Stem Cell Biology in Health and Disease

Stem Cell Biology in Health and Disease Thomas Dittmar · Kurt S. Zänker Editors Stem Cell Biology in Health and Dise...

Author: Thomas Dittmar | Kurt S. Zanker

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Stem Cell Biology in Health and Disease

Thomas Dittmar · Kurt S. Zänker Editors

Stem Cell Biology in Health and Disease

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Editors Dr. Thomas Dittmar Witten/Herdecke University Institute of Immunology Stockumer Str. 10 58448 Witten Germany e-mail: [email protected]

Prof. Dr. Kurt S. Zänker Witten/Herdecke University Institute of Immunology Stockumer Str. 10 58448 Witten Germany e-mail: [email protected]

ISBN 978-90-481-3039-9 e-ISBN 978-90-481-3040-5 DOI 10.1007/978-90-481-3040-5 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009935391 © Springer Science+Business Media B.V. 2009 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Within the last decade there has been a dramatic increase in the understanding and application of biological principles within stem cell therapies, which has made it necessary to produce a book which intends to summarize much of the body of knowledge concerning Stem Cell Biology in Health and Disease. Although some of the treatments have been suggested for many years, knowledge and technology have now progressed sufficiently to allow us to test many of the different concepts with human embryonic, induced pluripotent, organ-specific and resident, cancer and mesenchymal stem cells in animal models and clinical settings – alone or in combination with other therapies in cardiovascular and neurodegenerative diseases, in diabetes and against cancer. Studies on stem cells have been hampered in the past by the ethical and biological difficulties in preparing sufficient cell numbers in a reasonable characterized and pure form. In stem cell research we are now on the threshold of a revolution; a revolution that will have major ramification for human medicine. Giant strides in our understanding of stem cell biology and the elements that control the biological behavior of the different traits of stem cells have made it possible to intervene directly with regenerative life processes and to open a novel chapter in the fight against cancer. Chapter 1 shortly summarizes the historical hall marks of stem cell research in biology; Chapter 2 describes the hematopoietic stem and progenitor cells in clinical use; Chapter 3 describes the protocols to expand hematopoietic stem cells ex vivo; Chapter 4 highlights one important feature of hematopoietic stem/progenitor cells, namely cell migration; Chapter 5 opens the books on properties of mesenchymal stem cells for cancer cell therapy; Chapter 6 reviews intensively alternative embryonic stem cell sources to solve both ethical concerns and the allogeneic nature of human embryonic stem cells for therapeutic use; Chapters 7 and 8 describe the role of stem cell therapy in Multiple Sclerosis and Parkinson’s Disease; Chapters 9, 10 and 11 introduce novel perspectives on cancer stem cells stimulating a provocative discussion of the complexity of cancer origin, and their niches of existence either in a tumor mass or in chronically inflamed microenvironment, e.g. inflamed periodontium (Chapter 12); Chapters 13 and 14 directly address hematopoietic and solid cancer stem cells and Chapter 15 embarks on a novel role of the diversity of cancer stem cells in tumor relapse and metastases formation. Chapter 16 describes v

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new therapeutic approaches to eliminate cancer stem cells and Chapter 17 puts the focus on a molecular target family in cancer stem/progenitor cells - the ATP-binding cassette membrane transporters - which are promising therapeutic entities. Multiple key references are provided by the authors at the end of each chapter, and the reader is encouraged to consult these sources as well, because due to the limited space of a monograph the technical details cannot be presented in a survey of this type. Again, we would like to thank all distinguished authors for their valuable contributions to provide with this book a robust ground for the avalanche of discoveries that will deluge the field of stem cell research in the years to come. Summer 2009

Witten, (Germany) Thomas Dittmar Kurt S. Zänker

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomas Dittmar and Kurt S. Zänker Part I

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Bone Marrow-Derived Stem Cells

2 Hematopoietic Stem and Progenitor Cells in Clinical Use – Transplantation and Mobilization . . . . . . . . . . . . . . . Michael Punzel

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3 Ex Vivo Expansion of HSPCs . . . . . . . . . . . . . . . . . . . . . Yaming Wei and Xin Ye

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4 Modulation of Hematopoietic Stem/Progenitor Cell Migration . . . Thomas Dittmar, Susannah H. Kassmer, Benjamin Kasenda, Jeanette Seidel, Bernd Niggemann, and Kurt S. Zänker

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5 Properties of Mesenchymal Stem Cells to Consider for Cancer Cell Therapy . . . . . . . . . . . . . . John Stagg and Sandra Pommey Part II

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Embryonic Stem Cells

6 Alternative Embryonic Stem Cell Sources . . . . . . . . . . . . . . Tomo Šari´c, Narges Zare Mehrjardi, and Jürgen Hescheler

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7 Cell Therapy in Parkinson’s Disease . . . . . . . . . . . . . . . . . R. Laguna Goya and R.A. Barker

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8 Transplantation of Stem Cells and Their Derivatives in the Treatment of Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . Eric C. Larsen and Ian D. Duncan

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Part III Cancer Stem Cells 9 Cancer: A Stem Cell-based Disease? . . . . . . . . . . . . . . . . . James E. Trosko

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Contents

Stem Cell Niche Versus Cancer Stem Cell Niche – Differences and Similarities . . . . . . . . . . . . . . . . . Bruce C. Baguley and Graeme J. Finlay The Chronically Inflamed Microenvironment and Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hanchen Li, Calin Stoicov, Xueli Fan, Jan Cerny, and Jean Marie Houghton Does the Chronically Inflamed Periodontium Harbour Cancer Stem Cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . Wolf-Dieter Grimm, Wolfgang H. Arnold, Sebastian Becher, Aous Dannan, Georg Gassmann, Stathis Philippou, Thomas Dittmar, and Gabor Varga

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Leukemia Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . Markus Müschen

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Cancer Stem Cells in Solid Tumors . . . . . . . . . . . . . . . . . . Melia G. Nafus and Alexander Yu. Nikitin

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“One for All” or “All for One”? – The Necessity of Cancer Stem Cell Diversity in Metastasis Formation and Cancer Relapse . Thomas Dittmar, Christa Nagler, Sarah Schwitalla, Kathrin Krause, Jeanette Seidel, Georg Reith, Bernd Niggemann, and Kurt S. Zänker

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Elimination of Cancer Stem Cells . . . . . . . . . . . . . . . . . . . A. Sagrera, J. Pérez-Losada, M. Pérez-Caro, R. Jiménez, I. Sánchez-García, and C. Cobaleda

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Potential Molecular Therapeutic Targets in Cancer Stem/Progenitor Cells: Are ATP-Binding Cassette Membrane Transporters Appropriate Targets to Eliminate Cancer-Initiating Cells? . . . . . . . . . . . . . . . . . . . . . . . . Murielle Mimeault and Surinder K. Batra

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors

Wolfgang H. Arnold Institute of Anatomy, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, [email protected] Bruce C. Baguley Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand, [email protected] R.A. Barker Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge, CB2 2PY, UK; Department of Neurology, Addenbrookes Hospital, Cambridge, CB2 2QQ, UK; Edith Cowan University, Perth, Australia, [email protected] Surinder K. Batra Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA, [email protected] Sebastian Becher Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, basti [email protected] Jan Cerny Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected] C. Cobaleda Departamento de Fisiología y Farmacología, Edificio Departamental, Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain, [email protected] Aous Dannan Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, [email protected] Thomas Dittmar Institute of Immunology, Faculty of Medicine, Witten/Herdecke University, Stockumer Str. 10, 58448 Witten, Germany, [email protected]

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Contributors

Ian D. Duncan Department of Medical Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA, [email protected] Xueli Fan Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected] Graeme J. Finlay Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand, [email protected] Georg Gassmann Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, [email protected] R. Laguna Goya Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge, CB2 2PY, UK, [email protected] Wolf-Dieter Grimm Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, [email protected] Jürgen Hescheler Institute for Neurophysiology, Center for Physiology and Pathophysiology, Medical Center; Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany, [email protected] JeanMarie Houghton Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected] R. Jiménez Departamento de Fisiología y Farmacología, Edificio Departamental, Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain, [email protected] Benjamin Kasenda Department of Hematology and Oncology, University of Freiburg Medical Center, D-79106 Freiburg, Germany, [email protected] Susannah H. Kassmer Department of Laboratory Medicine, Yale Stem Cell Center, Yale University, New Haven, CT, USA, [email protected] Kathrin Krause Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected] Eric C. Larsen Department of Medical Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA, [email protected] Hanchen Li Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected]

Contributors

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Narges Zare Mehrjardi Institute for Neurophysiology, Center for Physiology and Pathophysiology, Medical Center, University of Cologne, 50931 Cologne, Germany; Department of Stem Cells, Royan Institute, Tehran, Iran, [email protected] Murielle Mimeault Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA, [email protected] Markus Müschen Leukemia Research Program, Childrens Hospital Los Angeles; Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90027, [email protected] Melia G. Nafus Department of Biomedical Sciences, Cornell University, Ithaca, New York, 14853, USA, [email protected] Christa Nagler Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected] Bernd Niggemann Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected] Alexander Yu. Nikitin Department of Biomedical Sciences, Cornell University, Ithaca, New York, 14853, USA, [email protected] M. Pérez-Caro OncoStem Pharma, Salamanca, Spain, [email protected] J. Pérez-Losada Departamento de Medicina, Facultad de Medicina, Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain, [email protected] Stathis Philippou Institute of Pathology, Faculty of Medicine, Ruhr University Bochum, 44801 Bochum, Germany, [email protected] Sandra Pommey Department of Medicine, Immunology Research Centre, St. Vincent’s Hospital, University of Melbourne, Melbourne, Victoria, Australia, [email protected] Michael Punzel Institute of Transplantation Diagnostic and Cellular Therapeutics, Universitätsklinikum Düsseldorf, 40225 Düsseldorf, Germany, [email protected] Georg Reith Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected] A. Sagrera OncoStem Pharma, Salamanca, Spain, [email protected] I. Sánchez-García Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain, [email protected]

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Contributors

Tomo Šari´c Institute for Neurophysiology, Center for Physiology and Pathophysiology, Medical Center; Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany, [email protected] Sarah Schwitalla Second Department of Medicine, Klinikum rechts der Isar, Technical University of Munich, 81675 Munich, Germany, [email protected] Jeanette Seidel Medizinische Klinik II m. S. Hämatologie/Onkologie, Charité Campus Mitte, 10117 Berlin, Germany, [email protected] John Stagg Cancer Immunology Program, Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia, [email protected] Calin Stoicov Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected] James E. Trosko Department of Pediatrics/Human Development, College of Human Medicine, Michigan State University, East Lansing, Michigan 48824, MI, USA, [email protected] Gabor Varga Department of Oral Biology, Semmelweis University, Budapest, Hungary, [email protected] Yaming Wei Guangzhou Institute of Clinical Medicine, Guanzhou Municipal First People’s Hospital, Guangzhou Medical College, Guangzhou, China, [email protected] Xin Ye Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, China, [email protected] Kurt S. Zänker Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected]

Chapter 1

Introduction Thomas Dittmar and Kurt S. Zänker

Within the past years our knowledge about stem cell biology in health and disease has changed dramatically. What rather sounded like Science Fiction 10–15 years ago, namely that e.g., stem cells from bone marrow or from adipose tissue can be used for regenerative medical approaches, or that it is possible to create donor specific stem cells (so-called induced pluripotent stem cells (iPS cells), exhibiting embryonic stem cell (ESC) properties) simply by transducing 2–4 transcription factors, has now become reality. Likewise, the knowledge that cancer tissues are hierarchically organized like normal tissues, namely comprising of a small amount of tumorigenic cancer stem cells (CSCs) and a huge mass of non-tumorigenic cancer cells will play a crucial role in the development of novel anti-cancer strategies. It is remarkable what has been achieved in the field of regenerative medicine within the past 10–15 years. In summary, this is an exciting story of what is possible in stem cell-based regeneration strategies, but it is also a story about a long and stony way with lots of unknown pitfalls. In 1999/2000 first data have been published demonstrating that bone marrowderived stem cells (BMDCs) can develop into hepatocytes [1, 2]. These original studies, being performed in rodents, were the first hints that stem cells of the bone marrow do not only give rise to cells of the blood lineage, but can also differentiate into cells of a different germ layer, a phenomenon, which has been referred to as “transdifferentiation” [3]. Till then (and to date), BMDCs were/are commonly used for bone marrow reconstitution after high-dose chemotherapy of patients with malignant hematopoietic disorders, such as multiple myeloma [4] or acute leukemias [5], or solid tumors [6]. The finding that BMDCs, and later on other types of adult stem cells, e.g., adipose-derived stem cells (ASCs) or neural stem cells (NSCs), are capable to transdifferentiate into various tissues, thereby restoring tissue integrity [7], offered perspectives for novel therapeutical approaches to heal various severe diseases, such as heart attack, liver cirrhosis, and neuronal degenerative disorders (stroke, T. Dittmar (B) Institute of Immunology, Faculty of Medicine, Witten/Herdecke University, Stockumer Str. 10, 58448, Witten, Germany e-mail: [email protected]

T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_1,

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Parkinson Disease, etc.). Among adult stem cells, particularly BMDCs and ASCs raised (and still raise) great expectations for stem cell-based tissue regeneration strategies. Both stem cell types are easily accessible (BMDCs from bone marrow via aspiration or apheresis from mobilized donors, ASCs from liposuction) and possess an enhanced transdifferentiation capacity as verified by a plethora of excellent animal studies (for review see [7–9]). BMDCs can give rise to liver, skeletal muscle, gastric mucosa, and small intestinal epithelial cells [7]. The differentiation potential of ASCs includes adipocytes, cardiomyocytes, chondrocytes, endothelial cells, myocytes, neuronal-like cells, and osteoblasts [8]. However, there are some concerns about the overall pluripotency of adult stem cells. In contrast to ESCs and iPS cells, it is not possible to transdifferentiate adult stem cells functionally in certain tissues, like cardiomyocytes and dopaminergic neurons, in-vitro. In addition to that, even in vivo studies presented inconsistent data concerning the transdifferentiation capacity of adult stem cells. For instance, in 2001, Orlic and colleagues reported that transplanted adult bone marrow cells repaired myocardial infarcts in mice [10]. Examination of the infracted region after a period of 9 days following transplantation demonstrated that newly formed myocardium, comprising of proliferating myocytes and vascular structures, occupied about 68% of the infracted region [10]. Moreover, the functional competence of the left repaired ventricle was improved for several hemodynamic parameters [11] suggesting that efficient myocardial repair by application of BMDCs is conceivable. Only one year later, in 2002, Strauer et al. already reported about the repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans [12]. After standard therapy for acute myocardial infarction (AMI), 10 patients were transplanted with autologous BMDCs via a balloon catheter placed into the infarct-related artery during balloon dilation [12]. After 3 months of follow-up, patients of the cell therapy group showed a significantly decreased infarct region, a significantly increased infarction wall movement velocity, and a significant improvement in stroke volume index, left ventricular end-systolic volume and contractility [12]. At a first glance, these data might tell a successful “form bench to bedside” story. However, in 2004, two independent studies demonstrated that BMDCs do not undergo transdifferentiation into cardiomyocytes in myocardical infarcts [13, 14]. Murry and colleagues showed that only 1–3 cells per 100,000 cardiomyocytes were of bone marrow origin [14], which is in clear contrast to 68% as reported by Orlic et al. [10]. Likewise, data of Balsam and colleagues provided evidence that BMDCs rather adopted mature hematopoietic fates in ischemic myocardium than to transdifferentiate into cardiomyocytes [13]. Balsam and colleagues speculated that there may be differences in their anesthetic and/or surgical technique and that these may resulted in a different outcome [13], whereas Murry and colleagues assumed subtle differences in the protocols, e.g., differences in trace components in the stem cell preparation or different assays used to detect cardiomyogenic differentiation, which might explain the discrepant results [14]. In a long-term study Meyer and colleagues were able to show that a single dose of intracoronary bone marrow-derived HSPCs did not provide long-term benefit on left ventricular systolic function after acute

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myocardial infarction (AMI) as compared with a randomized control group [15]. Similar results were reported recently by Choi and colleagues demonstrating a lack of additional benefit of intracoronary transplantation of autologous peripheral blood stem cells in AMI patients [16]. However, both studies reported that after 6 months the left ventricular ejection fraction was significantly improved in the cell therapy group [15, 16], which may point to a stem cell specific effect. Further disadvantages of most adult stem cells are (i) that they do not remain in a stem cell state under in vitro conditions and (ii) that they can only expanded for limited passages. Both disadvantages omit long-term cultures of adult stem cells, which is in contrast to ESCs and iPS cells that could be cultivated nearly unlimited. For instance, bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) can be cultured for 5–7 days without a significant decrease of CD34/CD133 expression. Longer cultivation periods is associated with a decrease of these two HSPC marker molecules indicating induction of differentiation. To delay the autologous differentiation capacity of HSPCs, e.g., for ex vivo expansion approaches optimized culture medias have been developed, which mostly vary in the choice of supplemented cytokines. Using optimized culture conditions it is possible to expand HSPCs ex vivo without a noteworthy level of differentiation. On the other hand, these optimized culture condition might have different effects on the expanded cells. We have recently demonstrated that the stromal cell-derived factor-1α (SDF1α) induced migratory activity of cultivated murine HSPCs strongly depended on the used cytokine combinations [17]. For instance, cultivation of murine HSPCs in the presence of stem cell factor, thrombopoietin and Interleukin-11 yielded in the third highest expansion rate of all tested cytokines and cytokine combinations [17]. However, analysis of the migratory behavior revealed that these cells did not react to SDF-1α stimulation with an increased locomotory activity [17], which could be a severe side-effect if such cells would be used for HSPC transplantation for bone marrow reconstitution. In contrast to adult stem cells, ESCs remain in their stem cell state in vitro and can be propagated nearly unlimited. Moreover, these cells possess an unlimited differentiation capacity in vitro and in vivo. However, human ESCs are still a subject to controversial and ethical discussions since isolation of human ESCs prerequisites the destruction of a human embryo (or the killing of a putative human life). Another disadvantage of ESCs is that they could not be administered directly in degenerated tissues while this would result in teratoma formation (which nicely illustrates their unrestricted differentiation capacity). Thus, these cells could only be implanted after in vitro pre-differentiation. However, pre-differentiated ESCs exhibit an overall lesser survival rate when removed from culture and being transplanted. Ultimately, transplantation of pre-differentiated ESCs prerequisites immunosuppression of the patients to avoid the risk of graft rejection, which, however, is associated with other risks and concerns. The latter problem could be overcome by generating “patient/custom-made embryonic cell lines”, so-called therapeutic cloning. Even if this technique would be feasible one day the other two problems (ethical debate and risk of tumor formation) would remain.

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Within the past two to three years a novel embryonic stem cell-like type has emerged, so-called iPS cells. These cells can be generated by viral transfection of two or four transcription factors into adult stem cells or adult somatic cells, respectively [18, 19], which ultimately leads to a redirection of this cell types towards and embryonic-like, undifferentiated state. In fact, induced pluripotent stem cells possess several ESC characteristics, such as morphology, proliferation, gene expression, telomerase activity, epigenetic status, and the capacity of unrestricted differentiation. Like ESCs, the latter property is associated with teratoma formation in-vivo if iPS cells are transplanted undifferentiated. However, even if iPS cells will be pre-differentiated prior implantation, they might bear potentially tumorigenic risks since these cells were generated by using the proto-oncogene c-myc and viral vectors, which integrate randomly into the host genome. Whether human iPS cells, either generated without the use of c-myc [18, 20] or without viral integration [21] using plasmids, will find their way into clinical use has to be elucidated in future studies. Nonetheless, the benefit of such cells would be that they behave like ESCs, thus being capable to differentiate into various tissues, and “patient/custom-made iPS cells” can be generated, which supersedes immunosuppression. A severe side-effect of most, if not all, stem cells is their potential tumorinitiation capacity. It is well recognized that ESCs and iPS cells induce teratomas in-vivo if implanted in a undifferentiated state. Pre-differentiation of both ESCs and iPS cells could minimize this risk, whereby iPS cells might still bear potentially tumorigenic risks if such cells were generated by the use of the proto-oncogene c-myc and viral vectors, which integrate randomly into the host genome. With prolonged passage for >4 months, human ASCs have been observed to undergo malignant transformation, which was correlated with karyotypic abnormalities, tumor formation in immunodeficient mice [22], and epithelial-mesenchymal transition [23]. Nearly 4 years ago, Houghton and colleagues demonstrated that gastric cancer originates from BMDCs, which have been recruited and transformed malignantly by chronically inflamed gastric mucosa tissue [24]. In addition to gastric cancer there is compelling evidence that also other epithelial cancers, such as benign and malignant tumors of the skin, Kaposis sarcoma, and Barretts’ adenocarcinoma of the esophagus might originate from BMDCs (for review see [25]). The inherent tumorigenic capacity of stem cells points to another type of stem cells, which has gained much of attention within the last decade: cancer stem cells (CSCs) (for review see [26]). CSCs have been described as a rare population of cancer cells exhibiting stem cell properties such as self-renewing, differentiation, tissue reconstitution, and multiple drug resistance. Because of their tumor initiation capacity and resistance against cytotoxic drugs and radiation CSCs [27–29] have not only been linked to primary tumor formation, but also to metastases and cancer relapses. The knowledge that a tumor is organized hierarchically like normal tissue, namely comprising of a small number of stem cells, which give rise to differentiated cells, thereby maintaining tissue integrity and organ function, is of crucial interest for our understanding how to treat cancer in future times. The dilemma of current cancer therapies (conventional chemotherapy, radiation therapy, hormonal therapy, humanized monoclonal antibodies, and/or inhibitors) is that although most cancer

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patients respond to therapy, only few are definitely cured [30]; a matter, which applies to both solid tumors as well as hematological disorders. This phenomenon, which has been entitled as “the paradox of response and survival in cancer therapeutics” [30] has been compared to “cutting a dandelion off at ground level” [30, 31]. Current cancer therapies are designed to target highly proliferating tumor cells and determination of tumor shrinking concomitant with mean disease free survival of patients are commonly used as read-outs for the efficacy of the appropriate therapy. While such strategies eliminate the visible portion of the tumor, namely the tumor mass, they mostly fail to eliminate the unseen root of cancer, namely CSCs. Thus, elimination of the unseen root of cancer, CSCs, would mean to have a chance to cure disease. However, there is increasing evidence that both metastases and cancer relapses might be initiated by specific CSCs, referred to as metastatic CSCs (mCSCs) [32] and recurrence CSCs (rCSCs) [33]. Quite recently, Hermann and colleagues identified a specifically metastatic CSC subpopulation in pancreatic cancer [34], whereas Shafee et al. demonstrated that the cisplatin resistance of murine mammary CSCs was associated with genetic aberrations in the platinum resistant cells [35]. These findings suggest that different cancer stage specific CSCs exist, which might play a role in the development of anti-CSC strategies. Is it possible to eliminate distinct CSC subtypes with a single anti-CSC strategy or demand distinct CSC subtypes distinct anti-CSC strategies? The answer to this question can not be given yet since only a handful of data exist for mCSCs and rCSCs so far. In summary, it is remarkable what has been achieved in only 10–15 years in the field of stem cell biology in health and disease. Even if still some problems, being associated with stem cell-based regeneration strategies (e.g., choice of the stem cell type (adult stem cells, ESCs, or iPS cells), how to apply them (by injection, by infusion etc.), exist, we know from several animal studies that stem cell-based regeneration strategies are feasible and that it will be only a matter of time when such approaches will become reality in humans. Likewise, the knowledge that CSCs exist has changed our understanding of the disease cancer and will help us to develop novel anti-cancer strategies. There is a growing list of CSC specific target molecules/pathways, which might be used for selective CSC elimination or which could be used to drive CSCs from their stem cell state into a more differentiated state, thereby making these cells susceptible to conventional cancer therapy. So, we the scientists, physicians, and patients should be optimistic what the future will bring in the field of stem cell biology in health and disease. We are glad that so many internationally recognized experts accepted our invitation to contribute to this exciting book. We sincerely thank them all for their interest in this important topic and that they, despite other duties and responsibilities, found the possibility to present excellent and comprehensive overviews of the most important recent findings in their field of scientific engagement within this topic. We would also like to thank Cristina Aves dos Santos, Sara Huisman, and Peter Butler from Springer Publishers for their kind assistance and excellent collaboration on this project, as well as for giving the opportunity to realize this book project.

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We hope that this book may encourage new scientific approaches within the field of stem cell biology in health and disease as well as closer interdisciplinary collaborations on this fascinating and important issue in the future.

References 1. Petersen BE, Bowen WC, Patrene KD, et al. (1999) Bone marrow as a potential source of hepatic oval cells. Science 284: 1168–1170 2. Theise ND, Badve S, Saxena R, et al. (2000) Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31: 235–240 3. Eisenberg LM and Eisenberg CA (2003) Stem cell plasticity, cell fusion, and transdifferentiation. Birth Defects Res Part C Embryo Today 69: 209–218 4. Nau KC and Lewis WD (2008) Multiple myeloma: diagnosis and treatment. Am Fam Physician 78: 853–859 5. Niederwieser D, Gentilini C, Hegenbart U, et al. (2005) Allogeneic hematopoietic cell transplantation (HCT) following reduced-intensity conditioning in patients with acute leukemias. Crit Rev Oncol Hematol 56: 275–281 6. Banna GL, Simonelli M, and Santoro A (2007) High-dose chemotherapy followed by autologous hematopoietic stem-cell transplantation for the treatment of solid tumors in adults: a critical review. Curr Stem Cell Res Ther 2: 65–82 7. Dittmar T, Seidel J, Zaenker KS, et al. (2006) Carcinogenesis driven by bone marrow-derived stem cells. Contrib Microbiol 13: 156–169 8. Gimble JM, Katz AJ, and Bunnell BA (2007) Adipose-derived stem cells for regenerative medicine. Circ Res 100: 1249–1260 9. Mimeault M and Batra SK (2006) Concise review: recent advances on the significance of stem cells in tissue regeneration and cancer therapies. Stem Cells 24: 2319–2345 10. Orlic D, Kajstura J, Chimenti S, et al. (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410: 701–705 11. Orlic D, Kajstura J, Chimenti S, et al. (2001) Transplanted adult bone marrow cells repair myocardial infarcts in mice. Ann NY Acad Sci 938: 221–229; discussion 229–230 12. Strauer BE, Brehm M, Zeus T, et al. (2002) Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106: 1913–1918 13. Balsam LB, Wagers AJ, Christensen JL, et al. (2004) Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 428: 668–673 14. Murry CE, Soonpaa MH, Reinecke H, et al. (2004) Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 428: 664–668 15. Meyer GP, Wollert KC, Lotz J, et al. (2006) Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months follow-up data from the randomized, controlled BOOST (BOne marrOw transfer to enhance ST-elevation infarct regeneration) trial. Circulation 113: 1287–1294 16. Choi JH, Choi J, Lee WS, et al. (2007) Lack of additional benefit of intracoronary transplantation of autologous peripheral blood stem cell in patients with acute myocardial infarction. Circ J 71: 486–494 17. Kassmer SH, Niggemann B, Punzel M, et al. (2008) Cytokine combinations differentially influence the SDF-1alpha-dependent migratory activity of cultivated murine hematopoietic stem and progenitor cells. Biol Chem 389: 863–872 18. Kim JB, Zaehres H, Wu G, et al. (2008) Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454: 646–650 19. Takahashi K, Tanabe K, Ohnuki M, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872

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20. Yu J, Vodyanik MA, Smuga-Otto K, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920 21. Okita K, Nakagawa M, Hyenjong H, et al. (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322: 949–953 22. Rubio D, Garcia-Castro J, Martin MC, et al. (2005) Spontaneous human adult stem cell transformation. Cancer Res 65: 3035–3039 23. Rubio D, Garcia S, De la Cueva T, et al. (2008) Human mesenchymal stem cell transformation is associated with a mesenchymal-epithelial transition. Exp Cell Res 314: 691–698 24. Houghton J, Stoicov C, Nomura S, et al. (2004) Gastric cancer originating from bone marrowderived cells. Science 306: 1568–1571 25. Li HC, Stoicov C, Rogers AB, et al. (2006) Stem cells and cancer: evidence for bone marrow stem cells in epithelial cancers. World J Gastroenterol 12: 363–371 26. Wicha MS, Liu S, and Dontu G (2006) Cancer stem cells: an old idea – a paradigm shift. Cancer Res 66: 1883–1890; discussion 1895–1886 27. Eyler CE and Rich JN (2008) Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. J Clin Oncol 26: 2839–2845 28. Rich JN (2007) Cancer stem cells in radiation resistance. Cancer Res 67: 8980–8984 29. Shervington A and Lu C (2008) Expression of multidrug resistance genes in normal and cancer stem cells. Cancer Invest 26: 535–542 30. Huff CA, Matsui W, Smith BD, et al. (2006) The paradox of response and survival in cancer therapeutics. Blood 107: 431–434 31. Blagosklonny MV (2005) Why therapeutic response may not prolong the life of a cancer patient: selection for oncogenic resistance. Cell Cycle 4: 1693–1698 32. Li F, Tiede B, Massague J, et al. (2007) Beyond tumorigenesis: cancer stem cells in metastasis. Cell Res 17: 3–14 33. Dittmar T, Nagler C, Schwitalla S (2009). Recurrence cancer stem cells - made by cell fusion? Med Hypotheses: doi: 10.1016/j.mehy.2009.05.044 34. Hermann PC, Huber SL, Herrler T, et al. (2007) Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1: 313–323 35. Shafee N, Smith CR, Wei S, et al. (2008) Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors. Cancer Res 68: 3243–3250

Chapter 2

Hematopoietic Stem and Progenitor Cells in Clinical Use – Transplantation and Mobilization Michael Punzel

Abstract It took exactly 100 years from the original discovery of the blood formation within the bone marrow until the first successful clinical bone marrow transplantation has been performed. Today, the transplantation of hematopoietic stem cells from various sources, such as bone marrow, mobilized stem cells as well as umbilical cord blood has become a routine procedure, reaching currently more than 10,000 transplantations per year in the allogeneic setting and over 40,000 autologous transplantations. Although, the number of transplantations is increasing every year, the field is constantly changing in terms of conditioning procedures and clinical indications. In addition, the increase in the availability of multiple graft sources for allogeneic transplantation, such as related or unrelated living donors versus frozen umbilical cord blood as well as the choice between mobilized peripheral blood versus steady state bone marrow is challenging not only for transplant physicians but also for the donors. This chapter provides an overview about the history of stem cell transplantation, current procedures and future developments in terms of donor selection and graft choices for hematopoietic stem cell transplantation. Keywords Hematopoietic stem/Progenitor cells · Bone marrow · Peripheral blood stem cells (PBSC) · Umbilical cord blood (UCB) · Stem cell transplantation · Stem cell mobilization · G-CSF · AMD3100 · Graft-versus-host-disease (GVHD) · CD34

Contents 2.1 Historical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Stem Cell Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Stem Cell Mobilization and Autologous Transplantation . . . . . . . . . . . . .

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M. Punzel (B) Institute of Transplantation Diagnostic and Cellular Therapeutics, Universitätsklinikum Düsseldorf, 40225 Düsseldorf, Germany e-mail: [email protected] T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_2,

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2.4 Allogeneic Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.1 Historical Aspects The origin of blood formation within the bone marrow was discovered in 1868 independently by Ernst Neumann [1] and by Giulio Bizzozero [2]. The German hematologist Arthur Pappenheim postulated in 1898 a monophyletic basophil mononuclear precursor for all blood cells, followed by the “common stem cell” concept of Alexander Maximow which suggested a common stem cell among the small blood lymphocytes [3, 4]. Although the interest in this field had been present since these initial observations, research efforts took another step after the first atomic bomb explosions in the wake of world war II in attempts to prevent the lethal effects of irradiation. One of the most important discoveries at that time was the observation that marrow failure and subsequent lethality of photon beam irradiation in mice could be reduced by shielding the spleen and femur with lead [5]. In the following years experimental evidence from animal experiments in rodents could demonstrate that intravenous infusion of bone marrow protected them from lethal irradiation [6]. Although there was a long controversy about the origin of the protective effects of marrow infusions, in the mid-1950s it was well accepted that not humoral factors but transplantable hematopoietic stem cells are responsible for marrow protection [7, 8]. In 1957 the pioneer of clinical stem cell transplantation, E. Donnall Thomas, published results on infusing unrelated bone marrow into six patients. Although all patients died and only one of them had transient engraftment, this particular report is considered as the seminal paper of modern hematopoietic stem cell transplantation. Thomas and colleagues showed for the first time that human bone marrow could be collected in significant quantities and could be administered safely after cryopreservation [9]. Two years later, Thomas s team performed the first successful bone marrow transplantation in a 3-year-old girl with leukemia using marrow donated from her identical twin. The girl did well for six months until her leukemia relapsed [10]. At this time it became evident, that alloreactivity is one of the most crucial factors for this therapeutic concept in two ways: On one hand the alloreactivity is directed against the tumor cells and protects the patient from relapse but on the other hand it caused fatal graft-versus-host disease (GVHD) if no identical twin has served as bone marrow donor. Doubts were raised if the “allogeneic barrier” could ever be passed since it turned out that the graft-versus-host (GVH) reaction in man was much more violent compared to inbred rodents [8]. The fatalities of allogeneic marrow infusions in the clinic setting caused most investigators to abandon such studies in the 1960s.

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However, under the impetus of accumulating knowledge of the human histocompatibility system, researchers laid the foundation for modern bone marrow transplantation. The Seattle group around ED Thomas developed matching strategies for bone marrow transplantations in dog experiments and related their results to the human leukocyte antigen (HLA)-system [11, 12]. Important knowledge to the field was added by Till and McCulloch in a series of experiments, which are generally considered as the beginning of the modern area of hematopoietic stem cell biology. Starting in 1961 the group demonstrated clonogenic colony formation of all hematopoietic lineages in the spleen (colonyforming-unit-spleen; CFU-S) in lethally irradiated mice after transplantation with bone marrow cells from healthy donor animals [13–15]. Thus, for the first time evidence was provided for the dose dependent, clonal repopulation, differentiation and self-renewing capacity of hematopoietic stem cells. The area of modern clinical bone marrow transplantation began in November 1968 when Robert Good from the University of Minnesota, USA carried out the first marrow transplantation in a 5-month-old boy with hereditary immunodeficiency that had killed 11 male members of his extended family with marrow from his 8-yearold matched sister [16]. Only 4 months later the Seattle group performed the first successful adult bone marrow transplantation in a patient with advanced leukemia using bone marrow from an HLA-matched sibling [17]. In the early years bone marrow transplantation was still restricted to patients with end-stage or refractory disease status and most patients were in poor condition at the time of transplantation, which resulted in a high proportion of deaths related to this therapy. Due to the myeloablative conditioning regimen that consisted of chemotherapy and total body irradiation various efforts had been made in the 1970s to decrease this transplant related mortality. On the one hand, a continuous improvement in the supportive therapy of blood cell substitution, antifungal, antimicrobiotic and antiviral chemoprophylaxis as well as nutritional supportive care could be achieved. On the other hand, the introduction of effective immunosuppressive agents in the GVHD-prophylaxis regimen, i.e. methotrexate and cyclosporine A improved the outcome of transplantation continuously [18, 19]. Important observations on the road to common practice for stem cell transplantation were published in the mid 1970s by the Seattle group: (i) Patients that were in better clinical condition at the time point of transplantation had a better long-term survival than those in poor condition, (ii) 75% of patients with advanced hematological disease relapsed after HSC-Tx, and (iii) the general proof of significant disease-free long-term survival in the first large cohort of patients with leukemia/lymphoma and aplastic anemia after failure of conventional therapy was encouraging to the field [17, 20]. Consequently, the number of patients referred for bone marrow transplantation at earlier stage of disease and in good clinical condition improved the field of allogeneic stem cell transplantation to full recognition as a clinical routine procedure in hematologic malignancies. As all subsequent studies confirmed the success of this treatment, E. Donnall Thomas received the Nobel Price for his pioneering work in clinical bone marrow transplantation in 1990.

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2.2 Stem Cell Donors While the number of transplants involving related donors increased continuously and proved to be successful, only 25–35% of patients had a matched sibling donor available. Further advances in histocompatibility typing technologies made it possible to include unrelated donors. In the beginning, serological matching for HLA-A, HLA-B and HLA-DR-loci and a non-reactive mixed lymphocyte culture (MLC) was required for donor selection and proved to be feasible in early clinical studies [21, 22]. In 1974 the initiative of recruitment of unrelated volunteers willing to donate bone marrow for anybody was started by Shirley Nolan in the United Kingdom in the search for bone marrow donors for her son, Anthony [23]. The Anthony Nolan Trust was the first active donor registry in the world. Today in almost every developed country registries with HLA-typed volunteers have been established, which have raised the chance for patients to find a suitable unrelated donor. Per November 2008 more than 12.5 [24] million donors have been registered world wide, of those more than 25% are registered in Germany [25]. This corresponds to more than 10% of all Germans between the age of 18–60 who have volunteered for a possible bone marrow donation. In Germany there are currently 29 national and local donor registries [25]. Since one third of all transplants worldwide requires a graft from a foreign country, searching all the national and local registries in the world step by step separately is virtually impossible and only at considerable expense and time [26]. Thus, several platforms and networks have been established to provide an easy accessible listing of all donors nationwide as well as worldwide. Beginning in 1988 the Bone Marrow Donors Worldwide (BMDW) database has been summarizing the data of most registries in the world [27]. The World Marrow Donor Association (WMDA) has defined policies and procedures for international data exchanges [26]. Since more than 95% of all unrelated transplants are facilitated through the pool of complete HLA-typed donors, it was of great importance that the number of donors which have been typed for HLA-A, -B and -DR increased up to 9.6 million. This relates to approximately 75% of all available donors worldwide [27]. However, due to the diversity of HLA-allele and haplotype frequencies in human populations, the vast majority of patients that can be provided with a full matched donor belong to the Northern European (Caucasian) ethnicity only. Therefore, many efforts have been undertaken to establish ethnic minority programs within most of the registries, i.e. within the largest single registry worldwide, the National Marrow Donor Program (NMDP) in the USA. This has resulted in a significant increase of donor availability especially for the Afro-American population within the NMDP [28]. Currently, the optimal choice for an unrelated donor is a full allele-match for HLA-class I (HLA-A, -B, -C) as well as two matched gene loci of HLA-class II (HLA-DRB1, -DQB1). This requires expensive high resolution DNA-typing. Challenges in terms of transplantation outcome still remain in undetected variations of the human major histocompatibility complex (MHC) as well as in non-genetic factors such as the disease status of the patient.

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During the 1980s, umbilical cord blood, which is collected from the umbilical cord and placenta of healthy newborns, has emerged as an alternative clinical source for hematopoietic stem cell transplantation. Elaine Gluckman performed in 1988 in Paris the first successful clinical transplantation in a six-year old boy suffering from Fanconi-anemia using umbilical cord blood (UCB) from a sibling [29]. In 1992, the first public UCB-bank was established in the New York Blood Center followed by institutions in many countries. In 1993, the first unrelated UCB-transplantation was performed at Duke University in the USA. Today, there are more than 330,000 UCB-units stored and available through the BMDW database and it is estimated that more than 14,000 unrelated UCB-transplantations have been performed so far [27, 30]. There are major differences between stem cell transplantations using grafts from adult donors or alternatively from UCB. UCB-transplants require fewer nucleated cells/kg body weight (>2.5×107 /kg) than bone marrow grafts (>2×108 /kg) and only 3 HLA-loci (HLA-A, -B, -DRB1) are relevant for transplantation at allelic level. Due to the lower alloreactivity of cord blood derived immune cells grafts with a limited HLA-disparity (1–2 allele mismatches) are suitable for transplantation [31, 32]. Over the last years it became evident that the nucleated cell dose, which correlates directly with the number of hematopoietic stem and progenitor cells in the UCB-transplant, is of higher priority than a full HLA-match [31–33]. This is significant since in the early years of UCB-banking many UCB-grafts were stored with only limited cell numbers [34, 35]. For this reason UCB-transplantations had been performed almost exclusively in children until the end of the last century [34, 35]. To overcome these limitations and to provide sufficient cell doses for adult patients novel graft selection strategies are under investigation. One attempt is the simultaneous transplantation of two UCB-units if the cell number of one single cord is insufficient, called “double cord blood” transplantation. Both of the two UCB-units must be matched to each other as well as to the patient appropriately, at least with 5/6 relevant alleles [36]. Another strategy has been the use of purified haploidentical stem and progenitor cells in conjunction with one UCB-unit. The haploidentical stem cells provide rapid engraftment and serve temporarily as “bridging cell unit” until the UCB engrafts and finally rejects the haploidentical cells from the patient’s relative [37, 38]. Based on these encouraging results and the increasing availability of suitable UCB-units in the BMDW-database, UCB-transplantation will become a valid alternative in the field of adult stem cell transplantation also for adults [31, 35, 39, 40].

2.3 Stem Cell Mobilization and Autologous Transplantation Encouraged by the rapid clinical development in the field of allogeneic bone marrow transplantation along with the feasibility of harvesting, processing, cryopreserving and reapplication of bone marrow cells, the concept of high dose chemotherapy

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with subsequent autologous transplantation has been proven safe and feasible for lymphohematologic malignancies as well as certain immune disorders [40]. Unlike allogeneic transplantations high dose chemo- and radiation therapy with autologous stem cell support can be performed in elderly patients as well without the significant mortality of transplantation related complications. Between 2002 and 2006 sixtytwo percent of all autograft recipients were older than 50 [41]. Since 1962 it has been known that peripheral blood leukocytes fully reconstituted lethally irradiated mice of the same genetic strain [42]. In humans, hematopoietic stem cells in the peripheral blood were reported in the early 1970s [43, 44]. An increase in the amount of human hematopoietic stem cells in the peripheral blood was observed after chemotherapy for the first time in 1976 [45]. The amount of hematopoietic stem and progenitor cells in the peripheral blood was determined by the number of colonies that could be generated in semisolid methylcellulose cultures. These colonies have been defined as Colony-Forming Units (CFUs) at different stages of maturation. The numbers of CFUs for granulocytes and macrophages (CFU-GM), CFUs for erythroid colonies (CFU-E) as well as the number of CFUs for more primitive CFU-GEMM (mixed colonies for granulocytes, erythroid cells and monocytes/macrophages) directly relates to the amount of vital stem and progenitor cells with repopulating capacity in the peripheral blood [43, 44, 46–48]. Thus, such colony assays are still in place as quality control measurement of cryopreserved stem cells. Finally, the technical development of cell separators made it possible to collect clinically relevant amounts of stem cells from the peripheral blood [49]. The disadvantage of time delay inherent in the methylcellulose assays lead to the application of immunophenotyping for stem and progenitor cell determination. One of the most important discoveries in the field was the establishment of the CD34-membrane glycoprotein as a surrogate marker for the clinical enumeration of human stem and progenitor cells for transplantation [50, 51]. Initial mobilization regimens and proof of principle for the feasibility of autologous transplantations were pioneered in 1979 by Goldman and colleagues in 6 patients with myeloproliferative disorders [52]. The first successful clinical transplantation after myeloablative radiochemotherapy with large numbers of chemotherapy mobilized peripheral blood stem cells (PBSC) being transplanted was performed in 1985 in Heidelberg, Germany. The rapid hematopoietic reconstitution within 9 days suggested an advantage over bone marrow and paved the way for the preferred use of mobilized PBSC as stem cell source today [53]. To et al. established the modern chemotherapy based mobilization regimen in the autologous transplantation setting as single infusion of cyclophosphamide (4 g/m2 ) that is still the gold standard, despite minor modifications [54–56]. The discovery and clinical development of human hematopoietic growth factors such as Granulocyte-colony stimulating-factor (G-CSF) and GranulocyteMacrophage colony-stimulating factor (GM-CSF) allowed the collection of larger amounts of hematopoietic stem cells compared to chemotherapy alone [57]. Since the mobilizing effect of G-CSF was better than GM-CSF the latter did not make it to a widespread clinical use. The addition of G-CSF to chemotherapy based mobilization regimens led to the favorable use of mobilized PBSC as autologous grafts [58].

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Today, the use of mobilized peripheral blood accounts for 90% of all autotransplants in children and for more than 95% in adults [41]. As minimal required cell dose, 1– 2×106 CD34+ cells per kg body weight have been established without any clinical benefit using CD34+ cell doses >8×106 /kg [55, 59]. Clinical indications and frequencies of high dose therapies with autologous stem cell transplantation have changed over the past decade. The number of autologous transplantations that is performed annually had risen from approximately 5,000 in early 1990 to almost 40,000 in 1999 worldwide [41]. This was mainly due to the introduction of high dose chemotherapy in solid tumors, such as malignant melanoma, small cell lung cancer, colon cancer and in particular breast cancer. The initial enthusiasm about preliminary results turned into disappointment after the first randomized studies did not show any significant survival differences compared to conventional treatment. The latter data together with the disclosure of scientific misconduct in one of the breast cancer trials [60] has virtually abandoned autologous transplantations in the treatment of most non-hematologic malignancies. However, high dose therapy in other diseases, such as multiple myeloma or systemic amyloidosis has emerged as preferred treatment modality and thus, the number of autologous transplantations is on the increase again since 2002. Today, multiple myeloma is the most common indication for high dose therapy and autologous transplantation with a 3-year survival probability of 68% [41]. Similar results could be obtained for relapsed diffuse large cell B-cell lymphoma (DLBCL) with a 3-year survival probability of 61% in chemosensitive disease as well as for relapsed or aggressive follicular lymphoma (FL) with a 3-year survival probability of 73% in chemosensitive disease [41]. Several major studies have shown the advantages of mobilized peripheral blood over bone marrow as stem cell source for autologous transplantation [61–63]. Patients that have received autologous mobilized PBSC-transplantations showed a more rapid granulocyte and platelet recovery, enhanced immune reconstitution and subsequently a reduced transplant related morbidity [64–66].

2.4 Allogeneic Transplantation The emergence of mobilized PBSC as preferred autologous stem cell source has sparked the use of G-CSF in healthy donors to obtain allogeneic PBSC-grafts with similar advantages as has been shown for the autologous setting [67, 68]. Studies that compared G-CSF-mobilized PBSC with bone marrow as graft source in related allogeneic HLA-identical transplantations demonstrated similar results for hematopoietic recovery as observed in the autologous setting: more rapid engraftment, less infectious complications and a lower transplantation related mortality were advantages of the PBSC-group [69–71]. Except one study, the rate of acute GVHD was not different in both graft sources but chronic GVHD was more frequent in patients that received PBSC-transplants [70–72]. Subsequently, the use of mobilized PBSC as preferred graft source for allogeneic transplantation has

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increased markedly in the last decade. With the exception of pediatric transplantation procedures, mobilized PBSC has been the most common source of allogeneic grafts from 2002 to 2006 in patients older than 20 years, with the use of PBSC twice as much as bone marrow [41]. The number of allogeneic grafts collected in Germany went over 3,000 in 2006, more than in any other country of the world. The data show that already 80% of these grafts were collected from mobilized PBSC and the numbers are rising [25]. Despite the preferred use of mobilized peripheral stem cells in allogeneic transplantations controversies still exist about long-term outcome from both adult graft sources [73]. Since most studies have demonstrated an increased risk of chronic GVHD in mobilized PBSC-transplantation, it is not yet clear whether this will result in higher late mortality or in a decrease of the relapse rate due to a prolonged graft versus malignancy effect. A meta-analysis of several randomized trials that compared the outcome of PBSC versus marrow as graft source in full matched sibling transplantations showed significant improvement in disease-free survival at 5 years (54–47%) which was associated with increased chronic GVHD (51–35%) and decreased relapse rate (24–32%) in favor of PBSC-grafts [74]. However, a recent study that had the longest follow up for matched sibling transplants so far could not confirm the improved 5-year disease-free survival from the metaanalysis after 6 years, despite confirmation of the increased chronic GVHD incidence [75]. Since the patient cohorts in both analysis were different, the advantage of mobilized PBSC in matched sibling transplants remains unclear. The first comprehensive analysis that compared bone marrow transplantations with mobilized PBSC allografts in matched sibling transplantations in the pediatric setting demonstrated a significant increased mortality of PBSC-transplants clearly attributed to the higher incidence of GVHD in the PBSC-group [76]. First data on long-term follow up in unrelated donor transplantations demonstrated an expected higher incidence in extensive chronic GVHD in the PBSC group (85 vs. 59%, p<0.01) compared to bone marrow grafts [77–79]. The differences in GVHD, however, did not relate to any differences, neither in disease-free and overall survival, nor in relapse rates [79]. Depending on the underlying disease, it has been shown that in contrast to acute myeloid leukemia (AML), the use of unrelated mobilized PBSC as graft source in acute lymphatic leukemia (ALL) is associated with an increased transplant-related mortality (TRM) [80]. Additional data analysis, long-term follow up of the studies and the first prospective randomized clinical trial that compares unrelated bone marrow transplantation versus mobilized PBSC may finally solve these questions. The gold standard of stem cell mobilization is currently the subcutaneous application of 10 μg G-CSF per day per kg body weight for 4–5 days followed by the apheresis collection. Although there is great practical experience in the use of GCSF, the biological effects of mobilization have not been fully understood. G-CSF binds to its receptor, which is present on almost all cells of the myeloid lineage; from very few receptors on the most primitive progenitors in the bone marrow up to high density expression on neutrophil granulocytes in the peripheral blood [81, 82]. Upon

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G-CSF stimulation in the bone marrow, certain proteases, released from neutrophil granulocytes, such as the neutrophil elastase (NE) and cathepsin G (CG) as well as metalloproteinases, such as Matrix Metalloproteinase-9 (MMP-9) will be released to cleave adhesion molecules that are important for hematopoietic stem cell trafficking and mobilization. In particular, the disruption of Very Late Antigen-4 (VLA-4) with its receptor Vascular-adhesion molecule-1 (VCAM-1) as well as effects on stroma cell derived factor-1 (SDF-1) and its chemokine-receptor-4 (CXCR-4) may play an important role in the release of primitive stem and progenitor cells from the microenvironment and their trafficking into the blood stream. Other molecules that have been shown to be involved in mobilization and trafficking of human stem and progenitor cells are CD44 and L-selectin [83–85]. Due to the widespread use of G-CSF with more than 10,000 allogeneic donors that receive G-CSF for clinical mobilization every year, safety issues have to be taken in consideration for healthy individuals [85, 86]. Few reports have shown that G-CSF can affect the genomic stability in hematopoiesis of healthy individuals, however long-term consequences remain largely speculative [87, 88]. Acute leukemias have been observed in siblings, stimulated with G-CSF [89, 90] while major concerns of long-term consequences of G-CSF application, i.e. in children had been already present [91]. Since there is no evidence that G-CSF causes any long-term effects in normal donors, the reported cases of leukemia in matched siblings may have occurred by chance – due to the fact of a generally higher risk of leukemia in first degree relatives. Thus, long-term follow up of all healthy donors that have undergone G-CSF mobilization is necessary. About 4% of healthy individuals do not mobilize sufficient numbers of CD34+ stem and progenitor cells into the peripheral blood [92]. The reasons why this small cohort of “poor mobilizers” fail to release the CD34+ cells from the bone marrow into the blood remains unclear. Donor age >38 years, low baseline levels of CD34+ cells and single daily application instead of two applications per day were identified as predictors for poor mobilization [93–95]. In autologous patients that receive chemotherapy (cyclophosphamide) followed by G-CSF the mobilization failure rate is much higher and depends on previous chemotherapy, i.e. the cumulative dose of alkylating agents [92]. Recently, the better understanding of mechanisms in stem cell mobilization led to the discovery and subsequent clinical development of new mobilizing agents. The diversity and large number of hematopoietic growth factors, chemokines and cytotoxic agents that induces the release of hematopoietic stem and progenitor cells into the peripheral blood is somewhat surprising. Besides the clinically approved G-CSF and GM-CSF several cytokines, such as interleukin-3, interleukin-8, recombinant human growth hormone and stem cell factor, had been tested but did not make it to clinical use [84]. The introduction of a pegylated G-CSF molecule (Pegfilgrastim) with prolonged half-life into clinical use resulted in a more convenient single dose application but did not change the poor mobilization responses in some patients [96]. Clinical trials are currently underway to determine the efficacy of Pegfilgrastim as mobilizing agent in patients for autologous transplantations as well as in healthy donors.

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A specific CXCR-4 antagonist, called AMD3100, that reversibly inhibits the binding of SDF-1 to its receptor, is probably the most promising mobilizing agent of a new kind that has successfully passed clinical phase III studies and is expected to get clinical approval in Europe by 2009 [97–100]. Most importantly, in combination with G-CSF this drug allowed the mobilization of sufficient numbers of CD34+ cells into peripheral blood in poor mobilizers that previously failed G-CSF-mobilization [101, 102]. A single dose of AMD3100 causes a rapid and significant release of CD34+ cells from the bone marrow within 1 h. The number of peripheral progenitors peaks after 9 h and declines to baseline levels within 24 h, which allows stem cell harvest on the same day of application [84, 98, 103]. Although a single injection of AMD3100 results in a lower yield of CD34+ cells, it acts synergistically with G-CSF [101, 102]. Recently, two reports have demonstrated that a clinical grade antibody (natalizumab) approved to treat multiple sclerosis, was able to release clinically significant amounts of CD34+ stem and progenitor cells into peripheral blood by blocking VLA-4 [104, 105].

2.5 Outlook The increased availability of registered unrelated stem cell donors as well as suitable umbilical cord blood units has remarkably improved the outcome of allogeneic stem cell transplantations over the recent years and opens the perspective to choose from several available graft sources according to the specific conditions of each individual patient. This also includes the use of related haploidentical donors in various clinical settings. These donors are only partially HLA-matched relatives of the patients that are usually immediately available for transplantation workup. Based on initial results that have shown the feasibility of this approach despite the risk of graft failure and severe GVHD, modern concepts of haploidentical transplantations have incorporated reduced intensity conditioning (RIC) in the transplant procedure combined with high dose enrichment of CD34+ stem and progenitor cells [106–113]. Instead of purification of CD34+ cells by positive selection, the depletion of selected lymphocytes that leaves monocytes, Natural Killer cells (NKcells) and/or T-cell-subsets within the graft, has opened the perspective of targeted allogeneic immunotherapy by choosing stem cell donors that exhibit specific graft versus tumor/leukemia alloreactivity in the NK-cell repertoire but does not show significant graft versus host reactivity [112, 114–119]. The clinical introduction of AMD3100 and other possible mobilizing agents could change the field in many ways due to their different biological properties compared to G-CSF. Chemokine-receptor inhibitors release primitive hematopoietic cells into the blood stream that have differential cell cycle properties than G-CSF mobilized cells. The immunomodulatory effects of AMD3100 in the hematopoietic system differing from those observed after G-CSF treatment, the significant increase of circulating endothelial and angiogenic progenitor cells in the peripheral blood as

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well as additional (still unknown) properties open the exciting perspective of novel therapeutic approaches using mobilized peripheral stem cells. In addition, the release of stem cells from the niche by chemokine receptor inhibitors or antibodies against certain adhesion molecules, such as VLA-4, may lead to novel approaches to treat hematologic malignancies by releasing leukemic stem and progenitor cells from the niche into the peripheral blood that results in cell cycle entry and subsequently enhanced susceptibility to chemotherapy.

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46. Udomsakdi C, Lansdorp PM et al. (1992) Characterization of primitive hematopoietic cells in normal human peripheral blood. Blood 80: 2513–2521. 47. Spitzer G, Verma DS et al. (1980) The myeloid progenitor cell – its value in predicting hematopoietic recovery after autologous bone marrow transplantation. Blood 55: 317–323. 48. Fauser AA and Messner HA (1979) Identification of megakaryocytes, macrophages, and eosinophils in colonies of human bone marrow containing neurtophilic granulocytes and erythroblasts. Blood 53: 1023–1027. 49. Weiner RS, Richman CM et al. (1977) Semicontinuous flow centrifugation for the pheresis of immunocompetent cells and stem cells. Blood 49: 391–397. 50. Civin CI, Strauss LC et al. (1984) Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 133: 157–165. 51. Berenson RJ, Andrews RG et al. (1988) Antigen CD34+ marrow cells engraft lethally irradiated baboons. J Clin Invest 81: 951–955. 52. Goldman JM, Catovsky D et al. (1979) Cryopreserved peripheral blood cells functioning as autografts in patients with chronic granulocytic leukaemia in transformation. Br Med J 1: 1310–1313. 53. Korbling M, Dorken B et al. (1986) Autologous transplantation of blood-derived hemopoietic stem cells after myeloablative therapy in a patient with Burkitt’s lymphoma. Blood 67: 529–532. 54. To LB, Davy ML et al. (1989) Autotransplantation using peripheral blood stem cells mobilized by cyclophosphamide. Bone Marrow Transplant 4: 595–596. 55. To LB, Roberts MM et al. (1992) Comparison of haematological recovery times and supportive care requirements of autologous recovery phase peripheral blood stem cell transplants, autologous bone marrow transplants and allogeneic bone marrow transplants. Bone Marrow Transplant 9: 277–284. 56. To LB, Shepperd KM et al. (1990) Single high doses of cyclophosphamide enable the collection of high numbers of hemopoietic stem cells from the peripheral blood. Exp Hematol 18: 442–447. 57. Welte K, Platzer E et al. (1985) Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor. Proc Natl Acad Sci USA 82: 1526–1530. 58. Stadtmauer EA, Schneider CJ et al. (1995) Peripheral blood progenitor cell generation and harvesting. Semin Oncol 22: 291–300. 59. Buscemi F, Indovina A et al. (1995) CD34+ cell subsets and platelet recovery after PBSC autograft. Bone Marrow Transplant 16: 855–856. 60. Bezwoda W (1999) Randomized, controlled trial of high dose chemotherapy (HD-VNVp) versus standard dose (CAF) chemotherapy for high risk, surgically treated, primary breast cancer, JCO, American Society of Clinical Oncology meeting (ASCO) Prcoeedings 1999 and related ASCO-statement letter February 4th, 2000. ASCO. 61. Beyer J, Schwella N et al. (1995) Hematopoietic rescue after high-dose chemotherapy using autologous peripheral-blood progenitor cells or bone marrow: a randomized comparison. J Clin Oncol 13: 1328–1335. 62. Hartmann O, Le Corroller AG et al. (1997) Peripheral blood stem cell and bone marrow transplantation for solid tumors and lymphomas: hematologic recovery and costs. A randomized, controlled trial. Ann Intern Med 126: 600–607. 63. Schmitz N, Linch DC et al. (1996) Randomised trial of filgrastim-mobilised peripheral blood progenitor cell transplantation versus autologous bone-marrow transplantation in lymphoma patients. Lancet 347: 353–357. 64. Bensinger W, Singer J et al. (1993) Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor. Blood 81: 3158–3163.

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65. Blume KG and Thomas ED (2000) A review of autologous hematopoietic cell transplantation. Biol Blood Marrow Transplant 6: 1–12. 66. Welte K, Gabrilove J et al. (1996) Filgrastim (r-metHuG-CSF): the first 10 years. Blood 88: 1907–1929. 67. Schmitz N, Dreger P et al. (1995) Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor). Blood 85: 1666–1672. 68. Korbling M, Przepiorka D et al. (1995) Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 85: 1659–1665. 69. Champlin RE, Schmitz N et al. (2000) Blood stem cells compared with bone marrow as a source of hematopoietic cells for allogeneic transplantation. IBMTR Histocompatibility and Stem Cell Sources Working Committee and the European Group for Blood and Marrow Transplantation (EBMT). Blood 95: 3702–3709. 70. Cutler C, Giri S et al. (2001) Acute and chronic graft-versus-host disease after allogeneic peripheral-blood stem-cell and bone marrow transplantation: a meta-analysis. J Clin Oncol 19: 3685–3691. 71. Couban S, Simpson DR et al. (2002) A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 100: 1525–1531. 72. Mohty M, Kuentz M et al. (2002) Chronic graft-versus-host disease after allogeneic blood stem cell transplantation: long-term results of a randomized study. Blood 100: 3128–3134. 73. Koca E and Champlin RE (2008) Peripheral blood progenitor cell or bone marrow transplantation: controversy remains. Curr Opin Oncol 20: 220–226. 74. Stem-Cell-Trialists’-Collaborative-Group (2005) Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: an individual patient data meta-analysis of nine randomized trials. J Clin Oncol 23: 5074–5087. 75. Schmitz N, Eapen M et al. (2006) Long-term outcome of patients given transplants of mobilized blood or bone marrow: A report from the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation. Blood 108: 4288–4290. 76. Eapen M, Horowitz MM et al. (2004) Higher mortality after allogeneic peripheralblood transplantation compared with bone marrow in children and adolescents: the Histocompatibility and Alternate Stem Cell Source Working Committee of the International Bone Marrow Transplant Registry. J Clin Oncol 22: 4872–4880. 77. Ringden O, Remberger M et al. (1999) Peripheral blood stem cell transplantation from unrelated donors: a comparison with marrow transplantation. Blood 94: 455–464. 78. Remberger M, Ringden O et al. (2001) No difference in graft-versus-host disease, relapse, and survival comparing peripheral stem cells to bone marrow using unrelated donors. Blood 98: 1739–1745. 79. Remberger M, Beelen DW et al. (2005) Increased risk of extensive chronic graft-versus-host disease after allogeneic peripheral blood stem cell transplantation using unrelated donors. Blood 105: 548–551. 80. Garderet L, Labopin M et al. (2003) Patients with acute lymphoblastic leukaemia allografted with a matched unrelated donor may have a lower survival with a peripheral blood stem cell graft compared to bone marrow. Bone Marrow Transplant 31: 23–29. 81. Demetri GD and Griffin JD (1991) Granulocyte colony-stimulating factor and its receptor. Blood 78: 2791–2808. 82. Hernandez-Bernal F, Garcia-Garcia I et al. (2005) Bioequivalence of two recombinant granulocyte colony-stimulating factor formulations in healthy male volunteers. Biopharm Drug Dispos 26: 151–159. 83. Cashen AF, Lazarus HM et al. (2007) Mobilizing stem cells from normal donors: is it possible to improve upon G-CSF? Bone Marrow Transplant 39: 577–588.

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84. Nervi B, Link DC et al. (2006) Cytokines and hematopoietic stem cell mobilization. J Cell Biochem 99: 690–705. 85. Pelus LM (2008) Peripheral blood stem cell mobilization: new regimens, new cells, where do we stand. Curr Opin Hematol 15: 285–292. 86. Horowitz MM and Confer DL (2005) Evaluation of hematopoietic stem cell donors. Hematology Am Soc Hematol Educ Program 469–475. 87. Hernandez JM, Castilla C et al. (2005) Mobilisation with G-CSF in healthy donors promotes a high but temporal deregulation of genes. Leukemia 19: 1088–1091. 88. Nagler A, Korenstein-Ilan A et al. (2004) Granulocyte colony-stimulating factor generates epigenetic and genetic alterations in lymphocytes of normal volunteer donors of stem cells. Exp Hematol 32: 122–130. 89. Makita K, Ohta K et al. (2004) Acute myelogenous leukemia in a donor after granulocyte colony-stimulating factor-primed peripheral blood stem cell harvest. Bone Marrow Transplant 33: 661–665. 90. Bennett CL, Evens AM et al. (2006) Haematological malignancies developing in previously healthy individuals who received haematopoietic growth factors: report from the Research on Adverse Drug Events and Reports (RADAR) project. Br J Haematol 135: 642–650. 91. Shpilberg O, Modan M et al. (1994) Familial aggregation of haematological neoplasms: a controlled study. Br J Haematol 87: 75–80. 92. Moncada V, Bolan C et al. (2003) Analysis of PBPC cell yields during large-volume leukapheresis of subjects with a poor mobilization response to filgrastim. Transfusion 43: 495–501. 93. de la Rubia J, Arbona C et al. (2002) Analysis of factors associated with low peripheral blood progenitor cell collection in normal donors. Transfusion 42: 4–9. 94. Suzuya H, Watanabe T et al. (2005) Factors associated with granulocyte colony-stimulating factor-induced peripheral blood stem cell yield in healthy donors. Vox Sang 89: 229–235. 95. Lysak D, Koza V et al. (2005) Factors affecting PBSC mobilization and collection in healthy donors. Transfus Apher Sci 33: 275–283. 96. Molineux G, Kinstler O et al. (1999) A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans. Exp Hematol 27: 1724–1734. 97. Flomenberg N, Devine SM et al. (2005) The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood 106: 1867–1874. 98. Devine SM, Flomenberg N et al. (2004) Rapid mobilization of CD34+ cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J Clin Oncol 22: 1095–1102. 99. Devine SM, Vij R et al. (2008) Rapid mobilization of functional donor hematopoietic cells without G-CSF using AMD3100, an antagonist of the CXCR4/SDF-1 interaction. Blood 112: 990–998. 100. Cashen A, Lopez S et al. (2008) A phase II study of plerixafor (AMD3100) plus G-CSF for autologous hematopoietic progenitor cell mobilization in patients with Hodgkin lymphoma. Biol Blood Marrow Transplant 14: 1253–1261. 101. DiPersio J, Micallef I et al. (2007) A Phase III, multicenter, randomized, double-blind, placebo controlled, comparative trial of AMD3100 (Plerixafor)+G-CSF vs. Placebo+G-CSF in Non-Hodgkin’s Lymphoma (NHL) patients for Autologous Hematopoietic Stem Cell (aHSC) transplantation. Blood 110: 601a. 102. Micallef I, Stiff P et al. (2007) Successful stem cell mobilization rescue by AMD3100 (Plerixafor) + G-CSF for patients who failed primary mobilization: rescue from phase III (3101-NHL) study. Blood 110: 602a. 103. Hendrix CW, Flexner C et al. (2000) Pharmacokinetics and safety of AMD-3100, a novel antagonist of the CXCR-4 chemokine receptor, in human volunteers. Antimicrob Agents Chemother 44: 1667–1673.

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104. Bonig H, Wundes A et al. (2008) Increased numbers of circulating hematopoietic stem/progenitor cells are chronically maintained in patients treated with the CD49d blocking antibody natalizumab. Blood 111: 3439–3441. 105. Zohren F, Toutzaris D et al. (2008) The monoclonal anti-VLA-4 antibody natalizumab mobilizes CD34+ hematopoietic progenitor cells in humans. Blood 111: 3893–3895. 106. Aversa F, Martelli MM et al. (1997) Use of stem cells from mismatched related donors. Curr Opin Hematol 4: 419–422. 107. Aversa F (2001) New forms of transplantation: haploidentical transplants. Bone Marrow Transplant 28 (Suppl 1): S16–17. 108. Liso A, Tiacci E et al. (2004) Haploidentical peripheral-blood stem-cell transplantation for ALK-positive anaplastic large-cell lymphoma. Lancet Oncol 5: 127–128. 109. Aversa F, Reisner Y et al. (2008) The haploidentical option for high-risk haematological malignancies. Blood Cells Mol Dis 40: 8–12. 110. Passweg JR, Kuhne T et al. (2000) Increased stem cell dose, as obtained using currently available technology, may not be sufficient for engraftment of haploidentical stem cell transplants. Bone Marrow Transplant 26: 1033–1036. 111. Martelli MF and Reisner Y (2002) Haploidentical ‘megadose’ CD34+ cell transplants for patients with acute leukemia. Leukemia 16: 404–405. 112. Reisner Y and Martelli MF (2008) From ‘megadose’ haploidentical hematopoietic stem cell transplants in acute leukemia to tolerance induction in organ transplantation. Blood Cells Mol Dis 40: 1–7. 113. Handgretinger R, Klingebiel T et al. (2003) Megadose transplantation of highly purified haploidentical stem cells: current results and future prospects. Pediatr Transplant 7 (Suppl 3): 51–55. 114. Velardi A (2008) Role of KIRs and KIR ligands in hematopoietic transplantation. Curr Opin Immunol 20: 581–587. 115. Ruggeri L, Mancusi A et al. (2008) NK cell alloreactivity and allogeneic hematopoietic stem cell transplantation. Blood Cells Mol Dis 40: 84–90. 116. Ruggeri L, Mancusi A et al. (2006) Natural killer cell alloreactivity and haplo-identical hematopoietic transplantation. Cytotherapy 8: 554–558. 117. Kolb HJ, Guenther W et al. (2003) Tolerance and chimerism. Transplantation 75: 26S–31S. 118. Rocha V and Locatelli F (2008) Searching for alternative hematopoietic stem cell donors for pediatric patients. Bone Marrow Transplant 41: 207–214. 119. Koh LP and Chao N (2008) Haploidentical hematopoietic cell transplantation. Bone Marrow Transplant 42 (Suppl 1): S60–S63.

Chapter 3

Ex Vivo Expansion of HSPCs Yaming Wei and Xin Ye

Abstract Transplantation of hematopoietic stem cells (HSCs, the cells that can give rise to all blood and most immune cell types) is a life-saving procedure for patients with hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. However, the wide application of this procedure is always limited either by availability of suitably HLA-matched adult donors or obtaining enough stem cell for a successful transplant. Over the past years, the results of ex vivo stem/progenitor cell expansion have been promising, numerous studies have described the effects of combinations of a variety hematopoietic growth factors on hematopoietic stem and progenitor cells (HSPCs) expansion in vitro. Most experimental evidence indicated that a combination of several cytokines such as stem cell factor (SCF), FLT-3/FLK-2 Ligand (Flt3-ligand), thrombopoietin (TPO) seems to be essential for progenitor amplification. Among these growth factors, SCF is unanimously agreed to be indispensable for stem and progenitor expansion and even shows to be a key factor for hematopoietic progenitor cell survival. With this cytokine cocktail, CD34+ cells can be expanded ex vivo about 10–1000-fold over pre-expanded values [1–3]. These kinds of expansion protocol provided sufficient numbers of hematopoietic progenitor cells to rapidly restore blood formation in patients undergoing high-dose chemotherapy or/and irradiation treatment. The development of ex vivo culture systems that facilitate the expansion of HSCs is crucial to stem cell research and clinical application. In this chapter, we describe the protocols to expand HSPCs ex vivo and analyze their population ability. This information is beneficial for successful use of stem cells in therapeutic studies. Keywords hematopoietic stem cell · progenitor cell · umbilical cord blood · ex vivo expansion · transplantation · cytokines · CD34

Y. Wei (B) Guangzhou Institute of Clinical Medicine, Guanzhou Municipal First People’s Hospital, Guangzhou Medical College, Guangzhou, China e-mail: [email protected] T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_3,

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Contents 3.1 The Sources of HSPCs . . . . . . . . . . . . . . . 3.1.1 Bone Marrow HSPCs . . . . . . . . . . . 3.1.2 Peripheral HSPCs . . . . . . . . . . . . . 3.1.3 Umbilical Cord Blood HSPCs . . . . . . . 3.2 The Expansion of HSPCs . . . . . . . . . . . . . . 3.2.1 Cytokines . . . . . . . . . . . . . . . . 3.2.2 Ex Vivo Expansion of HSPCs . . . . . . . 3.2.3 Regulation of HSPCs Expansion . . . . . . 3.2.4 Free Radical Regulation on HSPCs Expansion 3.2.5 Megakaryocytic Progenitor Cells Expansion . 3.2.6 Red Cells Expansion . . . . . . . . . . . 3.2.7 T-Cell Expansion . . . . . . . . . . . . . 3.2.8 NK Cell Expansion . . . . . . . . . . . . 3.2.9 DC Expansion . . . . . . . . . . . . . . 3.2.10 HSPC Ex Vivo Expansion and Gene Therapy 3.3 Expansion Bioreactor . . . . . . . . . . . . . . . 3.4 The Application of Expanded HSPCs . . . . . . . . 3.4.1 Transplantation of HSPCs in Animal Model . 3.4.2 Transplantation of HSPCs in Human . . . . 3.5 The Future of HSPCs Expansion . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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28 28 29 29 31 31 31 34 35 36 36 37 38 39 41 41 44 44 47 48 48

3.1 The Sources of HSPCs Hematopoiesis in mammalian systems is initiated in the yolk sac (YS) and then migrates into embryo as the initial source of stem cells [4]. The bone marrow develops hematopoiesis at 11 week in human embryo [5]. After that time, bone marrow keeps the hematopoiesis capability in ones all life until the life ends or it is inhibited by disease or extra factors. All mature blood cells come from bone marrow stem/progenitor cells, include peripheral blood, and umbilical cord blood.

3.1.1 Bone Marrow HSPCs Hematopoietic stem/progenitor cells (HSPCs) reside in specific niches in the bone marrow and give rise to either more stem cells or maturing hematopoietic progeny depending on the signals provided in the bone marrow microenvironment. This microenvironment is comprised of cellular components as well as soluble constituents called cytokines. Therapeutic agents interrupt a stem and progenitor cell tethering to matrix molecules and stromal cells in the bone marrow environment. Stem cells and progenitor cells are released from their attachment to stromal cells

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when a cytokine-receptor axis such as SCF or CXCR4-stromal derived factor-1 is interrupted. The number of circulating peripheral blood HSCs can be increased in response to treatment with drugs and chemokines. Once released from its attachment, a stem cell undergoes transmural membrane migration through the opening in the basement membrane and endothelial lining of a blood sinus and enters the circulation. Bone marrow HSPCs were once used as the main source of clinical transplantation for a long time, but now, with the development of mobilization and collection of peripheral stem cells and cord blood, its application has become less and less.

3.1.2 Peripheral HSPCs The HSPCs in BM and blood are the ancestors of all mature blood cells. The initial report found that bovine fraternal twins sharing a common placenta and blood supply were each endowed with chimeric BM and lymphhematopoietic cells from its sibling after birth [6]. Experiments in 1940s, 1950s and 1960s demonstrated the existence of HSC in the circulation [7]. In the middle of 1980s, peripheral blood was found to be a stem cell resource to rescue patients following high-dose chemotherapy or chemoradiotherapy [8–11]. In the 1990s, with the realization that the yield of circulation primitive hematopoietic cells could be greatly increased during recovery from chemotherapy and/ or hematopoietic cytokines treatment, mobilized peripheral blood progenitor cells replaced BM as the source of stem cells for autologous and allogeneic transplantation [12, 13].

3.1.3 Umbilical Cord Blood HSPCs Umbilical cord blood (UCB) is a valuable source of the rare but precious primitive HSCs and progenitor cells, UCB stem cell transplantation (CBSCT) has approached significant success in treatment of lethal congenital or malignant disorders. UCB from sibling with more than one human leukocyte antigen (HLA) loci mismatches or unrelated partially mismatched donors has been increasingly used to reconstitute the hematopoietic system in patients after myeloablative therapy. UCB cells possess an enhanced capacity for progenitor cell proliferation and self-renewal in vitro. Moreover, CBSCT shows a relatively low incidence and severity of graft-versushost disease (GVHD) [14]. UCB has advantages of easy collection and storage, no risk to donors, low risk of transmitting infections, immediate availability and immune tolerance allowing successful transplantation despite HLA disparity. UCB is usually discarded, and it exists in almost limitless supply. Cord blood stem cells can be collected only once, an average 50–100 ml of blood containing stem cells is obtained from the umbilical cord and the placenta after birth. The predominant collection procedure currently practiced involves a relatively simple venipuncture, followed by gravity drainage into a standard sterile anti-coagulant-filled blood bag, using a closed system, similar to the one utilized on whole blood collection. After

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aliquots have been removed for routine testing, the units are cryopreserved and stored in liquid nitrogen. UCB banks have being established throughout the world. UCB units are collected for allogeneic unrelated and related HSC transplantation. In unrelated cord blood banks, donated UCB units are collected and stored for allogeneic use in patients who do not have an identified HLA matched relative. UCB banks report available units to national and international donor registries. The second model of UCB banking is referred to as family banking, where UCB is stored for the benefit of the donor or their family members. According to the National Marrow Donor Program (NMDP), more than 6,000 men, women and children are searching the NMDP registry on any given day. After more than one decade of clinical experience, it is currently accepted that UCB transplants, related and unrelated, are equivalent to or might compare favorably with bone marrow (BM) transplants, especially in children. Initial studies of long-term survival in children with both malignant and non-malignant hematologic disorders, who were transplanted with UCB from a sibling donor, demonstrated a comparable or superior survival rate to the children who received BM transplantation [15]. Since the first cord blood transplantation was performed in 1988 [16], the UCB transplantation program was established nearly all over the world. Up to 2005, two large groups from European and North American retrospective studies demonstrated that UCB is an acceptable alternative source of HSCs for adult recipients who lack HLA-matched adult donors, over 10,000 UCB transplant procedures in children and adults have been performed worldwide using UCB donors [17]. The greater the number of umbilical stem cells used, the better the prospects for healing will be. UCB cells are showing their unique qualities and potential, and consequently UCB banks might dramatically increase the scope of their clinical application [15]. UCB is anticipated to address needs in both transplantation and regenerative medicine fields. One factor that limits the use of UCB transplantation in adult patients is the relatively limited number of HSC that may be harvested from umbilical cord, resulting in a more time to engraftment and higher transplant related mortality, mainly due to the long aplasia period after transplantation and susceptibility to viral and fungal infections. To allow for multiple uses and also to increase the capacity for transplantation in adolescents and adults, researchers are developing methods to stimulate stem cells to divide and increase in number while retaining their primitive state. This has prompted intensive research on ex vivo expansion of UCB stem cells and UCB graft-engineering including accessory cells able to improve UCB engraftment and reconstitution and for tissue regenerative potential. Expanding the volume of stem cells would allow more patients to be treated, including adults. It would also allow families who have privately banked their cord blood stem cells to use them for multiple treatments and even potentially donate a portion of their cord blood sample to patients in need. The current strategies are focused on the development of much more efficient technologies for ex vivo expansion of HSPCs, such expanded stem cells have been proposed as elements suitable for cellular therapy and regenerative medicine.

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3.2 The Expansion of HSPCs Due to the low yield of HSC from typical sources, HSC transfusion has historically been most effective in children with limited applications and marginal success in adults. In recent years, the development of media used to expand and mature adult stem cells has greatly increased the number of candidates eligible as well as the success rate of adult stem cell therapy. In order to obtain sufficient numbers of cells for applications of this therapeutic approach in adults, ex vivo expansion has been utilized to ensure successful engraftment and minimize the short-term effects of neutropenia and thrombocytopenia. The following shows main cytokines and their functions which include media optimized for the expansion and maturation of various adult stem cell types.

3.2.1 Cytokines When the hematopoiesis system feels signal changes come from inflammation and cytopenia, increased levels of hematopoietic growth factors (HPGFs) induce in vitro mobilization and proliferation of HSC and hematopoietic progenitor cells, resulting in spatial and quantitative in vivo expansion of the hematopoietic tissue. HPGF are also known as colony stimulating factor (CSF), it was originally given to agents recognized to stimulate the growth of colonies containing differentiated myeloid cells from a single bone marrow-derived precursor cell plated in semisolid agar. CSF glycoproteins considered to include: granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF); macrophage colony-stimulating factor (M-CSF); interleukin-3 (IL-3); IL-5; erythropoietin (EPO), and TPO [18]. In later of 1990s, cytokines were introduced to ex vivo expand human umbilical cord blood HSPCs cells and to elucidate its capacities of self-renewal potential and reconstitution in mice. Exogenous administration of recombinant HPGFs, followed by collection and transplantation of autologous or allogeneic stem cells is routine for mobilization of stem cells. In animal experiments, recombinant SCF was injected into mice for 7 day inducing a 10-fold increase in HSPCs in the absolute number of HSC in total blood volume from a baseline value of 10–100, and a decrease in the number of HSPCs in bone marrow from 2,400 to 900, the overall increase in HSPC was three fold [19]. Most of these growth factors and cytokines have already been used both in research and clinical treatment. An overview of cytokines and its stimulated cells is given in Table 3.1.

3.2.2 Ex Vivo Expansion of HSPCs The expansion of UCB stem cells at differing stages of maturity has been successfully repeated in recent years. Depending on the composition of the experiment, an expansion fold of 10 to more than 1,000 has been achieved. What is most important

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Y. Wei and X. Ye Table 3.1 Cytokines and their target cells

Recombinant cytokines

Stimulated cells

Erythropoietin (EPO)

Erythroid progenitor cells. EPO receptors are lost during cell differentiation. Primitive hematopoietic (multi-lineage) progenitor cells that express the FLT-3 receptor. Myeloid and pro-B cells. Proliferation, differentiation, survival and activation factor for hematopoietic restricted granulocyte lineage cells. Neutrophils, myeloid leukemia cells, neutrophilic granulocytes. Growth, differentiation, and essential survival factor for granulocyte, macrophage and eosinophil lineage cells from progenitor stage to maturity. Most types of myeloid progenitor cells, mature monocytes, neutrophils, eosinophils, basiophils, dendritic cells and epithelial cells and osteoclasts. Multi-potential hematopoietic progenitor cells: macrophage, neutrophils, mast cells and megakaryocytes from bone marrow, and Stimulate T-cells and induce IgG secretion from activated B-cells. Wide range of cell types, such as fibroblasts, myeloid progenitor cells, T-cells, B-cells, and hepatocytes. Primarily targets macrophages and stimulates multiple responses, such as proliferation, cytokine and inflammatory modulator release, cytotoxicity and pinocytosis; osteoclast differentiation and placental trophoblasts. Broad activities on hematopoietic, pigment and primordial germ cell lineages, increase myeloid, erythroid, and lymphoid lineage colonies. Primary regulatory factor of growth and maturation of megakaryocytes and their progenitors for megakaryocytopoiesis and thrombopoiesis.

FLT-3/FLK-2 Ligand

Granulocyte Colony-Stimulating Factor (G-CSF)

Interleukin-3 (IL-3)

Interleukin-6 (IL-6)

Macrophage Colony-Stimulating Factor (M-CSF) Stem Cell Factor (SCF)

Thrombopoietin (TPO)

is not the expansion of all (both differentiated and undifferentiated) cells in the cord blood but the expansion of undifferentiated stem cells. Bone marrow stem cells were successfully expanded and then subsequently transplanted, the expanded stem cells regenerated the immune system in all case following chemotherapy. Mobilized peripheral blood (PB) is another important resource of HSPCs. Kawano et al. [20] assessed the efficacy of PB CD133+ cells in a coculture system (contained SCF, THP TPO and Flk-2/Flt3-ligand) using human telomerized stromal (HTS) cells. PB CD133+ cells proliferated efficiently above the stromal layer, while maintaining the characteristics of CD133+ cells, even after long-term hematopoietic-stromal interaction. In clinical trials, PB stem progenitor mobilization is always carried out and collected from donors, which is much more convenient, efficient, and economical than ex vivo expansion.

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UCB is a promising source of HSCs for allogeneic transplantation. Many published research indicates that CB stem cells can be successfully expanded under certain combination of cytokines and preserve the activity of transplanted cells [21, 22]. Others have evaluated the possibility of increasing the number of competitive repopulating units in a NOD-SCID murine recipient [23, 24]. Guenechea et al. [25] found that these cells retained their capacity to support long-term repopulation with delayed engraftment as compared to fresh cells, whereas Piacibello et al. [26] reported that CB CD34+ cells expanded for up to 10 weeks maintained their in vitro repopulating potential. Cord blood has also been proved to prompt the recovery of immune function in children who underwent CBT and this reconstitution was favored by the reduced incidence and severity of GVHD observed [27, 28]. Mohamed et al. [29] defined optimal conditions composed with SCF, GMCSF, IL-3, TPO for ex vivo expansion of CB stem cells, the culture expanded for 7 days was better than 11 days, if more cytokines added (IL-6 and Flt3L), the fold expansion of CD34+ cells were not significantly increased or even decreased, even apoptotic cells (CD95+ cells) were observed. Yao et al. [30] optimized another serum-free and cytokines-limited medium using statistic methodology for UBC-derived HSC expansion. After a 7-day culture, the average absolute fold expansions were CD133+ cells 21-fold, CD34+ CD133+ cells 20-fold, CD34+ CD38+ cells 723-fold, CD133+ CD38- cells 618-fold, CD34+ CXCR4+ cells 160-fold, CD133+ CXCR4+ cells 384-fold and long-term culture-initiating cells 8fold, respectively. In terms of telomere length and telomerase activity compared to adult HSCs, the expansion of human CB HSCs is instrumental in obtaining a large number of “good quality” cells, these expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo [30, 31]. Madkaikar et al. [32] combined different cytokines and other support factors, assayed the mean CD34+ cell count, fold expansion, viability, clonogenic assays and immunophenotypic characterization at 7, 12 and 14 culture day, the maximum expansion was achieved using cytokines cocktail (SCF + IL-3 + GM-CSF) with stromal cell support, the mean CD34+ cell expansion on day 7 and 12 was 16.25- and 21.4-fold respectively, and the mean nucleated cell expansion was 15.1- and 21-fold, CFU-GEMM showed a 20.4-fold increase after 12 days. These cells can provide enough cells from a single cord blood unit to reduce the period of cytopenia after single unit cord blood transplantation. Wei et al. [33, 34] defined 7 groups of incubation conditions for ex vivo expansion and amplification CD34+ cell from UCB MNCs, all groups contained basic combination of SCF, IL-3, IL-6 for stimulating CD34+ cells expansion. Each test group showed significant increasing results of CD34+ cell either in percentage or expanded fold manner on 3, 7, 14 culture day compared to the decreasing in control group, which contains medium alone. The fold expansion rates of CD34+ cell numbers ranged from 10 to 50-fold as compared to the fresh UCB. The addition of IL-7, IL-2 or IL-4 into basic cytokine cocktails probably improved the expression of CD34 antigens on cells and increased CD34+ cell ratio respectively. On 7 expansion day, the least expansion of CD34+ cells number in basic

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combination with SCF, IL-3, IL-6 group was about 10 fold of that in fresh UCB, and enough for an adult transplantation. The expanded cells were able to engraft in the SCID recipients and reconstitute their hematopoiesis. Human hematopoietic cells could be detected in the marrow of the recipients 6 weeks after transplantation [35]. It seems possible to expand hematopoietic cells ex vivo efficiently and maintain concomitantly their self-renewal and hematopoietic reconstitution capacities by the combination of cytokines [36].

3.2.3 Regulation of HSPCs Expansion Although HSCs cycle and expand provide compelling evidence for a positive and dynamic regulation of HSC self-renewal [37, 38], the physiological regulators of HSC self-renewal and expansion remain largely unknown. However, besides cytokine genes commonly relevant to stem/progenitor cell expansion, there are many signal molecules involving in stem cell renew and regeneration. A few intrinsic cues – including the transcriptional repressor BMI-1 [39, 40], the protooncogene MYC [41], and the transcription factor C/EBP [42], overexpression of HoxB [43] promotes extensive HSC expansion ex vivo. TPO may act primarily to induce HSC apoptosis [44], LNK acts as a broad inhibitor of growth factors and cytokines TPO, KITL, EPO, IL-3, and IL-7 signaling pathways [45–49]. Uncovering the molecular mechanism underlying expansion of HSPCs is critical to extend current therapeutic applications. HOXB4 is known to be involved in stem cell maintenance and had shown some promise for stem cell expansion in mice. Zhang [50] showed that HOXB4 over-expression in populations of cells enriched for stem cells for 6–9 days prior to transplantation greatly improved their subsequent engraftment in radiated monkeys. Beslu et al. [51] proved HOXB4 gene can instruct stem cells into divide cell cycle and make more stem cells, these expanded cells cold reconstitute the monkeys’ immune and blood systems. AMD3100 is a small molecule initially developed as a highly potent and selective inhibitor of human immunodeficiency virus (HIV)-1 and HIV-2 replication. AMD3100 showed a binding-specificity to CXCR4, it can induce 1.5–3.1 fold WBC count, and 5-fold CD34+ increase in circulation as well as 18-fold CFU-GM [52–54]. SB-251353 is a truncated form of human chemokine GROB that binds specially to the CXCR2 receptor, SB-251353 combined with G-CSF could increase HSPCs in the circulation compared to G-CSF alone [55]. Some novel stem cell expansion factors were identified as part of pathways associated with mesodermal induction, or as factors produced by supportive stroma. These new factors showed their potential in CB HSC ex vivo expansion [56]. Okamoto [57] expanded ex vivo CD34+ CD133+ progenitor cells from human umbilical cord blood and analyzed gene expression changes using microarrays covering up to 55,000 transcripts. Several new genes and signaling pathways not previously associated with ex vivo expansion of CD133+ CD34+ cells were identified, most of which associated with cancer. Regulation of MEK/ERK and Hedgehog signaling genes in addition to numerous proto-oncogenes were detected during

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conditions of enhanced progenitor cell expansion. DOCK4 and SPARCL1 tumor suppressors, were confirmed down-regulation in CD133+ CD34+ cells. These findings suggest that there is a common source of stem cells and cancer stem cells, and some of them might be used both in stem/progenitor expansion and potential molecular targets for malignant treatment. Most of the hematopoietic cytokines promote either survival or differentiation or both in HSC ex vivo, whereas extracellular morphogens (Wnt, Notch, Hedgehog, bone morphogenetic protein 4, and Tie2/angiopoietin-1) signaling pathways, and intracellular mediators (phosphatase and tensin homolog and glycogen synthase kinase-3) have been signified a class of HSC regulators that support expansion of the HSC pool by a combination of survival and induced self-renewal in vivo, but these pathways alone does not result in substantive expansion of HSCs ex vivo. Bcl-2 gene family, which regulates cell apoptosis, may play an important role in inducing survival in HSCs both in vivo and ex vivo. Correctly understanding the effect of these unique signaling pathways and their relationship will be essential to achieve successful ex vivo expansion and make UCBT available to more patients, decrease engraftment times and allow more rapid immune reconstitution post transplant [58, 59].

3.2.4 Free Radical Regulation on HSPCs Expansion Series studies about reactive oxygen species (ROS) on stem cell expansion were investigated recently. Hypoxia favored the preservation of progenitor characteristics of HSPCs in bone marrow. Fan reported that NADPH oxidase activity and ROS generation were reduced in hypoxia with respect to normal oxygen tension. The NADPH oxidase inhibitor diphenyleneiodonium, or the ROS scavenger N-acetylcysteine could inhibit this procedure. Hypoxia effectively maintained biological characteristics of CD34+ cells through keeping lower intracellular ROS levels by regulating NADPH oxidase [60]. In another study, they investigated the effect of regulating intracellular ROS with antioxidants on the ex vivo expansion of cord blood CD34+ cells. The generation of ROS was increased markedly by the cytokine combination, and these ROS could be eliminated by antioxidant effectively. The percentage of CD34+ cells and CD34+ CD38- cells, the colony growth of colony-forming cells (CFC) and the re-expansion capability of CD34+ cells were enhanced by low concentration of antioxidant such as 2,000 U/mL SOD, 200 U/mL CAT or 2 mmol/L NAC. When increasing antioxidant to high concentration of 8,000 U/mL SOD, 1,000 U/mL CAT or 5 mmol/L NAC, the expansion of the cells was inhibited [61]. Copper (Cu) is known to generate oxidative stress in cells which in turn affects proliferation, differentiation and apoptosis. Prus [62] showed that Cu chelator tetraethylenepentamine (TEPA) reduces the free Cu content of HPCs and stimulates cord blood-derived CD34+ CD38- cells ex vivo expansion by lowering their oxidative stress. Srp et al. [63] reviewed oxygenation level as a physiological regulator of HSC maintenance: very low oxygen concentrations (0.1%) enable the preservation of the quiescent (G0 ) stem cell pool; low oxygen concentrations (1%) are compatible with the proliferation of primitive stem cells (self-renewal)

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but inhibit their differentiation; moderately low oxygen concentrations (3%) allow a balance between differentiation and self-renewal, permitting the simultaneous amplification of progenitors and the maintenance of stem cell activity; and very high oxygen concentrations (20–21%), like those in the air, enhance the differentiation of primitive stem cells, abrogating their self-renewal capacity.

3.2.5 Megakaryocytic Progenitor Cells Expansion Megakaryocytic progenitor cells (MKPC) infusion is a treatment selection for thrombocytopenia after HSC transplantation. Xia et al. [64] described some culture system composed of various cytokine combinations (TPO, SCF, Flt3-ligand, IL-1, IL-3, IL-6) on ex vivo expansion of megakaryocytic progenitors from CD34+ cells of peripheral blood. The content of CD41+ cells increased 94-fold at day 5 and 131-fold at day 10 and then decreased obviously, the CFU-MK were 93 and 121 respectively at day 5 and 10. The cytokine combination TPO/FL/IL-6/IL-3 was optimal for expansion ex vivo of megakaryocytic progenitors from mobilized PB. Boyer [65] designed a two-phase culture strategy to induce megakaryocyte (MK) differentiation from CD34+ -enriched CB cells. They optimized two functionally divergent cocktails to significantly increase the final yield of both MKs and HPC. Lin et al. [66] reviewed growth factors, including TPO, megakaryocyte growth and development factor (MGDF), IL-1, IL-3, IL-6, IL-11, platelet-derived growth factor (PDGF), and serotonin (5-HT) on the regulation of megakaryocyte/platelet development, and the efficient conditions for the expansion of the MK progenitors from HSPC. TPO alone could produce a high proportion of MK progenitors but a low total cell count. IL-1β, IL-3, IL-6 and Flt3-ligand improved the expansion outcome. PDGF also enhanced the ex vivo expansion of CD61+ CD41+ cells and CD34+ cells in combination with TPO, IL-1β, IL-3, IL-6 and Flt3-ligand, as well as engraftment of human stem and progenitor cells in NOD/SCID mice, but without promoting their in vitro maturation. The combination of three to five cytokines produced more efficient expansions of hematopoietic stem and MK progenitors.

3.2.6 Red Cells Expansion It is difficult in obtaining adequate supplies of all blood components, especially great numbers of red blood cells (RBCs). Douay et al. [67] described a methodology permitting the massive ex vivo production of mature human RBCs having all the characteristics of native adult RBCs from hematopoietic stem cells of diverse origins: blood, bone marrow, or cord blood. This protocol allows both the massive expansion of HSPCs and their complete differentiation to the stage of perfectly functional mature RBCs. The levels of amplification obtained 1×105 to 2×106 are compatible with an eventual transfusion application. Even if this is a considerable advance in blood transfusion, we do not think we can afford its luxury in clinical practice.

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3.2.7 T-Cell Expansion Potential advantages of using CB relate to the high proportion and quality of HSPCs. CBSCT requires less stringent HLA matching and results in less GVHD. This may be attributed to the immature neonatal immune system, which shows more tolerant to alloantigens, compared with the corresponding cells from adult. More studies have been reported that cord blood T-lymphocytes are immature in phenotype and function and that little cytotoxic is generated after allogeneic transplantation [68–70]. Since mature T-cells play an important role in GVHD pathogenesis and GVL effect process. Interleukins are essential for immune cells [71–73]. Immune cells decreased in expanded cells supplemented with SCF, Flt3-ligand, G-CSF without adding interleukins, even no T, B, NK cell [74] were detected when incubating over 3 weeks. Ballen et al. [75] incubated the cord blood with a cytokine mixture of IL-3, IL-6, IL-11 and SCF, and resulted in increased survival of irradiated NOD-SCID recipients posttransplantation of the expanded cord blood. Robinson [76] reported that ex vivo combination of IL-2, IL-12, anti-CD3, and IL-7 significantly enhances the proliferation, activation, maturation, and cytotoxic potential of UCB T-cells of both fresh and thawed UCB MNC. The four cytokine combination significantly induced expression of CD45 RO in both the CD4+ and CD8+ T-cells expressing CD25 respectively and increased the production of IFN-γ. The combination also significantly increased the killing of K562 target cells. For an adoptive immunotherapy of cancer, autoimmunity, and infectious disease, Skea et al. [77] developed a new method involving the use of a conditioned medium (XLCM) that consistently results in levels of UCB T-cell expansion. From initiation of the UCB or adult PB low-density LDMNC/XLCM cultures up to approximately 2 weeks, the cultures were dominated by CD4+ T-lymphocytes. By 4 weeks, more than 80% of the cultured cells bear the CD8+ phenotype, it permits the selective expansion of different T-lymphocyte subsets from a single source. Li et al. [78] has proved that CB CD34+ cells were cultured for 5 days in the presence of human cytokines and the murine stromal cell line HESS-5, and transplanted into irradiated NOD/SCID mice, functional capacity of B cells marker CD19+ cells appeared at 6 weeks. Enrichment of donor grafts with CB T-cells expanded ex vivo might facilitate improved T-cell immune reconstitution post-transplant. We studied UCB-derived T-cell amplification under the cytokines combinations. The CD3+ T-cells could be expanded to higher level in the combination 50 ng/mL SCF + 2 ng/mL IL3, 20 ng/mL IL-6 cocktail with 5 ng/mL IL-7 or 10 ng/mL IL-2, or 10 ng/mL IL-4. Moreover, if IL-2 or IL-4 concentration were increased to 5 times, more effective expansion of CD3+ cells exhibited [78]. D’Arena et al. [67] compared the difference between HUCB and adult PB lymphocytes in their immunophenotypic profile. Significant differences in percentage were found between cord and adult T-cells, respectively (CD3+ : 59.9 vs. 74.9%), CD3- CD16+ and/or CD56+ NK cells (23.8 vs. 10.8%) and CD3+ CD16+ and/or CD56+ cytotoxic T-lymphocyte subset (0.3 vs. 10.7%). There was no difference in CD4/CD8 ratio (1.7 vs. 1.6%) between the two groups. Szabolcs et al. [79] and Guo et al. [80] utilized FACS

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to characterize surface and intracellular protein expression on lymphocyte subsets from fresh unmanipulated UCB and adult PB, similar results were acquired. Parmer [81] successfully expanded CB T-cells using paramagnetic microbeads covalently linked to anti-CD3 and anti-CD28 Ab in the presence of 200 IU/mL IL-2. A mean 100-fold expansion (range 49–154) of total nucleated cells was observed in the CD3+ magnetically enriched fraction. The expanded CB T-cells retained a naive and/or central memory phenotype and contained a polyclonal TCR diversity. This in particular showed that HUCB T-lymphocytes appeared to be phenotypically immature. Cycling UCB T-cells retain a naive immunophenotype that may represent homeostatic expansion rather than antigen-driven proliferation. Although still in its infancy, human CB progenitor cells hold considerable potential for in vitro expansion and to transplant the adult recipients with genetic inherited diseases, cancer and some immunodeficiencies [82].

3.2.8 NK Cell Expansion NK cells are important as the first line of the host defense, and as one of the final effectors cells in resistance to tumor, metastases, and viral infections. Allogeneic NK cells are known to show a high cytotoxic activity against HLA-nonidentical residual leukemia or tumor cells and relapse, and to reinduce remission after bone marrow transplantation, but its application has been limited by the inability to obtain sufficient numbers of pure NK cells. It is possible to effectively expand cord bloodderived CD56+ cells ex vivo, while maintaining their high lymphokine activated killer activity. NK cells can be induced by various stimuli, in particular IL-2, from bone marrow, cord blood and peripheral blood purified CD34+ stem cells and exhibit similar phenotype and functions [83, 84]. Cytokine IL-2 has been proved to enable to activate in vitro antitumor cytotoxic of HSCs even at a low-dose [85]. Li et al. [86] isolated NK cells from human peripheral blood and cultured them in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium) combinations with IL-2, IL-12, IL-15 for 15 days, 50.5 and 52.4-fold cells were expanded in IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group respectively. All expanded cells showed over 94% CD3- CD56+ NK cells purity, and a significantly higher cytotoxicity were observed compared to starting population. Koehl et al. [87] purified and activated CD56+ CD3- NK cells with IL-2, a five-fold expansion of NK cells was observed, which showed a highly increased lytic activity against the MHC-I deficient K562 cells and a medium cytotoxicity against patients’ leukemic cells. UCB is a rich source of cytotoxic CD56+ cells including fetal NK cells (CD16- CD56+ ) with high lytic capabilities. NK T-cells in human CB are very small populations (<1.0%). When lymphocytes in cord blood were cultured with rIL-2 for 14 days in vitro, CD56+ T-cells could be expanded up to 25% of T cells [88]. Azuma et al. [89] has achieved approximately 1000-fold ex vivo expansion of cord blood lymphocytes for two weeks, and the phenotype and function of

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expanded CB lymphocytes (CBL) were essentially equivalent to those of expanded PB lymphocytes (PBL). At an effector to target ratio (E/T) of 40:1, the natural killer activity of expanded CBL was significantly higher than that of expanded PBL. In addition, coculturing the expanded NK cells with fresh acute lymphocytic leukemia (ALL) blasts resulted in 85% inhibition of colony growth in methylcellulose [90]. Joshi et al. [91] additionally reported that IL-2-activated mononuclear cells from umbilical cord blood cells (UCBCs) had been shown increased cytotoxicity against K562 and Raji hematopoietic malignant cells compared with peripheral blood cells (PBCs). Human NK cells from HSC transplanted NOD/SCID mouse origin showed cytotoxicity against HLA class 1-deficient K562 targets either in vitro or in vivo [92]. Cavazzana-Calvo and colleagues [93] studied NK cell differentiation from cord blood CD34+ cells in the presence of SCF, IL-2, and IL-7, this cytokine combination efficiently induced CD34+ cord blood cells to proliferate and mature into NK cells. NK cells expressed CD56 and efficiently killed K562 target cells. CB cells were shown more efficient at generating CD56hi NK T-cell fractions than bone marrow. It has been demonstrated that UCB MNCs stimulated with IL2 for 72 h have an increased cytotoxicity of K562 cells in vitro [91]. Moreover, UCB CD56+ CD3- NK cell and CD56+ CD3+ NK T-cells could be expanded in vitro with IL-2 from sorted CD56- CD3- cell and mediated cytotoxicity [83]. The mean cytotoxic activity level of IL-2-activated natural killer was higher in UCB than in maternal PB (MPB) [94]. The expanded NK cells manifested potent lytic capabilities not only against K562 cell lines but also inhibition of colony growth of fresh ALL blasts [95]. Wei et al. [34] expanded UCB MNCs in vitro in response to cytokines combined with or without IL-2. When 10 ng/mL IL-2 was added to incubation medium with SCF, IL-3, IL-6, IL-7 cytokine cocktail, CD3- CD56+ cell numbers were up-regulated significantly compared to the cytokine cocktail medium group without IL-2 addition. Moreover, the CB derived ex vivo expanded CD56+ cytotoxic cells demonstrated killing and apoptosis induction ability against fresh leukemia cells [74, 84, 95]. These results and other findings suggest that UCB natural killer cells can be generated in vitro as a useful source of cellular therapy for patients with hematological malignancies [74].

