal subarachnoid space at the atlanto-occipital junction [110, 111]. In this study, baseline responses to acute noxious stimuli (hot plate, tail pinch, neuromuscular electrical stimulation) were elevated at 2 and 4 weeks postimplantation. This group also obtained similar results using another b-endorphin-producing cell line, Neuro2A, a mouse neuroblastoma cell line transfected with the pro-opiomelanocortin (POMC) gene [110, 147]. The antinociceptive effects were blocked by naloxone treatment and morphological analysis of the capsules after retrieval revealed healthy cells aggregating in the outer layers with a necrotic core typical of capsules containing rapidly dividing cells. In addition to producing b-endorphin, some AtT-20 cells have been transfected with the human proenkephalin gene (AtT-20/hENK) and thus also produce enkephalins [19]. These cells were reported to produce antinociception when implanted intrathecally in mice following stimulation by isoproterenol, and this could be blocked by naloxone [148]. More recent approaches have attempted to control POMC gene expression and b-endorphin secretion using an inducible promoter (a Tet-on system), and reports indicate inducible gene expression in correlation with enhanced analgesic activity when cells are transplanted [46, 109]. Similarly, engineered embryonic carcinoma cell line P19 has been engineered with dexamethasoneinducible expression of b-endorphin [62]. When transplanted into the rat spinal subarachnoid space, these cells produced naloxone-reversible antinociception on hot plate and formalin tests, which was further enhanced by dexamethasone administration. Other agents can also potentially be delivered using tumor cells lines. To supply catecholamines, B16 F1C29 melanoma cells have been implanted in mouse and rat spinal subarachnoid space [46]. Results showed reduced substance-P-induced biting and scratching behaviors compared to control implants, and this was reversible by a2-adrenergic antagonists. In addition, catecholamineopioid synergism was suggested by findings indicating a tenfold increase in morphine potency in mice with B16 F1C29 implants.
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CONDITIONALLY IMMORTALIZED CELL LINES Conditionally immortalized cell lines are another approach that retains some of the advantages of tumor cell lines (ability to grow large quantities of homogeneous cell populations, transfectability), while theoretically limiting the potential detrimental effects of unregulated growth. The earliest reported use of this approach for pain management was a derivation of a serotonergic neuronal cell line from the embryonic rat medullary raphe nucleus [146]. To generate these cells, the thermolabile variant of the SV40 large T antigen (the temperature-sensitive derivative) was used. This allows for continuous cell division at the “permissive” temperature (e.g., 33◦ C), during which the T-antigen is expressed. However, when raised to “nonpermissive” temperatures (e.g., 38◦ C), cells stop expressing T-antigen, cease division, and undergo differentiation. The resultant cell line RN46A [36] required the addition of brain-derived neurotrophic factor (BDNF) to develop the serotonergic phenotype [142]. Thus, BDNF was transfected into these cells to autoregulate serotonin production [37]. When transplanted into the spinal subarachnoid space of rats (1 × 106 cells) with chronic constriction nerve injury within 1 week following peripheral nerve ligation, thermal hyperalgesia, cold allodynia, and mechanical hyperalgesia were reversed, in contrast to continuing pain symptoms in animals implanted with the non-serotonergic parent line [38]. Other genes that have been tested for pain alleviation using this cell backbone include the GAD gene for synthesis of GABA, the preproenkephalin gene for met-enkephalin synthesis, and the preprogalanin gene for the peptide galanin synthesis [17, 31, 35]. The RN33-GAD67, which synthesizes and secretes GABA after differentiation in vitro, has also been transplanted in a spinal cord injury pain model — with reduced thermal hyperalgesia and tactile allodynia were observed [28].
HUMAN EMBRYONIC CARCINOMA LINES A human embryonal carcinoma cell line that exhibits stem-cell-like properties and can be differentiated into postmitotic neuron-like cells has been explored for clinical use. Ntera2 (NT2) cells, originally isolated from a human teratocarcinoma, terminally differentiate to postmitotic neurons when treated in vitro with retinoic acid,
and can maintain their neuronal characteristics. Their apparent safety and preclinical evaluations have led to the use of these cells in clinical transplantation trials in stroke victims [64, 88]. Subpopulations of these cells have recently been explored for potential use in pain management. Two phenotypes abundantly present within the NT2 population are those that synthesize the inhibitory neurotransmitters GABA and serotonin (5HT), which have been recently subcloned at the Miami Project (Dr. Mary Eaton, personal communication). These subclones have been characterized by HPLC and shown to synthesize and release the expected neurotransmitters (GABA or 5HT) after differentiation. Preliminary results indicate that intrathecal grafts of these cells can reduce thermal hyperalgesia and tactile allodynia when tested in an excitotoxic spinal cord injury pain model [27, 32].
CONDITIONALLY IMMORTALIZED CHROMAFFIN CELLS Because chromaffin cells are a known entity, and possess numerous valuable properties that include secretory capabilities and production of numerous analgesic molecules, they may be considered a “gold standard” for use as transplantable cells for pain therapy. However, because chromaffin cells are largely post-mitotic their availability in large quantities is limited. Thus, an ideal solution may be the generation of conditionally immortalized chromaffin cell lines. Several attempts have been made to generate immortalized chromaffin cell lines, including the use of v-myc immortalized sympathoadrenal progenitor cell lines from embryonic rat adrenal glands [6] and generation of adrenal cell lines from tyrosine hydroxylase-SV40 T antigen transgenic mice [129]. However, similar to PC12 cells these immortalized cell lines appeared to retain an immature phenotype, producing primarily dopamine and lacking the synthetic enzyme PNMT. In our laboratory, we attempted to use the tsTag construct to confer temperature sensitivity, as described previously for rat neural cells. This approach yielded both embryonic rat and neonatal calf chromaffin cell lines [29]. Results indicated that expression of catecholamine synthetic enzymes was low during proliferation (when cells are dividing under Tag control), but tend to increase as Tag protein shuts down during differentiation at nonpermissive temperatures. When transplanted, some neuropathic pain behaviors are
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attenuated [33]. Nevertheless, even with up-regulation of enzyme expression that accompanies differentiation these conditionally immortalized chromaffin rat and bovine cells do not synthesize appreciable catecholamine levels in vitro [29]. This may be due to a continued low level of Tag expression that may suppress some normal cellular functions, including neurotransmitter synthesis. Thus, a second generation of conditionally immortalized chromaffin cells was sought in order to completely remove the Tag gene prior to transplantation. For this purpose, an approach is to perform site-specific DNA recombination and Cre recombinase/loxP (Cre/lox) excision that have been used in vitro and in vivo for removal of deleterious genes including Tag [94, 114]. Eaton and colleagues recently explored this approach in immortalized embryonic rat adrenal chromaffin cells where the tsTag construct (tsA-TN) was flanked by loxP sequences, which included a positive/negative neo/HSV-TK sequence for selection with either G418 or ganciclovir, respectively [30]. After neomycin selection, the cells were further infected with a retrovirus expressing the CrePR1 gene encoding a fusion protein that combines Cre activity plus the mutant human steroid receptor, hPRB891. Cells that are not successfully disimmortalized are removed by their continued expression of the TK, which is toxic in the presence of ganciclovir. Findings indicated that when the cells were disimmortalized in vitro the Tag protein was completely and efficiently removed by 10 days of treatment with RU486, followed by incubation with the antibiotic ganciclovir [30]. This approach appeared to allow for increased catecholamine synthesis (fivefold increase in norepinephrine after disimmortalization) and a more normal phenotype than previously attempted methods. Preliminary findings suggested that grafts of these cells could reduce neuropathic pain symptoms in a peripheral nerve injury model. An additional advantage of working with disimmortalizable chromaffin cells is the potential ability to genetically manipulate the cells to produce increased levels of desired neuropeptides or neurotransmitters for pain alleviation. Toward this end, Duplan and colleagues [26] infected the disimmortalizable rat chromaffin cell line with constructs for the synthesis and secretion of the opioid Met-enkephalin. The transgene also contained an NGF sequence for secretion of synthesized nascent protein. These transgenic rat chromaffin cell lines expressed high levels of
Met-enkephalin, in comparison with cells expressing the empty vector alone. When these disimmortalizable high-Met-enkephalin chromaffin cells were grafted prior to formalin testing, a significant reduction both in hindpaw flinching responses and in dorsal horn c-fos expression was observed.
Chromaffin Cells Revisited USEFULNESS OF LENTIVIRAL VECTORS FOR NON-MITOTIC CELLS Because chromaffin cells already possess secretory cellular machinery and can process a cocktail of potentially synergistic analgesic agents, albeit at possibly subtherapeutic levels, another alternative strategy would be to increase the levels and/or breadth of analgesic molecules produced by the cells. This approach has been limited in the past by the nature of differentiated chromaffin cells that like differentiated neurons are mainly non-mitotic and hence traditional cell transfection methods cannot be utilized. This limitation has been overcome with the advent of lentiviral-based vectors. Gene transduction using lentiviral vectors provides an attractive approach for gene delivery in the context of nondividing or slowly dividing cells. In contrast to oncogenic retroviruses, which are dependent on mitotic cells, lentiviruses complete their replicative cycle independent of cell division. Safe and stable systems for high-titer lentivirus vector production have been developed, efficient delivery of genes into target cells is becoming customary, and the application of these vectors to deliver therapeutic genes is emerging. Our preliminary findings reveal that lentiviral vectors can also be used to transduce chromaffin cells (see Figure 26.5).
SELECTION OF CANDIDATE THERAPEUTIC PEPTIDES The selection of appropriate therapeutic molecules to be delivered by transduced chromaffin cells with potential for the treatment of persistent pain will need to be carefully considered and may vary depending on the etiology and pharmacologic responsiveness of various chronic pain conditions. Although this can be theoretically varied depending on future applications, our laboratory has focused recently on two promising classes
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Figure 26.5. Conofocal image of GFP-transduced bovine chromaffin cells. Twenty-four hours following transduction with control-GFP lentivirus, cells were immunoreactive for GFP (green) and chromogranin (red) — showing that lentiviral vectors can also be used to transduce nondividing chromaffin cells.
of neuropeptides that may also be complementary as an initial approach and proof of concept.
THE ENDOMORPHINS The first class of neuropeptides for gene transduction using lentiviral vectors into chromaffin cells is the endomorphins [endomorphin-1 (EM1) and endomorphin-2 (EM2)]. These are potent selective m-opioid agonist peptides originally isolated from the bovine brain and described by Zadina et al. [159]. They have the highest affinity and selectivity for the m-opioid receptor of all known mammalian opioids, and have potent antinociceptive activity in a variety of animal models of acute, neuropathic, and inflammatory pain [20, 59, 65, 85, 96, 97, 112, 124, 126, 132, 158]. The endomorphins are widely distributed throughout the nervous system and concentrated near m-opioid receptors in pain modulatory regions. The distribution of the two peptides is somewhat distinct, with EM2 predominating in the spinal cord associated with primary afferents and their terminals in the dorsal horn, and EM1 more prevalent in the brain [44, 85, 95]. Nevertheless, both peptides demonstrate antinociceptive activity when injected intrathecally. Dose-dependent elevations in tail flick latencies and to a lesser extent paw pressure latencies were observed using either peptide,
comparable to the effects of morphine. These elevations can be antagonized by the nonselective opioid antagonist naloxone and selective m-opioid receptor antagonists b-funaltrexamine and cyprodime but not d- or k-selective antagonists [96, 126]. Interestingly, whereas the endomorphins were reported to be less potent than morphine in experimental inflammatory pain models both EM1 and EM2 appear to be effective in neuropathic pain. They display dose-dependent m-opioid antagonist reversible inhibition of cold and mechanical allodynia when injected intrathecally, in marked contrast to morphine [96, 97]. Both EM1 and EM2 antinociceptive effects appear to be short acting, most likely due to rapid degradation by peptidases. Repeated or continuous intrathecal administration may overcome these limitations to some extent. Such administration has also been effectively used to reduce thermal hyperalgesia in the carrageenan model without decrement during the 1-hr infusion, followed by a gradual return of hyperalgesia after its cessation [20]. Thus, this limitation is likely to be overcome by cell transplants that can continually synthesize and secrete new peptides. THE HISTOGRANINS The second class of potential therapeutic molecules we are exploring is histogranin. This is a 15-amino acid neuropeptide that is naturally occurring and has been detected in the chromaffin granules of the adrenal gland, pituitary, brain, and other tissues [71]. In vitro binding assays have revealed high-affinity sites in the CNS with characteristics of membrane-bound receptors [99]. This peptide and a chemically stable analog, [Ser1]histogranin (SHG), demonstrate inhibitory properties at the NMDA receptor (NMDAR) in both in vitro and in vivo assays [72, 116]. Previous findings in our laboratory have shown that intrathecally injected SHG can block NMDA-induced hyperalgesia and allodynia with no apparent adverse motor effects [55]. In another study, Ruan et al. reported antinociceptive effects of high doses of icv histogranin in the mouse writhing test, without any significant effect on motor coordination in the rotorod assay [100]. SHG has been demonstrated in our laboratory to have efficacy in the chronic constriction injury neuropathic pain model and has selective and modest effects on inflammatory pain [49, 118, 122]. No attendant motor dysfunction was observed using a battery of tests, in contrast to findings with MK-801 and AP-5 [49, 50]. These findings suggest that this peptide
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may be a useful adjuvant in the management of a variety of persistent pain syndromes. An important consideration for any potential pain management therapy is that of tolerance that can result in decreased antinociceptive potency over time. Reduced tolerance development to morphine and other m-opioids has been obtained by combination with NMDA receptor antagonists [4, 39, 82–84]. Thus, chromaffin cells — particularly when engineered to produce additional SHG and EMs — in conjunction with their catecholamines may provide an ideal synergistic antinociceptive combination.
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As an initial screen for potential candidates, adrenal medullary transplants were evaluated in combination with exogenously administered SHG, which was shown to attenuate pain symptoms in some animal models in a dose-related fashion. The formalin and CCI models were used to evaluate the combined effects of chromaffin cell transplantation and intrathecal SHG injections. Animals were implanted with either adrenal medullary or control striated muscle tissue in the spinal subarachnoid space. For evaluation of formalin responses, animals were pretreated with SHG followed by an intraplantar injection of formalin, and flinching responses were quantified [90]. Pretreatment with SHG had no significant effect on flinching behavior in control animals at lower doses, with incomplete attenuation only at the highest dose. In contrast, 0.5 mg SHG significantly reduced flinching responses in animals with adrenal medullary transplants, and 1.0 mg nearly completely eliminated flinching in these animals in the tonic phase. For evaluation of effects on neuropathic pain, animals received transplants 1 week following CCI, and were tested for thermal and mechanical hyperalgesia and cold allodynia before and following SHG treatment. Effects on thermal hyperalgesia are shown in Figure 26.6. The addition of low doses of SHG nearly completely eliminated neuropathic pain symptoms in adrenal medullary transplanted animals, whereas in control transplanted animals only thermal hyperalgesia was attenuated, and only at the highest dose of SHG. These results suggest that SHG can augment adrenal medullary transplants, and the combination may result in improved effectiveness and range in the treatment of chronic pain syndromes [90].
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Dose SHG Figure 26.6. Effect of [Ser1]histogranin supplementation and adrenal medullary or striated muscle transplants CCI-induced thermal hyperalgesia. Baseline latencies (BL, open bars) of rats were evaluated prior to CCI, after CCI (cross-hatched bars), and before (Pre-SHG, solid bars) and after (Post-SHG, diagonal bars) intrathecal treatment with saline vehicle or SHG (0.5, 1.0, or 3.0 mg). Mean latencies S.E.M. in adrenal medullary (top panels) or striated muscle (bottom panels) transplanted animals are shown. (Taken from Nasirinezhad and Sagen [90].)
GENERATION OF TRANSDUCED CHROMAFFIN CELLS FOR ANALGESIA Therefore, using a recombinant-DNA-based approach we combined synthetic EM1, EM2, and SHG genes with the nerve growth factor-beta (NGF-b) amino-terminal signal peptide [15, 42]. The NGF-b/E1/E2/SHG constructs were then individually subcloned into a lentiviral vector, and recombinant lentiviruses encoding the constitutively secretable EM1, EM2, or SHG genes were generated. RT-PCR analysis of the transduced
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M 1 2 3 4 5 M 6 7 8 9 10 M
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Figure 26.7. Behavioral data from animals transplanted with chromaffin cells transduced with EM1, EM2, and SHG recombinant genes. All chromaffin cell groups attenuated the formalin response, particularly in phase II, when compared to control neural stem cells (NSC) transduced with GFP. Suboptimal numbers of control bovine chromaffin cells (100,000) were transplanted to detect potential increased antinociceptive effects of transduced chromaffin cells.See color insert.
Figure 26.8. Expression of EM1, EM2, SHG, and GFP cDNA in genetically modified bovine chromaffin cells. Using primers flanking recombinant endomorphin-1 (EM1) gene, a 393-bp message corresponding to preproNGF-EM1 was detected in the BCC-EM1 cDNA (lane 4). the same primer pair amplified an identical size product from the positive-control EM1/pRRL plasmid (lane 5). No bands were detected in control conditions such as PCR with water alone (lane 1), cDNA from Naïve BCCs (lane 2), or BCC-EM1 sample with no reverse transcriptase added (lane 3). Similarly, for EM2 specific bands could be seen only in PCR reactions with BCC-EM2 cDNA (lane 9) or positive-control plasmid (lane 10) but not the negative controls (lanes 6–8). For SHG, the 425-bp SHG cDNA band was seen in BCC-SHG cDNA only (lane 14), and the positive-control plasmid (lane 15) but not the negative controls (lanes 11–13). For GFP, a message corresponding to GFP (700 bp) could be detected in BCC-GFP cDNA only (lane 19), and the positive-control plasmid (lane 20) but not in the negative controls (lanes 16–18).
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chromaffin cells showed expression of the recombinant gene mRNAs for EM1, EM2, and SHG (Figure 26.7). Chromaffin cells transduced with control GFP lentivirus and double-stained for GFP and chromogranin show the expression of GFP (green) and chromogranin (red) (Figure 26.5). Preliminary evaluations of these cells in the formalin model have been conducted [14, 41]. Behavioral data from animals transplanted with chromaffin cells transduced with EM1, EM2, and SHG recombinant genes showed that all chromaffin cell groups attenuated the formalin response, particularly in phase 2, when compared to control neural stem cells (NSCs) transduced with GFP (Figure 26.8). These findings are similar to previous findings using control chromaffin cells. The SHG cells alone did not appear to further reduce the formalin response, whereas the EM2 cells only had marginal effects. However, a combination graft containing a mix of both SHG- and EM2-transduced chromaffin cells attenuated the formalin response, both in phase I and phase II. Together, these data suggest that this combined cellular approach may be a promising strategy for reducing pain.
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27 Cell Grafting for Spinal Cord Injury Repair: Cell Replacement and Bridging Strategies D. D. Pearse and D. J. Barakat The Miami Project to Cure Paralysis, Department of Neurological Surgery, The Neuroscience Program, University of Miami Miller School of Medicine, Miami, Florida
Acknowledgments We thank David Mawson, Bethany Milliron and Dr. Maneesh Garg for their help with the manuscript.
Introduction The brain and spinal cord are inter-connected by a dense network of nerve fibers responsible for the constant transmission of electrical impulses encoding a myriad of sensory and motor information that is critical to the daily functioning of the organism. The processes involved in the development of this complex circuitry, the mechanisms responsible for the integration of extraordinary amounts of accumulated information, and the roles of the multifarious structural, cellular, and molecular interactions that produce meaningful communication with an equally intricate environment are still largely unknown and remain major challenges to scientific endeavor. Trauma to these highly organized central structures, such as the spinal cord, produces interruptions and abnormalities that can often lead to either death or permanent paralysis. In the least of cases, injury to the spinal cord results in the disruption of axonal integrity, death of cells at the site of the insult, neurological dysfunction, and infiltration into the spinal cord of elements from various peripheral and systemic systems including peripheral nerve, immune, and musculature components, which remodel the injured spinal cord into a foreign and hostile environment. The neuron is at the top of the cellular hierarchy of the central nervous system and is one of the few cells present Cellular Transplantation: From Laboratory to Clinic
within an organism that is incapable of self-renewal. Furthermore, central neurons appear to lack both the appropriate intrinsic programming and the growth supportive cues within the injury milieu to facilitate their regeneration, reconnection, and restoration of function. In order to provide a favorable environment within the injured spinal cord for regeneration or to replace lost nervous tissue, the grafting of cells native to the nervous system or that are either capable of, or have been modified to possess, functions analogous to those endogenous to the CNS have been employed. The success and future of these cellular strategies in repairing the complex networks of the spinal cord following trauma will be discussed in the following chapter.
Spinal Cord Injury Epidemiology and Pathophysiology Recent progress in the development of novel treatments for neurological injury and disease in experimental models has made it seem plausible that therapies for spinal cord injury (SCI) repair will soon find success in the clinical setting. It is apparent, however, that this achievement will likely involve a number of smaller steps that improve various aspects of the quality of life for SCI persons rather than a single treatment facilitating complete anatomical and functional recovery.
EPIDEMIOLOGY Traumatic SCI affects up to 250,000 people in the United States alone, with more than 10,000 new cases
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reported annually. Almost half of these injuries result from motor vehicle accidents, with males accounting for greater than 75% of SCI individuals [84]. The majority of those injured is young adults, who will collectively require more than 7 billion dollars in healthcare expenses per year over their near-normal life spans [84, 221].
PATHOPHYSIOLOGY The pathophysiology of spinal cord injury can be generally divided into two phases: a primary mechanical injury that is usually instantaneous and irreversible and a secondary injury phase characterized by progressive tissue damage that occurs over hours to days to weeks following the initial insult and that is amenable to therapeutic intervention. The primary injury results from external forces that drive either vertebral or foreign bodies to compress, contuse, or transect the spinal cord. This mechanical insult shears cellular and axonal membranes, ruptures blood vessels, and disrupts cerebrospinal fluid flow. This provokes the release of toxic metabolites, neurotransmitters, and enzymes, and the formation of microhemorrhages and spinal cord edema [12, 254, 346, 377]. These events lead to the cessation of adequate blood flow and ischemia, systemic hypertension, and a shift in the balance of electrolytes within the injury epicenter and its penumbra that produces progressive secondary injury to nearby tissue by hypoxic [69, 90, ], excitotoxic [65, 132, 258, 271], or oxidative mechanisms [17, 315, 340, 386]. The relatively immune privileged status of the spinal cord is also compromised early after injury to the systemic components of the immune response, which can further exacerbate tissue damage as a consequence of their production of a multitude of proinflammatory cytokines, degradative enzymes, and free radicals that are used to clear debris from the injury site and to ward off infection [217]. The immediate and delayed loss of spinal cord tissue frequently results in the cavitation of the central gray matter with varying degrees of sparing of surrounding white matter.
OBSTACLES TO SPINAL CORD SELF-REPAIR The cavities formed following injury are often fluidfilled cysts, engorged with immune cells and surrounded by scar tissue. The scar is constructed by reactive glial cells and meningeal fibroblasts, which produce a number
of extracellular matrix (ECM) molecules to wall off and protect the contiguous healthy spinal cord tissue from the injury [97, 133, 329]. Astrocytes also detoxify the lesion by regulating ionic homeostasis [81, 247, 330] and removing excitatory and oxidative molecules, such as glutamate and reactive oxygen species [7, 88, 112, 262]. Microglia and fibroblasts produce a number of growth factors and cytokines that are important for wound healing and neovascularization [115, 350], and both microglia and macrophages phagocytose tissue debris and enable lipid recycling for the reconstruction of membranes [208]. The physical incompleteness of the injured spinal cord caused by cyst formation also limits the substratum upon which severed axons can regrow. The ECM produced by reactive glia within the scar contains a number of axon growth inhibitory factors, including chondroitin sulfate proteoglycans (CSPGs) [97, 329], collagen IV [133], tenascins [398], ephrins [45, 239], and semaphorins [78]. These require removal by physical debridement [145, 361], degradative enzymes [35, 103, ], or masking antibodies [102, 233] to permit axon growth. In addition, the inability of resident microglia and systemic immune cells to coordinate the removal of debris efficiently after injuries to central versus peripheral structures [281, 282] results in a chronic immune response and incomplete removal of tissue debris. This debris comprises the remnants of destroyed cellular and axonal components of the spinal cord, many of which are potent inhibitors of axon growth. Myelin and myelin-associated proteins — including NI250/Nogo [54, 116, ], myelin-associated glycoprotein (MAG) [79], and oligodendrocyte myelin glycoprotein (OMgp) [125] — are present within fragmented myelin at the lesion site and are major inhibitors of axon growth after SCI [42, 44, 194]. The growth inhibitory milieu of the injured spinal cord is further complicated by the lack of appropriate growth-promoting molecules or neurotrophic factors [123, 243], which are important to the survival and growth of neurons during development and to neural plasticity in the adult [87, 210, 231]. Supplementation of a large number of growth factors — including neurotrophin3 (NT-3), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial-derived neurotrophic factor (GDNF) — have been shown to promote axon growth after SCI [31, 244, 321]. In addition to these extrinsic obstacles to axon growth present within the hostile environment of the traumatized spinal cord, injured neurons must also overcome
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cell can no longer maintain itself. Cell death after spinal cord injury can be triggered by mechanical, hypoxic, excitotoxic, oxidative, and inflammatory insults. Mechanical trauma to the spinal cord, the primary injury, causes the instantaneous death of many spinal cord neurons and glia at the epicenter of the impact. The physical forces transferred to the spinal cord by trauma result in the shearing of cell membranes and release of the cell’s content into the extracellular space [23]. The rapid onset of cell death by this process means that it is not amenable to therapeutic prevention. The mechanisms of hypoxia-induced cell death are not known but appear to encompass purine nucleotide depletion, mitochondrial dysfunction, and Ca2+ influx [140, 356]. Oxygen deprivation hampers the ability of the cell to generate sufficient levels of the energy-rich Repairing the Injured Spinal Cord molecule adenosine triphosphate (ATP) [333]. By reducThe complex pathological sequelae of SCI mean that ing amounts of ATP, the cell can no longer produce repair of the spinal cord will likely require a combi- proteins required for maintaining the structural integrity nation strategy to target the various components of the and functional output of the cell. Furthermore, loss of injury process. These various stages are described in ATP forces energy metabolism within the cell toward anaerobic glycolysis, which generates inorganic phosgreater detail in the sections following. phate and lactic acid and thus lowers intracellular pH [355]. This acidosis hinders the function of enzymes INHIBITING EXCITOTOXIC, HYPOXIC, and produces DNA damage [138]. Excitotoxic cell death occurs largely through the AND OXIDATIVE CELL DEATH exposure of cells within the immediate vicinity of the Trauma to the spinal cord leads to the death of cells by initial trauma to excitatory amino acids released after two mechanisms: a type of inadvertent cell death termed the disruption of synaptic vesicles [22]. Primary among necrosis [23, 248] and a form of programmed cell death these are the excitatory neurotransmitters glutamate and called apoptosis [22, 23, 169]. During the primary insult, aspartate [132, 258]. Glutamate incapacitates cells by many of the cells within the spinal cord die by necrosis opening Ca2+ channels, either by triggering depolar[23, 248], whereby the cells swell following mechanical ization of voltage-gated channels or by binding and agitation. That is, their intercellular processes become activating ligand-gated channels such as the N-methylunconstrained due to unregulated ion exchange, leading D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4to organelle damage and dysregulation of metabolism. isoxazolepropionic acid (AMPA), and kainate receptors Finally, the cells undergo lysis and release their internal [65, 132, 271]. Opening of these channels leads to excescontent into the extracellular space that in turn induces sive Ca2+ influx and the overactivation of degradative inflammation. Apoptosis is the more predominant mech- intracellular enzymes such as proteases, phospholipases, anism responsible for cell death that occurs during the and endonucleases — which cause considerable damage secondary injury phase of SCI [22, 169]. Apoptosis to proteins, lipids, and DNA and thereby trigger cell involves the cell killing itself in response to endoge- death [22, 23, ]. nous or exogenous signals, leading to shrinkage of the Oxidative stress by exposure to free radicals such as cell (in particular, its nucleus) and subsequent DNA O2− , OH-, and NO — which are reactive by-products of fragmentation. Apoptotic cells commit suicide through cell metabolism — can also trigger cell death [17, 203, the production of proteases that auto-digest their con- 234, 273]. Levels of free radicals increase dramatically tent for ensuing clearance of the cell’s remnants by after SCI due to lipid peroxidation of polyunsaturated phagocytosis. Apoptosis is a process that requires the fatty acids from mechanically disrupted cellular and expenditure of energy. Therefore, necrosis appears to be axonal membranes [127] and due to the loss of endogea default pathway for cell death when a compromised nous antioxidants, including superoxide dismutase the impediment of a reduction in their intrinsic capacity for regrowth that occurs during their development from embryo to adult [24]. Adult neurons lack the intrinsic regenerative programs that allow younger neurons to extend neurites over inhibitory substrates such as myelin [48]. This intrinsic capacity can be restored through the activation of specific signal transduction cascades that appear to be down-regulated in neurons following the development of the central nervous system [48, 111, 251, 275, 289]. Therefore, a number of intrinsic and extrinsic factors must be overcome to achieve successful regeneration of central neurons and functional restitution after SCI.
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(SOD), glutathione peroxidase, and catalase [340]. The enzyme, inducible nitric oxide synthase (iNOS), is also activated after SCI in neurons and immune cells and is capable of generating large amounts of NO, and from NO the highly reactive peroxynitrite radical [17, 273]. These free radicals cause severe damage to proteins, lipids, and nucleic acids through nitrosylation, leading to cell death [17, 273].
MODULATING THE IMMUNE RESPONSE AND INFLAMMATION Injury to the relatively immune privileged spinal cord permits both the infiltration of cellular components from the systemic immune system as well as the activation of endogenous CNS mononuclear phagocytes called microglia. At 1 week post-injury the immune response reaches a peak. After a transient (1 to 3 days) infiltration of neutrophils the injury site becomes filled with activated systemic macrophages and microglia [160]. Of the large number of factors involved in regulating this immune response, a number of proinflammatory molecules and free radicals produced by immune cells can exacerbate the initial trauma by triggering progressive cell death. These proinflammatory molecules include tumor necrosis factor-a (TNF-a) [186, 277, 395], Fas ligand (FasL) [82, 193], interleukin-1b (IL-1b) [369], arachidonic acid derivatives [143], and nitric oxide synthase [60, 273]. These molecules alone, or in concert with one another or with glutamate, can cause damage to cellular and axonal membranes, promote vascular permeability, and/or induce apoptosis [38, 132, 391].
REDUCING SCAR FORMATION Within days to weeks following SCI, a glial scar develops around the injury site. This inhibitory environment is a major determinant of abortive axon regeneration in the injured spinal cord [97, 329]. The glial scar appears to be a physical and chemical barrier composed of a number of cell types, including astrocytes, meningeal fibroblasts, oligodendrocytes plus their precursors, and microglia — as well as the extracellular matrix proteins they produce [97, 329]. Of the extracellular matrix molecules present within the scar, CSPGs appear to be among the most abundant and inhibitory [97, 329]. The CSPG family includes a number of proteins — such as neurocan, phosphacan, brevican, versican, aggrecan,
and NG2 — which normally play important roles in establishing discrete boundaries to guide axon growth during development [56]. Tenascin-C is another highly inhibitory protein found within the glial scar in close association with astrocytes and meningeal fibroblasts [226]. Adding to its own inhibitory nature, tenascin-C has also been reported to bind CSPGs and may anchor and retain them within the scar [288]. Tenascin-R — which is present on oligodendrocytes and in the surrounding extracellular matrix [283] — is also inhibitory to axon growth and has been shown to interact with a number of proteoglycans [288] and to be up-regulated following injury [199]. Finally, several forms of collagen are also present within the scar and can contribute to axon growth inhibition. For example, the removal of collagen IV using specific antibodies has been shown to provide modest relief of axon growth inhibition after SCI [133].
NEUTRALIZING INHIBITORS OF AXON GROWTH IN MYELIN Disruption of myelinated axons either by the mechanical trauma or following Wallerian degeneration of axotomized axons leads to the deposition of fragmented myelin debris. Myelin debris, like the glial scar, contains numerous inhibitory molecules — including NI250/Nogo, MAG, and OMgp [224, 323]. The poor ability of mononuclear phagocytes to remove myelin debris ensures that these inhibitory molecules remain present within the injured spinal cord from immediately post-injury to many months or years after trauma [76]. NI250/Nogo is present within myelin and possesses axon growth inhibitory activity both in vitro [224, 323] and in vivo [102, 233]. MAG, a member of the Siglec family of sialic acid-binding lectins [368], is found on oligodendrocytes and is also released in a soluble form [320]. Although MAG is thought to play a critical role in the interaction of oligodendrocytes with axons during myelination [320], it is a potent inhibitor of axon growth of specific neuronal populations [345].
PROVISION OF MOLECULES THAT STIMULATE AXON GROWTH Proteins that function as important chemoattractant and guidance cues during development — such as neurotrophic factors, transmembrane or soluble guidance cues, or matrix molecules — can be administered to
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encourage axon growth within and beyond the area of spinal cord injury [31, 131, 174, 309]. These growth-promoting molecules are down-regulated after axons have established contact with their appropriate targets in many regions of the adult CNS and are present at even lower amounts within the injured spinal cord [123, 243]. Among these molecules, neurotrophic and growth factors have been the predominant candidates used for SCI repair [31, 174, 309]. Introduction of various neurotrophins and growth factors — including NT-3, BDNF, NGF, and basic fibroblast growth factor (bFGF) — has been shown to enhance the growth of sensory or spinal cord neurons after injury and to subsequently enhance function [174, 309]. These neurotrophic factors can also rescue axotomized CNS neurons after injury from retrograde cell death [34, 113, 353] and atrophy [34, 353] and prolong the expression of regeneration-associated genes (which are normally only transiently up-regulated after SCI) [177].
REACTIVATING INTRINSIC PROGRAMS FOR AXON GROWTH Embryonic neurons exhibit a high intrinsic capacity for neurite extension and are able to grow over inhibitory substrates during the development of the nervous system [61, 357]. Following development, however, the adult neuron loses these intrinsic abilities for growth [24]. It has been shown that this loss of growth potential may be caused by alterations in specific intracellular signaling pathways that are responsible for activating genetic programs for regeneration [48]. One intracellular signaling cascade involving cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and the transcription factor cAMP binding protein (CREB) has been shown to reinstate these intrinsic growth abilities upon activation [49, 272, 275, 289]. Furthermore, this phenomenon has been recapitulated in vivo through the use of synthetic membrane-permeable cAMP analogs, phosphodiesterase inhibitors to prevent cAMP hydrolysis, or the gene delivery of constitutively activated CREB to promote extensive sensory axon growth across the injured spinal cord [111, 251, 253, 275].
Cell Grafting for SCI Repair Transplantation of a heterogeneous population of peripheral and central nervous system, epithelial,
immune, and undifferentiated stem or restricted precursor cells has been effective in SCI repair [196, 223, 274d, 295, 300, 342]. Cell grafts from these disparate sources have been shown to reduce progressive tissue loss [274], retard axonal dieback [342], promote sensory, proprio-, and/or supraspinal axon regeneration [196, 275, 295], facilitate myelination [182, 275], and improve functional outcome [196, 223, 275, 295, 300] in experimental models of SCI.
CELL TYPES EMPLOYED FOR SPINAL CORD GRAFTING Cellular therapies for SCI repair can be classified into three broad categories: (1) cells endogenous to the organism that can be modified or recruited in vivo, (2) cells endogenous to the organism that can be harvested, purified, and/or altered ex vivo and can subsequently be autologously implanted, and (3) cells that can be harvested from one organism, purified and/or modified ex vivo, and transplanted into the injured organism as in allograft or xenograft [318]. To date, cellular interventions for CNS injury have largely focused on the final two cell categories. The following is a discussion of the major cell grafting strategies that have been employed for repair of the injured spinal cord.
PERIPHERAL NERVES One of the earliest reports that described the use of a cellular repair strategy for the injured spinal cord was that by Ramón y Cajal in 1928, in which he transplanted a peripheral nerve graft (PNG) into the spinal cord [50]. The transplanted PNG readily survived, and as opposed to the hostile milieu of the injured CNS supported the growth of axotomized central neurons — confirming the theory that some central neurons do retain the intrinsic ability to regenerate if given an extrinsic environment conducive to axon growth. Later studies by a number of groups confirmed the potential of PNGs to support central axon growth [75, 190, 307, 339]. PNGs for spinal cord repair have been derived from a number of peripheral nerve sources, including saphenous or sciatic [392, 393], femoral [385], peroneal [256, 336], intercostal [63, 104, 188], tibial [135, 371], and sural [99] nerves. PNGs has been employed as either a fresh nerve isolate [63, 104, 187] or following a week of degeneration [99, 135, 371] when macrophage infiltration and Schwann cell activation within the nerve
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have made it a more conducive substrate for axon growth [192]. Several different surgical procedures have been developed to transplant PNGs into the spinal cord — methods that have been largely dependent on the type of nerve and the injury paradigm. PNGs have been grafted as a single nerve placed into a gap following excision of the dorsal columns with subsequent dural repair [372], as a single nerve placed between the rostral and caudal host stumps following spinal cord hemisection or complete transection that is then sutured to the stumps [392], as multiple fine nerve implants after complete spinal cord transection that are juxtaposed from proximal white matter to distal gray matter and suspended in a fibrin matrix [63, 104, ], and as a minced nerve suspension that is injected into the lesion site after dorsal hemisection [99]. Although it has been important to ensure that the PNG is a stable conduit for axon growth, the use of fibrin glue is preferred over the suturing of the PNG to the spinal cord — as the latter invasive procedure causes additional damage, including increased retrograde cell death that may reduce the efficacy of the intervention [308]. It also appears that the transplantation of multiple smaller nerve fibers in precise white-to-gray matter orientation gives a better result than the use of fewer, thicker nerves placed across the site of excised spinal cord [63, 104, 187]. A major advantage of PNGs, dependent on the source of the nerve, is that they offer the potential for autologous transplantation without the need for immune suppression [95]. PNGs, by themselves, have been shown to support axons largely originating from spinal cord interneurons and dorsal root ganglion neurons located within 10 mm of the graft/cord interface [372]. Many of the axons present within the PNG are myelinated [101], and have been demonstrated to retain their ability to spontaneously discharge action potentials [80]. When transplanted into the upper cervical cord, the additional growth of various brainstem populations — including the red nucleus, reticular formation, raphe, and vestibular nuclei — is observed [3]. However, growth of these axons from the PNGs into the contiguous cord is rarely reported without the use of additional strategies [100], as is the growth of corticospinal tract (CST) axons into PNGs [372]. Together, these are major impediments to the independent use of PNGs, as well as many other cell types, for SCI repair. Except for a study by Ferguson and colleagues [99], in which peripheral nerves were minced and injected into the spinal cord lesion, the employment of PNGs
for SCI has generally required a highly invasive surgical procedure. A significant percentage of people with SCI have residual connections and function. Even “functionally complete” injuries are very rarely “anatomically complete” [163]. The grafting of peripheral nerves using a highly invasive procedure is thus more risky for potentially damaging intact axons and leading to functional loss than intraspinal cord injections of cell suspensions. Despite these putative dangers, a report has shown successful use of a PNG in an SCI person with a functionally incomplete ASIA C injury [64]. The growth supportive ability of PNGs has been largely attributed to the Schwann cells (SCs) within them and to the neurotrophic molecules and extracellular matrix these cells produce [291]. The use of purified SCs for SCI repair is the focus of the next section.
SCHWANN CELLS Endogenous to peripheral nerves, SCs are the myelinating glial cells of the PNS [291]. The ability of SCs to direct successful axon regeneration in the PNS and to guide and support regrowing axons after the transplantation of a PNG into the injured spinal cord [291] makes them ideal candidates for SCI repair. The axon growth promoting ability of SCs has been proposed to arise from their production of a number of neurotrophic factors, including BDNF [2], NGF [16], and ciliary neurotrophic factor (CNTF) [306]; secretion of extracellular matrix molecules such as laminin and collagen [47, 257]; and expression of cell adhesion molecules, such as L1, on their surfaces [74, 252]. Purified rat [43, 380] and human [57] SCs are obtainable from peripheral nerves and have been employed in a number of SCI paradigms, including spinal cord hemisection, complete transection, and contusion [18, 46, 218, 277, 342, 387, 388, 390]. Initial experiments investigating the ability of SCs to promote axon regeneration after SCI employed the complete transection paradigm in which a piece of thoracic spinal cord (3 to 4 mm) was removed and replaced by a PAN/PVC biopolymer tube containing a solidified mixture of SCs and the ECM composite matrigel [387, 388]. The examination of SC guidance channels demonstrated that these bridges, like the PNGs, supported the growth and myelination of proprioceptive and sensory (but not supraspinal) axons [387, 389]. In addition, like PNGs a relatively distinct border was observed between host spinal cord astrocytes and the Schwann cell bridge
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as delineated by glial fibrilary acidic protein (GFAP) staining [342]. Similar results have been obtained after the transplantation of SCs as a suspension graft into the contused spinal cord [46, 218, 277, 342]. In a study by Martin and colleagues [218] in which SCs were grafted immediately — 3 or 10 days after an inflatable ballooninduced thoracic compression — only SCs that were grafted immediately or 10 days after injury formed a substantive graft that supported the growth of numerous neurofilament (NF) positive axons that were identified to be largely calcitonin-gene-related peptide (CGRP) and substance P immunoreactive sensory axons from the dorsal roots. No growth of serotonergic (5HT), tyrosine hydroxylase (TH) positive, or wheat germ agglutininhorseradish peroxidase (WGA-HRP) traced CST axons were observed in the grafts. A subsequent experiment by Takami et al. [342] employed SC transplantation at 7 days following a thoracic contusion produced by the Multicenter Animal Spinal Cord Injury Study (MASCIS) weight drop impactor at a height of 12.5 mm. In this study, grafts were found to produce significant tissue preservation and supraspinal axon sparing, as determined by retrograde tracing. The results of this study corroborated the earlier experiments by Martin and colleagues [218], in that the SC grafts largely supported sensory and proprioceptive axon in-growth. Furthermore, limited growth of serotonergic, but not adrenergic, vestibulospinal or reticulospinal axons was seen in the grafts and many SC-myelinated axons were present within the transplants. Although anterogradely traced CST axons failed to penetrate the SC grafts, these transplants did reduce the dieback of these fibers from the rostral edge of the injury/graft. Last, SC-grafted animals demonstrated an improvement in open-field locomotion (BBB score of 12 versus 10.5 in nongrafted controls) that may have been mediated either through increased supraspinal axon sparing or by the regrowth of local spinal cord axons. In all of these studies using SC-only grafts, the same problems seen with PNGs arise: (1) a lack of a response from supraspinal axons to the grafts and (2) an extensive growth of sensory and proprioceptive axons into the grafts that are abortive at the caudal graft-host cord interface. A major factor that appears to contribute to this failure of axon growth from SC grafts into the contiguous cord is the presence of high levels of axon growth inhibitory CSPGs at the caudal interface [284]. To overcome this barrier, a number of combination strategies with SCs have been employed
(see the section “Provision of a Substrate for Axon Regeneration”). Growth from an SC-only transplant, however, has been observed under specific conditions. The grafting of SC minichannels into a unilateral dorsal spinal cord hemisection with subsequent dural repair was shown to facilitate the growth of rostral spinal cord axons into and then from the channel for up to 3.5 mm into the caudal contiguous spinal cord, where terminal bouton-like structures were identified [390]. In addition, these grafts also supported supraspinal axon growth as determined by retrograde tracing from the center of the SC cable and by 5HT and DbH immunochemistry. It was not determined, however, whether these fibers were able to surmount the caudal graft-cord interface. Finally, in a more recent study by our group [18] SCs were grafted into a chronically injured spinal cord at 2 months following a weight-drop (25.0 mm) contusion injury. Similar to acute transplantation, the SC grafts supported the growth of numerous NF+ and sensory axons. In a first for SC grafting, we were able to quantify the number of SCs that survived following spinal cord transplantation by pre-labeling the cells by lenti-viral vector introduction of the enhanced green fluorescent protein (EGFP) gene (95 to 98% efficiency). We determined that less than 20% of the grafted SCs survived at 11 weeks after transplantation, raising the possibility that poor SC graft survival may be a factor that reduces their ability to support axon growth. In this study, we also found that unlike the transplantation SCs at 1 week SCs grafted into a chronic spinal cord contusion injury could support the growth of many supraspinal serotonergic axons, although these fibers failed to exit the grafts. Importantly, the grafting of SCs into the chronically contused spinal cord produced modest (though significant) improvements in open-field locomotion (BBB score of 10.2 versus 8.7 in the nontransplanted controls) and foot positioning. Like PNGs, purified SC transplants have demonstrated an ability to reduce secondary tissue damage and support substantial sensory and proprioceptive fiber in-growth that leads to modest improvements in function [277, 342, 387]. Although axons within SC-only grafts generally fail to exit the graft toward their appropriate targets, and supraspinal axons do not respond to SC transplants [277, 342, 387], recent combination studies employing genetic modification of SCs [15, 365] or additional pharmacological strategies [10, 274, 275] have demonstrated the significant potential of SC grafts for SCI repair (see the section “Provision of a Substrate for Axon Regeneration”). Furthermore,
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these cells have immediate clinical potential because human SCs can be obtained from a biopsy of an SCI person, expanded to large numbers in culture, and then autologously transplanted back into the same individual for SCI repair [46].
MACROPHAGES AND MICROGLIA Macrophages are another cell type that is integral to the success of PNS regeneration and the efficacy of PNGs following implantation into the injured spinal cord [280]. Macrophages are mononuclear phagocytes that following injury to the organism: (1) invade the site of injury in response to specific chemotactic signals to scavenge tissue debris by phagocytosis and mediate the presentation of specific antigens to lymphocytes to focus the targeting of the immune response, (2) produce various proteases, oxidative enzymes, and ECM molecules that are important for tissue remodeling and lipid recycling, and (3) secrete various cytokines and growth factors that promote cell proliferation and differentiation, initiate angiogenesis, and facilitate wound healing. After injury to the PNS, macrophages are responsible for removing axon and myelin debris from the site of injury and distally (in a process called Wallerian degeneration) and then recycling these lipids for the formation of new membranes so that subsequent nerve regeneration can occur [149, 208, 335]. This process involves a rapid infiltration of the peripheral nerve within 24 hours by macrophages that remove debris. In the CNS, this process is significantly slower and very inefficient by comparison. Tissue debris may be present within the injury for many months to even years after trauma [282]. The CNS also contains resident mononuclear phagocytes called microglia, which are thought to behave similarly to systemic macrophages after injury [122]. The poor reparative response of the immune system in the CNS, however, suggests either an inability of microglia to act similarly to their systemic counterparts [292] or the presence of various factors within the CNS that inhibits the immune response [139, 396]. Several groups have employed the grafting of specifically activated macrophages or microglia to overcome this immune response failure in the CNS and facilitate SCI repair [32, 107, 290, 300]. Prior to their implantation into the injured spinal cord, macrophages had been shown to promote regeneration in another CNS structure inherently incapable of selfrepair — the optic nerve [183]. For the macrophages
to promote axon growth, however, they required exposure to a peripheral nerve for activation — in a process analogous to that which facilitates PNS regeneration. Endogenous macrophages or those harvested and exposed to optic nerve prior to transplantation into the injured optic nerve could not mediate repair. Therefore, this group previously proposed that an immune regulatory factor in the CNS suppresses the ability of macrophages to successfully repair the CNS [139, 396]. In the SCI study by Rapalino and colleagues [300], peripheral blood monocytes were harvested and exposed to excised peripheral nerve for 24 hours prior to their injection into both the center of a complete thoracic transection injury and the caudal parenchyma immediately beyond. They found that macrophage transplantation facilitated the growth of neurofilamentpositive and anterogradely traced rostral spinal cord axons across the transection site. Discontinuity of axon profiles was seen in the controls. Behaviorally, animals demonstrated improvements in BBB score. The macrophage transplanted group, however, was subdivided into two groups based on either their dramatic (two-thirds, BBB score 7.1) or modest (one-third, BBB score 3.4) improvements in BBB score compared to controls (BBB score 1.1). Improvements in open-field locomotion following macrophage grafting were first observed at 6 weeks, and animals continued to improve over the course of 19 weeks post-injury. Macrophage-transplanted rats also exhibited a higher frequency of hind limb movements and showed motorevoked potential (MEP) responses in hind limb muscles after repetitive cortical stimulation. Subsequent retransection of the spinal cord abrogated these responses as well as the functional improvements, demonstrating that axon growth across the lesion (not changes in caudal cord plasticity) was responsible for this recovery. Subsequent experiments by Bomstein et al. [32] extended these findings to a severe spinal cord contusion model (MASCIS impactor, 50.0 mm weight drop) and the use of macrophages exposed to skin as opposed to peripheral nerve. Skin is another region of the body that is capable of mounting a successful wound healing response [229], and skin can be harvested using a less invasive procedure and without deleterious effects compared to peripheral nerve. In this study, macrophages were injected into the caudal border of the lesion at 4 to 9 days post-injury. At end point (5 to 6 months), treated rats showed significantly less cavitation of the spinal cord and improved behavioral recovery as demonstrated by a greater percentage of treated rats obtaining a BBB
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score of 6 (extensive movement of two joints) and above as compared to controls. Protection of the spinal cord and improved function of the macrophage treated rats were attributed to the improved ability of the skinexposed macrophages to function as antigen-presenting cells (higher levels of CD54, CD80, CD86, and MHC II) and to modulate the profile of the cytokines [increased interleukin-1b (IL-1b) and reduced TNF-a] and growth factors (increased BDNF) that they secreted. Macrophage transplantation by Franzen and colleagues [107] showed less dramatic improvements when peritoneal macrophages were harvested, activated by exposure to latex beads for 30 minutes, and then transplanted into a 2.5-mm rostral and caudal to a 5-minute balloon-induced thoracic compression injury. Macrophage grafts (compared to controls) contained a higher density of NF+ axons (which were largely CGRP+) but no 5HT fibers were shown to have penetrated the transplants. No behavioral testing was performed in this study. Differences in the results of these studies, although difficult to assess due to the disparity in outcome measures, may be attributable to the lack of an appropriate stimulus to activate the macrophages (i.e., a peripheral nerve or a difference in the severity of injuries performed). Transplantation of enriched populations of CNS resident microglia for spinal cord repair has also been attempted, although their ability appears to be inferior to that of macrophages. In a study by Rabchevsky and Streit [290], microglia harvested from neonatal rat brain were purified and transplanted into a dorsolateral lesion of the spinal cord with or without prior exposure to the immunomodulator lipopolysaccharide (LPS) [184], a bacterial cell wall component. It was found that grafts of microglia in gelfoam, independent of LPS stimulation, were capable of supporting significant in-growth of NF+ axons and contained large amounts of the growthpromoting ECM molecules thrombospondin [265] and laminin [227] — which may have been produced by either the microglia or infiltrating endogenous Schwann cells. The close association of laminin with blood vessels also indicated that the grafts were likely well vascularized. Factors produced by microglia, such as the cytokine tumor necrosis factor-a (TNF-a) [18], have been shown to promote angiogenesis after injury [172]. The grafts also reduced the intensity of host GFAP immunoreactivity and supported the ingress of CGRP+ axons. This increase in sensory axon growth by microglia grafts is supported by the recent observation that microglia can enhance DRG outgrowth in
vitro [147], although the mechanism responsible for this growth is unknown. Therefore, although microglia are thought to be a primary component of secondary injury pathology through the production of cytokines, degradative enzymes, and oxidative molecules [72, 173, 272, 322], they are also capable of supporting an environment conducive for axon growth — which may result from their secretion of a number of growth factors (NGF, bFGF, GDNF, HGF, TGF-b, BDNF, and NT-3) known to support the survival and regeneration of CNS neurons [70, 77, 93, 94, 242, 341]. The use of specifically primed macrophages as a cellular therapy for SCI is a relatively new direction and is therefore a relatively small field. As yet, there has been limited histological examination of supraspinal responses to these grafts, with the exception of the finding that 5HT axons fail to enter macrophage transplants [107]. Indirect evidence from the recording of hind limb MEPs after cortical stimulation in macrophage grafted completely transected rats suggests that the growth of corticospinal tract axons across these grafts could have occurred [300]. Furthermore, macrophage transplant studies have been restricted predominantly to severe injuries and their efficacy in lesser injuries is currently unknown. Also to date there are no beneficial combinatory strategies that have been reported with macrophage grafts. The study by Rabchevsky and colleagues [290] did explore the efficacy of combining grafts of microglia and astrocytes but found these grafts inferior to microglia alone. Similarly, in the study by Rapalino [300], aFGF was combined with macrophage transplants, but no additional improvements (histologically or behaviorally) were observed over macrophageonly grafts. Of the promising types of purified cell transplants for SCI repair, macrophages are the least studied (yet closest to implementation as a treatment for human SCI repair) currently being tested in phase II clinical trials in several centers in the United States (see the section “The Clinical Use of Cell Transplantation for SCI Repair”). Many questions remain regarding the role of macrophages in SCI repair, and in light of a large body of literature pointing to the cytotoxic role of macrophages in progressive injury to the spinal cord [160] and our limited understanding of the complexity of the immune response after CNS injury [160], many believe their clinical use to repair the injured CNS is premature. Nevertheless, macrophages are a highly advantageous cell type for transplantation, as they (1) can be rapidly and easily harvested in significant numbers using routine procedures that involve the drawing
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of blood and the subsequent separation of the monocytic cell fraction for autologous grafting in a person with SCI, (2) are efficacious even when delivered up to 9 days after injury, and (3) have been shown in a phase I clinical trial to be relatively safe in humans following intraspinal cord injection.
FIBROBLASTS Fibroblasts have been a popular cell for transplantation into the injured spinal cord, even though this choice is not largely based on the inherent functionality of the cells. Instead, their attractiveness stems from their ability to yield high numbers from culturing and the ease of their genetic modification for the secretion of neurotrophic and growth factors within the spinal cord to stimulate axon growth [27]. Fibroblasts are closely linked with tissue repair and wound healing in the periphery [310], whereas in the CNS invasion of fibroblasts is commonly associated with their excessive cellular proliferation, formation of a limiting barrier called the glia limitans, enhancement of astrocyte reactivity, and production of potent axon growth inhibitory molecules (including semaphorins, collagen IV, NG2, and tenascins) [317]. Both dermal and meningeal fibroblasts have been employed as sources for transplantation into the experimentally injured spinal cord. Dermal fibroblasts do produce axon growth-permissive extracellular matrix molecules (including laminin, fibronectin, and collagen I [166, 168]) and secrete NGF [270]. Despite these positive effects, dermal fibroblasts have only promoted marginal axon growth of neurofilament immunoreactive axons and fail to improve behavioral outcome when transplanted into the injured spinal cord [118, 240]. In contrast to dermal fibroblasts, meningeal fibroblasts express a wide range of growth factors — including b-NGF, NT-3, aFGF, and bFGF [106] — and support the growth of modest numbers of neurofilamentpositive axons, largely peptidergic from the dorsal roots. However, only limited penetration of 5HT axons into these grafts, was observed. The potent inhibitory nature of fibroblasts for the growth of many axonal types may limit their potential for SCI repair, even when genetically modified (see the section “Provision of a Substrate for Axon Regeneration”). In addition, many other cell types discussed in this chapter have the natural ability to support the growth of various axonal types and are still amenable to genetic modification ex vivo.
OLFACTORY ENSHEATHING GLIA The olfactory neuroepithelium is one of the few places in the organism where there is turnover of neurons. Sensory neurons are replaced every one to two months [55]. Newly generated sensory neurons must then extend an axon across connective tissue and the cribriform plate, through the pia mater, to gain entry to the CNS — where they traverse along the route of the olfactory nerve to synapse with appropriate target neurons [19]. Olfactory ensheathing glia (OEG) are a unique type of macroglia that are found within the fila olfactoria and the first two layers of the olfactory bulb. They appear to play a critical role in the ensheathment and guidance of the axons growing along this path. By enfolding bundles of unmyelinated axons, OEG prevent the contact of the growing axons with the inhibitory environment of the CNS, which would otherwise prevent them from reaching their targets [318]. In addition, OEG also express a wide range of axon growth-promoting cell adhesion molecules: L1, neural cell adhesion molecule (N-CAM) and polysialylated neural cell adhesion molecule (PSAN-CAM) [86, 238], ECM molecules, laminin [162] and neurotrophins, BDNF, NGF, and NT-4/5 [383]. These unique properties of OEG and their ability to also facilitate axon regeneration in the olfactory system after axotomy or neurotoxic insults [117, 118, ] have drawn attention to them as putative candidates for promoting axon regeneration and repair in other regions of the CNS, such as the spinal cord, following injury. In an initial experiment to examine the ability of OEG to guide axons growing into the injured CNS, a thoracic dorsal root was transected and anastomized back to its original entry position. OEG were then transplanted into the dorsal region of the spinal cord near the dorsal root entry zone (DREZ) [296]. It was found that OEG were able to support the growth of GAP-43− and CGRP-positive axons across the DREZ and into laminae 1 through 5, the target laminae they normally innervate. In a pivotal study by Li and colleagues [198], transplantation of OEG into the C1-2 cervical spinal cord 6 days following electrolytic lesion of the CST resulted in regeneration of these CST axons across the graft/lesion for at least 2 to 3 mm into the caudal spinal cord for restoration of directed paw reaching — a CST-dependent task absent in injury-only controls. In a follow-up study to further elucidate the mechanism of CST regeneration, Li et al. [197] proposed that two morphologically distinct OEG were present within the grafts: S cells (which expressed p75 and
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were responsible for ensheathing axons and forming peripheral-type myelin analogous to SCs) and A cells, which expressed fibronectin and assembled tubular sheaths that wrapped around bundles of myelinated axons. In this fashion, both OEG types associated with CST axons and cooperated to facilitate their regeneration and myelination. In an additional study, Li and colleagues [195] examined the ability of retrovirally infected GFP-OEG to improve breathing function after a removal of one side of the upper cervical spinal cord. In a new procedure, the OEG were transplanted as pieces within the ECM they had produced in culture. It was found that at 2 months post-lesion the majority of the transplanted animals had a clear respiratory rhythm in the phrenic nerve during spontaneous breathing as measured electrophysiologically. This response was not detected in injury-only controls that had received a complete injury. It was unclear, however, if ipsilateral axons or contralateral spouting was responsible for the restoration of breathing function [245]. In addition, OEG-treated rats showed extensive growth of NF+ axons within the graft and improvements in their climbing ability. Last, it was reported by the same group that grafting of OEG at 8 weeks after a heat-induced dorsal column lesion, which partially ablated the CST and prevented rats from performing the pellet retrieval task, was able to restore forelimb function [171]. In another cervical spinal cord lesion model (a crush of the dorsal funiculus at C4), Ramer and colleagues [293] implanted olfactory ensheathing glia derived from the lamina propria (LP-OEG) of GFP transgenic mice immediately after injury for SCI repair. Unlike olfactory bulb nerve-fiber-derived OEG, LP-OEG in the olfactory epithelium ensheath olfactory receptor axons that are growing toward the olfactory bulb. The location of LP-OEG within the periphery of the olfactory system means that their extraction from the nasal mucosa rather than the olfactory bulb is a much less invasive and arduous procedure and thus more amenable to clinical application for autologous transplantation [25, 211, 212, 293]. The greater heterogeneity of the nasal mucosa and the increased propensity for culture contamination, however, require more rigorous methods for their purification and culture [25, 211, 212]. Following their transplantation as a xenograft into rat, GFP LP-OEG failed to survive at 60 days post-injury but were present at 28 days. At 28 days following grafting, transplanted GFP LP-OEG were restricted to the lesion and failed to migrate beyond the host glial
barrier. A change in the morphology of the glial scar was observed after LP-OEG grafting, with GFAP and CSPG immunoreactivity more diffuse than in injuryonly controls and possibly indicating a more axon growth-permissive environment. The grafting of LP-OEG also led to significant endogenous SC in-migration and the production of higher levels of laminin within the lesion site compared to injury-only controls. Quantification of the growth of various axon populations was performed at 60 days post-lesion when grafted LP-OEG were absent, and thus analysis were performed in relation to the lesion. It was found that NF+, CGRP+, 5HT+, and TH+ axons grew both into and from the lesion site into the caudal spinal cord in LP-OEG-grafted rats. Biotinylated dextran amine (BDA)-traced rubriospinal tract axons, however, exhibited very little growth into the lesion site and did not extend caudally. In addition to cervical injury models, the transplantation of OEG — derived from either the olfactory bulb or lamina propria — has been employed in a number of studies with complete and incomplete thoracic SCI [18, 211, 212, 285, 293, 294, 297, 342]. Ramón-Cueto and co-workers [297] used the injection of OEG both rostral and caudal to a PAN-PVC tube containing an SC bridge to guide supraspinal axon growth both into and from the bridge. The SC bridge in these experiments was implanted after complete removal of the T8 spinal cord. At 6 weeks after injury, SC bridges contained a large number of NF+, GAP-43+, and WGA-HRP-traced fibers from the rostral spinal cord. Unlike SC bridges alone, with the addition of OEG WGA-HRP spinal cord axons were able to grow past the caudal interface into the contiguous spinal cord. It was demonstrated that OEG, which were prelabeled with Hoechst, had migrated for significant distances from their original site of deposition and were associated with the growing axons. The use of Hoechst as a marker for grafted cells is now known to be unreliable due to its ability to be taken up by host cells, in particular macrophages, should the grafted cells die after transplantation. In light of the poor use of Hoechst as a cell marker and recent findings using GFP that have not shown OEG to migrate, the migratory ability of OEG has been brought into question [18, 82, ]. It was also found that 5HT axons responded positively to the presence of OEG. However, these axons did not find the SC graft very permissive. Regenerating 5HT axons were found growing along the outside of the SC bridge in a connective tissue/OEG
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environment and were able to cross the transection site and continue caudally in the contiguous spinal cord for up to 1.5 cm. Changes in hind limb function were not analyzed. In a subsequent study by Ramón-Cueto et al. [294], OEG were used by themselves in a complete thoracic spinal cord transection model in which a segment of the cord was not removed. The Hoechst-labeled OEG were injected at four sites 1 mm rostral and caudal to the transection injury. At 8 months post-injury and implantation, histological evaluation of the spinal cord showed that BDA-traced corticocospinal tract and 5HT and DbH immune immunoreactive axons had grown across the transection site and into the contiguous caudal spinal cord for up to 3 cm. No growth of these axon populations was observed in the caudal stump of nongrafted controls. At 7 months after injury, the OEG transplanted rats also demonstrated improved motor function — as shown by their ability to climb a grid slanted at a 45-degree angle — and restored sensorimotor reflexes, the ability of the rat to respond to light touch (contact placing) and joint bending (proprioceptive) stimuli of the hind limbs. Untreated rats were unable to perform the motor task and did not respond to the sensory stimuli. Linear regression analysis comparing length of axon growth and behavioral scores indicated a strong correlation. In a similar study by Lu and colleagues [211], LPOEG were grafted either as pieces of lamina propria into the site of a complete thoracic transection injury or were placed in culture and then injected as mixed cell suspensions at three midline locations 2 mm rostral and caudal to the transection. Examination of axon systems at 10 weeks post-injury showed that only in animals receiving LP-OEG did 5HT immunoreactive axons grow across the transection/graft and enter the contiguous caudal cord. Retrogradely traced neurons in the raphe nuclei were also identified when tracer was placed caudal to the lesion. Behavioral improvements in openfield locomotion were observed after LP-OEG grafting (BBB score 5 to 6 versus < 2 in injury-only controls), as well as grid walking along a wire mesh positioned at a 45-degree angle. Increased motor function was lost following retransection of the spinal cord, indicating that supraspinal input was involved in the observed recovery. In a follow-up experiment, Lu et al. [212] demonstrated efficacy of LP-OEG in a more chronic SCI paradigm in which the cells were grafted at 4 weeks after the lesion. Similar histological results were observed. However, the improvement of open-field locomotion was less dramatic (BBB score 4 versus 1 in injury-only controls). A reduction in the efficacy of cell grafts for SCI repair
in chronic injuries has been reported previously with a number of different cell types [145]. OEG have also been employed in a more “clinically relevant” contusion injury model. Takami and colleagues [342] implanted OEG 7 days after a moderate thoracic contusion injury (12.5 mm) created using the MASCIS impactor. The OEG were injected in medium into the center of the lesion and were compared to SC grafts that were used similarly. It was shown that the SC grafts contained many more myelinated axons than the OEG grafts and were the only grafts that demonstrated increased sparing/regeneration caudal to the lesion/graft and improved open-field locomotor ability. In a subsequent study, the transplantation of OEG at 7 days after contusion in medium was determined to be a better transplant protocol than immediate injection or grafting in a fibrin matrix [285]. Furthermore, it was shown in this study that OEG could improve open-field locomotion after spinal cord contusion (BBB score 13.9 versus 11.7 in injury-only controls). In a study by our group [18], OEG were transplanted into a moderate (25.0-mm) chronic contusion injury (8 weeks) created by the MASCIS impactor using the previously described procedure [342]. In this paradigm, through the use of lentiviral vector transduced EGFP cells we found that OEG survival was very poor, with less than 5% of the cells present after 3 months and no significant improvement in function observed. Future studies are planned with a rigorous immune suppressive regimen. In conclusion, OEG — unlike most other purified cell populations — have demonstrated an ability to promote regeneration of long-tract supraspinal axons and facilitate functional restitution in experimental models of both acute and chronic and complete and incomplete SCI [211, 212, 294, 297]. The debate on their ability to extensively migrate and myelinate after transplantation into the injured spinal cord is still ongoing, and with the advent of specific cell markers [18] should be answered in the near future. It is also evident that OEG will likely need to be grafted in relatively normal tissue surrounding — not directly within larger spinal cord lesions to ensure graft survival [18] — and thus imaging techniques to ensure the injection procedure will not injure the spinal cord during transplantation will be important for clinical application [302]. In addition, the source of OEG for transplantation is another decision for clinical application of these cells for SCI repair. The majority of studies to date have employed OEG harvested from the olfactory bulb, which as a source for cells does not appear as
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advantageous as the lamina propria of the nasal mucosa for autologous transplantation due to the requirement for a more invasive procedure [20, 211, 212, 293]. Nonetheless, OEG do exhibit great potential for SCI repair and much optimism has been placed on them for the human SCI clinical trials that have recently commenced outside the United States and for those planned to commence within the United States over the coming years (see the section “The Clinical Use of Cell Transplantation for SCI Repair”).
