Frontiers in Brain Repair (Advances in Experimental Medicine and Biology, Volume 671)

Frontiers in Brain Repair

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Editorial Board: NATHAN BACK, State University of New York at Buffalo IRUN R. COHEN, The Weizmann Institute of Science ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research JOHN D. LAMBRIS, University of Pennsylvania RODOLFO PAOLETTI, University of Milan Recent Volumes in this Series Volume 663 STRUCTURE AND FUNCTION OF THE NEURAL CELL ADHESION MOLECULE NCAM Edited by Vladimir Berezin Volume 664 RETINAL DEGENERATIVE DISEASES                Volume 665 FORKHEAD TRANSCRIPTION FACTORS Edited by Kenneth Maiese Volume 666 PATHOGEN DERIVED IMMUNOMODULATORY MOLECULES Edited by Padraic G. Fallon Volume 667 LIPID A IN CANCER THERAPY Edited by Jean François Jeannin Volume 668 SUPERMEN1 Edited by Katalin Balogh and Attila Patocs Volume 669 NEW FRONTIERS IN RESPIRATORY CONTROL Edited by Ikuo Homma and Hiroshi Onimaru Volume 670 THERAPEUTIC APPLICATIONS OF CELL MICROENCAPSULATION Edited by José Luis Pedraz and Gorka Orive Volume 671 FRONTIERS IN BRAIN REPAIR Edited by Rahul Jandial

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Frontiers in Brain Repair Edited by Rahul Jandial, MD, PhD Division of Neurosurgery City of Hope Comprehensive Cancer Center & Beckman Research Institute Duarte, California, USA

Springer Science+Business Media, LLC Landes Bioscience

Springer Science+Business Media, LLC Landes Bioscience Copyright ©2010 Landes Bioscience and Springer Science+Business Media, LLC All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechani $ $% '$'  $ %   # &  %   "  &  ' &   % # &  '    *$' #  &  '' '$$ #  ' ' # %    *$   $&'  &< # *$"    ! $ #   = Printed in the USA. >' % >$$?@   QXX >' % >  Z \ = Z \ = ^__^X `> 'jqq ' % $& Please address all inquiries to the publishers: Landes Bioscience, 1002 West Avenue, Austin, Texas 78701, USA Phone: 512/ 637 6050; FAX: 512/ 637 6079 'jqq $$ $& The chapters in this book are available in the Madame Curie Bioscience Database. 'jqq $$ $&q$  Frontiers in Brain Repair, edited by Rahul Jandial. Landes Bioscience / Springer Science+Business Media, LLC dual imprint / Springer series: Advances in Experimental Medicine and Biology. ISBN: 978 1 4419 5818 1 |       ' "   % $  %   '$$  % # }'&  "$   #    =    $$   $  $&&  ' $$   & # '$  &=     *'   &'  '$  &  $    = ~ " #  %%  $ }'& "'& $%  governmental regulations and the rapid accumulation of information relating to the biomedical sciences,     %  $ # "  "  # & ' "  

Library of Congress Cataloging-in-Publication Data Frontiers in brain repair / edited by Rahul Jandial. p. ; cm. (Advances in experimental medicine and biology ; v. 671) Includes bibliographical references and index. ISBN 978 1 4419 5818 1 1. Brain Regeneration. 2. Stem cells Transplantation. I. Jandial, Rahul. II. Series: Advances in experimental medicine and biology ; v. 671. [DNLM: 1. Nervous System Diseases therapy. 2. Gene Therapy methods. 3. Neurons cytology. 4. Stem Cell Transplantation methods. 5. Stem Cells cytology. W1 AD559 v.671 2009 / WL 140 F9345 2009] RD594.12.F76 2009 616’.02774 dc22 2009048362