3.2.9 DC Expansion Dendritic cells (DCs) are the most potent antigen presenting cells (APCs) in the initiation of an immune response and immunological tolerance, as well as for the regulation of T-helper 1 (Th1) and 2 (Th2) immune responses, and may be one of the earliest cell types exposed to pathogens [96]. DCs may also play a critical role in the induction of peripheral immunological tolerance, which could have important implications in the treatment of autoimmunity or in the outcome of clinical transplantation [97]. This well-known function of DCs has offered the possibility of developing clinical protocols for their use in immunotherapy

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to tumors. A wide range of hematopoietic cells have the capacity to acquire the phenotype and function of DCs. Human DCs can differentiate from CD34+ hematopoietic progenitor cells in the cord blood, the adult bone marrow or blood [98], and also from other blood precursors cells [99]. Very little is known about DC in cord blood, and whether they are involved in the low incidence and severity of GVHD following CBT. UCB MNCs were reported to be hardly express DCs markers, such as CD1a, CD11b, CD83, CD86, but resemble immature DC in peripheral blood [100]. IL-4 was demonstrated to play a key role in CB CD34+ cells differentiation and maturation of DCs [85]. Human UCB CD34+ stem cells could be induce to differentiate into dendritic cells using GM-CSF, IL-4, TNF-α, Differentiated DCs were CD80+ , CD86+ , CD83+ , CD54+ , CD1a+ , CD11b+ [101]. Liu et al. [102] and Wang et al. [103] have reported that DCs cells could generate from cord blood after culture for 7 day with IL-4 and GM-CSF, and thereafter its percentage decreased. Liu et al. [104] used a two-step method to generate a large number of functional and mature dendritic cells from CB or normal human bone marrow CD34+ progenitor cells. Culturing CB CD34+ cells with GM-CSF and TNF-α for 12 days, and SCF for 5 days, resulted in a 40-fold expansion in cell numbers, with 38% DCs. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL3, IL-6, G-CSF, and 7–11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-α. By the two-step culture, total nucleated cells were increased 208-fold in the culture of CB. Out of the total nucleated cells, 23% of cells in CBCs culture acquired DC characteristic phenotypes, which showed marked expression levels of CD1a and HLA-DR. Wei et al. [34] demonstrated that UCB MNCs expressed very low level of DCs markers, when incubated in the medium with cytokine cocktail containing IL-4 over one week. CD1a, CD80, CD83, and CD86 antigen expression increased very sharp to a higher level then reduced slowly. It is concluded that mature DCs could be obtained from human CB MNCs cells. Both NK cells and dendritic cells are important in the innate host defense. DCs appeared to be essential in initiating the activation of NK cells in response to pathogens. Some clues showed that DCs could help NK cells differentiation and maturation and enhance NK cell-dependent antitumor effects [85, 105]. Ueda and colleagues [106] reported that auto-DCs were prove to have the capability to enhance NK cell cytotoxicity against K562 and to produce a high level of IFN-γ than T-cells alone. Coculture of the NK cells and DCs generated from same donor human CB CD34+ showed a similar result by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways [85]. Furthermore, direct interaction between DCs and NK cell is necessary for DC-mediated enhancement of NK cell cytotoxicity [107]. In a recent study, Wei et al. did not observe a more increase of NK cells number under the culture condition with IL-2 and IL-4 together compared to that with IL-2 alone [34]. However, these results may not deny DCs the coeffective cytotoxicity to NK cells against tumor cells [108].

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3.2.10 HSPC Ex Vivo Expansion and Gene Therapy Ex vivo expansion of human HSCs is an important issue in gene therapy. Some researches of gene therapy based on ex vivo expansion of HSC have been achieved to success. Zubler et al. [109] developed HIV-1-derived bicistronic lentiviral vectors that could be efficiently transferred into immature hematopoietic cells and primary human B lymphocytes. Before larger clinical trials, clinical gene therapy still faces a variety of problems, especially the risks of insertional mutagenesis.

3.3 Expansion Bioreactor UCB stem cells, which are used in humans treatment, are considered pharmaceuticals. A safe and replicable process is required for routine expansion of stem cells. In many cases, the stem cells are expanded in culture dishes in an open processing system. These processes are not the implementation as a good manufacturing process (GMP), which ensures safety for the patient and required by the FDA. Three-dimensional culture conditions can therefore provide an ex vivo mimicry of bone marrow, recapitulate the desired niche, and provide a suitable environment for stem cell expansion and differentiation. The use of bioreactors for cultivation of hematopoietic cells will allow for culture control, optimization, standardization, predictable, free of contaminants, and therefore suitable for human therapeutic applications [110]. An emerging number of expansion techniques succeeding in vitro and in animal models suggest that one or many of these methods will eventually be available. Galan et al. [111] seeded CB stem cells in platforms containing different cocktails of cytokines, which mimics the bone marrow microenvironment, and resulted in a double cell number after 1 week, whereas a 3D biomatrix does not enhance cell proliferation. Zhou and colleagues [112] developed a new-type bioreactor by combining superiorities of static and stirred culture models. SCF, TPO, Flt-3L were used as the cytokines cocktails, after 7 day cultures, The effects of the expansion of total cells in the static culture was 13.86-fold, higher than that in the cyclic culture (7.23-fold). Both the static culture and the cyclic culture could be used in ex vivo expansion of CD34+ cells. In static culture HSCs differentiated into progenitor cells, whilst the cyclic culture favored the expansion of primary HSPCs. Astori et al. [113] tested a Dideco ‘Pluricell System’ to expand CBSC. The mean CD34+ cell expansion on day 7 and 12 was 7-fold and 12-fold respectively, the mean NC expansion was 69-fold and 180-fold. After 12 days, the mean NC viability was 96.9%, GMCFU showed a 20-fold increase. Fan et al. [114] used rotating wall vessel (RWV) for the ex vivo expansion of UBC stem cells. The MNCs from UCB were cultured in T-flasks for 24 h, and then inoculated in RWV to culture for 200 h. Nucleated cells and CD34+ cells had a 435-fold expansion and a 32.7-fold expansion respectively in 197 h, and CFU-GM cells had a 21.7-fold expansion. In the whole course

Bioreactor (closed auto-matic system) and therapy kits

Aastrom Biosciences (USA): AastromReplicellTM

Cytomatrix (USA): The CytomatrixTM

CytomatrixTM Bioreactor (Frame for cell growth)

R culture bag CellGenix VueLife R R (Germany)VueLife and CellGro R and CellGro Medium

Process

Product

2- 4-fold expansion of UCB SC

No information on CB-I Therapy Kit. CB-II Kit offers 8,3-times more nucleated cells as well as 9.5-times more CD34+ cells as compared to CB-I Kit No data

Expansion factor

Expansion of different cell types with the CytomatrixTM Bioreactor has been described numerous times in scientific literature.

Unknown

Unknown

Unknown

Children and adults with leukemia and other blood diseases, severe osteopetrosis, lymphoma

Phase I/II Study on expansion of UCB SC • Completed for CB I-Kit • Ongoing for CB II-Kit Phase III Study for expanding UCB SC • Ongoing for CB I-Kit

Closed system and CB-I Therapy Kit in Phase III Study and CE certified in Europe

R culture bag VueLife is CE-certified. Medium only intended for research CytomatrixTM Bioreactor only used clinically but not commer-cially

Applications

Studies

Introduction

Table 3.2 Development status of stem cell expansion bioreactor

42 Y. Wei and X. Ye

Process

Use of low molecular Chelate for copper binding

Culture system

Culture medium, removal of differentiated cells

Product

Gamida (Israel) StemExTM

MainGen (Germany)GMPExpansion

ViaCell (USA): Selected Amplifica-tionTM Up to 40-fold expansion of stem cell populations

18-fold expansion of stem cells within 7 days

100–1000-fold expansion of stem and precursor cells within several weeks

Expansion factor

Unknown

Production permit for stem cell expansion received (01/2001)

2006

Introduction

Table 3.2 (continued)

Pre-Clinical Study on expansion of UCB SC • Completed Clinical Study on expansion of UCB SC • Planned Pre-Clinical Study on the expansion of UCB SC • Completed Clinical Studies on expansion of UCB SC • Launched end of 2003

Phase I Study for expansion of UCB SC • Study initiated 02/2003

Studies

None

10 persons age 55 with leukemia, non Hodgkin’s and Hodgkin’s lymphoma Unknown

Applications

3 Ex Vivo Expansion of HSPCs 43

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of culture, the pH was kept from 7.2 to 7.4, and the osmolality was kept from 290 to 310 mmol/kg to ensure HSPC expansion. Bone marrow stromal cells promote and regulate the self-renewal, commitment, differentiation, and proliferation of stem cells and progenitors through their secreted extracellular matrices and cytokine environment in the hematopoietic microenvironment. To avoid expensive cytokines, some clonal stromal cell lines have been established from the bone marrow cells in order to simplify and stimulate the ex vivo expansion of hematopoietic cells. A novel membrane-separated coculture system, in which stromal cells adhere onto the lower surface of a porous membrane and hematopoietic cells are incubated on the upper surface of the membrane, was invented to ex vivo expand HSPC for clinical application [115]. In the industry, several systems and test conditions are being developed. A bioreactor was developed to settle and expand stem cells in the hollow spaces of the collagen spheres just as in the niches of bone marrow. The collagen spheres are subsequently dissolved through the addition of an enzyme, allowing the stem cells to be harvested. Bone marrow stem cells, even cryopreserved UBC were successfully expanded in this kind of bioreactor and subsequently transplanted. Another closed processing system are designed through the addition of a set medium to expand stem cell. Some of them is currently undergoing Phase III clinical trials in the U.S. and is CE certified in Europe. Table 3.2 shows the bioreactor of stem cell expansion development status [116–118].

3.4 The Application of Expanded HSPCs HSCs supply all blood cells throughout life by making use of their self-renewal and multilineage differentiation capabilities. Stem cell expansion is an important tool both for improving transplant outcomes and enabling individuals to use their own cord blood samples for more than one treatment. This is particularly important for given advances in regenerative medicine. Regenerative medicine always use the patient own cells to repair or replace damaged tissues and organs by increasing the number of HSPCs. Over the last few years, transplantation of HSPCs from mobilized peripheral blood stem cells, UCB bone marrow cells has been used as source of cellular reconstitution for the treatment of malignancy. The techniques have become available and allow the extensive proliferation, orderly differentiation, functional activation and gene transfer of HSPCs in ex vivo culture systems. These techniques have now developed to the point at which clinical trials are now underway in a variety of settings for the applications of HSC transplantation, hematopoietic support after high-dose chemotherapy, immunotherapy of cancers, and gene therapy [119].

3.4.1 Transplantation of HSPCs in Animal Model While expanded stem cells are not yet approved for medical use in humans, several expansion studies and clinical trials are underway. To establish a more appropriate animal recipient for xenotransplantation, immunodeficiency mice are found to be a

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small animal model as hosts for the in vivo analysis of normal and malignant human pluripotent HSCs. Over the past decade the human-immunodeficient mouse chimera has become a well-established in vivo model for studying the human immune system and hematopoiesis. Ballen et al. [120] analyzed irradiation dose, cell dose, age of mice, and maternal and neonatal characteristics of the cord blood donor on the effect of engraftment in non-obese diabetic-severe combined immunodeficient (NOD–SCID) mice. Engraftment was a higher level in 400 cGy-irradiated mice than 350 cGy treated ones. Successful engraftment was detected up to 6 months, with skewing to B lymphocytes. Mean engraftment was 31% (range 0–67%) of human cells in recipient bone marrow, but the radiation dose of 350 cGy was superior in survival of the murine recipients compared with 400 cGy. Moreover, the female of the NOD/SCID recipients showed a significant effect on survival than male ones did. These mice are a suitable model for studying the reconstitution of human hematopoiesis and immune system. The BALB/C nude mice are immunodeficient due to atrophy in thymus, this characteristics prevents normal T cells differentiation and maturation, the infancy nude mice kept a low level of NK cells activity, while increasing to high level of NK cells activity when they grew up. Comparing to T- and B-cell deficient SCID mice [1, 121, 122], which has been used as recipient of human hematopoietic engraftment, the BALB/C nude mice are much cheaper either in price or requirement for breeding conditions than NOD/SCID mice, thereafter, widely used in most laboratories for transplantation analysis of tumor cells and HSCs. Ding et al. [123] and Duan et al. [124] both reported that BALB/C mouse were used to generate a transplantable leukemia model and established a useful tool for the study of graft-versus leukemia. Pretreatment with CTX and irradiation could deplete host BALB/C mouse natural killer cell and enhance human HSC engraftment in early stages [125]. Lan et al. [126] previously reported that BALB/C nude mice were used to establish a murine model to evaluate the hematopoietic potentiality and the migration and homing route of separated UCB hematopoietic cells. Chronic myeloid leukemia is a clonal myeloproliferative expansion of transformed primitive hematopoietic progenitor cells characterized by high-level expression of BCR/Abl chimeric gene. Baron and colleagues [127] demonstrated that BCR/Abl high expression transfectants UT-7/9 cells were lysed by NK cells with a higher efficiency than its parental. It seems that expression of oncogene BCR/Abl may increase susceptibility of leukemic progenitors to NK cell cytotoxicity. Recently, Wei et al. [128] reported a simple method to establish a mouse model of leukemia. 2×105 K562 cells injection could produce leukemic mice in 4–5-week-old female BALB/C nude mice, K562 cells human antigen CD13 and BCR/Abl gene were detected in mice PB on 10 day post transplantation as markers of success of mice model establishment. The injection methods i.v. or i.p. did not show difference in K562 tumor cells burden in mice peripheral blood, the pretreatment with 2 mg/mouse CTX for 2 days showed a little promotion effect on inoculation of K562 cell into BALB/C mice. The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when CB is used as a source of hematopoietic graft. Mice receiving purified CD34+ cells survived up to 207

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days in good health with more than 95% human cells in the bone marrow. In those mice, all lineages (B and T lymphocytes, monocytes, granulocytes, erythrocytes and thrombocytes) were demonstrated in the bone marrow and peripheral blood [129]. Expanded CB cells were tested to transplant into sublethally irradiated NOD/SCID mice, and the engraftment of human CD45+ cells and subsets in the bone marrow, spleen, and peripheral blood was determined. When compared with fresh CB, samples stimulated for 6 days with IL-3/ IL-6/ SCF contained increased numbers of hematopoietic progenitors. The ex vivo expanded transplantation showed an impairment in the long-term repopulation of recipients BM at 120 days post transplantation [26]. Azuma et al. [89] and Pei et al. [130] reported that cord blood HSCs could be expanded ex vivo efficiently by the combination of cytokines including Flt3ligand, SCF, TPO, IL-6 + IL-3, and smoothly engrafted into SCID recipients and reconstitute their hematopoiesis. In recent report, PBPCs cells obtained from four patients with hematological malignancies and one patient with Ewing’s sarcoma were expanded in the Dideco Pluricell system for 12 days, then transplanted into irradiated NOD/SCID mice. Eighty percent mice transplanted with expanded cells survived compared to 20% in mice transplanted with non-expanded cells [131]. In another research sublethally irradiated K562 leukemic BALB/C nude mice were transplanted with expanded CB containing various levels of immune cells, which were stimulated by different cytokines combination ex vivo for 7 days, CD45+ and CD3+ cells were found in all survival mice bone marrow at 6 weeks post transplantation. BCR/Abl gene remarked recipient leukemia disappeared in most of survival mice PB and BM, while the human β-actin band appeared as a UCB engraft marker in all transplantation mice [132]. Denning-Kendall et al. [133] and Lam et al. [134] confirmed that the engraftment potential of SCID repopulating cells (SRCs) in sublethally irradiated NOD/SCID model was preserved after CB CD34+ cell in vitro expansion. Denning-Kendall et al. [133] and Kobari et al. [135] proved that in vitro expansion of CB could retain their ability to support long-term hematopoiesis. In another expanded transplantation, an average of 45% human CD45+ was found in bone marrow at 6 weeks after transplantation [136]. Ando et al. [137] used limiting dilution analysis (LDA) to demonstrate reliability of current ex vivo expansion for transplantation. Except for cytokines, MSC are prove to be benefit for longterm engraftment of hematopoietic cells as well as gene therapy [138–140]. It is clear that ex vivo expanded HSPCs not only reconstitute hematopoiesis, but also improve mouse model cardiac function and remodeling post myocardial infarction (MI) [141]. Under certain experimental conditions, the recipient mice may develop a xenogenic graft-versus-host-disease (GVHD). Depending on the ratio of lymphocytes to HSCs, proliferation of human T-cells, hematopoiesis or a combination of the two GVHD is observed in widely varying proportions. When the graft contains a preponderance of lymphocytes, fatal protracted discordant xenogenic GVHD develops. Gorin et al. [121] found that sublethally irradiated NOD/SCID mice, which received graded human CB or BM hematopoietic cells (5–500×106 MNCs, containing 2–325×106 CD3+ T-cells, per mouse) from individual donors, developed

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no GVHD. Conventionally, GVHD are thought to be associated with graft-versusleukemia (GVL) effect after allogeneic HSC transplantation. For example, stem cells eliminated T-cell transplantation decreased the risk of severe GVHD, but increased the rate of relapse. On the contrary, donor leukocyte infusion (DLI) has been used as antitumor immunotherapy to induce remission of patients with relapse of patients with transplantation of SCT or keep GVL effects for transplanted patients [142]. Locatelli et al. [143] demonstrated a 10–20% incidence of leukemia relapse after UCBT in children treated for acute leukemia, and DLI has been used successfully following UCBT for CML because of the availability of DLI from the sibling donor peripheral blood [144]. Although the pathophysiologic mechanisms of GVL effects remains unclear, it is believed that various immune cells, such as CD4+ and CD8+ lymphocytes, NK cells, monocytes and lymphokine-activated killer (LAK), are involved in this process. Cytokines are reported not only to play an important role in the pathogenesis of GVHD, but also to contribute to GVL effects [145]. Some clues have shown that donor derived cytotoxic T-lymphocytes with anti-leukemic activity, but without normal host tissue reactivity (GVL without GVHD) could be isolated in vitro and this implicated that they were two separately phenomena [146]. These suggest that it is possible to avoid GVHD while to keep or even to enhance GVL effects at same time.

3.4.2 Transplantation of HSPCs in Human The successful transplantations of expanded stem cells from bone marrow, peripheral blood, as well as UCB in humans are being documented and assessed in a number of clinical trials. As early as in 1992, there was a first case report of clinical transplantation with a proportion infusion of allogeneic bone marrow HSPCs expanded in GM-CSF and IL-3 [147]. In 1997, US physicians conducted a phase I study on the expansion of UCB stem cells with the AastromReplicel system, 47 months following the transplant the patient’s physician certified that was a permanent engraftment of the stem cells [148]. At Hackensack University Medical Center, USA, two adults with chronic myelogenous leukemia exhibited rapid engraftment after receiving expanded cord blood transplants [149]. UCB is now widely accepted as a source of stem cells in patients with malignant hematologic and genetic disorders. Brahmi et al. [150] have reported that in a series of 30 pediatric UCB transplant recipients will give comparable outcomes to that anticipated with other unrelated stem cell sources. David et al. [151] summarized nearly 300 patients experienced expanded HSPC transplantation in 2004. Although no toxicity or serious adverse events were found, there is wide discrepancy in the documented effects on hematopoietic recovery attributed to the infused cells. A common feature of these studies is the use of combinations of recombined human cytokines, such as G-CSF, GM-CSF, SCF, Flt3-ligand, MGDF, EPO, MIP-Iα and IL-1, IL-3, IL6, IL-11, their main objectives are to generate a cellular product of neutrophil or platelet recovery posttransplant or after high dose chemotherapy. In November 2007, the Gamida

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Cell/Teva Joint Venture announced that the first patient in its international, multicenter trial received a transplant of stem/progenitor CB stem cells in combination with non-expanded cells from the same unit. The trial will assess the efficacy and safety of expanded CB transplantation as a treatment for hematological malignancies, including leukemia and lymphoma [152]. Shpall et al. [153] reported patients received a median of 0.99×107 total nucleated cells (mixed with expanded 40– 60% plus 60–44% unexpanded) per kilogram. The median time to engraftment of neutrophils was 28 days and of platelets was 106 days. At a median follow-up of 30 months, 35% patients survived. Grade III/IV acute GVHD was documented in 40% and extensive chronic GVHD in 63% of patients. More recently, a phase I/II trial was performed to test the feasibility and safety of transplantation of CD133+ CB hematopoietic progenitors cultured in media containing SCF, Flt3-ligand, IL-6, TPO. Ten patients with advanced hematological malignancies were transplanted with a CB unit originally frozen in two fractions. The smaller fraction was cultured ex vivo for 21 days and transplanted 24 h after infusion of the larger unmanipulated fraction. Pre-expansion total nucleated cells (TNCs) per kilogram contained were less than 2×107 cells, the average TNCs fold expansion was 219, mean increase of CD34+ cell count was 6-fold. Total 9 patients were beyond first remission, median time to neutrophil and platelet engraftment was 30 and 48 days. No case of grades 3–4 acute GVHD was observed and 100-day survival was 90% [154]. These studies partially demonstrated the hematopoiesis reconstitution capability of ex vivo expanded HSPCs, but widely use of ex vivo expanded HSPCs in clinical treatment needs more information, especially proof of the directly hematopoietic recovery from expanded HSPCs alone.

3.5 The Future of HSPCs Expansion New information is published on a daily basis promising improved results in the expansion of stem cells. It can be expected that in the coming years additional expansion processes will be developed and used in more patients. With the increasing information on the number, quality, and characteristics of HSPC, the transplantation of ex vivo expanded HSPCs is a promising approach to restore the required bone marrow function of patients with hematological disorders and other non-hematopoietic disease.

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4. Moore MAS, and Metcalf D. (1970) Ontogeny of the hemopoietic system:yolk sac origin of in vivo and in vitro colonyforming cell in the developing mouse embryo. Brit J Haematol 18:279–296. 5. Robert L, John G, Brigid H et al. (2004) Hand Book Stem Cells. In: MAS Moore (ed.) Ontogeny of the Hematopoietic System. Elsevier Inc. 6. Jacobson Lo, Marks EK, and Gaston E (1955). Observations on the effect of spleen shielding and the injection of cell suspensions on survival following irradiation. In: ZMA Bacq and P Alexander (eds.), Radiobiology Symposium, pp. 122–133, Academic Press, New York. 7. Owen RD (1945) Immunogeneitic consequences of vascular anastomoses between bovine twins. Science 102:400–401. 8. Juttner CA, LB T, Haylock DN et al. (1985) Circulating autologous stem cells collected in very early remission from acute non-lymphoblastic leukaemia produce prompt but incomplete haemopoietic reconstitution after high dose melphalan or supralethal chemoradiotherapy. Br J Haematol 61:739–745. 9. Kessinger A, Armitage JO, Landmark JD et al. (1986) Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells. Exp Hematol 14: 192–196. 10. Korbling M, Dorken B, Ho AD et al. (1986) Autologous transplantation of blood-derived hemopoietic stem cells after myeloablative therapy in a patient with Burkitt’s lymphoma. Blood 67:529–532. 11. Reiffers J, Bernard P, David B et al. (1986) Successful autologous transplantation with peripheral blood hemopoietic cells in a patient with acute leukemia. Exp Hematol 14: 312–315. 12. Bensinger WI, Martin PJ, Storer B et al. (2001) Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 344: 175–181. 13. To LB, Haylock DN, Simmons PJ et al. (1997) The biology and clinical uses of blood stem cells. Blood 89:2233–2258. 14. Wanger J, Rosenthal J, Sweetman R et al. (1996) Succsessful transplantation of HLAmismatched umbilical cord blood from unrelated donors analysis of engraftment and acute graft-versus-host disease. Blood 88:795–802. 15. Bojani´c I and Golubi´c Cepuli´c B (2006) Umbilical cord blood as a source of stem cells. Acta Med Croatica 60:215–225. 16. Gluckman E, Broxmeyer HA, Auerbach AD et al. (1989) Hematopoietic reconstitution in a patient with Fanconi anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 321: 1174–1178. 17. Tse W, Bunting KD and Laughin MJ (2008) New insights into cord blood stem cell transplantation. Curr Opin Hematol 15:279–284. 18. Möhle R and Kanz L (2007) Hematopoietic growth factors for hematopoietic stem cell mobilization and expansion. Semin Hematol 44:193–202. 19. Bodine, DM, Seidel NE, Zsebo KM et al. (1993) in vitro administration of stem cell factor to mice increases the absolute number of pluripotent hematopoietic stem cells. Blood 82, 445–455. 20. Kawano Y, Kobune M, Chiba H et al. (2006) Ex vivo expansion of G-CSF-mobilized peripheral blood CD133+ progenitor cells on coculture with human stromal cells. Exp Hematol 34:150–158. 21. Bruno S, Gammaitoni L, Gunetti M et al. (2001) Different growth factor requirements for the ex vivo amplification of transplantable human cord blood cells in a NOD/SCID mouse model. J Biol Regul Homeost Agents 15:38–48. 22. Denning-Kendall PA, Evely R, Singha S et al. (2002) in vitro expansion of cord blood does not prevent engraftment of severe combined immunodeficient repopulating cells. Br J Haematol 116:218–228.

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23. Conneally E, Cashman J, Petzer A et al. (1997) Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitive assay of their lymphmyeloid repopulating activity in nonobese diabetic scid mice. Proc Natl Acad Sci USA 94:9836–9841. 24 Bhatia M, Wang JCY, Kapp U et al. (1997) Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci USA 94: 5320–5325. 25. Guenechea G, Segovia JC, Albella B et al. (1999) Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34+ cord blood cells. Blood 93:1097–1105. 26. Piacibello W, Sanavio F, Severino A et al. (1999) Engraftment in non-obese diabetic severe combined immunodeficient mice of human CD34+ cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 93: 3736–3749. 27. Moretta A, Maccario R, Fagioli F et al. (2001) Analysis of immune reconstitution in children undergoing cord blood transplantation. Exp Hematol 29:371–379. 28. Novitzky N and Davison GM (2001) Immune reconstitution following hematopoietic stemcell transplantation. Cytotherapy 3:211–220. 29. Mohamed AA, Ibrahim AM, El-Masry MW et al. (2006) Ex vivo expansion of stem cells: defining optimum conditions using various cytokines. Lab Hematol 12:86–93. 30. Yao CL, Feng YH, Lin XZ et al. (2006) Characterization of serum-free ex vivo-expanded hematopoietic stem cells derived from human umbilical cord blood CD133+ cells. Stem Cells Dev 15:70–78. 31. Piacibello W, Gammaitoni L and Pignochino Y (2005) Proliferative senescence in hematopoietic stem cells during ex vivo expansion. Folia Histochem Cytobiol 43:197–202. 32. Madkaikar M, Ghosh K, Gupta M et al. (2007) Ex vivo expansion of umbilical cord blood stem cells using different combinations of cytokines and stromal cells. Acta Haematol 118:153–159. 33. Wei YM, Lan JC, Meng FY et al. (2005) Ex vivo expansion of T, NK and CD34+ cells from umbilical cord blood. J Exp Hematol 13:1076–1081. 34. Wei YM, Lin XM, Mao P et al. (2006) Ex vivo expansion of Cd34+ cells and immunocytes from umbilical cord blood. Chin-German J Clin Oncol 5:412–415. 35. Liu Y, Pei X, Lu S (1999) Studies on hematopoietic reconstitution by ex vivo expanded human cord blood hematopoietic stem/progenitor cells in SCID mice. Chin J Hematol 20:634–636. 36. Bruno S, Gammaitoni L, Gunetti M et al. (2001) Different growth factor requirements for the ex vivo amplification of transplantable human cord blood cells in a NOD/SCID mouse model. J Biol Regul Homeost Agents 15:38–48. 37. Pawliuk R, Eaves C, Humphries RK (1996) Evidence of both ontogeny and transplant doseregulated expansion of hematopoietic stem cells in vitro. Blood 88:2852–2858. 38. Iscove NN and Nawa K (1997) Hematopoietic stem cells expand during serial transplantation in vivo without apparent exhaustion. Curr Biol 7:805–808. 39. Molofsky AV, Pardal R, Iwashita T et al. (2003) Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425:962–967. 40. Iwama A, Oguro H, Negishi M et al. (2004) Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1. Immunity 21:843–851. 41. Wilson A, Murphy MJ, Oskarsson T et al. (2004) c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes Dev 18:2747–2763. 42. Zhang P, Iwasaki-Arai J, Iwasaki H et al. (2004) Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha. Immunity 21:853–863. 43. Antonchuk J, Sauvageau G and Humphries RK (2002) HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109:39–45.

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Chapter 4

Modulation of Hematopoietic Stem/Progenitor Cell Migration Thomas Dittmar, Susannah H. Kassmer, Benjamin Kasenda, Jeanette Seidel, Bernd Niggemann, and Kurt S. Zänker

Abstract The ability to migrate is an innate and fundamental function of hematopoietic stem/progenitor cells (HSPCs) enabling them to leave and to return to the bone marrow, as well as to be recruited to injured tissues. The latter property of HSPCs concomitantly with their ability to transdifferentiate, thereby restoring the integrity of damaged tissues, raised great expectations for regenerative medicine purposes. It is well recognized that the migration of HSPCs is initiated and maintained by the chemokine stromal cell-derived factor-1α (SDF-1α). SDF-1α is expressed by bone marrow stroma cells, thereby generating a gradient, which directs the HSPC homing to the bone marrow. Likewise, SDF-1α is released by endothelial cells in close proximity of damaged organ tissue, thereby attracting HSPCs to sites of injury, which thereupon participate in tissue repair. It is of interest that most studies deal with the investigation of the SDF-1α-mediated induction of HSPC migration concomitantly with the decipherment of signal transduction cascades engaged by the SDF-1α receptor CXCR4. By contrast, considerably less is known about factors, conditions, and mechanisms that modulate the SDF-1α induced HSPC migration. Here we will give an overview about our research dealing with this topic and will show that the SDF-1α mediated migratory activity of cultured HSPCs strongly depends on the cytokines/cytokine combinations being used for HSPC cultivation. In fact, the removal of only one factor from a cytokine cocktail, which give rise to highly SDF-1α susceptible HSPCs, will yield in cells, which migratory activity is inhibited by SDF-1α. Additionally, we will also summarize our results concerning factors that might act as stop-signals for the SDF-1α induced migration of HSPCs, which may play a role in the termination of HSPC migration. Keywords Hematopoietic stem/Progenitor cells · Cell migration · Homing · SDF-1α · CXCR4 · Signal transduction · Termination of cell migration · Culture conditions · Cytokines · Chemokines

T. Dittmar (B) Institute of Immunology, Witten/Herdecke University, D-58448, Witten, Germany e-mail: [email protected]

T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_4,

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Contents 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 The SDF-1α/CXCR4 Axis . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Stromal Cell-Derived Factor-1α (SDF-1α) . . . . . . . . . . . . . . . . 4.2.2 CXCR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3 SDF-1α/CXCR4 Signaling . . . . . . . . . . . . . . . . . . . . . . . 4.3 Homing of HSPCs to Bone Marrow and Organs . . . . . . . . . . . . . . . . . 4.4 Influence of Culture Conditions on the Migration of Human CD34+ /CD133+ Cord Blood HSPCs . . . . . . . . . . . . . . . . . 4.5 Influence of Culture Conditions on the Migration of Murine Lin- c-kit+ HSPCs . . 4.6 Termination of the SDF-1α Induced Migration of HSPCs . . . . . . . . . . . . 4.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4.1 Introduction Hematopoietic stem/progenitor cells (HSPCs) are of increasing interest due to accumulating evidences of their therapeutic potential for bone marrow transplantation purposes as well as for their putative use in autologous stem cell-based regeneration strategies of damaged/injured tissues such as liver [1–4], neuronal tissue [5–7], skeletal muscle [8–10] and heart muscle [11, 12]. The self-renewal and transdifferentiation capacity of HSPCs is however worthless, if these cells will loose their capacity to migrate to regions of interest. Because transplantation protocols utilize intravenous injections, diseases that require HSPC transplantation would fail to rescue lethally irradiated recipients if their homing potential will be impaired. This also suits to HSPC-based tissue regeneration strategies if cells will be applied intravenously. However, in addition to the cells’ susceptibility to respond to promigratory factors, which initiate and attract HSPCs to home to the appropriate tissue of interest, it is also crucial that the cells do not loose their susceptibility towards factors that terminate cell migration. In the present chapter, we will give an overview about factors/factor combinations that modulate the migratory behavior of HSPCs in response to SDF-1α stimulation.

4.2 The SDF-1α/CXCR4 Axis 4.2.1 Stromal Cell-Derived Factor-1α (SDF-1α) SDF-1 belongs to the large family of chemokines being a group of small peptides that by definition initiate the migration of effector cells. To date more than 50 chemokines and 18 chemokine receptors are known, whereby chemokines bind to several receptors and vice versa [13, 14]. An exception is SDF-1, which preferentially binds to CXCR4, also known as CD184. However, a recent study of

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Balabanian and colleagues indicated that SDF-1α also binds to and signals through the orphan receptor RDC1 in T-lymphocytes [15]. The SDF-1 gene was mapped to chromosome 10q, in contrast to other members of the intercrine family, which are localized on chromosome 4q and 17q. It has thus been hypothesized that SDF-1α may have important functions distinct from those of the other members of the intercrine family [16]. At present, six human SDF1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1∂, SDF-1ε, and SDF-1φ are known [17]. SDF-1α and SDF-1β are generated by alternative splicing [16], whereby SDF1β differs from SDF-1α due to additional 4 amino acids at the C-terminal end. Sequence alignment analyses revealed that SDF-1α/β are highly conserved between species. For instance, murine SDF-1α and SDF-1β sequences are more than 92% identical to those of human origin [16]. The new SDF-1 isoforms (γ to φ) are also splice variants. They all share the same first three exons, but contain different four exons [17]. SDF-1α/β share similar expression patterns and tissue distribution. Their highest expression levels are detected in liver, pancreas and spleen [17]. The human SDF-1γ isoform is only expressed in the heart and seems to be the human orthologue of rat SDF-1γ [18], which, however, is predominantly expressed in neurons and Schwann cells in these animals. All human SDF-1 isoforms can stimulate cell migration in a CXCR4-dependent manner suggesting that the novel SDF-1 splice variants encode functional proteins [17]. However, the role of human SDF-1γ in heart tissue or SDF-1 in the fetal liver has not yet been elucidated and thus remains ambiguous. In endothelial cells, SDF-1α/β gene expression is regulated by the transcription factor hypoxia-inducible factor-1α (HIF-1α) resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension [19].

4.2.2 CXCR4 The SDF-1 receptor CXCR4 was independently described by several authors, thus explaining the various alternative names, such as neuropeptide Y (NPY) Y3 receptor [20], D2S201E [21], leukocyte-derived seven-transmembrane domain receptor (LESTR) [22], or fusin [23]. All studies revealed a single open reading frame (ORF) of 352 amino acids that shared features common to many other 7-transmembrane G protein-coupled receptors (also known as serpentine receptors), such as the human interleukin-8 (IL-8) receptor (36–37% homology), and bovine NPY receptor (92–93% homology). Bleul et al. and Oberlin et al. reported that SDF-1α is a ligand for this receptor, which they referred to as CXCR4 [24, 25]. Both groups showed that binding of SDF1α to it’s receptor CXCR4 blocked the infection by lymphocyte-tropic HIV-1 strains in vitro. Similar results were obtained by Feng et al. showing that blocking of the CXCR4 receptor using anti-CXCR4 antibodies strongly inhibited HIV-1 infection of normal human CD4+ target cells [23]. Moreover, transient expression of the CXCR4

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gene allowed non-human cells co-expressing recombinant CD4 to undergo EnvCD4-mediated cell fusion and productive HIV-1 infection, revealing that CXCR4 acts as a cofactor for HIV-1 infection in CD4+ T cells. CXCR4 is expressed in a variety of tissues, including brain, endothelial and epithelial cells, tissues of hematopoietic origin [21], various tumor cells [26] and even cancer stem cells [27]. Studies performed with CXCR4 knock-out mice revealed that CXCR4 is required for several physiological processes during late embryonic and early postnatal stages, as well as in the adult. CXCR4-deficient mice exhibited hematopoietic and cardiac defects [28], or died perinatally with defects in both the hematopoietic and nervous system [29]. Tachibana et al. showed that CXCR4 is essential for vascularization of the gastrointestinal tract [30]. Similar observations were made with SDF-1α deficient mice, highlighting the importance of the SDF-1α/CXCR4 signaling. Additionally, a reduced B-lymphopoiesis and myelopoiesis were observed in the fetal liver, whereby bone marrow myelopoiesis was completely ablated in these mice. Both groups showed that fetal cerebellar development was markedly different compared to wild type animals. For instance, numerous proliferating granule cells abnormally invaded within the cerebellar anlage.

4.2.3 SDF-1α/CXCR4 Signaling The interaction between SDF-1α and its receptor CXCR4 plays a pivotal role in regulating the retention, migration, and mobilization of HSPCs during steady state homeostasis and injury [31]. Administration of anti-CXCR4 antibodies prevented the engraftment of murine bone marrow by human SCID repopulating stem cells [32], whereas cytokine mediated CXCR4 up-regulation led to increased SDF-1α mediated migration, in vivo homing, and repopulation of HSCs [32–34]. Similarly, CXCR4 over-expression by human cord blood and mobilized peripheral blood CD34+ cells by lentiviral gene transfer resulted in an increased proliferation, SDF-1α mediated migration, and bone marrow engraftment of these cells [35, 36]. In addition to HSPCs, activation of the SDF-1α/CXCR4 axis also initiates the migration of lymphocytes [37]. Moreover, within the past years it became evident that the progression and organ-specific metastatic spreading of various cancer types is associated with the SDF-1α/CXCR4 axis [38, 39], which acts a navigation system for circulating tumor cells [26]. Cell migration is an essential component of both successful mobilization and homing of HSPCs. However, cell migration is also a complex process, which is directed by the interplay of several signal transduction pathways initiated by various ligands, such as cytokines, chemokines, and extracellular matrix components that activate growth factor receptors, chemokine receptors and integrins [40]. The SDF-1α induced chemotaxis in HSPCs is inhibited by pertussis toxin, indicating that CXCR4 is associated with a Gαi -protein subtype [41–43]. Binding of SDF-1α to CXCR4 activates several signal transduction cascades including the PI3-kinase (PI3K)/Akt pathway, the phospholipase C-γ (PLC-γ)/protein kinase C

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(PKC) pathway and the MAPKp42/44 (ERK-1/2) pathway [44, 45]. Studies on human T cell lines indicated that SDF-1α triggers CXCR4 dimerization and activates the JAK/STAT pathway, which suggests gene regulation [46]. Likewise, Ganju and colleagues reported that SDF-1α treatment led to increased NF-κB activity in nuclear extracts of CXCR4 transfectants, indicating that changes of the gene expression level can be initiated via two independent signal transduction pathways downstream of the SDF-1α/CXCR4 axis [44]. By contrast, in factor-dependent MO7e cells NF-κB did not appear to be involved in SDF-1α actions [47]. The actin cytoskeleton is one of the central mechanical components responsible for the motility of cells, and it’s analysis is an effective method of determining a migratory phenotype. Actin polymerization in migration is induced by the PI3K/Akt signaling and the PLC-γ/PKC cascade in a variety of cell types [48–51], including HSPCs. So far several groups have convincingly demonstrated that SDF-1α induces actin polymerization [42, 52] as well as tyrosine phosphorylation of several components of focal adhesion complexes such as paxillin, the related adhesion focal tyrosine kinase (RAFTK/pyk2), p130cas , Crk-II, and Crk-L [44, 45]. Inhibition of PI3K signaling using wortmannin partially inhibited the SDF-1α induced migration and tyrosine phosphorylation of paxillin [44], further underpinning the role of PI3K/Akt signaling in cell migration. In a recent study by Petit and co-workers, the SDF-1α mediated cell polarization, adhesion to bone marrow stromal cells, and chemotaxis of human CD34+ progenitor cells were all shown to be PKC-ζ-dependent [53]. PKC-ζ belongs to the group of atypical PKC isoforms, which activation does not depend on calcium or diacylglycerole (DAG) [54, 55]. Petit and colleagues identified PI3K as an activator of PKC-ζ, and Pyk-2 and MAPKp42/44 (ERK-1/2) as downstream targets of PKC-ζ [53]. In vivo studies showed that the engraftment, but not homing, of human CD34+ HSPCs was also PKC-ζ dependent [53]. Cancelas and colleagues were recently able to demonstrate that a post engraftment deletion of the Rho GTPases Rac1 and Rac2 led to a massive mobilization of HSPCs from the bone marrow. Rac1(-/-) mutants were not able to rescue hematopoiesis after transplantation, but a deletion of Rac1 did not prevent steady state hematopoiesis. The authors were thus able to demonstrate that Rac proteins regulate HSC motility in both homing and mobilization [56].

4.3 Homing of HSPCs to Bone Marrow and Organs Lapidot defined “homing” as a descriptive term used for the crossing of circulating HSPCs across the blood/bone endothelial barrier into the bone marrow compartment within a fairly short time span of hours to days. Thereby, the successful reconstitution of hematopoiesis is used as a read-out for successful homing [57]. In addition to homing to the bone marrow, also the recruitment of HSPCs to injured tissue is referred to as “homing” [33].

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Homing of HSPCs is a multi-step process requiring the interplay of adhesion molecules, cytokines and chemokines, and extracellular matrix degrading proteases. Extravasating cells have to adhere to the vascular endothelium, to transmigrate across the endothelial lining and the underlying basement membrane, and migrate into the surrounding tissue [26]. In fact, HSPC extravasation is similar to the transendothelial migration of leukocytes/lymphocytes [58] and metastasizing tumor (stem) cells [26, 59]. The capturing of circulating cells by the endothelium and the subsequent rolling phase is facilitated by E- and P-selectins [60, 61]. Subsequent firm adhesion is mediated through intercellular adhesion molecule-1 (ICAM-1)/leukocyte function-associated antigen-1 (LFA-1) and vascular adhesion molecule-1 (VCAM1)/very late antigen-4 (VLA-4; (α4 β1 integrin) ligand pairs [61]. The importance of VCAM-1/VLA-4 interactions have been demonstrated by the use of a blocking anti-α4 -antibody; results showed that HSC homing was inhibited by more than 90% [62, 63]. Similar results were achieved with hypomorphic VCAM-1 mice with domain 4 deletion (D4D) and low expression of VCAM-1. Experiments with the latter showed that homing was virtually abrogated when the animals were treated with anti-VCAM-1 antibody and were given anti-CD11a-treated cells [63]. However, β2 integrins and selectins may be used as a fall-back if the dominant VLA-4/VCAM-1 (α4 β1 /VCAM-1) interaction is compromised [63]. Furthermore, α4 -integrins have been shown to selectively mediate the homing of cells to the bone marrow, but not to the spleen (as was demonstrated in transplantation studies using α4 -integrin (-/-) cells) [64]. VLA-4 does not only play a role during extravasation of HSPCs (and other cells), but is also a key adhesion molecule, which facilitates the cell-to-cell contact between HSPCs and bone marrow stroma cells [65]. Thus, inhibition of VLA4 function by use of the blocking anti-α4 -antibody does not only impair HSPC homing [62, 63], but also result in increased numbers of circulating HSPCs [66]. For instance, patients with multiple sclerosis (MS) who were treated with the anti-VLA-4 antibody natalizumab had a significantly higher amount of circulating CD34+ cells (median proportion of 7.6 CD34+ cells/μl) as compared with healthy volunteers (median proportion of 1.4 CD34+ cells/μl) and untreated MS patients median proportion of 1.0 CD34+ cells/μl) [67]. Whether natalizumab might be an alternative mobilizing compound for patients with a poor response to granulocytecolony stimulating factor (G-CSF)-based protocols [67] is currently under investigation. A key molecule in the process of HSPC homing is SDF-1α, which is expressed by bone marrow stroma cells as well as by endothelial/epithelial cells in close proximity to injured tissue. The latter was demonstrated by Kollet and coworkers showing that both irradiation and inflammation led to elevated SDF-1α expression in liver bile and duct epithelial cells, which was correlated with a SDF-1α dependent recruitment of HSPCs to the liver [33]. As mentioned above, the recruitment of CXCR4-positive HSPCs to regenerating tissues is mediated by the hypoxic gradient, namely via HIF-1 induced expression of SDF-1α in endothelial cells [19]. The up-regulation of SDF-1α in ischemic tissue is directly proportional to reduced

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oxygen tension, and was correlated with increased adhesion, migration, and homing of circulating HSCs to ischemic tissue. In addition to its function as a chemoattractant, thereby recruiting circulating HSPCs from peripheral, SDF-1α also triggers HSPCs’ firm adhesion to the endothelium by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1 [68]. Likewise, several studies have shown that also various cytokines such as granulocyte/macrophage-colony stimulating factor (GM-CSF), Interleukin-3 (IL-3) and stem cell factor (SCF) temporarily increase the adhesiveness of HSCs by activating the β1 -integrins VLA-4 and VLA-5. Furthermore, cytokines (Flt3-ligand, SCF, IL-3, IL-6, and hepatocyte growth factor (HGF)) up-regulate CXCR4 expression on HSPCs in vitro and in vivo [33, 68, 69], thereby enhancing the intracellular signals generated through the SDF-1α/CXCR4 axis [47]. Ultimately, the expression and secretion of extracellular matrix-degrading enzymes, especially those capable of degrading type IV collagen such as MMPs [70], is stimulated by various chemokines and cytokines. Degradation of the basement membrane is a prerequisite for transendothelial migration. Both MMP-2 and MMP-9 are known to be expressed by all extravasating cell types including leukocytes [71], tumor cells [70, 72], and HSPCs [31, 73]. Two recent studies by Zheng and colleagues showed that CD34+ HSC from cord blood exhibited significantly lower expression levels of CD49e, CD49f, CXCR4 as well as MMP-2 and MMP9, compared to their counterparts from peripheral blood and bone marrow, which may be a reason for delayed hematopoietic reconstitution after umbilical cord blood transplantation [74, 75]. However, upon incubation with recombinant human SCF, these cells gained increased expression of homing related molecules, including CXCR4 and MMP-2/-9, and showed an increased ex vivo transmigratory and in vivo homing potential [74, 75].

4.4 Influence of Culture Conditions on the Migration of Human CD34+ /CD133+ Cord Blood HSPCs In a previous work we have already demonstrated that human CD34+ /CD133+ cord blood HSPCs showed a differential migratory behavior if cultivated for up to five days in the presence of Flt3-ligand alone or in the presence of a combination of Flt3-ligand and Interleukin-6 (IL-6) [76]. Cell migration of such cultured cells was analyzed by using the 3-dimensional (3D) collagen matrix migration assay, which allows for a detailed analysis of cell movement on a single cell level [76–78]. Due to the continuous monitoring of migrating cells, various cell migration parameters (the so-called migration pattern) could be estimated, such as the time of active movement, the number of moving cells, the speed of migrating cells, as well as the number and the length of pauses the cells’ have made [76–78]. Our previous data showed that the SDF-1α induced migratory activity of solely Flt3-ligand cultured human CD34+ /CD133+ cord blood HSPCs was attributed to an increased number

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of moving cells, whereas the SDF-1α induced locomotory activity of Flt3-ligand/IL6 cultured human CD34+ /CD133+ cord blood HSPCs was attributed to both an increased number of moving cells and an altered migration pattern, e.g., due to an increased time of active movement [76]. The expression of CXCR4 on HSPCs is very dynamic and known to be regulated by both cytokines and chemokines. For instance, exposure of cord blood derived and mobilized peripheral blood derived CD34+ HSPCs to SCF or a combination of SCF and IL-6 led to a CXCR4 up-regulation, concomitant with increased motility of SCID repopulating cells (SRC) [32]. Similar results were achieved with HGF, alone or in combination with SCF [33]. Sorted CD34+ CXCR4– HSPCs express intracellular CXCR4, which can be up-regulated and expressed on the cell surface in response to stimulation with 5 cytokines (SCF, Flt3-ligand, IL-6, IL-3, and G-CSF), thereby converting these cells into definitive SRC with high CXCR4 expression levels (both intra-cellular and cell surface) and SDF-1α induced migration [79]. In contrast, our findings showed that incubation of human CD34+ /CD133+ cord blood HSPCs with Flt3-ligand alone or in combination with IL-6 for 5 days resulted in a comparable CXCR4 down-regulation in both populations [78]. Because IL-6 signaling induces the transcription of target genes, via the JAK/STAT pathway [80], we assumed that the differential SDF-1α mediated migratory activity of solely Flt3-ligand and Flt3ligand/IL-6 cultivated CD34+ /CD133+ cord blood HSPCs might be attributed to an altered gene expression level. In a first set of experiments we investigated the role of PKC-α in the SDF-1α dependent migration of cord blood CD34+ /CD133+ HSPCs cultured either in the presence of Flt3-ligand or Flt3-ligand/IL-6. PKC-α was chosen since this PKC isoform has been implicated in a variety of cellular functions including cell migration [81]. Cell migration data revealed that the migratory activity of both Flt3-ligand and Flt3-ligand/IL-6 cultivated cord blood CD34+ /CD133+ HSPCs was markedly inhibited by Gö6976 (a specific PKC-α inhibitor [82]) treatment on day 1 (Fig. 4.1a). This is in agreement with confocal laser scanning microscopy data showing that in day 1 cells PKC-α is co-localized with collagen fibers (Fig. 4.1d). By contrast, analysis of the locomotory behavior of cord blood CD34+ /CD133+ HSPCs cultured for 5 days with either Flt3-ligand alone or in combination of Flt3-ligand/IL-6 revealed a different migratory phenotype. For solely Flt3-ligand cultured cord blood CD34+ /CD133+ HSPCs we noticed only a slightly decreased migratory activity in the presence of the PKC-α inhibitor Gö6976 (Fig. 4.1b), whereas for day 5 Flt3ligand/IL-6 cultured cord blood CD34+ /CD133+ HSPCs cells no inhibitory effect of Gö6976 was observed (Fig. 4.1b). In fact, the migratory activities of untreated and Gö6976 treated as well as SDF-1α and SDF-1α/Gö6976 treated Flt3-ligand/IL-6 cultured cord blood CD34+ /CD133+ HSPCs were virtually identical. Interestingly, Western Blot analysis revealed comparable PKC-α expression levels in both Flt3-ligand and Flt3-ligand/IL-6 cultured cord blood CD34+ /CD133+ HSPCs on both day 1 and day 5 (Fig. 4.1c). These findings indicate that the involvement of PKC-α in the process of cell migration is altered by the prolonged culture period of 5 days. Although PKC-α expression is clearly detectable in both Flt3-ligand and Flt3-ligand/IL-6 cultured cord blood CD34+ /CD133+ HSPCs after

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Fig. 4.1 Migratory activity and PKC-α expression of Flt3-ligand and Flt3-ligand/Interleukin-6 cultured cord blood CD34+ /CD133+ HSPCs. (a) day 1, (b) day 5. Flt3-ligand (F) cultured cells appear in white; whereas Flt3-ligand/Interleukin-6 (FI) cultivated cells appear in grey. Statistical significance (paired t-test): n.s. = not significant, ∗ = p<0.05, ∗∗ = p<0.01, ∗∗∗ = p<0.001. (c) PKC-α expression level of Flt3-ligand (F) and Flt3-ligand/Interleukin-6 (FI) cultured cells is shown in relation to the β-actin control. (d) Confocal laser scanning microscopy data of day 1 cord blood CD34+ /CD133+ HSPCs embedded within a 3D-collagen matrix environment. Green: PKC-α, red: β-actin, blue: collagen fibers. Colocalization of PKC-α and collagen fibers are marked by arrows

5 days of culture (Fig. 4.1c), it seems that this molecule does not longer play a key role in the migratory activity of these cells. Otherwise, one would expect a more profound inhibitory effect of Gö6976. In any case, the molecular processes causing the differential Gö6976 dependent migratory phenotype of cultured cord blood CD34+ /CD133+ HSPCs remain unknown. Fukuda and colleagues demonstrated recently that cultivation of cord blood CD34+ cells for 48 h with Flt3-ligand leads to a differential phosphorylation kinetics of intracellular signaling molecules such as MAPKp42/p44 or Akt after SDF-1α stimulation [83]. Thus the observed differences in the migratory activity of cultured cord blood CD34+ /CD133+ HSPCs might be attributed to a differential kinetics of PKC-α and/or signal transduction molecules/pathways downstream of PKC-α. It is also conceivable that other signaling molecules than PKC-α mediate cell migration of 5 days F and FI cultured CD34+ cells. For instance, Petit et al. reported recently that the SDF-1α mediated migration and development of human CD34+ progenitor cells is regulated by the atypical PKC-ζ [53].

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4.5 Influence of Culture Conditions on the Migration of Murine Lin- c-kit+ HSPCs In addition to studies with human cord blood HSPCs we also performed cultivation and migration experiments with murine HSPCs, which can be isolated from murine bone marrow and cultivated by supplementation of a variety of hematopoietic cytokines, such as Flt3-ligand, SCF, thrombopoietin (TPO), IL-3, IL-6, and Interleukin-11 (IL-11) [84–89]. Previous studies already showed that supplemented cytokines/cytokine combinations might have severe effects on the homing capacity of cultured murine HSPCs. For instance, cultivation of murine Lin- Sca-1+ HSPC in the presence of IL-3, IL-6, IL-11 and SCF for 48 h resulted in reported a cytokine induced defect in homing to the bone marrow and engraftment potential [90]. Similar results were obtained for human CD34+ HSPC that have been exposed for 48 h to Flt3-ligand, SCF, IL-3, and IL-6 and which then showed an irreversibly impaired homing to the bone marrow [91]. In a more comprehensive study, von Drygalski and coworkers performed transplantation studies using murine bone marrow cells that have been cultured with three different cytokine cocktails ([Flt3ligand, SCF, TPO], [Flt3-ligand, SCF, TPO, IL-11], and [Flt3-ligand, SCF, TPO, IL-11, IL-3]) [92]. Compared to freshly isolated cells all cultured murine bone marrow cells showed a poorer donor engraftment concomitant with markedly decreased 7 months survival rates [92]. To analyze the influence of cytokines/cytokine cocktails on the migratory behavior of murine Lin- c-kit+ HSPCs these cells were cultured for up to five days in the presence of twelve combinations of the hematopoietic cytokines Flt3-ligand, SCF, TPO, and IL-11 [93]. As expected, each cytokine/cytokine combination had a distinct effect on the proliferation as well as the migratory activity of murine Lin- c-kit+ HSPCs. For instance, cells cultivated with all four cytokines showed the highest proliferation rate concomitant with the second highest response to SDF1α (Fig. 4.2). By contrast, the removal of only one factor, in this case Flt3-ligand, gave rise to cells exhibiting a totally different migratory phenotype. Murine Linc-kit+ HSPCs cultivated in the presence of SCF, TPO, and IL-11 showed the third highest proliferation rate of all tested cytokine/cytokine combinations, but did not respond to SDF-1α stimulation with an increased locomotory activity (Fig. 4.2). The supposed non-responsiveness of SCF, TPO, and IL-11 (and Flt3-ligand and IL-11) cultured murine Lin- c-kit+ HSPCs (as well as the low SDF-1α susceptibility of so-called low-responder murine Lin- c-kit+ HSPCs) was not attributed to different CXCR4 expression levels (Fig. 4.2). Comparable CXCR4 expression levels (both intracellular and surface bound) concomitant with detectable increased cytosolic calcium levels after SDF-1α stimulation were detectable in all murine Lin- c-kit+ HSPCs irrespective of used cytokines/cytokine combinations (Fig. 4.2). Moreover, the migration pattern of SCF, TPO, and IL-11 cultured murine Lin- c-kit+ HSPCs showed that both the amount of moving cells and the time of active movement were significantly decreased upon SDF-1α stimulation (Fig. 4.2). This indicates that SCF, TPO, and IL-11 cultured murine Lin- c-kit+ HSPCs were able to respond to the SDF1α signal, but that the intracellular processing of the SDF-1α signal did not result in cell migration activity.

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Fig. 4.2 Modulation of murine Lin- c-kit+ HSPCs by culture conditions. Lin- c-kit+ HSPC were cultivated for 5 days in the presence of the indicated cytokines/cytokine combinations. Subsequently, the proliferation rate, the migratory activity and CXCR4 expression of cultured cells was determined. The cytokines/cytokine combinations were grouped according to the differences ( migration rate [MR]) between the spontaneous and SDF-1α-induced migration of murine Lin- c-kit+ cells. Cytokines: F: Flt3-Ligand, S: SCF, T: TPO, I: IL-11. Significance of differences between spontaneous and SDF-stimulated migration was analyzed using paired Student’s t-test. Statistical significance: n.s. = not significant, ∗ = p<0.05, ∗∗ = p<0.01. (a) Proliferation rate of cultured cells. (b) Migratory activity of cultured HSPC with (grey) and without (white) SDF-1α-stimulation. (c) Percentage of moving cells. (d) Time of active movement (time active [%]). Extracellular and intracellular expression levels of CXCR4 were determined by flow cytometry. Total cellular levels of CXCR4 expression were determined by western blot analysis. After densitometric evaluation, CXCR4 expression was calculated in relation to expression of β-actin

In analogy to the above mentioned PKC-α phenomenon we also found that the differences in the migratory SDF-1α response of cultured murine Lin- c-kit+ HSPCs were not related to differential expression levels, but rather to the differential engagement of the CXCR4 dependent MAPKp42/44 and PI3K signal transduction pathways [93]. This suggests a strong influence of different cytokines and cytokine

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combinations on the kinetics of signal processing downstream of CXCR4. Cell migration data revealed that in cells exhibiting an intermediate to low increase of migratory activity in response to SDF-1α, the PI3K and MAPKp42/44 pathways were not involved in the SDF-1α induced migration, while in cells exhibiting a high response to SDF-stimulation, PI3K and MAPKp42/44 were crucial for SDF1α directed migration (Fig. 4.3). However, Western Blot data clearly showed that both Akt and pAkt, as well as MAPKp42/44 and phosphorylated MAPKp42/44 were clearly detectable in untreated and SDF-1α stimulated cells (data not shown). Thus, cultivation of murine Lin– c-kit+ HSPCs with various cytokines/cytokine combinations likely caused alterations in signal transduction cascades rather than causing alterations on a gene expression level. This would be in accordance with the above presented data that cultivation of cultured cord blood CD34+ /CD133+ HSPCs for 5 days resulted in a switch from a PKC-α dependent migration to a PKC-α independent migration although PKC-α expression remained unchanged in these cells [78]. Moreover, Fukuda and colleagues demonstrated previously a differential phosphorylation of key signal transduction proteins induced by Flt3-ligand treatment in human CD34+ HSPC [52]. Thereby, SDF-1α and Flt3-ligand synergistically enhanced the phosphorylation of MAPKp42/44 , cyclic adenosine monophosphate response element binding protein (CREB) and Akt, which was correlated with an increased migratory activity of these cells [52]. By contrast, a prolonged exposure of CD34+ HSPCs to Flt3-ligand was associated with a CXCR4 down-regulation concomitant with a reduced SDF-1α-mediated phosphorylation of MAPKp42/44 , CREB and Akt, as well as an impaired SDF-1α mediated migration [52]. In summary, cytokines/cytokine combinations commonly used for HSPC cultivation seem to influence the migration of hematopoietic cells in many different ways, which might depend on the combination with other cytokines, cell types, incubation time, etc. Although our data suggest that cytokines/cytokine combination rather alter the kinetics of signal transduction pathways, we can not rule out that also the gene expression pattern of cultured HSPCs is altered since most cytokines, if not all, do also lead to the activation of transcription factors. Nonetheless, the finding that cytokines/cytokine combinations markedly influence the SDF-1α-mediated migratory behavior of HSPCs should be considered if such cells will be expanded for transplantation purposes since the therapeutic success is fundamentally dependent on the migration and homing capacity of transplanted HSPCs to bone marrow.

4.6 Termination of the SDF-1α Induced Migration of HSPCs It is remarkable that in cell migration studies mostly factors are investigated that induce cell migration. While this is definitely of interest for several purposes, such as the role of growth factors and chemokines in tumor cell migration, which is a prerequisite for metastasis formation [26] as well as leukocyte/lymphocyte and HSPC migration [51, 65], considerably less is known about molecules that terminate the migration of these cells. Nonetheless, the success of e.g., bone marrow repopulation after HSPC transplantation or immune responses does not only depend on

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Fig. 4.3 Cytokines influence CXCR4-related signal transduction pathways. (a) PI3K inhibitor Wortmannin 100 nM, (b) MAPK inhibitor PD98059 50 μM. Data are means of at least 3 independent experiments. Cytokines: F: Flt3-Ligand, S: SCF, T: TPO, I: IL-11. Statistical significance (paired Student’s t-test): n.s. = not significant, ∗ = p<0.01 (in relation to control), † = p<0.01 (in relation to SDF-1α)

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the induction of cell migration, which is a prerequisite to recruit cells to the particular tissues, but also to the inhibition of migration once the cells have reached the desired tissues. For instance, once HSPCs have homed to the bone marrow or injured tissue, respectively, they have to stop their migratory activity to settle down in their niche or to initiate tissue regeneration, respectively. Likewise, immunocompetent cells, such as neutrophil granulocytes and/or monocytes/macrophages have to terminate their migratory activity once they have reached inflamed tissue in order to eliminate invaded pathogens. In a previous report Lang and colleagues demonstrated that the chemokine Interleukin-8 (IL-8) is not only a potent promigratory compound, but that this factor is also an inhibitor of cell migration [94]. Thereby, IL-8 dose dependently increased the frequency and the duration of stop-periods of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophil granulocytes [94]. Because IL-8 has also been shown to be stimulatory chemoattractant for HSPCs [95] the question arose whether IL-8 in conjunction with SDF-1α do also inhibit the locomotory activity of adult CD34+ /CD133+ HSPCs. In fact, both IL-8 and SDF-1α alone stimulate the migratory activity of adult CD34+ /CD133+ HSPCs (Fig. 4.4a). However, compared to SDF-1α stimulation alone the locomotory activity of adult CD34+ /CD133+ HSPCs was markedly decreased in the presence of both SDF-1α and IL-8 (Fig. 4.4a). This effect was not attributed to a reduced number of moving cells, but rather to a decreased time of active movement (or an increased average pause length), which is similar to findings of Lang and colleagues. Whether IL-8 inhibits the SDF-1α induced migration of adult CD34+ /CD133+ HSPCs by a similar mechanism as IL-8 blocks the fMLP induced migration of neutrophil granulocytes is not yet clear. Nonetheless, these data show that IL-8 is capable to impair the SDF-1α induced migration of adult CD34+ /CD133+ HSPCs, which might play a role in the termination of these cells. In addition to IL-8, the migration of adult CD34+ /CD133+ HSPCs is also markedly blocked by the neurotransmitter γ-aminobutyric acid (GABA), which is mainly associated with neuronal tissues (Fig. 4.4b). Unfortunately, considerably less is known about GABA receptor expression and function in non-neuronal tissues. However, in a recent study Rane and colleagues demonstrated that GABAB receptors stimulate the chemotaxis of neutrophil granulocytes via PI3K/Akt signaling during ischemia reperfusion [96]. Whether this process might also play a role in the recruitment of neutrophil granulocytes into non-neuronal tissues is not clear. Nonetheless, these data suggest an additional function for GABA. In accordance with neutrophil granulocytes, GABAB -receptor expression was also detected on adult CD34+ /CD133+ HSPCs [77, 97], whereas GABA markedly blocked both the spontaneous and SDF-1α induced migration of these cells [77]. Functional characterization of distinct migration parameters revealed that the inhibitory effect of GABA on the spontaneous and SDF-1α-stimulated migration of HSPC was primarily attributed to a decreased time of active movement (Fig. 4.4b) [77]. We also noticed a slightly, but not significantly reduced amount of moving cells in the presence of GABA (Fig. 4.4b). Mechanistically, GABA impairs the SDF1α induced calcium influx most likely via inhibition of calcium release activated calcium (CRAC) channels [77]. However, whether GABAB -receptor signaling

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Fig. 4.4 Termination of SDF-1α induced HSPC migration by IL-8 and GABA. (a) Cell migration data for SDF-1α and IL-8. (b) Cell migration data for SDF-1α and GABA. Both, IL-8 and GABA, inhibits the SDF-1α induced migration of human adult CD34+ /CD133+ HSPC by affecting the time of active movement, but not the number of moving cells. Shown are the means of at least three experiments. Statistical significance (paired Student’s t-test): n.s. = not significant, ∗ = p<0.01 (in relation to control), † = p<0.01 (in relation to SDF-1α)

directly interacts with CRAC channels, thereby inhibiting it or whether GABAB receptor signaling indirectly impairs CRAC channel function due to interaction with the CXCR4 signal transduction cascade remains unknown. As indicated above, considerably less is known about GABAB -receptor induced signal transduction cascades.

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In summary, these data show that factors exist that inhibit the SDF-1α induced migration of adult CD34+ /CD133+ HSPCs. IL-8 is a well-known chemokine, which recruits leukocytes to areas of inflammation. However, in combination with other compounds, such as fMLP and SDF-1α, IL-8 has an inhibitory effect on locomoting cells suggesting that IL-8 might function as a migration terminating factor, thereby regulating the motility of immunocompetent cells (and possibly HSPCs) in inflamed tissues. Likewise, the neurotransmitter GABA is a potent inhibitor of HSPC migration, whereby the role of GABA in non-neuronal tissues still remains ambiguous. However, the finding of Rane and colleagues that GABA recruits neutrophil granulocytes [96] might suggest a putative role for GABA in inflammatory conditions.

4.7 Conclusion The ability to migrate is a prerequisite for HSPCs to successfully home to both bone marrow and injured tissue. Our data show that the inherent migratory activity of HSPCs is modulated by cytokines and chemokines, which are used for HSPC cultivation. Thereby, cytokines/cytokine combinations exist, which might have a favorable effect on the cells’ migratory behavior. On the other hand, the removal of only one factor from a “high-responder” cytokine cocktail can yield in a “non-responder” cytokine mix. Together with the findings that cytokines/cytokine combinations rather alter the kinetics of signal transduction cascades instead of its expression levels our data substantiate the complexity of the cellular and systemic regulation of the migration of HSPCs. We conclude from our data that they are useful for those who are still working in the field of HSPC ex vivo expansion. As shown here, a good proliferation rate does not necessarily correlate with a good SDF-1α responsiveness. Our findings are also of interest if HSPC specific signal transduction cascades and its kinetics are analyzed. For instance, PKC-α is expressed on both day 1 and day 5 cultured cord blood HSPCs, but only the migration of day 1 cells depends on this PKC isoform. Likewise, the PI3K/Akt pathway is involved in the SDF-1α induced migratory activity of high-responder murine HSPCs, but not in the SDF-1α induced migratory activity of low-responder murine HSPCs, although phosphorylated Akt is clearly detectable upon SDF-1α stimulation in both responder types. The knowledge of factors that terminate HSPC migration might be of interest for regenerative medicine purposes. It is conceivable that co-application of HSPCs with the well-known GABAB -receptor agonist baclofen to areas of interest (e.g., injured myocardium) might enhance the number of HSPCs that remain in the organ, thereby possibly increasing the HSPC mediated tissue regeneration efficacy. However, this has to be investigated in further studies. In conclusion, the elucidation of HSPC migration mechanisms is of crucial interest for the future use of this cell type in the field of regenerative medicine. Acknowledgments This work was supported by the Fritz-Bender-Foundation, Munich, Germany.

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Chapter 5

Properties of Mesenchymal Stem Cells to Consider for Cancer Cell Therapy John Stagg and Sandra Pommey

Abstract Ex vivo cultured mesenchymal stem cells (MSCs) are being investigated for regenerative and cell-based therapy. The observation that human MSCs possess tumor-homing properties has generated a great deal of interest in using MSCs as carriers of anti-cancer biotherapeutics. However, MSCs possess intrinsic properties that may significantly affect the nature of developing tumors. Therefore, understanding these interactions between MSCs and tumor cells will be essential if MSCs are to be used for cancer therapy. In this chapter, we firstly review the cell surface antigens expressed by MSCs and discuss a newly described method to reduce the risk of emboli associated with MSCs infusion. Secondly, we review the literature on the identified molecular pathways governing MSCs migration, including the role of tolllike receptors and death receptors. Thirdly, we present an overview of the biological properties of MSCs that affect tumor survival, metastasis and immune responses. Finally, we describe various approaches to engineer MSCs in order to generate efficient anti-cancer cell therapies, including gene modification to express anti-cancer cytokines and infection with oncolytic viruses, and means to redirect the tropism of MSCs to specific microenvironments. Keywords Mesenchymal · Stem cell · Cancer · Gene therapy · Cell therapy · Tumor homing · Chemokine · Oncolytic virus · Immunosuppression

Contents 5.1 5.2 5.3 5.4 5.5

Identifying MSCs . . . . . . . . . . . . . . Tissue Origin of MSCs . . . . . . . . . . . . Induced Pluripotent Stem Cells and MSCs . . . MSC Homing in Tissue Repair and Oncogenesis MSCs Tumor Tropism and Pro-metastatic Effects

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80 Chemokine Receptors Regulating MSCs Homing . . . . . Growth Factor Receptors Regulating MSCs Homing . . . . Toll-Like Receptors and Death Receptors in MSC Migration Oncogenic Potential of MSCs . . . . . . . . . . . . . . Immunosuppressive Effects of MSCs . . . . . . . . . . . 5.10.1 T Cells . . . . . . . . . . . . . . . . . . . . . 5.10.2 NK Cells . . . . . . . . . . . . . . . . . . . . 5.10.3 DCs . . . . . . . . . . . . . . . . . . . . . . 5.10.4 B Cells . . . . . . . . . . . . . . . . . . . . . 5.11 Modulation of MSCs Immunosuppression . . . . . . . . 5.12 MSCs for Cellular Gene Therapy . . . . . . . . . . . . 5.13 Retroviral Vectors for MSC Gene Transfer . . . . . . . . 5.14 Electroporation for MSC Gene Transfer . . . . . . . . . 5.15 Ex Vivo Reprogramming of MSCs Without Gene Transfer . 5.16 Cytokine-Producing MSCs for Cancer Cell Therapy . . . . 5.17 Oncolytic Virus-Infected MSCs . . . . . . . . . . . . . 5.18 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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5.1 Identifying MSCs Mesenchymal stem cells (MSCs) are non-hematopoietic progenitor cells involved in the maintenance and regeneration of connective tissues. MSC can be isolated from adherent bone marrow cell cultures following Friedenstein’s technique and can be induced to differentiate into a variety of cell lineages including osteocytes, chondrocytes, adipocytes, myocytes, astrocytes, neurons, endothelial cells and lung epithelial cells [1–4]. Although MSCs are routinely isolated from the bone marrow, most adherent cells from bone marrow aspirates do not meet the criteria of a stem cell. As such, unfractionated adherent cells from bone marrow aspirates should be referred to as mesenchymal stromal cells in accordance with the consensus recommendation of the International Society for Cellular Therapy [5]. Other reviews have extensively described techniques to isolate and expand primary MSCs [6–10]. MSCs are generally identified based on their ability to differentiate into mesenchymal lineage cells (e.g. adipocytes, chondrocytes and osteocytes). It is important to note that each individual bone marrow-derived MSC colony can display variable degree of plasticity [2]. However, as a whole, bone marrow-derived MSCs express specific membrane-bound surface antigens, including CD44 (hyaluronate receptor), CD73 (SH3/SH4; ecto-5 -nucleotidase), CD105 (SH2; endoglin) and CD166 (ALCAM). The absence of CD45, CD34, CD31, Mac1 (CD11b), CD19 and glycophorin A expression distinguishes MSCs from hematopoietic cells, endothelial cells, endothelial progenitors, monocytes, B cells and erythroblasts, respectively. Although the monoclonal antibody Stro-1 has been historically used to enrich for MSCs, the Stro-1 antigen can also be found

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expressed on hematopoietic cells [11]. Recently, Gang et al. [12] identified the globo-series glycolipid SSEA-4 [13] as a new marker for MSCs. Purification of SSEA-4-expressing bone marrow cells was shown to give rise to MSCs cultures devoid of hematopoietic cells. Upon IFN-γ stimulation, mouse and human MSCs upregulate MHC class I and class II molecules but do not upregulate the costimulatory molecules CD80 and CD86 associated with professional antigen presenting cells [11]. Upon expansion, two morphologically distinctive adherent cell types can be observed in bone marrow cultures: small spindle-shaped and rapidly self-renewing cells referred to as RS-MSCs, and more mature slowly replicating larger cells referred to as SR-MSCs [14, 15]. Recently, the same group that described the presence of RS-MSCs and SR-MSCs identified six cell surface proteins selectively expressed on rapidly self-renewing RS-MSCs [16]. Using microarray analysis, they compared the mRNA levels of cell surface proteins expressed in low density MSC cultures (enriched in RS-MSCs) to those expressed by confluent MSC cultures (enriched in SR-MSCs). Podocalyxin-like protein (PODXL), alpha6-integrin (CD49f), alpha4-integrin (CD49d), c-Met, CXCR4, and CX3CR1 were all shown to be selectively upregulated on rapidly small self-renewing SR-MSCs. Most interestingly, the study demonstrated that MSCs sorted for PODXLhi /CD49hi expression were more efficient in generating single-cell derived colonies and engrafted more efficiently following intravenous infusion into mice. Another important aspect of the study is the observation that expression of PODXL prevented MSCs from aggregating, both in vitro and in vivo. Since MSCs aggregation and the ensuing risk of emboli is a major concern with MSC-based cell therapy, these studies suggest that MSCs enriched for PODXLhi /CD49hi expression may be safer to administer, especially via the intravenous route, than MSCs isolated from confluent cultures.

5.2 Tissue Origin of MSCs As we have discussed, MSCs have been traditionally isolated from bone marrow aspirates and, accordingly, have been considered to be essentially bone marrow resident progenitor cells [17]. However, since the original report that pluripotent MSCs can be isolated from the bone marrow, several studies have described the isolation and expansion of MSCs or MSC-like cells from various tissues including white adipose tissue, skin, pancreas, placenta, cord blood and skeletal muscle [18–21]. An interesting concept that has arisen is that all these multipotent MSC-like cells may actually share a common ancestor. A recent Cell Stem Cell paper by Crisan et al. [22] has shed some new light on this question. The authors demonstrated that human pericytes possess MSC-like progenitor potential irrespective of their tissue of origin. Pluripotent pericytes with MSC-like properties can indeed be sorted from diverse tissues in order to give rise to multilineage cells. Accordingly, tissue cultured perivascular cells sorted from diverse human tissues shared the cell surface markers associated with MSCs. When placed in appropriated conditions, cultures of perivascular cells were shown to differentiate into osteocytes, chondrocytes

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and adipocytes. This study proposes that MSCs, or ancestors thereof, are natively associated with blood vessel walls. These new observations might explain why multilineage progenitor cells with MSC-like properties can be isolated from multiple organs.

5.3 Induced Pluripotent Stem Cells and MSCs While the isolation and expansion of unmanipulated adult stem cells such as MSCs may allow for patient-specific stem cells to be generated for the study of human diseases, current protocols have failed to generate unmanipulated adult stem cells with embryonic stem (ES)-cell properties. In 2006, a groundbreaking study showed that mouse skin cells can in fact be reprogrammed back to the equivalent of ES cells by the transient expression of four transcription factors: Oct4, Sox2, Kfl4 and Myc [23]. This landmark study has since generated a lot of hype and opened the door to a new field of research: induced pluripotent stem cell (iPS cells). Through direct reprogramming, it is proposed that iPS cells will avoid the ethical controversies surrounding the use of embryos for deriving stem cells for medical research. In recent months, four studies have now reported that the introduction and expression of the same transcription factors, or a slightly modified combination of them, can also trigger the reprogramming of differentiated human cells into iPS cells [24–27]. In one of these studies [24], the authors tested whether primary MSCs could be efficiently reprogrammed into iPS cells. Surprisingly, in contrast with their results with fibroblasts, introduction of the transcription factors into MSCs resulted in slowed proliferation and cellular senescence and failed to generate iPS. However, when the catalytic subunit of human telomerase hTERT and SV40 large T were also introduced into MSCs, the cultures developed colonies with ES-cell-like morphology. Using a similar reprogramming strategy, the same group recently reported the generation of iPS cells from adult MSCs from a patient with Swachman-Bodian-Diamond syndrome, a congenital disorder characterized by exocrine pancreas insufficiency, skeletal defects and bone marrow failure [28]. Thus, while the generation of unmanipulated multipotent adult stem cells with ES properties has yet been reported, transcriptional reprogramming of human fibroblasts and MSCs into iPS cells has recently been achieved and constitutes an important new source of immortal human cells for medical research, thereby circumventing the controversial use of human embryos.

5.4 MSC Homing in Tissue Repair and Oncogenesis If primary MSCs are to be used for regenerative medicine or as cellular vehicle for the delivery of therapeutic payloads, it is important to understand the mechanisms that regulate their migration and tissue-homing properties. Proper harnessing of these processes will be paramount to the development of effective MSC-based

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therapies. It has long been hypothesized that MSCs can be recruited in response to tissue damage. However, the molecular pathways governing MSCs homing in response to tissue damage have only recently started to emerge. In the context of skin wound repair, a recent paper by Sasaki M. et al. [29] identified the release of the chemokine CCL21 by injured keratinocytes and the expression of its receptor CCR7 on MSCs as a major pathway governing the recruitment of intravenously injected MSCs to wounded skin. The study demonstrated that intradermal injection of recombinant CCL21 can significantly enhance the recruitment of intravenously injected MSCs to wounded skin area. Intradermal injection of CCL21 thus significantly accelerated wound closure following MSCs cell therapy. After being recruited to the site of injury, it appeared that MSCs could further differentiate into multiple skin cell types. Notably, it was observed that up to 33% of pericytes constituting the injury site were derived from green-fluorescent protein (GFP)-labelled MSCs. Given the study by Crisan et al. [22] suggesting a perivascular origin for MSCs, it is tempting to speculate that pericyte-derived MSCs actively contribute to skin wound repair. This study by Sasaki et al. thus suggests that MSCs may be recruited from their normal niche to participate in skin repair; however the actual demonstration that endogenous MSCs – in contrast to ex vivo expanded MSCs – behave in a similar fashion is still pending. Solids tumors can be considered like “wounds that never heal”, a concept introduced by Dvorak and colleagues more than two decades ago. Akin to wounds, tumors are often associated with continuous production of inflammatory cytokines and chemokines. In some cancer patients, this chronic inflammation significantly contributes to tumor growth. In other cancer patients, the immune response induced by cellular transformation can restrict tumor growth and shape the nature of the developing tumor, a concept known as cancer immunoediting [30]. Because the microenvironment of a tumor closely resembles the microenvironment of an injured tissue, and given the involvement of MSCs in tissue repair, it was hypothesized that MSCs may be recruited by growing tumors. Several independent groups have now clearly established that tumors can indeed recruit blood-circulating MSCs through the release of endocrine and paracrine factors. We will focus hereafter on recent developments in the field of MSCs tumor homing.

5.5 MSCs Tumor Tropism and Pro-metastatic Effects Since the initial observation that ex vivo expanded MSCs exhibit tumor tropism, several independent studies have now shown that human MSCs can be recruited to experimental human tumors, including breast carcinomas, colon carcinomas, ovarian carcinomas, gliomas, melanomas and Kaposi’s sarcomas, following intravenous injection in mice [31–35]. In few studies, MSCs have been shown to trigger antitumor effects. For instance, intravenously injected human MSCs were shown to home to human Kaposi s sarcomas in immunodeficient mice and to significantly delay tumor growth [36]. The anti-tumor effect of MSCs against Kaposi’s sarcomas is unclear but was shown to be partly dependent on E-Cadherin and the inhibition

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of AKT activation. Recently, Qiao L. et al. [37] reported that human MSCs can also inhibit the proliferation of human cancer cell lines through the downregulation of anti-apoptotic Bcl-2, possibly via the Wnt pathway. These anti-tumoral properties of MSCs are still not clear and warrant further investigation. In contrast to the few studies reporting anti-tumoral effects of infused MSCs, several studies have demonstrated that intravenously injected MSCs can enhance tumor growth and metastasis [32, 38–41]. Understanding – and perhaps developing means to inhibit – these pro-tumoral and pro-metastatic properties of MSCs will be of great importance if MSCs are to be used for cell therapy. One of the effect of MSCs on tumor cells may actually be to promote a proglycolytic phenotype (also known as the Warburg effect), giving them a survival advantage in hypoxic and inflammatory conditions. Indeed, Samudio et al. [42] recently demonstrated that MSCs can promote a metabolic shift in cancer cells mediated in part via the activation of mitochondrial uncoupling proteins. Such metabolic shift induced by the interactions between MSCs and tumor cells may enhance tumor growth and promote their resistance to chemotherapy. In addition, MSCs can promote tumor cell survival through the activation of integrin-linked kinase (ILK) on tumor cells. ILK links cell adhesion receptors to downstream prosurvival signaling pathways, such as AKT activation, and is significantly up-regulated in several types of cancers. Tabe et al. [43] demonstrated that the ILK-AKT pathway is a critical prosurvival pathway activated both leukemic cells upon interaction with MSCs. A recent research article by the Weinberg laboratory [32] demonstrated that human MSCs are particularly potent at enhancing the metastatic ability of human breast tumor cells in xenografted mice. Consistent with what had been previously reported, they observed that human MSCs migrated up to 11-fold more efficiently towards conditioned media from cancer cells than conditioned media from nontransformed cells. The most striking observation of the study was that out of 4 different human breast cancer lines tested, all showed a significant increase in metastatic potential to the lungs and one showed accelerated primary tumor growth when co-injected with human MSCs. In contrast, the co-injection of tumor cells with other types of cells such as human fibroblasts did not enhanced tumor growth or metastasis. The authors hypothesized that MSCs might favor the outgrowth of rare breast cancer cells that exhibit high metastatic potential, or that MSCs might cause otherwise weakly metastatic tumor cells to acquire enhanced metastatic abilities. The fact that lung metastases isolated from mice co-injected with tumor cells and MSCs no longer displayed a pro-metastatic phenotype when re-injected in the absence of MSCs suggested that MSC-induced metastasis was acquired by tumor cells and thus reflects a reversibly trait. These observations suggested that MSCs produce paracrine factors that induce breast cancer cells to metastasize. To understand this, in vitro co-cultures of breast cancer cells and MSCs were established and their conditioned media were screened for the levels of various cytokines, chemokines and growth factors. Strikingly, the chemokine RANTES (CCL5) was upregulated to levels up to 60-fold higher when breast cancer cells were co-cultured with MSCs and MSCs were demonstrated to be the source of CCL5. Although CCL5 was required in MSC-induced metastasis in two human breast cancer model,

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it was not involved in the MSC-induced metastasis of two other models, revealing that other factors can be at play. Taken together, this landmark study suggests that the ability of a tumor to form distant metastases is not necessary intrinsic to the tumor but rather can be induced in response to signals that tumor cells receive from their stromal microenvironment, or from signal afforded by intravenously injected MSCs. Thus, in order for MSCs to be used for cancer cell therapy, these pro-metastatic properties shall be constrained.

5.6 Chemokine Receptors Regulating MSCs Homing The spectrum of factors governing the tropism of MSCs to tissue damage and tumors has only recently started to emerge and remains to be fully elucidated. Some groups have proposed that MSCs might actually share chemotactic properties with immune cells in response to injury and inflammation [44]. With this in mind, it is perhaps not be surprising that nearly every chemokine receptor has been reported to be expressed on human MSCs. One of the difficulties in studying chemotaxis of MSCs stems from the fact that ex vivo expanded MSCs often loose expression of chemokine receptors and responsiveness to chemokines during long term culture [45]. The following chemokine receptors have been reported to be expressed on MSCs: CCR1 to CCR10, CXCR1 to CXCR6, CX3CR1 and XCR1 [44]. However, some studies have reported that chemokine receptors are heterogeneously expressed on MSCs, with a small fraction expressing CCR1 and CCR7, and a higher fraction expressing CXCR6, CX3CR1 and CXCR4 [46]. A study by Dwyer et al. [47] described a particularly important role for CCR2 expression by human MSCs in controlling their chemotaxis to primary breast cancer cells. While investigating factors mediating homing of human MSCs to breast cancer primary cultures, Dwyer et al. observed that blocking CCL2/CCR2 interactions with an antibody significantly decreased the ability of human MSCs to migrate to tumors. Consistent with this, Klopp et al. [48] recently reported that the receptor CCR2 is up-regulated in human MSCs exposed to irradiated tumor cells and that inhibition of CCR2 leads to a marked decrease of MSCs migration to tumors. In addition of confirming an important role for CCR2 in regulating human MSCs tumor tropism, this latter study demonstrated that clinically relevant doses of irradiation increased the tropism for and engraftment of MSCs in the tumor microenvironment. However, it should be pointed out that the study also revealed that in the presence of irradiated tumor cells, MSCs increased their release of pro-angiogenic factors, which could be detrimental in the context of cancer therapy. New evidence suggests that the receptor CCR2 may play a broader role in controlling MSCs homing than previously thought. Belenm-Bedada et al. [49] recently demonstrated that CCR2 expression is necessary for the heart-specific homing of MSCs into damaged hearts following ischemia/reperfusion. Of interest, the authors demonstrated that migration of MSCs was dependent on the intracellular adaptor molecule FROUNT, which interacts with CCR2. Upon recruitment by CCR2 activation, they showed that MSCs secreted SDF-1, which can attract additional MSCs

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via activation of its receptor CXCR4 to contribute to tissue repair. The activation of the chemokine receptor CXCR4 by its ligand SDF-1 may play a similarly role in tumor homing of MSCs, perhaps via the presence of carcinoma-associated fibroblasts (CAFs). A recent study by Mishra et al. [50] demonstrated that human MSCs exposed to tumor-conditioned medium assume a CAF-like phenotype and exhibit functional properties of CAFs, including sustained expression of stromal-derived factor-1 (SDF-1). The release of SDF-1 by CAFs may thus act as a feed-forward mechanism promoting the recruitment of additional MSCs via CXCR4. In support of a role for CXCR4 in tumor homing of MSCs, Orimo et al. [51] demonstrated that the release of SDF-1 by CAFs extracted from human breast tumors promoted the recruitment of stromal elements and stimulated angiogenesis.

5.7 Growth Factor Receptors Regulating MSCs Homing In addition to chemokines, several growth factors can enhance MSCs migration. Basic fibroblast growth factor, vascular endothelial growth factor and plateletderived growth factor can all act as chemoattractants, sometimes synergistically, in order to induce MSCs migration [44, 52]. Li et al. [53] demonstrated that insulingrowth factor (IGF)-1 can also enhance MSCs migration, albeit by an indirect mechanism. Indeed, IGF-1 was shown to increase the expression of CXCR4 on MSCs, thereby enhancing their response to the chemokine SDF-1. In addition to CXCR4, IGF-1 may also upregulate CCR5 expression and may thus enhance MSCs migration in response to CCL5 [54]. Finally, tumor necrosis factor (TNF)-α has also been shown to enhance MSCs migration [55]. TNF-α was shown to upregulate CCR3 and CCR4 expression on MSCs and to increase their ability to migrate towards RANTES and macrophage-derived cytokine (MDC/CCL22).

5.8 Toll-Like Receptors and Death Receptors in MSC Migration Toll-like receptors (TLRs) play a vital role in the activation of innate immune cells, enabling them to recognize conserved patterns expressed on pathogens. MSCs have been shown to express several TLRs, including TLR-1 to TLR-6 [56–59]. TLR activation triggers the release of chemokines and regulates cell migration. Tomchuck et al. [58] reported that human MSCs make use of TLR signaling to regulate their migratory potential. Specifically, they demonstrated that TLR-3 activation was a potent inducer of MSCs migration. Accordingly, blocking TLR-3 with a neutralizing antibody decreased by 50% the ability of MSCs to migrate in response to TLR-3 agonist. Notably, TLR-3 recognizes double-stranded RNA, suggesting that endogenous MSCs may play an important role in regulating innate immune responses to viral infections. Activation of TLR-2 (which recognizes lipopeptides and peptidoglycans) was shown to enhance proliferation and IL-6 production of MSCs. Furthermore, MSCs derived from MyD88 (an adaptor protein essential for several

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TLR signaling)-deficient mice failed to differentiate effectively into osteogenic and chondrogenic cells. Taken together, these studies suggested that TLR activation is an important regulator of MSCs biology. A recent study by Secchiero et al. [60] suggests that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might also act as a chemoattractant to MSCs. TRAIL is a type II membrane protein and a soluble cytokine that induces programmed cell death (apoptosis) via the activation of the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) [61]. TRAIL is the subject of intensive investigation since various cancer cells have been shown to be more susceptible than normal cells to TRAIL-induced apoptosis. The authors found that in spite of the expression of detectable surface levels of TRAIL-R2, MSCs showed limited levels of apoptosis when exposed to recombinant soluble TRAIL. Most intriguingly, they reported that recombinant TRAIL promoted MSCs migration with an efficiency higher than SDF-1. On one hand, these findings open the door to the possibility of using MSCs overexpressing TRAIL for cancer cell therapy. On the other hand, this study suggests a previously undescribed role for TRAIL in regulating cell migration, possibly via activation of the ERK pathway.

5.9 Oncogenic Potential of MSCs A potential major drawback with the use of MSCs for cell therapy is the observation that MSCs can spontaneously transform following ex vivo culture. In tissue cultures, human and mouse MSCs are susceptible to spontaneous mutations, including loss of p53 function. Rubio et al. [62] reported that human adult MSCs can spontaneously transform after long-term culture; virtually all human MSC preparations were shown to bypass cellular senescence. Normally, when cells bypass senescence, they continue to grow until telomeres become too short and then enter a crisis phase, characterized by chromosome instability followed by cell death. However, Rubio et al. [62] observed that 50% of human MSC preparations can bypass this crisis if maintained 4–5 months in culture. Most importantly, MSCs that bypass senescence crisis were shown to form tumors when injected into immunodeficient mice. A recent study reported the occurrence of age-related fibrosarcomas in mice injected with MSCs [63], possibly involving acquisition of p53 mutations, while others have reported that MSC-derived tumors are poorly differentiated carcinomas, perhaps suggesting a mesenchymal-epithelial transition [64]. Recently, Berman et al. [65] demonstrated that the combined loss of Rb and p53 in MSCs is sufficient to induced osteosarcoma formation in mice. MSCs may also be linked to soft-tissue sarcomas. Accordingly, when expressing the fusion gene PAX-FKHR – though to be the initiator of alveolar rhabdomyosarcomas (ARMS; a soft-tissue sarcoma) – together with p53 loss-of-function, MSCs can give rise to ARMS in mice [66]. It should be pointed out that in contrast to these aforementioned studies, Bernardo et al. [67] failed to observe malignant transformation of human MSCs using genomic hybridization, karyotyping, subtelomeric fluorescent in situ

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hybridization and telomerase activity assays. Since Bernardo et al. limited their study to MSCs with less than 25 passages, it may suggest that human MSCs expanded for less than 25 passages may be safer to use for cell therapy.

5.10 Immunosuppressive Effects of MSCs An intriguing feature of ex vivo expanded MSCs is their ability to suppress on-going immune responses. Several studies have now described the immunosuppressive effects of ex vivo expanded MSCs in animal models as well as human patients. Although not fully characterized, the immunosuppressive properties of MSCs have been described to affect the function of a broad range of immune cells, including T cells and regulatory T cells, B cells, natural killer (NK) cells and antigen-presenting cells. A comprehensive review on the immuno-modulatory properties of MSCs was recently published by Uccelli et al. [17]. In brief, the expression of inhibitory ligands such as B7-H1 (PD1-L) and the release of soluble factors such as hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), transforming growth factor (TGF)-β1, indoleamine 2,3-dioxygenase (IDO), nitric oxide and IL-10 have all been identified as important factors in the immunosuppressive effects of MSCs [68–74]. We hereafter summarize the effects of MSCs on various immune cells.

5.10.1 T Cells MSCs can suppress T cell proliferation [70, 74, 75] and have been reported to induce split T cell anergy, an anergic state that is only partly reversed by exogenous IL-2 [76]. MSCs can also promote the expansion of regulatory T cells. Regulatory T cells are a subset of T cells that suppress immune activation in order to prevent autoimmune diseases and maintain homeostasis. MSCs have been shown to induce the production of IL-10 by plasmacytoid dendritic cells (DCs), which in turn induces the generation of regulatory T cells [68, 77]. In a heart transplant mouse model, it was recently shown that infusions of donor-derived MSCs can prolong cardiac allograft survival via the expansion of regulatory T cells [78]. Of interest, this new study reported that hematopoietic stem cells can negatively affect MSC-mediated immunosuppression. Activation of TLR-3 and TLR-4 on MSCs have also been shown to block the ability of MSCs to inhibit T cell proliferation [56]. These recent studies thus suggest that fail-safe mechanisms might exist to control MSC-mediated immunosuppression.

5.10.2 NK Cells MSCs inhibit IL-2- and IL-15-induced proliferation of NK cells and are highly susceptible to NK cell-mediated lysis [79]. Accordingly, MSCs express a broad

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range of surface ligands involved in NK cell activation, including the NKG2D ligands MICA and ULBPs and the DNAM-1 ligands PVR and Nectin-2. Blocking NKG2D and DNAM-1 using monoclonal antibodies results in the partial inhibition of MSCs killing by NK cells and exposure to IFN-γ totally protects MSCs from NK cell-mediated killing.

5.10.3 DCs MSCs also suppress APCs. MSCs cocultured with blood monocytes significantly inhibit their differentiation into DCs, and MSCs cocultured with matured DCs causes a significant decrease in MHC class II molecules, CD80 and CD86 expression on DCs [80]. Zhang et al. [81] recently demonstrated that MSCs actually drive DCs to differentiate into Jagged-2-dependent regulatory DCs, thereby limiting their antigen presenting function.

5.10.4 B Cells MSCs can in addition suppress B cell function. Corcione et al. [82] reported that MSCs cocultured with purified CD19+ B cells inhibited B cell proliferation. Recently, Rafei et al. [83] investigated the capacity of MSCs to modulate immunoglobulin (Ig) production. The study demonstrated that secreted factors produced by MSCs have the capacity to decrease the number of antibody secreting cells following ovalbumin injection in mice. At least two chemokines, namely CCL2 and CCL7, were involved in MSC-mediated suppression of Ig production. More specifically, this new study suggests that the proteolytic processing of MSC-derived CCL2 (and possibly CCL7) by matrix metalloproteinases (MMPs) are key B cell regulatory molecules. MSCs can indeed secrete multiple MMPs, including MMP-1, -3 and -8, involved in chemokine processing. In support of a role for CCL2-CCR2 interactions in MSC-mediated immune suppression, spleen-derived plasma cells from CCR2 knockout mice were shown to be refractory to MSC-mediated suppression and MSCs isolated from CCL2 knockout mice were shown to be less suppressive. The study further demonstrated that infusion of MSCs to hemophilic mice with antifactor VIII antibodies decreased by nearly two logs antibody titers. This new study thus opens up the possibility of exploiting MSCs for the treatment of autoimmune diseases characterized by auto-antibodies.

5.11 Modulation of MSCs Immunosuppression Cytokines such as TNF-α and IFN-γ play a crucial role in regulating MSCs immune functions. For instance, TNF-α can enhance by as much as 100-fold the production of immunosuppressive factors by MSCs [68]. In contrast IFN-γ upregulates MHC class I and class II molecules on MSCs and induces MSCs to behave as conditional

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APCs [84]. The antigen presenting function of MSCs appears to be tightly regulated by IFN-γ levels. Accordingly, upregulation of MHC expression and antigen presentation is progressively lost as IFN-γ levels increase. Thus, MSCs may function as conditional APCs in the early phase of an immune response and later switch to immune-suppressive function.

5.12 MSCs for Cellular Gene Therapy The accessibility, plasticity, tissue-homing and immunomodulatory properties of MSCs make them ideal candidates for regenerative medicine and cell-based therapy. Clinical studies based on the infusion of MSCs have already generated promising results for the treatment of several diseases. In the context of cancer, the observation that MSCs possess tumor-homing properties has generated great interest in using gene-modified MSCs as delivery vehicles of therapeutic gene products. Some of the advantages of using tumor-homing carrier cells for the delivery of biotherapeutics include: (i) the efficient delivery of biotherapeutics difficult to synthesize; (ii) the continuous production of biotherapeutics with short half-lifes; (iii) the protection of biotherapeutics from inactivation by host defence systems; and (iv) the targeted delivery of biotherapeutics to the tumor microenvironment. We hereafter review the current literature on the various means to gene-modify primary MSCs and on the nature of the biotherapeutics currently under investigation in the context of cancer cell therapy.

5.13 Retroviral Vectors for MSC Gene Transfer Because primary MSCs readily undergo cell division when placed in culture, retroviral vectors have been traditionally used in order to gene-modify MSCs. However, while retroviral gene transfer offers stable transgene expression, it also presents the risk of insertional oncogenesis. The risk of oncogenesis following gene-modification with integrating retroviral vectors has been clearly established in clinical trials testing the use of transduced CD34+ hematopoietic cells for the treatment of genetic severe-combined immunodeficiencies (SCID). The clinical benefit of such treatment has been overshadowed by the occurrence of leukemia in 4 SCID patients [85]. In the first 2 cases initially described, the vector used for transduction integrated near a proto-oncogene, the LIM domain-only 2 (LMO2), activating its transcription and promoting clonal T cell proliferation. Some have proposed that the nature of transgene being expressed, namely the common γ-chain, was directly responsible to the occurrence of leukemia in these trials. However, studies have since indicated that the common γ-chain is not an oncogene per se. A recent study suggested that some retroviral constructs may be more prone than others to insertional oncogenesis. It was observed that stable gene transfer into MSCs with the retroviral vectors pBABE-puro, PINCO-puro and the lentiviral vector pSICO PGK-puro, exhibited a higher risk of insertional oncogenesis than the use

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of the same vectors harboring another selection marker in place of the puromycin resistance gene [86]. The puromycin-expressing vectors were shown to enhance the production of reactive oxygen species as a result of mitochondrial dysfunction. Recently, a variety of improvements in retroviral vector design have been proposed in order to further reduce the risk of insertional oncogenesis [87–89].

5.14 Electroporation for MSC Gene Transfer The use of cationic lipids complexed with DNA has been extensively used for the transfection of established cell lines. However, primary cells such as MSCs are in general difficult to transfect using cationic lipid complexes. As an alternative, recent studies have described protocols of electroporation in order to efficiently genemodify primary MSCs. Electroporation uses a pulsed electrical field to make the cell membrane permeable to DNA plasmids. In some cases, electroporation appears to be superior to cationic lipid complexes. Of interest, Helledie et al. [90] recently reported a simple and highly efficient method of DNA electroporation into MSC that does not involve prolonged incubation. This electroporation method resulted in transfection efficiencies up to 90% into MSCs. Perhaps as important was the observation that in contrast to the use of cationic lipids, electroporation maintained the pluripotentiality of the cells. Thus, the use of electroporation may be an efficient means to transiently gene-modify primary MSCs.

5.15 Ex Vivo Reprogramming of MSCs Without Gene Transfer Recent studies have provided new indications that MSCs homing can be reprogrammed without the need for gene transfer. Specifically, Sackstein et al. [91] recently demonstrated in a Nature Medicine paper that human MSCs could be reprogrammed to specifically home to the bone microenvironment using a simple preparation of enzymatic conditions designed to convert native CD44 on MSCs into the glycoform HCELL (hematopoietic cell E-selectin/L-selectin ligand). The study was based on previous work that revealed a key role for vascular E-selectin in the homing of hematopoietic cells to the bone. For instance, it was known that hematopoietic stem cells (HSCs) use two E-selectin ligands, i.e. P-selectin glycoprotein ligand-1 (PSGL-1) and a sialofucosylated glycoform of CD44 known as HCELL, to direct their migration to the bone. While MSCs express CD44, they do not express the sialofucosylated glycoform of CD44 and thus have poor osteotropism. Sackstein et al. reported a simple method of engineering HCELL expression on MSCs using conditions able to drive surface α-1,3-fucosylation without detrimental effects to the cells. While this specific engineering can enhances the potential of using infused MSCs for the treatment of skeletal diseases, the study also suggests that similar approaches could be undertaken in order to enhance the tissue homing properties of MSCs to navigate to other locations, for instance, toward tumors.

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5.16 Cytokine-Producing MSCs for Cancer Cell Therapy The tumor-homing properties of MSCs have led to studies investigating their potential as targeted delivery vehicles for therapeutic genes. The selective engraftment of MSCs to tumors may thus constitute a new therapeutic avenue to target anti-tumor gene products such as cytokines to the tumor microenvironment. Michael Andreeff and is team were pioneers in this field when they demonstrated that intravenously injected human MSCs gene-modified to secrete IFN-β preferentially engrafted into xenografted tumors and significantly delayed tumor growth in immunodeficient mice [33, 35, 92]. MSCs were found to have significant tumor-tropism, which was required in order to achieve optimal anti-tumor effects. Accordingly, no beneficial effects were observed when the IFN-β-producing MSCs were administered distally. Our group has investigated whether MSCs could be used in immunocompetent hosts for the delivery of immunostimulatory proteins such as IL-2 and IL-12. We have demonstrated that gene-modified IL-2-producing MSCs can generate CD8dependent and NK-mediated anti-tumor immune responses in a B16 mouse model of melanoma [93]. Recently, Eliopoulos et al. [94] reported the effective anti-tumor effects of implanting IL-12 gene-modified MSCs in immunocompetent mice with breast cancer or melanoma. Other studies using IL-12-producing MSCs have also been conducted [95, 96]. Taken together, these studies suggest that IL-2 or IL-12 producing MSCs could be used, for instance, against locally advanced tumors where high-dose administration of the cytokines has shown some promises but is limited by the toxicity associated to its systemic administration. Targeted delivery of such potent cytokines directly to the tumor microenvironment may allow for a greater therapeutic effect while limiting the systemic side-effects.

5.17 Oncolytic Virus-Infected MSCs Oncolytic viruses are currently being investigated as a new strategy for cancer therapy. Oncolytic virus therapy is based on the notion that various wild type or engineered viruses preferentially replicate and kill tumor cells. Accordingly, oncolytic viruses have been shown to have great potential for the treatment of cancer and a variety of strategies are being developed to restrict oncolytic viruses to tumor microenvironment. However, several studies have demonstrated that humoral immune responses against oncolytic viruses greatly limit their anti-tumor potential, highlighting the need for new approaches to oncolytic virus therapy [97]. The administration of ex vivo infected cells has been suggested has an efficient alternative. Several groups have now demonstrated that cellular carrier cells can shield oncolytic viruses from neutralizing antibodies during delivery. Given their tumortropism, MSCs could constitute a source of carrier cells for the delivery of oncolytic viruses. Recently, Sonabend et al. [98] investigated whether human MSCs could be used to deliver a replication-competent oncolytic adenovirus in a model of glioma.

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Following injection of infected MSCs, the cells efficiently migrated to the tumor and delivered a significantly greater viral load to the tumor than injection of the virus alone. In another study, Komarova S. et al. [99] demonstrated that injection of MSCs infected with another oncolytic adenovirus increased the survival of ovarian cancer-bearing mice compared with direct viral injection. Since MSCs can be readily expanded and gene-modified, MSCs could further be engineered to have limited innate antiviral response or gene-modified to overexpress specific chemokine receptors thus increasing the output of therapeutic virus at the tumor site.