FETAL TISSUE Fetal CNS tissue transplantation is another extensively studied cellular therapy for repair of the injured spinal cord [39, 144, 181, 268, 305]. Fetal tissue for spinal cord grafting is usually taken from a number of mixed neuroanatomical regions of brain or spinal cord of the developing embryo at a gestational age of 14 days (E14). In the majority of reports, embryonic spinal cord has been harvested for use in transplantation [40, 71, 144], but sometimes different regions of brain [366] have also been used as candidate tissue grafts for spinal cord implantation. Like PNGs, fetal tissue grafts are a mixture of cellular components and ECM intended to prevent cavitation and provide a bridge for the support of the growth of host axons across the injury site/graft. In addition, fetal tissue grafts supply donor neurons that may act as mediators for the transmission of information between the rostral and caudal spinal cord elements [41]. The differentiation of the implanted fetal tissue into CNS neuronal and glial phenotypes is observed in these transplants [114]. The pursuing discussion of studies utilizing fetal tissue grafts for spinal cord repair is restricted, for the most part, to qualitative analysis due to the limitation of advanced stereological techniques at the time these studies were performed. Moreover, the results of studies using fetal tissue grafts in the current review will be limited to the discussion of their transplantation into the injured adult spinal cord. In a study by Bregman and colleagues [41], E14 embryonic rat spinal cord tissue was transplanted into the adult rat thoracic spinal cord immediately following a dorsal “over hemi-section” injury. It was shown that in this acute grafting paradigm like PNG and SC grafts fetal tissue alone was unable to support significant growth of supraspinal axons onto the transplants as measured at 4 weeks posttransplantation.
Fetal tissue grafts, however, supported the growth of sensory CGRP+ axons and enhanced the ability of treated rats to traverse a grid walk as measured by their crossing time. Mori et al. [240] employed the grafting of fetal tissue in a C3-4 cervical hemisection paradigm. The E14 fetal tissue grafts were implanted into the injury epicenter immediately post-injury. At 2 to 4 months after grafting, significant decreases in the size of the lesion and the degree of retrograde neuronal cell death within the red nucleus contralateral to the injury were observed. No decreases in perikaryal atrophy of red nucleus neurons or growth of rubrospinal axons into the grafts were seen. With the injection of the retrograde tracer fluorogold two segments caudal to the injury/graft, it was demonstrated that fetal neurons were able to extend axons from the transplant for significant distances into the caudal host spinal cord. This study showed that the main benefits of fetal tissue grafts appear to be the limitation of secondary tissue damage, prevention of retrograde cell death, and the establishment of novel circuits across the site of injury. In a chronic injury model, Houle and Reier [144] examined the transplantation of E14 embryonic spinal cord into a thoracic lateral hemisection at 2 to 7 weeks post-injury. At 6 months post-transplantation, the fetal grafts were viable and even expanded beyond the cavity. The grafts were well integrated into host gray matter and observed to be fused with host white matter. Anterograde and retrograde tract-tracing results demonstrated that axon growth into the grafts was limited to projections from host neurons located immediately adjacent to the host-graft interface. Limited penetration of host serotonergic axons into the fetal grafts was observed. Giovanini and colleagues [114] used two different transplantation and injury paradigms. Human embryonic spinal cord (gestational age of 6 to 9 weeks) was transplanted between 14 and 40 days following either a thoracic midline myelotomy or a thoracic contusion. It was shown that in both models graft survival was high (> 80%) whether fetal tissue was used either as a solid or a suspension transplant and that significant axon growth from neurons within the grafts into the host spinal cord was observed. Akesson and colleagues [4] also examined human xenograft transplantation into both acute and chronic (6 to 7 weeks) thoracic aspiration injuries. The grafts were found to support 5HT+, TH+, and CGRP+ axons, and these fibers were found to exit the grafts for short distances, (1–2 mm). However, no significant differences, were observed.
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Fetal tissue grafts are the only cellular therapy that has progressed through completion of clinical trials for SCI repair in the United States [96, 351, 379]. The use of fetal tissue grafts in the trial was not intended to promote regeneration and restore function after SCI but to abate progressive cavitation associated with syringomyelia. Following the transplantation of fetal tissue grafts, it was found that no significant functional improvements occurred, as assessed using the American Spinal Injury Association (ASIA) impairment scale. Importantly, however, the syrinx appeared to close at the site of fetal tissue transplantation and this reduction in the cavity has been observed to persist for 18 months post-grafting. It was unclear in this study whether any donor fetal tissue survived, and thus it was difficult to predict the long-term consequences of this procedure. The clinical studies with fetal tissue transplantation did demonstrate that the surgical procedures of cell grafting did not worsen patients’ status and thus appear to be a safe treatment option. Information from this clinical study, however, has shed important light on how future cell therapies for SCI repair should be conducted, and its demonstrated safety is a promising sign for future clinical trials for cell transplantation in humans (see the section “The Clinical Use of Cell Transplantation for SCI Repair”).
BONE MARROW STROMAL CELLS Bone marrow stromal cells (MSCs) are a relatively heterogeneous population of pluripotent mesenchymal cells, endothelial cells, and macrophages found in the bone marrow. Their heterogeneity is further exemplified by their growth in culture in which they comprise wideranging phenotypes but are usually selected based on positivity for CD90 and/or fibronectin [26]. MSCs are easily extracted from raw bone marrow and are readily expandable in culture, although procedures for their isolation, propagation, and characterization are highly diversified [250]. This nonuniform methodology for culturing MSCs has resulted in the experimental use of cells with markedly different populations and phenotypes, and has led to some debate about what these cells can become and what they are capable of doing in vitro and in vivo [178, 279]. The ease with which MSCs are extracted and cultured, their ability to differentiate into a wide variety of cell types (including those from neural lineage), and their potential to improve outcome in a number of
experimental models of disease have enticed researchers to further investigate their biology and function [250]. Importantly for neurological disease, particularly neuroreplacement strategies, MSCs have been shown to have the potential to differentiate into neuronal cells in vitro [83, 155, 316, 381, 382] and in vivo [235, 236, 375, 399] — albeit with much controversy in the literature as to whether [58, 249, 349] and why this phenomenon occurs (i.e., cell culture artifacts, transdifferentiation, differentiation from a selective pool of pluripotent cells within the cultures, or cell fusion of MSCs with host neuronal cells) [134, 158, 334]. In SCI models, MSCs have been shown to reduce progressive tissue damage [9, 202, 261], support axon growth [9], promote remyelination [5, 6, 180], and enhance functional recovery [66, 141, 202, 261, 400]. These effects of engrafted MSCs to promote spinal cord repair may be related to their ability to produce a wide variety of growth factors and cytokines [62, 195, 250]. In the first study that examined the utility of MSCs in an SCI model, Chopp and colleagues [66] found that transplantation of MSCs into the epicenter of a moderate (25.0-mm) rat contusion injury 6 days postinjury produced significant improvements in open-field locomotion over the course of the subsequent 4 weeks (BBB score 15.3 versus 11.5 in injury-only controls). Through the use of BrdU labeling, it was shown that the transplanted cells had survived and were present throughout the lesion and for up to 5 mm rostral and caudal. In addition, scattered BrdU cells also expressed the neuronal specific marker NeuN, indicating possible differentiation of MSCs into neurons after transplantation. In 2002, a study by Hofstetter [141] using the same injury paradigm supported the earlier observations that MSCs could survive and enhance functional recovery following SCI. Retroviral transduced GFP MSCs, however, were transplanted not only within the lesion epicenter but at 2 mm rostral and caudal to the central injection. The transplanted MSCs were found to be fibronectin-positive and to exhibit weak but distinct NeuN immunoreactivity, but were not nestin positive. Robust numbers of neurofilament and 5HT-positive axons were found predominantly at the interface of the MSC graft and scar tissue. Importantly, unlike delayed grafting transplantation of MSCs immediately after the injury resulted in limited cell migration and no functional improvements. In contrast to these findings of weak NeuN positivity in grafted MSCs after SCI, Lee and colleagues [184] found after spinal cord contusion injury and
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transplantation of mouse MSCs that the cells did not express NeuN but did express GFAP, indicating possible glial cell differentiation. The disparity in these studies may indicate that MSCs from various species may exhibit a different propensity for cell type differentiation in the injured spinal cord. In contradiction to the inability of MSCs to improve functional recovery after immediate transplantation into a contusive SCI lesion, Wu and co-workers [384] found that immediate grafting of GFP MSCs after a severe (50-mm) contusion in rat could lead to functional improvements as measured by open-field locomotion at 4 weeks post-injury (BBB score of 10 versus 7 in injury-only controls). Remarkably, however, all transplanted GFP MSCs had died by this time. The beneficial effects of MSCs in this model appeared related to an initial reduction in the amount of progressive tissue damage post-injury. Differences in outcome between this study and that of Hofstetter [141] could be explained by either the more severe injury (50 mm versus 25 mm) or the use of juvenile rats in the latter study (70 to 90 g versus 250 to 260 g SD rats). The same group later reported [261] that the immediate administration of GFP MSCs via cerebrospinal fluid could be tissue protective in juvenile rats after a moderate contusion (both 12.5 and 25 mm) and promote functional recovery (12.5 mm, BBB score 13.9 versus 10.1 in injury-only controls, and 25 mm, BBB score 10.3 versus 8 in injury-only controls). Again, GFP MSCs may not have survived well in this study, as they had disappeared from the spinal cord by 3 weeks after the injection. The protective ability of MSCs after spinal contusion injury was further substantiated by a study by Ankeny and colleagues [9] in which significant reductions in lesion area and improved white matter sparing was observed. In addition, BrdU-labeled MSCs survived up to 8 weeks post-injury, were able to partially fill the lesion cavity at the injury site, and supported increased numbers of NF+ axons. Improvements in open-field locomotion as assessed by the BBB score, however, were not seen. Finally, Zurita and Vaquero [400] demonstrated that MSC transplants could also bridge a chronic (3-month) spinal cord injury site and improve functional outcome (BBB score 8 versus 0 in injury-only controls) after a severe contusion injury. Grafted cells were reported to survive and differentiate into both astrocytes and neurons. In light of these initial studies, MSCs (a relatively novel cell type for SCI transplantation) demonstrate a number of properties that would make them worthy
candidates as a cellular therapy. It is evident, however, that more studies are needed to identify the cell fate of MSCs following spinal cord transplantation and why different extraction and culture procedures can generate MSCs with disparate differentiation potential. In addition, experiments have shown that MSCs can improve functional outcomes after SCI, but more detailed behavioral testing and histological parameters are needed to identify the mechanisms by which MSCs produce these effects. The ability of these cells, however, to differentiate into multiple cell lineages coupled with the ease of their extraction from bone marrow, a relatively routine medical procedure, and capacity to be used autologously without the necessity of immune rejection drugs ranks them among the cell types with the greatest potential for SCI repair.
STEM CELLS AND PROGENITOR CELLS In recent years, stem cells have received the attention of the public and scientific community alike because of ethical concerns in their use, their intrinsic quality for pluripotency (enabling them to potentially replace any cell type in the adult organism), and their demonstrated efficacy to restore function in a number of experimental models of disease [200, 347]. Stem cells are by differentiation unspecialized cells that possess the ability to self-renew indefinitely without aging and have the potential to differentiate into any cell type. During development, embryonic stem cells are known to respond to different cues within their environment that ensure their differentiation into the diversity of cell types found in the mature animal [36, 89, ]. Differentiation is the basic principle that defines stem cells and their progenitors. Progenitor cells are similar to stem cells, but have a more restrictive potential for differentiation than a stem cell [225]. Neural progenitors, for example, can conceivably differentiate into oligodendrocytes, astrocytes, or neurons and are found in neonates, adults, and fetal tissue [216]. Another level of hierarchy is precursors, cells that are earlier in the developmental cascade than another. Glial- or neuronal-restricted precursors, for example, are more confined in their capacity for differentiation — being restricted to the generation of astrocytes and oligodendrocytes or neurons, respectively [298]. When transplanted, stem cells are observed to differentiate by site-specific mechanisms [206, 264, 327], with different regions and different states (uninjured
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versus injured) of the CNS being either more neuroor more glio-genic [314]. Furthermore, stem cells and their progenitors and precursors can be categorized by the region of their origin, their expression of different receptors, their response to various cytokines or growth factors, and their methods of isolation [59, 164]. EMBRYONIC STEM (ES) CELLS ES cells obtained from fetal tissue or neonates can be identified easily with antibodies against the nestin protein, and these cells can be differentiated into normally unobtainable cultures of glia or neurons, making them a pragmatic candidate for cell therapy [8]. ES cells, which do not age through division, have the best chance of producing a genetically normal cell type after differentiation [222]. They are also able to divide for multiple passages without the loss of their differentiation potential [52]. ES cells were originally transplanted into the injured spinal cord in an undifferentiated state with the hope that the cells would then be directed by the host to differentiate into the particular cell types required for rebuilding and reconnecting the spinal cord. In a landmark study by McDonald and colleagues [223], mouse ES cells were xeno-transplanted into the injured rat spinal cord at 9 days post-contusion injury (25 mm), as performed by the MASCIS impactor. Although many of the cells died, enough of them survived to fill the injury site, and these cells were then able to differentiate into all neural cell types, neurons (8%), astrocytes (19%), and oligodendrocytes (43%). In addition, no evidence of tumor formation was observed. Importantly, these cells were able to restore function as assessed by open-field locomotion scoring (BBB score of 10 versus 8 in injury-only controls), leading to an explosion in the usage of various stem, progenitor, and precursor cells in cellular transplantation strategies for SCI repair over the course of the subsequent 5 years. PLURIPOTENT STEM CELLS (NSCS) AND NEURAL PROGENITOR CELLS (NPCS) NPCs are more restricted in the types of cells they can differentiate into, generally being limited to neurons, astrocytes, and oligodendrocytes [298]. NPCs form the largest area of cellular transplantation research, using stem cells and their progenitors for SCI repair. Use of NPCs for SCI repair may limit unwanted side effects that can be envisioned with the transplantation of ES cells, such as the formation of inappropriate tissue types that include bone and muscle within the injured spinal
cord. In one of the earliest uses of NPCs, Chow and co-workers [67] transplanted E14 spinal-cord-derived NPCs with and without BDNF immediately after a C4 partial spinal cord hemisection. Animals were immunosuppressed with cyclosporin A (CsA). However, survival after 1 week was poor in SD rats but was good (50%) in Fischer rats. In transplanted animals that did not receive BDNF, the NPCs became GFAP+ or RIP+, and BDNF MAP1B+ cells were also observed. The transplants were shown to support modest in-growth of neurofilament-positive 5HT+ and CGRP+ axons. In contrast with this study, Ogawa et al. [260] — using the transplantation of E14 spinal-cord-derived NPCs — found that a small percentage of the cells could differentiate into neurons without BDNF following transplantation into a C4-5 spinal cord compression if the cells were cultured as neurospheres and then grafted at 9 days post-injury. Using enhanced yellow fluorescent protein (EYFP)-labeled cells in addition to prelabeling with BrdU, it was found that 5.9% of the cells became neurons (Hu+), 4.4% oligodendrocytes (CNP+), and 32.6% astrocytes (GFAP+). In addition, the differentiated EYGP neurons after maturation were able to integrate into the host, extend processes, become myelinated, and form presynaptic structures. The transplanted cells also improved functional outcome as demonstrated using a forelimb reaching test. Differences between this study and the former may have resulted from the use of neurosphere cultures, different cell-specific markers, delayed grafting, or a disparate injury model. Lu and colleagues [214] examined the ability of murine neonatal cerebellar NSCs (C17.2 cell line) to survive, differentiate, and promote axon growth after immediate xenotransplantation into a rat C3 dorsal column lesion. Grafted NSCs with CsA immunosuppression survived well and filled the lesion site at 2 weeks post-injury, exhibited poor migration from their site of deposition (1 to 2 mm), and displayed little morphological evidence of differentiation. NSCs remained small, rounded, and nestin-positive, and did not label for a wide variety of neural cell markers. NSC grafts supported growth of NF+ axons, CGRP+, and choline acetyl transferase positive (ChAT+), but very little graft penetration by TH+, 5HT+, and trace dorsal column sensory or CST axons was observed. In addition, NSCs were shown to be capable of secreting a number of neurotrophic molecules, including NGF, BDNF, and GDNF. Cao and colleagues [53] transplanted NPCs derived from either E14 cerebral cortex or the adult rat
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subventricular zone into one segment rostral or caudal to a moderate (12.5-mm) contused spinal cord at 10 days post-injury. It was found at 2 months posttransplantation that grafted cells survived well, but almost all had differentiated into GFAP+ cells with some remaining undifferentiated and nestin positive. No differentiated neurons or oligodendrocytes were observed. Studies have begun to compare different sources of NPCs for their ability to differentiate, migrate, promote axon growth, and enhance recovery after SCI. In a study by Watanabe and colleagues [373], NPCs derived from either the E14 spinal cord or forebrain were transplanted as neurospheres 9 days after a moderate (25-mm) contusion injury performed using the MASCIS impactor. At 12 weeks post-injury, both NPCs had survived well and had integrated into the host spinal cord. It was found using cell-specific markers (Hu, GFAP, and APC) that forebrain-derived NPCs differentiated into more neurons and fewer oligodendrocytes than spinal-cordderived NPCs. Spinal-cord- or forebrain-derived NPC grafts, however, supported the growth of NF+ axons and led to improvements in open-field locomotion [BBB score 11.2 (spinal cord) and 12 (forebrain) versus 8.1 in injury-only controls] and on the inclined plane test. Adult NPCs have also been examined for their ability to differentiate and promote axon growth after SCI [367]. In this study, adult NPCs were derived from adult rat cervical spinal cord, retrovirally infected to express GFP and transplanted as neurospheres immediately following a C3 dorsal column transection. At 3 weeks post-injury, grafted NPCs were found predominantly in a dense rim around the lesion site with some migrating up to 3 mm rostral and caudal via the central canal. Adult NPCs were found to differentiate into glial precursors (NG2), radial glia (BLBP), astrocytes (GFAP), and oligodendrocytes (APC) — but not neurons. It was also found that neurofilament-positive and traced CST axons appeared to align with the grafted cells, although the functional relevance of this association was not eluded to or further investigated in the study. A major advantage of NPCs realized in the past year was their ability to migrate to the site of a spinal cord lesion following the less invasive procedures of intravenous [108] or intrathecal [189] injection. In the study by Fujiwara and colleagues [108], E14 hippocampal-derived NPCs were injected intravenously after a rat contusion injury via the penile vein. Grafted cells migrated to the injured spinal cord parenchyma, survived for up to 8 weeks post-injury, and became NeuN+ neurons (4.7%), GFAP+ astrocytes (47.1%),
and Gal-C+ oligodendrocytes (48.2%). Lepore et al. [189] demonstrated that following a lumbar puncture E13.5 spinal-cord-derived NPCs could migrate to the site of a C4 lateral funiculus injury and differentiate into all three neural cell types. In addition, multiple lumbar punctures of the NPCs at weekly intervals increased the efficacy of cells that had migrated to the injury site. It was additionally found that the transplanted NPCs had also migrated to a number of other CNS structures, which may potentially lead to undesirable effects associated with this route of administration. Last, the grafting of NPCs has recently been extended to the preclinical evaluation of human NPCs in a nonhuman primate SCI model by Iwanami and co-workers [152]. In this study, NPCs were derived from fetal spinal cord and cultured as neurospheres. Human NPCs were grafted at 9 days after a C5 spinal cord contusion injury in marmosets, and subjects received daily CsA injections for 8 weeks post-injury to prevent rejection. At this end point, NPCs were found around the original graft site and for up to 8 mm rostral and caudal. The cell fate of the grafted NPCs was 20.6% bIII-tubulin+ neurons, 46.2% GFAP+ astrocytes, and 5.3% Olig2+ oligodendrocytes — with 25.2% remaining undifferentiated nestin+ cells. NF+ axons were found to closely associate with human NPCs, and the transplanted animals demonstrated significant functional improvements — including increased bar grip strength and the number of spontaneous limb movements. In attempts to better control the cell fate of grafted NSCs, however, other groups have attempted either transplantation of a single type of precursor cell or ex vivo differentiation of NPCs to neurons and/or glia before transplantation to obtain more predictable results [67, 110, 136, 259]. These precursor cells, neuronalrestricted precursors (NRPs), and glial-restricted precursors (GRPs) are discussed in the following section. NEURONAL-RESTRICTED PRECURSOR CELLS (NRPS) NRPs, as their name implies, differentiate into neuronal phenotypes. They have been found to express the embryonic neuronal cell adhesion molecule (E-NCAM) and thus can be isolated using immunopanning techniques [220]. These cells are also mitotically active and express NT-3 and FGF [220]. The capacity of these cells to renew and differentiate into mature neurons has made them a promising candidate for transplantation [298]. Cao and colleagues [52] examined an isolated culture
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of NRP from E14 spinal cord and found that the majority of the NRPs expressed gamma-aminobutyric acid (GABA), with lesser numbers being glutaminergic, glycinergic, and ChAT+. After transplantation into the contused thoracic spinal cord, the NRP cells did not express nestin but also failed to express mature neuronal markers, indicating that the environment of the injured spinal cord can even restrict the differentiation of NRPs. Other NRPs include RN33B cells, which are derived from the embryonic raphe nucleus [264]. These NRPs have not been extensively studied in SCI repair. RN33B NRPs are an interesting cell type in that they undergo cell division at 33◦ C, doubling in number every 48 hours. At higher, nonpermissive temperatures (> 37◦ C), such as the body temperature of a grafted host, these cells stop their mitotic activity and undergo neuronal differentiation [378]. In addition, these cells are advantageous for cell transplantation, as they do not express major histocompatability complex (MHC) I or II antigens and are resistant to T-cell lysis [376]. RN33B NRPs are also known to survive and integrate well within the uninjured rat brain [215]. When transplanted into the completely transected thoracic spinal cord, however, RN33B cells showed little differentiation, exhibited a bipolar morphology, and did not express ChAT, suggesting that there was a lack of neuronal differentiation [263]. This phenomenon, as described previously, has been seen with a number of NPCs following transplantation into the nonneurogenic zone of the injured spinal cord. GLIAL-RESTRICTED PRECURSOR CELLS (GRPS) GRPs have also been examined in spinal cord injury transplantation paradigms [136, 189]. These cells are able to differentiate into oligodendrocyte type-2 astrocyte (O2A) precursors, astrocytes, or oligodendrocytes [299]. In one of their first uses in SCI repair, Hill and colleagues [136] transplanted PLAP-labeled GRP cells into a thoracic contusion immediately after the injury. In this experiment, methylprednisolone was administered to alleviate inflammation and CsA to prevent immune rejection. GRP cell transplantation was shown to significantly reduce CSPG expression around the lesion and to retard glial scarring. However, 5HT+ and traced CST axon sprouting into the grafts were not observed. Of note, the astrocytes that had differentiated from the GRPs were not altered to the reactive phenotype associated with the glial scar based on their morphology and CSPG expression.
O2A cells are another type of glial precursor that have been used for regeneration and myelination of the injured spinal cord [185]. O2A precursors are similar to GRPs but have a more restricted differentiation potential [105]. They can differentiate into oligodendrocytes or into two distinct types of astrocytes. Lee et al. [185] transplanted these cells at 1 week postinjury into the lesion epicenter of a moderate (25-mm) thoracic contusion. O2A precursors survived within the lesion environment and differentiated predominately into oligodendrocytes (CNPase+) or remained as precursors. No differentiation into astrocytes or neurons was observed. Retrograde tracing with fluorogold from lumbar L1 demonstrated that there were more spared/regenerated axons caudal to the graft/injury site from neurons of the red, raphe, and reticular nuclei in O2A transplanted animals compared to injury-only controls. Significant electrophysiological and behavioral improvements as assessed by motor-evoked potentials and open-field locomotion (BBB score of 12.2 versus 9.8 in injury-only controls), respectively, were additionally observed. Oligodendrocyte precursor cells (OPCs) constitute another type of restricted glial lineage cell that has been employed for SCI repair. OPCs are derived and enriched under specific culture conditions, usually involving retinoic acid and the supplementation of SATO medium, from ES cells or NPCs and grown as oligospheres. OPCs, as their name implies, are immature cells that have a restricted differentiation to oligodendrocytes. OPCs have been successfully used to remyelinate the demyelinated spinal cord following injury [13, 170, 204]. Bambakidis and Miller [9] investigated the transplantation of OPCs either alone or with recombinant sonic hedgehog protein (Shh) into the injury epicenter at 5 days after a moderate (12.5-mm) contusion performed using the MASCIS impactor. Shh was added because it was previously shown to induce the proliferation of endogenous nestin-positive cells [14]. The grafting of OPCs, independent of Shh use, reduced white matter damage, improved open-field locomotion (BBB score of 18.2 versus 13.6 in injury-only controls), and enhanced MEP recordings. Further discussion of OPCs and their ability to remyelinate the injured spinal cord can be found in the section “Remyelination of Axons.” The use of stem cells and their progenitors and precursors for spinal cord repair is still in its infancy. Significant investigation into the following is warranted: (1) the most appropriate and efficacious sources of these
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cells, (2) the best way to direct their differentiation to obtain the correct proportions of each required neural cell type by ex vivo or in vivo manipulations, (3) the extent to which these cells integrate into the host and associate with or direct axon growth and myelination, or perform as adequate replacements for lost neural cells, (4) the determination of the potential adverse effects of these transplants, including aberrant migration, differentiation, and tumorigenesis, and (5) whether combination with molecular or pharmacological strategies will be needed to ensure axon growth across the grafts and functional restoration. The multifunctional capabilities of stem cells, however, place them at the forefront of prospective candidates for SCI repair.
OTHER CELL TYPES Numerous cell types have been employed to repair the injured spinal cord, and thus not all cells can be presented in depth in this review. The following cell types have been employed in a limited number of studies with varying degrees of success in SCI repair: choroid plexus ependymal cells [148, 175], embryonic astrocytes [161, 370], enteric glia [153, 154], and dendritic cells [130, 237]. Further investigation into the functionality of these cell types to promote regeneration and functional restoration after SCI is likely underway.
ENHANCING GRAFT CELL FUNCTIONALITY A great number of studies have now demonstrated that grafts of diverse cell types have the potential to (1) limit the spread of progressive tissue damage, (2) promote axonal growth, (3) promote remyelination, and/or (4) improve functional outcome after SCI. The histological repair and functional improvements produced by cellular transplants alone, however, have been far from the goal of near-normal recovery. To enhance the efficacy of cellular therapies, researchers have employed grafting in combination with a variety of pharmacological and molecular strategies. COMBINATION WITH PHARMACOLOGICAL OR BIOLOGICAL AGENTS The efficacy of cellular transplants can be increased through combination with a variety of pharmacological or biological agents. These compounds can be classified
into a number of categories: (1) immune suppressive drugs (these agents inhibit the cellular immune response to prevent rejection of grafted cells such as CsA and FK506), (2) anti-inflammatory compounds (these drugs can aid in the reduction of secondary tissue damage and suppress the proinflammatory cytotoxic milieu of the injury site prior to grafting, such as methylprednisolone and interleukin-10), (3) biopolymer scaffolds (nerve conduits or matrices composed of various biopolymers can be used to support and orient grafted cells and allow directed rostral-caudal axon growth, such as PAN-PVC tubes and PLA-PLG matrices), (4) growth factors (proteins that exhibit growthpromoting or neurotrophic activities can be combined with cell grafts to increase the diversity and number of axons that grow upon them, such as NGF, NT3, BDNF, GDNF, bFGF, and aFGF), (5) masking antibodies (antibodies that bind and mask the inhibitory ability of various proteins within tissue debris can be used to enhance the growth promoting ability of cell grafts, such as anti-Nogo or anti-MAG antibodies), (6) degradative enzymes (enzymes can be employed to reduce scarring and remove inhibitory extracellular matrix to enhance axon growth into and from cell grafts, such as chondroitinase ABC), and (7) intracellular signaling molecules or inhibitors thereof (the use of synthetic analogues, inhibitors of hydrolytic enzymes, or antagonistic enzymes can be used to modulate intracellular signaling programs of axon growth and enhance the intrinsic ability of neurons to grow across cell grafts, such as cyclic AMP, rolipram, C3-transferase, or the ROCK inhibitor Y27632). The use of these compounds in combination with specific cellular transplantation strategies is discussed in detail in the section “The Merit of Cell Grafting for Restoration of the Spinal Cord.” GENETIC MODIFICATION To enhance the protective or regenerative potential of transplanted cells, they can be genetically modified ex vivo by viral or nonviral vector systems prior to spinal cord grafting [29–31, 128, 312, 313]. Vector systems enable the delivery and overexpression of genes or sequences capable of inhibiting the expression of a specific gene, such as small-interference RNA (siRNA). Ex vivo delivery of genes or siRNA can be used to express or suppress secreted proteins that can affect the injured spinal cord environment after cell transplantation, or they can be used to alter intracellular signaling and functioning of the cells themselves.
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Genetic modification of cells ex vivo overcomes the need for cell-specific promoters to drive gene expression through the use of purified cell cultures. Furthermore, the amount and length of administration of the secreted protein in vivo may be controlled through regulatable gene promoters and the grafting of different numbers of cells, respectively. Ex vivo gene therapy also allows the infection or transfection protocols of the cell type being transplanted to be delineated relatively quickly in vitro, and permits the use of nonviral transfection procedures that are challenging or not feasible for in vivo gene therapy, such as electroporation. Last, multiple infections or transfections can be performed using a number of the same or different genes to increase the amount or diversity of the gene(s) being expressed. The utility and success of ex vivo gene therapy has been demonstrated with a number of viral vector systems (including adenoviral, lentiviral, and adenoassociated viral vectors) in various cell types (including OEG, SCs, fibroblasts, and NPCs) for the expression of secreted factors (such as neurotrophic factors) within the injured spinal cord [29–31, 51, 128, 157, 312, 313, 319, 353, 361]. The ex vivo gene therapy approach as a combination strategy, increases or enhances the beneficial effects of the exogenous cell type used for transplantation.
THE MERIT OF CELL GRAFTING FOR RESTORATION OF THE SPINAL CORD The transplantation of a number of neural and nonneural cells has demonstrated their potential to repair the injured spinal cord in a diverse array of experimental models of SCI in animals. The complexity of cells and their multifarious interactions with their environment make them ideal candidates for overcoming the complex nature of the obstacles facing successful protection and restoration of the injured spinal cord. The highly developed functional attributes of cells make them more suitable for spinal cord repair than either pharmacological or molecular strategies that are generally highly specific in their target choice and thus more limited in efficacy. The following section focuses on the major impediments facing SCI repair and the cellular strategies that have shown the greatest potential in overcoming these obstacles. The majority of the presented therapies is combination approaches using cellular, pharmacological, and/or molecular strategies — as these have demonstrated the highest efficacy in restoring
the complex environment of the spinal cord after injury and in producing functional restitution. PREVENTION OF PROGRESSIVE INJURY The use of cellular therapies during the period of progressive cellular and axonal damage after SCI relies on the cell’s capacity to secrete neuroprotective molecules — including growth factors [16, 207, 286, 318], anti-inflammatory cytokines [300], and free radical scavenging antioxidants [344] — either to accelerate or enhance revascularization [142] or to modulate the immune response to impart protective autoimmunity [130]. The transplantation of many cell lineages relatively acutely post-injury has demonstrated their potential to restrict secondary damage, suggesting that other mechanisms involving the prevention of cavitation through tissue supplementation could be physiologically advantageous in reducing the stress effects of mechanical shear — especially in cases of continual progressive tissue loss such as syringomyelia [1, 191]. SC grafts transplanted immediately following transection injury or within 7 days after contusion injury, by themselves [342] or with methylprednisolone and interleukin-10 [274], cyclic AMP analogs, phosphodiesterase inhibitors [275], or bFGF [230] have been shown to retard tissue damage, increase axon sparing, and retard neuronal cell loss. SCs by themselves may reduce secondary injury either by preventing the stress of mechanical shear after filling the injured spinal cord cyst and/or preventing cavitation or by secretion of neuroprotective growth factors [2, 16, 306]. Like SCs, OEG in combination with SCs, methylprednisolone, and/or IL-10 have been shown to improve the ability of OEG to retard secondary tissue loss and enhance axonal sparing after spinal cord contusion [274]. Not only is there significant and progressive cell death at the injury site but retrograde cell death of those neurons axotomized by the primary injury, which is detectable as far rostral as the neurons of the motor cortex [126]. Neurotrophins, delivered by a number of methods, have been shown to attenuate both retrograde neuronal cell death and atrophy after SCI [40, 137, 205, 352]. Cellular delivery of NT-3 by fibroblast grafts is one method that has been shown to prevent retrograde cell death in the Clarke’s nucleus after a unilateral funiculus lesion [137]. The normal reduction in largesize neurons at 2 months post-injury was ameliorated by 50%. Application of this cellular delivery strategy to other regions of the CNS has not yet been examined.
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Like NT-3 fibroblasts, BDNF-secreting fibroblasts have also been shown to retard retrograde neuronal cell death and atrophy after SCI [205]. In this study, BDNF fibroblasts in gelfoam were grafted following a unilateral hemisection injury at the C3-4 cervical level in which the dorsal columns were left intact. Examination of the red nucleus at 1 to 2 months post-injury demonstrated that compared to fibroblast and injuryonly controls (in which 55% of the estimated original number of rubrospinal neurons survived after SCI) rats grafted with BDNF fibroblasts exhibited survival of more than 85% of rubrospinal neurons. BDNF fibroblasts also prevented the degree of atrophy by 55% as determined by mean somal size of neurons from the injured red nucleus. Finally, the combination of NT-3- and BDNF-secreting fibroblasts was able to prevent rubrospinal neuron death and atrophy even after a chronic injury (6 weeks) [352]. In injury-only controls, 73% of neurons from the red nucleus had survived at 5 months post-injury compared to 82% in the NT-3 and BDNF fibroblast group. In addition, compared to the control group in which surviving neurons had atrophied to 51% of their original size the NT-3 and BDNF fibroblast group had neurons that atrophied less — to only 59% of their original size. Fetal tissue grafts have also been used with neurotrophin delivery to retard retrograde neuronal cell death. Bregman and colleagues [40] used NT-3 or BDNF added in gelfoam with transplants of embryonic spinal cord tissue grafted immediately into a thoracic spinal cord hemisection. Assessment of neuronal cell atrophy in the red nucleus demonstrated that although neurotrophins alone partially prevented neuron atrophy, the combination of fetal grafts and neurotrophins completely reversed atrophy. PROVISION OF A SUBSTRATE FOR AXON REGENERATION The removal of damaged tissue by activated immune cells results in the formation of one or more fluid-filled cysts termed a syrinx, which are teaming with monocytic phagocytes and encased by the glial scar to isolate them from the contiguous healthy spinal cord [394]. These syrinxes can often completely encompass the circumference of the spinal cord, encircled by peripheral white matter, the breadth of which is proportional to the severity of the injury [21]. Cell grafts can be transplanted into the injury syrinx to provide a bridge or permissive substrate for the growth of axons from the
rostral to caudal spinal cord. To achieve this function, the cells need to be able to (1) endure the cytotoxic environment of the injured spinal cord [28, 324], (2) have the ability to secrete growth-promoting extracellular matrix, including laminin and collagen for the promotion of axon extension [16, 207, 318], (3) orient themselves for the guidance of axon growth in a direction parallel to the rostral-caudal axis of the spinal cord [18, 276, 278], and (4) encourage axons to reenter the contiguous cord on the opposite side of the injury by guiding them across the inhibitory glial scar [103, 295]. PNGs have been shown to support the growth of many axonal populations, in particular proprioceptive and sensory axons [372]. The inabilities of PNGs alone to encourage growth of these axons from the grafts into the contiguous spinal cord or to support corticospinal axon growth have detracted from the clinical use of this strategy. In a landmark study, however, Cheng and colleagues [63] performed a complex surgical procedure in rats in which 18 fine intercostal nerves were grafted into the completely transected thoracic spinal cord in such a way that the nerves redirected descending and ascending pathways from proximal white to distal gray matter. The nerves were stabilized in place using fibrin glue and by fixing the vertebral column in dorsiflexion by wiring. The growth factor aFGF, shown previously to increase neuronal survival and axon growth after injury [187, 348], was also added to the graft. Rats were examined histologically at 3 to 12 months after treatment using anterograde and retrograde tracing with WGAHRP. They found that numerous supraspinal axons (from the motor cortex, red nuclei, reticular nuclei, and raphe nuclei) had grown both into and beyond the PNG and were present in the lumbar spinal cord, where appropriate connections with hind limb motor neurons could have been made. Axon regeneration beyond the PNG was accompanied by improvements in hind limb function, which included movements of three hind limb joints, partially supported body weight, and four-limbed stepping (features not present in the controls). In a repetition of this experiment, Lee et al. [188], at 6 months after grafting, reported extensive NF+ axon growth across the PNG, improvements in open-field locomotion (BBB score of 7 versus 1 in controls), and contact placement responses, as well as the presence of SSEPs and MEPs — demonstrating evidence of both sensory and motor information crossing the injury/PNG. In 2004, Fraidakis and co-workers [104] extended these findings to the successful implementation of
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this intervention in a chronic (4- to 6-month-old) injury. In this paradigm, the combination of white-to-graymatter-placed PNG, fibrin glue with aFGF, and wiring of the vertebrae exhibited a similar efficacy for histological and behavioral improvements. Interestingly, they also found that aFGF was not required for the functionality of the therapy in chronic SCI. Last, Tsai and colleagues [358] repeated the combination strategy and identified that although multiple systems regenerated after treatment the CST was the motor pathway that correlated with the functional improvements in the hind limbs. The collective results of these studies are dramatic and this therapy has since progressed to the successful treatment of human SCI [64], despite less dramatic results obtained in nonhuman primate SCI models [190]. The diversity and number of axons that grow into purified SC grafts can be significantly enhanced through the addition of neurotrophic and growth factors, either by protein delivery methods such as intraspinal canulae [15] or embedded matrices [146] or by genetic modification of the SCs to enhance their secretion of these factors [232, 365, 374]. The supplementation of SC bridges with NT-3 and BDNF delivered by a distal in-dwelling catheter after spinal cord transection facilitated the growth of both 5HT- and DbH-positive axons onto the grafts [388]. Retrograde tracing further demonstrated that this treatment also encouraged the growth of numerous vestibulospinal axons onto the grafts. The use of a caudal close-ended channel in which the neurotrophins were delivered to the caudal end prevented the assessment of the growth of these axons from the bridges back into the host cord and of hind limb function. A subsequent study by Menei et al. [232] employed retroviral vector delivery of BDNF to SCs in culture. These genetically modified SCs were then introduced by injection into the site of a complete spinal cord transection and as a 5-mm-long trail in the caudal cord stump. Analysis of supraspinal axon growth by immunochemistry and retrograde tracing showed that both the transplant and trail of BDNF-SCs contained 5HT- and DbH-positive axons, as well as fibers from the reticular and raphe nuclei. These supraspinal axons, however, failed to leave the trails — which like a PNG were encased by reactive host astrocytes. No behavioral testing was performed in this experiment. In addition to this study, several other neurotrophic or growth factors — such as NGF [365, 374] and aFGF [120] — have been shown to increase the diversity and/or amount
of axon growth into SC grafts. Other growth factors, however, have been shown to either be ineffective in altering axon growth into SC grafts — such as bFGF [230] — or to actually reduce axonal regeneration into SC transplants [266]. In addition to neurotrophins and degradative enzymes, the elevation of cAMP to enhance the intrinsic capacity of neurons to regrow has been shown by our group to enhance the efficacy of SC grafts for supporting a greater number and diversity of axons within the transplants and to facilitate their growth from the grafts into the contiguous caudal spinal cord [275]. In this study, the phosphodiesterase IV inhibitor rolipram was given for 2 weeks after a thoracic moderate contusion (12.5 mm) to prevent cAMP hydrolysis. It was found that SCI produces dramatic reductions in cAMP in many spinal-cord-projecting supraspinal structures. At 1 week after injury, SCs were grafted into the injury epicenter, and the cAMP cell-membrane-permeable analog dibutyryl cAMP was injected rostral and caudal to the graft. At 10 weeks posttransplantation, the combination grafts exhibited significantly more myelinated and unmyelinated axons in the grafts, 5HT+ axons grew not only into but exited the SC grafts to reenter the host spinal cord, and greater numbers of neurons from the reticular formation and raphe nuclei had axons projecting beyond the transplants (as identified by retrograde tracing). In addition, animals receiving SC grafts and cAMP elevation demonstrated improvements in openfield locomotion (BBB score of 15 versus 10.4 in injury-only controls) and positioning of the hind paw during conditioned locomotion and when traversing a grid walk. Similar to SC grafts, fibroblasts have been transduced with viral vectors to deliver various neurotrophic and growth factors (BDNF, NT-3, aFGF, bFGF, NGF, or combinations thereof) to the injured spinal cord to promote regeneration and repair [119, 157, 206, 328, 352, 353, 360–364]. Initial experiments employed NGF-secreting fibroblasts that had been transduced with retroviral vectors and transplanted into the uninjured spinal cord [364]. These grafts supported the growth of numerous NF+ axons, which were predominantly peptidergic fibers (CGRP+). The grafts did not support the growth of cholinergic, dopaminergic, serotonergic, or noradrenergic axons. Of concern, the grafts expanded almost fourfold in size over the course of 1 year, highlighting that these grafts could in the long term produce compression of the spinal cord — leading to undesirable outcomes. In a subsequent study
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by Tuszynski and colleagues [360], these cells were grafted into a microaspirated dorsal hemisection lesion. Slight changes to the methodology of the grafting paradigm in this study included the suspension of grafted fibroblasts in a collagen matrix and artificial dural repair using durafilm. It was observed that the transplantation of NGF fibroblasts, like the previous study, supported the growth of a large number of NF+ and CGRP+ axons. In addition, cholinergic axons were found penetrating the grafts, and both TH+ and DbH+ axons were found extensively throughout the grafts at later time points (> 6 months). The finding that this also occurred in uninjured spinal cords grafted with NGF fibroblasts points to the beneficial effects of the additional grafting parameters. In 1997, a follow-up study by Tuszynski and co-workers [363] using NGF fibroblast grafts reported that after SCI fibroblast grafts did not stimulate growth of the CST axons. The axons that did grow into the grafts failed to exit the grafts and thus could not reestablish appropriate connections. Behavioral recovery, as measured by the number of footfall errors over a wire mesh, was not improved by this strategy. In the same year, this group introduced NGF fibroblasts into a chronic spinal cord hemisection lesion (1 to 3 months post-injury) after scar debridement [119]. Results similar to the acute paradigm were observed, including an extensive penetration of the grafts by cerulospinal and sensory axons (but not other) supraspinal fiber populations. Behavioral outcomes were not analyzed in this study. These findings were later extended to the use of NGF fibroblast grafts in a nonhuman primate SCI model [362]. In addition, in this study the grafted NGF fibroblasts were shown to induce robust production of the axon growth inhibitors CSPG and collagen IV by endogenous SCs which had migrated both within and around the graft, which could account for the absence of a supraspinal axon growth response to the grafts. Using grafting methods and SCI models comparable to those of NGF fibroblasts, NT-3-secreting fibroblasts (transduced by retrovirus) have demonstrated more impressive results. In an initial study by Grill [119], NT-3-secreting fibroblasts were shown to promote significant CST axon growth around the lesion/graft and for up to 8 mm beyond — as compared to a lack of CST response in NGF fibroblastor control-grafted rats. The growth of BDA-traced CST axons was observed in host cord gray matter but not white matter, likely due to the inhibitory nature of myelin against growth of this axon population [323].
Although NT-3 fibroblast transplanted animals showed no improvements in hind paw positioning as measured by footprint analysis, their ability to walk across a wire mesh was enhanced by measurement of a reduction in the number of footfall errors. These studies were subsequently repeated using a chronic injury paradigm in which the cells were introduced at 3 months after injury following scar debridement [361]. A second graft of NT-3-secreting fibroblasts was placed 6 mm caudal to the original lesion site. In response to the treatment, significant growth of BDAtraced CST axons around the lesion and for up to 15 mm caudal in host cord gray matter was observed. The further extension of CST growth may have been prevented, as the authors suggest, by termination of these axons on neuronal targets as visualized by the formation of clusters of varicosities along the labeled axons. In addition to CST growth, NT-3 fibroblast grafted animals demonstrated a modest improvement in open-field locomotion (12.1 versus 10.6 in the controls). The end-point BBB score, however, was significantly less than what had been observed in dorsal hemisected controls in previously performed studies (BBB score ∼ 15) [362]. This lower score was attributed to the additional damage caused to the spinal cord by the scar resection procedure. Fibroblast delivery of BDNF has also been examined as another cellular therapy for SCI repair. Liu and colleagues [206] first examined this strategy in a unilateral hemisection injury paradigm at cervical level C3-4 in which the dorsal columns were left intact. Cells were transplanted immediately post-injury in a gelfoam matrix and rats examined up to 2 months post-injury. BDNF-modified fibroblasts were shown to promote the growth of BDA-traced rubrospinal tract CGRP+ and 5-HT+ axons into the grafts. Traced rubrospinal fibers also extended significant distances caudal to the graft, as far as the mid-thoracic spinal cord. This growth was interpreted as regeneration due to the completeness of the lesion to ablate the rubrospinal tract, as determined by anterograde and retrograde tracing beyond the lesion in controls. Last, animals receiving BDNF fibroblast exhibited functional improvements, as demonstrated by increased use of the affected forelimb when rearing in the cylinder test. This lesion paradigm and treatment strategy with additional scar debridement were repeated in a subsequent study by Jin et al. [159] to examine efficacy in a chronic injury (4 weeks). The use of BDNF fibroblasts in a chronic injury proved less effective. Although growth of traced rubro-, reticulo-, and vestibulo-spinal
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axons was seen onto BDNF fibroblast grafts, only a few vestibulo-spinal axons was able to exit the grafts onto the caudal cord. Despite a poorer supraspinal axon growth response, chronically injured rats treated with BDNF fibroblasts did exhibit functional improvements (i.e., an increased usage of the affected forelimb when rearing in the cylinder test). The use of a combination of NT-3 and BDNF fibroblasts in a chronic (6 weeks) injury [352] led to a similar growth response profile as BDNF fibroblasts alone [157]. However, supraspinal axons were not seen leaving the grafts. Subsequent behavioral analyses demonstrated that there was no significant improvement in motor behavior between NT-3 and BDNF fibroblast transplants and injury-only controls. This was attributed primarily to an effect of the damage produced by the debridement of the injury site prior to grafting, which reduced BBB scores by almost 5 points compared to preintervention values [328]. OEG grafts have been employed with a number of pharmacological and molecular strategies to enhance their capacity to support axonal regeneration after SCI. The use of OEG in combination with the neuroprotective glucocorticosteroid methylprednisolone immediately after ablation of the dorsal hemisection by selective lesion has been shown to enhance CST axon regrowth across the lesion/graft and to improve performance on the directed reaching task [246]. Growth of CST axons was seen up to 13 mm beyond the graft and was confirmed by retrograde tracing in which neurons were found in the motor cortex (layer V) after injection of the tracer caudal to the lesion/graft. OEG have also been employed with SCs and enzymes capable of degrading the inhibitory ECM of scar to enhance growth into and from SC bridges implanted into the completely transected thoracic spinal cord [103]. In an experimental paradigm analogous to that of Ramón-Cueto and co-workers [297], chondroitinase ABC (cABC) — an enzyme capable of degrading CSPGs and reducing their axon growth inhibitory ability [35] — was delivered by infusion catheter to the rostral and caudal SC cable/host cord interfaces. The OEG were injected 2 mm from the SC cable/host cord interface. Application of the combination treatment significantly increased the number of myelinated axons (likely sensory and proprioceptive axons) within the SC bridges and led to the growth of 5HT axons both onto the SC bridge and from the bridge into the caudal contiguous spinal cord. Two other supraspinal tracts, CST and reticulospinal axons, did not respond to the intervention. Behaviorally, animals treated with SC bridges, OEG, and cABC exhibited
improvements in open-field locomotion (BBB score of 6.6 versus 2.1 in injury-only controls) and forelimb/hind limb coupling. They also had an increased response to tactile stimulation. Of note, unlike the study by Ramón-Cueto and colleagues [295] improvements in open-field locomotion were not shown to correlate with serotonergic axon growth beyond the grafts, but rather to the number of myelinated axons within the SC bridges. This may highlight the importance of intraspinal cord connections via proprioceptive neurons for the observed motor recovery. OEG, as with SCs and fibroblasts, have been genetically modified ex vivo prior to spinal cord transplantation to enhance their ability to secrete various growth factors and to increase their capacity for axon regeneration. Ruitenberg and colleagues [313] examined the efficacy of OEG transduced with adenoviral vectors for NT-3, BDNF, or both neurotrophins to alter the growth of rubrospinal tract (RST) axons after a C4 unilateral dorsal funiculus lesion that completely ablates the RST. It was found that all OEG grafts reduced progressive tissue loss after the injury and that OEG-only and NT-3 OEG grafts supported very little BDA-traced RST axon growth. The use of BDNF OEG alone or in combination with NT-3 OEG, however, increased RST axon growth into, around, and caudal to the grafts. Improvements were observed behaviorally on the horizontal rope walk test with all OEG grafted groups. However, BDNF and NT-3 OEG grafted rats exhibited significant increases over OEG grafts alone. The degree of functional recovery on the test was shown to correlate with the amount of tissue sparing provided by the various OEG graft types. In a subsequent experiment by Ruitenberg [311], the use of NT-3 OEG grafts was extended to their employment in a unilateral microlesion of the C4 CST. Unlike previous studies [195, 197, 198], OEG-only grafts did not support extensive CST growth across the lesion/graft. It was only when OEG were genetically modified to secrete NT-3 that there were more CST axons present within the caudal contiguous cord for distances in excess of 1 cm. No improvements in open-field locomotion were observed in treated rats, likely due to the inability of this type of microlesion to produce gross locomotor deficits, but rather deficiencies in fine motor control were reduced [209]. In contrast to Nash et al. [246], this study reported no differences between experimental groups using a directed forelimb reaching task. Improvements were seen in all experimental groups over time and were identified as compensatory movements rather than the restitution of
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the original action. This compensatory function was suggested to occur largely independently of the observed CST regrowth and to involve other supraspinal or spinal pathways and learning adaptation. Cao and colleagues [51] examined the ability of genetic modification of OEG with another neurotrophic factor, GDNF, to enhance supraspinal regeneration and functional recovery after complete thoracic spinal cord transection. Using the same injury and transplantation paradigm as Ramón-Cueto et al. [113], GDNF OEG were injected into the rostral and caudal spinal cord stumps immediately following transection. Using retrograde tracing, in which HRP was injected 3 to 4 spinal cord segments caudal to the lesion at 8 weeks, numerous labeled cortico- and rubro-spinal neurons were identified with axons projecting for significant distances caudal to the lesion/graft in rats receiving OEG-only compared to none in lesion-only controls. The use of GDNF OEG significantly enhanced the amount of supraspinal axon growth caudal to the lesion/graft. In addition, anterograde tracing confirmed that a number of CST axons had grown for up to 4.5 mm caudal to the lesion in the OEG and GDNF groups. Behavioral assessment of open-field locomotion demonstrated that both OEG and GDNF OEG dramatically improved gross locomoter ability over controls with GDNF OEG rats exhibiting significantly higher BBB scores than OEG alone (BBB scores of almost 10 in GDNF OEG rats versus 7 in OEG grafted and 2 in injury-only controls). Like other cell types, fetal grafts have also been used with pharmacological agents or neurotrophic molecules to enhance their efficacy for axon regeneration. In a C3-4 lateral hemisection, Nikulina and co-workers [253] examined if elevation of cAMP by the administration of the phosphodiesterase inhibitor rolipram could increase the regenerative potential of immediately grafted fetal spinal cord tissue transplants. It was shown that this combination strategy led to the growth of many serotonergic axons onto the grafts, as well as to functional improvement of the affected forelimb as assessed using analysis of paw preference when the animals reared in a glass cylinder. Fetal spinal cord transplants have also been combined with the delivery of the neurotrophins NT-3 and BDNF to the injury/graft site by mini-pump after acute (immediate) or chronic (2 to 4 weeks) thoracic T6-8 complete spinal cord transection in which the grafts were placed within the transection site to form a substantive bridge for axon growth [71]. In this study it was shown that supraspinal axons failed to grow into and beyond the
injury/graft site when either fetal grafts or neurotrophins were employed by themselves. When NT-3 or BDNF administration, however, was combined with fetal grafts after chronic injury (but to a lesser degree in acute) not only were serotonergic, adrenergic, and traced CST axons found traversing the injury/graft but many axons were present within target regions of the lumbar spinal cord far caudal to the injury/graft site. In addition, animals receiving the combination of fetal spinal cord grafts and neurotrophins after chronic SCI demonstrated improvements in locomotion, including recovery of weight-supported plantar stepping on both treadmill and in over-ground tasks such as stair climbing. Unlike other cell types, a limited number of combination strategies using MSC transplantation to promote axon regeneration after SCI have been performed. In a recent study, Lu and colleagues [213] performed a retroviral transduction of MSC to increase BDNF secretion. Prior to transplantation into the bilaterally transected dorsal columns of the cervical C3 spinal cord, MSCs were chemically induced to become “neural-like” in vitro according to previous methodology [382]. It was found that both MSCs and BDNF MSCs exhibited good survival after transplantation and fully filled the extent of the lesion. BDNF MSC grafts were larger than MSC grafts and progressively increased in size over time, which as suggested by the authors likely resulted from increased infiltration of endogenous SCs into the lesion/graft. The transplanted cells failed to express any markers that would identify them as differentiated neurons, astrocytes, or oligodendrocytes, and all MSCs in both groups remained as undifferentiated stromal cells that were CD90 and fibronectin positive. These cells also exhibited limited migratory ability and rarely penetrated the limitations of the lesion. Modest NF+ axon growth was observed into MSCs. This growth was increased in BDNF MSCs. Sparse to modest growth of cholera toxin B (CTB)-labeled ascending dorsal column axons and TH+, 5HT+, CGRP+, and ChAT+ axons were observed in MSC grafts. The growth of all of these axon populations was increased in BDNF MSCs, but once in the grafts the axons failed to exit. BDA-labeled CST axons rarely penetrated any MSC grafts, and behavioral improvements — as assessed by forelimb function and adhesive tape removal tests — were not observed in any transplant groups. To promote axon outgrowth from the grafts, an additional pharmacological strategy (elevation of cAMP) was employed in combination with MSC grafting and neurotrophin delivery [213]. In the same C4 lesion paradigm, a cAMP
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analogue was injected bilaterally into the L4 DRGs five days prior to injury. MSCs were transplanted immediately after the lesion along with NT-3 protein into the injury epicenter. Then, at 1 week post-injury, NT-3 protein was injected into the dorsal columns 1.5 mm rostral to the lesion/graft to entice CTB-labeled ascending sensory axons from L4 DRG that had entered the graft to exit into the contiguous host spinal cord. It was found that long-tract ascending axons grew into and beyond the grafts in the contiguous rostral spinal cord for distances of 1.5 to 2 mm only in the full combination treatment group. No improvements in function, however, were observed with this treatment. It is evident that additional combination strategies and the targeting of supraspinal axon populations are needed to test the efficacy of MSCs to promote axon regeneration and to improve function following SCI. Very few combination studies using stem cells, progenitors, or precursors have been reported — due largely to their infancy in the field in SCI repair. Some early work using the genetic manipulation of neural stem cells (NSCs) demonstrates that these cells can also be used as a vehicle for the delivery of therapeutic proteins to the injured spinal cord [214]. NSCs by themselves have been shown to secrete various growth factors known to enhance axonal regeneration, including NGF, BDNF, and GDNF [214]. In a study by Lu and colleagues [214], murine neonatal cerebellar NSCs (C17.2) were modified to secrete NT3 using retroviruses and underwent immediate xenotransplantation into a rat C3 dorsal column lesion. The secretion of NT3 by the NSCs did not increase graft size, enhance cell migration, promote differentiation, or induce CST axon growth into the grafts. NT3 NPCs, however, did support greater numbers of NF+ and long-tract dorsal column ascending sensory axons than NSC-only grafts. REPLACEMENT OF ENDOGENOUS CELLS Significant loss of neurons and glia occurs after SCI, which in the case of neurons is endogenously irreparable due to the incapability of terminally differentiated neurons to divide and replace those that have perished [255]. When spinal cord injury affects the motor pools responsible for the functioning of the upper and lower limbs at the cervical and lumbar enlargements of the spinal cord, the replacement of lost neurons becomes a limiting factor for the restitution of locomotion. Without intact spinal cord motor targets to coordinate descending inputs from the brain to the muscles,
regeneration of axons alone cannot restore function. In this scenario, the depletion of appropriate motor targets to which regenerating axons must reestablish connections requires either the introduction of exogenous cells that exhibit neuronal function [108, 129, ] or the promotion of endogenous precursor cell populations to differentiate into cells with neuronal function [201, 237]. MSCs have demonstrated the potential to differentiate into cells of a neural lineage, including neurons and astrocytes both in vitro and in vivo — making them candidates for neuroreplacement therapies [83, 155, 235, 236, 316, 375, 381, 382, 399]. This ability has, however, come into question by other groups [58, 249, 349], and it is not yet clear whether this is an artifact, cell fusion, or true differentiation [134, 158, 334]. It has also not yet been shown if MSCs are able to differentiate into functional oligodendrocytes, although they can form peripheral myelinating cells that have many properties of Schwann cells [85, 397]. In addition, it has been reported that although MSCs can differentiate into cells that express various neuronal specific markers they do not seem to possess the correct Na+ and K+ channels for neurons and are unable to depolarize [267]. Further investigation of the pluripotency of MSCs in vitro and in vivo is needed to clarify under what conditions MSCs can differentiate into cells of a neural lineage and whether these differentiated cells can function as the natural mature phenotype in vitro and in vivo after SCI. Stem cells and their progenitors and precursors are also able to differentiate into all CNS cells, offering the greatest potential for neuro-replacement therapies for SCI repair. There are now numerous reports on how various stem cells and their neural progenitors can be specifically driven toward a neuronal, astrocytic, or oligodendrocytic cell fate using various factors and under certain culture conditions in vitro [83, 156, 235, 236, 316, 375, 381, 382, 399]. The environment of the injured spinal cord, however, is very difficult to control compared to culture conditions. Thus far, the transplantation of these cells has led largely to glial cell differentiation; in particular, astrocytes (GFAP+) [179, 367]. To overcome this obstacle, researchers are currently attempting to manipulate NPCs prior to spinal cord implantation, either through genetic modification or by culture procedures that commit them to a single cell fate (e.g., OPCs) and thus restrict their further differentiation [13, 170, 204, 326]. In one example, Setoguchi et al. [326] used retroviral vectors to introduce BMP
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inhibitors noggin, smad 6, and smad 7 into adult mouse spinal-cord-derived NPCs prior to transplantation as small aggregates into a mouse thoracic spinal cord compression (30 g, 2 min) at 8 days post-injury. Introduction of these genes permitted the differentiation of the cells, as analyzed at 10 weeks post-injury, into bIIItubulin-positive neurons (6.4%) and GST p-positive oligodendrocytes (7.5%) rather than purely astrocytes (GFAP). Furthermore, they could substantially improve functional outcome in the open field (BBB score of 10 versus 8 in NPCs only and 4 in injury-only controls) by directing the fate of grafted adult NPCs. REMYELINATION OF AXONS To ensure that the transmission of certain electrical signals can be propagated at fast speeds, a lipid called myelin insulates some axons formed by specialized glial cells: Schwann cells in the PNS and oligodendrocytes in the CNS. Following SCI, significant demyelination of axons can occur [121, 354], either when the myelin sheath is destroyed directly by the mechanical injury or after its accompanying myelinating glial cell is lost when subjected to the cytotoxic mediators of the secondary injury. Either of these processes produces a demyelinated and dysfunctional axon. Axons that are severed following SCI and are then encouraged to regenerate across the injury site may later need to be myelinated in order to function properly. To accomplish remyelination of these axons, a number of alternative approaches have been investigated: (1) the grafting of exogenous glial cells that are innately capable of myelination, including Schwann cells or olfactory ensheathing glia [167, 276, 342], (2) the transplantation of precursor cells that have been differentiated ex vivo into cells that are, or function as, oligodendrocytes [5, 13, 204], and (3) stimulating endogenous precursor cell populations within the injured spinal cord to differentiate into myelinating glial cells [228]. The implantation of a PNG into the transected spinal cord supports supraspinal axon in-growth and the myelination of these axons by SCs, as demonstrated by electron microscopy and P0 immunostaining [101]. The inability of the SCs within the PNG to cross into the GFAP-positive regions of the host cord and myelinate demyelinated axons, as seen through an absence of P0 immunoreactivity in this area [101], limits the potential of PNGs as a remyelination strategy for SCI. Purified grafts of SCs are capable of facilitating the remyelination of central axons after SCI [365, 389] or
in models of chemically induced demyelination [182]. After SCI, SC grafts (like PNGs) are generally only able to myelinate those axons able to enter the transplants. The restriction of grafted SCs to the lesion site by the glial scar [387, 389] prevents them from accessing regions of demyelinated axons outside the area of the primary lesion. The enhancement of the ability of SCs to myelinate axons, indicated either by an increase in the number of myelinated axons and/or a decrease in the ratio of myelinated:unmyelinated axons, has been observed following increases in levels of various growth factors [15, 389] or cAMP [275, 398]. OEG have also been shown to myelinate axons in models of chemically induced demyelination and after SCI. Imaizumi and co-workers [151] grafted neonatal OEG into an electrolytic lesion of the dorsal columns. They observed extensive Schwann-cell-like myelin throughout the lesion and enhanced conduction velocities of dorsal column axons at 21 to 25 days after grafting. Subsequent studies demonstrated OEG myelination when both neonatal and adult OEG were employed in a dorsal column transection injury [150] or human OEG were used following an electrolytic lesion within the dorsal columns [167]. Other groups, however, have shown a lack of OEG association with and myelination of central axons [33]. In the study by Boyd and colleagues, embryonic OEG were labeled with b-galactosidase and transplanted at 7 days after a clip compression injury for examination by immunoelectron microscopy at 3 weeks posttransplantation. The myelination found near grafted labeled OEG was suggested to be formed instead by endogenous SCs. MSCs have been shown to remyelinate axons after demyelinating electrolytic lesions of the spinal cord when grafted either directly into the site of the lesion [5, 180] or even when given intravenously [6]. This ability of MSCs to myelinate after intravenous administration is not shared by other peripheral cells employed to promote SCI remyelination, such as SCs and OEG [6]. It has not yet been reported, however, if MSCs are capable of myelinating axons after traumatic SCI. The myelinating characteristics of MSC are very similar to SCs, and whether these cells differentiate in order to myelinate is still unknown. Indeed, MSCs have demonstrated the ability to differentiate into SCs. Stem cells and their progenitors and precursors have also been employed for remyelination of the injured spinal cord. McDonald and colleagues [223] found that transplantation of mouse ES cells into a spinal cord contusion injury resulted in the differentiation of close
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to 50% of the cells into oligodendrocytes with their subsequent remyelination of central axons. To enhance the myelinating ability of stem cells, however, precursor cells with a more restricted differentiation (OPCs) have achieved oligodendrocytic differentiation to greater than 90% of the cells after spinal cord grafting. In an early study with ES-derived OPCs by Liu and colleagues [204], cultures of oligospheres were first obtained using the 4+/4− protocol (4 days with and 4 days without retinoic acid), as described by Bain et al. [11]. To produce oligospheres, the cells were then grown in SATO medium. In this study, dissociated oligospheres of OPCs were grafted as either a xenotransplant into a chemically induced demyelinating lesion of the rat spinal cord at 3 days post-injury or into the spinal cord of shiverer mice, which lack the gene required to produce myelin basic protein (MBP). In the milieu of the demyelinated rat spinal cord, OPCs differentiated into oligodendrocytes (APC+), but not into astrocytes (GFAP+) or neurons (NeuN+). In the dysmyelinated shiverer mouse spinal cord, grafted OPCs induced the production of MBP at 2 weeks after implantation, indicating that myelin formation had occurred. In another study, human-derived OPCs were transplanted into the injured rat spinal cord at 7 days and 10 months after a thoracic moderate contusion (200 kdyn force) performed using the Infinite Horizons Impactor [170]. In the delayed transplantation paradigm (7 days), OPCs survived, migrated over short distances, differentiated into mature oligodendrocytes, myelinated axons (based on myelin sheath diameter), and significantly improved open-field locomotion. When transplanted in the chronic paradigm (10 months), however, although the transplanted cells were found to survive and differentiate into mature oligodendrocytes, no myelination of axons or improvements in locomotor function were observed — indicating that although OPCs can remyelinate the injured spinal cord and restore function there is a critical window during which grafted OPCs can remyelinate after SCI. PITFALLS AND LIMITATIONS OF CELL TRANSPLANTATION FOR SCI REPAIR The use of cell transplants for spinal cord injury repair does not come without various limitations and potential risks to their application for clinical practice. Primary among the risks of cell transplantation are graft versus host disease (GVHD) and tumorigenesis. GVHD, which is a rejection response to the implanted cells by the
host immune system, is a common complication when donor cell transplants are employed but is not observed when the cells are grafted autologously [219, 332]. GVHD occurs in up to 50% of allogeneic and nearly all unrelated donor transplant patients [219]. Moderate to severe GVHD can lead to a persistent disabling disease involving liver dysfunction, lung disease, and infections that can last for years and that can be fatal [165, 219, 332]. An immunosuppressive drug regimen is employed to try to prevent or reduce the symptoms of GVHD [98, 176, 359]. Another concern regarding the transplantation of cells capable of proliferating or that have been undergoing long-term propagation in vitro is that these cells may generate tumors following grafting within the spinal cord, or in other regions of the body should the cells be able to migrate and/or access the systemic circulation. It has been shown that extensive culturing of rodent neural stem cells, for example, produces genetic changes that modify their cell growth and differentiation [269]. In contrast, the formation of tumors in vivo following grafting into animals has not yet been reported for cells used in SCI repair strategies [109]. An additional concern for allografts is the potential of transferring diseases from the donor to host as has been reported for interchange of other bodily products such as blood [37, 331]. Primary among these transferable diseases are human immunodeficiency virus (HIV) and hepatitis, although lesser instances of other pernicious diseases have been reported. Thus, as an example, although the ability of stem cells from one donor to be expanded in vitro to large numbers for the treatment of many individuals is advantageous, should the donor line be infected with a communicable disease this could lead to widespread transfer and seriously affect the lives of transplanted individuals. The performance of rigorous testing of donor cells should, however, minimize the risks of disease transfer. Adverse host reactions to the grafted cells could also include aberrant axon growth that produces sensory abnormalities and pain, spasticity, and/or autonomic dysfunction. A number of regenerative strategies have been shown to increase the growth of axons involved in nociceptive transmission [159, 174, 175, 210, 233, 236, 276, 285, 287, 347, 348]. In such cases, additional treatments to ensure that these alterations to sensory, motor, or autonomic systems do not lead to a worsening of the quality of life of the SCI individual need to be employed in conjunction with grafting (e.g., anti-NGF antibodies to reduce nociceptive fiber in-growth and
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pain) [68, 124]. Diligence in the assessment of potential alterations in the normal patterns of axon growth and the production of aberrant function needs to be undertaken in the preclinical evaluation of any novel cellular therapy to ensure that proper steps can be undertaken for their clinical use. Last, the employment of undifferentiated cells or cells capable of de-differentiating — such as stem cells, neural progenitors, and precursors or MSCs — may lead to the formation of inappropriate tissue/cell types within the spinal cord, such as muscle, bone, or adipose tissue. Embryonic stem cells have been reported to differentiate into bone and chondrocytes when transplanted into immunosuppresed mice [222]. Although possible for a number of cell types, to date reports of aberrant cell differentiation within the CNS of grafted cells are rare. THE CLINICAL USE OF CELL TRANSPLANTATION FOR SCI REPAIR Several hundred spinal cord injured people have been reported to have received cell grafts worldwide, yet to date few controlled studies have been performed to examine the safety and efficacy of cell transplantation for SCI repair [301]. Trials of intraspinal grafting of human fetal CNS tissue for posttraumatic syringomyelia, initiated in the United States in 1997, are currently the only clinical trials involving cell transplantation in SCI that have been completed and that have provided reliable and peer-reviewed scientific data on the feasibility and safety of this therapeutic direction [351, 379]. Furthermore, this study has highlighted the need for: (1) substantive preclinical characterization, independent replication, and scientific dialogue prior to clinical implementation, (2) reliable and quantitative outcome measures for clinical evaluation, and (3) appropriate control groups for determination of safety and efficacy of any future clinical trial of a cell transplantation procedure [301, 303, 304, 351, 379]. In light of these important criteria for critical clinical evaluation of any novel cellular therapies for SCI, numerous groups outside of the United States continue to offer various cell therapies as strategies for SCI repair without the prior rigorous evaluation of the safety and efficacy of these procedures. They also fail to provide peer-reviewed reports on their progress. Without reporting of adverse effects, the long-term monitoring of patients post-treatment or supervising data collection by independent researchers and governmental regulators, these procedures merely take financial advantage
of a population of individuals who yearn to be treated. Moreover, not only are there significant deficiencies in clinical outcome measures associated with these practices but the actual surgical procedures performed and the techniques used for the procurement and culture of the cells used for transplantation are often poorly detailed or hidden in obscurity. Examples of these inadequately detailed, improperly evaluated, and unregulated forays into the clinical use of cell transplantation for SCI include (1) the transplantation of embryonic shark cells in Tijuana, Mexico, (2) the employment of peripheral nerve grafts in Ecuador and in Brazil, and (3) the use of OEC and/or various OEC combination strategies in China and in Portugal. Clinical testing of cell transplantation strategies for SCI repair under the auspices of governmental regulation has, however, become more prevalent in recent years. A number of trials have begun to test the safety and/or efficacy of several different cell types for SCI in the United States, Europe, and Australia. Although the results of these trials have not yet been reported in peer-reviewed publications, there is optimism that these controlled studies will provide valuable information about the feasibility of cell therapies for SCI. Proneuron Biotechnologies, after completing small phase I safety trials in Israel and Belgium investigating the use of autologously transplanted macrophages, has initiated a multi-center phase II trial employing randomized control subjects and blinded assessment of outcome in five centers, including several in the United States. Macrophages are harvested from the injured individual’s own blood, activated through exposure to a biopsy of their skin tissue, and then transplanted back into the spinal cord both within and caudal to the lesion. The initial phase I trial demonstrated that this procedure was safe in humans. This work is an extension of the endeavors of Dr. Schwartz and colleagues, who reported efficacy of macrophage transplantation in promoting protection and axon growth in an experimental SCI model [300, 325]. A group of neurobiologists from Griffith University and spinal surgeons from The Princess Alexandra Hospital, headed by the scientist Dr. Phil Waite, have initiated a phase I clinical trial in Australia to examine the safety — and to some degree the efficacy — of the spinal cord transplantation of OEG autologously harvested from the individual’s own nasal mucosa. The OEG are harvested from individuals with functionally complete injuries from 6 months to 3 years post-injury, purified (to 95%), expanded over 6 weeks (to yield
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15 to 20 million cells), and then transplanted at 40 sites immediately rostral and caudal to the injury by injection. Half of the patients are scheduled to undergo a placebo procedure, and then all patients will be assessed according to a number of neurological and functional outcome measures. This work is an extension of the many reports on the ability of OEG to promote SCI repair [171, 196, 287, 295] and of Dr. Mackay-Sim’s own laboratory findings [211, 212]. Diacrin, now GenVec, in collaboration with Albany Medical Center and Washington University Medical Center, has performed phase I clinical trials to examine the safety and efficacy of fetal porcine neural cells for SCI. The cells are obtained from fetal pigs that have undergone genetic modification to make these cells less likely to be rejected by the host immune system following transplantation. In the transplantation procedure, the porcine cells were injected into the spinal cord, assisted by magnetic resonance imaging (MRI) to identify the lesion so that a total of 14 million cells could be deposited into the injury using seven separate injections. Previously, fetal porcine neural cells have been used in humans for Parkinson’s disease, Huntington’s disease, and other neurological conditions relatively safely and with some degree of success [91, 92]. Concerns have been raised, however, regarding the exposure of transplanted humans to porcine viruses, which has dramatically slowed progress in this field over recent years [73, 337, 338, 343]. The results of the Diacrin/GenVec clinical trials in SCI repair with porcine cells are expected to be available soon [301]. THE FUTURE OF CELL GRAFTING FOR SCI REPAIR The last three decades have seen a rapid evolution in cellular transplantation therapies for repair of the injured spinal cord. Initial implementation of tissue grafts comprising heterogeneous cell types, peripheral nerves, or fetal tissue demonstrated the intrinsic capacity for regeneration in the CNS when axons were supplied with a more permissive environment than the hostile milieu of the injured spinal cord, confirming the earlier pioneering work of Ramón S. Cajal in the 1920s. In various SCI paradigms, it was concluded that these grafts by themselves supported local spinal cord and sensory axon growth (but not supraspinal axons) unless the grafts were placed within the cervical spinal cord, closer to the neuronal cell bodies. In addition, axons that grew into the grafts were largely unable to leave the grafts to reenter the contiguous host spinal cord resulting in limited
functional improvements. During the past 10 years, the employment of purified cell grafts, SCs, macrophages, OEG, MSCs, and (more recently) NSCs has yielded greater efficacy for SCI repair. Numerous studies have reported that purified cell grafts alone can support significant supraspinal and local axon growth both into and beyond the grafts — as well as produce improvements in functional recovery. Despite these achievements, however, it is unlikely that a cellular transplantation strategy alone will attain the goal of complete or even substantial spinal cord restoration, highlighting the need for combination therapies. Genetic modification of transplanted cells or their use in conjunction with various pharmacological or biological agents has been shown to enhance the regenerative capacity of cell transplants and the associated degree of functional improvement they facilitate. It is evident that the use of cellular therapies in SCI repair has an auspicious future. The coming years will see both the initiation and reporting of the results from a number of clinical trials investigating the safety and therapeutic efficacy of several cell types for the restoration of motor, sensory, autonomic, or sexual function after human SCI. One of the most promising cell types, the stem cell, has also emerged in recent years and appears to have the potential to overcome many of the obstacles associated with spinal cord repair, including the replacement of lost spinal cord circuitry. The use of stem cells for the treatment of SCI, however — apart from ethical concerns — will require extensive scientific endeavor to ensure that these highly plastic cells can be properly controlled and integrated into the host CNS without aberrant differentiation, migration, or tumorigenesis. Cellular transplantation strategies, in light of the complexity of the CNS and our limited understanding of its inner workings, have proven to be successful in repairing various components of the intricate network of the spinal cord following trauma — and as therapies offer considerable promise as a means of overcoming the inability of this system to mediate self-repair and restitution of function.