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PREFACE In the rapidly-evolving landscape of neurosciences, it is not an easy task to select a limited array of topics to present in a text such as this. The purpose of this volume is to provide a representative survey of the current science of brain repair for those seeking to establish a #   #   '  '  =%   & " '   $ $ ~  '  ## %   remains elusive to our collective investigations, defining the “frontiers” of brain repair for those that are currently immersed in the exciting intersection of biological advances and neuroscientific discoveries. In Chapter 1 the fundamentals of imaging transplanted cells is $  &'  & &       # clinical trials. Then, detailed methods on the culture of neural stem $  "   # # '' $%  '$ % Chapter 3 presents the broad scope of animal models that serve as the  # "'&  ' ‚$$ "%  & of recent genetically engineered mouse models that represent the best models for studying disease development and treatment. Chapter 4 provides background on the delivery techniques to animals and patients that are available, providing vital information on the subtleties of technique necessary for optimal cellular grafting. Chapters 5 and 6 $   ""% # &    % #   &  and the indelible role of stromal and microenvironmental influences on oncogenesis and tumor progression. Subsequently, the utility of neural stem cells as cellular vehicles to deliver chemotherapeutics to broad  '%  " ~ ' ƒ  $' #  %   tumors is expanded beyond stem cells, to present the best biological interventions to improve upon current treatment options for brain vii

&%$ „   $'  '   $&' " "  stem cell and gene therapy options for treating cerebrovascular and neurovascular pathology. In amassing this collection, my intention has been to provide        $  &$ &%% & cell biology, cell therapy, animal models, central nervous system malignancies, stroke, and neurodegeneration. My hope is that Frontiers of Brain Repair    $  # & $  '   ‚  "% #  $$    Rahul Jandial, MD, PhD Division of Neurosurgery City of Hope Comprehensive Cancer Center and Beckman Research Institute Duarte, California, USA

ABOUT THE EDITOR...

RAHUL JANDIAL, MD, PhD, is an Assistant Professor in the Division of Neurosurgery, Department of Surgery at City of Hope Comprehensive Cancer Center and the Beckman Research Institute in Los Angeles, California. His clinical practice #$    %$  &$ $  # '     ' &  ~ the laboratory his team investigates the intersection of stem cell biology and neuro $%  '$#$ # %  $ # & $   &      treatment resistant cells can be selectively targeted to help patients live longer. A prolific author, Jandial has authored/edited seven books and over 35 papers. He lives   # …      €   =

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PARTICIPANTS Pragathi Achanta Department of Neurosurgery Johns Hopkins University School of Medicine Baltimore, Maryland USA Lissa Baird Division of Neurosurgery UCSD Medical Center San Diego, California USA Justin Chan UCSD School of Medicine San Diego, California USA Joseph Ciacci Division of Neurosurgery UCSD Medical Center San Diego, California USA Vincent J. Duenas Del E. Webb Neuroscience, Aging and Stem Cell Research Center Burnham Institute for Regenerative Medicine La Jolla, California USA

Tomas Garzon Muvdi Department of Neurosurgery Johns Hopkins University School of Medicine Baltimore, Maryland USA Allen L. Ho Harvard Medical School Boston, Massachusetts USA Samuel A. Hughes Department of Neurological Surgery Oregon Health and Sciences University Portland, Oregon USA Rahul Jandial Division of Neurosurgery City of Hope Comprehensive Cancer Center & Beckman Research Institute Duarte, California, USA Jennifer Katz Del E. Webb Neuroscience, Aging and Stem Cell Research Center Burnham Institute for Regenerative Medicine La Jolla, California USA

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Participants

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Bryan Keenan Del E. Webb Neuroscience, Aging and Stem Cell Research Center Burnham Institute for Regenerative Medicine La Jolla, California USA Sassan Keshavarzi Division of Neurosurgery UCSD Medical Center San Diego, California USA Michael L. Levy Pediatric Neurosurgery Rady Children’s Hospital San Diego, California USA Rohit Mahajan College of Medicine University of Toledo Toledo, Ohio USA Jayant P. Menon Division of Neurosurgery UCSD Medical Center San Diego, California and UCSD Jacobs School of Engineering La Jolla, California USA @ Z& Division of Neurosurgery UCSD Medical Center San Diego, California USA Alfredo Quiñones Hinojosa Neuroscience and Cellular and Molecular Medicine Johns Hopkins University Department of Neurosurgery Baltimore, Maryland USA