5.18 Conclusion As we have discussed, despite their tumor-homing capacity, MSCs possess biological properties that may significantly enhance the growth and metastasis of tumors. Among those properties, it has been clearly demonstrated in mice that infused human MSCs significantly enhanced the metastatic potential of breast cancer cells. Another important effect of MSCs is their ability to suppress a broad range of immune cells, including DCs, T cells, B cells and NK cells. Given the wellestablished importance of the immune system at controlling and shaping developing tumors, and the importance of immune effector cells in the efficacy of various forms of treatments, infusion of immunosuppressive MSCs may be detrimental to conventional cancer therapies. Correspondingly, irradiation of tumor cells can stimulate MSCs to release pro-angiogenic factors, perhaps reflecting the ability of MSCs to perform tissue repair. Furthermore, human MSCs have been shown to be susceptible to spontaneous transformation upon extensive ex vivo culture and have been suggested as progenitors of various forms of malignancies. The oncogenic potential of MSCs is of particular concern where retroviral vectors are uses. Indeed, while retroviral gene transfer offers stable transgene expression, it also presents the risk of insertional oncogenesis. Notwithstanding these major limitations, ex vivo expanded MSCs offer a great deal of therapeutic potential. As we have discussed, the selective engraftment of MSCs to tumors offers the unique possibility to target biotherapeutics such as cytokines and oncolytic viruses directly to the tumor bed. In our view, the future of targeted cancer therapy using MSCs will depend on the efficient exploitation and enhancement of these tumor-homing properties coupled to the development of new engineering strategies aimed at limiting the pro-tumorigenic, pro-metastatic, pro-angiogenic and immunosuppressive effects of MSCs. Acknowledgments J.S. is supported by a post-doctoral fellowship from the Canadian Institutes of Health Research.

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2. Pittenger MF, Mackay AM, Beck SC, et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147 3. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM (2001) Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98: 2615–2625 4. Wang G, Bunnell BA, Painter RG, et al. (2005) Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: potential therapy for cystic fibrosis. Proc Natl Acad Sci USA 102: 186–191 5. Horwitz EM, Le Blanc K, Dominici M, et al. (2005) Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 7: 393–395 6. Laitinen A, Laine J (2007) Isolation of mesenchymal stem cells from human cord blood. Curr Protoc Stem Cell Biol Chapter 2: Unit 2A 3 7. Kumar S, Chanda D, Ponnazhagan S (2008) Therapeutic potential of genetically modified mesenchymal stem cells. Gene Ther 15: 711–715 8. Delorme B, Ringe J, Gallay N, et al. (2008) Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells. Blood 111: 2631–2635 9. Muller I, Kordowich S, Holzwarth C, et al. (2006) Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM. Cytotherapy 8: 437–444 10. Anjos-Afonso F, Bonnet D (2008) Isolation, culture, and differentiation potential of mouse marrow stromal cells. Curr Protoc Stem Cell Biol Chapter 2: Unit 2B 3 11. Stagg J (2007) Immune regulation by mesenchymal stem cells: two sides to the coin. Tissue Antigens 69: 1–9 12. Gang EJ, Bosnakovski D, Figueiredo CA, Visser JW, Perlingeiro RC (2007) SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood 109: 1743–1751 13. Kannagi R, Cochran NA, Ishigami F, et al. (1983) Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J 2: 2355–2361 14. Colter DC, Sekiya I, Prockop DJ (2001) Identification of a subpopulation of rapidly selfrenewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA 98: 7841–7845 15. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ (1999) Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 107: 275–281 16. Lee RH, Seo MJ, Pulin AA, Gregory CA, Ylostalo J, Prockop DJ (2009) The CD34-like protein PODXL and {alpha}6-integrin (CD49f) identify early progenitor MSCs with increased clonogenicity and migration to infarcted heart in mice. Blood: 113: 816–826 17. Uccelli A, Moretta L, Pistoia V (2008) Mesenchymal stem cells in health and disease. Nat Rev Immunol 8:726–736 18. Zuk PA, Zhu M, Ashjian P, et al. (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13: 4279–4295 19. Baertschiger RM, Bosco D, Morel P, et al. (2008) Mesenchymal stem cells derived from human exocrine pancreas express transcription factors implicated in beta-cell development. Pancreas 37: 75–84 20. Metcalfe AD, Ferguson MW (2008) Skin stem and progenitor cells: using regeneration as a tissue-engineering strategy. Cell Mol Life Sci 65: 24–32 21. Markov V, Kusumi K, Tadesse MG, et al. (2007) Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles. Stem Cells Dev 16: 53–73 22. Crisan M, Yap S, Casteilla L, et al. (2008) A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3: 301–313

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23. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676 24. Park IH, Zhao R, West JA, et al. (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451: 141–146 25. Dimos JT, Rodolfa KT, Niakan KK, et al. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321: 1218–1221 26. Wernig M, Lengner CJ, Hanna J, et al. (2008) A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol 26: 916–924 27. Kim JB, Zaehres H, Wu G, et al. (2008) Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454: 646–650 28. Park IH, Arora N, Huo H, et al. (2008) Disease-specific induced pluripotent stem cells. Cell 134: 877–886 29. Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H (2008) Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol 180: 2581–2587 30. Smyth MJ, Dunn GP, Schreiber RD (2006) Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol 90: 1–50 31. Hung SC, Deng WP, Yang WK, et al. (2005) Mesenchymal stem cell targeting of microscopic tumors and tumor stroma development monitored by noninvasive in vivo positron emission tomography imaging. Clin Cancer Res 11: 7749–7756 32. Karnoub AE, Dash AB, Vo AP, et al. (2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449: 557–563 33. Nakamizo A, Marini F, Amano T, et al. (2005) Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 65: 3307–3318 34. Nakamura K, Ito Y, Kawano Y, et al. (2004) Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther 11: 1155–1164 35. Studeny M, Marini FC, Dembinski JL, et al. (2004) Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst 96: 1593–1603 36. Khakoo AY, Pati S, Anderson SA, et al. (2006) Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi’s sarcoma. J Exp Med 203: 1235–1247 37. Qiao L, Xu Z, Zhao T, et al. (2008) Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model. Cell Res 18: 500–507 38. Ame-Thomas P, Maby-El Hajjami H, Monvoisin C, et al. (2007) Human mesenchymal stem cells isolated from bone marrow and lymphoid organs support tumor B-cell growth: role of stromal cells in follicular lymphoma pathogenesis. Blood 109: 693–702 39. Hall B, Dembinski J, Sasser AK, Studeny M, Andreeff M, Marini F (2007) Mesenchymal stem cells in cancer: tumor-associated fibroblasts and cell-based delivery vehicles. Int J Hematol 86: 8–16 40. Sasser AK, Sullivan NJ, Studebaker AW, Hendey LF, Axel AE, Hall BM (2007) Interleukin-6 is a potent growth factor for ER-alpha-positive human breast cancer. FASEB J 21: 3763–3770 41. Zhu W, Xu W, Jiang R, et al. (2006) Mesenchymal stem cells derived from bone marrow favor tumor cell growth in vivo. Exp Mol Pathol 80: 267–274 42. Samudio I, Fiegl M, McQueen T, Clise-Dwyer K, Andreeff M (2008) The warburg effect in leukemia-stroma cocultures is mediated by mitochondrial uncoupling associated with uncoupling protein 2 activation. Cancer Res 68: 5198–5205 43. Tabe Y, Jin L, Tsutsumi-Ishii Y, et al. (2007) Activation of integrin-linked kinase is a critical prosurvival pathway induced in leukemic cells by bone marrow-derived stromal cells. Cancer Res 67: 684–694 44. Spaeth E, Klopp A, Dembinski J, Andreeff M, Marini F (2008) Inflammation and tumor microenvironments: Defining the migratory itinerary of mesenchymal stem cells. Gene Ther 15: 730–738

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45. Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM, Silberstein LE (2006) Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells 24: 1030–1041 46. Sordi V, Malosio ML, Marchesi F, et al. (2005) Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 106: 419–427 47. Dwyer RM, Potter-Beirne SM, Harrington KA, et al. (2007) Monocyte chemotactic protein1 secreted by primary breast tumors stimulates migration of mesenchymal stem cells. Clin Cancer Res 13: 5020–5027 48. Klopp AH, Spaeth EL, Dembinski JL, et al. (2007) Tumor irradiation increases the recruitment of circulating mesenchymal stem cells into the tumor microenvironment. Cancer Res 67: 11687–11695 49. Belema-Bedada F, Uchida S, Martire A, Kostin S, Braun T (2008) Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell 2: 566–575 50. Mishra PJ, Humeniuk R, Medina DJ, et al. (2008) Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells. Cancer Res 68: 4331–4339 51. Orimo A, Gupta PB, Sgroi DC, et al. (2005) Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121: 335–348 52. Schmidt A, Ladage D, Schinkothe T, et al. (2006) Basic fibroblast growth factor controls migration in human mesenchymal stem cells. Stem Cells 24: 1750–1758 53. Li Y, Yu X, Lin S, Li X, Zhang S, Song YH (2007) Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells. Biochem Biophys Res Commun 356: 780–784 54. Mira E, Lacalle RA, Gonzalez MA, et al. (2001) A role for chemokine receptor transactivation in growth factor signaling. EMBO Rep 2: 151–156 55. Ponte AL, Marais E, Gallay N, et al. (2007) The in vitro migration capacity of human bone marrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activities. Stem Cells 25: 1737–1745 56. Liotta F, Angeli R, Cosmi L, et al. (2008) Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling. Stem Cells 26: 279–289 57. Pevsner-Fischer M, Morad V, Cohen-Sfady M, et al. (2007) Toll-like receptors and their ligands control mesenchymal stem cell functions. Blood 109: 1422–1432 58. Tomchuck SL, Zwezdaryk KJ, Coffelt SB, Waterman RS, Danka ES, Scandurro AB (2008) Toll-like receptors on human mesenchymal stem cells drive their migration and immunomodulating responses. Stem Cells 26: 99–107 59. Yu S, Cho HH, Joo HJ, Bae YC, Jung JS (2008) Role of MyD88 in TLR agonist-induced functional alterations of human adipose tissue-derived mesenchymal stem cells. Mol Cell Biochem 317: 143–150 60. Secchiero P, Melloni E, Corallini F, et al. (2008) TRAIL promotes migration of human bone marrow multipotent stromal cells. Stem Cells 26: 2955–2963 61. Takeda K, Stagg J, Yagita H, Okumura K, Smyth MJ (2007) Targeting death-inducing receptors in cancer therapy. Oncogene 26: 3745–3757 62. Rubio D, Garcia-Castro J, Martin MC, et al. (2005) Spontaneous human adult stem cell transformation. Cancer Res 65: 3035–3039 63. Li H, Fan X, Kovi RC, et al. (2007) Spontaneous expression of embryonic factors and p53 point mutations in aged mesenchymal stem cells: a model of age-related tumorigenesis in mice. Cancer Res 67: 10889–10898 64. Rubio D, Garcia S, De la Cueva T, et al. (2008) Human mesenchymal stem cell transformation is associated with a mesenchymal-epithelial transition. Exp Cell Res 314: 691–698 65. Berman SD, Calo E, Landman AS, et al. (2008) Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage. Proc Natl Acad Sci USA 105: 11851–11856

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66. Ren YX, Finckenstein FG, Abdueva DA, et al. (2008) Mouse mesenchymal stem cells expressing PAX-FKHR form alveolar rhabdomyosarcomas by cooperating with secondary mutations. Cancer Res 68: 6587–6597 67. Bernardo ME, Zaffaroni N, Novara F, et al. (2007) Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms. Cancer Res 67: 9142–9149 68. Aggarwal S, Pittenger MF (2005) Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105: 1815–1822 69. Beyth S, Borovsky Z, Mevorach D, et al. (2005) Human mesenchymal stem cells alter antigenpresenting cell maturation and induce T-cell unresponsiveness. Blood 105: 2214–2219 70. Djouad F, Plence P, Bony C, et al. (2003) Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 102: 3837–3844 71. Le Blanc K, Rasmusson I, Gotherstrom C, et al. (2004) Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes. Scand J Immunol 60: 307–315 72. Sato K, Ozaki K, Oh I, et al. (2007) Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells. Blood 109: 228–234 73. Sheng H, Wang Y, Jin Y, et al. (2008) A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1. Cell Res 18: 846–857 74. Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC (2003) Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75: 389–397 75. Krampera M, Glennie S, Dyson J, et al. (2003) Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 101: 3722–3729 76. Glennie S, Soeiro I, Dyson PJ, Lam EW, Dazzi F (2005) Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood 105: 2821–2827 77. Maccario R, Podesta M, Moretta A, et al. (2005) Interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of CD4+ T-cell subsets expressing a regulatory/suppressive phenotype. Haematologica 90: 516–525 78. Casiraghi F, Azzollini N, Cassis P, et al. (2008) Pretransplant infusion of mesenchymal stem cells prolongs the survival of a semiallogeneic heart transplant through the generation of regulatory T cells. J Immunol 181: 3933–3946 79. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L (2006) Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 107: 1484–1490 80. Jiang XX, Zhang Y, Liu B, et al. (2005) Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 105: 4120–4126 81. Zhang B, Liu R, Shi D, et al. (2009) Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2 dependent regulatory dendritic cell population. Blood 113: 46–57 82. Corcione A, Benvenuto F, Ferretti E, et al. (2006) Human mesenchymal stem cells modulate B-cell functions. Blood 107: 367–372 83. Rafei M, Hiseh J, Fortier S, et al. (2008) Mesenchymal stromal cell derived CCL2 suppresses plasma cell immunoglobulin production via STAT3 inactivation and PAX5 induction. Blood 112: 4991–4998 84. Stagg J, Pommey S, Eliopoulos N, Galipeau J (2006) Interferon-gamma-stimulated marrow stromal cells: a new type of nonhematopoietic antigen-presenting cell. Blood 107: 2570–2577 85. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302: 415–419

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86. Piccoli C, Scrima R, Ripoli M, et al. (2008) Transformation by Retroviral Vectors of Bone Marrow-Derived Mesenchymal Cells Induces Mitochondria-Dependent CAMPSensitive ROS Production. Stem Cells 26: 2843–2854 87. Aker M, Tubb J, Groth AC, et al. (2007) Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. Hum Gene Ther 18: 333–343 88. Montini E, Cesana D, Schmidt M, et al. (2006) Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol 24: 687–696 89. Zychlinski D, Schambach A, Modlich U, et al. (2008) Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors. Mol Ther 16: 718–725 90. Helledie T, Nurcombe V, Cool SM (2008) A simple and reliable electroporation method for human bone marrow mesenchymal stem cells. Stem Cells Dev 17: 837–848 91. Sackstein R, Merzaban JS, Cain DW, et al. (2008) Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone. Nat Med 14: 181–187 92. Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M (2002) Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res 62: 3603–3608 93. Stagg J, Lejeune L, Paquin A, Galipeau J (2004) Marrow stromal cells for interleukin-2 delivery in cancer immunotherapy. Hum Gene Ther 15: 597–608 94. Eliopoulos N, Francois M, Boivin MN, Martineau D, Galipeau J (2008) Neo-organoid of marrow mesenchymal stromal cells secreting interleukin-12 for breast cancer therapy. Cancer Res 68: 4810–4818 95. Chen X, Lin X, Zhao J, et al. (2008) A tumor-selective biotherapy with prolonged impact on established metastases based on cytokine gene-engineered MSCs. Mol Ther 16: 749–756 96. Elzaouk L, Moelling K, Pavlovic J (2006) Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model. Exp Dermatol 15: 865–874 97. Power AT, Wang J, Falls TJ, et al. (2007) Carrier cell-based delivery of an oncolytic virus circumvents antiviral immunity. Mol Ther 15: 123–130 98. Sonabend AM, Ulasov IV, Tyler MA, Rivera AA, Mathis JM, Lesniak MS (2008) Mesenchymal stem cells effectively deliver an oncolytic adenovirus to intracranial glioma. Stem Cells 26: 831–841 99. Komarova S, Kawakami Y, Stoff-Khalili MA, Curiel DT, Pereboeva L (2006) Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses. Mol Cancer Ther 5: 755–766

Chapter 6

Alternative Embryonic Stem Cell Sources Tomo Šari´c, Narges Zare Mehrjardi, and Jürgen Hescheler

Abstract Pluripotency refers to the ability of a cell to differentiate in vivo or in vitro to practically all cell types of an adult organism. In vivo, pluripotent stem cells exist only transiently in early embryos. However, when explanted the in vitro counterparts of these cells, known as embryonic stem (ES) cells, can be maintained indefinitely in culture in undifferentiated state and used for generation of various mature cell types. While this broad differentiation potential, easy accessibility and possibility for large expansion of ES cells in culture classifies them as a most promising source of cells for regenerative medicine, unresolved ethical concerns and allogeneic nature of human ES cells seriously hampered their therapeutic use. Therefore, the goal of autologous and ethically uncontroversial alternative sources of human pluripotent stem cells has been intensively pursued by a number of laboratories. Here we review the tremendous progress made in recent years in this very dynamic area of research and discuss implications that these new developments may have for biomedical research and regenerative medicine in near future. Keywords Pluripotency · Embryonic stem cells · iPS cells · Reprogramming · Cell fusion · Nuclear transfer · Transdifferentiation · Tissue repair · Regenerative medicine · Drug development · Disease modeling

Contents 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Embryonal Carcinoma (EC) Cells . . . . . . . . . . . . . . 6.3 Embryonic Stem (ES) Cells . . . . . . . . . . . . . . . . . 6.3.1 Hallmarks of ES Cells . . . . . . . . . . . . . . . . 6.3.2 Regulation of Self-Renewal and Pluripotency of ES Cells 6.4 Alternative Sources of Pluripotent Stem Cells . . . . . . . . .

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˘ c (B) T. Sari´ Institute for Neurophysiology, Center for Physiology and Pathophysiology, Medical Center, University of Cologne, 50931, Cologne, Germany e-mail: [email protected]

T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_6,

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102 6.4.1 Derivation of Pluripotent Stem Cells by Reprogramming of a Somatic Nucleus . . . . . . . . . . . . . . . . . 6.4.2 Pluripotent Stem Cells Derived from Germ Cells . . . . 6.5 Future Prospects and Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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6.1 Introduction Murine embryonic stem (ES) cells have been instrumental in studying early embryonic development and clarifying the function of many genes utilizing transgenic or knock-out animals. With the derivation of first human ES cell lines 10 years ago their potential as a source of cells for tissue repair, gene therapy, drug discovery and toxicological testing began to be intensively explored [1, 2]. Proof of principle experiments have demonstrated that specific ES cell-derived mature cells such as cardiac myocytes, neurons, endothelial cells, insulin-producing β-Langerhans cells, hematopoietic precursors and hepatocytes exhibit phenotypic properties comparable to that of corresponding fetal or adult cells and can be successfully used for tissue repair in experimental animals [3–5]. Despite these successes, development of human ES cell-based therapies has been hampered by unresolved legal, ethical and political issues, which are based on the argument that derivation of ES cell lines from human blastocysts destroys potential human life and can therefore not be used for scientific or therapeutic purposes [6, 7]. Further major obstacle to clinical translation of human ES cell derivatives was lack of autologous cells that can be transplanted without the risk of immune rejection [8]. To circumvent this problem and obviate the use of genetically mismatched blastocyst-derived ES cells, major effort in the last decade has been put into development of methods for derivation of autologous pluripotent stem cells without destroying human embryos [9]. In this review we describe different ways for derivation of pluripotent stem cells with special emphasis on recent groundbreaking achievements in nuclear reprogramming of adult somatic cells by ectopic expression of “stemness” factors and discuss how these new developments may affect the progress in biomedical research and regenerative medicine in the near future.

6.2 Embryonal Carcinoma (EC) Cells The history of pluripotent stem cells began more than 50 years ago with the observation that males of mouse strain 129 develop spontaneous testicular teratocarcinomas with high incidence of about 1% [10]. These tumors arise from abnormal germ cells within the seminiferous tubules and belong to a group of non-seminomatous germcell tumors, which are characteristically composed of randomly distributed areas of differentiated structures resembling various adult tissues and, in rare cases, even organs. Teratocarcinomas sometimes also contain structures that closely resemble an early embryo at the gastrulation stage, so called embryoid bodies (EB). This same term has been later adopted to describe solid or cystic spherical cell aggregates into which ES cells differentiate in in vitro suspension cultures. Following

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the description of spontaneous teratocarcinomas in 129 mouse strain the methods were developed for producing murine teratomas and teratocarcinomas experimentally by explanting to extrauterine sites genital ridges of fetuses between 11 and 13.5 days of development [11] or mouse egg-cylinders at about 7 days of development [12]. Although teratocarcinomas could not be generated by such techniques in other animal species for unknown reasons, these transplantation experiments provided the first hint that the intact embryo contains a population of cells that may become pluripotent in appropriate microenvironment. The availability of spontaneous teratomas and teratocarcinomas and the ability to experimentally induce teratoma formation in mice stimulated further developments in this field. Among the most important ones was the demonstration that a single cell derived from a retransplantable tumor can differentiate into diverse cell types found in teratocarcinomas, which could be repeatedly transplanted in successive recipients without loosing their pluripotent character [13]. This finding firmly established that these tumors contain pluripotent stem cells, so called embryonal carcinoma (EC) cells, which can divide indefinitely and possess the ability to differentiate into multiple somatic cell types. Subsequent establishment of permanent murine [14] and human [15–17] EC cell lines greatly facilitated the experimentation with this model system. Mouse EC cells are regarded as an in vitro counterpart of the pluripotent cells present in the inner cell mass (ICM) of the pre-implantation blastocyst, because these cell types express similar markers [18–20]. However, the utility of both murine and human EC cells as an experimental model for developmental studies was greatly limited due to their restricted developmental potential in vitro and inability of most mouse EC cell lines to contribute to chmieric mice after injection into the blastocyst, most likely as a consequence of genetic alterations that these malignant cells frequently harbour [21]. Nevertheless, work with EC cells led to discoveries that were later crucial for generation of murine and human ES cell lines from early blastocysts. For example, the finding that some EC cell lines required culture on feeder layers of mouse fibroblasts to maintain their pluripotency [22] and that their differentiation can be induced by removal from feeder cells and formation of EB in suspension cultures [23] are still routinely utilized today for generation, propagation and differentiation of mouse [24–26], primate [27, 28] and human ES cells [29, 30]. In addition, cell surface antigens that are today broadly used to assess the undifferentiated status of ES cells have been first identified on either undifferentiated mouse (e.g. stagespecific embryonic antigen 1, SSEA1) or human (e.g. SSEA3, SSEA4, TRA-1-60 and TRA-1-81) EC cells.

6.3 Embryonic Stem (ES) Cells Building up on the experience gained from studies on EC cells, culture conditions have been developed that enabled in vitro derivation of first pluripotent ES cells from the ICM of mouse blastocysts in 1981 [24, 25]. Initially, these ES cells were of interest almost exclusively to developmental and cell biologists and mostly served to elucidate the function of specific genes in vivo through generation of transgenic or knock-out animals. Establishment of in vitro pluripotent cell lines from human

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blastocysts significantly lagged behind their murine counterparts due to their different cell culture requirements. However, the successful derivation of primate ES cell lines [27, 28] and improvements in culturing human embryos obtained by in vitro fertilization (IVF) [31] led to subsequent isolation of human ES cell lines in the late 1990s [30]. After this high-profile discovery the interest in ES cells literally exploded and their basic properties and potential for cellular replacement therapy began to be extensively explored.

6.3.1 Hallmarks of ES Cells Human and murine ES cells differ in morphology, cell culture requirements, cell surface marker expression, signaling pathways and differentiation ability. However, they share the two fundamental properties of stem cells: self-renewal (ability to remain undifferentiated by symmetrically dividing into the same non-specialized cell types over long periods of time) and pluripotency (ability to differentiate into cells of different lineages). The pluripotency of murine ES cells is typically assessed, in the order of increasing stringency, by their ability to (a) differentiate in vitro into lineages of all three primary germ layers, (b) form teratomas, (c) contribute to chimera formation, (d) germ line transmission, and, (e) allow tetraploid complementation [32]. In chimera formation a single putative pluripotent cell (genetically labeled to permit tracking of daughter cells) is injected into a mouse blastocyst. If the labeled injected cell is truly pluripotent, labeled daughter cells will be seen throughout the embryo and in adult organism in tissues derived from all germ layers. In germ line contribution test, the ability of ES cells to generate functional germ cells is assessed by the ability of chimeric or “all ES” animals to give rise to offspring carrying genetic and phenotypic marks of a donor ES cell. Tetraploid complementation is the most stringent test for pluripotency and involves injection of ES cells into 4n host blastocyst. Animals derived from these blastocysts are composed exclusively of donor ES cell derivatives because 4n blastocyst can not contribute to somatic lineages. Measure of pluripotency based on blastocyst manipulation can not be applied for obvious ethical reasons on human or primate ES cells. Consequently, developmental potential of these ES cells is typically demonstrated by in vitro differentiation and by formation of all three germ layers in teratomas generated by transplantation of pluripotent stem cells into immunodeficient animals [30, 33]. In a more practical way, the quality control of ES cells in culture is routinely performed by much faster cytochemical, flow cytometry or RT-PCR detection of various ES cell-specific proteins and transcripts (Table 6.1)

6.3.2 Regulation of Self-Renewal and Pluripotency of ES Cells The understanding of how the self-renewal and pluripotency of ES cells are regulated at the molecular level is crucial for understanding the principles that maintain differentiated state and for devising strategies to reprogram this differentiated state

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Table 6.1 Commonly used markers of murine and human embryonic stem cells Expression in ES cells Marker

Name

SSEA1

Location

Stage-specific embryonic CS antigen 1 SSEA3 Stage-specific embryonic CS antigen 3 SSEA4 Stage-specific embryonic CS antigen 4 TRA-1-60 Tumor rejection CS antigen-1-60 TRA-1-81 Tumor rejection CS antigen-1-81 Oct4 POU domain, class 5, IC (POU5F1) transcription factor Nanog Nanog homeobox IC Sox2 Sex determining region IC Y – box 2 Rex1 Zinc finger protein 42 IC (Zfp42) FoxD3 Forkhead box D3 IC TDGF1 Teratocarcinoma-derived IC growth factor 1 UTF1 Undifferentiated cell IC transcription factor 1 LIN28 Lin28 homolog (C. elegans) IC ZIC3 Zinc family member 3, IC heterotaxy 1 HESX1 Homeobox (expressed in IC ES cells) 1 SLD5 SLD homolog IC GDF3 Growth differentiation IC factor 3

Human

Murine

References

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[20]

+

–

[235, 236]

+

–

[235, 236]

+

–

[235–237]

+

–

[235–237]

+

+

[53]

+ +

+ +

[56] [55]

+

+

[238]

+ +

+ +

[73] [239]

+

+

[240, 241]

+ +

+ -

[138] [138]

+

+

[138]

+ +

– +

[138] [242]

Abbreviations: IC – intracellular, CS- cell surface; CNS – central nervous system; HSPC – hematopoietic stem/progenitor cells.

back to the full or intermediate state of pluripotency. Self-renewal and pluripotency in ES cells are strictly controlled by various extrinsic and intrinsic (genetic and epigenetic) factors. 6.3.2.1 Extrinsic Factors The requirements for extrinsic factors are substantially different for murine and human ES cells [34–36]. The most important extrinsic factor for murine ES cells is the leukemia inhibitory factor (LIF), which is widely used as a component of murine ES cell culture media [37]. LIF, member of the interleukin-6 (IL-6) cytokine family, exerts its effects through binding to a heterodimeric cytokine receptor complex that

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Fig. 6.1 Simplified scheme of signaling pathways regulating self-renewal of murine ES cells. Detailed description of individual pathways and their interactions are described in the text. Outlined substances are inhibitors of indicated signaling molecules. PD325901 is inhibitor of ERK1/2, BIO and CHIR99021 are inhibitors of GSK3 and ICG-001 is inhibitor of CBP

consists of two subunits, the LIF receptor β (LIFRβ) and the gp130 (Fig. 6.1). The tyrosine kinase Janus kinase (JAK) binds constitutively to the intercellular domain of this receptor complex in its inactive form. Upon LIF binding, JAK phosphorylates tyrosine residues of both gp130 and LIFRβ. This phosphorylation recruits the latent transcription factor signal transducer and activator of transcription 3 (STAT3) to the receptor and causes the phosphorylation of the STAT3 by activated JAK tyrosine kinases [38]. Phosphorylation of STAT3 induces homodimerization of these molecules, nuclear translocation and subsequent binding to STAT-binding elements in the promoter/enhancer regions of target genes involved in the regulation of cell growth. Among downstream targets of STAT3 signaling are the transcription factor c-Myc [39] and the ligand Wnt5a, which is a member of the Wnt/β-catenin signaling pathway [40]. Besides the JAK-STAT3 axis, LIF is also capable of inducing a PI3K-dependent phosphorylation of protein kinase Akt (also known as PKB) and glycogen synthase kinase 3β (GSK3β). The later phosphorylates a number of downstream signaling components such as c-Myc and β-catenin, which marks them for degradation by the ubiquitin-proteasome pathway. However, inactivation of GSK3β by phosphorylation or pharmacological inhibition with BIO leads to c-Myc accumulation but does not change β-catenin levels in murine ES cells, indicating that GSK3β plays β-catenin independent role in defining murine ES cell properties. The transcription factor c-Myc is well known regulator of cell proliferation and transformation and is involved in epigenetic chromatin modifications [41]. In murine ES cells, overexpression of c-Myc leads to their LIF-independent self-renewal [39].

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In contrast, in human ES cells c-Myc induces apoptosis and differentiation into extraembryonic endoderm and trophectoderm lineages [42]. Differentiation induced by c-Myc was associated with downregulation of pluripotency markers Oct4, Nanog and α6-integrin. Additional extrinsic factor known to support mouse ES cell pluripotency is the bone morphogenetic protein 4 (BMP4) (Fig. 6.1). BMPs bind their cell surface receptors and initiate downstream signaling via DNA-binding transcriptional modulators called SMADs to regulate gene expression. The BMP-triggered signaling through SMADs involves the R-SMAD family members SMAD1, SMAD5, and SMAD8, which, once activated, bind the same co-SMAD, SMAD4. The resulting SMAD complex then assembles with other transcription factors in the nucleus and activates members of the inhibition of differentiation (Id) gene family and support the self-renewal together with LIF-STAT3 pathway [43]. Recent data from Austin Smith’s laboratory provide another view on requirements for self-renewal of murine ES cells and suggest that ES cells have an intrinsic self-renewing capacity, which is not dependent on extrinsic stimuli [44]. Using specific inhibitors of FGF receptor tyrosine kinases and ERK and GSK3 signaling cascades, they show that extrinsic LIF-STAT3 and BMP-SMAD-Id signaling actually do not directly instruct the self-renewal of murine ES cells but rather act in unrefined culture conditions to shield the pluripotent ES cell state from differentiation signals emanating from FGF receptor and extracellular signal regulated kinase 1 and 2 (ERK1/2) signaling. In addition, this study also questions previous conclusion that upregulation of c-Myc mediates ES cell self-renewal downstream of LIF [39] and suggests that elevated c-Myc is not necessary for ES cell propagation in undifferentiated state. This conclusion is based on observation that simultaneous inhibition of FGF receptor, ERK- and GSK3-signalling, which allows for ES cell self-renewal in the absence of feeders, serum and LIF, leads to suppression of c-Myc transcript and protein expression in ES cells [44]. The secondary role of LIF-STAT3 signaling in self-renewal of murine ES cells is clearly demonstrated by the ability to establish the STAT3-null ES cells in the absence of LIF, serum and feeder cells given that the above mentioned inhibitors are present. These ES cells are morphologically indistinguishable from wild-type mES cells, express Oct4 and Nanog and can differentiate into multiple lineages in EB cultures. Therefore, absolute requirement for STAT3 in the derivation and self-renewal of ES cells is rendered dispensable by inhibition of FGF receptor, ERK and GSK signaling. According to these data, ES cells have an innate program for self-renewal and extrinsic factors only serve to suppress differentiation signals. In contrast to mouse ES cells, the BMP pathway has opposing role in human ES cells and it promotes their differentiation to throphoblast instead of pluripotency [45]. Moreover, human ES cells also do not require LIF for propagation in an undifferentiated state. Instead, most feeder-free culture conditions for human ES cells require the supplementation of the basic fibroblast growth factor (bFGF/FGF-2) and transforming growth factor-β (TGF-β)/Nodal/Activin [35, 36, 46, 47]. Activin A is a pleiotropic cytokine member of the TGF-β superfamily, it is expressed both in human ES cells

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and in murine embryonic fibroblast feeders, and it seems to be the most important exogenous factor for maintaining the pluripotency of human ES cells [48–50]. The TGF-β/Activin signaling is transmitted by the phosphorylation of SMAD2/3, which are abundant in undifferentiated human ES cells. Upon binding of Activin A to its cell surface receptors, phosphorylated SMAD2 associates with SMAD4, which translocate to the nucleus to regulate expression of genes such as Oct4, Nanog, Wnt3, bFGF and FGF8 [36]. Both the TGF-β-responsive SMAD2/3 and the BMP-responsive SMADs (SMAD1/5/8) were found to bind directly to the NANOG proximal promoter in human ES cells [51]. However, the former appeared to enhance while the later decreased the NANOG promoter activity. This indicates that an intricate balance of SMAD2/3 and SMAD1/5/8 could modulate the expression of NANOG and potentially determine the choice between undifferentiated and lineage-committed fates. Recent studies have shown that bFGF cooperates with Activin/Nodal [35, 50] and synergizes with TGF-β1 to support the prolonged selfrenewal of human ES cells by inhibiting BMP signaling and sustaining expression of pluripotency genes Nanog, Oct4 and Sox2 [51, 52]. 6.3.2.2 Transcriptional Regulation Preservation of pluripotency in ES cells and their differentiation into somatic cells are governed by two kinds of intrinsic mechanisms, genetic and epigenetic. The key genetic regulators of self-renewal and pluripotency in both murine and human ES cells are the homeodomain transcription factors Oct4/Pou5f1 [53, 54], Sox2 [55] and Nanog [56]. Utilizing genome-scale location analysis recent studies with human and mouse ES cells revealed that Oct4, Sox2 and Nanog together occupy promoter regions of several hundred active ES cell-specific genes and, surprisingly, also the promoters of developmental genes that are inactive in ES cells. Each of these factors alone occupies the promoters of more than 1,000 genes [57, 58]. Among genes that were bound by these factors in ES cells were the genes of BMP and JAK/STAT signaling components [59, 60]. Interestingly, Oct4, Sox2 and Nanog also bind to their own promoters as well as promoters of the other two genes, indicating that these three factors together tightly control their own expression in an autoregulatory circuity [61]. The best-characterized intrinsic regulator of pluripotency is the homeodomain protein Oct4 (also known as Oct3/4), a member of the POU class of transcription factors (POU5F1). Oct4 derives its name from its ability to activate the transcription of genes containing the octameric consensus-binding motif ATGCAAAT within their promoter or enhancer regions. Oct4 is broadly expressed throughout the prepluripotent phase of embryo development, in the post-implantation egg cylinder as well as in epiblast stem cells (EpiSC) derived from E8.5 egg cylinder. Oct4 regulates cell fate in a dosage dependent fashion. Niwa and coworkers demonstrated that the level of Oct4 activity specifies three distinct fates of ES cells: (1) a less than twofold increase in expression of Oct4 turns ES cells into primitive endoderm and mesoderm; (2) repression of Oct4 induces the formation of trophectoderm; and (3) only a optimal amount of Oct4 can sustain stem cell self-renewal [54]. Recently,

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expression studies of Oct4 in human ES cells have shown that only one of the isoforms of POU5F1, namely POU5F1-iA, plays a role in stemness [62]. Sometimes, Oct4 pseudogene may be confused for the real Oct4 expression, which must be taken into account when Oct4-specific primers are designed for gene expression analyses [63]. However, alternative transcripts that lack exon 1 and are transcribed from regions between intron 1 through exon 3 of the Oct4 gene were recently found to be expressed in somatic tissues [64]. The expression of these alternative transcripts is regulated by novel promoter regions and one of the two encoded truncated proteins was capable of transforming non-tumorigenic fibroblasts in vitro. However, the role of alternative Oct4 transcripts in somatic cells and cancer stem cells remains to be determined. Nanog is one additional marker for pluripotency. Nanog was discovered based on its ability to sustain human ES self-renewal in the absence of LIF [56, 65]. This transcription factor behaves as a strong activator of Oct4 promoter, thus, participating in the regulation of Oct4 expression in ES cells. Overexpression of Nanog in murine ES cells was shown to increase the efficiency of fusion-mediated reprogramming of a somatic nucleus [66]. However, recent genetic deletion of Nanog revealed that ES cells can self-renew indefinitely in the permanent absence of Nanog and remain pluripotent [67]. However, Nanog-negative cells were more susceptible to differentiation. In chimeric mice Nanog-deficient cells colonize embryonic germ layers and exhibit multilineage differentiation but primordial germ (PG) cells lacking Nanog fail to mature. Thus Nanog appears to be dispensable for pluripotency but is specifically required for formation of germ cells, prevents differentiation of ES cells and may ensure their survival in an epigenetic plastic state [67]. Sox2, a high-mobility group DNA-binding domain transcription factor, is expressed in the pluripotent lineage of the mouse embryo. Sox2 also plays a critical role in the pituitary, brain, stomach and eye during embryonic development. In ES cells, Sox2 is involved in regulating its own expression and expression of other pluripotency factors thus, stabilizing ES cells in a pluripotent state by maintaining the requisite level of Oct4 and Nanog expression [68, 69]. In vitro, Sox2 is highly expressed in both murine and human ES cells. In murine ES cells, reduction of Sox2 expression is associated with a loss of the pluripotent state and a propensity for differentiation [70]. Recently, Fong et al showed that reduction of Sox2 by RNAi resulted in a loss of the human ES cell state as demonstrated by altered cell morphology, loss of stem cell antigen expression, and differentiation of stem cells primarily into trophectoderm [71]. Downregulation of Sox2 also reciprocally decreased Oct4 and Nanog expression, suggesting the coordinated role of all three transcription factors in regulating pluripotency. These results established that Sox2 is not merely a synergistic factor in the regulatory network but a factor whose requirement, alone, is needed to preserve self-renewal and pluripotency in human ES cells. In addition, reduction of Sox2 expression in turn reduced the expression of several new ES cell-associated genes, including transcription factors regulating stemness and factors involved in chromatin remodeling and senescence signaling. In addition to these three “master regulators” several additional factors have been implicated to play a role in ES cell biology. These factors include, among

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others, Foxd3, Sall4, Klf4 and Lin28. Foxd3, a member of the forkhead transcription factors, also activates Oct4 expression [72]. FoxD3 is also required for epiblast formation in blastocysts and stem cell pluripotency in mouse [73]. Sall4 is a member of Spalt family of transcription factors that was originally identified in Drosophila as a homeotic gene required for head and tail development. In mammals it is essential for early embryo development and for the establishment of the ICM [74]. Sall4 has been reported in mouse ES cells as a new component of the transcriptional regulatory network and may form a regulatory circuit together with Nanog similar to that of Oct4 and Sox2 [75, 76]. The Kruppel-like factor 4 (Klf4) is abundantly expressed in ES cells and cooperates with Oct4 and Sox2 to activate downstream stem cell specific genes [77]. Although ES cells lacking Klf4 do not loose their stem cell properties [77], simultaneous knock down of several Klf family members in ES cells causes their differentiation and down-regulation of a small group of ES cells specific genes [78]. This indicates that there is a certain degree of functional redundancy among various Klf proteins. It is also of note that Klf family members and Nanog bind to similar target genes, suggesting their participation in the core transcriptional regulatory network in ES cells [78]. Lin28 is a small, highly-conserved and developmentally regulated RNA-binding protein, which is specifically expressed in the cytoplasm and nucleus of EC cells as well as mouse and human ES cells. Lin28 is down-regulated upon differentiation of ES cells [79]. However, its downregulation neither impaired human ES cell proliferation nor led to their differentiation or apoptosis, indicating that this factor is not involved in control of ES cell self-renewal. It has been recently shown that Lin28 blocks the processing of primary microRNA (pri-miRNA) transcripts in ES cells (see below in 6.3.2.4). 6.3.2.3 Epigenetic Regulation In addition to autoregulatory transcriptional control described above, the expression of Oct4 and other pluripotency genes is subject to epigenetic regulation as well. It has been recently shown that Oct4 promoter/enhancer region is hypomethylated and hyperacetylated in murine ES cells, indicating that the DNA methylation and chromatin structure status are important regulatory elements of Oct4 gene expression [80]. Not only in the Oct4 locus, but also globally has the chromatin of pluripotent stem cells unique properties. In ES cells large number of genes important for development harbor, so called, bivalent domains containing at the same time both the repressive (tri-methylated histone H3 at lysine 27; H3-K27me3) and activating (H3-K4me3) histone modifications on lysine side chains [81, 82]. A large number of repressed developmental genes that contained bivalent domains in their promoter regions were found to be associated in ES cells with Polycomb group (PcG) proteins [83, 84]. These proteins function in two distinct multiprotein complexes, termed Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2), and play essential roles in concert with master transcriptional regulators Oct4, Sox2 and Nanog in repression of gene expression during embryogenesis and in preventing premature differentiation of ES cells. H3-K27 methylation serves as a binding surface for PRC1, which is responsible for condensation of chromatin

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structure and gene silencing. Recently, Guenther and coworkers revealed that silent developmental genes that are occupied by Oct4, Sox2, and Nanog and PcG proteins also have the transcriptional initiation apparatus bound to their promoters. However, the RNA polymerase is not able to fully transcribe these genes, presumably because of repressive action of PcG proteins. Apparently, this combination of modifications keeps developmental genes in a silenced state in ES cells and may enable their quick transcription as soon as the ES cells start to differentiate and repressive epigenetic marks are erased.

6.3.2.4 MicroRNAs MicroRNAs (miRNAs) are a family of small non-coding RNA molecules that are involved in post-transcriptional control of steady-state levels of multiple mRNA targets in the cell. Specific miRNAs have been shown to participate in mammalian cellular differentiation, developmental patterning and morphogenesis [85–88]. Mouse and human ES cells express specific groups of miRNAs that are not found in other types of cells [89]. miRNAs belonging to miR-290 and miR-302 clusters have been shown to be specific for ES and EC cells as well as for the pluripotent stem cells derived from adult mouse testis [90, 91]. miRNAs are transcribed as immature precursors, which must be enzymatically processed in order to exert their regulatory function in development. The processing of miRNAs occurs in two steps. The first step is mediated by RNase III enzyme Drosha that acts together with the RNAbinding protein DGCR8 in a complex called Microprocessor. The second step is mediated by a complex composed of TRBP and Dicer proteins. DGCR8-deficient mouse ES cells are devoid of all canonical miRNAs. Analysis of these ES cells showed that they proliferate slowly and accumulate in G1 phase of the cell cycle [91]. However, these cells still express many pluripotency markers and retain ES cell morphology in culture [92]. These data indicate that miRNAs function in suppressing the self-renewal and enabling proliferation of ES cells. Later assumption is corroborated by data showing that members of the miR-290 family of ES cellspecific miRNAs promote the G1-S transition of the ES cell cycle [91]. miR-302 family of miRNAs is expressed most abundantly in slow-growing human ES cells and quickly decreases after cell differentiation and proliferation [93]. Transgenic expression of miR-302 into several human cancer cell lines converted these cells into an ES cell-like pluripotent state in which the reprogrammed cells shared over 86% similarity in genome-wide gene expression pattern with human ES cells und could differentiate into several distinct cell types in vitro [93]. Lee and co-workers demonstrated that miR-302b in human EC cells plays an important role in maintaining the pluripotency of EC cells by post-transcriptional regulation of Cyclin D2 expression [94]. The production of Let-7 family of miRNAs also involves Drosha-mediated processing of immature Let-7 primary transcripts [95]. The ES cell-specific protein Lin28 exerts its function by inhibiting Drosha and pri-let-7g miRNA processing in ES cells [96]. Unprocessed pri-let-7g miRNA transcripts are readily detectable in ES cells and in EBs in the course of differentiation. However, mature let-7g is

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Fig. 6.2 Different strategies for production of pluripotent ES cell-like stem cells. Pluripotent stem cells can be generated from early embryos, fetal, newborn or adult somatic cells as well as from fetal primordial or adult germ cells. In a single-cell biopsy concept a single totipotent blastomere is removed from an eight-cell embryo (morula) to derive a new ES cell line and the remaining sevencell embryos are transferred into surrogate mothers, in which they develop into normal organism. The second approach is based on extracting pluripotent cells from the inner cell mass (ICM) of poor-quality embryos generated in the process of in vitro fertilization (IVF). These embryos are unsuitable for uterine transfer or cryopreservation and would therefore be discarded. Somatic cell nuclear transfer (SCNT) involves the transplantation of a somatic cell nucleus into an enucleated unfertilized oocyte. Pluripotent ES cell lines can be establish from cloned blastocysts (therapeutic cloning) and used to generate cells for autologous cellular therapies. Reprogramming of an adult nucleus to a pluripotent state can be also achieved by fusion with an ES cell. The resulting cells have all characteristics typical of ES cells but the disadvantage of being tetraploid. Nuclear reprogramming can be also achieved by incubating streptolysin O-permeabilized somatic cells with crude protein extract of ES cells. This method does not lead to generation of fully reprogrammed pluripotent stem cells. Complete reprogramming of somatic cells can be induced by viral overexpression of a defined combination of transcription factors associated with ES cells. This approach holds great promise for science and medicine because it circumvents the use of oocytes as well as ES cells and may allow for patient-specific autologous therapies and establishment of human in vitro disease models. Unipotent spermatogonial stem cells from adult testis can be converted to a pluripotent state by prolonged culture. Mammalian oocytes can be artificially induced to undergo parthenogenesis in vitro by a two-step protocol involving electroporation and/or treatment with a chemical agent (ionomycin, ethanol, or inositol 1,4,5-triphosphate) to elevate calcium levels transiently, followed by application of an inhibitor of protein synthesis (cycloheximide) or protein phosphorylation (6-dimethylaminopurine). These agents mimic the calcium wave induced by sperm during normal fertilization

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undetectable in ES cells but is strongly induced at later differentiation stages [96]. By blocking miRNA processing Lin28 inhibits miRNA-mediated differentiation of ES cells and plays an important role in maintenance of the undifferentiated state. Recent study identified several naturally occurring miRNAs (miR-296, miR-470 and miR-134) that target coding regions of known pluripotency factors Nanog, Oct4 and Sox2 and modulate ES cell differentiation [97]. Recent comprehensive analysis of transcriptional regulatory circuitry in ES cells that also involved the analysis of miRNA promoter regions revealed that the key ES cell transcription factors are also associated with promoters for ES cell-specific miRNAs [98]. These data suggest that miRNAs exert an important role in regulating self-renewal and differentiation of ES cells, but the exact mechanism of their participation in this complex process in concert with other factors is not yet understood.

6.4 Alternative Sources of Pluripotent Stem Cells To obviate the use of ES cells, adult stem cells have been proposed as an alternative source of cells for regenerative medicine. However, adult stem cells are difficult to culture for extended periods of time and they seem to possess only limited ability to differentiate into a variety of tissue-specific cells. Therefore, adult stem cells can hardly surpass the pluripotential nature of ES cells so that the derivation of many scientifically and clinically useful cell types may still be dependent on the availability of well characterized ES cell lines. Virtually all existing human ES cell lines have been derived from the ICM of the healthy blastocyst-stage embryos. Due to ethical objections to the use of human ES cells, many investigators and legislative bodies examined the alternative ways for producing ethically, scientifically and therapeutically acceptable pluripotent stem cells [9]. Various approaches for generation of pluripotent stem cells differ in their technical complexity as well as in the functionality of established cells, their safety, ethical acceptability and therapeutic applicability. These approaches can be grouped into three categories depending on the source of cells used for derivation of pluripotent stem cells (Fig. 6.2). In the first category, adult somatic cells are the source of pluripotent stem cells. Approaches in this category have the capability for derivation of autologous stem cells and include nuclear reprogramming by somatic cell nuclear transfer (SCNT), altered nuclear transfer (ANT), cell fusion, cell extracts or ectopic expression of pluripotency factors. In the second category, pluripotent stem cells are derived from early embryos in a way that may be considered ethically acceptable. This concept includes derivation of ES cells from single blastomeres or from poor-quality IVF embryos that would otherwise be discarded. With this technique only allogeneic stem cells can be generated. Methods in the last category make use of adult germ cells (e.g. oocytes or spermatogonial stem cells) for obtaining pluripotent stem cells.

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6.4.1 Derivation of Pluripotent Stem Cells by Reprogramming of a Somatic Nucleus 6.4.1.1 Somatic Cell Nuclear Transfer (SCNT) Development of techniques to generate autologous pluripotent stem cells has a long history. It began at the end of 19th century with the question whether cells acquire reversible or irreversible changes in their genome in the course of development and differentiation. Contrary to earlier believe that during differentiation cells irreversibly loose those genes no longer needed to be expressed, we know today that in this process the physical integrity of the genome is not altered and that the genetic material remains identical in nearly all cells of an organism regardless of their type or maturational status. This conclusion has been derived from a series of nuclear transfer (NT) studies pioneered by Briggs and King using frog embryos in the 1950s [99–101]. In this procedure, also termed the somatic cell nuclear transfer (SCNT), the nucleus of a differentiated cell is transferred into an enucleated unfertilized oocyte with the purpose to determine the potential of adult somatic cell nucleus to support embryonic development and give rise to a live animal (Fig. 6.2). These experiments led several decades later to the generation of the sheep Dolly from an adult mammary gland cell [102] and were followed few years ago by unequivocal demonstration of nuclear totipotency of terminally differentiated cells by cloning mice from B and T lymphocytes [103], post-mitotic olfactory neurons [104], natural killer T cells [105], peripheral blood granulocytes [106] and even malignant melanoma [107] and embryonal carcinoma cells [108]. The successful conversion of a fully differentiated cell into a totipotent state through SCNT implies that epigenetic, and not genetic, mechanisms are responsible for this plasticity. Epigenetic mechanisms establishing cellular identity during differentiation and dedifferentiation include (a) DNA cytosine methylation, (b) covalent histone modifications such as acetylation methylation and ubiquitylation, (c) remodeling of other chromatin associated proteins such as polycomb group proteins and transcription factors, and (d) pre- and post-transcriptional gene regulation by small non-coding RNAs, such as miRNAs. During epigenetic reprogramming of the nucleus of a differentiated cell, these modifications, in particular DNA methylation must be reset from a fully differentiated to a pluripotent or totipotent state. Especially the promoter regions of key pluripotency factors Oct4, Sox2 and Nanog must be converted into the replication permissive state characterized by hypomethylation and hyperacetylation. The exchange of nuclear factors between the donor cell nucleus and the enucleated egg cytoplasm is considered to be important for this process. Until recently, SCNT was the only technique to accomplish complete nuclear reprogramming and was used not only to clone live animals, such as Dolly (reproductive cloning) but also to establish SCNT-derived ES cell lines from cloned murine [109] and, recently, primate blastocysts [110] for the purpose of therapeutic cloning. These cells appear to be indistinguishable in their proliferative, developmental and therapeutic potential from ES cells derived from fertilized embryos and are regarded as a potential source of patient-specific cells for custom-tailored tissue repair or gene therapy [111]. In a first proof of principle experiment, SCNT ES

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cells were derived from an immunodeficient mouse and then used in vitro to correct the original genetic defect by homologous recombination. Subsequently, the repaired SCNT ES cells were differentiated into hematopoietic stem cells and transplanted back to the immunodeficient donor mouse to restore lymhopoiesis [112]. In one another example, mouse SCNT-derived stem cells were differentiated into ckit-positive fetal liver stem cells and injected in the border zone of infarcted mouse heart. One month after transplantation injected stem cells formed new myocytes and blood vessels and improved the heart function [113]. Recently, Tabar and coworkers have generated 187 SCNT ES cell lines from 24 parkinsonian mice with a minimum of one line per mouse [114]. These multiple pluripotent cell lines were then subjected to neural induction and differentiation into the midbrain dopamine neurons. When these cells were transplanted into genetically matched parkinsonian mice, cells engrafted permanently and the animals showed significant improvements in all behavioral scores. In contrast, cells injected into allogeneic mice were rejected without any therapeutic benefit to transplanted animals. These data suggest that in the future human SCNT ES cells may have similar therapeutic potential. Recently, French and coworkers have generated human cloned blastocysts by SCNT with adult fibroblast nucleus but they did not succeed in establishing human ES cell lines from these blastocysts [115]. Although, no human SCNT ES cell lines have been created yet, the successful recent generation of SCNT ES cell lines in primates [110] suggests that therapeutic cloning in humans using SCNT may be feasible in future. Despite the advantage that autologous patient-tailored stem cells can be generated by this approach without the need for genetic modification of cells, the need for a large number of donated human oocytes, ethical concerns and extremely low cloning efficiency by SCNT in mammals render this method impractical for derivation of pluripotent cells for therapeutic purposes. 6.4.1.2 Cytoplasmic Hybrids One possibility to overcome ethical concerns and the shortage of human oocytes is by using enucleated animal oocytes to reprogram human somatic nuclei in a form of, so called, cytoplasmic hybrids or “cybrids”. After long debate this approach has been approved by the UK government in 2007 but it is highly controversial due to unpredictable influences that egg cytoplasmic components (mitochondria and mitochondrial DNA, proteins) may have on the developmental potential of the resulting entity and due to the risk of transmission of mitochondrial diseases, genetic mutations or infectious agents from the animal oocyte to newly generated stem cells. Up to date, only one laboratory could obtain human ES cells from a cybrid, but they have not been able to repeat the result, apparently due to difficulty in reaching late stage embryos required for generation of ES cell lines [116]. 6.4.1.3 Altered Nuclear Transfer (ANT) One another attempt of getting around the ethical concerns related to SCNT approach was the suggestion to derive pluripotent ES cell lines from blastocysts generated by a so-called altered nuclear transfer (ANT) [117]. This technology

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involves temporal inactivation before nuclear transfer into an oocyte of a gene required for normal development, such as Cdx2. This gene is essential for trophectoderm formation in mouse. Blastocysts with disabled Cdx2 lack trophectoderm and cannot implant but can serve as a source of normal ES cells after removal of a transgene producing the Cdx2-interfering RNA [118]. Murine ANT-derived stem cells can self-renew in culture and are pluripotent as demonstrated by teratoma formation and tetraploid complementation. According to proponents of this concept, blastocysts, which lack the full developmental potential, should be ideologically acceptable for derivation of stem cells. However, the validity of the ANT for production of ideologically acceptable human ES cell lines has been strongly questioned [119, 120]. Critics of the conditional Cdx2-knockdown strategy argue that reversible inactivation of Cdx2 is ethically not distinct from destroying the embryo by the immunosurgical method that is routinely used to derive human ES cells. Moreover, it is not clear if the Cdx2 would have the same function in human development as it has in a mouse and testing this by using human embryos would not be allowed for ethical reasons. Even if this could be verified, the method for knocking down the gene by RNA interference would never be in the position to insure that absolutely every embryo produced by this technology is incapable of normal development. In addition, this method would be technically very demanding and thus too inefficient for practical purposes. Furthermore, the need for removing and reinserting genes in the process of ANT could produce genetic errors and such ES cells may not be useful for scientific or therapeutic applications. Heated debate instigated by this proposal illustrates that it is elusive to solve ethical questions by scientific means. One possible solution to this problem would be to completely abandon the idea of deriving cells for replacement therapy from blastocysts but to derive the patient’s own pluripotent cells in vitro from germ cells or by nuclear reprogramming of her or his own adult somatic cells to a pluripotent state. Since these approaches do not require destruction of embryos or donation of human oocytes, they would be more practical and ethically acceptable. 6.4.1.4 Nuclear Reprogramming by Cell Fusion In addition to SCNT, other methods have been used to convert adult cells to a less differentiated or pluripotent state. The method of cell fusion is based on polyethylene glycol-induced or spontaneous heterokaryon formation between adult somatic cells and undifferentiated ES cells (Fig. 6.2). Fusions between murine or human ES cells and lymphocytes [121, 122], neurosphere cells [123], fibroblasts [124, 125] or myeloid precursor cells [126] have been reported to promote the epigenetic reprogramming of the adult genome to a pluripotent embryonic phenotype. The resulting fusion hybrids were morphologically indistinguishable from normal ES cells, had the potential to differentiate into multiple lineages, contributed to all three primary germ layers of chimeric embryos and exhibited transcriptional activity and epigenetic profile similar to that of ES cells. The method of cell fusion might be regarded superior to SCNT, as it is technically less challenging and does not

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require the use of oocytes and preimplantation embryos. However, it’s utility for the generation of cells for tissue repair is very limited by the tetraploid character of hybrid cells and their inability to separate the ES cell genome from a somatic partner. Studies examining segregation of parental chromosomes in ES cell-somatic cell hybrids using microsatellite analysis and in situ hybridization revealed that hybrid clones between murine ES cells and splenocytes or fibroblasts were stable but exhibited variable chromosome numbers ranging from 40 to 85 due to extensive loss or gain of individual chromosomes indicating that “clean” spontaneous segregation of parental genomes does not occur even after prolonged cell passaging [121, 127]. Even though a sophisticated method to remove single chromosomes from ES-somatic hybrid cells has been reported [128] it is unlikely that separation of all chromosomes of the somatic and the ES cell fusion partner apart from each other will be possible with this technique. Because of this difficulty, the cell fusion approach has low potential for use in human therapy but will remain useful as a tool for investigating the mechanism of cellular plasticity and reprogramming. Indeed, this method has proven very powerful for investigating factors required for reprogramming of somatic nucleus into a pluripotent state [129–131]. Ma and coworkers used murine ES cell-somatic cell fusion approach to enable direct visualization of rare fusion and transient reprogramming events at a single cell level. They have determined that up to 95% of all fused cells reactivated endogenous Oct4 in somatic cells and that it takes approximately 2–4 days for reprogramming to complete. Overexpression of histone demethylase Jhdm2a and knock-down of histone methyltransferase G9a by short hairpin RNAs in a somatic cell fusion partner (neural stem cells, NSCs) increased the reprogramming frequency and efficacy as assessed by Oct4-EGFP reactivation after fusion of NSCs with ES cells. Jhdm2a is known to regulate ES cell self-renewal and G9a plays critical roles in silencing Oct4 during differentiation of ES cells. These data suggest that early phase of reprogramming may involve extensive epigenetic remodeling (e.g. demethylation at the Oct4 and other promoters), while transcription factors may be required for recruiting epigenetic regulators and stabilizing the pluripotent epigenome [129]. With similar approach, Wnt/β-catenin pathway [130] and Sall4 [131] have been identified to enhance somatic cell reprogramming after fusion with ES cells. This data vividly illustrate how one approach without direct utility for derivation of therapeutically relevant cells may still provide important cues for understanding molecular basis of reprogramming and be used for optimization of reprogramming strategies based on other principles (see below). 6.4.1.5 Nuclear Reprogramming by Cell Extracts One approach that circumvents the use of intact ES cells and embryos is reprogramming of adult cells with cell-free extracts (Fig. 6.2). In this technique, the plasma membrane of adult cells to be reprogrammed is reversibly permeabilized with the bacterial toxin Streptolysin O. Permeabilized cells are then incubated with a nuclear and cytoplasmic extract derived from another type of cell in the presence of an ATP-regenerating system and GTP to promote nuclear import of reprogramming

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factors. After transient exposure to extract, cells are resealed and cultured. During incubation, factors in the cell extract required for reprogramming diffuse into permeabilized cells and activate genes that are typically expressed in cells from which the extract has been prepared and downregulate the acceptor cell-specific genes. The principle of this approach has been demonstrated in several different systems. In one of them, the kidney epithelial 293T cells have been reprogrammed to express T lymphocyte or neuronal markers by incubating them with an extract prepared from T cells or neuronal precursor cells, respectively [132]. However, although reprogrammed fibroblasts expressed T cell-specific surface markers, such as CD3, CD4, CD8, CD45 and T cell receptor (TCR) αβ chains, and a T cell-specific function (induction of high affinity IL-2Rα by stimulation of the TCR-CD3 signaling pathway), the reprogramming of the 293T cells into T cells was not complete, as the reprogrammed cells did not express a pure T cell-specific phenotype and the expression profile of many genes did not match that of T cells. In addition, no evidence was provided in this study for the genomic rearrangement of the TCR locus, which is required for the expression of a TCR on the cell surface, thus making it difficult to determine the source of TCR molecules. In similar approach, 293T cells were dedifferentiated by exposure to an extract of undifferentiated human teratocarcinoma cells leading to transition of epithelial 293T cell culture to an ES cell-like morphology, to activation of endogenous Oct4 gene associated with the DNA demethylation of its promoter, and to global changes of gene expression, which lasted over several weeks and were characterized by downregulation of 293T cell-specific genes and up-regulation of embryonic, germ cell and stem cell genes, including Nanog, Rex1, Sox2 and Utf1 [133, 134]. These reprogrammed cells could be induced to differentiate toward lineages unrelated to 293T cells such as neurogenic, adipogenic, osteogenic and endothelial lineages. However, extracts of undifferentiated teratocarcinoma cells were incapable of reprogramming 293T cells completely and not all changes were stable and heritable. In addition to human 293T cells, cell extract-based reprogramming approach was also applied to other mammalian cells such as human adipose tissue stem cells, which adopted cardiomyocyte properties following transient exposure to rat cardiomyocyte extract [135], to primary rat fetal fibroblasts, which were changed to β-cell phenotype after treatment with rat insulinoma cell extract [136] and to primary human leukocytes, which up-regulated pluripotency markers Oct4 and germ cell alkaline phosphatase but failed to express other surface antigens characteristic of pluripotent cells after exposure to frog Xenopus laevis egg extracts [137]. At present it is unclear if complete and stable reprogramming to a pluripotent state can be achieved with crude cell extracts and if reprogramming efficiencies would be different with different types of cells. However, this method is very attractive because cell extract-derived factors are presumably not permanently active in target cells but turn over at kinetics corresponding to their half-lives. In addition, by circumventing the use of whole cells, the difficulties associated with removal of extra chromosomes are eliminated. This technique has also a lot of room for improvement. Instead of crude extracts, defined combinations of known recombinant pluripotency factors in pure form could be used. The advantage of this approach would be that the

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composition and concentrations of reprogramming factors could be easily changed and optimized for different types of cells, that undesired effects mediated by whole cell extracts would be eliminated, and that the efficiency of reprogramming could be modulated by designing factors with lower or higher stabilities. Search for the identity of factors involved in maintenance of pluripotency was major focus of many studies in the past decade [57, 58, 138–140] and this knowledge was instrumental for generation of induced pluripotent stem (iPS) cells by stable or transient expression of defined stemness factors, which is currently the most efficient and promising way of reprogramming somatic cells to pluripotency.

6.4.1.6 Nuclear Reprogramming by Defined Transcription Factors Generation of Murine iPS Cells As discussed above, Oct4, Sox2 and Nanog are important transcription factors involved in the maintenance of pluripotency in ES cells (Table 6.1). However, they do not act in isolation but cooperate with many other regulatory proteins to exert this function. Gene expression profiling of six different human ES cell lines identified 92 genes that showed increased expression in ES cells [141]. Similarly, meta-analysis of 38 original studies on transcriptomic profile of human ES cells derived a consensus list containing 40 human ES cell specific genes [138]. Ectopic expression of either Oct4, Sox2 or Nanog alone in differentiated cells was not sufficient to convert them to a fully pluripotent state supporting the view that these factors exert their action in concert with others [142, 143]. In order to find the minimal combination of “stemness” factors capable of inducing an ES cell-like phenotype in somatic cells, Shinya Yamanaka’s group initially screened a group of 24 gene candidates known to be critical for pluripotency. They found that just four genes Oct4, Sox2, Klf4 and c-Myc are sufficient for reprogramming of mouse embryonic fibroblasts into, so called, induced pluripotent stem (iPS) cells when transduced with retroviral vectors (Fig. 6.2) [144]. This combination of factors is usually abbreviated as O-S-K-M (Oct4-Sox2-Klf4-c-Myc). Although slightly different combination of genes (Oct4, Sox2, Nanog and Lin28) has also been shown to work for reprogramming human fibroblasts [145], almost all murine and human iPS cell lines generated so far were produced with the Yamanaka’s OS-K-M combination in different perturbations. The initial strategy for obtaining iPS cells was based on selection of cells in which antibiotic resistance was driven by the ES-specific Fbx15 gene promoter, which is the downstream target of Oct4 [144]. These first iPS cells were not completely identical to ES cells since they had different gene expression and DNA methylation patterns and generated abnormal lethal chimeric embryos after injection into blastocysts. However, when the iPS cells were selected based on the reactivation of marked endogenous Oct4 or Nanog genes in murine fibroblasts isolated from transgenic mice, the resulting cells were highly similar to ES cells as demonstrated by highly comparable gene expression profiles, DNA methylation status and chromatin configuration [146–148]. Furthermore, when injected into immunodeficient mice these cells formed teratomas composed of

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various mature somatic tissues of all three germ layers including neurons, astrocytes, skeletal and smooth muscle cells, various types of epithelial cells, endothelium and cartilage. Moreover, these Oct4- and Nanog-selected iPS cells could form viable adult chimeras when injected into diploid blastocysts and were germ linecompetent as demonstrated by derivation of retrovirus positive blastocysts from matings between iPS chimeras and normal females. In a strictest test for developmental potency, iPS cells injected into tetraploid blastocysts generated live midand late-term embryos composed entirely of injected iPS cells [147, 148]. However, live animals could not be obtained yet from iPS cells by tetraploid complementation, most likely due to adverse effects of inserted proviral sequences. Efficient epigenetic reprogramming was also demonstrated in iPS cells by tracking the reactivation of the inactive X chromosome during reprogramming of female fibroblasts into iPS cells and its random inactivation during iPS cell differentiation into somatic cells, thus recapitulating the behavior of X chromosomes during normal development [146]. Over the past months, besides fibroblasts, many other cell types such as mature B lymphocytes [149], liver and stomach cells [150], primary hepatocytes [151], pancreatic β-cells [152], as well as mesenchymal and neural stem cells [153–157] have been successfully converted to iPS cells. Generation of Human iPS Cells Successful conversion of murine somatic cells to an ES cell-like state was a sensation for itself. However, it was initially not clear if such a simple reprogramming strategy will ever work in human cells using the same complement of genes. These concerns were laid to rest soon after discovery of murine iPS cells by Shinya Yamanaka’s and James Thomson’s laboratories [158, 159]. Yamanaka’s group succeeded in converting fibroblasts isolated from a 36-year-old woman and a 69year-old man into an ES cell-like state by using the same O-S-K-M combination of factors and retroviral vectors that were successfully employed in murine models [158]. Interestingly, Thomson’s group obtained similar result by using a different set of factors, namely Oct4, Sox2, Nanog and Lin28 (so called, O-S-N-L combination), which were transduced into fibroblasts isolated from fetal skin and the foreskin of a newborn boy using lentiviral vectors from which these factors are constitutively expressed from the elongation factor 1α (EF1α) promoter [159]. Recently, iPS cell lines have been also obtained from primate fibroblasts by retrovirus-mediated introduction of monkey transcription factor combination O-S-K-M [160]. Like murine iPS cells, their human and primate counterparts also have all essential properties typical of human ES cells and differentiate into derivatives of all three primary germ layers in vitro and form teratomas in vivo [145, 158]. Retroviruses Versus Lentiviruses Most iPS cells generated up to date were made with Molony-based retroviral vectors. The retrovirally introduced genes are expressed in transduced cells only early upon transduction and silenced in fully reprogrammed iPS cells due to methylation

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of retroviral long-terminal repeat sequences [161]. The retroviral vector silencing is indication that cells reached the fully reprogrammed pluripotent state in which active endogenous pluripotency genes stabilize and maintain the ES cell-like state. Prolonged expression of transgenic sequences can be detrimental to production of a stable pluripotent state and proper differentiation of iPS cells [156, 162]. Therefore, efficient silencing of retroviruses is an important prerequisite for establishment of iPS cells. Surprisingly, fully reprogrammed human [145] and mouse [163] iPS cells were also generated with lentiviral vectors that constitutively express reprogramming factors under the control of EF1α and CMV promoters, which are only poorly silenced in pluripotent cells. Recently, iPS cell lines were generated from murine postnatal fibroblasts and human keratinocytes using a single constitutive as well as inducible EF1α-driven lentiviral vectors expressing all four factors (O-S-KM) in a “stem cell cassette” [164, 165]. It remains to be clarified how continued transgene expression allows for establishment of iPS cells. However, since persistent expression of transgenes in established iPS cells is not desired, the great majority of current protocols for derivation of murine or human iPS cells utilize retroviral vectors that are spontaneously silenced in fully reprogrammed iPS cells or doxycyclin-inducible lentiviral vectors that express transgenes only in the presence of the inducer. However, while delivery of reprogramming factors with viral vectors enabled initial establishment of iPS cells and use of inducible lentiviruses was instrumental for clarifying the temporal requirements for ectopic factor expression, their further use for research and therapy will require replacing this strategy with those based on transient expression of factors without stable genetic modification of cells (see section Generation of iPS Cells Without Permanent Genetic Modification). Partially and Fully Reprogrammed Cells Early studies used transgenic adult cells expressing antibiotica resistance markers specifically from endogenous promoters of ES cell specific genes such as Oct4 and Nanog. This strategy enabled direct visualization and positive selection of pluripotent iPS cell clones. In order to make this approach applicable to human system, where transgenic cells expressing selection markers from endogenous promoters are not available, a method that allows for creation of iPS cells without requirement for antibiotic selection from wild type adult cells was developed [163, 166, 167]. In this strategy, murine embryonic or adult fibroblasts were transduced with four retroviral vectors encoding Oct4, Sox2, c-Myc and Klf4 and the iPS cells were selected based on morphological criteria (while fibroblasts grow in a monolayer, murine ES cell-like iPS cells form compact, domed colonies). These iPS cells were also indistinguishable from ES cells based on criteria listed above. This selection strategy has been adopted and refined by many other groups and is today an integral part in the process of selection of iPS cell colonies. However, morphologybased selection is unreliable, because besides derivations of true iPS cells it often results in selection of partially reprogrammed cells, which only rarely attain full pluripotency after long passaging. Studies using a doxycycline-inducible lentiviral

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systems have shown that the reprogramming induced by ectopic expression of pluripotency factors is a gradual process. Within two to three days of infection, mouse embryonic fibroblasts (MEFs) repress the expression of the fibroblastspecific Thy1 gene, which is accompanied by activation of alkaline phosphatase and followed by expression of SSEA1. Expression of endogenous pluripotency markers (such as Oct4, Sox2, Nanog and telomerase) and the reactivation of the silent X chromosome occur late in the reprogramming process [162, 168]. These studies have also determined that virally transduced factors must be expressed for at least 8–16 days for generation of iPS cells capable of self-sustained propagation in a pluripotent state [157, 162, 164, 168–170]. Cells that remain trapped in a stable, partially reprogrammed state in the process of reprogramming are morphologically indistinguishable from ES cells, stain positively for alkaline phosphatase and cell surface marker SSEA1 [162]. Nevertheless, these cells do not silence the expression of retrovirally transduced factors, express only a subset of endogenous stem-cell-related genes and show DNA hypermethylation at pluripotency-related loci, which are normally hypomethylated in ES cells. In addition, partially reprogrammed cells upregulate some lineage-specific transcription factors and show particularly high expression of proliferative genes [171]. Small fraction of these cells can even reactivate the transgenic Oct4 promoterdriven GFP expression, which is usually associated with fully reprogrammed cells, but this is unstable and lost within few passages [156]. The incomplete reprogramming of these cells is also evident by their inability to yield chimeras after blastocyst injection and failure to reactivate the inactive X chromosome in female cells [156, 171]. Interestingly, the transition of partially reprogrammed cells into fully reprogrammed iPS cells could be achieved by treating cells with the DNA methyltransferase inhibitor 5-aza-cytidine (AZA) [171] or by modulation of intracellular signaling with ERK1/2 and GSK-3 inhibitors in serum-free medium containing LIF [156]. These data show that conversion to full pluripotency that normally occurs only at very low frequency (about 0.001 to 0.1% of the infected cells) and at slow kinetics (2–8 weeks) can be significantly enhanced by affecting the chromatin state or by culture conditions that promote pluripotency (LIF) and suppress differentiation-inducing signaling from ERK1/2 (PD0325901) and GSK3 (CHIR99021) (Fig. 6.1). Optimization of Reprogramming Several additional studies have demonstrated that certain epigenetic modifiers, small-molecule compounds and signaling molecules can enhance the efficiency and the kinetics of reprogramming of somatic cells and that these improved conditions can reduce the number of oncogenic transcription factors to be introduced by transduction. Shi and coworkers have shown that small molecule inhibitor of the G9a histone methyltransferase, BIX-01294, can improve the reprogramming efficiency of murine neural progenitor cells (NPCs) transduced with only Oct4 and Klf4 to a level comparable with the O-S-K-M combination of factors [172]. Most likely, BIX-01294 facilitated the activation of endogenous Oct4 gene through inhibition

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of histone H3 dimethylation at lysine 9 (H3K9me2), which has been implicated in Oct4 inactivation. In addition, the treatment of cells with a specific synthetic inhibitor of ERK1/2, PD0325901, at later stages of reprogramming facilitated the selection of reprogrammed iPS cell colonies. Adult murine neural stem cells have been earlier identified as a more suitable source for iPS cell production than MEFs [154, 173, 174]. The fact that they already express two of the required pluripotency factors, Sox2 and c-Myc, reduced the requirement for exogenous factors to only Oct4 and Klf4 for successful establishment of iPS cells from NSCs [154]. This data indicate that careful selection of a somatic cell type and use of small molecules can dramatically reduce the number of viral vectors required for reprogramming and enhance its efficiency. Under these conditions, the risk for insertional mutagenesis and oncogenesis associated with stably integrated proviral sequences can be reduced. In another study from Jaenisch laboratory, conditioned media containing Wnt3a were found to enhance the reprogramming of MEFs transduced with O-S-K combination by almost 20-fold over control cells cultured without Wnt3a [175]. This enhancing effect could be reduced by disruption of β-catenin and CREBbinding protein (CBP) interaction using small molecule inhibitor ICG-001, which suggests that Wnt-induced effects were mediated by stabilized β-catenin affecting the transcription of target genes, the identity of which is not yet clear (Fig. 6.1). The ability of soluble Wnt3a to enhance reprogramming is in agreement with the recent study by Lluis and coworkers who used ES cell-somatic cell fusion-based assay to show that β-catenin accumulation in ES cells enhances reprogramming of a somatic cell nucleus [129]. Efficiency of mouse and human somatic cell reprogramming using retroviral transduction could be also greatly increased through addition of valproic acid (VPA), a histone deacethylase (HDAC) inhibitor [176, 177]. VPA improves reprogramming efficiency of MEFs by more than 100-fold and enables about 1% reprogramming efficiency of human neonatal fibroblasts infected with OS-K retroviruses. Moreover, in the presence of VPA, the reprogramming of human fibroblasts was possible also with only two exogenous factors Oct4 and Sox2. However, under this condition the efficiency was low, 0.001 to 0.004% [177]. The effects of the chromatin-modifying agents trichostatin A (TSA) and AZA that block histone acetylation and DNA methylation have been also investigated in murine neurosphere cells by global gene expression profiling [178]. This study showed that several stem cell (e.g. CD34, CD133) and pluripotency-associated genes (e.g. Oct4, Nanog and Klf4) are induced by TSA/AZA treatment in neurosphere cells. These data indicate that chromatin remodeling induced by these factors may be an important permissive part of the reprogramming process. Zhao and coworkers showed that downregulation of the tumor suppressor protein p53 and overexpression of the stem-cell-specific transcription factor Utf1 greatly improves the efficiency of human iPS cell generation [179]. Others have derived human iPS cells with increased efficiency by adding one or two additional factors (TERT and/or large T antigen) to the traditional O-S-K-M combination [180, 181]. By using the simian virus (SV) large T antigen (SVTag), Mali and coworkers achieved more than seven fold higher reprogramming efficiency of human fibroblasts with O-S-SVTag (Oct4-Sox2-Simian virus large T antigen) as compared to

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usual O-S-K-M combination of factors [181]. The most recent effort in reducing the number of viral reprogramming vectors resulted in successful generation of murine and human iPS cells with a single inducible lentiviral vector using 2A "self-cleaving" peptides, which support efficient polycistronic expression of four reprogramming factors (O-S-K-M) from a single promoter [164, 165]. With this approach, human iPS cells were generated from keratinocytes and single proviral copy was sufficient to generate iPS cells from mouse embryonic fibroblasts [165]. Human keratinocytes have been identified as a particularly suitable cell type for reprogramming [165, 170, 182]. The efficiency of iPS cell generation from keratinocytes with O-S-K-M factors has been reported to be at least 100-fold more efficient and twofold faster as compared to human fibroblasts [182]. The practicability of this approach has been underscored by generation of iPS cells from keratinocytes isolated from a single adult human hair [182]. Generation of iPS Cells Without Permanent Genetic Modification Despite these advances, the most suitable iPS cells for scientific and therapeutic use will be those produced without permanent genetic modification. As discussed above, the original reprogramming technique makes use of retroviral or lentiviral vectors to generate iPS cells. Infection with retroviruses results in their stable integration at multiple random sites in the genome [183], which may disrupt genes important for normal cellular homeostasis and differentiation due to insertional mutagenesis. Multiple studies have dismissed the proposal that activation of genes triggered by retroviral insertions may be required to induce reprogramming [151, 164, 165, 183, 184]. However, insertions could lead to aberrant expression of an oncogene and may cause leukemia or other malignancies. In addition, the use of known oncogenes, such as c-Myc, for the purpose of reprogramming carries the risk for tumor formation on its own [147]. The retrovirally introduced genes including c-Myc were expressed in transduced cells only early upon transduction and silenced in fully reprogrammed cells due to methylation of retroviral long-term repeat sequences [146–148]. However, in iPS chimeric mice the viral c-Myc oncogen got reactivated and caused tumor formation in about 20% of F1 generation of chimeric mice [147], strongly indicating that use of oncogenes should be avoided in the process of generation of clinically acceptable iPS cells. Different groups have already shown that c-Myc is not indispensable for generation of both human and murine iPS cells and that the combination of only two factors also could work but sacrifices the efficiency [145, 154, 155, 175, 177, 185–187], unless supporting factors or more reprogrammable cell types are used for reprogramming [175, 177, 185]. Despite reduction in number of factors required for reprogramming and recent generation of iPS cells with only a single integration of a polycistronic vector per cell [165], it would be desirable to achieve reprogramming without having to resort to viruses and without stable integration of foreign genes. Therefore, alternative approaches that permit transient factor delivery into somatic cells are under development. They are based on gene expression from non-integrating vectors or delivery of transducible versions of transcription factors in form of active cell-permanent proteins

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[188, 189]. While the later method has not yet been successfully used for generation of iPS cells, two recent studies in a murine system showed that traditional factor delivery through retro- or lentiviral transduction of somatic cells can be replaced by non-integrating adenoviral vectors [184] or by transient plasmid transfection [151]. iPS cell generated by these two approaches were morphologically, functionally and molecularly indistinguishable from classical iPS and ES cells, suggesting that viral integrations are not required for successful reprogramming of somatic cells to pluripotency. Adaptation of this strategy to human cells will be crucial for generation of therapeutically acceptable iPS cell lines that do not harbor any permanent genetic modifications and thus do not carry risk of insertional mutagenesis. Instead of using proteins or non-integrating vectors, even more elegant way of inducing reprogramming would be entirely with small molecules that mimic the effects of pluripotency factors. Creating this kind of reagents will facilitate translation of this technology to clinical use. First successes of this approach combined with viral factor delivery have been summarized above. Therapeutic Potential of iPS Cell-Derivatives The most important prerequisite for therapeutic use of iPS cell derivatives is their functional integrity and capacity to replace diseased or damaged cells in the body (Fig. 6.3). Initial studies have already demonstrated that diverse types of mature cell derivatives of all three embryonic germ layers can be differentiated from mouse and human iPS cells including cardiomyocytes, smooth muscle cells (SMC), endothelial cells, hematopoietic cells, neural precursor cells and insulin-producing β-cells [190–193]. Detailed electrophysiological analyses of murine iPS cell-derived cardiomyocytes demonstrated that they have similar properties to cardiomyocytes derived from conventional murine ES cells [190, 194, 195], suggesting that they may be suitable for cardiac tissue repair. However, it remains yet to be shown whether these cardiomyocytes are capable of improving the heart function in animal models of heart infarction and whether cardiomyocytes generated from human iPS cells are functionally intact. Another cell type that may be useful for cardiac repair are SMCs. Xie and coworkers have generated SMCs from murine iPS cells by treatment with retinoic acid [192]. This group showed that after retinoic acid treatment, 40% of iPS cell derivatives expressed SMC-specific markers and acquired functional characteristics of SMCs including contraction and calcium influx in response to pharmacological stimuli. However, gene expression analyses revealed certain differences in SMC-specific gene expression patterns between SMCs derived from different iPS cell clones as well as normal ES cells. It is unclear whether these differences are the result of clonal differences between different cell lines or whether they reflect aberrant gene expression specific to reprogrammed cells. One of the disease that may be amenable to cell replacement therapy is the type I diabetes mellitus. Comparable to successful derivation of insulin-secreting cells from murine and human ES cells [3, 196–198], Tateishi and coworkers recently reported generation of insulin-producing islet-like clusters from human iPS cells [191]. These iPS-derived clusters contained C-peptide- and glucagon-positive cells

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Fig. 6.3 Therapeutic and scientific potential of iPS cells. Different adult somatic cells can be converted to a pluripotent state by ectopic expression of pluripotency factors. iPS cells can be than differentiated into multiple derivatives of all three embryonic germ layers and used for cell replacement therapy, in vitro disease modeling, drug screening and toxicological analyses. In future, there may not be necessary to revert to a pluripotent stem cell state in order to generate required progenitor or mature cell types. Using the same strategy of ectopic expression of defined transcription factors or signaling molecules, it may be possible to directly transdifferentiate one cell type into another (dotted lines), as has been shown by converting liver cells into insulin-producing beta cells or exocrine pancreatic cells into endocrine ones

and released C-peptide upon glucose stimulation in vitro. It remains to be shown whether these cells can protect against streptozotocin-induced hyperglycemia when implanted to diabetic animals, as it has been demonstrated for hES cell-derived cells [3]. Two recent studies in these remarkable series of reports have provided proof of principle for the applicability of iPS cells for customized gene and cell replacement therapy [193, 199]. In the report from Hanna and coworkers, iPS cells have been derived from a humanized knock-in mouse suffering from sickle cell anemia. In this model, the mouse α-globin genes are replaced with corresponding human α-globin genes and the mouse β-globin genes were replaced with human Aγ- and βS (sickle)-globin genes. After stable iPS cell clones had been obtained from this mouse, the genetic defect was repaired in vitro by homologous recombination. Subsequently, iPS cells were differentiated in vitro into hematopoietic progenitors and transplanted back into irradiated donor mice. Treatment of diseased mice with iPS cell-derived hematopoietic progenitors resulted in significant improvement of all pathological features and symptoms of disease [199]. Wernig and coworkers showed that murine iPS cells can be efficiently differentiated into neural precursor cells in vitro [193]. Upon transplantation into fetal mouse brain,

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these cells migrated into various brain regions and differentiated into glia and different neuronal subtypes. Electrophysiological measurements demonstrated that these cells functionally integrated into the host tissue. In a rat model of Parkinson’s disease, transplanted iPS cell-derived dopamine neurons of midbrain character could ameliorate the symptoms of the disease. These data hold great promise that similar strategy may be applicable for treatment of genetic and degenerative disorders in humans. However, before any clinical use of iPS cell derivatives the safety issues must be carefully addressed. Like ES cells, the undifferentiated iPS cells also form teratomas when injected into mice. Therefore, the achievement of safe engraftment and function of iPS cell-derived transplants without teratoma development represents a major challenge. Even after purifying the desired mature cells prior to transplantation, the risk of teratoma formation from few contaminating undifferentiated iPS cells will still be present because, under appropriate conditions, the injection of only few ES cells can lead to teratoma formation [200]. Another concern is the possibility that deregulation of certain regulatory pathways due to faulty reprogramming/differentiation may lead to uncontrolled proliferation of iPS cell derivatives upon transplantation. As delineated above, comparisons between human ES and iPS cells revealed that they have highly similar growth characteristics, gene expression profiles, epigenetic modifications and developmental potential. However, the inability of iPS cells to yield live animals by tetraploid complementation suggests that iPS cells are not fully identical to ES cells. This raises the highly relevant question whether iPS cells can be regarded as promising and safe therapeutics. Disturbances in epigenetic regulatory mechanisms are known to contribute to malignant transformation [201, 202], and incomplete reprogramming by SCNT frequently causes health problems in cloned animals, which range from large offspring/obesity, placental problems and arthritis to premature death [203]. However, the question whether slight differences in gene expression profiles and epigenetic status between iPS and ES cells and their derivatives are extensive enough to promote malignant transformation or malfunction remains to be determined. It is certainly possible that small aberrations from a physiological state will be less detrimental in the context of cell replacement therapy with specialized cell types as compared to those observed with cloned animals because mechanisms required for proper functioning of a cell are less complex than those necessary for proper embryonic development or homeostasis of an adult organism. This notion is supported by studies demonstrating that stem cells derived from SCNT embryos are sufficiently normal to repair damaged tissue in vivo [112, 113]. It is, nevertheless, still possible that faulty epigenetic reprogramming could negatively affect specialized functions of iPS cell derivatives or diminish efficiency of iPS cell differentiation toward a particular cell lineage. Indeed, murine iPS cells have been reported to exhibit delayed differentiation into cardiomyocytes [194], but this could not be confirmed by independent studies [195]. In any case, careful comparisons of developmental potential of iPS cells with ES cells and other pluripotent cell types and functional properties of their differentiated derivatives will be needed to address issues raised above.

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Disease-Specific iPS Cells In addition to their potential significance for regenerative medicine, iPS cells derived from patients suffering from specific diseases, may allow establishment of permanent tissue culture models of these diseases and thus offer an unprecedented opportunity to investigate the molecular mechanisms of human diseases in vitro, develop new drugs or perform toxicological screens. While more time will be required to overcome all the obstacles for the safe therapeutic use of iPS cell derivatives, their potential for establishment of in vitro human disease models can be immediately realized. Tissue culture of human cells is today largely limited to tumor cell lines or transformed derivatives of native tissues. With the iPS cell technology it is possible now to derive permanent cell lines from patients with a variety of genetic diseases with either Mendelian or complex inheritance. Tissuespecific cells resembling those in diseased organs can be differentiated in vitro from iPS cells and used for studying the disease pathophysiology, development of new drugs and, eventually, autologous cell replacement therapies. In addition, iPS cell lines from patients with monogenic diseases could be used for repairing gene defect ex vivo prior to transplantation. Many complex genetic diseases have familial and sporadic forms. iPS cells derived from patients with complex sporadic diseases would have the unique advantage of carrying the precise patientspecific constellation of genetic factors responsible for the disease in that person. Since mid 2008 a number of disease-specific iPS cells have been generated. These include iPS cells from adenosine deaminase deficiency-related severe combined immunodeficiency, Shwachman-Bodian-Diamond syndrome, Gaucher disease type III, Duchenne and Becker muscular dystrophy, Parkinson disease, Huntington disease, juvenile-onset type 1 diabetes mellitus, Down syndrome/trisomy 21, the carrier state of Lesch-Nyhan syndrome [204], familial form of amyotrophic lateral sclerosis (ALS) [205], and spinal muscular athrophy (SMA) [206]. These cells have been generated from individuals of different age ranging from 3 months to 82 years, demonstrating that patient’s age or his/her health status do not represent an obstacle to iPS cell derivation. Furthermore, similar to iPS cells derived from healthy individuals, patient-specific iPS cells also possess all defining properties of ES cells and can be successfully differentiated into cell types derived from all three embryonic germ layers including somatic cells affected by the disease. For example, ALS is characterized by the progressive degeneration of spinal cord motor neurons and motor neurons could be generated from ALS-iPS cells upon treatment of EBs with sonic hedgehog and retinoic acid [205]. These data demonstrate that patient-specific iPS cells appropriately respond to specific differentiation signals to produce large numbers of motor neurons. Studies in a mouse model of ALS have shown that astrocytes secreting toxic factors are involved in degeneration of motor neurons in this disease. This finding has been corroborated in experiments using coculture of murine ES cell-derived motor neurons with ES cell-derived astrocytes [207], indicating that derivation of these cells from ALS-iPS cells can help identify drugs that prevent astrocytes from secreting these toxic factors or directly block motor neuron degeneration in their presence. However, it must be kept in mind that independent iPS cell lines derived from each patient will be heterogeneous in

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terms of their differentiation potential, transcriptional and epigenetic profiles, or response to various substances and environmental conditions. Therefore, it will be important to distinguish this heterogeneity from specific functional and molecular abnormalities of iPS cell derivatives that are affected by the disease. Another common genetic disease in which motor neurons are affected is the spinal muscular atrophy (SMA). In this disease, patients have selective loss of lower motor neurons resulting in muscle weakness, paralysis and often death. iPS cell were recently generated from skin fibroblasts taken from a child affected with SMA and shown to have all the characteristics of typical human ES cells and capacity to differentiate into motor neurons [206]. Interestingly, these motor neurons showed selective deficits compared to normal cells, suggesting that human iPS cells can be used to model the specific pathology seen in a genetically inherited disease. It remains to be seen to what extent can the clinical disease phenotype in other genetic diseases be recapitulated in vitro in specific populations of isolated somatic cell types. 6.4.1.7 Alternative Ways for Obtaining Pluripotent Stem Cells from Early Embryos An ethically acceptable procedure for generation of human ES cell lines is a single-cell biopsy of early embryos similar to that used in preimplantation genetic diagnosis (Fig. 6.2). In this concept a single totipotent blastomere is removed from an eight-cell embryo to derive a new ES cell line and the remaining seven-cell embryos are transferred into surrogate mothers, in which they develop into normal organism. Proof-of-principle studies with mouse and human blastocysts demonstrated that stable ES cell lines can be established from single blastomeres [208, 209]. The second approach is based on extracting still viable pluripotent cells from poor-quality IVF embryos that are unsuitable for uterine transfer or cryopreservation and would therefore be discarded as medical waste after 3–5 days of culture (Fig. 6.2) [210]. Since these poor-quality embryos are discarded every day in large numbers (hundreds of thousands) in the course of IVF, it is believed that pluripotent ES cell lines derived from them would be ethically acceptable. In recent study, Lerou and coworkers demonstrated that creation of ES cell lines from IVF embryos at different stages of development and of different quality is feasible. However, the efficiency of generating stable stem cell lines strongly depended on the stage of the embryo’s development. Only 1 out of 171 (0.6%) poor-quality embryos that were discarded on day 3 post fertilization yielded a stem cell line, while the efficiency was much higher (8.5%) with poor-quality embryos that were discarded at day 5 post fertilization at the blastocyst-stage [210]. Because this efficiency is comparable to that obtained with normal frozen embryos, by using poor-quality embryos many new ES cell lines could be generated without sacrificing the output. The validity of this approach has been questioned. One of the critics’ strongest arguments against the proposal by Klimanskaya and coworkers, which can also be applied for the strategy with poor-quality embryos, is the lack of evidence that a single blastomere from a human embryo at the eight-cell stage has no capacity

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to develop into a normal human being. If this cell would be capable of normal development, as is the case with single blastomeres isolated from a rabbit or sheep, the ES cells derived from such blastomeres would also not be ethically acceptable according to the opponents of this approach. The difficulty of this concept is that the developmental potential of human blastomeres, similarly as the developmental potential of Cdx2-deficient embryos in ANT approach (see above), can never be tested in humans for ethical reasons since it would require the transfer of a single blastomere into the uterus. It remains to be seen, whether any of these two methods will become acceptable for derivation of human pluripotent stem cells.

6.4.2 Pluripotent Stem Cells Derived from Germ Cells 6.4.2.1 Pluripotent Stem Cells from Testis In teratocarcinoma transplantation experiments it has been already shown in 1960s that these tumors are derived from primordial germ (PG) cells. PG cells are the progenitors of mature gametes (eggs and sperm) in early embryonic development. When PG cells are explanted from embryos and cultured in vitro they give rise to pluripotent stem cells, called embryonic germ (EG) cells that are morphologically and antigenically similar to conventional ES cells (Fig. 6.2) [211]. These cells also contribute to chimeric mice but, unlike ES cells, retain genome-wide demethylation, erasure of genomic imprints and reactivation of X-chromosomes [212]. More recently, unipotent spermatogonial stem cells isolated from neonatal and adult mouse testis were also successfully converted in culture to ES cell-like pluripotent cells [213, 214]. These cells are capable of contributing to chimeras and generating entire progeny (reviewed in [215]. The latest breakthrough in this field was the derivation of the first pluripotent cells from adult human testis [216]. These cells were generated from pre-selected spermatogonial stem cells by using CD49f (α6-integrin) as a marker. Morphologically distinguishable pluripotent stem cell colonies appeared spontaneously after 10–15 days in culture. Established pluripotent cell lines were morphologically, molecularly, structurally very similar to human ES cells, karyotipically normal, differentiated in vitro into various types of somatic cells of all three germ layers and produced teratomas after implantation into immunodeficient animals. These cells have clear advantages over stem cells derived from early embryos. No embryos or oocytes are required for their generation and they are genetically identical to the donor. Therefore, these cells may be suitable for individualized cell replacement therapies without ethical objections and risk of immune rejection. 6.4.2.2 Pluripotent Stem Cells from Oocytes Unfertilized oocytes can be used for derivation of pluripotent stem cells by a process called parthenogenesis. Parthenogenesis is an asexual form of reproduction found in females where growth and development of embryos occurs without fertilization by

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a male. Lower organisms such as snakes, fish, ants, flies, lizards, amphibians may routinely reproduce by parthenogenesis, with a single egg that starts developing without the intervention of the male counterpart. This form of reproduction does not spontaneously occur in mammals, but mammalian oocytes can be artificially induced to undergo parthenogenesis in vitro (Fig. 6.2) [217, 218]. Mouse parthenogenic ES (pES) cells have been established only two years after derivation of mouse ES cells [219, 220]. These cells are pluripotent as demonstrated by their ability to form teratomas. However, due to faulty imprinting, pES cells are unable to produce viable mice after tetraploid complementation. Chimeric mice produced using parthenogenic blastomeres manifested significant growth retardation that was directly correlated with the degree of parthenogenic contribution [221]. Based on these results, it is tempting to conclude that parthenogenesis will never be a viable form of mammalian reproduction, but could serve as an alternative source of pluripotent ES cells und as an instrument to investigate imprinted gene function. Different groups have also established nonhuman primate pES cells [222–224] and in 2007 several human pES cells have been generated [225–228]. These cells were immunoreactive for typical pluripotency markers, showed broad differentiation potential in vitro and formed teratomas and thus appear to have properties similar to those of conventional ES cells. Interestingly, first human pES cells are believed to be generated earlier by Hwang Woo-Suk who was accused of fabricating data as his laboratory claimed to have cloned first human embryos by SCNT and generated first human SCNT stem cells. It now appears that these SCNT cell lines were actually derived from a parthenote [229, 230]. pES cells contain genetic material exclusively from the oocyte donor. However, due to segregation of homologous chromosomes during the first meiotic division of the oocyte, pES cells are actually not genetically identical to the donor. In addition, most of the pES cells show a loss of heterozygosity in the MHC and thus may be rejected by natural killer cells that recognize the lack of one set of histocompatibility antigens [231]. Recently, patient specific pES cells that are homozygous in the HLA region have been generated [227]. In case, these cells will be found therapeutically acceptable, the bank of different HLA homozygous pES cells will be applicable to many more people than conventional allogeneic ES cells. The potential of pES cells to serve as a source of cells for cell replacement therapy has been demonstrated in studies that showed that pES cells differentiate into mature cells with expected functional properties. For example, monkey pES cells grown on fibronectin/laminin-coated plates and in neural progenitor medium assumed a nestin-positive neural precursor phenotype, and when transferred to differentiation conditions these cells showed neuronal and epithelial morphologies and acquired electrophysiological characteristics of functional neurons [224]. In one another study, dopaminergic neurons obtained from primate pES cells displayed persistent expression of midbrain regional and cell-specific transcription factors, and after transplantation restored motor function in hemi-parkinsonian, 6hydroxy-dopamine-lesioned rats [232]. Exposure to Wnt5a, FGF20 and FGF2 at the final stage of in vitro differentiation enhanced the survival of dopamine neurons and, correspondingly, the extent of motor recovery of transplanted animals.

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Similar to pluripotent stem cells derived from spermatogonial stem cells of adult testis, parthenogenic ES cells derived from oocytes may serve as an ethically acceptable source of patient-specific pluripotent stem cells for research and therapy.

6.5 Future Prospects and Conclusions Since the first nuclear transfer experiments in the 1950s and even more so since the birth of the cloned sheep Dolly in 1997 it was clear that various types of adult somatic cells could be reverted back to their embryonic state but the method of doing it in morally acceptable and efficient way was not available until recently. Human pluripotent stem cells can now be derived not only from scarce and ethically controversial early embryos and oocytes but also from adult germ and somatic cells. Recent discovery that full reprogramming of cultured adult somatic cells into a pluripotent ES cell-like state can be achieved simply by overexpression of only few transcription factors represents the last major advance in this field with great potential for research and medicine. However, this major step forward could not be realized without previous achievements in the fields of developmental and stem cell biology. In only one year since the iPS cells have became fully established, tremendous progress has been made toward generation of iPS cells that are more suitable for scientific and clinical use. The molecular mechanisms of reprogramming have begun to be elucidated, efficiency of reprogramming has been greatly increased by combining viral expression systems and small molecules, newly designed compact vectors have reduced the number of viral insertions in iPS cells, somatic cell types that are more susceptible to reprogramming have been identified, repositories of disease-specific iPS cells started to be established, and most recently, iPS cells have been generated with non-integrating viral and non-viral vectors. In future, it will be necessary to further develop techniques for generation of human iPS cells without their permanent genetic modification by replacing integrating viruses with plasmid- or protein-based transient delivery methods and small molecules. There is also a great need to increase the efficiency and the kinetics of reprogramming. The full reprogramming of a somatic cell nucleus after SCNT or fusion with an ES cell occurs within few cell divisions in the first 2–4 days upon induction, while the generation of iPS cells requires weeks. Understanding the molecular basis for this difference, especially the role of factors involved in epigenetic control of gene expression, will allow for the development of improved reprogramming protocols for generation of iPS cells. Once the key molecular culprits of reprogramming are known, small molecules could be developed to enhance the reprogramming by exogenous transcription factors or to induce the reprogramming on their own completely eliminating the need for other delivery systems. iPS cells generated with current techniques could be immediately used to investigate the pathophysiology of simple and complex human diseases in vitro, develop new drugs, and test the toxicity of substances or response to various environmental conditions. However, significant effort must be directed toward removal of obstacles that currently hinder

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the clinical use of pluripotent stem cells. First, all types of pluripotent stem cells, including iPS cells, form teratomas in vivo. Therefore, the achievement of safe engraftment and function of iPS cell-derived transplants without teratoma development represents a major challenge. Second, yield of many desired cell types from undifferentiated pluripotent stem cells is still very variable and inefficient. Hence, defining optimal conditions for reproducible differentiation of human pluripotent stem cells toward a required cell type at sufficient, clinically useful quantities and at satisfactory purity and functionality represents another big challenge. Third, there is a great need for improving the long-term engraftment of therapeutic cells. With current approaches, the survival of transplanted cells is very poor even if injected into healthy organ of a genetically identical recipient. Forth, adoption of standardized procedures based on good manufacturing practice (GMP) guidelines and stringent quality and cryopreservation control protocols will insure that cells at highest quality free of animal products and infectious agents are reproducibly obtained and given to prospective patients. Finally, development of iPS cell-based therapies for clinical use will require use of scalable processes, which will allow proliferation of undifferentiated cells as well as their differentiation toward a desired cell population on a large scale in bioreactors. However, high costs of production of each individual patient’s cell line may limit their practical use in human medicine. Therefore, alternative sources of HLA-matched pluripotent stem cells (e.g. conventional ES cells or pluripotent stem cells derived from testis) will still be needed in future, especially in the case when autologous stem cells will be derived from donors suffering from a genetic disease that affects the cell type that would be needed for therapy. Human ES cells will continue to serve as a gold standard for the determination of a stem cell status of new iPS cell lines as well as other types of pluripotent stem cells. However, pluripotent stem cells may even not be needed for generation of lineage restricted progenitors or mature cells for cell replacement therapy (Fig. 6.3). In future, the same technique used to generate iPS cells could be adopted to directly reprogram somatic cells of one lineage into desired tissues-specific cells of an another lineage. It has been already demonstrated in the past that this approach may be successful. Ectopic expression of pancreatic transcription (Pdx1, NeuroD1) and differentiation factors (e.g. betacellulin) in liver cells lead to their transdifferentiation into functional insulin-producing cells that could correct hyperglycemia in an animal model of diabetes mellitus [233]. Recently, Zhou and coworkers in Dough Melton’s laboratory identified three transcription factors (Ngn3, Pdx1 and Mafa) that upon adenoviral delivery into the pancreas of living mice can induce transdifferentiation of pancreatic exocrine cells into cells that closely resemble beta-cells in their morphology, ultrastructure and function [234]. These studies clearly demonstrate that nuclear reprogramming using defined factors could be adapted in future for shifting cell phenotypes across lineages without need to generate pluripotent stem cells. This would eliminate the risk for teratoma formation and may be closer to clinical application. Future research in this extremely dynamic and exciting field will determine which reprogramming strategy and which cell types will be most suitable for treatment of particular disease. Most likely, there will be no single answer to this question.

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Acknowledgments Work in our laboratories is supported by grants from The Federal Ministry of Education and Research (to T.S. and H.J.), European Community (to H.J.), Center for Molecular Medicine Cologne (to H.J. and T.S.), Else-Kröner-Fresenius Stiftung (to T.S.) and Köln Fortune Program (to T.S. and H.J.). N.Z.M. is supported by the stipend from the Royan Institute, Tehran, Iran.