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28 Human Embryonic Stem Cells, Dopaminergic Neurons, and Pathways for Developing a Parkinson’s Disease Therapy William J. Freed (1), Joseph F. Sanchez (1), Chun-Ting Lee (1), Tandis Vazin (1), Rose Amable (1), Cynthia Schoen (1), Catherine Schwartz (2) Mahendra Rao (2) Xianmin Zeng (1) (1) Development and Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland (2) Laboratory of Neuroscience, National Institute on Aging, National Institutes of Health, Department on Health and Human Services, Baltimore, Maryland
Acknowledgments We thank Cindy Ambriz for editing and preparing the manuscript. This research was supported by the IRPs of NIDA and NIA, NIH, DHHS.
Introduction It has now been 25 years since it was first reported that symptoms believed to be representative of Parkinson’s disease (PD) in animal models can be alleviated by the transplantation of fetal tissue. For a number of reasons, however, fetal tissue transplantation remains at best an experimental approach. There are both practical and theoretical limitations that remain. Although a great deal of progress has been made, it has become apparent that even for those who were relatively conservative about the possibilities for clinical use in the early 1980s, the difficulties were underestimated. Recently, there has been a tremendous interest and research investment in the possibility that human embryonic stem cells (hESC) can be used for therapy, and at times a degree of overenthusiasm about developing dopaminergic neurons and a PD therapy from hESC. It is possible that hESC-derived therapeutic transplantation approaches will ultimately succeed, but this is by no means assured. The idea that hESC can be Cellular Transplantation: From Laboratory to Clinic
used for transplantation in PD essentially rests on the assumption that we can overcome a number of longstanding problems that are probably inherent to neural transplantation therapy, regardless of the source of the cells used. How these problems might be overcome by the use of hESC is rarely mentioned or discussed. (Figure 28.1). The purpose of this chapter is to examine the possibilities for employing hESC for treating PD. We first examine some of the limitations of fetal cell transplantation therapy, and the possible reasons for these limitations. We also discuss the possibilities for the use of hESC-derived dopaminergic neurons in transplantation therapies, potential issues that may be common to both fetal and hESC-derived transplantation approaches, and the potential for future development of technology to overcome these issues.
“Traditional” Transplantation Using Fetal Cells The general notion of transplanting fetal mesencephalic tissue into the brain was developed using animal models of PD. Dopaminergic neurons in the ventral mesencephalon (VM) of the rat can be destroyed in animals with the neurotoxin 6-OHDA, and these lesions can be measured in animals by a number of techniques,
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Freed et al.
Human fetus
Immature dopaminergic neurons in mesencephalon (5–7 weeks)
Dissected block of tissue
Human embryonic stem cells in vitro
Cultured blastocyst
Cells in suspension
Differentiation of dopamine neurons in vitro
Cells in suspension
Isolated inner cell mass
Inner cell mass Figure 28.1. Obtaining dopaminergic neurons from fetal material or from hESC.
ranging from motor asymmetry (as indicated by rotation induced by amphetamine) or apomorphine, to various indicators of motor coordination [10, 36, 51, 118]. Beginning in 1979, many studies showed that under appropriate conditions of tissue preparation, fetal tissue age, and transplantation parameters, some alleviation of certain measures of behavioral impairment after 6-OHDA-induced lesions could be obtained by transplantation of dopaminergic cells [48, 53]. On the basis of these studies, transplantation has been performed in a considerable number of human patients with PD. Nonetheless, there are limitations in the effects of fetal tissue transplants that are apparent even from animal studies. The three principal limitations are: (1) the limited behavioral effects of transplants, (2) the limited survival of transplanted dopaminergic neurons, and (3) the relatively limited scope of reafferentation of the brain that transplanted dopaminergic neurons can produce. The behavioral and functional improvements seen in animals after transplantation of fetal VM are very limited. This is often not appreciated because the most common behavioral test of transplant efficacy, amphetamine-induced rotation, does show large effects. Because of particular properties of this behavioral test (i.e., amphetamine can release dopamine from the transplants themselves, and thus this test does not reflect transplant efficacy under all other conditions when
amphetamine is not present), results of this test are somewhat misleading. All other tests, ranging from rotation induced by apomorphine [47, 50, 51] to more complex tests of motor coordination [99], show that fetal tissue transplants produce very modest effects [37, 98, 101]. By some measures of motor coordination, and aphagia and adipsia after complete bilateral lesions, no positive effects of transplants can be detected in adult animals [35, 38, 101, 141]. The second limitation is that transplanted dopaminergic neurons survive quite poorly. Typically, when fetal tissue (VM) is transplanted into animal brains — and the transplants are subsequently examined — usually no more than 10% of the transplanted dopaminergic neurons survive, and often less [15, 87, 93, 151]. Thus, the percentage of dopaminergic neurons — among the various other cell types present in transplants that survive more robustly — is small. Most of the dopaminergic neurons that die do so within the first few hours after transplantation [7, 41]. In addition, most dopaminergic neurons in transplants are found to have differentiated prior to transplantation, as opposed to differentiating from precursor cells following transplantation [145]. A number of manipulations that can to some degree increase survival of transplanted dopaminergic neurons have been identified [63, 72, 73, 87, 93, 150, 151]. Although these effects are relatively modest, in some (but not all) cases multiple treatments
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are additive when combined with each other [16, 63, 120]. The reasons for the limited survival of transplanted dopaminergic neurons are not well understood. Finally, transplanted dopaminergic neurons have a limited ability to become integrated into the adult host brain circuitry. Such cells, transplanted into adult hosts, can produce some connections with host neurons. However, the extent of synapse formation between transplanted dopamine neurons and host neurons is minimal as compared to the normal connections of these cells [55, 89, 96]. It is not likely that grafted dopaminergic neurons can produce functional effects unless they make connections with host neurons. Xenografts of human mesencephalic dopaminergic neurons have also been found to form synapses with target neurons in the rat striatum [25]. Functional effects were seen between approximately 8 and 11 weeks after grafting, the time when a dopaminergic fiber network had begun to extend into the host striatum [25]. Most transplantation studies do not involve transplantation of cells into their normal location in the substantia nigra. Cells are instead transplanted to target areas (e.g., the putamen), in which case complete restoration of normal circuits does not occur. In some studies, transplants are placed into multiple sites — such as both neostriatum and substantia nigra — which does produce some improvements but still does not entirely reproduce normal neuronal circuitry [7, 70, 98, 99, 107, 130]. The few synapses that form between transplants and host are far from the level of integration that takes place when these neurons develop normally, as an integral part of the brain. Penetration of efferents from transplanted cells is also minimal, and these efferents extend relatively short distances into the host brain. Thus, transplanted dopamine neurons become only minimally integrated into the host brain circuitry, and the integration that does occur is not the same as that seen for normal dopamine neurons. It may be possible to increase the penetration of processes from transplanted cells into the host brain. For example, certain types of brain injury and transplantation techniques increase the growth of neurites from transplanted dopamine neurons [49, 106, 108, 156, 166, 183]. It is, however, very difficult to conceptualize a solution to the problem of reconstructing the normal dopaminergic neuronal circuitry through the use of transplants. Finally, it should be mentioned that these three previously cited “problems” might be to some extent multiple manifestations of a single problem. When dopaminergic
neurons are transplanted into the brain, they do not form connections with host neurons in the same manner as normal developing neurons. This could contribute to the poor cell survival observed through programmed cell death, and to the limited behavioral effects. Manipulations such as antioxidant treatments, which increase cell survival without increasing integration of transplanted cells into the host brain, may be associated with increased development of abnormal cellular connections rather than to improved connectivity and behavioral efficacy. Transplanting large numbers of cells to overcome limited cell survival may have a limited ability to improve efficacy, because dopaminergic cell survival may be limited by connectivity, not only by the number of cells implanted. Transplanting larger numbers of cells could perhaps result in larger numbers of abnormal interconnections among transplanted neurons as well. It is, therefore, possible that improving transplant performance will require improved graft connectivity, which could necessitate increasing the ability of neurites of transplanted dopaminergic neurons to extend into the adult host brain (see the section “Beyond Stem Cells: Cell Lines and Other Possibilities”).
Clinical Studies There have now been a considerable number of reports on fetal tissue transplantation for PD, including early studies of one or two patients at a time, several studies of groups of five to ten subjects, and more recently two reports of large-scale controlled clinical trials. In general, nearly all studies have observed at least some improvements. For the most part, although the degree of improvement has varied somewhat there have been modest improvements that developed over a time course of several months after transplantation. It should also be noted that several prior studies of adrenal medulla transplantation employed transplantation techniques that usually did not even result in chromaffin cell survival, but nonetheless substantial improvements were also observed, in some cases similar in degree to the improvements seen after fetal mesencephalic tissue transplants, and usually developing with a similar time course [52, 58] of several months. This latter observation emphasizes the importance of controlled clinical trials [46, 113] for interpretation of studies on transplantation of fetal dopaminergic neurons.
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There is one clear and notable difference between effects of adrenal medulla transplants in human subjects and effects of fetal transplants: essentially all of the improvements seen after adrenal medulla transplants had disappeared by 2 years after transplantation. Improvements reached maximum by about 6 months, and beginning 6 to 9 months after transplantation showed a gradual decline. There were no reported additional improvements beyond 1 year in any of the studies [48, 52, 58, 59]. In contrast, for a minority of patients who received transplants of fetal substantia nigra, there were improvements that developed gradually, over a time course more than 1 year. In addition, improvements observed after a few months did not necessarily fade rapidly. Among others (e.g., possibly [82]), these cases included one reported by C. Freed in which longterm improvement was especially apparent [44, 45], transplants in two subjects with parksonsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [171], and one case (case 4) studied by Lindvall et al. [85]. In the overview of the first six transplant cases by Wenning et al. [167], case 4 described earlier by Lindvall et al. [85] was the one that showed by far the greatest improvement, whereas improvement in subsequent cases was smaller and in some cases very minor. A recent review by Winkler et al. [172] discussed the possibility that optimization of transplantation and patient selection parameters could lead to more consistent and uniform clinical improvements. Factors related to the degree of improvement seen have been suggested to be immunosuppression, number of cells transplanted and transplantation technique (dissociated cells versus tissue fragments and tissue storage prior to transplantation), inclusion of serotonergic neurons in the transplants, and patient variables including age and L-dihydroxyphenylalanine (l-dopa) responsiveness [46, 113, 172]. These factors, or other unspecified factors, may account for some of the variation in the levels of improvement that have been reported. However, the factors responsible for the very long-term improvements have not been identified, and it seems likely that additional factors (as yet uncertain) account for the few individual cases that showed marked long-term improvement. Two additional recently reported cases have also shown long-term improvement, good graft survival, and no motor side effects [100]. This certainly highlights the importance of controlled trials for interpreting the effects of fetal transplantation, despite the possibility that conditions employed
might not have been ideal. Overall, there were some improvements in both of the controlled trials [46, 113]. However, the improvements were not remarkable, and the major outcome measures did not reach statistical significance. In addition, development of dyskinesias in some of the patients was seen in both studies [46, 62, 113]. These dyskinesias did not disappear when l-dopa treatment was discontinued. The reason for the frequent occurrence of dyskinesias is unknown, although this has been suggested to be due to “patchy” innervation of the host brain by the transplants [88]. Although the clinical studies are somewhat discouraging overall, there remain sufficient indications that long-term improvements can occur and that it is likely, or at least possible, that transplantation can work in principle. It is likely that we need to both understand the factors that differentiate very efficacious transplants from those that are only marginally effective, in addition to finding ways to fundamentally improve effects of transplants.
Why We Need Cultured Cells Although a great emphasis has been placed on the functional efficacy of fetal dopaminergic neuronal transplants in animals, the degree of success is in fact very limited. Likewise, for human clinical trials there is reason for cautious optimism, but again the level of success is very limited and the reasons that marked long-term improvement has occurred in a few cases are unknown. A potential alternative to employing tissue dissected from fetal material is the generation of cells in vitro, from cells such as hESC (Figure 28.1). There are many reasons to believe that in the longterm a practical transplantation therapy for PD will require the use of cells that can be propagated in vitro. Obtaining fetal neuronal tissue is a complex and difficult procedure and requires the use of aborted fetal material, which is not likely to ever be readily available. Fetal tissue is inherently inconsistent, which complicates the comparison of clinical trials and interpretation of results. Cell lines that can be propagated in vitro, whatever source is eventually used, have many potential advantages for clinical transplantation:
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Cultured cells can, conceivably, be produced as pure dopamine neurons, or as mixtures of dopamine neurons with other types of neurons and glia in optimal proportions for transplantation. If efficacy, for example, is entirely related to numbers of dopamine neurons transplanted, a preparation of pure dopamine neurons would provide many more useful neurons per injection, as compared to the mixed population of cells present in fetal tissue. Cultured cells can be pretreated with drugs or growth factors in vitro, or combined with a supportive matrix for whatever period of time is necessary to improve posttransplant cell survival. Cultured cells can, conceivably, be genetically modified to improve transplant performance. Multiple genetic alterations, including targeted gene deletions, could in principle be combined in a single cell line.
progeny. Progenitor cells do not, in theory, have the capacity for self-renewal, although they may divide many times prior to differentiation. In recent years, however, the terms progenitor and stem cell have been used somewhat loosely in reference to neuronal precursors, and in many cases there is not a clear distinction. Embryonic stem cells are, by definition, immortal in that they can give rise to all cells of the body including germ cells. Cellular immortality is the ability to divide indefinitely, without undergoing the phenomenon of cellular senescence or crisis [20, 142, 173]. Neural stem cells may not be immortal, although they may be capable of many cell divisions prior to undergoing cellular senescence. One of the attractions or advantages of embryonic stem cells as a source of material, therefore, is that they can, in theory, be propagated indefinitely in culture without deterioration or loss of differentiation capacity.
Development of Dopaminergic Neurons
Stem Cells and Cells at Other Stages of Development Embryonic stem cells are usually obtained from the inner cell mass of the embryo, subsequent to the differentiation of the trophoblast, which occurs after three or four cell divisions. Derivation of embryonic stem cells from earlier pre-blastocyst stages has also recently been described [157]. These cells have the potential to differentiate into all cells of the body, and are thus termed pluripotent. By comparison, prior to differentiation of inner cell mass during the first two or three cell divisions cells of the developing embryo can differentiate into trophoectoderm (which gives rise to the placenta) as well as all cells of the body, and are thus termed totipotent. Following subsequent divisions, cells become more differentiated and produce cell types, such as neural stem cells, that are in theory limited in their differentiation — being able to produce only neural cells. Such cells are termed multipotent, being able to give rise to a limited number of differentiated cell types. A contrasting term, progenitor cell, is used to describe a cell type that is limited in that it is the precursor of a specific type of cell and may be capable of cell division but not self-renewal. That is, stem cells in theory can divide asymmetrically, leading to copies of themselves in addition to differentiated
Development of mesencephalic dopaminergic neurons from neural precursors follows a number of stages that are regulated by signaling molecules and specific transcription factors [175]. The process of mesencephalic dopaminergic neuron differentiation is summarized and discussed in the following sections (Figure 28.2).
DOPAMINERGIC NEURON INDUCTION The identity of early proliferating dopaminergic progenitors is specified by two secreted signaling proteins: sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8). These two growth factors can induce the formation of dopaminergic neurons in the ventral midbrain. The transcription factor Otx2 is required for positioning control and regulation of Shh and FGF8 expression [163]. Early midbrain markers — Pax2, Pax5, and engrailed-1 and -2 (En-1 and En-2) [2], maintained by expression of the signaling molecule Wnt1 — are expressed at this stage. Nestin — a marker for central nervous system (CNS) progenitor cells [27] — Sox1 (defining the dividing neural precursors [121]), and neural cell adhesion molecule (NCAM), are also expressed [24, 144]. NCAM is widely expressed during all stages
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DA neuron induction
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Figure 28.2. Markers expressed during neuronal induction, differentiation, specification, and development of the dopaminergic neuronal phenotype.
of CNS development, whereas expression of the polysialylated form (PSA-NCAM) is generally restricted to immature or migrating neural precursor cells.
transcription factor, Ptx3 [111, 146], which is crucial for survival and development of mesencephalic dopaminergic neurons [111].
PHENOTYPE DEVELOPMENT
DIFFERENTIATION Dopaminergic progenitor cells stop proliferating and enter the differentiation process, expressing the neuronspecific marker class III beta-tubulin (TuJ1). Activation of a Lim homeodomain-containing gene, Lmx1b, takes place during this phase [146]. The cellular morphology is now bipolar with neurite formation [175, 179].
SPECIFICATION The transcription factor genes Lmx1b and Nurr1 are involved in specification of mesencephalic dopaminergic neurons [119, 182]. Nurr1 is a member of the nuclear receptor superfamily of transcription factors. Nurr1 expression in the ventral midbrain is required for dopaminergic neuron development [12], and precedes transcriptional activation of the tyrosine hydroxylase gene (TH), which is the rate-limiting enzyme for dopamine synthesis [148]. Lmx1b contributes to the specification of mesencephalic dopaminergic neuronal progenitors and regulates the expression of another
The final steps involved in maturation of the dopaminergic phenotype include expression of genes directly involved in synthesis of dopamine, and in development of complete machinery for process extension, synaptic, and neuronal function. Markers associated with the mature dopaminergic neural phenotype include TH, the enzyme aromatic amino acid decarboxylase, the vesicular monoamine transporter type 2, and the dopamine transporter [119, 179]. Morphologically THexpressing neurons exhibit multipolar morphology with fine axons and dendrites, and additional markers of synaptic function such as synaptic vesicle proteins and synapsin can be detected [119, 179].
Dopamine Neurons from Neural Stem Cells and Other Sources There is a fairly large literature on the use of various types of cells, including cells obtained from non-neural sources for transplantation [84, 140]. For example, Ling and co-workers [86] were able to produce dopaminergic cells from rats by treating progenitor cells obtained
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from the VM with a complex series of growth factors and cytokines — including interleukin (IL) 1, IL11, glial-cell-line-derived neurotrophic factor, and tissue extracts — resulting in differentiation of a high percentage of dopaminergic neurons [86]. The percentage of dopaminergic neurons produced in this manner from human tissue using similar methods was fairly small [86, 152]. Another method of producing dopaminergic neurons involves expression of the transcription factor Nurr1 in neural stem cells combined with astrocyte co-culture [164]. Neuronal progenitor cells within germinal ventricular zones of the developing VM are able to proliferate, self-renew, and generate dopaminergic neurons when induced by mesencephalic specific signals, especially Shh (Figure 28.3, J. Sanchez, unpublished data [80, 154]). These cells also express mesencephalon-specific transcription factors, including Nurr1 and Ptx3 [134, 147]. During neurogenesis, midbrain dopaminergic neurons are generated from dividing neuronal progenitors in the ventricular zones of the developing VM. Once these ventricular neuronal progenitors withdraw from the cell cycle, they start to differentiate to dopaminergic neurons and migrate to their specific destinations in the ventral tegmental area and substantia nigra [75]. Most strategies using these self-renewing multipotential neuronal
progenitor cells involve expanding them from a small quantity of fetal VM tissue. Previous studies have shown that mesencephalic progenitor cells can differentiate to dopaminergic neurons in vitro when exposed to various cytokines, hormones, or conditioned media [26, 81, 129, 133]. Sawamoto et al. [136] transplanted mouse nestinGFP-sorted mesencephalic progenitors to a rat PD model, generating sufficient dopaminergic neurons to decrease amphetamine-induced rotational behavior. In other studies, however, transplantation of human CNS progenitor cells using the same rat model of PD failed to restore behavioral defects, apparently because of limited dopaminergic neuron production [153, 154]. This discrepancy may be because the sorted mouse progenitors were transplanted to animals without expansion, whereas in contrast the human CNS progenitors were expanded in vitro before they were transplanted. Therefore, long-term culture of neuronal progenitors may result in the loss of neuronal differentiation potential. Eagleson et al. [40] have demonstrated that in the absence of the native environmental signals in vivo cultured progenitors retain the memory of a phenotypespecific inductive signal received in vivo after passing through about two cell cycles. The laminar identity
Undifferentiated
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Figure 28.3. Human neural progenitor cells were isolated from 20-week whole brain and cultured in proliferative media in ultralow attachment flasks. Left panel: undifferentiated cells are positive for the neural precursor protein, nestin (red). Nuclei (blue) are stained with DAPI. Right panel: differentiation was induced by removal of mitogens and addition of serum for 3 weeks, resulting in efficient glial and neuronal differentiation, as well as phenotypic specification. This image shows indirect immunofluorescence staining for neuron-specific b-III-tubulin (red) and tyrosine hydroxylase (green). See color insert.
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of newly generated post-mitotic neurons appears to be modulated by cell cycle kinetics (i.e., the time of a cell’s terminal mitotic division) of progenitor cells in the germinal zones before they migrate to their final destinations [29, 124]. Expansion of neuronal progenitors by mitogenic stimulation may result in loss of the potential to differentiate to dopaminergic neurons, because environmental factors encountered during terminal mitotic division play a critical role in determining neuronal fate [40, 97]. Sinclair et al. [145] have shown that transplanted post-mitotic neurons have more potential to yield surviving dopamine cells in nigral grafts as compared to cycling neuronal progenitor cells. However, Sawamoto et al. [136] found that dividing mesencephalic progenitors without in vitro expansion are able to produce dopaminergic neurons in the adult striatum as well. For development of clinical therapeutic applications, it will probably be essential to better understand the molecular mechanisms controlling the generation and maintenance of mesencephalic dopaminergic progenitors as well as their migration properties.
Mouse and Subhuman Primate Embryonic Stem Cells Production of dopaminergic neurons from embryonic stem cells was first reported using mouse cells. The neural-inducing activity of certain stromal cell lines (PA6, OP9, S17, and MS5) is known as stromalcell-derived inducing activity (SDIA). SDIA accumulates on the surface of stromal cell lines and induces mid/hindbrain neuronal differentiation of co-cultured embryonic stem cells from mouse and subhuman primates. The co-culture method was first described by Kawasaki et al. [76] for mouse embryonic stem cells (mESCs). In the absence of exogenous BMP4, mESCs efficiently differentiate into neural precursors and neurons. More than 90% of cells become NCAM positive when cultured on PA6 cells for 1 week. After 2 weeks of co-culture with PA6 cells under such conditions, mESCs differentiate into midbrain dopaminergic neurons at a high frequency as much as 30% of neurons derived from mESC can become dopaminergic and produce significant amounts of dopamine [76]. Kawasaki et al. [77] reported that a similar method could be employed to induce dopaminergic differentiation of primate embryonic stem cells by co-culture with the stromal cell line PA6. An alternative method
for obtaining dopaminergic neural differentiation from mESCs, described by Lee et al. [83] and Kim et al. [78], involves a five-stage method for differentiation from embryonic stem cells to dopaminergic and serotonergic neurons. Stage 1 consists of expansion of undifferentiated embryonic stem cells in embryonic stem cell medium (LIF present). Stage 2 consists of the generation of embryoid bodies in suspension cultures for 4 days. Stage 3 consists of the use of defined medium to select for CNS stem cells. Stage 4 consists of selection of nestin-positive cells and the proliferation of CNS stem cells in the presence of Shh, FGF8, ascorbic acid, and basic fibroblast growth factor. Stage 5 consists of differentiation of the expanded neuronal precursor cells by withdrawing the previously cited factors from the medium. mESC have also been transplanted into rats, without prior modification or differentiation, using a PD model [11]. A significant number of cells were found to differentiate into dopaminergic phenotypes, with more dopaminergic cells found in animals transplanted with a lower density of cells. Teratoma formation was also observed. In addition, transplantation studies of predifferentiated mESCs have reported functional improvements in PD models [78, 109].
Human Embryonic Stem Cells (hESC) Once hESCs became available, speculation that these cells might be used for PD began to appear well before conversion of hESCs to dopaminergic neurons was reported. There have now been several reports that hESCs can be differentiated into dopaminergic neurons, mainly using techniques involving co-culture with other cell types or cell extracts [8, 18, 115, 179]. Currently, several methods have been described to induce dopaminergic neuronal differentiation of hESCs [18, 116, 119, 138, 175, 179, 180], especially involving co-culture with the mouse stromal cell lines PA6 and MS5 [18, 116, 119, 179]. This co-culture method is particularly effective in inducing dopaminergic neuronal differentiation (Figure 28.4). Because attempts to identify the factors responsible for the differentiation-inducing effect of PA6 cells have not been entirely successful [174, 179], this remains the most viable method of obtaining dopaminergic neurons from hESC. Other approaches for generation of TH-positive neurons have also been reported, including the use of an
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hESC on amphetamine-induced rotation in animals. Ben Hur et al. [9] found that differentiated neuronal precursors, derived from hESC, can produce behavioral improvement in a rat model of PD — and observed modest numbers of surviving TH-positive neurons after transplantation. Whether the limitations of fetal tissue transplantation that have been observed in animals and in human subjects can be overcome by the use of hESC is, however, unknown (Figure 28.5).
Genetic and Epigenetic Stability Figure 28.4. Efficient generation of TH-positive neurons from hESC by co-culture with the mouse stromal cell line PA6. Immunostaining of BG01 cells with a TH antibody after 3 weeks of differentiation on PA6 cells. See color insert.
aggregated serum-free suspension culture system [138] and a multistep protocol via embryoid body formation followed by selection of neural precursors and exposure to growth factors including Shh and FGF8 [175]. Initial attempts to transplant dopaminergic neurons derived from hESC yielded small numbers of surviving dopaminergic cells [179], or none at all [115]. One recent study has reported effects of differentiated
A critical issue for the use of hESC for any clinical purpose is the necessity of growing cells for a long period of time so that it can be ensured that the effects of the cells are predictable over an extended period of time. This requires that cells be stable in terms of karyotype, epigenetic regulation, mitochondrial genome and in terms of their ability to maintain appropriate telomere biology. It is generally recognized that cell culture in itself exerts a selection pressure in favor of fastergrowing cells. Current studies suggest that although hESC are relatively stable, these cells must nevertheless be monitored routinely to ensure that stability is maintained. Results from several groups have shown that cells can be maintained for over one hundred passages while retaining stability, which provides some
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Figure 28.5. Limitations of fetal dopaminergic neuronal transplants may or may not be overcome by the use of hESC.
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encouragement that long-term expansion of hESC is possible. There have been several reports, however, that karyotype abnormalities can develop in hESC cultured for long periods [14, 19, 180]. Karyotype analysis, although relatively simple to perform, does not detect all mutations that can occur. Smaller abnormalities such as single nucleotide substitutions would not be detected by karyotype analysis. Recently, our group has found that hESC maintained for a number of passages developed genomic alterations commonly observed in human cancers, such as attenuations in gene copy number and mitochondrial gene sequences, including promoter methylation [90]. Therefore, it will probably be advantageous to develop additional tests for long-term stability of hESC. Because it may not be practical to routinely perform sufficiently fine-grained tests to detect the development of mutations over long-term culture, other tests may need to be developed. For example, it may be possible to develop a test that can accurately measure cell cycle length, so that shortening of the cell cycle can be routinely measured in hESC cultures. Any shortening of the cell cycle or development of subpopulations of cells that have a growth advantage would be an indication of instability and possible unsuitability for transplantation in human subjects. Additional modifications (e.g., suicide genes such as thymidine kinase) could be introduced into cells, so that if tumors developed they could be killed with ganciclovir, for example [137]. Such measures would, of course, be only an insurance against unexpected catastrophe, and could not be the primary means of controlling against growth of transplanted cells.
An Alternative to hESCs: NTera2 Due to the demanding cell culture procedures involved in maintaining hESC lines, “model” cell systems may be valuable for examining issues such as transplantation, self-renewal, and mechanisms of differentiation. Several groups have proposed using teratocarcinoma embryonal carcinoma cells (ECs) as an alternative to hESCs based on earlier observations that germ cells are multipotent, share similar properties with hESC [3, 5, 6, 33, 64, 105], and undergo processes of self-renewal and differentiation when exposed to appropriate stimuli [3, 4, 92, 123, 158, 178]. NTera2, a human EC cell line, is similar to hESC in the expression of pluripotency markers that include
OCT4, Nanog, UTF1, SSEA4, Tra-1-81, and Wnt and Notch signaling pathways [34, 132, 139, 159, 165]. Unlike mESC and hESC, NTera2 cells can be grown indefinitely without feeders, recover rapidly from freezing, can easily be produced in large quantities while sustaining a consistent undifferentiated state, and maintain a stable karyotype over extensive passaging [39, 161]. NTera2 cells are capable of differentiation into neural cell types in vivo and in vitro [39, 42, 131, 161]. Earlier results using retinoic acid-induced differentiation paradigms have shown that a subpopulation of NTera2-derived neurons are dopaminergic [67, 68, 114, 135, 184–186]. Recently, NTera2 cells have been found to differentiate into dopaminergic neurons via PA6 coculture, with efficiency and time course similar to that of hESCs (Figure 28.6 [140]). To date, dopaminergic differentiation and transplantation paradigms involving hESC have not been entirely successful, in part due to the presence of undesired nonCNS cellular phenotypes in PA6-differentiated hESC cultures. Sorting or enrichment of cells prior to transplantation may therefore be essential to remove nonneuronal cells as well as undifferentiated cells that could potentially form teratomas. Flow cytometry and fluorescence-activated cell sorting (FACS) are utilized across many disciplines in biomedical and clinical research for selecting cells based on expression of cell surface markers, and have recently been used to isolate glial progenitor, neuronal progenitor, and neural stem cells [94]. NTera2 and hESC undergoing dopaminergic differentiation by PA6 cell co-culture were enriched by FACS at a partially differentiated PSA-NCAMpositive stage and retained their ability to differentiate into dopaminergic neurons. PSA-NCAM-sorted cells produced a higher percentage of dopaminergic neurons as compared to nonsorted cells (Figure 28.7, C. Schwartz, unpublished data, [139]). Although currently it is unclear whether pure dopaminergic progenitors, neural stem cells, or a mixture of glial and neuronal restricted precursors would be the best material for transplantation, FACS can be used to obtain specific cell populations until it is possible to generate pure phenotypes directly from hESC. For certain applications, it may therefore be advantageous to employ alternatives to hESC that are more easily maintained in vitro. Possible alternative cell lines include NTera2, mESC, and the variant hESC line BG01V [180]. These alternative systems may be especially useful for studying issues such as differentiation protocols, cell sorting and enrichment
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Figure 28.6. A schematic of the NTera2-PA6 co-culture system for generating dopaminergic neurons. Undifferentiated NTera2 cells and PA6 cells were grown separately. Once PA6 cells were confluent, undifferentiated NTera2 cells were seeded at a density of 2,000 cells/cm2 on top of the PA6 cells. Neuronal markers began to appear shortly after PA6 induction. By day 5, NCAM (neural cell adhesion molecule)-positive cells appear; by day 7, TuJ1 (Beta-III-tubulin)-positive cells appear; and by day 8 cells expressing TH are present (tyrosine hydroxylase). Cells were cultured for a total of 3 weeks, at which time 85% of colonies expressed TH and approximately 8% of the total cells were TH positive. Alternatively, cells were grown for 12 days, FACS sorted for PSA-NCAM (polysialic acid-neural cell adhesion molecule), and then grown for an additional 4 weeks in medium conditioned by PA6 cells. Cells sorted for PSA-NCAM and grown further in vitro displayed electrical excitability, responded to neurotransmitters, and expressed characteristic dopaminergic markers that include TH, AADC, DAT, Nurr1, Ptx3, and Lmbx1.
methods, transplantation techniques, and cell types to be transplanted for behavioral recovery in PD models.
Designer Stem Cells
Figure 28.7. PSA-NCAM sorted cells differentiated in vitro for 4 weeks with PA6-conditioned medium differentiated into TH (red) positive cells. Calibration bar = 100 mM.
Notwithstanding the partial immune privileged nature of the brain with respect to allotransplantation, it has generally been agreed that at least some level of immunosuppression is necessary for fetal transplantation success. Immunosuppression in itself involves risk, and matching donor tissue to the recipient can minimize the requirement for immunosuppression. Such strategies have been successful as evidenced by the large number of organ and bone marrow transplants performed each year. Major organ transplantation probably requires a greater level of immunosuppression than transplantation of neural cells into the brain, often requiring lifetime immunosuppressive therapy. One of the potential reasons for attempting to employ adult-derived stem cells obtained from tissues such as bone marrow or cord blood is that using SC from adults would allow for personalized stem cells that could
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bypass hESC immune issues altogether. In contrast, if a sufficient number of hESC lines were available one could match donor hESC with the host, as is done routinely for blood transfusion or organ banks. Another possibility is to generate personalized embryonic stem cell lines by somatic cell nuclear transfer (SCNT) using techniques that are currently available although not applicable for federal funding in the USA and that are banned in some countries. Such cells would, in theory, be matched to the DNA profile of the donor, although because the blastocysts used would be derived from different individuals mitochondria would not be matched and the cells would contain genetic information from more than one individual. It would thus be important to determine if cells with mismatched mitochondria behave differently from other cells in transplant procedures. Issues such as aging and its reversal can be addressed by transplanting differently aged nuclei into blastocysts and examining self-renewal, telomere length, and pluripotency. The process of epigenetic remodeling can be examined by transplanting germ cells and somatic nuclei and examining factors that regulate reactivation and suppression of genes in different tissues and organs. This also allows the process of homologous recombination to be performed in hESC using cell lines that are patient specific, thereby examining pathways that are disease specific and allowing methods to treat specific genetic defects to be evaluated. Having patient-specific cell lines would allow for development of individualized drug therapies, and for testing environmental and allelic variability in drug response.
Beyond Stem Cells: Cell Lines and Other Possibilities Are stem cells, in fact, the ultimate source of cells, or can something better ultimately be found? Certainly, embryonic stem cells have the capacity to generate any cell type found within the body, including dopaminergic neurons. However, there is a possibility that a cell type farther along the differentiation pathway toward becoming dopaminergic neurons — for example, neural stem cells or a dopaminergic progenitor cell line — would require less manipulation in culture prior to use and would thus be preferable. The complexities of culturing hESCs present some difficulties, including potential mutation pressure, which could eventually provide reasons to employ other cell types. The problems that have
become apparent with fetal dopaminergic neurons may not be overcome simply through the use of hESCs. Therefore, it may be that an alternative cell type that can be propagated in culture, perhaps derived from hESC, or perhaps obtained by other means, will be preferable. Cell lines consisting of partially differentiated cells that are induced to proliferate in culture by genetic modification or some other means present difficulties related to the possibly increased risk of tumor formation. On the other hand, the use of genetic alteration to enhance cell growth may remove some of the selection pressure for growth-enhancing mutations that could otherwise be present. In any case, use of cell lines for transplantation in human subjects may be many years away. Nevertheless the use of cell lines made from cells that have differentiated beyond the stem cell phase may have certain advantages with respect to consistency and manipulation of the cells in culture. In the long term, the major limitations of transplantation may not be in the realm of simply obtaining dopaminergic neurons. Rather, it may be necessary to create an entirely new type of cell that does not now exist in nature. Perhaps we will find it essential to alter the manner in which engineered dopaminergic neurons interact with other cells, so that they express cell surface proteins that enhance their interactions and process extension within the mature brain — with the result that they are able to better form synapses with host neurons. Perhaps these cells will contain modifications that permit them to avoid apoptosis when synaptic connections with other appropriate target cells do not develop. Possibly, these engineered neurons will have genetic modifications so that their ability to produce dopamine, and perhaps other substances such as cholecystokinin, can be regulated by administration of specially designed drugs or hormones. The development of technologies to produce cell lines that are appropriate for transplantation may in the long term make such manipulations a possibility. Immortalized cell lines have been very valuable as experimental models for both in vitro and in vivo studies; for example, as models for ovarian cancer [56], prostate cancer [117], and studies on neuroprotection [23]. There are, however, significant obstacles to the therapeutic use of immortalized cell lines, which invariably harbor mutations or express molecules designed to disrupt normal cell cycle control. Although some cell lines (such as the carcinoma line NT2) have been used for clinical trials, concerns about tumorigenicity pose major constraints [104]. Nevertheless, it may be
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possible to develop an adequate control of the possibility of tumor formation through a combination of judicious choice of oncogene, promoter, and immortalizing vector, and by incorporating additional control measures [65, 168, 181] into cells designed for therapeutic use. Several immortalizing methodologies exist, each of which involves a different set of issues concerning biosafety and clinical efficacy. One “classical” immortalizing molecule is immortalization by passaging [160], which generally results in loss of p53 per se — or loss of p53 function by mutation [71, 187] — and in genetic instability [60]. This technique is not effective for human cells, and loss of p53 function would in any case be undesirable for cells to be used therapeutically. Another “classical” technique involves expression of full-length T antigen, which can be accomplished by transfection [91] or retroviral and adeno-associated virus vectors [22, 57, 122, 170]. SV40 large T antigen is a potent transforming molecule, and is effective for both human cells and neural cells [17, 91, 169]. Its use for the establishment of cell lines meant for therapeutic transplantation is, however, not ideal due to its potential tumorigenicity. Other isoforms of large T antigen have been assessed for their immortalizing capabilities and have met with some success. A temperature-sensitive isoform, nonpermissive at body temperature, has been used to conditionally immortalize several tissue types, but the control of tumor formation is incomplete [28, 95, 155]. Our laboratory has developed a minimal isoform of large T antigen, T155, encoding the first 155 amino acids of the full-length molecule. T155 lacks the ability to bind and sequester p53 tumor-suppressor protein, which appears to allow for relatively normal cell function and differentiation [162]. To date, T155 expression in immortal cell lines has not generated tumors posttransplantation [32, 54]. Another possible candidate for the generation of transplantable cell lines, including neural progenitor cells, is v-myc [79, 103]. Expression of v-myc has been used to generate cell lines from several tissue origins, and also has not produced tumors posttransplantation [43, 149]. The mechanisms whereby either v-myc or T155 exert their conditionally immortalizing nontransforming effect have, however, yet to be elucidated. Immortalization of human and rodent cells differs to some degree in that telomerase expression is generally required for immortalization of human but not rodent cells [142]. This difference is thought to be due to the
longer telomeres of rodent cells and to the phenomenon of telomeric shortening, ultimately leading to senescence of human cells after short-term culture. Forced expression of telomerase activity in human cells has shown limited promise for compensating for this phenomenon and allowing longer-term propagation [13, 176]. Interestingly, however, only a few telomeraseimmortalized cell lines are commercially available, as the use of increased telomerase activity alone has not consistently been sufficient to generate cell lines from human cell cultures [31, 74, 110]. Telomerase activity is nonetheless an important candidate for human cell immortalization, as it does not appear to potentiate tumor formation in vivo [69]. Perhaps the combination of telomerase activity and an attenuated oncogene such as v-myc or T155 may be a viable approach for generating cell lines from hESC or neural progenitor material. Using these activities to “capture” and immortalize a developing dopaminergic neuron, for example, could be an important step toward the generation of unlimited homogeneous phenotype-specific material for neurotransplantation and cellular therapy.
Engineering Stem Cells This brings us to the topic of how to improve cells for future use. What hESC can provide, at least potentially, is a virtually unlimited source of material that can be further engineered for later therapeutic purposes. For example, as discussed previously it is likely that the limited ability of fetal cells to survive, to extend processes into the host brain, to form synapses with host neurons, and to produce behavioral amelioration in animal models of PD is related. In other words, if the integration of transplanted dopamine neurons into the host brain can be improved, cell survival and behavioral efficacy may also be concomitantly improved. Although we do not currently know how to accomplish this, we might at least think of modifying cells for transplantation so that they express a molecule that enhances the interaction of dopaminergic axons, extended from transplanted cells, with the host brain. A molecule along the lines of the adhesion molecule L1, or perhaps an altered or composite form of L1, would be a reasonable first guess [1, 30, 66, 93, 112, 125–128, 143]. L1 appears to play an important role in the growth and development of dopaminergic axons, and can enhance survival of dopaminergic neurons in vitro [30, 66, 93, 112, 125–127, 143, 177]. L1 expression
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also appears to be largely responsible for the favorable properties of Schwann cells in supporting axonal growth [1]. Yue et al. [177] have suggested that ephrin-B2, a membrane-anchored ligand of the Eph family of receptor tyrosine kinases, and its receptor EphB1 are candidate molecules for providing axonal guidance of dopaminergic neurons in developing neural pathways. EphB1 was found to be strongly expressed in dopaminergic neurons of the substantia nigra (which project to the caudate putamen) but not in those of the ventral tegmental area, whereas the EphB1-negative dopaminergic neurons of the ventral tegmental area project to the nucleus accumbens and olfactory tubercle [177]. Furthermore, ephrin-B2 was expressed at high levels in the nucleus accumbens and olfactory tubercle but only at low levels in the caudate putamen [177]. Yue et al. [177] propose that the negative interaction between ephrin-B2 and EphB1 may regulate the development of the midbrain dopaminergic pathways (the mesolimbic and mesocortical pathways comprised of dopaminergic neurons originating in the ventral tegmental area and projecting to the nucleus accumbens and olfactory tubercle, and to the cortex) and the development of the nigrostriatal pathway, comprised of dopaminergic neurons originating in the substantia nigra and projecting to the caudate putamen. A third possible manipulation to enhance the integration of transplanted dopaminergic neurons into the adult host brain would be to improve their ability to penetrate the brain extracellular matrix. Chondrotin sulfate proteoglycans play a particularly important role in the inhibition of axonal growth and in limiting axonal regeneration [21]. Enzymes that can digest brain extracellular matrix and allow increased regeneration of axons in the CNS have been identified, including chondroitinase and xylosyltransferase-1 [61, 102]. Incorporation of a strategy to allow for improved navigation of the mature host brain by growing dopaminergic axons might, therefore, be an achievable goal. Perhaps, it would be possible to construct an altered cell type that can extend neurites through mature brain tissue, containing a growth cone molecule interacting more strongly than that of normal immature dopamine neurons with the mature brain terrain and with potential synaptic sites. It might be possible to engineer cell surface proteins that have properties different from those of native proteins, to allow for navigation through a mature brain tissue matrix. Engineered
Figure 28.8. Possibilities for “improvement” of dopaminergic neurons for use in transplantation.
cells might also include genetic modifications to allow for regulated dopamine release, resistance to cell death during transplantation, resistance to environmental toxins or other causative factors in PD, production of growth factors to improve function of associated host brain neurons, and production of mechanisms to allow for cell destruction in the event of tumor formation (Figure 28.8).
Conclusions It has become apparent that a therapeutically useful transplantation therapy for PD will require more than simply the transplantation of material from human fetuses into the brains of patients. First, the logistical difficulties associated with using material from human fetuses would probably render this impractical for general use as a therapy, even under ideal circumstances. Moreover, it seems likely that simple transplantation of cells into the brain will not ultimately be sufficiently efficacious. Additional measures may eventually be needed to improve cell survival, or to augment the functional properties of transplanted cells — either through genetic alteration or by measures such as pretreatment of cells with drugs or growth factors. Such alterations and treatments are most readily performed in cell culture systems. In addition, the use of cultured cells allows for consistency, predictability, and quality control. Although in some sense the results of controlled clinical trials of fetal tissue transplantation for PD have been disappointing in terms of overall efficacy and
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the high frequency of dyskinesias, there is reason for optimism. First, it seems likely that some beneficial clinical effects have occurred, and the magnitude of these effects is sufficient that the effects would be of treatment value if they could be obtained consistently. Second, some of these improvements were found to persist over a very long timescale. Third, there are a number of readily identifiable routes through which efficacy of transplants might conceivably be improved. Finally, methods of obtaining human dopaminergic neurons in a cell culture system, perhaps derived from hESC, are now becoming available. These methods render manipulations to improve the performance of transplants quite feasible.
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29 Concepts in Cell Therapy: From Cord Blood to Sertoli Cells Paul R. Sanberg (1), Denis K. English (1), Amal Hakki (1), Don F. Cameron (2), Svitlana Garbuzova-Davis (1), Alison E. Willing (1), Cesario V. Borlongan (5), Robert Henning (3), Stephen K. Klasko (4), and Nagwa S. El-Badri (1) Center for Excellence for Aging and Brain Repair, Departments of Neurosurgery (1), Anatomy (2), and Internal Medicine (3), University of South Florida College of Medicine (4), USF Health, Tampa, Florida, Department of Neurology, Medical College of Georgia (5), Augusta, Georgia
Introduction Stem cell therapy has roots in the restoration of the hematopoietic system. Impressed by the particular and unusual radiosensitivity of the hematopoietic system, Thomas et al. pondered the genesis of circulating blood cells, and examined the effect of shielding of various tissues during radiation. The results were clear and stunning. Shielding of the femur of an adult dog completely abrogated the ability of bone marrow to cause a subsequent and irreversible decline in levels of all circulating blood cells after lethal total-body radiation [121]. The implications were obvious: within the bone marrow resided cells of a type that produced all circulating blood cells (the hematopoietic stem cell). This study was quickly followed by a number of investigations, many conceived with the premise that a singular hematopoietic stem cell existed and that isolation of this cell could result in a way of restoring hematopoiesis in radiated hosts. Indeed, studies quickly provided evidence consistent with this hypothesis and the notion of the pluripotential hematopoietic stem cell was born. These studies provided a clear mechanism underlying the results of a previous study by Donnall Thomas and colleagues [122] in which an intravenous infusion of bone marrow was administered to patients receiving radiation and chemotherapy. Thus, these farsighted individuals reasoned that incurable diseases of
Cellular Transplantation: From Laboratory to Clinic
the hematopoietic system (such as leukemia) may in fact be cured by changing the hematopoietic environment with hematopoietic stem cells from a person free of disease. However, they correctly assumed, the diseased cells present would have to first be removed, and the immune system of the recipient dampened so that the infused allogeneic cells would not be rejected. This was accomplished by radiation and delivery of the chemotherapeutic radiometric drug cyclophosphamide in doses that were fatal. However, if hematopoietic stem cells were delivered after the drugs were given, hematopoiesis and immune function were restored. In the first report of a cure of leukemia in 1967, Storb et al. used allogeneic hematopoietic stem cells to restore a functional and nondiseased hematopoietic system to a dying leukemic patient [116]. The patient was first treated with 1,200 rads of radiation and lethal doses of cyclophosphamide to remove leukemic cells. However, the patient was now fated. Absent intervention, he would die of irreversible aplastic disease. However, the transplant worked. The patient was infused with hematopoietic stem cells from the bone marrow of a relative and fully recovered. Dr. Thomas would later receive the Nobel Prize for this work, but is quick to point out that the credit belongs to a collection of individuals who were vigorous and productive at this time, including Drs. Malcolm Moore, Robert Good, Donald Metcalf, Eddie Boise, and other pioneers in this field of dreams.
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Cord Blood Transplants Subsequent studies examined other sources of hematopoietic stem cells and isolated stem cells with various degrees of success. For many years, the initial technique developed in Thomas’ lab was used as the only cure for leukemia and other irreversible diseases of the hematopoietic system. In 1989, Hal E. Broxmeyer and colleagues at Indiana University faced an unusual challenge. A young patient presented with Fanconi’s anemia, and the hematopoietic pathology of this boy was progressive. Clearly he needed a bone marrow transplant, but his brother did not provide matching cells and he had no other siblings. The child’s mother was eager to have another child for reasons unrelated to this boy’s health, and she asked when the new arrival could provide cells for a transplant of his anemic brother. The answer was not known, but it was thought to be at least two years — a time that would render the pathological progression irreversible and devastating. These facts came to the attention of Dr. Broxmeyer, who thought of other ways of isolating hematopoietic stem cells from the baby. He quickly examined the potential of stem cells of the umbilical cord and found these to be present in high number and to be extremely active in colonyforming assays. He developed a method of freezing cord blood stem cells for later infusion, and formulated a carefully designed plan to collect and freeze stem cells of the umbilicus when the new baby arrived. Careful and rigorous preliminary experiments demonstrated the rationality of the plan [13], which was implemented with great success when the baby was born [42].
Cord Blood Hematopoietic Stem Cells (HSCs) In subsequent years, the number of cord blood transplants has increased dramatically, in part as a result of the remarkable characteristics of cord blood stem cells that were not anticipated by the original team. After the initial success with curing Fanconi’s anemia [42], it became evident that transplantation of human umbilical cord blood (HUCB) elicited a lower incidence of graft rejection and graft-versus-host-disease (GVHD) than bone marrow hematopoietic stem cells [43, 98]. Similar to bone marrow, HUCB is enriched for the radioprotective hematopoietic stem cells (HSCs). However, the immunogenic properties of HUCB are different from those of adult bone marrow. The low immunogenicity
of HUCB is substantiated by observation that much less stringent MHC-matching criteria between transplant donor and recipient are necessary when HUCB cells are used compared to bone marrow. Furthermore, less immunosuppressive pretransplant conditioning is required for successful cord blood transplants. The ability of HUCB cells to induce less GVHD may be related to the low frequency of GVHDcausing mature lymphocytes and a high proportion of CDRA+ /CD45RO− CD62 L+ phenotype-naive T lymphocytes [118]. In addition, pluripotential HSCs in cord blood have a higher cloning efficiency than bonemarrow-derived stem cells and are more proliferative after stimulation with hematopoietic growth factors and cytokines [48]. Accordingly, fewer cord blood HSCs are required for cord blood versus bone marrow transplantation, and fewer GVHD effectors need be infused for successful engraftment [44]. Studies have shown that HSCs from cord blood are pluripotential on the single-cell level and can rescue lethally irradiated mice and reconstitute their bone marrow in preclinical studies [14, 15]. Clonogenic assays have shown colony-forming cells such as erythroid burst-forming unit (BFU-e), granulocyte macrophage colony-forming cells (CFU-GM), and granulocyteerythroid macrophage megakaryocytic colony-forming units (CFU-GEMM) in the cord blood [12, 13]. HSCs are frequently isolated from HUCB mononuclear cell fraction as CD34+ /CD38− /HLA-DR− cells. HSCs from HUCB transplanted into NOD/SCID mice engrafted their bone marrow. Curiously, these cells generated endothelial cells as well as T lymphocytes [22]. To date, more than 5,000 clinical trials using cord blood stem cell transplantation have been carried out worldwide. Most transplants have been effective in children, in that the stem cell content was not known to be high enough to affect engraftment of adults. Therefore, it is clearly a high priority for hematologists to explore various techniques for expanding the HSC population in the HUCB samples [43, 141]. Co-culture of cord blood HSCs with feeder cell layers such as mesenchymal cells from the bone marrow and peripheral blood supported the expansion of the HSC population. More practical protocols employed the use of cocktails of cytokines and hematopoietic growth factors such as stem cell factor (SCF) and interleukin 3 (IL-3), members of a group of glycoprotein growth factors called hematopoietic cytokines (HCs). These factors take part in the regulation of developmental processes of hematopoietic progenitor cells.
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HSCs that express the CD34 antigen and c-kit ligand proliferate in response to growth factors and produce several progeny as well as more hematopoietic precursors. CD34+ c-kit− cells are believed to be more immature hematopoietic progenitors that establish longterm culture-initiating cells (LTC-IC) and have high levels of telomerase activity [100]. A population of the CD34+ HSCs in the cord blood also expresses the progenitor marker 133. It is not fully understood, however, how the expression of this marker, which also identifies endothelial progenitor cells (EPCs), affects the hematopoietic reconstitution capacity of the cord blood transplant. Other techniques for accelerating clinical trials in adults include using two or more units of HUCB, either sequentially or combined [5]. Infusing adults with multiple mismatched units did not seem to increase the risk of immune rejection [131]. Successful engraftment, however, was consistently correlated with the percentage of CD34+ HSCs. It appears that storing cord blood cells by freezing does not compromise the percentage or the quality of the HSCs. In fact, it may relatively increase the proportion of the CD34+ cells because the process of cryopreservation typically damages the more mature terminally differentiated cells. These qualities of easy access and possibility of in vitro expansion of the CD34+ stem cell populations made HUCB the ideal choice for patient populations in which matched bone marrow donors are not available, or for patients with an urgent need for a stem cell transplant (such as patients with acute leukemia). The curative utility of HUCB for disease is believed to be related to enrichment for stem and progenitor cells. It is not clear, however, whether there are multiple progenitors present within the HUCB and what their defining features may be. In addition to the CD34+ hematopoietic stem cells (HSCs), whose presence has been well described within the HUCB, other proposed progenitors include endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and a stem cell similar to a neural stem cell that is capable of giving rise to neural progeny. Neural stem cells are self-renewing cells that proliferate and differentiate into neuronal, astrocytic, and oligodendrocytic lineages. They were first isolated from the adult mouse subventricular zone [97], but have also been isolated from human fetal brain and spinal cord [120, 124]. Similar cells that are capable of multipotential differentiation into neuronal and glial cells have been isolated from the HUCB mononuclear fraction.
Cord Blood Mesenchymal Stem Cells (MSCs) Mesenchymal stem cells refer to a population of progenitors originally described in the bone marrow that provides a supportive microenvironment for maturation and differentiation of HSCs. Progeny of bone-marrowderived MSCs include adipocytes, reticular cells, osteogenic cells, and other connective tissue cells [18, 37, 94]. This appealing multipotentiality renewed interest in MSCs as a highly desirable therapeutic tool for cell transplant because of ease of culture, expansion, and genetic manipulation. In addition to contributing to the bone marrow stromal and connective tissue cells, MSCs were shown to differentiate into cells of all three germ layers — such as hepatocytes [104, 130], myocytes [24, 138], and neuroectodermal cells [67, 112, 113]. This plasticity in an adult stem cell is especially attractive because it represents a practical alternative to embryonic stem cells for replacement of nonfunctional tissues in diseases such as myocardial infarction, advanced autoimmune, and neurodegenerative diseases. Compared to embryonic stem cells (ESCs), MSCs represent a safe, versatile, and noncontroversial cell source for cell-based therapies. In addition, MSCs are known to be immunosuppressive [26], a particularly valuable quality for transplantation between mismatched donors and recipients. Unlike the bone marrow, it has been challenging to isolate MSCs from HUCB. The observations of multiple research groups that HUCB cells can differentiate into cells of all three germ cell layers have provided anecdotal evidence that an MSC must exist in the HUCB [7, 16, 45, 52, 74, 77, 102]. Recently, however, several laboratories (including our own) have isolated MSC-like cells from the HUCB with varying degrees of success. Cord blood MSCs share phenotypic and culture criteria with marrow-derived MSCs, and they display high potential to differentiate into cells of all three germ layers — such as osteoblasts, adipocytes, hepatocytes, and neuroglial cells [38, 56, 70]. A rare CD45− population termed unrestricted somatic stem cells (USSCs) was described in HUCB. This adherent population showed long-lasting pluripotency over an extended culture period and differentiated with significant homogeneity into osteoblasts, chondroblasts, adipocytes, neural cells, and hematopoietic cells [66]. We have recently isolated a population of an adherent nonhematopoietic cord blood MSC population that is enriched with multipotent progenitors
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that differentiate with relative abundance into cells with neural and glial morphology [30]. It is noteworthy that the connective tissue of the cord blood, referred to as Wharton’s jelly, is a potentially useful source for MSCs that express c-kit and show high telomerase activity. In vitro, Wharton’s-jelly-derived MSCs could be induced to differentiate into neural and glial cells [79]. When these MSCs were transplanted into rats, they did not induce immune rejection and differentiated into cells that expressed neural markers [132]. In vivo studies have shown that cord blood MSCs engraft immunodeficient mice [35]. Mesenchymal stem cells from bone marrow have also demonstrated usefulness as co-graft facilitators. Cells obtained from one HUCB are usually insufficient to reconstitute adult patients. It has been shown that pooled cells from multiple HUCB units increase the absolute number of cells engrafting adult patients. This approach, however, has shown that engraftment of cells from one donor usually predominates over other donors. We and others have previously shown that cells of stromal origin promote engraftment of allogeneic donor HSCs [31, 55]. Recently, studies by Kim et al. [63] have shown that marrow stromal cells have a similar engraftment-promoting effect on donor cord blood cells. More balanced representation of both donors of HUCB cells was detected in NOD/SCID mice after co-transplantation of third-party marrow stromal cells. Although the role of mesenchymal cells as potent modulators of the bone marrow microenvironment is well established, this is the first report that is supportive of the role for mesenchymal cells facilitating engraftment of HSCs from HUCB origin. Further, Noort et al. [2002] have since shown that co-transplantation of mesenchymal stem cells from HUCB has facilitated engraftment of cord blood HSCs in various mouse models [1, 86].
Cord Blood Endothelial Progenitor Cells (EPCs) Endothelial progenitor cells constitute a population of cells that contribute to angiogenesis, a process in which new capillaries are formed in response to tissue ischemia [125]. When circulating EPCs migrate to the sites of ischemia and initiate new capillary growth, this process is described as neovascularization. The vascularizationpromoting effect of stem cell transplants has often been suggested as a potent mechanism for tissue repair.
Injection of a preparation enriched in stem cells at the site of ischemia is believed to stimulate the collaboration among HSCs, EPCs, and other progenitors necessary for initiating and maintaining the process of healing. In fact, EPCs were first identified in the bone marrow as progenitors that express both the CD34 HSC marker and the progenitor marker CD133 [3, 41]. The capacity of EPCs to induce blood-vessel-like structures prompted the search for cells with similar capacity in other tissues. Angiogenic cells were later isolated from the CD14+ myeloid cells [106, 126], suggesting that CD14+ blood monocytes could act as progenitors for endothelial cells when cultured under favorable conditions [142]. Other studies suggest that MSCs may also contain progenitors for endothelial cells [88, 96]. These progenitors, however, express a different phenotype and more versatile differentiation potential than CD34+ cell-derived endothelial progenitors. For example, EPCs isolated and described by the Verfaillie group [96] could be induced to differentiate into cells of the three types of germ layers in addition to demonstrating angiogenic potential in vivo. Recent studies suggest that HUCB is a rich source for EPCs. In a study by Murohara, EPCs were obtained from the CD45− , CD34+ mononuclear fraction of HUCB. These cord-blood-derived EPCs were KDR+ , VE-cadherin+ , CD31+ , and von Willebrand factor+ , and promoted neovascularization when injected into the ischemic hind limbs of immune-deficient nude rats [82]. CD34+ (but not CD34− ) EPCs could differentiate in vitro and in vivo into endothelial and myogenic cells [93]. In another study, two subpopulations of HUCB mononuclear cells contributed to the formation of capillary-like structures in vitro and migrated to the tumor site in tumor-bearing severe combined immune deficiency mice (SCID). These cord-blood-derived cells included the CD34+ cobblestone-forming EPCs, as well as spindle-shaped cells the authors described as smooth muscle cells (positive for alpha smooth muscle actin, smooth muscle heavy chain, SM22, and calponin). It was the interaction between these two types of cells that produced capillary-like structures in type-I collagen culture [69]. These experiments attest to the current uncertainty to whether vascularization is maintained by local progenitors, or is in fact an orchestrated process that involves many resident cells, activated progenitors, and perhaps an intricate balance of cytokines and growth factors. The encouraging in vivo data after transplantation of EPCs led to studies examining their potential as a
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target for gene therapy. This potential was evident when fluorogenically labeled cord-blood-derived EPCs were injected in SCID mice with orthotopic brain glioma. EPCs migrated to the gliomas in tumor-bearing mice, whereas few EPCs were found in matched brain regions of nontumor-bearing mice [80]. Acute ischemia is the most common cause of mortality due to myocardial infarction and stroke. Chronic ischemia, on the other hand, is responsible for the high morbidity associated with diseases such as diabetes and peripheral vasculopathies. To test the therapeutic potential of EPCs in models of induced ischemia, culture-expanded CD133+ cord blood EPCs were transplanted intravenously into nude mice. Improved neovascularization was observed in the mice ischemic hind limbs [139]. Similar effects in regard to significant increase in neovascularization were observed in nude rats transplanted with EPCs isolated from the mononuclear fraction of HUCB. In vitro, these cells exhibited the markers of EPCs, AC 133, KDR, CD34, CD31, and von Willebrand factor [72]. The kinetics of migration and engraftment of cord blood cells were investigated after intravenous administration of HUCB-derived HSCs in mice. In these experiments, sublethal irradiation to induce injury was used to stimulate migration and differentiation of donor HSCs to the injured tissues. Human hematopoietic cells and endothelial cells were detected in the transplanted NOD/SCID mice. Donor-derived endothelial markers (CD31+ , CD45− , and VE-cadherin+ cells) were detected regularly in the bone marrow but not in other tissues of the transplanted mice [28]. In view of these data, it remains inconclusive to suggest the extent of contribution of transplanted donor stem cells to the observed revascularization of the ischemic tissues. A recent study by the Naruse group, however, demonstrates that cord blood EPCs contributed directly to the therapeutic neovascularization of diabetic neuropathy [85]. Culture-expanded cord blood EPCs injected into the skeletal muscle of the hind limb of streptozotocininduced diabetic nude rats ameliorated the impaired sciatic motor nerve conduction velocity, stimulated revascularization, and increased blood flow in the sciatic nerve. Because revascularization in diabetic patients is a cause for severe complications such as diabetic retinopathy, the approach of local administration of EPCs to stimulate revascularization only in the tissues affected by ischemia is clever. However, long-term follow-up of the injected rats is required to exclude the contribution of donor EPCs to further revascularize at
undesired sites before the safety of this form of therapy is confirmed. Clearly, a myriad of factors contribute to tissue repair and revascularization initiated by stem cell therapy. Hematologists have led the way in application of stem cell therapy with high clinical efficacy using HSCs. No doubt these advances were facilitated by the availability of the clonal analyses and colony assays used to evaluate the phenotype and function of HSCs. Similar assays are being developed for other progenitors, including MSCs and EPCs. A clonogenic population of cord-blood-derived EPCs was defined based on high proliferative-potential endothelial colony-forming cells (HPP-ECFCs). These cells achieved at least a hundred population doublings and maintained high levels of telomerase activity [54]. This high proliferation and relatively easy in vitro manipulation enabled scientists to bioengineer tissues for autografting. In this regard, Schmidt et al. [107] showed that EPCs isolated from fresh HUCB could be expanded in vitro on 3D biodegradable vascular scaffolds with relative ease and abundance. Autografts could thus be engineered to replace or patch a defective organ or tissue (for example, in congenital heart defects). Similarly good patches could be engineered by addition of HUCBderived myofibroblasts (desmin+ and alpha SMA+ ) to the EPCs [108].
Cord Blood Neural Progenitor Cells Early studies by Sanchez Ramos et al. demonstrated that culture of mononuclear fraction of HUCB in a proliferating medium supplemented with all-trans-retinoic acid (RA) and nerve growth factor (NGF) promoted the expression of neural cell markers. Neural markers Musashi-1 and TuJ-1 together with the astrocytic marker glial fibrillary acidic protein (GFAP) were expressed, as was the mRNA for the neuronal markers nestin and necdin [102]. These results suggested that a stem cell existed in cord blood that could generate neural cells. Moreover, HUCB cultured in beta-mercaptoethanol differentiated into cells with a neural phenotype [45]. When cultured for 15 days, most of the cells died, but those that survived formed floating cell clusters reminiscent of neurospheres. Replating these cell clusters produced a monolayer of cells that expressed neuronal nuclear antigen (NeuN), neurofilament, and GFAP as assessed by immunocytochemistry and mRNA for nestin, neurofilament, and microtubule-associated protein (MAP2).
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In a later study, these researchers showed that these stem-like cells co-express CD133, a marker of a primitive stem cell and nestin [46]. It has been recently demonstrated that HUCB cells could expand — in liquid culture supplemented with thrombopoietin, flt-3 ligand, and c-kit ligand (TPOFLK) — into both hematopoietic and neuroglial progenitors [77]. Buzanska and colleagues [16] have taken the field one step further by immunodepleting the HUCB original mononuclear fraction of CD34+ cells and then plating the depleted fraction in long-term culture, which depleted the CD45+ cells. The resulting cells were then cloned, producing highly proliferative cells that expressed neural markers but that did not have hematopoietic or angiogenic properties. Consistent with these observations, our group has shown by in vivo studies that the mononuclear cell fraction of HUCB transplanted in the subventricular zone of neonatal rat pups (a highly neurogenic region) could be detected in the subventricular zone overlying cortex and corpus callosum one month posttransplantation [143]. Cells positive for both human antigens and for glial and neuronal phenotype (GFAP and TUJ-1) were detected in the developing brain, indicating neural differentiation of human cord blood cells.
Cord Blood Therapy for Acute Ischemic Diseases MYOCARDIAL INFARCTION Acute myocardial infarction and congestive heart failure are leading causes of morbidity and mortality in the population of the Western hemisphere. Myocardial ischemia and infarction cause cardiac myocytes necrosis and eventual replacement of necrotic tissue by noncontractile collagen scar tissue. This process decreases the ability of the heart to eject blood to the body and predisposes affected individuals to congestive heart failure. Consequently, cardiovascular scientists have sought for the past 40 years to limit the damage from myocardial infarction and necrosis. Initial therapies have been directed toward pharmacological treatment to limit myocardial infarction size and toward myocardial revascularization with coronary artery bypass grafts. More recently, cardiovascular scientists have sought to replace injured/infarcted myocardium with stem cells that might limit myocardial injury and possibly increase contractile myocytes and the number of myocardial
blood vessels. Initial efforts with the use of exogenous cells for heart repair involved the direct injection into the myocardium of skeletal myoblasts or of bone marrow stem cells [83]. Subsequently, granulocyte colony-stimulating factor and stem cell factor have been utilized to mobilize stem cells from bone marrow to the site of myocardial injury in order to initiate myocardial repair [87]. In most of these studies, myocardial “repair” has been demonstrated by limitation of myocardial infarction size and improvement in myocardial contractile function. Closely following these early studies in research animals, clinical trials have been initiated using either skeletal myoblasts or bone marrow stem cells. Skeletal myoblasts have proven useful in limiting cardiac dilation after myocardial infarction [78], but unfortunately do not establish a functional muscle syntium with the remaining normal cardiac myocytes. Subsequently, the normal electrical depolarization-repolarization of the heart is interrupted and cardiac arrhythmias can occur. As a consequence, skeletal myoblasts are currently implanted primarily in the hearts of patients who have automatic cardiac defibrillators in place. An alternative source of progenitor cells is bone marrow aspirate that has been filtered and then directly injected into the myocardium of patients with severe heart disease. The bone marrow aspirate — which contains hematopoietic, mesenchymal, and endothelial stem cells — improves heart function and increases myocardial perfusion [4, 92]. Research groups have also begun to use specific hematopoietic, mesenchymal, or endothelial stem cells for transplantation for heart repair [23, 87, 115]. These cells have been injected directly into myocardium or administered into the coronary arteries of research animals or patients with heart disease. To date, the optimal dose and time for cell transplantation for maximal therapeutic effect has not been determined. In addition, caution has been recommended with the use of CD34+ stem cells in patients with myocardial infarction that undergo coronary angioplasty because coronary artery restenosis may occur [59]. Moreover, the use of autologous bone marrow stem cells is restricted by the fact that progenitor cells from sick patients exhibit impaired proliferation, adhesion, and incorporation [25, 95, 105]. In that more than three million births occur every year in the United States, and umbilical cord blood contains relatively high numbers of CD133+ and CD34+ progenitor cells that have myogenic and angiogenic potential, umbilical cord blood mononuclear cells are applicable
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for myocardial repair [71, 82]. HUCB mononuclear cells have been used for the first time for the treatment of acute myocardial infarction in rats in our laboratories [49]. None of the rats received immune suppression therapy at any time during the study. When the rats were examined at 1 to 4 months after the direct HUCB cell injection into the ischemic/infarcted myocardium, the left ventricular function was significantly better — as measured by left ventricular (LV) ejection fractions and changes in LV dP/dt — than the LV function in the untreated infarcted rats. The superior ventricular function in the HUCB-cell transplanted rats was due to infarction sizes that were three to seven times smaller than the infarctions in untreated rats. Moreover, myoblasts were identified in the infarcted myocardium of the transplanted rats. Similar results have been reported in a subsequent study in which HUCB CD34+ hematopoietic cells, injected into injured/infarcted rat myocardium, improved rat ventricular function in comparison with untreated controls when measured at one month after infarction [51]. In this study, the HUCB CD34+ cells persisted in the injured myocardium for at least one month. HUCB hematopoietic CD34+ progenitor cells have also been administered intravenously to NOD/SCID mice with acute myocardial infarction [73]. The HUCB CD34+ cells migrated to infarcted myocardium in 10 of 19 mice as determined by polymerase chain reaction (PCR) for human DNA, contributing to neoangiogenesis in the infarct border zone and reduction in infarct size in comparison with untreated infarcted mice. The cord blood CD34+ KDR+ cell subpopulation is thought to contain hematopoietic and endothelial (hemangioblast) progenitor cells. The therapeutic potential of these hematopoietic-endothelial progenitors in the treatment of myocardial infarctions has been examined [11]. Intramyocardial transplantation of cord blood CD34+ KDR+ cells significantly improved the function of infracted myocardium of immunocompromised NOD-SCID mice. In this study, cord blood CD34+ KDR+ progenitor cells released vascular endothelial growth factor (VEGF) — which exerted antiapoptotic and angiogenic effects — and approximately 1% of the CD34+ KDR+ cells expressed the cardiomyocyte protein troponin, which contributed to myocardial recovery. In a recent study by Ott et al. [90], culture-expanded EPCs from HUCB have been used to limit myocardial infarction size and promote myocardial repair in immunocompromised rats. In this study, the use of immunomagnetic beads permitted isolation of CD34+
cells from cord blood and from G-CSF mobilized cells in peripheral blood. The cord blood cells, when expanded in culture with endothelial growth media, showed phenotypic and mRNA similarities with human umbilical vein endothelial cells (HUVEC). Transplantation of these cells into rat ischemic/infarcted hearts resulted in improvement in left ventricular function, determined by echocardiography, which was attributed to increased vascularization that occurred at the site of myocardial injury/infarction. Cellular therapy for the treatment of acute myocardial injury/infarction is an extremely active area of basic and clinical cardiovascular research. Clinical trials are in progress despite the absence of consensus about the precise mechanisms whereby stem cells limit the size of myocardial infarction and improve myocardial function. Currently bone marrow stem cells are most commonly used in basic research and in clinical trials. Cord-blood-derived progenitor cells appear to be an important alternative to bone marrow stem cells and have been proven to be beneficial in the treatment of acute myocardial infarction in research animals. Although stem cells are most frequently directly injected into the myocardium at the time of heart surgery, vascular catheters are being developed that permit at the time of heart catheterization the direct injection of stem cells into the myocardium, as well as the intracoronary injection of stem cells. However, the most optimal time for stem cell injection, the most optimal route for administration, and the precise dose of stem cells for transplantation remain to be determined. The role of cell therapy in the treatment of cardiovascular disease in the future will not be limited to myocardial repair because the use of stem cells for tissue grafts to replace defective cardiac valves and for biologic cardiac pacemakers is an active area of research [61, 117].
STROKE Stroke is produced by localized loss of blood flow and nutrients (ischemia) in the brain with subsequent neural degeneration and damage. Stroke is one of the leading causes of death worldwide. Unfortunately, curative treatment is not available, and prevention is considered the main defense against the pernicious ischemic attack. Significant progress has been achieved in understanding the dynamics and pathophysiology of neuronal cell death after stroke. However, designing medications to
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curtail its immediate and long-lasting complications has met with limited success. Recombinant tissue plasminogen activator (rtPA) has been shown effective at treating embolic stroke, but only when it is administered within a very limited time frame after the stroke. This means that even those patients that could benefit from this treatment rarely receive it. Brain ischemic damage is thus considered a prime target for cell therapy, in part due to the imperfect ability of neurogenic regions of the brain to induce adequate regeneration of the damaged area and thereby induce functional repair. Studies from several laboratories, including ours, suggest that cell therapeutic approaches to stroke will lead the way in regenerative cell therapy. The original hypothesis was that HUCB cells could replace neurons, astrocytes, or oligodendrocytes lost to injury or disease. In the first study to report the use of HUCB cells for the treatment of stroke, Chopp’s laboratory [19], showed that rats subjected to middle cerebral artery occlusion (MCAO) demonstrated specific motor deficits. After intravenous administration of HUCB, motor function improved. The best improvements were observed when the cord blood cells were administered 24 hours after the MCAO, although some improvements were also observed when HUCB were delivered at 7 days following transient MCAO. Cells were found in the affected cortex, subcortex, and striatum of the damaged brain. Further, immunohistochemical examination of the brains showed positive staining of the donor cord blood cells for neuronal markers such as NeuN and MAP-2, astrocyte marker (GFAP), and endothelial marker FVIII. However, less than 10% of the transplanted cells were found in the injured brain and even fewer cells expressed neural proteins. Subsequent studies have demonstrated that the therapeutic effects of HUCB administration after stroke in the rat MCAO model are robust. Since that first study in stroke, we have shown that route of transplantation is an important factor in determining functional recovery [134], with systemic administration in the femoral artery producing more long-lasting motor improvements than direct transplantation into the striatum. However, in both groups immunohistochemical analysis conducted 2 months after transplantation failed to detect donor HUCB cells at the site of injury in recipient rats. We have since shown (using PCR) that human DNA that could only be derived from the HUCB cells was detected in the injured and not the healthy hemisphere [127]. In this study we also showed that there was a dosedependent improvement in motor function 4 weeks
after the MCAO, as well as a significant decrease in infarct size. From these studies it is clear that HUCB cells do improve functional outcome when administered after a stroke and probably do so by a local mechanism, in that the cells can be found in the infarcted hemisphere [19, 127]. This is supported by in vitro studies using migration assays that show that extracts of ischemic tissues promoted more migration of HUCB cells than did normal tissues [19]. Furthermore, a migration study by Newman et al. [85a] suggested that it might be possible to administer the HUCB cells even later than 24 hours to obtain maximum therapeutic benefit. This could be a very promising outcome when the currently available treatment must be delivered by 3 hours post-stroke in order to be effective. More recent studies would suggest that even if some of the HUCB cells do become neurons or astrocytes it is more likely that the HUCB may be especially effective because they act through multiple pathways manipulating the complex network of growth factors and inflammatory mediators to provide neuroprotection, stimulate vascular reconstruction, and increase neurogenesis. Borlongan’s group has transplanted HUCB indirectly via intra-arterial injection and directly by intrastriatal injection into the brains of rats subjected to transient MCAO. Mannitol was co-infused in some experimental groups along with the HUCB cells to permeabilize the blood-brain barrier [10]. Despite the fact that engrafted HUCB cells were not detected in the injured brain, reduced cerebral infarct size and increased levels of neuroprotectant factors such as GDNF were observed. Improved behavioral functions were observed in rats that received striatal HUCB transplants regardless of whether they received mannitol. Mannitol administration was necessary, however, for induction of similar behavioral benefits in the groups that received the cord blood cells via the intra-arterial route. The investigators thus suggested that the presence of donor HUCB cells at the site of damage was not necessary for the observed recovery after stroke. The mechanism of recovery was possibly mediated by trophic factors and soluble molecules secreted by the cord blood cells. Growth factors that are present in the cord blood cells include brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), plateletderived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), stromal-derived factor (SDF)1b, granulocyte colony-stimulating factor (G-CSF),
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glial-derived neurotrophic factor (GDNF), stem cell factor (SCF), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), and transforming growth factor (TGF-b1) — all of which have been reported to be secreted by cells within the mononuclear fraction from HUCB [50, 62, 77, 123, 133, 137]. Neovascularization is another mechanism by which recovery may be induced after administration of HUCB in stroked animals. This mechanism was investigated by the Taguchi group. They transplanted immunocompromised mice with CD34+ HUCB cells 48 hours after induction of stroke [119]. Neovascularization was observed on the border of the ischemic zone, and the width of the cortex was greater in the HUCB treated animals. By itself, this revascularization of the ischemic zone would enhance recovery by providing nutrient-laden blood to cells in this nutrientpoor environment. However, an interesting observation was made when these researchers administered the antiangiogenic agent endostatin. This agent impaired neurogenesis, suggesting that systemic administration of cord blood CD34+ cells stimulated endogenous neural stem cells to proliferate and differentiate into neurons and astrocytes in order to rebuild the disrupted neural circuitry. It has been shown by multiple research groups that stroke itself can enhance neurogenesis in both neurogenic regions of the adult brain — the dentate gyrus and the subventricular zone [2, 57, 58, 60, 91] — but without additional support or stimulation these new cells have not been convincingly shown to integrate into the neural circuitry to effect repair. The therapeutic potential of HUCB treatment is not limited to the embolic stroke. Two studies were recently published that demonstrate the versatility of the HUCB cells. In the first, HUCB cells were systemically delivered after a hemorrhagic stroke. Hemorrhagic stroke associated with hypertension is another major form of stroke for which no current treatment is available. The rtPA cannot be administered to hemorrhagic stroke because it will aggravate the bleeding. In a recent study [84], the mononuclear cell fraction of HUCB was administered by intravenous injection 24 hours after induction of hemorrhagic brain injury in rats. Transplanted rats demonstrated improvement of neurological deficits associated with intracerebral hemorrhage such as limb placement functions and stepping tests. In the second study, HUCB cells were administered either systemically or into the cerebral ventricles of animals suffering heat stroke. The therapeutic effect of the cord
blood transplant contributed to reducing the circulatory shock, cerebral ischemic injury, and extended the survival of the treated rats. Unlike most other studies, however, they found that intracerebroventricular administration produced the best recovery [21]. Clearly, HUCB is a more globally effective treatment option for all strokes than is currently available.
Cord Blood Cells for Neurological Diseases Advances in cell therapy have fueled the recent surge in research for curative therapy of diseases of the central nervous system. Studies have shown that embryonic stem cell, bone marrow, and HUCB transplants are all promising therapies for neurological diseases. Studies showed that embryonic stem (ES) cells could be induced in vitro to differentiate into neurons and glial cells [75], generating excitement about the potential role of ES cells to replace degenerating neural cells. Considering the controversy regarding therapeutic usage of ES cells in the United States as an ethical issue, researchers have investigated the use of alternative sources for stem cells such as the HUCB. Reports from our laboratories and others showed that HUCB stem cells could differentiate across tissue lineage boundaries into endoderm, mesoderm, and ectoderm.
Cord Blood as a Treatment for Inborn Errors of Metabolism with Neurologic Consequences Neurological disorders are especially challenging because of the current lack of curative therapies. However, the limited success in clinical trials to treat metabolic disorders affecting the neural tissues such as Krabbe’s disease and Hurler’s syndrome have provided encouragement to researchers, practitioners, and patients alike. Krabbe’s disease is an enzymatic disorder caused by deficiency of galactocerebrosidase (GAL-C) that results in defective myelin formation in the central and peripheral nervous system. Cord blood transplantation has shown moderate potential by alleviating the usually devastating symptoms of the disease [128]. Better outcome was achieved when cord blood from unrelated donors was used to treat patients affected with Hurler’s syndrome. This is a severe form of mucopolysaccharidosis that affects children and causes a progressive and fatal deterioration of the central
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nervous system. Affected children transplanted with cord blood after non-myeloablative preparative regimen showed an impressive increase in survival and cord blood engraftment was achieved [114]. Another neurodegenerative disease is Sanfilippo syndrome type B. This disease, also known as mucopolysaccharidosis III B (MPS III B), is caused by a deficiency of the a-N-acetylglucosaminidase enzyme — leading to accumulation of undegraded heparan sulfate, a glycosaminoglycan (GAG). Results of a recent study from our laboratories [39] are the first supporting enzyme replacement by the administered human umbilical cord blood cells. These cord blood cells were shown to contain and extracellularly release the deficient enzyme in vitro. The cells, transplanted into the lateral cerebral ventricle of mice modeling Sanfilippo syndrome type B, migrated into the parenchyma of the brain, expressed neural antigens, and exhibited neuron and astrocyte-like morphology. The administered cells were also found in peripheral organs. Transplant benefits were demonstrated by stable cytoarchitecture in the hippocampus and cerebellum, and by reduced GAGs in the livers of treated mutant mice. These results showed that the ability of cord blood cells to substitute the deficient enzyme might be considered a potent therapeutic tool for the treatment of Sanfilippo syndrome type B. However, systemic administration of enzymatically rich cord blood cells may prove more effective.
Cord Blood Therapies for Neurodegenerative Diseases Cord blood transplantation has shown promise in treatment of another group of formidable diseases, neurodegenerative diseases. These include (but are not limited to) Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). A common feature shared by this group of disorders is that nerve cell function is lost as specific regions of the brain degenerate. Depending on the specific neuronal population that is affected, cognitive function or motor systems may deteriorate. Clinical symptoms usually start in middle life or later, and treatment is for the most part symptomatic. Unlike cerebral ischemia or spinal cord injury, this group of disorders is characterized by slow onset and a progressive course. Undoubtedly, advances in the treatment of these illnesses have dramatically improved the quality of life and extended life span of afflicted
patients. However, because no cure is currently available, the vigorous search for therapeutic alternatives has turned to cell-based therapies. Cell therapy for such group of disorders has faced the challenges of finding appropriate animal models and providing effective forms of therapy that aim at neural regeneration. The development of transgenic animal models has helped in this respect, although even these models may not reflect the total spectrum of disability that affects the afflicted individual. An SOD1 transgenic (B6SJL-[TgN SOD-G93A] 1GUR) mouse is one such model that has helped us better understand the development of amyotrophic lateral sclerosis (ALS). ALS is characterized by progressive degeneration of the motor neurons causing muscle frailty and paralysis. Patients with ALS suffer weakness and progressive wasting of the muscles that affect their ability to move, speak, swallow, and ultimately to breathe. Progressive paralysis always leads to death, usually within 5 years of disease onset. Currently, treatment for ALS is limited to alleviating the symptoms of the disease to improve the quality of life and delay the onset and severity of complications. This grave prognosis and the availability of an animal model situated ALS as an ideal candidate for cell-based therapies. The first study to demonstrate the therapeutic benefit of HUCB delivery was performed by Ende and associates [20, 34]. They showed that systemic administration of large doses of the mononuclear cell fraction of HUCB cells into irradiated SOD1 mice resulted in a prolongation of life span in SOD1 mice. A study from our laboratories employing a low dose of these cord-blood-derived cells injected into the jugular vein of presymptomatic G93A SOD 1 mice resulted in a significant delay in onset of disease symptoms, with a longer life span than the diseased nontransplanted mice [40]. The transplanted cells entered regions of motor neuron degeneration in the brain and spinal cord. Infused cord blood cells were also widely distributed in peripheral organs, but mainly in the spleen. Advantages of cord blood cell transplantation in ALS are still controversial, as it is unclear if the HUCB cells replaced damaged motor neurons. However, it is most likely that cord blood efficacy is partially a result of neuroprotection. Although HUCB cells have not been employed in a clinical trial, autologous bone-marrow-derived mesenchymal stem cells were injected into the spinal cords of ALS patients [76]. The transplantation did not result in any detrimental effects on neurological function or any major adverse effects. Thus, such an approach
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would indicate a reasonable clinical safety and tolerance of the protocol by ALS patients. Meanwhile, more research is required to optimize the procedures applied in cell therapy.
Therapeutic Potential of Blood-derived Cells
develop from mature cells in the graft. Despite the low immunogenicity of cord blood cells, there is a need for isolating defined populations of stem cells. Transplantation of isolated stem cells alone or in conjunction with trophic factors may represent a valuable alternative to whole cord blood cell grafts.
Sertoli Cells and Transplantation Tolerance The therapeutic potential of the reliable induction of bone marrow or cord blood cells to differentiate into cells of the neural lineage after incubation with appropriate neural differentiation factors has been a largely controversial subject. On the one side, accumulating evidence from the literature suggested that the capacity of stem cells to transdifferentiate into cells of functional organs and tissue is crucial for the regenerative therapy initiated by stem cells. Recent reports, however, suggest that the effective therapeutic benefit of stem cell sources such as bone marrow and HUCB may be achieved via release of various growth and trophic factors, or by stimulation of intrinsic progenitors. Transdifferentiation will remain an in vitro phenomenon, achieved only after stimulation with specific growth factors, sometimes for prolonged periods. How faithfully these in vitro data translate to the in vivo situation is not clear. Animal data remain sketchy, especially when clonally purified stem cells are not the source for the stem cell graft. It has been argued that multipotentiality on the single-cell level is of minimal consequences in clinical transplantation even when the outcome of the transplant is beneficial. This argument is supported by the data that showed successful engraftment and disease amelioration when the mononuclear fraction of the HUCB cells has been the source of transplantation and not a specifically selected or highly purified population of stem cells. One complicating issue has been that identification of various stem cell populations typically relies on surface markers, a criterion that has been elusive for most stem and progenitor cells — perhaps with the exception of the CD34+ HSCs. Whether stem cells structurally replace defective cells and promote neuronal regeneration in neurodegenerative diseases is a question that still requires more assessment. Despite the promising effect of cell therapy, the evidence that trophic factors secreted by cord blood cells may be the primary effectors in promoting neural repair demands reevaluating the cell types involved in the repair process. Many of the transplant-associated complications in the form of graft rejection and GVHD
Specific immunological tolerance of transplant recipients to foreign organ or tissue grafts is a goal that has been sought by transplant biologists for more than 50 years. Since the original description of the phenomenon in experimental animals by Medawar and colleagues [8, 9], a wealth of experimental data has accumulated relating to strategies for extending allograft survival and function. Over the past few decades, advances in tissue and organ transplantation have made it possible to treat many devastating illnesses by means of cell therapy. The revived interest in cell-based therapies has been largely due to the promising success of transplantation regimens such as the Edmonton protocol. Using a modified immunosuppressive protocol with islet allotransplantation, a 100% cure rate for type-I diabetics was achieved [53]. Despite this success, many obstacles delayed broader applications of cell-based therapies, such as the inability to provide immediate trophic support to the newly grafted cells and to prevent acute and/or long-term graft rejection triggered by the host’s natural immune defense mechanisms. To understand mechanisms of allograft rejection and engineer cell transplants to overcome these impediments, investigations have aimed at identifying the role of regulatory T-cells and the impact of the immune mediators that determine the ultimate balance between Th1 and Th2 profiles. In this regard, Fowler and Gress have demonstrated that functionally defined donor Th1 and Th2 populations play an important role in the regulation of GVHD [36], whereas Wang’s group showed that the distinct cytokine profiles expressed by different mouse strains play an essential role in regulating the pattern of rejection and efficacy of cell therapy [129]. To overcome graft rejection and to improve the cell transplantation outcome, scientists sought to induce specific transplantation tolerance to tissue and organ grafts using co-transplantation of “tolerizing” cells, such as bone marrow cells and more recently Sertoli cells. Sertoli cells are a population of terminally differentiated
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cells that provide an immunoprivileged microenvironment for the growth and maturation of sperm cells in the mammalian testis. They are believed to play an important role in preventing rejection of the highly antigenic spermatids by secreting trophic factors and exhibiting immunosuppressive properties. These qualities have prompted researchers to investigate whether this intratesticular immunoprotective effect could be extended when Sertoli cells are co-transplanted with other cells in tissue or organ grafts for therapeutic purposes. For specific reviews on Sertoli cell immunoprotection, see Emerich and Halberstadt [33, 47]. Testicular Sertoli cells confer local immunoprotection for co-transplanted cells, and can apply this protection to specific pathological conditions. In various studies [33, 47], a product(s) secreted by Sertoli cells inhibited Con-A-stimulated lymphocyte proliferation in a dose-dependent manner. The mechanism of inhibition of lymphocyte proliferation by Sertoli cell-enriched media was via inhibition of IL-2 production. Such inhibition was not reversed even with the addition of exogenous IL-2, indicating that the Sertoli cell-enriched media inhibit both IL-2 production and IL-2 responsiveness of T lymphocytes. Dufour et al. showed that co-transplantation of neonatal porcine Sertoli cells (NPSCs) protected grafted aortic endothelial cells via immunosuppression [29]. In an in vitro cytotoxicity assay, co-transplanted SCs showed more than 60% inhibition of the cellular lysis observed when cultured aortic endothelial cells were incubated with human antibody serum plus complement. These data demonstrated that despite expression of alphaGal binding of xenoreactive antibodies and the activation of complement NPSCs survived human antibody and complement-mediated lysis. Although the complement cascade was activated, as indicated by deposit of C3 and C4 on the surface of the NPSC membrane, the membrane attack complex (MAC) was not formed. This suggested that NPSCs might be able to survive humoralmediated rejection in a clinical situation and protect co-transplanted cells from allogeneic and xenogeneic rejection. The ability of extra-testicular Sertoli cells to cause a significant reduction or even elimination of allo- or xenograft rejection appeared to be related to their close proximity to the co-transplanted cells and tissues. This has prompted the suggestion that Sertoli cells provide local immunosuppression at the graft site [17, 33, 47, 110, 136, 140]. This tolerizing ability was experimentally applied to the treatment of diseases of the central nervous system and for diabetes.
Co-transplantation of isolated Sertoli cells (iSCs) with neurons has demonstrated promise for the treatment of neurodegenerative diseases and brain injury [17, 27, 33, 89, 101, 103]. Likewise, cell transplant therapy using iSCs in the 3-nitropropionic acid (3-NP) rat model of Huntington’s disease (HD) has been shown to provide immunoprotective effects similar to those obtained using systemic treatments with nonsteroidal anti-inflammatory medications. Grafted SCs in the CNS can locally deliver molecules with known trophic and anti-inflammatory effects on the surrounding tissue. When SCs were transplanted into the striatum of rats following two injections of 3-nitropropionic acid (3-NP), there was significant reduction in the locomotor hyperactivity induced by 3-NP, with some behaviors returning to baseline. The 3-NP-Sertoli cells survived in the striatum without systemic immunosuppression and some formed tubule-like structures. These results showed that Sertoli transplants could ameliorate locomotor abnormalities in a 3-NP model of early HD, and thus should be further evaluated as a possible treatment strategy for the early stages of HD [32, 99]. The other system in which Sertoli cell transplantation has been especially promising is pancreatic islet transplantation for the treatment of diabetes. Kin et al. [64] indicated that transplantation of islets into an immunoprivileged ectopic site created by pre-engrafting SC did not affect islet function. Moreover, the SC transplant provided a means of developing an immunoprivileged ectopic site that permits prolonged islet allograft survival without systemic immunosuppression [65]. It was suggested that Sertoli cells are effective in providing this local immunoprotection to co-grafted discordant cells, including those from xenogeneic sources [33]. In addition to protection of grafted allogeneic and xenogeneic cells from immune destruction, Sertoli cells enabled survival and function of dopaminergic neurons [135] and reversed diabetes in animal models [109–111]. These data attest to the potentially beneficial effect of SCs in the clinical setting. The question of how extra-testicular Sertoli cells impart their “immunosuppressive” or “immunoprotective” properties remains open to speculation. Bellgrau and Selawry first presented reports that mechanisms other than local devices are involved in this tolerizing effect of extra-testicular Sertoli cells. In these experiments, tolerance to hamster islets was examined by co-transplantation of rat cryptorchid testes with hamster islet xenografts. Hamster islets were subsequently transplanted either beneath the kidney capsule or in the liver
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of the host rat. This resulted in successful islet engraftment in the experimental rats, whereas in control rats (who did not first receive hamster islet xenografts in their cryptorchid testes) rejection of the hamster islet transplants was observed [6]. These data suggested that Sertoli cells might induce some type of systemic tolerance in the transplanted host. However, this hypothesis of systemic immune modulation was later challenged by Korbutt and co-workers, who tested induction of immune tolerance by transplanting islet allografts and allogeneic Sertoli cells under the rat kidney capsule [68]. This was followed by removal of the grafted kidney and the additional transplantation of rat islets in the contralateral kidney. If the initial transplant had induced systemic tolerance, the subsequent islet transplants should have been accepted. This was not the case. Instead, the secondary transplants resulted in a hyperimmune response and islet rejection. Since then, little research has been directed toward elucidating the effects of transplanted extratesticular Sertoli cells on the systemic immune system and the mechanism(s) by which these testis-derived cells result in immunoprotection of co-transplanted cells and tissues. It became interesting to test the hypothesis that isolated Sertoli cells could induce systemic tolerance by a mechanism that involves generalized immune suppressive regimens. We have initiated experiments in which isolated rat Sertoli cells were injected via the lateral tail vein into normal mice without the use of immune suppression. The mice tolerated the graft well, lived a normal life span, and showed a vigorous primary humoral antibody response. Recipient lymphocytes were tolerant to donor cells in a mixed lymphocyte reaction, but appropriately alloreactive to third-party cells. These in vitro data indicating that a state of immune tolerance was generated were confirmed by in vivo data. The mice tolerated skin grafts from donor rats and the graft rejection was delayed for almost 1 month compared to 2 weeks in control mice that did not receive the Sertoli cell injection [112a, 27]. These data attest to the systemic immune tolerizing effect of Sertoli cells and suggest that this approach could be employed for allowing other tissue and organ transplantation without immune suppression, such as islet transplantation for diabetes. The immune suppressive properties of Sertoli cells have been frequently linked to secretion of immune modulatory cytokines, primarily TGF-beta. TGF-beta1 is a polypeptide growth modulatory and differentiation
factor involved in many biological processes, including immune homeostasis and self-tolerance. An earlier report indicated that TGF-beta1 secreted by cultured SCs attenuates the action of follicle-stimulating hormone (FSH). Such inhibitory action is potentially exerted through a decrease in cAMP levels. Because of the local production of TGF-beta1, it is suggested that the effects of this peptide might be exerted in the context of the testicular paracrine mechanisms [81]. Theories that explain the immunosuppressive properties of Sertoli cells include the release of TGF-beta leading to suppression of activated T-cells. Other mechanisms determining the effect of Sertoli cells on suppressor and regulatory T lymphocytes remain to be elucidated.
Future Directions in Stem Cell Therapy The search for a cure for intractable diseases and the hope for replacing defective tissues with healthy or younger ones has been the driving force for stem cell research. Therapeutic cloning using embryonic stem cells seems to be the most versatile approach. However, the ban on embryonic stem cell research has accelerated the use of adult stem cells that showed high therapeutic potential, not only in efficacy but also in safety. Adult stem cells could also be manipulated for purposes of gene therapy or drug delivery. This necessitates a certain degree of controlled delivery, in order to prevent uncontrolled cell division that may result in tumor formation. Accelerated cell senescence, on the other hand, will result in failure of the therapeutic medications to reach their target tissues. These issues and other challenges still face stem cell research and cell therapy in general. Adult stem cell collection from mobilized autologous peripheral blood has recently been the standard of care for many hematological diseases to avoid histocompatibility mismatching and problems of graft rejections that represent the most important and long-standing challenges in transplantation immunity. Finding a compatible donor has been made easier by establishing bone marrow donor and cord blood donor programs. Finding stem cell or other cells that have immunosuppressive qualities, or that facilitate engraftment of other co-transplanted tissues, is another dynamic research direction. The most promising candidates are MSCs from bone marrow and cord blood, and recently, from isolated Sertoli cells. Finding and isolating the cells of interest is perhaps the most active field of investigation, and ironically the
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most disappointing. The elusive nature of stem cells, their rare frequency among other more mature cells, and the reliance on evolving surface markers has limited the progress of stem cell translational research — with the exception of the HSC. In addition to the low numbers of stem cells that could be obtained from certain adult tissues, culture expansion of these populations in vitro without losing their multipotentiality has been for the most part unsatisfactory. After several passages in culture, most stem cells lose some or more of their identifying surface markers, and their potential to differentiate or transdifferentiate is often compromised. Despite the large volume of published literature that describes the multilineage differentiation potential of various stem cells, this capacity is not consistently reproducible. With very few exceptions, one cannot predict the precision and efficacy of inducing stem cells to differentiate into the desired progeny, such as generating suppressor lymphocytes from marrow HSCs, or neurons from MSCs. Identifying those cues that initiate division into appropriately functioning progenitors is another challenge still considered a work-in-progress. In most of the reports that describe differentiation of stem cells into neural tissue, heart tissues, or pancreatic tissues, compelling evidence that shows production of fully functioning neurons, cardiac myocytes, or insulinsecreting pancreatic islet cells is still lacking. In our view, the most promising application for stem cell therapy involves a highly orchestrated process in which transdifferentiation plays a minor role if any. The dramatic potential of cell therapy involves replacing diseased cells and a diseased environment with healthy cells and a healthy environment comprised of more accessory cells in addition to a myriad of growth and trophic factors. This intricate composition initiates a cascade of events that involve release of intrinsic factors and stimulation of healthy cells to proliferate and initiate the desired process of repair. This premise, along with the advantages of high stability and non-tumorigenicity of cord blood cells compared to ES cells, warrants the search for the fountain of youth in the young cells of the cord blood.
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111. Selawry, H. P., Whittington, K. B., Bellgrau, D. Abdominal intratesticular islet-xenograft survival in rats. Diabetes 38 Suppl 1:220–223; 1989. 112. Seshi, B., Kumar, S., King, D. Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis. Blood Cells Mol Dis 31:268–285; 2003. 112a. Shamekh, R., El-Badri, N., Saporta, S., Pascual, J., Sanberg, P. R., Cameron, D. F. Sertoli cells induce system donor-specific tolerance in xenogenic transplantation model. Cell Transpl 15:45–53; 2006. 113. Silva, G. V., Litovsky, S., Assad, J. A., Sousa, A. L., Martin, B. J., Vela, D., Coulter, S. C., Lin, J., Ober, J., Vaughn, W. K., Branco, R. V., Oliveira, E. M., He, R., Geng, Y. J., Willerson, J. T., Perin, E. C. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 111:150–156; 2005. 114. Staba, S. L., Escolar, M. L., Poe, M., Kim, Y., Martin, P. L., Szabolcs, P., Allison-Thacker, J., Wood, S., Wenger, D. A., Rubinstein, P., Hopwood, J. J., Krivit, W., Kurtzberg, J. Cord-blood transplants from unrelated donors in patients with Hurler’s syndrome. N Engl J Med 350:1960–1969; 2004. 115. Stamm, C., Westphal, B., Kleine, H. D., Petzsch, M., Kittner, C., Klinge, H., Schumichen, C., Nienaber, C. A., Freund, M., Steinhoff, G. Autologous bone-marrow stemcell transplantation for myocardial regeneration. Lancet 361:45–46; 2003. 116. Storb, R., Epstein, R. B., Ragde, H., Bryant, J., Thomas, E. D. Marrow engraftment by allogeneic leukocytes in lethally irradiated dogs. Blood 30:805–811; 1967. 117. Sutherland, F. W., Perry, T. E., Yu, Y., Sherwood, M. C., Rabkin, E., Masuda, Y., Garcia, G. A., McLellan, D. L., Engelmayr, G. C. Jr., Sacks, M. S., Schoen, F. J., Mayer, J. E. Jr. From stem cells to viable autologous semilunar heart valve. Circulation 111:2783–2791; 2005. 118. Szabolcs, P., Park, K. D., Reese, M., Marti, L., Broadwater, G., Kurtzberg, J. Coexistent naive phenotype and higher cycling rate of cord blood T cells as compared to adult peripheral blood. Exp Hematol 31:708–714; 2003. 119. Taguchi, A., Soma, T., Tanaka, H., Kanda, T., Nishimura, H., Yoshikawa, H., Tsukamoto, Y., Iso, H., Fujimori, Y., Stern, D. M., Naritomi, H., Matsuyama, T. Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J Clin Invest 114:330–338; 2004. 120. Tamaki, S., Eckert, K., He, D., Sutton, R., Doshe, M., Jain, G., Tushinski, R., Reitsma, M., Harris, B., Tsukamoto, A., Gage, F., Weissman, I., Uchida, N. Engraftment of sorted/ expanded human central nervous system stem cells from fetal brain. J Neurosci Res 69:976–986; 2002. 121. Thomas, E. D., Ashley, C. A., Lochte, H. L. Jr., Jaretzki, A., III, Sahler, O. D., Ferrebee, J. W. Homografts of bone marrow in dogs after lethal total-body radiation. Blood 14:720–736; 1959.
122. Thomas, E. D., Lochte, H. L. Jr., Lu, W. C., Ferrebee, J. W. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med 257:491–496; 1957. 123. Tucci, A., Mouzaki, A., James, H., Bonnefoy, J. Y., Zubler, R. H. Are cord blood B cells functionally mature? Clin Exp Immunol 84:389–394; 1991. 124. Uchida, N., Buck, D. W., He, D., Reitsma, M. J., Masek, M., Phan, T. V., Tsukamoto, A. S., Gage, F. H., Weissman, I. L. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA 97:14720–14725; 2000. 125. Urbich, C., Dimmeler, S. Endothelial progenitor cells: Characterization and role in vascular biology. Circ Res 95:343–353; 2004. 126. Urbich, C., Heeschen, C., Aicher, A., Dernbach, E., Zeiher, A. M., Dimmeler, S. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation 108:2511–2516; 2003. 127. Vendrame, M., Cassady, J., Newcomb, J., Butler, T., Pennypacker, K. R., Zigova, T., Sanberg, C. D., Sanberg, P. R., Willing, A. E. Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke 35:2390–2395; 2004. 128. Wagner, J. E., Kernan, N. A., Steinbuch, M., Broxmeyer, H. E., Gluckman, E. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 346:214–219; 1995. 129. Wang, H., Hosiawa, K. A., Min, W., Yang, J., Zhang, X., Garcia, B., Ichim, T. E., Zhou, D., Lian, D., Kelvin, D. J., Zhong, R. Cytokines regulate the pattern of rejection and susceptibility to cyclosporine therapy in different mouse recipient strains after cardiac allografting. J Immunol 171:3823–3836; 2003. 130. Wang, P. P., Wang, J. H., Yan, Z. P., Hu, M. Y., Lau, G. K., Fan, S. T., Luk, J. M. Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction. Biochem Biophys Res Commun 320:712–716; 2004. 131. Weinreb, S., Delgado, J. C., Clavijo, O. P., Yunis, E. J., Bayer-Zwirello, L., Polansky, L., Deluhery, L., Cohn, G., Yao, J. T., Stec, T. C., Higby, D., Anderzejewski, C. Transplantation of unrelated cord blood cells. Bone Marrow Transplant 22:193–196; 1998. 132. Weiss, M. L., Mitchell, K. E., Hix, J. E., Medicetty, S., El-Zarkouny, S. Z., Grieger, D., Troyer, D. L. Transplantation of porcine umbilical cord matrix cells into the rat brain. Exp Neurol 182:288–299; 2003. 133. Welker, P., Grabbe, J., Gibbs, B., Zuberbier, T., Henz, B. M. Human mast cells produce and differentially express both soluble and membrane-bound stem cell factor. Scand J Immunol 49:495–500; 1999. 134. Willing, A. E., Lixian, J., Milliken, M., Poulos, S., Zigova, T., Song, S., Hart, C., Sanchez-Ramos, J., Sanberg, P. R.
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30 Immunologically Privileged Environments Mark P. Hedger Monash Institute of Medical Research, Monash University, Melbourne, Australia
Acknowledgments The author is supported by the National Health and Medical Research Council of Australia through the award of a Senior Research Fellowship (#143788) and Program Grant (#143786).
Background: What Is “Immunological Privilege”? The term immunological privilege (also referred to as “immunoprivilege” or “immune privilege”) was originally employed to describe organs and tissues that showed some advantage as a site for graft survival in transplantation studies [16]. It was noticed that tissue from immunoprivileged sites also tended to show increased survival when engrafted into conventional or nonprivileged sites. Such tissues were already well known to transplantation biologists long before the establishment of the clonal selection theory of lymphocyte development [27], and before the classical experiments of Billingham, Brent, and Medawar demonstrated that tolerance was a specific failure of immune responsiveness [21]. In spite of its extensive history, however, the intriguing phenomenon of immunological privilege still remains incompletely understood. In addition to its historical meaning, the expression “immunologically privileged” has been used (or perhaps more accurately, misused) to describe tissues that were believed to be inaccessible to circulating immune cells, due to the existence of blood-tissue “barriers” and/or deficient lymphatic drainage to local lymph nodes. Examples of such tissues notionally included the central nervous system (CNS), the structures of the eye, and the placenta within the pregnant uterus.
Cellular Transplantation: From Laboratory to Clinic
However, as is discussed later, it is quite evident that immune cells do gain access to these tissues (even under normal conditions) and that drainage of antigen to local lymph nodes occurs as well. Immunological privilege has been used more recently to describe tissues for which there is either real or indirect evidence that localized antigen-specific immune responses against endogenous antigens (e.g., spermatogenic cells and fetal cells) and exogenous antigens (e.g., grafts and tumors) are actively suppressed. These alternative appropriations of the expression “immunological privilege” often lead to confusion about what researchers actually mean when they use this term. As a result, it may be unclear whether immunological privilege is meant to refer to the functional definition of a site where grafts enjoy extended survival, a theoretical definition of reduced immune responses at a site, or the presence of specialized endothelial and epithelial barriers to immune cell traffic. For the purposes of this chapter, the terms immunological privilege and immunoprivilege are used to refer to the extended survival of cells expressing antigens that under normal circumstances should provoke an immune response, as well as to the mechanisms that contribute to this survival. The better-known and studied organs that display immunological privilege include the eye, the brain, the testes, and the pregnant uterus and placenta. Less well known is the fact that immunological privilege also has been attributed to subcutaneous fatty tissue, the hair follicle matrix, the prostate, the adrenal gland, the liver, and even the bone marrow space and scar tissue [16, 85]. Animal-specific sites such as the hamster cheek pouch and artificially created sites with deficient or damaged lymphatics also display immunoprivilege. Moreover, certain tumors exhibit immunoprivilege by effectively evading the host immune response [279, 303]. It is
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evident that these tissues are all quite different in morphology and functional characteristics. What features then, if any, do immunologically privileged tissues have in common that might explain the phenomenon? Moreover, given that a functional immune system is essential to survival and bearing in mind that transplantation and graft rejection are artificial conditions, what might be the physiological significance of immunological privilege? Potential drawbacks of immunoprivilege would include a reduced capacity to deal with infections and greater susceptibility to certain types of tumors; for example, gliomas [275] and relapsing acute lymphocytic leukemia in the testes [97]. Given such obvious potential disadvantages, what advantages does immunoprivilege confer, and how might the inherent disadvantages be overcome? Finally, what are the implications of immunological privilege for transplantation medicine? These questions are addressed in this chapter. It should be noted that the views expressed herein are those of the author, and that other interpretations and opinions are entirely feasible. The scope of this chapter is confined to the historical list of immunologically privileged tissues that has been best studied in this context. The discussion particularly focuses on the reproductive tissues because (1) there are already many excellent reviews on immunoprivilege in the brain and eye and in tumors and (2) the testis, ovary, and uterus represent immunologically privileged sites where entirely novel antigens appear after the period of neonatal tolerance — which, in theory at least, more closely resembles the situation experienced during grafting and transplantation rejection.
Immunologically Privileged Tissues: The Evidence It is generally considered that immunological privilege operates in tissues with critical function, where overt inflammatory and immune events might cause irreparable harm to the function of the organ and to the host organism as a result. Immunity and inflammation is invariably associated with cellular destruction, tissue remodeling, and fibrosis [209]. Unrestrained inflammation within the eye could lead to impaired vision, whereas inflammation in the CNS could cause permanent alterations in vital brain connections. In the case of the testis, a unique situation exists, where only the
most primitive spermatogenic stem cells are present at birth. Current concepts of tolerance require that antigens should be expressed within the thymus during fetal and neonatal life to facilitate the editing of the lymphocyte repertoire. This central tolerance due to editing of the developing immune system is backed up by induction of antigen-specific regulatory lymphocytes throughout life, providing peripheral tolerance [154]. The details of how the antigen sampling process occurs, and how the system deals with lymphocytes directed against antigens that are not normally expressed in the thymus, remain incompletely understood. However, the process appears to involve a transcription factor that regulates thymic expression of many organ-specific genes: the autoimmune regulator (AIRE) [134, 243]. As a consequence of the fact that so many of the genes expressed by the developing male gamete are unique to the spermatogenic process and are completely absent from the antigenic repertoire until the time of puberty, it appears that many spermatogenesis-specific antigens escape tolerance — making these cells particularly susceptible to autoimmune attack. Activation of an immune response and consequent loss of spermatogenic cells when they first appear at the time of puberty would be disastrous for the species, if not for the particular individual. A similar life-threatening conundrum faces the developing embryo and fetus, which express paternal alloantigens. In all of these tissues there is an inherent benefit derived from inhibiting antigen-specific immunity in a localized manner. In spite of the potential drawbacks of such inhibition, in general these tissues seem to cope relatively well with the increased level of threat, having developed alternative strategies for immunological protection — possibly by relying more on the innate arm of the immune system [11]. There are certain morphological and functional features shared by the various immunologically privileged tissues, although there is no single set of characteristics common to all such tissues (Table 30.1). Deficient or altered lymphatic drainage seems to play a role in many, but certainly not all, of the tissues. Restricted access by certain immune cells, most notably lymphocytes, appears to be a feature of the brain, eye, and uterus, but not of the testis (the blood-testis barrier represents a special case, which is discussed in more detail later). Immunoprivilege, however, does parallel the immunoregulatory environment that exists within the mucosal immune system of the respiratory and gastrointestinal tract. These tissues face the problem of constant exposure to inhaled and ingested foreign
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TABLE 30.1 Characteristics of the immunologically privileged tissues.
Eye Brain Testis Uterus and placenta Tumors
Deficient Lymphatics
Blood-tissue Barrier
Evidence for Acquired Reduced MHC FasL Expression Immune Deviation Expression
Yes Yes No Yes Yes/no
Yes Yes No* Yes No
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes
Yes Yes No* Yes Yes
∗ See text for explanatory details.
antigens and responses to these antigens are very tightly controlled, partly through ongoing local induction of tolerance [101, 262]. The epithelia of the eye and reproductive tract also are considered by many researchers part of the common mucosal system [6, 36, 302]. Before examining in detail the evidence that immunological privilege involves a specialized immunoregulatory environment, however, it is worth briefly considering the evidence that these tissues are special sites for grafts and the studies that have been performed to explore the parameters and limitations of this survival.
THE BRAIN AND EYE There is no doubt that allografts and xenografts can enjoy extended survival in the brain, although it is equally clear that immunoprivilege in the brain is conditional on factors such as the inherent immunogenicity of the graft and the actual site of engraftment [86, 150, 193, 284]. At least part of the immunoprivilege is due to the existence of the blood-brain barrier, comprising specialized interendothelial cell junctions and molecular shuttling systems that limit access of cells and macromolecules from the blood into the brain tissue (Figure 30.1) [188]. The brain also lacks conventional lymphatic drainage. However, introduced antigens can escape from the brain into the circulation, eventually reaching local lymph nodes and activating the peripheral immune system [71, 276, 295]. Moreover, the brain parenchyma itself contains numerous specialized resident macrophages, or microglia, which possess low inherent antigen-presenting activity but have the capacity to support ongoing immunological responses [274]. Finally, exogenous sensitization of the host to
graft antigens will cause rejection of neural grafts, and activated lymphocytes can traverse the blood-brain barrier without serious impediment [86, 93, 120]. The eye is the best studied of all of the immunologically privileged sites, and indeed the earliest successful grafts involved transplantation of corneal tissue. There have been a number of excellent recent reviews on this topic, and the interested reader is directed to these for a much more detailed treatment of this area [74, 171, 239]. The main regions of the eye that possess immunoprivilege are the cornea, the anterior chamber, the vitreous space, the subretinal space, and the lens. These sites will support the survival of allografts as well as tumors. Clearly, these tissues have deficient vascularity and lymphatics, and there are specialized endothelial tight junctions that limit passage of immune cells from the blood to the tissue, but these barriers are not absolute [22, 94]. There is a substantial body of evidence to indicate that the immune environment of the eye is quite different from that found at conventional sites, and that tissues from the eye also possess privilege as donor tissue [239]. The strongest evidence that the tissues of the CNS are immunologically privileged due to a specialized immune environment comes from studies in which antigens are introduced via these sites. Injection of soluble antigen into the anterior chamber or other compartments of the eye or into the brain leads to development of antigen-specific suppression of cell-mediated immunity, specifically Th1/delayed-type hypersensitivity reactions [116, 277, 278]. This is called anterior-chamberassociated acquired immune deviation (ACAID) and brain-associated acquired immune deviation (BRAID), respectively. These phenomena clearly indicate that there is something unique about these sites with respect to the development of immune responses.
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Figure 30.1. Diagrammatic comparison of the general organization of the blood-tissue barriers in (A) the brain, (B) the human placenta, and (C) the testis. The blood-brain barrier comprises endothelial tight junctions that prevent ready access of lymphocytes in particular from entering the brain parenchyma under normal conditions, although resident mononuclear phagocytes (microglia) are found throughout the central nervous system. In the placenta, the interface between the maternal circulation and the fetal tissues occurs at a multinucleated syncitium of fused trophoblastic cells, called the syncytiotrophoblast that surrounds the maternal blood spaces. The cellular cytotrophoblast comprises the interface between the fetal tissues and the maternal decidua (not shown here) in the basal plate. In the testis, circulating leukocytes (monocytes, dendritic cells, and lymphocytes) have access to the interstitial compartment, which is more or less continuous with the lymphatic drainage of the organ, but have no access to the seminiferous epithelium proper due to the highly specialized tight junctions between adjacent Sertoli cells.
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THE UTERUS, PLACENTA, AND FETAL ALLOGRAFT
to the implanting embryo and ultimately to the fetus itself.
The pregnant uterus provides a unique model of immunoprivilege, as it represents the closest parallel to a naturally occurring tissue transplant. Although the fetal tissues express the foreign antigens of the father (i.e., the fetal allograft), most women sustain pregnancy without catastrophic immunological disturbance. The preimplantation embryo is protected by a dense glycoprotein coat, called the zona pellucida, which is poorly immunogenic and appears to protect the embryo from immune attack [56]. After implantation, the placenta forms the main interface between the mother and fetus and is composed of the trophoblast originating from the embryo and the decidua, which is maternal in origin. The trophoblast invades the uterine endometrium at the time of implantation, and the fetal circulation and maternal circulation are physically separated by the syncytiotrophoblast (Figure 30.1b). All exchange between the mother and fetus occurs across this barrier, where maternal immune cells actually come into contact with the fetal trophoblast cells. There is no conventional lymphatic drainage of the feto-placental unit [83]. Clinical and experimental studies have confirmed that the maternal immune system recognizes and responds to paternal major histocompatibility complex (MHC) antigens during pregnancy [3, 157, 162]. In fact, there is evidence that some cases of spontaneous abortion or preterm labor are the result of immunological events [157, 178]. However, there is also evidence that tolerance to paternal alloantigens develops during pregnancy [248] and that successful pregnancy is dependent on maternal immunological deviation [197]. An event that usually receives much less attention is the response of the nonpregnant uterus to paternal alloantigens when sperm are deposited in the female tract during coitus. Sperm also express paternal alloantigens, but this does not result in an immune or allergic response, except in a very small number of cases. On the contrary, it has been suggested that the female response to sperm antigens may be important for ensuring the survival of the pregnancy after it is established [205, 206]. Seminal plasma contains specific immunoregulatory cytokines, such as transforming growth factor-b (TGFb), that may be involved in inducing a tolerizing response to the antigens expressed on the sperm. There is evidence that this priming of the immune response in the female tract leads to tolerance
THE GONADS While there is considerable body of evidence for immunoprivilege of the testis, including transplantation studies that go back more than 100 years, the evidence for immunological privilege in the female orthologue of the testis is largely inferential. The testis comprises two structural and functional compartments: the seminiferous tubules (where spermatogenesis occurs) and the interstitial tissue, which also contains the vasculature and lymphatics of the testis. In the ovary, the eggs develop singly inside follicles, which are found throughout the cortex. Between the follicles lies a complex and highly vascularized stromal tissue. Sex steroids, which are essential for spermatogenesis and oogenesis, are produced by steroidogenic cells in the testicular interstitial tissue and ovarian stroma and by the cells lining the ovarian follicles. The gonads are the only organs in the body where meiotic division occurs, producing the uniquely haploid cells of the male and female germ-lines. In the testis, spermatogenic cell development occurs post-pubertally, long after the temporal window of tolerance induction, and involves considerable structural differentiation to produce the sperm. As a consequence, the developing male gamete is highly immunogenic, and autoimmune reactions to sperm in the form of antisperm antibodies are a significant cause of infertility in men [145]. Autoimmune responses against the testis, by comparison, are extremely rare [246]. In contrast to the male, the developing eggs arrest at a late meiotic stage during fetal life and do not resume development until after ovulation. Moreover, as the egg develops in the follicle the egg surrounds itself with the antigenically inert zona pellucida. In spite of this relative quiescence, however, the egg can be a target of “spontaneous” autoimmune oophoritis [67, 169]. In experimental rodents, allografts and xenografts placed into the testicular interstitial tissue are preserved for extended periods of time, even indefinitely [60, 84, 87, 283]. This enhanced survival is not simply due to the reduced ambient temperature of the testis, as grafts to the equally hypothermic skin of the ear are not preserved [84], whereas grafts continue to survive in testes that have been translocated to the abdominal cavity [84, 223]. Notwithstanding some reports
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of direct drainage of some lymphatic vessels to the thoracic duct, the efferent lymphatics of the testis do not show any evidence of structural or functional deficiency [88, 158]. Indeed, allogeneic cells injected into the rat testis induce typical immune responses in the draining lymph nodes, and intratesticular inoculation of hybrid offspring with parental lymphoid cells results in graft-versus-host (GVH) reactions in the regional lymph nodes [88]. In spite of the apparently intact lymphatic drainage of the testis, there is evidence that testicular-associated immune deviation (TAID) can be induced at this site. Several studies have shown that it is possible to inhibit experimental induction of autoimmune uveoretinitis, adjuvant-induced arthritis, and autoimmune encephalomyelitis by prior injection of the appropriate dominant antigens into the testis [48, 131, 179, 199, 270]. The most common misconception regarding immunoprivilege in the testis centers on the role of the bloodtestis barrier in this process. The blood-testis barrier was originally identified as a physiological barrier restricting the passage of lipid-insoluble molecules from the blood into the seminiferous tubular fluid, including immunoglobulins, complement, and immune cells. Unlike the blood-brain barrier, the actual location of the barrier lies in tight junctional specializations between adjacent Sertoli cells in the seminiferous epithelium (Figure 30.1c) [54, 177, 299]. These junctions are assembled at the time of puberty and form a complete seal separating the basal and adluminal compartments of the epithelium. Their primary role is to create a unique physiological environment for the meiotic and postmeiotic germ cells. Loss of the integrity of this barrier is a major factor in several forms of testicular failure, including spermatogenic damage during inflammation and infection. Although there are other cell layers separating the spermatogenic cells from the circulation, specifically the vascular endothelium and the peritubular cell layer, these appear to provide little or no barrier to circulating cytokines, immunoglobulins, complement components, or even immune cells [4, 15, 192, 299]. They do not comprise functional components of the blood-testis barrier. As far as this author is aware, there have been no published systematic studies of the immunological determinants of grafts to the ovary or grafts of ovarian tissue. Although ovarian transplantation is quite commonly performed, this is usually autologous, or employs immunosuppressed or immune-deficient animals as the
host. However, the egg is clearly a target for autoimmune attack, and immune cells and antibody have access to the developing follicle, suggesting that there should be local regulation of immune responses as there exists in the testis. It also should be noted in this context that, although the testis is clearly an immunologically privileged site for grafts the success of testis tissue engraftments to other sites has proven to be quite variable, to say the least [17, 18, 49, 96, 118, 122, 214, 237]. This suggests that technical issues may impact upon such studies, and the implications of this variability are discussed in more detail later.
OTHER TISSUES: TUMORS, PROSTATE, ADRENAL GLAND, AND LIVER In spite of the fact that mononuclear cells frequently infiltrate tumors, it is widely recognized that many tumors are able to inhibit the activity of immune cells in their local environment, thereby protecting themselves from attack by immunosurveillant lymphocytes [279, 303]. This apparent immunoprivilege of tumors has been suggested to be the reason otherwise promising immunotherapies for tumors tend to be ineffective. Apart from the well-characterized sites already discussed, several other immunologically privileged sites are often cited in the literature that have received relatively much less attention. The prostate was originally included in the list due to the high incidence of prostate carcinoma in humans and the erroneous suggestion that there were no draining lymphatics in the organ, but the evidence that grafts enjoy extended survival in the prostate is relatively weak [69, 102, 167, 280]. Recent reports of the existence of a blood-prostate barrier restricting the movement of inflammatory cells into the ductal lumen of the rat prostate [65], however, suggest that some reassessment may be worthwhile. The inclusion of the liver is largely based on the high rate of successful transplants of this organ and deficiency of several immune responses involving the liver [29, 51, 72, 207]. It appears highly likely that the liver is an immunologically privileged organ and that this may involve mechanisms in common with those of other immunoprivileged sites. Finally, parathyroid allografts have shown extended survival in the rat adrenal gland [124], which is the site of production of the major endogenous regulators of inflammation, the corticosteroids.
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Unifying Principles: Mechanisms Contributing to Immunological Privilege The fact that the immunologically privileged tissues display such a range of structural and functional properties suggests that immunoprivilege depends on a number of mechanisms, which may have varying prominence in different tissues. These mechanisms and the evidence for their operation are discussed in this section (see Figure 30.2 and Table 30.2 for summary).
At the extreme lies the testis, which possesses both a normal vascular supply and efficient and effective lymphatic drainage to local lymph nodes [88], yet clearly displays immunoprivilege. Consequently, although there is no doubt that a deficient or specialized blood supply or lymphatic drainage plays some role in immunoprivilege — and in some tissues (such as the hamster cheek pouch) may be the principle explanation [16] — this does not appear to be the primary mechanism of immunological privilege in most tissues.
DEFICIENT OR SPECIALIZED LYMPHATIC DRAINAGE
IMMUNE IGNORANCE 1: RESTRICTED ACCESS BY IMMUNE CELLS
Originally considered the most likely mechanisms contributing to immunoprivilege, it seems that, although some of the classical immunologically privileged organs lack conventional lymphatic drainage, antigens introduced at these sites almost invariably escape and gain access to local lymph nodes [48, 71, 86, 88, 116, 131, 179, 199, 248, 270, 276–278, 295]. As already noted, however, the primary response to these antigens generally appears to be acquired immune deviation.
Figure 30.2. Summary of the immunoregulatory mechanisms operating at sites of immunological privilege. Membrane-bound ligands and secreted proteins, cytokines and hormones produced within the tissue direct the activity of local dendritic cells (DC), macrophages (Mf), natural killer (NK) cell subsets and T-cells (T) to regulate antigen-specific responses by promoting tolerance through the development of regulatory T-cells (Tr), or by active deletion of antigen-specific T-cells. See Table 2 and the text for a more detailed description of the function of the various components.
Blood-tissue barriers include the highly specialized structures of the blood-brain barrier, trophoblast, and blood-testis barrier, as well as more pervasive endothelial and epithelial cell barriers (Figure 30.1). These barriers involve tight junctions between endothelial or epithelial cells, as well as a range of more specialized junction types such as the complex tight junctions of the Sertoli cells in the testis [34, 177]. There is certainly some restriction involved. For example, immune cells never appear in the seminiferous epithelium of the testis and do not cross the blood-testis barrier into the seminiferous epithelium. However, cells of the mononuclear phagocyte lineage, as well as small numbers of lymphocytes, are found throughout the parenchyma of the CNS and in the testis interstitial tissue — and maternal immune cells appear to be able to cross the placental barrier [90, 168, 274]. Finally, adoptive transfer of immunity experiments and the existence of acquired immune deviation do not support immune cell exclusion as the main explanation for immunological privilege [48, 140, 199, 275, 277, 278]. In the case of the testis, which possesses the most elaborate immunological barrier, there are several lines of experimental evidence that this barrier is not fundamentally responsible for immunoprivilege in this organ. Spermatogenic cell autoantigens are not completely confined behind the Sertoli cell tight junctions, and there is clear evidence that the barrier is incomplete in the epithelium of the rete testis, where spermatozoa are collected after they are released from the seminiferous epithelium. Significantly, orchitis can be passively transferred to naive mice by lymphocytes from mice with active autoimmune orchitis, with the initial reaction concentrating in the interstitium surrounding the
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Mark P. Hedger TABLE 30.2 Immunoregulatory molecules associated with immunological privilege. Molecules
Principal Immunoregulatory Roles
Immunoregulatory cytokines TGFb family (TGFb, activin)
Inhibit T-cell and B-cell function; regulate APC activity; regulate production and activity of inflammatory cytokines Type 2 cytokines; inhibit cell-mediated immunity Inhibits NK and cytotoxic T-cell-killing activity Blocks proinflammatory actions of IL-1
IL-10 (and IL-4) MIF IL-1ra
Membrane-bound ligands FasL (CD95L) HLA-G and HLA-E B7-H1
Induces apoptosis of T-cells Inhibit NK cell and T-cell functions; promote Th2 responses Inhibits T-cell activation
Complement inhibitors (CD46, CD55, CD59, Crry)
Inhibit complement activation and stability of the membrane attack complex
Indoleamine 2,3 dioxygenase (IDO)
Inhibits T-cell activity and survival; inhibits macrophage activation
Peptides a-MSH VIP CGRP
Suppresses neutrophil activity; regulates T-cell function Inhibits T-cell activation and differentiation Inhibits NO production by macrophages
Bioactive lipids Prostaglandins (E and J series) Other bioactive lipids
Anti-inflammatory Various immunosuppressive actions
Steroids
Suppression of APC and lymphocyte activity; stimulation of type 2 cytokine production
rete testis [140, 267]. A similar initial pattern of development of orchitis around the rete testis region has been observed in mice immunized with viable spermatogenic cells [103]. In many seasonally breeding species, annual regression of both the later spermatogenic cells and blood-testis barrier occurs without inducing overt inflammation or autoimmunity [177, 265]. Finally, the blood-testis barrier cannot explain the enhanced survival of grafts within the interstitial tissue, undeniably outside the blood-testis barrier [84, 87]. On balance, there is a body of evidence that endothelial cell barriers (such as the blood-brain barrier) and epithelial barriers such as the trophoblast do play some role in immunoprivilege, but not the major role. It can be considered that these purely structural mechanisms play a supportive role only, and possibly are too unreliable and susceptible to breach by mechanical or inflammatory processes to provide the main mechanism of immunoprivilege.
IMMUNE IGNORANCE 2: ALTERED MHC ANTIGEN EXPRESSION Reduced expression of MHC antigens, especially class Ia (HLA-A, HLA-B, and HLA-C) and class II antigens, appears to be an important feature of the brain, eye, testis, zona pellucida, and trophoblast [37, 53, 78, 84, 235, 241, 247, 267, 269, 274]. Selective loss of MHC class Ia expression is also a feature of tumor cells [66]. A reduction in class I and II expression reduces the likelihood of immune-activating events in the tissues, as such cells are able to avoid direct recognition by regulatory CD4+ and cytotoxic CD8+ T-cells, and reduces the likelihood of rejection when transplanted. Lack of class Ia expression, however, tends to make the cells more susceptible to attack by natural killer (NK) cells [136, 174], which means that control of NK cell activity becomes more important in the maintenance of immunological privilege.