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Chapter 7

Cell Therapy in Parkinson’s Disease R. Laguna Goya and R.A. Barker

Abstract Parkinson’s disease (PD) is a common neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons in the substantia nigra. It is typically treated symptomatically by dopamine replacement using either levodopa or dopamine agonists, however, cell therapies that aim to repair and replace these lost neurons and their projections to the striatum represent a very promising strategy to help cure people of this condition. Several cell sources have been considered for this use including fetal ventral mesencephalon, embryonic stem cells and induced pluripotent stem cells. Some of them have gone into the clinical arena with mixed results, whilst others still remain purely experimental. In this chapter we will discuss the stage of development of each of them and the pros and cons of their use for the treatment of PD. Keywords Parkinson’s disease · Cell therapy · Neural precursor cell · Fetal ventral mesencephalon · Embryonic stem cells · Induced pluripotent stem cell · Graftinduced dyskinesia

Contents 7.1 Introduction . . . . . . . . . . . . . . 7.2 Fetal Ventral Mesencephalon . . . . . . 7.3 Fetal Neural Precursor Cells . . . . . . 7.4 Embryonic Stem Cells . . . . . . . . . 7.5 Induced Pluripotent Stem Cells (iPS Cells) 7.6 Adult Stem Cells . . . . . . . . . . . 7.7 Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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7.1 Introduction In recent years, cell replacement therapies have been pursued as a potential curative treatment for a number of central nervous system (CNS) diseases, such as Parkinson’s disease (PD), Huntington’s disease, multiple sclerosis, traumatic brain injury and stroke. However, the relatively circumscribed pathology that lies at the heart of PD has meant that this disease has gained prominence as the prototypical neurodegenerative disorder for treatment with cell therapies. PD is the second commonest neurodegenerative condition, after Alzheimer’s disease, with a worldwide overall prevalence of around 1%. It is characterized by the slow progressive loss of dopaminergic nigrostriatal neurons, which leads to classical motor triad off bradykinesia, resting tremor, and rigidity. By the time these motor symptoms are manifest, at least 50% of these neurons have been lost. However, the pathology is not just localized to this pathway, but involves a number of other CNS and non-CNS sites which may account for some of the non-motor symptoms of PD, i.e. the cognitive, affective and autonomic deficits which are commonly seen. The cause of the neuronal degeneration in PD is not fully understood, but the pathological hallmark is the formation of α-synuclein positive Lewy bodies, in the remaining substantia nigra pars compacta neurons and in numerous other sites within the brain. This core loss of striatal dopamine secondary to the demise of the dopaminergic nigral neurons has meant that its treatment has focused on dopamine replacement. This normally takes the form of the administration of levodopa and/or dopamine agonists in the first instance, which have significant clinical benefits especially in early stages of the disease. However, as the condition progresses, the efficacy of this therapy wanes and many patients develop drug-induced motor complications including “on-off” fluctuations and levodopa-induced dyskinesias (LID). At this stage of disease more aggressive drug therapies are considered R infusions into the small bowel) along with (e.g. apomorphine pumps; DuoDopa surgical procedures, of which deep brain stimulation of the subthalamic nucleus has proven to be the most effective with the advantage of being adjustable and reversible. However, none of these therapies are curative but merely symptomatic and as such these treatments produce only temporary clinical benefit, albeit for several years in some cases, but the pathological process causing neuronal degeneration continues. There is therefore a pressing need to find disease modifying strategies that can either slow down the course of the disease or even cure it. In this respect, the delivery of neurotrophic factors to rescue the surviving dopaminergic neurons is an attractive approach to prevent further disease progression and has been successfully used with GDNF in some [1, 2], but not all studies [3]. Alternatively, disease progression could potentially be reversed by cell replacement of the lost dopaminergic neurons and several different cell sources have been investigated in this respect. These include fetal ventral mesencephalic (VM) tissue, fetal neural precursor cells (NPCs), embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells, as well as some adult sources of cells such as the bone marrow and even adult brain (Table 7.1).

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Table 7.1 Advantages and disadvantages of the different cell sources being considered for cell replacement therapy in PD Cell sources

Advantages

Disadvantages

Fetal VM

No tumor formation Proven efficacy in patients

Fetal NPCs

Homogeneous cell population Larger numbers of donor cells can be obtained from a limited source Homogeneous cell population Potentially plenty of cells available at any one time Homogeneous cell population Potentially plenty of cells available at any one time No immune rejection No use of human embryos

Graft induced dyskinesias in some patients Immune rejection Use of human embryos (ethical and logistical problems) Use of human embryos Immune rejection

ESCs

iPS cells

Risk of tumor formation Use of human embryos Immune rejection Risk of tumor formation Inefficient process for their production

In this chapter we will discuss the different cell sources being considered for the treatment of PD and their level of development as a cell replacement therapy. This involves an evaluation of how well such cells survive post-transplantation, their ability to differentiate into functional dopaminergic neurons and to produce behavioral recovery and their capacity not to have unregulated proliferative cells that could form tumors in the long term.

7.2 Fetal Ventral Mesencephalon One of the first cell sources to be considered for the treatment of PD was the fetal ventral mesencephalon (VM), the developmental region that contains the emerging dopaminergic neurons of the SN. In the late 1970s and early 1980s it was shown for the first time that both rodent and human fetal VM could survive in the adult brain in rat PD models, and this led to behavioral recovery [4–6]. As a result, trials of patients with PD undergoing human fetal VM transplantation were undertaken in the 1980s and 1990s. These open-label clinical trials showed that VM from 6 to 9 weeks old human embryos transplanted into the striatum of PD patients resulted in improved clinical state as evidenced by reductions in motor scores on the Unified Parkinson’s Disease Rating Scale (UPDRS) and activities of daily living (ADL) scores, increased fluorodopa uptake on PET scanning and decreased levodopa requirements [7–10]. These encouraging results, in the absence of major side-effects, led to two double-blind trials to compare striatal implantation of fetal VM versus sham surgery [11, 12] in cohorts of patients with moderate and advanced PD. These two trials used two different primary outcome measures, a subjective

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global rating of clinical improvement one year after transplantation in the study of Freed et al. [11] and a significant improvement in the ‘off’ UPDRS score two years post-grafting in the study of Olanow et al. [12]. In neither case though was there a significant improvement in the grafted patients. Nevertheless, embedded in these patient cohorts, were individuals who had done well with their transplant, and this taken together with the open-labeled trials, suggested a proof of principle that cell replacement could be a very effective therapy in the treatment of PD in some cases. These studies have also helped to define which subset of PD patients may benefit most from cell therapy- and this currently appears to be patients under the age of sixty with idiopathic PD at an early stage of the disease and with dopaminergic loss restricted to the dorsal striatum on fluorodopa PET scanning. In addition it is important to also recognize that a considerable number of patients in these trials of Freed et al. and Olanow et al. developed graft induced dyskinesias, which in a few cases required corrective additional neurosurgical intervention. However factors that may be important and which may help prevent these in future transplant trials include: (i) transplanting earlier in the course of the disease before irreversible postsynaptic changes have occurred downstream of the dopaminergic receptors; (ii) grafting a cell population as homogeneous as possible with exclusion of significant numbers of serotoninergic neurons; (iii) and ensuring that the cells are evenly delivered over the whole dorsal striatal complex. In summary, fetal VM transplantation is so far the only cell therapy that has shown significant effects in some patients with PD. However, the heterogeneous population of donor cells that are grafted in these patients coupled with the logistical (e.g. scarcity of donor tissue) and ethical problems (i.e. use of human embryos) makes it likely that alternative sources of stem cells will be preferred as the treatment of choice in the future in PD.

7.3 Fetal Neural Precursor Cells Neural precursor cells (NPCs) have the capacity to differentiate into all the different neural cell types (neurons, astrocytes and oligodendrocytes) and can be found in both the developing and adult brain. In the developing VM there exist NPCs that have already been specified to mature into dopaminergic neurons and they can be selectively expanded in vitro prior to transplantation, and by so doing generate larger numbers of appropriate donor cells. This could therefore provide a solution to the logistical problems of fetal VM tissue availability. In the case of murine VM NPCs, they have been cultured with fibroblast growth factor 2 (FGF-2) [13] as a way to promote cell proliferation, which can then be differentiated in vitro into dopaminergic neurons upon withdrawal of mitogens and addition of serum. These cells so generated have also been shown to produce behavioral improvements upon transplantation in animal models of PD [13]. Similarly, human fetal NPCs can be expanded long-term in vitro in the presence of FGF-2 and epidermal growth factor (EGF) and then differentiated into dopaminergic neurons

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using factors like FGF-8 and glial-derived neurotrophic factor (GDNF) [14, 15]. However, long expansion times in vitro do though seem to reduce the potential of VM NPCs to adopt a dopaminergic phenotype [16, 17]. Furthermore, human fetal expanded NPCs have been transplanted into parkinsonian rats with variable results in terms of survival and behavioral benefit [17, 18], suggesting that this approach might be less robust with human than with murine VM NPCs. However, the advantage of expanding fetal VM NPCs in culture before the grafting (apart from helping with issues of tissue availability) is that they would represent a more defined/homogeneous population than primary tissue. Thus other attempts have been made to purify this population and so more recent refinements to increase the yield of dopaminergic cells have involved culturing them in low oxygen atmosphere [19] and transfection with transcription factors, e.g. Wnt5a [20]. However, the expansion behavior of these cells in culture has yet to be optimized and the survival of such cells after transplantation has to be further proven before these cells can be taken into clinical trials.

7.4 Embryonic Stem Cells Embryonic stem cells (ESCs) are derived from the inner cell mass of the embryo and have the capacity to proliferate indefinitely and to give rise to differentiated cells from all three germinal layers. Mouse ESCs have been grown in vitro and differentiated into dopaminergic neurons using protocols that involve culturing on feeder layers, addition of soluble factors like FGF-2, fibroblast growth factor 8 (FGF-8), sonic hedgehog (SHH) and ascorbic acid, and/or transfection with genes known to have an impact on dopaminergic differentiation during development, like Nurr1 [21–24]. Although poor survival after transplantation has been generally observed, grafted tyrosine hydroxylase positive (TH+ ) cells have been found in the host brain in some cases with motor improvement in the parkinsonian animals. Very similar protocols to the mouse ones have now been used for dopaminergic induction of human ESCs [25] and again shown to survive upon transplantation and produce functional recovery in animal models of PD [26]. Optimized differentiation protocols for human ESCs could represent a desirable and reproducible source of defined dopaminergic neurons for use as a cell therapy in PD. Compared to fetal NPCs, ESCs proliferate and adopt a dopaminergic phenotype more readily (up to 90% of TH+ neurons [24]) and by so doing provide a more homogeneous source of dopaminergic neurons. However, there are several problems to overcome before ESCs can be taken into the clinic (reviewed in [27]). First, the differentiation protocols for clinical use should not include genetic manipulation or animal products. In this respect, Roy et al. [28] have developed a protocol using human fetal midbrain astrocytes as a feeder layer, obtaining nigrostriatal dopaminergic neurons that survived transplantation and produced motor benefits. However, they also found undifferentiated dividing neuroepithelial cells within the grafts, which leads to the second concern with using such cells, namely

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the risk of tumor formation. Extensive in vitro differentiation prior to grafting reduces this risk but, unfortunately, cells cultured for long periods of time in such a way present a more mature phenotype and their survival is poorer when removed from culture and transplanted. An alternative strategy to minimize the mitotic cells in the transplant could be to negatively select the undifferentiated cells or positively select the already specified non-mitotic cells by fluorescence-activated cell sorting (FACS). In addition to the above concerns, a third problem with using ESCs for cell replacement is the risk of graft rejection. Immunosuppression of the patients can be used to reduce this but it brings with it other risks and concerns. However, a bank of genotyped human ESCs could be created, reducing the possibility of immune rejection by partially matching the HLA genotype of the graft recipients [29]. Alternatively, cells for replacement therapy in PD could be obtained from the own patient by nuclear reprogramming as is now being explored with induced pluripotent stem (iPS) cells (see below).

7.5 Induced Pluripotent Stem Cells (iPS Cells) Obtaining patient-specific pluripotent stem cells from adult differentiated somatic cells is a new exciting strategy that has gained great impetus recently. This approach as a source of cells for grafting has the advantages of not producing any immune rejection as well as circumventing the ethical and practical problems associated with other types of stem cells. The strategy adopts a process called nuclear reprogramming, in which the nuclei of differentiated cells can be transformed into a pluripotent state and from there the cells can be directed into the desired cell type for transplantation, in the case of PD dopaminergic neurons. Somatic cell nuclear transfer into oocytes and cell fusion with ESCs were two early techniques that allowed this reprogramming of differentiated somatic cells, however, the use of embryos was still required in the process. More recently, reprogramming of differentiated somatic cells has been achieved through the viral transfection of four transcription factors Oct4, Sox2, c-Myc, Klf4 into mouse [30] and human [31] adult fibroblasts, obtaining iPS cells. These cells are similar to ESCs in morphology, proliferation, telomerase activity, gene expression, epigenetic status and potential to differentiate into the three germ layer lineages and to give rise to adult chimeras. However, there are two major concerns in the clinical use of iPS, the transfection with the proto-oncogene c-Myc and the use of viral vectors that integrate into the genome, both of these manipulations have potentially tumorigenic consequences to the cells. Recently though the induction of human iPS cells has now been achieved without c-Myc, using Oct4, Sox2, and Klf4 [32] or alternatively, Oct4, Sox2, Nanog and Lin28 [33]. Also, mouse iPS cells have been obtained by transfecting with Oct4, Sox2, c-Myc, Klf4 but without viral integration, using either adenoviruses [34] or plasmids [35].

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Mouse iPS cells can be driven to a neural stem cell phenotype by culturing with FGF-2, and then by adding sonic hedgehog and FGF-8 some of them differentiate into dopaminergic neurons [36]. These cells are functionally integrated when transplanted into the developing mouse brain, and produce functional recovery in parkinsonian rats upon transplantation. Teratomas appeared in some of the transplanted animals, probably due to remaining undifferentiated cells within the grafted tissue but when the iPS derived dopaminergic neurons were purified from undifferentiated cells by FACS prior to transplantation, they produced the same behavioral recovery without teratoma formation. Nuclear reprogramming of the adult cells of the patients themselves and subsequent differentiation into a specific cell type represents a very promising methodology to obtain cells for cell therapy. They would not produce immune rejection and would neither entail the ethical issues that ESCs and fetal tissue do nor would there be practical problems of tissue availability. However, these cells have nevertheless been reprogrammed to an undifferentiated state, so there is the risk of tumor formation when they are grafted into patients. This could, and has been addressed by removing the undifferentiated cells by FACS. At the present time though further work still needs to be done to better define the protocols to obtain neural cells from adult human fibroblasts, avoiding the transfection with a proto-oncogene and the use of viral vectors that integrate into the genome. Once this is achieved or in parallel with these studies, the functionality of these cells needs to be proven both in vitro and in vivo, showing long term behavioral recovery in PD animal models, before they can be used in the clinical arena.

7.6 Adult Stem Cells A range of other stem cells have been considered for use in the treatment of PDthese include those in the carotid body and bone marrow [37, 38]. In all cases though, the problem remains of obtaining sufficient numbers of true nigral dopaminergic neurons, and until such time as this can be done robustly and reliably these cells remain very much in the domain of experimental possibilities rather than therapeutic realities. An alternative source of cells for treatment of PD is the adult substantia nigra (SN) itself. An obvious and intuitively attractive way of treating PD would be to recruit endogenous dopaminergic neural precursor cells in the adult SN to effect intrinsic repair. This of course presupposes that this structure contains such cells and this has proven controversial with some groups reporting adult nigral neurogenesis whilst others do not (reviewed in [39]). At the present time it is still unclear the extent to which dopaminergic neurons can be generated by adult nigral neurogenesis in the mammalian brain- and even if this process does occur, it is unproven whether it is unaffected by the PD disease process. Thus, for the moment, whilst attractive, endogenous repair through local dopaminergic neurogenesis remains a long way off.

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7.7 Conclusions Cell replacement therapy is a very attractive therapeutic approach for PD, as it currently lacks a curative treatment and its main symptomatology derives from the degeneration of a specific cell type in the brain, namely the dopaminergic nigral neurons. Clinical trials over the last twenty years using fetal VM in patients with PD has shown a proof of principle that this therapy can be very effective in improving motor deficits in some cases. However, not all patients have benefited from this approach and indeed some have had significant side-effects. Thus there is a need to more carefully select the patients that are most likely respond to this kind of therapy, in particular young patients, early in the course of the disease. The now established side effects in some patients of graft induced dyskinesias (GIDs) could also be minimized by improving the purity of the donor cells and the delivery of those cells to the striatal complex. The heterogeneous nature of fetal VM tissue, together with the logistic and ethical problems inherent in their use, has led to the search for alternative sources of dopaminergic neurons. Of all of them, iPS cells are the most promising, as they could represent a more homogeneous autologous source of dopaminergic neurons, without major immunological and ethical concerns. Nevertheless there still exist a number of technical difficulties in obtaining sufficient numbers of dopaminergic neurons from these cells and as such their ability to be employed safely and effectively in PD remains unproven. Thus we live in exciting times with cell therapies and PD, although it must be stressed that all these strategies work around dopaminergic cell replacement even though this is only a part, albeit a very important one, of the pathology of PD. As such the search for a cure for PD must continue as dopaminergic cell therapies will only ever be able to treat and cure a part of this complex and all too common neurodegenerative disorder. Acknowledgments Our own work is supported by the Medical Research Council and Parkinson’s Disease Society.

References 1. Gill SS, Hotton GR, O’Sullivan K et al. (2003) Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat Med 9:589–595. 2. Patel NK, Plaha P, Svendsen CN et al. (2005) Intraputamenal infusion of glial cell line-derived neurotrophic factor in PD: a two-year outcome study. Ann Neurol 57:298–302. 3. Lang AE, Patel NK, Lozano A et al. (2006) Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson’s disease. Ann Neurol 59: 459–466. 4. Perlow MJ, Freed WJ, Hoffer BJ et al. (1979) Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 204:643–647. 5. Bjorklund A and Stenevi U (1979) Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res 177:555–560. 6. Brundin P, Nilsson OG, Strecker RE et al. (1986) Behavioral effects of human fetal dopamine neurons grafted in a rat model of Parkinson’s disease. Exp Brain Res 65:235–240. 7. Freed CR, Breeze RE, Rosenberg NL et al. (1992) Survival of implanted fetal dopamine cells and neurologic improvement 12 to 46 months after transplantation for Parkinson’s disease. N Engl J Med 327:1549–1555.

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8. Hagell P, Piccini P, Jahanshahi M et al. (1999) Sequential bilateral transplantation in Parkinson’s disease: Effects of the second graft. Brain 122:1121–1132. 9. Hauser RA, Snow BJ, Nauert M et al. (1999) Long-term evaluation of bilateral fetal nigral transplantation in Parkinson’s disease. Arch Neurol 56:179–187. 10. Brundin P, Hagell P, Piccini P et al. (2000) Bilateral caudate and putamen grafts of embryonic mesencephalic tissue treated with lazaroids in Parkinson’s disease. Brain 123:1380–1390. 11. Freed CR, Breeze RE, Tsai WY et al. (2001) Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N Eng J Med 344:710–719. 12. Olanow CW, Kordower JH, Stoessl AJ et al. (2003) A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson’s disease. Ann Neurol 54:403–414. 13. Studer L, McKay RD (1998) Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1:290–295. 14. Storch A, Csete M, Boehm BO et al. (2001) Long-term proliferation and dopaminergic differentiation of human mesencephalic neural precursor cells. Exp Neurol 170:317–325. 15. Mukhida K, Baghbaderani BA, Hong M et al. (2008) Survival, differentiation, and migration of bioreactor-expanded human neural precursor cells in a model of Parkinson disease in rats. Neurosurg Focus 24:E8. 16. Jensen P, Pedersen EG, Zimmer J et al. (2008) Functional effect of FGF2- and FGF8expanded ventral mesencephalic precursor cells in a rat model of Parkinson’s disease. Brain Res 1218:13–20. 17. Jain M, Armstrong RJ, Tyers P et al. (2003) GABAergic immunoreactivity is predominant in neurons derived from expanded human neural precursor cells in vitro. Exp Neurol 182: 113–123. 18. Sanchez-Pernaute R, Bankiewicz KS, Major EO et al. (2001) In vitro generation and transplantation of precursor-derived human dopamine neurons. J Neurosci Res 65:284–288. 19. Studer L, Csete M, Lee SH et al. (2000) Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J Neurosci 20:7377–7383. 20. Parish CL, Castelo-Branco G, Rawal N et al. (2008) Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice. J Clin Invest 118:149–160. 21. Kawasaki H, Mizuseki K, Nishikawa S et al. (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28:31–40. 22. Lee SH, Studer L, Auerbach JM, et al. (2000) Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotech 18:675–679. 23. Kim JH, Rodriguez-Gomez JA, Velasco I et al. (2002) Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418:50–56. 24. Kim DW, Chung S, Hwang M et al. (2006). Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells. Stem Cells 24:557–567. 25. Perrier AL, Tabar V, Barberi T et al. (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA 101:12543–12548. 26. Yang D, Zhang ZJ, Oldenburg M et al. (2008) Human embryonic stem cell-derived dopaminergic neurons reverse functional deficit in parkinsonian rats. Stem Cells 26:55–63. 27. Li JY, Christophersen NS, Hall V et al. (2008) Critical issues of clinical human embryonic stem cell therapy for brain repair. Trends Neurosci 31:146–153. 28. Roy NS, Cleren C, Singh SK et al. (2006) Functional engraftment of human ES cellderived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med 12:1259–1268. 29. Taylor CJ, Bolton EM, Pocock S et al. (2005) Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching. Lancet 366:2019–2025. 30. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. 31. Takahashi K, Tanabe K, Ohnuki M et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872.

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32. Nakagawa M, Koyanagi M, Tanabe K et al. (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26:101–106. 33. Yu J, Vodyanik MA, Smuga-Otto K et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920. 34. Stadtfeld M, Nagaya M, Utikal J et al. (2008) Induced pluripotent stem cells generated without viral integration. Science 322:945–949. 35. Okita K, Nakagawa M, Hyenjong H et al. (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322:949–953. 36. Wernig M, Zhao JP, Pruszak J et al. (2008) Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci USA 105:5856–5861. 37. Minguez-Castellanos A, Escamilla-Sevilla F, Hotton GR et al. (2007) Carotid body autotransplantation in Parkinson disease: A clinical and PET study. J Neurol Neurosurg Psychiatry 78: 825–831. 38. Dezawa M, Hoshino M, Cho H et al. (2004) Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest 113: 1701–1710. 39. Borta A, Hoglinger GU (2007) Dopamine and adult neurogenesis. J Neurochem 100:587–595.

Chapter 8

Transplantation of Stem Cells and Their Derivatives in the Treatment of Multiple Sclerosis Eric C. Larsen and Ian D. Duncan

Abstract Multiple sclerosis (MS) is a debilitating disorder of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal degeneration. Chronic demyelination is believed to result in axon degeneration, leading to long-term disability in the majority of MS patients. However, there are currently no therapies available that will promote myelination. The therapeutic potential of exogenous stem cells in the treatment of a MS is the subject of intensive investigation. The pluripotency and self-renewal properties exhibited by stem cells offer a potentially limitless source of cells that can differentiate into myelinating oligodendrocytes following transplantation into the damaged CNS. Transplanted precursor cells derived from stem cells have been shown to myelinate axons in genetic models of myelin disease and in models of chemically-induced demyelination. However, studies are still ongoing to determine whether stem cell-derived precursor cells are capable of myelinating axons in animal models of MS, an important step in demonstrating the therapeutic potential of transplanted cells in MS. In addition, the method of cell delivery to patients and the selection of MS patients for cell-based repair therapy are issues that are being explored. Keywords Multiple sclerosis · Demyelination · Axon degeneration · Remyelination · Stem cells · Precursor cells

Contents 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 The Pathology of Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . . . 8.3 Current Treatments for Multiple Sclerosis . . . . . . . . . . . . . . . . . . .

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I.D. Duncan (B) Department of Medical Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA e-mail: [email protected]

T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_8,

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8.4 The Need for Repair-Oriented Therapies for Multiple Sclerosis . . . . . . . . . . 8.4.1 Transplantation of Exogenous Stem Cells . . . . . . . . . . . . . . . . 8.4.2 Mobilization of Endogenous Stem Cells . . . . . . . . . . . . . . . . . 8.5 Which Exogenous Stem Cells Can Be Used for Transplantation? . . . . . . . . . 8.5.1 Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.2 Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . 8.5.3 Neural Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.4 Hematopoietic Stem Cells (HSCs) and Mesenchymal Stem Cells (MSCs) . . 8.6 Stem Cells and Stem Cell-Derived Precursors are Capable of Myelination in Animal Models . . . . . . . . . . . . . . . . . . . . . . . 8.6.1 Remyelination in Genetic Disease Models . . . . . . . . . . . . . . . . 8.6.2 Remyelination Following Chemically-Induced Demyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7 Studies of Stem Cells in Experimental Autoimmune Encephalomyelitis Models . . 8.7.1 Survival and Migration of Stem Cell-Derived Progenitors in EAE . . . . . . 8.7.2 Immunomodulatory Effects of Stem Cell-Derived Progenitors in EAE . . . . 8.7.3 Remyelination by Stem Cell-Derived Progenitors in EAE . . . . . . . . . 8.8 Initial Investigations into Exogenous Cell-Based Therapy in Multiple Sclerosis . . 8.9 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9.1 Establishing the Myelinating Capacity of Stem Cells in MS . . . . . . . . 8.9.2 Inflammation and Astrocytes . . . . . . . . . . . . . . . . . . . . . . 8.9.3 Method of Cell Delivery to MS Patients . . . . . . . . . . . . . . . . . 8.9.4 The Use of Genetically Modified Cells for Transplantation . . . . . . . . . 8.9.5 Selection of MS Patients for Transplantation Therapy . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8.1 Introduction With an estimated 2.5 million cases diagnosed worldwide [1], multiple sclerosis (MS) is a leading cause of disability in young adults. MS is an immune-mediated, possibly autoimmune, neurological disease in which myelin in the central nervous system (CNS) is lost. Myelin, which is synthesized by oligodendrocytes, ensheaths and insulates the axons of the CNS and in turn enables faster nerve conduction. Myelin also protects and likely provides trophic support to axons. The loss of myelin in the CNS of MS patients results in a delay or disruption in conduction and possible loss of axons, leading to neurologic symptoms related to the site of demyelination. Common symptoms include fatigue, numbness, paresis, imbalance, bowel and bladder dysfunction, and vision problems. Cognitive deficiencies are also frequently observed [2]. Relapsing-remitting MS (RRMS) is the most common form of MS, accounting for approximately 85% of all cases. Patients with RRMS experience acute attacks, or relapses, followed by periods of remission with at least partial recovery. Over 50% of RRMS patients will subsequently develop secondary-progressive MS (SPMS), a more progressive disease without remissions [3].

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There is no clear-cut single factor that makes an individual more susceptible to MS than another; in all likelihood a number of factors come into play to trigger disease. Women are twice as likely to develop the disease as men [4]. There is evidence that MS patients may have a genetic predisposition to disease, with the human leukocyte antigen (HLA) gene cluster having the strongest evidence for such a role [5, 6]. MS is more common in certain ethnic groups, with individuals of Northern European descent having a higher incidence of the disease [5]. Environmental conditions may also play a role, as the disease occurs more frequently with increasing distance from the equator, although this trend appears to be decreasing [7]. While some of this equator-to-pole gradient can be explained by differences in disease frequency among different ethnicities, there is growing evidence that exposure to sunlight and subsequent vitamin D production may influence MS susceptibility [8]. Exposure to bacteria and viruses may also play a role, albeit in potentially contrasting ways. A number of viruses, in particular Epstein-Barr virus, have been suggested to act as the trigger for MS development, although there is no concrete evidence for any one single virus being involved [9, 10]. Alternatively, the “hygiene hypothesis” postulates that exposure to infectious agents early in life is protective against MS; this hypothesis may also help to explain the latitude difference in MS frequencies [9]. In all likelihood, development of MS in a patient is dependent on a combination of these factors, and perhaps some that have yet to be isolated. Regardless of the mechanism by which an individual develops MS, the result in most cases is long-term progressive disability. Therapies exist that lessen the severity and frequency of relapses and improve the quality of life of many MS patients. However, there are currently no therapies available to repair the damage caused by the disease that typically leads to progressive disability. The development of therapies that can repair MS-related damage and prevent long-term disability is under intense investigation. One of the most promising repair-oriented therapeutic approaches involves the transplantation of stem cells and their derivatives into the CNS of MS patients. The goal is that these cells will differentiate into myelinating oligodendrocytes following transplantation. The myelin sheaths generated by these oligodendrocytes will in turn restore normal nerve conduction and prevent axon degeneration, thereby attenuating the progressive disability that many MS patients suffer. This chapter will review the types of stem cells that may be used in repairoriented therapies, the evidence in animal models of demyelination that support the potential of stem cell-based repair therapy, and the challenges that will need to be resolved before this therapeutic approach can be used to treat MS patients.

8.2 The Pathology of Multiple Sclerosis The pathology and disease progression of multiple sclerosis greatly affects the therapeutic approach used to treat the disease, a situation complicated by the heterogeneity of MS observed in patients. Multiple sclerosis is a disease characterized by immune cell infiltration of the central nervous system (CNS) and inflammation, demyelination of axons, and axon degeneration. However, the sequence in which

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these events occur may vary from patient to patient and may be responsible for the heterogeneity of lesions observed in MS patients. MS is traditionally thought of as a white matter disease, as the primary targets of the disease are myelin and oligodendrocytes. However, there is growing evidence that gray matter is also targeted [11], although gray matter lesions exhibit reduced inflammation and more efficient remyelination compared to white matter lesions [12, 13]. The acute stages of the RRMS disease course are marked primarily by CNS inflammation and demyelination followed by remyelination. During these early stages of the disease, the use of immunomodulative and immunosuppressive drugs can limit the extent of demyelination and axon damage mediated by inflammation. However, there are situations in which inflammation may have positive effects on CNS repair, and as such the timing of the use of such drugs may have to be adjusted in order to maximize the beneficial effects and minimize the deleterious effects of inflammation. Over time, the extent of inflammation decreases, and the extent of neurodegeneration increases. As the disease enters a more chronic phase, the primary concerns become chronic demyelination and axon degeneration. Under these conditions, the chief therapeutic approaches will focus on restoration of myelin in chronically demyelinated areas so as to prevent axon loss. Much of the pathological sequence of events in MS has been determined and extrapolated from post-mortem analysis and biopsies from MS patients. Using this information, a correlation between disease pathology and progression and CNS imaging (in particular by magnetic resonance imaging) has emerged that is aiding physicians in diagnosing and monitoring MS patients. Four patterns of demyelination have been observed in lesions from MS patients [14]. In type I and type II patterns, demyelination is mediated either by antibody/complement-dependent mechanisms or by inflammatory processes. In type III and type IV patterns, oligodendrocyte apoptosis is the primary mechanism of demyelination [15]. In these patterns, inflammation was a secondary response designed to clear myelin debris. However, recent autopsies of lesions from patients with established MS suggest that these four heterogeneous patterns of demyelination may converge into a common mechanism of demyelination [16]. Regardless of the mechanism, demyelination leads to the generation of myelin debris, some of which is cleared by phagocytosis, and exposure of axons to the inflammatory environment of the MS lesion. During the early stages of MS, demyelination is at least partly counterbalanced by endogenous remyelination [17]. In addition to a population of oligodendrocyte progenitor cells (OPCs) that are present throughout the CNS [18], OPCs can also be recruited from the sub ventricular zone to sites of inflammation in animal models of MS [19], as well as in MS lesions in humans [20]. This population of OPCs, whether already present in a site of inflammation or mobilized to a site of inflammation, is capable of differentiating into mature, myelinating oligodendrocytes [19, 20]. In addition to its role in stimulating recruitment of potential myelinating cells, acute inflammation has been shown to be beneficial in endogenous remyelination by macrophage removal of myelin debris [21, 22]. Endogenous remyelination results in myelin sheaths that are disproportionally thin for the size of the axon [23, 24], yet restore conduction [25] and function [26]. Regions of remyelination can be

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identified in post-mortem tissue by reduced myelin staining (so-called “shadow plaques”) and may be correlated with hyperintense T2-weighted images generated by MR imaging [27]. As patients progress to a more chronic disease state, endogenous remyelination becomes less efficient or ceases altogether. Like most biological processes, endogenous remyelination becomes less efficient with age [28, 29]. There is evidence that chronic demyelination depletes both oligodendrocytes and OPCs [30]. Furthermore, the environment of the chronic MS lesion has a significant deleterious effect on remyelination. OPCs differentiate and mature poorly in chronic MS lesions [31], a deficit that may be in part due to production of a high-molecular form of hyaluronan by astrocytes [32]. Even in those cases where OPCs can differentiate into pre-myelinating oligodendrocytes, a loss of axon-oligodendrocyte signaling, which may be in part due to PSA-NCAM expression on demyelinated axons [33], prevents remyelination in chronic lesions [34]. In spite of this, extensive endogenous remyelination can still be observed in a subset of MS patients with chronic disease [27, 35, 36]. It should be noted that, much in the same way that it may hinder endogenous precursor cell migration and differentiation, the milieu of the chronic MS lesion may also interfere with the ability of exogenous transplanted cells to migrate, differentiate, and myelinate. During acute phases of MS, some axon loss has been observed [37–39]. However, the primary mechanism of axon degeneration and subsequent disability in MS patients is thought to be the result of chronic demyelination during later stages of the disease. There are several mechanisms by which chronic loss of myelin leads to axon damage. Although redistribution of sodium channels along the length of the demyelinated axon can temporarily restore conduction, increased energy demands coupled with impaired ATP production eventually leads to intracellular accumulation of calcium ions, which triggers a fatal cascade of axons within the axons [40]. Demyelinated axons, having been stripped of myelin, are especially vulnerable to a host of environmental insults. The inflammatory environment of the MS lesion generates a number of potentially deleterious compounds, such as proteolytic enzymes, cytokines, oxidative products, and free radicals. Furthermore, there is significant evidence that oligodendrocytes and the myelin sheaths that they generate provide trophic support to the axon [41]. These factors, either by themselves or in combination, frequently result in axon damage and degeneration. While neurogenesis can occur in a subset of demyelinated white matter lesions in MS patients [42], in many cases axon loss is irreversible and leads to long-term disability.

8.3 Current Treatments for Multiple Sclerosis The majority of drugs currently used to treat MS are immunomodulatory or immunosuppressive in nature [43]. Many of these drugs act to prevent immune cell activation and/or infiltration of the CNS. These therapies have been effective in reducing the frequency and severity of relapses in many RRMS patients, as

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well as reducing the number of enhancing lesions in the brain visualized by MR imaging. However, the efficacy of immunosuppression diminishes as the disease shifts into a more chronic disease state. In this chronic state, lesions display little or no inflammation, compared to acute MS lesions, and as a result the actions of anti-inflammatory drugs are reduced. Furthermore, the efficacy of certain immunosuppressive therapies varies from patient to patient, with some drugs working better than others or not at all. These differences in drug efficacy from patient to patient may be in part due to the heterogeneity of MS and differences in disease pathology and progression from one patient to the next.

8.4 The Need for Repair-Oriented Therapies for Multiple Sclerosis There are at present no treatments available that promote remyelination. The importance of remyelination is supported by findings that remyelination protects demyelinated axons from degeneration following cuprizone-mediated demyelination [44] and in MS [39]. Chronic demyelination in MS lesions leads to disruption of normal nerve conduction and eventual axonal loss, which is widely thought to be responsible for the progressive disability observed in MS patients. Therefore, the development of therapies aimed at remyelination in MS lesions should limit the extent of long-term disability by restoring nerve conduction and preserving axons from degenerative processes. Stem cells are characterized by their ability to differentiate into multiple cell lineages (multipotency) and their ability to self-renew, almost indefinitely in some cases, over multiple passages. Two potential therapeutic approaches using stem cells are currently being explored as a means of remyelinating axons in MS lesions. As shown in Fig. 8.1 [45], one approach involves the transplantation of exogenous stem cells, while the other approach involves the mobilization of endogenous stem cells. In both cases, stem cells must be able to overcome the environmental barriers that may be present within the MS lesion that hinder remyelination. For example, if the milieu of the MS lesion is such that migration of stem cells into the lesion is altered, then exogenous stem cells can be directly injected into the MS lesion in order to bypass this migratory block.

8.4.1 Transplantation of Exogenous Stem Cells The approach that we will focus on in this chapter involves the transplantation of exogenous stem cells into the CNS. Stem cells can either be transplanted directly into the CNS of an MS patient, or pre-differentiated in vitro into a precursor or progenitor cell population prior to transplantation. Cells can either be injected directly into a MS lesion; alternatively, cells can be injected adjacent to a lesion and subsequently migrate to the lesion. One potential advantage of this approach, compared

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to the recruitment of endogenous stem cells, is the capability of genetically engineering stem cells prior to transplantation. These genetically modified stem cells could be engineered in such a way that, for example, they secrete growth factors that make the milieu of the MS lesion more conducive to remyelination. Alternatively, stem cells could be modified such that their differentiation to oligodendrocytes is made more efficient by altering the expression of a transcription factor regulating oligodendrocyte differentiation, as has been demonstrated by over expression of the transcription factor Olig2 in neural stem cells in vitro [46].

8.4.2 Mobilization of Endogenous Stem Cells As previously noted, endogenous repair mechanisms are capable of remyelinating axons in MS patients during acute stages of the disease and, in some patients, even during chronic disease. However, in most cases, over time the endogenous repair process fails and axons remain demyelinated, leaving them susceptible to degeneration [17]. Efforts are underway to enhance the endogenous repair system in MS patients by modifying the milieu of the MS lesion as to enable enhanced proliferation, migration, and differentiation of endogenous stem cells into myelinating oligodendrocytes.

8.5 Which Exogenous Stem Cells Can Be Used for Transplantation? As shown in Fig. 8.1, there are several potential sources of transplantable stem cells that have the potential to be used in repairing MS-related damage. Each of these stem cell populations has their own advantages and disadvantages with regards to their potential utility in MS repair therapy.

8.5.1 Embryonic Stem Cells While there is considerable interest in the use of stem cells in disease therapies, this interest is especially high regarding the use of embryonic stem cells (ESCs). ESCs are isolated from the inner cell mass of blastocysts derived by in vitro fertilization [47, 48]. ESCs display an almost infinite ability to self-renew, thereby providing a potentially limitless source of transplantable cells. ESCs are also notable among stem cells in being pluripotent, thus having the capability to differentiate into virtually any mature cell type in the body. In order for ESCs to have therapeutic potential in MS repair, however, ESCs need to be able to differentiate into myelinating oligodendrocytes. ESCs derived from both mouse and human blastocysts have been shown to differentiate into myelinating oligodendrocytes in vitro and in vivo

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Fig. 8.1 Two approaches for the usage of stem cells in the treatment of neurological diseases. Exogenous stem cells derived from a variety of sources (from the blastocyst, the central nervous system (CNS), or tissues such as bone marrow or umbilical cord) can be either be directly transplanted into the CNS of affected individuals, or transplanted following in vitro predifferentiation and/or genetic modification. Alternatively, endogenous stem cells already present in the CNS can be triggered to proliferate and/or migrate to sites of neurological damage. (Reprinted by permission [45])

following transplantation into the spinal cord of the myelin-deficient (md) rat [49] and the shiverer (shi) mice [50, 51]. While there is evidence that transplanted ESCs can differentiate into myelinating oligodendrocytes in animal models, direct injection of ESCs into MS patients for therapeutic purposes is not considered to be a feasible approach due to the likely development of teratomas. Rather, the approach currently favored involves the predifferentiation of ESCs in vitro into neural precursors (NPs), glial precursors, or oligodendrocyte precursor cells (OPCs) prior to transplantation, a process which has been achieved from mouse ESCs [49, 52] and human ESCs [53, 54]. While NPs, glial precursors, and OPCs derived from ESC pre-differentiation demonstrate

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the ability to differentiate into myelinating oligodendrocytes, it is not clear as to which stage of pre-differentiation will result in the optimal development of large numbers of myelinating oligodendrocytes following transplantation. Human ESCderived NPs are capable of differentiating into neurons and glial cells in vitro and in vivo following transplantation (Fig. 8.2). NPs derived from human ESCs differentiate into dopaminergic neurons and partially reverse behavioral deficits in a rat model of Parkinson’s disease [55]. Glial precursor cells derived from mouse ESCs differentiate into myelinating oligodendrocytes in the md rat following transplantation [49]. Oligodendrocyte precursors generated by in vitro pre-differentiation of human ESCs are capable of differentiating into mature, myelinating oligodendrocytes upon transplantation into animal models [56, 57].

8.5.2 Induced Pluripotent Stem Cells A recent and exciting possible approach to circumvent the issue of graft rejection of cells transplanted as allografts would involve the usage of patient-specific induced pluripotent stem (iPS) cells. iPS cells have been produced from both human and mouse somatic cells via retroviral expression of transcription factors [58, 59], which reprogram the somatic cells to an ESC-like state. From there, iPS cells can proliferate ad infinitum and potentially be differentiated into a number of different somatic cell types. iPS cells derived from mouse fibroblasts have already been demonstrated to be capable of differentiating into neural precursor cells and generating mature neuronal cell types, such as neurons and glia [60]. The possibility of tumor formation as a result of random integration of the retroviral genome has prompted the development of alternative approaches for the reprogramming of somatic cells into iPS cells that circumvent the need for retroviral vectors. Given the rapid pace at which this field is moving, significant technical improvements have already been made in iPS methodology. Recently, iPS cells have been generated from mouse somatic cells through the use of non-integrating adeno-associated viral vectors and plasmids, which potentially eliminate the risk of tumor formation due to genomic integration [61, 62]. Efforts are also underway to generate iPS cells by the direct delivery of transcription factors through transduction and the use of small molecules [63]. However, generation of iPS cells from somatic cells has thus far proven to be a slow and inefficient process, and this is a hurdle that shall need to be overcome before iPS cell-based therapies can become readily available. Furthermore, much like ESCs, iPS cells carry the risks of teratoma formation following transplantation. Therefore, as with ESCs, transplantation therapies with iPS cells will likely require in vitro differentiation into neural-restricted or glial-restricted precursor cells prior to transplantation. While continuing studies are needed, iPS cells could in theory be derived from an MS patient’s fibroblasts and then differentiated in vitro into neural or glial precursor cells. These precursor cells in turn could be injected into the patient without concern of immunorejection of the graft.

Fig. 8.2 Human embryonic stem cells (hESCs) differentiate into mature neural and glial cell types both in vitro and in vivo following transplantation. (a) An attached embryoid body (EB) grown in the presence of FGF-2 for five days shows flattened cells at the periphery and small elongated cells congregated in the center. (b) By seven days, many rosette formations (arrows) appear in the center of the differentiating EB. Inset: 1 μm section of the rosette formation stained with toluidine blue, showing columnar cells arranging in a tubular structure. Bar, 20 μm. (c, d) Cells within a cluster of rosettes (lower left) and a small evolving rosette (center) are positive for nestin (c) and Musashi-1 (d), while the surrounding flat cells are negative. Bar, 100 μm. (e) After 45 days of differentiation, GFAP+ astrocytes (green) appear along with NF200+ neurites (red, yellowish due to overlapping with green GFAP). Bar, 100 μm. F-I) hESCderived neural precursors were injected into the lateral ventricles of newborn mice. (f) hESCderived neuron in the cortex of a two-week-old host, exhibiting a polar morphology and long processes. The cell is double labeled with antibodies to a human-specific nuclear marker (green) and βIII -tubulin (red). (g) Network of donor-derived axons in the fimbria of the hippocampus, identified with an antibody to human neurofilament. (h) Donor-derived multipolar neuron, double labeled with antibodies recognizing the a and b isoforms of MAP2 (red) and human nuclei (green). (i) ES cell derived astrocyte in the cortex of a four-week-old animal, double labeled with the human nuclear marker (green) and an antibody to GFAP (red). (Reprinted with permission [54])

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8.5.3 Neural Stem Cells Neural stem cells (NSCs) are a population of stem cells located in particular niches of both the fetal and adult CNS that give rise to neurons, oligodendrocytes, and astrocytes [64, 65]. As opposed to ESCs and iPS cells, NSCs are far more restricted in their ability to differentiate into mature cell types, as well as far more limited in their ability to self-renew. In the fetal CNS, NSCs are typically concentrated in the olfactory bulb, the ventricular and subventricular zones near the lateral ventricles, the hippocampus, the spinal cord, the cerebellum, and the cerebral cortex. Adult NSCs can be detected in and isolated from the subventricular zone (SVZ), subgranular zone (SGZ) and the spinal cord [66, 67]. NSCs in the subventricular zone of the adult brain are of particular interest in the field of MS repair as there is evidence that these cells can proliferate, migrate from the SVZ, and differentiate into oligodendrocytes in response to chemically-induced demyelination [68], inflammation in an animal model of MS [19], and lesion formation in MS patients [20]. NSCs are also capable of migrating to sites of inflammation in the spinal cord in an animal model of MS [69]. After isolation of CNS tissue, NSCs can be pre-differentiated into OPCs [70, 71], which can give rise to oligodendrocytes or type-2 astrocytes in vitro [72]. As shown in Fig. 8.3, NSCs isolated from human fetal brain tissue give rise to OPCs and oligodendrocytes in vitro. OPCs from fetal tissue and from adult tissue possess similar intrinsic properties; both OPC populations are capable of differentiating into myelinating oligodendrocytes [73]. However, differences do exist between the two precursor cell populations. Fetal OPCs from the rat optic nerve divide, migrate, and differentiate more rapidly than do adult OPCs [74]. However, OPCs obtained from adult human subcortical white matter samples myelinated axons in the brain of the shi mouse more rapidly and more efficiently than did OPCs isolated from the SVZ of fetal human brain [75]. In many cases, the NSCs and OPCs used in transplantation studies are isolated from the SVZ of the fetal brain.

8.5.4 Hematopoietic Stem Cells (HSCs) and Mesenchymal Stem Cells (MSCs) Hematopoietic stem cells (HSCs) give rise to all constituents of blood. HSCs have traditionally been obtained from bone marrow, although increasingly they are isolated from umbilical cord blood. HSCs are currently used in the treatment of cancers of the blood (leukemia and lymphoma) and inherited blood disorders. Mesenchymal stem cells (MSCs; also known as bone marrow stromal cells) can also be isolated from bone marrow, cultured and expanded in large quantities, although their ability to self-renew is not indefinite as with ESCs. MSCs are traditionally known to differentiate into cell types such as osteoblasts and adipocytes both in vivo and in vitro. MSCs display immunomodulatory properties in vitro and in vivo, and it is these properties that have been exploited in the use of MSCs in the treatment

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Fig. 8.3 Neural stem cells isolated from fetal human brain can be differentiated in vitro into mature glial cells. (a) Neural stem cells [cultured as neurospheres that positively stain for nestin (inset)] were cultured under differentiating conditions and stained with O4, showing that O4+ oligodendroglia emerged from the neurosphere (NS). Note the morphological transition from bipolar cells adjacent to the sphere, to more branched oligodendroglia in the periphery. (b) A similar culture was stained with O1 (in red) and GFAP (in green). (c, d) A 23-day-old differentiating culture was stained with O4 (c) and MBP (d) showing that the well-branched (arrows) but not the morphological simple (arrowheads) oligodendroglia expressed MBP. (e–g) An oligodendrocyte that grew outside of the astrocytic layer and exhibited membrane-like structure. (e) stained with O4, (f) with MBP and (g) with both O4 and MBP. Cell nuclei in (a) and (g) were stained with DAPI (in blue). Bars, 100 μm. (Reprinted with permission [71])

of disease [76]. HSCs and MSCs have an advantage over other stem cell types in that the patient can be the donor of cells to be transplanted, thereby eliminating the need for the use of immunosuppressive drugs to prevent graft rejection. The only stem cell-based therapeutic studies that have been performed thus far in MS patients have involved autologous HSC transplantation [77–80]. However, the potential of HSCs and MSCs in repair-oriented therapies for MS has not been as well established as it has for ESC-derived precursor cells or neural stem cells. There are studies that suggest that MSCs can remyelinate axons in the spinal cord

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following chemically-induced demyelination [81–84]. However, in a recent study, MSCs injected into a demyelinated spinal cord lesion failed to differentiate into myelinating oligodendrocytes [85]. These findings, along with the axonal damage associated with transplanted MSCs, suggest that this cell population may not be suitable for exogenous repair therapies.

8.6 Stem Cells and Stem Cell-Derived Precursors are Capable of Myelination in Animal Models The myelinating capacity of cells that may be used therapeutically in MS can be tested in different model systems that represent some of the varying pathological milieus of MS lesions. These model systems allow for the generation of either a focal region of demyelination, as is frequently the case with chemically-induced demyelination models, or widespread demyelination, as is typically the case with genetic disease models. Both model systems provide a means to determine the myelinating ability of transplanted cells. However, the differences in the host environment between a genetic disease model and a chemically-induced demyelination model can result in the same pool of precursor cells giving rise to different final populations of differentiated cells [86]. It should be noted that the environment in these models to which cells are transplanted is frequently not as complex as the milieu of the MS lesion, which is marked by inflammation and repeated bouts of demyelination and remyelination, nor is there a model system that truly mimics chronic MS lesions.

8.6.1 Remyelination in Genetic Disease Models Animal models of genetic myelin disease, which were initially used to study the pathology of human leukodystrophies such as Pelizaeus-Merzbacher disease, have provided valuable model systems in which to test the myelinating capacity of cells. Many of these animal models fail to myelinate due to a mutation in a protein component of the myelin sheath. For example, the md rat [87] has a mutation in the proteolipid protein (PLP) gene, while myelination failure in the shi mouse [88] and the Long Evans Shaker (les) rat is due to mutations in the myelin basic protein (MBP) gene [89, 90]. Due to the short lifespan of the md rat (approximately 21 days), longer-lived models such as the shi mouse and the les rat are valuable for testing the long-term myelination capabilities of transplanted cells. Other genetic disease models, such as the taiep rat, develop progressive demyelination as the animal ages [91]. NSCs and OPCs have been shown to myelinate axons following transplantation into the md rat [65, 92], as have mouse ESC-derived glial precursors [49]. Myelination of axons in the md rat by transplanted cells resulted in the enhancement of action potential conduction [93]. As demonstrated in Fig. 8.4, OPCs isolated from GFP-transgenic mice migrate across and myelinate extensive areas of

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Fig. 8.4 Transplanted OPCs differentiate into oligodendrocytes and myelinate the spinal cord of the shiverer(shi) mouse. (a, b) Myelin basic protein (MBP) immunolabeling of spinal cord from (a) a 4-month old wild-type mouse and (b) a 45-day old moribund shi mice. Note the complete absence of MBP staining in the shi spinal cord. (c–e) OPCs expressing EGFP under the control of the CNP promoter were transplanted into the spinal cord of 3-week old shi mice and exhibited 4 months post-transplant. Donor cells spread over the majority of whole white matter, as indicated by EGFP fluorescence (C), and expressed MBP (d). (e) Merge of (c) and (d). (f–h) Toluidine blue staining of 1 μm sections from the spinal cord of (f) wild-type mice, (g) shi mice, and (h) shi mice that received transplanted OPCs. Myelin sheaths are completely absent in the untransplanted shi mouse; however, OPC transplantation results in widespread axon ensheathment. (Reprinted with permission [94])

the spinal cord in the shi mouse following transplantation [94], a property duplicated by adult rat NSCs [86]. Both fetal and adult human OPCs display extensive myelination of axons in the shi forebrain [75]. Transplanted OPCs can myelinate axons in the les rat, although microglial activation must be suppressed to facilitate OPC survival [95]. Restoration of nodes of Ranvier and improved conduction can be observed following exogenous cell transplantation into the shi mouse [96, 97]. Although there was abundant evidence that transplanted cells can myelinate axons in genetic animal models of demyelination, the question remained as to whether a sufficient degree of myelination by transplanted cells would be capable of rescuing the disease phenotype in these animals. However, recent studies have demonstrated that human-derived NPCs transplanted into five sites in the neonatal shi mouse were able to migrate across almost the entire brain and spinal cord and replace the defective oligodendrocyte population in a percentage of transplanted

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mice [97]. By myelinating axons and restoring nerve conduction in these transplanted shi mice, premature death in these animals was prevented. These results clearly demonstrate that, by injecting a sufficient number of precursor cells into multiple sites of a demyelinated CNS, transplanted cells can sufficiently migrate and differentiate into myelinating oligodendrocytes in myelin disease models so as to least partially reverse the disease course.

8.6.2 Remyelination Following Chemically-Induced Demyelination The remyelinating potential of transplanted stem cell-derived precursors has also been reproducibly shown in animal models in which demyelination is induced chemically. When the myelinating ability of transplanted cells is to be studied in these models, endogenous remyelination is frequently inhibited by prior focal X-irradiation of the demyelinated area [98]. One of the most frequently used models involves feeding mice a diet containing the copper chelator cuprizone. Ingestion of cuprizone results in progressive demyelination in specific white matter tracts. Transplantation of cells into these affected white matter tracts results in remyelination [30, 44, 46]. Similarly, direct injection of chemical agents such as ethidium bromide, lysolecithin, and antibodies against galactocerebroside into white matter has also been used to generate focal regions of demyelination [99–101]. Remyelination of axons in these lesions have been demonstrated following injection of NSCs [86], OPCs [102–104], and white matter progenitor cells [105]. ESC-derived glial precursors are also capable of myelinating axons in an antibody/complement-mediated model of demyelination [106]. Targeting of focal demyelinating lesions to specific axonal tracts in the spinal cord can result in observable behavioral phenotypes, most notably motor deficiencies [26]. While axonal tract targeting of demyelinating lesions was originally used to demonstrate repair and restoration of locomotor function by endogenous remyelination processes, the use of such focal demyelinating lesions have demonstrated that remyelination by exogenous cells can repair a lesion and restore normal motor function [103].

8.7 Studies of Stem Cells in Experimental Autoimmune Encephalomyelitis Models Experimental autoimmune encephalomyelitis (EAE) is the most widely used animal model of MS. EAE is typically induced in animals either by the injection of an emulsion containing a fragment of a myelin membrane protein or a homogenate of spinal cord tissue, or by the injection of T cells reactive towards a myelin-based antigen. Following disease induction, activated T cells cross the blood-brain barrier (BBB) into the CNS and initiate a disease course similar in many respects

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to what is observed in MS (inflammation, demyelination, and axonal degeneration) [107]. Depending on the animal used and the means of disease induction, the disease course may either be acute or chronic, mimicking at least in part some aspect of the disease course in MS patients. In particular, chronic EAE models can exhibit a disease progression similar to those observed in relapsing-remitting disease and progressive MS disease, and as such these models tend to be used more frequently given their relevancy to human disease. It should be noted that concerns have been raised about just how closely EAE resembles MS [108–110]. Despite discrepancies between the two diseases, EAE continues to be the principal means of testing potential MS therapies, among them the potential therapeutic effects of transplantation of stem cell-derived neural precursors. While a majority of MS patients display a relapsing-remitting disease progression featuring several relapses, few EAE models displays a similar relapsing-remitting course, and many of these models only exhibit one relapse. One EAE model that shows great potential as a model of relapsing-remitting MS is in the Biozzi ABH mouse [111]. In this mouse, immunization with spinal cord homogenate leads to a disease course with at least three to four bouts of disease with significant remission, with mice eventually developing disability associated with axon loss. It therefore provides a promising model system to study myelin repair and neuroprotection through transplantation of stem cells and their derivatives. Transplantation of stem cell-derived precursors into EAE models can provide the means to determine some important questions about the feasibility of stem cell-based therapy in MS: (1) can transplanted precursor cells survive in a chronically inflamed environment, such as that observed both in EAE models and MS; (2) can transplanted precursor cells offer therapeutic value in the absence of differentiation into oligodendrocytes; and (3) are transplanted precursors capable of differentiating into mature, myelinating oligodendrocytes?

8.7.1 Survival and Migration of Stem Cell-Derived Progenitors in EAE In order for transplanted cells to be useful in a remyelination therapy, they must be capable of surviving in the inflammatory environment found in many MS lesions. To address this issue, stem cell-derived precursors have been grafted into EAE animal models to determine their ability to survive and migrate. OPCs transplanted into the spinal cord of rats with EAE could be detected up to 50 days post-transplantation and were found to migrate as far as 5 cm from the site of injection [112]. Similarly, neural precursors introduced directly into the inflammatory environment of chronic EAE mice by intraventricular injection were capable of surviving up to 74 days post-transplantation [113]. NPCs can migrate through the inflamed white matter of the brain and spinal cord in rats with acute EAE [114]. NPC migration into white matter lesions in EAE mice can be monitored by MR imaging [115]. These findings suggest that stem cell-derived precursors can indeed survive and migrate in areas of prolonged inflammation.

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8.7.2 Immunomodulatory Effects of Stem Cell-Derived Progenitors in EAE Pluchino and colleagues [116] demonstrated that both the intraventricular and intravenous injection of adult-derived neural precursors into mice with chronic EAE resulted in functional improvement. A similar attenuation of the severity of disease was observed following the intraventricular injection of fetal-derived NPCs into rats with acute EAE [117]. The reduction in the severity of EAE following injection of neural precursors was subsequently found to be the result of reduced CNS inflammation, which in turn resulted in reduced demyelination and acute axonal damage [113, 118, 119]. Decreased CNS inflammation in EAE animals that received NPC transplants was largely due to reduced infiltration of activated T cells either by direct effects at the site of infiltration in the case of intraventricular injection [113], or peripheral effects such as induction of apoptosis of T cells [118] and inhibition of T cell activation and proliferation in the lymph nodes [113] in the case of intravenous injection. Similar immunomodulatory effects have been observed following both the intravenous and intraventricular injection of mesenchymal stem cells obtained from mouse bone marrow into EAE mice [120, 121]. Recent studies have begun to explore the therapeutic potential of precursor cells derived from human ESC (hESC) in rodent EAE models. Intraventricular transplantation of hESC-derived NPCs into mice with chronic EAE resulted in attenuation in disease severity [122]. Transplanted NPCs were detected up to 50 days posttransplantation, again confirming that exogenous cells can survive in chronically inflamed white matter. The reduction in EAE severity was the result of diminished neuroinflammation, which in turn reduced axonal damage and demyelination in affected mice receiving NPC transplants. As with similar studies using mousederived NPCs, transplanted cells were able to migrate through the white matter of the brain. Previously, the progress of hESC-derived NPC migration through the white matter of EAE-affected mice was tracked by MRI and shown to be dependent on inflammatory signals [123].

8.7.3 Remyelination by Stem Cell-Derived Progenitors in EAE Of particular importance in the field of MS repair is the question of whether transplanted stem cell-derived precursors are capable of remyelinating demyelinated axons in MS lesions. To address this issue, the myelinating capability of transplanted cells in EAE models has been studied. Neural precursor cells from adult mice displayed the ability to remyelinate axons in a chronic EAE model [116], although the myelin formed by these cells was poorly compacted. More recent studies have addressed the ability of human-derived neural precursors to remyelinate axons in EAE models. However, transplanted hESC-derived NPCs largely failed to differentiate into more mature cell types, such as astrocytes or oligodendrocytes, in the brain of EAE-affected mice [122]. Furthermore, no effect on remyelination

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was observed in animals that received hESC-derived NPCs compared to untreated animals. In summary, there is significant evidence from EAE models that transplanted stem cell-derived precursor cells can survive and migrate in the inflammatory environment of the EAE CNS. Furthermore, these cells are capable of reducing the severity of EAE by attenuating the autoimmune response and thereby decreasing the degree of demyelination and axonal degeneration. However, questions remain as to whether these exogenous precursor cells are capable of directly remyelinating demyelinated axons in EAE models.

8.8 Initial Investigations into Exogenous Cell-Based Therapy in Multiple Sclerosis Thus far, the only studies on the effects of exogenous cell transplantation in MS patients have involved the intravenous administration of autologous HSC. Such transplants show positive effects on inflammation and immune cell response [78], and a subset of MS patients that received HSC transplants exhibited slowed disease progression [79]. However, they have failed to demonstrate any effect on the progression of demyelination or neurodegeneration [77, 80]. Furthermore, while some MS patients that received HSC grafts showed improvement, the myeloablative conditioning regime required prior to grafting was associated with a 5-8% mortality rate [79], making this a high risk procedure [124].

8.9 Future Directions 8.9.1 Establishing the Myelinating Capacity of Stem Cells in MS While stem cell-derived precursor cells have consistently shown the ability to remyelinate axons in a number of other models, thus far the same ability has not been definitively shown in EAE models. Part of the issue may lie with the nature of the EAE model itself. In models of genetic myelin disease, the entire white matter of the CNS is affected; as such, transplantation of cells into any site will elicit remyelination. Similarly, the use of chemically-induced demyelination models allows for the demyelination of specific regions; this allows for the targeted introduction of exogenous cells into or in the vicinity of these regions of demyelination. However, lesions in EAE models are typically widespread throughout the CNS, and without a means to properly locate these lesions, the ability to specifically target transplantation of cells into or near an EAE lesion is compromised. A focal EAE model, in which localized MS-like lesions can be generated by immunization of rats and subsequent cytokine injection into the white matter [125], may prove to be a better model system for demonstrating that transplantation of exogenous cells can result in remyelination of axons in the focal lesion. In this model, the proper placement of

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the focal lesion can result in changes in motor function; consequently, transplantation of stem cells either directly into the focal lesion or adjacent to the lesion may result in repair that results in restoration of normal motor function.

8.9.2 Inflammation and Astrocytes Another issue to consider is the dual effects of CNS inflammation on cell transplantation therapy. Some studies have demonstrated beneficial effects of inflammation on migration, differentiation, and myelination, while other studies have shown conflicting results. Both endogenous and exogenous precursor cells migrate to sites of inflammation [19, 20, 112]. Acute inflammation aids in remyelination via macrophage-mediated removal of myelin debris [21, 22]. Similarly, myelination by transplanted precursor cells is augmented by acute inflammation in the myelin mutant taiep rat [126] and in adult rat retina [127]. On the other hand, chronic inflammation negatively affects migration, proliferation, and differentiation of endogenous precursors [31, 128], and it is possible that exogenous precursors would similarly be affected. Therefore, while acute inflammation may be beneficial to remyelination in the short term, exposure to a chronic inflammatory environment may limit precursor cell proliferation, migration, and differentiation. Cell transplantation therapies may well have used in concert with immunosuppressive or immunomodulatory drugs in such a way that the administration of these drugs is timed as to maximize the beneficial aspects of inflammation while minimizing its deleterious aspects. In a similar fashion, astrocytes can be either a benefit or a detriment to remyelination, depending on the situation. Chemokine secretion by astrocytes can induce OPC migration to sites of demyelination, and secretion of interleukins and growth factors by astrocytes can promote OPC survival and maturation [129]. Conversely, astrocytes can be inhibitory to remyelination, both by endogenous and exogenous precursor cells. The glial scar can act as a physical barrier to the remyelination process [129]. The amount of remyelination produced by transplanted OPCs was reduced in demyelinating lesions when astrocytes were introduced into such lesions [130]. Astrocytes produce a high-molecular form of hyaluronan that inhibits OPC maturation [32], and FGF-2 secretion by astrocytes is likewise capable of inhibiting the maturation of OPCs [131, 132].

8.9.3 Method of Cell Delivery to MS Patients Lesions that are surgically accessible in the brain and spinal cord could be targeted by direct injection. This may be clinically useful if lesions are at strategically important sites that can be correlated with clinical disability, such as in the spinal cord. Direct injection would remove the requirement for the migration of cells to the site of interest and allow remyelination to occur, provided axons are preserved and gliosis is not severe. Extensive transplantation experiments in multiple models suggest

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that such focal repair is indeed possible. However, it remains to be shown whether inhibitory signals to OPC differentiation and myelination that may be present at the site of interest, would prevent such focal repair. The long-term goal, however, is to use exogenous cells to remyelinate multiple lesions. Hence their dissemination in perhaps a less invasive manner than direct injection would be preferable. One such approach could involve intrathecal injection of exogenous cells. Neural precursor cells introduced by intrathecal injection at the lumbar cord can migrate to sites of inflammation in the spinal cord of rats with acute EAE [114], as well as to sites of spinal cord injury in the cervical spinal cord [133]. A similar approach may allow precursor cells to be delivered to MS lesions in the spinal cord. Neural stem cells injected into the ventricles of mice with EAE have been shown to migrate into areas of inflammation in the white matter [114, 123]; a similar migration of exogenous cells throughout diseased white matter following intraventricular injection may therefore be possible in MS patients. As an alternative to this approach, cells injected intravenously might also target multiple lesions. The intravenous injection of NPCs and MSCs has been shown to attenuate the severity of disease in EAE models, primarily through immunomodulatory processes [113, 116, 118, 121]. NPCs introduced intravenously can migrate to the inflamed white matter of mice with EAE [115]. However, the ability of exogenous cells to differentiate into myelinating oligodendrocytes following intravenous injection into EAE models has not been conclusively demonstrated. It has been shown, however, that intravenous delivery of MSCs can result in the myelination of axons in a focal demyelinating lesion generated by ethidium bromide [83, 84]; therefore, it is possible that intravenously introduced cells may be capable of differentiating into oligodendrocytes in EAE models and in MS patients.

8.9.4 The Use of Genetically Modified Cells for Transplantation As previously described, the environment into which exogenous cells will be introduced presents potential obstacles for repair-oriented therapies. One possible means to circumvent these obstacles involves the genetic engineering of stem cells prior to transplantation. Using this approach, a gene or genes of interest would be stably integrated into the genome of transplanted cells. Upon expression, the integrated gene product(s) could be secreted by transplanted cells to modulate the local environment and make it more conducive to remyelination. Bone marrow stem cells have been engineered to deliver interferon-β (IFN-β) and brain-derived neurotrophic factor (BDNF) and have demonstrated the ability to reduce EAE severity in mice [134, 135]. Alternatively, the integrated gene product(s) could be retained by transplanted cells to enhance their differentiation into oligodendrocytes following transplantation. For example, overexpression of the transcription factor Olig2 has been shown to augment NSC differentiation in vitro [46]. However, the potential side effects of such genetic modification, such as inadvertent tumor generation due to genomic integration, raise questions about the feasibility of such an approach. In addition,

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the modification of cells in such a fashion will instigate a greater need for proof of their safety by regulatory agencies.

8.9.5 Selection of MS Patients for Transplantation Therapy The selection of patients who may benefit from the exogenous delivery of cells either focally or by global delivery is the subject of careful consideration. A prerequisite for such therapy will be the presence of lesions that have a significant preservation of axons. The anatomical site of the lesion must also be considered, both in regard to its surgical accessibility and its proximity to “sensitive” sites such as the brain stem nuclei. A major factor that will also need to be considered is the stage of disease. In acutely demyelinated lesions, endogenous remyelination will occur, and therapies should not interfere with this process. In chronic disease, there will likely be axonal loss and little or no inflammation, both of which may be required for remyelination to occur. The continued refinement of current imaging techniques is therefore important in identifying lesions which meet the criteria for exogenous cell treatment. The imaging of lesions using diffusion tensor imaging and positron emission tomography may provide critical information regarding the degree of axon survival and the level of inflammation in a lesion, respectively. These imaging techniques, in conjunction with the use of MRI to detect gadolinium-enhancing lesions that are indicative of ongoing disruption of the blood-brain barrier, will aid greatly in locating repairable lesions in MS patients. Acknowledgments The authors would like to thank the National Multiple Sclerosis Society for their support (grant TR-3761), Yoichi Kondo for his helpful advice, and Kristin Boswell for her support.

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Chapter 9

Cancer: A Stem Cell-based Disease? James E. Trosko

Abstract To understand the complex origin of human cancer, one cannot ignore the adage, “Nothing in biology makes sense except in the light of evolution” [1]. In view of the fact that current understanding of carcinogenesis involves genetic, developmental, gender, dietary, nutritional-caloric restriction, environmental, life style, and cultural factors, what seems to be missing is not the amassing of more facts but a unifying conceptual framework to integrate all of these factors. (“. . .those researching the cancer problem will be practicing a dramatically different type of science than we have experienced over the past 25 years. Surely much of this change will be apparent at the technical level. But ultimately, the more fundamental change will be conceptual [2].”) The major concept presented in this review is that carcinogenesis involves the reversible alteration of an embryonic stem cell’s ability to divide asymmetrically will lead to teratomas, whereas the stable irreversible alteration of an adult stem cell’s ability to proliferate asymmetrically will lead carcinomas and sarcomas. An attempt will be made to integrate the (a) multi-stage/multi-mechanism hypothesis; (b) mutation/epigenetic theories, (c) stem cell /de-differentiation or “reprogramming” hypotheses; (d) genetic and environmental interaction hypothesis of carcinogenesis; (e) oncogene/tumor suppressor theories; and (f) the homeostatic cell communication mechanisms with the stem cell hypothesis of carcinogenesis and with the biological and cultural evolutionary theories of carcinogenesis. Keywords Cancer stem cells · Adult stem cells · Re-programming · Barker hypothesis · Gap junctional intercellular communication · Evolution and cancer · Epigenetics · Side-population cells · Immortalizing viruses

J.E. Trosko (B) Department of Pediatrics/Human Development, College of Human Medicine, Michigan State University, East Lansing, Michigan, 48824, MI, USA e-mail: [email protected] T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_9,

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution of Homeostatic Control of the Hierarchical and Cybernetic Nature of Human Health . . . . . . . . . . . . . . . . . . . 9.3 The Concept of Stem Cells in the Evolutionary Transition from Single Cell Organisms and the Metazoan . . . . . . . . . . . . . . . . 9.4 Gap Junctional Intercellular Communication as the Evolutionary “Biological Rosetta Stone” for Understanding the Homeostatic Regulation of Cellular Functions in Metazoans . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Cancer as the Result of a Disease of Homeostatic Control of Cell Communication 9.6 Role of Gap Junctions in the Multi-Stage/Multi-Mechanism Hypothesis of Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . 9.7 What Is Source of the Initiated Cell? . . . . . . . . . . . . . . . . . . . . . 9.8 Adult Stem Cells, “Immortalizing” Viruses and Cancers: . . . . . . . . . . . . 9.9 Tumors and Tumor Cell Lines: A Mixture of Cancer Stem Cells and Cancer Non-stem Cells . . . . . . . . . . . . . . . . . . . . . . 9.10 Characteristics of Normal Adult Stem Cells, Cancer Stem Cells and Cancer Non-stem Cells . . . . . . . . . . . . . . . . . . . . . . 9.11 Cancer Stem Cells, Drug-Resistant Cancer Cells and “Side-Population” Cells . . 9.12 Two Types of Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . 9.13 Stem Cells, Hypoxia, Drug Resistance and Oct-4A . . . . . . . . . . . . . . 9.14 Dietary Modulation of Cancer: The Barker Hypothesis, Stem Cell Frequency and Risk to Cancer . . . . . . . . . . . . . . . . . . . 9.15 Does the Adult Stem Cell or the Differentiated Somatic Cell Act as the Target Cell for the Initiation of Cancer . . . . . . . . . . . . . 9.16 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9.1 Introduction While cancers have always been a part of the human condition (even the prehuman condition), patterns and frequencies of different cancers continue to change. Historically, pre-scientific explanations to explain cancers helped to explain ways to treat or “cure” the disease. It has always been a tragically physically-painful disease, but, in addition, a terribly psychologically-fearfully disease. In spite of our current scientific understanding of the factors contributing to cancers today, the fear of this disease resides high on the list of those diseases in the minds of the non-scientific population that can end our life. Accumulation of more recent views of how cancers can be influenced, from environmental factors (chimney sweep soot exposure); viral infections, inherited genes (i.e., retinoblastoma), caloric restriction; diet and nutrition (high fat; anti-oxidants); environment (asbestos); life style (smoking; exercise);

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cultural (dietary changes; postponement of pregnancy), have not altered the state of fear that comes from one’s knowledge of our risk to cancer and the consequences, once diagnosed. Today, due to many advances from disciplines of epidemiology to sophisticated technology in molecular biology, the stage seems to be set for integrating all of these discipline-dependent observations and explanations into a larger integrative concept. In addition, specific conceptual advances (e.g., oncogenes; DNA damage; isolation of cancer-predisposing genes; identification of the mechanisms of action of environmental and dietary agents contributing to the induction or prevention of the carcinogenic process; and the multi-stage, multi-mechanism nature of carcinogenesis), force an integration to be made. However, to focus only on a reductionalistic view, that is, by delineating the sequence of altered nucleotides in an oncogene or tumor suppressor gene between a normal and cancer cell, or by detecting only the genes expressed in normal versus cancer tissues, will not allow one to understand the origin of cancer. That this might seem to be an important statement reflects an assumption that, only by understanding the complex carcinogenic process, can one have a chance to reduce the risk of cancer by efficacious prevention and therapeutic strategies. Conceptually, as Potter has stated: “The cancer problem is not merely a cell problem, it is a problem of cell interaction, not only within tissues, but also with distal cells in other tissues. But in stressing the whole organism, we must also remember that the integration of normal cells with the welfare of the whole organism is brought about by molecular messages acting on molecular receptors.” [3], one can see that “cancer is not simply a disease of a single cell”. A cell is classified as a cancer cell simply because it has no growth control; it is “Immortal”; it can not terminally differentiate and accrues the “hallmarks of cancer” [2]. Single cell organisms, which are only growth-controlled by nutrient restriction, temperature, and high doses of radiation, are not thought of as having a cancerous condition. Therefore, once the multi-cellular metazoan appeared via evolutionally changes, new homeostatic mechanisms had to evolve to (a) control growth of cells, (b) generate specialized or differentiated cell types, (c) allow for immortality of a few specialized cells (e.g., germ and somatic stem cells) while inducing terminally differentiated or “mortalized” cells (e.g., red blood cells; neurons, keratinocytes) [4]. Two philosophical concepts emerged to help explain the complex regulation that was needed to explain this new organization of cells, namely, the hierarchical view of a human being (“the whole is greater than the sum of its parts” [5]) and the cybernetic view (positive and negative information feedback between levels of the units of the hierarchy of organization [6, 7]).

9.2 Evolution of Homeostatic Control of the Hierarchical and Cybernetic Nature of Human Health The fundamental assumption to be made in this review of the origin of cancer is that, to ignore both biological and cultural evolutionary theories, is to miss a

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possible over-arching concept that might integrate all that we do know about the carcinogenic process. The conversion of a normal cell in a metazoan to a cancer cell has to be viewed from the vantage point of observing the differences of single cell organisms, living as a loose collection of individual cells, to those cells living in a tightly controlled social colony. The metazoan is a tightly organized collection of highly specialized cells, communicating via a number of delicately regulated communication mechanisms, designed to regulate, homeostatically, cellular functions not found in the repertoire of a single cell organism. While the process of carcinogenesis has been viewed within the biological process of evolution before [8, 9] rarely has carcinogenesis been viewed within a larger concept of both biological and cultural evolution. This means that the mechanisms of biological evolution, namely the production of mutations and disruptive alterations of epigenetic expression of normal genes, as well as natural selection in an inevitable changing environment, yet, one cannot ignore how cultural evolution, which emerged from the biological evolution of the human being, influenced behavioral changes of the human being. These behavioral changes, including dietary changes, influenced the carcinogenic process. Together, that interaction of biological and cultural evolutional factors help to explain current observations on the process of carcinogenesis. In addition, the concept of evolution, especially how it provides insights to the transition from the single cell organism, where biological phenomenon of cancer does not exist, to a multi-cell organism, where new biological cell types, communication mechanisms, and cell functions emerged [8–10]. The collision of the slow process of biological evolution with the very rapid process of cultural evolution creates the disruption of the biologically-evolved delicate homeostatic regulatory processes to maintain health. With the appearance of living single cell organisms utilizing oxidative metabolism to generate energy for life [7, 11], a potentially detrimental by-product, namely oxygen free radicals or reactive oxygen species (ROS), had to be dealt with, in order to protect the genome from DNA damage and mutagenesis. Both genetically-determined enzymes to deal with protecting the DNA from these ROS and to repair the DNA damage once it did occur had to be co-selected. However, one must note that the primary cellular function of a single cell organism was to reproduce (to divide, symmetrically), in order to maintain the species that had originated to be adaptive to the extant environment. The fact is that the environment never stays static. Therefore, if the genetic information of any single cell organism is totally resistant to change (mutation) by both protecting DNA from damage or always repairing the damaged DNA perfectly (error-free DNA repair) or always replicated its DNA perfectly (prevented error-prone DNA replication), then this species would not survive the inevitable environmental change because the genome would not be adaptive. In addition, if the organism always replicated its DNA in an error-free fashion, it also would not survive inevitable environmental change. On the other hand, if a single cell organism is extremely inefficient in protecting the DNA from damage or inefficient in repairing the DNA damage or in replicating DNA, then this species would also be prone to rapid extinction.

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If a species strikes a balance between perfect DNA maintenance (no mutations) and too many mutations (no stability of the genome), such that, in a population of these single cell organisms, at least a few individual organisms will have specific new mutations that might enable the individual to survive and reproduce, when the new environment challenges the adaptability of the current species genome. In effect, a single cell organ divides by symmetrical cell division. They communicate with each other via secreted factors to control their primary functions and are environmentally influenced by temperature, nutrient availability and by environmental agents, such as atmospheric composition, radiation, and chemical toxicants [12]. When the first multi-cellular metazoan appeared via biological evolution, several new phenotypes, coded by new genetic information and new communication processes [13], new biological processes (asymmetrical cell division) and new cell types (embryonic, germ line and adult stem cells) appeared. While, in effect, cells of a multi-cell organism are also in a social community, it is a pluralistic community, rather than a monolithic community. New phenotypes that had to emerge during this first major biological evolutional transition to a metazoan. Growth control, the formation of specialized cells, namely, the embryonic, germ line and adult stem cells (cells that had the ability to divide both symmetrically or asymmetrically), the process of differentiation via the new cell process of asymmetrical cell division, programmed cell death or apoptosis; and “mortalization” of the differentiated cells were the new genetically-determined processes (Fig. 9.1). Clearly, many new genes had to appear during the transition from the single cell species to the multi-cellular metazoan species. The formation of these new genes occurred by various mechanisms of mutagenesis, the grist of biological evolution.

Fig. 9.1 This diagram illustrates that, from a stem cell, which can divide both symmetrically to produce two stem cells, the process of asymmetrical division can occur to lead to a daughter cell that has a finite life span (a progenitor or transit amplifying cell) and can terminally differentiate, apoptose or senesce. Each cell state is a reflection of the differential expression of the total genome

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However, with the appearance of differentiated cells, the metazoan had to restrict the process of “immortalization” of the single cell organism (its most adaptive means, namely symmetrical division, to survive as a species in an ever-changing environment). Clearly, the appearance of differentiated cells in this new society of cells in a metazoan conferred some new strategies for survival, namely, neuronal cells for sensing change; muscle cells for movement; liver cells for detoxification; etc. In addition, restriction of cell growth had to be a critical part of this evolutionary transition. It had to exist for both the undifferentiated embryonic, germ and adult or somatic stem cells. Usually the development of the metazoan goes through a series of transitional stages, needed for transient adaptive purposes (such as in a butterfly’s larval stage for food consumption, which is needed for pupae transition to the single adult’s adaptive phenotypic features of flight and sexual reproduction). For this to occur, the process of apoptosis or programmed cell death had to be genetically programmed. It is important to remember that, intrinsic to the appearance of differentiation, growth control and apoptosis, differential gene expression of the total genome required complex new mechanisms to regulate directing a cell’s choice of function. In addition to the continuing to maintain the process of communication by secreted factors that occurs to control single cell organisms in their societies, the metazoans had to accrue new processes to communicate, not only between itself and the external environment, but with its identical sibs or like type cells. It did so with new extra-cellular structures (extra-cellular matrices [14] and nichemicroenvironments [15, 16]) and with its differentiated sibs (differentiated cells of its lineage or of other differentiated cells of other lineages). A new highly integrated hierarchy of cybernetically-controlled communication mechanisms had to emerge [17].

9.3 The Concept of Stem Cells in the Evolutionary Transition from Single Cell Organisms and the Metazoan While the concept of stem cells existed in the disciplines of the embryology of both plants and animals for many decades, only recently, since the cloning of Dolly [18], and the isolation of human embryonic stem cells [19–21], did the concept of stem cells resurrect the old hypothesis of the stem cell theory of cancer. The idea of “cancer as a disease of differentiation” [22, 23]; of a “stem cell disease” [24]; or of “oncogeny as partially blocked ontogeny” [25] was conceived before direct evidence could be generated showing that a stem cell could actually be a target cell for initiating the carcinogenic process [26]. As with most hypotheses, an alternative hypothesis to the stem cell hypothesis was offered to explain the origin of a cancer cell, namely, the theory of “de-differentiation” [27]. In this hypothesis, any or, at least, most differentiated cells could de-differentiate to an embryonic-like cell and then progress to become carcinogenic.

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This, then, gets to the two major concepts that must be defined, namely, (a) the nature of stem cells and (b) the prevailing concept of the biological processes involved during the transition of the normal “target cell” to the malignant cancer cell. A generic stem cell is defined as a cell that can divide symmetrically to produce two identical stem cells or that can divide asymmetrically to produce one daughter to maintain “stemness” and the other which can ultimately differentiate (Fig. 9.2). One important feature of a stem cell is that it is “immortal” until it is induced to differentiate or to become “mortal”. Therefore, it is absolutely critical to view a stem cell as being intrinsically immortal. In the evolutionary transition to the metazoan, the formation of the stem cell had to occur, in order (a) to provide germ cells to pass on the species’ genome for species survival; (b) to provide transit or progenitor cells for growing tissues; (c) to replace dying or injured cells; and (d) for generating lineage-specific differentiated cells. In a metazoan, there are different classes of stem cells, namely, the “toti-potent” stem cell or the fertilized diploid egg. These totipotent stem cells can give rise to all the cell types for the ultimate generation of a complete organism. In the early developing embryo, embryonic stem cells or pluripotent stem cells, derived from the toti-potent stem cell, are those that have the ability to give rise to all the specific organs, tissues and cell types found in adult organism (in the case of the human being, the estimate is about two hundred different cell types). As the embryo further develops into the fetal stage, more “restriction” of the pluri-potent stem cell to a multi-potent stem cell occurs, as the specific genes are regulated to give rise to different differentiated cells within a specific organ or tissue. Upon further restriction of the multi-potent stem cell, bi-polar cells, such as the liver oval cell [28, 29], and uni-polar stem cells, which give rise to a single lineage offspring, occurs. The transit-amplifying cell or progenitor cell is now defined as a cell with a limited life span potential [30]. In other words, these cells can symmetrically divide to build up the cells of a tissue mass before it terminally differentiates, senesces or undergoes apoptosis. In the context of a metazoan, such as a mouse or a human being, the embryonic stem cell is responsible for generating the germ cell lineage for maintaining the survival of the species’ survival. These embryonic stem cells also give rise to the somatic or adult stem cells that are needed for growth, differentiation of all the organs and for tissue replacement or repair. However, while the germ line stem cells can, conceptually, be viewed as being “immortal” in the broadest sense, the adult or somatic stem cells are “contextually immortal” only as long the individual organism, a “mortal” being, remains alive. The bulk of the cells of a metazoan are transit – amplifying, progenitor and terminally differentiated or “mortal” cells in interacting tissues and organs that eventually “ages” and dies of total organ failure (aging as a failure of function). Alternatively, cells can affect the quality of function of one of a number of organs and lead to chronic diseases when they become dysfunctional (cancer, diabetes, cardiovascular, neurological dysfunction). It is known that, when an individual dies, cells, both adult stem cells and transit or progenitor cells, can still stay alive ex vivo. This illustrates the philosophical concept of the hierarchy principle, in that conscious life of the human being is the result of the

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Fig. 9.2 This diagram illustrated a hypothesis that regulates whether a stem cell will proliferate, symmetrically or asymmetrically. In the case of symmetrical cell division, the stem cell in its niche, affected by the micro-environmental factors, particularly, low oxygen tension, will divide symmetrically to produce to stem cells because the net effect of all the signals received from the extra-cellular matrix, nutrients, and mitogens causes a division plane that is vertical to the plane of the substrate. On the other hand, if the stem cell receives signals from the micro-environment, such that the net effect causes a division plane that is parallel to the substrate, the two daughters are not receiving the same identical signals. Since the stem cell has no functional gap junctional communication, the specific signal from the niche matrix is only received by the one daughter having a receptor linkage. The other daughter is now “free” to differentiate. Oct-4+ is expressed only in the stem cell, while the stem cell is not expressing its connexin gene (Cx-). Upon differentiation, the progenitor cell represses its Oct-4 (Oct4-) and transcriptionally and functionally expresses Cx+

cybernetic communication between the different levels or organization of molecules, cells, tissues, organs and organ systems, with not only genetic, gender and stagedependent endogenous signals, but also with exogenously-dependent signals from diet, pollutants, drugs, and life styles.

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9.4 Gap Junctional Intercellular Communication as the Evolutionary “Biological Rosetta Stone” for Understanding the Homeostatic Regulation of Cellular Functions in Metazoans Now to link the concepts of stem cells with the homeostatic control of a cell’s decision to respond to either or both endogenous and exogenous communicating signals, a major hypothesis is suggested, namely, gap junctional intercellular communication (GJIC) is responsible for maintaining homeostatic control of cellular behavior. This form of cell communication appeared when growth control, differentiation and apoptosis had to emerge during the biological evolution of the early metazoan [13]. The leap of faith, which suggests that the 20 highly evolutionary-conserved genes found in metazoans, such as the rat and human being [31, 32], are responsible, in large part, for maintaining homeostatic control of growth control, differentiation, and apoptosis comes from comparing the role of gap junctions in these species (Fig. 9.3). It might be legitimately argued that the gap junction, a membrane-associated protein channel, composed of two co-joined hemi-channels, the connexons, which are composed of six connexin gene-coded proteins, “connexins”, should not be singled out as the most important metazoan organelle responsible for the evolution of a differentiated metazoan. Why would not a nucleus, mitochondrion, endoplastic reticulum, or lysosome be considered as more important? There might be several major sources of the answer to this question. The first is all the indirect evidence that gap junctions are correlated with the control of growth of progenitor cells [33, 34] and the phenomenon of “contact inhibition” [35]. In addition, stem cells seem to be devoid of expressed connexin genes or functional gap junctions [36–39]. While stem cells are usually growth controlled, it is implied that either or both communication signals from extracellular molecules in the nichemicro-environments and secreted extracellular molecules keep these stem cells from excessive cell proliferation. This might be an evolutionary strategy to help to minimize the risk of errors of replication in the somatic stem cells. The transit-amplifying cells, which are committed to senesce or terminally differentiate because of its limited life span might, have been the beneficial by-product of differentiation to limit cancer [40, 41]. Gap junctions have also been correlated with development and differentiation of cells [42]. Even in cells of the metazoan that are not coupled but are able to freely migrate (no need for gap junctions at this stage of these differentiated cell type), there is evidence of transient coupling with nurse cells during their early stages of differentiation [43, 44]. There is evidence, such as that from the study of gap junction function in the skin that more extensive gap junction-mediated communication occurs between cells of the basal layer. This suggests the ability of the keratinocytes to intercommunicate is dependent on their state of differentiation [45]. While the process of apoptosis can be triggered by secreted factors, in solid tissues, apoptosis seems to be dependent on the transfer of some death signal via gap

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Fig. 9.3 Diagram of the details of gap junction structures. The connexin proteins are coded by a highly conserved series of connexin genes. A hexamer of connexins form a connexon (either homomeric or heteromeric). Selective uniting of connexins can form homotypic or heterotypic gap junction channels, allowing a direct transfer of ions and small molecules from the cytoplasm of one cell to that of its neighbor. From Mehta [141]; Permission granted by Springer Publisher, New York

junctions [46–49]. More will be said later when the process of apoptosis is examined during the carcinogenic process. Also, while apoptosis can occur in stem cells [50], which do not seem to have functional gap junctions, this important biological functional of metazoan cells in solid tissues seems to be dependent, in part, on gap junctions. The concept of moralization (terminal differentiation or apoptosis) or senescence also seems to be dependent on gap junctions, since stem cells that are immortal do not have functional gap junctions, while those that are mortal can have gap junctions. The second reason that gap junctions might be considered the “Biological Rosetta Stone” comes from studying the role of gap junctions during the carcinogenic process [51].

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9.5 Cancer as the Result of a Disease of Homeostatic Control of Cell Communication Within the over-arching concept of biological evolution, we can now look at the nature of cancer cells and compare them to normal single cell organisms. One of the interesting observations that come out this analysis is that the normal metazoan stem cell is (a) immortal, (b) growth-controlled, (c) can differentiate, (d) can be induced to mortalize or senesce, and (e) can apoptose. On the other hand, after the complete carcinogenic process, a malignant cancer cell has no growth control, can not terminally differentiate, apoptose, or senesce. It maintains or restores its ability to be immortal. Most interestingly, cancer cells lack functional gap junctional intercellular communication [52], either because of (a) the non-expression of the connexin genes, as in HeLa and MCF-7 carcinomas cells [53, 54], or (b) the expressed connexin genes and proteins are rendered non-functional by activated oncogenes, such as ras, src, neu [51] (Fig. 9.4). In effect, the cancer cell, by losing the function of gap junctional intercellular communication, “reverts” back, evolutionarily, to a single cell-like organism, having no growth control, no ability to terminally differentiate or to apoptosis, and it is now immortal. The current paradigm in carcinogenesis has been based on the idea that a normal, mortal cell must be first “immortalized” and then neoplastically transformed to acquire the “hallmarks of cancer” [55].

9.6 Role of Gap Junctions in the Multi-Stage/Multi-Mechanism Hypothesis of Carcinogenesis While the observation that cancer cells do not “contact inhibit” their growth is very old [35], the link of cell growth inhibition of normal cells to the main phenotype of a cancer cell to the lack of functional gap junctional intercellular communication has not been widely accepted. However, this has not been due to the lack of a wide range supporting evidence. Starting with one of the oldest observations that cancer cells lacked functional gap junctions [56], normal progenitor cells, on the other hand, contact inhibited growth and had functional gap junctional intercellular communication. In order to understand the following supporting evidence, one of the strongest concepts of the complex carcinogenic process is the initiation/promotion/ progression hypothesis [57, 58]. This hypothesis must be viewed as a framework to integrate the subsequent research observations. Clearly, the current evidence indicates that all cancers start from a single cell, which, when exposed to a physical, biological or chemical agent, is irreversibly altered, so as not to terminally differentiate or to undergo apoptosis, easily (Fig. 9.5). This “initiated” cell is essentially “immortal” {more to be said about this state later}. However, it is surrounded by, and communicating with, normal cells that suppress the mitogenesis of that initiated cell by

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Fig. 9.4 Diagram illustrating the potential origin of two types of non-gap junctional communicating cancer cells, either due to the original target cell being an adult stem cell that did not transcriptionally express its connexin genes (HeLa and MCF-7-type tumors). Many cancer cells have expressed connexins, but they have non-functional gap junctions. This could be due either to mutations or posttranslationally modified connexin proteins, caused by expressed oncogenes. These tumor cells might have been derived from adult stem cells that expressed connexins before the loss of Oct-4A expression, due to induced partial differentiation by micro-environment changes

either (a) negative growth suppressing signals via gap junctions from surrounding and direct contiguous cells or (b) negative secreted growth factors that trigger receptor anti-mitogenesis signaling [59] (Fig. 9.4). This implies that there might be two kinds of “initiated” cells, one with no expressed connexin genes and no functional gap junctions, whose growth must be suppressed by secreted factors. The other type

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Fig. 9.5 In this diagram, a normal adult stem cell is shown dividing asymmetrically to form one daughter that is committed to ultimately terminally differentiate. The other daughter is designated to be identical to its mother adult stem cell (Oct-4+). If that adult stem cell is exposed to some condition that prevents asymmetrical cell division, but does not suppress the Oct-4 expression, it is operationally an initiated cell. That is, if mitotically stimulated to divide, it divides symmetrically to form two initiated, non-terminally differentiated cell. Initiation is, then, defined as the process that prevents an “immortal” normal adult stem cell to terminally differentiate or become “mortal”. These adult initiated stem cells are still Oct-4 positive or benign cancer stem cells. As these initiated Oct-4+ cells are stimulated to proliferate and resist apoptosis, the growing benign tumor microenvironment changes, some of these initiated Oct-4 + cells can partially differentiate into “cancer non-stem cells”[Oct-4 negative]. Evening, additional stable mutational or epigenetic events occur, providing the benign Oct-4+ cancer stem cells to become invasive, metastatic “cancer stem cells”

of initiated cell has functional gap junctions, through which signals are directly transferred from neighboring normal cells. The promotion process of carcinogenesis involves the clonal expansion of the initiated cell by (a) the mitogenic stimulation and (b) the inhibition of the apoptosis of these initiated cells [60]. Multiple mechanisms have been associated with the chronic clonal expansion of these initiated cells, including wounding of initiated tissue, death of cells by all kinds of cytotoxicants, normal growth factors on initiated tissue, chronic inflammation by inert particles, infectious bacteria, fungi or viruses, and exogenous chemicals [61]. What do all of these various modes of promotion have in common, including the wide range of chemical promoters, such as phorbol esters, polybrominated biphenyls, phenobarbital, saccharin, TCDD, etc.? [62] The answer comes from direct observations of how a mitogenic-suppressed contact-inhibited cell starts to proliferate. What was observed was that the inhibition of contact-inhibited cells was correlated with the transient down-regulation of

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Fig. 9.6 This diagram depicts the homeostatic interaction of extra- and intercellular signaling between the three types of cells within a tissue, triggered by either or both endogenous and endogenous factors, which can alter, differentially, intracellular signaling and gene expression in the stem, progenitor and terminally differentiated cells

gap junctions [63, 64]. Subsequently, wounding, inflammatory agents, inert objects, and growth factors also were related to the down regulation of gap junctions. The down regulation of gap junctions is usually done indirectly via the triggering of various intracellular signaling [65] (Fig. 9.6). This signaling has two major functions. The first effect, which takes within minutes, is the down regulation of gap junction function at the posttranslational level, either by inhibiting the gating of the gap junction channel or by physical disruption of the gap junction’s assembly. This uncoupling of the cell with its neighbors now prevents ions and small regulator molecules from either escaping the cell or from entering the cell (“sink” or “source” [66]). As a result this change of the ground state of this “Go cell” is changed, with the extant posttranslational proteins/enzymes subject to change (phosphorylated or de-phosphorylated, etc.). This can now trigger transcription factor activation and altered gene expression. Many, if not most, of these promoting agents and conditions are not mutagenic to the nuclear DNA. They are “epigenetic toxicants” [67]. This observation is extremely relevant to the current use of DNA micro-array technology to study the effect of agents on tissues. Measuring altered expression

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Fig. 9.7 Restoration of GJIC by crude and EtOH extract from psyllium seed husk in WB-Haras cells treated for 48 h, as measured by Lucifer yellow dye transfer. The cells were treated for 48 h with the ethanol extract from the seed husk of psyllium with: vehicle (a); 1.5 mg/ml crude powder (b); 50 μg/ml EtOH extract (c). GJIC was measured using the scrape loading dye transfer technique. Bar inset = 50 μm. From Nakamura [188]. Permission granted by Elsevier Publisher, Amsterdam

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of genes in tissues takes place well after the agent has already altered the signaling process in three types of cells (the adult stem cells, the contact-inhibited progenitor cells and the terminally-differentiated cells). In the progenitor cells, gap junctions were affected within minutes after chemical exposure, while altered gene expression occurred later. Those initiated cells that might not have expressed connexins and functional gap junctions [see below], promoters would have to (a) suppress the production of, or inhibit, the secreted negative growth regulators, such as TGF-beta, or (b) down regulate the receptors or the signaling from those receptors of the negative growth regulators. Finally, the progression step of carcinogenesis is that point where, after the promotion of the initiated cell to a critical mass, one cell has now accrued all the so-called “hallmarks” of cancer, such that this cell is able to break away from the mass of initiated cells, invade the surrounding tissue and metastasize to distal organ. From studies of the “seed-soil” idea of the metastatic process, it appears that these break-away, invasive cells can find their way to multiple organ sites, but only a few organ sites allow their continued growth, whereas other sites either induce growth arrest, induce apoptosis, senescence or differentiation. The role of gap junctions in the carcinogenesis process has also been demonstrated by (a) the transfection of connexin genes into cancer cells; (b) the transfection of anti-sense connexin genes into normal cells which changed the phenotype of normal contact-inhibited cells to cancer-like cells; (c) knock out connexin 32 mice having enhanced risk to spontaneous and chemically-induced cancers; and (d) chemopreventive and chemotherapeutic agents enhancing GJIC [51] (Fig. 9.7).

9.7 What Is Source of the Initiated Cell? The fundamental question in the area of cancer research today is, “Is the target cell that is ‘initiated’ the adult stem cell?” or “Is the target cell any (or most) adult somatic differentiated cell?” Let us return to the current paradigm that suggests that a normal “mortal” cell must first be “immortalized” to allow this cell to proliferate long enough to accrue enough genetic/epigenetic changes to fulfill the requirements needed for have all 6 phenotypes or “hallmarks” of a cancer cell [2]. This paradigm would then suggest the first step of carcinogenesis of a normal, mortal and specific differentiated cell of the target organ (liver, brain, lung or breast cancer) “re-program” its genes, such that it regain the gene expression of a cell that, at least, restores the ability to proliferate in an unlimited fashion. The restored gene expression would probably include those genes associated maintaining “stemness” in adult stem cells. The recent demonstration that adult somatic mouse, non-human primate, and human skin fibroblast cells could be isolated after viral-transfected cells carrying a

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few embryonic-associated “stemness” genes, including Oct-4, Sox-2, that were “reprogrammed” to have embryonic stem cell-like properties support this interpretation [68–76]. However, there is an alternative hypothesis to the interpretation of the iPS cells (“induced pluripotent stem cells”) [77]. In brief, since adult stem cells do exist and those that have been examined, express Oct-4 gene but do not have functional gap junctional intercellular communication [78]. There is a possibility that the iPS cells isolated in the reported studies were actually adult stem cells of the skin, since adult skin stem cells have been reported [79–83]. The major argument used to support the idea that these iPS cells were “reprogrammed” adult differentiated skin fibroblast is because in one the reports the isolated iPS had multiple copies of the exogenously-introduced Oct-4 gene. In short time, these exogenous Oct-4 genes were transcriptional suppressed and the endogenous Oct-4 was expressed [69]. The interpretation is that multiple copies of the exogenous Oct-4 were needed to re-program the genes of the differentiated fibroblast, including repressing the genes for the fibroblast phenotype and transcriptionally turning on the endogenous Oct-4 gene. The alternative explanation favored here is that the lentivirus carried copies of Oct-4 integrated into all primary skin fibroblast cells, including the few adult fibroblast skin stem cells. The only cells that survive their selection strategy would be the few stem cells that had already expressed its endogenous Oct-4. At this time these cells would have both the endogenous and exogenous Oct-4. However, as has been shown before in cells infected with various viruses [84], the cell suppressed the viral genes. If that occurred in this case, the iPS-cells were actually not “re-programmed” adult differentiated skin fibroblasts but adult skin fibroblast stem cells. To test the hypothesis that Oct-4 could re-program adult skin differentiated cells (or any other kind of differentiated somatic cell, highly differentiated skin keratinocytes should be used. Alternatively, the frequency of recovery of iPS cells should be measured if Oct-4 is transfected or infected into cultures of pure adult skin fibroblast stem cells. One would predict that in this latter case, the frequency would be much higher than when tested on a primary culture of skin fibroblast cells, in which the number of skin fibroblast stem cells would be extremely much lower. Additionally, other reports have questioned the interpretation of these iPS cells [85, 86]. The most recent report of mouse liver cells having been “reprogrammed” to iPS cells, the authors do admit that: “The mechanisms of iPS cell induction, however is unknown. Low efficacy of iPS induction suggests that their origins may be of undifferentiated stem cells co-existing in fibroblast culture” [75, 76]. The concept of the stem cell theory, while conceived of many decades ago, and with, possibly, the exception of the idea of cancer having been derived in the case of leukemia [87], was based on quite indirect evidence. The background to our lab’s approach to test the stem cell theory of cancer had quite a long, but rational, series of observations and ideas spliced together. Studying the role of gap junctions in the tumor promotion phase [88] provided some clues to the carcinogenic process. In addition, the field of gap junction biology yielded information about its role in growth control and in apoptosis [46] and

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in differentiation. All of these cell functions are dysfunctional in a cancer cell. Mutated connexins, which are responsible for multiple inherited human diseases [89, 90], also, indirectly, provided clues to the role of gap junctions in cancer. A major insight came from the unexplained phenomenon of the relative ease by which the neoplastic transformation of rodent cells occurs in vitro, compared to the almost impossible task of neoplastically transforming primary human fibroblasts and human epithelial cells [91–93]. In addition, even in the case of rodent cell in vitro transformation, the ability to obtain consistent results was not possible with the same cells and same carcinogen. However, when the T’sao group set out to resolve this problem, using baby Syrian hamster embryo cells, a remarkable observation was made [94]. The observation was that, from experiment to experiment, the only time one obtained neoplastic transformants after exposure to the chemical carcinogen was when the population of primary embryonic-derived cells had a special class of cells, namely cells that were “contact-insensitive”. Since these cells were not exposed prior to the exposure to the experimental carcinogen, these “contact-insensitive” cells had to be assumed to be normal. Since previous studies by Loewenstein and Kanno showed that cancer cells were not “contact-inhibited” [56] or they were “contact-insensitive” [94], it seemed these normal “contact-insensitive” Syrian Hamster embryo cells behaved very much like neoplastic cells, in the that they crawled over or piled up on themselves. Therefore, our lab group assumed these “contact insensitive” primary Syrian hamster embryo cells must not have functional gap junctional intercellular communication. We, therefore, designed a “kiss of death” selection assay to isolate from normal human kidney tissue any potential cells that might not have functional gap junctional intercellular communication. To do this, we used lethally-irradiated human fibroblasts to form a contiguous cell mat, onto which we placed disassociated cells from human kidney tissue. We assumed that the tissue would contain a few adult stem cells, the many transit-amplifying or progenitor cells and the terminallydifferentiated cells. We also assumed that the few adult kidney stem cells would have no expressed connexins or functional gap junctions, because we knew cells with gap junctions contact inhibited or differentiated. We also knew the lethallyirradiated human fibroblast mat were gap junctionally-coupled. Therefore, any adult kidney stem cell, when landing on the lethally-irradiated human fibroblasts, would not couple with these cells. On the other hand, the progenitor kidney cells had gap junctions and they would couple with the lethality irradiated human fibroblasts and would be “contact-inhibited” and not grow or possibly die by apoptosis. Clearly, any terminally differentiated cell landing on the mat would not grow. Our results showed that the only cells that grew on the mat were a small number from the millions we placed from the tissue. These cells had no expressed connexins nor did they have functional gap junctions. As a control, we tested 10 known human carcinomas cells, which had no functional gap junctions, and all of these cells grew on the irradiated cell mat. In brief, cells with functional gap junctions died via the “kiss of death”, whereas, those with no functional gap junctions, grew

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on the mat [95]. We, subsequently, showed these cells were adult stem cells and expressed Oct-4 gene [77]. The easiest explanation of these results is that adult stem cells, expressing Oct-4 and having no functional gap junctions, are the target cells for initiating the carcinogenic process. Our lab has subsequently isolated adult human stem cells from differentiated tissues from the kidney, breast, pancreas, mesenchyme, liver and intestine. They all expressed Oct-4A in the nuclei.