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This is not to say that these vital transplantation and T-cell activation molecules are completely absent from immunoprivileged tissues. Small numbers of antigenpresenting cells (APC) expressing MHC class II (dendritic cells and macrophages) are found throughout the tissues of the eye, the brain parenchyma, and the placenta [63, 73, 79, 93, 148, 238, 247]. These cells increase in number and play a role in the onset of inflammation in these tissues. Although most studies have reported a characteristic absence of expression of both MHC class I and class II proteins on the cells of the seminiferous epithelium under normal conditions [7, 55, 78, 84, 190, 267, 299], both are expressed in human spermatozoa [59, 143]. This later expression of MHC molecules may play a role in protection of the sperm against infection in the reproductive tract. In contrast to the seminiferous epithelium, moreover, both MHC class I and II proteins are expressed throughout the testicular interstitial tissue. As would be expected, MHC class I expression is found on most interstitial cells, including the Leydig cells [78, 189]. Studies on MHC class II cell expression, however, indicate that there are significant species differences in the number and distribution of these cells in the testis under normal conditions [78, 84, 189, 267]. In light of all the observations, it appears unlikely that reduced MHC class II-positive APCs is a contributing factor in testicular immunoprivilege, although differences in the distribution of such cells in the testis may help to explain differences in susceptibility and development of autoimmune reactions between species and strains. Significantly, up-regulation of MHC expression in the trophoblast does not cause a significant compromise of fetal survival in mice [208]. In addition to their role in inflammation, it is likely that MHC class II-positive macrophages and dendritic cells in the immunologically privileged tissues actually play a role in controlling the immune response to intratesticular antigens by regulating tolerance induction. One of the mechanisms by which peripheral tolerance is believed to be induced involves an ongoing process of T-cell down-regulation by low-dose exposure of T-cells to their antigen, accompanied by either modified costimulatory or specific immunoregulatory signals from the APCs or other regulatory lymphocytes in the vicinity [154, 213, 261]. Interaction between an APC and an antigen-specific CD4+ T-cell can lead to development of either a Th1 or Th2 cell, largely depending on the presence of type 1 or type 2 cytokines, respectively [39]. Engagement of the TCR in the absence of linkage by the co-stimulatory molecule B7 on immature
APCs via CD28 on the T-cell leads to T-cell inactivation, followed by either functional anergy or apoptosis [31, 95, 219]. Engagement of the TCR and CD28 in the presence of immunosuppressive cytokines, such as interleukin-10 (IL-10) or TGFb — or in the absence of signalling via CD40L — can lead to induction of regulatory/suppressor T-cell activity [186, 259]. Similarly, the cytotoxic T lymphocyte antigen (CTLA-4) expressed by regulatory/suppressor T-cells can bind to CD86 to control autoimmune responses and graft rejection responses [31, 194]. Significantly, deficient expression of the classic B7 isoforms (CD80 and CD86) has been reported on APCs from the mouse testis [212] and eye [79, 80, 147] and in the human trophoblast [181]. More recently, it has become clear that novel immunoregulatory members of the B7 family, such as the variant B7-H1 expressed on the placental trophoblast [181, 182], on microglia in the CNS [139, 290], and in tumors [42, 50] may contribute to immunoprivilege. As opposed to the classical MHC molecules, the nonclassical MHC class Ib molecule HLA-G is associated with the placenta and is expressed on the nonvillous cytotrophoblast, and its soluble form has been implicated in apoptosis of alloreactive CD8+ cytotoxic T-cells [64, 128]. HLA-G inhibits the cytolytic activity of maternal NK cells, suppresses T-cell proliferation, and deviates the activity of decidual leukocytes from type 1 to type 2 responses [12, 111, 114, 203]. This molecule has also been found in the rhesus monkey testis [210, 228], human eye [129, 204, 228], and in various tumors [28]. Recently, yet another MHC class Ib molecule HLA-E has been implicated in the control of NK cells at the maternal-fetal interface [25, 119], and is also expressed on germ cells [61].
IMMUNOREGULATION AND PERIPHERAL TOLERANCE There is little doubt that allografts come to the attention of the immune system when they are placed into immunologically privileged sites. As already mentioned, evidence from studies in the eye, brain, and testis indicates that exposure to antigens within these immunologically privileged tissues induces tolerance (or acquired immune deviation) rather than autoimmune reactions. Fundamentally, peripheral tolerance involves deletion, anergy, or induction of regulatory/suppressor T-cells. This phenomenon has been studied most extensively in
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the anterior chamber of the eye, although the situation in the brain and testis has also been investigated. Extensive studies by Streilein, Niederkorn, and colleagues have established that introduction of antigen within the eye leads to a deviant immune response characterized by the production of regulatory T-cells, CD8+ cytotoxic T-cells (but not CD4+ helper T-cells) and B-cells that preferentially secrete noncomplementfixing antibodies [239]. In ACAID, trafficking of the resident APCs directly via the blood to the spleen (thereby bypassing local lymph nodes) appears to be important [58, 240, 277, 286]. The deviant response develops in the spleen, involving regulatory T-cells (both CD4+ and CD8+ ), immunoregulatory NK T-cells, and gd T-cells, as well as the cytokines thrombospondin, TGFb, and IL-10 [144, 227, 233, 234, 253]. Similar mechanisms appear to operate in the creation of BRAID [278], but direct passage to the spleen does not appear to be essential and the deviated response may occur in the draining cervical lymph nodes instead. Similarly, data suggest that introduction of soluble antigen into the testis leads to development of TAID [48, 179, 270], even though the draining lymphatics of the testis connect directly to conventional pelvic and abdominal lymph nodes [88, 104, 158]. The development of regulatory T-cells also appears to be important in maintaining immunoprivilege during pregnancy [41, 92, 232, 300] and in the survival of tumors [225, 229, 287]. Another mechanism for creating tolerance at immunologically privileged sites involves inactivation and/or deletion of antigen-specific T-cells within the immunoprivileged site itself. The mechanisms proposed involve the T-cell death ligand FasL or CD95L (discussed in more detail in the next section) and the tryptophan metabolizing enzyme indoleamine 2,3 dioxygenase (IDO). Inhibition of IDO activity compromises fetal survival in allogeneically unmatched pregnancies [152]. Data suggest that degradation of tryptophan by IDO, which is expressed by the trophoblast and by interuterine macrophages, inhibits T-cell activity and thereby controls autoimmune attack — although the actual mechanism responsible for IDO protection of pregnancy remains somewhat contentious. There is evidence that this mechanism may be relevant in other immunologically privileged sites [109, 126]. There is considerable evidence that T-cell-mediated tolerance plays an essential role in controlling autoimmunity to organ-specific antigens in the testis and ovary. The cluster of autoimmune diseases induced by thymectomy at age day 3 in mice and rats, which is attributed to
the elimination of regulatory T-cells, frequently includes both orchitis and oophoritis [169, 249, 266]. Moreover, testicular and ovarian autoimmunity and subsequent hypogonadism occur in a subset of sufferers of the human polyglandular autoimmune (PGA) syndromes, caused by a defect in regulatory T-cell function (type 2) or mutation in the AIRE gene that controls thymic expression of organ-specific genes and susceptibility to autoimmunity (type 1) [2, 137, 170, 243]. Significantly, PGA type 1 also involves two other less well-characterized immunologically privileged tissues: the adrenal and the liver [2, 180, 230]. Similarly, mice in which the AIRE gene is inactivated display lymphocytic infiltration and/or autoantibodies to the liver, eyes, gonads, and adrenals (as well as a number of other tissues), leading to reduced fertility and adrenal atrophy [107, 198]. These autoimmune disease models in humans and experimental rodents are linked to a shift in the balance between autoreactive T-cells and specific regulatory, or suppressor, T-cells. For example, the isolation of a CD4+ T-cell line that was able to down-regulate the development of adoptive transfer of EAO in mice has been reported [105]. Exposure to testicular antigens during maturation of the immune system has been shown to reduce the severity of orchitis in adult immunodeficient SCID mice, which have had their immune system reconstituted [273]. Evidence from studies in the testis indicates that activated and/or memory T-cells, which would normally effect an immunological response, are instead targeted for destruction when they enter the gonadal environment [45, 166]. Significant populations of cells expressing NK markers, both NK and NK T-cells, are found in the interstitial tissue of the testis and in the decidua in the absence of inflammation [24, 98, 264] (Hedger, unpublished data). Recent studies show that NK cells are able to modulate dendritic cell function and survival in order to control adaptive immune responses, in addition to their role in innate immunity to viruses and tumors [8, 156, 185]. NK T-cells are T-cells with natural killer activity, which possess unique restriction to glycolipid antigens presented by the MHC-like molecule CD1d [19, 70]. These cells play a role in promoting graft survival [13, 14], immunotolerance to tumors [229], and in the generation of CD8+ regulatory/suppressor T-cells in ocular immunoprivilege [58, 164]. Consequently, there is strong evidence that NK and NK T-cells are central to the maintenance of immunoprivilege, either at the tissue itself or within the immune tissues. Finally, γ δ T-cells have been implicated in
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the suppression of autoimmune reactions in a bilateral model of bacterial-induced autoimmune orchitis, in part through production of IL-10 and TGFb [160, 161]. This lymphocyte subset also appears to be involved in maintaining immunological privilege in the eye [227].
FAS LIGAND
On the weight of the evidence, it appears unlikely that FasL on its own is the fundamental explanation for immunoprivilege, although the importance of interaction with other immunoregulatory factors such as TGFb needs to be taken into consideration [33]. It is reasonable to conclude, nonetheless, that FasL plays a role at some level in at least some immunologically privileged sites.
Fas ligand (FasL, CD95L) is a cell death signal for LOCAL IMMUNOSUPPRESSION OF activated T-cells, and interactions between FasL and ADAPTIVE AND INNATE IMMUNITY its receptor (Fas on T-cells) are involved in regulation of the immune response [163]. In 1995, Bellgrau and colleagues presented data that FasL was consti- Fluids collected from immunologically privileged tutively expressed by mouse Sertoli cells, and that sites — including testicular fluid, ovarian follicular fluid, testis cells from mice deficient in FasL or trans- amniotic fluid, and the aqueous humor — are potent planted into mice deficient in Fas no longer exhibited inhibitors of T-cell activation and survival, as well as evidence of testicular immunoprivilege [18]. It was regulators of several specific lymphocyte functions [57, suggested that Fas-FasL interaction was responsible 91, 110, 272]. This indicates that soluble immunosupfor the prevention of antigen-specific responses within pressive factors are a principal influence on lymphocytes the testis, and, by extension, other immunoprivi- entering these tissues. Some of these factors have been leged sites. This hypothesis received further support identified, and are discussed in the material following, from contemporaneous studies on Fas/FasL expression although the list may yet be incomplete. and tolerance to viral infection in the eye by Griffith and Several cytokines with immunoregulatory properties colleagues [75, 76], and observations that corneal allo- have been implicated in the local control of immune grafts that did not express FasL were twice as likely responses in various immunologically privileged tisto be rejected as normal corneal tissues [242, 296]. sues. The TGFb family is a family of potent immunoregFasL expression by tumor cells has also been implicated ulatory proteins that includes TGFb1, 2, and 3 and in tumor survival [279], and expression on vascular the activins A and B [130, 183, 215]. The TGFbs in endothelial cells in brain immunoprivilege [35]. Evi- particular are extremely effective immunosuppressive dence for a crucial role for FasL in pregnancy is and anti-inflammatory cytokines. TGFb isoforms are ambivalent: FasL is expressed on the trophoblast and present in the aqueous humor (TGFb2), testicular fluid gld mice lacking FasL are prone to leukocyte-mediated (TGFb1), and amniotic fluid (TGFb1 and 2), and have placental failure, but mice lacking Fas have normal been directly implicated in the maintenance of immunopregnancies [100, 208]. privilege in the anterior chamber of the eye, testis, and Unfortunately, the role of FasL expression in immuno- feto-placental unit [40, 127, 146, 172, 175, 191, 244, logical privilege remains a topic of considerable contro- 285]. Activin A is a product of several testicular somatic versy at the present time [173, 201]. Several studies have cells, including the Sertoli cells, and is found at relareported that expression of FasL fails to confer immuno- tively high levels in testicular and amniotic fluids [46, protection in either the testis or other tissues, and in fact 47, 172]. Compared with the TGFbs, the role of activin induces quite virulent neutrophilic inflammatory reac- in immune regulation is less well understood, but activin tions [5, 113, 220]. FasL also appears to be expressed at A has several immunosuppressive actions, including relatively high levels in the epithelia of several human inhibiting T-cell and B-cell function and blocking the tissues that lack any evidence for immunological privi- activity of proinflammatory cytokines [183, 298]. Other lege [293]. In addition, the methods used to detect FasL immunoregulatory cytokines involved in immunoregexpression — particularly the mature protein — in sev- ulation produced in the immunologically privileged eral immunoprivileged sites have been subject to some tissues are IL-10 [133, 172, 225] and the IL-1 receptor criticism [44, 118, 201]. Finally, the absence or inhibi- antagonist (IL-1ra) [117, 291, 301]. Notwithstanding its tion of Fas or FasL does not automatically cause failure largely proinflammatory functions, macrophage migraof immunoprivilege in many systems [208, 244, 271]. tion inhibitory factor (MIF) also appears to play a vital 577
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role by inhibiting the cell-killing activity of cytotoxic T-cells and NK cells at immunoprivileged sites [1, 10, 151, 200]. This is vital, in that NK cells and T-cells are a common feature of several of the immunoprivileged tissues, particularly the testis and decidua [98, 264]. Apart from the cytokines, various bioactive peptides and small proteins possessing immunoregulatory activity are associated with immunoprivilege in the eye. These include the neuropeptides vasoactive intestinal peptide (VIP) and somatostatin [256, 257], the calcitonin gene-related peptide (CGRP) [258], and the pro-opiomelanocortin (POMC) peptide a-melanocytestimulating hormone (a-MSH) [165, 255]. Several POMC-derived peptides, including a-MSH, are also produced in the testis, ovary, and placenta [32, 132, 187]. In addition, particularly in the eye and placenta, a local regulatory role is indicated for soluble forms of the immunoregulatory membrane-bound ligands, FasL and the MHC class Ib antigens, HLA-G and HLA-E [81, 125, 128, 155, 210, 245]. Corticosteroids are potent immunosuppressives, and are part of the body’s normal regulation of the inflammatory process [115]. These steroid hormones are produced almost exclusively by the adrenal gland, and no doubt contribute to immunoprivilege in that organ [124]. However, the sex steroids — particularly testosterone and progesterone — also have a number of immunoregulatory activities, including suppression of APC and lymphocyte activity and stimulation of type 2 cytokine production [9, 43, 52, 77, 141, 184, 218, 254]. The mechanisms of action remain uncertain, in that peripheral leukocytes do not express conventional steroid receptors [20, 38, 135]. Although testosterone concentrations are extremely high within the testicular interstitial tissue, the evidence for a role for androgens in immunoprivilege in the testis has been equivocal. Manipulation of local and circulating androgen levels had no effect on survival of parathyroid allografts in the normal rat testis [281–283] or the survival of pancreatic cell allografts in the abdominal testis model of Selawry and colleagues [30, 224]. In the absence of any comprehensive study of this issue, it remains difficult to say whether or not androgens do play an important local role in immune regulation in the testis. In pregnancy, production of progesterone by the placenta has been postulated to exert immunosuppression at the maternal-fetal interface [52], and progesterone has been implicated in controlling proinflammatory cytokine production by cervical cells, which can lead to premature labor [176, 263].
The potential role of prostaglandins and other bioactive lipids in controlling immunological privilege appears to have been almost completely ignored by researchers in this field. Acting via specific receptors, E-series prostaglandins exert both proinflammatory and immunoregulatory effects on T-cells, macrophages, and dendritic cells [68, 82, 159, 216], whereas prostaglandins of the D and J series as well as lipoxins possess specific anti-inflammatory actions [82, 106, 159, 250]. Moreover, phosphatidylcholine-containing lipids, produced by the cleavage of arachidonic acid from phospholipids by the action of phospholipase A2, posses both anti-inflammatory and immunoregulatory actions [108, 251]. The enzymes responsible for production of these lipids (and the lipids themselves) are found in all tissues, but their potential role in controlling immunoprivilege has been investigated in only a handful of studies [153, 195, 202]. Finally, inhibition of complement is an important mechanism for reducing transplant rejection, particularly in the early phase. Cell surface complement inhibitors — such as CD46, CD55, CD59, and the complement receptor related protein (Crry) — are expressed in the eye membranes, on the trophoblast, and in the testis, and have been implicated in suppression of intraocular and placental inflammation in particular [23, 26, 149, 231, 236]. The source of local immunosuppression can be from circulating and resident immune cells, but just as often somatic cells (and even germ cells in the case of the reproductive tissues) can be responsible. The Sertoli cell of the testis provides a particularly interesting example of a somatic cell with specialized immunoregulatory properties, and indeed there have been a number of studies on the use of Sertoli cells as supporting cells in engraftment studies. Sertoli cells from immature rat, murine, or porcine testes display extended survival as allografts or xenografts, and co-transplantation of Sertoli cells or testis cell mixtures containing these cells confers increased survival on neural cell xenografts, and pancreatic islet allografts, and xenografts [18, 122, 221, 244, 252, 289, 297]. The number of Sertoli cells transplanted is a critical determinant of success [123], but the exact mechanisms are poorly understood. Sertoli cells are major testicular sites of production of immunosuppressive factors, including both TGFb and activin A [47, 222, 260, 292]. Production of TGFb1 by co-transplanted Sertoli cells has been implicated in protection of pancreatic islets implanted under the kidney capsule of syngeneic recipients [244]. Studies on a role
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for FasL expression, however, have been equivocal [18, 244, 252]. Other protective mechanisms have also been suggested, including the ability of the Sertoli cell to form a physical barrier through reformation of inter-Sertoli tight junctions within the mixed graft [214], the inherent ability of the Sertoli cell to provide a fully supportive environment for cell growth and differentiation [288], and the fact that Sertoli cells express no MHC class II and comparatively low levels of MHC class I, enhancing their potential to avoid detection by T-cells and subsequent immune activation [78, 121, 268]. Finally, Sertoli cells possess an enormous capacity for phagocytosis of senescent cells, cell debris, and other potentially antigenic complexes. Some or all of these characteristics no doubt contribute to the unique graft-protecting abilities of the Sertoli cell, and may play a role inhibiting adaptive immune responses in the intact testis. Resident macrophages are found in all immunologically privileged tissues, and a role for these cells under the influence of the local environment is also likely to be important. There is compelling evidence that macrophages from immunoprivileged sites such as the testis, uterus, and brain (microglia) — and at the sites of tumors — possess a characteristic immunosuppressive phenotype characterized by reduced Toll-like receptor (TLR) expression, diminished inflammatory cytokine production (IL-1b, tumor necrosis factor-a, and IL-6) in response to stimulation, and the loss of capacity to produce reactive oxygen and nitrogen products [89, 99, 142, 294]. Instead, these macrophages display increased production of anti-inflammatory cytokines such as IL-1ra, IL-10, and/or TGFb. Because of their central position in the inflammatory and immune response, by producing immunoregulatory cytokines instead of supporting an inflammatory response these cells could be a major determinant of immunoprivilege at these sites.
METHODOLOGICAL ISSUES One factor in studies of immunoprivilege that does not receive enough attention is the influence of technical and methodological factors on the success or otherwise of transplantation, which is the key definition of immunological privilege. The early detailed surveys of the field noted considerable variability in the success of various types of grafts across various studies [16, 85]. The very different results that have been obtained from studies on the testis provide a good example of this issue. In contrast to the results of studies using
laboratory rodents, allograft survival in the monkey and sheep testies has proved difficult to establish, although this conclusion is based on a very small sample of studies [138, 226]. Moreover, highly variable success rates have been reported for the transplantation of germ cell and testis fragments across allogeneic barriers in various species using various protocols [17, 18, 49, 96, 112, 118, 122, 214, 237]. This variability suggests that differences in procedure or other technical issues may be involved. Other contributing factors could be due to differences in the effectiveness of local immunoregulatory mechanisms, species differences in systemic immunity overall, differences in testicular architecture, or physical features of the graft tissue itself such as which cells are present in the graft. In other words, successful grafts across allogeneic and xenogeneic barriers are a proof of immunological privilege, but the failure to achieve grafts is not evidence that privilege does not exist. This variability in graft success also suggests that immunological privilege is not an all-or-none phenomenon. In fact, it might be considered that immunological privilege is an arbitrary term that refers to one extreme end of a continuum in which immune responses in all tissues are under some degree of localized regulation. After all, organspecific autoimmunity such as thyroiditis and diabetes could be thought of as a failure of local mechanisms providing immunoprivilege for local autoantigens. Ultimately, understanding immunological privilege may simply involve understanding the fundamental principle involved in immunoregulation throughout the body.
Relevance of Immunological Privilege to Transplantation Medicine One of the most obvious potential benefits from understanding the principles of immunoregulation that operate in immunologically privileged sites such as the testis is the possibility that the same mechanisms might be used to control immune responses at other sites. As has been outlined in this chapter, many of the immunoregulatory mechanisms that operate in the testis (for example) are not specific to this organ. However, if there are immunoregulatory mechanisms that are unique to the testis, where extended graft survival has already been shown to be feasible in spite of ostensibly normal lymphatic drainage, discovery of these mechanisms has the potential to lead to the development of entirely novel
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methods of treating graft rejection in general. The current methods used in transplantation medicine involve broad spectrum immunosuppressives, principally cyclic peptides of fungal origin that interfere with T-cell signaling and prevent proliferation of these immune cells in a nonselective manner [62, 217]. These drugs have considerable limitations and significant side effects, such as generalized immune suppression, nephrotoxicity, and inherent tumorigenicity [196, 211]. The treatment also requires close monitoring and adjustment for the remainder of life. Viable alternatives or adjuncts to these agents would definitely be desirable. The use of Sertoli cells in transplantation provides an interesting proof of principle. This obviously seems to work in certain experimental protocols, but it is by no means clear why it works. Incorporating Sertoli cells into grafts is not going to be a practical solution for therapy in humans, and thus reaching an understanding of the exact properties of the Sertoli cell that provide protection is essential. Furthermore, the concept that certain features of immunoprivilege can only be explained by localized immunosuppression is most attractive for development of novel therapies. Although physical barriers, altered lymphatic drainage, systemic immunoregulation, and local immunosuppression all play a role in immunological privilege, it is the mechanisms responsible for local immunosuppression that provide the best opportunity for new drug development.
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31 Converging Cell Therapy with Biomaterials Omar A. Ali and David J. Mooney Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts
Introduction Cell therapies are designed to restore or replace tissue function or to remove dysfunctional tissues by regulating the presence and function of cell populations. These approaches have the potential to treat a broad range of disease states (including organ failure, cancer, and autoimmunity) that stem from sources that include trauma and genetic defects [122]. The ultimate goal is to create a dynamic living therapy that appropriately interacts in concert with the host response and environmental signals. The idea of using cells for therapy has been proposed and implemented for a variety of pathological conditions that require either the replacement of tissue function or the removal of dysfunctional tissue. Clinical use of cellular therapies began with bone marrow transplants that were introduced to reconstitute the immune systems of cancer patients who had undergone chemotherapy and radiation treatment [108]. Technological advancements have allowed cell therapy to branch into other areas where the restoration of tissue function is needed. Initial clinical trials involved the direct injection of cells into injured tissue sites, such as skeletal myocytes into the scarred tissue of the heart and neurons into the brain of patients suffering from Parkinson’s disease [25, 50, 72, 84]. The direct application of skin grafts along with stem cell therapies for bone and cartilage reconstruction have also been implemented in clinical trials, and some have progressed through FDA approval [23, 84, 131]. Other efforts have targeted the development of cancer vaccines that remove harmful cancerous tissue with immunotherapeutic approaches that modify a patient’s own tumor-infiltrating leukocytes or dendritic cells (DCs) [42, 132, 140]. It is estimated that novel cell
Cellular Transplantation: From Laboratory to Clinic
therapies could be applicable to more than 67 million Americans (Table 31.1). Administering cell therapies with biomaterials may greatly enhance their efficacy due to mechanical and chemical cues delivered by the materials [36]. Synthetic and naturally derived biodegradable materials are often utilized to transplant or locally recruit the specific cell types targeted for therapy, as they mechanically support and guide the function of these cells. Material surfaces can be modified with bioactive peptides to control cellular adhesion, functionality, and subsequent tissue growth [68, 74]. Soluble growth factors and plasmid DNA-encoding bioactive factors and drugs have also been incorporated and delivered from biomaterials to control cellular processes such as migration, adhesion, proliferation, and differentiation [36, 68, 74]. These approaches may be especially important in addressing the widespread loss of viability and function of transplanted cells, as biomaterial-mediated delivery can spatially and temporally maintain control over the transplant microenvironment. Similarly, injecting solutions of growth factors to stimulate various host cell populations has been plagued by the inability to sustain signaling, as these molecules have short half-lives and are susceptible to degradation [27, 36]. Biomaterial delivery may address this challenge by stabilizing these factors and prolonging tissue exposure. The immune response to biomaterials must be characterized in these applications, as it may affect therapeutic signaling to cellular targets through the accumulation of phagocytic cells and inflammatory cytokines, and the up-regulation of specific immune responses is the intention of cancer vaccines and other cellular therapies [42, 68, 140, 157]. Autologous and allogeneic cell transfer initiates immune mechanisms that lead to inflammatory responses that compromise long-term
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Omar A. Ali and David J. Mooney TABLE 31.1 Number of American patients who could potentially benefit from cell therapies [1–10]. Condition
Number of People Affected
Diabetes Ischemic heart disease1 Cancer Osteoporosis Autoimmune diseases2 Alzheimer’s Parkinson’s Burns (severe) Spinal cord injuries Birth defects Total
18,000,000 13,000,000 12,000,000 10,000,000 7,500,000 4,500,000 1,500,000 650,000 400,000 120,000 67,670,000
A second approach is to modify the surface of inert biomaterials with bioactive peptides that control the adhesion and function of specific cell types. A third approach involves incorporating growth factors or DNA into scaffold constructs that temporally control and sustain the release of these factors over an extended period of time. These approaches may also be utilized to target immune cells and modulate their functionality to anti-inflammatory tissue-regenerative states or to proinflammatory tissue-destructive states.
The Immune Response to Biomaterials
1. Ischemic heart disease includes acute myocardial infarction, other acute ischemic (coronary) heart disease, angina pectoris, atherosclerotic cardiovascular disease, and all other forms of heart disease [4]. 2. Autoimmune diseases include rheumatic diseases, AIDS, and multiple sclerosis [5].
graft survival and function [35]. Immune responses to biomaterials may increase material degradation, resulting in device failure or may inappropriately modulate cell adhesion and tissue reconstruction [35, 159]. Activated immune cells will release cytokines and other bioactive molecules that have been shown to affect the differentiation and functionality of endothelial cells, neuronal cells, smooth muscle cells, and fibroblasts [158, 171]. Inflammatory cells may also intercept bioactive molecules released from biomaterials, inhibiting their ability to act on target cells. This chapter reviews the development of sophisticated biomaterials used to mediate host and transplanted cell responses in order to facilitate one of the two basic objectives of cell therapy — to develop functional cells and tissue or to eliminate harmful cells and tissue. The basics of the immune response to materials are first reviewed, and the design of biomaterials to either suppress or up-regulate this response for therapy is subsequently reviewed. There are several approaches to designing biomaterial cell therapies so that they may integrate with host responses and minimize the detrimental effects of the immune response. One approach involves local cellular transplantation into disease sites by seeding the appropriate cells onto biodegradable polymeric scaffolds, or encapsulating the cells into biologically inert materials.
Effective administration of specific biomaterial cell therapies will require an understanding of how the host’s immune system will respond. An unplanned or unintended immune response to biomaterial cell therapies may complicate control of the phenotype and function of transplanted cells, which is the key to effective therapy. The host response to an implanted biomaterial proceeds by the foreign body reaction, which is initiated by nonspecific protein adsorption and denaturation that manipulates host cellular adhesion and can cause immunogenicity [37]. This results in inflammation, and inflammatory cells produce chemical mediators that locally guide the body to isolate the implant within a capsule of fibrous tissue [35, 37, 158, 177]. Evaluation of the biological responses to biomaterials must be characterized by identifying the specific players involved at the phases of protein adsorption, inflammation, and encapsulation and how they may affect the appropriate integration of cellular therapies within the host environment.
PROTEIN ADSORPTION AND IMMUNE RECOGNITION The foreign body reaction is initiated by nonspecific protein adsorption followed by immune recognition of denatured proteins that mediate platelet and macrophage adhesion at the surface of implanted biomaterials [13]. Biomaterial surfaces may nonspecifically adsorb proteins within seconds after implantation, and these proteins are most likely to be soluble proteins, or blood proteins (such as complement or fibrinogen) — in that tissue and structural proteins such as collagen do not freely diffuse to the implant surface [37]. Upon surface deposition, a relatively inert biomaterial
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can become bioactive, as individual proteins undergo conformational changes that expose binding sites that facilitate platelet, complement, or macrophage adhesion and activation [13, 37]. Immune recognition begins with the activation of platelets and the complement pathway via adherence to the adsorbed protein layer, and in some cases directly to the biomaterial surface [80, 157]. Platelets that adhere and coagulate to the adsorbed protein layer will release a variety of cytokines, including PDGF and TGF-b, which are chemotactic for neutrophils, monocytes, and fibroblasts [80, 177].
which up-regulate the expression of adhesion molecules on inflammatory cells and facilitate the recruitment of a large number of cells to the implant site [63, 117, 171]. Infiltrating macrophages and dendritic cells (DCs) are the antigen-presenting cells that are most responsible for generating immunocompetent responses to a particular antigen, but in reactions involving biomaterials the lack of proper antigen and activation signaling may induce these cells to continually produce reactive oxygen molecules and proteases in attempts to destroy and break down the biomaterial [88, 110, 114, 140]. (See Table 31.2.)
INFLAMMATION
FIBROUS ENCAPSULATION
Inflammatory responses to biomaterials are characterized by immune cell infiltration and adhesion to the materials and their subsequent production of inflammatory cytokines. In its acute state, inflammation is characterized by swelling as blood pours into the injured site along with the emigration of neutrophils and other leukocytes from the circulation into the implant site [95]. Neutrophils, attracted by the soluble factors (e.g., PDGF and TGF-b) released by platelets and complement, are responsible for engulfing microorganisms, releasing degradative enzymes and reactive oxygen intermediates ROIs, and the clearance of bacteria and foreign materials [95]. Monocytes are recruited following the initial influx of neutrophils, and may differentiate into macrophages. Macrophages are very important cells in repairing injury [13, 63]. These leukocytes will adhere to the protein monolayer through ligandreceptor interactions. Monocytes and then macrophages attempt to phagocytose the foreign biomaterial while releasing a variety of growth factors — including TGF-b, PDGF, and GM-CSF — that promote the migration, proliferation, and the activation of additional macrophages [13, 171]. The inability of the immune system to clear biomaterial implants is hypothesized to result in “frustrated phagocytosis,” which causes chronic inflammation as macrophages may actively persist throughout the lifetime of the biomaterial [13]. The accumulation and activation of macrophages will continue as long as they are unable to phagocytose and rid the body of the biomaterial, and macrophages will increase their production of degradative enzymes and later fuse to form foreign body giant cells (FBGCs) to increase their capacity to engulf the material [14]. The macrophages continue to produce the proinflammatory cytokines TNF-a, IL-1, and IL-6,
The inability of the immune response to clear implants leads to the development of fibrotic tissue that encapsulates the biomaterial and isolates it from local tissue. While neutrophils and macrophages attempt to disinfect and degrade the implant they produce stimulatory cytokines and chemoattractants (such as PDGF and EGF) that induce fibroblasts and endothelial cells to deposit matrix and form granulation tissue [13, 63]. The production and deposition of collagen and fibronectin act to seal the biomaterial off from the rest of the body [13, 37, 63, 177]. Ultimately, fibrous tissue — which consists of collagenous connective tissue (including fibroblasts, macrophages, and capillary structures) along with foreign body giant cells FBGCs result from this process [13, 14, 37, 63, 177]. The development of fibrotic tissue may interfere with the proper integration of cellular therapies with the host by reducing appropriate cell-cell contact, vascularization, and nutrient transport, and may lead to the lack of tissue functionality or transplanted cell death [110].
Biomaterial Design To develop successful cellular therapies, it is important to develop systems that regulate the fate of cell populations and that may preferentially minimize or up-regulate the host immune response. Biomaterials are now being modified to modulate the migration, adhesion, and proliferation of specific cell populations through the inclusion of various mechanical and chemical cues. Materials may provide the necessary structural support as scaffolds for cell transplantation and/or incorporate bioactive peptides, growth factors,
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Omar A. Ali and David J. Mooney TABLE 31.2 The important phases, cells, and chemical mediators in the foreign body response to biomaterials. Phase
Activated Cells
Protein adsorption
Platelets
Chemical Mediators
Biological Activity
PDGF, TGF-a
Promotes immune recognition: recruits neutrophils, monocytes, fibroblasts Platelet coagulation and clotting; facilitates neutrophil and monocyte adhesion to biomaterial Recruits monocytes; disinfects and kills bacteria; material degradation Monocyte accumulation, proliferation, and activation; phagocytosis and biomaterial degradation “Frustrated phagocytosis” macrophage chemotaxis and accumulation; production of inflammatory mediators up-regulated Fibroblast and endothelial cell recruitment and activation; connective tissue and blood vessel formation Produce and deposit collagen and ECM components; fibroblast proliferation; formation of fibrotic tissue to isolate biomaterial Promotes neovascularization; fibronectin and collagen synthesis; fibrous encapsulation Increased capacity for phagocytosis and material degradation; promotes ECM synthesis
C3a, C5a, C3b, IgG
Acute inflammation
Neutrophils
PDGF, ROI
Monocytes/macrophages
PDGF, GM-CSF, ROI, TGF-a, IL-1 ROI, IL-1, IL-6, TNF-a
Chronic inflammation Macrophages/FBGC PDGF, FGF, EGF, HB-EGF, TGF-a, TGF-b
Granulation tissue fibrous encapsulation
Fibroblasts
PDGF, IGF, EGF
Endothelial cells
IGF-1, TGF-a , TGF-b
Foreign body giant cells
IL-1, TNF-a, TGF-b, ROI
and DNA. These approaches may be used to create a microenvironment that modulates the immune response and may produce better integration between the cell therapy and the host. (See Figure 31.1.)
MATERIALS AS DELIVERY VEHICLES AND SCAFFOLDS Transplanting cells in materials that have optimized physical properties — including degradation rate, stiffness, and porosity — helps to maintain cellular viability and function and may enhance tissue regeneration. Biodegradable scaffolds have been synthesized from natural materials (including collagen and alginate), as well as from a variety of synthetic polymers, including polyglycolide (PGA), polylactide (PLA), and their
co-polymers polylactide-co-glycolide (PLG) — which degrade into the natural metabolites lactic and glycolic acid [68, 74]. Recent studies indicate that matching the degradation rate of the scaffold with the rate of tissue formation can increase the amount and quality of bone formation, demonstrating that matching degradation rates to tissue regeneration is important for proper tissue formation [11, 12]. Conversely, inappropriate material degradation in cellular therapies has been shown to promote the production of ROI and degradative enzymes by inflammatory cells and the formation of FBGC [13, 95, 177]. The stiffness of the materials to which cells adhere also affects cellular proliferation [92] and cellular receptiveness to signaling molecules such as DNA in gene therapy approaches [91]. The introduction of appropriately differentiated cells and tissues as biodegradable material-cellular
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a. Biomaterial
Unmodified biomaterial
Protein adsorption and immune cell adherence
b.
Biomaterial
Fibrous encapsulation
d.
TISSUE
c.
e.
Figure 31.1. Modifying biomaterials to control cell responses. (a) Schematic of the typical foreign body response to unmodified biomaterials, including nonspecific protein adsorption, immune cell adherence (during inflammation), and fibrous encapsulation. (b) Cellular transplantation in tissue defect sites using degradable biomaterials may reduce fibrous tissue formation and enhance host tissue integration. (c) Bioactive peptides may be immobilized into biomaterials to control and specify cell adhesion. (d) Growth factors and other proteins are incorporated into biomaterials and released to manipulate cell migration, proliferation, and differentiation. (e) Plasmid DNA is incorporated into biomaterials and released to cells that take up the DNA and locally express its protein product.
constructs may enhance the quality of tissue regeneration and reduce the formation of fibrotic tissue. Many cell types — such as endothelial cells, hepatocytes, chondrocytes, and osteoblasts — require anchorage to extracellular matrix (ECM) for survival and to maintain their phenotypic state, a function that may be mimicked by biomaterials but missing in defect sites [49, 114, 149, 163, 167]. Autologous cells may be cultured in 3D porous PLG scaffolds or in collagen gels, in vitro, as model systems or to optimize tissue development before local implantation, and to reduce fibrotic tissue formation in favor of proper host tissue integration [114, 149]. Skin substitutes that place autologous dermal fibroblasts and keratinocytes onto biopolymers highlights the advantage of optimizing 3D cell cultures into differentiated and functional tissues that can appropriately interact with the adjacent cells of the host without the formation of fibrotic tissue [28, 29]. The porosity of materials may also be optimized to control cell-cell contact and tissue infiltration into the materials. Materials with an interconnected macroporous structure enhance tissue penetration, and
may promote the appropriate cell-cell contact needed for tissue regeneration [128]. In contrast, semipermeable membranes formed from polycations and alginate (a bioinert semipermeable hydrogel) are porous enough to allow for the passage of growth factors but resist cellular adhesion and immune responses and have been used to immunoisolate transplanted cells [60, 130, 162]. For example, encapsulated rat islets implanted in subcutaneous tissue were shown to restore normoglycemia in diabetic mice [130]. Similarly, encapsulated fibroblasts that were genetically modified to produce BDNF survived transplantation into the injured spinal cords in murine models, in the absence of immune suppression, and provided a permissive environment for host axonal growth [162]. Generally, manipulations of the physical properties of materials have focused on enhancing transplanted cell viability and function, but transplanted cells can also secrete factors and mediate signaling that inhibit proinflammatory processes and direct the desired host immune response to promote tissue regeneration. Co-cultivation of fibroblasts with macrophages has been
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shown to increase the cell-mediated synthesis of a matrix that may be favorable for tissue remodeling and repair [150]. Similarly, co-cultivation of endothelial cells with macrophages treated to down-regulate the production of proinflammatory factors produced a proangiogenic environment that promoted endothelial cell proliferation [90, 110, 113]. Macrophages, in particular, have been implicated in the vascularization of polymer meshes used to transplant endothelial cells in rat models, which were not able to form vascular networks in vitro [79, 110]. Transplantation of endothelial progenitor cells into mice was shown to enhance revascularization, which coincided with an increase in the numbers of recruited monocytes and macrophages [155].
BIOACTIVE PEPTIDES Biomaterials are being designed to promote the adhesion of specific cell types by incorporating bioactive peptides with amino acid sequences specific for the cellular receptors called integrins. Cells normally bind to the ECM through receptor-ligand interactions, which modulate cell proliferation and differentiation in numerous cell types [21]. An important criterion in creating adhesive materials that are cell specific is that the materials exhibit little intrinsic host cell adhesion, and a number of polymers — including alginate and polyethylene glycol (PEG) — meet this criterion [77, 147]. Peptides immobilized in biomaterials commonly contain the amino acid sequence Arg-Gly-Asp (RGD), the cell-binding domain of fibronectin and other adhesive proteins [15]. RGD and other peptides have been immobilized onto biomaterial surfaces or incorporated into the bulk of biomaterials to facilitate cell adhesion, and promote the proliferation and differentiation of various cell types — including fibroblasts, osteoblasts, neuronal cells, macrophages, and endothelial cells [15, 27, 77, 147]. Because different cell populations express various integrin receptors, it is possible to use distinct peptide sequences in order to bind specific cell types to biomaterials. The amino acid sequence REDV (derived from fibronectin), for example, has been reported to specifically bind endothelial cells (ECs) and thus may be useful in designing vascular grafts [82, 103, 104, 147]. The sequences YIGSR and KQAGDV have been shown to mediate the adhesion of neuronal and smooth muscle cells [102, 147]. One may be able to promote the adhesion and proliferation of specific cell populations and
organize tissue growth by also changing the structure and orientation of immobilized ligands. Peptide incorporation into biomaterials can organize cellular adhesion and subsequent tissue growth, but it has also been shown to modulate the immune response by manipulating macrophage adhesion and function. Incorporating specific peptide sequences could render the material resistant to macrophage adhesion and minimize immune responses. The formation of FBGC was shown to be highly dependent on the relative orientation between PHSRN and RGD domains incorporated onto polyethyleneglycol-based networks [87]. Although RGD domains have been demonstrated to increase monocyte and macrophage attachment to biomaterials, cyclic RGD treatment has been shown to significantly reduce the recruitment of macrophages and repress the production of proinflammatory cytokines [55, 69, 86]. RGD incorporating poly(L-lysine)-g-poly(ethylene glycol) (PLL-gPEG) microspheres enhanced ligand-specific targeting of monocytes and dendritic cells over other peptides, and may aid in the therapeutic targeting of these cells [54]. YEILDV represents a sequence that may be more specific for monocytes macrophages than are RGD sequences, and may be utilized to control their adhesion and response to biomaterials [170].
PROTEIN DELIVERY Soluble proteins, or growth factors, may be incorporated into biomaterials and utilized to transmit signals that modulate cellular function. For example, PDGF, BMP, and VEGF have been delivered from materials in order to induce the migration, proliferation, and differentiation of smooth muscle, bone, and endothelial cells, respectively [27, 36, 64, 97]. To effectively administer these molecules, one must optimize factor delivery to the target cells while minimizing signal interception by nontarget tissues and cells, which can lead to a lack of appropriate signaling and undesired side effects. These molecules are commonly injected in solution form locally or systemically, but the therapeutic effect is lost quickly because these molecules have short half-lives and are quickly degraded [27, 36, 64, 96, 97]. Biomaterial implants can be modified to locally sustain the delivery of growth factors to target cells over an extended time course [27, 36, 96]. Natural polymeric materials, including albumin gels and collagen, have been simply mixed with growth factor to create a
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protein carrier [94, 96]. Synthetic polymers, including PLG, have been utilized to fabricate protein-loaded microspheres or gas-foamed scaffolds [97, 127, 145]. In these systems, growth factor release is controlled by diffusion and/or material degradation. These systems can be utilized to deliver two or more growth factors with distinct kinetics, and this increases the potential to mimic developmental biology by delivering numerous growth factors that regulate tissue formation at various stages. Although current efforts focus largely on signaling specific target cells for regeneration, biomaterialmediated growth factor delivery may also be utilized to minimize or modulate immune cell responses. Exposing macrophages to IL-4 in the absence of antigenic stimulation has been shown to create a matrix-secreting and proangiogenic macrophage phenotype, and to decrease FBGC fusion and the production of proinflammatory cytokines [90, 113]. The immunoregulatory cytokines TGF-b and IL-10 play an important role in dampening macrophage activation and inflammation, and may be used to suppress immune responses to biomaterials or to counteract rheumatic autoimmune diseases [65, 90, 113]. IFN-g and TNF-a can induce the proinflammatory phenotype of macrophages [152], and a recent study has shown that sequentially regulating the exposure of macrophages to IFN-g , IL-12, IL-4, or IL-10 and antigen stimulus can reversibly modulate the development of proinflammatory and anti-inflammatory subsets [152]. Similarly, although the delivery of GMCSF has been shown to expand dendritic cell populations in vivo the activation state of these cells depends on secondary signals, which may include antigen or cytokines such as IL-4 [33, 135]. The ability to create local biomaterial depots that can control the release of multiple growth factors may be particularly useful in manipulating immune cell migration and activation.
GENE DELIVERY Biomaterials may be utilized to locally deliver DNA, which encodes for growth factors and other bioactive proteins, to target cells. The stability of plasmid DNA, as compared to proteins, and the prospect of having a more bioactive protein product being produced by genetically modified cells in vivo make it an attractive approach for regulating cell populations with growth factors or cytokines. Plasmid DNA delivery currently results in low transfection rates in vivo [100], but these limitations
could be bypassed by the development of biomaterial vectors that can sustain the local delivery of the gene without systemic side effects [144]. In addition, biomaterial carriers may potentially deliver the gene directly to local target cells and tissues. In gene therapy, production of a biologically active protein requires that exogenous cDNA penetrate the cell membrane, avoid lysosomal degradation, and enter the nucleus to undergo transcription [100]. Plasmid DNA and adenoviral vectors have been widely used to express the target gene transiently without incorporation into the chromosome. These methods, however, are not suitable to induce long-term gene expression — which may be required in certain diseased states because they exhibit low transfection efficiencies and the copy number of the administered gene decreases through dilution by cell division and intracellular degradation [89, 107]. To overcome this shortcoming, repeated administration of the plasmid or virus vector is required, but this imposes a heavy burden on the patients because the delivery of supraphysiological amounts of genetic material is inconvenient and may cause side effects [100, 107, 144]. Macroporous scaffolds that allow for the infiltration of host cells have recently been designed to immobilize DNA and to sustain its delivery to native cells for extended periods [144]. These materials are able to enhance DNA transfection over plasmid delivery alone, apparently without the side effects and safety concerns associated with viral vectors. PLG scaffolds have since been modified to incorporate DNA that has been condensed with polycations to form electropositive nanoparticles that improve transfection efficiency and extend the duration of gene expression [81]. The delivery of plasmid DNA-encoding PDGF from PLG scaffolds was found to enhance matrix deposition and blood vessel formation [89], and previous studies demonstrated that delivery of plasmid DNA-encoding BMP-4 and PTH could enhance the bridging of bone fractures [53]. Immune cells may also be targeted to express desirable genes using biomaterials to deliver plasmid DNA. Similar to the use of adenovirus for gene therapy, the host immune response to biomaterials may limit gene expression in immunocompetent animals due to immune cell uptake and degradation [81, 107, 120]. DNA that is intercepted by infiltrating macrophages will likely not result in significant gene expression and protein production, as the transfection efficiency of these cells is very poor due to the barriers of phagosomal and endosomal degradation [121]. However, continuously
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stimulating macrophages and other immune cells with DNA for extended time frames via biomaterial-mediated delivery may result in biologically relevant gene expression that may be especially attractive for vaccine formulations.
Promoting Therapeutic Immune Responses Although many biomaterial systems are currently being developed to target nonimmune cells, it may also be critical for cellular therapies to target immune cells that can aid in regenerating or removing cell populations. It is important to note that all of the biomaterial modifications that have been designed to date have had effects, perhaps unintentionally, on the immune response. All tissues in our body are homeostatically maintained by interactions with the immune system that contribute to cellular turnover, tissue remodeling, and bacterial clearance, and these processes deserve consideration in designing biomaterial cell therapies. This section addresses the current focus of biomaterial research in both tissue regenerative and destructive applications, and discusses the possibility of promoting the appropriate function of macrophages and dendritic cells DCs to serve as mediators. Using biomaterials as cell therapy vehicles provides the interesting possibility to locally target signaling molecules to macrophages and DCs that innately migrate to the implant site as part of the inflammatory response. The activities of macrophages are diverse, and the conflicting nature of certain activities of these cells appears to be dependent on the microenvironment. These cellular activities can be proinflammatory, immunogenic, and tissue destructive, or anti-inflammatory, tolerogenic, and tissue restorative [90, 113]. For tissue regeneration, it may be possible to harness some of the beneficial functions of macrophages to remodel tissues and promote the expression of angiogenic and other stimulatory growth factors while avoiding some of their detrimental behavior, including fusion into FBGCs and the production of inflammatory cytokines. Conversely, DCs may be exploited to generate specific immune responses against unwanted cell populations. Controlled macrophage and DC function could have wide therapeutic potential, including mediating angiogenesis or tissue remodeling, regulating tumor cells, or inducing the release of specific cytokines and growth factors to direct host healing and immune responses [65, 151, 152].
REGENERATING TISSUE Conventionally, immune cells and inflammatory respon ses are associated with immune surveillance and the clearance of foreign bodies, but macrophages are especially important in maintaining tissue growth and homeostasis. A subset of macrophages, termed the alternatively activated macrophage, has been associated with immunosuppression and tissue repair [65, 90, 113, 151, 152]. Macrophages that are present in the early inflammatory response display cytotoxic activities, whereas those present at later times can produce matrix and promote tissue regeneration and angiogenic activity via the production of growth factors [113]. These cells also remodel tissue by the regulated production of a family of matrix metalloproteinases (MMPs) that are involved in proteolysis of the ECM and can regulate cell adhesion, chemotaxis, and other cellular responses important in tissue repair [66]. However, dysfunctional inflammation and tissue remodeling can lead to scarred, ischemic, and nonfunctional tissue [44]. These findings suggest that macrophages play an important role in tissue regeneration by disinfecting, clearing, and remodeling the space required for host cell infiltration and by synthesizing growth factors that facilitate this process. Macrophages have been associated with the degradation and clearance of ECM and scarred tissue that allows for the growth of blood vessels and the regeneration of nerves [44]. Immune cells can also produce growth factors that stimulate and mediate the recruitment, proliferation, and maturation of angiogenic and nerve cells [57, 66, 113]. The absence of immune cells that can appropriately remodel scarred tissue resulting from myocardial infarction or spinal cord trauma has been associated with dysfunctional vascularization, ischemia, and the loss of cardiac and motor function [17, 57, 98]. Biomaterials may potentially be utilized to actively recruit immune cells to disease sites, and promote the regenerative properties that induce vascularization and regeneration. THERAPEUTIC ANGIOGENESIS Angiogenesis involves the formation of new blood vessels that sprout from the already present vasculature, and is an integral part of many physiological and pathological processes — including wound healing, tissue regeneration, and cancer [17, 57, 98]. The goal of therapeutic angiogenesis is to deliver factors that drive blood vessel formation in ischemic tissue, which could
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aid the 13 million Americans that suffer from ischemic heart disease resulting from obstructed or blocked coronary arteries (Table 31.1). Moreover, inducing angiogenesis could aid in the engineering of new tissues and organs, which are limited by a lack of vasculature that mediates nutrient and waste transport. The general cascade of events associated with angiogenesis includes multiple cell types such as endothelial and smooth muscle cells, and growth factors such as VEGF, PDGF, angiopoietin-1 and 2 (ang-1 and ang-2), and bFGF [57, 61]. The first step in angiogenesis involves the vasodilation of existing vessels, an event that is mediated by VEGF and that increases vascular permeability. The subsequent degradation of the ECM is necessary for endothelial cell migration, and may be mediated by macrophages and monocytes through the production of MMPs. EC proliferation and migration are followed by the assembly of immature vessel networks — and VEGF, ang-1, and ang-2 are key players in the steps of these processes [147]. Immature blood vessels are later stabilized by smooth muscle cells (SMCs), whose recruitment is regulated by PDGF [57]. Biomaterials have been shown to improve the formation and maturation of vascular networks by sequentially delivering multiple growth factors at different stages of blood vessel development [127]. In this study, embedded VEGF recruited endothelial cells in order to play an active role in new blood vessel formation, and then subsequent delivery of PDGF from the material-enhanced vessel maturation. Monocytes and macrophages are key components of the angiogenic machinery, as they first contribute to tissue remodeling and set the foundation for endothelial cell migration and proliferation. Angiogenesis and artereogenesis are often preceded or accompanied by an inflammatory infiltrate composed mainly of monocytes and macrophages [40, 112]. Removal of macrophages reduces angiogenesis and the wound-healing process in animal models [46]. Macrophages participate in tissue remodeling through the secretion of ECM-degrading enzymes (MMPs), which may contribute to neovascularization by staging a pattern for the development of new capillaries [22, 112]. In this role, monocytes and macrophages may first “tunnel” to provide a conduit for endothelial cell infiltration and revascularization, and then produce factors such as VEGF, angiopoietin-2 (ang-2), basic fibroblast growth factor (bFGF), and tumor necrosis factor-a (TNF-a) to promote the angiogenic process [111, 121, 138, 156]. For example, macrophages
contribute to the progression of neovascularization in the developing eye and other organs by degrading the ECM and paving the way for native cellular migration, and providing various growth factors that modulate their phenotype [66, 111]. It may be advantageous to mimic this natural process by incorporating macrophages into cellular therapies that seek to create viable and functionally mature blood vessels. Biomaterial cell therapies may enhance angiogenesis at the sites of trauma and scarring by coordinating immune cell infiltration and up-regulating their angiogenic function. Precedence for this concept is found in the many tumors that recruit monocytes and macrophages to their necrotic regions and subsequently induce their pro-angiogenic function to facilitate growth [115]. In some applications, the recruitment of macrophages at the site of biomaterial implantation has been correlated with the increased production of MMPs and vascularization [67, 129]. The chemotactic molecule chemokine ligand-2 (CCL2) has been delivered from biomaterials to increase the recruitment of monocytes and macrophages to the implant site, but has also been found to participate in FBGC fusion and proinflammatory reactions and these effects coincided with a lack of epithelization and angiogenesis [45, 62]. Interestingly, CCL2-mediated recruitment of macrophages into the tumors and the corneas of animal models promoted vascularization [40, 46, 115]. These findings suggest that for biomaterials to utilize and control macrophages to induce angiogenesis it may be necessary to subsequently direct them to a pro-angiogenic and non-cytotoxic phenotype, and this may involve signaling through IL-4, 1L-10, and TGF-b [24, 51, 113, 124, 146]. Biomaterials could potentially also be designed to genetically modify the function of macrophages to secrete VEGF and other angiogenic factors via DNA delivery. In addition, creating biomaterials that are sensitive to MMPs and degrade in a controlled fashion to create space for vascularization [129] may enhance the remodeling efficacy of macrophages that are recruited into scarred and ischemic regions.
NERVE REGENERATION Functional nerve regeneration is a complex biological process that often occurs after minor peripheral injury, but central nervous system damage and bridging nerve gaps created by major injury require therapeutic intervention [139, 166]. Cell therapies that can attenuate
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or restore the loss of neuronal function could be used to treat a variety of diseases, such as Parkinson’s, or aid in the recovery from traumatic damage. Biomaterials may be particularly useful in facilitating the creation of local environments that inhibit scarring and guide nerve regeneration. The central nervous system (CNS) includes the brain and spinal column, whereas the peripheral nervous system (PNS) consists of the nerves arising from the brain and spinal cord that innervate and transmit sensory and excitatory inputs into muscle tissue [139, 166]. Neurons are the basic functional element of the nervous system and consist of the cell body, or the soma, and its extensions — axons that direct electrical impulses away from the cell and dendrites that direct electrical impulses to the cell. In many cases, a myelin sheath of Schwann cells surrounds the axons of peripheral nerves and support their function, impulse propagation, and regeneration. In the CNS, this myelin sheath of nerve fibers consists of oligodendrocytes. Nerve regeneration can occur in the PNS if the damage does not involve the cell body and proceeds as macrophages, and monocytes remove myelin debris and release neurotrophins [139]. Nerve growth factor (NGF), which is produced by activated macrophages, is the best characterized neutrophic factor and can promote neuronal survival, growth, and branching [118, 164]. Schwann cells support regeneration via production of ECM, cell adhesion molecules, integrins, and neurotrophins that lead axons toward their synaptic targets [173]. In the CNS, however, attempts at regeneration are characterized by neuronal sprouting that is subsequently blocked by glial scarring composed of myelin and cellular debris with limited remodeling capacity due to the local lack of macrophages and proteinases [58, 139]. Biomaterial cell therapies are now being designed to provide physical and chemical pathways that guide neuronal regeneration. Guidance channels or nerve conduits have been fabricated from synthetic biomaterials that can physically direct axonal regeneration to their synaptic targets via architectural cues, and the channel content may be modified by the addition of various cellular and/or chemical elements to enhance neurite extension [52]. Sustained delivery of NGF from synthetic guidance channels has also been shown to increase regeneration in the PNS and CNS of adult rats [26]. Similarly, simultaneous BDNF and GDNF delivery promoted neuronal survival in a rat model of motor neuron damage [168]. Transplanted Schwann cells promote axonal regeneration, and this effect may be enhanced
when combined with protease treatments that degrade scars that inhibit proper regeneration and reconnection in spinal cord injuries [70, 141]. Biomaterials that activate macrophages and antigenpresenting cells may enhance functional nerve regeneration by facilitating myelin degradation and the clearance of scar tissue. Recent studies indicate that controlling cell-mediated immunity can lead to the regrowth of the injured spinal cord [59, 73]. Transplantation of macrophages can decrease myelin-associated glycoproteins and scarring, and increase angiogenesis, Schwann cell infiltration, and axonal regeneration [31, 123]. Similarly, autologous macrophages and DCs — activated against myelin antigens ex vivo and retransplanted at the site of completely transected spinal cords — supported the partial recovery of motor function in rodents [73, 142]. Effective nerve regeneration using biomaterials and macrophages will likely have to be accompanied by other signals that facilitate cellular growth and extension. Although activated macrophages and DCs produce NGF, this singular action may not be sufficient to promote nerve regeneration [32]. VEGF administration from biomaterials may be utilized to promote vascular permeability and increase macrophage recruitment and vascularization at wounded nerve sites, and thus serve as an indirect neutrophic factor [38, 78]. In addition, cell adhesion peptides, laminin, and N-cadherin have been immobilized in fibrin gels that can be degraded and remodeled by proteases released by macrophages, and these materials enhance neurite extension and the number of regenerated axons in animal models [134, 137].
REMOVING DYSFUNCTIONAL CELL POPULATIONS Immune cells regulate many processes that mediate autoimmunity and cancer, and could be targets to therapeutically remove dysfunctional cells. Biomaterials may be utilized to create antigen-specific tolerogenic DCs or antigen-specific activated DCs, by recruiting and targeting antigen delivery to these cells (in vivo), in tandem with the appropriate maturation signals. The inherent host immune response to biomaterials causes an influx of antigen-presenting cells, such as macrophages and DCs that may be targeted with specific antigenic and maturation signals. DCs are of particular interest because of their potent ability to initiate and
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modulate adaptive antigen-specific immune responses [19]. Immature DCs scavenge the environment for antigens, and upon uptake of antigen and maturation travel to the draining lymph nodes. They have an extraordinary capacity to stimulate naive T-cells, and control the quality of the T-cell response through antigen presentation on MHC complexes and expression of co-stimulatory molecules [109]. Appropriate antigenic signaling, mediated by DCs, to T-cells creates specific populations that can suppress, destroy, and clear undesirable cell populations from the body. Fully matured DC cells express the co-stimulatory molecule, CD86 (B7-2), and MHC-II concurrently with the secretion of the proinflammatory cytokines IL-12, IL-6, and TNF-a [18, 19, 109]. Maturation may be triggered via microbial and viral products such as LPS, CpG DNA, or dsRNA, and by inflammatory cytokines such as TNF-a. DCs maturation results in their migration to the draining lymph nodes, where they signal and stimulate T-cells. Tolerance to foreign and auto-antigens results from DC antigen presentation in an environment that either suppresses or lacks maturation signals [18, 71]. Biomaterial cell therapies may be designed to target signals to the immune system that produce tolerized or activated DCs and T-cells that may suppress or remove harmful cell populations in autoimmune diseases and cancer. AUTOIMMUNITY Autoimmune diseases characterized by rheumatic inflammation are usually driven by autoimmune lymphocytes that induce or sustain these processes. Currently about 7 million Americans suffer from rheumatic diseases (Table 31.1), and almost all current therapeutic concepts are based on systemic suppression of immune functions through blocking antibodies (e.g., anti-CTLA-4 and TNF) [125]. Curative intervention for autoimmunity will likely have to target the suppression and elimination of dysfunctional immune cells that secrete inflammatory mediators. Regulatory CD4+ T-cell (T-reg) lymphocyte subsets have been identified as the central controller of autoimmunity in a variety of experimental animal models, and their development and expansion can be regulated by DCs [165, 172, 174]. T-reg therapies can effectively delay and cure mice of a variety of immunological and inflammatory diseases, including type I diabetes, colitis, and gastritis [99, 125, 126, 136, 148, 160]. Several studies have suggested
that the T-regs are antigen specific, requiring cell-cell contact, and appear to function preferentially at the site of inflammation to effect proliferation, differentiation, and/or the cytokine production by the pathogenic T-cells [20, 75, 125, 153, 154]. Whether the suppressive pathway of T-regs is antigen specific or soluble, it acts to suppress the development and action of pathogenic T-cells, thus removing them from the body. The isolation and expansion of T-regs in vitro are still a technically intensive and difficult process, limiting their utility in therapies [160, 161]. It has been reported that T-regs preferentially respond to antigen-loaded presenting dendritic cells by proliferating in vitro and in vivo, but the in vitro T-reg expansion induced by dendritic cells is still very limited. In vivo, T-reg growth requires DC-T-cell contact and is partially dependent on the production of small amounts of IL-2 by the T-cells, and a lack of co-stimulation by the DCs [160]. Biomaterial vehicles may be able to bypass limitations to ex vivo T-reg expansion by targeting the delivery of autoantigens to DCs in vivo, and enriching the sites of autoimmunity with concentrated immunosuppressive DCs and T-regs. Large numbers of tolerogenic DCs and T-cells may be produced locally by delivering cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-16 and autoantigens from biomaterials in the absence of maturation signals [39, 41, 83, 101, 160]. High local production of inflammatory mediators, such as GM-CSF, has also been shown to induce immunosuppressive DC differentiation that can inhibit T-cell responses and proliferation [30, 106, 143]. This protocol may be manipulated to deliver other immunosuppressive factors, such as IL-10 and TGF-b, to enhance the efficacy of tolerogenic DC vaccines [16, 20]. An effective immunosuppressive vaccine would need to carefully coordinate delivery of tolerogenic factors both in magnitude and sequence to develop the appropriate immunosuppressive function in DCs and T-cells and limit the proinflammatory reactions associated with autoimmunity. CANCER VACCINES Cancer vaccines seek to activate the immune system to destroy tumor cells and achieve a memory of this interaction to prevent tumor occurrence [42, 56]. Ordinarily, the ability of tumor cells to stimulate an immune response is limited. This poor excitation of the immune system has been attributed in part to the weak expression of MHC-antigen complexes and
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co-stimulatory signals by antigen-presenting cells that stimulate T-cell activation [43, 56]. Many vaccines make use of DCs and their potent abilities to initiate immune responses and activate naive T-cells. Relatively large numbers of DCs can now be generated from human peripheral blood monocytes or other progenitors, and can be efficiently modified to express tumor-associated antigens [105]. Thus, current vaccines are manufactured by pulsing cultured DCs with tumor-associated antigens (TAA), DNA encoding these antigens, or using tumor lysates in the hope of eliciting a specific antitumor response from these cells following transplantation [34, 85, 119, 133, 175]. Although initial results are promising, they require technically intensive cell isolations and in vitro DC modifications, and the DC modification may be transient and lose effectiveness with time. The in vivo recruitment of DCs to a biomaterial that delivers inflammatory adjuvants serially with tumorassociated antigens may allow for the in vivo local expansion and activation of DC populations in the presence of antigens, leading to enhanced tumor-specific responses without the need for in vitro DC manipulation. Recent studies have shown that biomaterials can be modified to locally recruit DC populations and deliver TAAs that are effective in promoting T-cell responses. PLG microspheres that sustain the release of macrophage inflammatory protein (MIP-3b) were chemoattractive for murine dendritic cells in vitro [176]. Biomaterials can also be efficient carriers for antigen delivery, as illustrated by the prolonged antigen expression by DCs following PLG microsphere delivery of the antigen [169]. Polymer rods have also been used to locally co-deliver MIP-3b with tumor lysates or TAA, and induced the recruitment of dendritic cells that were able to induce antigen specific cytotoxic T-lymphocyte activity that yielded antitumor immunity [93]. Intratumoral injection of GM-CSF-and IL-12-loaded microspheres was also shown to eradicate disseminated disease in a murine surgical metastasis model [76]. (See Figure 31.2.) Cancer vaccines that use DNA encoding for tumor associated antigens (TAAs) and/or immunostimulatory molecules to stimulate DCs could benefit from the sustained delivery associated with biomaterial carriers. The most common route of DNA administration, intramuscular injection, results predominantly in keratinocytes and myocytes presenting the antigen and CD4+ T helper responses. T helper cells may secrete cytokines that lead to tumor destruction or promote the
Dendritic cells
Systemic anti-tumor immunity
Chemoattractant T-cells Tumor antigen Biomaterial
1) MHC-antigen 2) Costimulation
Activated dendritic cells Figure 31.2. Schematic of cancer vaccine using biomaterial delivery vehicles. Chemoattractants or growth factors may be delivered from biomaterials to induce the recruitment and maturation of DCs. The biomaterial is also incorporated with tumor antigens in the form of proteins or DNA that encodes for these proteins. Activated DCs then leave the implant site and stimulate T-cells with MHC-antigen complexes and costimulatory molecules. Tumor-specific T-cell populations develop and can mediate systemic anti-tumor immunity.
expansion of CTL populations [48]. But, direct gene targeting of DCs to express TAA is of interest because it leads to endogenous synthesis of the protein antigen and antigen presentation, processing, and presentation of antigen on MHC class I molecules — an efficient pathway in generating CD8+ T-cells to directly eliminate tumors [48, 175]. A DNA cancer vaccine prepared by efficiently transducing DCs ex vivo with a gene encoding a melanoma-associated antigen, gp100, was shown to enhance anti-tumor responses mediated by NK cells and gp-100-specific CTLs [116]. A useful feature of a DNA vaccine is that it already carries its own immunostimulatory adjuvant in the form of unmethylated cytidine phosphate guanosine motifs (CpG) in the plasmid region of most vectors. CpG motifs may activate DCs through Toll-like receptor ligation and further enhance vaccine effectiveness [47]. Although both the endogenous and exogenous pathways are utilized to mediate anti-tumor responses, the specific targeting of DCs by recruiting them to DNA-releasing biomaterials may provide an avenue to continuously pulse host DCs with DNA-encoding TAA and enhancing CTL responses to tumors.
Conclusions and Future Directions Typically, the immune response to biomaterials is considered a hindrance to developing effective therapies, but given the importance of immunity in the body’s
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homeostatic processes it is crucial that this response is controlled and directed in cellular therapies. To date, the design of biomaterials has largely focused on the manipulation of target cells used in tissue regeneration and the minimization of immune responses. These designs may have had a profound effect on the immune response, but the effect has been ignored in many cases. In addition, disease states are often associated with the coordinated regulation of immune cell phenotypes, as exhibited by the immunosuppressive environments of tumors or the proinflammatory environments of autoimmunity. Future cell therapies will likely include cotransplantation of multiple cell types, including immune cells, and the temporally and spatially regulated provision of multiple signals to facilitate the removal and regeneration of specific cell populations. The current paradigm that focuses on single factors and single cell types is shifting as the dynamic nature of multiple regulatory factors and processes is increasingly appreciated. Multiple-factor delivery from biomaterials may be used to induce the proper migratory and developmental action in immune cells at the appropriate time frames for a specific therapy. Mimicking natural developmental pathways provides rational design criteria for new biomaterial cell therapies, and as our understanding of immunological regulation and maintenance grows so will our ability to develop clinically relevant biomaterial formulations and cellular therapies.
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32 Nanotechnology’s Impact on Cell Transplantation Dwaine F. Emerich (1) and Craig Halberstadt (2) (1) LCT Biopharma, Providence, Rhode Island (2) Carolinas Medical Center, Charlotte, North Carolina
Introduction Nanotechnology — defined as the development of materials designed, constructed, and functional at the nanometer scale — is an emerging field in many scientific disciplines. In 1959, Dr. Richard Feynman described nanotechnology [35] as a futuristic approach to the development of materials atom by atom. It has taken approximately 40 years to realize this basic fundamental approach toward developing materials at this size scale. The successful manipulation of atoms to create exact self-assembling molecules at 10 to 100 nm has been based on the development of technologies such as lithography (deep UV, e-beam, X-ray, and microstamping), self-assembly molecules, and detection systems (scanning, tunneling, and ballistic energy electron microscopy) and to manipulation of the material surface (scanning tunnel, atomic force, and surface force microscopy) [49, 82, 90]. The development of carbon nanotubes, rods, and dots; self-assembly molecules with cell adhesion peptides; and materials developed with nanostructures on their surface has led to the creation of new materials and their applications. The unique properties of these materials — such as non-quenching fluorescence, controlled charge density, hollow cores for drug delivery, increased tensile strength, and defined nanostructures on material surfaces — are providing the impetu for the development of applications such as cancer therapy, in vitro and in vivo diagnostics, gene delivery to cells, nanomachines, localizing cell function, and cell transplantation. How does the field of nanotechnology impact cell transplantation? The ability to create nanoparticles at the size scale of 10 to 100 nm allows us to easily deliver Cellular Transplantation: From Laboratory to Clinic
these particles to cells. As a point of reference, a protein such as an antibody is approximately 10 nm and a virus particle is roughly 100 nm [3]. A 20 nm particle can freely transport across the endothelial barrier of a capillary, and a particle of approximately 50 nm can transport across most cell membranes [3]. In addition, adhesive cells form bonds primarily through an integrinligand interaction that forms clusters at approximately the 20- to 50-nm scale [62]. Hence, for cell transplantation applications nanotechnology can be used for gene delivery using a nonviral transport mechanism, live cell tracking using fluorescent quantum dots [2], control of mechanical and cell signaling at the nanometerscale using highly controlled nanostructured materials, and complex 3D cellular structures using cell printing (nanometer to micrometer scale). This field is very broad in its applications and there have been several review articles that have dealt with this subject [2, 23, 44, 54, 74, 81, 95, 98]. For conciseness, this chapter focuses on the application of nanotechnology for gene delivery, cell tracking, and cellular transplantation (primarily on immunoprotection).
Cell Biology Nanoscale: What’s the Big Deal? The delivery and engraftment of a functional cell are an enormous challenge in the fields of cell transplantation and tissue engineering. A large data pool has been generated over the last three decades on fundamental cell biology that has led to several successful cellular transplants ranging from allogeneic skin grafts
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Dwaine F. Emerich and Craig Halberstadt
and autologous chondrocyte transplants to allogeneic islet transplants. The research supporting this success has focused on both the macro- and nanoscale. The macroscale has concentrated on engineering a wellvascularized bed for cellular engraftment, biocompatible materials designed at the micro- and macroscale to enhance or support cellular engraftment, and materials to create immunological barriers (see material following). Cell and molecular biologists have also been working at the nanoscale, albeit not identifying it as nano. In these cases, the focus has been the subcellular dimensions that influence cell-to-cell and cell-to-matrix interactions such as the integrin (cell membrane protein) and ligand interaction functions [62]. One avenue of controlling cellular behavior at the nanoscale is via cell-adhesion receptors. Some of the primary cell receptors associated with mammalian cells include the integrins, selectins, Ig-superfamily, cadherins, syndecans, and ADAMS [47]. These transmembrane proteins provide a link between the extracellular and intracellular domains. Depending on the ligand, signals can be transmitted to the cellular machinery to form adhesions to the extracellular matrix, cell-cell adhesions, and tight junctions; control cell proliferation and migration; provide biomechanical cues; or maintain a cellular differentiated state. For example, the tri-peptide RGD (arginine, guanine, and aspartic acid) was identified in the 1980s as a specific ligand for integrin binding [72, 73], and has been used to control cellular behavior on 2D and 3D surfaces [40, 41, 66, 75, 77]. Depending on the concentration and spacing [41, 62] of this ligand, it is possible to control a range of cell behaviors, including cell adhesion, proliferation, and differentiation. The knowledge of ligand presentation coupled with biomaterial processing allows the generation of nanoscale structures that can control cell behavior. The majority of these biomaterials has been designed as 2D planes with 3D nanofeatures. Hence, utilizing these approaches for cell transplantation and tissue engineering has not been fully utilized. In the long term it should be possible to engineer specific patterns in materials to create small implantable tissue structures.
and colleagues at Northwestern University. They have developed self-assembled amphiphiles that form cylindrical nanostructures [82]. By utilizing specific peptide sequences, they have developed molecules that form hydrophobic and hydrophilic cylinders. The longchain nano-peptides form rods of varying length that can be tailor made for supporting cellular adhesion or other functions such as maintaining cellular differentiation. The conceptual basis of this strategy is to use self-assembling materials to deliver cells in a precisely defined orientation into specific sites of the body. When injected in vivo, the nanofibers reorganize into more complex 3D macrostructures that can entrap cells and provide signaling cues for the cells to either proliferate or differentiate. The authors envision the day when these nanomaterials are used to create new islets, skin, cartilage, neurons for spinal cord injury, and muscle [82].
Mechanical Influences Biomechanical forces and environmental cues at the nanoscale play an important modulating role in cell behavior and function. Ingber has proposed a model called tensegrity [50, 97], describing the impact of the local biomechanical forces on cell function. Through cellular adhesions to the cell via the extracellular matrix, transmembrane proteins interact with the cytoskeleton to influence DNA transcription with eventual protein production. These forces imparted on the cell are influential at both the micro- and nanoscale. For example, Wang et al. [97] coupled the RGD peptide to small magnetic beads. By applying a magnetic force to the beads, the twisting impacted the stiffness of the cytoskeleton. The ability to manipulate cells at the most basic level of cytoarchitecture illustrates the great potential to optimize cell behavior at the nanoscale and will lead to a greater control of cell aspects such as cell proliferation or differentiation.
Contact Guidance Self-assembled Nanostructures An approach to using protein chemistry to create nanostructures for cell transplantation is through the development of self-assembled materials. One group that has focused in this is area Dr. Samuel Stupp
The nanotopography of a surface plays a significant role in cell behavior [18, 19, 23–25]. Using photolithography, Curtis and colleagues have demonstrated that the micron-size channels on a material surface are controlled by nanotopographical cues such as the depth and pitch of the channels. These cues appear to effect
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cell behavior such as cell adhesion, migration, and spreading. Because a cell’s focal contact point is in the area of 5 nm [24], further improvements in controlling material structures at the nanometer scale may make it possible to one day lay out an exact “road map” to control cell behavior. An example may be expanding stem cells using one defined surface material and then specifically driving those expanded cells to differentiation using a different cell topography.
Drug and Gene Delivery DRUG DELIVERY In the last decade, nanotechnology and nanofabrication have significantly impacted the field of drug delivery. Techniques have shifted from microfabrication and micromachining (e.g., the osmotic pump) to designs ranging from secondary constructs on the nanometer level (e.g., microspheres) to delivery systems composed entirely of submicron components. The engineering of nanodelivery systems for small molecules, proteins, and DNA is opening new therapeutic opportunities for agents that cannot be used effectively as conventional drug formulations due to poor bioavailability or drug instability. Microsphere formulations have been successfully used to protect agents susceptible to degradation or denaturation while prolonging the duration of action of a drug by increasing systemic exposure or retention of the formulation [8, 12, 45, 89, 94]. Nanoparticles have the added advantage of being able to cross membrane barriers, particularly in the absorptive epithelium of the small intestine [48]. Several groups have used both nonresorbable and biodegradable nanospheres to deliver small molecules, proteins, and other therapeutics. The attraction of biodegradable nanospheres is based on enhanced bioavailability through uptake, followed by degradation and disappearance of the vehicle from the system.
INITIAL CELL TRANSPLANT STUDIES USING MICROSPHERES Cell therapy is promising for the treatment of several diseases, including neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease (PD). Clinical trials with fetal dopaminergic cells for PD are limited by the low survival rate and poor integration
of transplanted cells, as nearly 90% of the cells die in the first two weeks. Progress in neurotransplantation, as in cell therapy in general, requires improvement in the preparation and the delivery of the cell product in order to increase the survival rate and cell integration in the host environment. Associating nerve cell transplantation and neurotrophic factor-releasing microspheres is an interesting strategy to overcome this problem. One approach uses pharmacologically active microcarriers (PAMs) that can provide a cell adhesive surface while delivering an active molecule. PAMs can be designed to be biocompatible and biodegradable microparticles coated with cell adhesion or extracellular matrix molecules — providing a mechanism for a controlled delivery of a growth factor. Thus, the combined effect of the growth factor and the coating may influence the transplanted cells by promoting their survival and differentiation and favoring their integration in the host tissue after their complete degradation. Initially, nerve growth factor (NGF)-releasing PAMS conveying PC12 cells were transplanted in a rat model of PD [86]. When PC12 cells that express tyrosine hydroxylase (TH) are exposed to NGF, they stop cell division, extend long neurites, and become excitable. After depolarization, they can release large amounts of dopamine, the missing neurotransmitter in a rodent model of PD. Transplanted PC12 cells have been studied in animal models with encapsulated biomaterials. Tatard and colleagues evaluated their efficacy using NGF-releasing PAM and PC12 cells in a PD paradigm. After implantation of NGFreleasing or control (unloaded) PAM-conveying PC12 cells, or PC12 cells alone, the authors studied cell survival, differentiation, and apoptosis, as well as behavior of the treated rats. NGF-releasing PAMS coated with two synthetic peptides (poly-D-lysine and fibronectinlike) induced PC12 cell differentiation and reduced their death and proliferation. After transplantation, the animals receiving this implant were also behaviorally normalized, as shown by improved amphetamine-induced rotational behavior. These PAMs represent a very interesting tool for cell therapy, as the cells may be cultured and sorted in the same support before implantation. Following transplantation, they can induce cell differentiation and limit cell death and proliferation essential when stem cells are employed. Depending on the molecule released, PAM may also modify the microenvironment (favor angiogenesis, local immunodepression, or any interaction with the host cells), favoring the integration of grafted cells in the host tissue. In this regard, a recent
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article reported that the implantation of neural stem cells seeded on polymer scaffolds augmented the constitutive repair response in the injured brain [70]. Other practical advantages of PAMs are that they can be produced in advance and stored as a freeze-dried powder and that the cell adhesion protocol may be easily adapted to many cell types and neurological diseases.
have good biocompatibility and low toxicity and are under investigation as antiviral agents [79], antibacterial agents [11, 91, 92], anticancer therapies [69, 84], antioxidants, and anti-apoptosis agents for potentially treating amyotrophic lateral sclerosis and Parkinson’s disease [30].
NANOSHELLS MICROCHIP-CONTROLLED RESERVOIRS Controlled-release drug reservoirs with microchips [78] have unlimited potential for modulation of release, with the ability to combine modalities within each reservoir as well as the control of each individual pulse of drug release. As pioneers of drug delivery therapies and devices, Dr. Robert Langer and colleagues have consistently provided the field with a wealth of innovation and palpable technologies. One recent approach by this group has focused on the integration of controlledrelease drug reservoirs with a microchip [78], with operational elements of both disciplines. The chip is composed of a silicon wafer containing up to 34 reservoirs, each occupying a volume of 25 nl, sealed with a gold membrane anode 300 nm in thickness. Drug is released from a reservoir when a current is applied between the gold membrane and an embedded cathode. Although only in its early stages, the chip has shown extremely promising results with fluorescent tracers and is in continued development.
A third drug delivery approach mentioned here uses “nanoshells,” or dielectric-metal (gold-coated silica) nanospheres. One exciting area of potential use for nanoshells is the delivery of chemotherapeutics to tumors. These nanoshells, embedded in a drugcontaining polymer and then injected into the body, accumulate near tumor cells. When heated with an infrared laser, the nanoshells selectively absorb a specific infrared frequency, melting the polymer and releasing the drug at a specific site. Researchers have also developed an immunoassay using antibody-nanoparticle conjugates of gold nanoshells and have successfully detected nanogram/ml quantities of immunoglobulins in saline, serum, and whole blood within 10 to 30 minutes of sample acquisition [46]. As these drug delivery systems become more refined it will be possible to integrate them into cell constructs, providing temporally controlled delivery of single or multiple compounds for cell graft integration, growth, and function.
GENE DELIVERY NANOTUBES A second rapidly developing drug delivery approach uses nanotubes. Nanotubes have large relative internal volumes, can be functionalized on either the internal or external surface, and can be constructed using a variety of techniques [64]. One fabrication technique used self-assembling lipid microtubes to deliver testosterone in rats [64]. Testosterone was covalently bound with an ester linkage to a glutamide core lipid to form nanotubes with a biphasic release profile consisting of an initial burst followed by a more sustained release. Another method of fabrication used for drug delivery involves synthesizing carbon nanotubes using fullerenes. These nanotubes range from 1 to 10 nm in diameter, and their length can range from several to hundreds of microns. Recent studies confirm that drugs can be covalently attached to functional groups on the external surface of the nanotubes [15]. Soluble derivatives of fullerenes
Gene therapy is dependent on the entry of the vector into the cell while protecting the content from the harsh environment of the cytoplasm. Next, transport of the gene into the nucleus must occur without imparting damage from the vehicle itself. Gene therapy can either be attempted through in vitro or in vivo means. The traditional approaches to gene delivery have included naked DNA coupled with calcium ionophores to permeabilize the cell membrane, electroporation, and viral delivery systems. Recently, several investigators have looked into utilizing nano-derived materials for the targeted delivery of specific genes to the targeted cell.
LIPOSOMES One of the most investigated approaches to gene therapy uses liposomes as submicron delivery vehicles.
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Liposomes consist of a lipid shell surrounding a core containing a therapeutic molecule or gene. They are particularly useful as gene therapy devices due to their ability to pass through lipid bi-layers and cell membranes, and several groups have recently reported convincing results following local delivery. Composite liposomes containing poly(cationic lipid) and cholesterol showed much higher transfection in the liver than naked DNA alone [60]. This work focused on lipoplexes consisting of poly(cationic lipid), cholesterol, and DNA injected directly into portal circulation following a partial hepatectomy. The reporter gene expression (luciferase) was much higher in these lipoplexes than in naked DNA alone. Targeted therapy can also be achieved using liposomes. Pegylated liposomes linked to a monoclonal antibody for the human insulin receptor led to the widespread reporter expression in the brain of rhesus monkeys [105]. Plasmids encoding luciferase or galactosidase were administered via the “immunoliposome” through the common circulation and were localized primarily to the brain. Such complexes give a hint at the future of targeted therapy and the importance of nanometer-sized constructs for the advancement of molecular and cellular medicine.
POLYMER NANOSPHERES Gene delivery using a polymeric vehicle has also been explored recently. Polymeric nanospheres can translocate into the cytoplasm of a cell, but transport to the nucleus has not been established. Cohen-Sacks et al. [20] have characterized the delivery of 300 nm-diameter nanospheres loaded with platelet-derived growth factor beta-receptor antisense for the treatment of restenosis. This group observed cell internalization as well as inhibition of smooth muscle cell proliferation and roughly a 22% reduction in restenosis compared to the control. Berton et al. [9] have also demonstrated successful delivery of oligonucleotides. This group characterized the ability of PLGA nanospheres to deliver phosphorothioate oligonucleotides, with subsequent inhibition of viral HIV-1 production. Plasmid DNA can also be delivered by biodegradable nanospheres. Perez et al. [71] constructed poly(lactic acid)/poly(ethylene glycol) composite nanospheres with mean diameter < 300 nm and showed that modulation of release could be achieved with the addition of either poly(vinyl alcohol) or poly(vinyl pyrrolidine), or both.
DENDRITIC POLYMERS Dendritic polymers provide another avenue for delivery of genes. They can form extremely small particles, on the order of angstroms, and are effective as DNA conjugates. The resulting dendrimer-DNA complex differs from encapsulation in that the primary interaction causing gene retention is caused by electrostatic interactions between the negatively charged phosphate groups on the DNA backbone and the positively charged amino groups on the polymer. Mixing the components in an aqueous solution forms the complex. Polyamidoamines (PAMAM), are the most often used and characterized dendrimers for gene delivery, and several groups have recently demonstrated their efficacy. PAMAM dendrimers complexed to the pCF1CAT plasmid for intravascular and endobronchial delivery of chloramphenicol acetyl-transferase (CAT) are being investigated as a potential treatment for cystic fibrosis [53]. Intravenous administration of the complex showed transgene expression in the lung, with peaks at 12 to 24 hours and at 3 to 5 days. The dendrimer-plasmid complex was completely localized to the lung, whereas the naked plasmid was randomly distributed. In comparison, endobronchial delivery of naked plasmid was more effective than the dendrimer complex. Significant improvements in transfection using PAMAM conjugates have also been achieved [63]. The transfection of several cell lines by pCMV a-Gal plasmiddendrimer complexes was enhanced by the presence of anionic oligomers, including oligonucleotides or dextran sulfate. The degree of enhancement was dependent on the size and charge of the additive, with 35 to 50 phosphate group oligonucleotides providing the highest efficiency. The development of these DNA carriers using defined chemistries may one day overcome the current limitations of viral vectors; namely, the potential inflammatory responses and the lack of specificity for delivery of the gene to specific cells. However, much work still needs to be done in this area to ensure that these new chemistries are nontoxic and can be delivered to specific targeted cells.
Nanoparticles for Screening and Monitoring of Transplanted Tissue One of the first applications of nanotechnology is the use of fluorescent markers for diagnostic and screening purposes. These applications have already demonstrated
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their utility for preclinical cellular transplant strategies [2], and may find a real use for clinical cellular transplantation. Specifically, there are currently no reliable methods for quantifying in vivo cell survival in a clinical setting. Fluorescent nanoparticles may overcome some of the obstacles encountered by conventional markers such as the requirement for color-matched lasers, the fading of fluorescence even after a single use, and the lack of discriminatory capacity of multiple dyes due to “bleeding.”
QUANTUM DOTS AND PEBBLES Perhaps the most recognizable form of this technology is the “quantum dot.” These nanocrystals are several nanometers in diameter and can be manufactured in a nearly unlimited range of sharply defined colors [5]. Because these particles are excitable using white light and can be linked to specific biomolecules, separate biological events can be monitored by simultaneously tagging each biological component (e.g., different proteins or DNA sequences) with specifically colored nanodots. Together with their clinical potential, the research value of quantum dots includes the possibility of simultaneously tagging multiple biomolecules both on and inside cells to monitor the complex cellular changes and events associated with disease, providing valuable clues to the development of future pharmaceuticals and therapeutics. A related technology called PEBBLES (probes encapsulated by biologically localized embedding) allows dye-tagged nanoparticles to be inserted into living cells to monitor metabolism or disease conditions [17, 83]. This system was used to quantify zinc levels within living cells [83]. The sensing components were entrapped within a polymer matrix using a microemulsion polymerization process that produced spherical sensors with a size of 20 to 200 nm. The system was sensitive, rapid, and reversible, and was immune to interference from intracellular or extracellular proteins. A similar application uses superparamagnetic nanoparticles as magnetic resonance (MR) contrast agents for imaging specific molecular targets [102]. Wunderbaldinger et al. characterized the physical and biological properties of MION-47 and amino-CLIO nanoparticles that serve as precursors for the synthesis of targeted MR contrast agents — as well as of Tat-CLIO, a nanoparticle used to label cells. Following intravenous injection, the plasma half-lives of MION-47 and amino-CLIO were greater than 10 hours.
The attachment of Tat peptides to amino-CLIO significantly reduced the circulating half-life to less than 1 hour and resulted in the greatest accumulation of MION47, amino-CLIO, and Tat-CLIO in the liver, spleen, and lymph nodes. Furthermore, 24 hours after injection of amino-CLIO, the nanoparticles were concentrated in the endothelial and Kupffer cells surrounding hepatic blood vessels — whereas Tat-CLIO was present throughout the parenchyma. Future approaches using peptides as components of nanoparticles might make it possible to design sensors to detect macromolecules present in specific intracellular compartments. Other applications include using gold nanoparticles for DNA diagnostics [22] and DNA microarrays for genotypic analysis for both diagnostic purposes and assessment of drug responses [7]. Fluorescent nanoscale devices might also allow transdermal monitoring of changes in interstitial fluid constituents. Electrostatic self-assembly processed solid nanoparticles coated with fluorescent enzymecontaining thin films and hollow micro/nanocapsules containing fluorescent indicators and enzymes or glucose-binding proteins are being used to monitor glucose concentrations [68]. Nanoengineering of the coated colloids and microcapsules allows precision control over optical, mechanical, and catalytic properties to achieve sensitive responses using a combination of polymers, fluorescent indicators, and glucose-specific proteins.
TISSUE TARGETING BY NANOSPHERES Polymeric nanospheres can be used to selectively target and image different tissues, which also has enormous implications in cell therapy. Lanza et al. [56, 57, 101] have convincingly demonstrated the evolution of one of these nanoparticle systems from use as a contrast agent to the more advanced application of targeted local drug delivery. Through MRI imaging, ligand-linked perfluorocarbon nanoemulsions (250-nm diameter) have been localized to various sites, including thrombi and neovascularization during tumor genesis both in vitro and in vivo [5, 36, 55, 56, 58, 103]. By directing the delivery vehicle to a site-specific target, the formulation can locally deliver the drug without the need for internalization (e.g., contact-facilitated drug delivery). A similar product has been brought through phase III clinical trials in the form of biodegradable microspheres. Grayburn et al. [39] used poly(lactide-co-glycolide) particles to encapsulate a low-solubility gas as a contrast agent for
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echocardiography. This technique, which uses a bolus IV dose to monitor myocardial perfusion and ventricular wall motion over the course of 10 minutes, may provide a first detailed look at real-time hemodynamics in the human heart. The potential impact of nanotechnology in these areas is enormous. In many, if not most, chronic diseases, it takes considerable time (weeks to months) for both the clinician and patient to discern if a given therapy (including transplanted cells) is functioning or not. These delays can extend the duration of clinical trials, delay the introduction of newer (perhaps more effective) treatments because of additional time between preclinical and clinical evaluation, and likely render subsequent therapies for patients ineffective. Nanotechnology has the potential to markedly alter the time frame for determining the first signs of efficacy or a lack thereof. Nanoscale constructs have the potential for measuring real-time efficacy, monitoring and obviating potential dose-limiting side effects, and providing preliminary indications that an experimental drug is reaching its target.
Tissue Engineering Designing a biological system capable of reproducing a physiologic event requires not only that the system provide the desired outcome but that the outcome be reached in a suitable and repeatable fashion. Historically, this is one of the great challenges of tissue engineering, but a new level of control is being gained with advancements in biohybrid processing techniques and nanoscale fabrication and modification. Scaffolds capable of sustaining dense populations of cells for extended periods are being developed, new materials are being used in a variety of matrices, and new constructs are being engineered that will lead to the eventual development of the artificial cell/organ. The response of cell motility and metabolism to changes in substrates has been thoroughly studied in the past decade. Size, structure, geometry, integrin binding, and other factors have all been investigated. Various techniques have been employed to create micropatterned surfaces of different materials to study cell behavior, including microlithography [88], microcontact printing [59], hydrolytic etching [76], and many others. Thompson et al. [88] demonstrated that in the presence of patterned stripes of bovine serum albumin and laminin, Schwann cells aggregate preferentially on the laminin
regions. Lee et al. [59] showed that using microcontact printing to create shapes composed of inhibitory molecules affects the morphology and growth of retinal pigmented epithelium. He et al. [42, 43] demonstrated that fibroblast cell alignment and behavior can be controlled by changing the hydrophobic and hydrophilic surface properties using photolithography on a novel photoresist that does not require harsh chemical treatments. The material’s properties are ideal for coupling cell adhesion peptides to the surface and both microand nanofeatures can be controlled using the appropriate photomasks [93]. The surface depth and pitch at the nanometer scale may also play an important factor in cellular behavior [23, 65]. This form of contact guidance was proposed by Curtis et al. and has been extensively studied by this group [18, 19, 24–26, 96]. Such forms of surface modification have evolved into even smaller-scale attempts to control cell culture. Irvine et al. [51] have developed a novel material of poly(methyl methacrylate-r-polyoxyethylene methacrylate) comb polymers that present RGD peptides in nanoclusters on the surface. The RGD adhesion ligand was clustered with a spatial distribution of 50 nm at varying densities, and the murine NR6 fibroblast cell line was cultured onto the surface. The required density of RGD was significantly reduced compared to a standard RGD peptide on a 2D surface for cell migration to occur in this arrangement. Osteogenic cells have been cultured in a 3D nano-hydroxyapatite/collagen matrix by Du et al. [29]. This matrix is precipitated such that hydroxyapatite crystals are uniformly distributed in a matrix of collagen, seemingly ideal for bone construction. In this system, bone-derived mesenchymal cells reached confluence at 3 weeks with a tissue morphology consisting of a mass of polygonal cells over which spindle-shaped cells migrated — similar to the 3D construction of bone. Nano-fibrillar extracellular matrices have also been created using a novel technique employing porogenic materials and polymers [104]. Sugar particles were melted and extruded into nanofibers, which were subsequently suspended in an organic solution of poly(lactic acid) in tetrahydrofuran. The solution was dripped into the dissolved porogen and gelation was induced with freezing. After leaching and lyophilization, the resulting porous matrices were extremely homogeneous and contained a true 3D interconnecting nanoporous matrix. Folch et al. [37] have also explored inverse patterning in recent studies. In this method, a polymeric membrane is applied to a substrate and cells are allowed
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to spread across the surface. After a confluent layer has been achieved, the membrane is removed and the resulting cell pattern is left on the surface. Patterned cells were created on polystyrene, glass, and collagen, with either flat or curved geometry. This technique has the potential to facilitate the development of a variety of cell constructs as well as the creation of biohybrid organs using multidimensional applications. The techniques described previously are extremely relevant for the development of artificial tissues and organs. Still, new advances are leading to models for individual cells, and Cans et al. [13] have cleverly characterized the process of exocytosis using liposomes and nanotubes. In an effort to understand and reproduce neuronal transmission, this group has effectively simulated natural physiology using a system composed of liposome-nanotube networks controlled by electroinjection. A small vesicle was introduced into a liposome connected to a nanotube, which together resembled a neurotransmitter vesicle and elongated fusion pore. Measured with fluorescence microscopy and amperometry, this model was compared to dopamine release from PC12 cells and was found to be slower but qualitatively similar in magnitude. The system can be further controlled to investigate individual phases of this process.
Cellular Immunoisolation Transplanting a failing organ or specific cell type is a logical means of restoring function for secretory diseases. While inherently appealing, the lack of suitable donor organs or tissues makes the widespread use of cell transplantation impractical. To overcome this limitation, allogeneic (derived from stem cells) or xenogeneic cells can be encased within a selectively permeable polymeric membrane known as immunoisolation. Immunoisolation owes much of its foundation to investigators focused on diabetes [16] and Parkinson’s disease [100], showing that islets and dopamine-secreting cells are protected from host rejection by encapsulating them within a semipermeable membrane. Single cells or clusters of cells can be enclosed within a selective semipermeable membrane barrier that admits oxygen and required nutrients and releases bioactive cell secretions but restricts passage of larger cytotoxic agents from the host immune defense system [31, 33]. Immunoisolation eliminates the need for chronic immunosuppression and allows the implanted cells to be obtained from human or nonhuman
sources, thus avoiding the constraints associated with cell sourcing — which limit the clinical application of unencapsulated cell transplantation. Submicron pore sizes are generally incorporated into the material barrier for isolation of the cells from the host immune system. The following sections introduce the reader to material structures and how nanotechnology has played a role in this field.
IMMUNOISOLATORY MEMBRANES True immunoisolation requires restricting the membrane transport of all relevant molecules belonging to the host and the encapsulated cells. Although a molecular weight cutoff of 50 to 100 kDa likely prevents infiltration of IgG, greater size restrictions must be employed to achieve a complete barrier. Growth factors, cytokines, complement, and smaller metabolites several logs lower in molecular weight exist within immunoisolated systems that are immunogenic and can lead to graft rejection. Limiting passage of these smaller molecules would retain the cell-secreted molecule of interest, and thus such a system would not be practical. For example, membranes with pore diameters that range between 30 and 50 nm could block key molecules such as IgG and complement [21]. However, this level of protection is not always necessary — particularly in allogeneic or syngeneic transplantation — and immunosuppressants can be adminstered to overcome this effect. The polymer membrane of an immunoisolatory device is a thin barrier that restricts transport between adjacent phases [28, 38, 67]. Most membranes are produced from thermoplastics such as polysulfone or acrylic co-polymers cast into hollow fiber membranes less than 1 mm in diameter, with a wall thickness of 10 to 15% of the total fiber diameter. The majority of thermoplastic ultrafiltration and microfiltration membranes used to encapsulate cells is manufactured from homogeneous polymer solutions by phase inversion. Ultrafiltration membranes have pore sizes ranging from 5 nm to 0.1 µm, whereas microfiltration (or microporous) membranes have pores ranging from 0.1 to 3 µm. Phase inversion forms membranes with a wide variety of nominal molecular weight cutoffs, permeabilities, and morphologies. The polymer is first dissolved in an appropriate solvent and then cast as a flat sheet or extruded as a hollow fiber. As part of the casting or extrusion procedure, the polymer solution is precipitated by a phase transition brought about by temperature
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change or solution composition. This process involves the transfer of a single-phase liquid polymer solution into a two-phase system consisting of a polymer-rich phase that forms the membrane structure and a second liquid polymer-poor phase that forms the membrane pores. Any polymer that forms a homogeneous solution that will separate into two phases can be used. Thermodynamic and kinetic parameters, such as the chemical potential of the components and the free energy of mixing of the components, determine the manner of the phase separation. There are generally two categories of encapsulation, micro- and macro-, each with some benefits and limitations. Several detailed reviews are available [28, 31, 33, 38, 61, 67, 99]. In addition, Desai et al. recently proposed a nanoporous encapsulation device that combines both the micro- and nanoscale [27]. The design criteria have focused on building the material at the molecular level in order to create specific pore structures that will provide specificity for blocking specific cell types and proteins.
MICROENCAPSULATION Microencapsulation involves surrounding cells with a thin, spherical, semipermeable polymer film [99]. The small size, thin wall, and spherical shape of microcapsules is structurally optimal for diffusion, cell viability, and release kinetics [21]. The material components of microcapsules vary from polysaccharides to thermoplastics. There are potential benefits associated with each choice, however, during the manufacture of certain thermoplastic cell-encapsulated systems because exposure to organic solvents is not easily avoided and is a potential liability risk. Thus, work has primarily focused on the polyelectrolytes, which are nontoxic during processing but are generally mechanically fragile and chemically unstable, as well as difficult to retrieve. For the polyelectrolytes, microcapsule membranes are formed by ionic or hydrogen bonds between two weak polyelectrolytes with opposite charges. Microcapsules can be prepared by gelling droplets of a polyanion/cellular suspension (e.g., alginate) in a mixing divalent cationic bath — which immobilizes the cells in a negatively charged matrix — and then coating the immobilized cells with a thin film of a polycation such as poly (L-lysine). The poly(L-lysine)-alginate bond creates a perm-selective membrane whose molecular mass cutoff is on the order of 30,000 to 70,000 daltons. Because
poly(L-lysine) is not biocompatible, a second layer of alginate is generally added to the capsule surface. Alternatively, polyanions such as polyornithine have greater reactivity during capsule formation and are biocompatible. Polyelectrolyte-based techniques avoid organic solvents, and with proper permeability control, microcapsules represent a very effective configuration for cell viability and small-molecule diffusion.
MACROENCAPSULATION Macroencapsulation involves filling a hollow, usually cylindrical, perm-selective membrane with cells, generally suspended in a matrix, and then sealing the ends to form a capsule [33]. Polymers used for macroencapsulation are biodurable, with a thicker wall than that found in microencapsulation. Although thicker wall and larger implant diameters can enhance long-term implant stability, these features may also impair diffusion, compromise the viability of the tissue, and slow the release kinetics of desired factors. In theory, macrocapsules can be retrieved from the recipient and replaced if necessary. Macroencapsulation is generally achieved by filling preformed thermoplastic hollow fibers with a cell suspension. The hollow fiber is formed by pumping a solution of polymer in a water-miscible solvent through a nozzle concurrently with an aqueous solution. The polymer solution is pumped through an outer annular region of the nozzle while the aqueous solution is pumped through a central bore. Upon contact with the water, the polymer precipitates and forms a cylindrical hollow fiber with a perm-selective inner membrane. Further precipitation of the polymer occurs as the water moves through the polymer wall, forcing the organic solvent out and forming a trabecular wall structure. The hollow fiber is typically collected in a water bath, allowing complete precipitation of the polymer and dissolution of the organic solvent. The ends of the hollow fiber are sealed using a biocompatible fast-curing adhesive manually applied to the ends of the device. A second method of macroencapsulation, called coextrusion, avoids the sealing problem by entrapping cells within the lumen of a hollow fiber during the fabrication process. Pinching the fiber before complete precipitation of the polymer causes fusion of the walls, providing closure of the extremities while the cells are inside.
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CELLS AND EXTRACELLULAR MATRICES USED IN ENCAPSULATION Successful cell encapsulation involves the choice of the cells to be encapsulated, the type of intracapsular matrix used, and the ability to control membrane geometry, morphology, and transport [28, 31, 33, 38, 61, 67, 99]. Cells placed within encapsulation devices generally fall into one of three categories (Table 32.1), including primary post-mitotic cells, immortalized (or dividing) cells, and cell lines genetically engineered to produce specific factors. Dividing tissue has advantages over post-mitotic tissue. It can be expanded and banked, and thus more easily tested for sterility and contaminants. However, dividing tissue is also constrained by the potential for overgrowth within the capsule environment, resulting in an accumulation of necrotic tissue that could diminish the membrane’s permeability characteristics, further reducing cell viability and protein output. In vivo, extracellular matrices (ECMs) control cell function through the regulation of morphology, proliferation, differentiation, migration, and metastasis. Within a capsule, ECMs are used to prevent aggregation of cells (immobilization) and resultant central necrosis, but are also beneficial to the viability and function of cells that require immobilization. For example, adrenal
TABLE 32.1 Essential components of an immunoisolatory device. Component
chromaffin cells have been immobilized in alginate to prevent aggregation and the formation of central necrotic cores. The chromaffin cells thrive in alginate, whereas mitotically active fibroblasts do not. In this case, the use of alginate is essential to the optimal functioning of this device because some anchoragedependent cells (such as fibroblasts or endothelial cells) are present with the adrenal chromaffin cells. In the absence of alginate or similar immobilizing matrices, the fibroblasts expand and overgrow the encapsulated milieu, resulting in a device deficient of bioactive factors produced from the chromaffin cells. In contrast, BHK cells (a fibroblastic cell line) prefer collagen — whereas PC12 cells exhibit a preference for distribution within precipitated chitosan, which provides a scaffolding structure on which the cells anchor. Polymeric matrices can also be used as cell scaffold material. Poly(ethylene oxide) (PEO)-star co-polymers have been fabricated as a potential synthetic extracellular matrix. The star co-polymers provide many hydroxyl groups where various synthetic oligopeptides can be attached to desired specifications. Other more resilient polymers — such as poly(ethylene terephthalate) (PET) polymers — can be extruded into unique geometries that facilitate a 3D arrangement of cells within the device, such as cylinders, braids, or other geometries. Polyesters such as this can be modified to have a surface microtexture for improved cell anchoring, or attachment of peptides or other components desirable for cell attachment.
Form
ONGOING CLINICAL TRIALS Cells
Primary
Dividing Genetically engineered
Islets (diabetes), hepatocytes (liver failure), chromaffin cells (pain and Parkinson’s disease) PC12 cells (Parkinson’s disease) Fibroblasts producing neurotrophic factors Extracellular Matrix
Hydrogels Scaffolds
Alginate and collagen Foamy materials, including urethane Membranes
Dialysis membranes Polyelectrolytes Combinations
PAN-PVC, polysulfone, polyethersulfone Alginate, polylysine, polyornithine Conformal coating on surface combined with reinforcing structures
As shown in Table 32.2, there are extensive ongoing efforts to develop cell encapsulation across a wide range of preclinical and clinical applications. Here we highlight the ongoing clinical efforts using encapsulated cells for treating CNS diseases. AMYOTROPHIC LATERAL SCLEROSIS Amyotrophic lateral sclerosis (ALS) is a progressive degeneration of spinal motor neurons. Systemic delivery of ciliary neurotrophic factor (CNTF) has therapeutic potential but is limited clinically due to systemic side effects, short half-life, and the inability to cross the BBB [4]. Continuous intrathecal delivery of CNTF proximal to the nerve roots in the spinal cord could result in fewer side effects and better efficacy of CNTF in ALS patients [1]. After safety, toxicology, and preclinical 620
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TABLE 32.2 Diseases to which cell encapsulation is being applied. Disease/Model
Encapsulated Cell/Experimental Paradigm
Results
Hormonal and Whole Organ Diseases
Diabetes Hypoparathyroidism Kidney failure Growth hormone deficiency
Islets in rodents and dogs Parathyroid tissue in rats Orally delivered E. coli bacteria to rats Growth-hormone-producing cells in dogs
Normoglycemia for 2 years Normocalcemia for 30 weeks Normalized urea metabolism Hormone secretion for 1 year
Single-gene Diseases
Hemophilia Lysosomal storage disease
Factor 9 cells in rats Beta-glucuronidase cells
Cell survival and secretion Behavioral normalization in mice
Age-related/Neurodegenerative Diseases
Age-related motor decline Amyotropic lateral sclerosis Alzheimer’s disease cholinergic Huntington’s disease Parkinson’s disease Retinitis pigmentosa Spinal cord damage
Catecholamine and GDNF cells in rats CNTF-producing cells in mice NGF cells in rat and primates NGF and CNTF cells in rat and primates Catecholamine and GDNF cells in rat and primate brain Human cells secreting hCNTF into the vitreous BDNF cells in rats
Improvement in motor function Protection of motor neurons Protection of neurons, improved memory Protection of neurons, improved behavior Improved behavior, protection of dopaminergic neurons Rod preservation Outgrowth of neurites
Oncology
Colon cancer Glioblastoma HER-2/neu-positive tumors Leukemia Ovarian cancer
iNOS cells (Tet-regulated system) in mice Endostatin cells in mice and rats IL-2 fused with anti HER-2/neu antibody in mice Hybridoma-producing antibodies to p15E in mice iNOS cells (Tet-regulated system) in mice
Enhanced survival Reduced tumor growth, enhanced survival Modest survival benefit Enhanced survival Enhanced survival, cures
Other
Acute and chronic pain
Chromaffin cells in rats
Reduced pain Clinical Trials
Amyotropic lateral sclerosis Chronic pain
CNTF cells intrathecally Chromaffin cells in subarachnoid space
Diabetes Huntington’s disease Hypoparathyroidism Pancreatic cancer
Human islets intraperitoneally CNTF cells into ventricles Parathyroid tissue CYP2B1 cells in tumor vessel
Retinitis pigmentosa
CNTF cells in eye
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Sustained delivery, no toxicity Prolonged cell survival, no pain reduction in phase II trials Insulin independence for 9 months Delivery for 6 months Successful in 2 patients Local tumor growth controlled, well tolerated Phase I completed
Dwaine F. Emerich and Craig Halberstadt
evaluation, a clinical trial was performed to establish the safety of encapsulated CNTF-producing cells in ALS patients. Six ALS patients with early-stage disease received BHK cells that were encapsulated into 5-cm-long by 0.6-mm-diameter hollow membranes and implanted into the lumbar intrathecal space. CNTF from the cells was found in all six patients at 3 to 4 months postimplantation. All six explanted devices had viable cells and detectable CNTF secretion. More recently, a phase I/II clinical trial was initiated in 12 ALS patients using the same approach. CNTF was detectable for several weeks in the CSF of 9 out of 12 patients, and only a very weak antigenic immune response was detected with bovine fetuin as the main antigenic component [106]. HUNTINGTON’S DISEASE Huntington’s disease (HD) is an inherited neurological disorder characterized by an intractable course of mental deterioration and progressive motor abnormalities, with death usually occurring within 15 to 17 years. There are no treatments for HD. Recent studies have suggested that CNTF might slow the neural degeneration in HD [32, 34, 52]. Recently, clinical trials were completed to determine the safety and tolerability of CNTF-producing cells (0.15 to 0.50 µg CNTF/day) implanted into the lateral ventricle of HD patients [6, 10]. HD symptoms were also analyzed using neuropsychological, motor, neurological, and neurophysiological tests — and the striatal pathology monitored using MRI and PET-scan imaging. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle. The capsule was retrieved and exchanged for a new one every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed. However, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous cell survival, however, stresses the need for improving the technique. RETINITIS PIGMENTOSA Retinitis pigmentosa (RP) is an inherited disorder of the retina and is the sixth leading cause of blindness.
Although current clinical strategies are primarily palliative in nature, research in animal models demonstrates that neurotrophic factors inhibit retinal degeneration caused by RP. Studies in the rd model of retinal degeneration demonstrated that CNTF gene transfer maintains retinal morphology [14], and later studies in the rcd1 dog model [85] using intra-vitreal implants of encapsulated CNTF-secreting cells demonstrated a dose-dependent preservation of rods. In the dog studies, untreated eyes showed on average three layers of the outer nuclear layer, whereas treated eyes showed between three and six layers. The distribution of retinal preservation was homogeneous throughout the retina. Pharmacokinetic analysis in rabbits was carried out on two variants of the NT-201 cell line that were engineered to produce two different doses [87]. Devices secreting approximately 20 or 60 ng CNTF per day were implanted in normal rabbits, and at time points between 1 and 365 days were removed and assayed for protein analysis along with extracted vitreous. Cell viability within the device was characterized histologically. Over the course of one year, both device types secreted CNTF and maintained linear pharmacokinetic profiles. Over the one-year period, devices with the lower dose had an associated vitreous half-life of 88 days, whereas high-dose devices had a vitreous half-life of 126 days. High-output devices secreted roughly tenfold higher levels of CNTF to the vitreous. Histology confirmed cell viability at the study termination. These experiments paved the way for the recently initiated clinical trials by Neurotech USA. Patients diagnosed with RP have received intra-vitreal implants of an immunoisolatory membrane containing a human epithelial cell line engineered to secrete CNTF. Neurotech is currently testing their device in a phase I clinical trial in patients with RP [80]. Ten patients were implanted with either 6-mm or 11-mm devices and were followed for 6 months. There were no significant adverse events reported, and it was reported that some patients showed an increase in the amount of lines read in the visual examination chart [80].
Cell Encapsulation Conclusion There is an abundance of work in the field of immunoisolation to highlight the importance of creating a synergistic system capable of maintaining the production and delivery of secreted factors while promoting device viability and rejecting cells and molecules of
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the immune system. An effective device relies on the ability of the cells to withstand transplant conditions, and likewise the encapsulated cells must be in a favorable immunoisolatory environment to optimally perform their function. Immunoisolation can be achieved using a variety of materials and techniques, but the secreted component must be able to diffuse outward while inward transit of immune components is halted. The ability to control these transport behaviors may be enhanced by our increasing knowledge of controlling polymer chemistry at the nanometer level. The potential of building encapsulating structures at this dimension may one day allow us to directly control the transport of nutrients and metabolic by-products while completely masking the transplanted cells from the host immune system. Ongoing research and the products being investigated in clinical trials will provide the field of cell transplantation with a tangible picture of the true importance of immunoisolation.
efficiently delivering genes to the cells (both in vitro and in vivo), or creating 3D tissue structures with an associated vascular bed ready to be anastamosed with the surrounding vasculature. All of these techniques have a potential and are going to be reliant more and more on our understanding and control of the nanoscale cellular environment.
Future of Nanotechnology There are many avenues the field of nanotechnology will play in the field of cell transplantation. This field, although proposed almost 50 years ago, is still in its infancy. There is a great deal of potential. However, there are also potential hazards with this field. The ability for certain nanoparticles to easily transport across barriers such as skin and easily transport into the cell membrane makes these materials both powerful but also potentially dangerous. It has been demonstrated that some nanoparticles (cadmium) have caused cell death in in vitro studies. Because of these concerns, several regulatory bodies have begun to create policies to further study the effects of nanotechnology on the environment and potential health risks. However, this field will also provide great advances in cell transplantation. Understanding and controlling cell-cell and cell material interactions at the nanoscale will provide a greater opportunity for in vivo cell survival. In addition, the ability to create technologies using nanodetectors for noninvasive means of detecting transplanted tissue will provide clinicians the necessary tools to assess a successful graft. The ultimate goal will be to control cellular behavior by engineering surfaces at the nanoscale coupled with the micro- and the macroscale to create environments for successful cellular engraftment. This may include engineering a vascular bed prior to cell transplantation, 623
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33 Design and Implementation of a Cell Processing Facility Tatsuya Kin, Mohammadreza Mirbolooki, and Jonathan R. T. Lakey Clinical Islet Transplant Program, University of Alberta and Capital Health Authority, Edmonton, Alberta, Canada
Introduction A new form of therapy based on biological rather than pharmacological intervention has grown dramatically in power and scope in the past decade. This novel therapeutic option, cell therapy, utilizes living cells to rebuild or replace damaged organs and tissues. Hematopoietic stem cell transplantation in cancer therapy, mesenchymal stem cell transplantation in orthopedic repair, and pancreatic islet cell transplantation in diabetes are some examples of cell therapy currently being used or currently under development. Preparing cellularbased products often requires complex procedures. For example, harvested human cells are sometimes implanted onto a scaffold material, which enhances the cells’ structural properties, delivers biochemical factors or cell nutrients, or exerts mechanical or biological influences to modify the behavior of the cells or tissue. Regardless of the complexity in process, it must be recognized that consistency, potency, and purity are characteristics of optimal cellular-based products. To manufacture these products, cell-processing facilities require highly controlled process environments that must not only protect the products from biological and environmental contamination but protect their personnel. This chapter discusses several issues related to the design and construction of a cell-processing facility, the implementation of cell manipulation process, quality control of the final products, and regulation.
Clean Room Construction The construction of the clean room is the most important step for a cell-processing facility and is a highly Cellular Transplantation: From Laboratory to Clinic
specialized activity. Clean rooms are defined as specially constructed environmentally controlled enclosed spaces with respect to airborne particulates, viable microorganisms, temperature, humidity, air pressure, and airflow patterns. Facilities usually consist of multiple clean rooms with different requirements for cleanliness. Positive differential pressures should be maintained between the rooms to ensure airflows from the cleanest space to the least clean space. The cleanest room or space should be kept at the highest pressure, with pressure levels decreasing as cleanliness levels decrease. Regarding classification of air cleanliness, most of the industrialized countries have set their own standards, usually modifying the American Federal Standard 209E to their local needs [1]. These standards are based on measuring the presence of particles of varying sizes and number. They are not intended to characterize the viable nature of the particles. A more recent standard, International Standards Organization (ISO) 14644, considers not only the levels of nonviable particles as specified in Federal Standard 209E but viable particulates (i.e., microorganisms) (Table 33.1) [2].
Air-filtration System Particle filters are critical to creating and maintaining clean environments. Most modern cell-processing facilities have ventilation systems with filtered air — using high-efficiency particulate air (HEPA) filters, a technology developed in the 1940s. HEPA filters have a minimum particle collective efficiency of 99.97% for particles of 0.3 mm diameter or more. A HEPA filter typically lasts three to five years, depending on hours
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Tatsuya Kin, Mohammadreza Mirbolooki, and Jonathan R. T. Lakey TABLE 33.1 Clean room classification. ISO Classification Class Class Class Class Class Class Class Class Class
I II III IV V VI VII VIII IX
Superior Limits in Measured Particle Size per Cubic Meter (equal to or greater than stated size) 0.1 mm
0.2 mm
0.3 mm
0.5 mm
1.0 mm
5 mm
10 100 1,000 10,000 100,000 1,000,000
2 24 237 2,370 23,700 237,000
10 102 1,020 10,200 102,000
4 35 352 3,520 35,200 352,000 3,520,000 35,200,000
8 83 832 8,320 83,200 832,000 8,320,000
29 293 2,930 29,300 293,000
of operation, cleanliness of the laboratory, and type of work being performed. Most are inspected and certified on an annual or semiannual basis, depending on the standard of the facility.
Biological Safety Cabinet Biological safety cabinets (BSCs) are designed to provide biological protection for user, environment, and cellular-based product when appropriate practices and procedures are followed. Three types of BSCs — designated classes I, II, and III — have been developed to meet varying clinical needs. Class I BSCs have an open front and provide good protection for the user but not for the product. Class II BSCs protect the material being manipulated inside the cabinet from external contamination. These also have the capacity to protect the user. Class III BSCs are used when absolute containment of highly infectious or hazardous materials is required. Therefore, class II BSCs are the most useful type for a cell-processing facility. We have nine class II BSCs in our clinical islet isolation facility at the University of Alberta (Figure 33.1).
(VCT), or epoxy. Unless a very high level of cleanliness is required, VCT flooring is totally acceptable and much easier to maintain and repair. Plastic laminate, epoxy, and stainless steel are the preferred ceiling types. Plastic laminate is not a chemical-resistant material, but may be good enough for a cell-processing facility that does not heavily use chemicals.
Change Room The change room has a vital role as the primary interface between the clean room and the outside world. The change room air system must be designed to remove the potential for particulate generated by un-gowned personnel contaminating the gowned individual and the clean room. The change room is an ideal candidate for a unidirectional horizontal flow design, with the flow from the clean room entry to the change room entry. The room should be long and narrow, providing a gradual change from dirty to clean with traffic flow in one direction only. An air shower may be useful at the entry to the change room, where the removal of loose particles on street clothing can reduce the incidence of transported contamination.
Selection of Furniture
Microbiological Monitoring in Controlled For the interior surfaces of walls, floors, and ceiling, Environments a water-resistant material must be integrated into the design and construction of any clean room facility. Floors can be covered with vinyl, vinyl composition tile
Maintaining a controlled environment for manufacturing of cellular products is essential to minimize
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Design and Implementation of a Cell Processing Facility
Figure 33.1. Class II biological safety cabinets placed in a clean room with background condition ISO class VII.
the potential for introduction of contaminants into the product. The objective of microbiological monitoring is to assess the effectiveness of cleaning practices and of personnel that could have an impact on the bioburden of the controlled environment. A microbiological environment plan usually incorporates airborne microbial sampling and surface microbial sampling. There are two categories of air sampling device: (1) passive air sampling devices such as settle plates and (2) active air sampling devices such as impact, impingement, and filtration. Sampling methods used to capture airborne microorganisms include inertial, impact, centrifugal, filtration, and sedimentation. There are many factors to consider when selecting an air sampler. The sampling rate, duration of sample, and type of sampling device can strongly influence the viability of the microorganisms collected. Numerous reports of air sampling devices and techniques have presented data on the efficacy of these procedures [3, 4]. In our clinical islet isolation facility, we use the portable Reuter centrifugal air sampler (Folex-Biotest, Schlussner Inc., Moonachie, NJ) because it is easily disinfected and practical to transport and use (Figure 32.2). Surface microbial sampling may be accomplished by the use of contact plates or by means of swabs. There is no international consensus on
Figure 33.2. Reuter centrifugal air sampler.
the methods, types of sampling, frequency and timing of sampling, or tolerable limits of bioburden in cellprocessing facilities. Each facility should be responsible for establishing its own environmental microbial levels based on historical data, individual facility operations, and specific product considerations.
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Regulation As human cellular engineering is increasing in volume and complexity, problems stemming from a lack of regulatory oversight grow more acute. Many countries have moved to introduce specific regulations/guidelines for cellular-based products rather than continuing to rely on the traditional model of regulation for medicines and medical devices (Table 33.2). Although each country has its own regulatory system, the key is to bear in mind that it is really all about control. Because the complexity of cell therapies has been accompanied by an increased risk of adverse events, regulatory requirements are usually stratified according to the degree of cell or tissue manipulation in process. In the USA, the final guideline “Current Good Tissue Practice for Human Cell, Tissue, and Cellular and Tissue-based Product Establishments; Inspection and Enforcement” issued by the Food and Drug Administration took effect on May 25, 2005. This covers all aspects of production, including cell/tissue recovery; donor screening and testing; donor eligibility determinations; processing and process controls; supplies and reagents; equipment; facilities; environmental and labeling controls; storage conditions; product receipt, predistribution shipment, and distribution; advertisement/deviation reporting; and tracking from donor to product consignee. Although it may be a significant challenge for cell-processing facilities to
implement the elements required under the regulations, the regulations impact patients who have an expectation that the therapies are safe, effective, and of high quality.
Islet Isolation Facility at the University of Alberta Our islet isolation facility has two clean rooms in which the main procedure of islet isolation is conducted, allowing us to simultaneously process two pancreases at all times. However, the entire process for the pancreas is kept separate from any other pancreatic tissue, ensuring that no cross-contamination will occur. The facility also has its own dedicated supporting laboratories, including tissue culture laboratory and quality control laboratory. The islet products are processed under environmental conditions ISO class V in a class II BSC placed in a clean room with background condition ISO class VII. Separate air locks for personnel and material lead to clean rooms ISO class VII that are used as a tissue culture laboratory and a quality control laboratory. A consecutive air lock, representing clean room condition ISO class VIII, leads to a laboratory corridor with restricted access. The clean room clothes consist of surgical gown, cap, gloves, shoe covers, eye cover, and face mask. Sterile gloves and gown are worn after hands are scrubbed with povidone-iodine for all
TABLE 33.2 Regulation/guideline in various countries. Country
Agency/Body
Document
USA
Food and Drug Administration
Human Cells, Tissues, and Cellular and Tissue-based Products; Establishment Registration and Listing Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-based Products Current Good Tissue Practice for Human Cell, Tissue and Cellular and Tissue-based Product Establishments; Inspection and Enforcement Biologics and Genetic Therapies Directorate Compliance and Enforcement Strategy for the Safety of Cells, Tissues, and Organs Intended for Transplantation A Code of Practice for the Production of Human-derived Therapeutic Products A Code of Practice for Tissue Banks Guidance on the Microbiological Safety of Human Organs, Tissues and Cells Used in Transplantation Australian Code of Good Manufacturing Practice: Human Blood and Tissues
Food and Drug Administration Food and Drug Administration Canada
Health Canada Health Canada
United Kingdom
Medicines and Healthcare Products Regulatory Agency Department of Health Department of Health
Australia
Therapeutic Product Agency
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Figure 33.3. An alarm system for controlling temperature, air pressure, and humidity in clean rooms.
critical procedures. All materials are transferred into the clean room ISO class VII through a transfer hatch. An alarm system is implemented to control temperature, air pressure, and humidity in the clean rooms (Figure 33.3).
and personnel flow, process controls, and staff training and competency all help to achieve the goal that cellular-based products are safe, consistent, and of high quality.
REFERENCES Conclusions Cell-processing facilities should have an environment such that safe, pure, and effective products can be manufactured. There are challenges inherent in this task due to a number of factors in the cell-processing environment, including facility design issues such as clean room construction; personnel issues such as training, compliance, and supervision; and issues concerning the development and implementation of the microbiological monitoring program. The inherent variability of cellular-based products adds further challenges. Facility design, materials
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1. Airborne particulate cleanliness classes in cleanroom and clean zones. Institute of Environmental Sciences, 1992 (revised). 2. Cleanrooms and associated controlled environments, Part 1: Classification of air cleanliness. International Organization for Standardization Technical Committee (ISO TC 209), 1999. 3. Parks, S. R., Bennett, A. M., Speight, S. E., Benbough, J. E. An assessment of the Sartorius MD8 microbiological air sampler. J Appl Bacteriol 80:529–534; 1996. 4. Placencia, A. M., Peeler, J. T., Oxborrow, G. S., Danielson, J. W. Comparison of bacterial recovery by Reuter centrifugal air sampler and slit-to-agar sampler. Appl Environ Microbiol 44:512–513; 1982.
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34 Developing Cell Therapy Products Alfred V. Vasconcellos, MEM LCT Biopharma
Introduction Living cell therapies that allow the replacement or regeneration of function lost to organ damage or cellular-based diseases have the potential to significantly change the way medicine is practiced. This ability to treat the underlying cause of many diseases versus the masking or minimization of a disease’s symptoms promises to create a new class of products that harnesses the capabilities of transplantable cells, tissues, and eventually organs. The most important step responsible for making these technologies a reality is careful and insightful research. The most critical step to making these technologies bear fruit in the form of clinically relevant products is product development. Product development is the critical step that transforms a great idea that has been shown to work into a manufacturable and clinically relevant product that works in a reproducible and predictable manner. Whether that living cell product is provided as a service using autologous cells or an inventoriable aliquot of encapsulated pancreatic islets, the goal of product development is to allow a unique therapeutic concept to reach its altruistic and commercial goals.
Product Development Irrespective of taking place in an academic or industry setting, a properly executed research program should result in an initial description of a technology and confirmation of the technology’s functional capabilities. This description will be used to define the starting intellectual property, and if desired form the foundation of patents that will provide protection of commercial opportunities associated with the technology. This
Cellular Transplantation: From Laboratory to Clinic
description will also form the basis of the product definition that will be a critical tool for the product development process. Product development is the point where a technology has shown its potential in limited experiments and must now demonstrate its capability on a larger scale. These larger-scale preclinical studies will predict, as much as possible, the product’s safety and efficacy when used in a clinical trial (more stringent models, larger study populations, validated characterization, and end points). During the product development phase, the technology must also be transformed into a manufacturable product with reproducible and predictable performance that will support the larger-scale preclinicals. The process is a complex orchestration of hundreds and sometimes thousands of milestones that can further build a foundation of understanding of the base technology. This understanding provides the design controls that enable one to predict the functional outcome of a properly designed and manufactured product and to establish a minimized core of quality control tests that when satisfactorily completed ensure that end product will function as expected when used within a set of functional parameters. Accordingly, these design controls will be an important part of the regulatory submissions filed with the U.S. Food and Drug Administration and its international counterparts. Traditional methods of product development are well defined and documented. They have been the cornerstone tool responsible for turning innovation into commercial opportunity. Product development (PD) has historically been passed on in an interestingly oldfashioned manner. PD skills have been traditionally cultivated by gaining experience through “apprenticing” with others who have brought similar products to market. Although academic institutions provide excellent
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exposure to the tools necessary to develop today’s medical products, they offer only entry-level educations in the science of PD. This methodology has not kept pace with new technologies. In March of 2005, the U.S. Department of Health and Human Services (FDA, NIH, CMS, CDC, AHRQ) established a task force to address this problem. The Secretary of HHS stated: “Despite notable advances in basic biomedical research there has been a downward trend in the number of new drug and biologic marketing applications being submitted to the FDA for review” [1]. The comments and findings of the task force underscore the necessity and importance of PD in transferring the fruits of research to the clinic. As difficult as the PD of medical products is, the development of living cell products adds additional challenges. Traditional programs must be upgraded to take into account added complexity and the necessity for a multidisciplinary team of experts who usually do not work together. Combining biologists (who are comfortable with a raw material that is living, dynamic, and ever-changing with time and environmental conditions but who are uninterested in limiting the polydispersity of a polymer’s molecular weight) with engineers whose base desire to definitively characterize every aspect of a product’s raw material is thwarted by the complexity of a “living, breathing” cell requires additional communication and program management efforts. There are several thorough and updated primers on the development of medical products or drugs [2], but none specifically focus on cell therapy products. This chapter cannot detail the process but will attempt to identify some of the key issues to be addressed in the course of bringing cell-based therapeutics to the clinic and marketplace. This chapter focuses on issues important to early development programs dealing with the initial products developed from novel cell therapy technologies. Many of the issues identified herein undergo diminished importance in large established organizations with a significant project portfolio and are replaced with team overall program management issues.
is developed in an academic/clinical setting, an industrial environment, or a hybrid of both, the program is subjected to an ever-changing environment of constraints and requirements. The PD phase is unique in that it is where most promising technologies are abandoned due to their inability to cost effectively become a viable product offering. Department heads, technology transfer departments, or senior managers responsible for providing resources (money, people, facilities) for the program look to PD for confirmation that proceeding with the program is justified. This justification is often constrained by specific time frames. It is not uncommon to hear “We’ve got 24 months to demonstrate that this works in the clinic.” Right or wrong, such is the nature of the environment under which most cell therapy products are developed. Investors and financiers often correctly equate the initial demonstration of clinical safety and efficacy with confirmation that further investment is justifiable. This is a position that has evolved out of the historical ability of R&D organizations to justify program delays or lack of progress to nonscientific investors and management using technical terms that obfuscate the fact that the technology is in trouble. As a result, the demands on PD are quick turnaround and cost-effective confirmation of a clinically viable product. The responsibilities of PD include the management of expectations (don’t make promises you can’t keep) and a clearly defined and documented program that highlights the difficult spots as soon as possible so that they can be resolved quickly. The following points arise from these observations. •
•
•
Organizational Life Cycles Before proceeding into specific issues associated with expeditious and effective PD, it is important to recognize how important it is to optimize the efficiency of the process. Irrespective of whether the technology 636
•
Decisions are best made by people with the most information and individuals at the right level. Hire good people. Do not hide important information from them, and let them do their jobs. In addition, good managers understand the technology they are managing. Be honest about the capabilities of your technology and what it takes to develop it into a product. It is not always possible to determine this up front, but leave enough room to handle unforeseen difficulties. Understand that the appetite for risky investment in cell-based therapies has waned. The best response to risk-adverse decision makers is the confirmation of a product in the clinic. Getting there is PD’s priority. There is often a disconnect between the necessary time and money needed to develop a product and
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what is actually available. This means that wasted or noncritical path efforts need to be minimized. This also means that choosing the right product definition or application may simplify the development process, which is almost always a critical decision. Whether it is because a certain application has a shorter development program, simplified regulatory pathway, or faster clinical trials program going after “low-hanging fruit” first is often the easiest way to confirm that successful products can be derived from a technology (e.g., Genzyme and the development of Ceredase [3]) and is usually the correct strategic decision.
miscommunication and ensures continuity. Similar transition teams will also smooth the transfer of projects as they move from PD to clinical and commercial stages.
MULTIDISCIPLINARY PROJECT TEAMS Establishing multidisciplinary project teams adds to the completeness and efficiency of technology transfer. Engineering, product development, or manufacturing personnel occasionally participating in research project team meetings injects a sense of what defines a mature technology and what that technology may need to do in the course of becoming a product.
Technology Transfer
Product Definition
The transfer of cell therapy technology from research to the development teams has traditionally been responsible for delays and intercompany aggravation. It is not uncommon for a research group that has taken a good idea and made it work in a limited setting to transfer such a project to the development scientists and engineers without sufficient documentation and confirmation of therapeutic reproducibility. Efforts spent on developing a project that has missing procedures and poorly defined protocols more often than not will result in wasted time and resources during the crucial PD phase. Forward-looking organizations have developed methods for transferring a project without this disconnect.
As with most complex tasks, having a plan that identifies the many tasks required to meet critical milestones is a necessity for the product development process. The establishment of an initial product definition is required to properly plan the product development process, and establishing the initial version of the product definition is one of the first important milestones to be achieved in the PD process. Establishing a product definition is, however, a dynamic process and the definition will change as environmental design constraints and considerations (developmental pressures from the product’s commercial or clinical environment) change and data from the development process are analyzed. Getting started is difficult. In most cases a good technology can be converted into a final product in a number of ways. Many times a strong technology platform can result in a large family of different products or a product that has a number of different therapeutic applications. Integrating all factors that determine the likelihood of a product making it to the clinic and then becoming a therapeutic or commercial success is challenging but can greatly simplify the development process.
DOCUMENTATION Good scientific documentation of even the most mundane procedures used to culture cells or prepare polymer solutions is important. These steps will often prevent key pieces of information (common knowledge to advanced researchers) from being lost in the transfer to the PD team.
PROJECT OWNERSHIP
IF POSSIBLE, START WITH AN “EASY” PRODUCT
A project manager or a small dedicated team, intimately involved in the research phase of a program, should be entitled with ownership of the project. Transferring this team along with the project from research to product development demonstrates the ability to greatly reduce
Products developed via paths of least resistance, identified by data from early research studies, can build organizational credibility and a financial foundation upon which future products can be developed. If therapeutic transplantation of a novel human cell line stretches
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the budget, start by generating cash flow using the cell line as a screening tool for novel drugs. If a unique immunoisolatory polymer encapsulation struggles to protect xenogeneic cells in larger animals, start by marketing its use for transplanting allogeneic tissues with minimized immunosuppression. Successful introduction of a product often allows subsequent product offerings, which are more difficult to bring to the clinic, to be developed using more significant and readily available resources.
BUILD THE BRIDGE FROM BOTH SIDES OF THE RIVER The development process occurs under a wide range of ever-changing design constraints and considerations, and the final product will have to integrate with regulators, the clinic, and the marketplace. The initial product definition should take into account how the new product will fit into the overall picture. If done properly, the results of the PD process will capitalize on opportunities in FDA and international regulatory requirements, successfully predict market need, enhance business development and strategic partnering discussions, and enjoy short clinical trials with succinct patient populations. Unfortunately, these constraints sometimes conflict with each other and good modeling tools of the business (see “Product Development Tools”) are needed to provide an optimal resolution.
responsibilities such as manufacturing, regulatory, clinical, business development, distribution, and marketing is very important. The benefit of these additional voices is clear when the primary design constraints and considerations are examined during product definition. (See Table 34.1.)
Product Development Plan and Budget Establishing the product development plan and budget is a critical and conflicted task. Without exception, the lack of careful planning results in a program that is longer and more expensive than anyone imagined. Experienced product development specialists are invaluable at recognizing areas where identification of critical path events and combination studies can reduce time and expense. As important as it is to be thorough in the planning process, financial and time constraints will require an additional level of analysis to determine critical path milestones that will result in additional resources such as time, money, and personnel. The members of your organization charged with the responsibility to obtain these resources will have important information as to which milestones investors, granting agencies, or management deem worthy of additional time and money. Require them to provide insight into setting these goals. At the expense of possibly being redundant, the most efficient plans cannot be developed in a vacuum. •
DESIGN CONSTRAINTS AND CONSIDERATIONS It is a rare organization that has the ability to accurately gather information on all design constraints and considerations at the time of the initial product definition. Most of the time a seasoned PD team will acknowledge (document) the existence of these criteria, make educated experientially driven assumptions, and then schedule a task to confirm or update the information. Remembering that the product definition and the product development plan it helps define will be a dynamic and constantly refined process, a periodically convened task force that evaluates whether the plan or the product definition needs updating is commonly used. The multidisciplinary philosophy that works so well in a research team once again has value in this development task force. The inclusion of individuals with experience or 638
•
Get a well-documented transfer package from research. Educate them about the development process. In addition, hybrid products will be subjected to regulatory requirements that demand formal documented design controls. The process of creating the product must be carefully detailed and documented. Build a good team and maintain the team’s culture. Ask any venture capitalist, the first criterion to identifying a winning investment is the people — the team. There are many demands on the product development team, and every team will suffer from a failure in culture and communication. ◦
◦
Carefully select the members of the team. Where possible, let members of the team have some involvement in the hiring process. Promote risk-taking behavior and creative problem solving. Do not condone or reward mistakes but do not penalize when people
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Developing Cell Therapy Products
TABLE 34.1 Design constraints and considerations that shape cell therapy product development programs. Constraint/Consideration Description
Technology capability
Select a product definition for the first product attempts that is well within the technology’s capabilities based on existing performance data. For the initial product definition, no one knows this better than members of the research or transition team. Selecting and successfully developing the "low-hanging fruit" will allow the later development of more difficult or time-consuming products with greater available resources.