9.8 Adult Stem Cells, “Immortalizing” Viruses and Cancers: One of the oldest hypothesis of a cancer-causing agent has been the viral hypothesis of cancer. The hypothesis has gone through periods of support and rejection [96]. However, together with the idea that the first step of carcinogenesis must be to “Immortalize” a normal “mortal” cell and the observation that stem cells, which are naturally “immortal” until they are induced to terminally differentiate or “mortalized”, a real contradiction of a paradigm or hypothesis and experimental findings has appeared. It now appears that the adult stem cell is immortal and is the target cell to start the carcinogenic process. Therefore a new paradigm has emerged, in that, the first step or initiation of carcinogenesis is the blockage or “immortalization” of a normal, immortal adult stem cell, rather than the induction of immortalization of a normal mortal cell. In effect, this view rejects the idea that “re-programming” of “stemness”- genes occurs during the first step of carcinogenesis. In the last few decades, various primary cell strains were treated with viruses, such as SV40 and human papilloma viruses. Out of these studies, rare “immortal” cells were isolated. The general observations made in these studies were that, after the primary cells were exposed to the viruses, most of the cells senesced or went through “crises”. A few cell survived this treatment and were shown to be “immortalized”. Clearly, the old paradigm suggested that the viruses, after genomic integration, “re-programmed” the normal, mortal cells to become “immortal”. However, the new interpretation is that in the population of normal, primary cells, there existed a few adult immortal stem cells, which when exposed to these viruses, lost ability to differentiate or to divide asymmetrically by the viral genes (i.e., large T –antigen; E-6, E-7 antigens). The progenitor and terminally differentiated cells did not become “immortal” because they were already committed to senesce or die by apoptosis. In the epidemiological cases, where exposures to several viruses has been linked to cancer, the virus probably acted as an “initiator” by infecting the few adult stem cells of the targeted organs. These viral-inhibited adult stem cells are not tumorigenic. However, they can be promoted by a number of factors, whereupon, additional genetic/epigenetic changes in these cells could neoplastically transform them. In a specific experimental example [77], normal human adult breast stem cells, which expressed Oct-4A and had no expressed connexins, were exposed to SV40 large T gene. The isolated “immortalized” clones were not tumorigenic, but still

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expressed Oct-4A and did not express functional gap junctions. Only after exposure to X-ray radiation did clones of weakly tumorigenic clones appear. These cells still expressed OCT-4 but did not express connexins or have functional gap junctions. When these cells were transfected with the activated neu oncogene, highly tumorigenic clones, which still expressed Oct-4A and had no functional gap junctions, were obtained. It should be pointed out that, when the experiment was done on cultures of pure differentiated breast epithelial cells, having no expressed Oct-4A but having functional gap junctions, and which were derived from the breast stem cells, no “immortalized cells” and no tumorigenic cells were obtained. In this latter case, clearly, SV40 large T gene was unable to “re-program” their differentiated gene pattern of the differentiated breast epithelial cells. To illustrate the point that the phrase, “immortalizing” viruses, is a misnomer, is the observation that an SV40 “immortalizing” human brain cell line [97] differentiated into various neuronal cell types when exposed to agents that increase cAMP levels in the cells [98]. This indicated that that the SV40-“immortalized” brain cell was a stem cell that was blocked in its ability to differentiate in the presence of the expressed large T-antigen. However, whatever the cellular increase of cAMP did, the SV-40 large T was no longer able to block differentiation. In brief, there is now evidence supporting the role of various viruses in contributing to the multi-stage process of carcinogenesis. Its role is most likely the blockage of “mortalization” of the few adult immortal stem cells. In effect, these tumor-contributing viruses probably affect the “initiation” phase of carcinogenesis. In the case where a virus might induce massive cell death in an organ, the consequence of killing cells might lead to the induction of compensatory hyperplasia of any surviving adult stem cell that might have been initiated by other factors. The role of these “immortalizing” viruses in cancer is unlikely via the “re-programming” of differentiated somatic cells. It would be an indirect tumor promoter.

9.9 Tumors and Tumor Cell Lines: A Mixture of Cancer Stem Cells and Cancer Non-stem Cells Starting with the fact that (a) all of the cells of a pre-malignant lesion were derived from an initiated single cell that was promoted to a large mass [61] and (b) the malignant tumor were derived from a single cell [99, 100], this implies that, at least, the initiated cell started out with an unlimited proliferative potential. The jury is still out on resolving the problem as to whether the original cell that was initiated was (a) an immortal stem cell that was blocked in its ability to terminally differentiate or (b) a differentiated somatic cell (in the case of carcinomas, a differentiated epithelial cell) that became “re-programmed” to have an unlimited proliferative potential. As this initiated cell was stimulated to divide, the genotypes and phenotypes of the cells of the tumor started to deviate from each other. Genomic and epigenomic instabilities [101] probably were the consequence of the “initiation” event and by the constantly changing micro-environment. This created a new selective pressure to survive in a

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new type of environment for a cell that was designed to exist either in a stem cell niche microenvironment or a highly differentiated niche microenvironment. If, as it was implied in the original stem cell theories of cancer [22, 23, 102, 103], a growing tumor was dependent on the original initiated stem cell, then it follows that, in this growing tumor, a “cancer stem cell” must exist. In the case of leukemia, this could be strongly supported [104]. However, until it was demonstrated that not all cells of the tumor could perpetuate the growth of the tumor, except for a few, the term “cancer stem cell” emerged in the field of cancer research [105]. Subsequently, many reports have claimed the isolation or characterization of cells that are the “cancer stem cells” [106–125]. Included were surface antigens that were claimed to identify these cancer stem cells. Another approach was used to identify presumptive cancer stem cells. It was initiated by the isolation of human adult breast stem cells, which was reported 2 years before the isolation of human embryonic stem cells [95]. After the demonstration these adult human breast stem cells did not have functional gap junctions and could be differentiated into breast epithelial cells with functional gap junctions, a completely different pattern of expressed genes [126], it was later shown to express an important “stemness” gene, Oct-4A [77]. The rationale for this study was based on the original observation that Oct3/4 was a marker for embryonic stem cells but not for normal differentiated tissues [127, 128]. Later, when Oct3/4 was soon to be expressed in several tumors [129– 131], it was claimed that the Oct3/4 was “re-expressed” or “re-programmed” during the carcinogenic process [129], it stimulated our laboratory to believe that there is another potential interpretation. Given that the stem cell theory suggests that a tumor, which is derived from adult tissues, there must exist adult stem cells in all adult organs. Since our laboratory had already isolated adult human kidney, breast, pancreas, intestinal mesenchyme and liver stem cells [37, 95, 132–136], it was clear that we could test the hypothesis that tumors in adult organs started from existing stem cells. If these adult stem cells could give rise to tumors, then they probably expressed the Oct-4A stemness gene. Further, it was previously shown that the adult normal breast stem cell could be “immortalized” or blocked from “mortalization”. They could, then, be neoplastically transformed [133]. It was a simple matter to demonstrate whether all these human adult stem cell expressed Oct-4A. The results clearly showed that all the normal adult human stem cells did express a nuclear Oct-4A protein and message for Oct-4A [77]. This was the foundation supporting the hypothesis that Oct-4A is a good marker for adult stem cells and that this gene remained expressed during the initiation of a breast stem cell into an immortal but non-tumorigenic human breast cell, as well as during its neoplastic transformation. It was then predicted that malignant tumors would contain Oct-4A positive cells and that these Oct-4A positive cells were probably the presumptive “cancer stem cell”. After testing 83 canine tumors from 21 organ sites, it was shown that 100% of these different tumors had Oct-4A positive cells. What was interesting, although not surprising, was the observation that the frequency of Oct-4A cells in each tumor varied widely. While the explanation for this frequency difference is not known, it implies that as the tumor grows

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in the individual, the Oct-4A positive cells divide either symmetrically or asymmetrically depending on the diet/physiology of the individual. As a tumor grows, the micro-environment of the tumor changes and this alters the gene expression of individual cancer stem cells. Those changes that induce the Oct-4A transcriptional down-regulated would cause these cancer stem cells to partially differentiate to cancer non-stem cells, which now have a finite life span. Recent papers, repeating this same observation in human bladder cancer [137–139] and human oral cancers [140], showed that almost all these tumors expressed Oct-4 gene. These observations lead to some important questions: (a) “Does the initiate cell (either an adult stem cell or a de-differentiated somatic cell) give rise to a cancer stem cell?”; (b) “How does the cancer stem cell differ from the initiated stem cell and normal stem cell?”; and (c) “How does the cancer non-stem cell differ from the cancer stem cell and how does it arise?”.

9.10 Characteristics of Normal Adult Stem Cells, Cancer Stem Cells and Cancer Non-stem Cells The lack of gap junctional intercellular communication was the first characterization of cancer cells postulated by Lowenstein [33]. This was assumed to explain both their inability to “contact inhibit” as did normal cells and to terminally differentiate. A bit later it was shown that tumor promoters could inhibit gap junctional intercellular communication of normal cells [64]. In vivo, tumor promoters caused initiated cells to clonally expand as though the initiated cell was contact inhibited by surrounding normal cells [51]. The real problem with the past and current explanations is how to integrate the use of mechanistic studies in vitro with the empirical studies, particularly with the in vivo initiation/promotion/progression studies and the genetic engineering studied with various cancer-related genes. Since the Loewenstein observation, it has been generally demonstrated that all cancers cells lack functional gap junctions. In other words, lacking gap functional gap junctions, the cell could not contact-inhibit or terminally differentiate. Since that time, we now know that (a) cancer cells can be defective in their ability to communicate because the cell do not express its connexin genes or because the connexin genes that are expressed could be mutated, or rendered non-functional by activated oncogenes. In addition, the expressed connexin could be incompatible with its neighboring cell because not all connexons will couple with all other connexons [141]; or (b) normal stem cells, which do not have functional gap junctions [36], are, however, growth controlled either or both by the stem cell niche [16] and by the secreted factors from their terminally-differentiated lineage daughter cells [59] or by a stromal factor [142]. It has been generally assumed that cells, such as HeLa or MCF-7, derived from a tumor, represented the whole tumor from which it was originated. Secondly, tumor cells that were derived by genetically engineering with various oncogenes, etc, would represent similar tumors derived in vivo that expressed the same activated

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oncogene, especially when it had to be tested in an immune-compromised animal. However, it is now known that tumors represent a mixture of genotypically and phenotypically different cells. The new concept of cancer stem cells is that they are the driving force perpetuating the tumor. The ability to identify normal adult stem cells and cancer stem cells, both in vitro and in vivo, is critical to the eventual development of effective therapy. Several new observations have appeared. The first is that, while primary cell strains, under normal contemporary tissue culture conditions, soon senesce (a) under high oxygen tension [143]; (b) in traditional culture media; and (c) when grown on plastic [144]. Immortalized and cancer cell lines, grown under identical conditions, are, by definition, immortal. However, when one examines normal embryonic or adult stem cells in vitro, one sees that it is not easy to maintain these normal stem cells indefinitely. We know that growing stem cells in low oxygen tension and in the presence of anti-oxidants allows them to have longer life spans in vitro [133, 143]. The second observation is that, while primarily cultures senesce, cancer cell lines are immortal. That implies that the immortal line lines must contain “cancer stem cells”. If it did not, the culture or cell line would not be able to be passed indefinitely. The third observation of these immortal cancer cell lines is that they are heterogeneous with regard to the Oct-4A marker. Even the HeLa and MCF-7 cell are heterogeneous in vitro [77]. This suggests that, even in vitro, the micro-environment can influence gene expression as it can in vivo. In other words, cancer cells in vitro contain “cancer stem cells”, which can partially differentiate. These cells cancer non-stem cells, while not expressing Oct-4A, can divide a finite number of times but would not be able to perpetuate the cell line indefinitely. By down regulating the Oct-4A gene, the cell could partially differentiate. The point of this analysis should make one cautious of directly extrapolating results on whole population of cells in vitro to whole populations of cells in vivo.

9.11 Cancer Stem Cells, Drug-Resistant Cancer Cells and “Side-Population” Cells Tumors are a mixture of cancer stem cells and cancer non-stem cells. This has, presumably, been the reason that past radiation and chemotherapies have been less than successful in treating all cancers. If, as it seems, the past strategies to develop cancer therapies, was to kill cancer cells before they killed normal cells, there was no idea that, in the tumor, there was differential sensitivities to the chemotherapeutic drug or radiation, with the exception of the stage of the cell cycle the cell might be in. In the recent years, in an independent series of experiments to identify drug resistant cancer cells, another series of observations have been made to support the stem cell hypothesis of cancer. It had been generally assumed that treating cancers with toxic agents lead to initial killing of many cells of a tumor, but, in many cases, the tumor re-established itself by becoming resistant to the toxic agent. Therefore, it

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was assumed that the chemotherapeutic agent induced mutations that lead to the appearance of multi-drug resistance genes. However, with studies on leukemia, and then on other solid tumors, when the tumor cells were treated with a fluorescent toxic agent, and separated with a cell sorter, two populations of cells appeared, the majority population, which was fluorescent, and a smaller population, the so-called “side population”, which was not fluorescent [122, 145–164]. In addition, these “side population” cells tended to have stem-like properties. These side population cells also expressed the drug transporter gene [165, 166]. In other words, in a cancer, there were two populations of cells, the cancer stem cells and cancer-non-stem cells. The cancer-stem cells apparently expressed both Oct-4A and a drug transporter gene, making them resistant to toxic agents [165–167]. Therefore, multi-drug resistance cancer cells seen in cancer-treated patients were the result of selection of pre-existing cancer stem cells, rather than new cancer resistant cells induced by the cancer therapy [75, 76]. If this interpretation is correct, then one obvious strategy to treat cancer would be to use a bi-phasic approach. First, try to remove the cancer non-stem cells with tradition treatments. After, treat the surviving cancer stem cells with either inhibitors to drug transporters and then treat with cytotoxic agents or agents to induce transcription of the connexin genes and down regulate transcription or functioning of the Oct-4A transcription factor protein [168].

9.12 Two Types of Cancer Cells As was previously noted, while all cancer cells seem to lack gap junctional intercellular communication, there are two major causes of the disrupted communication, lack of transcription of the connexin genes or the posttranslational function of the expressed connexin gene and protein. There have been several important observations that might provide clues as to the ultimate explanation of the target for cancer stem cells. The first interesting observation comes from the generation of the connexin 32 knock-out mouse. These mice survive, unlike the knock-out 43 and knock-out 26 mice, to actually form a “normal” functioning liver [169]. It should be noted that connexin 26, connexin 43 and connexin32 are all expressed in different cell types in the liver. The oval cell, a presumptive bi-polar stem cell of the liver [170], expresses Cx43. The hepatocyte, derived from the oval cell, expresses Cx26 and Cx32, while the oval cell represses Cx32. Spontaneous and induced tumors are much higher in these knock-out Cx32 mice. This suggests that important differentiation and liver function signals can be transmitted via the Cx26 gap junction channels, but that tumor suppressing signals can not be transferred through the Cx26 channel. In an amazing series of experiments with conditional KO 26 and 32 mice, it was shown that total lack of gap junctional intercellular communication did not drastically alter basal hepatocytic function and did not lead to increased spontaneous liver

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tumor formation [171], including no formation of pre-malignant foci. This creates a potential dilemma for the hypothesis that gap junctions play a role in carcinogenesis. Alternatively, these observations might provide support for the hypothesis that the oval cell, which expressions Cx43 and which is the presumptive adult bipolar stem cell, can allow the differentiation of the hepatocyte, albeit with a slight increase in the number of hepatocytes and some altered morphological states, such as amyloidosis. More importantly, it might suggest that the hepatocellular carcinoma are derived from the oval cell, not from pre-existing hepatocytes. Other kinds of studies, especially the classic initiation/promotion studies with rodents, the formation of “enzyme-altered foci” has been used to detect liver tumor promoters, such as phenobarbital, DDT, TCDD. One assumption that has been made is that these tumor promoting chemicals worked by reversibly inhibiting gap junctional intercellular communication of the cells in the enzyme- altered foci, which are morphologically hepatocytes [62]. That these cells do seem to have functional gap junctions comes from direct tests and from a study that indicated that dye transfer can occur within the foci but not between the surround normal hepatoctyes [172]. The fact that carcinomas can eventually appear in some of these pre-existing enzyme altered foci leads to the conclusion that a malignant, non-communicating hepatocellular carcinoma arises from these pre-malignant foci. These hepatocarcinomas would be expected to express either mutated gap junctions or gap junctions that are rendered dysfunctional by oncogenes, such as ras [173]. However, since there are carcinomas that have no expressed connexins, could they arise from differentiated somatic cells, such as the HeLa or MCF-7 cells from cervical or breast tissues, respectively, by a total re-programming back to a stem cell-like state? Alternatively, could they arise, not from a pre-malignant foci of cells that have expressed their connexins, but directly from the adult stem cell that never expressed their connexins [60]? Many colon polyps, which are pre-malignant lesions, similar to the pre-malignant skin papillomas or enzyme-altered foci of the liver, seem to be the origin of many colon carcinomas. However, the existence of “flat” lesions of the colon might suggest another origin of these colon carcinomas [174]. If the colon adult stem cell was initiated before any connexin was expressed and before any partial differentiation could appear, then these lesions might be similar to the breast stem cell that was initiated by the SV40 large T antigen, keeping it in the stem cell-like state. Eventually during its carcinogenic process, when it losses responsiveness to secreted negative growth suppressing effects, it would become tumorigenic. If these observations prove to give clues to the two kinds of non-communicating cancer cells (those with no expressed connexins and those with expressed but nonfunctional gap junctions), the next question is: “Is the non-connexin-expressing cancer cell derived directly from the adult stem cell?” These cancer cells would be expected to express Oct-4A gene, at least at some frequency in a tumor amidst their partially differentiated cells due to changing micro-environments in the tumor. “Are the expressing-, but non-functional, connexin tumor cells also derived from the adult stem cell but ones, in which the connexins were expressed, but that the Oct-A was not, transcriptionally suppressed.?”

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9.13 Stem Cells, Hypoxia, Drug Resistance and Oct-4A It was noted earlier that normal stem cells, in their in vivo niche, appears to exist in a hypoxic micro-environment [143]. Growing cells in lower oxygen environments and with anti-oxidants appears to extend their in vitro lifespan [132, 133]. The hypoxic inducible factors (HIF-1α, HIF-2α) have been implicated as critical genes helping to regulate genes needed for survival under different toxic conditions. Interesting, Oct-4 gene has been designated as a redox-regulated gene [175]. Moreover, HIF-2α has been shown to be involved in the regulation of Oct-4 [176]. With Oct-4 being associated with maintaining “stemness” in cells, the implication that the genes, which this transcription factor protein regulates, include those positively associated with stem cell maintenance and negatively with the differentiation of that particular adult stem cell. While, in those stem cells that have been tested, expressed Oct-4A was associated with no expressed connexins [36]. In the report that embryonic stem cells being associated with connexins, there was no data showing functionality of the gap junctions or that all the embryonic stem cells were expressing the connexin gene [177]. Completing the circle about the role of evolution with all of these disparate observations, one must again consider the appearance of the gap junction in the early evolution of the metazoan and the absence of functional gap junctions in cancer cells. The need was to sequester the stem cell in a low oxygen micro-environment, which keeps the stem cell from proliferating and differentiating. In addition, these stem cells must be kept relatively safety from toxins/toxicants. Using the metaphor that the stem cell is the queen bee in the hive of with her progeny nurse bees and drones, when a tissue of a few stem cells and the many progenitor and differentiated cells is exposed to a toxicant, the differentiated and progenitor cells could died because the toxicants can penetrate these cells and be metabolized to electrophiles. These cells could die. These cells, however, can be replaced, because the stem cells have expressed drug transporters and are resistant to the toxicant. If the drones kill invaders in protecting the queen bee, the queen, having survived, can re-establish the hive, in the same manner the stem cell can replace dead or removed differentiated cells.

9.14 Dietary Modulation of Cancer: The Barker Hypothesis, Stem Cell Frequency and Risk to Cancer From the initiation/promotion/progression hypothesis to explain the multi-stagemulti-mechanism basis of carcinogenesis, it is clear that, in principle, that diet and abnormal nutrient exposure could affect any one of the three steps. While the initiation of a single cell can be affected by exposures to true mutagens, such as UV light, and viruses, it is unlikely that diet can reduce the risk of the initiation process to zero, since there is always a risk of an error of replication of DNA in any gene and cell replication must occur during growth and wound healing. However, given

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that initiation of a single cell in any organ is possible, the long period of time it takes to promote an initiate cell so as it accrued all the “hallmarks of cancer”, the promotion phase would appear to be that phase of carcinogenesis most amenable for either enhancing or reducing the risk to cancer as long as the agent/condition of promoters and chemopreventive agents meet all the characteristics of promoters or anti-promoters (threshold levels; species, gender, organ and cell-type specific promoters/anti-promoters [9]). Assuming for the moment that either the adult stem cell or a “re-programmed” adult differentiated somatic cell could be the target cell for the initiation phase, rarely has the idea that one can influence the initiation phase by either increasing or decreasing the adult stem cell pool by diet and nutrition. However, in principle, it should be possible to increase or decrease the risk of initiating a cell if the stem cell pool is significantly increased or decreased, all other things being equal. Small changes in numbers of stem cells would lead to major differences in differentiated cell numbers. The insight by Barker [178, 179] is that adult diseases can be linked to pre-natal and early post-natal life. Specifically, as has been hypothesized [26], increasing or decreasing the stem cell pool in specific organs during embryogenesis and fetal development by dietary/drug/environmental epigenetic factors could alter chronic stem-cell based diseases later in life. In fact, it has been suggested that there is a correlation of umbilical cord blood hematopoietic stem and progenitor cell levels with birth weight and cancer risk [180]. In addition two other examples, one human epidemiological study of the survivors of the atomic bombs and one experimental carcinogenesis study, could support the Barker hypothesis. Japanese women, exposed to the atomic bombs between 10 and 14 years of age, were more susceptible to breast cancer [181]. When one examines the reason that the radiation expose caused a statistically-significant increase over the non-exposure population of Japanese women is because the control background frequency was extremely low. One very reasonable explanation for this low background frequency of breast cancer is diet, as seen when Japanese women in Western cultures demonstrate frequencies shared by the endogenous non-Japanese women. Clearly the genetics of the Japanese women can be ruled out in this case. Because the diet of the Japan women, both the mothers and the exposed daughters, were exposed to high levels of a soy diet (as well as low caloric restricted diet [182]), the nutrients of this diet have been suggested as causing a reduction of breast cancers. Animal studies, although contradictory, in large part suggests that soy can help to reduce the risk to certain cancers [183]. More relevant to human breast cancer, it has been shown that human adult breast stem cells, when exposed to genistein, a component of soy, induced differentiation of these human breast stem cells [184]. In addition, the other anti-cancer agent in soy would be the Bowman-Birk inhibition [185]. If by eating soy, pregnant women might be causing the breast stem cells to differentiate in the developing female fetus. Upon reaching puberty, these daughters would have few stem cells to make breast tissue and there would be fewer breast stem cells as “targets” for the radiation exposure. Those few initiated breast stem cells in these exposed daughters would have also been exposed to caloric restriction for years after the atomic bomb exposure, thereby, affecting the promotion phase of

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these initiated cells. Currently, Japan, with the diet changing to the Western diet, the background frequency of breast cancer is increasing. If ever there might be an example where cultural evolution interacts with biological evolution and impact on the cancer process it is through the global dietary patterns and the dramatic change in global diets. With another experiment example, rodent prostate cancer has been shown to be influenced by a pregnant rat having been exposed to a non-genotoxic or nonmutagenic chemical, biphenyl A, even though the male offspring was not exposure to the toxic chemical after birth [186]. Moreover, if the pregnant rats were coexposed to a soy diet and the bisphenyl A, the frequency of prostate cancers in the offspring was dramatically reduced even though neither the soy nor bisphenyl A was given to the ale offspring after birth. This implies, clearly, that if stem cells are the target cells for cancer, then the bisphenyl A might have increased the number, possibly the quality, of the prostate stem cells [26], By the same reasoning, if the soy diet caused differentiation of the prostate stem cells, the frequency of prostate cancer would have been predicted to be reduced. In this case, the initiation phase would be predicted to have been affected by this environmental/dietary history, not the promotion phase of carcinogenesis. Here, again, as we affect the environment by our pollution, our cultural evolutionary practices, we can impact the biological factors of the carcinogenesis process. Lastly, with the emergence of the role of epigenetic changes occurring after dietary or environment chemical exposures, the mechanism, underlying these prenatal, Barker-type effects, has been shown to correlate with altered gene expression. The classic case of altered gene expression of a genetic-determined coat color of rodents provides strong molecular bases for the Barker hypothesis. It must be emphasized that changes in gene expression of organs and tissues, as well as tumor tissues, can be the result, not only because the epigenetic chemical altered gene expression in target cells, but that the ratio of stem cells, their progenitor daughter cells and the terminally-differentiated linage could be altered by the chemical. That is the chemical might increase or decrease any one of the three cell types (all of which express different gene patterns) in the tissue.

9.15 Does the Adult Stem Cell or the Differentiated Somatic Cell Act as the Target Cell for the Initiation of Cancer To be scientifically rigorous, it must be stated that the answer to this question is still an open question. However, that is not to say that there is equal support for either opposing hypothesis. One of the ultimate experiments that has to be done is to neoplastically transform both true human adult stem cells and true human differentiated epithelial and fibroblastic cells to give rise to carcinomas and sarcomas. The example of a human breast

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stem cell being neoplastically transformed, but that the differentiated daughter of that stem cell being resistant to immortalization and to neoplastic transformation, supports the stem cell hypothesis. Although all the reports of neoplastically transforming human epithelial and fibroblastic cells, which under normal conditions are very resistant to immortalization and neoplastic transformation [91, 92, 94], they can be immortalized with various viruses. Although there are two explanation of the immortalization step (the virus blocks terminal differentiation of the adult stem cells or it causes “re-programming” of a differentiated somatic cell [27]), these results cannot be unequivocally support either hypothesis. However, the studies of the multi-stage, initiation/promotion/progression hypothesis seems to support the stem cell theory. Classic initiation studies of mouse skin with carcinogens, which, by themselves, do not lead to tumors, have shown that one can expose the animal to the initiator and wait up to a year to promote and one obtains tumors. This suggests that the initiation event is irreversible and that the initiated cell remains in the skin during that year before being promoted. In the case of rodent liver promotion, the mature hepatocytes are probably not the target cell for hepatocarcinomas, because they are very polyploidy (tetra- or octoploid). When one views the liver pre-malignant lesions as being primarily diploid [187]. Therefore, if the hepatocyte were the target cell for initiation, the first step of initiation would be the de-diploidization of a polyploidy cell. It then must be re-programmed to become immortal or stem-like. This pre-programming hypothesis, then, has one more step in the process of carcinogenesis than the stem cell hypothesis. In the case of colon carcinogenesis, in which the colon tumor arrives from the polyp, it seems unlikely that the terminally differentiated colon epithelial cell, which normally apoptoses or gets sluffed off the crypt, would be “re-programmed” near the top of the crypt and then stop terminally differentiation to remain only to be promoted to form a polyp and subsequently a colon carcinoma.

9.16 Conclusion While it might seem academic to a cancer patient as to whether a cancer is derived from a stem cell or a de-differentiated or “re-programmed” somatic differentiated cell, there might be practical implications of one or the other hypothesis that could lead to (a) preventing or lowering the risk to get a cancer or (b) treating the tumor. With the ability to isolate adult human stem cells and their ability to produce differentiated daughters, and with improved methods of culturing these cells, it should be possible to perform neoplastically transformed cells with both cell types as the target cells for initiation. In addition, with more improved techniques to identify the adult stem cell in vivo with both universal adult stem cell and organ-specific markers, identifying the in situ origin of the tumor could help to resolve this problem.

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Chapter 10

Stem Cell Niche Versus Cancer Stem Cell Niche – Differences and Similarities Bruce C. Baguley and Graeme J. Finlay

Abstract The organization of normal tissues and organs is thought to be based on stem cells, which are present as a small proportion of the total tissue. Stem cells in turn absolutely require supporting structures to maintain their self-renewal properties and such structures are termed niches. A large amount of evidence now supports the concept that tumors are also maintained by tumor stem cells, and by implication such cells will be contained within tumor stem cell niches. In this review we explore the hypothesis that the transition of the normal niche to the tumor niche occurs over a significant period of time and that different intermediate stages, termed here the “inflammatory niche” and the “immunological niche”, can be discerned. The inflammatory niche provides a chronic stress stimulus which causes an increased rate of stem cell proliferation while the immunological niche provides mechanisms to inhibit the proliferation of potential tumor cells that have formed in the inflammatory niche. The tumor cell niche therefore represents the ultimate breakdown of such proliferation control. Keywords Stem cell · Niche · Chronic inflammation · Tumor fibroblasts · Lymphocytes · Macrophages · Cell cycle arrest · Tumor dormancy · TGF-β · Interferon-γ

Contents 10.1 Introduction . . . . . . . . . . . . 10.2 The Stem Cell Niche in Normal Tissue 10.3 The “Inflammatory” Niche . . . . . . 10.4 The “Immunological” Niche . . . . . 10.5 The Tumor Cell Niche . . . . . . . . 10.6 Conclusion . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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10.1 Introduction The human body appears to utilize two main strategies to regulate the proliferation of cells and to maintain the appropriate cellularity of tissues. In the first, the majority of cells in a tissue are mature and unable to undergo further proliferation, while a small number of cells exist predominantly as a non-dividing population but are potentially able to undergo self renewal and thus maintain the tissue as a whole. These cells, termed stem cells, are thought to be located within a specialized microenvironment comprising fibroblasts, endothelial and other cells, called the niche. Proliferation of stem cells is constrained by the niche microenvironment but when cells leave the niche they are free undergo proliferation and terminal differentiation, while continuing to be supported by fibroblasts and blood vessels. In the second strategy, represented by fibroblasts in connective tissue, the majority of cells normally exist in a non-proliferating state but have the potential to undergo proliferation under appropriate conditions, such as following tissue damage. Once the damage is repaired, these cells either die or resume a nonproliferating state. Human tissues are largely organized as a partnership between two populations that adopt these two strategies. Interestingly, this partnership exists even at the level of isolated embryonic stem cells where, at least in culture, some cells undergo a transition to a fibroblast-like phenotype that can then support stem cells and maintain their properties [1]. The functional partnership between stem cells and fibroblasts, together with macrophages, vascular endothelial cells and other stromal cells, provides a good framework for understanding the nature of the niche. Increasing evidence supports the hypothesis that human cancer growth is driven by a small population of tumor cells, called tumor stem cells, which are sustained in a niche-type microenvironment by fibroblasts or fibroblast-like cells. As is the case for stem cells in normal tissues, tumor stem cells are maintained in a selfrenewing state within the niche. When tumor cells leave this niche, they continue to be supported by fibroblasts and a blood supply but most cannot sustain their selfrenewal capacity and eventually die. However, some may have the ability to return to the niche and retain self-renewal properties. Alternatively, they may enter the circulation and migrate to another niche, perhaps at a distant site, where their selfrenewal capacity can be sustained. Thus, the niche is essential for the survival of the tumor in much the same way as it is essential for normal tissue. The aim of this review is to focus not on the biology of stem cells but rather on the microenvironment in which they are maintained. Discussion on the tumor cells is necessarily speculative because little direct evidence is available. It is hypothesized here that modification of the niche microenvironment is important not only in the maintenance of tumor stem cells but also in the period, which may extend over many years, in which potential tumor stem cells develop. Therefore, the review commences with a discussion of the normal stem cell niche then describes how this might be disturbed during the development of neoplasia. The likely properties of the tumor stem cell niche, and how they are modified by tumor cells themselves, are then discussed.

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10.2 The Stem Cell Niche in Normal Tissue The essential nature of the stromal microenvironment in the maintenance, structure and function of normal tissues has been confirmed in many studies. In glandular organs, stromal cells comprise cells of the fibroblast lineage as well as vascular endothelial cells, and this principle is well characterized in hematopoetic tissue [2] and extends to brain, muscle and other tissues. Stromal cells provide not only a supporting structural network comprising fibrous proteins and other macromolecules, but also provide survival factors and differentiation factors essential for the correct development of the organ as a whole. The stromal cells and their products thus seem to determine the overall dimensions and the structural framework of an organ, providing microenvironments in which epithelial cells can proliferate, differentiate and perform their specialized function. Only a small proportion of stromal cells appear to be specialized to form niches in which they can maintain the stem cell populations necessary for maintenance of the organ. The development of culture methods for bone marrow cells, together with observations on the cellular composition of bone marrow under normal and pathological conditions, originally led to the concept of hematopoietic pluripotent stem cells that are maintained within the bone marrow for the life of the individual and can that give rise to multiple lineages [2, 3]. The niche is lined by endostomal and endothelial cells, which maintain the stem cells in a slowly proliferating but self-renewing state. Later studies with other tissues, including the gut and the brain, supported the concept that many, and perhaps all, organs have similar stem cell organization. In a typical normal glandular organ, fibroblasts derived from the mesenchyme must accomplish different tasks depending on whether they are in the stem cell niche or in the remaining part of the organ (Fig. 10.1). In the niche, these cells produce factors that suppress proliferation and differentiation of the stem cells while maintaining their self-renewal properties. They must also provide homing signals that can recruit and retain circulating stem cells arising from the bone marrow or other sites. The factors required for this process are thought to include members of the TGF-β superfamily as well as the Notch, Wingless (Wnt) and Hedgehog (Hh) pathways. The TGF-β superfamily includes TGF-βs, activin, inhibins, nodal/cripto and bone morphogenetic proteins (BMPs). A feature of the normal niche is that stem cells are normally in a quiescent phase [4]. Proteins maintaining low proliferation within stem cells include p16CDKN2a (p16INK4a ), p15CDKN2b (p15INK4b ), p21CDKN1A and p27CDKN1 (Fig. 10.1). Members of the TGF-β superfamily are thought to act through receptors on stem cells through Smad proteins to increase p15CDKN2b expression. In hematopoietic tissue, p16CDKN2A acts in concert with p53 and p14ARF to limit self-renewal [5]. Products of the forkhead FOXO gene act to preserve regenerative potential [6] by inducing expression of p19ARF [7], p15CDKN2b and p19INK4d [8]. FOXO also protects from the effects of reactive oxygen species (ROS), which increase proliferation [9]. Phosphatase and tensin homologue (PTEN) suppresses proliferation of stem cells, at least in hematopoiesis [10] and negatively regulates the activity of phosphatidylinositide-3-kinase (PI3K). PI3K is strongly negatively regulated in

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Fig. 10.1 A selection of control mechanisms that operate in the niche. Stromal products in the niche act on stem cells to maintain a state of quiescence under normal conditions, to prevent differentiation, senescence and cell death, and to maintain self-renewal capacity. Stromal components in other parts of the tissue act to promote proliferation and differentiation, to ensure cell survival and to promote senescence in cells that have undergone incomplete senescence

normal stem cells and since PI3K negatively regulates FOXO, low PI3K leads to increased FOXO activity. The niche has mechanisms to suppress differentiation and the TCF-4, MAP kinase and GSK3β pathways may be involved in this process [11]. In colons of mice that lack the TCF-4 transcription factor, stem cells are driven in the direction of differentiation, leading to progressive depletion the population [12]. The stem cell niche also has mechanisms to induce proliferation, as required for self-renewal and response to demands. These involve the polycomb family member BMI1 [13, 14], which acts by repressing p16CDKN2a and p14ARF expression, as well as the transcription factor c-myc, which acts on p21CDKN1A and p15CDKN2b expression, and the transcription factor ID1/3, which acts on p16CDKN2a . The growth factor independence-1 gene (Gfi1) may also be involved in maintaining self-renewal in some tissues [15]. Several other genes, such as TP53 and Mel18 [16] may also contribute. Thus, there is a finely balanced complex of gene products that maintain low stem cell proliferation under normal conditions but that stimulate proliferation under demanding conditions. Under normal conditions stem cells divide infrequently and often asymmetrically, so that one daughter cell remains in the niche and the other leaves the niche (Fig. 10.2a) [17]. Such cells escape the inhibitory environment of the niche and come under the influence of a variety of stromal factors whose identities overlap with those produced in the niche. However, the effects of these factors contrast with those occurring in the niche; proliferation is now promoted whereas self-renewal

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Non-proliferating normal stem cells Proliferating normal stem cells Non-proliferating tumour stem cells Proliferating tumour stem cells

Fig. 10.2 Hypothetical scheme depicting changes linking normal and tumor cell niches. (a) Proliferation of stem cells in the normal niche is maintained at a low rate by factors released by stromal cells, particularly by fibroblasts, which act through surface receptors and intermediate proteins to inhibit cyclin dependent kinases. (b) Under conditions of chronic stress in the inflammatory niche, the negative effect of the stroma on proliferation is offset by the proliferative stimuli of locally produced ROS and cytokines. This leads to both genetic and epigenetic (changed promoter methylation) changes. Depletion of stem cells can lead to recruitment of further cells from the bone marrow. (c) Proliferation of tumor stem cells that have lost proliferation control through genetic and epigenetic changes is constrained by immune cells, particularly by IFN-γ. (d) Tumor products (e.g. apoptotic tumor cells) suppress immune cell mediated control of proliferation and thus maintain tumor stem cells predominantly in a proliferative state

is not (Fig. 10.2b). Induction of proliferation is tightly coupled with induction of differentiation, which eventually leads to cessation of cell division.

10.3 The “Inflammatory” Niche Transition from normal tissue to cancer tissue is a complex one, involving multiple genetic and epigenetic changes and occurring over a time period of several years. Chronic inflammation produces changes, some of which resemble those occurring during wound healing [18] and can be linked to the development of many different cancer types [19–21]. Genetic changes can be produced as the result of inflammatory processes such as the release ROS. Epigenetic changes can also be produced in a chronic inflammatory environment [22], probably involving the EMT (epidermal to mesenchymal transition) and activation of DNA methylases [23, 24]. ROS arising from inflammatory processes also produce, which increase the proliferation rate of stem cells. At least in hematopoietic tissue, stress induction may be mediated by local production of inflammatory cytokines and changes in intracellular signaling such as those in the Wnt pathway [25]. The classic concept of

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cancer causation in experimental animals is provided by a combination of a carcinogen followed by a promoter, and there is good evidence that an important effect of promoters such as phorbol esters is to induce an inflammatory response [26]. Human colorectal cancer provides one of the best studied examples of the role of inflammation in carcinogenesis, and clinical trials have demonstrated that tumor incidence is reduced by administration of anti-inflammatory drugs [19, 27]. The gastrointestinal mucosa is a complex microenvironment in which immune responses can be mounted to pathogens while tolerance to commensal bacteria and other benign dietary antigens is maintained. In some cases this balance is disturbed, causing chronic inflammatory conditions such as ulcerative colitis and Crohn’s disease and is associated with increased amounts of the enzyme cyclooxygenase II. The resulting increased incidence of colorectal cancer could be related firstly to genetic and epigenetic changes to the stem cells in the mucosa and secondly to the induction of excessive proliferation by cytokines or other components of the inflammatory microenvironment. Cyclooxygenase II over-expression has been observed in invasive breast carcinoma and in preinvasive ductal carcinoma in situ [28], and there is also evidence for inflammatory processes in the development of prostate cancer [29]. There is interesting evidence that the process of breast tissue involution, which involves a series of changes occurring at the end of lactation, is associated with both inflammatory changes and increased incidence of breast cancer [30]. Chronic inflammation can also be induced by microbial or viral infection. Helicobacter pylori infection is known to be associated with an increased incidence of gastric cancer in humans and an interesting experimental study has been carried out in mice with gastric Helicobacter pylori. Gastric tumors that form have been found to originate from progenitor cells in the bone marrow [31, 32]. The likely explanation is that chronic inflammation leads to progressive depletion of normal stem cells for the gastric epithelium, leading eventually to recruitment of circulating mesenchymal stem cells originating the bone marrow that have a high propensity to become tumor stem cells. Sunlight (UV radiation exposure) is another agent that can induce inflammatory changes as well as increasing the incidence of non-melanocytic and melanocytic tumors [33–35]. Pioneering studies in mice [36] established that the induction of papillomas by UV irradiation involves UV-induced changes in the skin microenvironment including inflammation and immunosuppression. In the case of basal cell carcinoma, sustained signaling, such as that involving Hh, may contribute to development [37].

10.4 The “Immunological” Niche A consequence of genetic damage to stem cells is the formation of mutations. Generally, mutations that result in functional changes to the stem cells can lead to changes in their ability to differentiate once they have left the niche, but they can

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also affect the control of proliferation within the niche. Recent large scale genetic studies have delineated mutations that lead to loss of function of protein such as p16CDKN2A , p15CDKN2B , PTEN and pRb, often from large deletions, that potentially lead to reduced control of proliferation, as well as numerous other mutations that might affect differentiation potential and resistance to the induction of apoptosis [38]. These changes may also lead to triggering of responses that lead to additional mechanisms of proliferation control. There is evidence, particularly based on melanoma, that some tumors can be maintained for many years in a dormant state, with activation occurring in response to immunosuppressive treatment [39, 40]. A model for such dormancy is provided by studies in which mice were monitored following treatment with a carcinogen. Most mice did not develop tumors but examination revealed a life-long presence of microscopic groups of tumor cells associated with T lymphocytes [41]. Treatment of such mice with antibodies to T lymphocytes or to γ-interferon led a high proportion to develop tumors. The likely explanation of these results is that the microscopic groups of tumor cells in these mice were maintained in a dormant state by IFNγ and other cytokines produced by surrounding T lymphocytes (Fig. 10.2c) [42, 43]. The likely mechanism involves binding of IFNγ to the IFNGR1 receptor, inducing dimerization and recognition by the extracellular domain of IFNGR2 to form a receptor complex. Subsequent binding of Jak1 and Jak2 kinases lead to IFNGR1 phosphorylation and the formation of docking sites for Stat1, which is subsequently phosphorylated and translocated to the nucleus. Stat-1 dimerizes and binds to GAS (gamma-activated sequences) in gene promoters and one of the products of its transcription, p21, induces cycle arrest [42]. Transcription factors such as ATF6α, signaling through the mTOR pathway, may also be important [44]. The production of IFNγ can also stimulate the activity of tumor associated macrophages, potentially leading to the elimination of the tumor cells [45]. Other host mechanisms, such as the induction of senescence by means of the p14ARF -p53-p21 axis, may also contribute to the removal of potential cancer stem cells [46, 47].

10.5 The Tumor Cell Niche In view of the discussion in the previous sections, it is possible to envisage a sequence where the persistence of a chronic inflammatory stimulus within a normal tissue leads to increased proliferation of the stem cell population. When this is combined with the induction of mutations, some of which lead to loss of the normal mechanisms of proliferation control, potential tumor stem cells are formed. The niche seems to be able to control the proliferation of such cells, for instance through local induction of IFNγ and other cytokines. The development of overt cancer may therefore reflect the failure of these secondary mechanisms to limit the proliferation of potential tumor stem cells. Similarly, the appearance of metastases may not be simply a result of recent relocation of cancer cells to distant sites but rather

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the escape of the corresponding pre-existing tumor stem cells in these sites from host-mediated suppression of proliferation. Once tumor stem cells have escaped host-mediated suppression, they or their progeny may act to suppress the mechanisms that constrained their proliferation within the “immunological” niche. For instance, tumor populations are continuously turning over [48] and the consequent released apoptotic tumor cells can be taken up by tumor macrophages and dendritic cells, limiting their ability to mount responses to the tumor cells [49]. Tumor signaling may also block the induction of senescence [50]. Tumor cells may also release products that “educate” surrounding macrophages to modify the stromal microenvironment and thereby to protect the tumor stem cell compartment (Fig. 10.2d) [51]. Genetic alterations that result in increased entry of stem cells into the cell cycle affect centrosome replication [52, 53] and defective centrosomal control leads in turn to abnormalities in mitosis, chromosome instability and aneuploidy [54]. Importantly, this also leads to genetic and phenotypic heterogeneity [55]. Individual tumor cells can then take on new functions, including mimicry, whereby they take on some of the characteristics of host cells within the tumor tissue. Vasculogenic mimicry, whereby tumor cells take on some of the functions of vascular endothelial cells, is a good example of such changes [56]. Thus, factors released by tumors, as well as contributions by tumor cells that carry out niche functions, can have profound effects on the function of the tumor stem cell niche.

10.6 Conclusion The concept developed here in which the normal cell niche evolves over a period of many years to a tumor cell niche remains highly speculative. Evidence is provided that modifications to the niche occur firstly as a result of effects of associated inflammatory and immune cells, and secondly as a result of products of tumor cells themselves. Tumors might therefore contain a diverse set of niches that have evolved at different times or at different rates. The heterogeneity of the niche also reflects the likely heterogeneity of the tumor stem cells that are contained within the niches. The protection of tumor stem cells by both intrinsic resistance of the cancer stem cells and survival pathways induced by the niche could both contribute to resistance to cancer therapy. The model suggests, as an alternative strategy to elimination of cancer stem cells, that therapy-induced re-establishment of the “immunological” niche is a worthwhile consideration. While it is imperative that we learn more about the nature of the tumor stem cell niche, the concepts of the “inflammatory” niche and the “immunological” niche also require further exploration.

References 1. Bendall SC, Stewart MH, Menendez P et al. (2007) IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448:1015–1021.

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2. Meads MB, Hazlehurst LA, Dalton WS (2008) The bone marrow microenvironment as a tumor sanctuary and contributor to drug resistance. Clin Cancer Res 14:2519–2526. 3. Moore KA, Lemischka IR (2006) Stem cells and their niches. Science 311:1880–1885. 4. Orford KW, Scadden DT (2008) Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation. Nat Rev Genet 9:115–128. 5. Akala OO, Park IK, Qian D et al. (2008) Long-term haematopoietic reconstitution by Trp53/-p16Ink4a-/-p19Arf-/- multipotent progenitors. Nature 453:228–232. 6. Tothova Z, Gilliland DG (2007) FoxO transcription factors and stem cell homeostasis: insights from the hematopoietic system. Cell Stem Cell 1:140–152. 7. Bouchard C, Lee S, Paulus-Hock V et al. (2007) FoxO transcription factors suppress Mycdriven lymphomagenesis via direct activation of Arf. Genes Dev 21:2775–2787. 8. Katayama K, Nakamura A, Sugimoto Y et al. (2008) FOXO transcription factor-dependent p15(INK4b) and p19(INK4d) expression. Oncogene 27:1677–1686. 9. Arden KC (2007) FoxOs in tumor suppression and stem cell maintenance. Cell 128:235–237. 10. Yilmaz OH, Valdez R, Theisen BK et al. (2006) Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 441:475–482. 11. Ying QL, Wray J, Nichols J et al. (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–523. 12. Korinek V, Barker N, Moerer P et al. (1998) Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet 19:379–383. 13. Lessard J, Sauvageau G (2003) Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 423:255–260. 14. Grinstein E, Wernet P (2007) Cellular signaling in normal and cancerous stem cells. Cell Signal 19:2428–2433. 15. Duan Z, Horwitz M (2005) Gfi-1 takes center stage in hematopoietic stem cells. Trends Mol Med 11:49–52. 16. Lee JY, Jang KS, Shin DH et al. (2008) Mel-18 negatively regulates INK4a/ARF-independent cell cycle progression via Akt inactivation in breast cancer. Cancer Res 68:4201–4209. 17. Fuentealba LC, Eivers E, Geissert D et al. (2008) Asymmetric mitosis: Unequal segregation of proteins destined for degradation. Proc Natl Acad Sci USA 105:7732–7737. 18. Schafer M, Werner S (2008) Cancer as an overhealing wound: an old hypothesis revisited. Nat Rev Mol Cell Biol 9:628–638. 19. Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420:860–867. 20. Balkwill F (2004) Cancer and the chemokine network. Nat Rev Cancer 4:540–550. 21. Hagemann T, Balkwill F, Lawrence T (2007) Inflammation and cancer: a double-edged sword. Cancer Cell 12:300–301. 22. Lee JM, Yanagawa J, Peebles KA et al. (2008) Inflammation in lung carcinogenesis: new targets for lung cancer chemoprevention and treatment. Crit Rev Oncol Hematol 66:208–217. 23. Morel AP, Lievre M, Thomas C et al. (2008) Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS ONE 3:e2888. 24. Dumont N, Wilson MB, Crawford YG et al. (2008) Sustained induction of epithelial to mesenchymal transition activates DNA methylation of genes silenced in basal-like breast cancers. Proc Natl Acad Sci USA 105:14867–14872. 25. Congdon KL, Voermans C, Ferguson EC et al. (2008) Activation of Wnt signaling in hematopoietic regeneration. Stem Cells 26:1202–1210. 26. Gebhardt C, Riehl A, Durchdewald M et al. (2008) RAGE signaling sustains inflammation and promotes tumor development. J Exp Med 205:275–285. 27. Wang D, DuBois RN (2008) Pro-inflammatory prostaglandins and progression of colorectal cancer. Cancer Lett 267:197–203. 28. Howe LR (2007) Inflammation and breast cancer. Cyclooxygenase/prostaglandin signaling and breast cancer. Breast Cancer Res 9:210. 29. De Marzo AM, Platz EA, Sutcliffe S et al. (2007) Inflammation in prostate carcinogenesis. Nat Rev Cancer 7:256–269.

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30. Schedin P (2006) Pregnancy-associated breast cancer and metastasis. Nat Rev Cancer 6: 281–291. 31. Houghton J, Wang TC (2005) Helicobacter pylori and gastric cancer: a new paradigm for inflammation-associated epithelial cancers. Gastroenterology 128:1567–1578. 32. Takaishi S, Okumura T, Wang TC (2008) Gastric cancer stem cells. J Clin Oncol 26: 2876–2882. 33. Ruiter DJ, Bhan AK, Harrist TJ et al. (1982) Major histocompatibility antigens and mononuclear inflammatory infiltrate in benign nevomelanocytic proliferations and malignant melanoma. J Immunol 129:2808–2815. 34. Hart PH, Grimbaldeston MA, Finlay-Jones JJ (2001) Sunlight, immunosuppression and skin cancer: role of histamine and mast cells. Clin Exp Pharmacol Physiol 28:1–8. 35. Wilgus TA, Koki AT, Zweifel BS et al. (2003) Inhibition of cutaneous ultraviolet light Bmediated inflammation and tumor formation with topical celecoxib treatment. Mol Carcinog 38:49–58. 36. Kripke ML (1988) Immunoregulation of carcinogenesis: Past, present, and future. J Natl Cancer Inst 80:722–727. 37. Hutchin ME, Kariapper MS, Grachtchouk M et al. (2005) Sustained Hedgehog signaling is required for basal cell carcinoma proliferation and survival: conditional skin tumorigenesis recapitulates the hair growth cycle. Genes Dev 19:214–223. 38. Ding L, Getz G, Wheeler DA et al. (2008) Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455:1069–1075. 39. MacKie RM, Reid R, Junor B et al. (2003) Fatal melanoma transferred in a donated kidney 16 years after melanoma surgery. N Engl J Med 348:567–568. 40. Aguirre-Ghiso JA (2007) Models, mechanisms and clinical evidence for cancer dormancy. Nat Rev Cancer 7:834–846. 41. Koebel CM, Vermi W, Swann JB et al. (2007) Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450:903–907. 42. Muller-Hermelink N, Braumuller H, Pichler B et al. (2008) TNFR1 signaling and IFNgamma signaling determine whether T cells induce tumor dormancy or promote multistage carcinogenesis. Cancer Cell 13:507–518. 43. Kortylewski M, Komyod W, Kauffmann ME et al. (2004) Interferon-gamma-mediated growth regulation of melanoma cells: Involvement of STAT1-dependent and STAT1-independent signals. J Invest Dermatol 122:414–422. 44. Schewe DM, Aguirre-Ghiso JA (2008) ATF6à-Rheb-mTOR signaling promotes survival of dormant tumor cells in vivo. Proc Natl Acad Sci USA 105:10519–10524. 45. Duff MD, Mestre J, Maddali S et al. (2007) Analysis of gene expression in the tumorassociated macrophage. J Surg Res 142:119–128. 46. Kuilman T, Michaloglou C, Vredeveld LC et al. (2008) Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 133:1019–1031. 47. Acosta JC, O‘Loghlen A, Banito A et al. (2008) Control of senescence by CXCR2 and its ligands. Cell Cycle 7:2956–2959. 48. Baguley BC (2006) Tumor stem cell niches: a new functional framework for the action of anticancer drugs. Recent Pat Anti-Cancer Drug Discovery 1:121–127. 49. Fonseca C, Dranoff G (2008) Capitalizing on the immunogenicity of dying tumor cells. Clin Cancer Res 14:1603–1608. 50. Weinberg RA (2008) Twisted epithelial-mesenchymal transition blocks senescence. Nat Cell Biol 10:1021–1023. 51. Pollard JW (2004) Tumor-educated macrophages promote tumor progression and metastasis. Nat Rev Cancer 4:71–78. 52. Kramer A, Lukas J, Bartek J (2004) Checking out the centrosome. Cell Cycle 3: 1390–1393. 53. McDermott KM, Zhang J, Holst CR et al. (2006) p16(INK4a) prevents centrosome dysfunction and genomic instability in primary cells. PLoS Biol 4:e51.

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Chapter 11

The Chronically Inflamed Microenvironment and Cancer Stem Cells Hanchen Li, Calin Stoicov, Xueli Fan, Jan Cerny, and Jean Marie Houghton

Abstract The notion that tumors contain a population of cells termed cancer stem cells, or cancer initiating cells has been gaining momentum over the past several years. It is postulated that much like a normal organ in the body, tumors contain a population of stem cells which are responsible for driving tumor growth, invasion and metastasis. This becomes of paramount clinical importance because our present therapies often times shrink tumors, but do not prevent their recurrence or metastatic spread. This behavior of tumors suggests that a population of cells within the tumor capable of driving tumor growth is insensitive to our current therapies and survives to reestablish disease. Therefore, understanding the biology of the cancer stem cell which is believed responsible for this tumor re-growth, will allow targeted therapy to better treat cancers. This chapter reviews the cancer stem cell hypotheses, and investigates the potential sources for the cancer stem cell. Keywords Stem cell · Stem cell niche · Stroma · Inflammation · Cancer associated fibroblasts

Contents 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . 11.2 The Association of Inflammation and Cancer . . . . . . . . 11.3 What Does the Chronic Inflammatory Environment Look Like? 11.4 What are the Long Term Effects of Chronic Inflammation? . . 11.5 Inflammation and Tumors – Inflammations Many Roles . . . . 11.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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11.1 Introduction The last several years has seen a dramatic shift in the way we view the role of inflammation in the initiation and progression of cancer. Initially felt to be a beneficial response, we now recognize that in many situations inflammation is far from an advantageous response and may in fact drive malignancy. Another prominent shift in our understanding of cancer biology involves the cellular origins of cancer. Cancer was originally felt to be a homogenous collection of abnormal cells; each of which was capable of independent growth and metastasis. We have changed our thinking 180 degrees to realize that cancer is composed of a hierarchy of cells and cell types interacting very much like an organ. Similar to an organ, it is now felt that at the heart of cancer is a population of stem cells which give it life and decide its fate. Here we will explore the interaction of the inflammatory environment with the cancer stem cell from initiation through metastasis. To do this, we much draw on what has been learned from studies on the inflammatory environment present in tissues at risk for inflammation mediated cancers, and combine findings from many solid and liquid tumors. We must also look to the tissue stem cell for clues on how the cancer stem cell responds to the inflammatory environment. We must infer effects of inflammation on the cancer stem cell, which for many types of cancer remains an elusive player.

11.2 The Association of Inflammation and Cancer The association of inflammation and cancer was first made by Rudolf Virchow when he noted in 1863 that cancer arose in areas of chronic inflammation (reviewed in [1]). He further speculated that cancer arose from a mature cell type within the peripheral tissue. During this time period, the exact mechanism of the association between inflammation and cancer was debated, as was the cell or origin of tumors. Early arguments for the cell of origin of cancer came from Julius Cohnheim and Wilhelm Waldeyer. These two researchers proposed opposing views of the cancer cell of origin with Julius Cohnheim proposing tumors derived from “embryonal cell rests”, residual embryonic cells “left behind” in the adult organism [2]. Wilhelm Waldeyer on the other hand proposed that the cell of origin of cancer was a differentiated cell within the epithelial or endothelial compartments. The idea that a peripheral differentiated cell could become malignant and form a tumor remained a popular concept for many years. While this concept is still helped by some, emerging evidence supports the notion that a more primitive cell type is likely responsible for tumors. Tissue stem cells which have a wide differentiation potential and tissue progenitor cells which have a more limited repertoire of differentiation potential are emerging as the likely candidates as the cancer initiating cell. The concept of a cancer stem cell finds its origins in the notion that peripheral organs rely on stem cells for growth and renewal. These stem cells are a minority of

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cells as not every cell in the organ is capable of division or multiple lines of differentiation. In the 1950s radiation biologists discovered that lethally irradiated mice could be rescued by reintroduction of whole bone marrow cells. The cells within the heterogenous population of BM responsible for repopulating the bone marrow formed colony units in the spleen, and could be cultured. These colony forming units could be used to repopulate other recipients [3–8] and were found to give rise to all the formed units of the blood, verifying that one cell had the potential to differentiate down multiple lineages and maintain the organ. Since that time, the identity of the hematopoietic stem cell has been clearly defined and this system serves as the gold standard for stem cell identification in other organs. Based on the concept of an organ specific stem cell, and with the recognition of cancer as a complex multi cellular “organ like” structure the concept of a stem cell initiating and propagating cancer emerged. The notion of a less differentiated, more pluripotent (stem) cell forming cancer was taken a step further by G. Barry Pierce [9], who studied mouse teratocarcinomas. His studies were instrumental in forging the notion of the cancer stem cell as we embrace it today. He demonstrated that individual tumor cells were capable of forming normal, non-malignant daughter cells when transplanted into another host. These cancer stem cells maintained stem cell function yet gave rise to daughter cells with varying degrees of differentiation and function which themselves were not capable of propagating tumor growth. Recent work has upheld the notion of a stem cell fueling cancer, and the search is on to identify this cell type in different tumors. Elegant and careful work by several laboratories has provided a phenotype of the cancer stem cells based on surface marker expression. Isolation of the presumed stem cell population by fluorescence activated cell sorting (FACS) or magnetic bead separation has certainly enriched for these elusive cells [10–16], however the prospective identification of a single cell capable of recapitulating the original complex tumor is still not possible for most types of cancer. While it is widely accepted that the cancer stem cell derives from a peripheral tissue stem cell pool, there is accumulating evidence from mouse and human studies that mobilized pluripotent stem cells originating in the bone marrow may seed distant sites of injury and act as the cancer stem cell [17–23], and may contribute to tumor stroma as fibroblasts, myofibroblast and endothelial cells [24–28] and provide crucial signals to the tumor stem cells. What is it about the inflammatory environment that encourages the malignant growth of peripheral organ stem cells and calls in circulating pluripotent stem cells, allowing them to reside within injured tissue? Just as we have experienced an evolution in our views of the cell(s) responsible for propagating tumors, we have also greatly altered our views on the role of inflammation and cancer. Initially the presence of inflammation at sites of cancer was interpreted as the bodies defense against the foreign invader, with leukocytes attacking neoplastic cells. However more recent data indicate that far from a protective role, inflammatory cells participate and drive initiation and promotion of many cancers. Cancer has long been viewed as “the wound that will not heal”. Epidemiologically, chronic inflammatory disorders show a strong association with cancer risk. Many malignancies are

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Cancers associated with infection Epstein barr virus Helicobacter pylori infection Hepatitis B and C virus Human papilloma virus Schistosomiasis Liver fluke infestation Chronic osteomyelitis

Burkett’s lymphoma Gastric adenocarcinoma/MALT lymphoma Hepatocellular carcinoma Cervical and anal carcinoma Bladder cancer Cholangiocarcinoma Osteosarcoma

Cancers associated with inflammation/injury Chronic skin irritation Sunburn Smoking Inflammatory bowel disease Pancreatitis Asbestosis Esophageal reflux

Squamous cell carcinoma Melanoma Lung cancer Colon cancer Pancreatic cancer Mesothelioma Barrett’s adenocarcinoma of the esophagus

initiated by tissue injury or chronic inflammation, which can be linked to known bacterial, viral or parasitic infections [29]. The most well known of these infectious linked cancers include Helicobacter pylori infection and gastric cancer, viral hepatitis and hepatocellular carcinoma, bladder cancer due to schistosomiasis, cervical cancer due to human papilloma virus (Table 11.1). Overall, approximately 15% of malignancies worldwide can be attributed specifically to chronic infections. Many other cancers are initiated by chronic inflammation initiated by agents other than infection and include esophageal adenocarcinoma due to gastroesophageal reflux, colon cancer due to inflammatory bowel disease, and lung cancer due to smoking. These examples likely represent the “tip of the iceberg”, since most cancers do not arise in a normal tissue environment but require some initial degree of tissue alteration which may ultimately tie these cancers to inflammation as well.

11.3 What Does the Chronic Inflammatory Environment Look Like? The chronic inflammatory environment is composed of cells, matrix, secreted cytokines and chemokines. Macrophages, dendritic cells, lymphocytes and to a lesser degree neutrophils chronically infiltrate tissue and secrete a host of inflammatory mediators, enzymes for tissue remodeling and pro-growth factors, creating an environment similar to wound healing but with the notable difference that this wound does not heal. Stem cells are thought to be relatively quiescent. Their progeny – the transient amplifying (TA) cell – is mitotically active and participates robustly in the repair process of most tissues. These cells are longer lived

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than terminally differentiated cells, however they are not immortal, and need to be periodically replaced by the tissue stem cell. There is accumulating evidence that stem cells- even in tissues which are thought to be relatively inactive- can be coaxed back into the cell cycle when needed to replenish the TA population and as such are vulnerable to damage. Therefore, death of normal or injured cells leads to a compensatory increase in proliferation ultimately driven by the resident tissue stem cell. While this increased proliferation heals injury, sustained proliferation predisposes the stem cell population and the proliferating cell pool within the tissue to DNA damage. Damage occurring within a “wound healing” environment can be sustained and cumulative because cells bypasses many of the safety checks which normally would remove these cells. In this situation, damaged cells are allowed to survive and avoid elimination by apoptosis. All inflammatory cells have been implicated in the growth alterations seen in tissue affected by chronic inflammation. Lymphocytes, neutrophils, macrophages and dendritic cells are found in chronic inflammation. Their number and proportions may depend upon the tissue involved as well as the stimulus for inflammation. Cells within chronically inflamed tissues produce a variety of factors linked to oncogenesis. Macrophages are a pivotal player in the chronic inflammatory response because of the vast arsenal of bioactive products they release (Table 11.2). Lymphocytes, endothelial and epithelial cells also release a storm of biologically active agents responsible for growth alterations within inflamed tissue. Further determining the effects of chronic inflammation on the tissue is the quality of the immune response. Host genetic factors dictate the individual cytokine and chemokine response which in turn orchestrates additional homing of inflammatory cells to the area and provides signaling molecules to the epithelium. Several large epidemiological studies demonstrate that polymorphisms within cytokine genes which create a more pro-inflammatory environment increase the risk of cancer in the setting of inflammation, but not in the absence of inflammation [30–32]. Other cellular components of the inflammatory environment include fibroblasts, myofibroblasts and endothelial cells. These cells respond to the inflammatory environment by secreting their own repertoire of growth factors, chemokines and matrix modifying factors, act to lay down new extracellular matrix and participate in angiogenesis

Table 11.2 Factors released by macrophages Cytokines and chemokines (IL-1β, TNF, IL-8) Growth factors (TGF-β, EGF, FGF, PDGF) Reactive oxygen species Nitric oxide Collagenase Elastase Neutral proteases Enzyme lipase Coagulation factors Components of complement Eicosanoids

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and fibrosis. Architectural distortion interrupts crucial cell-cell signaling further disrupting the tissue homeostasis. Overall, this altered environment has dramatic impact on the growth differentiation and maturation of cells. In most tissues, actively proliferating cells within the transit amplifying population give rise to post mitotic cells, which have a limited life span and are replaced thus eliminating them from concern. The transit amplifying cells themselves are longer lived – but they too are eventually replaced. It is the stem cell population which is felt to be continually exposed to the abnormal environment of inflammation and bears the brunt of the adverse effects. To protect themselves, stem cells possess many mechanisms not shared by other cells to maintain genomic stability [33–35]. However, these protective mechanisms are a double edged sword. Mechanisms used to protect the stem cell genome appear to limit the life span of the stem cell pool and contribute to aging. In the face of chronic injury and inflammation, this process is greatly accelerated and the life span of the stem cell appears to be shortened. The consequences of this, is a predisposition to cancer. In the face of DNA damage, stem cells with malignant potential are marked for senescence or apoptosis. Removing these cells leads to a compensatory reaction of remaining stem cells as demonstrated by elegant work from Ruzankina et al. [36]. In their mouse model, the DNA damage response gene Atr was targeted in the adult mouse. Loss of Atr is toxic to proliferating cells resulting in a loss of affected TA cells and stem cells. Due to incomplete recombination, elimination of proliferating cells in this model was not absolute; a small number of proliferating cells did not recombine the Atr allele and therefore survived. The resulting phenotype was both predictable and surprising. Predictably, initial loss of Atr lead to a severe atrophy of the intestinal tract due to the loss of stem cells and TA cells which repopulate this rapidly renewing epithelium. After approximately 4 weeks however, the mice regained normal intestinal histology with the formerly atrophic epithelium repopulated by the remaining Art- competent cells. These data demonstrate that the peripheral tissue stem cell can divide symmetrically to expand the stem cell pool, and these stem cells can migrate to populate adjacent areas within the epithelium and reconstitute dwindling stem cell pools which have been injured or removed. The surprise came when these mice were followed for longer times. Interestingly, the mice rapidly aged with graying of the fur, osteopenia and decline in hematopoietic function suggesting that the forced replication of the remaining stem cells compromised their long term function and viability. If we compare this animal model to chronic inflammation, we can see how the constant push to replicate in order to repair damaged epithelium may deplete stem cell function and stem cell pools. Initially, adjacent stem cells can take over this function, however as inflammation progresses, this compensatory function may be overwhelmed and the stem cell compartment unable to continue multilineage differentiation. This loss of function and number likely underlies atrophy of the tissue so commonly seen with longstanding inflammation. This puts the peripheral tissue at risk of inadequate healing, and also may set the stage for recruitment of more pluripotent stem cells (i.e. from the bone marrow) to repopulate the “empty niche” [37]. Stem cells native to the tissue

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or recruited to the niche to replace dead or damaged stem cells are subjected to the inflammatory environment. Overall, the progression of several types of cancer is determined by the severity of the inflammatory response which is regulated by NF-κB. Many of the key pro-inflammatory cytokines, such as IL-6, IL-8, IL-1β are TNF-α, are encoded by target genes of the IKKb-dependent NF-κB-activation pathway. Recent studies have demonstrated in vivo the importance of macrophage-derived NF-κB expression [38]. Using an ulcerative colitis model of colorectal cancer, they showed that disruption of the NF-κB pathway in myeloid cells resulted in a marked reduction in expression of IL-1β, IL-6, and MIP-2 and a significant reduction in colon cancer. A similar reduction in tumor incidence was seen when IKKb was deleted in the myeloid lineage in a mouse model of hepatocellular cancer, further supporting these findings [39]. Proinflammatory factors act alone and in concert to create an environment conducive to perpetuating the inflammation and creating a pro-neoplastic niche. Two specific factors stand out as central players. TNF is a major mediator of inflammation stimulating both tissue destruction and repair. TNF mediated apoptosis removes injured cells from areas of damage while the same signaling stimulates fibroblast growth geared toward tissue remodeling. There is evidence that while terminally differentiated cells undergo apoptosis through TNF signalling – TNF may protect some stem cell populations from apoptosis [40]. In addition to this protection, mesenchymal stem cells are directly stimulated by TNF to produce VEGF, FGF2, HGF, and IGF-1 by an NF-κB dependent mechanism [41]. As part of this double edged sword of injury/repair, TNF both devastates and rebuilds blood vessels via induction of angiogenic factors [42–44]. However, newly formed vessels are often not able to provide adequate oxygen supplies to the tissue and a situation of relative hypoxia is created and perpetuated. Once cancer is established, TNF appears to act directly on cancer cells to affect the migration, adhesion and invasion of cells [45, 46]. IL-1 is secreted by macrophages, neutrophils and epithelial cells within the chronic injured/inflamed tissue and synergizes with TNF for its multiple functions including its proinflammatory effects and effects on vascular permeability. Polymorphisms within the IL-1β gene which are associated with relatively higher tissue levels of IL-1β have been linked with a more severe inflammation and tissue damage secondary to agents such as Helicobacter pylori, and are also linked with a higher incidence of gastric cancer in infected patients [30]. In a similar fashion, Japanese patients with chronic hepatitis C have a higher incidence of hepatocellular carcinoma if they have a specific IL-1β polymorphism, which confers a greater proinflammatory profile. IL-1β induces PGE2 production, hepatocyte growth factor and increased Th1 type cytokines; all linked with higher incidence of hepatocyte transformation [32]. TNF, IL-1, IL-6, and TGF-β induce production of the angiogenic factor VEGF from stromal cells (which may be bone marrow derived) from resident stem cells and from tumor cells. Neovascularization into damaged and repairing tissues alters the oxygen tension of tissues and can set up relative hypoxia. Tissue hypoxia

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induces a host of cellular alterations and regulates survival programs which may further subvert growth of cells in favor of allowing genetically damaged cells to survive [47]. Indeed, in vitro studies evaluating the effects of relative hypoxia on mesenchymal stem cell function show that oxygen concentrations affects many aspects of stem-cell physiology, and may be a critical parameter during wound healing as expansion and differentiation of stem cells continues in the presence of hypoxia long after normal constraints of contact inhibition and cell number would have induced growth arrest [48].

11.4 What are the Long Term Effects of Chronic Inflammation? Chronic inflammation dramatically alters the local cytokine network, influences apoptotic and proliferative programs, directs differentiation and remodeling. This environment directly, and via impact on cell-cell signaling induce epigenetic changes, and also directly lead to heritable genetic changes of cells (Table 11.3). As discussed previously, this abnormal milieu promotes apoptosis of cells leading to a compensatory proliferative response by the remaining tissue. Chronic injury and inflammation over extended periods of time results in a sustained expansion of tissue proliferative zones and predisposes to neoplastic progression [49]. Interestingly, the architectural changes that occur during the progression of chronic inflammation share striking similarities between tissues. Atrophy of the tissue is a common precursor of inflammatory mediated cancer in organs such as the stomach [50], pancreatic [51] and prostate [52]. Atrophy, which is the loss of the normal architecture and a drop out of mature cell types, represents the severe blow to the resident stem cells that inflammation delivers. Evidence of damage to the stem cell in the form of abnormal differentiation can be seen as metaplasia- the replacement of one adult cell type with another. Metaplasia can be the result of environmental influences on cellular differentiation and may be reversible when the environment is returned to

Table 11.3 Cellular effects of chronic inflammation Reactive oxygen species leading to DNA damage NO production leading to DNA mutations, alterations in DNA repair Tissue hypoxia leading to alterations in gene regulation and angiogenesis Inactivation of tumor suppressor genes Upregulation of anti-apoptotic genes Induction of pro-proliferation programs Activation of tumor promoters Inactivation of tumor suppressors Increased vascular permeability Angiogenic factors leading to increased neovascularization Tissue remodeling secondary to collagenases, MMPs Alteration in cell – cell adhesion Alterations in cell mobility

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its usual state. Many times however, metaplasia is incomplete with cells partially taking on characteristics of another cell type, and these changes may be irreversible with removal of the stimulating environment. This permanent change in tissue architecture and cellular differentiation may represent permanent changes to the stem cell. This evidence of a presumed preneoplastic permanent change to the stem cell compartment further supports the resident stem cell as the cancer stem cell, and demonstrates the effect of the inflammatory environment on this cell type. If we look to the stomach as an example, Helicobacter infection causes a chronic gastritis, which places the mucosa at risk for developing gastric cancer. The sequence of events and the histological alterations are well described in man and in mouse models [53–56]. Chronic inflammation of the gastric mucosa progresses through stages of hypertrophy, atrophy, metaplasia, dysplasia and finally adenocarcinoma. It is not clear if one stage leads to the next- for example, is metaplasia a precursor lesion of dysplasia, and is dysplasia a precursor of adenocarcinoma? Conversely, these changes may mark a field of genetic alterations and signaling conducive to cancer induction, but the cellular changes themselves may not be directly premalignant [53].

11.5 Inflammation and Tumors – Inflammations Many Roles Recent work suggests that inflammation may contribute to all phases of cancer; initiation, promotion, progression and cancer metastasis. We have addressed initiation of cancer via the pro-proliferative inflammatory environment combined with the DNA and protein damaging effects. Progression and metastasis can be viewed in a similar fashion. Mesenchymal stem cells localize to tumors as they form and progress, contributing to the local growth of cells. Studies addressing the role of BM derived MSC in breast cancer show that these bone marrow derived stem cells enhance mobility and invasiveness of cancer cells through production of RANTES [27]. Though themselves not malignant, the stromal stem cells are intimately involved in the signaling and fate of the cancer stem cells through the local environment they create. The importance of the niche created for the cancer stem cells is further stressed when one looks to sites of metastasis. Contrary to the commonly held notion that cancer cells gain access to the circulation and bully their way into peripheral tissues to form metastatic disease; new data suggest that these circulating cancer cells are invited in. Elegant work from the laboratories of Rafii and Lyden demonstrate that sites of metastatic tumors are set up before the arrival of any tumor cells [26]. The details are still being worked out. It is likely that tumors arising in different organs secrete a unique signature of factors which act on peripheral tissues to orchestrate the specific pattern of metastasis peculiar to each tumor type (for example, melanoma metastasizes widely, colon tumors metastasize to liver and prostate cancer metastasizes preferentially to bone). Factors such as VEGF-A, TGF-β and TNF-α elaborated by primary tumors [57–59] act distally to activate local inflammatory and mesenchymal cells to upregulate expression of the chemoattractants S100A8 and S100A9.

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Coincident with this, bone marrow derived VEGFR1+ VLA-4+ [26] or Mac1+ cells [57] are recruited to set the stage for recruiting in S100-responsive tumor cells. While the precise details are still not clear, it looks as if our understanding of and approach to metastatic disease will be very different than it has been in the past. Based on these new findings, metastasis can be viewed as an active process whereby local tumor cells or tumor niche cells elaborate factors which circulate and effect changes in peripheral organs. Environments conducive to growth of metastatic tumors are set up long before tumor cells arrive providing targets which may be exploited for anti- tumor treatments. Many tissue stem cells express CXCR4, a G-protein-coupled seven span transmembrane receptor. CXCR4 is responsible for regulating trafficking of normal hematopoietic stem cells and their mobilization and homing to the bone marrow [60–62]. Additionally, it is thought that CXCR4-SDF-1α is used during embryogenesis for appropriate cell migration [63]. The ligand for CXCR4 is the alpha-chemokine stromal derived factor (SDF-1α). This ligand-receptor pair is unusual in that they bind each other exclusively. It is well known that HSC depend upon an SDF-1α gradient to home back to the marrow cavity for circulation. Indeed, agents used to mobilize BM cells for transplant purposes do so by disrupting the SDF-1α/CXCR4 axis [64, 65]. In like fashion, successful engraftment of the bone marrow transplant relies heavily on an intact SDF-1α/CXCR4 signaling axis. Similar to their non-malignant stem cell counterparts, many cancer cells express CXCR4. Like the hematopoietic stem cells, it is believed that cancer stem cells use the CXCR4 receptor to mobilize, invade and metastasize [66] further supporting that cancer stem cells derive from a tissue stem cell source. The function of CXCR4 receptor on normal or malignant cells appears to be multifactorial and is modulated by several components of the inflammatory environment. CXCR4 is regulated at the molecular level by factors related to stress and tissue damage including NF-κB [67], HIF-1 [68], TGF-β [69], VEGF [70], IFNα [71] and IL-2, IL-4 and IL-7 [72, 73] leading to increased surface expression. CXCR4 and Rac1 incorporate into lipid rafts [74]. Factors which affect the cholesterol content of the cell membrane may therefore significantly affect the response of the CXCR4 receptor to SDF-1α. This may explain the seeming decrease cancer incidence associated with the statin drugs [75, 76] which inhibit intracellular cholesterol synthesis and therefore membrane cholesterol levels. Proteases may cleave SDF-1α to an inactive form and antagonize CXCR4-SDF1α activities [77] and acts to retain CXCR4 bearing cells in tissues. SDF-1α does more than act as a chemotactic factor for CXCR4 bearing cells. SDF-1α has been shown to induce adhesion, and induce secretion of MMPs and VEGF in cells bearing CXCR4 [78–81] and increase interaction with several integrins [79]. SDF-1α may also stimulate proliferation of several cell types - including cancer cells [82]. SDF-1α levels are highest in bone, lung, liver and lymph nodes and may direct metastasis of CXCR4 bearing cancer cells to these locations [82–89]. SDF-1α is increased in inflamed tissues and may act to attract marrow derived stem cells to the area to participate in neovascularization, stroma formation and engraftment into the stem cell niche for repair [90].

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11.6 Summary The inflammatory environment dramatically impacts the cancer stem cell at many levels from the environment which fosters the initiation of cancer through its contribution to metastatic disease (Fig. 11.1). Early on, chronic inflammation exhausts

Fig. 11.1 Role of the inflammatory environment in cancer stem cell activity. (a) An environment of chronic inflammation stimulates proliferation of the involved tissue. Over time however, the tissue stem cell pools (green cells) are exhausted and atrophy results. Migration of adjacent stem cells, or recruitment of BMDC to the stem cell niche (red cells) allows recovery of stem cell function. In the presence of inflammatory mediators, abnormal signaling may occur as evidenced by metaplastic differentiation of cells. (b) Activated fibroblasts contribute to the stem cell niche. Continued exposure of cells to the inflammatory environment leads to DNA damage and epigenetic changes. (c) Once established, cancer stem cells orchestrate their own migration and metastasis by the release of soluble factors which are responsible for recruiting BMDC to peripheral sites to set up a pre-metastatic niche conducive to incoming cancer cells

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tissue stem cells, forcing remaining stem cells work overtime, and calling in replacement cells from marrow sources. Marrow derived stromal cells orchestrate growth and remodeling through secreted factors and cell-cell communication. Once cancer is present, the inflammatory environment is responsible for the continued growth signals to the cancer stem cells and to the stromal cells which become a vital part of the cancer niche as well as the premetastatic niche which will effectively lure cancer cells into peripheral organs for distant growth. This understanding of the inflammatory environment and its many effects on cancer throughout its natural history provides intervention targets directed at unique aspects of cancer behavior.

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Chapter 12

Does the Chronically Inflamed Periodontium Harbour Cancer Stem Cells? Wolf-Dieter Grimm, Wolfgang H. Arnold, Sebastian Becher, Aous Dannan, Georg Gassmann, Stathis Philippou, Thomas Dittmar, and Gabor Varga

Abstract Trials aiming at isolating cultures and subcultures of cells from the periodontal ligament to uncover special features of such tissue-residing population are related to the past decade [1]. Moreover, the idea of cell-delivery systems, and especially with stem cells, to initiate periodontal regeneration is not new. However, describing suitable animal models for such new techniques is less documented in the literature and sometimes underestimated. The wide range of animal species allows appropriate selection of bio-models for different investigations. Each species has unique similarities and dissimilarities to humans. While many studies could ensure the initiation of periodontal regeneration using stem cells extracted from the periodontium, seeding of human periodontium-derived stem cells (pdSCs) on collagen carriers could induce major features of periodontal regeneration when implanted in experimental periodontal defects in the athymic immunodeficient rat as could be shown in our studies. However, remarkable notifications regarding the results obtained in our studies were the induction of malignant tumors (squamous cell carcinoma) in the majority of investigated animals. Considering the data presented in the literature, our studies seems to be the first that demonstrates the initiation of malignant tumors when using human pdSCs. The patients from whom the pdSCs had been extracted, the animal model used and a possible oncogenic alteration of the pdSCs themselves might all be factors behind the tumors’ initiation. In our present studies, the animal model used and the related experimental periodontal defect, as a heterologous model, could successfully present two important biological features after implantation of pdSCs; namely periodontal repair and tumorigenicity. Although not fully histo-morphometrically assessed in our studies, these features are very important for further investigations and for future more-controlled studies. However, further studies using advanced histological, immunological and genetic techniques are required to assure different supposals presented in the current studies regarding stem cell-based periodontal regeneration and stem cell-tumorigenesis.

W.-D. Grimm (B) Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448Witten, Germany e-mail: [email protected] T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_12,

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Keywords Periodontium · Animal models periodontium-derived stem cells (pdSCs) · Tumorogenicity neural stemness markers · Minimally-invasive periodontal surgical cells · Osteogenic differentiation

W.-D. Grimm et al.

in periodontal research · Human Athymic immunodeficient rat · Periodontal tissue regeneration · therapy · Mesenchymal stem

Contents Introduction . . . . . . . . . . . . . . . . . . . . Stem Cells and Periodontal Tissue Regeneration . . . . Animal Models in Periodontal Research . . . . . . . Tumor Induction Mediated by pdSCs? . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . 12.5.1 Stem Cells in the Periodontium . . . . . . . . 12.5.2 The Periodontal Defect . . . . . . . . . . . 12.5.3 Source of pdSCs . . . . . . . . . . . . . . 12.5.4 Special Features . . . . . . . . . . . . . . . 12.5.5 Considering the Nude Rats . . . . . . . . . . 12.5.6 pdSCs and Tumor Initiation . . . . . . . . . 12.6 Conclusion . . . . . . . . . . . . . . . . . . . . 12.6.1 Periodontal Stem Cells as Functional Elements of Regenerative Periodontology . . . . . . . 12.6.2 Possible Periodontal Tissue Regeneration . . . References . . . . . . . . . . . . . . . . . . . . . . .

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12.1 Introduction Stem cells are one of the most fascinating areas of biology today. But like many expanding fields of scientific inquiry, research on stem cells raises scientific questions as rapidly as it generates new discoveries. Stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. A stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell); it cannot carry molecules of oxygen through the bloodstream (like a red blood cell); and it cannot fire electrochemical signals to other cells that allow the body to move (like a nerve cell). The term “adult stem cell” refers to any cell which is found in a developed organism that has two properties: the ability to divide and create another cell like itself and also divide and create a cell more differentiated than itself. Also known as somatic (from Greek ωματικóζ, “of the body”) stem cells and germline (giving rise to gametes) stem cells, they can be found in children, as well as adults [2]. Pluripotent adult stem cells are rare and generally small in number but

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Fig. 12.1 Histological section (Azan staining) of the periodontal structures depicting acellular-fibrillar cementum, connective tissue fibres containing blood vessels and alveolar bone

can be found in a number of tissues including umbilical cord blood [3]. Most adult stem cells are lineage-restricted (multipotent) and are generally referred to by their tissue origin (mesenchymal stem cell, adipose-derived stem cell, endothelial stem cell, etc.) [4, 5]. Recently, it has been found that specific populations of stem cells and/or progenitor cells could be isolated from three main dental resources, namely the dental follicle, the dental pulp and the periodontal ligament. The periodontal ligament is a group of specialized connective tissue fibers that essentially attach a tooth to the alveolar bone within which it sits. These fibers help the tooth withstand the naturally substantial compressive forces which occur during chewing and remain embedded in the bone (Fig. 12.1). Another function of the PDL is to serve as a source of proprioception, or sensory innervations, so that the brain can detect the forces being placed on the teeth and react accordingly. To achieve this end, there are pressure sensitive receptors within the PDL which allow the brain to discern the amount of force being placed on a tooth during chewing, for example. This is important because the exposed surface of the tooth, called enamel, has no such sensory receptors itself. In addition to the PDL fibers, there is another set of fibers, known as the gingival fibers, which attach the teeth to their adjacent gingival tissue. Both the gingival fibers, as well as the PDL fibers, are composed primarily of type I collagen. The periodontal ligament is derived embryologically from the ectomesenchymal tissue of the dental follicle that surrounds the developing tooth in its bony crypt. At the time of tooth eruption the cells and collagen fibers in the dental follicle, i.e. the future periodontal ligament, are orientated primarily with their long axis parallel to the root surface. Remodeling of the follicle into a periodontal ligament begins at the cemento-enamel junction and proceeds in an apical direction. The periodontal ligament contains a unique assortment of cells that are capable of generating and

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maintaining three distinct tissues, namely the ligament itself as well as the mineralized tissues on either side of it, i.e. the cementum and the alveolar bone. The major cell types of the periodontal ligament include: Fibroblasts, macrophages and undifferentiated ectomesenchymal cells, cementoblasts and cementoclasts, osteoblasts and osteoclasts, cell rests of Malassez, vascular and neural elements. PDL contains heterogeneous cell populations [6, 7] that can differentiate into either cementumforming cells (cementoblasts) or bone-forming cells (osteoblasts) [8–11]. Recent findings suggest that PDL cells have many osteoblast-like properties, including (1) the capacity to form mineralized nodules in vitro, (2) expression of the boneassociated markers alkaline phosphatase and bone sialoprotein, and (3) response to bone-inductive factors such as parathyroid hormone, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein 2 (BMP2) and transforming growth factorβ1 (TGF-β1) [6, 12–16]. The presence of multiple cell types within PDL has led to speculation that this tissue might contain progenitor cells that maintain tissue homoeostasis and regeneration of periodontal tissue [8, 17–19]. Using a methodology similar to that utilized to isolate mesenchymal stem cells (MSCs) from deciduous and adult pulp, multipotent postnatal stem cells from human periodontal ligament or Periodontal Ligament Stem Cells (PDLSCs) have also been described [20–29]. Cultured cells were expanded from single cell suspensions derived from periodontal ligament tissue and the presence of stem cells was determined using

Fig. 12.2 Morphology of isolated PDLSC culture, passage 2. Upper right: first day, immediately after passage with trypsin-EDTA solution and followed by replating the cells. Upper right: 3 h later the cells are already attached to the surface. Lower left: 3 days old subconfluent state. Lower right: after 5 days of culture the cells are totally confluent. 200x magnification

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antibodies such as STRO-1 and CD146. Under defined cultured conditions, PDLSCs differentiated into cementoblast-like cells, adipocytes and collagen-forming cells (Fig. 12.2). Immunohistochemical staining and Western blot analysis showed that cultured PDLSCs express an array of cementoblastic/osteoblastic markers, including alkaline phosphatase, bone sialoprotein, osteocalcin and TGF-β1 receptor. The cells also expressed scleraxis, a tendon transcription factor and under certain conditions become adipogenic. These PDLSCs were transplanted into artificially created periodontal defects in the mandibular molars in rats. Histological evaluations 6–8 weeks after implantation showed that these cells had the capacity to generate a thin layer of cementum-like tissue on the surface of the hydroxyapatite/tricalcium phosphate ceramic particles carrier, along with condensed collagen fibers that resembled Sharpey’s fibers [27]. The presence of MSCs in the periodontal ligament is also supported by other findings where a population of MSCs from the periodontal ligament has been isolated and characterized showing the ability to express a variety of stromal cells markers (CD90, CD29, CD44, CD166, CD105, CD13) [26, 30]. The clinical potential for the use of periodontal ligament-derived stem cells has been further enhanced by the demonstration that these cells can be isolated from cryopreserved periodontal ligaments, thus providing a ready source of MSCs [28]. Moreover, putative stem cells in both healthy and diseased periodontal ligament could be identified [20]. They were mainly located in the paravascular region and small clusters of cells were also found in the extra-vascular region. Wider distributions of these cells were detected in sections of diseased ligament. In a recent study, our research group described a method for serum-free culture, rapid expansion and subsequent efficient induction of neuronal phenotype in the

Fig. 12.3 Blow-up image of pdSCs’ neurosphere

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periodontium-derived stem cells (pdSCs) [31] (Fig. 12.3). The isolated, cultured, highly proliferative cells were positive for the neural stemness markers Nestin and Sox2 and negative the for the differentiation markers β-III-tubulin for neurons and GFAP for glial cells. In addition, the pdSCs migrated when exposed to chemokines that are reported to be migration-inducing in neural stem cells isolated from subventicular zone. Retinoic acid treatment efficiently induced neuronal fate in pdSCs as shown by the high levels (>90%) of neuron-specific markers such as β-III-tubulin, MAP2 and neurofilaments M, H and L. When they were cultured in the presence of 10% FBS, pdSCs differentiated into a glial lineage, as demonstrated by glial morphology and robust GFAP expression after four days. However, the capacity of these periodontium-derived neural stem cells to function in vivo remains unresolved.

12.2 Stem Cells and Periodontal Tissue Regeneration The immune-inflammatory response that develops in the gingival and periodontal tissues in response to the chronic presence of plaque bacteria results in destruction of structural components of the periodontium, leading, ultimately, to clinical signs of periodontitis [32]. This condition induces the breakdown of the tooth-supporting structures until teeth are lost [33]. To stop the progression of the disease and to regenerate the lost tissue, it may be necessary to intervene surgically. A number of surgical techniques have been developed to regenerate periodontal tissue, including guided tissue regeneration [34–36], bone grafting [37, 38] and the use of enamel matrix derivative [39–42]. However, recent developments in the field of tissue engineering and regeneration suggest that it may eventually be possible to replace or repair damaged tissue [43, 44]. Periodontal regeneration requires new connective tissue attachment to the root surface, a process that involves the regeneration of periodontal fibers and the insertion of these fibers into newly formed cementum [45, 46]. Unfortunately, currently available regeneration techniques are clinically unpredictable, resulting in only partial regeneration at best [47–49]. Yet another approach, known as guided-tissue regeneration, has been developed to achieve periodontal regeneration. This utilizes barrier membranes to guide and instruct the specialized cellular components of the periodontium to participate in the regenerative process. The guided-tissue regeneration concept was founded on sound scientific research and is based on the premise that the periodontal ligament contains all the progenitor cells required for the formation of bone, cementum and periodontal ligament [47–49]. Tissue engineering is defined as the reconstruction of living tissues to be used for the replacement of damaged or lost tissue/organs of living organisms and is founded on the principles of cell biology, developmental biology and biomaterials science [50, 51]. However, this process may require a team effort engaging the expertise of biomaterials engineers, cell biologists, matrix biologists, molecular biologists, microbiologists, immunologists, pharmacologists, and nanotechnologists [52].

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Fig. 12.4 Tissue engineering determinants in periodontics

Successful periodontal tissue engineering requires the interplay among three components: the implanted and cultured cells that will create the new tissue; a biomaterial to act as a scaffold or matrix to hold the cells; and biological signaling molecules that instruct the cells to form the desired tissue type [53] (Fig. 12.4). From a biological perspective, current and future prospects for improved regeneration of periodontal tissues are dependent on the ability to facilitate the repopulation of the periodontal wound by cells capable of promoting regeneration. From this perspective, the periodontal ligament has been shown to be of critical importance in the regenerative process. It has been demonstrated that only the periodontal ligament, but not the gingival connective tissue or bone, contains cells capable of establishing new attachment fibers between cementum and bone [54, 55]. It has also been shown that gingival fibroblasts grown in vitro failed to contribute to reformation of periodontal ligament around teeth implanted in dogs, while periodontal ligament fibroblasts were observed to either contribute to regeneration or at least not to inhibit regeneration [18]. The ability of periodontal ligament cell populations to achieve regeneration has implied that progenitor cells and possibly stem cells exist within the periodontal ligament. Although it is clear that cells residing in the periodontal ligament can achieve regeneration, this population is heterogenous [56] and it is not known which subpopulations are capable of achieving regeneration. Indeed, cells derived from regenerating defects were found to have specific properties, such as increased proliferation rates, representative of a regenerative phenotype, and distinct from periodontal ligament cells [57]. In order to understand the cellular origins of the developing periodontal attachment apparatus, transplanted tooth buds were used to show that the mesenchymalderived dental follicle surrounding the developing tooth root is the source of

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progenitor cells for cementum, alveolar bone and periodontal ligament [58, 59]. Further evidence, that cells from the dental follicle are precursors to cementoblasts, has been shown by direct cell migration in the rat molars [60, 61]. More recently, the dental follicle associated with third molars has been shown to contain precursor cells, which are clonogenic and which have the ability to differentiate under in vitro conditions to a membrane-like structure containing calcified nodules [62]. The source of post-natal progenitor cells which may be capable of regenerating the periodontium has been investigated for a number of years. Cell kinetic experiments in mice and rats [63, 64] have shown that periodontal ligament fibroblast populations represent a steady-state renewal system, with the number of new cells generated by mitosis equal to the number of cells lost through apoptosis and migration [11]. This capacity for self-renewal, which is further evidenced by the rapid turnover of the periodontal ligament, supports the notion of progenitor/stem cell populations. Furthermore, a significant number of periodontal cells do not enter the cell cycle [65], suggesting that these cells may act in a similar manner to quiescent, self-renewable and multipotent stem cells. The relationship between progenitor cells in regenerating tissues and normally functioning (steady-state) tissues has been investigated in studies performed in normal mouse periodontal ligament [66], wounded mouse periodontal ligament [8], normal rat gingiva [67] and inflamed monkey gingiva [68]. These studies have identified a common paravascular location for fibroblast progenitors. These cells exhibit some of the classical cytological features of stem cells, including small size, responsiveness to stimulating factors, and slow cycle time [9, 11, 66]. Furthermore, these paravascular cells exhibit spatial clustering, which suggests a possible clonal distribution of progenitors and their progeny [66]. Other possible sources of osteoblast and cementoblast precursors are the endosteal spaces of alveolar bone from which cells have been observed to adopt a paravascular location in the periodontal ligament of mice [69]. Through a combination of transplanted biomaterials containing appropriately selected and primed stem cells, together with an appropriate mix of regulatory factors and extracellular matrix components to allow growth and specialization of the cells, new therapies of significant clinical potential in periodontal research are emerging [70]. However, finding a standard animal model to better investigate periodontal tissue regeneration and other biological features in the periodontium is always an important priority.

12.3 Animal Models in Periodontal Research Animal models have been used to evaluate the pathogenesis of periodontal diseases and various periodontal treatment modalities. Human longitudinal studies of periodontal diseases pose many problems such as determining the level of disease activity, individuals at risk, and susceptibility to disease progression.

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From the viewpoint of comparative biology, non-human primates are similar to humans, having comparable periodontal tissue structures and healthy and diseased periodontal states, as observed in humans [71]. However, most non-human primates used for research purposes are large, expensive, and difficult to handle. Furthermore, the genetic background of many of these animals has not been established, because animals used in research are often wild-captured animals, with heterogeneity in age, body weight, and oral and general health conditions [71]. Among the species of non- human primates, squirrel monkeys and marmosets are small in size and relatively easy to handle, but unfortunately do not exhibit an inflammatory profile characteristic of human periodontal disease. Periodontal tissue specimens from these animals, unlike humans, exhibit very limited numbers of lymphocytes and plasma cells [72–74]. Rodents, belonging to the cohort Glires, such as mice, rats, and hamsters, have been used widely for periodontal research because of specific advantages such as small size, low cost, known age and genetic background, controllable microflora, and ease of handling and housing [75]. However, anatomical structures of periodontal tissues and histopathological features of periodontal disease of rodents are different from those of humans [75]. For example, oral sulcular epithelium is keratinized in rodents, but not in humans [76]. Neutrophil Granulocytes appear to be the only infiltrating cells in periodontal lesions of rodents. In contrast, periodontally involved human tissues show a complex infiltrate of lymphocytes, plasma cells, macrophages and Neutrophil Granulocytes [75]. Suggested reasons for these histological variances include the possibility of some fundamental differences in host responses, or at least in part, some divergence in the reaction of tissues to specific challenges between rodents versus humans [75, 77]. It is clear that efforts to find a standard animal model, which better represents the periodontal disease state in humans, the periodontal regeneration actions and other histological and morphological features would be advantageous for researchers focusing in this area. Recently, many studies investigated deeply the location of stem cells in the periodontium [20, 22, 24, 26–28, 31, 78–80]. Moreover, using different animal models like immunocompromised mice, immunodeficient rats, sheep and mini-swine, the ability of periodontium-derived stem cells to regenerate periodontal tissues has also been investigated [21, 23, 27, 28, 41, 81–84]. Collectively, all these studies demonstrate the feasibility (and potential) of using a combination of periodontium-derived stem cell populations for functional periodontal regeneration. However, in these studies, detailed description of the animal model in particular was somehow of less importance. Moreover, other species (e.g. athymic rats) were less used as animal models in the field of stem cell-based periodontal regeneration.

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Fig. 12.5 Minimallyinvasive isolation of periodontal tissues during periodontal regenerative surgical therapy (Areas of tissue containing pdSCs are marked with black arrows)

12.4 Tumor Induction Mediated by pdSCs? In our studies we isolated pdSCs from patients with periodontal disease by minimally-invasive regenerative surgical therapy as described previously [31, 85]. Periodontal tissues were scraped from the exposed root surfaces. This was done in different parts of the mouth (upper or lower jaw) according to every case separately wherever an open flap surgery is indicated. The size of the removed tissues from the periodontal area (i.e. marginal periodontal tissues), which supposedly contain pdSCs was approximately 2 × 3 × 3 mm per patient. The stem ness of pdSCs was verified by RT-PCR analysis (determination of several stem cell marker, such as Sox2), flow cytometry and differentiation studies (e.g., into neuronal cells, such as Glia cells) [31]. Isolated pdSCs were expanded for 10–14 days under serum-free conditions. For implantation of pdSCs into athymic rats, bilateral artificial defects in the root of the first mandibular molar were prepared via an extraoral access to minimize oral bacterial penetration (Fig. 12.6). The artificial bone defect was prepared under sterile irrigation using a micro drill with round dental bur (2.3 mm Ø) on both sides of the mandible, whereby the right mandible served as the test side (implantation R ]) and the left of 1 × 105 pdSCs embedded within a collagen sponge [Opti Maix mandible was used for control purposes (only application of an collage sponge). At the time point of sacrificing the animals, the first group (sacrificed at week 2) did not show any distinctive features, whereas two animals from the second group (sacrificed at week 6) and all the animals of the third group (sacrificed at week 8) presented remarkable tissue enlargements exactly at the right mandible (test side). These tissue enlargements were sub-cutaneous, a little bit hard when palpated and, in some animals, distributed toward the neck area with different sizes (Fig. 12.7). Conjointly, animals with these tissue enlargements suffered from weight loss (about 40% of the original weight) and seemed to be fatigued.

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Fig. 12.6 Minimally-invasive isolation of periodontal tissues during periodontal regenerative surgical therapy (Areas of tissue containing pdSCs are marked with black arrows). Initiation of the surgical incision along with the inferior edge of the mandible (marked with white lines). The transmuscular incision through the masseter exposing the mandibular boneregion (1) Skin layer, (2) Muscular layer, (3) Bone surface, (4) Mental nerve (Nervus mentalis). (a–d) Bone defect reaching the distal roots of the first molar (black arrow), (e–f) Application of the collagen sponge in the artificial defect

Analysis of decalcified paraffin sections of test side mandibles of animals without tissue enlargements (animals of the first group (week 2) and two animals of the second group (week 6)) revealed a reformation of PDL-like tissues, elements of bone and osteocyte-lacunae in the bone tissue concomitant with blood vessels and collagen fibers (Fig. 12.8). However, analysis of decalcified paraffin sections of test side mandibles of animals with tissue enlargements (two animals of the second group (week 6) and all animals of the third group (week 8)) revealed a tissue that contained remarkable invasion of undefined cell types of epithelial-origin, very slight bone elements and

Fig. 12.7 Rats presented tumors on the test sides at W6 and W8 after pdSCs implantation (in this figure, only 2 rats are demonstrated)

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Fig. 12.8 (a) Histological section taken from a test site which clinically showed no tumor (∗ ) (orig mag ×2.5), (b) Area “B” under higher magnification showing bone tissue elements (BT), osteocyte-lacunae features (white triangles), callus of cells (CCs), periodontal ligament-like tissue (PDL), collagen fibers (black triangles) and blood vessels (black arrow) (orig mag ×40) (DHT: Dental Hard Tissue, DP: Dental Pulp, GT: Gingival Tissue, BT: Bone tissue elements) and Area “B”: The area where collagen sponge with pdSCs was transplanted

some collagen fibers. Moreover, a detailed pathologic analysis demonstrated that tissue enlargements could be identified of being a type of anaplastic squamous epithelial-cell carcinoma (Fig. 12.9). The weight of dissected tumor tissues of the third group varied from 0.5 g (rat #3.2) to 6.0 g (rat #3.3) (see also Fig. 12.7). Immunohistochemical analysis clearly identified human mitochondria in rat tumor tissue (Fig. 12.10) suggesting that the cancer tissue might have originated from implanted pdSCs. Whether expanded pdSCs possess tumor initiation capacity remains unclear since tumors have only arisen from cells derived from one donor. Karyotypic analysis of putative tumorigenic pdSCs revealed an aneuploidy karyotype with chromosome counts peaking at 70 chromosomes (Fig. 12.11). It is well recognized that aneuploidy has been correlated to tumorigenesis [86–88], whereas other data indicate that aneuploidy not only acts oncogenically, but also as an tumor suppressor [89], thus preventing cancer formation.

12.5 Discussion 12.5.1 Stem Cells in the Periodontium In spite of the advances in the knowledge of adult stem cells (ASCs) during the past few years, their natural activities in vivo are still poorly understood. Mesenchymal

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Fig. 12.9 (a) Histological section taken from a test site which clinically showed tumor formation (∗ ), (b) Area “C” under higher magnification showing little bone tissue elements (BT), osteocyte-lacunae features (white triangles), area of dense infiltration with epithelial-like cells (EC) interrupted by fillets of collagen fibers (black triangles), (c) Part of area “C” under higher magnification showing dense diffuse infiltration of undefined malignant, non-small epithelial-like cells"(arrows) and collagen fibers (triangles) (DHT: Dental Hard Tissue, DP: Dental Pulp, GT: Gingival Tissue, MFs: Muscular Fibers) and (Area “C”: The area where collagen sponge with pdSCs was transplanted)

stem cells (MSCs), one of the most promising types of ASCs for cell-based therapies, are defined mainly by functional assays using cultured cells. The identification of the MSC niche is necessary to validate results obtained in vitro and to further the knowledge of the physiological functions of this ASC. Here we show an analysis of the evidence suggesting a periodontal wound regeneration for pdSCs and present an animal model in which the created periodontal defect is the MSC niche in vivo, where local cues coordinate the transition to progenitor and mature cell phenotypes. This view connects the pdSCs to the immune and vascular conditions of the periodontium, emphasizing its role as a physiological integrator and its importance in periodontal tissue repair/regeneration.