Analytical tools
Regulatory requirements and good manufacturing practices both demand that cell therapy products be characterized as completely as possible. The limitations often arise from the unavailability of antibodies, markers, and analytical methods to do the job accurately (to begin with) or to set up cost-effective QA/QC procedures for product manufacture. Thinking ahead can sometimes delay or avoid bottlenecks.
Preclinical models
Foundational to the completion of the product development phase is the confirmation of safety and (if possible) efficacy of the proposed product. The selection of clinical indications that have well-developed and readily accessible preclinical models can greatly simplify and shorten the process.
Developmental resources
In conjunction with the initial product definition and program plan, a budget tied to milestones is important to ensure that the development challenges associated with the selected product are adequately supported by available resources or the plan is capable of generating “fundable” milestones along the way.
Strategic partnerships
Selecting initial development opportunities with a potential corporate strategic partner can significantly improve the success rates of the product development cycle. Corporate and strategic “large pharma” deals are often looked upon as a source of money and validation. These potential partners may also have internal capabilities to offer, such as manufacturing, assay development, regulatory affairs, preclinical studies, clinical affairs, and market analysis. Keep in mind, however, to establish limited product- or indication-specific partnerships if possible. Giving away large portions of the technology’s rights at so early (and undervalued) a time point will greatly minimize the valuation of your efforts during clinical and commercialization phases. It is a trade-off to be carefully evaluated. Irrespective of the decision, never totally depend on a strategic partner. Plan for success, and, if possible, always have a mechanism for survival should the partnership dissolve.
Raw material availability
Raw material selection opportunities may not always be available. However, if possible, the use of raw materials generally regarded by regulatory agencies as safe and well characterized or materials readily available from multiple sources (single-sourced supply is a risk factor for commercialization) can significantly simplify the development process. Usually a unique technology based on novel materials or cells still depends on mundane materials such as packaging, catheters, polymers, media, growth factors, or feeder cells to some extent. Using materials that are well known to regulatory agencies or that are pre-characterized (validated QC procedures) will save time and effort. continued
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Alfred V. Vasconcellos TABLE 34.1 —cont’d Constraint/Consideration Description
Clinical indication
In cases where a technology is capable of addressing multiple clinical indications, comparative analysis of the developmental, regulatory, clinical, and commercial impact of the product application can expedite product approval. Compassionate use or orphan drug regulatory applications can positively impact the clinical and commercialization phases of a product’s life cycle. Selection of indications where the therapeutic molecule or mechanism is well known or already has a significant clinical history can minimize study populations, simplify preclinical studies, and shorten timelines.
Regulatory requirements
The regulatory agencies of most developed countries have provided detailed guidance designed to help with the product development and clinical development phases of cell therapy products. For the most part, these guidances have been modeled after the FDA programs, and with few exceptions the more developed the regulatory system the more efficient it is to obtain allowances. The FDA has committed significant effort to properly staff its agency with knowledgeable experts who have spent a lot of time looking at and trying to understand the challenges of cell therapy products. Most cell therapy efforts have found recent experiences with FDA helpful and instructive. It is important that this process start early and that the requirements of the regulatory process are carefully integrated into the development plan. Long-term studies take a long time. Plan for them and do them with materials and product prototypes that are representative of clinical product.
Clinical environment and competitive products
An important part of the product definition must be integrated from the existing marketplace. The involvement of clinicians and sales/marketing personnel is an important parameter in establishing critical product characteristics and functionality. Whether for altruistic or commercial reasons, or both, the end product must favorably position itself into the clinical setting — taking into account parameters such as current therapies and product, product end users (surgeons, interventional radiologists, endocrinologists), distribution demands, available sales forces, and so on. Do not assume that a good product will always succeed even if it is not needed or is cumbersome to integrate into patient care paradigms.
Distribution
Living cell products have the added challenge that they must get to the patient alive and functional. (Alive and functional are two related but different criteria. Remember that damaged or hypoxic cells may be alive at transplantation but fail to function as a product.) Shipping methods exist to deliver product overnight upon demand, but they are expensive and occasionally unreliable in geographic outskirts. Cryopreservation helps solve the problem but adds a level of complexity to the development process that must be integrated early into the plan. Remember that initially pretransplant assays may be required at the time of implantation. Tissue retains from the patient and the product may be required (especially with xenogeneic products) and that the complexity of the procedure determines the level of training required to separate user error from the functional behavior of the product. It may be that the first cell therapy products for complex applications are done in "franchised" hospital centers of excellence or standalone clinics, where specialized patient care and product inventories can be carefully controlled. continued
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Developing Cell Therapy Products TABLE 34.1 —cont’d
Constraint/Consideration Description Market demand and market size
Cost of goods/profit margins
Freedom to operate
Manufacturing
◦
•
•
•
In selecting first applications of novel cell therapy products, it is important to analyze marketing demands and constraints. Raw material availability and product scale-up issues are simpler to resolve when incorporated early in the development process. Although it is difficult to effectively integrate product costing early in the product development process (and care should be taken to prevent costing constraints from limiting creativity in the R & D process), the necessity of being able to profitably produce product at a price that fits the marketplace must be recognized at the beginning of the product development process and addressed as soon as possible when freezing product designs. Know the intellectual property landscape. As important as reading the scientific literature, read the patent estates of the competition. To actually bring a product to fruition you need freedom to operate (no infringement of existing patents) or a license. Licenses are easier and less expensive to obtain prior to the added value your product development program will create. Without limiting the development process by avoiding novel designs that demand new manufacturing techniques, evaluate existing manufacturing methods first. Producing product using existing validated methods simplifies the development program and often provides increased similarity between product prototypes used in early experiments and clinical or commercial grade product.
Gretsky (one of ice hockey’s all-time greats) once said that his father taught him to “Go to where the puck is going, not where it has been.” Everyone from investment bankers to refrigerator salesmen has used that quote. It applies to product development as well. Development cycles can be years in length. In the course of that process, assumptions and constraints may change. Testing if those changes impact the development program and updating the product definition when necessary are important.
extend themselves. In creative problemsolving sessions, no idea is stupid. Wild, seemingly inappropriate, ideas often lead to the most creative solutions. If at all possible, never restrict information. Empower the doers. Do not allow any politics that equate maintaining job security with hiding crucial facts.
Gather information. Do not reinvent the wheel. Use scientific and trade publications, patents, regulatory guidance, summaries, and scientific meetings. Integrate independent and external resources into your programs. Develop clinical and scientific advisory boards, and when appropriate hold clinical focus panel meetings. Get independent evaluation of plans, milestones, progress, and decisions. Use the clinic as a touchstone. Talk to the caregivers sooner rather than later. In regard to product design in an ever-changing world and compensating for change, Wayne
When developing the plan, demand and allow appropriate resources for robust and reproducible results, particularly when scaling preclinical results from small to large models. Although smaller preclinical models may have their idiosyncrasies most of the time, it only gets more difficult as you scale up to larger more clinically relevant disease models. If your product works variably or marginally in rodents, it is unlikely therapeutic results appropriate for the clinic will occur in
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nonhuman primate studies. Assemble a plan that each milestone results in a sound basis for the next step. Product development is a time-consuming process. If the foundation is not correct, the superstructure is doomed to failure. Do not spend precious limited funds and time building something that will have to be redone. There are a number of key activities identified in a cell therapy development plan. They are derived from the requirement that cell therapy products be safe, efficacious, and reproducible in their effect. These activites are outlined in Tables 34.2 through 34.5.
Intellectual Property Intellectual property (IP) and the patents/trade secrets that arise from the IP are crucial to provide the freedom to operate and eventual product protection. Most earlystage cell therapy companies are valued on the strength
of their intellectual property portfolio as a criterion second only to the strength of their team. Throughout this discussion, a significant effort has been placed on carefully defining the product being developed and planning a very focused and efficient development process designed to bring that product into the clinic. This focus is absolutely necessary for the development process to work well. This focus and indepth definition, the designs developed to create the product, and the experimental confirmation of those designs are the substance of which the IP is created. A strong patent portfolio often results from the novel enablement of a unique technology. There is, however, a small exception to be noted here. Good development organizations are able to maintain this focus but still take a periodic look at the longterm potentials of their technology. These periodic looks sometimes take the form of “blue sky” development meetings in which potential future products to fill the
TABLE 34.2 Cellular product components. Component
Characterization
Shipping and packaging
Isolation
Culture
Quality control/quality assurance
Description Establish a baseline that ensures that the cells used for product development experiments are substantially equivalent to the materials used for GLP (good laboratory practices) studies. These often include validatable determination of viability, identity, functionality (metabolism), potency, purity, and sterility. Eventually, these studies must also include latitude analysis to confirm that the range of acceptance criteria (75 to 100% viable and 80 to 100% potency) does not cause product failure when combined in their extremes. Confirmation and eventual validation of a reproducible transport method that ensures that materials shipped for use in preclinical, clinical, and commercialization applications are functionally the same material that passed quality control specifications at the manufacturing site. SOPs (standard operating procedures) and QA/QC characterization of all in-process materials, including media and digestive enzymes (if applicable). In cases where donor materials (such as human organs) vary greatly in content and condition, nonsubjective mechanisms to ensure substantially equivalent cell preps are obtained from each isolation or from the combining of multiple lots. Media, validation of serum-free substitutes or serum removal, and antibiotic removal. Such changes often require confirmation of the resulting cell preps using the characterization tools previously cited. In conjuction with all product development tasks, careful confirmation of traceability and accuracy of the work is required. Often a QC/QA representative is part of the development team. At some point in time prior to obtaining a BLA, system and process validations must occur—including traditional IQs, OQs, and PQs.
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Developing Cell Therapy Products TABLE 34.3 Biomaterials (if applicable).
Component
Characterization
Biocompatibility
Quality control/quality assurance
Description Establish a baseline to ensure that the introduction of the required biomaterials to the cellular component of the product does not change the safety efficacy or reproducibility of the final product. These often include validating vendors and establishing responsible raw material sourcing, establishing and validating incoming QA/QC specifications and procedures, GLP process validation, material purity, lot reproducibility, mechanical and functional analysis (both raw material and processed materials), and manufacturability. Regulatory agencies have defined a critical set of tests to be performed on biomaterials to be used in medical devices and hybrid medical products. It is a reasonable list and there are many "for-hire" GLP labs capable of performing these studies. They may include cytotoxicity, sensitization, irritation, acute systemic toxicity, subchronic toxicity, genotoxicity, implantation biocompatibility, hemocompatibility, chronic toxicity, carcinogenicity, reproductive/developmental toxicity, and biodegradation. As was necessary with the celluar component of these products, careful confirmation of traceability and accuracy of the product development effort is required. At some point in time prior to obtaining a BLA, system and process validations must occur — including traditional IQs, OQs, and PQs.
product pipeline are brainstormed into being. Mature development organizations (with available resources) will often retain a small portion of their budget to play with these ideas just enough to generate intellectual property. The resulting broad enablement can provide patents that protect future uses of the platform technology. Such protection adds value to the organization and may justify additional developmental resources. It is also a defensive strategy preventing other skilled organizations from reaching and patenting similar inventive conclusions based on published research. The process of using laboratory notebooks and carefully documenting and witnessing inventions is a common concept available from legal counsel or tech transfer personnel and is not discussed herein.
Product Development Tools There are a number of well-defined project planning and management tools and aids. The use of project management tools becomes increasingly important due to the complexity of cell therapy products. Many of these tools are primarily mechanisms to simplify and
visualize large numbers of tasks and their complex interrelationships. They are crucial to prevent critical path steps in the development process from being missed. Equally important, good program management techniques and tools improve communication and establish commitment from individuals and subproject teams. Plan visualization is also critical for determining and justifying the resources needed to complete a project and to simplify the presentation of project progress to senior management. The need for these tools and their benefits is so obvious that alternatives abound. If you search the Web for “product development tools” you will obtain in excess of a million links to sites offering documentation products, software, consultants, and training. Training is important, and conservatively priced programs are available for key personnel or for the entire team. Reasonably experienced product development programs will rely on one or more of the following.
FLOWCHARTS Flowcharts provide simple visualization of tasks and milestone interactions.
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Alfred V. Vasconcellos TABLE 34.4 Final product. Component
Characterization
Functional analysis
Product-specific in vivo analysis
Packaging
Shipping
Culture
Quality control/quality assurance
Description Establish a baseline that ensures that the final product used for product development experiments is substantially equivalent to the materials used for GLP (good laboratory practices) studies and clinical studies. These often include validatable determination of viability, identity, functionality (metabolism), potency, purity, and sterility. Eventually, these studies must also include latitude analysis to confirm that the allowable range of acceptance criteria does not cause product failure when combined in their extremes. Methods to identify, quantitate, and validate the in vitro and in vivo functionality of the product. These methods may be different for the preclinical and clinical settings. If they are different, they must be validated and correlated to each other. Often there is a required diagnostic that must be developed in parallel for the clinic in order to properly evaluate clinical trials and to support product commercialization. Dependent on the product configuration, site of implantation, or therapeutic indication, special analysis may be required to support product safety and efficacy. If possible, trafficking or migration of the product, product degradation timing, and resulting degradation products may be analyzed. If appropriate, methods of product retrieval or visualization should be determined. If possible, certain traditional preclinical evaluations (such as pharmacokinetics, dosing, biodurability, therapeutic product life, counter indication, and drug interaction studies) may be required. Validation of final product packaging to confirm that nothing associated with final product contact with the packaging or the packaging process itself does not alter the product’s safety, efficacy, or reproducibility. In addition, validation of the sterilization process that will demonstrate either inactivation of or elimination of microbial contaminants, endotoxin levels lower than regulatory requirements, and a package that can maintain sterility during shipping and handling will be crucial. Aseptically packaging cell-based products can be a difficult task and should be addressed early in the PD cycle. In addition, understanding the customer’s needs will be important in designing the package, including whether the package needs to be opened in a sterile field and will the final material (for example, a cell-filled syringe) be handled by the surgeon. This should also include confirmation of shelf life and the acquisition of data to support product dating and expiration dates to the regulatory authorities. Confirmation and eventual validation of a reproducible transport method that ensures that product shipped for preclinical, clinical, and commercial use is functionally the same material that passed product specifications as part of the manufacturing process. Media, validation of serum-free substitutes or serum removal, and antibiotic removal. Such changes often require confirmation of the resulting cell preps using the characterization tools previously cited. Special efforts must be taken in hybrid products due to many biomaterials’ tendency to bind foreign proteins. In conjunction with all product development tasks, careful confirmation of traceability and accuracy of the work is required. Often a QC/QA representative is part of the development team. At some point in time prior to obtaining a BLA, system and process validations must occur — including traditional IQs, OQs, and PQs.
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Developing Cell Therapy Products TABLE 34.5 Additional components.
Component
Description
Product instrumentation
Characterization and validation of instruments, tools, and product accessories required to introduce the product into a sterile field, thaw or prepare the product for use, implant or deliver the product, confirm functionality, or retain cell and tissue samples.
PERT OR GANTT CHARTS
PROGRAM MANAGER
PERT or GANTT charts are timelines that have the ability to analyze and display visualized tasks and their relationships, milestone interaction, resource planning and loading, task-specific development budgets, program variable definitions and alternative developmental pathways, manpower requirements, and distribution (a commonly used tool is Microsoft’s Project Program).
Updating and managing the plan is time consuming. Having an individual or small team (dependent on the size of the program) dedicated to keeping the plan and documenting accomplishments against the plan is one of the better scenarios for ensuring the availability of timely and accurate progress data.
BUSINESS MODELING IPR ESTATE MAPPING IPR estate mapping is graphic representation of your patents and IP rights as they fit into the competitive landscape. This is an important tool to plan patent strategy and prevent wasted product development activities. This is a complex process and requires access to patent databases and search engines. Even then, the data may not reflect patents undergoing prosecution and not in the public domain. Most patent lawyers can assist with this analytical tool.
REGULAR PROJECT TEAM MEETINGS AND PROGRESS PRESENTATIONS Updating and tracking progress against the plan on a regular basis improves inter- and intrateam communication and reduces the long-term effects of missed short-term milestones. If missed milestones are caused by poor planning or incorrect priorities, real-time adjustments can be made. If missed milestones are caused by technical challenges, team meetings have the ability to focus multidisciplinary resources on the problem.
In the course of an organization’s technology or product life cycle there are a number of pathways that must be evaluated. Identifying the best pathway is difficult because it often requires the decision maker to consider not just the near-term impact of the alternative but the long-term effect on the business itself. Well-run organizations often use financial modeling to analyze that long-term effect to the product, department, company operations, or shareholders. These business models, typically created and manipulated by finance departments, play an important role in major product development decisions. The effect of controlling budgets and milestone timing using strategies such as corporate partnering, in-licenses, alternative product designs, and choice of clinical indications can be defined and quantified using financial modeling as a tool.
Window of Opportunity Live cell therapies, cellular medicine, cell therapy, and cell-based therapeutics have the potential to significantly revolutionize the way we treat a number of serious diseases. This holds however you might choose to define
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or label the use of cells, such as the following. • • • • • •
To create an ex vivo physical construct (skin) To create a scaffold for autologous in-growth (vascular grafts) To replace a missing biological or mechanical function (islets) To create an extracorporeal device that provides a functional scaffold to prolong life (hepatocytes) To localize or enhance naturally occurring therapeutic capabilities (choroid plexus transplants) To enable long-term delivery of therapeutic proteins
These provide us with the tools to actually treat the disease, not just the symptoms. Although we have seen limited use of cell therapies in the clinic, the full promise of cell therapy has yet to be delivered. Documented scientific efforts to transplant cells date back centuries. There are filed (and expired) patents from the 1800s that describe the use of fine knit silk stocking material sewn into a drawstring bag to contain transplanted cells. In the latter part of the twentieth century, limited success in the academic and clinical research environment combined with the tremendous commercial desire to make living cell products a reality
resulted in the creation of companies focused on the commercialization of such cellular transplants. The majority of these pioneering efforts was unable to establish clinically or commercially robust product offerings with the technology and resources available to them at the time. They did, however, create a significant foundation of knowledge that is available to us today. In the first decade of the twenty-first century we have a unique window of opportunity. Never before has there been such an array of technology, cells, animal models, manufacturing methods, or analytical tools at our disposal. More importantly, there is now a cadre of experienced professionals with hands-on experience using and developing therapeutic live cell technologies. Success will depend on the ability of these torch-bearers to combine technology, expertise, resources, and determination into the critical mass needed for successful product and clinical development.
REFERENCES 1. Moving medical innovations forward: New initiatives from HHS, http://www.hhs.gov/reference/medicalinnovations.shtml. 2. Development of FDA-regulated medical products: Prescription drugs, biologics, and medical devices, 2nd ed. Ed by Whitmore, E., ASQ Quality Press, 2003. 3. http://www.genzyme.com/corp/structure/timeline_genz.asp.
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Index
A AAV (adeno-associated) viral vectors, 290, 365 AAV-GDNF (glial-cell-line-derived neurotrophic factor), 335 Abatacept (CTOA4-Ig), 23–24 ABO-compatibility, 23, 66 AC133, 264, 267 ACAID (anterior-chamber-associated acquired immune deviation), 571 ACE inhibitors, 259 Acellular matrices, 228, 231 Acinus, liver, 216–217 Activin A, 579 Acute rejection etiology of, 155 signs of, 45 treating with monoclonal antibodies, 21–23 treating with polyclonal preparations, 20 treating with steroids, 19–20 Acute renal failure. See ARF (acute renal failure) Acute tubular necrosis (ATN), 164, 180–182 AD (Alzheimer’s Disease) cell type therapy for, 35 choroid plexus in, 403, 404 cord blood cell therapies for, 556–557 NGF-producing transplants for. See NGF-producing cell transplants AD (autoimmune diseases), stem cell transplantation for, 241–257 ablative therapy without HSCT, 248 allogenic HSCT, 247 immune reconstitution, 247–248 introduction, 241 mesenchymal stem cell immunomodulation, 248–250 overview of, 241–242 in patients receiving HSCT for another indication, 242
references, 252–257 treating Crohn’s disease with HSCT, 246 treating MS with HSCT, 243–244 treating phase I/II JIA with HSCT, 246 treating RA with HSCT, 245–246 treating SLE with HSCT, 246 treating SSC with HSCT, 244–245 treating with HSCT, 242–243 ADCC (antibody-dependent T-cellular cytotoxicity), 316 ADEMEX (ADEquacy of PD in MEXico), 171 Adenosine, 18 Adenosine diphosphate (ADP), 112 Adenosine triphosphate (ATP), 112, 479 Adenovirus, 187 ADEquacy of PD in MEXico (ADEMEX), 171 Adhesion with alginate. See Alginate microcapsules in nanotechnology, 614–615 ADP (adenosine diphosphate), 112 Adrenal gland immunoprivilege of, 574 steroids produced by, 580 Adrenal medullary chromaffin cells. See Chromaffin cells Adrenal medullary transplants for acute pain, 458 for chronic pain, 458–462 for PD, 444, 525–526 Adult-derived stem cells ban on ESCs leading to research on, 559 ESC-derived graft rejection in CNS and, 310 mesenchymal bone marrow cells, 190 overview of, 219–220 parenchymal bone marrow cells, 189–190 reasons for using, 533–534 647
Index Advanced glycated end (AGE) products, 73 AdvaSeal, 147 aFGF, 498 AGE (advanced glycated end) products, 73 Age-related macular degeneration. See AMD (age-related macular degeneration) Aging choroid plexus in, 403–404 transplanting aged nuclei, 534 Agranulocytosis, 14 Air-filtration systems cell processing facility design, 631–632 for change room, 632 microbiological monitoring in controlled environments, 632–633 Albumin gels, 598 Albuminuria, 73 Alemtuzumab, 23 Alginate microcapsules biomaterial adhesion with, 598 for brain tumors, 348–349 cell encapsulation using, 141–142 characterization of, 407 creating, 621 for encapsulated transplants, 224–225 limitations of, 142 for tissue engineering, 231 for xenogeneic CP transplants, 406–407 Alginate-polylysine-alginate-bovine chromaffin cells (APA-BCC), 462 Aliquots DTZ staining using, 89 morphological analysis using, 89 as weak link in characterizing islet preparations, 98 Allogenic CNS grafts, 309, 311 Allogenic HSCT, 247, 248 Allogenic human tissue, 220 Allografts, CNS, 315–316 Alloimmume response, 13–14 Allophenes, 44 Allopurinol, 14 Allotransplantation defined, 3 of embryonic kidneys, 159–161 ethical and immunological barriers to, 3, 8 secondary diabetes using, 76–77 type 2 diabetes using, 77 xenotransplantation vs., 4 /f2a-/f01,3-Gal (/f2a/f0–1,3 galactosyltransferase) CNS transplant rejection and, 311 defined, 4 immune response of host to xenografts and, 315 /f2a/f04/f2b/f01 integrin, 242 ALS (amyotrophic lateral sclerosis) BHK cells for, 392 cord blood cell therapies for, 556–557 encapsulated cells for, 622–624 Alternatively activated macrophages, 600 AMD (age-related macular degeneration)
cell therapy in, 338–339 PEDF and, 335 reported cases of, 327 American Federal Standard 209E, 631 Amino-CLIO, 618 Amniotic cell transplantation, for LSD, 210 Amyotrophic lateral sclerosis. See ALS (amyotrophic lateral sclerosis) Analgesia adrenal medullary chromaffin cells and. See Adrenal medullary transplants cell lines for delivery of, 463–465 Anastomosis, 275 Anemia antilymphocyte sera side effect, 21 azathioprine side effect, 14 curing with cord blood cells, 548 MMF side effect, 18 sirolimus side effect, 17 Ang-2 (angiopoietin-2), 601 Angiogenesis defined, 600 therapeutic, 600–601 tissue engineering vasculature and, 233 Animal models of AD, 241, 373–375, 379 adrenal medullary transplants in chronic pain, 460–461 alginate microcapsules, 142 anti-T-cell monoclonal antibodies, 20 bioartificial kidney in ARF, 181 cell therapy in eye, 337 CNS transplants, 309, 319 cord blood cell injection, 264 CP transplants, 408–411 of DMD, 290 encapsulated human islet allografts, 145–146 GDNF in, 334 glioblastoma transplants, 345–346 of HD, 336–337, 386–387, 391–392, 418–420 hepatocyte transplants, 215–216 vs. human models, 36 islet transplantation for diabetes, 48, 61, 64 mesenchymal cell transplants, 209 of PD, 440–444, 523–525 polyclonal preparations, 20 translating results from, 37 Animals creating. See Xenotransplantation, creating animals for as donors. See Xenotransplantation, immunology of humane care of laboratory, 276–277 Annexin V, 111 Anterior-chamber-associated acquired immune deviation (ACAID), 571 Antibodies clinical islet xenotransplantation and, 50–51 enhancing SCI grafts by masking, 495 limiting on cell and tissue grafts, 8
648
Index removing antispecies, 47 risk of antilymphocyte sera, 21 Antibody-dependent T-cellular cytotoxicity (ADCC), 316 Anti-Cd-3, 21–22, 138 Anti-Cd-20 (rituximab), 23 Anti-Cd-25, 22–23 Anticoagulation therapy, 174 Anti-DNA-POD (peroxidase), 111 Antigen-presenting cells. See APCs (antigen-presenting cells) Antigens, tumor-associated (TAAs), 604 Antihypertension medications, renal transplants, 16 Antilymphocyte sera, 20–21 Antimacrophage activity, of DSG, 24 Antithymocyte globulin, 67 AO (Acridine orange), 107 APA-BCC (alginate-polylysine-alginate-bovine chromaffin cells), 462 APCs (antigen-presenting cells) alloimmume response and, 13 cell replacement for kidney failure and, 158 CNS graft rejection and, 310–316 CNS immunity and, 305–308 immune response to xenografts and, 5–7 islet encapsulation and, 136–138 Aplastic anemia, 14 Apoptosis after SCI resulting in DNA fragmentation, 479 decrease in MMP during, 120 GDNF reducing, 363 and islet tissue, 110–112 Aqueous humor, eye anatomy, 327–328 ARF (acute renal failure) bioartificial kidney in, 180–182 current therapies for, 171 development of SIRS with, 173 high mortality rate and, 171 statistics for, 169 Arg-Gly-Asp. See RGD (Arg-Gly-Asp) Arthopathy, in hemophilia, 187 Articular cartilage formation, 295 Aspartate, 479 Assays, 63 ASTIC (Autologous Stem Cell Transplantation International Crohns) trial, 246 ASTIMS (Autologous Stem Cell Transplantation International Multiple Sclerosis) trial, 244 ASTIS (Autologous Stem Cell Transplantation International Scleroderma) trial, 245 Astra AB Sweden, clinical trial, 462 Astrocytes CNS immunity and, 306 produced after SCI, 478, 480 Atgam (polyclonal antilymphocyte sera), 20–21 ATN (acute tubular necrosis), 164, 180–182 ATP (adenosine triphosphate), 479 measuring concentration for cell viability, 113–114 in mitochondrial function, 112 AtT-20 cell line, 463
Autoimmune diseases. See AD (autoimmune diseases), stem cell transplantation for Autologous Stem Cell Transplantation International Crohns (ASTIC) trial, 246 Autologous Stem Cell Transplantation International Multiple Sclerosis (ASTIMS) trial, 244 Autologous Stem Cell Transplantation International Scleroderma (ASTIS) trial, 245 Autologous tissue grafts CNS, 309 engineered trachea, 282–283 HSCT for liver constructs, 228–229 HSCT for treatment of autoimmune disease, 242–243 limitations as cell source, 220 Autotransplantation of islets, 76 Avascular (dry) AMD, 339 Avascular necrosis (AVN), 19 Avitene, 68 Axokine, 333 Axon growth, and spinal cord injury repair neutralizing inhibitors of, 480 obstacles, 478 OEG promoting, 486–488 peripheral nerve grafts promoting, 482 providing substrate for, 497–502 reactivating intrinsic programs for, 481 reducing scar formation, 480 remyelination, 503–504 Schwann cells promoting, 482–484 stimulating, 480–481 Axon growth, with biomaterials, 602 Azathioprine clinical use of, 14 islet allograft immunosuppression with, 138 rejection with MMF vs., 18–19
B B7 family, 13, 23–24 B16 F1C29 implants, 463 Baboons, 3–4, 51–52 Baby hamster kidney (BHK), 390–393 Bachoud-Lévi, Anne-Catherine, 425–427 Bacillus laterosporous, 24 BAHF (bioartificial hemofilter), 174–175 Basal ganglia degeneration of in PD, 439 motor disorders associated with, 418 Basic fibroblast growth factor (bFGF) associated with angiogenesis, 601 for SCI axon growth, 498 Basiliximab (Simulect), 22 BBB (blood-brain barrier) in administering NGF, 374–375, 379 in CNS graft rejection, 312–315 in CNS immunity, 305–306, 308 in CNTF delivery, 389–390 in ERT therapy for LSD, 206 viral vectors for GDNF gene therapy, 365
649
Index B-cells, 59 in CNS graft rejection, 311–314 relapses in allogenic HSCT from, 247 rituximab directed to CD-20 antigen of, 23 in survival of cell and tissue grafts, 5, 9 /f2b/f0-cells, pancreatic islet comparison of literature values for content in islets, 97–98 EM analysis of islet purity and, 89 EM vs. LM analysis of islet purity, 92 measuring viability of using MMP, 121 quantifying in islet preparations, 98 quantifying with PCV islet measurement, 100–101 BDNF (brain-derived neurotrophic factor) CNTF and, 333 for HD, 387 measuring in intraocular grafts, 376 nerve regeneration with biomaterials and, 602 overview of, 332–334 preventing progressive SCI, 497 promoting axon growth after SCI, 478 providing substrate for SCI axon growth, 498–501 Schwann cells producing, 482 BEAM therapy, 244 Belatacept, 24 Beta blockers, 259 beta-GAL (beta-galactosidase) gene, 362 Beta score, 71–72 bFGF (basic fibroblast growth factor) associated with angiogenesis, 601 for SCI axon growth, 498 BFU-e (burst-forming unit), 548 BHK (baby hamster kidney), 390–393 Bilateral lesion syndrome, in PD, 440 Bile, 215–217 Bioactive peptides adhesion of biomaterials with, 598 administering cell therapies with, 593 Bioartificial hemofilter (BAHF), 174–175 Bioartificial kidneys, 172–176 bioartificial renal tubule, 175–176 extracorporeal. See Extracorporeal bioartificial kidneys hemofilter, 173–175 implantable, 176 requirements for engineering, 172–173 Bioartificial livers, 215 Bioartificial renal tubule assist devices. See RADs (bioartificial renal tubule assist devices) Biocompatibility Novocell PEG encapsulation, 144–145 scaffolding material for tissue-engineered liver, 230 Biodegradable microspheres, 284–285 Biodegradable scaffolds, 596–598 Biogen antibody, 138 Biological safety cabinets (BSCs), 632 Biomaterial cell therapies, 593–611 bioactive peptides in, 598 as delivery vehicles and scaffolds, 596–598 design overview, 595–596
future directions, 604–605 gene delivery in, 599–600 immune response to, 594–596 introduction, 593–594 producing NGF, 377–378 protein delivery in, 598–599 references, 605–611 therapuetic immune responses in, 600–604 Biondi ring tangles, 403–404 Biopsy-proven rejection, 18–19 Bioreactors (cell-growth systems) extracorporeal bioartificial kidneys, 177–178 tissue-engineered livers and, 231 Biventricular pacemakers, 259 Blastocysts, ESCs from, 219 Blood-brain barrier. See BBB (blood-brain barrier) Blood-tissue barriers, and immunoprivilege, 575–576 BMECs (brain microvascular endothelial cells), 305, 308 BMPs (bone morphogenetic proteins), 295 BMSCs. See MSCs (bone marrow-derived mesenchymal stem cells) BMT (bone marrow transplantation) for cancer, 189 inducing graft-specific tolerance in CNS, 318 LSD therapy with, 205–207 Bone healing, 295–296 Bone marrow aspirate, 552 Bone marrow cells adult mesenchymal, 190 adult parenchymal, 189–190 mesenchymal stem cells derived from. See MSCs (bone marrow-derived mesenchymal stem cells) therapeutic potential of, 557 using adult-derived stem cells from, 533–534 Bone marrow stromal cells, 284–285, 490–491 Bone marrow transplantation. See BMT (bone marrow transplantation) Bone morphogenetic proteins (BMPs), 295 Bovine serum albuminum (BSA), 179 Bowman’s capsule, 170 BRAID (brain-associated acquired immune deviation), 571 Brain blood-brain barrier. See BBB (blood-brain barrier) CNS graft rejection and, 315 CNTF delivery to, 389–390 CP epithelial cell transplants for. See CP (choroid plexus) functional/structural differences in, 30 GDNF protein delivery in, 364 immunoprivilege of, 571–572 NT2N cell grafts in normal host, 355–356 NT2N cell grafts in stroke host, 355–356 repair of, 29–30 Sertoli cell transplants for injury to, 558 therapeutic effects of GDNF on, 363 tissue grafts, 375–376 transplants for glioblastoma, 345–352 Brain-associated acquired immune deviation (BRAID), 571
650
Index Brain-derived neurotrophic factor. See BDNF (brain-derived neurotrophic factor) Brain microvascular endothelial cells (BMECs), 305, 308 BrdU-retaining cells, 165 Broxmeyer, Hal E., 548 BSA (bovine serum albuminum), 179 BSCs (biological safety cabinets), 632 Budget, product development, 640–644 Burst-forming unit (BFU-e), 548 Business modeling, product development, 647
C C (calcein AM), 107 C3bBb convertase, 45 C5b678, 45 CABG (coronary artery bypass grafting), 266–267 CAG triplets, 386 Calcein AM (C), 107 Calcineurin inhibitors, 14–16, 17 Calcitonin-gene-related peptide (CGRP), 483 Calcium-calcineurin pathway, 13 cAMP (cyclic adenosine monophosphate), 481, 498 Campath-1H (alemtuzumab), 23 Canals of Hering, 220 Cancer bone marrow transplants for, 189 MPA inhibiting, 18 stem pools, 349 vaccines using biomaterial delivery, 603–604 CAPIT-HD (core assessment protocol for transplantation in HD), 425–427, 428 Carbohydrate metabolism, 218 Cardiac arrhythmias, 552 Cardiac cell transplantation, 259–274 background, 259–260 cell delivery timing and techniques, 264–266, 268 challenges for future improvement, 268–270 clinical studies, 266–268 contractile cells, 260–261 differentiating progenitor cells into cardiomyocytes, 269 electrical integration in, 269–270 with MDSCs, 296 noncontractile cells, 262–264 optimizing cell type, 268 references, 270–274 survival of transplanted cells in, 268–269 Cardiac myocytes necrosis, 552 Cardiac stem cells (CSCs), 260 Cardiomyocytes cardiac repair with, 260–261 differentiating progenitor cells into, 269 ESCs differentiating into, 262 HSCs differentiating into, 264 vs. skeletal myoblasts, 261 Cardiovascular disease cell transplants for. See Cardiac cell transplantation islet transplants for diabetes and, 74–75 Cartilage tissue engineering
composite chondrocyte and epithelial cells, 279–280 hyaline vs. ear, 276 nasal septum as source of, 276 for tracheal equivalents, 275–276 tracheal vs. nasal chondrocytes, 276–279 Caspase activity, 110–111 Catecholamine neurons, 442 Cats, creating with nuclear transfer, 45 CCI (chronic constriction injury), of sciatic nerve, 456 CCL2 (chemotactic molecule chemokine ligand-2), 601 CD4 T-cells, 8, 21, 307, 603 CD8 T-cells, 8, 21, 307 CD14 T-cells, 264 CD-20 T-cells, 23 CD-25 T-cells, 22 CD34+ T-cells, 264, 548, 552–553 CD-52 T-cells, 23 CD133+ T-cells, 552 CelCept, 138 Cell-adhesion receptors, 614 Cell-based delivery systems, 348–350 Cell composition of islets. See Islet preparations, purity and cell composition Cell grafting approach to HD, 387 CNTF delivery to brain with, 390 for spinal cord injury. See SCI (spinal cord injury) repair, cell grafting for Cell lines for analgesic delivery, 463–465 encapsulation of, 330 generating dopamine neurons from, 534–535 propagation in vitro, 526–527 as source for cells, 220 Cell membrane integrity, 107–110 live/dead stains, 107–108 quantitative membrane integrity measurement, 108–110 Cell processing facility, designing, 631–635 air-filtration system, 631–632 biological safety cabinet, 632 change room, 632 clean room construction, 631 furniture selection, 632 introduction, 631 islet isolation facility at University of Alberta, 634–635 microbiological monitoring in controlled environments, 632–633 references, 635 regulation, 634 Cell quantity, predicting islet transplant, 122–124 Cells death of in spinal cord injury, 479–480 delivery techniques, 265–266 survival of transplanted, 36–37 suspension methodology, 442–444 used in encapsulation, 622 Cell transplant therapy antibodies and, 8–9 barriers to xenografts, 5–6
651
Index Cell transplant therapy (continued) cellular rejection in, 8 chronic pain management and, 455–456 humoral rejection and, 156 immune response to, 5–7 limitations of, 156 liver disease. See Liver disease cellular transplants LSD and, 207–210 nanotechnology and. See Nanotechnology preventing rejection of, 196–197 primary nonfunction in, 7–8 product development. See PD (product development) risk of rejection in, 188 structurally complex organs and, 4 therapeutic approaches to, 9 treating kidney failure with, 176 Cellular immortality, 527 Cellularized constructs, 228 Cellular mini-pumps, 457 Cellular niches of liver, 221–222 Cellular rejection, 8 Cellular uromoyplasty, 295 Central nervous system. See CNS (central nervous system) Cerebral ventricles, 385 Cerobrospinal fluid. See CSF (cerobrospinal fluid) CFU-GEMM (granulocyte-erythroid macrophage megakaryocytic colony-forming units), 548 CFU-GM (granulocyte macrophage colony-forming cells), 548 CGRP (calcitonin-gene-relatead peptide), 483 Chang, Thomas, 139 Change room, cell processing facility, 632 Chemical islet xenotransplantation, 50–51 Chemotactic molecule chemokine ligand-2 (CCL2), 601 Chemotherapy, brain tumors, 345 Children, islet transplantation for diabetes, 77–78 Chimeras, 44 Chimpanzees, as transplant donors, 3–4 Chloramine T method, 277 Chondrocytes autologous engineered trachea, 282–283 composite, 279–280 tracheal vs. nasal, 276–279 Chondroitin sulfate proteoglycans. See CSPGs (chondroitin sulfate proteoglycans) Choroidal epithelial cells. See CP (choroid plexus) Choroid plexus. See CP (choroid plexus) Chromaffin cells adrenal medullary transplants and, 457–458 adrenal medullary transplants in chronic pain, 458–460 clinical trials using transplants of, 460–462 conditionally immortalized cell lines and, 464–465 selection of candidate therapeutic peptides, 465–467 transduced, 467–469 using lentiviral-based vectors, 465 Chronic constriction injury (CCI), of sciatic nerve, 456 Chronic kidney disease (CKD), 169, 171 Chronic pain, transplants for, 455–475 adrenal medullary transplants, 457–460
cell lines for analgesic delivery, 463–465 cell transplantation, 455–457 cellular pumps, 457 chromaffin cells and, 465–469 clinical trials, 460–463 introduction, 455 references, 469–475 Chronic renal failure (CRF), 169, 171 CIA (collagen-induced arthritis), 251 Ciliary neurotrophic factor. See CNTF (ciliary neurotrophic factor) Circulating progenitor cells (CPCs), 267 CKD (chronic kidney disease), 169, 171 Class I antigens, 158–159 Class II antigens, 158–159 Clean room construction air-filtration system, 631–632 biological safety cabinets, 632 change room, 632 furniture selection, 632 microbiological monitoring in controlled environments, 632–633 overview of, 631 Clinical neural xenotransplantation, 51 Clinical trials adrenal medullary or chromaffin cell transplants, 460–462 cardiac cell transplantation, 266–268 cell therapy in retinitis pigmentosa, 337–338 cell transplantation for SCI repair, 505–506 encapsulated cells, 622–624 encapsulated CNTF-producing cells in HD, 392–394 for encapsulated human islets, 143 fetal tissue vs. adrenal medulla transplantation for PD, 525–526 neural transplantation in HD, 423–427 neural transplantation in PD, 444–448 with NGF-producing human fibroblasts, 380 not recommended in hemophilia, 198 NT2N cells in stroke patients, 360–361 treating autoimmune diseases with HSCT, 243–248 Cloning generating histocompatible tissues, 165 producing Dolly the sheep/other animals, 44–45 Clonogenic human CNS stem cells (hCNS-SC), 310 Cloquet’s canal, of eye, 328 CMV (cytomegalovirus) infection, 18, 75 CNS (central nervous system) cellular therapy for diseases of, 51 immunoprivilege of, 574 macrophage activity in, 484 neurotrophic encapsulated cell therapy and, 336–337 neurotrophic factors in eye and, 331–332 CNS (central nervous system) transplants, 305–326 factors involved in immunity, 305–306 introduction, 305 mechanisms of graft rejection, 311–316 mechanisms of immunity, 307–309 nerve regeneration with biomaterials, 602 overcoming rejection with graft-specific tolerance, 318–320 overcoming rejection with nonspecific immune tolerance, 316–318
652
Index references, 320–326 role of graft in, 309–310 role of host in, 310–311 CNTF (ciliary neurotrophic factor) clinical trials using encapsulated cells, 392–394, 622–624 discovery of, 388 overview of, 332–333 potential in retinitis pigmentosa, 337–338 Schwann cells producing, 482 CNTF-producing cells, for treating HD, 385–398 animal models of HD, 336–337 approach, 387–388 cellular vectors for delivery, 390–392 clinical features of HD, 385 clinical trials for encapsulated cells in HD, 392–394 discovery of, 388 etiology of HD, 386 introduction, 385 models of HD, 386–387 neuropathological features of HD, 385–386 neuroprotective effects of, 388–389 references, 394–398 strategies for delivery to brain, 389–390 therapeutic approaches to HD, 387–388 Coagulation, liver, 215 Coatings, 142–143 COBE cell separator, 63 Co-culturing interactions, 222, 229 Colchicine, 15 Collaborative Islet Transplant Registry report, 135 Collagen for encapsulated hepatocyte transplants, 224 for protein delivery in biomaterial implants, 598 for tissue engineering, 230–231 Collagenase, 63, 87 Collagen-induced arthritis (CIA), 251 Collection duct, 170, 173 Committed progenitors (late progenitors), 219, 220 Complement activation in hyperacute rejection, 45 inhibition of in xenotransplantation, 46–47 regulators of, 46 Complement regulatory proteins, 47–48 Conditionally immortalized cell lines, 464–465 Cones, retinal anatomy, 328–329 Conezyme Q10, for HD, 387 Conformal coatings, encapsulation, 142–143 Continuous venovenous hemofiltration (CVVH), 171, 182 Contractile cells cardiomyocytes, 260–261 replacing damaged myocardium, 260 skeletal myoblasts, 261–262 Convective-based therapies, 171–172 Cord blood transplants for acute ischemic diseases, 264, 552–555 banking procedures, 38 cord blood endothelial progenitor cells, 550–551 cord blood hematopoietic stem cells, 548–549
cord blood mesenchymal stem cells, 549–550 for inborn errors of metabolism with neurologic consequences, 555–556 for neurodegenerative diseases, 556–557 for neurological diseases, 555 overview of, 548 therapeutic potential of cells, 557 using adult-derived stem cells from, 533–534 Core assessment protocol for transplantation in HD (CAPIT-HD), 425–427, 428 Cornea, eye anatomy, 327–328 Coronary artery bypass grafting (CABG), 266–267 Corticosteroids avoiding use in islet transplantations, 67 clinical use of, 19–20 as potent immunosuppressives, 580 Co-stimulation inhibitors, 23–24 Coulter Multisizer II, 102 COV (coefficient of variation), 116 COX (cyclooxigenase), 318 CP (choroid plexus), 399–416 in aging, 403–404 basic structure of, 399–401 characterization of alginate/encapsulated choroidal epithelial cells, 407–408 in CNS early development, 402–403 encapsulated xenogeneic transplants in animal models, 408–411 first line of defense for brain, 401–402 introduction, 399 medical conditions from changes in, 399–400 and neurodegeneration, 404–405 and neurogenesis, 405–406 as pathway of entry for infected cells into brain, 402 receptors in, 402 references, 412–416 secretory role of, 302 traditional roles of, 401 for transplantation therapy, 406 xenogeneic transplants, 406–407 CPCs (circulating progenitor cells), 267 C-peptide levels, 70 C-peptide secretion, 72 C-reactive protein (CRP), 173, 183 Creatine, 387 CREB, 481 Cre/lox, 465 CrePRI gene, 465 CRF (chronic renal failure), 169, 171 Crigler-Najjar syndrome, 192–193, 223 Crohn’s Disease, 246 Cross collection, 205 CRP (C-reactive protein), 173, 183 Cryropreservation, of N2TN neurons, 358 CSCs (cardiac stem cells), 260 CSF (cerobrospinal fluid) choroid plexus producing, 401 depositing cells in, 30 in ischemia, 405
653
Index CSF (cerobrospinal fluid) (continued) production decreasing with age, 404 separating CNS from peripheral system, 305–306 CSPGs (chondroitin sulfate proteoglycans) barriers to SC grafts, 483 GRPs for spinal cord injury transplants and, 494 scar formation after SCI and, 480 Cultured cells, 526–527 Culture of islets, 63–64 Cushingoid facies, 19 CVVH (continuous venovenous hemofiltration), 171, 182 Cy-based protocols, 244–246 Cyclic adenosine monophosphate (cAMP), 481, 498 Cyclooxigenase (COX), 318 Cyclophilin, 14 Cyclosporine biopsy-proven rejection with MMF vs., 18–19 clinical use of, 14–16 combination therapy with sirolimus, 17–18 conformal coatings and, 142–143 drug interactions with, 15 enhancing NT2N cell graft survival, 358 islet allograft immunosuppression, 138 tacrolimus side effects vs., 16–17 as therapy for xenografts, 9 Cylindrical cartilage transplantation, 275–288 autologous engineered trachea, 282–283 cylindrical tube: composite chondrocyte and epithelial cells, 279–280 cylindrical tube: tracheal vs. nasal chondrocytes, 276–279 development of tracheal replacement, 275–276 discussion, 285–286 helical and Y-shape, 280–281 marrow stromal cells with TGF-/f2b/f02, 284–285 references, 287–288 CyQUANT dye, 105 Cysts, after SCI, 478 Cytochrome P450 inhibitors, 15, 18 Cytokines, 8, 579 Cytomegalovirus (CMV) infection, 18, 75 Cytotherapeutics, Inc., 143, 462
D DA (dopaminergic) neurons, 309 in animal models, 440–441 from cell lines and other possibilities, 534–535 degeneration of in PD, 439 from designer cells, 533–534 development of, 527–528 from engineered stem cells, 535–536 experimental transplantation of, 442 fetal VM transplantation for PD, 524–526 from hESCs, 530–531 from mouse and subhuman primate ESCs, 530 from neural stem cells and other sources, 528–530 from NTera2 cells, 532–533 Daclizumab (Zenapax), 22, 67, 138 Danon disease, 205
DAPC (dystrophin-associated protein complex), 289 Dark neurons, 455, 459 Davol, 68 DBS (deep brain stimulation), 440, 448 DCs (dendritic cells) autoimmunity and, 603 cancer vaccines and, 603–604 overview of, 602–603 decay-accelerating factor (hDAF), 48–49, 155–156 Deep brain stimulation (DBS), 440, 448 Degradative enzymes, 495 Dendritic cells. See DCs (dendritic cells) Dendritic polymers, 617 Density gradient centrifugation, 87 Deoxyribonucleic acid. See DNA (deoxyribonucleic acid) Deoxyspergualin (DSG), 24 Dermal fibroblast transplantation, 486 Designer stem cells, 533–534 Determined multipotent stem cells, 219 Dextrose, 69–70 Diabetes. See also Encapsulated human islet allografts diabetes. See also islet preparations Diabetes. See also Islet transplants for diabetes causing progressive kidney damage, 171 characteristics of, 59 corticosteroids increasing risk of, 19 Edmonton protocol and, 48 insulin therapy limitations, 60 pathogenesis of, 59–60 quantity of islets needed to cure, 64–65 Sertoli cell transplantation for, 558 tacrolimus side effect, 16–17 Diabetic nephropathy, 59, 73–74 Diabetic retinopathy, 59, 73 Dialysis, 155, 171 Diarrhea, 18 Dielectric-metal nanospheres, 616 Diet, after islet infusion, 70 Differentiation, 31, 36–37 Dilitiazem, 15 Dimethylsufoxide (DMSO), in DTZ staining, 89 Direct antigen presentation, 6 Direct injection, for liver diseases, 223 Disease-modifying antirheumatic drugs (DMARDs), 245 Dithizone (DTZ) staining assessing islet purity with, 89 vs. morphological analysis, 97 visual counting of islets using, 98–99 Diuretics, for heart failure, 259 Dividing tissue, encapsulation of, 330–331 DMARDs (disease-modifying antirheumatic drugs), 245 DMD (Duchenne’s muscular dystrophy) adult-derived stem cell therapy for, 293–294 animal model of, 290 current treatment and limitations, 290–293 overview of, 289–290 DMEM (Dulbecco’s modified Eagle’s medium), 280, 284–285 DMSO (dimethylsufoxide), in DTZ staining, 89
654
Index DNA (deoxyribonucleic acid) apoptosis after SCI resulting in fragmentation of, 479 apoptosis in islets and, 111–112 azathioprine and, 14 cancer vaccines using encoding of, 604 fragmentation of in ischemia, 404–405 measuring quantity of islet tissue, 105 microinjection of, 43 nanotechnology gene delivery, 616–617 utilizing biomaterials to deliver, 599–600 Documentation, product development, 639 Dogs, creating with nuclear transfer, 45 Dolly the sheep, 44–45 Dopaminergic neurons. See DA (dopaminergic) neurons Dor procedure, 259 Dorsal root entry zone (DREZ), 486 Dorsal root ganglion (DRG) neurons, 405 DREZ (dorsal root entry zone), 486 DRG (dorsal root ganglion) neurons, 405 Drug therapies for heart failure, 259 nanotechnology drug delivery approaches, 615–617 Dry (avascular) AMD, 339 DSG (deoxyspergualin), 24 DTZ (dithizone) staining assessing islet purity with, 89 vs. morphological analysis, 97 visual counting of islets using, 98–99 Duchenne’s muscular dystrophy. See DMD (Duchenne’s muscular dystrophy) Dulbecco’s modified Eagle’s medium (DMEM), 280, 284–285 Dyes measuring MMP, 120–122 used in Annexin V, 111 used in DTZ staining, 89 used in live/dead stains, 108 used in nuclei counting of islet cells, 101–104 Dyskinesias, 447–448, 526 Dystrophin-associated protein complex (DAPC), 289 Dystrophin gene, 289, 290
E EAE (experimental allergic encephalomyelolithis), 251 Ear cartilage, 276 Early progenitors, 219 EB (ethidium bromide), 107 EBMT (European Group for Blood and Marrow Transplantation) autoimmune diseases and, 241 patients receiving HSCT for AD, 242–243 patients receiving HSCT for SLE, 246 phase I/II studies, 243–246 SSC patients receiving HSCT, 244–245 EBV (Epstein-Barr) virus, 18 EC (embryonic carcinoma) cells, 34, 175, 464 ECB (encapsulated cell biodelivery), 379 ECCO (European Chrohns Colitis Organization), 246 ECMs (extracellular matrices) cell encapsulation engineering and, 331
DMD and, 289 hemofilter design and, 175 liver disease transplant requirements, 222 macrophages’ role in tissue repair, 600 overview of, 222 recreating in scaffolds for tissue engineering, 232 scar formation after SCI and, 480 spinal cord injury compromising, 478 used in encapsulation, 622 ECS (extracapillary space), 179 ECT (encapsulated cell technology), 338 Edmonton protocol, 48, 557. See also Islet transplants for diabetes EGF (epidermal growth factor), 222, 232 eGFP gene, 362 EGFR, 346 Electrical integration biventricular pacemakers for, 259 cardiac cell transplantation improvements, 269–270 of cardiomyocytes, 260–262 safety of skeletal myoblasts, 267 skeletal myoblasts and, 261–262 Electron microscopy analysis. See EM (electron microscopy) analysis Elion, Gertrude, 14 EM (electron microscopy) analysis of islet purity, 89–90 vs. LM, 91–97 with nuclei counting, 104 with PCV islet measurement, 100–101 Embolic strokes, 553–555 embryonic carcinoma (EC) cells, 34, 175 Embryonic neuronal cell adhesion molecule (E-NCAM), 493 E-NCAM (embryonic neuronal cell adhesion molecule), 493 Encapsulated cell biodelivery (ECB), 379 Encapsulated cell technology (ECT), 338 Encapsulated human islet allografts, 135–152 biocompatibility and, 144–145 clinical trials of, 143 development of, 139–143 human use of PEG coatings, 146–148 immune protection with, 136–139 immunosuppression complications, 136 nonhuman primate safety and efficacy studies, 146 Novocell PEG encapsulation technology, 143–148 references, 148–153 restricted acceptance of clinical islet transplantation, 135–136 small animal studies, 145–146 treating many diseases with, 148 Encapsulation, cell cells and ECMs for, 622 clinical trials, 622 CNTF delivery in HD, 390–391 coextrusion method, 330 delivery systems for brain tumors, 348–349 engineering of, 330–331 immune responses to, 197 for liver diseases, 224–225 macroencapsulation, 328–330, 621
655
Index Encapsulation, cell (continued) microencapsulation, 407, 621 safety in neurotrophic cell transplants and, 328–330 xenogeneic CP transplants, 406–407 Endogenous cells, 502–503 Endomorphins, 466 Endothelial cells cultivating for biomaterial cell therapies, 598 as factor in CNS graft rejection, 314–315 fibrous encapsulation from biomaterial implants, 595 treating hemophilia A with, 194 Engineered cells CNTF delivery in HD, 390–391 producing NGF, 377–378 Engraftment, intrahepatic islet, 68–69 ENU (ethylnitrosourea), 345 Environmental cues, cell-based therapies, 221–222 Enzyme replacement therapy (ERT), 206 Enzymes human islet isolation process, 86–87 SCI grafts, 495 EPCs (endothelial progenitor cells) from cord blood, 550–551 treating ischemic heart disease, 263–264 treating myocardial infarction, 552–553 EphB1, 536 Ephrin-B2, 536 Epibatidine, 458–459 Epidermal growth factor (EGF), 222, 232 Epigenetic stability, hESCs, 531–532, 534 Epithelial cells adding to engineered trachea, 279–280 of choroid plexus, 399–400 EPNP (embryonic porcine expanded neural precursors), 310 EPO (erythropoietin), 309 Epstein-Barr virus (EBV), 18 ERT (enzyme replacement therapy), 205–206 Erythromycin, 15 Erythropoietin (EPO), 309 ES (embryonic stem cells). See ESCs (embryonic stem cells) ESCs (embryonic stem cells) aberrant cell differentiation in, 505 advantages of, 527 and knockouts, 43–44 liver regeneration and, 219 MSCs vs., 549 overview of, 33–35 replacing degenerated neural cells, 555 role of graft in CNS, 309 source of, 527 in spinal cord injury, 502–504 therapies for liver diseases, 225–228 tissue engineering using, 164–165 treating ischemic heart disease with, 262–263 ESRD (end-stage renal disease) chronic inflammation state in, 173 extracorporeal RAD in, 182–183 human hemodialysis for treating, 169
kidney transplants for, 171 not reversible, 171 organ transplantation for, 210–211 statistics for, 169 Etancercept, 67 Ethics of fetal tissue donation, 444–445 of human donors for transplantation, 3 of human neural transplantation, 427–428 Ethidium bromide (EB), 107 Ethylnitrosourea (ENU), 345 Euglycemia, 69–70, 73 EULAR (European League Against Rheumatism) autoimmune diseases and, 241 patients receiving HSCT for AD, 242–243 patients receiving HSCT for SLE, 246 European Chrohns Colitis Organization (ECCO), 246 European Group for Blood and Marrow Transplantation. See EBMT (European Group for Blood and Marrow Transplantation) European League Against Rheumatism. See EULAR (European League Against Rheumatism) Evans syndrome, 247 Excitotoxicity inducing cell death after SCI, 479 metabolic toxins and, 418–419 striatal excitotoxins, 418 Excretion excretory process of, 170 kidney physiology, 169–171 Experimental allergic encephalomyelolithis (EAE), 251 Extracapillary space (ECS), 179 Extracellular matrices. See ECMs (extracellular matrices) Extracorporeal bioartificial kidneys, 177–183 in ARF, 180–182 bioartificial tubule assist device (RAD), 177 characterization of RAD, 178–180 characterization of single hollow-fiber bioreactor, 177–178 circuit for, 180 clinical experience with human RAD, 182 in ESRD, 182–183 references, 183–186 EY (eosin Y), 143–144 Eye anatomy of, 327–328 cell transplants for diseases of. See Neurotrophic cell transplants diseases, 327 immunoprivilege of, 571–572 immunoregulation and peripheral tolerance in, 577–579 immunoregulation in, 580
F Fabry disease, 205, 210–211 Fanconi’s anemia, 548 FasL (Fas ligand) CNS immunity and, 305–306 immune-modulatory functions of, 319–320 role in immunoprivilege, 577–578
656
Index Fasting, 70 Fat metabolism, liver function, 218 FDA (Food and Drug Administration), 37–39, 146–147 Fetal allograft, 573 Fetal porcine neurons, 51 Fetal tissue transplants collection and preparation of, 429 ethics of human neural transplantation, 427–428, 444–445 as experimental approach, 523 hibernation of, 446 initial studies in CNS, 354 limited availability of, 428–429, 445–446 traditional, 523–525 VM transplantation, 445 Fetal tissue transplants, SCI repair clinical trials, 505, 506 overview of, 489–490 preventing progression of injury, 497 providing substrate for axon growth, 501 FGF (fibroblast growth factor), 222, 232, 527 Fibroblasts cultivating for biomaterial cell therapies, 597–598 fibrous encapsulation in biomaterial implants, 595 Fibroblast transplantation for hemophilia, 191 preventing progressive SCI, 496–497 providing substrate for SCI axon growth, 498–500 SCI repair and, 486 treating ischemic heart disease, 262 Fibrous encapsulation, 594–595 Filtration artificial kidney hemofilter, 173–175 bioartificial kidneys with, 173 recapitulating liver, 215 First-dose response, 21 5HT (serotonin), 464 FIX (hemophilia B) expression of FVIII vs., 195–196 fibroblast transplants for, 192 keratincocyte transplants for, 190–191 overview of, 187–189 FK506. See Tacrolimus Flowcharts, and product development, 645–646 Flow cytometry, 99–100, 103 Fluorescence microscopy, 91 Fluoroquinolones, 15 FocalSeal-L, 147–148 Food and Drug Administration (FDA), 37–39, 146–147 Free radicals, 479–480 Furniture selection, cell processing facility, 632 Fusion, liver regeneration, 227 FVIII (hemophilia A) expression of FIX vs., 195–196 fibroblast transplants for, 191–192 overview of, 187–189
G GABA (/f2g/f0-aminobutyric acid), 456 GABAergic spiny projection neurons adrenal medullary transplants and, 460 chronic pain management and, 456 neuropathological features of HD, 386 GABA-IR (GABA-immunoreactivity), 456 GAD gene, 464 GAGs (glycosaminoglycans) cord blood cells treating, 556 in native vs. tissue-engineered trachea, 283 in tissue-engineered liver, 232 GAL (galactocerebrosidase), 51, 555 Gal/f2a/f01–3Gal, 4, 8–9 Galactosyl pycosine, 206 Gal-KO (galactosyl transferase gene), 51 /f2g/f0-aminobutyric acid (GABA), 456 Ganglion cells, 328–329 GANTT charts, 647 Gaucher disease, 206 GBM (glioblastoma multiforme), transplants for, 345–352 animal models, 345–346 cell-based delivery systems, 348–349 future prospects, 349–350 introduction, 345 new treatment principles for, 346–347 references, 350–352 stem cell therapy, 347–348 G-CSF (granulocyte colony stimulating factor), 260 GDNF (glial-cell-line-derived neurotrophic factor) approach to HD, 387 combined with NT2N cell transplantation, 366 influencing benefits of NT2N cell grafts, 359 nerve regeneration with biomaterials, 602 overview of, 334–335 potency for strokes and PD, 363–364 promoting axon growth after SCI, 478 protein delivery in brain, 364 strike pathology as guide for optimizing, 366 GDNF family ligands (GFL), 363 Gelation sponge (gelfoam), 68 Gender, in hemophilia, 187 Gene therapy delivering CNTF to brain in vivo, 390 delivering DNA with biomaterials, 599–600 delivering genes with nanotechnology, 616–617 DMD research focused on, 290, 294 NT2N cells and, 362–363 treating brain tumors with, 347, 349 treating hemophilia with, 187–188 treating human AD brains, 378 vector choice and design of, 194–196 viral vectors for GDNF, 364–366 Genetic engineering enhancing SCI grafts with, 495–496 overview of, 3–5
657
Index Genetic HD models neural transplantation and, 419–420 neuroprotective effects of CNTF in, 388–389 overview of, 386–387 Genetics, hESCs and, 531–532 Germ line modification, glioma animal studies, 346 GFAP (glial fibrillary acidic protein) choroid plexus and neurogenesis, 405 cord blood neural progenitor cells and, 551–552 repairing SCI with Schwann cells, 483 GFL (GDNF family ligands), 363 GFR alpha, 363–364 GFRs (glomerular filtration rates), 160–161 GI Bleeding, 75 Gingival hyperplasia, 16 GI tract, 18 Glaucoma, 327 Glial-cell-line-derived neurotrophic factor. See GDNF (glial-cell-line-derived neurotrophic factor) Glial cells as factor in CNS graft rejection, 312 loss of after SCI, 502 NT2N cell grafts and response of, 357–358 olfactory ensheathing glia, 486–489 reducing scar formation after SCI, 480 using progenitor cells from, 32 Glial fibrillary acidic protein. See GFAP (glial fibrillary acidic protein) Glial progenitors, 31 Glial-restricted precursor (GRP) cells, 494–495 Glioblastoma multiforme. See GBM (glioblastoma multiforme), transplants for Gliomas animal studies for treatment of, 345–346 gene therapy treatment for, 346 immunotherapy for, 346 overview of, 345 Globoid-cell leukodystrophies, 206 Glomerular filtration rates (GFRs), 160–161 Glomerulus artificial kidney hemofilter and, 173–175 defined, 170 in excretory process of nephron, 170 Glucagon, 69, 71 Glucocorticoid response elements (GREs), 19 Glucose insulin secretion and control of blood, 72 intolerance after islet transplants, 85 monitoring graft function, 70 peritransplant management of, 69–70 posttransplant monitoring of, 70 Glucose-potentiated arginine test, 71 Glucose tolerance test, 70–71 Glutamate, 479 Glutamate agonist quisqualic acid (QUIS) injections, 456 Glycemic liability (lability index), 72–73 GM (genetically modified) animals. See Xenotransplantation, creating animals for
GM-CSF (granulocyte macrophage colony-stimulating factor), 603, 604 GMP (good manufacturing process), 38 Goats, creating with nuclear transfer, 45 Gonads, immunoprivilege of, 573–575 Good manufacturing process (GMP), 38 Graft rejection, 548, 557–559 Graft-specific tolerance, CNS transplants, 318–320 Graft-versus-host-disease. See GVHD (graft-versus-host-disease) Gram stains, 63 Granulocyte colony stimulating factor (G-CSF), 260 Granulocyte-erythroid macrophage megakaryocytic colony-forming units (CFU-GEMM), 548 Granulocyte macrophage colony-forming cells (CFU-GM), 548 Granulocyte macrophage colony-stimulating factor (GM-CSF), 603, 604 GREs (glucocorticoid response elements), 19 Gretsky, Wayne, 643 Growth factors corticosteroids adverse effects, 19 enhancing SCI grafts with, 495 ESC-derived graft rejection in CNS expressing, 309 identifying early dopaminergic progenitors with, 527 providing substrate for SCI axon growth, 498 sirolimus side effects, 17 used for SCI repair, 481 GRPs (glial-restricted precursor) cells, 494–495 GTP (guanosine triphosphate), 18 Guanosine, 18 Guanosine triphosphate (GTP), 18 Guava PCA flow cytometer, 103 Guidance channels, in nerve regeneration, 602 Guide for the Care and Use of Laboratory Animals (National Academy of Science), 276–277 GVHD (graft-versus-host-disease) after HSCT, 247, 248 cell transplantation risk, 504 cord blood HSCs and, 548 using mesenchymal stem cells for acute, 248–250
H HAMA (human anti-mouse antibodies), 22 Hank’s Balanced Salt Solution (HBSS), 87 HB-EGF (heparin-binding epidermal growth factor-like growth factor), 232 HBSS (Hank’s Balanced Salt Solution), 87 hCNS-SC (clonogenic human CNS stem cells), 310 HD (human hemodialysis) current treatment for renal failure, 171 first use of, 169 future improvements in, 174 vs. kidney transplants, 171–172 treating acute renal failure with, 171 HD (Huntington’s disease), 385–398 cell type therapy for, 35 clinical features, 385 clinical trials with encapsulated cells, 624 CP transplants in monkey model of, 410–411
658
Index CP transplants in rat model of, 409–410 etiology of, 386 identifying presymptomatic individuals, 409 introduction, 385 models of, 386–387 neuropathological features, 385–386 primate studies, 336–337 rodent studies, 336 symptoms of, 336 therapeutic approaches to, 387–388 HD (Huntington’s disease), neural transplantation in, 417–437 animal models, 418–420 clinical trials, 423–427 ethics of, 427–428 experimental transplants, 420–423 future directions, 429–430 limitations of tissue supply, 428–429 pathology of disease, 417–418 references, 430–437 surgical and postoperative issues, 429 tissue collection and preparation, 429 HD (Huntington’s disease), transplants of CNTF-producing cells for cellular vectors for delivery, 390–392 clinical phase I trial for encapsulated, 392–394 conclusions, 394 discovery of, 388 neuroprotective effects of, 388–389 references, 394–398 strategies for delivery to brain, 389–390 hDAF (decay-accelerating factor), 48–49, 155–156 HDF (hemodiafiltration), 171–172, 174 Heart failure, 259 Helical template, for engineered trachea, 280–282 Hemacytometer, 102 Hematologic side effects, 14, 17 Hematopoietic stem cells. See HSCs (hematopoietic stem cells) Hematopoietic stem cell transplantation. See HSCT (hematopoietic stem cell transplantation) Hematopoietic system, curing diseases of, 547 HEMO (Hemodialysis) study, 171 Hemodiafiltration (HDF), 171–172, 174 Hemofilter, artificial kidney, 173–175 Hemofiltration (HF), 171–172, 174 Hemolytic anemia, 247 Hemophilia, transplants for, 187–203 encapsulation, 197 endothelial cells, 194 fibroblasts, 191–192 future directions, 197–198 hepatocytes, 192–193 human keratinocytes, 190–191 myocytes, 193–194 overview of, 187–189 preventing immune-mediated rejection of, 196–197 references, 198–203 stem cell transplantation, 189–190 vector choice and design of gene expression, 194–196
Hemophilia A. See FVIII (hemophilia A) Hemophilia B. See FIX (hemophilia B) Hemorrhagic strokes, 555 HEPA (high-efficiency particulate air) filters, 631–632 Heparin, 68 Heparin-binding epidermal growth factor-like growth factor (HB-EGF), 232 Hepatic growth factor (HGF), 222, 232 Hepatic stimulatory substance (HSS), 232 Hepatic toxicity, 16 Hepatitis, 504 Hepatoblasts, 218, 220 Hepatocytes capacity for regeneration, 219 direct injection into host, 223 as encapsulated transplants, 224–225 ESC differentiation into, 225 fully differentiated, 220 injected as microcarrier, 223–224 liver anatomy, 216–217 liver development, 218 source of donor tissue for tissue engineering, 228–229 transformed, 220 Hepatocyte transplantation advantages of, 215 for hemophilia, 192–193 limited long-term functioning of, 215–216 for liver diseases, 222–223 hESCs (human embryonic stem cells) engineered, 535–536 genetic and epigenetic stability, 531–532 NTera2 as alternative to, 532–533 possibilities for treating PD, 523 reasons for using cultured cells, 526–527 using designer cells and, 533–534 HF (hemofiltration), 171–172, 174 HFHD (high-flux hemodialysis), 171–172 HGF (hepatic growth factor), 222, 232 Hibernation, of fetal neuronal tissue, 446 High-flux hemodialysis (HFHD), 171–172 Hippocampal grafts, 376–377 Histocompatibility system (HLA), 196 Histocompatible tissues, 165 Histogranins, 459, 466–467 Hitchings, George, 14 HIV (human immunodeficiency virus), 504 HLA (histocompatibility system), 196 HLAs (human leukocyte antigens), 307, 308. See also MHC (major histocompatibility complex) Homeostasis, 600 Homologous recombination, 44 Horses, creating with nuclear transfer, 45 Hosts adverse reactions to SCI transplants, 504–505 encapsulation of xenogeneic cells for immunoisolation in, 406–407 risk of transferring disease in SCI transplants to, 504 role in CNS graft rejection, 310–311
659
Index HPTCs (human proximal tubule cells), 179–180 HRMAS 1H NMR (NMR), 110, 112 HSCs (hematopoietic stem cells), 548 from cord blood vs. bone marrow, 548 defined, 31 features of, 219 introduction, 547 as mechanism for liver regeneration, 225–227 reported benefits using, 38–39 transdifferentiated cell therapy vs., 32 using progenitor cells vs., 31–32 vector choice and, 195 HSCT (hematopoietic stem cell transplantation) ablative therapy without, 248 allogenic, 247 immune reconstitution after, 247–248 transfer of AD through, 242 treating Crohn’s Disease with, 246 treating hemophilia, 189–190 treating human AD with, 242–243 treating ischemic heart disease, 264 treating JIA with, 246 treating LSDs with BMT, 206 treating MS with, 243–244 treating myocardial infarction, 552 treating RA with, 245–246 treating severe AD with, 241 treating SLE with, 246 treating SSC with, 244–245 HSS (hepatic stimulatory substance), 232 Htt (huntington) protein, 386 HUCB (human umbilical cord blood) neural progenitor cells, 551–552 for neurodegenerative diseases, 556–557 for neurological diseases, 555–556 as rich source of EPCs, 550–551 as source of HSCs, 548–549 as source of MSCs, 549–550 therapeutic potential of, 557 treating myocardial infarction, 552–553 treating strokes, 553–555 Human anti-mouse antibodies (HAMA), 22 Human donors. See also Allotransplantation decline in number of, 3, 65 islet transplantation problems, 85 limitation of BMT for LSD, 205, 207 limited for cardiac transplantation, 259 limited for HD, 428–429 limited for OLT, 215 selection and harvesting of pancreas islets, 62 Human hemodialysis. See HD (human hemodialysis) Human leukocyte antigens (HLAs), 307, 308. See also MHC (major histocompatibility complex) Human Ntera-2 cells. See NT2 (human Ntera-2) cells Human proximal tubule cells (HPTCs), 179–180 Human umbilical cord blood. See HUCB (human umbilical cord blood) Human umbilical endothelial cells (HUVECs), 194
Humoral immune responses cell and tissue grafts provoking, 5 cell and tissue vs. organ grafts, 8–9 CNS transplant rejection and, 311 Humoral rejection cell and tissue grafts less susceptible to, 155–156 transplantation of pig kidneys into nonhuman primates, 155–156 transplantation of pig organs into humans, 155 Hunter syndrome, 207 Huntingtin protein, 417 Huntington (Htt) protein, 386 Huntington, George, 417 Huntington’s disease. See HD (Huntington’s disease) Hurler’s syndrome, 555–556 HUVECs (human umbilical endothelial cells), 194 Hyaline cartilage, 276 Hydrogel microcapsule technology, 141–143 Hyperacute rejection control of thrombosis, 52 inducing in pig-to-primate transplantation, 51 organ transplants from pigs to humans, 155 signs of, 45 Hyperglycemia diabetes characterized by, 59 islet function causing, 70–71 linked to cardiovascular disease, 74 type 2 diabetes with, 59 Hyperinsulinemia, 59 Hyperlipidemia, 15, 16 Hypertension causing progressive kidney damage, 171 cyclosporine side effect, 16 tacrolimus unlikely to cause, 16 Hypertrichosis, 16 HYPO (hypoglycemia) glycemic liability and, 72–73 insulin therapy for diabetes and, 60 peritransplant management of, 69–70 Hypoxia, 68 islet transplantation and, 85–86 Hypoxia-induced cell death, in SCI, 479
I IBMIR (instant blood-mediated inflammatory reaction), 68 IBMTR (International Bone and Marrow Transplantation Registry), 241, 242–243 Ibotenic acid, 418 IE (islet equivalence), 64 IFN-/f2g/f0, 599 IGF (insulin-like growth factor), 222 IL-2 (interleukin-2), 22–23 IL-3 (interleukin-3), 548 IL-4 (interleukin-4), 599 IL-6 (interleukin-6), 222 IL-10 (interleukin-10), 599 IL-12 (interleukin-12), 604 Immortalized cell lines, 534–535, 622 Immune recognition, 159, 594–595
660
Index Immune reconstitution, 247–248 Immune responses to biomaterials, 594–596 biomaterials modulating, 599 biomaterials promoting tissue regeneration, 600–602 biomaterials removing dysfunctional cells, 602–604 cell and tissue grafts, 5–7 cellular rejection, 8 liver disease cell transplants, 233–234 organogenesis and, 159 primary nonfunction, 7–8 regulating after SCI, 480 spinal cord injury compromising, 478 Immune Tolerance Network, 71 Immunohistochemical analysis, 91 Immunoisolation cells and extracellular matrices in encapsulation, 622 immunoisolatory membranes, 620–621 macroencapsulation, 621 microencapsulation, 621 ongoing clinical trials, 622–624 overview of, 620 xenogeneic CP transplants, 406–407 Immunoisolatory membranes, 620–621 Immunological barriers to allotransplantation, 3 type of transplant and, 5–6 to xenotransplantation, 3, 5 Immunologically privileged environments, 569–592 altered MHC antigen expression in, 576–577 deficient or specialized lymphatic drainage in, 575 defining, 569–570 Fas ligand in, 579 immunoregulation in, 577–579 local immunosuppression of immunity in, 579–581 methodological issues in, 581 peripheral tolerance in, 577–579 references, 582–592 relevance for transplantation medicine, 581–582 restricted access by immune cells in, 575–576 in tissues with critical function, 570–574 Immunophilin binding drugs, 17 Immunoprivilege, 569–570 Immunosuppression of alternatively activated macrophages, 600 as effect of MSCs, 250–251, 549 ESC transplantation into host heart requiring, 262 NT2N cell grafts and, 358–359 Sertoli cell, 557–559 Immunosuppressive drugs, 13–28 alemtuzumab, 23 alloimmume response and, 13–14 azathioprine, 14 for CNS transplants, 316–318 combination therapies for, 13 complications related to, 136 corticosteroids, 19–20 co-stimulation inhibitors, 23–24
cyclosporine, 14–16 deoxyspergualin (DSG), 24 development of, 13 enhancing SCI grafts with, 495, 504 islet allograft, 138 islet transplantation and, 67, 75, 78, 135–136 for liver disease cellular transplants, 234 mycophenolate mofetil (MPA), 18–19 neural transplantation in HD, 429 not inducing state of transplantation tolerance, 196 protein drugs, 20–23 references, 24–28 sirolimus, 17–18 tacrolimus, 16–17 for whole-pancreas transplantation, 60 for xenografts, 9 Immunotherapy, for brain tumors, 347 IMPDH (inosine 5’-monophosphate dehydrogenase), 18 Imuran (azathioprine), 14 Inflammation allogenic vs. autologous HSCT, 248 corticosteroids increasing risk of, 19 immunological privilege and, 570 regulating after SCI, 480 response to biomaterial implants, 595 SCI grafts using anti-inflammatory compounds, 495 Inheritance, LSD and, 205 Inhibition of complement, 46–47, 580 Ink4a-Arf loss, 346 Inner limiting membrane, retinal anatomy, 328–329 Inosine 5’-monophosphate dehydrogenase (IMPDH), 18 Instant blood-mediated inflammatory reaction (IBMIR), 68 Insulin assessing graft function, 70 blood glucose control and, 72 defined, 59 hypoglycemia and glycemic liability, 72–73 independence, after islet transplantation, 62, 64–65, 71–72, 85, 135–136 liver as key site of, 69 pathogenesis of diabetes, 59–60 peritransplant glucose management, 69–70 posttransplant monitoring, 70 role in glucose homeostasis, 59 Insulin-like growth factor (IGF), 222 Integration, noninvasive measures of, 36–37 Intellectual property (IP), 644–645 Interleukin-2 (IL-2), 22–23 Interleukin-3 (IL-3), 548 Interleukin-4 (IL-4), 599 Interleukin-6 (IL-6), 222 Interleukin-10 (IL-10), 599 Interleukin-12 (IL-12), 604 International Bone and Marrow Transplantation Registry (IBMTR), 241, 242–243 International Standards Organization (ISO), 631 Interstitial fibrosis, 15–16 Intracellular signaling molecules, 495
661
Index Intrahepatic islet engraftment, 68–69 Intraocular grafting system, 375–377 Intraventricular infusion, 379 Intrinsic regrowth, in SCI, 478–479, 481 Intrinsic sphincter deficiency (ISD), 294–295 Inverse patterning, 619–620 Investments, biotechnology, 38 IP (intellectual property), 644–645 IPR estate mapping, 647 Ischemia choroid plexus and, 404–405 cord blood therapy for acute, 552–555 islets susceptible to, 63 potential of cord blood EPCs, 551 repurfusion, in hepatocyte transplantation, 223 therapeutic angiogenesis for, 600–601 ISD (intrinsic sphincter deficiency), 294–295 Islet equivalence (IE), 64 Islet isolation facility at University of Alberta, 634–635 human, 86–87 inefficiency of, 65 optimization of, 77 overview of, 63–64 Islet preparations, 85–133 introduction, 85–86 islet isolation, 86–87 list of symbols, 124–126 references, 126–133 what we want to know, 88 Islet preparations, purity and cell composition, 88–98 comparison among various studies, 97–98 DTZ staining, 89 DTZ staining vs. morphological analysis, 97 EM vs. LM morphological analysis, 91–97 morphological analysis, 89–91 other approaches using immunohistochemical analysis, 91 overview of, 88 sampling issues, 98 what we want to know, 88 Islet preparations, quantity of tissue, 98–106 DNA measurements, 105 estimating islet equivalents in pancreas, 105–106 large particle flow cytometry, 99–100 nuclei counting, 101–104 overview of, 98 packed cell volume islet measurement, 100–101 ultrasound pulsed doppler measurements, 101, 103 visual counting of DTZ-stained islets, 98–99 Islet preparations, viability of, 106–124 apoptosis, 110–112 cell membrane integrity, 107–110 membrane integrity vs. mitochondrial function tests, 122 mitochondrial function. See Mitochondrial function OCR and cell quantity predicting transplant outcome, 122–124 overview of, 106–107 what we want to know, 88
Islet rest, 65 Islets of Langerhans, 59 Islet stress, 65 Islet transplants for diabetes, 59–84 allotransplantation for secondary diabetes, 76–77 allotransplantation for type 2 diabetes, 77 assessment of islet function, 70–71 autotransplantation of islets, 76 cross-matching, 66 current clinical islet transplantation, 63–65 encapsulated. See Encapsulated human islet allografts future directions, 77–78 historical perspectives on, 61–62 hypoglycemia and glycemic liability, 72–73 immunosuppression, 67 individuals with high PRA, 66–67 inefficiency of islet isolation/transplantation, 65 insulin independence and, 71–72 insulin secretion and blood glucose control, 72 intrahepatic islet engraftment, 68–69 microvascular complications, 73–74 pathogenesis of diabetes, 59–61 peritransplant glucose management, 69–70 posttransplant monitoring, 70 procedure, 67–68 recipient selection, 65–66 risks of, 75–76 Sertoli cells, 558 xenotransplantation of porcine islets for, 50–51, 78 Islet xenotransplantation chemical, 50–51 overview of, 48–50 ISO (International Standards Organization), 631 Isotransplantation, 159–161
J JAK/STAT (Janus kinase pathway), 332 JAMs (junctional adhesion molecules), 305 JC-1 dye, MMP, 120–121 JIA (juvenile idiopathic arthritis), 246
K Kainic acid, 418 Karyotype analysis, 138 Keratincocyte transplantation, 190–191 Ketoconazole, 15 Kidney failure, cell replacement strategies, 155–168 availability of source material, 161–162 integrating new nephrons, 156–157 isotransplantation/allotransplantation of embryonic kidneys, 159–161 nuclear transplants/therapeutic cloning, 165 organogenesis, 158–159 overview of, 155–156 perspectives and challenges, 166 references, 166–168
662
Index stem cells, 164–165 xenotransplantation of embryonic kidneys, 162–164 Kidney failure, extracorporeal devices for, 169–186 cell therapy/tissue engineering approach, 172 current status of renal support, 171–172 engineering bioartificial kidneys, 172–176 extracorporeal bioartificial kidneys, 177–183 implantable bioartificial kidneys, 176 overview of, 169 Kidneys acute renal failure. See ARF (acute renal failure) current status of renal support, 171–172 GDNF found in high concentration in fetal, 363 history of treating, 169 normal function of, 169–171 Killer immunoglobulin-like receptor (KIR), 248 KIR (killer immunoglobulin-like receptor), 248 Knockouts, 43–44, 51–52 Kopyov, Oleg, 425 KQAGDV, 598 Krabbe’s disease BMT treating, 206 cord blood cells treating, 555 umbilical cord transplants treating, 207 Kupffer cells, liver, 217, 218
L Label-retaining cells (LRCs), 164–165 Lability index (glycemic liability), 72–73 Lacy, Dr. Paul, 61 Lamina propria-OEG (LP-EOG), 486 Langer, Dr. Robert, 616 Large particle flow cytometry, 99–100 Large T antigen, 535 Laser photocoagulation, 73 Laser scanning microscopy, 91 Laser therapy, 275 Late progenitors, 219, 220 L-dopa, for PD, 439 LEDGF (lens epithelium-derived growth factor), 335 Left ventricular assist devices (LVADs), 259 Lens, of eye, 327–328 Lens epithelium-derived growth factor (LEDGF), 335 Lentiviral-based vectors, 195–196, 465 Leukemia, 547 Leukemia inhibitory factor (LIF), 309 Leukemia inhibitory factor receptor (LIFR), 332 Leukocytes, 595 Leukopenia, 14, 18 Lewy bodies, in PD, 439 LGE dissection, 423 Liberase, 63, 87 LiberaseRI, 87 LIF (leukemia inhibitory factor), 309 LIFR (leukemia inhibitory factor receptor), 332 Light microscopy analysis. See LM (light microscopy) analysis Lineage-restricted precursors, 35 Lipoproteins, 17
Liposomes, 616–617 Lithography, 613 Live/dead stains, membrane integrity, 107–108 Liver anatomy, 216–217 development, 218–219 function, 217–218 immunoprivilege of, 574 intrahepatic islet engraftment and, 69 lobules, 216 regeneration, 219 risk of islet transplants for diabetes to, 75 Liver disease cellular transplants, 215–240 with direct injection, 223 with encapsulated transplants, 224–225 future directions, 234–235 with hepatocyte transplantation, 192–193, 215–216, 222–223 immune response modulation and tolerance, 233–234 with implantable tissue-engineered liver, 228–233 with microcarrier transplants, 223–224 overview of, 215–216 references, 235–240 with stem cell therapies, 225–228 Liver disease cellular transplants, cell types cell sources, 221 cellular niches of liver, 221–222 co-culturing interactions, 222 committed progenitor cells, 220 extracellular matrix, 222 fully differentiated hepatocytes, 220 soluble signals, 222 stem cells, 219–220 Living donor islet transplantation, for diabetes, 77–78 L-lysine (poly-alginate) bond, 621 LM (light microscopy) analysis vs. EM, 91–97 of islet purity, 90–91 with nuclei counting, 104 with packed cell volume islet measurement, 100–102 Lmx1b gene, 528 Long-term proliferating (LTP) cells, 292–293 LP-EOG (lamina propria-OEG), 486 LRCs (label-retaining cells), 164–165 LSD (lysosomal storage disease), 205–213 background, 205–206 cell type therapy for, 35 organ transplantation for, 210–211 other cell-based therapies for, 207–210 references, 211–213 treating with BMT, 206–207 LTP (long-term proliferating) cells, 292–293 LVADs (left ventricular assist devices), 259 Lymphatic drainage in brain, 306 role in immunoprivilege, 575 Lymphocytes, 314–315, 347 Lysosomal storage disease (LSD), 205–213
663
Index M M6PR (mannose 6-phosphate receptor), 205 MAb (monoclonal antibody) preparations ANTI-CD-3, 21–22 ANTI-CD-20, 23 ANTI-CD-25, 22–23 Macroencapsulation cell immunoisolation by, 328–330 coextrusion method, 330 immunoisolation and, 621 overview of, 141–142 Macroglia, 486 Macrophage migration inhibitory factor (MIF), 579 Macrophages alternatively activated, 600 associated with angiogenesis, 601 for biomaterial cell therapies, 597–598, 602 DSG inhibiting several, 24 immunosuppression of, 581 inflammatory responses to biomaterial implants, 595 NT2N cell grafts and response of, 357–358 role in tissue repair, 600 Macrophage transplantation clinical trials for SCI repair, 505 as possible therapy for LSD, 210 SCI repair with, 484–486 Macroscale research, for transplants, 614 Macula, eye, 328 Macular degeneration cell therapy in, 338–339 PEDF and, 335 reported cases of, 327 MAG, 480 MAGIC (Myoblast Autologous Graft in Ischemic Cardiomyopathy) trial, 266–267 Magic angle spinning 1H NMR, 110 Magnetic resonance (MR), 618 Major histocompatibility complex. See MHC (major histocompatibility complex) Mannose 6-phosphate receptor (M6PR), 205 Manual counting of islets, 98–99 MAPK (mitogen-activated protein kinase) pathway, and GDNF, 363 Markers, 225 MASCIS (Multicenter Animal Spinal Cord Injury Study), 483 Matrix metalloproteinases (MMPs), 600, 601 Maturity onset diabetes, 59–60 MCAO (middle cerebral artery occlusion) and neurodegeneration, 404–405 preclinical studies of NT2N cell grafts in, 356–358 treating strokes, 554 MDSC (muscle-derived stem cell) transplantation, 289–302 for articular cartilage healing, 295–296 for bone healing, 295–296 for cardiac repair, 296 combined with gene therapy, 294 defined, 293 for DMD, 293–294
introduction, 289 other therapeutic applications of, 294–296 for peripheral nerve regeneration, 296–297 references, 297–302 treating DMD. See DMD (Duchenne’s muscular dystrophy) for urologic dysfunction, 294–295 Measurements, success of transplantation, 36–37 Membrane integrity tests live/dead stains, 107–108 vs. mitochondrial function tests, 122–123 quantitative membrane integrity measurement, 108–110 Membranes, immunoisolatory, 620–621 Menasche, Phillippe, 266 Menigeal fibroblast transplantation, 486 Mesenchymal stem cells. See MSCs (bone marrow-derived mesenchymal stem cells) Metabolic disorders affecting neurologic tissues, 555–556 Metabolic tests, 70 Metabolic toxins, 418 Metabolism in liver function, 217–218 recapitulating liver, 215 Metachromatic leukodystrophy (MLD), 206–207, 209–210 Metallothioneines (MT), 43 Metanephric kidneys, 156–157 Metanephroi, 183–186 Metaplasia, 226 Methodological factors, and transplantation, 581 Methynitrosourea (MNU), 345 MGE dissection, 423 MHC (major histocompatibility complex) CNS graft rejection and, 310–311, 315–316 CNS immunity involving, 307–308 NRPs for spinal cord injury transplants, 494 overview of, 5–8, 307 role in immunoprivilege, 576–577 MHP36, 310 MI (myocardial infarction), 259, 552–553 Microbiological monitoring in controlled environments, 632–633 Microcarrier transplants, for liver diseases, 223–224 Microchip-controlled drug reservoirs, 616 Microencapsulation, 407, 621 Microenvironment NT2N cell grafts in normal host brain, 355–356 NT2N cell grafts in stroke host brain, 356 Microglia CNS immunity and, 306 role in T-cell activation in CNS, 318 SCI repair with transplantation of, 485–486 Microinjection of DNA, 43, 44 Microspheres, in cell transplants, 615–616 Microvascular complications, islet transplants, 73–74 Middle cerebral artery occlusion (MCAO), 404–405, 554 MIF (macrophage migration inhibitory factor), 579 Migration, 36–37 Mitochondrial function, 112–122 ATP/ADP ratio in, 112 membrane integrity tests vs., 122–123
664
Index MMP (mitochondrial membrane potential), 120–122 oxidative phosphorylation, 113–120 redox state of cell, tetrazolium salts, 112–113 Mitochondrial membrane potential (MMP), 120–122 Mitogen-activated protein kinase (MAPK) pathway, and GDNF, 363 Mixed-meal test, 70, 72 MLD (metachromatic leukodystrophy), 206–207, 209–210 MMF (mycophenolate mofetil) for islet transplantations, 67 side effects in islet transplants, 75 MMP (mitochondrial membrane potential), 120–122 MMPs (matrix metalloproteinases), 600, 601 MNU (methynitrosourea), 345 MODS (multiorgan dysfunction syndrome), 169 Modulation, 233–234 Monoclonal antibodies. See MAb (monoclonal antibody) preparations Monocytes associated with angiogenesis, 601 inflammatory responses to biomaterial implants, 595 Morphological analysis assessing islet purity with, 89 vs. DTZ staining, 97 EM (electron microscopy) analysis, 89–90 EM vs. LM analysis of islet purity, 91–97 LM (light microscopy) analysis, 90–91 MPA (mycophenolate acid), 18 MPS (mucopolysaccharidosis) amniotic cell transplantation for, 210 BMT treating, 206 cord blood cells treating, 556 as lysosomal storage disease, 205 neural stem cell transplantation in, 207–209 MPTP (1-methyl-4-phenyl-tetrahydropyridine), in PD, 18–19, 441 MR (magnetic resonance), 618 MRI, 425–427 mRNA, 333, 334 MS (multiple sclerosis), 243–244, 556–557 MSCs (bone marrow-derived mesenchymal stem cells) as co-graft facilitators, 550 developmental biology of, 36 FDA review and, 38 features of, 219–220 future prospects for, 38–39 for hemophilia, 190 immunomodulation of AD, 248–251 for liver regeneration, 226 therapy for LSD, 209–210 transdifferentiated cell therapy and, 32 treating brain tumors, 347–348 treating ischemic heart disease, 263, 267–268 treating myocardial infarction, 552 treating renal repair, 164 treating SCI repair, 490–491, 501–503 MSCs (cord-blood derived mesenchymal stem cells), 549–550 MSCV (murine stem cell virus), 196 MT (metallothioneines), 43
MT (myoblast transplantation), 193–194, 266–267, 291–293 MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium, inner salt), 112–113 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tretrazolium bromide), 112–113, 116 Mucopolysaccharidosis. See MPS (mucopolysaccharidosis) Multiorgan dysfunction syndrome (MODS), 169 Multiple sclerosis (MS), 243–244, 556–557 Multipotent cells, 527 Murine stem cell virus (MSCV), 196 Muscle-derived stem cell transplantation. See MDSC (muscle-derived stem cell) transplantation Muscular dystrophy. See DMD (Duchenne’s muscular dystrophy) Mutant huntingtin, 417 Mutated Huntington protein (NuHtt), 386 Mycophenolate acid (MPA), 18 Mycophenolate mofetil. See MMF (mycophenolate mofetil) Myelin inhibiting axon growth after SCI, 478 nerve regeneration with biomaterials, 602 neutralizing inhibitors of axon growth in, 480 remyelination of axons after SCI, 503–504 Myoblast Autologous Graft in Ischemic Cardiomyopathy (MAGIC) trial, 266–267 Myoblasts, skeletal, 261 Myoblast transplantation (MT), 193–194, 266–267, 291–293 Myocardial infarction (MI), 259, 552–553 Myostatin, 292
N N1250/Nogo, 480 Nanoparticles overview of, 617–619 quantum dots and PEBBLES, 618 tissue targeting by nanospheres, 618–619 Nanoshells, 616 Nanospheres, 618–619 Nanotechnology, 613–629 cell biology nanoscale in, 613–614 cell encapsulation with, 624–625 cellular immunoisolation with, 620–624 contact guidance in, 614–615 defined, 613 drug and gene delivery with, 615–617 future of, 625 impact of, 613 mechanical influences in, 614 nanoparticles in, 617–619 references, 625–629 self-assembled nanostructures in, 614 tissue engineering with, 619–620 Nanotubes, 616 Nasal chrondocytes, 276–280 Nasal septum cartilage, 276 NASH (nonalcoholic steatohepatitis), 75–76 National Heart, Lung, and Blood Institute (NHLBI), 38
665
Index National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), 38 Natural killer cells. See NK (natural killer) cells Naturally derived materials, polymers from, 230–231 NCAM (neural cell adhesion molecule), 363, 527–528, 530 Necrosis, 479 NECTAR (Network of European CNS Transplantation and Restoration), 427 Neokidneys, 165 Neoplasia, 75 Neoral (microemulsion cyclosporine), 14–15 Neovascular (wet) AMD, 339 Neovascularization, 555 Nephrons cell types in kidneys, 156 as functional elements of kidneys, 170 integrating into kidney, 156–157 Nephrotoxicity, cyclosporine use and, 15–16 Nerve conduits, 602 grafts, 296 regenerating with biomaterial implants, 601–602 Network of European CNS Transplantation and Restoration (NECTAR), 427 NeuN-ir (NeuN immunoreactive) neurons, 410–411 Neural cell adhesion molecule (NCAM), 527–528, 530 Neural progenitor cells (NPCs), 492–493, 502–503 Neural progenitor cells, cord blood, 551–552 Neural stem cells. See NSCs (neural stem cells) Neural transplantation for PD. See PD (Parkinson’s Disease), neural transplantation in stem cell therapy for LSD, 207–209 Neurobiology of Aging, 380 Neurodegeneration choroid plexus and, 404–405 cord blood cell therapies for, 556–557 Sertoli cell transplantation for, 558 Neurogenesis, 405–406 Neurological diseases, 555–556 Neurologic toxicities cyclosporine side effect, 16 tacrolimus side effects, 16 Neuronal progenitors, 31 Neuronal-restricted precursor (NRP) cells, 493–494 Neurons CNS cells, 29 defined, 602 replacing after SCI, 502–503 retinal anatomy, 328–329 significant loss after SCI, 502 Neurotech, 338 Neurotophrin-3. See HD (Huntington’s disease), neural transplantation in; NT-3 (Neurotophrin-3) fibroblasts Neurotrophic cell transplants, 327–343 anatomy of eye, 327–328 brain-derived neurotrophic factor, 333–334 cell encapsulation engineering, 330–331 cell encapsulation for immunoisolation, 328–330
ciliary neurotrophic factor, 332–333 clinical trials, 337–339 conclusions, 339 glial-cell-line-derived neurotrophic factor, 334–335 neurotrophic encapsulated cell therapy, 336–337 neurotrophic factors in eye, 331–332 opthalmic diseases and, 327 other neurotrophic factors, 335–336 references, 340–343 Neurotrophic factors. See NT (neurotrophic) factors Neutropenia, 17 Neutrophils, 595 NF-ATc, 14 NGF (nerve growth factor) approach to HD, 387 nerve regeneration with biomaterials, 602 promoting axon growth after SCI, 478 providing substrate for SCI axon growth, 498–499 Schwann cells producing, 482 using microspheres, 615 NGF-producing cell transplants, 373–384 brain tissue grafts, 375–377 clinical applications, 379–380 engineered cells or biomaterials producing NGF, 377–378 NGF levels in AD and, 373–375 past concerns/future promises of, 380–381 peripheral tissue grafts, 377 references, 381–384 rodent studies of HD, 336 NHLBI (National Heart, Lung, and Blood Institute), 38 NI (non-islet) cells, 89 NIDDK (National Institute of Diabetes and Digestive and Kidney Diseases), 38 Niemann-Pick disease, 209 NIH (National Institute of Health), 241, 290 NK (natural killer) cells causing primary nonfunction in cell/tissue grafts, 7–8 effects of FasL on, 319–320 as factor in CNS graft rejection, 315–316 NMR (HRMAS 1H NMR), 110, 112 NOGA catheter, 266–267 Nonalcoholic steatohepatitis (NASH), 75–76 Non-/f2b/f0 endocrine cells, 89, 92 Noncontractile cells, 262–264 Noninsulin-dependent diabetes, 59–60 Noninvasive measurements, and researchers, 36–37 Non-islet (NI) cells, 89 Nono-fibrillar extracellular matrices, 619 Nonspecific immune tolerance, in CNS, 316–318 Nonsteroidal anti-inflammatory drugs (NSAIDS), 318, 455 Novocell PEG encapsulation technology, 143–148 NPCs (neural progenitor cells), 492–493, 502–503 NRPs (neuronal-restricted precursor) cells, 493–494 NSAIDS (nonsteroidal anti-inflammatory drugs), 318, 455 NSCs (neural stem cells) future prospects for, 38 generating dopamine neurons from, 528–530
666
Index as multipotent cells, 527 vs. other cell sources, 35 overview of, 29–30 predifferentiating, 32 problems with, 30–31 treating brain tumors with, 347–348 treating certain diseases with, 35 treating SCI axon growth, 502 NT (neurotrophic) factors approach to HD, 387 BDNF (brain-derived neurotrophic factor), 332–334 in biologic regulation of CNS, 327 CNTF (ciliary neurotrophic factor), 332–333 encapsulated, 336–337 in eye, 331–332 GDNF (glial-cell-line-derived neurotrophic factor), 334–335 influencing benefits of NT2N cell grafts, 359–360 other types of, 335–336 potential for neurodegenerative disorders, 327 used for SCI repair, 478, 481, 496, 498–500 NT (nuclear transfer) of ESCs, 33 producing Dolly the sheep from, 44 producing other animals with, 45 NT2 (human Ntera-2) cells and gene therapy, 362–363 NT2N cells generated from, 357 overview of, 354–355 NT2N cell grafts clinical trials in stroke patients, 360–361 combining with GDNF therapy, 366 immunosuppression and, 358–359 neurotrophic factors influencing, 359–360 normal host brain microenvironment, 355 NT2N cells and gene therapy, 362 preclinical studies in stroke, 356–358 producing neurons, 354 recent advances in transplantation therapy, 361–362 references, 366–371 stroke host brain microenvironment, 355 NT2N neurons, 354–355 NT-3 (Neurotophrin-3) fibroblasts preventing progressive SCI, 496 promoting axon growth after SCI, 478 providing substrate for SCI axon growth, 498–501 NTera2 cells, 464, 532–533 Nuclear factor-K/f2b /f0pathway, 13 Nuclear transfer. See NT (nuclear transfer) Nuclear transplantation, 165 Nuclei counting of islet cells, 101–104 quantitative membrane integrity measurements using, 109 Nucleosome detection, in islets, 111 NuHtt (mutated Huntington protein), 386 Nurr1 gene, 528–529 n-VP (N-vinylpyrrolidone), 143–144
O O2A precursor cells, 494 Obesity, 59 OBS (oxygen biosensor system), 119–120 OCR (oxygen consumption rate) measurement of, 114 measuring with perifusion bioreactor, 114 measuring with static culture, 118–120 measuring with stirred chamber, 114–118 predicting islet transplant outcome in mice, 122–124 OEG (olfactory ensheathing glia) grafts clinical trials for SCI repair, 505–506 overview of, 486–489 providing substrate for SCI axon growth, 500–501 remyelination of axons after SCI, 503 Olfactory ensheathing glia. See OEG (olfactory ensheathing glia) grafts Oligodendrocyte precursor cells (OPCs), 494, 504 OLT (orthotropic liver transplantation), 215 Oncoretroviral genome, 195 1-methyl-4-phenyl-tetrahydropyridine (MPTP), in PD, 441 ONL (outer nuclei layer), 328 OPCs (oligodendrocyte precursor cells), 494, 504 Opioids, 455 Opthalmic diseases anatomy of eye, 327–328 cell transplants for. See Neurotrophic cell transplants overview of, 327 Optic nerve, 484 Organ donors, 135 Organizational life cycles, 638 Organogenesis antigen presentation, 158 expression of class I and class II antigens, 158–159 immune response and recognition, 159 overview of, 158 vascularization of transplanted renal anlagen, 159 xenotransplantation with, 4 Organs barriers to xenografts, 5–6 cellular immunoisolation overcoming lack of suitable donor, 620–624 displaying immunological privilege, 569–570 new technology for replacing, 4 potential sources for xenografts, 5 tissue engineering using nanotechnology, 620 Organ transplantation, 62, 210–211 Orthotropic liver transplantation. See OLT (orthotropic liver transplantation) Osmotic pumps, 389–390 Osteoarthritis, 295 Osteoporosis, 19 Outer nuclei layer (ONL), 328 OV-6 marker, 225 Oval cells, 220 Oxidative phosphorylation, 113–120 ATP concentration, 113–114 oxygen consumption rate, 114–120
667
Index Oxygen biosensor system (OBS), 119–120 Oxygen consumption rate. See OCR (oxygen consumption rate) Oxygen partial pressure (pO2), 119–120
P P19 cell line, 463 Packed cell volume (PCV) measurements, 100–102 PACT (Production Assistance for Cellular Therapies), 38 PAH (P-aminohippurate), 161 PAMAM (polyamidoamines), 617 P-aminohippurate (PAH), 161 PAMs (pharmacologically active microcarriers), 615–616 Pancreas encapsulated islet allografts. See also Encapsulated human islet allografts estimating islet equivalents in, 105–106 human islet isolation process, 86–87 islet transplants. See Islet transplants for diabetes rat islet isolation process, 87–88 transdifferentiation potential of cells of, 226 Pancreas (whole organ) transplantation. See also Encapsulated human islet allografts complications related to, 136 donor selection for islet programs vs., 63 insulin independence following, 65 microvascular complications after, 73 for type 1 diabetes, 60 Pancytopenia, 14 Panel reactive antibody (PRA), 66 Parenchymal cells, 229 Parkinson’s Disease. See PD (Parkinson’s Disease) Particle filters, air-filtration, 631–632 Patents, in product development, 644–645 Paternal alloantigens, 574 PC12 cell line, 463 PCNA (proliferating cell nuclear antigen), 165 PCV (packed cell volume) measurements, 100–102 PD (Parkinson’s Disease) animal research on NT2N transplants for, 362 cell type therapy for, 35 cord blood cell therapies for, 556–557 ESCs producing DA neurons for, 309 genetically engineered pigs for treating, 51 glial-cell-line-derived neurotrophic factor and, 334–335 symptoms of, 439 tolerance in CNS transplants for, 316–318 PD (Parkinson’s Disease), dopaminergic fetal and hESC transplants, 523–544 cell lines/other possibilities, 534–535 clinical studies, 525–526 designer stem cells, 533–534 developing dopaminergic neurons, 527–528 dopamine neurons from stem cells/other sources, 528–530 engineering stem cells, 535–536 genetic and epigenetic stability, 531–532 hESC, 530–531 introduction, 523 mouse/subhuman primate embryonic stem cells, 530
need for cultured cells, 526–527 NTera2, 532–533 references, 537–544 stem cells/other cells, 527 traditional transplantation using fetal cells, 523–525 PD (Parkinson’s Disease), neural transplantation in, 31, 439–454 animal models, 439–441 clinical trials, 444–445 comparison with DBS, 448 dyskinesias, 447–448 experimental transplants, 441–444 future prospects, 448–449 limited tissue availability, 445–446 overview of, 439 references, 449–454 trial design, 447 unreliable benefit and recovery, 446–447 PD (peritoneal dialysis), 169, 171 PD (product development), 637–648 defining, 637 intellectual property and patents, 644–645 organizational life cycles, 638–639 overview of, 637–638 plan and budget, 640–644 product definition, 639–640 references, 648 technology transfer, 639 tools for, 645–647 unique window of opportunity for, 647–648 PDGF (platelet-derived growth factor), 346, 601 PDGFR, 346 PEBBLES (probes encapsulated by biologically localized embedding), 618 PEDF (pigment epithelium-derived growth factor), 335, 339 PEG (polyethylene gylcol) adhesion of biomaterials and, 598 biocompatibility and, 144–145 human use of, 146–148 nonhuman primate safety and efficacy studies, 146 Novocell encapsulation technology, 143–144 overview of, 142–143 small animal studies, 145–146 using in tissue-engineered liver, 232 Penicillium brevicompactum, 18 PEO (polyethylene oxide)-star co-polymers, 331 Perflourocarbon, 63 Perifusion bioreactor, measuring OCR, 114 Peripheral nerve grafts. See PNGs (peripheral nerve grafts) Peripheral nerve regeneration, with MDSCs, 296–297 Peripheral nervous system (PNS), 602 Peritoneal dialysis (PD), 169, 171 Peritransplant glucose management, 69–70 Permeability, of microcapsules, 142 PERT charts, 647 Peschanski, Marc, 425–427 PET scans, 360–361, 425 PGS (poly[glycerol-sebacate]), 231 Pharmacologically active microcarriers (PAMS), 615–616
668
Index Pharmacology, approach to HD, 387 PHC (polycyclic hydrocarbons), 345 Phenotypic HD models, 386, 388 Phenytoin, 15 Photolithography, 619 PHSRN, 598 Pigment epithelium-derived growth factor (PEDF), 335, 339 Pigs complement regulatory proteins and transgenic, 47–48 creating with nuclear transfer, 45 favored as source of xenografts, 4–5 generating for clinical neural transplantation, 51 genetically altering, 45 transplantation of kidneys from, 155–156, 162–164 as transplant donors, 3–4 xenotransplantation of islets, 78 PKS (protein kinase A), 481 Placebo trials designing neural transplantation in PD, 447 ethics of, 428 Placenta immunoprivilege of, 573 immunoregulation in, 580 PLA General Hospital, Beijing, 462 Plan, product development, 640–644 Plasma, and hemophilia, 187 Plasmid DNA delivery, 599–600 Platelet-derived growth factor (PDGF), 601 PLGA (polylactic-co-gylcolic acid) advanced tissue engineering uses, 233 as polymer for microcarrier beads, 224 for protein delivery in biomaterial implants, 599 for tissue engineering scaffolds, 230–231 PLG microspheres, 604 PLL (polylysine microcapsules), 141–142 Pluripotent cells, 527 PMN (polymorphic mononucleocytes), 319 PNGs (peripheral nerve grafts) as growth factor source in animal models, 377 providing substrate for SCI axon growth, 497–498 remyelination of axons after SCI, 503 SCI repair with, 481–482 PNS (peripheral nervous system), 602 pO2 (oxygen partial pressure), 119–120 Poisson statistics, 103 Poly (L-lysine)-alginate bond, 621 Poly[glycerol-sebacate] (PGS), 231 Polyamidoamines (PAMAM), 617 Polyclonal antilymphocyte sera (Atgam), 20–21 Polyclonal antilymphocyte sera (Thymoglobulin), 20–21 polycyclic hydrocarbons (PHC), 345 Polyethylene gylcol. See PEG (polyethylene gylcol) Polyethylene oxide (PEO)-star co-polymers, 331 Polylactic-co-gylcolic acid. See PLGA (polylactic-co-gylcolic acid) Polylysine microcapsules (PLL), 141–142 Polymeric matrices, 622 Polymeric nanospheres, 617, 618–619 Polymers, in macroencapsulation, 621
Polymers, in tissue engineering classes of, 230–231 configurations and modifications of, 232 hemofilter design, 175 producing NGF, 377–378 Polymorphic mononucleocytes (PMNs), 319 Polyomavirus nephropathy, 16 Polypeptides, in CP, 403 Polysaccharide membranes, 621 Polyvinyl alcohol (PVA), 233 Porcine CD55, 47 Porcine islets, isolation of, 87–88 Porcine proximal tubule cells (PPTCs), 179–180 Pore size, hemofilter design, 175 Porosity, biodegradable scaffolds, 597 Portal vein, islet transplantation, 67–68 Posterior chamber, of eye, 327–328 Post-mitotic cells encapsulation of, 330–331 using dividing tissue vs., 622 Posttransplant lymphoproliferative disorder (PTLD), 18, 21–22 Posttransplant monitoring, islets, 70 PPTCs (porcine proximal tubule cells), 179–180 PRA (panel reactive antibody), 66–67 Precursor cells mature kidney originating from, 156 overview of?, 164–165 possibility of renal, 156 risk of aberrant cell differentiation in, 505 SCI repair with, 493–495, 502–504 Prednisolone, 19, 20 Prednisone, 19, 138 Pregnancy, immunoprivilege of, 574 Primary nonfunction, 7–8 Primates, nonhuman neurotrophic encapsulated cell therapy and, 336–337 Novocell PEG encapsulation studies in, 146 transplantation of pig organs into, 155–156 as transplant donors, 3–4 xenogeneic CP transplants for HD, 410–411 Principles of Laboratory Animals Care (National Society for Medical Research), 276 Probes encapsulated by biologically localized embedding (PEBBLES), 618 Product definition, 639–640 Product development. See PD (product development) Production Assistance for Cellular Therapies (PACT), 38 Progenitor cells for cardiac cell transplantation, 269 committed, 220 cord blood neural, 551–552 defined, 219 generating dopamine neurons from, 528–529 for heart failure, 260, 263–264 overview of, 527 risk of aberrant cell differentiation in, 505 SCI repair with, 493–495, 502–504 Progesterone, 580
669
Index Prograf, 138 Program manager, in product development, 647 Project ownership, 639 Project teams planning and budgeting for, 640–642 product development and, 639 regular progress presentations for, 647 Proliferating cell nuclear antigen (PCNA), 165 ProNectin-L, 177 Proneuron Biotechnologies, 505 ProNGF, 380 Prostaglandins, 580 Protein adsorption, in biomaterial implants, 594–595 delivery of, for biomaterial implants, 599–600 metabolism in liver, 217–218 microcapsule limitations, 142 Protein drugs clinical use of, 20–21 monoclonal antibody preparations, 21–23 polyclonal antilymphocyte sera, 20–21 Protein kinase A (PKS), 481 Proximal tubules bioartificial, 175–176 bioartificial kidneys with, 173 excretory process of nephron and, 170 Psoriasis, 23–24 PTLD (posttransplant lymphoproliferative disorder), 18, 21–22 Ptx3 gene, 528–529 Purity of islets. See Islet preparations, purity and cell composition PVA (polyvinyl alcohol), 233
Q QA (quinolinic acid) characterizing striatal damage using, 418 CP transplants in monkey model of HD, 410–411 CP transplants in rat model of HD, 409–410 HD and, 336–337, 386 Quality control tests, 63 Quantitative membrane integrity measurement, 108–110 Quantum dots, 618 QUIS (glutamate agonist quisqualic acid) injections, 456
R RA (rheumatoid arthritis), 23–24, 245–246 Radial glia, 29 Radiotherapy for brain tumors, 345 for SSC patients receiving HSCT, 244–245 RADs (bioartificial renal tubule assist devices) bioartificial kidney in ARF and, 180–182 characterization of, 178–180 extracorporeal bioartificial kidney circuit and, 180 overview of, 177 Phase I/II clinical experience with human, 182 Rapamune, 138 Rapamycin (sirolimus), 17–18 RAS-MAP (mitogen-activated kinase pathway), 13
Rat islets, isolation of, 87–88 RCA (regulators of complement activation), 46, 47 Reactive oxygen species, islet viability and, 85 Receptors, in choroid plexus, 402 Receptor tyrosine disease (RET), 334 Receptor tyrosine kinases (RTKs), 346 Recipient selection, islet transplantation, 65–66 Recombinant cells, for hemophilia, 187–188 Recombinant proteins, for hemophilia, 187 Recombinant tissue plasminogen activator (rtPA), 554 REDV, 598 References animals for cell xenotransplantation, 52–56 ASTIC trial for treatment of Crohn’s Disease, 246 ASTIMS trial for treatment of MS, 244 ASTIS trial for treatment of SSC, 245 available embryonic stem cell lines, 33 cardiac cell transplantation, 270–274 cell processing facility design, 635 chronic pain transplants, 469–475 CNS transplants, 320–326 CP epithelial cell transplants for brain repair, 412–416 dopaminergic fetal transplants, 537–544 encapsulated human islet allografts, 148–153 extracorporeal bioartificial kidneys, 183–186 glioblastoma transplants, 350–352 hESC transplants, 537–544 immunosuppressive drugs, 24–28 islet preparations, 126–133 islet transplants for diabetes, 78–84 kidney failure, 166–168 liver disease cellular transplants, 235–240 lysosomal storage disease, 211–213 nanotechnology, 625–629 neurotrophic cell transplants, 340–343 NGF-producing cell transplants for Alzheimer’s, 381–384 NT2N cell transplants/GDNF treatment in stroke, 366–371 product development, 648 SCI repair grafts, 506–521 stem and precursor cells, 39–42 stem cell therapy concepts, 560–566 xenotransplantation immunology, 9–11 Regeneration, liver, 219 Regulation cell processing facility design, 634 cell transplants for SCI repair, 505 fetal neuronal tissue collection, 427–428 future of stem and precursor cells, 37–39 islet transplantation with immunosuppression, 135–136 xenotransplantation, 9 Rejection acute. See Acute rejection as main risk of cell therapy, 188 Remacemide, 387 Renal allotransplants cyclosporine nephrotoxicity in, 15–16 present treatment for renal failure, 155 tacrolimus side effects in, 17
670
Index Renal anlagen antigen presentation in, 158 availability of source material for, 161–162 isotransplantation/allotransplantation of, 159–161 overview of, 157 vascularization of transplanted, 159 xenotransplantation of, 162–164 Renal function acute renal failure. See ARF (acute renal failure) bioartificial kidneys, 172–176 cell replacement for failure of. See Kidney failure, cell replacement strategies chronic renal failure, 169, 171 extracorporeal devices for failure of. See Kidney failure, extracorporeal devices for islet transplants impacting, 73 Renal plasma flow, 160 Restricted precursor cells, 31–32 RET (receptor tyrosine disease), 334 Retina anatomy of eye, 328–329 reported cases of degeneration, 327 Retinal-pigmented epithelium (RPA), 328–329 Retinitis pigmentosa. See RP (retinitis pigmentosa) Retrovirus, for hemophilia, 187 Reuter centrifugal air sampler, 632 RGD (Arg-Gly-Asp) adhesion of biomaterials with, 598 cell encapsulation engineering with, 331 scaffolding in tissue engineering with, 232 tissue engineering with nanotechnology using, 619 Rheumatoid arthritis (RA), 23–24, 245–246 Ribonucleic acid (RNA), 14 Ricordi chamber, 63 Riluzole, 387 Rituximab (anti-Cd-20), 23 RN33B cells, 494 RNA (ribonucleic acid), 14 Robotic-assisted cell delivery, 265–266 Rods, retinal, 328–329 ROS (reactive oxygen species), islet viability and, 85 RP (retinitis pigmentosa) cell therapy in, 337–338 encapsulated cells for, 624 potential for neurotrophic cell transplants, 327 RPE (retinal-pigmented epithelium), 328–329 RST (rubrospinal tract) neurons, 500 RTKs (receptor tyrosine kinases), 346 rtPA (recombinant tissue plasminogen activator), 554 Rubrospinal tract (RST) neurons, 500
S Safety, islet preparations, 88 Safety, of NGF, 380 Safranin-O staining, 277, 279 Sandimmune, 14–15 Sanfilippo syndrome, 556 Satellite cells, 261
Scaffolding advances in, 233 biomaterial cell therapies, 596–598 biomaterials and DNA delivery, 599 implantable tissue-engineered livers, 229–230 polymer configurations and modifications, 232 polymers for, 230–231 SCI grafts, 495 SCF (stem cell factor) graft rejection in CNS expressing, 309 increasing HSC in HUCB using, 548 treating heart failure, 260 Schwann cells nerve regeneration with biomaterials, 602 SCI repair with, 482–484 SCI (spinal cord injury) repair cell type therapy for, 35 epidemiology of, 477–478 inhibiting cell death, 479–480 introduction, 477 modulating immune response/inflammation, 480 neutralizing inhibitors of axon growth in myelin, 480 obstacles to self-repair of, 478–479 pathophysiology of, 478 reactivating axon growth, 481 reducing scars, 480 stimulating axon growth, 480–481 SCI (spinal cord injury) repair, cell grafting for bone marrow stromal cells, 490–491 cell types for, 481 enhancing functionality, 495–496 fetal tissue, 489–490 fibroblasts, 486 future of, 506 macrophages and microglia, 484–486 merits of, 497–504 olfactory ensheathing glia, 486–489 other cell types, 495 peripheral nerves, 481–482 pitfalls and limitations of, 504–506 preventing progressive injury, 496–497 references, 506–521 Schwann cells, 482–484 stem cells and progenitor cells, 491–495 Sclera, eye, 328 SCNT (somatic cell nuclear transfer), 534 sCRIs (soluble complement inhibitors), 46–47 SCs (stem cells). See Stem cells SDIA (stromal-cell-derived inducing activity), 530 Sehgal, Suren, 17 Self-assembled nanostructures, 613, 614 Self-renewal, neural stem cell problems, 31 Ser1 (histogranin), 466–468 Sertoli cells clinical islet xenotransplantation using, 50 relevance for transplantation medicine, 582 as source of local immunosuppression, 580–581 transplantation tolerance and, 557–559
671
Index Serum triglycerides, 17 7-AAD dye, 102–103, 109 Sham surgery, ethical issues, 428 Sheep, as transplant donors, 3 SHG ([Ser1]histogranin), 466–468 Shh (sonic hedgehog protein) generating dopamine neurons from, 527, 529 OPCs for spinal cord injury transplants and, 494 Signal transducer and activation of transcription proteins (STATS), 332 Simulect (basiliximab), 22 Simultaneous pancreas and kidney transplantation (SPK), 73 Single-donor islet transplantation, 77 Single hollow-fiber bioreactor, 177–178 Sinusoids, 217, 223 siRNA (small-interference RNA), 495 Sirolimus cautious use of MMF with, 18 clinical use of, 17–18 in islet transplantations, 67, 75 Sirolimus (Rapamycin), 175–176 SIRS (systemic inflammatory response syndrome), 171, 173 6-OHDA (6-hydroxydopamine), 440, 442 Skeletal myoblast transplantation cardiac repair with, 261–262, 296 treating myocardial infarction, 552 Skin grafts, 593 SLE (systemic lupus erythematosus), 246 SLEDAI (SLE Disease Activity Index), 246 Small hepatocyte cells, 229 Small-interference RNA (siRNA), 495 SMCs (smooth muscle cells), 601 Smooth muscle cells (SMCs), 601 Soluble complement inhibitors (sCRIs), 46–47 Soluble factors, 231 Soluble signals, 222 Somatic cell nuclear transfer (SCNT), 534 Somatic modification, glioma animal studies, 346 Sonic hedgehog protein. See Shh (sonic hedgehog protein) Soon-Shiong, Patrick, 143 Space of Disse, liver, 217 Sperm antigens, 574 Spinal cord, chronic pain management and, 455–457 Spinal cord injury repair. See SCI (spinal cord injury) repair SPK (simultaneous pancreas and kidney transplantation), 73 SSC (systemic sclerosis), 244–245 Standard fluorescence microscopy, 91 Standards, clean room, 631 Static culture, measuring OCR, 118–120 STATS (signal transducer and activation of transcription proteins), 332 Steatosis, 75–76 Stem cells, 29–42 adult-derived. See Adult-derived stem cells basic facts, 29–30 designer, 533–534 difficulties researchers face, 36–37 embryonic. See ESCs (embryonic stem cells)
future prospects, 37–38 hematopoietic. See HSCs (hematopoietic stem cells) liver regeneration and, 219–220 neural. See NSCs (neural stem cells) overview of, 492 potential in HD therapy, 428–429 and progenitor cells, 491–492 references, 39–42 restricted precursor cells, 31–32 role of graft in CNS, 309–310 structurally complex organs and, 4 therapies for liver diseases, 216 therapy for DMD, 293–294 tissue-specific, 30 transdifferentiated cells, 32–33 transplantation in hemophilia, 189–190 transplants for glioblastoma, 347–348 treating LSD with, 207 Stem cell therapy with biomaterials. See Biomaterial cell therapies transplantation for AD. See AD (autoimmune diseases), stem cell transplantation for Stem cell therapy, concepts, 547–566 cord blood transplants. See Cord blood transplants future directions in, 559–560 overview of, 547 references, 560–566 Sertoli cells and transplantation tolerance, 557–559 Stents, 275 Stereological principles, 64 Stereotaxic implantation of injection needle, 429 Steroids, 19 Stirred chambers, measuring OCR, 114–118 Streptomyces hygroscopicus, 17 Streptomyces tsukubaensis, 16 Stress-strain curves, for native trachea, 279 Striatal neurons, 418–429 in animal models of HD, 418–420 ethics of fetal neuronal tissue collection, 427–428 experimental transplantation of, 420–421 limitations of tissue supply, 428–429 neural transplantation in HD, 423–427 recovery after striatal transplants, 421–423 Striatum, in HD, 385 Stroke cell type therapy for, 35 cord blood therapy for, 553–555 initial cell transplants using fetal tissues, 354 regenerative stroke brain, 353–354 xenogeneic CP transplants in animal models, 408–409 Stroke, and GDNF treatment combining with NT2N cell transplants, 366 GDNF protein delivery in brain, 364 overview of, 363–364 references, 366–371 stroke pathology as guide for optimizing, 366 viral vectors for GDNF gene therapy, 364–365 viral vectors for GDNF gene therapy, limitations of, 365–366
672
Index Stroke, and NT2N cell grafts clinical trials in stroke patients, 360–361 combining with GDNF therapy, 366 immunosuppression a, 358–359 neurotrophic factors, 359–360 normal host brain microenvironment, 355 NT2N cells, 354–355 NT2N cells and gene therapy, 362 preclinical studies of, 356–358 recent advances in transplantation therapy, 361–362 references, 366–371 stroke host brain microenvironment, 355 Stromal-cell-derived inducing activity (SDIA), 530 Stupp, Dr. Samuel, 614 Supermouse, creating, 43 Surgery for brain tumors, 345 for heart failure, 259 neural transplantation in HD, 429 for PD, 439–440 spinal cord repair with PNGs, 482 SVZ (subventricular zone), 29 Symbols, characterizing islet preparation, 124–126 Syngeneic grafts, CNS, 309 Synthetic polymers, 231 Systemic inflammatory response syndrome (SIRS), 171, 173 Systemic lupus erythematosus (SLE), 246 Systemic sclerosis (SSC), 244–245 SYSTO-13, 107
T TAA (tumor-associated antigens), 604 Tacrolimus cautious use of MMF with, 18 clinical use of, 16–17 for islet transplantations, 67, 75 Tag protein, 464–465 TAID (testicular-associated acquired immune deviation), 574 T antigen, 535 Target of rapamycin (TOR), 17 Tat peptides, 618 T-cells allogenic HSCT relapses caused by, 247–248 allogenic MSCs and, 250–251 allograft rejection and, 557–558 alloimmume response and, 13 APCs and, 307–308 autoimmunity and, 603 azathioprine and, 14 cellular rejection caused by, 8 CNS graft rejection and, 309–316 immunoprivilege and, 578–579 infiltration into brain, 402 MHC-encoded proteins and, 6–7 primary nonfunction caused by, 8 TdT (terminal deoxynucleotidyl transferase), 111–112 Teams, project development planning and budgeting, 640–642
product development and, 639 regular progress presentations for, 647 Technology, project development planning and budgeting, 640–641 product development and, 639 Telomerase-immortalized cell lines, 535 Tenascin-R, 480 Tensegrity, 614 TEOA (triethanolamine), 143–144 Teratocarcinoma lines, 34 Terminal deoxynucleotidyl transferase (TdT), 111–112 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), 111–112, 207 Testicular-associated acquired immune deviation (TAID), 574 Testis immunoprivilege of, 574 immunoregulation in, 580 restricted access by immune cells, 575–576 Testosterone, 580 Tetramethylrhodamine ester (TMRE), 120–121 Tetraparentals, 44 Tetrazolium salts, 112–113 TETs (tissue-engineered tracheas). See Cylindrical cartilage transplantation TGF-/f2a/f0 (transforming growth factor)/f0/f2, 222 TGF-/f2b/f0 (transforming growth factor) biomaterials, modulating immune response, 599 CNS immunity and, 305–306 GDNF as member of, 334–335, 364–365 in hepatic profileration/differentiation, 222 in marrow stromal cells, 284–285 role in immunoprivilege, 579 Sertoli cell immunoprotection linked to, 559 TH (tyrosine hydroxylase), 362, 528 Thermoplastic membranes in macroencapsulation, 621 in microencapsulation, 621 overview of, 620–621 Thomas, Donnall, 547 3D scaffolding, 229–230 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tretrazolium bromide (MTT), 112–113, 116 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt (MTS), 112–113 Thrombocytopenia antilymphocyte sera side effect, 21 azathioprine side effect, 14 MMF side effect, 18 sirolimus side effect, 17 Thrombosis control of, 52 resulting from IBMIR, 68 risk of islet transplantation, 75 Thymidine kinase, 43 Thymocytes, 20 Thymoglobulin (polyclonal antilymphocyte sera), 20–21 Thyroglobulin, 138
673
Index Timing, of cell therapy, 264–265 Tipping points, 242 Tissue engineered liver, 228–233 bioreactors and implantation, 231 cell typing and sourcing, 228–229 classes of polymers, 230–231 critical factors/advances in, 232–233 overview of, 216, 228 polymer configurations and modifications, 231 scaffold design and manipulation, 229–230 soluble factors, 231 Tissue engineering less promising for structurally complex organs, 4 with nanotechnology, 619–620 for tracheal replacement. See Cylindrical cartilage transplantation tracheas. See Cylindrical cartilage transplantation treating kidney failure with, 172 Tissues obtaining for cell-based therapies, 220 regenerating with biomaterial implants, 600–602 Tissues, immunologically privileged adrenal gland, 574 brain, 571–572 eye, 571–572 fetal allograft, 573 gonads, 573–575 liver, 574 overview of, 570–571 placenta, 573 tumors, 574 uterus, 573 Tissue-specific stem cell (TSC), 30–31 Tissue transplantation antibodies and, 8–9 barriers to xenografts, 5–6 cellular rejection in, 8 generation of histocompatible tissues, 165 immune response to, 5–7 less susceptible to humoral rejection, 156 nanoparticles screening and monitoring, 617–619 primary nonfunction in, 7–8 therapeutic approaches to, 9 using embryonic stem cells, 164–165 T-lym cell, 136–138 TMRE (tetramethylrhodamine ester), 120–121 TNF (tumor necrosis factor) autoimmune diseases and, 242 CNS immunity and, 305 MMF and, 18 TNF-/f2a/f0 (tumor necrosis factor-/f2a/f0), 599, 601 Tolerance CNS transplantation, 316–320 immunoregulation and peripheral, 577–579 inducing transplantation, 196–197 islet transplantation, 78 liver disease cell transplantation, 233–234 MSC transplantation, 249–250
myoblast transplantation for DMD and, 292 Sertoli cells and, 557–559 Tolypocladium inflatum, 14 Tools, product development, 645–647 TOPCARE-AMI study, 267 TOPCARE-HF study, 268 TOR (target of rapamycin), 17 Tracheal chondrocytes, 276–279 Tracheal replacements. See Cylindrical cartilage transplantation Transdifferentiation for liver regeneration, 226–227 vs. other cell sources, 35 overview of, 32–33 treating certain diseases, 35 Transduced cells administering NGF via, 379–380 generating chromaffin, 467–469 Transformed hepatocytes, 220 Transforming growth factor (TGF-/f2a/f0), 222 Transforming growth factor (TGF-/f2b/f0). See TGF-/f2b/f0 (transforming growth factor) Transgenic animals creating, 43 models of human PD, 441 Translational medicine, 289 Transplant-related mortality. See TRM (transplant-related mortality) Trauma, and choroid stem cells, 405 TRECs, 248 Triethanolamine (TEOA), 143–144 TrkA receptors, 373–375 TRM (transplant-related mortality) patients receiving HSCT for AD, 243 patients receiving HSCT for JIA, 246 patients receiving HSCT for MS, 244 patients receiving HSCT for RA, 245 patients receiving HSCT for SLE, 246 patients receiving HSCT for SSC, 244–245 Trophoectoderm cells, 527 Truncal obesity, and corticosteroids, 19 TSC (tissue-specific stem cell), 30–31 Tubule cells bioartificial kidneys with, 173 defined, 170 excretory process of nephron and, 170 roles of, 173 Tumor-associated antigens (TAAs), 604 Tumor cell lines, 463–464 Tumorigenesis, 504, 534–535 Tumor necrosis factor. See TNF (tumor necrosis factor) Tumors cancer vaccines seeking to destroy, 603–604 immunoprivilege of, 574 TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), 111–112, 207 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylam)carbonyl)2H-tetrazolium hydroxide (XTT), 112–113
674
Index Type 1 diabetes allotransplantation for, 76–77 insulin therapy limitations, 60 pathogenesis of, 59 whole-pancreas transplantation for, 60 Type 2 diabetes allotransplantation for, 77 pathogenesis of, 59–60 prevention of, 60 Tyrosine hydroxylase (TH) gene, 528
Viral vectors for GDNF gene therapy, 364–365 treating brain tumors with, 347 treating hemophilia with, 187 Visual Analog Scale (VAS) pain ratings, 461–462 Vitreous chamber, of eye, 327–328 VM (ventral mesencephalon) transplantation fetal tissue transplantation for PD, 523–525 generating dopamine neurons from, 529–530 rejection of CNS transplants, 315–318 VPCs (vascular progenitor cells), 264
U UF (ultrafiltrate) of plasma, 170 Ultrasonic pulsed Doppler (USPD) measurements, 101, 103 Umbilical cord blood transplantation, 207 Unilateral lesion syndrome, in PD, 440–441 UPS (ubiquitin-proteasome-system), 307–308 Urea, in liver function, 217–218 Uremia, 180 Ureteric buds, 165 Urinary incontinence, 294–295 Urine volumes, 161 Urologic dysfunction, 294–295 USPD (Ultrasonic pulsed Doppler) measurements, 101, 103 Uterus, immunoprivilege of, 573 UW solution, 87
V Vaccines, cancer, 603–604 VAS (Visual Analog Scale) pain ratings, 461–462 Vascular endothelial growth factor. See VEGF (vascular endothelial growth factor) Vascularization diseases in organ grafts, 8–9 primary nonfunction after, 8 of transplanted renal anlagen, 159 Vascular progenitor cells (VPCs), 264 Vasculature, for tissue-engineered constructs, 233 VCT (vinyl composition tile) flooring, 632 Vector choice, in gene therapy, 194–196 VEGF (vascular endothelial growth factor) age-related macular degeneration and, 339 angiogenesis and, 601 bone healing and, 295 CNS immunity and, 308 immunosuppression of MSC in vivo and, 251 inhibiting with PEDF, 336 nerve regeneration with biomaterials and, 602 as soluble factor in tissue engineering, 232 for tissue engineering vasculature, 233 VEGF-R2, 264 Ventral mesencephalon. See VM (ventral mesencephalon) transplantation Viability. See Islet preparations, viability of Vinyl composition tile (VCT) flooring, 632 Viral infections, and xenotransplantation, 4
W Waite, Dr. Phil, 505 Wait list, islet transplantation, 66 Wet (neovascular) AMD, 339 Wharton’s jelly, 550 Whole-pancreas transplantation donor selection for, 63 reducing microvascular complications, 73 sustained insulin independence after, 65 for type 1 diabetes, 60 Wild-type Htt, 386 Wound healing, 17, 19
X Xenogeneic CNS grafts antigen presentation and rejection of, 311 defined, 309 ESC rejection in, 309 immune response of host to, 315 mechanisms of graft rejection, 315–316 Xenogeneic CP transplants alginate/encapsulated choroidal epithelial cells, 407–408 in animal models of stroke, 408–409 encapsulated alginate microcapsules enabling, 406–407 in rat model of HD, 409–410 Xenogenic tissues for cell-based therapies, 220 encapsulating hepatocytes, 225 Xenosensitization, 22 Xenotransplantation, creating animals for, 43–56 chemical islet xenotransplantation, 50–51 clinical neural xenotransplantation, 51 control of thrombosis, 52 development of microinjection, 43 Dolly the sheep and other animals, 44–45 embryonic stem cells and knockouts, 43–44 future directions, 52 inhibition of complement activation, 46–47 islet xenotransplantation, 48–50 knockout pigs, 51–52 making pigs, 45 pigs transgenic for complement regulatory proteins, 47–48 references, 52–56 regulators of complement activation (RCA), 46
675
Index Xenotransplantation, immunology of, 3–11 antibodies, 8–9 barrier to, 8–9 B-cells, 8–9 defined, 3 early attempts at, 3 immune response to, 5–7 immunological barriers to, 5 impact of immunity, 7–8 rationale for, 4 references, 9–11 therapeutic approaches to, 9 type of transplant and barrier to, 4
XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5((phenylam)carbonyl)-2H-tetrazolium hydroxide), 112–113
Y YEILDV, 598 YIGSR (Tyr-Ile-Gly-Ser-Arg), 232, 331, 598 Y-shaped prothesis, for engineered trachea, 280–282
Z Zenapax (daclizumab), 22, 67, 138 Zona pellucida, 574
676