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Fig. 12.10 Immunohistochemical staining of the tumor extracted from the rat#3.3 showing the presence of human mitochondria antigens (stained red) among the rat cells (stained green)

The periodontium is an unusually complex tissue comprised of two hard (cementum and bone) and two soft (gingiva and periodontal ligament) tissues. Once damaged, the periodontium has a limited capacity for regeneration. During the early phases of periodontal disease some minor regeneration of the periodontium may be seen. However, once periodontitis becomes established, only therapeutic intervention has the potential to induce regeneration [70]. The complex series of events associated with periodontal regeneration involves recruitment of locally

Fig. 12.11 Karyotype analysis of PLdSCs showing aneuploidy karyotype with chromosome counts peaking at 70 chromosomes

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derived progenitor cells to the site which can subsequently differentiate into periodontal ligament-forming cells, mineral-forming cementoblasts, or bone-forming osteoblasts [90]. A further class of dental ectomesenchymal stem cells are PDL stem cells, which were isolated from the root surface of extracted teeth and described firstly by [27]. These cells could be isolated as plastic-adherent, colony-forming cells, but display a low potential for osteogenic differentiation under in vitro conditions [27]. PDL stem cells differentiate into cells or tissues very similar to the periodontium [27]. Moreover, PDL stem cells transplanted into immunocompromised mice and rats demonstrated the capacity for tissue regeneration and periodontal repair. However, little is still known about other side effects which may reside within the periodontal ligament-derived neural stem cells’ colony when considered for periodontal tissue regeneration [90]. Thus, a suitable animal model that can present both regenerative features as well as other unusual features is always a need in periodontal research. Our current studies aimed at investigating the athymic immunodeficient nude rat as an animal model that could be used in the field of periodontal research, which is related to stem cell-based tissue regeneration. For this investigation, we chose periodontal ligament-derived stem cells, or pdSCs, which were previously isolated during a minimally-invasive periodontal surgery and well characterized in vitro according to [31].

12.5.2 The Periodontal Defect It was the choice to design a split-mouth model in order to compare what happens when collagen carriers with or without pdSCs are implanted. That is why we considered the right side of the rat’s mandible as a test side (collagen with pdSCs) and the left side as a control side (collagen without pdSCs). Various methods have been used to establish the periodontal disease animal model, such as suture ligatures, bacteria [91, 92], bone defect [93], and orthodontic elastics [94]. The purposes of these methods were to generate clinical manifestations similar to those seen in human periodontitis. On the other side, many studies described different forms of periodontal defects for stem cell transplantation in beagle dogs [81, 95], in miniature pigs [83, 84], in Wistar rats [96] and in immunodeficient rats [27]. Additionally, some of these studies and others concerning dental stem cells had also implanted the cells subcutaneously into the dorsal surface of immunocompromised mice for further investigations [21, 23, 27, 28, 82, 84, 97]. In our studies, and concerning the area in which pdSCs should have been transplanted, it was the choice to create “acute” periodontal defects at the level of the distal root of the first molars. By eliminating small part of the bone and, probably, slight parts of cementum, these defects were considered as “acute” because they were created in one time point using an external instrument (i.e. rotating dental burs) and not over a long time with which ligatures, tubes or other devices are used

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to induce a “ligature-induced periodontitis” [83]. Because of the small size of the oro-facial region in rodents, it was difficult to use “chronic” periodontal defects by means of ligatures in our studies. The current model of periodontal defect looks similar in design with the defect model described in a past study related to periodontal healing [98]. Moreover, [27] used a similar animal model (immunodeficient rats) with similar periodontal defect to investigate the effects of multipotent postnatal stem cells from human periodontal ligament. In this model, a 2 mm2 bone defect had been used whereas a 2.5 × 2.5 × 2 mm defect was created in our studies. While Seo and co-workers created the periodontal defect intraorally, we chose an extra-oral entrance. However, the results showed the ability to create acute defects from an extra-oral entrance in this animal model. This has been shown to be easier, and could be also of benefit against possible bacterial invasion comes from the intra-oral cavity. As cell carriers, Seo and co-workers used a 40 mg-mixture of Hydroxyapatite (HA) and Tricalcium Phosphate (TCP) while, in our study, collagen matrix was chosen for this purpose. Collagen, being a major protein of connective tissues in animals, is widely used in medical application. It plays an important role in the formation of tissues and organs, and is involved in various cells in terms of their functional expression. Collagen exhibits low antigenicity, excellent biocompatibility, biodegradability and bioresorbability and becomes a superior drug carrier to control sustain release of proteins or other active products [99]. Moreover, [100] showed that a collagen sponge supporting live human allogeneic skin cells could stimulate accelerated skin regeneration and wound healing of burn patients. Collagen scaffolds have been investigated as a cell delivery device for many years [99]. Collagen sponges offer particular features for cell integration and tissue engineering. Cells can readily be seeded onto collagen sponges or membranes, cultured and then introduced into a tissue defect site, where they can affect tissue repair and regeneration [101]. We believe that collagen sponges as cell carriers are more stable, and reside perfectly in the periodontal defects. Moreover, collagen might have a better possible enhancement of wound healing. Another special feature in the current studies relates to the fact that a minimallyinvasive surgical intervention was applied to operate the rats. This was achieved by means of special surgical instruments similar to those used in the minimally-invasive periodontal surgery [85]. Minimally invasive surgery should have less operative trauma than an equivalent invasive procedure. Operative time is longer, but it causes less pain and scarring, speeds recovery, and reduces the incidence of post-surgical complications. Different features of the animal model used in the current study compared to other studies which used pdSCs are summarized in Table 12.1. Of these studies, however, [96] used a periodontal defect to investigate the potential of adiposederived stem cells and not of pdSCs. The same should be considered regarding the study of [95] who used bone marrow-derived stem cells to investigate possible periodontal tissue regeneration.

Mixture of human PLDSCs and DPSCs and stem cells from the apical papilla (SCAP) from extracted third molars, expanded in vitro to a density of 4 × 106 cells, mixed with HA/TCP

Autologous adipose-derived stem cells from wistar rats, expanded in vitro to a density of 1 × 107 cells, mixed with 1 ml platelet-rich plasma gelatin

Human PLDSCs line, expanded in vitro to a density of 1 × 106 cells, mixed with 40 mg of (β-TCP)

Human PLDSCs from extracted premolars (scraped from the root surface), expanded in vitro to a density of 1 × 105 cells, multilayered and placed onto dentin blocks

Human PLDSCs from extracted third molars (scraped from the root surface), expanded in vitro to a density of 2 × 106 cells, mixed with 40 mg of ceramic bovine bone Autologous PLDSCs from extracted cuspids (scraped from the root surface) of miniature pigs, expanded in vitro to a density of 2 × 107 cells, mixed with HA/TCP

10-week-old athymic Experimental extra-oral immunodeficient nude rat acute periodontal defect on the buccal cortex of the mandibular first molar (2.5 × 2.5 × 2 mm) 6-week-old Subcutaneous pockets on immunocompromised the backs of mice mice 12-month-old inbred Experimental chronic miniature pigs periodontal defect in the mesial region of the maxilla and mandibular first molars (3 × 7 × 5 mm) covered with gelatin membrane 8-week-old athymic rats Direct implantation subcutaneously at the muscle surface on the dorsa 5-week-old Direct implantation immunodeficient (SCID) subcutaneously on the mice dorsa 10-week-old inbred wistar Experimental intraoral rats acute periodontal defect on the palatal side of the upper first molar (1 × 1 × 1 mm) 10-week-old Direct implantation immunocompromised subcutaneously on the mice dorsa

Human PLDSCs isolated during minimally-invasive periodontal surgery (scraped from the root surface), expanded in vitro to a density of 1 × 105 cells, seeded onto collagen sponges

Defect description

Animal model

Characteristics of cells and carriers

[84]

[96]

Autologous

Heterologous

[21]

Heterologous

[82]

[83]

Autologous periodontitis model

Heterologous (cell sheet technique)

[97]

Current study

Heterologous

Heterologous

Study

Type of model

Table 12.1 Features of the animal model used in the current study compared to other studies

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Inbred male mini-pigs

Mixture of (SCAP+PLDSCs) from the swine, loaded into root-shaped HA/TCP block with artificial dental crown

Human PLDSCs from extracted third molars (scraped from the 10-week-old root surface), expanded in vitro to a density of 4 × 106 cells immunocompromised beige mice Human PLDSCs from extracted third molars (scraped from the Immunodeficient rats root surface), expanded in vitro to a density of 2 × 106 cells, mixed with 40 mg HA/TCP

6-week-old NOD/SCID PLDSCs from extracted premolars (scraped from the root mice surface) of mature merino ewes (sheep), expanded in vitro to a density of 5 × 106 cells, mixed with 40 mg HA/TCP Human PLDSCs from extracted third molars (scraped from the 10-week-old root surface), expanded in vitro to a density of 2 × 106 cells, immunocompromised mixed with 40 mg HA/TCP beige mice Autologous periodontal ligament cells from extracted Beagle dogs premolars of beagle dogs, expanded in vitro to a density of 1 × 105 cells, fabricated on biological sheet

Autologous bone marrow-derived stem cells from beagle dogs, 12–20-month old female expanded in vitro to a density of 2 × 107 cells, mixed with beagle dogs 1 ml atelocollagen

Animal model

Characteristics of cells and carriers

Table 12.1 (continued)

Direct insertion into alveolar socket where an incisor had been extracted Experimental class III furcation defects on mandibular second, third and forth premolars Direct implantation subcutaneously on the dorsa Direct implantation subcutaneously on the dorsa Experimental dehiscence defect on the mesial root of the mandibular first molar Direct implantation subcutaneously on the dorsa Experimental acute periodontal defect on the buccal cortex of the mandibular molar (2 mm2 )

Defect description

[27]

Heterologous

[81]

Autologous (cell sheet technique)

[27]

[28]

Heterologous

Heterologous

[23]

[95]

[84]

Study

Heterologous

Autologous

Autologous

Type of model

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12.5.3 Source of pdSCs In the current studies, human pdSCs were used and transplanted. These cells had been extracted during a minimally-invasive periodontal surgery from patients suffered a chronic type of periodontitis with severe degree of inflammation. However, Seo and co-workers collected PLdSCs from the root surfaces of extracted third molars under undefined conditions (i.e. whether the area of extraction was inflamed or not) [27]. Similarly, in other studies, PLdSCs were collected directly from the root surfaces of extracted teeth and not from teeth subjected to a surgery [21, 28, 82, 84, 97]. Our current studies demonstrate for the first time that extracting pdSCs from patients undergoing open flap periodontal surgery seems to be a simple method and a suitable resource of collecting pdSCs since it is not related to specific time points where premolars or third molars should firstly be extracted. Moreover, this method based on the fact that postnatal stem cells reside in both healthy and periodontitis-affected human periodontal ligament [20].

12.5.4 Special Features Another discrepancy between the methods of our current studies and the methods used elsewhere is that our pdSCs were expanded in vitro in a serum-free medium, whereas almost all other studies used a cell culture medium consisting primarily of fetal calf suspension [23, 27, 28, 84]. Serum-free media are media designed to grow a specific cell type or perform a specific application in the absence of serum. The use of serum-free media represents an important tool that allows cell culture to be done with a defined set of conditions as free as possible of confounding variables. Other advantages of using serum-free media are: (1) Increased definition, (2) More consistent performance, (3) Easier purification and downstream processing, (4) Precise evaluations of cellular function, (5) Increased growth and/or productivity, (6) Better control(s) over physiological responsiveness and (7) Enhanced detection of cellular mediators.

12.5.5 Considering the Nude Rats In the current studies, the survived rats were sacrificed at three different time points; at 2 weeks, at 6 weeks and at 8 weeks after pdSCs implantation. Normal healing of bone fractures in the rat is accomplished almost exclusively by the external periosteal callus 4–5 weeks post fracture with very little involvement of medullary callus [102]. Primitive (mesenchymal) repair tissue in skeletal injuries must differentiate into either osteoblastic or chondroblastic lineages for osseous repair of the lesion to occur. The former results in direct new bone formation (analogous to intramembranous ossification in the developing animal) and the latter forms new bone by endochondral ossification [103]. So, the whole observation period of the study (i.e. 8 weeks) was considered suitable to explore – histologically – any features of tissue regeneration and/or other abnormalities which might be shown.

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12.5.6 pdSCs and Tumor Initiation Sixty percent of investigated rats presented remarkable tissue enlargements (later referred to as tumors) at 6 and 8 weeks after pdSCs implantation exactly at the right side of the mandible which was considered the “test” side. However, the initial visual and manual examination could not tell if those external tissue alterations were just inflammatory reactions or considerable as tumor initiation. The weight loss and fatigue that accompanied those distinctive features have led to the thinking that a local neoplastic alteration within the involved tissues has been occurred, since in late-stage cancer, it is believed that tumor- or stromal cell – derived molecules disturb the stringent control of appetite and weight control, leading to wasting, debility and often death [104]. Under light optical microscope, the histological sections taken from the test sides of the rats, which presented the tissue enlargements showed a tissue with dense infiltration of epithelial-like cells interrupted by fillets of collagen fibers. Those features were considered to present a classical histological screen of carcinoma. By definition, a carcinoma is any malignant cancer that arises from epithelial cells. Carcinomas invade surrounding tissues and organs and may metastasize, or spread, to lymph nodes and other sites. Severely anaplastic tumors might be so undifferentiated that they do not have a distinct histological appearance. In our current studies, the histological screening of the tumors initiated showed undifferentiated features so that the tumor type was considered as undifferentiated type of carcinoma. Malignant neoplasms that are composed of undifferentiated cells are said to be anaplastic. Lack of differentiation, or anaplasia, is considered as a hallmark of malignancy. The term anaplasia literally means “to form backward.” It implies dedifferentiation, or loss of structural and functional differentiation of normal cells. In our studies, the tumors detected were considered to be anaplastic type of carcinoma and might be malignant because they were less differentiated and – under optical light microscope – not clearly characterized.

Fig. 12.12 The hypothesis of tumor initiation presented in our current studies

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We hypothesize that 3 major factors could be responsible for this malignant initiation; (1) the patients who played the role of pdSCs donors, (2) the rat model used and (3) the pdSCs themselves (Fig. 12.12). The pdSCs used in the current studies were extracted from adult patients (27– 56 years old) who visited the Department of Periodontology at the University of Witten/Herdecke seeking a complete periodontal treatment, of which surgery was an important part. All the patients were suffering a severe type of chronic periodontitis which means that the pdSCs were extracted from severe inflamed tissues. Inflammation can play a role in tumor suppression by stimulating an antitumor immune response, but more often it appears to stimulate tumor development. Interestingly, inflammation functions at all three stages of tumor development: initiation, progression and metastasis. Inflammation contributes to initiation by inducing the release of a variety of cytokines and chemokines that alert the vasculature to release inflammatory cells and factors into the tissue milieu, thereby causing oxidative damage, DNA mutations, and other changes in the microenvironment, making it more conducive to cell transformation, increased survival and proliferation. The link between infection, chronic inflammation and cancer has long been recognized [105], a prime example being infection with Helicobacter pylori (H. pylori) and gastric cancer [106]. Chronic gastric inflammation, which develops as a consequence of H. pylori, leads over time to repetitive injury and repair resulting in hyper-proliferation, an increased rate of mitotic error, and progression to adenocarcinoma. Houghton and colleagues work [107] suggested an alternative to the inflammation theory. The research group focused on the idea that bone-marrowderived cells move into areas of chronic injury or inflammation to effect repairs. A strain of mice (C57BL/6) and a relative of H. pylori (H. felis) had been used so that, together, they formed a well-established model of gastric cancer in humans. It has been shown that the implanted bone marrow-derived stem cells migrated first into the stomach lining to repair the damage caused by the bacteria; by 20 weeks, the labeled cells were differentiating into cells with the characteristics of stomach epithelial cells. These differentiating cells showed a high rate of growth, and after 52 weeks the mice were in the early stages of developing gastric cancer, and the tumors that subsequently formed stained positively for markers that indicated that the cells indeed came from the bone marrow. Other studies showed that in chronically inflamed skin, or in an immunodeficient patient, malignant transformation of extra-cutaneous stem cells is more likely to occur [108]. All these data suggest that an inflamed environment could change the manner of stem cells and subsequently enhanced the (inflammation/infection- intotumor) cascade. A genetic mutation might have played a role at this level. The rat model used in the current study may be a hidden reason that explains the tumors’ initiation. The notion that the immune system could protect the host from neoplastic disease was initially proposed by [109] and formally introduced as the cancer immunosurveillance hypothesis nearly 50 years later by [110]. Tumor development in mice had been shown to be controlled by components of the immune system like interferon-γ (IFN-γ), Perforin, Natural Killers (NK) and Lymphocytes.

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Girardi and co-workers examined the relative contributions of different T-cell subsets in blocking primary tumor formation in mice lacking different types of T cells and showed that those mice have more tendencies to initiate tumors [111]. Moreover, it has been suggested that the fate of developing pre-cancer stem cells (pre-CSCs) is determined by the status of the host immune system and the environmental cues (the site of cell colonization or route of inoculation) [112]. In their study, the pre-CSCs appeared to be scrutinized by the mechanism of tumor immune surveillance because they had different fates in three animal models with different levels of immune surveillance; they developed into neither solid nor leukemic tumors in native immunocompetent mice. However, they developed into tumors or leukemia in the NK cell-sufficient but T- and B-cell deficient mice with a variable latency of tumor initiation and tumor incidence in separate experiments [112]. Since the rats used in our study are T-cell deficient and show depleted cell populations in the thymus-dependent areas of peripheral lymphoid organs, it could be stated that this immunodeficiency might be the reason why the tumors occurred. The presence of pdSCs within the solid tumor may give a sign that those stem cells could be responsible for the initiation of the neoplasias detected in the rats. Whether those tumors can be considered as “stem cell-induced tumors” is not clear and needs further immunological examinations. However, the presence of human mitochondria antigens in the solid tumor may lead to the supposal that the implanted human pdSCs might have played a role at this level. According to the Cancer Stem Cell (CSC) theory, both adult stem cells and progenitor cells are proliferating cells, and therefore go through sufficient cell cycles to accumulate oncogenic mutations during their life. The adult stem cells are longlived cells going through relatively few cell cycles, and may be the primary targets of accumulation of oncogenic mutations [113–116]. Adult stem cells and tissueuncommitted stem cells might be more susceptible to developing into pre-cancer stem cells and CSCs than other progenitors [117], and the pre-CSCs might be more plastic than CSCs. Our results, showing that pdSCs exhibited an aneuploidy karyotype with chromosome counts peaking at 70 chromosomes, and that the human cells (i.e. the pdSCs) reside within the tumor tissue, might also be of great importance. Two forms of genomic instability have been described for cancers: chromosomal instability (CIN) and micro-satellite instability (MSI) [118]. CIN in tumor cells invariably leads to aneuploidy, while cells with MIN are usually nearly diploid [119]. Analysis of karyotype of pre-CSCs and cancer cells from the same mouse revealed that all the pCSC clones analyzed were pseudo-diploid with multiple chromosomal translocations, whereas cancer cells were usually aneuploid [112]. Certainly increased genomic instability is believed to be correlated with the increased probability of oncogenic mutations. In our study, the aneuploidy karyotype of pdSCs shown may give a sign that those cells might have been at the level of carcinogenetic alteration. According to all these data, and because we used human adult stem cells in this study which have been previously shown to be highly proliferative [31], it might be stated out that the pdSCs and/or their progenitors have underwent critical

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alteration(s) which has (have) redirected their development into tumor initiating cells. We also hypothesize that the method used to expand pdSCs in vitro in our studies might be a factor that altered the proliferation form of those cells when implanted in vital tissues. However, deep investigation of the tumors observed in the current results as well as analyzing their characteristics were not the definite priorities of this work. Further genetic analyses in this term are needed to uncover the real chromosomal instability presented in our model.

12.6 Conclusion 12.6.1 Periodontal Stem Cells as Functional Elements of Regenerative Periodontology Periodontal stem cells are the functional elements of Regenerative Periodontology, and the use of living periodontal stem cells as a therapy presents several challenges. These include identification of a suitable periodontal source, development of adequate transplantation methods, and proof of safety and efficacy in an adequate animal model. The true potential of periodontal stem cells will only be realized through continued effort to increase basic scientific understanding at the level of induced pluripotent periodontal stem cells, the development of adequate methods to achieve a functional phenotype from inflamed or from non-inflamed periodontium, and attention to safety issues associated with careful control of cell localization, proliferation, and differentiation (GMP). The cells or tissues to be constructed in periodontal regeneration will not behave as a whole-tooth transplant. It is therefore important that we understand the periodontal stem cell transplantation’s ability to react and interact within the local inflammational situation (“non-shedding” root surfaces) and the immune host response, since clinical effectiveness has proven to be one of the most difficult milestones to achieve. The extent to which we are able to achieve effective periodontal stem cell therapies will depend on assimilating a rapidly developing base of scientific knowledge with the practical considerations of design, delivery, and host response of periodontal tissue regeneration (excellently reviewed in [24]). Trials aiming at isolating cultures and subcultures of cells from the periodontal ligament to uncover special features of such tissue-residing population are related to the past decade [1]. Moreover, the idea of cell-delivery systems, and especially with stem cells, to initiate periodontal regeneration is not new. However, describing suitable animal models for such new techniques is less documented in the literature and sometimes underestimated. The wide range of animal species allows appropriate selection of bio-models for different investigations. Each species has unique similarities and dissimilarities to humans. While many studies could ensure the initiation of periodontal regeneration using stem cells extracted from the periodontium, seeding of human periodontium-derived stem cells (pdSC) on collagen carriers could induce major features of periodontal

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regeneration when implanted in experimental periodontal defects in the athymic immunodeficient rat as could be shown in our studies, see also chapter “Possible periodontal tissue regeneration”. However, remarkable notifications regarding the results obtained in our studies were the induction of malignant tumors (squamous cell carcinoma) in 60% of all investigated animals. Considering the data presented in the literature, our studies seems to be the first that demonstrates the initiation of malignant tumors when using human periodontal ligament-derived stem cells. The patients from whom the pdSCs had been extracted, the animal model used and a possible oncogenic alteration of the pdSCs themselves might all be factors behind the tumors’ initiation. However, further studies using advanced histological, immunological and genetic techniques are required to assure different supposals presented in the current studies regarding stem cell-based periodontal regeneration and stem cell-tumorigenesis.

12.6.2 Possible Periodontal Tissue Regeneration While all of the animals showed a primary reformation of PDL-like tissue on the control sites, where only collagen sponges have been implanted, several animals showed primary features of periodontal regeneration on the test sites where pdSCs on collagen sponges have been implanted. This could be shown by comparing the histological sections from the control rat (normal periodontium) with other experimental sections. However, whether considerable periodontal tissue regeneration has been induced and presented within the artificial periodontal defects in the current studies was not yet evaluated by histo-morphometrically analyzes. This may be questionable for future studies regarding the same animal model and the same resource of stem cells used [120].

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Chapter 13

Leukemia Stem Cells Markus Müschen

Abstract Normal hematopoiesis develops hierarchically from a hematopoietic stem cell, which is defined by both extensive self-renewal capacity and multi-lineage potential, i.e. the ability to give rise to fully differentiated cells of all hematopoietic lineages. Since leukemia can be considered as malignant hematopoiesis, the existence of a developmental hierarchy in leukemia with a malignant stem cell at its apex was postulated almost three decades ago. Recent data questioned the presence of a unique leukemia stem cell population in some subtypes of leukemia. In other leukemia subtypes, however, leukemia stem cell populations have been extensively characterized with respect to their self-renewal capacity, multilineage potential, their phenotype and the signaling pathways that are required for their maintenance. Importantly, recent data show that leukemia stem cells are not only required for initiation of leukemia at its onset. They also represent the rare leukemia cell population that gives rise to relapse and drug-resistance of the disease in patients. Here, current data on the cellular origin, phenotype and central signaling pathways of selfrenewal in acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) subtypes of leukemia are being reviewed. Keywords Acute lymphoblastic leukemia (ALL) · Acute myeloid leukemia (AML) · Chronic myeloid leukemia (CML) · Leukemia stem cell (LSC) · SCIDrepopulating cell (SRC) · SCID leukemia-initiating cell (SL-IC)

Contents 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 Stem cells in acute myeloid leukemia are defined by their ability to reconstitute leukemia in serially transplanted NOD/SCID mice . . 13.3 Phenotype of Leukemia Stem Cells in AML . . . . . . . . . . . 13.4 Leukemia Stem Cells in CML . . . . . . . . . . . . . . . . .

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13.1 Introduction The presence of a stem cell population in leukemia with a similar function as in normal hematopoiesis was first suggested more than 25 years ago [1]. Since then, leukemia stem cells have been phenotypically characterized in some leukemia types and central signaling pathways that are required for leukemia stem cell self-renewal and maintenance have been elucidated [2]. Given that recent work implicated leukemia stem cells in drug-resistance and relapse of leukemia [3], the identification of mechanisms of leukemia stem cell self-renewal are of particular interest because they offer potential targets for innovative drug-therapies. Despite significant advances in the treatment of leukemia over the past four decades, the rate of longterm survival has reached a plateau and still large numbers of leukemia patients die, mostly because of relapse and drug-resistance, which was recently attributed to the persistence of leukemia stem cells. If a therapy succeeds in eradicating leukemia stem cells, de novo initiation of the disease (relapse) is no longer possible. Therapeutic progress in recent clinical trials has likely been stalled, partly because current cytotoxic therapy approaches target proliferating bulk leukemia cells rather than quiescent leukemia stem cells. Leukemia arises as malignancy from hematopoietic progenitor cells that are developing in the bone marrow. Depending on in which cell type the decisive transforming event occurs, normal hematopoiesis is disrupted by the overwhelming growth of a malignant clone in one hematopoietic lineage. Therefore, leukemias are typically subdivided based on the lineage of their cell of origin. Among leukemias of myeloid origin, acute myeloid leukemia (AML) is characterized by a large number of known genetic lesions that cause transformation, whereas chronic myeloid leukemia (CML) is defined by one unifying abnormality, namely the Philadelphia chromosome, which encodes the oncogenic BCR-ABL1 kinase. Leukemias of lymphoid origin (acute lymphoblastic leukemias, ALL) are derived from B cell or T cell precursors or a common lymphoid progenitor (cALL) and are characterized by a wide spectrum of recurrent and sporadic genetic lesions. Here, current data on the cellular origin, phenotype and central signaling pathways of self-renewal in AML, CML and ALL leukemia subtypes are reviewed.

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13.2 Stem cells in acute myeloid leukemia are defined by their ability to reconstitute leukemia in serially transplanted NOD/SCID mice The initial concept of a developmental hierarchy in leukemia with leukemia stem cells at its apex was established in landmark studied by Dick et al. in acute myeloid leukemia (AML [4, 5]). This was based on experiments, in which patient-derived AML cells were divided into more primitive (CD34+ CD38– ) and more mature (CD34+ CD38+ ) subpopulation by flow cytometry sorting. Xenotransplantation of CD34+ CD38– and CD34+ CD38+ AML cell populations into immunocompromised NOD/SCID mice demonstrated that only primitive CD34+ CD38– but not more mature CD34+ CD38+ AML cells were able to re-initiate leukemia in serial transplant NOD/SCID recipient mice. Of note, AML initiated from primitive CD34+ CD38– AML cells contained a pool of more mature CD34+ CD38+ AML cells at the same percentage as the initial leukemia. This functional difference between primitive and more mature AML cells provided direct evidence for the existence of leukemia stem cells that were enriched in the CD34+ CD38– but missing in the CD34+ CD38+ AML cell population. Interestingly, the enriched [NOD/] SCID leukemia-initiating cell (SL-IC) is defined by the same phenotype as the hematopoietic stem cell population that reconstitutes normal human hematopoiesis in NOD/SCID mice (CD34+ CD38– ), the so-called SCID-repopulating cell (SRC). Hence, leukemia stem cells in AML are defined by their SL-IC potential, i.e. the ability to reconstitute a heterogeneous full-blown leukemia in serial NOD/SCID transplants [4–9]. For instance, 20 AML leukemia stem cells are sufficient to reconstitute leukemia in a transplant recipient as compared to 100,000 unsorted leukemia cells [7].

13.3 Phenotype of Leukemia Stem Cells in AML The phenotypic parallel between SRC in normal hematopoiesis and and SL-IC in AML suggested that leukemia stem cells are restricted to a small pool of hematopoietic stem cells that carried the initial transforming lesion. According to this scenario, the cell of origin (i.e. the cell that acquired the decisive transforming event at the onset of leukemia) of leukemia would invariably be identical with the leukemia stem cell (i.e. the leukemia cell with SL-IC potential). While indeed SRC-like populations in a wide range of AML subtypes have SL-IC potential, SL-IC potential and, hence, the pool of leukemia stem cells is not always restricted to leukemia cells with an SRC-like phenotype in all subtypes of AML [10, 11]. In AML with MOZ-TIF2, MLL-ENL and MLL-AF9 gene rearrangement, the oncogenic fusion transcription factors conferred SL-IC potential also to committed progenitor cells that would otherwise lack stem cell self-renewal capacity. Conversely, BCR-ABL1 failed to promote self-renewal capability in committed progenitors ([10, 11] Fig. 13.1).

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MOZ-TIF2, MLL-ENL, MLL-AF9

B

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SRC

Committed progenitor cells

Fig. 13.1 SL-IC potential of leukemia cells depends on the degree of differentiation and the ability of the oncogene to promote self-renewal. (a) AML populations with SCID repopulating cells (SRC)-like phenotype (e.g. CD34+ CD38– ) function as leukemia stem cells in a wide range of AML subtypes and are defined by their SL-IC potential. However, the pool of leukemia stem cells is not always restricted to SRC-like AML cells: In AML with MOZ-TIF2, MLL-ENL and MLL-AF9 gene rearrangement, the oncogenic fusion transcription factors conferred SL-IC potential also to committed progenitor cells that would otherwise lack stem cell self-renewal capacity. (b) Conversely, BCR-ABL1 failed to promote self-renewal capability in committed progenitors [10, 11]. Therefore, SL-IC potential does not only depend on the degree of differentiation of AML blasts but also by the ability of the transforming oncogene to promote self-renewal capacity

Therefore, it is likely that presence or absence of SL-IC potential is not only determined by the degree of differentiation of individual leukemia cells but also by the ability of the transforming oncogene to promote self-renewal capacity (Fig. 13.1). From the initial definition of AML stem cells based on an SRC-like phenotype, the phenotypic characterization of AML stem cells has been further refined based on additional antigens including CD90 (CD34+ CD38– CD90– [12]), and the interleukin 3 receptor (IL3Rα+ [13]). Also more recent definitions of AML stem cells closely resemble the normal hematopoietic stem cells (Table 13.1). However, while expression of CD13 [14], CD96 [15] and CLEC12A (CLL1 [16]) are specifically expressed on AML stem cells but not on normal SRC, expression of CD90 and CD117 (c-Kit) is found on SRC cells but not on AML stem cells (Table 13.1).

Table 13.1 Phenotype of normal hematopoietic and leukemia stem cells in AML Cell type

SRC

LSC(AML)

Lineage CD13 CD33 CD34 CD38 CD71 CD90 CD96 CD117/c-kit CD123/IL3Rα CLEC12A HLA-DR

– – + + – + + – + – – –

– + + + – + – + – + + –

SRC: SCID-repopulation hematopoietic stem cell; LSC: leukemia stem cell

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13.4 Leukemia Stem Cells in CML Chronic myeloid leukemia (CML) is thought to be initiated from the transformation of a hematopoietic stem cell by the oncogenic BCR-ABL1 kinase. BCR-ABL1 is encoded by the Philadelphia chromosome [t(922)(q34q11)], which is present in virtually all cases of CML. As a characteristic feature, CML a tri-phasic clinical course and begins with a chronic phase, which may last for up to 15 years, followed by an accelerated phase of beginning blastoid transformation, which leads to terminal and fatal blast crisis. Even though chronic phase CML is virtually in all instances a myeloid disease, in many cases blast crisis develops under a B lymphoid phenotype and reason for lineage switch in these cases is not understood. CML has typically multi-lineage involvement, with the BCR-ABL1 gene rearrangement present in myeloid, erythroid, megakaryocytic and B cell progenitors developing from a transformed hematopoietic stem cell. While this suggests a developmental hierarchy and the transformed hematopoietic stem cell being the leukemia stem cell in CML, previous work implicated that granulocyte-macrophage progenitors function as leukemia stem cells in blast crisis CML [17]. On the other hand, Huntly and Gilliland [2, 11] demonstrated that BCR-ABL1 lacks the ability to confer SL-IC potential to committed progenitor cells, such as granulocyte-macrophage progenitors. Therefore, it is not clear whether leukemia stem cells in CML phenotypically resemble an SRC as in AML or a committed myeloid progenitor cell. Of note, in CML leukemia cells were identified and enriched based on a functional criterion, namely their ability to resist therapeutic BCR-ABL1 kinase inhibition using Imatinib (Gleevec, STI571 [18]). Consistent with the concept of stem cell quiescence, leukemia stem cells were flow sorted as a quiescent population that was resistant to Imatinib-mediated BCR-ABL1 inhibition. This functional characteristic of leukemia stem cells in CML was consistent with the subsequent finding that leukemia stem cells in CML exhibit very low levels of BCR-ABL1 expression and hence a very low level of “oncogene addiction” to BCR-ABL1 [19]. This concept is further supported by the activity of BCR-ABL1-independent signaling pathways of self-renewal that are active in leukemia stem cells in CML [20, 21]: Of note leukemia stem cells in CML exhibit high levels of WNT/β-catenin activity and Smo/Sonic Hedgehog signaling [20, 21], which will be discussed in more detail below.

13.5 Stem Cells in Acute Lymphoblastic Leukemia (ALL) Acute lymphoblastic leukemia (ALL) typically develops as the malignant outgrowth of B cell (and less frequently T cell) precursors and represents the most frequent type of malignancy in children. While the natural course of leukemia in both AML and CML exhibits substantial phenotypic heterogeneity and hierarchic development from a hematopoietic stem cell, the phenotype of leukemia cells in ALL is typically uniform and “hierarchic” progenitor-progeny relationships among individual subpopulations are less obvious in ALL. In addition, whereas AML and CML

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originates from a transformed hematopoietic stem cell, in most cases of ALL, the leukemia is derived from progenitor cells that are committed to the B cell lineage. As a consequence, leukemia stem cells with SL-IC potential in ALL express the B cell-specific CD19 antigen in many cases, whereas leukemia stem cells in AML and CML typically lack expression of lineage-specific antigens (Lin– ).

13.6 Identity of Leukemia Stem Cells in ALL Is Controversial While earlier studies on leukemia initiating or stem cells propose a CD19-negative phenotype in ALL (CD34+ CD19– and CD34+ CD10– [22, 23]), more recent work demonstrated that also leukemia cells with a committed progenitor phenotype CD34+ CD38low CD19+ have SL-IC potential [24]. While these findings question the existence of a clear-cut hierarchy among leukemia cell subpopulations, findings by Kelly et al. [25] challenged the leukemia stem cell concept based on a fundamental observation related to technical aspects of the SL-IC assay and the ability of leukemia stem cells to reconstitute leukemia in serial transplant recipients. In serial transplants of murine leukemic cells from three different transgenic mouse models, they showed that as few as one single cell from the bulk of the leukemia was able to cause leukemia in recipients. In view of their own results, they argue that the transplants of human cancer cells into immunodeficient mice suggests that only few of the cells in the total population give rise to leukemia because the mouse system may not support the growth of all the cells in the total leukemia cell population. However, in this context it should be noted, that this would still imply that human leukemias are not homogenous, warranting investigation into why certain more primitive subpopulations have higher engraftment capabilities and are more difficult to eradicate. The interesting results by Kelly, Strasser and colleagues [25] underscores the urgent need for further rigorous experiments to investigate if there is a minority population within the bulk of cancer cells, the putative “leukemia stem cell” or a “leukemia-propagating cell” that is a major contributor to relapse.

13.7 Leukemia Stem Cells and Drug-Resistance in ALL The acquisition of drug resistance and relapse of leukemia was recently attributed to the persistence of so-called leukemia stem cells in acute myeloid leukemia (AML [2]). Importantly, leukemia stem cells in both AML and CML acquire drug resistance through expression of multidrug resistance proteins at high levels [3, 18] and ATP-binding cassette (ABC) drug transporters. For example, HSC express high levels of ABCG2, which is not active in most committed progenitor and mature blood cells [26, 27]. LSCs are in addition less sensitive to cell-cycle dependent drugs reflecting their quiescent phenotype [18]. For these reasons, efficient targeting of leukemia stem cells appears to be the key to further improvement of clinical treatment of leukemia. Since their first description in 1994 [5], leukemia stem cells are

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now well characterized in AML and CML. Recent research has developed novel agents for the specific targeting of stem cells in AML and CML. Such agents are currently being investigated in clinical trials [28–30]. Compared to AML, the clinical prognosis for patients with ALL is generally better, and this is the case particularly for children. Despite substantial advances in the treatment of ALL during the past four decades, the rate of long-term survival has reached a plateau, currently at approximately 80% for children and 55% for adults [31]. The main challenge in the therapy of ALL results from acquired drug resistance and relapse [32, 33]. Therefore, specific targeting of stem cells in ALL would represent an attractive concept to improve current treatment options for ALL. Recently, [34] reported serial transplants of three different primary ALL patient samples into an improved strain of immunocompromised (NOD/SCIDγc–/– ) mice, and the characterization of the cell surface phenotype of the leukemia initiating cells using CD19, CD34, and CD38 markers. These data strongly support the existence also in ALL of a leukemic initiating cell/ALL stem cell, but a functional and phenotypic definition of such cell is currently incomplete. BCR-ABL1 and MLL-AF4 gene rearrangements define “very high” risk groups of ALL. For risk stratification of ALL, besides initial white blood cell counts at diagnosis and the age of the patients, the assignment to a cytogenetic subgroup carries substantial weight. While BCR-ABL1 represents the most frequent recurrent genetic aberration in adult leukemia (including 30% of cases of adult ALL and >95% of cases of CML), the chimeric MLL-AF4 transcription factor is typically found in a highly aggressive form of childhood ALL and is particularly frequent in infant ALL. The oncogenic MLL-AF4 transcription factor induces upregulation of the stem cell antigen Prominin1 (CD133 [35]), which is aberrantly expressed on cancer stem cells in a variety of malignancies. Whereas MLL-AF4 is only common in infants and early childhood, BCR-ABL-ALL is frequently found in adults and accounts for up to 30% of all cases of ALL [36]. The BCR-ABL kinase inhibitor Imatinib (Gleevec, STI571) is the standard of treatment for CML. Imatinib and more recent BCR-ABL kinase inhibitors such as Nilotinib (AMN107) have also been used to treat BCR-ABL-ALL patients. Unfortunately, response to treatment in BCR-ABL1ALL is generally transient [37] and it is likely that the persistence of leukemia stem cells is one of the factors that contribute to drug-resistance in this case [18, 19].

13.8 Mechanisms of Leukemia Stem Cell Self-renewal 13.8.1 The PTEN/PI3K/AKT/FOXO Axis For this reason, pathways that are required for the maintenance of stem cells in leukemia but not normal hematopoiesis are of particular interest, because they potentially represent a target for drug-treatment. Recent “achilles heel-screens” have focused on the identification of leukemia-specific factors of stem cell selfrenewal. One such example represents the PTEN phosphatase (phosphatase and

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tensin homologue), which antagonizes the enzymatic activity of PI3K. Loss-offunction mutation of the PTEN tumor suppressor leads to excessive PI3K-mediated accumulation of PIP3 in lipid rafts. As a consequence, hematopoietic stem and progenitor cells are transiently activated, which leads to massive expansion of committed progenitor cells at the expense of hematopoietic stem cells and subsequent stem cell exhaustion [38, 39]. Therefore, PTEN serves a critical role in stem cell maintenance in normal hematopoiesis. Importantly, however, leukemic stem cells do not share this requirement and PTEN deletion predisposes to the onset of acute leukemia [38]. Conversely, unopposed PI3K activity is not only tolerated in acute leukemia cells, it also represents a growth requirement for leukemia stem cells, since inhibition of PI3K downstream signaling by rapamycin not only reinstated normal hematopoietic stem cell quiescence, but also prevented the onset of PI3K-dependent acute leukemia [40].

13.8.2 Deregulation of CDX2/HOX Genes HOX genes are required for self-renewal in normal hematopoietic stem cells [41] and are aberrantly expressed in a wide array of acute leukemias (e.g. AML and ALL with MLL gene rearrangement [42]). The relevance of HOX genes was further demonstrated in a genetic model, which identified HOXA7 and HOXA9 as critical requirements for MLL-mediated leukemogenesis [43]. Importantly, CDX2 (Drosophila caudal-like 2 homologue) represents a developmental regulator of HOX gene expression and is aberrantly expressed in most if not cases of human AML, where it promotes abnormal self-renewal capacity [44]. While the precise components of the CDX2/HOX pathway remain to be identified, it is obvious that aberrant HOX function plays a fundamental role in self-renewal signaling of leukemia stem cells in AML and MLL-related ALL.

13.8.3 Nuclear Reprogramming by OCT4 Oct4 (Pou5f1) is a transcription factor which, in the context of normal embryonic stem cells, has been called a “master regulator” for stem cell characteristics including self-renewal signaling and pluripotency. Remarkably, a quartet of genes including Oct4, Klf4, Sox2 and Myc, was recently identified that is capable of reprogramming normal mouse skin fibroblasts and pro-B, pre-B and even mature B cells into mouse stem cells (induced-pluripotency stem cells, or iPS cells) that can participate in normal embryonic development [45–48]. Given that three of the four factors needed for nuclear reprogramming, namely KLF4, SOX2 and MYC are readily expressed in bulk ALL cells, it appears possible that leukemia stem cells are distinguished from bulk leukemia cells in ALL by expression of the fourth missing factor OCT4. Interestingly [49], analyzed biopsy material from 14 ALL patients with the MLL-AF4 fusion gene and, using RT-PCR, found OCT4 transcripts, as well

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as those of a second stem cell marker, NANOG, in these leukemic cells. In addition, a second report identified a primitive “leukemia stem cell-like” population with extensive self-renewal capacity that expressed OCT4 [50]. However, a role of OCT4 in normal hematopoiesis has not been defined. OCT4 is normally expressed during embryonic development, where it is indispensable [51]. There is increasing interest in also studying OCT4 in adult tissues, specifically in adult stem cells: recent publications reported OCT4 expression in stem cell-like progenitors in pancreas [20] and in neonatal and adult lung stem cells [52, 53]. Ling et al. were able to culture the neonatal lung epithelial cells and show staining for Sca-1, SSEA-1 and nuclear OCT4. The existence of a multipotent adult progenitor cell in the bone marrow, that is characterized by low-level OCT4 expression was also reported [54, 55] listed 64 different studies in which OCT4 expression was reported in somatic stem cells of different origin, in transformed cells or in amniotic and umbilical cord-derived cells. The finding of OCT4 expression in the ALL cells raises several questions. Why does a subpopulation of lymphoblastic leukemic cells express OCT4? What is the significance of this expression? These cells could be leukemic primitive hematopoietic progenitors that normally express OCT4, implying that normal hematopoietic progenitors exist that express OCT4, which is supported by previous reports [55–57]. Alternatively, it is possible that the leukemia cells show a de novo activation of OCT4 transcription. As reviewed by [58] for other cancer models, it is possible that cancer cells dedifferentiate when they lose repression of the OCT4 locus, for example as a consequence of disruption of epigenetic programming. Therefore, it is currently not clear why or through which mechanism the ALL cells express OCT4. Since OCT4 is a transcription factor that may regulate the expression of over 1,000 genes in stem cells [59], even low or moderate levels may have an amplified downstream effect in terms of gene expression and differentiation pathways. The profound effect of aberrant activation of Oct4 in adult tissues was strikingly demonstrated by [60] who generated mice with a doxycycline-inducible OCT4-transgene. Induction of OCT4 resulted in a fulminant proliferation of stem cells in the intestinal crypts, contributing to the death of the mice within days of OCT4 induction. In this model, OCT4 had a very pronounced effect by inhibition of differentiation, through activation of the WNT/β-catenin pathway.

13.8.4 WNT/β-Catenin Signaling Pathway The hypoplasia caused by OCT4 expression in the intestinal crypt cells of the OCT4 transgenics was clearly mediated by increased expression and activation of β-catenin [60]. Wnt signaling is critical in numerous events in development including both the proliferation and differentiation of stem cells [61, 62]. Previously published data support the concept that this pathway in particular is directly relevant to the self renewal/differentiation balance of leukemia initiating/stem cells in BCR-ABL1 caused leukemias. Coluccia et al. demonstrated that BCR-ABL1 stabilizes β-catenin through tyrosine phosphorylation [63]. Also, the BCR gene loses its function as a negative regulator of WNT/β-catenin signaling when it becomes part of the fusion

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with ABL1 [64]. Finally, a recent landmark study implicated WNT/β-catenin signaling as a requirement for the self-renewal capacity of BCR-ABL1-transformed CML stem cells [17]. Moreover, our own data provide evidence for the regulation of OCT4 gene transcription by the β-catenin pathway and stimulation of transcription of Tcf4/Lef responsive genes by OCT4. β-catenin is stabilized, translocates to the nucleus and functions as a transcriptional activator in complex with Lef1/Tcf4 transcription factors upon activation of the WNT pathway Interestingly, constitutively active β-catenin signaling in hematopoietic stem cells leads to exaggerated “stemness” properties in these cells [65–67]. Moreover, hematopoietic stem cells with constitutively active β-catenin signaling not only have accentuated stem cell features but also fail to differentiate normally into more mature hematopoietic cell types [66, 67]. Many WNT signaling related genes are upregulated in CML [68]. However, less is known about ALL. One ALL subtype is characterized by the leukemogenic fusion gene E2A-PBX1. WNT16B expression is upregulated by E2A-PBX1, as are a number of other WNT-related genes in cells that carry this gene rearrangement. Furthermore, siRNA knockdown of WNT16B in these cells inhibited canonical WNT/β-catenin signaling, initiated apoptosis and reduced the expression of the WNT/β-catenin regulated gene survivin (BIRC5 [69]).

13.9 Perspective In addition to CDX2/HOX, PI3K/FOXO and WNT/β-catenin pathways of selfrenewal in leukemia stem cells, recent data implicate a major role of BMI1 and related polycomb group proteins, NOTCH, and Smoothened (SMO)/Sonic Hedgehog pathways. For this reason, pharmacologic inhibition of WNT/β-catenindependent self-renewal signaling [70, 71], γ-secretase inhibitors to interfere with NOTCH signaling [50] and Cyclopamine to disrupt Smoothened/Sonic Hedgehog signaling [21] represent promising approaches for leukemia stem cell eradication in combination with standard chemotherapy (see Table 13.2). One such agent, the NF-κB inhibitor Parthenolide is currently being tested for leukemia stem cell eradication in AML [29]. In addition, a fusion molecule (DT388IL3) between diphtheria toxin and interleukin 3 as ligand for the IL3Rα expressed on leukemia Table 13.2 Signaling pathways of leukemia stem cell maintenance Pathway

Target

Agent

Reference

NF-κB WNT/β-catenin

NF-κB CBP β-catenin NOTCH-IC Smoothened CD123/IL3Rα CD13 CD96 CLEC12A

Parthenolide ICG-001 PKF115-584 γ-secretase inhib. Cyclopamine DT388IL3

[28] [70] [71] [50] [21] [72]

NOTCH Smo/Shh Surface antigen

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stem cells is currently tested in clinical trial [72]. While a number of these novel selfrenewal pathways still await validation as useful therapeutic targets, Parthenolide and DT388IL3 serve as example to demonstrate that the leukemia stem cell as such is already part of the clinical reality and a central consideration in the design of current and future clinical trials.

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Chapter 14

Cancer Stem Cells in Solid Tumors Melia G. Nafus and Alexander Yu. Nikitin

Abstract Confirmation and assessment of general applicability of cancer stem cell concept towards solid tumors greatly depends on development of reliable approaches to selectively identify populations of neoplastic cells carrying “stemness” features, such as extensive capacity for self-renewal and ability to undergo a range of differentiation events. This chapter describes such assays as sphere formation, side population isolation and cancer stem cell marker detection and addresses their potential pitfalls. Also discussed are the cell of origin of stem cells and remaining challenges in solid tumor stem cell research. Keywords Brain · Breast · Cell of origin · Challenges · Colon · Head and neck · Kidney · Liver · Ovary · Pancreas · Prostate · Side population · Skin · Solid tumor · Sphere assay · Stem cell markers

Contents 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Finding the Needle in the Haystack: Identifying Phenotypically Distinct Prospective Cancer Stem Cells . . . . . . . . . . . . . . . . . . . 14.2.1 Sphere Assays . . . . . . . . . . . . . . . . . . . . . . 14.2.2 Side Population Cells . . . . . . . . . . . . . . . . . . . 14.2.3 Cancer Stem Cell Markers . . . . . . . . . . . . . . . . . 14.3 Cell of Origin of Cancer Stem Cells . . . . . . . . . . . . . . . . 14.4 Perspectives and Challenges . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A.Yu. Nikitin (B) Department of Biomedical Sciences, Cornell University, 14853, Ithaca, New York, USA e-mail: [email protected] Requests for reprints: Alexander Yu. Nikitin, Department of Biomedical Sciences, Cornell University, T2 014A VRT Campus Road, Ithaca, New York 14853-6401. Phone: (607) 253-4347. Fax: (607) 253-4212. E-mail: [email protected].

T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_14,

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14.1 Introduction The term "cancer stem cell" (CSC, often referred to as the tumor-initiating cell) is used to denote a cancer cell subpopulation demonstrating characteristics similar to those of normal stem cells. Normal somatic stem cells have two intrinsic properties: (1) an extensive capacity for self-renewal thereby allowing the maintenance of a stem cell pool over the life-time of any given organism; and (2) a multipotent ability to undergo a range of differentiation events. Simply put, stem cells have the ability to divide and create daughter cells identical to themselves as well as progeny with more restricted potentials. Accordingly, the CSC is a population of neoplastic tumorigenic cells that is able not only to self-renew, thereby giving rise to another tumorigenic cells, but also to generate a heterogeneous population of more differentiated neoplastic cells. The CSC concept dates back to the "embryonal rest" theory formulated by Cohnehein and Durante almost 150 years ago [reviewed in 1]. According to the modern version of this concept, tumors consist of a heterogeneous cell population in which only a limited number of phenotypically distinct cells, CSCs, are functionally able to initiate the tumor. While their number may be comparatively small, the CSC subset should be extremely efficient at tumor initiation. It is possible that CSC population either persists from the earliest stages of carcinogenesis (Fig. 14.1a) or is specific for its particular stages (Fig. 14.1b). Both variants would be consistent with expected clonality of the majority of neoplasms [2], as well as with observations that not every cell within a tumor is capable of tumor regeneration [3]. The presence of CSCs in hematopoietic malignancies has been firmly established [reviewed in 4] and significant advances have been made towards our understanding of CSC biology in some solid tumors. However, there is still a great deal of

Fig. 14.1 Possible scenarios for cancer stem cell (CSC) appearance in solid tumors. (a) CSC population persists from the earliest stages of carcinogenesis (yellow cells). (b) CSCs are specific for particular stages of carcinogenesis (light cells), such as dysplasia (traits associated with cell proliferation and survival, blue), invasion (green) and metastasis (red)

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uncertainty regarding whether or not CSCs present in all solid tumor types, because of limitations of currently available technologies to detect, isolate and characterize individual cells.

14.2 Finding the Needle in the Haystack: Identifying Phenotypically Distinct Prospective Cancer Stem Cells Common approaches currently used by the scientific community to identify the putative CSC are based on the notion that the CSC should have conserved stem and progenitor cell function and phenotype. For instance, the cell should demonstrate an ability to generate spheres in non-adhesive cell culture conditions and initiate tumors in vivo, even after serial passage, thus indicating that the cell has an extensive capacity for self-renewal. Furthermore, the cell should be able to not only generate a heterogeneous tumor cell population, indicating multipotent potential, but also should have an unlimited capacity to reconstitute the primary tumor morphology, thus demonstrating that heterogeneity is not simply a result of genetic instability. Serial transplantation has long been the golden standard for determining stem cell properties, in that it determines the regenerative potential of a cell population. For example, serial transplantation was used to determine that adult mouse hepatocytes contain stem-like cells [5]. Serial transplantation has also been used to determine the presence of stem-like cells in the prostate [6] and the mammary gland (Shackleton et al. 2006; Stingl et al. 2006). Therefore, demonstration of an ability to undergo serial transplantation by one solid tumor population and not another is indicative that the population has stem-like characteristics. A final feature often proclaimed to be indicative of a CSC is expression of common stem cell associated genes, particularly in that they should be upregulated compared to other tumor cell populations. If the cell is able to demonstrate several of the latter characteristics, then it is often labeled a CSC. The following sections examine the various techniques that have so far been used in an attempt to identify CSCs in solid tumors, and discuss current limitations.

14.2.1 Sphere Assays The sphere assays is relatively simple yet robust method used to isolate and expand stem cell populations in a number of different tissue types. Original sphere assays were developed in the 1990s using neural stem cells (NSC). In these assays neurosphere forming cells demonstrated an ability to undergo multilineage differentiation, generating the three neural cell lines, self-renewal and maintenance of their stem cell phenotype even after serial passage [7]. Since then the sphere assay has been adapted to isolate stem cells of other tissue types, and more recently putative CSCs in solid tumors.

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The identification of putative CSC in brain tumors originally relied heavily on the use of sphere assays. In fact, an initial indication that neuroblastomas may contain CSCs was gleaned from their repeated ability to generate neurospheres that resembled those formed by normal NSC. For instance, glioblastoma multiforme (GBM) tumors form neurosphere-like colonies appear after one week of culture in stem cell maintaining growth conditions [8]. Furthermore, in a separate study, 20–40 days after plating GBM cells, colonies resembling classical neurospheres formed by normal NSC were detected. Interestingly, the frequency of sphere-initiating cells was between 0.5 and 31% of total cells plated for GBM and 50–80% for medulloblastomas. Conversely, sphere-initiating cells were not observed to occur in gliomas or oligodendromas, confirming previous work showing that EGF/FGF2-responsive precursors are specific to GBM and medulloblastomas [9]. A further study showed that subspheres generated from original sphere cultures form with an efficiency between 3 and 5%, suggesting tumor neurosphere colony cells can undergo serial passage. Strikingly, sphere forming GBM cells are able to form tumors in mice at cell numbers of 5,000, whereas non-sphere cells are unable to do so, even at much higher numbers [8]. Finally, the frequency of the prospective stem cell population within a given brain tumor has been noted to range between 0.3 and 25.1%, as determined by the frequency with which plated cells are able to generate spheres. Intriguingly, the frequency with which one tumor sphere will proliferate to form new spheres varies according to tumor subtype. More aggressive medulloblastomas often demonstrated much higher self-renewal capacities than the less aggressive pilocytic astrocytomas [10]. These results could be an indication that sphere forming ability, hence presence of CSC subpopulation, correlates with tumor aggression due to enhanced self-renewal capacities. Ultimately, however, these data do demonstrate that tumor neurosphere initiating cells are able to undergo self-renewal, have increased tumorigenicity, and represent a relatively rare cell type, which would be expected of a stem cell population. Interestingly, neurosphere initiating cells have also demonstrated multipotency and conserved expression of nestin, a cytoskeleton protein whose expression has high correlation with NSC and the developing central nervous system (CNS) [11]. For example, dissociated GBM tumor cells have been observed to form neurospheres that express progenitor marker nestin, but not neuronal or astrocyte differentiation markers. However, growth in differentiation-inducing conditions results in slowed proliferation in conjunction with induction of expression of differentiation markers of neuronal and glial lineages and loss of nestin expression [12]. Other studies have confirmed the presence of nestin mRNA in GBM spheres, but not in differentiated tumor progeny [8, 13]. Conversely, expression of lineage markers is elevated in differentiated progeny but not in the neurospheres [8, 13]. Intriguingly, comparisons between neurosphere colonies derived from normal brain tissue and gliomas, demonstrate that both populations express nestin, as well as phenotypic markers of neuronal (β-III tubulin) and glial (glial fibrillary acidic protein, GFAP) lineages [14], suggesting that there are similarities between tumor neurosphere initiating cells and the NSC. Separate studies have also confirmed that GBM spheres are able to differentiate into the three neural cell types, though admittedly different

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from normal neurospheres due to altered proportions of progeny lineages [8, 13]. These studies provide strong evidence that neural tumor spheres have properties and characteristics similar to those of normal neural spheres. There are, however, notable differences between neural tumor spheres and normal neural spheres. For example, in GBM a small number of cells in the tumor spheres stain positive for both neuronal marker β-III tubulin and astrocyte marker GFAP, a phenomenon that does not occur in normal tissue-derived spheres [9, 14]. Additionally, after exposure to differentiation-inducing environment, medulloblastoma and astrocytomas preferentially express lineage markers associated with their respective tumor of origin [10]. These results strongly suggest that while sphere forming cells originating from tumor specimens are able to activate some latent attributes of the neural developmental program, they are also aberrant as compared to normal brain-derived spheres. The sphere assay has also been used by at least one group to isolate and expand putative CSCs in melanoma. Melanoma sphere-initiating cells were found to have the capacity to persist in long-term culture for up to 8 months with no significant decrease in growth efficiency. Additionally, sphere-generating cells cultured in stem cell growth medium maintained their growth potential; however, under differentiation-inducing conditions, cells slowed their growth dramatically and completely stopped after 18 days. Furthermore, melanoma spheres could readily differentiate into numerous mesenchymal lineages including, melanogenic, adipogenic, chondrogenic and osteogenic lineages. Of the six clones tested, three could differentiate into all three of the later lineages demonstrating clear multipotency; although, the three other clones were only tripotent, bipotent or unipotent [15]. Thus, the sphere assay appears to be relatively successful in enriching for cells with enhanced similarities to CSC in melanomas. A further solid tumor type in which the sphere assay has been used to search for stem cell characteristics is the ovary. A single tumorigenic clone was isolated from among a mixed population of cells derived from the ascites of a patient with advanced ovarian cancer [16]. The clone possessed stem-like cell characteristics and was able to grow in anchorage-independent culture to generate spheres. Furthermore, the clone was able to establish tumors in immunodeficient mice that resembled the original human tumor in their histopathology. These tumorigenic clones were able to continue to establish tumors even after serial transplantation, which, as already discussed, is one of the golden standards used to identify stem cells. While the sphere assay has demonstrated strong potential as a method to culture and increase stem-like cell numbers, some caution should be kept in mind when using this system. Recently, it was observed that fusion of neurospheres is in actuality a common and rapid event, regardless of whether the culture is composed of primary or secondary spheres. This would suggest that the behavior is not just a result of culture conditions. Furthermore, sphere merging appears to be independent of the tissue, age, species of origin (e.g., human vs. rat) or culture plate size (e.g., 24vs. 96-well). Additionally, absorption of single free floating cells or cells adhered to the plate has been observed [17]. The fact that spheres are noted to absorb free cells

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raises a number of alarms. First, variable cellular component may not be a reflection of differentiation capacity of the initial sphere-initiating cell. Second, the size of the sphere is not necessarily an accurate representation of proliferation activity or self-renewal. Third, unless specifically tested to be so, the sphere is not necessarily of clonal origin. Notably, there is also considerable variability in the "established protocols" typically used in sphere assays. Factors, such as length of time cultured, depth of medium and size of cell plate, may be responsible for unknown, and important, variability. Study results also indicate that many dividing cell from various tissues, if grown in serum-free medium on a non-adherent substrate form floating cell clusters [17]. In support of this, as noted in the melanoma cells tested, spheres were formed by cells which proved to be only bipotent and unipotent [15]. Without verified self-renewal potential such cells could be progenitor or transit-amplifying cells rather than stem cells. Therefore, while the sphere assay is useful for expanding putative stem-like cell populations in tumors, it may not necessarily be an indication that such cells are CSCs.

14.2.2 Side Population Cells The population of cells termed side population (SP) cells due to a unique ability to prevent DNA binding dye, Hoechst 33342, from accumulating have so far demonstrated a great deal of promise in their capacity to enrich for prospective CSCs in solid tumors. SP cells were originally identified in bone marrow stem cells, in which the SP cells were shown to not only have stem cell characteristics, but also enrich for a stem cell population [18]. Though the mechanism whereby the SP phenotype occurs remains unclear the most popular theory proposes that ATP-binding cassette (ABC) transporters are able to pump out the fluorescent dye. This is due to a strong correlation between the SP phenotype and ABCG2 expression in bone-marrow cells [19]. In support of this theory, SP cells in several solid tumor tissue types have been demonstrated to have higher expression of several ABC drug transporters, including BCRP/ABCG2, as compared to non-side population (non-SP) cells [20–22]. In one extreme example, ABCG2 expression was observed to be increased 30-fold in Cal-51 breast carcinoma cells as compared to non-SP cells [23]. Alternatively, the phenotype could be generated due to limited uptake of the dye as a result of relative quiescence. There is appreciable evidence to suggest that the majority of solid tumors contain SP cell populations, though some cell lines do appear to be lacking them. In neuroblastoma cell lines examined, all lines contained SP cells with values ranging from 4 to 37% of the total viable cell population [21]. Additionally, SP cells have been identified in small-cell lung cancer, breast adenocarcinoma, Ewing sarcoma, teratocarcinoma, hepatocellular and ovarian carcinoma cell lines in various percentages [20, 21, 24, 25]. Therefore, that there is a key role of the SP cell in solid tumor initiation and progression is an intriguing notion, and one which many researchers have begun to investigate in an attempt to elucidate it more fully.

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Impressively, the SP phenotype has been linked to increased tumorigenicity, a poorly differentiated phenotype and sphere forming abilities in many solid tumors examined so far, strongly suggesting SP cells possess numerous cancer stem-like abilities. For example, in the human breast cancer cell line MCF7, SP cells demonstrate higher colony-formation efficiency (6.0%) in cell culture than non-SP cells (0.5%) [26]. Additionally, they also show higher tumorigenicity in vivo than do non-SP cells, forming tumors, respectively, in 70% vs. 20% of mice inoculated with 2,000 cells. Furthermore, these same cells show typical pathological features of cancer, including poorly differentiated cells, whereas non-SP MCF7 cells demonstrate a different pathology with a higher frequency of differentiated cells and fewer poorly differentiated cells. The MCF7 SP population is also able to reconstitute original tumor pathology. Higher tumorigenic potential of SP cells has also been observed in several ovarian carcinoma and at least one human hepatocellular carcinoma (HCC) cell line [20, 25]. In fact, in xenograft transplant experiments with the HCC cell line, 1 × 103 SP cells was sufficient to cause tumor growth in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice whereas twice that number of non-SP cells was unable to initiate such growth [20]. HCC SP cells also demonstrated high proliferative potential and anti-apoptotic properties compared to non-SP cells [20]. Further examination of the SP cell population in four HCC demonstrated that in all cell lines the SP cells showed similar characteristics of self-renewal, high clonogenicity, high expression of ABCG2 and notable chemoresistance. Furthermore, as few as 2,000 SP cells were able to initiate tumors in NOD/SCID mice [27]. In the murine C6 glioma cell line SP cells are able to form floating, neurosphere-like cell aggregates when grown under conditions similar to those that stimulate neurosphere growth in CNS NSC. Strikingly, this ability was found to be limited to the SP cell phenotype [24]. These results demonstrate that SP cells probably have multilineage potential, but that non-SP cells do not; therefore, SP cells in many solid tumors demonstrate many of the characteristics that are used to identify CSCs. SP cells have also repeatedly shown an ability to undergo asymmetric division in multiple solid tumor tissue types. MCF7 SP cells were able to generate both SP and non-SP cells, but non-SP cells were only able to generate more non-SP cells [26], suggesting the ability to undergo asymmetric division is limited to the SP cell subpopulation. In support of this, serial sorting of ovarian SP cells demonstrated enrichment of SP cells and the presence of non-SP cells [25]. Additionally, at least one glioma and three other neuroblastoma cell lines have demonstrated an ability to undergo rapid and discernable asymmetric division, which generate SP and non-SP subpopulations [21, 24]. In a human HCC cell line, SP cells were also able to generate both SP and non-SP cells. Additionally, the HCC SP cells contained a large number of cells that marked positive for the hepatocyte-specific marker, alpha-fetoprotein, and the cholangiocyte-specific marker cytokeratin 19 (CK19). Conversely, most non-SP cells were labeled with either one marker or the other [20]. Therefore, in the majority of solid tumors tested SP cells consistently demonstrate the stem cell characteristic of asymmetric division.

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Notably, the SP cell phenotype has been correlated to the expression of “stemness genes.” For example, in the C6 glioma cell line SP cells were positive for nestin, but not for neural lineage markers, MAP2 and β-III tubulin, or for GFAP, perhaps indicating that C6 SP cells represent undifferentiated NSC-like cells. Supporting this notion, growth in differentiation inducing conditions led to loss of nestin expression and resulted in cells labeled positive for MAP2, β-III tubulin and GFAP. Therefore, C6 SP cells are apparently able to generate both neurons and glial cells in culture and in vivo [24]. Furthermore, neuroblastoma SP cells demonstrate an increased number and intensity of cells expressing CD117 (stem cell factor receptor) [21]. Additionally, HCC show an upregulation of numerous genes associated with stem cell characteristics as compared to non-SP cells [20]. Finally, genes associated with regulation of cell-cycle quiescence are also upregulated in SP cells compared to non-SP cells, a phenomenon that would be expected of cells having stem cell characteristics. Flow cytometry has demonstrated that indeed a greater proportion of SP cells are in a quiescent state [25, 26]. Therefore, SP cells have a stronger resemblance to stem cells in their gene expression profiles than do non-SP cells. SP cells appear to share a number of notable characteristics with normal stem cells; however, there are also some notable limitations and concerns in regards to use of this technique to identify putative CSCs. First, there are a number of solid tumor cell lines that have not been found to contain SP cells, which indicates that this phenotype is not necessary for clinically relevant tumor initiation and progression. Furthermore, a relatively recent study found that Hoechst die is able to induce differentiation in tumorigenic PC12 and C2C12 cell lines [28], indicating that there may be unknown confounding errors being introduced when using the method. Finally, the manner in which the die works, AT specific minor grove DNA binding, could result in altered gene expression. For instance, the dyes have been demonstrated to have an ability to inhibit topoisomerases I and II, which play important roles in damage response pathways. Human glioma cell lines have been shown to be more sensitive to cell death upon Hoechst exposure following irradiation. Additionally, cells experience a growth inhibition and significant cell cycle delay [29]. Therefore, Hoechst die may have significant impacts on tumor cells, and exposure to the die may result in selection of a population of cells, which appears to have stem-cell like characteristics after exposure, but may not in fact be CSCs in the primary tumor.

14.2.3 Cancer Stem Cell Markers Biological markers, particularly antigenic markers have been used with reasonable success in recent years to identify, isolate and characterize normal stem cells in numerous tissues. Previously, it has been shown that the phenotype of CSCs in acute myelogenous leukemia is similar to that of early hematopoietic stem cells [reviewed in 4]. Similarly, recent studies have been successful in using normal antigenic stem cell markers for isolation of putative CSCs in solid tumors (Table 14.1).

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Table 14.1 Isolation and characterization of human and mouse cancer stem cells Organ/Location

Markers

Isolated cells (%) Assays

Brain [10, 35]

CD133+

3.5–46.3

Breast [38, 44]

CD44+ CD24–/low ; CD44+ CD24–/low ESA+ ; CD133+ (Mouse: CD29high CD24high ) CD133+

Colon [45, 46]

Head and neck [49] CD44+ Kidney [50] CD105+ Liver [51] Ovary [52]

CD133+ CD44+ CD117+

Pancreas [53]

CD44+ CD24+ ESA+

Prostate [56, 68]

CD44+ /α2β1high /CD133+ (Mouse: Sca-1+ )

Skin [62, 63]

ABCB5+ (Mouse: CD34+ )

Sphere formation; xenograft (brain); serial transplantation 2–4 Sphere formation; xenograft (mammary fat pad); serial transplantation 2.5±1.4 Sphere formation; xenograft (subcutaneous; renal); serial transplantation 0.1–41.7 Xenograft (subcutaneous) 8.0±3.3 Xenograft (subcutaneous); serial transplantation 50.8 Xenograft (subcutaneous) <0.2 Sphere formation; xenograft (subcutaneous); serial transplantation 0.2–0.8 Xenograft (subcutaneous; tail); serial transplantation 0.1 Colony-forming and long-term serial culture assays 9.0±3.5 (Mouse: Xenograft (subcutaneous); 17.2±4.8) serial transplantation

14.2.3.1 Brain A promising marker that has been gaining popularity for identification of tumorigenic brain tumor cells is CD133, also known as Prominin 1. CD133 is a 120-kDa cell surface protein compromising five transmembrane domains and two glycosylated extracellular loops, originally described as a hematopoietic stem cell marker [30]. The protein has also been used to mark normal human neural stem cells [31]. In the CNS single cells sorted for CD133+ CD34– CD45– were able to initiate neurosphere cultures and the progeny of clonogenic cells were able to reestablish neurosphere cultures, demonstrating self-renewal potential in the CD133 expressing population. Additionally, the CD133+ population was able to produce the three main mature cell classes of the CNS: neurons, astrocytes and oligodentrocytes [32]. As discussed in subsequent sections, CD133 also appears to enrich for CSCs in a number of tissues, especially those of epithelial origin [33]. In many brain tumor cell lines and primary cultures examined so far there is a strong correlation between CD133 and sphere-initiating ability. For example, one study on medulloblastomas and pilocytic astrocytomas found that all tumor

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spheres from examined samples showed immunoreactivity for CD133, and that CD133 expression ranged from 3.5 to 46.3% [10]. GBM spheres have also been found to be positive for CD133 expression [8]. Furthermore, cells sorted to be CD133+ were able to grow as non-adherent tumor spheres capable of continual expansion. In contrast, CD133– cells adhered to culture dishes and neither proliferated nor formed spheres. Interestingly, low-grade astrocytomas typically have fewer CD133+ cells than high-grade medulloblastomas [10], suggesting a possible correlation between tumorigenicity and CD133 expression. CD133+ cells are clearly enriched for sphere-initiating cells in numerous brain tumor types. Elaborating on the possibility that CD133 expression is associated with tumorigenicity several studies have positively linked its expression to tumor initiation potential and progression. By examining the first and second resection of tumor tissue from the same patient, it was found that CD133 expression was significantly higher in recurrent tumor tissue than in primary tissue [34], suggesting that increased number of CD133+ cells correlates with tumor progression. Additionally, implantation of CD133+ cells in immunodeficient mice demonstrates that as few as 100 CD133+ cells are needed for tumor initiation, whereas 105 –106 cells are typically needed for successful bulk tumor xenografts, and much larger numbers of CD133– cells do not consistently form tumors. Furthermore, in cell culture CD133+ cell fractions were observed to be proliferating while demonstrating clear self-renewal capacity, an ability that was lacking in CD133– cells [35]. Clearly, CD133-based isolation has the capacity to enrich for cells capable of initiating tumor growth in multiple brain tumor types. Interestingly, CD133 expression correlates with stem cell associated gene expression, such as nestin, implicating conservation of the NSC phenotype in putative brain tumor CSCs. Postnatally nestin expression is restricted to the subventricular zone and vascular endothelium. Nestin is additionally present in early ENU-induced neural neoplasia in the undifferentiated, proliferating cell populations [36]. GBM cells in culture were almost all CD133+ , in addition to being positive for CXCR4 and nestin [13]. CD133 has been further correlated to nestin expression in a number of neuroblastoma types, including further confirmation of it’s expression in GBM spheres [8, 10]. Additionally, the CD133+ phenotype has been associated with an increased expression of genes such as CD90, CD44 and Musashi-1, as compared to cells lacking CD133 expression [34]. The importance of these genes in the NSC phenotype is exemplified by Musashi-1, a neural RNA-binding protein strongly expressed in both fetal and adult mammalian NSC [37]. Furthermore, CD133+ cells expressed oncogenes such as Bmi-1, Sonic hedgehog (Shh), Oct-4 and Snail mRNA, also common regulators of the stem cell. In contrast, their expression was not detected in the counterpart negative cells. CD133+ cells also had significantly higher levels of stem cell-associated proteins Gli1 and Patched [34]. All of the evidence presented indicates that there is a stem-like cell population in many neural carcinomas, and that they can be enriched by using CD133 as a marker. However, evidence does not disprove that the above described phenotype cannot be reacquired by CD133– cells, nor does it necessarily demonstrate that the origin cell is a NSC

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stem cells, as many of these pathways might be activated via genetic or epigenetic events in more differentiated cells. 14.2.3.2 Breast Initial pioneering work in the identification and isolation of stem-like cells in solid tumors was done in breast cancer cells [38]. In this study, non-epithelial lineages (Lin), such as hematopoietic, endothelial and fibroblast cells were eliminated from dissociated breast tumor tissue and the remaining cells were selected for a CD44+ CD24–/low phenotype. CD24 is widely recognized as a marker for differentiation in several lineages, while CD44 is a transmembrane protein cell adhesion molecule originally described as a lymphocyte-homing receptor [39]. Results suggested that the Lin- CD44+ CD24–/low population was 10- to 50-fold enriched for the ability to form tumors in NOD/SCID mice, relative to unfractionated tumor cells. Upon this discovery, further enrichment was done using epithelial-specific antigen (ESA), which probably functions in cell-to-matrix adhesion. As few as 200 ESA+ CD44+ CD24–/low cells were needed to consistently form tumors after injection, which suggests a remarkable enrichment for tumor initiating cells, heretofore unknown in solid tumors. These cells constituted a mere 2–4% of the tumor cell population, as would be expected of a stem cell population. Furthermore, they were able to self-renew, generating more ESA+ CD44+ CD24–/low cells, in addition to giving rise to a phenotypically diverse population of non-tumorigenic cells. Furthermore, new tumors were morphologically similar to the primary tumor from which the ESA+ CD44+ CD24–/low cells had been derived [38]. Therefore in human breast tumors sorting for CD44+ CD24–/low cells enriches for tumor-initiating cells able to undergo self-renewal. The CD44+ CD24– phenotype has further been demonstrated to have a clear ability to be propagated in culture. After sorting for CD44+ CD24–/low phenotype cells were plated and mammosphere growth was observed after 10–15 days. Serial passages demonstrated a continuous ability of these cells for self-renewal. In fact, when plated as single cells this subpopulation could still give rise to mammospheres. These spheres did not appear to express lineage-specific differentiation markers CK14/18, CD10 or ESA [40], suggesting that they were relatively undifferentiated cells. This seems to be contradictory to the previous study in which ESA was a marker found to enrich for the CSC. Intrinsic variations in cell lines could be a possible explanation. The ability of the CD44+ CD24–/low cells to grow in culture was further confirmed and elaborated in Brca-1 deficient mouse tumor cell lines. CD44+ CD24– Brca-1 deficient cells formed significantly more viable proliferating spheres that were able to grow in non-adherent conditions after numerous passages, compared to CD44– cells. The frequency of sphere formation was between 2 and 4%, with spontaneous enrichment of those cells expressing the putative cancer stem cell markers occurring after multiple passages. CD44+ CD24– Brca-1 deficient spheres also had distinct CD24–/low cell populations, indicating asymmetric division occurred.

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Furthermore, as few as 50–100 cells were needed to form tumors in NOD/SCID mice, whereas 50- to 100-fold more cells were required of parental or putative CSC depleted subpopulations in order to form a few slow growing tumors [41]. However, it is notable that tumors could still grow even in the absence of CD44+ CD24– cells. Further investigation of the CD44+ CD24– phenotype was done to determine if this population possesses three of essential characteristics of cells with metastatic potential, including expression of invasion/metastasis-associated genes, invasion, and homing and proliferation to sites of metastasis. While it was found that these cells do indeed express higher levels of proinvasive genes and are able to exclusively invade matrigel in cell culture, as compared to CD44– cells, the CD44+ CD24– phenotype was not found to be sufficient for homing and proliferation at sites of metastasis [42]. Interestingly, CD44+ CD24– cells from MCF-S cell line were found to overexpress neoangiogenic and cytoprotective factors [40]. Therefore, though sorting for the CD44+ CD24– phenotype appears to enrich for cancer stem-like cell population with angiogenic properties, it is not sufficient to isolate prospective metastatic CSC. The expression of CD44 and its correlation to CD133 expression was also investigated in several breast cancer cell lines. All cell lines contained cells expressing CD44, though the degree of expression varied by cell line. However, no overlap was found between CD133 expression and the CD44+ CD24– phenotype. Furthermore, only one tumor-derived cell line was found to have a significant CD133+ population, which was between 2 and 6%. Similar to the CD44+ CD24– phenotype CD133+ cells were able to recapitulate tumor growth at numbers as low as 50–100 cells, whereas CD133 depleted populations took 50 to 100-fold more cells to initiate slow tumor growth. Furthermore, CD133– cells were unable to reconstitute the parental population, a problem not experienced by CD133+ cells [41]. Therefore, the data demonstrate that non-overlapping CD133+ and CD44+ CD24– cell populations have similar capacities for self-renewal and are able to repopulate cell fractions found in the respective parental cells in breast tumor cell lines. Interestingly, the overlapping expression of stem cell genes, such as Notch1, Fgfr1, and Sox1, occurs in both the Brca-1 deficient CD44+ CD24– and CD133+ phenotypes. Additionally, both CD44+ CD24– and CD133+ spheres are also positive for Oct4 expression, which is a common marker for pluripotency. Cells expressing the putative cancer stem cell markers (CD44+ CD24– /CD133+ ) were also enriched for expression of ABC drug transporters, already discussed to be upregulated in the putative SP CSC, perhaps indicating that these phenotypes also correlate to the SP phenotype. CD133 expression also overlapped with expression of Numb, a marker associated with asymmetric division, in one cell line examined [41]. Therefore, it can be asserted that though there is no correlation between CD44+ CD24– and CD133+ cells, both subpopulations represent a cancer stem-like cell population in Brca-1 deficient cells. Another approach to identify prospective breast tumor-initiating cells is detection of aldehyde dehydrogenase activity (ALDH), which is significantly higher in stem and progenitor cells and can be detected by such commercial reagent

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as ALDEFLOUR (StemCell Technologies Inc.). Of 14 different patient samples examined, an average of 8% of the cell population was found to be ALDEFLOURpositive (AP) [43]. When subjected to suspension culture growth assays the AP subpopulation isolated from fresh mammoplasty samples was able to generate mammospheres when plated at a density of 5,000 cells/ml, whereas the negative cell subpopulation was not. Multiple passages demonstrated that AP cells could continually reform tumors even at low cell numbers. Additionally, AP cells were enriched for bilineage progenitor cells capable of generating mixed lineage, ESA+ CD10+ , colonies at much higher frequencies than negative cells, 67 and 9% respectively. Furthermore, size and latency of tumor formation was correlated to the number of AP cells injected, where increasing AP numbers resulted in decreasing latency and negative cells failing to generate tumors. Intriguingly, negative cells sorted for the Lin- CD44+ CD24–/low phenotype were not tumorigenic even when implanted at numbers as high as 50,000 cells/mammary pad. Contrastingly, cell subpopulations bearing both phenotypes had high tumorigenic capacity and generated a tumor from as few as 20 cells [43], suggesting that the AP phenotype further enriches for CSC in breast tumors. Stem cell markers were also used to identify CSC in mammary tumors associated with p53 deficiency in mice. Tumors formed after orthotopic transplantation of mouse p53 null mammary cells are heterogeneous, demonstrating expression of myoepithelial (CK5, CK14) and luminal markers (CK8, ERα), as determined by immunohistochemical analysis. Coexpression of CK8 and CK14 occurs, indicating the expansion of a putative bipotent progenitor, previously suggested being potential target in Wnt-1 tumors. The Lin– CD29high CD24high population, representing approximately 5–10% of the total cell population, displayed significantly increased tumorigenic potential compared with Lin– CD29high CD24low , Lin– CD29low CD24high , Lin– CD29low CD24low populations. As few as 100 Lin– CD29high CD24high cells were needed for tumor initiation in 8 of 14 transplants, whereas no tumors were observed from 100 cells of the other three populations, indicating these cells had markedly increased tumorigenic potential. However, the Lin– CD29high CD24low subpopulation also displayed increased tumorigenicity when compared with the Lin– CD29low CD24low and Lin– CD29low CD24high subpopulations which represented the bulk (>90%) of the tumor cells, suggesting that this subpopulation may represent a sort of transitamplifying cell in this model. Tumors generated by the Lin– CD29high CD24high subpopulation had comparable morphology to original tumors from which they were derived, suggesting that this tumor initiating population is able to regenerate the heterogeneous characteristics of the original tumor. Additionally, the Lin– CD29high CD24high population was also able to form mammospheres, which were larger in size and number compared to other antigenic marker combinations [44]. All of these results suggest that the CD29high CD24high population is enriched for the putative CSC in mouse mammary carcinomas associated with p53 deficiency. However, it should be noted that all of the above experiments were performed on tumors derived by transplantation of p53-null mammary epithelium cells into cleared mammary fat pads. Thus, it remains to be demonstrated if observed

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results can be reproduced in an autochthonous sporadic mouse model of mammary carcinogenesis. 14.2.3.3 Colon Interestingly, the CD133 phenotype also appears to be associated with cancer stemlike cells in colon cancers. Human colon cancer cells demonstrated presence of rare CD133+ cells in areas of high cellular density, with a calculated average of one per 5.7 × 107 tumor cells [45]. One-fourth of mice with xenograft implantation of 100 CD133+ colon cancer cell fractions develop tumors. From these results the authors calculate that the average CSC population in CD133+ cells is one in 262, representing a 216-fold enrichment of putative CSC as compared to the unfractionated colon cancer cells [46]. A similar study confirmed CD133+ cells had enhanced ability to form tumors, in that 105 CD133– primary colon cancer cells were unable to induce tumor formation, whereas the injection of 106 unseparated cells or 3,000 primary CD133+ cells generated visible tumors within 4–5 weeks after transplantation. All tumors generated from CD133+ cells were not only phenotypically similar to original tumors, but also able to generate CD133– cells. Additionally, during in vivo passage CD133+ cells did not lose their tumorigenic potential, but instead increased in aggressiveness. Furthermore, CD133+ cells did not express CK20, an intermediate filament protein whose expression is limited to differentiated cells of the gastric and intestinal epithelium and urothelium [45, 47]. CD133+ tumor spheres grown in culture before being xenografted were able to form tumors in mice at cell counts of 50–500 and continued to express negligible amount of CK20. Strikingly, CD133+ cells grown under differentiation inducing effects did express CK20, and also acquired a morphology closely resembling the major colon cancer cell population that was present in the original tumor. Differentiated cells were unable to form a tumor when implanted at numbers even as high as 106 cells [45], clearly demonstrating that CD133 expressing colon cancer cells are enriched for a relatively undifferentiated, tumor-initiating cell population. 14.2.3.4 Head and Neck In head and neck squamous cell carcinoma distinct populations of CD44+ and CD44– cancer cells are identifiable. CD44+ cells are able to generate new tumors much more efficiently than CD44– cells. Interestingly, CD44 expression was detected in the basal layer, but not in the more differentiated cells, indicating a tumor hierarchy in which CD44 expression correlates to less differentiated cells. Furthermore, CD44 costained with CK5 and CK14, which are both markers of progenitor cells. Finally, CD44 and involucrin, a differentiated keratinocyte marker, stainings were mutually exclusive [48, 49], supporting the role of CD44 as a putative marker of stem-like cells in squamous cell cancer. 14.2.3.5 Kidney Interestingly, the promising CD133 putative CSC marker, which has been found to enrich for normal stem cells in the kidney, was not found to enrich for tumorigenic cells in renal carcinomas. However, sorting for CD105 led to marked

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enrichment for cells possessing a number of characteristics associated with CSCs. For instance, the CD105+ cell population induced tumors with 100% incidence, whereas CD105– cells only induced tumors at a 10% incidence. CD105+ cells also expressed a number of stem cell associated markers, such as nestin, Nanog, Musashi and Oct4, in addition to the renal embryonic marker Pax2. However, when grown in differentiation inducing conditions, cells lost expression of nestin, and began expressing endothelial markers VEGFR3, CD31, KDR and CK7. Growth in anchorage-independent conditions in sphere-generating media demonstrated that CD105+ cells are able to generate spheres. Furthermore, single cells from enzymatically digested primary spheres are able to propagate as spheres for up to 10 passages, indicating extensive self-renewal capacities. Subcutaneous injection into SCID mice demonstrated that cell clones have tumorigenic potentials similar to other putative solid tumor CSC, such that only 100 CD105+ cells are needed to initiate tumors. Furthermore, primary tumors in mice were found to contain both CD105+ and CD105– cells, indicating that CD105+ cells are able to generate tumor heterogeneity. Additionally, serial passages demonstrate that cells from primary tumors are able to generate secondary and tertiary tumors that are essentially morphologically indistinguishable from primary tumors. In contrast, CD105– cells were unable to do so [50]. Therefore, CD105 appears to enrich for stem-like characteristics in renal tumor cells. 14.2.3.6 Liver The ability of CD133 to enrich for HCC has been briefly explored. Out of three HCC cell lines tested only one (Huh-7) was found to stain positive for CD133. However, in the Huh-7 cell line CD133+ cells performed higher on cell culture proliferative potential assays, and also had lower expressions of mRNA’s typically associated with differentiated hepatocytes, such as glutamine synthetase and cytochrome P450 3A4, than did the CD133– population. Therefore CD133 cells appear to have a less differentiated phenotype. Furthermore, they also had higher tumorigenic potential, and were able to generate significantly larger tumors when grafted into mice than CD133– cells [51]. Therefore, CD133 expression appears to be associated with a cancer stem-like phenotype in HCC. 14.2.3.7 Ovary A recent study indicates that CD44+ CD117+ cells have enhanced stem-like characteristics [52]. Both CD44 and CD117 are overexpressed in advanced ovarian malignancies. Sphere-initiating ovarian carcinoma cells demonstrate that greater than 80% of the cells coexpress CD44 and CD117. However, showed CD44+ CD117+ cells constitute less than 0.2% of the total tumor cell population, but are fully able to recapitulate the tumor’s original phenotype upon engraftment of only 100 cells. Serial transplantation demonstrates that the double positive cells are able to maintain a consistent population size. In contrast, double negative cells are non-tumorigenic. Therefore, in ovarian carcinoma CD44+ CD117+ population is likely enriched for a CSCs.

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14.2.3.8 Pancreas Markers that have been used in an attempt to isolate putative pancreatic CSCs in human cells include those that were used successfully to enrich for breast CSCs, CD44, CD24 and ESA, as well as CD133. In pancreatic tumors, bulk injection of tumor tissue requires at least 104 cells in order for tumor growth to occur [53]. However, once sorted for the CD44+ CD24+ ESA+ phenotype as few as 100 cells are necessary to initiate tumor growth, in approximately half the cases. Furthermore, primary and secondary tumors derived from CD44+ CD24+ ESA+ cells are morphologically similar to the patient’s primary tumor, maintaining the CD44+ CD24+ ESA+ cell population, as well as generating a phenotypically diverse non-tumorigenic population. Therefore, this tumor cell population has multipotent tumorigenic potential even after multiple passages. Similarly, CD133+ pancreatic tumor cells were able to form tumors with as few as 500 cells, whereas CD133– cells did not form tumors even at cellular numbers as great as 106 . CD133+ primary tumor xenografts serial transplanted into secondary and tertiary mice grew rapidly, and continued to be negative for cytokeratin, a marker of differentiation. However, the developing tumors did express cytokeratin and formed tumors with morphologies indistinguishable from tumors generated by unfractionated cells [54], suggesting that CD133+ cells are able to reconstitute original tumor morphology. To our knowledge there have been no studies yet examining the correlation between the CD133+ phenotype and the CD44+ CD24+ ESA+ phenotype. It would be interesting to know if these markers are typically coexpressed, or if, as it has been observed in breast carcinoma, they indicate distinct types of CSC populations. Notably, CD133+ pancreatic cancer cells were not only able to form spheres in known sphere forming conditions, but also showed preferential resistance to chemotherapeutic drugs. When pancreatic cancer cells were cultured in serum-free medium containing EGF and FGF-2, spheres aggregates of undifferentiated CD133+ cells formed within 2–3 weeks. Conversely, CD133– cells invariably died when grown under such conditions. Furthermore, addition of differentiation inducing variables resulted in expression of cytokeratin and the acquiescence of a morphology closely resembling the major pancreatic cancer cell population. Finally, CD133+ cells showed dramatic resistance to chemotherapeutic agent gemcitabine compared to CD133– cells both in cell culture and in vivo [54]. Interestingly, CD133+ pancreatic tumor cells have also been correlated to an ability to undergo cell migration, a correlation that has not been directly made with markers of putative CSC in other solid tumors types. For example, staining for receptor CXCR4, specific to stromal cell-derived factor 1, an important mediator in cell migration, demonstrated strong costaining with CD133+ cells in the invasive front. Notably, L3.6pl pancreatic cancer cell linederived CD133+ putative CSCs predominately expressed the CXCR4 receptor. Furthermore, flow cytometric analysis of portal vein blood samples following development of CD133+ CXCR4– and CD133+ CXCR4+ xenograft tumors indicated that only the CXCR4+ group was able to generate circulating CD133+ CXCR4+ cells. Contrastingly, in the CXCR4– group no CD133+ cells or CXCR4+ cells

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could be detected in circulating blood. Additionally, liver metastases were only detected in mice receiving CD133+ CXCR4+ xenografts, and not in mice receiving CD133+ CXCR4– cells. Finally, a CXCR4 inhibitor, AMD3100, resulted in a significant reduction in tumor metastasis [54]. These results are some of the first that directly link putative CSCs to an ability to metastasize to other locations.

14.2.3.9 Prostate In the normal prostate epithelium sorting for α2 β1 high CD133+ markers enriches for stem cells [55]. Consistently, in the tumorigenic prostate, α2 β1 high CD133+ CD44+ sorting enriches for cells that have markedly enhanced proliferative potential and greater capacity for survival in anchorage-independent media. In fact, the α2 β1 high CD133+ CD44+ population gave rise to 3.7-fold more colonies than did the total population. Strikingly, 30-fold fewer colonies were observed to be initiated by the by the CD44+ /α2 ß1 low subpopulation, and the majority of these could not maintain renewal after three passages [56]. Therefore, CD44 tumorigenic prostate cell populations are enriched for putative CSC. The ability of CD44 to mark for putative prostate CSCs was further examined in the LNCaP, Du145 and PC3 cell lines, which demonstrate increasing tumorigenicity and malignancy respectively [57]. Interestingly, CD44 expression was observed in 100% of PC3 cells, approximately 28% of Du145 cells and was virtually undetectable in LNCaP cells, suggesting that CD44 expression correlates with tumor cell malignancy. Furthermore, CD44+ cells proliferate more extensively and form significantly more colonies than do CD44– cells. Additionally, CD44+ cells are both more tumorigenic and more invasive than CD44– cells, though both subpopulations could generate tumors in mice. Interestingly the variance between CD44+ and CD44– tumorigenicity was dependant on cell line. However, CD44 expression does appear to correlate with tumorigenic potential and aggression in prostate carcinoma. CD44+ prostate tumor cells also appear to have a more primitive phenotype with higher expression of a number of stem cell associated genes. For example, CD44 staining was not observed in more differentiated luminal epithelial prostatic cells expressing androgen receptor (AR). However, CD44 expression did colocalize with BrdU staining, demonstrating CD44+ cells represent a population of proliferating cells with intermediate cycling properties. CD44+ cells also expressed higher mRNA levels of Bmi-1, β-catenin, Smoothened (Smo) as well as Oct-3/4. Finally, approximately 15 of the clonally plated CD44+ cells examined in this study appeared to undergo asymmetric division, in that CD44 expression was only detected in one of two daughter cells [57]. Therefore, the evidence strongly indicates that CD44 serves to enrich for cells demonstrating cancer stem-like cell characteristics in human prostate tumor cells. As reviewed in [58] and Section 14.3 a number of studies have also identified CSC in mouse models of prostate cancer.

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14.2.3.10 Skin There is indirect evidence to support the presence of CSCs in melanomas, though relatively little work has been done on identifying markers that might be used to enrich for these cells in humans. For example, melanomas often show phenotypic heterogeneity both in vivo and in cell culture, indicating a cell with multilineage differentiation abilities is present in the tumor [59, 60]. Additionally, melanomas express a wide range of developmental genes. Interestingly, a small population of sphere cells express CD20, demonstrating that a subpopulation of melanoma cells express the hematopoietic marker CD20. Also of notable interest is that multipotent putative stem cells were enriched for in the CD20+ cell fraction of melanoma sphere cells isolated from fresh tumor lesions and established cell lines [15]. This could indicate that the hematopoietic marker CD20 enriches for CSC in melanoma. The finding that the hematopoietic marker CD20+ enriches for possible melanoma CSCs is consistent with observations in which markers originally characterized in the hematopoietic system also appear to have key roles in solid tumors. Interestingly, the ABC transporter ABCB5 is one of the first putative melanoma CSC markers to be thoroughly investigated. Characterization was initiated because ABCB5 is known to characterize progenitor cell subsets in normal skin [61]. Histologically, ABCB5+ cells correlate with non-melanized, undifferentiated regions of the tumor [62]. In contrast, melanized, more differentiated tumor areas are predominately ABCB5– . Furthermore, ABCB5 expression correlates to expression of stem cell markers nestin, BMPR1A and CD20, noted above to be a putative CSC marker for melanoma. Additionally, ABCB5+ cells are much more efficient at tumor initiation, with over half (14 out of 23) of the tumor cell injections resulting in tumor growth, than are the negative cells. ABCB5+ cells are able to generate both ABCB5 positive and negative cells, whereas ABCB5– cells are only able to generate negative cells. Finally, treatment of the tumor with an anti-ABCB5 monoclonal antibody, allowing for immune targeting of ABCB5+ cells, significantly inhibits tumor formation as compared to the control antibody. Therefore, this paper provides evidence for a link between prospective CSC and ABCB5 expression in solid malignancy. Chemically induced skin tumors have a 9-fold increase in CD34+ cell populations in mice. CD34+ tumor cells locate in close contact to stromal areas and also express additional markers of bulge skin stem cells, such as Gas3, Sox9 and Runx1, to name a few. Expression of CD34 was often found at the invading front, which is in line with another recent report identifying CSCs as the major source of tumor invasion [54, 63]. Additionally, the CD34+ subpopulation was over 100-fold more potent at initiating secondary tumors than unsorted tumor cells. Furthermore, secondary tumors maintain a stable population of CD34+ cells able to give rise to tertiary tumors, indicating maintenance of tumor-initiating potential. Conversely, CD34– cells do not produce tumors in this study. Notably, secondary tumors derived from CD34+ cells strongly resemble the parental tumor, such that they maintain a small population of CD34+ cells among the majority of more differentiated keratin 10-expressing cells [63]. An ability to maintain the CD34+ subpopulation and

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produce differentiated progeny further supports the stem-like nature of the CD34+ subpopulation. While many of the markers discussed above appear to have great promise in selecting for cells demonstrating tumor-initiating capabilities and perhaps representing CSCs in solid tumors, there are a number of concerns raised by experimental results that should be kept in mind. For example, recent work has demonstrated that CD44+ CD24/low cells can, in fact, originate from primary CD44low CD24+ human mammary epithelial cells following transformation with a limited number of oncogenes and cancer-associated genes [64]. Additionally, xenografted CD44– prostate tumor cells were found to contain CD44+ cells [57]. It is also should be noted that in many of the studies discussed in this section cells negative for the CSC marker being used were often able to generate tumors, though with admittedly reduced capacity. For example, tumors were observed when large number (>2,500) of Lin– CD29low CD24low and Lin– CD29low CD24high cells were injected, and Lin– CD29high CD24low cells were also able to form mammospheres [44]. While these observations may be a result of contamination, it is also plausible that non-tumorigenic neoplastic cells may reprogram towards stem cell properties. Additionally, the ability of the above markers to enrich for stem-like cells is highly variable depending on the tissue of origin, markers used and individual tumor. For instance, as discussed, putative CSC of the colon are proposed to represent approximately one in 262 cells positive for the putative CSC marker CD133 [46]. In HER2/Neu-induced mouse mammary tumors stem cell antigen-1 (Sca-1)+ CD24+ cells generate a calculated CSC frequency of one in 303 cells [65]. However, the putative renal CSC marker CD105 is only proposed to generate a clonal ability of one in 15,000 cells [50], and putative melanoma CSC are only enriched for by one in 158,170 cells using ABCB5 [62]. In fact, while using CSC markers appears to enrich for CSC-like cells, no study has successfully generated tumor growth from implantation of a single cell. Therefore, putative CSC markers in solid tumors are able to enrich for populations containing stem-like cells, but are unable to exclusively isolate them.

14.3 Cell of Origin of Cancer Stem Cells Since recent discoveries of multipotent progenitor cells with the capacity for selfrenewal in systems other than the hematopoietic system, there has been a flurry of activity in an attempt to not only identify CSCs in solid tumors, but also determine if their cellular origins are indeed adult somatic stem cells. Stem cells are particularly appealing candidates as the cell of origin for cancer (Fig. 14.2) for several reasons. In particular, there are a number of critical mutations that must occur in order to produce clinically significant cancers that include self-sufficiency for growth, insensitivity to antigrowth signals and limitless ability to replicate. Because of their pre-existing capacity for self-renewal and limitless replication abilities, stem

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Fig. 14.2 Cell of origin of cancer stem cell (CSC). The CSC theory predicts that malignant transformation (yellow bolts), results in the formation of a phenotypically distinct population of CSCs (dark red) reminiscent of normal stem cells. The CSC is able to self-renew and reconstitute tumor heterogeneity, perhaps creating a tumor hierarchy (varying shades of pink cells). The cell of origin for the CSC may be stem cells, progenitor cells and possibly even fully differentiated progeny (all blue). Additionally, it’s possible that non-tumorigenic bulk cells (pink) can transform into CSC

cells would need to undergo fewer mutational events in order to initiate carcinogenesis [4]. Furthermore, stem cells are relatively long-lived compared to other cells within tissues, giving them more opportunity to accumulate the multiple mutations necessary. There is accumulating evidence that the stem cell compartment is often the source of tumorigenic cells in solid tumors. Mice deficient for tumor suppressors p53 and Rb in the prostate epithelium develop metastatic cancer displaying features of both luminal and neuroendocrine differentiation [66], and early neoplastic cells often coexpress luminal epithelium markers CK8 and AR in addition to neuroendocrine markers synaptophysin and chromogranin A [22]. More importantly, malignant neoplasms are observed to arise from the proximal region of the prostatic ducts which is thought to be the location of prostate stem cells (reviewed in [58, 67]). Moreover, though inactivation of p53 and Rb also occurs in lineagecommitted transit-amplifying cells and/or differentiated cells in the distal region, tumor initiation does not occur in these areas. Additionally, cells of the earliest neoplastic lesions express Sca-1 and are not sensitive to androgen withdrawal. In murine prostate cells, Sca-1 enriches for cells capable of regenerating tubular structures containing basal and luminal cell lineages in a dissociated prostate regeneration system. Additionally, the Sca-1+ cell fraction contain increased percentages of cells with immature cell properties, including replication quiescence, androgen independence, and multilineage differentiation potential. Furthermore, Sca-1+ cells cluster at the proximal region [68].

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Interestingly, experiments on dissociated mouse prostate cells infected with a lentivirus mediating constitutive expression of AKT1, demonstrate that perturbations in the PTEN-AKT signaling axis in Sca-1+ cells can result in the initiation of prostate tumorigenesis. Notably, cancer progression was found to be associated with a concomitant increase in Sca-1+ cells [68]. This further suggest that Sca-1 enriched prostatic cell populations serve as targets for the initiation of prostate tumorigenesis. Immunohistochemical analysis demonstrates that regenerated prostatic intraepithelial neoplasia graft lesions contain both AR+ luminal cells and p63+ basal cells [68], which supports that lesions are derived from cells that possess the capacity for multilineage differentiation. Additionally, Pten has been shown to negatively regulate p63+ prostatic basal cell proliferation. In mice with prostate epitheliumspecific Pten deletion, basal cell proliferation is concomitant with expansion of prostate stem/progenitor-like subpopulation, as indicated by increased expression of Sca-1 and Bcl-2 positive cells [69]. These data strongly support that prostate carcinogenesis is often initiated in the regions of stem cell residence. In the brain there is also an accumulation of evidence suggesting that cells in the stem cell compartment are particularly susceptible to tumorigenic initiation. For instance, recent reports using Musashi-1 immunostaining have suggested that derivation of human pediatric CNS tumors commonly occurs from the subventricular zone [31]. Furthermore, the major regional distributions of some brain tumors overlap specific regions of the CNS that comprise glia-like neural stem cells and their progeny [70–73]. In animals, exposure to oncogenes or administration of carcinogens also appears to preferentially result in tumor initiation in regions of normal stem cell localization such as periventricular subependymal zone where there is rapid developmental proliferation of immature cells postnatally. Transgenic mice, in which SV40 T antigen is expressed under control of the GFAP promoter in astrocyte cells, demonstrate early neoplastic proliferation of cells in the periventricular subependymal zone, associated with strong expression of the transgene. Furthermore, levels of GFAP expression are low, which may indicate that SV40 T antigen targeted cells underwent transformation at an immature stage [74]. Transduction of constitutively active epidermal growth factor receptor, Ras and Akt or platelet derived growth factor into the forebrain nestin positive progenitor cells results in tumor formation [reviewed in 75]. Furthermore, deregulation of specific oncogenes such as Ink4A-Arf, epidermal growth factor receptor and c-Myc in GFAP or nestin expressing neural cells induces high-grade gliomas in brain areas that are typically considered to contain neural stem cells [74–77]. Moreover, intracerebellar injection of retroviruses carrying Shh and c-Myc can cause medulloblastoma in nestin-TVA mice. In this study, nestinexpressing neural progenitors appear to be the cell-of-origin for medulloblastoma [78]. In rats, transplacental exposure to ENU results in the development of tumors predominantly adjacent to the lateral ventricles in the subependymal plate [79]. A study on breast cell line HBEC indicates that variants with a more stem-like phenotype are more susceptible to transformation than variants with a more differentiated phenotype [80]. Type I HBEC cells are able to form budding and ductal structures in Matrigel when grown under a cyclic AMP differentiation inducing

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conditions. In contrast, Type II HBEC cells are unable to do so, which probably indicates that Type I cells have a stem cell population whereas Type II cells do not. Strikingly, Type I HBEC cells are much more susceptible to immortalization after transfection with SV40 large T antigen. The high potential of telomerase activation for Type I HBEC cells is one probable mechanism whereby Type I cells achieve more efficient immortalization than Type II cells [81]. Therefore, data not only provide evidence that a more immature cell phenotype of the same cell line is more susceptible to immortalization, but also indicates one likely mechanism by which the event occurs. In the liver, p53-null hepatic progenitor cells have been noted to undergo transformation into HCC [82]. These data would seem to implicate that progenitor cells are more susceptible to transformation. However, it remains unclear if there is a difference in the rates of transformation between progenitor and differentiated progeny. Additionally, these studies do not distinguish between lineage-committed progenitor cells and stem cells. In general, neoplastic cells appear to share many genetic similarities to normal stem cells and pathways important for stem cell regulation, such as Wnt, Shh and Notch, often demonstrate aberrant activity in cancer cells [4, 83]. However, the use of stem cell associated genes to define a cell population has proven to be tricky. Several studies have attempted to define a basic transcriptional program of “stemness” but results are still controversial [84–86]. Comparison of the shared microarray expression profiles of embryonic stem cells (ESC), NSC and hematopoietic stem cells, suggested that the groups only shared a single gene. While an inability to identify a consensus gene expression signature may indicate that stem cells in different tissues utilize unique pathways to achieve self-renewal and pluripotency, technical considerations such as variations in isolation techniques, cell purity or statistical analysis may also be at fault. In order to attempt to improve the organization and classification of stem cell transcriptional programs a gene module map method was proposed [87]. It was indicated that adult tissue stem cells are separated into two groups, one of which shares a core transcription program ESC. For instance, mouse ESC, NSC and retinal stem cells are clustered together by coordinate activation of a similar collection of gene sets, thereby demonstrating that a subset of adult tissue stem cells share a transcriptional program termed the “ESClike” gene set. The gene set included genes generally associated with pluripotency, such as Oct4, Nanog, Sox2 and c-Myc. In contrast, the majority of adult tissue stem cells, including neural crest stem cells, hair bulge stem cells and mammary stem cells clustered together by coordinate activation of a distinct collection of gene sets, termed the "adult tissue stem". Hair bulge stem cells from two independent studies were crossvalidated and demonstrated to cluster together, apparently indicating that the gene module map approach accurately clusters stem cell transcriptional patterns. Furthermore, those same genes were induced in mammary stem cells and neural crest stem cells relative to their differentiated counterparts. Essentially, the map confirms that there is no single “stemness” program shared across all stem cells, which may indicate that using stem cell associated genes as an identifying feature of the CSC may not be a strong validation test.

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However, the above study found that the ESC-like transcriptional program was frequently and significantly activated in aggressive human epithelial cancers, relative to their corresponding normal tissue but repressed in normal tissue relative to cancer. The opposite was true of normal tissue stem cells. Interestingly, c-Myc was found to be uniquely able to activate the ESC-like transcriptional program in adult epithelial cells, which in the appropriate genetic context confers cardinal properties of human cancer stem cells in vivo [87]. In summary, this may present evidence that activation of ESC-like pathways does not indicate a progenitor cell origin as the pathways that maintain stem cells in the majority of normal tissues appears to be quite different. Instead, data may support the generation of the CSC is purely a result, albeit an important one, of aberrant regulation of select oncogenes as a result of mutation or other genetic alteration. Intriguingly, further studies examined whether the ESC-like gene expression program is preferentially expressed in the CSC subpopulation. The expression of the ESC-like gene module in the CD44+ CD24 /low subpopulation of breast cancer cells in six independent breast cancers was examined. Notably, two of the CD44+ CD24 /low subpopulations has significantly upregulated ESC-like gene module relative to normal breast tissue, which would appear to suggest that the ESC-like signature is activated in CSC populations [87]. However, what of the other four cell lines in which a correlation was not observed? It seems significant that over half of the samples examined did not demonstrate strong correlation. Notably, the gene expression profile of CD44+ cells resembles that of stem cells. Furthermore, normal and tumor CD44+ cells are more similar to each other than to CD24– cells from the same tissue. Tumor CD44+ and CD24+ cells were found to be clonally related, though in some tumors CD24+ cells had additionally genetic alterations to the ones shared by the CD44+ cells. Intriguingly, genes involved in cell motility and angiogenesis were highly expressed in CD44+ cells, consistent with the demonstrations that CD44+ cells show a more mesenchymal, motile and less proliferative profile. Interestingly, cellular gene expression profiles reflect activation of distinct signaling pathways, some of which are specific for breast cancer CD44+ cells; a finding that could have significant therapeutic implications. In support of this, breast cancer CD44+ and CD24+ cell gene expression signatures correlated with clinical outcome [88]. Certainly, these results indicate that stem cell-like gene expression is important in CSC populations in breast tumors. Another argument that has been repeatedly used to suggest that stem cells are the cell of origin in many solid tumors is that studies in both the epidermis and hematopoietic system have demonstrated that cancers often express markers of the originating cells. Since numerous solid tumor cancer cells typically express stem and/or progenitor cell markers, it is proposed that this indicates that the majority of these cancers arise from multipotent stem cells [89, 90]. One example that will be discussed further here is the presence of stem or progenitor cell markers in mammary tumors. In this case, two genes, keratin 6 and Sca-1 appear to be preferentially expressed in mammary stem/progenitor cells. Furthermore, depletion of Sca-1+ cells results in a loss of functional stem cells in mammary gland reconstitution experiments [91]. Therefore, it is perhaps notable that keratin 6 and

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Sca-1 are observed in mammary tumors induced by the Wnt-1 signaling pathway. Furthermore, the authors found that the Wnt-1 induced tumors were composed of two predominant cellular components, including luminal epithelial and myoepithelial cells. Additionally, results indicated the Pten loss occurred in both lineages. This might suggest that loss of Pten occurred in a common precursor cell [92]. Therefore, expression of stem cell markers may indicate a stem cell origin, though substantially more evidence should be accumulated. There is some indication that a reduction in reproductive capacity of stem cells correlates with decreased propensity towards developing cancer. For example, continuous expression of TGF-β1 in the mouse mammary results in dramatically reduced tumor numbers, such that only one mouse out of 17 developed a tumor. In contrast, over half (15 out of 29) of the wild-type siblings developed tumor growth. Additionally, wild-type mice have much higher frequencies of hyperplastic alveolar nodules than TGF-β1 over-expressing mice. What is intriguing is that reproductive capacity of the mammary epithelial stem cell is reduced concordantly with the number of symmetric divisions it must perform, and ectopic expression of TGFβ1 results in premature aging of mammary epithelial stem cells [93]. These results strongly suggest that there is a positive correlation between the procreative life-span of mammary epithelial stem cells and the risk of neoplastic development, perhaps implicating the stem cell as the precursor to the CSC, especially in regards to the previously discussed notion that increased stem cell populations may result in increased tumor initiation probabilities. In contrast, there is also reasonable evidence to suggest that CSCs bear some resemblance to more differentiated progenitor cells. Transiently dividing progenitors are the immediate descendants of stem cells and therefore will inherit any mutations that were present in the stem cell. Therefore, it is arguable that these cells could accumulate many of the tumor-initiating mutations required, and would only need to further acquire the abilities of self renewal and limitless replication. In both mice and humans Shh pathway-associated medulloblastomas have a phenotype and gene expression profile that strongly resembles that of a committed granule cell precursor [94]. Furthermore, transgenic mice expressing v-erbB driven by an S100-β promoter, active in glial precursors, develop oligodendrogliomas [95], supporting that restricted progenitors may also be targets of transformation. Furthermore, nestin is highly expressed in multipotent stem cells of the developing CNS, but during neuro- and gliogenesis it is replaced with cell type-specific intermediate filaments like GFAP. Nestin expression has been demonstrated to be reactivated in reactive astrocytes in the adult brain, and its upregulation appears to be mediated by the same mechanisms that control nestin expression during CNS development [96]. Therefore, active stem cell pathways are not necessarily an indication that the cell of origin was a stem cell, only that pathway activation is probably necessary for tumorigenic initiation. Additionally, the most common forms of prostate cancer express luminal cellspecific markers, CK8, CK18 and PSA, but very low levels of basal cell marker p63, which may suggest that the disease typically arises from differentiated luminal cells [97, 98]. Melanomas have historically been thought to arise from a mature

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differentiate melanocyte, and sphere forming melanoma cell lines have been demonstrated to only be able to undergo melanogenic differentiation suggesting the cell of origin was lineage-committed cell type [15]. Taken together, it is likely that both stem cells and more differentiated cells are in fact able to become CSC given the right conditions. Interestingly, results generated from aberrant Wnt-1 signaling have indicated that expansion of the keratin 6 positive stem/progenitor cell population is highly dependent on this pathway. Keratin 6 staining was found to be pronounced heavy in MMTV-β-catenin, MMTV-c-Myc transgenic mice. These mice express β-catenin and c-Myc, which are known to be involved in the Wnt pathway, in the mammary epithelium. Due to the expression of stem cell associated markers (keratin 6 and Sca-1), the authors suggest that aberrant expression of Wnt-1 results in an arrest of differentiation in mouse mammary cells in an early phase. However, MMTV-H-Ras, MMTV-PyMT and MMTV-Neu transgenic mice did not experience a notable increase in the Keratin 6 cell subpopulation [92], suggesting tumorigenic activation of cells is dependent on the Wnt-1 pathway. Additionally, spheres formed from MMTV-Neu transgenic mouse mammary cells are enriched for CD24+ cells, whereas Wnt-1 spheres contain a substantial population of CD24+ Sca-1+ cells. Additionally, primary MMTV-Neu tumorspheres are enriched for luminal cells (87%), whereas MMTV-Wnt-1 tumorspheres are enriched for myoepithelial cell types (62%). Furthermore, secondary tumorspheres derived from MMTVWnt-1 mice demonstrated continuous capacity differentiate into both luminal and myoepithelial cell types. In contrast, secondary MMTV-Neu tumorsphere cells differentiated exclusively into K18+ luminal cells. Therefore, Wnt-1 tumorspheres are bipotent, whereas Neu tumorsphere cells are committed to the luminal cell fate [65]. These results indicate that the type of cell transformed can be highly dependent on the specific pathway that is aberrantly regulated. Similarly, in the prostate, inactivation of p53 and Rb leads to neoplasms consisting of cells with luminal and neuroendocrine differentiation [66], while neoplasms initiated by Pten deletion additionally contain cells with basal differentiation [99]. Taken together, the majority of evidence presented suggests that cancer stem cell arise from either stem cells or their immediate progeny. However, the plasticity of differentiated adult cells had been demonstrated by the fact that introduction of just four factors, Oct3/4, Sox2, c-Myc and Klf4 under embryonic stem cell culture conditions results in dedifferentiation of multiple adult cell types into pluripotent cells [100]. Consistently, observations in Drosophila demonstrate that prematurely differentiated spermategonia can be induced to regenerate male germline stem cells upon restoration of the JAK-STAT pathway in Drosophila testes [101]. Additionally, it is well known that in urodeles a differentiated limb muscle cell can de-differentiate and yield muscle, cartilage and connective tissue progeny [102, 103]. Finally, nuclear cloning results have demonstrated that differentiation and development do not require either loss or irreversible inactivation of genes [104, 105]. Therefore, there is also a strong possibility that cancer stem cells arise from more differentiated cells that have reactivated stem cell pathways at some stages of carcinogenesis.

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14.4 Perspectives and Challenges In many ways the cancer stem cell hypothesis offers explanations as to why current treatments targeting rapidly dividing cells fail to cure patients with some types of cancer. Stem cells are relatively quiescent cells, so one might expect that CSC would evade current treatment regimes. Furthermore, there is a reasonable amount of evidence linking many putative solid tumor CSCs to increased therapeutic resistance by additional mechanisms. For example, CD133+ GBM cells show dramatic drug resistance to four commonly used therapeutic agents, temozolomide, carboplatin, VP16 and Taxol as compared to CD133– cells [34]. Additionally, CD133+ neuroblastoma cells show marked increase in expression of DNA repair genes and O6-methylguanine–DNA methyltransferase, as well as significantly increased expression of anti-apoptotic genes and markedly higher activation of phosphorylation of a number of DNA damage checkpoints including Rad17, Chk1 and Chk2, in comparison to CD133- cells [34, 106]. Therefore, there is a great deal of appeal to detect and characterize a CSC subpopulation, because targeting such a population could allow for permanent eradicatoin of tumor cells responsible for solid tumor growth. Morphologic heterogeneity is often presented as evidence that a CSC not only exists in solid tumors, but also that the cell of origin is an immature precursor cell. However, malignant cells are well known for their genetic instability. Therefore, the ability of putative CSC to generate cellular heterogeneity does not necessarily indicate the cell has multipotent abilities, as the possibility that heterogeneity is purely a result of genomic instability cannot yet be ruled out. Furthermore, it is also possible that the relationship between stemness and differentiation may change in tumor cells, even over the lifetime of an individual tumor as a result of genetic instability. Expression of certain markers, such as CD133, could simply be a correlation to increased genomic instability, resulting in high levels of tumor cell heterogeneity. In fact, studies on transplantable tumors show that high rates of genetic or epigenetic changes in tumor may affect a number of properties including transplantability. For instance, cells with mismatch repair deficiencies, often observed in tumor cells, are known to have mutation rates 2–3 orders of magnitude above background (reviewed in [3, 107]). Additionally, there is a reasonable level of uncertainty regarding the effects of cellular adaptation to cell culture conditions [108]. Thus, ability of cultured cells to demonstrate stem-like characteristics may not be a very accurate representation of cell characteristics in primary tumors. Furthermore, as discussed elsewhere [83, 109] microenvironment or niche may play a strong role in the tumorigenic phenotype. Since current isolation techniques all rely on dissociation of the primary tumor preparation of cell suspensions may change the properties of the cells. Additionally, testing stem-like properties by injection of cells into a new tissue location is unlikely to recapitulate the microenvironment experienced by the original tumor. Given the fact that many tumorigenicity assays are done by implanting human cells, often being cell lines that have long been removed from primary tumor conditions, into immunocompromised mice, the new microenvironment is significantly different from the microenvironment experienced

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by primary tumor cells. Probable differences in the stroma and microenvironment between mice and humans may also have significant effects on tumor initiation and progression. Finally, it also should be acknowledged that CSC may represent very different populations in cancers associated with different initiating and secondary genetic and epigenetic alterations and arising from different cell lineages. Since current techniques have certain limitations in studying CSCs characteristics in their natural environment, researchers should be very cautious when making claims of identification of a CSC. It remains plausible that every cell within a tumor has the potential to function as a tumor-initiating cell, but the probability of any given cell to exhibit its stem cell-related properties is low in available biological assays [4]. Taken together, significant amount of knowledge about CSC has been accumulated during recent years. However, there is a strong need for developing new approaches allowing detection and tracing of CSC at different stages of carcinogenesis. This may be accomplished by identification and characterization of CSC in established autochthonous animal models of human cancer, as well as by development of new imaging approaches allowing detection and monitoring of CSC populations. These advances, together with improved understanding of intricacies of stem cell programs, should provide a solid basis for development of novel individualized diagnostic, therapeutic and preventive approaches based on the CSC concept. Acknowledgments This work was supported by the National Institutes of Health grants to AYN (CA096823 and CA112354) and to MGN (NIEHS Toxicology Training Grant ES007052), and by NYSTEM award C023050 to AYN.

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Chapter 15

“One for All” or “All for One”? – The Necessity of Cancer Stem Cell Diversity in Metastasis Formation and Cancer Relapse Thomas Dittmar, Christa Nagler, Sarah Schwitalla, Kathrin Krause, Jeanette Seidel, Georg Reith, Bernd Niggemann, and Kurt S. Zänker

Abstract Cancer stem cells (CSCs) are a rare population of cancer cells exhibiting stem cell properties, such as self-renewal, differentiation and tissue restoration. Because of the latter stem cell feature it is now assumed that (i) tumors originate from CSCs and (ii) that tumor tissues are organized hierarchically like normal tissues. Due to their tumor-initiating capacity as well as their inherent resistance towards radiation and cytotoxic compounds because of expression of multidrug resistance transporters and a highly efficient DNA repair system, CSCs have also been linked to metastatic spreading and cancer relapses. However, to initiate secondary lesions, CSCs must be capable to fulfill the hallmarks of metastasis formation and should be responsive to those factors facilitating organ-specific metastatic spreading. Additionally, cancer relapses exhibit an increased drug resistance and are often more aggressive than the original tumor. This suggest the necessity and existence of different CSC subtypes. In the present chapter, we will discuss whether different, cancer stage specific CSCs exist and if, how these particular cell types might originate. Keywords Cancer stem cells · Breast cancer cells · Breast stem cells · Metastatic cancer stem cells · Recurrence cancer stem cells · Oncogenic resistance · Cell fusion · Gene signature · Drug resistance

Contents 15.1 Introduction . . . . . . . . . . . . . . . . . . 15.2 The Necessity of Different CSC Subtypes . . . . . 15.3 The Origin of Primary Tumor CSCs . . . . . . . 15.3.1 Do pCSCs Originate from Adult Stem Cells? 15.3.2 Do pCSCs Originate from Progenitor Cells?

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15.1 Introduction Cancer as a disease has already been described in the earliest medical records found in the history of mankind, dating back to ancient Egypt [1, 2]. The Greek physician Hippocrates defined the term “cancer” by describing the bizarre “crablike” growth of tumors (“καρκινoμα” [karkinoma] is the Greek word for “crab”). To date, cancer is still the second most prevalent cause of death after cardiovascular diseases in the industrialized world and it is generally predicted that in 15–20 years cancer will be the leading cause of death. However, within the past 10–15 years our knowledge about cancer and how cancer cells originate has changed dramatically. What was once believed as a disease that has its origin in fully differentiated somatic cells, which have undergone malignant transformation due to accumulation of genetic aberrations (whether this is the result of genetic alterations (either activation or loss) of tumor suppressor genes and oncogenes, as proposed for the genetic multistep model of colon cancer [3], or due to aneuploidy [4–6] should not be discussed here) is now believed to have its origin in undifferentiated stem and/or progenitor cells [7, 8]. In fact, stem cells and cancer cells share several similarities such as immortality, telomerase activity, resistance to cytotoxic drugs/substances and radiation, activation of anti-apoptotic pathways, an intrinsic migratory activity (which is triggered by chemokines, cytokines, and growth factors), and a gradually un-differentiated phenotype [9, 10]. Moreover, stem cells are highly susceptible to accumulation of genetic aberrations since they represent a type of long-lived, slow-dividing, self-renewing cells. The hypothesis that cancer may originate from stem cells or germ cells was already postulated about 150 years ago [11] when Virchow [12], Cohnheim [13, 14] and Durante [15] proposed that adult tissues contain dormant embryonic remnants “lost” during developmental organogenesis and that this population of very primitive embryonic-like stem cells may give rise to malignancies [11–15]. Within the past 40–50 years several research groups provided evidences for the “stem cell theory of carcinogenesis” [9]. For instance, in 1967 Pierce already postulated his concept that a maturation arrest of stem cell differentiation might be a common pathway for the cellular origin of cancer [16, 17]. Likewise, Potter developed his

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concept of “oncogeny is blocked ontogeny”, which arose from numerous comparisons of the enzymology of transplantable liver neoplasms, whose patterns resemble those of fetal or immature liver, but never adult liver [18]. These studies were the background for the “stem cell theory of carcinogenesis” proposed by Trosko in 1989 [9]. Trosko hypothesized that a loss of gap junctional intercellular communication (GJIC), which is crucial for several cellular processes such as proliferation, differentiation, and apoptosis [9], plays a crucial role in the process of carcinogenesis [9]. Cancer cells lack either heterologous or homologous gap junctional intercellular communication, which is similar to human adult stem cells that neither have expressed connexins nor have functional gap junctional intercellular communication [19]. On the other hand, their normal differentiated, non-stem cell derivatives do express connexins and express gap junctional intercellular communication during their differentiation [19]. Chemical tumor promoters as well as oncogenes are capable to impair GJIC, thereby leading to a redirection of the cells’ fate, e.g., during differentiation processes, which ultimately can give rise to a cancerous phenotype. Likewise, the term cancer/tumor stem cell (CSC) can be dated back to the sixties and the seventies of the last century [20–22]. It is a well-known phenomenon that only a small subset of cancer cells is capable to form colonies in appropriate in vitro assays. For instance, only 1 in 10,000 to 1 in 100 mouse myeloma cells obtained from mouse ascites were able to form colonies in in vitro colony-forming assays [22]. Because of the similarities in the clonogenicity of leukemic cells and normal hematopoietic stem cells, the clonogenic leukemic cells were referred to as leukemic stem cells [22]. Similar data have been provided for solid cancer cells, of which solely a few (1 in 1,000 to 1 in 5,000) are capable to form colonies on soft agar [21] and in vivo [23], respectively. These observations led to the hypothesis that only a subset of cancer cells is tumorigenic and that these cells could be considered as CSCs [21]. Although these findings already pointed out to the existence of CSCs it long remained unclear whether all cancer cells have a low probability of proliferating extensively and thus all cancer cells behave as CSCs or whether most cancer have only a limited proliferative potential and cannot behave as CSCs, but only a small, definable subpopulation of cancer cells is enriched for the ability to proliferate extensively and form tumors, thus being CSCs [24]. This remaining question has been answered by Dick and colleagues in 1997 by demonstrating that only a small but variable population of acute myeloma leukemia (AML) cells, later entitled AML stem cells, were capable of transferring AML from human patients to a mouse model [25]. AML stem cells are exclusively CD34+ CD38– , which is virtually identical to normal hematopoietic stem/progenitor cells (HSPCs) [25]. Only this cell type was capable to initiate AML in a mouse model indicating that CSCs represent a small subpopulation of cancer cells. In 2003 Al-Hajj and colleagues reported on the prospective identification of tumorigenic breast cancer cells [26]. As few as 100 CD44+ CD24–/low ESA+ cells were capable to initiate tumors in mice, whereas tens of thousands of cells with alternate phenotypes failed to do so [26]. Moreover, tumorigenic breast cancer cells isolated from tumors that had been passaged once in NOD/SCID mice were capable to form tumors that contained phenotypically diverse cells similar to those observed

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in the original tumor [26]. These findings demonstrated that also solid cancers harbor a small subset of CSCs and that these cells were capable to self-renew, thereby giving rise to new CSCs, and to differentiate into phenotypically diverse tumor cells. Both, self-renewal and differentiation, are hallmarks of stem cells. Since then, CSCs have been identified in chronic myeloid leukemia (CML) [27] and several solid tumors, such as brain [28], melanoma [29], colon [30], colorectal [31], and pancreatic cancer [32]. Prospective tumorigenic CSC populations have also been reported for both prostate and lung cancer [33, 34], whereby in both studies serial transplantation assays – the to-date gold standard for CSCs, since this assay proves for both self-renewal and tumor propagation of CSCs [7] – have not been performed. Thus, the definite proof-of-principle for these cancers is still lacking. In any case, the knowledge that a tumor is organized hierarchically like normal tissues, namely comprising of a small number of stem cells, which give rise to differentiated cells, thereby maintaining tissue integrity and organ function, is of crucial interest for our understanding how to treat cancer in future times. The dilemma of current cancer therapies (conventional chemotherapy, radiation therapy, hormonal therapy, humanized monoclonal antibodies, and/or inhibitors) is that although most cancer patients respond to therapy, only few are definitely cured [35]; a matter, which applies to both solid tumors as well as hematological disorders. This phenomenon, which has been entitled as “the paradox of response and survival in cancer therapeutics” [35] has been compared to “cutting a dandelion off at ground level” [35, 36]. Current cancer therapies are designed to target highly proliferating tumor cells. Determination of tumor shrinking concomitant with mean disease free survival of patients are commonly used as read-outs for the efficacy of the appropriate therapy. While such strategies eliminates the visible portion of the tumor, namely the tumor mass, they mostly fail to eliminate the unseen root of cancer, namely CSCs. CSCs, like normal stem cells, possess an inherent resistance towards cytotoxic compounds and radiation [37–39], and a low cell cycle activity, thus being capable to survive therapy. Surviving CSCs are then the seed of relapses that could occur months to years later after therapy. This connection nicely illustrates why the CSC hypothesis is at the center of a rapidly evolving field that may play a pivotal role in changing how basic cancer researchers, clinical investigators, physicians, and cancer patients view cancer [7].

15.2 The Necessity of Different CSC Subtypes Because of their capacities to initiate tumor growth and, most likely, to survive conventional cancer therapy, CSCs have not only been linked to primary tumor formation, but also to metastases and relapses. The question that has to be addressed now is whether CSCs that initiate primary tumor growth, metastases, and relapses are phenotypically similar/identical, suggesting that only one CSCs phenotype exist, or whether primary tumor, metastases, and cancer relapse specific CSCs exist. So, one (CSC) for all (tumor stages) or all (tumor stage specific) CSCs for one (cancer disease)?

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Although it can not be ruled out that primary tumor initiating CSCs could also initiate metastases and relapses, which means that only one type of CSCs exist, there is compelling evidence that different types of CSCs must exist. Li and colleagues already postulated the existence of primary tumor CSCs (pCSCs) and metastatic CSCs (mCSCs) [40]. The first CSC subpopulation – pCSCs – induce primary tumor formation, whereas metastases originate from circulating mCSCs [40]. Recently, Hermann et al. identified two distinct populations of CSCs in human pancreatic cancer [41]. CD133+ pancreatic CSCs, which are exclusively tumorigenic and highly resistant to standard chemotherapy and CD133+ CXCR4+ pancreatic CSCs, which are present in the invasive front of the tumor and which determine the metastatic phenotype [41]. Depletion of CD133+ CXCR4+ pancreatic mCSCs virtually abrogated the metastatic phenotype of pancreatic tumors without affecting their tumorigenic potential [41] supporting the necessity and existence of a specific mCSCs phenotype for metastasis formation. A well-known observation in cancer relapses is the so-called “oncogenic resistance” [36], which describes the phenomenon that regrown tumors are often resistant to first line therapy and are generally more aggressive than the original cancer. Because of this diversity one can conclude that original tumor CSCs and CSCs that have initiated tumor regrowth after therapy should be different, which in turn points to the existence of cancer relapse specific CSCs. In this context, we would like to introduce recurrence CSCs (rCSCs). This CSC subtype defines the CSC population, which initiates tumor regrowth after cancer therapy and which exhibits an “oncogenic resistance” phenotype. In the following, we will give an overview about pCSCs [40], mCSCs [40], and rCSCs, will summarize models how these particular CSC types (most) likely originate and how diversity among CSCs could be achieved.

15.3 The Origin of Primary Tumor CSCs It is generally assumed that pCSCs originate either from adult stem cells or from progenitor cells [40, 42]. Evidences exist for both possibilities, whereby data published in the past two to three years strongly argue that progenitor cells are being the precursors to pCSCs. The third possibility that has been linked to pCSC origin is cell fusion [40, 42], whereby experimental proofs are still not available for the proposed third model.

15.3.1 Do pCSCs Originate from Adult Stem Cells? Tissue stem cells are long-lived, slow-dividing, self-renewing cells and might therefore be highly susceptible for an accumulation of genetic aberrations [9], which ultimately could give rise to a CSC phenotype. However, the definite proof-ofprinciple for this theory is still lacking. CSCs and their normal stem cell counterparts

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share similarities in cell surface marker expression and signal transduction cascades, which would support this theory. For instance, the universal stem cell marker CD133 is expressed by both human brain tumor stem cells (BTSCs) [28, 43] and human normal neural stem cells [44]. Conjointly, the Bmi-1 proto-oncogene is crucial in the regulation of self-renewal of both leukemia stem cells and hematopoietic stem cells (HSCs) [45, 46] as well as neural stem cells and brain tumor CSCs [47–49]. Otherwise, several data clearly indicate that CSCs differ from their normal stem cell counterparts, thus weakening this hypothesis. Prostate epithelial stem cells have been described to be CD133+ α2 β1 hi [50], which is not identical to the recently published prostate tumor initiating phenotype being CD44+ (α2 β1 +/hi /α2 β1 –/lo ) [51]. Indeed, α2 β1 +/hi cells are enriched in the cancer initiating population (about 70% of the cells are CD44+ α2 β1 +/hi ), but CD44+ α2 β1 –/lo prostate cancer cells (about 20–30% are CD44+ α2 β1 –/lo ) exhibit a similar tumorigenicity [51]. Bronchioalveolar stem cells (BASCs) have been suggested to be the putative cells of origin for lung adenocarcinoma [33]. BASCs were identified at the bronchioalveolar duct junction, which is assumed to be a possible site of lung tumorigenesis. Expression of oncogenic K-ras (G12D) in BASCs resulted in an increased expansion (2.5-fold) as compared to control. Conjointly, immunofluorescence studies revealed that BASCs numbers were increased in the earliest tumorigenic lesions in Lox-Kras mice supporting their role in tumorigenesis [33]. Similar findings have been reported recently for murine mammary stem cells (MaSCs) [52]. The absolute cell number of MaSCs, which are capable to generate a functional mammary gland from a single cell, was higher in MMTV-wnt-1 mice as well as particularly in premalignant MMTV-wnt-1 transgenic glands [52]. Moreover, the epithelial outgrowths arising from transplantation of MaSC MMTC-wnt-1 mammary cells were profoundly hyperplastic at 5 weeks post-transplantation suggesting a role for MaSCs in tumorigenesis [52]. However, the fact that tissue stem cell numbers were increased in premalignant tissue is not sufficient to conclude that CSCs originate from adult tissue stem cells. To date, the only known study providing evidence that tumor-initiating cells may originate directly from adult stem cells was provided by Houghton and colleagues showing that gastric cancer originates from bone marrow-derived stem cells (BMDCs) [53, 54]. Induction of chronic Helicobacter felis infection in female mice, which have received bone marrow transplants from male donors, gave rise to dysplatic gastric mucosa tissue comprising of male cells as indicated by Y-chromosome presence [53]. This indicates that chronically inflamed tissue has recruited BMDCs, which then, due to the chronically inflamed microenvironment, have undergone malignant transformation. An indirect hint indicating the tumor initiating potency of BMDCs was provided by Liu and colleagues [55]. The authors demonstrated that in vitro transformed BMDCs can give rise to multiple tumor types including epithelial tumors, neural tumors, muscular tumors, tumors of fibroblasts, blood vessel endothelial tumors, and tumors of poor differentiation in vivo [55]. Moreover, a single transformed BMDC had the ability to self-renew, to differentiate spontaneously into various types of tumor cells in vitro, expressed markers associated with multipotency, and formed teratoma in vivo [55]. However, Liu et al. transformed

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BMDCs artificially by cultivating them in the presence of 3-methycholanthrene for 1 week. Thus it remains unclear whether these findings might be of relevance for in vivo BMDC-derived tumors.

15.3.2 Do pCSCs Originate from Progenitor Cells? Within the past 2–4 years several data have been generated supporting the hypothesis that pCSCs most likely originate from progenitor cells, which have regained self-renewal capacity. Crossing of hMRP8p210BCR/ABL transgenic mice, in which BCR/ABL expression is absent in HSCs and targeted exclusively to myeloid progenitors and their myelomonocytic progeny, with apoptosis-resistant hMRP8BCL-2 mice gave rise to double-transgenic animals, of which 50% developed AML [56]. Thus AML can arise in mice without BCR/ABL expression in HSCs, whereby additional mutations inhibiting programmed cells death may be critical in the transition of this disease to blast-crisis leukemia [56]. Studies on CML patients, both CML in blast crisis and imatinib-resistant CML, revealed their granulocyte macrophage progenitor pool was expanded, expressed BCR/ABL, and had elevated levels of nuclear β-catenin as compared with the levels in progenitors from normal marrow [27]. Moreover, unlike normal granulocyte macrophage progenitors, CML granulocyte macrophage progenitors formed self-renewing, replatable myeloid colonies [27]. Because Wnt/β-catenin signaling plays a pivotal role in directing self-renewal of stem cells [57, 58] it might be concluded that activation of β-catenin in CML granulocyte macrophage progenitors appears to enhance the self-renewal activity and leukemic potential of these cells [27]. Krivtsov and colleagues reported that introduction of the MLL-AF9 fusion protein in committed granulocyte macrophage progenitors gave rise to leukemia stem cells (LSCs), which was correlated to a re-activation of a subset of genes in LSCs normally being highly expressed in HSCs [59]. These data indicate that the recovery of selfrenewal in LSCs might be attributed to the re-activation of normal self-renewal pathways. Vescovi et al. reported that also BTSCs, representing a type of solid tumor CSCs, might originate from transiently dividing progenitors [60]. For instance, progenitor cells within the subventricular zone express epidermal growth factor receptor (EGFR), whose activity is altered in more than 50% of human gliomas and which could cause glioma formation in the CNS if the receptor is constitutively activated [60]. In vivo application of EGF and TGF-α leads to formation of tumor-like structures protruding into the ventricle, concomitant with an aberrant and invasive pattern of neural cell proliferation [60]. Conjointly, in vitro data reveal that subventricular zone-derived transiently dividing progenitors can form neurospheres and show some proliferative ability suggesting that those cells could revert to a stem-cell-like phenotype [60]. A progenitor (adult stem) cell concept for breast cancer has also been proposed by Boecker et al., which allows for a rather uncomplicated interpretation of the complex morphological forms of tumor appearance (tubular carcinomas at

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one side and myoepithelial carcinomas on the other site of the spectrum) [61]. This model generally depends on the comparable differential expression of cytokeratins (CK; CK5 and CK8/18/19) and smooth muscle actin (SMA) [62] among breast stem/progenitor cells, their direct progenies, and fully differentiated cells, as well as different breast cancers [63, 64]. For instance, CK5+ putative breast progenitor (committed adult stem) cells have the potential to differentiate into either glandular (CK8/18+ ) or myoepithelial cells (SMA+ ) through intermediary cells (CK5+ and CK8/18+ or SMA+ ) [61]. Usual ductal hyperplasia (UDH) is characterized by CK5/14 expression, whereas CD8/18 is expressed in the overwhelming majority of ductal carcinoma in situ (DCIS) [61]. Comparison of breast stem/progenitor cells and breast carcinoma cells indicated that the CK expression pattern likely reflects the cellular state of the cell of origin at the time of tumor initiation. Thus, CK5/14 expression in UDH points to a cell of origin close to early breast progenitor cells as well as intermediary glandular cells, whereas DCIS might originate from CK5– but CK8/18+ glandular cells [61, 63]. However, whether CK5 is a suitable breast stem/progenitor marker remains unclear. Independent experiments failed to detect CK5/6 only cells in frozen sections of normal breast tissue, which brings into question the previously described profile of breast “stem cells” based on CK5/6 staining and hence the breast cancer progression model and classification based on this phenotype [65]. Moreover, Boecker and colleagues did not perform functional (stem cell) assays such as generation of mammospheres and subsequent in vitro and in vivo differentiation experiments. Stingl and colleagues identified three distinct types of human breast epithelial cell (HBEC) progenitors: luminal-restricted, myoepithelial-restricted and bipotent progenitors, but none of them were solely positive for CK5/6 expression [66]. Luminal-restricted progenitors are MUC1+ /CALLA(CD10)– /epithelial specific antigen (ESA)+ and express typical luminal epitopes (CK8/18/19, MUC1, ESA) as well as low levels of myoepithelial epitopes (CK14 and CD44v6) [67]. Myoepithelial progenitors are enriched in the MUC1– /CALLA+ /ESA– subpopulation [66]. Bipotent progenitors (MUC1– to ± /CALLA± to + /ESA+ ) generate mixed colonies of both luminal (CK8/18/19, MUC1) and myoepithelial cells (CK14 and CD44v6) when seeded in two-dimensional and three-dimensional cultures [66]. Dontu and colleagues demonstrated that the pattern of expression within secondary mammospheres was consistent with CALLA(CD10), α6-integrin and CK5 in earlier progenitors, and ESA and CK14, on later progenitors, which were still multipotent [68]. On the other hand, double staining for ESA and CK5 showed that all ESApositive cells also expressed CK5 [68] suggesting that ESA is also expressed by earlier progenitors and vice versa. Which of these breast stem/progenitor cells might be the precursor to breast CSCs remains unknown. Since the phenotype of AML leukemogenic cells is similar to that of early HSPCs, Al-Hajj et al. concluded that this may also be true for tumorigenic breast cancer cells (as mentioned above, breast CSCs are CD44+ CD24–/low ESA+ ), because early multipotent breast epithelial progenitor cells have been reported to express ESA and CD44 [26]. Luminal-restricted progenitor cells are ESA positive and express only low levels of the myoepithelial epitope CD44v6

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[67]. Likewise, ESA appears to be a marker of later breast progenitors, whereby double staining revealed that ESA is co-expressed with CK5, a marker for early breast progenitor cells [68]. Moreover, MUC1, CK18 and SMA are not expressed in mammospheres, but are expressed by mammosphere-derived cells transferred to collagen substrata, suggesting that these antigens are expressed only on more differentiated cells of ductal epithelial lineage (MUC1, CK18) and myoepithelial lineage (SMA) [68]. Since MUC1 and CK18 are expressed by luminal-restricted progenitors [66], it might be speculated that such cells represent more differentiated breast progenitor cells. Moreover, due to co-expression of ESA and CD44v6, luminal-restricted progenitor cells might be the precursors for breast CSCs, which would support the hypothesis that CSCs originate from progenitor cells. Comparison of normal and malignant human mammary stem cells revealed that in both stem cell types Hedgehog (Hh) signaling and Bmi-1 regulated self-renewal [48]. However, breast CSCs showed an up-regulation of Hh signaling components PTCH1, Gli1 and Gli2, as well as increased Bmi-1 expression levels [48] suggesting that deregulation of this pathway might play a key role in the origin of breast CSCs. This hypothesis is supported by findings that Bmi-1 induces telomerase transcription and activity, thereby immortalizing human mammary epithelial cells [69]. Moreover, Gli2 overexpression in mammospheres gave rise to ductal hyperplasia in NOD/SCID mice [48], further substantiating the importance of this pathway in self-renewal and malignant transformation. In addition to Hh signaling and Bmi-1, the deregulation of the selfrenewal pathways Notch and Wnt might also play a role in the carcinogenesis of the mammary gland [70].

15.3.3 Do pCSCs Originate from Cell Fusion Events? The third hypothesis postulates that pCSCs might originate from rare cell fusion events between stem cells and its fusion partners, which could be non-transformed somatic cells or other non-transformed stem cells [42]. Bjervig and colleagues postulated that hybrid cells might show large chromosomal aberrations and aneuploidy, which due to genetic and/or chromosomal imbalance could harbor unique cell-survival programs that are shared by normal stem cells and that drive tumor progression [42]. Aneuploidy is a common phenomenon in virtually all cancers and has been considered to be the primary cause of multilateral genomic instability of neoplastic and preneoplastic cells [71]. In fact, in vivo studies demonstrated that transgenic mice either overexpressing Mad2 (an essential component of the spindle checkpoint) [72] or the centromere-linked motor protein CENP-E [73] exhibited broken chromosomes, anaphase bridges, and whole chromosome gains and losses [72] or an increased aneuploidy rate [73], respectively, which was correlated to a higher tumor incidence [72, 73]. However, in examples of chemically or genetically induced tumor formation, an increased aneuploidy rate appears to be a more effective inhibitor, rather being an initiator of tumorigenesis [73]. This suggests a dual role for aneuploidy

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and chromosomal instability in the initiation and prevention of tumorigenesis. Likewise, “BMDC-mediated/myelomonocytic cell-mediated liver regeneration by cell fusion” studies revealed that hybrid cells exhibited an aneuploid karyotype [74–76]. Whether the aneuploid karyotype was attributed to unequal chromosome segregation in hybrid cells or was caused due to genomic instability because of FaH deficiency itself [77], remains unclear. Nonetheless, both FaH –/– wild type mice as well as transplanted animals did not show higher tumor incidences as compared to normal mice. Likewise, Kubota et al. demonstrated that BMDCs fused with hepatic oval cells, but that this cell fusion was not involved in hepatic tumorigenesis in the cholinedeficient ethionin-supplemented (CDE) diet rat model [78]. The CDE diet activates the replication of oval cells, a type of hepatic progenitor cells. Furthermore, this type of diet induces preneoplastic nodules and hepatocellular carcinomas (HCC) derived from oval cell progenitors. Transplantation of BMDCs from GFP transgenic female rats into male rat recipients, which were exposed to a CDE diet to induce hepatocarcinogenesis, revealed GFP expression and the presence of recipient-specific Y-chromosome in some host oval cells clearly indicating cell fusion [78]. However, preneoplastic nodules were GFP negative, suggesting that BMDC-fused oval cells might not have a malignant potential and thus did not gave rise to CSCs in the CDE diet rat model. In summary, these data indicate the pCSCs most likely do not originate from rare cell fusion events.

15.4 Metastatic CSCs The primary cause of death in cancer is not attributed to primary tumor formation, but rather to the growth of metastases at distant organ sites [1, 79]. Multiple cell-cell and cell-matrix interactions are necessary to allow tumor cells to detach from the primary tumor, get accession to the lymphatic or blood circulatory system, survive in the circulation, arrest at distant organ sites, transmigrate across the endothelial lining into the parenchymal tissue, and form secondary tumors [1, 79]. Thereby, most cancers metastasize in an organ-specific manner, e.g., breast cancers preferentially metastasize into the regional lymph nodes, bone marrow, lung, and liver [80], whereas liver and lung are the preferred organs for metastasizing colon cancer cells [81]. Within the past years it became evident that the organ-specific metastatic spreading of tumor cells does not only rely on heterotypic and homotypic adhesive interactions, but also on the interplay of chemokines and their receptors [1]. For instance, breast cancer metastasis to lung and bone has been associated with αv β3 integrin as well as CXCR4 and CCR7 expression [1]. Likewise, colon/colorectal cancer spreading to the liver is mediated by the selectins sialyl Lewisa and sialyl Lewisx , the integrins αv β3 and αv β5 , as well as the chemokine receptors CXCR4 and CCR7 [1]. These hallmarks of metastasis formation have to be fulfilled by mCSCs in order to initiate secondary lesions. This poses the question whether mCSCs are similar to pCSCs or whether mCSCs represent a distinct subtype of CSCs?

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Balic and colleagues reported recently that most early disseminated tumor cells detected in bone marrow of breast cancer patients possess the putative breast CSC phenotype [82]. Although the authors estimated that the mean prevalence of putative breast stem/progenitor was 72% and the median prevalence was 65% (range 33– 100%) among the overall disseminated tumor cells per patients [82], the presented data are less convincing. Balic and colleagues identified putative disseminated breast CSCs in bone marrow aspirates by double/triple-staining immunohistochemistry. However, only double-staining immunohistochemistry data are presented in their work making it difficult to conclude how putative breast CSCs were definitely identified. Abraham et al. reported that the prevalence of CD44+ CD24–/low cells in breast cancer may not be associated with the overall clinical outcome, but may favor distant metastasis, particularly osseous secondary lesions [83]. At a first glance this suggests that CD44+ CD24–/low cells might promote metastatic spreading to bones in breast cancer disease. While this can not be ruled our completely, the authors did not analyzed bone lesions of metastatic breast cancer patients and thus the phenotype of bone metastasis initiating CSCs remain unknown. It is well recognized that metastatic spreading of breast cancer is directed by the CXCL12/CXCR4 [1]. Thus it would be of interest to known if distinct breast cancer mCSCs exist and if they are expressing CXCR4 as was reported for pancreatic CSCs [41]. As mentioned above, Hermann and colleagues identified recently two distinct population of pancreatic CSCs [41]. Exclusively tumorigenic and chemotherapy resistant pancreatic pCSCs were CD133+ , whereas pancreatic mCSC were CD133+ CXCR4+ [41]. Depletion of CD133+ CXCR4+ pancreatic mCSCs virtually abrogated the metastatic phenotype of pancreatic tumors without affecting their tumorigenic potential [41] supporting the existence of mCSCs. Pancreatic cancer preferentially metastasize to multiple lymph nodes, liver, peritoneum, and lung [84]. Because the CXCL12/CXCR4 axis plays a pivotal role in directing metastatic spreading of various tumors, such as breast and melanoma, to liver, lung, and lymph nodes [1], it can be concluded that the metastatic spreading of pancreatic mCSCs is also directed by this chemokine/chemokine receptor interaction. In summary, the identification of two distinct CSCs types in pancreatic cancer – one responsible for primary tumor growth, the other for metastasis – strongly supports the hypothesis that distinct CSC subtypes exist. Because chemokine/chemokine receptor interactions play a crucial role in the organ-specific metastatic spreading of a variety of cancers [1] one can conclude that each metastatic cancer should harbor mCSCs.

15.4.1 The Origin of Metastatic CSCs The identification of two distinct population of pancreatic CSCs [41] suggests that mCSCs most likely originate directly from pCSCs. How mCSCs originate from pCSCs remains unknown. Hüsemann and colleagues demonstrated recently that

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systemic spread is an early step in breast cancer [85]. Hemizygous BALB-NeuT mice developed invasive mammary cancers within 23–30 weeks, whereby epithelial hyperplasia could already be detected microscopically in the mammary glands after 7–9 weeks [85, 86]. Progression to in situ carcinomas occurred between weeks 14 and 18 and at the same time tumors of the mammary gland became palpable or visible [85]. Investigation for CK and HER2 double positive breast cancer cells revealed that these cells became detectable in bone marrow at as early as 4–9 weeks when the most meticulous analysis of the mammary gland could detect areas of atypical ductal hyperplasia [85]. Likewise, single HER2 positive mammary tumor cells became detectable in lung tissue from week 9 on, and micrometastases were first visible around week 20 [85]. Resection of mammary glands of BALB-NeuT mice at week 18 neither prevented nor reduced the number of lung metastases, clearly indicating that dissemination of metastatic cancer cells had already occurred. If we conclude that only mCSCs are capable to seed secondary lesions at distant organ sites this would mean that the origin of mCSCs should also be an early event in (at least) breast cancer. Primary tumor CSCs and mCSCs have not been analyzed in detail so far, in fact, only one article dealing with this topic has been published yet, and thus it remains unknown whether mCSCs originate from pCSCs simply due to genetic/epigenetic alterations in the pCSC fraction, or by (a) different mechanism(s). It is conceivable that pCSCs are genetically/epigenetically instable, possibly due to alterations in intrinsic genetic/epigenetic regulation mechanisms (DNA repair, DNA synthesis, cell cycle checkpoints, histone modification by acetylation and/or methylation) caused by malignant transformation process itself. Likewise, tumor tissue resembles chronically inflamed tissue [87, 88] and because of that tumors are often referred to as “wounds that do not heal” [88]. Chronically inflamed tumor tissue is characterized by an undefined, aberrant mix of various cell types, such as monocytes/macrophages, neutrophil granulocytes, lymphocytes, mast cells, fibroblasts, epithelial cells, endothelial cells, tissue stem cells and recruited BMDCs, as well as tumor cells and CSCs, and the complex interplay among these cell types mediated by chemokines, cytokines, growth factors, reactive oxygen and nitrogen species, and proteases [89]. Reactive oxygen and nitrogen species can cause damages on both DNA and proteins, thereby causing genetic/epigenetic alterations in various cell types, including CSCs. As mentioned above, chronically inflamed gastric tissue can cause to malignant transformation of recruited BMDCs [53]. Thus, the intrinsic genetic/epigenetic instability of pCSCs, either due to intrinsic mechanism or being triggered by the chronically inflamed tumor microenvironment, might be one mechanism how pCSCs could give rise to mCSCs. 15.4.1.1 Cell Fusion and Metastatic CSCs It might also be conceivable that mCSCs originate from cell fusion events. Because of chronically inflammatory conditions inside tumor tissues various immunocompetent cells, such as monocytes/macrophages, neutrophil granulocytes, and

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lymphocytes, as well as BMDCs will be recruited. Hybrid cells that have been originated from the fusion between murine macrophages and weakly malignant human S91 melanoma cells revealed an enhanced metastatic capacity [90, 91]. Thereby, a striking characteristic was heterogeneity amongst hybrids, with some lines producing metastases in up to 80% in mice [91]. Moreover, with few exceptions, hybrids with the highest metastatic potential also had the highest basal melanin content whereas those with the lowest metastatic potential were basally amelanotic, as were the parental melanoma cells [91]. Furthermore, hybrids with the highest metastatic potential also exhibited markedly higher chemotaxis to fibroblast-conditioned media and demonstrated vascular invasion and spread to distant organs similar to that of metastatic melanomas in mice and humans [91]. Injection of weakly tumorigenic S91 melanoma cells into the tail of immunodeficient mice yielded in primary tumor, tail metastasis, and lung metastasis formation [92]. DNA restriction fragment analyses of mouse tissue, S91 melanoma cells, primary tumor tissue, tail metastasis tissue, and lung metastasis tissue indicated that the lung metastasis comprised chiefly of host × tumor hybrids [92]. Since metastasis initiation capacity has been linked to mCSCs, these data suggest that the fusion between macrophages and tumor cells, thereby giving rise to highly metastatic hybrid cells, could lead to the origin of a mCSCs phenotype. However, it should be emphasized that cell fusion is only one among other mechanisms that could promote the origin of mCSCs. For instance, karyotypic analysis of disseminated breast tumor cells from bone marrow of mice at week 9 and older than week 27 did not show an increased chromosomal number [85], which likely excludes cell fusion for early spreading breast mCSCs. Likewise, the fusion of BMDCs with neoplastic intestinal epithelium neither contributed to tumor progression nor to tumor initiation [93]. This indicates that BMDCs could fuse with tumor tissue, but that hybrid cells must not be necessarily converted into a more malignant phenotype. In summary, cell fusion between tumor cells and other cells, such as recruited macrophages and BMDCs, give rise to hybrid cells exhibiting certain properties. Whether such cells possess an enhanced metastatogenic behavior, suggesting the origin of a CSC phenotype, or not, will depend on the genetically/epigenetically modifications/reprogramming within the hybrid cell nucleus.

15.4.2 Do Organ-Specific Gene Signatures Point to the Existence of Organ-Specific mCSCs? In addition to adhesion molecules and chemokines, which serve as a navigation system for circulating tumor cells [1], there is compelling evidence suggesting the necessity of a defined set of genes, so-called “signatures”, for metastatic tumor cells to spread in an organ-specific manner [94–97]. Comparison of gene expression data obtained from organ-specific MDA-MB231 breast cancer metastases revealed that breast cancer metastasis-specific signatures vary among each other and are separate from the general breast tumor

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poor prognosis gene expression signature [98]. For instance, the lung metastasis gene-expression signature (LMS) mediates experimental breast cancer metastasis selectively to the lung and is expressed by primary human breast cancer with a high risk for developing lung metastasis [96, 97]. Interestingly, only four genes, the EGFR ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases 1 and 2 (MMP-1/-2), when expressed in human breast cancer cells, collectively facilitate the assembly of new tumor blood vessels, the release of tumor cells into the circulation, and the branching of lung capillaries by circulating tumor cells to seed pulmonary metastasis [94]. Genetic inhibition of EGFR, COX2, MMP1, and MMP2 activity by shRNA markedly abrogated lung metastasis growth [94]. Recent findings indicated that the LMS couples breast tumor size and metastatic spreading to lung [96]. Thereby, the LMS promotes primary tumor growth that enriches for LMS+ cells, which corresponds to LMS+ tumors being larger at diagnosis compared with LMS– tumors, and to a marked rise in the incidence of metastasis after LMS+ tumors have reached 2 cm in diameter [96]. Likewise, analysis of bone-specific metastatic breast cancer cells gave rise to the bone metastasis gene-expression signature (BMS), which differs from both the LMS and the overall poor-prognosis breast cancer gene signature. Genes that belong to the BMS include connective tissue growth factor (CTGF), Interleukin-11 (IL-11), CXCR4, MMP1, and osteopontin (OPN), whereby breast cancer cells overexpressing a combination of IL-11, OPN and either CXCR4 or CTGF were as much as aggressive as in vivo selected highly metastatic populations [95]. The necessity of organ-specific gene signatures is further supported by data showing that lung-specific metastatic breast cancer cells, particularly LMS+ cells, fail to colonize to bone [96] and vice versa [98]. This effect is most likely attributed to the fact that different organs will place different demands on the invading tumor cells [99]. Thus to successfully form a metastasis at a distant organ site, metastatic cells must adopt a signaling vocabulary that is appropriate for the extrinsic organ microenvironment [99]. But how do these findings relate to mCSCs? Do organ-specific gene signatures of circulating tumor cells point to the existence of distinct organ-specific mCSC subpopulations and if so, how would such cells have been originated? To decipher the specific metastasis gene-expression signature of circulating tumor cells, an in vivo selection procedure was applied. Thereby, human MDAMB-231 breast cancer cells were either inoculated into the left cardiac ventricle [95] or the tail vein [97] of immunodeficient mice. Subsequently, tumor cells were isolated form bone lesions and lung lesions, respectively, and were reinoculated after expansion in culture. To confirm the organ-specific metastatic phenotype, cells isolated from the second round of metastases were expanded in culture and reinoculated in immunodeficient mice [95, 97]. Both, tumor cells derived from lung lesions and bone lesions, respectively, were rapidly and efficiently metastatic to lungs and bone, respectively [95, 97]. These highly organ-specific metastatic cells, selected by repeating in vivo selection rounds, ultimately served as the cellular source for the resolution of the organ-specific gene signatures. Thus, already tissue-primed tumor

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cells were used for analysis and thus it remain unclear, which original cell type colonized to bone or lung tissue. It is conceivable that the organ-specific metastatic spreading of tumor cells might be attributed to one type of circulating mCSCs. Like BMDCs, which are capable to transdifferentiate in accordance to the blueprint being provided by the surrounding organ tissue [89], mCSCs might be able to adopt to the particular specific organ milieu, thereby changing their gene expression profile. However, it is also conceivable that different organ-specific circulating mCSCs exist, each of them already exhibiting organ-specific metastasis gene-expression signature. Which of these two possibilities will hold true is unclear.

15.5 Cancer Relapse and CSCs It is a well-known phenomenon that most cancer patients respond to therapy, but only a few are cured [35]; a matter, which applies to both solid tumors as well as hematological disorders. This phenomenon, which has been referred to as “the paradox of response and survival in cancer therapeutics” [35] has been compared to “cutting a dandelion off at ground level” [35, 36]. Although this will eliminate the visible portion of the weed, the unseen root also needs to be eliminated to prevent regrowth of the weed [35, 36]. In the context of cancer and CSCs, the tumor mass represents the visible portion of the weed, whereas CSCs are the unseen root. CSC have been linked to cancer relapses due to their inherent resistance towards radiation and cytotoxic compounds, such as common chemotherapeutic drugs like doxorubicin, 5-fluorouracil, cyclophosphamide, etc. [24, 37, 39, 100]. For instance, CD133+ glioblastoma (stem) cells preferentially survived radiation treatment at increased rates relative to the majority of cells, which were CD133– , and were able to repopulate both in vitro and in vivo [38]. Interestingly, radiation consistently induced DNA damage to similar degrees in both CD133+ and CD133– cells [38]. However, CD133+ cells repaired the DNA damage more efficiently and underwent less apoptosis due to activation of DNA damage and replication checkpoint responses such as ataxia telangiectasia mutated and the checkpoint kinases CHK1 and CHK2 [38]. Because CD133+ cells often showed a basal activation of rad17, a component of the DNA damage checkpoint, it might be assumed that CSCs, like normal stem cells, are already primed to genotoxic stresses [38]. Further mechanisms that may be involved in (CSC) radiation resistance are the Wnt/β-catenin signaling [101] and the Notch-signaling [102], respectively. Woodward and colleagues demonstrated that radiation of mouse mammary epithelial cells with clinically relevant doses yielded in an enrichment in both Sca-1+ and side population (SP) progenitors [101]. Thereby, irradiated Sca-1+ cells had a selective increase in active β-catenin and survivin expression levels as compared with Sca-1– cells [101]. Moreover, the colony formation ability of Sca-1+ progenitors remained unaffected by clinical relevant doses of radiation [101] suggesting an active role of Wnt/β-catenin signaling in radiation resistance. Radiation also

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induced an enrichment of human MCF-7 breast cancer SP cells [101]. SP cells are characterized by their ability to efflux Hoechst dyes such as Hoechst Blue and Hoechst Red [103] due to expression of various ATP-binding cassette (ABC) multidrug transporters such as ABCG2 (also named breast cancer resistance protein (BCRP)), ABCB1 (also named multidrug resistance transporter 1 (MDR1)), and ABCC3 [103]. These cells are more tumorigenic than non-SP cells [104, 105] and possess some intrinsic stem cell properties as they can give rise to non-SP cells in-vivo, can be further transplanted, and preferentially express some “stemness” genes, including Notch-1 and β-catenin [105]. However, whether SP analysis may be used to identify CSC populations remains unknown since it is not yet clear whether SP cells are CSCs or not. There is evidence that ABCG2 expression rather marks proliferating tumor progenitors, whereas primitive CSCs are present in the ABCG2– population [105]. Comparison of ABCG2+ and ABCG2– tumor cells revealed that both cell populations were similarly tumorigenic, whereby ABCG2– cancer cells formed more and larger clones in long-term clonal analyses [105]. Moreover, compared to ABCG2+ tumor cells ABCG2– cells expressed higher levels of “stemness” genes such as Notch-1, βcatenin, Smoothened (SMO), and Oct-4 [105]. Thus ABCG2 might be a marker for SP cells, but obviously not for CSCs suggesting that SP cells and CSCs are two distinct cell types both exhibiting tumorigenic potential. By contrast, chemoresistance of CD133+ glioblastoma CSCs has been correlated recently to a higher expression levels of MGMT and ABCG2 [106]. Likewise, stemlike neuroblastoma cells expressing high levels of ABCG2 and ABCA3 multidrug transporters had a greater capacity to expel cytotoxic drugs, such as mitixantrone, resulting in better cell survival [103]. Resistance of CSCs to cyclophosphamide has been linked to an increased expression of aldehyde dehydrogenase 1 (ALDH1), which confers resistance towards this chemotherapeutic compound in normal stem cells [107]. Leukemic CSCs revealed an amplified ALDH1 activity, which may correlate to cyclophosphamide resistance [108]. Likewise, in breast carcinomas, high ALDH1 activity identifies the tumorigenic cell fraction, capable of self-renewal, and of generating tumors that recapitulate the heterogeneity of the parental tumor [109]. Moreover, ALDH1 expression levels in primary breast cancer patient samples were correlated with poor prognosis [109], possibly due to an increased chemoresistance. Whether CSCs might survive chemotherapy because of their low cell cycle activity is not yet clear. The efficacy of chemotherapeutic agents is generally determined by their ability to induce lethal cellular damage in highly proliferating cells. It is well recognized that the stem cell niche plays a pivotal role in regulating stem cells’ homeostasis. Under normal physiological conditions the stem cell niche provides an environment that predominantly inhibits both proliferation and differentiation, thereby retaining stem cells in a quiescent state [57]. In case of tissue regeneration, the niche provides transient proliferating signals directing asymmetric cell division of stem cells, thereby giving rise to one stem cell and one already differentiated progeny [57]. However, for CSCs the role of the stem cell niche remains unclear. CSCs might be still niche-dependent, whereby the niche is converted into

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an environment with dominant signals favoring cell proliferation and growth [57], or have become niche-independent and self-renewal is cell autonomous [110]. CSCs isolated from acute myelogenous leukemia and chronic myeloid leukemia, respectively, are relatively quiescent [111, 112], which might contribute to therapeutic resistance. By contrast, ex vivo assays demonstrated a rapid proliferation of solid tumor CSCs [37]. However, this highly proliferatory activity of solid tumor CSCs is most likely attributed to the artificial in vitro culture conditions, e.g., due to the use of high levels of serum and growth factors in most assays [37]. In any case, due to various resistance mechanisms, such as ABC multidrug resistance transporters, a highly efficient DNA repair system, CSCs, like normal stem cells, are capable to survive conventional cancer therapy (chemotherapy/radiation). At a first glance, this may indicate that CSCs, which have survived cancer therapy might be phenotypically similar to original tumor CSCs. While this might be true, the question remains how CSC survival is related to the phenomenon of “oncogenic resistance”.

15.5.1 Oncogenic Resistance and Recurrence CSCs (rCSCs) It is a well-known phenomenon that recurrent cancers are often resistant to first line therapy and are often more aggressive than the original cancer. This phenomenon has been referred to as “oncogenic resistance” [36]. Thereby, an enhanced resistance towards DNA damage, either due to chemotherapy and/or radiation, can be attributed to increased expression levels of multidrug transporters, enhanced expression levels of anti-apoptotic pathways, or a combination of both [36]. How does the phenomenon of “oncogenic resistance” correlate to the inherent resistance towards cytotoxic agents of CSCs? If CSCs will survive first-line therapy and re-establish tumor growth, the recurrent cancer should be phenotypically similar to the original cancer, including growth characteristics and susceptibility to first line therapy (Fig. 15.1). But this is not the case if the recurring cancer exhibits an “oncogenic resistance” phenotype. Thus, to us the only reasonable explanation is that a new type of CSCs must have been originated during cancer therapy, which then give rise to a more aggressive and first line therapy resistant recurrent cancer. In this context, we would like to introduce a new subtype of CSCs, so-called recurrence CSCs (rCSCs). These cells defines that particular subtype of CSCs, which initiates aggressive and chemotherapy/radiation resistant “oncogenic resistance” tumors after conventional cancer therapy. Whether rCSCs already exhibit an “oncogenic resistance” phenotype or solely give rise to progenies exhibiting an “oncogenic resistance” phenotype is not clear. Both possibilities are conceivable.

15.5.2 How Do rCSCs Originate? The phenomenon of “oncogenic resistance” resembles Darwinian evolution. A selection pressure (first line therapy) is exerted to the system “cancer” and only those cells that can resist to the selection pressure will survive. As mentioned above,

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Fig. 15.1 CSC subtypes. Primary tumor formation is initiated by primary tumor CSC (pCSCs), which either originated from adult stem cells or progenitor cells. Due to genetic/epigenetic alterations and/or cell fusion metastatic CSCs (mCSCs) originate from pCSCs, which then initiate secondary tumor growth in distant organ sites. To do so, mCSCs must be able to fulfill the hallmarks of metastasis formation, e.g., cell migration, survival in the circulation, and extravasation. First line therapy either cause a decay of both tumor cells and mCSCs, or solely a decay of tumor cells, but not of mCSCs. Due to genetic/epigenetic alterations and/or cell fusion recurrence CSCs (rCSCs) originate from mCSCs. It is also conceivable that rCSCs originate from pCSCs if the primary tumor was only partially resected or a surgical removal was not feasible. RCSCs could exhibit an “oncogenic resistant” phenotype, thus being drug resistant, e.g., against first line therapy, and more aggressive than the original tumor

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CSCs possess inherent resistance mechanisms and thus CSCs are good candidates to be survivors of the selection pressure. If so, the regrowing cancer should be similar to the original cancer, which, however, is not the case in the context of “oncogenic resistance”. Thus it can be concluded that CSCs, which have survived the selection pressure (conventional cancer therapy) must have acquired genetic/epigenetic alterations during therapy, which have allowed these cells to resist much better to the selection pressure. This in turn suggest that rCSCs, exhibiting an “oncogenic resistance” phenotype, only originates due to exertion of the selection pressure (conventional cancer therapy). This, of course, is the well-known fatal side effect of conventional cancer therapies, namely that they might favor the evolution of a more aggressive recurrent cancer including rCSCs. But what is/are the cell type/s rCSCs stem from and how do conventional cancer therapies (chemotherapy/radiation) promote the evolution of rCSCs? Since the primary tumor, including its pCSCs, is generally removed surgically in cancer therapy we conclude that rCSCs most likely originates from mCSCs. However, it is also conceivable that rCSCs originate from pCSCs if the primary tumor was only partially removed or if surgically resection was not feasible. In any case, despite an enhanced multidrug resistance and DNA repair activity, first line therapy could induce chromosomal aberrations (mutations, strand breaks, rearrangements) as well as epigenetic alterations in remaining CSCs, which could lead to an altered gene expression level, e.g., up-regulation of drug transporters, increased expression of anti-apoptotic pathways, and/or cell cycle proteins in the evolving rCSC population and its progenies. Quite recently, Shafee and colleagues demonstrated that CSCs contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors [113]. Thereby, after 2–3 months of complete remission following platinum treatment, tumors relapsed and became refractory to successive rounds of treatment [113]. Conjointly, the amount of CD24med CD29hi mouse mammary tumor stem cells, raised from 5.9% in primary tumors to 8.8% in partially platinum-responsive primary transplants and to 22.8% in platinum-refractory secondary transplants [113]. Furthermore, refractory tumor cells had a greater colony-forming ability than the primary transplant-derived cells [113]. Most interestingly, the expression of the normal stem cell marker Nanog as well as the expression of Top2A were decreased in CD24med CD29hi populations in the secondary transplants [113]. Moreover, in one case Top2A down-regulation was accompanied by genomic deletion of Top2A [113]. These data clearly indicate that the selection for cisplatin resistant mouse mammary tumor cells went along with genetic alterations in the original CD24med CD29hi mouse mammary tumor stem cell population.

15.5.3 Cell Fusion and rCSCs Another possible mechanism that could promote the evolution of rCSCs might be cell fusion. Both chemotherapy and radiation kills rapidly proliferating (tumor) cells, thereby causing massive tissue destruction, which in turn causes inflammatory

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conditions, the recruitment of immunocompetent cells and BMDCs. Cell fusion is one mechanism how stem cells and cells of the myelomonocytic lineage restore tissue integrity [114–116]. Moreover, cell fusion could be linked to several characteristics of “oncogenic resistance”, such as increased malignancy [117], enhanced resistance to apoptosis, and drug resistance [118]. For instance, hybrid cells, originated from spontaneous cell fusion events between two mouse cell lines, grew faster than their parents [119]. Likewise, the polyethylene glycol (PEG)-mediated fusion between primary mouse mesenchymal stem cells (MSCs) and mouse fibroblasts generated hybrid cells with increased proliferation and altered differentiation [120]. Fusion of 5-fluorouracil resistant tumor cells with methotrexate resistant cancer cells gave rise to hybrid cells being resistant to both compounds [121]. Moreover, hybrid cells became also resistant to mephalan, a drug to which both parental tumor cell lines were sensitive [121]. Likewise, fusion of etoposide sensitive E1A expressing human fibroblasts with parental primary fibroblasts gave rise to etoposide (and apoptosis) resistant heterokaryons [122]. Similar results were achieved when primary fibroblasts were fused with etoposide sensitive cancer cell lines (HeLa and Jurkat). Those fibroblast/cancer cell line hybrids were resistant to both etoposide and apoptosis [122]. These data indicate that cell fusion can give rise to both drug resistance and apoptosis resistant hybrid cells, which are both characteristics of “oncogenic resistance”. Which cell types have to fuse with each other is not clear. Tumor cells, macrophages, and BMDCs are highly fusogenic [118, 123, 124] and thus various possibilities are conceivable. We presume that one fusion partner should be a stem cell (either a tissue stem cell, a recruited BMDCs, or a CSC), which brings the repertoire of stem cell characteristics into the hybrid cell. However, it is well recognized that cell fusion is associated with nuclear reprogramming. Thus, it can not be ruled out that nuclear reprogramming in hybrid cells might also cause the up-regulation of certain stem cell characteristics. Our own experiments show that human breast stem cells (M13SV1) [125] fuse spontaneously with human breast cancer cells (HS578T and MDA-MB-435) in vitro, thereby giving rise to stable heterokaryons [126]. Compared to their parental relatives, hybrid cells showed an enhanced mean chromosomal number, an increased proliferation rate, and an altered gene expression pattern. For instance, we observed Bcl-2 and BRCA1/2 up-regulation, as well as a differential expression of pro-invasive genes, such as Epiregulin, MMP-1 and MMP-2, in several M13SV1/HS578T and M13SV1/MDA-MB-435 hybrid clones [126]. Moreover, we also noticed an increased expression of members of the ABC multidrug transporter family, such as ABCG2 and ABCC6 in hybrid cells [126]. The chemotherapeutic drug etoposide is effluxed by ABCC6, whereas a variety of cytotoxic compounds, such as topotecan, doxorubicin, or methotrexate, are effluxed by ABCG2 [127]. Cytotoxicity experiments revealed that several M13SV1/MDA-MB435 hybrid clones possessed an enhanced resistance towards doxorubicin, 5-FU, and etoposide as compared to their parental relatives (Fig. 15.2). Whether breast stem cell/breast cancer cells exhibit an increased tumorigenicity is currently investigated. Flow cytometry analysis revealed that hybrid cells comprised of a higher fraction of CD44+ CD24–/low cells suggesting an increase in breast CSCs (Fig. 15.3).

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Fig. 15.2 Breast stem cell/breast cancer cell hybrids show an enhanced drug resistance against chemotherapeutic compounds. M13MDA435 hybrid clones, which originated from spontaneous fusion events between M13SV1-EGFP-Neo breast stem cells and MDA-MB-435-Hyg breast cancer cells, were cultivated in the presence of different concentrations of doxorubicin, 5-Fluorouracil, and etoposide. Compared to their parental relatives, the hybrid clone M13MDA435-2 showed an increased resistance to doxorubicin and 5-Fluorouracil, whereas the hybrid clone M13MDA4353 revealed a markedly resistance to etoposide. Even within the presence of 10 μM etoposide M13Sv1-EGFP-Neo cells displayed a nearly 100% viability

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In summary, cell fusion can give rise to cellular phenotypes exhibiting properties, such as increased malignancy, decreased apoptosis rate, and enhanced drug resistance, which are commonly associated with “oncogenic resistance” in recurrent cancers. Thus, cell fusion might be one mechanism how rCSCs could originate.

15.6 Conclusions In the present chapter we discussed the question whether the formation of the primary tumor, its metastases and cancer relapses demands the existence of distinct types of CSCs. One (CSC) for all (cancer stages), or all (CSCs) for one (cancer disease comprising of all stages)? Although the possibility that all three stages of a cancer disease will be initiated by one CSC type could not be ruled out completely, there are some evidences that each stage of a cancer disease in initiated by its own specific CSC type. For instance, two distinct types of CSCs have been identified in pancreatic cancer [41]. CD133+ pancreatic CSCs are tumorigenic and resistant to standard chemotherapy, whereas CD133+ CXCR4+ pancreatic CSCs determined the metastatic phenotype [41]. Likewise, cisplatin resistance in Brca1/p53-mediated mouse mammary tumors was associated with an increase in CD24med CD29hi mouse mammary tumor stem cells and alterations in the gene expression profile of platinum resistant cells, e.g., Top2A down-regulation due to genomic deletion of Top2A [113]. So, the answer is rather “all for one” than “one for all”. How cancer stage specific CSCs (mCSCs and rCSCs) originate is less clear. We presume that pCSCs as well as their derivatives are genetically/epigenetically instable, possibly due to alterations in intrinsic genetic/epigenetic regulation mechanisms, such DNA repair, DNA synthesis, histone modification by acetylation and/or methylation, caused by the malignant transformation process itself. This is supported by findings of Shafee and colleagues who provided evidence that selection of cisplatin resistant CD24med CD29hi mouse mammary tumor stem cells went along with alterations in the gene expression pattern of platinum resistant cells [113]. The other possibility that might play a role in the origin of cancer stage specific CSCs (mCSCs and rCSCs) is cell fusion. Several studies provided evidence that cell fusion can give rise to cell types possessing an enhanced metastatogenic potential, increased malignancy, an enhanced resistance to apoptosis, and drug resistance. If we conclude that metastases originates from mCSCs, because only CSCs possess the capacity to initiate tumor growth, than the observation that cell fusion of macrophages with weakly malignant melanoma cells gave rise to highly metastatic melanoma cells [91, 92, 128] suggests that mCSCs could originate from cell fusion

Fig. 15.3 The breast CSC phenotype CD44+CD24–/low is enriched in breast stem cell/breast cancer cell hybrids. Cells (M13SV1-EGFP-Neo, HS578T-Hyg, MDA-MB-435-Hyg, and hybrid cells) were analyzed for CD44 and CD24 expression by flow cytometry. Data show that the breast CSC phenotype CD44+CD24–/low is enriched in hybrid cells

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events. Likewise, the finding that hybrid cells possess an enhanced drug resistance and possibly increased malignancy [118, 121] suggest that mCSCs and rCSCs could originate from rare cell fusion events. Unfortunately, till now solely a handful of articles have dealt with this topic (CSC diversity) so far, and thus it might be difficult to conclude that CSC diversity could be a common finding in cancer. On the other hand, the prerequisites that CSCs have to fulfill in order to metastasize, e.g., to detach from the primary tumor, to migrate and to extravasate, or to re-establish tumor growth after therapy suggests that only CSCs exhibiting certain properties, e.g., being responsive to chemokines, are capable to do so. CSC diversity, either due to intrinsic genetic/epigenetic variability and/or cell fusion, would also explain, why some patients respond to therapy and are being cured and others not. Genetic/epigenetic alterations, both due to intrinsic mechanisms and/or cell fusion, always occur randomly and the results of such processes could not be predicted. Moreover, each individuals exhibit its own, like a fingerprint, specific mix of soluble factors (chemokines, cytokines, growth factors, proteases, etc.), cell-cell-contacts, cell-matrix-contacts, which also contribute to the ultimate result of genetic/epigenetic alterations in CSCs. For instance, in cancer patient “A” the fusion between a tumor (stem) cell and a macrophage promotes cancer due to generation of hybrid cells exhibiting an increased metastatogenic potential. On the contrary, the fusion between a tumor (stem) cell and a macrophage might lead to a spontaneous remission in cancer patient “B”. Macrophages belong to the group of antigen-presenting cells (APCs) and express major histocompatibility complex class I and II molecules as well as adhesion/co-stimulatory molecules. It has been shown that PEG-mediated dendritic cell/tumor cell hybrids present tumor cell specific antigens and are capable to activate primary helper and cytotoxic T-cells [129]. Such hybrid cells have been successfully tested in animal models as promising antitumor vaccines, which, e.g., are protective against tumor challenge and results in the regression of established metastatic disease [130]. Currently, dendritic cell/tumor cell hybrids are tested in phase I/II clinical trials [129, 130]. In summary, we conclude that CSCs possess genetic/epigenetic variability, which is crucial for the origin of mCSCs and rCSCs. On the one hand, CSC diversity, due to genetic/epigenetic variability, plays an elementary role for cancer metastasis and cancer relapse, particularly those associated with “oncogenic resistance”. On the other hand, the genetic/epigenetic variability of CSCs might be useful for the determination of CSC specific molecules/pathways, which could be used as targets for novel anti-CSC elimination strategies. From day-to-day, from monthto-month we learn more about the complexity of CSC biology. Thus, the challenge of future cancer research will be to identify and to characterize the distinct CSC subtypes, to compare them with normal stem cells to find the target molecules/ pathways where CSCs are vulnerable. Together, we will make it. One for all, all for one! Acknowledgments This work was supported by the “Verein zur Förderung der Krebsforschung e.V.”, Heidelberg, Germany and the Fritz-Bender-Foundation, Munich, Germany.

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Chapter 16

Elimination of Cancer Stem Cells A. Sagrera, J. Pérez-Losada, M. Pérez-Caro, R. Jiménez, I. Sánchez-García, and C. Cobaleda

Abstract The acceptance of the Cancer Stem Cell (CSC) concept has revolutionized all aspects of our understanding of cancer biology, from the cellular origin of cancer to its growth and expansion, shedding new light into the interrelations of all the cellular components of the tumour and their role in its progression. From the therapeutic point of view, the existence of CSCs also explains one of the most dramatic events in the clinical history of many cancer patients after diagnosis: the almost inevitable relapse after a period of variable clinical remission. The knowledge that tumours are maintained and propagated from just a small population of cells with self-renewal properties and resistant to current anti-proliferative treatments opens new avenues for therapeutic approaches. The development of new drugs targeting specifically these CSCs should allow us, in combination with current therapies, to eliminate entirely all the cellular components of the tumour, thus preventing relapse and completely curing the disease. Here we revise the most salient biological features of CSCs that are relevant for the development of new therapies and describe the molecular basis of the current approaches aimed at CSC elimination. Keywords Cancer stem cells · Targeted therapies · Differentiation therapies · Selfrenewal · Chemoresistance · Notch pathway · Hedgehog pathway · Wnt pathway · Polycomb pathway · PTEN pathway

Contents 16.1 The Cancer Stem Cell Hypothesis . . . . . . . . . . . . . 16.2 The Failure of Conventional Cancer Therapies . . . . . . . 16.3 Searching for a Valid Target: Knowledge of Stem Cell Biology 16.3.1 Self-renewal . . . . . . . . . . . . . . . . . . . 16.3.2 Differentiation Potential . . . . . . . . . . . . . .

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358 16.3.3 Quiescence/Long-Life . . . . . . . . . . . . . . . . . . . 16.3.4 Metastasis . . . . . . . . . . . . . . . . . . . . . . . . 16.3.5 Migration . . . . . . . . . . . . . . . . . . . . . . . . 16.3.6 Transplantation . . . . . . . . . . . . . . . . . . . . . . 16.4 Therapeutic Approaches Against Cancer Stem Cells: Eliminating CSCs 16.4.1 Differentiation Therapies . . . . . . . . . . . . . . . . . . 16.4.2 Destruction Therapies . . . . . . . . . . . . . . . . . . . 16.5 Imaging CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . 16.6 Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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16.1 The Cancer Stem Cell Hypothesis The traditional view of tumor development explains cancer as a disease of abnormal proliferation where cells divide in an uncontrolled manner, as a result of successive steps of symmetrical divisions. All daughter cells are identical, and each cell in the tumor mass should then be equally capable of regenerating a new tumor [1, 2]. However, this model of clonal expansion does not explain why, if every cell has the same proliferation capacity and can potentially give rise to a new tumor, it is necessary to inject an elevated number of cancerous cells in an empty host in order to transplant the tumor. In line with these findings, it is known that the tumor mass consists of a heterogeneous population of cells with different biological characteristics, among them the potential for self-renewal. The majority of the tumor mass is formed by differentiated cells, but there are a small percentage of non-differentiated cells. In the last years many evidences have accumulated showing that different types of tumors contain, indeed, a small population of cells with self-renewal and differentiation capacities: tumors of the hematopoietic system [3–5], of the breast [6], of the brain [7], pancreas [8], head and neck [9], colon [10–12] and many others. Due to the similarities between these tumor maintaining cells and stem cells, they have been named Cancer Stem Cells (CSCs). This term defines “a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineage of cancer cells that comprises the tumor” [13]. The percentage of CSCs in the global tumor mass is extremely low, and varies among different tumors [14, 15]. If these CSCs are the cells responsible for tumor regeneration (and the only ones with this capacity), this would explain why it is necessary to inject an elevated number of unsorted cancerous cells in order to re-generate a tumor. The fundamentals of cancer stem cell theory are not new: in the middle of 19th century, Virchow [16] and Cohnheim [17] found evidences accounting for the similarities between certain tumors and developing fetal cells; this was the base of the “Embryonal rest hypothesis” explaining cancer as a remnant of embryonic cells in the adult. The first documented evidences for the existence of CSCs came from leukemia studies (acute myeloid leukemias), where it was found that only a small subset of cancer cells, with the surface markers characteristics of hematopoietic progenitors (CD34+ CD38– ) was able to generate new tumors in immunodeficient mice. This finding was later expanded to many other tumor types, as mentioned

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above: with the progress of isolation techniques the identification of CSCs in blood, breast, brain, pancreas, head and neck, colon, lung, skin, liver, prostate and stomach tumors has been possible [2]. The percentage of these cells in the global tumor mass is extremely low. Thus, the CSC theory explains the tumor as a hierarchically organized tissue originated and maintained by CSCs. They could divide, being stem cells, either asymmetrically or symmetrically. The final result is a tumor containing two different pools of cells: Cells in the differentiated pool will expand, differentiate to a certain extent and constitute the majority of the tumor mass, while in the pool of CSCs, one single cell will be able to recapitulate all the tumor features and give rise to a complete tumor upon transplantation.

16.2 The Failure of Conventional Cancer Therapies Even with the great advances in the knowledge of cancer biology over the last decades, most cancers remains non-curable with current therapeutical approaches. The decreasing mortality is more a consequence of early diagnosis than actually due to better therapies [18]. Today, cancer treatments (surgery, chemotherapy, radiotherapy) are targeted to the main population of cells in the tumor: the differentiated, proliferating ones. Anti-proliferative agents effectively kill these cells, but the effect is usually transient, and tumor relapses occur in the majority of the cases. One characteristic of CSCs is their quiescent state, with low rates of division and proliferation, which make them resistant to the anti-proliferative agents [19, 20]. This fact is very likely one of the reasons for the failure of most current therapies: even if the majority of the cells in the tumor are killed, the small population of CSCs with self-renewal potential is still there, ready to regenerate the tumor in some months ´or years ´time. In fact the resistance of CSCs to therapy is higher than that of normal stem cells [21–24]. One of the main problems of current cancer therapy is that it is not selective, and it affects not only the proliferative cancerous cells but also the normal cells in the patient (hair, intestine, nails. . .), causing the well-known adverse effects. Therefore, and to avoid these problems in the future CSC-based therapies it will be crucial to obtain a better knowledge of the biology of CSCs in order to discover unique features that will not be shared by normal cells. This knowledge should allow us to design specific anti-CSC-based therapies, by aiming either at their differentiation or their direct destruction [25] to both eradicate their malignant effects and prevent tumor relapse. Thus, future therapies should implement conventional regimens aimed at reducing tumor size with anti-CSC-based strategies. This idea is reinforced by the fact that current therapies not only do not kill CSCs, but they favor the expansion of the resistant CSC clones, which in time could acquire new alterations that will increase their tumorigenic abilities and their resistance. An essential element that must be understood in order to learn how to kill CSCs is their cellular origin. The question about which is the origin of CSCs (i.e., the debate CSC vs. cancer-cell-of-origin) is an active field of research whose detailed

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discussion falls out of the scope of this Chapter (for a more detailed discussion of this point, see [2]). However, one fact that is important to mention is that, in order to elucidate this essential issue, accurate experimental models that closely mimic human disease must be generated. In humans, the cancer-cell-of-origin can not be identified because the cancers appear as already established diseases. This is why the most appropriated approach is the use of mouse models in which the disease can be “programmed” and its evolution can be followed clearly from healthy tissues to premalignant lesions to full cancers. The CSC point of view, of course, has also its detractors, that consider that the anti-drug resistance of CSCs is only relevant in the very initial stages of the disease, because in the later stages they play a negligible role. According to this classical point of view, in the advanced stages of cancer proliferating cells are the ones that determine disease progression, prognosis, therapeutic failures and resistance, simply due to the fact that they are not completely eliminated with the current therapies [26]. However, given the enormous amount of accumulating evidences favoring the CSC hypothesis, and the fact that with the previous paradigm no significant advances have been made towards achieving a cancer cure over the last 20 years, it is clear that a new way of addressing the cancer problem is mandatory.

16.3 Searching for a Valid Target: Knowledge of Stem Cell Biology Stem cells are undifferentiated cells defined by an indefinite potential for selfrenewal, multi-lineage differentiation, and long-life. Almost all adult tissues contain a small population (usually less than 1%) of tissue-specific stem cells than are responsible for tissue-homeostasis (renewal and damage repair) throughout the lifetime of an organism. But there are also these characteristics what make stem cells a serious threat to the organism if something goes wrong and, for some reason, their potential becomes uncontrolled [27]. Mutations within regulatory pathways could lead to the creation of stem cells with tumorigenic capabilities, the CSCs. It is important to understand in depth the characteristics of stem cells (shared by Cancer Stem Cells too), in order to search for a unique feature that would allow us to selectively target CSCs.

16.3.1 Self-renewal Self-renewal is the ability of a cell to divide and produce an exact copy of itself [28]. Stem cells can divide asymmetrically or symmetrically. Through symmetrical division, the cell gives rise to two daughter cells that either retain the same stemness properties of the progenitor or start the differentiation program, losing the ability of self-renewal. The stem cell would be in this case, lost. When the stem cell undergoes asymmetrical division, two different cells arise: one similar to the progenitor,

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which stays in the same microenvironment (the stem cell niche) and another cell that will move from the surroundings and will turn into a precursor/progenitor cell. These cells undergo a specialization program, meaning that at the same time they differentiate and proliferate in an active way. Progressively the amount of cancer cells grows and gives rise to the tumor mass. It has been proposed that the decrease in the number of cell divisions could be due to the loss of telomerase activity [29], since one singular feature of stem cells is an increased telomerase activity, which makes the length of telomeres to remain constant after cell division. This means that the cells are not subject to the aging effect and can acquire a theoretical infinite replication capacity [30, 31].

16.3.2 Differentiation Potential There are various levels of potentiality that can be used to classify stem cells [32]: – Pluripotentiality: a cell is capable of giving rise to any cell type of an organism, including some or even all extraembryonic lineages (i.e. embryonic stem cells). – Multipotentiality: a cell is capable of given rise to all the different cell types of a tissue or tissues. – Unipotentiality: a cell is capable of given rise to only a one cell type of a tissue (e.g., spermatogonial stem cells). CSCs are, by definition, the cells capable of giving rise to all the differentiated cells that compose the tumors mass. However, in different types of tumors also Cancer Stem Cells with different potentials can be found. Some tumors produce a broad range of differentiated types among their descendants (e.g. teratomas) thus making it more likely that their CSCs share more characteristics with pluripotent stem cells. Other tumors, however, have a much more limited type of descendants (e.g. lymphomas), suggesting that their supportive CSCs could be closer to more limited multi- or uni-potential progenitors. As mentioned before, the discussion about the cancer-cell-of-origin falls out of the scope of this Chapter.

16.3.3 Quiescence/Long-Life During quiescence some of the cellular activities stop, resulting in the activation of selective programs that make the cells refractory to differentiation. In a G0 –G1 arrest the cells enter in a phase of low metabolism, in which the relation between the cells and the microenvironment is minimal. The reduced energy cost would allow the stem cells to have a long life. It has been proposed that the quiescent cells are not subject to the aging effect, a point that is also supported by the fact that the length of telomeres in stem cells remains constant after cell division [31]. The quiescence process in stem cells is reversible, like in cells which are

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deprived of growth-promoting signals (i.e. serum withdrawal, contact inhibition). Stem cells are the only-long lived population of cells that is present in the tissues during all the time that is necessary for the accumulative mutations to cause the final oncogenic outcome. It is precisely their long life-span what makes them more susceptible to be the targeted by these fatal mutations. Quiescence of metastatic cancer stem cells is one of the postulated mechanisms to explain the dormancy of metastasis that allows tumor relapse to appear years after clinical remission was first achieved [33].

16.3.4 Metastasis At some point in tumor evolution, either at the beginning or during progression, a small population of cells will take off from the primary tumor, migrate through the organism after intravasating into the circulation or the lymph, and will end up in a new body location. In this secondary location, they will extravasate from the circulation and into the new tissue. Once there, in the first stages, the cells may stay quiescent, undetected by currently available diagnostic methods, forming what it is called a micrometastasis. Approximately 2% of the cells that took off from the initial tumor will give rise to micrometastasis [34]. At one point, depending on the abilities of the cells to modify the new matrix surrounding (among others to induce neovascularization, recruiting infiltrating cells) a small percentage (approx. 1%) of these cells will grow and maintain the new tumor mass, forming a macrometastasis [35]. In fact, given the complexity of the whole process, the efficiency of the metastatic process is very low. Not every cell from the tumor mass has metastatic properties and the metastatic potential of the cell depends on multiple factors that determine tumor cell growth, survival, neovascularization and invasion [34]. In the normal development of the organism, the phenomenon of Epithelial to Mesenchimal Transition (EMT) plays an essential role: the homeostasis of epithelial cells is loss, they acquire migratory capabilities and a mesenchimal phenotype. In cancer, EMT is also crucial in the metastatic progress and some of the pathways that play a role in the EMT (Wnt, TGF-β, see below) have been found to be activated during neoplasia. The EMT phenotype in cancer involves a decrease in tumor growth, increased motility and invasiveness, increased metastatic potential and more resistance to apoptosis. All these similarities point out to a link between the process of EMT and the acquisition of stem cell properties [36]. Even more, the precursors of cancer cells or CSCs with metastatic characteristics could be a cell undergoing EMT. Another possibility is that the metastases arise directly form CSCs, as it has been shown in studies with pancreatic carcinomas [37]. Since metastases are the predominant cause of lethality in cancer patients [38], therapeutic approaches based on drugs that target the metastatic CSCs could result in increased in patient survival, but further studies are still necessary to elucidate the precise function of the CSC in metastatic tumors (For more details about CSCs and Metastasis see Chapter 15).

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16.3.5 Migration It is known that there are preferential locations for metastatic cancers cells to migrate to, according to the origin of the primary tumor: breast cancers often metastasize to regional lymph nodes, bone, liver, brain and lung, while colorectal cancer favors metastasis to the liver or prostate cancer to the bone [39]. There are several theories, non mutually-excluding, to explain the dissemination of the cancerous cells all through the organism. It could be that the cancerous migrating cells, larger than the normal ones, “get stuck” in the capillary bed, and it is in that very same place where they form metastasis [39]. The “Seed and soil” theory [40] proposes that only when the secondary location (metastatic niche, “soil”) contains the appropriate growth factors (specific for each type of cells), the new cancer will grow. The “Homing theory” suggests that the migrating cells are attracted to the different locations due to the production of chemotactic compounds (for example, SDF-1) that are specific for particular cancerous cells [41]. The SDF-1/CXCR4 chemokine pathway plays a role in migration of stem cells. Among the biological effects of SDF-1 are motility induction and secretion of angiopoietic factors (VEGF). In tumor development the expression of both SDF-1 and CXCR4 are increased, leading to a more invasive and migratory phenotype, which allows the metastatic cells to migrate through the body using the SDF-1/CXCR4 axis (Ratajczak et al., 2006). Other works that support the importance of this axis come from the evidence that the treatment of breast cancer cells with a CXCR4 inhibitor impaired the metastatic ability [42].

16.3.6 Transplantation The main currently used criterion for CSC identification makes use to their ability to regenerate the tumor mass once they are transplanted into the proper recipient (so from humans into immunodeficient mice). Tumor transplantation, even in syngeneic cases (obviously, between mice) is extremely inefficient, and it is of course worse when talking about xenografts. For efficient tumor transplantation, in addition to the scarcity of CSCs in the tumor mass, it is now clear the important role that the recipient microenvironment (presence of certain cytokines and growth receptors. . .) plays in the final result. Although there are species-specific differences in the recognition of molecules that mediate the interactions between cancer cells and rest of the cells in the niche, it has been shown that some murine growth factors, such as EGF and FGF2 can interact with their human counterpart receptors (reviewed in [38]). Anyway, and given the diversity of the signals that are present in the microenvironment, it is important to mention that the changes in transplantation efficacy could be expected to be due not only to the different species involved but also to the different tissues in the same organism, and in different locations. As an example, a rate of 100% efficiency is achieved when glioma-derived cells are implanted into the cranium, but this efficiency is reduced to half when transplantation takes place subcutaneously [43].

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16.4 Therapeutic Approaches Against Cancer Stem Cells: Eliminating CSCs As it has been previously described, if CSCs are responsible for tumor growth and relapse, the elucidation of new cancer therapies targeting CSCs will undoubtedly contribute to cure the patients [44]. Therapies that kill all other cells in the tumor mass can shrink the tumor, but if CSCs remain unaffected, the relapse will eventually occur [25]. Figure 16.1 represents the possible therapeutic approaches that are being developed in an attempt to destroy CSCs. Since CSCs present alterations in the normal differentiation and self-renewal pathways (Notch, Wnt, Hh. . .), which are responsible for the persistence of the tumor, we should focus on these aspects in order to design proper anti-CSC agents. Also, other particular properties that can be altered in CSCs (like DNA repair mechanisms, altered expression of transporter proteins. . .) should be taken into account in order to evaluate all possible strategies to eradicate their malignant effect. The different approaches for the therapeutical treatment could be broadly classified into Differentiation therapies and Destructive therapies (Fig. 16.1).

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Fig. 16.1 Anti-CSC therapies. Conventional therapies destroy the more differentiated cells forming the majority of the tumour mass, but CSCs are left behind and they will eventually repopulate the tumour. CSC-targeted therapies are aimed at destroying them, either directly (destruction therapies targeting pathways or mechanisms essential for their survival) or more indirectly (differentiation therapies that force the CSCs out from their stem status into more differentiated, proliferating cells that can then be destroyed using more conventional therapeutic approaches)

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16.4.1 Differentiation Therapies They are aimed at the modification of CSCs in such a way that they will lose their abilities for self-renewal and multipotentiality [45]. This approach will convert CSCs into differentiated cells (alternatively the CSCs could die if differentiation cannot proceed normally). This kind of therapy tries to achieve the reversion of the malignant phenotype, and could make the CSCs susceptible to the current therapies that mainly affect the differentiated cells. The toxicity of the drugs used in this approach is often lower than that of the agents actually used for chemotherapy [46]. The drugs that could be used for differentiation therapy are either those that induce epigenetic changes in the CSC (methylation, phosphorylation, acetylation. . .) or vitamin A and retinoids. Retinoic acid and its analogous act in the modulation of the transduction signals. An increase in the cure rate of acute promyelocytic leukemia has been possible using a therapy based on analogous of vitamin A (all-trans-retinoic acid, ATRA) plus chemotherapy [47]. Unfortunately, no such effects have been found in solid tumors, although a positive effect of the retinoid group for head and neck, and lung cancer, acting as chemopreventing agents has been proposed [25]. Also, synthetic ligands activating the peroxisome proliferator-activated receptor-γ (PPAR-γ) have been shown to be able to induce terminal differentiation of human liposarcoma or breast cancer cells [48, 49]. Another group is the one of epigenetic drugs. Among the possible examples of targets that are involved in processes largely unrelated with the DNA sequence, one is the enzyme Histone Deacetylase (HDACs), which acts in the modulation of the heterochromatin, playing an important role in transcriptional control of several genes. It has been described an association between the development of several cancers and an increase in HDAC activity [50]. In this sense, the inhibition of HDAC activity with SAHA (suberoyanilide hydroxamic acid) has been suggested to induce differentiation, and so the use of this agent as well as other related HDACs inhibitors in clinical trials is already ongoing. These compounds interfering with or reverting epigenetic modifications, although hold a promise against cancer, obviously are not a therapy specifically targeted against CSCs, since they affect globally the tumor population, and also have important side effects since they attack basic common cellular machinery. However, their effects at the CSC level can be different from those at other cellular stages, and might be able to make the CSCs more prone to destruction by other agents. In a more targeted way, another protein that has been showed to induce differentiation of CSCs is BMP4 [51], which inhibits the tumorigenic potential of human brain tumor-initiating cells, promoting the glioma CSCs to differentiate into non-malignant cells.

16.4.2 Destruction Therapies 16.4.2.1 Targeted Therapy Against Self-renewal Signaling Pathways Targeting cell-fate modulation pathways has been considered one of the more promising ways in cancer therapy. The aim here would be to prevent the

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Fig. 16.2 Stem cell essential pathways as targets for therapy. Schematics of five pathways playing essential roles in stem cell survival and self-renewal. See details in the text. (a) NOTCH pathway; (b) HEDGEHOG pathway; (c) WNT signalling pathway; (d) PTEN/PI3K pathway; (e) Polycomb action mechanism

replacement/self-renewal of the CSCs pool. Nevertheless, to achieve long-standing therapeutic success, it would be most probably necessary to combine several kinds of approaches. We will discuss now some of the signaling pathways that are important for CSCs, and are under study to be used as targets for anti-cancer therapy (Fig. 16.2 and Table 16.1). NOTCH Signaling Pathway In normal conditions, NOTCH signaling is involved in the maintenance of selfrenewal and in the determination of stem cell fate in several tissues. It acts at different stages of differentiation, from the regulation of embryonic development to

– Self renewal – Proliferation – Differentiation – Migration

– Self Renewal – Proliferation – Cell fate determination Hox – Self renewal – Proliferation – Differentiation PTEN/PI3K – Proliferation – Apoptosis – DNA damage response – Development – Metabolism – Chemoresistance

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PTEN, PI3K, PDK1, AKT, RTK, RAS, p53, FOX family, BCL2, PAR4, BAD, GSK3β , p27, mTOR Kinase, EGFR, HER2, ERB, JNK

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-Inhibition of γ-secretase GIS -Inhibition of effectors, ligands, target genes – Inhibition of SMO Cyclopamine, SANTs – siRNA 1–4, Cur-61414, – Anti-HH antibodies compound-5, compound-Z, HhAntag, KAAD-cyclopamine, Antibody 5E1 PKF118-310, – Anti-WNT antibodies ZTM000990, – Disruption of Monoclonal antibodies Tcf-β-catenin anti-Wnt1, anti-Wnt2, complexes NSAIDS – Attacking Tcf-β-catenin target genes by RNAi or small molecules – Repression PRC2 DZNep

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HOXA9, HOXB6, HOX11, HOXB3, HOXB8, HOXA10

PRC1, PRC2, BMI1, EZH2, p16INK4, p19ARF

WNT, FZD, LRP,GSK3β β-Catenin, TCF, LEF, Cyclin D1, DKK1, Myc, FGF20, WISP1, CCND1

– Self Renewal Notch, γ -secretase – Cell fate determination – Differentiation – Developmental patterning SHH, IHH,DHH, PTCH, SMO, –Proliferation GLI, HRK4 – Self Renewal – Survival

Notch

Molecules involved in the pathway

Implicated in

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adult maintenance of homeostasis [52]. There are four different NOTCH transmembrane receptors in humans, which can bind with five ligands. After ligand binding, the intracellular domain of NOTCH is cleaved and translocated to the nucleus, where it activates a cascade of transcriptional regulatory events that ultimately affects the expression of numerous genes, and leads to different phenotypic characteristics [53]. In most cases the pathologic activation of NOTCH signaling causes increased proliferation, limited differentiation, and prevents apoptosis in cancer cells. NOTCH mutations have been detected in about 60% of T-cell acute lymphoblastic leukemias (T-ALL) [54] and, although to date mutations of NOTCH in solid tumors remain unknown [53], an augmented expression of NOTCH has been associated to aggressive types of prostate and breast cancer. Some characteristics of this pathway make it especially desirable as a target for anti-cancer therapy: (a) there is no enzymatic amplification step, meaning that the downstream effects of NOTCH activation are dose dependent. As result, therapeutic effects could be reached without completely turning off the pathway [55]; (b) the intracellular half-life of NOTCH is very short, so inhibition treatment does not need to be constant, but could be discontinuous; (c) the effects of NOTCH are context-dependent, therefore the effects of inhibition could be different depending on each cell type. Researchers are looking for NOTCH signaling inhibitors as potential therapeutic agents that could in theory act at different levels. At the early steps in the pathway it would be possible to interfere in the interaction between NOTCH and its ligands, and later on with the NOTCH activation that implies the release of the intracellular form of the receptor. Once the active form translocates into the nucleus, the interruption of protein-protein interactions that take place in the nucleus, would inhibit the signaling. The therapy could also target other components of the pathway (effectors, ligands, down-stream NOTCH target genes. . .). At the present time, inhibitors of the enzyme that cleaves NOTCH (γ-secretase; GIS) and some molecular antibodies against NOTCH receptor and ligands are in clinical trials.

Hedgehog Signaling Pathway There are three homologs of the Hedgehog (HH) gene: SHH, IHH and DHH. HH protein binds to its transmembrane ligand, Patched (PTCH), resulting in downregulation of Smoothened (SMO). In the nucleus, SMO activates the transcription factors GLI and HRK4. The Hedgehog pathway is involved in developmental patterning control, playing an important role in proliferation, and regulation of self-renewal and survival of CSCs [56]. There are evidences for the implication of the Hegdehog pathway (HH-GLI) in multiples forms of cancers affecting organs where this pathway plays a role in organogenesis, mainly brain, skin, esophagus, stomach, pancreas, breast, lung, biliary tract, bladder, oral, and prostate (reviewed in [57]). It has been described that in glioblastoma multiforme (GBM) self-renewal and survival of gliomasphere-forming cells relies on SMO and GLI activities [58, 59]. Alterations in some genes acting downstream of HH, such as PTCH, SMO, or GLI have been described in meduloblastoma, rhabdomyosarcoma and basal cell carcinoma.

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One potential therapeutic treatment points to the use of antagonists of the molecules, which act downstream from the components of HH pathway. Cyclopamine, a specific inhibitor of SMO is commonly used, and it has been shown to downregulate PTCH1 and GLI approximately 4 h after treatment, followed by recovery [59]. Some others inhibitors that remain to be tested for therapeutic use are, for example, SANTs 1–4, KAAD-cyclopamine, compound-5 and compound-Z, and Cur-61414. A SMO blocker, HhAntag, reduces the tumor volume of xenografts by 20–50% after approximately three weeks [60]. Another approach implies the use of siRNA that cause inactivation of SMO resulting in a reduction of growth of the gliomasphere-derived cells in GBM [59]. Recent studies in multiple myeloma have shown the potential use of anti-HH blocking antibodies, which reduce the clonogenicity of CSCs [61]. Since the expression of SHH has been shown to be necessary for the growth of some cancers, the use of specific antibodies against SHH could be valuable. Actually, it has been reported that 5E1, a specific anti-Shh antibody, blocks the tumor growth in small cell lung cancer cell lines [62]. Given the different origin of the organs affected in the several cancers in which the HH pathway is involved, and the disparity of response to blockade of HH pathway in epithelial cells, it has been proposed that this pathway acts only in the stroma [60]. Recent studies in mice revealed that blocking HH signaling could be an effective treatment for some cancers in adults. However, the inhibition of this pathway in pediatric tumors is not practical given the severe defects caused on bone growth [63]. Many aspects of HH signaling remain to be understood, and further studies will be necessary to throw some light into the mechanisms that regulate the developmental patterning control, and will help to design better targets against the HH pathway. Wnt Signaling Pathway Wnt signaling is involved in stem cell self-renewal, proliferation, differentiation and also in migration within the stem cell niche. In the canonical Wnt signaling pathway, extracellular Wnt proteins bind to the Frizzled (FZD) receptors in the membrane. After that, and together with LRP5 and LRP6 co-receptors, the Gsk3β and serine-threonine kinases are inhibited. Thus, β-catenin is not degraded and instead the unphosphorylated protein is first accumulated in the cytoplasm and then translocated to the nucleus. After that, the interaction of β-catenin with the transcription factors TCF/LEF leads to the activation of target genes such as Cyclin-D, DKK1, Myc, FGF20, WISP1 and CCND1, which are involved in regulation of cell proliferation and determination of cell-fate. Accumulation of β-catenin is associated with several solid tumors: melanoma, sarcoma, brain tumors and breast cancer, and mutated forms of the protein are found in several carcinomas (endometrial, prostate, hepatocellular) and colorectal cancer [64]. In fact, it has been suggested that the constitutive activation of Wnt signaling could be the initiation episode or at least an important player in the development of many solid tumors [65]. In addition, alterations in WNT cascade signaling are also found in different hematological malignances, such as myeloid and lymphoblastic leukemias, or multiple myeloma. In acute myeloid leukemia (AML), increased expression of β-catenin is associated

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with poor prognosis. Furthermore, many of the target genes of the fusion proteins created after chromosomal translocations leading to the disease play some part in the WNT signaling pathway [66]. In the terminal phase of chronic myeloid leukemia (CML), but not in early stages, WNT signaling is also activated. Experimental studies with T cell-lymphomas, and the analogy between NOTCH and WNT signaling in normal B- and T-cell development, suggest that constitutively active WNT signaling could contribute to Acute Lymphoblastic leukemia (ALL). Last but not least, alterations in this pathway have been described in multiple myeloma, where the WNT inhibitors DKK1 and secreted Frizzled-related protein 2 take part in the bone destruction process [67, 68]. The therapeutic approaches targeting WNT pathway include small molecules mainly targeted against the Tcf-β-catenin complex (e.g., PKF118-310 or ZTM000990) and monoclonal antibodies (anti-WNT1 and anti-WNT-2), which have been proved to have anticancer effects in vitro [69]. Also, non-steroidal antiinflammatory drugs (NSAIDS) like aspirin or indomethacin might contribute to the suppression of cancerous aberrant WNT signaling by a still unclear mechanism of action. Nowadays, many aspects of the WNT signaling pathway remain still unclear (the precise role of β-catenin in self-renewal, regulation of the dosage of WNT signaling. . .), and further studies will be necessary to elucidate the specific targets for therapeutic treatment. For a comprehensive review about the current drug development strategies against Wnt signally, see [70]. Polycomb Signaling Pathway Polycomb family genes (PcG) are involved in self-renewal and proliferation because they are essential for the epigenetic mechanisms that regulate cell fate determination and maintenance. PcG genes cause chromatin modifications that lead to the repression of the targeted genes. PcG proteins comprise the two multimeric Polycomb repressive complexes, PRC1 and PRC2, with different biochemical and functional properties [71]. Both present intrinsic histone modifying activities: ubiquitination of lysine 119 of histone H2A in case of PRC1, and trimethylation of lysine 27 of histone H3 in case of PRC2. PRC1 comprises, among other proteins, BMI-1, while PRC2 includes EZH2. It has been suggested that PRC2 inhibits transcription initiation, and then PRC1 maintains the conditions to allow the repression of the target genes, which may not necessary be the same for both complexes. BMI-1 induces telomerase activity in mammary epithelial cells [72], and is responsible for self-renewal in hematopoietic and neural stem cells, and also leukemic stem cells [73, 74]. In addition to its function in the proliferation of a variety of differentiated cells, BMI-1 has been shown to be essential for the proliferation and self-renewal of several adult stem cells, like the neural stem cells or the hematopoietic stem cells [74–76]. Moreover, BMI-1 is necessary for the propagation of leukemic stem cells and is expressed in glioma stem cells [73, 77]. Its oncogenic function has mainly been considered to be mediated by its repressive effect on the Ink4a/Arf tumor suppressor locus [78].

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BMI-1 has been demonstrated to be essential for the proliferation and selfrenewal of no only normal hematopoietic stem cells, but also of leukaemic stem cells [73, 79], as well as normal neural stem cells by repressing the Ink4a/Arf locus [75, 80]. Aberrant expression of PcG protein has been implicated in a broad type of tumors, where BMI-1 or EZH2 are amplified and/or overexpressed (prostate, bladder, breast, colon, kidney, lung, liver, leukemia. . .) (reviewed in [35]). BMI-1 and EZH2 promote aberrant proliferation of CSCs by repression of p16INK4A (an inhibitor of cell cycle progression, that also plays a role in stem cell senescence [81]) and p19ARF (a regulator of p53). The aberrant expression occurring in cancer results not only in changes in the protein composition but also in altered affinities of PRC complexes for their target genes. Balance between protooncogenes and tumor suppressor genes regulates stem cell function throughout life. Any deregulation (such as those which normally occur with aging) could fatally lead to development of cancer. In this sense, the stem cells in the tumor seem to be a good target for the epigenetic alterations driven by aberrant Polycomb proteins, which will promote the neoplastic transformation. From the therapeutical point of view, it has been shown that the interference of PRC2-dependent gene repression with the drug DZNep (S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A) induces apoptosis specifically in breast cancer cells but not in normal cells [82]. DZNep acts as a novel chromatin remodelling compound that induces reactivation of an important set of genes selectively repressed by PRC2 in breast cancer (among others a novel apoptosis effector, FBXO32). These results suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy. Hox Signaling Pathway Hox genes are involved in the process of self-renewal of stem cells, proliferation and differentiation of precursor cells. Aberrant expression of different members of the family is associated with leukemia. HOXA9 is involved in chromosomal translocations in acute myeloid leukemia [83], where its overexpression is associated to poor prognosis. HOXB6 is also overexpressed in AML [84], and overexpression of HOX11 is found in T cell acute lymphoblastic leukemias. In addition the use of mouse models has shown a role of HOXB3, HOXB8 and HOXA10 in leukemogenesis [31]. Until now, no drugs have been developed that can efficiently target this essential developmental pathway. PTEN/PI3K/Akt Signaling Pathway The phosphatase PTEN plays a role in numerous cellular processes such as cell cycle progression, proliferation, apoptosis, DNA damage response, among others, being important in the general process of development and metabolism. PTEN signaling is altered in almost every cancer analyzed, acting thus as a tumor suppressor gene. The preferential substrate for PTEN is phosphatidylinositol tri-phosphate (PIP3) that is converted in PIP2. On the contrary, the enzyme phosphatidylinositol 3 Kinase

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(PI3K), phosphorylates PIP2 to PIP3. PIP3 binds to and activates the kinases PDK1 and AKT, which regulates processes involved in cancer such as cell cycle progression, apoptosis and cellular growth. Levels of PIP3 in resting cells are normally low, and increase after growth factor activation, or under pathological conditions, as a result of the loss of activity of PTEN. The high levels of PIP3 lead to cell cycle progression, improved survival and increase in cell size. In contrast, overexpression of PTEN induces cell cycle arrest, apoptosis and reduction of the cell size. In the cancer scenario, the signaling cascade would start with the activation of PI3K by specific growth factor tyrosine kinases receptor and Ras, resulting in increased levels of PIP3. The aberrant activation of PTWN/PI3K pathway has been proved to be an essential step in the initiation and maintenance of the tumors. The whole range of mutations that promotes tumorigenesis through upregulation of the PTEN/PIK3 pathway includes PIK3 amplification, PTEN loss (by phosphorylation, acetylation, oxidation, ubiquitination and caspase cleavage), AKT mutations and receptor tyrosine kinase amplification [85]. The PTEN gene is frequently mutated in numerous cancers, affecting either in a monoallelic (gliomas, breast, colon, lung, prostate) or biallelic way (endometrial, glioblastoma, prostate and breast cancer). The reduced expression can also be due to post-transcriptional/translational modifications (reviewed in [86]). The transcription factor p53 transactivates PTEN expression [87], and their association affects the p53 acetylation state. The therapeutical approach could be targeted to different components of this complex PTEN pathway, but its broad functions make it necessary to carefully take all of them into account in order to decide which target therapy employ. It is important to mention that every member of the pathway is frequently altered in cancer, even when they serve the same purpose. It remains unclear if all of these alterations are redundant or not, depending on the type of tumor, although there is evidence indicating that individual mutations are not totally redundant. For example, in breast, endometrial and colon cancer coexist genetic alterations in PTEN and PI3K, while RAS and PI3K mutations in endometrial cancer seem to be mutually exclusive [85]. It is therefore reasonable to think that not all changes in the pathway are comparable and so different alterations will create different contexts that ultimately will play a role in the efficiency of the selected drug [86]. In recent years many PI3K pathway inhibitors have emerged, and several are already in clinical trials. The group of PI3K inhibitors include: Wortmannin (with very short half-life in serum) and its derivate PX-866 (Oncothyreon, WA, USA) which inhibit cancer cell motility and growth [88], LY294002 (Lilly, IN,USA) and its prodrug SF1126 (Semafore, IN,USA) with antitumor and antiangiogenic activity [89], ZSTK474 (Zenyaku Kogyo, Tokyo, Japan) with antitumorigenic effects on a broad type of xenografts [90], PI-103 (Piramed, Slough, USA) that in combination with radiotherapy is effective in the treatment of xenografts of glioblastoma cell lines [91], PIK-90 (Bayer, Leverkusen, Germany) and PIK-93 (Novartis, Basel, Switzerland). Some of the PI3K inhibitors that have already entered phase I clinical trials in patients with solid tumors, are L147 and Xl765 (Exelisis) which inhibits multiple PI3K isoforms, BGT226 and NVP-BEZ235 (Novartis) (reviewed in [92]).

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All the above-mentioned drugs are targeted at a specific pathway, but do not really take into account the CSC hypothesis and are rather focused at the global tumor destruction. However, once more, their effects in CSCs survival via PTEN pathway inactivation could be significant: with respect to CSC-targeted therapy, a key study [93] has identified different mechanisms for self-renewal in the maintenance of leukemic stem cells and normal HSCs pools, showing that deletion of the PTEN tumor suppressor gene results in generation of leukemic stem cells but depletion of normal HSCs. These data demonstrate that it is possible to identify – and to target therapeutically – pathways that have distinct effects on normal stem cells and cancer stem cells within the same tissue. This mechanistic distinction may enable the design of therapies to combat leukemia that target the PTEN pathway with the goal of killing leukaemic stem cells without damaging the normal stem-cell pool. Recently it has also been shown [94] that PTEN deletion in mouse hematopoietic stem cells leads to a myeloproliferative disorder, followed by an acute T-lymphoblastic leukemia (T-ALL) without alterations in Notch1 signalling but with aberrant overexpression of c-myc, thus suggesting that Pten inactivation and c-myc overexpression may substitute functionally for the Notch1 abnormalities so frequently found in T-ALLs. This last study indicates that multiple genetic or molecular alterations can contribute cooperatively to CSC transformation and that, therefore, it is very likely that several pathways have to be targeted in order to be able to destroy CSCs.

16.4.2.2 Modulation of Chemoresistance Mechanisms Processes related to the phenomenon of chemoresistance in stem cells could be classified in two groups: one group which includes events related with the drugs themselves, such as mutation or overexpression of the drug target, drug inactivation and drug elimination (i.e. by higher expression of efflux proteins), and a second group comprising the molecular events that take place in the cell in order to overcome the effects of the drugs: anti-DNA repair mechanism, and inhibition of antiapoptotic pathways. CSCs are more resistant to current therapeutic protocols than normal stem cells [23]. According to this, the pathways regulating chemorresistance are attractive targets for the modulation of drug efficacy against CSCs. So, although until now these groups of drugs have been studied more as a general type of global anticancer agent, targeted at the main bulk of the tumor mass, they might in the future also prove to be effective, to some degree and in specific combinations, against CSCs. High Levels of ABC-Transport Proteins (Efflux Proteins) One characteristic of stem cells is the high levels of expression of transmembrane ABC transporters (ATP-binding cassette transporters), such as Breast Cancer Resistance Protein (BCRP-ABCG2) and multi-drug resistance 1 (ABC1/MDR1). These proteins act as self-defense systems, and it is believed that they are also

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present in CSCs. The presence of ABC transporters in the cells of the Side Population (cells with stem cell activity in the tumor mass) would facilitate the elimination of anticancer drugs such as mitoxanthrone, gemcitabine, doxorubicin or 5-fluorouracil. The higher expression of ABCG2 protein could be behind the increased resistance of CSCs in brain tumors to several compounds, such as paclitaxel, carboplatin, etoposide and temozolomide (reviewed in [27]). Unfortunately, up to now, none of the clinical trials using inhibitors of ABC transporters have succeeded.

Anti-DNA Repair Mechanisms Current anticancer therapies are aimed at the elimination of proliferative cells, targeting mainly the cycling mechanisms, using drugs that cause DNA damage by various mechanisms. This damage results in cell cycle arrest and cell death, either directly of after the replication in S phase, due to the creation of replicationassociated DNA double-strand breaks (DSBs) that are highly toxic for the cell. However in certain cancerous cells, that are not responsive to these treatments, several mechanisms to repair the DNA damage are active, removing the lesions before they become toxic [95]. This fact could make DNA repair pathways an excellent target for anti-cancer treatments. There are evidences indicating that inhibitors of DNA repair pathways together with certain DNA-damaging anticancer drugs increase the efficiency of the cancer treatment, due to the inhibition of the pathways that lead to the elimination of the toxic effects. There are many agents under study whose full analysis falls out of the scope of this chapter, since they are not specifically targeted at CSCs. However, as mentioned above, it is very likely that this approach will uncover compounds that can interfere with mechanisms essential for CSC survival.

Inhibition of Antiapoptotic Mechanisms Apoptosis could be described as a cell suicide in response to signaling that tells the cell that something is wrong. The master players in the apoptotic pathways are the caspases. These proteases become activated after the right signaling is triggered by external or internal molecules and degrade their substrates leading to cell death. Inhibition of apoptotic processes allows the survival of the tumor cells and put them in an advantage situation when chemotherapy treatment is chosen. Alteration in apoptosis has been found in several cancers such as melanoma, myeloid leukemia, or lung cancer [96]. It has been shown that alterations in different pathways such as NOTCH and Polycomb pathways, involved in self-renewal, affect to the apoptotic process. For example, in most cases the pathologic activation of NOTCH signaling prevents apoptosis in cancer cells, and the interference of the multimeric Polycomb repressive complex PRC2 activity, by using the drug DZNep induces apoptosis specifically in breast cancer cells but not in normal cells [82].

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Nevertheless, the main group of proteins that contribute to the inhibition of apoptosis are the Inhibition apoptotic proteins (IAP), which block the pathways at different levels, and through several mechanisms, and what is more important from a therapeutic perspective, they have been found to be overexpressed in several cancers [97]. Some of the IAPs directly inhibit the caspases, binding to the enzyme, and by doing so, they interfere with the cell death process. One of the most potent inhibitor is XIAP, which binds to caspases 3, 7 and 9 and inhibits those [98]. In contrast, the Smac/DIABLO complex, in the cytosol, acts as antagonist of XIAP, and has proapoptotic effect. Some others IAP members, such as c-IAP1 and c-IAP2 develop their inhibition potential by binding to Smac subunits, so in a nondirect way improve the XIAP-mediated inhibition of caspases. c-IAP1 and c-IAP2 were initially identified through their ability to interact with tumor necrosis factor receptor-associated factor 2, and regulate the receptor mediated apoptosis [99]. In addition, some of these inhibitors promote ubiquitination of caspases and then proteosomal degradation. The role of IAP proteins goes further, and they also participate in the activation of certain signal transduction pathways involved in tumorigeneis (JNK1, PI3K/Akt. . .). The expression of some IAP (survivin and ML-IAP) is almost restricted to the tumor tissue, making them useful targets for anti-tumor therapy. There are several approaches targeting IAP, from siRNA to immunotherapy, going through physical inhibition (reviewed in [96]). The downregulation of the IAP proteins using siRNA techniques has also been used, in combination with chemotherapy, and lead to tumor growth inhibition in vivo and induction of apoptosis in vitro. The agents AEG-35156 (from Aegera Therapeutics, Inc.) against XIPA, and LY-2181308 (from ISIS Pharmaceuticals, Inc. and Eli Lilly) and the inhibitor YM-155 (from Astellas Pharma, Inc.) both against Survivin, are in phase I/II clinical trials. Another tempting approach implies the use of compounds with similar structure to the Smac protein, which interferes with the interactions between IAP/caspases and IAP/endogenous Smac, stimulating in this way the cell death. Some of these molecules, such as Compounds 8 and C (from Genentech), LBW242 and TWX024 (from Novartis), Compound 11 (from Abbot) . . . are already in preclinical trials, and some others have shown positive effect using in vivo models for breast cancer, melanoma, non-small cell lung cancer, multiple myeloma and glioma. Finally, the IAP proteins ML-IAP and Survivin could also be targeted with specific immunotherapy, given their antigenic potential.

16.4.2.3 Others Mechanism Affecting CSCs or Their Niche Telomerase Inactivation Chromosome stability is achieved because of the addition of nucleotides in the terminal region of the chromosomes (telomere) by the telomerase enzyme and because of the stabilization effect of telomere binding proteins. The role of telomerase is crucial for the appropriate DNA replication, since the polymerases themselves are not

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able to fully replicate the DNA in those regions. In the absence of the enzyme, the instability of the chromosomes will end in cell arrest or death [100]. A strict regulation of telomerase activity and its repression could be involved in tumor suppressor mechanisms, although the enzyme itself is not an oncogene. There are therefore some telomere-dependent pathways of cell mortality, which could be explored in terms of anti-cancer therapy. One of the main advantages of this kind of treatment would be its relative universality, critically and specificity for cancer cells. The level of expression of telomerase in normal cells, including stem cells, is very low in comparison to the one in CSCs [100, 101]; in addition, in cancer cells the telomere length is shorter than in normal cells. These two characteristics could be used as an advantage for designing specific agents against telomerase activity, since differential sensitivity for the two types of cells would be expected. Stem cells require the telomerase activity to keep the telomere length from shortening and therefore preventing the aging effect and acquiring an infinite replication potential [30]. For cancer cells the active presence of telomerase has been suggested to be an absolute requirement for the growth and maintenance of the tumor in many malignancies [102]. Several approaches are currently used as telomerase-based anticancer therapies: (a) blocking agents against telomerase activity, (b) telomere disrupting compounds, (c) therapy with suicide genes (using telomerase promoter driven-expression of a toxic gene itself, or of gene which lead to toxic actions), (d) telomerase inhibitors, and (e) immunotherapy against telomerase. Although all of them present certain advantages/disadvantages, up to now only the last two approaches have reached clinical trials [100]. The possibility of using small protein molecules as direct inhibitors of telomerase has been the focus of many drug screenings. Nevertheless, so far no candidate has reached clinical trials. Although the reason for the failure is not clear, the higher expression of efflux proteins in the stem cells could be behind this problem. The agent GRN163L, a lipidated oligonucleotide (13 nt), is a direct inhibitor of the human telomerase. This molecule binds to the hTR sequence (recognition site for RNA template) in the active region of telomerase and blocks telomere access to the enzyme, leading to telomerase inhibition. GRN163L and its non-lipidated version, GRN163, are currently in clinical trials for patients with different hematological malignances [103, 104], and solid tumors such as breast [105, 106], lung [107], brain [108], liver [109], bladder [110], and prostate [111]. The use of immunotherapy in cancer treatment is not very common given the fact that for most of the cancers there are not foreign proteins involved in the disease. In this aspect, the stem cells telomerase acts as an endogenous antigen, which could be targeted with the appropriate antibodies. The scarceness of telomerase is not only found in normal cells but also in the cancerous cells (about 100 molecules per cell). This could make it easier the treatment with immunotherapy agents. Up to now, several proofs of concept have been achieved using telomerase-based immunotherapy in cells and animal models: evidence of epitope presentations, generation of TERT (telomerase reverse transcriptase)-specific CD8+ and/or CD4+ cells in an HLA-specific way, and anti-tumor effects [112]. A comprehensive review about the

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products that have reached clinical trails can be found in Harley, 2008. Some of these molecules are: GV1001 is in phase II (for non small cell lung cancer treatment) and in phase III (for pancreatic cancer therapy); GRNVAC1 is in phase II (for Acute Myeloid Leukemia treatment); p540–548 is in phase I (for breast and prostate cancer treatment) and in phase I/II (for non small cell lung cancer therapy); TLI is in phase I (for prostate cancer treatment), and Vx01 is in phase I (for non small cell lung cancer therapy). Levels of ROS (Reactive Oxygen Species) Several studies with animal and human models involve ROS in the tumor initiation processes, and although the relation between intracellular ROS levels and CSC activity is still not clear, it has been suggested that the redox state plays a role in the balance between the process of self-renewal and differentiation. Also, it is known that stem cell niches tend to be hypoxic environments [113], and tumor masses also have a tendency to develop hypoxic regions due to the rapid cellular growth. Along these lines, the regulation of CSCs and niche ROS levels has been suggested as a potential way of therapy [114]. In this sense, enzymes that are involved in generation of reactive oxygen (such as superoxide dismutase) could be a possible target. Inhibition of Tumor Vasculature As it happens for the normal stem cells, there is a specific cell niche, providing an extrinsic microenvironment that promote CSCs maintenance and self-renewal properties, the “tumor cell niche” [115]. As previously mentioned, an appropriate niche is essential for CSC survival (for more details about the CSC-Niche see Chapter 10), meaning that alterations of any of the niche components could be potentially useful as therapy against CSCs. The inhibition of vasculature vessel formation within the niche would cease the nutrient supply to the CSCs environment and reduce their resistance to radio-chemotherapy.

16.5 Imaging CSCs We have previously mentioned that the population of CSCs in the tumor mass is extremely low (0.01–1%) and still, given their unique characteristics, these low fraction of cells is held responsible for tumor maintenance. The focus of this chapter has been to explain the approaches to eliminate CSCs, and we have gone through the main currently considered strategies to achieve this elimination. Nevertheless, even if a better knowledge of the CSC biology will improve the design of new therapeutic treatments, there is not doubt that being able to “see” where are the cells that we aim to target would improve enormously the therapeutic management. The imaging-based technology should allow us to follow-up the CSCs and locate them inside the organism, the tissue, and even within the tumor mass [116]. In recent years, many advances have been made in high resolution, in vivo imaging methods,

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including magnetic resonance (MRI), computer tomography (CT), nuclear imaging (positron emission tomography-PET; single photon emission tomography-SPECT), optical imaging (bioluminescence imaging, fluorescence imaging), and ultrasound technology. Imaging of gene expression has become progressively more important for diagnosis, prediction of tumor response to the therapy and monitoring the process of anti-cancer treatments [117]. The use of reporters of specific genes (such as PETreporter gene, that can be delivered inside the cell by viral infection, and codes for an enzyme that can be imaged with PET technology), and the possibility of quantification of the levels of expression of those genes and tumor volumes with imaging approaches, could undoubtedly improve the gene therapy strategies by providing with more accurate methods of measurement the toxicity and pharmacodynamics over the period of time that the treatment is used. Imaging techniques could be considered as real in vivo assays, in which cancer cells are not subjected to any modification in the microenvironment. We have already discussed the important relation between stem cells and their niche, so avoiding any alteration in this scenario, would give us a better idea about how biological processes occur. Employing specific imaging agents, hopefully it will be possible to detect the cancer cells anywhere in the body, since once the cancer has expanded more than one focus is expected in the body. In different stages of the disease, the phenotype of the cancerous cell may change, not only due to the differentiation process but also in response to the several drugs used in chemotherapy. In this sense, imaging techniques will provide evidences to decide if going on with the same therapy, change the dose or select another chemotherapy compound [118]. All these protocols will have first to be studied in suitable animal models, before they can be efficiently translated to humans (see below, Future Prospects). The identification of the right molecules that can be used as biomarkers for imaging studies will have an enormous impact in the detection, diagnosis, prognosis and monitoring of follow-up in cancer patients. It might be possible that in a not too distant future, with the combination of different techniques, we could trace the malignant cells in the tumor, including the small amount of CSCs.

16.6 Future Prospects Most of the anti-CSC therapeutic approaches discussed in this Chapter are mainly based on educated guesses supported by our pre-existing knowledge of all the mechanisms that are known, supposed or expected to play a role in the biology of cancer stem cells. However, clearly new strategies for target identification are needed, and they will most likely come from studies analyzing in a systematic and unbiased manner the differences between normal stem cells and cancer stem cells. There are, however, technological problems in studying these differences using samples directly obtained from human cancer patients. First, one would have to account for

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the enormous variability existing between patients in terms of genetic background; in fact, two patients with exactly the same diagnostic and staging can have completely different disease evolutions, even with the very same treatment. Also, using human samples we are not taking individual cancer susceptibility into consideration. The homogeneity of the starting samples in which to look for CSC-specific markers would then be a problem, introducing a high level of unpredictability when characterizing the CSC compartment in any given tumor. This implies the need for new approaches that involve animal models that can faithfully reproduce human cancer. Cell lines can help at later stages of drug screening, but in order to accurately identify the pathways responsible for cancer development, it is mandatory to perform experiments in an in vivo setting in which cancer arises in the appropriate microenvironment. Therefore, the generation of mouse models of human tumors is an unavoidable need for understanding the origin of the disease and developing new therapies. Also, the high attrition rate of drugs that fall from the development pipeline at late stages is mainly due to the lack of suitable systems for testing efficacy in a setting that is physiologically relevant for the human disease. Using mouse models the genetic variability is completely controlled, susceptibility to cancer can not only be controlled but also included in the studies, and homogeneous CSC samples can be obtained that can then be compared with their normal counterparts, all along the different stages of cancer progression. From the systematic comparison of normal versus cancerous stem cells in these models it should be possible to identify new pathways and molecules that can afterwards be used either as biomarkers, as targets for therapy or to follow up of the disease after treatment. This should open a new era in our understanding and treatment of cancer. Acknowledgments C.C. is a Spanish “Ramón y Cajal” investigator from the Spanish Ministerio de Educación y Ciencia. Research at C.C.’s lab is partially supported by Fondo de Investigaciones Sanitarias (PI04/0261; PI080164), Junta de Castilla y León (SA087A06) and Fundación de Investigación Médica MM. J.P.L. is an investigator of the “Programa Ramón y Cajal” from the Spanish “Ministerio de Educación y Ciencia”; his work is partially supported by the “Fondo de Investigaciones Sanitarias” (PI070057) and “MEC Consolider-Ingenio 2010” (Ref. CSD20070017). Research in ISG group is supported partially by FEDER and by MEC (SAF2006-03726), Junta de Castilla y León (CSI13A08 and GR15), FIS (PI050087), Federación de Cajas de Ahorro Castilla y León (I Convocatoria de Ayudas para Proyectos de Investigación Biosanitaria con Células Madre), CDTEAM project (CENIT-Ingenio 2010) and MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017).

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Chapter 17

Potential Molecular Therapeutic Targets in Cancer Stem/Progenitor Cells: Are ATP-Binding Cassette Membrane Transporters Appropriate Targets to Eliminate Cancer-Initiating Cells? Murielle Mimeault and Surinder K. Batra

Abstract Recent advances in the basic and clinical oncology have revealed that the malignant transformation of the tissue-resident adult stem/progenitor cells into leukemic or tumorigenic cancer stem/progenitor cells plays a central role in the etiopathogenesis and progression of the most human cancers. Cancer stem/progenitor cells endowed with a high self-renewal capacity and aberrant differentiation potential generally possess the high expression levels and activity of diverse ATP-binding cassette (ABC) transporters, DNA repair and detoxifying enzymes and anti-apoptotic factors. These phenotypic and functional features common to the most of cancer stem/progenitor cells have been associated with their intrinsic or acquired resistance phenotype to numerous chemotherapeutic drugs, disease recurrence and poor patient survival. Moreover, the sustained activation of distinct oncogenic signaling elements in these immature cancer-initiating cells during disease progression may also contribute to their acquisition of a more malignant behavior and development of metastatic disease state. Of particular interest, we describe the potential therapeutic targets to eradicate the cancer-initiating cells and their mature progenies in a variety of aggressive human cancers such as leukemias, melanoma and solid tumors including brain and epithelial cancers. The emphasis is on potential therapeutic molecular targets in cancer stem/progenitor cells and their progenies including ABC drug transporters, ceramide, telomerase, and diverse tumorigenic signaling elements. Keywords Cancer stem/progenitor cells · Leukemias · Solid tumors · ATP-binding cassette transporters · Multidrug resistance · Oncogenic signaling elements · Drug targets · Cancer therapies · Molecular targeting · Combination therapies

M. Mimeault (B) Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA e-mail: [email protected]

T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_17,

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Contents 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2 Phenotypic and Functional Properties of Cancer Stem/Progenitor Cells . . . . . 17.3 Intrinsic Properties Associated with the Treatment Resistance of Cancer Stem/Progenitor Cells . . . . . . . . . . . . . . . . . . 17.3.1 Structures of ABC Transporters and Their Functions in Multidrug Resistance 17.3.2 Therapeutic Strategies for Overcoming ABC Transporter-Mediated MDR . . . . . . . . . . . . . . . . . . . . . . 17.4 Implications of Cancer Stem/Progenitor Cells in Cancer Development, Treatment Resistance and Potential Molecular Therapeutic Targets . . . . . . . 17.4.1 Functions of Leukemic Stem/Progenitor Cells in Leukemias and Potential Therapeutic Targets . . . . . . . . . . . . . . . . . . . . . 17.4.2 Functions of Melanoma Stem Cells in Cutaneous Melanoma and Potential Therapeutic Targets . . . . . . . . . . . . . . . . . . . . . 17.4.3 Functions of Brain Tumor Stem Cells in Brain Cancers and Potential Therapeutic Targets . . . . . . . . . . . . . . . . . . . 17.4.4 Functions of Tumorigenic Stem/Progenitor Cells in Epithelial Cancers and Potential Therapeutic Targets . . . . . . . . . . . . . . . . 17.5 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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17.1 Introduction Recent advancements in the tissue-resident adult stem/progenitor cell biology have revealed that these immature cells endowed with a long-term self-renewal and multi-differentiation capacity provide critical physiological functions in cell replenishment during the tissue regenerative process in homeostatic conditions and after intense injuries along the lifespan of individuals [1–7]. Growing body of experimental evidence has also indicated that the genetic abnormalities may occur in adult stem/progenitor cells or their more committed progenies. These alterations may be associated with the etiology and development of diverse human pathologies, including certain hyperproliferative disorders and cancers [6, 8, 9]. In particular, the accumulation of genetic and/or epigenetic alterations in adult stem/progenitor cells may result in their malignant transformation into leukemic or tumorigenic cancer stem/progenitor cells also designated as cancer- or tumor-initiating cells [3, 4, 8–13]. In support with the major implication of cancer stem/progenitor cells in cancer initiation and progression, a small subpopulation of immature cancer stem/progenitor cells with stem cell-like properties has been identified in the most human leukemias, melanoma and solid tumors [3, 4, 8, 14–26]. It has been shown that the highly leukemic or tumorigenic cancer stem/progenitor cells can give rise in vitro and in vivo to the bulk mass of further differentiated cancer cells expressing phenotypes of the original patient leukemia or tumor [14, 15, 17, 18, 21, 23, 26, 27]. Similarly, small subpopulations of leukemic or tumorigenic cancer stem/progenitor

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cells expressing stem cell-like markers, which have been isolated from well established human cancer cell lines, were also able to give rise to leukemia or to form tumor and metastasize in animal models in vivo with a high incidence relative to their differentiated progenies [8, 28]. In addition, the cancer progression is generally associated with the activation of a complex network of oncogenic signaling pathways in cancer stem/progenitor cells and their progenies [8, 29–31]. Diverse growth factors, cytokines and soluble factors released by host stromal cells can confer to cancer cells a more aggressive malignant behavior. More specifically, the acquisition of a migratory phenotype by the tumorigenic cancer stem/progenitor cells during epithelial-mesenchymal transition (EMT) program, may lead to more invasive and metastatic cancer subtypes [8, 29– 34]. Importantly, the results from numerous recent studies have also revealed that the intrinsic or acquired resistance of leukemic and tumorigenic cancer stem/progenitor cells to current clinical therapies may result in their persistence after treatment initiation, and thereby be responsible for the tumor re-growth and disease relapse [6, 8, 19, 21, 24–26, 31, 35–50]. We describe here the phenotypic and functional properties of leukemic and tumorigenic cancer stem/progenitor cells and their implication in cancer development and treatment resistance. The emphasis is on the critical roles played by ABC multidrug transporters and distinct growth factor signaling pathways in drug resistance and novel molecular targeting approaches to eradicate the cancer-initiating cells and their differentiated progenies and improve the current cancer therapies. The provided information on potential molecular therapeutic targets in leukemic and tumorigenic cancer stem/progenitor cells and their progenies should help design new effective combination therapies against the most aggressive, recurrent and lethal human cancers.

17.2 Phenotypic and Functional Properties of Cancer Stem/Progenitor Cells A small subpopulation of cancer stem/progenitor cells, comprising about 0.1–3% of total cancer cell mass and which have generally a smaller size as compared to their differentiated progenies, has been identified and isolated from diverse human malignant tissues and metastatic neoplasms [3, 4, 8, 14–26, 51]. Among the human cancers harboring a very small subpopulation of cancer stem/progenitor cells, there are leukemias, lymphomas, sarcomas, melanoma, brain tumors and most epithelial cancers including skin, brain, gastrointestinal, colon, pancreas, liver, lung, prostate, breast and ovary cancers. Cancer stem/progenitor cells as their normal counterpart, tissue-resident adult stem/progenitor cells generally express several specific stem cell-like markers such as CD133, CD44, ATP binding-cassette (ABC) multidrug transporters and/or CXC chemokine receptor-4 (CXCR4) but lack differentiation marker expression [3, 4, 8, 14–26, 51]. In addition, the cancer initiation and progression is frequently associated with an up-regulation of telomerase activity as well as a down-regulation of distinct tumor suppressor gene products such as p16INK4A , retinoblastoma (pRb), p53 and/or phosphatase and tensin homolog

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deleted on chromosome 10 (PTEN) in cancer stem/progenitor cells and their progenies [8, 52–56]. Moreover, a sustained activation of distinct developmental cascades including hedgehog, epidermal growth factor (EGF)-EGFR system, Wnt/β-catenin, stromal cell-derived factor-1 (SDF-1)-chemokine receptor 4 (CXCR4) and/or polycomb group (PcG) protein signaling pathways in leukemic and tumorigenic cancer stem/progenitor cells may give to them a more malignant behavior [3, 8, 11, 12, 24, 29, 30, 32, 34, 42, 53, 57–60]. The stimulation of these oncogenic cascades by growth factors and cytokines through autocrine and paracrine loops may contribute to the sustained growth, survival, migration, invasion and/or treatment resistance of cancer stem/progenitor cells and their progenies. Thereby, these molecular transforming events may promote a transition from localized cancers into invasive, metastatic and refractory disease stages. Importantly, the EMT phenomenon, which occurs during embryonic development and wound healing, is also re-activated during the progression from numerous aggressive cancers such as brain, skin, prostate, ovarian, mammary, hepatic, gastrointestinal, pancreatic and colorectal carcinomas into locally invasive forms [8, 11, 12, 29, 30, 33, 34, 60]. The acquisition of a migratory phenotype by tumorigenic cancer stem/progenitor cells during EMT program concomitant with the changes in activated stroma may lead to their invasion, dissemination and formation of aggressive and metastatic cancers at distant sites [8, 11, 12, 29, 30, 33, 34, 60]. Hence, the multipotent and poorly differentiated leukemic or tumorigenic cancer stem/progenitor cells possessing aberrant proliferation and differentiation abilities may provide critical roles in leukemia and tumor development and metastases by giving rise to the bulk mass of further differentiated cancer cells. Additionally, these immature cancer-initiating cells typically possess the intrinsic properties that can give to them survival advantages relative to their mature progenies, and which may contribute to their high resistance to current clinical treatments and disease relapse.

17.3 Intrinsic Properties Associated with the Treatment Resistance of Cancer Stem/Progenitor Cells Numerous lines of experimental evidence indicated that the leukemic and tumorigenic cancer stem/progenitor cells may be intrinsically resistance to certain chemotherapeutic drugs at start of treatment and acquire an enhanced multidrug resistance (MDR) phenotype with cancer development [8, 24, 31, 35, 37, 41, 59–64]. Particularly, the cancer stem/progenitor cells often exist under a quiescent state and they are characterized by high expression levels and/or activity of diverse ABC multidrug efflux pumps, active DNA repair and intracellular detoxifying enzymes (ALDH and MGMT) and anti-apoptotic signaling factors (Bcl-2, survivin, and NF-kB) as well as deregulated apoptotic cascades (ceramide) [8, 24, 31, 35, 37, 41, 56, 59–72]. These distinctive features of cancer stem/progenitor cells may contribute to their resistance to current therapeutic treatments and disease recurrence. Consequently, the most patients who undergo potential curative treatments for locally advanced cancers and/or disseminated disease stages may subsequently relapse due to the persistence of highly leukemic or tumorigenic

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cancer stem/progenitor cells and/or their early progenies in primary neoplasms and/or micrometastases at distant sites. In this matter, we review the critical functions provided by ABC multidrug efflux pumps in the intrinsic and acquired MDR phenotype of cancer stem/progenitor cells and their progenies. We also discuss the results from prior studies carried on the development of potential ABC transporter inhibitors and recent accumulating data supporting the therapeutic interest of their molecular targeting.

17.3.1 Structures of ABC Transporters and Their Functions in Multidrug Resistance The ABC transporter superfamily comprises diverse members that have been divided in 7 subfamilies termed as A to G, based on their similarity in gene structure and sequence homology and order in their functional domains [73–77]. All of ABC transporters are integral membrane proteins containing one or two transmembrane spanning domains (TMDs) involved in substrate binding and one or two cytoplasmic nucleotide (ATP)-binding domains (NBDs). These efflux pumps actively translocate hydrophobic endogenous compounds and lipids across the cellular membrane with energy derived from the ATP hydrolysis (Fig. 17.1) [73, 74, 76–83]. Among the best characterized ABC transporters, there are multidrug resistance gene 1 (MDR1/ABCB1) encoding P-glycoprotein (P-gp), breast cancer resistance protein (BCRP/ABCB2) and multidrug resistance-related proteins (MRPs) (Table 17.1; Fig. 17.1) [74–76, 82, 84]. P-gp and MRPs, including MRP1 and MRP2, are large membrane proteins contain two TMDs made of six membrane-spanning segments and two NBDs [82, 85]. In contrast to P-gp and MRPs, BCRP/ABCB2 transporter is constituted from two half-molecules that must homodimerize to acquire transport activity [74, 76, 86]. The binding of hydrophobic substrates including chemotherapeutic drugs to these drug efflux pumps leads to a conformation change, ATP binding and release of substrates outward the formation of a pore-like structure. In generally, ABC transporters, which are expressed in a variety of tissues and organs, play an important role in the absorption, distribution and excretion of xenobiotics, chemical drugs and endogenous metabolic compounds [74–76, 82, 84]. In normal cells including primitive adult stem/progenitor cells, the ABC efflux pumps can participate to different vital mechanisms including detoxification by actively eliminating out of cells across the plasma membrane diverse intracellular cytotoxic compounds such as environmental toxic substances and highly reactive metabolic products resulting of oxidative stress [73, 75–77, 82, 84, 87]. Thereby, these efflux pumps can protect the cells against diverse adverse effects induced by the toxic substances. Numerous studies have revealed that these even types of ABC transporters are also expressed at high levels on the most primitive cancer stem/progenitor cells and their progenies, and thereby they can contribute to their intrinsic and/or acquired MDR phenotype and disease relapse by pumping outside of cells diverse toxic chemotherapeutic drugs [35, 75, 77, 82, 88–90]. Intrinsic or acquired MDR phenotype, which is characterized by cross-resistance to a wide range of structurally

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Fig. 17.1 Potential oncogenic signaling pathways involved in the sustained growth, survival, invasion, metastasis and multidrug resistance mechanisms of cancer stem/progenitor cells. The mitogenic and anti-apoptotic cascades induced through the activation of distinct growth factor signaling pathways, including receptor tyrosine kinase (RTK), sonic hedgehog SHH/PTCH/GLI, Wnt/β-catenin, hyaluronan (HA)/CD44, extracellular matrix (ECM) component/integrin and stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axes are shown. The up-regulation of the expression levels of numerous gene products including anti-apoptotic factors such as Bcl-2 and survivin, matrix metalloproteinase (MMPs), urokinase plasminogen (uPA), cyclooxygenase (COX-2), vascular epidermal growth factor (VEGF), transcriptional repressor of E-cadherin (snail, slug and twist) and ABC transporters mediated through the stimulation of these tumorigenic cascades are also indicated. Moreover, the efflux of the intracellular drug molecules out of cell mediated through ABC transmembrane multidrug efflux pumps such as P-glycoprotein (P-gp) and brain cancer resistance protein (BCRP/ABCB2) is illustrated. The possibility of an intracellular sequestration of drug molecules in lysosomes via the ABCA3 transporter is also indicated

and functionally unrelated chemical drugs represents one of the major obstacles to an effective chemotherapeutic treatment of cancer patients [8, 31, 35, 88]. Particularly, elevated expression levels of distinct ABC transporters including P-gp, BCRP/ABCG2, MRPs and/or ABCA3 as well as other MDR modulators such as lung resistance protein (LRP) have been detected in several cancer stem/progenitor cells and their progenies isolated from leukemias and solid tumors and a variety of well established cancer cell lines [35, 73–76, 78–83, 91]. Although that these different transporters may show an overlaps in their drug resistance pattern profiles, they also display certain distinctive substrate specificities (Table 17.1) [73–76, 78– 84]. For instance, P-gp and MRP1 can mediate the cellular export and resistance to similar anticancer drugs including anthracyclines, etoposide, methotrexate and

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Table 17.1 Cytotoxic drug resistance profiles of distinct multidrug resistance ATP-binding cassette transporter superfamily members and their potential inhibitors Type of ABC transporters

Cytotoxic drug resistance

ABCA3

Doxorubicin, methotrexate, vinblastine Anthracyclines (doxorubucin, daunorubicin, epirubicin), actinomycin D, colchicine, mitomycin C, epipodophyllotoxin derivatives (etoposide “VP-16” and teniposide), methotrexate, mitoxantrone, taxanes (paclitaxel “PXT or taxol”, “docetaxel taxotere”), Vinca alkaloids (vincristine, vinblastine)

MDR1 (P-gp)/ABCB1

MRP1/ABCC1

MRP2/ABCC2

BCRP/ ABCG2

Name of modulary agent

Anti-ABCA3 monoclonal antibody UIC2 monoclonal antibody, antisense oligodeoxynucleotide, verapamil, dexverapamil, cyclosporine A, valspodar (PSC-833), quinidine cinchonine, tamoxifen, toremifene, VX-710 and GF-120918, retinoid X receptor-selective agonist bexarotene (LGD1069,Targretin), dofequidar fumarate, MS-209, VX-710, flavonoids, gefitinib, erlotinib, CI1033, cyclopamine, dipyridamole Anthracyclines, colchicine, Anti-MRP1 antibody, NSAIDs etoposide, methotrexate, (indomethacin, sulindac, paclitaxel, camptothecine, tolmetin, acemetacin, vincristine, vinblastine zomepirac, mefenamic acid), quinidine, MS-209, VX-710 flavonoids, MK571, frusemide, dipyridamole Anti-MRP2 antibody, NSAIDs Irinotecan (camptothecin-11, (indomethacin, sulindac, CPT-11 or Camptosar), active tolmetin, ketoprofen, metabolite of irinotecan (7ethyl-10-hydroxy-camptothecin, ibuprofen, naproxen, and etodolac, salicylate, SN-38), cisplatin, doxorubicin, piroxicam), probenecid, etoposide, methotrexate, flavonoids, MK571, vincristine, vinblastine frusemide Fumitremorgin C analogue Anthracyclines, bisantrene, KO143, GF120918 9-amino-camptothecin, tamoxifen derivatives, irinotecan, SN-38, topotecan flavonoids, gefitinib, (TPT), mitoxantrone, erlotinib, CI1033, methotrexate, etoposide, cyclopamine epirubicin, flavopiridol, nucleoside analogues (cladribine and clofarabine)

Vinca alkaloids while MRP1 is usually less effective than P-gp at transporting taxanes such as paclitaxel (Table 17.1) [82, 85, 92]. BCRP/ABCG2 also designated as mitoxantrone-resistance protein (MXR) or ABCP, which has first been identified in doxorubicin-resistance MCF-7 breast cancer cell line, can also transport diverse

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chemical drugs such as mitoxantrone, anthracycline, irinotecan, SN-38, topotecan and nucleoside analogues (Table 17.1) [74, 76, 91, 93]. Moreover, certain members of ABC transporters including ABCB2 and ABCA3, are also expressed in the intracellular endolysosomal compartment in certain cell types including cancer cells (Fig. 17.1) [80, 94, 95]. Hence, these transporters can participate to intracellular membrane trafficking of distinct endogenous compounds, lysosomal detoxification and MDR mechanisms. Importantly, it has been shown that the cancer stem/progenitor cells isolated from malignant tissues and well established cancer cell lines can express higher levels of ABC transporters than their differentiated progenies [8, 35, 41, 70, 96]. In regard with this, the high capacity of cancer stem/progenitor cells to efflux Hoechst 33342 dye due to their high expression of ABC membrane transporters is notably at the basis of the Hoechst dye exclusion method which permits to isolate a very small cell fraction designated side population “SP” from the large cell mass [8, 41, 42, 96– 101]. It has been shown that the SP cells found in different cancer cell lines, which generally show the stem cell-like properties including their high expression levels of multidrug efflux pumps such as P-gp/ABCB1, BCRP/ABCG2 and/or ABCA3 relative to the non-SP cell fraction, may be more resistant to chemotherapeutic treatment [8, 41, 96, 99, 100, 102]. For instance, the subcellular drug sequestration into organelles through ABC transporters such as ABCA3 found in membranes of lysosomes may confer intrinsic MDR phenotype in certain SP cells from leukemia, lymphomas, neuroblastoma and breast cancer [41, 99, 100]. In addition to their functions as multidrug efflux pumps, ABC transporters can also be implicated in resistance of cancer cells to apoptosis via diverse other molecular mechanisms. For instance, P-gp can inhibit Fas-induced casapse-3 activation likely by inhibiting caspase 8 activation in a manner dependent of ATP hydrolysis [103]. In the same pathway, P-gp may also alter ceramide-induced apoptotic cell death by inducing the translocation of non-toxic glucosylceramide from the cytosolic to the luminal side of the Golgi apparatus, and thereby influence the metabolic pathway of ceramide synthesis and apoptosis resistance [104]. In this matter, we discuss here the different therapeutic strategies that have been developed in the last years in order to overcome the intrinsic and acquired MDR phenotype-mediated through ABC drug efflux pumps in cancer cells. The emphasis is on the new targeting strategies by using the specific inhibitors of ABC multidrug transporters, alone or in combination therapies, for eradicating the total cancer cell mass consisting of cancer stem/progenitor cells and their differentiated progenies.

17.3.2 Therapeutic Strategies for Overcoming ABC Transporter-Mediated MDR 17.3.2.1 Inhibitors of ABC Transporters Several chemical compounds and pharmacological agents have been shown to act as substrates and interact with one or several ABC drug transporters and inhibit their activity (Table 17.1; Fig. 17.2) [75, 83–85, 91, 105, 106]. Moreover, monoclonal

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Fig. 17.2 Potential cancer therapies by molecular targeting of distinct signaling pathways in cancer stem/progenitor cells. The inhibitory effects induced by diverse pharmacological agents such as the selective inhibitors of receptor tyrosine kinase activity (RTKI), smoothened (SMO) hedgehog signaling element (cyclopamine) and telomerase on cancer stem/progenitor cells are indicated. Moreover, the anti-carcinogenic effects induced by a monoclonal antibody (mAb) directed against integrin, CD44, Wnt ligand, SDF-1 or CXCR4 antagonist are shown. Particularly, the anti-proliferative, anti-invasive and apoptotic effects induced by these pharmacological agents in cancer stem/progenitor cells through the down-regulation of the expression levels of numerous gene products are indicated. In addition, the potent inhibitory effect mediated by the specific inhibitors of the ABC multidrug transporters on drug efflux concomitant with intracellular drug accumulation is also illustrated

antibody (mAb) directed against a specific ABC transporter and antisense (As) oligonucleotide or oligodeoxynucleotide have also been designed to inhibit the ABC transporter functions (Table 17.1; Fig 17.2) [75, 76, 105–108]. These modulators of ABC transporter activity have been shown to inhibit the cellular efflux of chemotherapeutic drugs, and thereby enhance the intracellular drug accumulation and drug-induced cytotoxicity [75, 76, 82, 83, 85, 91, 105–108]. Such inhibitory agents constitute the potential therapeutic tools to reverse MDR phenotype of cancer cells including cancer stem/progenitor cells. For instance, various compounds inhibiting P-gp activity and able to sensitize the cancer cells to certain chemotherapeutic drugs have been developed and some of them have also reached the clinical trials. Among the potential P-gp transporter inhibitors, there are the compounds that are currently used in other clinical therapeutic applications such as verapamil, cyclosporine A and quinidine as well as their analogues, dexverapamil, valspodar (PSC-833) and cinchonin. These agents have been observed to reverse MDR mediated via the P-gp transporter in a variety of cancer cells in vivo and in vitro [35, 109].

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Nevertheless, the results of clinical trials have indicated that these modulators of the P-gp activity generally show a less convincing response rate in clinical settings [35, 75, 77, 105, 106, 109]. The lack of clinical efficacy of these ABC transporters used as single modulatory agent to improve the cytotoxic effects of chemotherapeutic drugs has been associated, in part, with their low oral bioavailability, high cellular extrusion and metabolism, additional cellular targets and/or cellular systemic toxicity at effective doses [81, 110, 111]. Therefore, the use of different classes of P-gp modulators at low concentrations could represent a more promising approach for improving their efficacy and decrease the toxicity associated with the use of high concentrations of these individual agents. In support with this, the combined application of UIC2 monoclonal antibody directed against P-gp with cyclosporine A or valspodar, followed by their removal, resulted in nearly 100% inhibition of P-gp drug efflux pump activity and was more effective as single agent to reverse MDR in cancer cells in vitro and in vivo [112]. In view of the fact that the cancer cells including cancer stem/progenitor cells typically express diverse ABC transporter types that may actively contribute to drug efflux and MDR phenotype, the combination of the modulatory agents targeting distinct drug efflux pumps could be more effective for overcoming MDR. The results of clinical trials with the novel specific ABC transporter inhibitory agents acting on both P-pg and MRP1 drug efflux pumps such as quinoline derivative dofequidar fumarate (MS-209) and biricodar (VX-710) have notably given certain encouraging results for the treatment of the drug-resistant cancers including breast and non-small-cell lung cancers [109, 111, 113–115]. For instance, it has been reported that the combined use of orally active MS-209 plus cyclophosphamide, doxorubicin, and fluorouracil (CAF) was well tolerated and improved the progression-free survival in patients with advanced or recurrent breast cancer who had not received prior therapy in comparison with CAF treatment alone [113]. Additional data from more long-term randomized clinical trials should confirm the potent benefit of including these agent types in conventional chemotherapeutic regimens in order to reverse MDR and improve the overall survival of patients. 17.3.2.2 Modulatory Agents of the Transduction Signaling Elements Involved in the Regulation of ABC Transporter Expression and Functions Another promising approach to reverse MDR mediated by ABC transporters also includes the use of the agents that can modulate the signaling cascade elements that are involved in the regulation of the expression and/or functions of ABC transporters in resistant cancer cells [84, 105, 116]. For instance, several studies have revealed that the ABC transporter expression was enhanced after chemotherapy in leukemias and many solid tumors such as brain, breast, ovarian, prostate, pancreatic and cervical cancer through the modulation of diverse growth factors, PI3 K/Akt, NF-kB and/or ceramide signaling pathways [84, 116, 117]. Therefore, the use of modulatory agents of these cascades may represent an alternative strategy for overcoming MDR in certain cancer cells. Of particular interest, several dietary flavonoids, such as genistein, silymarin, quercetin, biochanin A, daidzein,

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naringenin and kaempferol may inhibit distinct ABC transporters including Pgp, MRP1 and BCRP/ABCB2-mediated cellular drug efflux, and thereby reverse MDR phenotype of cancer cells [118–120]. Recent lines of evidence have also revealed that the use of inhibitors of BCR-ABL tyrosine kinase (imatinib mesylate “ST1571”), EGFR tyrosine kinase (gefitinib, erlotinib and CI1033), hedgehog (cyclopamine) and estrogen signaling cascades (tamoxifen and its derivatives) can reverse MDR-mediated ABC transporters in cancer cell types including cancer stem/progenitor cell in vitro and/or in vivo [76, 121–132]. In this matter, we are reporting, in a more detailed manner, the accumulating body of experimental evidence indicating that cancer stem/progenitor cells provide critical functions in the development of leukemias, cutaneous melanoma and most of solid tumors, treatment resistance and disease relapse. Of clinical interest, we also describe the recent advances in the identification of new potential therapeutic targets in the cancerinitiating cells and their differentiated progenies for eradicating total cancer cell mass and overcoming the resistance to current cancer therapies.

17.4 Implications of Cancer Stem/Progenitor Cells in Cancer Development, Treatment Resistance and Potential Molecular Therapeutic Targets 17.4.1 Functions of Leukemic Stem/Progenitor Cells in Leukemias and Potential Therapeutic Targets Leukemias are a heterogeneous group of hyperproliferative hematopoietic disorders arising from the malignant transformation of bone marrow (BM)-resident primitive hematopoietic stem cells (HSCs) or their more committed lymphoid or myeloid progenitors into leukemic stem cells (LSCs) or leukemic progenitors also designated as leukemia-initiating cells [8, 15, 31, 60, 133–137]. LSCs endowed with a long-term self-renewal and aberrant differentiation ability are able to give rise to a heterogeneous population of malignant hematopoietic cells termed as leukemic blasts with abnormal hematopoietic functions that accumulate within BM and peripheral circulation [6, 137]. Among the types of leukemia, there are acute and chronic myeloid/myeloblastic leukemias (AML and CML), an AML subtype called acute promyelocytic leukemia (APL) and acute and chronic lymphoid/lymphoblastic leukemias (ALL and CLL). In regard with this, it is noteworthy that recent lines of evidence revealed that dysfunctions in BM-resident HSCs during chronological aging may lead to an enhanced commitment of these immature cells into myeloid progenitor cells, and thereby result in a high incidence of myeloid leukemogenesis with advancing age [138, 139]. Numerous investigations have indicated that the accumulation of distinct genetic and epigenetic alterations in HSCs or the pre-leukemic stem/progenitor cells (preLSCs) appear to be involved in their oncogenic transformation [31, 60, 140, 141]. Among the frequent transforming events, there are the chromosomal translocations

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resulting in the generation of chimeric fusion oncoproteins and aberrant expression and/or activation of telomerase and growth factor signaling cascades such as hedgehog, Wnt/β-catenin, Notch, FMS-like tyrosine kinase 3 (FLT 3) and/or polycomb group (PcG) ring finger protein, BMI-1 [8, 31, 60, 140–143]. For instance, APL is generally associated with the expression of the PML-RAR-α+ oncoprotein that is produced from the fusion of the promyelocytic leukemia (PML) gene to retinoic acid receptor-α (RAR-α) gene while the CML development implicates BCR-ABL fusion oncoprotein. Importantly, recent studies have also revealed that LSCs or more committed leukemic cells including malignant granulocyte-macrophage progenitors, which play a central role in the leukemogenesis, may be more resistant to current chemotherapeutic treatments than their differentiated progenies, and thereby contribute to the disease relapse. Consequently, the identification of drugs that can efficiently eradicate LSCs or their more committed leukemic progenitors is of the major clinical relevance for improving the current treatments and overall survival of the leukemic patients [144]. In regard with this, we describe here the phenotypic and functional attributes of LSCs that may contribute to their resistance to current clinical treatments, and which have mainly been elucidated from numerous studies concerning AML, APL and CML. We also describe the novel potential molecular therapies to eradicate the total cancer cell mass including LSCs and their more mature progenies.

17.4.1.1 Molecular Therapeutic Targets in AML and APL The identification of pharmacological agents that are able to induce the differentiation and/or apoptotic death of leukemic cells has led to a significant improvement in the clinical management of leukemic patients diagnosed with AML and APL [8, 31, 60, 140, 141, 144–149]. More specifically, a great majority of AML patients will achieve a complete remission (CR) with a treatment consisting of cytarabine (ara-C) and an anthracycline such as daunorubicin or idarubicin or the anthracenedione mitoxantrone [135, 143]. Moreover, the current treatment for APL patients consisting to the use of differentiating agent, all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy that differ to that for other forms of AML also result into a high rate of CR of about 90% [135, 136]. Unfortunately, several of these AML and APL patients diagnosed with a CR will ultimately relapsed due to the resistance of immature leukemic cells to these chemotherapeutic treatments [135, 136, 150]. In the case of patients diagnosed with refractory/relapsed APL, the combination of ATRA-based therapy with other anticancer drugs such as arsenic trioxide (ATO) may also constitute a more effective treatment [135, 136, 147, 150, 151]. In fact, arsenic trioxide can induce the anti-proliferative and/or pro-apoptotic effects on PML-RAR-α+ APL cells including LSCs. In addition, since frequent activating mutations in FLT3 leading to its constitutive activation may cooperate with other oncogenic events in AML and APL patients, the use of either anti-FLT3 antibody or inhibitor of its tyrosine kinase activity (PKC412, MLN 578, and CEP-701) also constitute the potential therapeutic

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strategies [152]. Importantly, the observations indicating that the CD33 cell surface antigen is expressed on CD34+ /CD38– /CD123+ AML stem cells but not on normal primitive CD34+ /CD38– HSCs found in BM also support the development of novel CD33 targeting-based therapies to selectively eradicate AML stem cells [153]. The antibody-targeted chemotherapy using Gemtuzumab ozogamicin (GO), which is a humanized anti-CD33 mAb conjugated to a cytotoxic antitumor antibiotic, calicheamicin g1 that targets CD33 leukemic blasts including AML stem cells, has given promising results for the treatment of relapsed/refractory AML patients [154–158]. The results from certain case studies have also indicated that GO can be successfully integrated into therapeutic regimens prior or after hematopoietic cell transplantation in children and adult patients with refractory/relapsed CD33+ ALL [159, 160]. It has also been noticed that the optimization of the GO dose in therapeutic regimen and administration schedule, is required to prevent the possibility of severe side effects such as hepatic toxicity [158, 159, 161]. In the same pathway, the radioimmunotherapy using (111)In-labeled anti-CD33 mAbs modified with peptides harboring nuclear localizing sequences (NLS) also constitutes an attractive strategy for treating chemotherapy-resistant AML patients [162]. It has been observed that (111)In-NLS-anti-CD33 mAbs were effective at killing HL60 and mitoxantrone-resistant HL-60-MX-1 cells as well as MDR leukemic cells from certain primary AML patient specimens expressing P-gp, BCRP1, or MRP1 transporter through the emission of Auger electrons [162]. Other promising adjuvant strategies to treat AML patients could also consist to targeting of ABC multidrug transporters. In support with this, numerous studies have indicated that the high expression levels of distinct ABC drug efflux pumps such as P-gp, MRPs, BCRP/ABCB2, ABCA3 and/or LRP on LSCs and their progenies from AML patients may contribute of a substantial manner to chemoresistance and disease relapse [89, 90, 99, 100, 163–165]. For instance, higher expression levels of P-gp, ABCB2, MRP-1 and LRP have been detected in the most immature, self-renewal and quiescent CD34+ CD38– CD123+ LSCs from 26 BM samples of AML patients as compared to their normal counterpart, CD34+ CD38– CD123– cells [163, 166]. Furthermore, a small SP subpopulation of human CD34low/– leukemic cells showing a higher intrinsic capacity to efflux daunorubicin and mitoxantrone, has been detected in the BM of more than 80% of 61 AML patients [167]. Similarly, an enhanced expression of MDR1 and ABCB2 was also detected in isolated subpopulations of highly enriched CD34+ CD38– putative LSCs from 8 of 10 AML patients who were refractory to induction chemotherapy (NR) as compared to 0 of 7 patients achieving CR [168]. Of therapeutic interest, it has also been observed that the ex vivo treatment of CD34+ CD38– cell fraction from NR patients expressing high levels of MDR1/ABCB2 with ABC transporter inhibitor, verapamil or PSC833 enhance their sensitivity to cytotoxic effect induced by daunorubicine [168]. Of particular interest, the results from a randomized controlled clinical trial have also indicated that the inclusion of first generation ABC inhibitor, cyclosporine A to an induction and consolidation chemotherapeutic regimen containing infusional daunorubicin led to a significant improvement in relapse-free and overall survival among poor-risk AML patients expressing moderate to high P-gp levels [169]. Thus,

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despite certain clinical trials using a specific ABC transporter inhibitor have given the discouraging results [170, 171], it will be important on the basis of these observations to re-consider the potential benefit to target distinct ABC transporters rather a single drug efflux pump for eliminating of LSCs in certain cases of AML patients. 17.4.1.2 Molecular Therapeutic Targets in CML The Philadelphia, Ph+ CML patients may now be successfully treated in early chronic phase with a tyrosine kinase inhibitor (TKI) such as imatinib mesylate (STI-571), dasatinib (BMS-354825) and/or nilotinib targeting BCR-ABL+ oncoprotein [6, 8, 60]. Although this TKI treatment will lead to a clinical CR of patients, the rapid CML progression to accelerated and terminal blast crisis phases is typically accompanied by the emergence of resistant disease and relapse [6, 8, 60, 172–175]. Experimental data suggests that imatinib mesylate and other TKIs may reversibly induce a growth arrest in CML stem/progenitor cells without trigger their apoptotic death. Therefore, the combination therapy by targeting BCR-ABL fusion oncoprotein plus other deregulated signaling elements in these leukemiainitiating cells could be more effective to eradicate the CML stem/progenitor cells, and thereby improve the current therapeutic regimens [176–178]. In particular, the development of resistance to TKI treatment occurring in blast crisis CML phase and disease relapse has been associated with the enhanced expression and/or activity of diverse signaling elements. These deregulated products include the ABC drug efflux pumps, low levels of cellular organic cation transporter OCT-1 which is involved in the intracellular uptake of imatinib mesylate, farnesyltransferase, anti-apoptotic factors (Bcl-2), Scr-related LYN kinase, Abelson helper integration site 1 (AHI-1), hedgehog and Wnt/β-catenin signaling elements (Tables 17.1 and 17.2 ) [8, 31, 60, 141, 174–177, 179–183]. Therefore, the molecular targeting of these altered signaling components may constitute a potential therapeutic strategy to more effectively eliminate CML stem/progenitor cells and prevent disease recurrence. Particularly, the use of inhibitors of ABC drug transporters including MDR-1 which may contribute to efflux of imatinib mesylate dasatinib and/or nilotinib is of great therapeutic interest [183–187]. Moreover, the use of inhibitors of Scr-related LYN kinase (PD180970), anti-apoptotic factor Bcl-2 (ABT-737) and/or farnesyltransferas (BMS-214662), alone or in combination with imatinib mesylate and/or other cytotoxic chemotherapy drugs such as interferon-α, also represent promising approaches (Table 17.2) [174–176, 179–181, 183, 188]. For instance, it has been reported that the farnesyltransferase inhibitor (FTI) designated as BMS-214662, alone or in combination with imatinib mesylate or dasatinib, potently induced apoptosis of both proliferating and quiescent CML blast crisis stem/progenitor cells in vitro [178]. Interestingly, a new antileukemic compound, 4-benzyl-2-methyl1,2,4-thiadiazolidine-3,5-dione (TDZD-8) also can provoke the death of leukemic stem/progenitor cells from primary blast crisis CML, AML, ALL and CLL specimens. The cytotoxic effects of TDZD-8 appear to be mediated through a rapid loss of membrane integrity, depletion of free thiols, and inhibition of both the PKC and FLT3 signaling pathways [189].

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Table 17.2 Potential therapeutic targets in cancer stem/progenitor cells and their malignant progenies Targeted signaling element Growth factor signaling element: EGFR (erbB1) antibody EGFR-TKI Anti-EGF antibody erbB1/erbB2/erbB3/erbB4-TKI Hedgehog Wnt/β-catenin Notch Flt3 Flt3-TKI HA/CD44 SDF-1/CXCR4 VEGF VEGFR VEGFR/EGFR ECM component/integrin Signal transduction element: BCR-ABL-TKI Telomerase

BMI-1 Bcl-2 PI3K mTOR kinase NF-κB

Farnesyltransferase Rac COX-2 ALDH MGMT Sphingosine kinase-1 GCS Ceramidase

Name of inhibitory agent mAb-C225, IMC-C225 Gefitinib, erlotinib, AG1478, EKB-569 ABX-EGF Cl1033 SMO inhibitor (cyclopamine), anti-SHH antibody Anti-Wnt antibody, WIF-1 γ-secretase inhibitor DAPT, MK-0752, GSI-18 Anti-Flt3 antibody CEP-701 (Lestaurtinib), PKC412 (everolimus) HA oligosaccharides, anti-CD44 antibody, soluble CD44 form Anti-SDF-1 or anti-CXCR4 antibody, CXCR4 antagonist (TC14012, TN14003 or AMD3100) Anti-VEGF antibody (bevacizumab) Anti-VEGFR antibody (DC101) ZD6474 Anti-integrin antibody Imatinib mesylate (ST1571), dasatinib, nilotinib G-quadruplex interacting agents (RHPS4), inhibitors of RT and telomerase phosphorylation or assembly, antisense-TERT or -TR, telomerase template antagonist Vorinostat, azacitidine decitabine Antisense-Bcl-2 (oblimersen sodium), ABT-737 LY294002 Rapamycin, CCI-779 IkBα inhibitor, sulfasalazine, bortezomib (PS-341), salinosporamides A (NPI-0052), parthenolide, dehydroxymethylepoxyquinomicin (DHMEQ) BMS-214662 NSC23766 NS-396, etodolax, celecoxib, rofecoxib Disulfiram, diethyldithiocarbamate or cyanamide O6-benzylguanine (O6-BG) N,N-dimethylsphingosine (DMS), F12509A, B-5354c D,L-threo-PDMP, PPMP, tamoxifen N-oleoylethanolamide (OE), B13, LCL204, D-e-MAPP

17.4.1.3 Stem Cell-Based Transplantation Therapies High-dose or intermittent systemic chemotherapy or ionizing radiation therapy plus stem cell transplantation also represents a potential therapeutic option to treat and even cure the high risk patients diagnosed with advanced and/or relapsed leukemias and other aggressive cancers [5, 6, 8, 31, 60, 139, 190]. Among them, there are multiple myelanomas, Hodgkin’s and non-Hodgkin’s lymphomas, melanoma,

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sarcomas, retinoblastoma, brain tumors as well as diverse epithelial cancers including kidney, brain, lung, pancreatic, prostate, breast and ovarian carcinomas [5, 6, 8, 31, 60, 139, 190]. In fact, the transplantation of BM-derived stem cells or HSCs may restore the hematopoietic and immune system after myeloablative effects induced by high-dose irradiation or chemotherapy following a treatment of cancer patients [191].

17.4.2 Functions of Melanoma Stem Cells in Cutaneous Melanoma and Potential Therapeutic Targets The cutaneous melanoma, which generally shows a high propensity to metastasize and treatment resistance, may also derive from the malignant transformation of skin stem cells like epithelial neural crest stem cells (eNCSCs) or their progenitors found in hair follicle bulge [18, 26, 63, 192–197]. In support with this, little subpopulations of melanocytic cells expressing different biomarkers [CD133, CD44, CD20, telomerase reverse transcriptase (TERT), Nanog and/or ATP-binding cassette (ABC) transporters such as [MDR1, ABCG2 and ABCG5] and possessing stem cell-like properties have been isolated from human melanoma specimens or human WM115 and murine B16F10 melanoma cell lines [18, 26, 63, 192–196]. These melanocyte stem cells endowed with a self-renewal potential were able to give rise to total cancer cell mass in vitro or in vivo [18, 194, 195]. Of therapeutic interest, it has also been reported that the molecular targeting of CD133+ melanoma stem cells expressing ABCB5 transporter using monoclonal anti-ABCB5 mAb significantly reversed resistance of G3361 melanoma cells to doxorubicin and inhibited the growth of melanoma stem cell xenograft-derived tumor in vivo [26]. In addition, the melanoma development is frequently associated with the occurrence of numerous transforming events including a decreased expression of tumor suppressor gene products such as pRb, p53, p21WAF1/CIP1 , p27KIP1 and proapoptotic factors, Bax and Bak [198]. Moreover, a sustained activation of Wnt/βcatenin, Notch, stem cell factor (SCF)/KIT tyrosine kinase receptor, hyaluronan (HA)/CD44 receptor, Cripto-1, BMI-1 and Ras/ERK signaling pathways often occur in invasive and metastatic melanocytic tumors [199–202]. Therefore, the molecular targeting of these oncogenic signaling elements may represent a potential therapeutic strategy against the aggressive melanoma. Consistently, it has been reported that the inhibition of KIT tyrosine kinase activity by imatinib mesylate or hyaluronan/CD44 tumorigenic cascade using soluble CD44 protein form or anti-CD44 mAb diminished the human melanoma growth and/or the incidence of metastases of melanocytic cancer cells in vitro and in mice models in vivo [203, 204]. In the same pathway, the pharmacologic suppression of microphthalmia-associated transcription factor (MITF) expression through a topical application of HDAC-inhibitor also reduced the skin pigmentation and melanoma formation in mouse xenografts in vivo [205]. On the other hand, the immunotherapy-based melanoma vaccines, chemoimmunotherapies with immunosuppressive agents such as interferon-α, gene therapy

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and anti-angiogenic therapy (thalidomine, angiostatin and endostatin), without or plus a stem cell-based transplant, also represent the attractive therapeutic strategies against invasive, metastatic and/or relapsed melanomas [54, 206, 207]. In regard with this, it has been observed that the melanoma stem cells can be efficiently targeted by cancer testis antigens (CTA)-directed immunotherapy [208]. A therapeutic regimen consisting to 5-fluorocytosine plus engineered neural stem cells (NSCs) expressing cytosine deaminase, which acts as a pro-drug activating enzyme, also resulted in a significant reduction in the tumor border in animal models with established melanoma brain metastasis in vivo [209].

17.4.3 Functions of Brain Tumor Stem Cells in Brain Cancers and Potential Therapeutic Targets Accumulating lines of experimental evidence have indicated that most of brain tumors, including medulloblastomas, astrocytomas, oligodendrogliomas, ependymomas and mixed gliomas, may arise of the malignant transformation of adult multipotent neural stem cells (NSCs) and/or more committed neuronal or glial cell lineage precursors localized in central nervous system (CNS) into brain tumor stem cells (BTSCs) [8, 9, 17, 34, 52, 60, 210–218]. In contrast, the neuroblastomas appear rather to derive from the malignant transformation of pluripotent neural crest stem cells during the embryonic development [219]. More specifically, the isolation of CD133/nestin positive-tumor cells from patients’ malignant brain tissues has revealed that these immature cancer progenitor cells can give rise in vitro and in vivo to different neural cell lineages, including neuron and glial cell-like cells but in different proportion in respect to the normal NSCs [8, 17, 52, 211–214, 220–222]. For instance, the secondary or progressive glioblastoma multiformes (GBMs) appear usually to arise from the malignant transformation of NSCs into CD133+ /nestin+ BTSCs possessing aberrant differentiation potential [17, 211–214]. GBM BTSCs can give rise to a heterogeneous population of the cancer cells including astrocytes, oligodendrocytes and/or ependymal cell-like cells in different proportions in vitro and in vivo [17, 211–214]. The results from a recent study have also revealed that certain primary glioblastoma subtypes may derive from a small subpopulation of CD133– tumor cells with stem cell-like properties showing a distinct gene expression pattern relative to CD133+ cancer stem/progenitor cells [223]. Moreover, the tumorigenic CD133+ /nestin+ BTSCs isolated from ependymomas possessing a phenotype resembling to radial glia-like cells, which are the neuronal precursor cells that may give rise to mature ependymal cells lining membrane of brain ventricles, were also able to form tumors when orthotopically transplanted in mice [210]. Additionally, a small SP population with the stem cell-like properties and expressing different markers such as MDR1, BCRP/ABCB2 and/or ABCA3 drug transporters has also been isolated from patient’s primary neuroblastoma tissues and established neuroblastoma and glioma cell lines [8, 224]. For instance, the FACS sorted SP fraction from the rat C6 glioma cell line expressing MDR1 and BCRP1 transporters can differentiate in vitro and in vivo into neurons and glias expressing

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the neuronal (low molecular weight neurofilament NF-L or βIII-tubulin) and glial (glial fibrillary acidic protein, GFAP) markers, respectively, and form the metastatic tumors when injected intraperitonally in nude mice in vivo [224]. In addition, the brain cancer development is usually associated with the accumulation of diverse genomic abnormality in NSCs or their early progenies as well as the changes in their local microenvironment, niche that may contribute to their acquisition of a more malignant behavior. The frequent oncogenic transforming events include the inactivating p53 mutations, sustained activation of distinct tumorigenic cascades such as EGF-EGFR, SHH-PTCH, Wnt/β-catenin, Notch and SDF-1/CXCR4 pathways and enhanced expression of ABC transporters and membrane type-1 matrix metalloproteinase (MT1-MMP) and MMP-9 [8, 9, 34, 52, 60, 213, 214, 223, 225– 231]. These cascades may contribute to the sustained growth and invasion of BTSCs as well as their resistance to chemotherapies and radiation therapies. In clinical settings, the therapeutic management of the patients with brain tumors largely varies with the cancer subtype, its anatomic localization and grade at the time of diagnosis and may include surgery, radiotherapy, chemotherapy and/or stem cell transplant [8, 232, 233]. The targeted therapy with new drug classes that are able to penetrate the blood-brain barrier such as temozolomide or nitrosoureas agents such as carmustine (also called BCNU) and lomustine (CCNU) may also be used for treating the patients with the aggressive and recurrent brain tumor types. Although the advances in the last years in the treatment of pediatric and adult brain tumors have led to increased cure rates, the advanced, metastatic and/or recurrent cancers, including GBMs, remain with a poor patient survival [8, 60, 234]. In regard with this, the BTSCs found in malignant brain tissues and cell lines, which express high levels of ABC multidrug efflux pumps and anti-apoptotic factor (Bcl-2 and survivin), could be more resistance than their differentiated progenies to the clinical treatments, and thereby they could be responsible for tumor re-growth and disease relapse [8, 59, 60, 68, 222, 234, 235]. In support with this, it has been observed that glioma tissues contained a higher fraction of CD133/MDR1/Bcl-2 positive cells relative to normal brain tissues [68]. The CD133+ BTSCs isolated from primary cell lines established from glioblastoma patients, also expressed higher levels of BCRP1/ABCB2, anti-apoptotic factors and CD90, CD44, CXCR4, nestin, Msi1 and MELK relative to CD133– cancer cells [24]. These CD133+ BTSCs were also resistant to diverse chemotherapeutic drugs such as temozolomide, carboplatin, etoposide and paclitaxel [24]. Moreover, the CD133+ BTSC population established from glioblastomas was also enriched after ionizing radiation treatment relative to the CD133– fraction [59]. This radioresistance phenotype of CD133+ BTSC cells may be associated with a preferential activation of the DNA damage checkpoint response concomitant with an increase in DNA repair capacity [59]. Therefore, the targeting of the ABC drug transporters and developmental signaling cascades such as hedgehog, EGFR, Wnt/β-catenin, Notch pathways and/or CDK1 and CDK2 checkpoint kinases by using the specific inhibitors could represent a promising strategy to eradicating BTSCs and their progenies, and thereby improving the current therapies [8, 59, 236, 237]. As a matter of fact, it has been observed that the blockade of the Notch signaling cascade by using γ-secretase inhibitors reduced the

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CD133-positive cell fraction and totally abolished SP population from medulloblastoma cell lines as well as tumor xenograft growth in vivo [236]. These data suggests that this treatment type could be effective for eradicating brain tumor-initiating cells [236]. In the same way, it has been observed that a selective inhibitor of smoothened (SMO) hedgehog signaling element, cyclopamine in combination with the current therapeutic drug, temozolomide induced an additive or synergistic anti-proliferative and apoptotic effects on the CD133+ gliomasphere cells established from human GBM tumor samples in vitro [237] Importantly, a long-term cyclopamine treatment of gliomasphere cells expressing a high expression of the stemness genes also eradicated all these BTSCs in culture, and induced the regression of glioma tumors established from the gliomasphere cells in nude mice in vivo, without systemic toxicity [237]. In the same pathway, it has also been reported that the blockade of HA/CD44 cascade using hyaluronan oligomers (o-HA) treatment of SP cell fraction from C6 rat glioma cell line down-regulated the activation of EGFR and Akt and expression of BCRP transporter [235]. These molecular events resulted in an increase of the cytotoxic effects induced in vitro by methotrexate on these immature cells [235]. o-HA was also effective to suppress the in vivo growth and invasiveness of parental C6 glioblastoma cells or SP cell fraction engrafted into rat spinal cord [235]. Interestingly, the treatment of the mice-bearing orthotopic U87 glioma cell xenografts with an anti-VEGF mAb, bevacizumab noticeably reduced the number of vessel-associated self-renewing CD133+ /nestin+ BTSCs and tumor growth in vivo [229]. Additionally, the transplantation of fetal-derived NSCs engineering to express interleukin-12 or tumor necrosis factor-α (TNF-α) related apoptosis inducing ligand (TRAIL) also resulted in their specific recruitment within intracranial glioma, release of therapeutic gene product concomitant with an inhibition of tumor growth [238–240].

17.4.4 Functions of Tumorigenic Stem/Progenitor Cells in Epithelial Cancers and Potential Therapeutic Targets The epithelial cancers represent the most common solid tumors and include skin, head and neck, thyroid, lung, cervical, renal, liver, esophageal, gastrointestinal, colon, bladder, pancreatic, breast, ovarian and prostatic cancers [8, 30, 60, 241]. It is now accepted that the most epithelial cancers appear to derive from the oncogenic events occurring in adult stem/progenitor cells resident within basal compartment near epithelial basement membrane in different tissues [8, 30, 60, 241]. The development of epithelial cancers generally implicates the accumulation of distinct genetic and/or epigenetic alterations in adult stem/progenitor cells that results in their malignant transformation into tumorigenic cancer stem/progenitor cells with abnormal proliferation and differentiation abilities [8, 29, 30, 53, 60, 241]. The acquisition of a migratory phenotype by tumorigenic cancer stem/progenitor cells during EMT program concomitant with the changes in their local microenvironment, niche may also lead to their invasion and metastasize at distant tissues and organs [8, 29, 30, 33, 60, 241]. In support with the critical functions of tumorigenic and migrating

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cancer stem/progenitor cells in the development of most epithelial cancers and metastases, small subpopulations of immature cancer cells have been isolated from different malignant epithelial cancer specimens and well established epithelial cell lines [4, 8, 12, 14, 19–23, 25, 27, 36, 41–51, 242–246]. These multipotent cancer stem/progenitor cells expressing different stem cell-like markers such as telomerase, ALDH, ABC transporters, CD133, CD44, BMI-1, CXCR4 and/or diverse transcription factors such as Nanog, Oct-4 and Sox-2, were able to give rise to total cancer cell mass in vitro and tumors in animal models in vivo resembling to the patients’ original epithelial cancers. Although a significant improvement in the diagnosis and management of localized epithelial cancers by tumor resection, radiotherapy, anti-hormonal therapy and/or chemotherapy has led to a successful treatment of cancer patients, the invasive and metastatic disease stages are typically associated with a poor prognosis and decreased overall survival of patients [8, 30, 60]. Importantly, some recent works have provided experimental evidence that the tumorigenic and migrating cancer stem/progenitor cells may be more resistant than their differentiated progenies to current therapies, and thereby play a major role in the disease relapse [8, 19, 21, 24, 25, 31, 35, 36, 38–50]. Consequently, the targeting of distinct oncogenic pathways in cancer stem/progenitor cells and their progenies appears to be essential to eradicate the total cancer cell mass including cancer-initiating cells, and thereby develop new effective combination therapies against aggressive and recurrent epithelial cancers. 17.4.4.1 New Therapies Against Epithelial Cancers by Molecular Targeting of Tumorigenic and Migrating Cancer Stem/Progenitor Cells and Their Progenies Recent advances in cancer stem/progenitor cell biology have provided important information on the signaling pathway elements that are often deregulated in tumorigenic and migrating cancer stem/progenitor cells and their progenies as well as in their microenvironment, niche during epithelial cancer progression, and which may contribute to their malignant behavior and treatment resistance [8, 29, 30, 60]. Therefore, the molecular targeting of these altered gene products in tumor-initiating cells and their differentiated progenies may represent a potential therapeutic strategy to improve the current cancer treatments and prevent disease relapse. Molecular Targeting of Tumorigenic Growth Factor Cascades Among the potential therapeutic targets in cancer stem/progenitor cells and their progenies, there are diverse growth factor cascades such hedgehog, EGFEGFR, Wnt/β-catenin, Notch, HA/CD44, interleukin-4 (IL-4)/IL-4Rα, BMI-1, SDF-1/CXCR4, SCF/KIT and/or extracellular matrix (ECM)/integrin (Table 17.2; Fig. 17.2) [8, 11, 12, 29, 30, 40, 45, 60, 225, 247–254]. Numerous investigations have revealed that the blockade of these tumorigeic cascades by using specific inhibitors or antagonists, mAbs or antisense oligonucleotides (As) led to a growth inhibition, apoptotic cell death and/or a reduction of invasiveness or metastatic spread of tumor-initiating cells and their progenies in vitro or in animal models in

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vivo (Table 17.2; Fig. 17.2) [8, 11, 12, 29, 30, 39, 60, 225, 247, 249–253, 255–258]. For instance, it has been reported that the inhibition of smoothened (SMO) hedgehog signaling element by cyclopamine eliminated the cancer stem/progenitor cells and improved the cytotoxic and anti-metastatic effects induced by the current therapeutic drug, gemcitabine on pancreatic cancer cell lines in vitro and/or in vivo [251]. The blockade of the HA-CD44 tumorigenic cascade with specific anti-CD44 antibody, soluble CD44 form, CD44 small interference RNA (siRNA), HA antagonists such HA oligosaccharides or hyaluronan synthase (HAS) siRNA also inhibited the survival, growth, mobility, invasion and/or metastases of diverse cancer cells in vitro and/or in vivo and enhanced their chemosensitivity [255–257]. Similarly, the inhibition of the SDF-1/CXCR4 axis, by using an anti-SDF-1 or anti-CXCR4 mAb or selective CXCR4 antagonists, such as 14-mer peptide (TN14003) and AMD 3100, was also effective to counteract the migration of cancer epithelial cells including cancer stem/progenitor cells to distant sites and metastases (Fig. 17.2) [8, 11, 12, 29, 30, 60, 247, 254, 259]. Molecular Targeting of Oncogenic Signal Transduction Elements and ABC Multidrug Transporters Other potential molecular targets also include the oncogenic signaling effectors such as telomerase, Cripto-1, tenacin C, NF-κB, PI3 K/Akt/mTOR, snail, slug and/or twist as well as ABC multidrug transporters, anti-apoptotic factors (Bcl-2, Bcl-xL and survivin) and deregulated apoptotic cascade elements (ceramide and caspases) [8, 11, 12, 29, 30, 35, 60, 72, 247, 260–262]. The changes in the expression and/or activity of these signal transduction elements during the cancer progression, and more particularly during EMT process, can stimulate the survival and invasion of cancer stem/progenitor cells and their progenies and/or to promote their chemoresistance and radioresistance (Table 17.2; Fig. 17.2). In view of the fact that the human cancer epithelial cells including cancer stem/progenitor cells typically express high levels of telomerase reverse transcriptase (hTERT) and telomerase activity, many new anti-telomerase strategies and telomerase-based gene therapies and immunotherapies have been designed to counteract the epithelial cancer progression [38, 247, 263–268]. The anti-telomerase strategies include the use of specific inhibitors or As strategies directed against hTERT or TR, which is a RNA primer sequence that acts as a template for hTERT and gene therapy vectors expressing the pro-apoptotic gene products such as Bax, constitutive active caspase-6 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in cancer cells (Table 17.2; Fig. 17.2) [38, 247, 263–268]. For instance, it has been shown that the tumor-specific replication competent oncolytic adenovirus constructs engineered with the specific promoters such as hTERT, cyclooxygenase-2 (COX-2) or MDR effectively eradicated CD44+ CD24–/low breast cancer stem/progenitor cells or SP cell fraction from radioresistant esophageal cancer cells in vitro and in vivo without major toxicity [38, 268]. The clinical trials consisting of human TERT-directed immunotherapies have also given promising results for the treatment of patients with lung, breast, prostatic and pancreatic cancers without major secondary effects [269–272].

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Importantly, some recent investigations have also indicated the potential benefit to inhibit the expression and/or activity of ABC transporters for overcoming MDR phenotype and radioresistance of cancer epithelial cells including cancer stem/progenitor cells [8, 31, 49, 96, 247, 258, 260]. In regard with this, it has been observed that the SP cells from malignant epithelial tissues or cancer cell lines, which display the stem cell-like properties and express high levels of distinct ABC drug efflux pumps and anti-apoptotic factors, were more resistant than the non-SP cells to the chemotherapeutic treatments or irradiation therapy [8, 40–50]. Of particular therapeutic interest, the treatment of SP cell fraction from human oral squamous cell carcinoma cell line H357 with a broad spectrum ABC transporter inhibitor, verapamil has been observed to block the mitoxantrone efflux-mediated via ABCG2 and MPR-1/ABCC1 pumps and restore the growth inhibitory effect induced by mitoxantrone on this stem cell-like population [49]. The inhibition of the Akt signaling element in SP cell fraction sorted from MHCC-97L cells also induced an intracellular translocation of ABCG2 transporter, attenuated the doxorubicin efflux and increased doxorubicin-induced cytotoxicity [96]. In the same way, the downregulation of MDR1 and ABCG2 expression by siRNA or inhibition of hedgehog cascade by cyclopamine partially reversed chemoresistance of highly tumorigenic PC3 prostatic cancer cells suggesting that the targeting of hedgehog pathway may represent a potential therapeutic approach to overcome MDR and increase chemotherapeutic response [258]. Additionally, the inhibition of NF-κB signaling pathway using parthenolide (PTL), pyrrolidinedithiocarbamate (PDTC) and its analog diethyldithiocarbamate (DETC) also preferentially inhibit the proliferation and colony formation of MCF7 mammosphere cell cultures and verapamil-sensitive SP cell population enriched in breast cancer stem-like cells [260]. On the other hand, the targeting of deregulated signaling elements involved in ceramide metabolism, including sphingosine kinase-1, glucosylceramide synthase (GCS) that catalyses the ceramide glycosylation and ceramidase also may constitute another therapeutic strategy to reverse MDR phenotype and promote ceramideinduced apoptotic death in certain cancer epithelial cells (Table 17.2) [31, 262, 273]. Moreover, the induction of the differentiation of cancer stem/progenitor cells by using agents such as retinoic acid and its synthetic analogues, interferons (IFNs) or histone deacetylase inhibitors, which can induce the differentiation, growth arrest and/or apoptotic death of cancer cells, also represent a promising therapeutic adjuvant strategy [274–278]. For instance, it has been reported that the IFN-α treatment caused a dramatic reduction in verapamil-sensitive SP cell fraction from diverse ovarian cancer cell lines [278]. Other Molecular Targeting Strategies Since the epithelial cancer development also depends of the change in activated stroma and tumor neo-angiogenic process, the targeting of the local microenvironment of cancer stem/progenitor cells including the host cells such as myofibroblasts and immune cells as well as the use of anti-angiogenic agents also may constitute an adjuvant treatment for counteracting the cancer progression to metastatic, recurrent

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and lethal disease states [8, 11, 12, 29, 30, 60, 247]. Among the anti-angiogenic strategies, the use of inhibitors of VEGF-VEGFR, COX-2, MMPs and uPA system may represent potential adjuvant treatments to counteract the angiogenic process, tumor formation and metastases at distant sites. Other anti-cancer strategies also include the specific delivering of therapeutic anti-carcinogenic agents including cytotoxic drugs or anti-angiogenic substances into tumors [5, 11, 60, 279–282]. The specific delivery of molecules in tumors may be accomplished by using the conjugation/fusion of drugs to tumor-specific antibodies, encapsulation of chemotherapeutic drugs in liposomes, or other carriers such as nanoparticules or genetically-engineered stem/progenitor cells as drug delivery vehicles [5, 279, 280, 282, 283]. For instance, it has been reported that a combination therapy consisting of the use of vinorelbine stealthy liposomes plus parthenolide stealthy liposomes induced the cytotoxic effects on both isolated SP and non-SP fractions from MCF7 breast cancer cell line and suppressed growth of MCF-7 cancer cell-derived xenografts in nude mice in vivo [282]. Additionally, the differentiated HSC-derived progenitors, such as dendritic cells, which are among the most efficient cells of the immune system in presenting an antigen to helper/cytotoxic T lymphocytes, also might be used as an adjuvant treatment in cancer immunotherapy to eliminate the neoplastic cells including cancer stem/progenitor cells that express immunogenic antigens at their surface [207, 284].

17.5 Conclusions and Perspectives Recent progresses in the cancer stem/progenitor cell research have revealed that these malignant cells endowed with stem cell-like properties can contribute of a substantial manner to leukemia and tumor development, metastases at distant sites, treatment resistance and disease relapse. The intrinsic and/or acquired MDR phenotype of leukemic and tumorigenic cancer stem/progenitor cells may be due, at least in part, to their high expression levels of distinct ABC transporters and antiapoptotic factors as well as the sustained activation of some developmental growth factor signaling cascades in these immature cancer cells. Therefore, the molecular targeting of the signaling elements involved in MDR may represent a promising therapeutic strategy to reverse treatment resistance and improve the current cancer treatments. Particularly, the high expression levels of different ABC transporters in cancer-initiating cells and their progenies that can actively participate in cooperation to drug elimination, MDR phenotype and treatment resistance support their molecular targeting. The use of different ABC transporter inhibitors or broad-specificity transporters may represent a more appropriate strategy than the targeting of a single ABC efflux pump. The combination of current chemotherapeutic drugs with low doses of different ABC transporter inhibitors could notably constitute the more effective and less toxic therapeutic regimens to treat the cancer patients. Since the normal primitive tissue-resident adult stem/progenitor cells also express high levels of the ABC drug efflux pumps, it will be important to develop the therapeutic strategies to selectively down-regulate the expression and/or activity of specific drug

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efflux pumps expressed in the cancer-initiating cells before their potential use in humans in safe conditions. The intervariability of patient responses to these treatment types due to cancer heterogeneity and differences between the expression and/or activity of potential molecular targets in cancer-initiating cells should also be considered. The establishment of the deregulated gene expression profiles of cancer patients, including ABC transporters and other altered products involved in MDR of cancer stem/progenitor cells, prior to therapy initiation could help identify the potential targets and predict the chemotherapeutic response. These gene expression signature patterns could help to establish the most appropriate therapeutic options for individualized treatment of cancer patients in the clinics. Hence, the development of combination therapies targeting the total cancer cell mass including leukemic or tumorigenic cancer stem/progenitor cells and their progenies should lead to more effective treatments for the patients diagnosed with locally advanced, high risk or refractory/relapsed cancers. Acknowledgments The authors on this work are supported by grants from the National Institutes of Health (CA78590, CA111294, CA133774 and CA131944). We thank Ms. Kristi L. Berger for editing the manuscript.

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Index

A Acute lymphoblastic leukemia (ALL), 282, 285–286, 368, 370, 371 Acute myeloid leukemia (AML), 18, 282, 283, 358, 369, 371, 377 Adult stem cells, 1, 2, 3, 4, 5, 15, 31, 82, 113, 151, 189, 191, 196, 197, 200, 201, 202, 203–204, 205, 206–207, 209, 210, 212–213, 252, 262, 272, 289, 329, 331–333, 344, 370 AMD3100, 20, 34, 311, 399 Animal models in periodontal research, 258–259 Athymic immunodeficient rat, 274 ATP-binding cassette transporters, 373, 391 Axon degeneration, 157, 158, 159 B Barker hypothesis, 210–212 Bone marrow (BM), 1, 2, 3, 12, 13, 14, 16, 17, 18, 19, 20, 28–29, 30, 31, 32, 35, 36, 38, 39, 40, 41, 44, 45, 46, 47, 58, 60, 61–63, 66, 68, 70, 72, 80, 81, 82, 151, 162, 165, 171, 174, 225, 227, 228, 237, 240, 241, 243, 244, 268, 271, 282, 289, 300, 332, 336, 337, 338, 339, 395 Brain, 60, 109, 115, 126, 127, 131, 146, 147, 148, 149, 151, 160, 165, 166, 168, 169, 170, 171, 173, 174, 175, 200, 204, 225, 253, 298, 299, 303–305, 315, 332, 358, 359, 363, 365, 368, 369, 374, 376, 387, 388, 390, 394, 400, 401–403 Breast, 83, 84, 86, 204, 205, 209, 211, 228, 305–308, 310, 335 Breast cancer cells, 84, 85, 93, 301, 305, 306, 317, 329, 334, 338, 340, 346, 347, 349, 363, 365, 371, 374, 407 Breast stem cells, 203, 204, 205, 209, 211, 346, 347, 349

C Cancer associated fibroblasts, 237, 239 stem/progenitor cells, 1, 4, 35, 60, 109, 183, 197, 204–208, 223–230, 235–246, 251–274, 287, 295–321, 327–350, 357–379, 401 therapies, 4, 5, 85, 92, 93, 207, 208, 230, 330, 331, 341, 343, 345, 359–360, 364, 365, 366, 368, 371, 374, 376, 377, 387, 393, 395 CD34, 3, 16, 80, 123, 287, 312 Cell cycle arrest, 372, 374 Cell fusion, 60, 113, 116–117, 123, 150, 331, 335–336, 338–339, 344, 345–349, 350 Cell migration, 57–72, 86, 87, 159, 244, 258, 310, 344 Cell of origin, 236, 282, 283, 313–319, 320, 334, 359, 361 Cell therapy, 2, 3, 31, 79–93, 145–152 Challenges, 14, 127, 133, 157, 189, 259, 273, 286, 287, 320–321, 350 Chemokines, 19, 20, 29, 34, 58, 60, 62, 63, 64, 68, 70, 72, 83, 84, 85–86, 89, 93, 173, 238, 239, 244, 256, 271, 328, 336, 337, 338, 339, 350, 363, 387, 388, 390 Chemoresistance, 301, 342, 361, 364, 373–375, 397, 405, 406 Chronic inflammation, 83, 173, 197, 227, 228, 236, 238, 239, 240, 242–243, 245, 271 Chronic myeloid leukemia (CML), 45, 282, 285, 330, 343, 370 Colon, 17, 83, 209, 213, 238, 241, 243, 303, 308, 313, 328, 330, 336, 358, 359, 371, 372, 387, 403 Combination therapies, 387, 392, 398, 404, 407, 408

T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5,

423

424 Culture conditions, 3, 40, 41, 63–65, 66–68, 103, 107, 122, 207, 297, 299, 319, 320, 343 CXCR4, 29, 34, 58–61, 63, 64, 66, 67, 71, 86, 244, 310, 311, 336, 337, 340, 363, 387, 388, 390, 393, 399, 402, 404, 405 Cytokines, 3, 19, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 44, 46, 47, 60, 62, 63, 64, 66, 67, 68, 69, 72, 83, 84, 86, 87, 89, 92, 105, 107, 159, 172, 227, 228, 229, 238, 239, 241, 242, 271, 328, 338, 350, 363, 387, 388 D Demyelination, 156, 157, 158, 159, 160, 165, 167, 168, 169, 170, 171, 172, 173 Differentiation therapies, 364, 365 Disease modeling, 126 Drug development, 370 Drug resistance, 4, 208, 210, 282, 286–287, 320, 346, 347, 349, 350, 360, 373, 387, 390, 391 Drug targets, 373 E Embryonic stem cells, 1, 4, 101–134, 146, 149–150, 161–163, 164, 190, 191, 201, 205, 210, 224, 288, 316, 319, 361 Epigenetics, 4, 105, 108, 109, 110–111, 114, 116, 117, 120, 122, 127, 129, 132, 150, 188, 197, 198, 200, 203, 211, 212, 227, 228, 242, 245, 289, 305, 320, 321, 338, 339, 344, 345, 349, 350, 364, 365, 370, 371, 386, 395, 403 Evolution and cancer, 187–190 Ex vivo expansion, 3, 27–48, 72 F Fetal ventral mesencephalon, 147–148 G Gap junctional intercellular communication, 193–194, 195, 201, 202, 206, 208, 209, 329 G-CSF, 16, 17, 18, 19, 20, 31, 32, 34, 37, 40, 47, 62, 64 Gene signature, 339–341 Gene therapy, 41, 44, 46, 90, 102, 114, 378, 400, 405 Graft-versus-host-disease (GVHD), 46 Graftinduced dyskinesias (GIDs), 147, 148, 152

Index H Head and neck, 303, 308, 358, 359, 365, 403 Hedgehog pathway, 290, 366, 368, 406 Hematopoietic stem cell, 12, 13, 15, 16, 19, 36, 38, 88, 91, 115, 165–167, 237, 244, 283, 284, 285, 288, 290, 302, 303, 316, 329, 332, 370, 371, 373, 395 Hematopoietic stem/Progenitor cells, 3, 28, 57–72, 105, 329 Homing, 45, 58, 60, 61–63, 66, 68, 82–83, 85–86, 90, 91, 93, 225, 239, 244, 305, 306, 363 Human periodontium-derived stem cells (pdSCs), 273 I Immortalizing viruses, 203–204 Immunosuppression, 3, 4, 88, 89–90, 150, 160, 228 Induced pluripotent stem cell (iPS cells), 1, 2, 3, 4, 5, 82, 119–120, 121, 122, 123, 124–129, 132, 133, 146, 147, 150–151, 152, 163, 165, 201, 288 Inflammation, 31, 62, 72, 83, 85, 157, 158, 160, 165, 167, 170, 171, 172, 173, 174, 175, 197, 227, 228, 236–238, 239, 240, 241, 242–244, 271, 273 Interferon-γ, 271 K Kidney, 118, 202, 203, 205, 303, 308–309, 371, 400 L Leukemias, 1, 19, 282, 286, 288, 289, 358, 368, 369, 371, 386, 387, 390, 394, 395–400 Leukemia stem cell (LSC), 281–291, 332, 333 Liver, 1, 2, 58, 59, 60, 62, 115, 120, 126, 133, 190, 191, 200, 201, 203, 205, 208, 209, 213, 238, 243, 244, 303, 309, 311, 316, 329, 336, 337, 359, 363, 371, 376, 387, 403 Lymphocytes, 12, 14, 20, 37, 38, 39, 41, 45, 46, 47, 58, 59, 60, 62, 68, 114, 116, 118, 120, 227, 229, 238, 239, 259, 271, 305, 338, 339, 407 M Macrophages, 16, 31, 32, 63, 70, 86, 158, 173, 224, 229, 230, 238, 239, 241, 254, 259, 285, 333, 338, 339, 346, 349, 350, 396 Mesenchymal, 4, 80, 87, 227, 243, 269, 299, 317

Index Mesenchymal stem cells, 79–93, 165–167, 171, 228, 241, 242, 243, 253, 254, 265, 346 Metastatic cancer stem cells, 362 Minimally-invasive periodontal surgical therapy, 265, 266, 269 Molecular targeting, 387, 389, 393, 398, 400, 404–407 Multidrug resistance, 286, 342, 343, 345, 388, 389–392 Multiple sclerosis, 20, 62, 146, 155–175 N Neural precursor cell, 125, 126, 146, 148–149, 151, 163, 171, 174 Niche, 21, 28, 41, 44, 70, 83, 165, 192, 205, 206, 210, 223–230, 240, 241, 243, 244, 245, 246, 263, 320, 342, 343, 361, 363, 364, 369, 375–377, 378, 403, 404 Notch pathway, 366 Nuclear transfer, 112, 113, 114–115, 132, 150 O Oncogenic resistance, 331, 343, 345, 346, 349, 350 Oncogenic signaling elements, 400 Oncolytic virus, 92–93 Osteogenic differentiation, 265 Ovary, 299, 303, 309, 387 P Pancreas, 59, 81, 82, 133, 203, 205, 289, 303, 310, 358, 359, 368, 387 Parkinson’s disease, 127, 145–152, 163 Periodontal tissue regeneration, 256–258, 265, 266, 273, 274 Periodontium, 251–274 Peripheral blood stem cells (PBSC), 3, 16, 44 Pluripotency, 2, 103, 104–113, 114, 118, 119, 121, 122, 123, 125, 126, 131, 288, 306, 316 Polycomb pathway, 374 Precursor cells, 31, 43, 116, 118, 159, 162, 163, 165, 166, 167, 169, 170, 171, 172, 173, 174, 258, 318, 320, 371, 401 Progenitor cell, 11–21, 28, 29, 32, 34, 35, 36, 38, 40, 41, 57–72, 81, 82, 160, 169, 191, 192, 193, 195, 200, 202, 210, 211, 228, 236, 253, 254, 256, 257, 258, 265, 272, 283, 284, 285, 286, 288, 289, 297, 306, 307, 308, 312, 313, 314, 315, 316, 317, 328, 331, 333–335, 361, 385–408 Prostate, 212, 228, 242, 243, 297, 303, 311, 313, 314, 315, 318, 319, 330, 332, 359,

425 363, 368, 369, 371, 372, 376, 377, 387, 388, 394, 400 PTEN pathway, 372, 373 R Recurrence cancer stem cells, 5, 331, 343, 344 Regenerative medicine, 1, 30, 44, 72, 82, 90, 102, 113, 128 Remyelination, 158, 159, 160, 161, 167–169, 170, 171–172, 173, 174, 175 Reprogramming, 82, 91, 102, 109, 112, 113, 114, 116–129, 132, 133, 150, 151, 163, 203, 204, 209, 213, 288–289, 339, 346 S SCID leukemia-initiating cell (SL-IC), 283 SCIDrepopulating cell (SRC), 46, 64, 283, 284 SDF-1α, 3, 58–61, 62, 63, 64, 65, 66, 67, 68–72, 244 Selfrenewal, 108, 224, 287, 290, 313, 333, 335, 360, 366, 371 Side population, 207–208, 300–302, 341, 374, 392 Side-population cells, 207–208 Signal transduction, 60, 61, 65, 67, 68, 69, 71, 72, 332, 375, 399, 405 Skin, 4, 81, 82, 83, 120, 129, 193, 200, 201, 209, 213, 228, 238, 261, 266, 271, 288, 303, 312, 359, 368, 387, 388, 400, 403 Solid tumors, 1, 5, 17, 208, 272, 295–321, 330, 333, 341, 343, 365, 368, 369, 372, 376, 386, 390, 394, 395, 403 Sphere assay, 297–300 Stem cell markers, 260, 289, 302–313, 318, 332, 345 mobilization, 15–17, 18, 19 niche, 205, 206, 223–230, 244, 245, 342, 361, 369, 377 transplantation, 12, 13, 15, 17, 20, 29, 265, 273, 399 Stroma, 3, 19, 28, 29, 32, 33, 34, 37, 44, 58–59, 61, 62, 80, 85, 86, 165, 206, 224, 225, 226, 227, 230, 237, 241, 243, 244, 246, 255, 270, 310, 312, 321, 369, 387, 388, 390, 406 T Targeted therapies, 364, 365, 373, 402 Termination of cell migration, 58, 68–72 TGF-β, 107, 108, 225, 226, 239, 241, 243, 244, 254, 255, 318, 362 Tissue repair, 82–83, 86, 93, 102, 114, 117, 125, 263, 266 Transdifferentiation, 1, 2, 58, 133

426 Transplantation, 2, 3, 11–21, 29, 30, 33, 34, 36, 37, 38, 39, 44–48, 58, 61, 62, 63, 66, 68, 103, 104, 112, 115, 126, 127, 128, 130, 131, 147, 148, 149, 150, 151, 155–175, 265, 273, 283, 297, 299, 303, 307, 308, 309, 330, 332, 336, 359, 363, 397, 399–400, 403 Tumor dormancy, 229 Tumor fibroblasts, 224, 237

Index Tumor homing, 83, 86, 90, 92, 93 Tumorogenicity neural stemness markers, 256 U Umbilical cord blood (UCB), 15, 20, 28, 29–30, 31, 39, 63, 165, 211, 252 W Wnt pathway, 84, 227, 290, 319, 370