Amol Shah UCSD School of Medicine San Diego, California USA Evan Y. Snyder Del E. Webb Neuroscience, Aging and Stem Cell Research Center Burnham Institute for Regenerative Medicine La Jolla, California USA Alexey Terskikh Del E. Webb Neuroscience, Aging and Stem Cell Research Center Burnham Institute for Regenerative Medicine La Jolla, California USA Kimberly D. Tran UCSD School of Medicine San Diego, California USA Klaudia Urbaniak Hunter University of Michigan Department of Radiation Oncology Ann Arbor, Michigan USA … |  Division of Neurosurgery UCSD Medical Center San Diego, California USA Chester Yarbrough Department of Neurological Surgery @   ' Washington University School of Medicine St Louis, Missouri USA

CONTENTS 1. IN VIVO IMAGING OF CELLULAR TRANSPLANTS ............................. 1 Justin Chan, Jayant P. Menon, Rohit Mahajan and Rahul Jandial Abstract................................................................................................................................. 1 Introduction .......................................................................................................................... 1 Florescent Imaging............................................................................................................... 1 Quantum Dots ...................................................................................................................... 3 PET and SPECT................................................................................................................... 5 MRI ....................................................................................................................................... 5 In Vivo Imaging and Tumors .............................................................................................. 6 In Vivo Imaging and Neurological Disease ........................................................................ 8 Conclusion .......................................................................................................................... 10

2. CULTURE AND MANIPULATION OF NEURAL STEM CELLS .......... 13 Jennifer Katz, Bryan Keenan and Evan Y. Snyder Abstract............................................................................................................................... 13 Introduction ........................................................................................................................ 13 Neural Stem Cells............................................................................................................... 14           ..................................................................... 15 Maintaining Neural Stem Cells In Vitro .......................................................................... 16 Materials and Methods ...................................................................................................... 17 Conclusion .......................................................................................................................... 21

3. ANIMAL MODELS OF NEUROLOGICAL DISEASE ............................. 23 Amol Shah, Tomas Garzon Muvdi, Rohit Mahajan, Vincent J. Duenas and Alfredo Quiñones Hinojosa Abstract............................................................................................................................... 23 Introduction ........................................................................................................................ 23 Parkinson’s Disease............................................................................................................ 23 Cerebral Ischemia .............................................................................................................. 29

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Huntington’s Disease ......................................................................................................... 32 Alzheimer’s Disease ........................................................................................................... 33 Conclusion .......................................................................................................................... 35

4. STEM CELL TRANSPLANTATION METHODS...................................... 41 Kimberly D. Tran, Allen L. Ho and Rahul Jandial Abstract............................................................................................................................... 41 Introduction ........................................................................................................................ 41 Factors................................................................................................................................. 43 Choosing the Ideal Cell Source ......................................................................................... 43 Adult Neural Stem Cells .................................................................................................... 43 Embryonic Stem Cells ....................................................................................................... 44 Method of NSC Isolation ................................................................................................... 45 Preparing NSCs before Transplant .................................................................................. 45 Choosing the Experimental Animal ................................................................................. 46 Choosing the Surgical Procedure ..................................................................................... 47 Potential Routes of NSC Administration ......................................................................... 47 Neonatal .............................................................................................................................. 49 Midgestational In Utero..................................................................................................... 49 Adult .................................................................................................................................... 49 Considerations during Surgery ........................................................................................ 51 Postoperative Care ............................................................................................................. 52 Optimization ....................................................................................................................... 52 Anticipated Results and Methods of Detection ............................................................... 53 Conclusion .......................................................................................................................... 54

5. STEM CELL ORIGIN OF BRAIN TUMORS............................................. 58 … |  @ Z&  $  " Abstract............................................................................................................................... 58 Introduction ........................................................................................................................ 58 Reappraising the Prevailing Theory of Tumor Genesis.................................................. 59 Evidence for NSC as the Cell of Origin............................................................................ 60 Conclusion .......................................................................................................................... 64

6. THE TUMOR MICROENVIRONMENT .................................................... 67 Lissa Baird and Alexey Terskikh Abstract............................................................................................................................... 67 Introduction ........................................................................................................................ 67 Prevailing Theory of Tumor Initiation ............................................................................. 68 Cancer Stem Cells Discovery and Evidence of BTSC ................................................. 68 Caveats to BTSC ................................................................................................................ 70 The Tumor Microenvironment ......................................................................................... 71 Conclusion .......................................................................................................................... 72

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7. EXPLOITATION OF GENETICALLY MODIFIED NEURAL STEM CELLS FOR NEUROLOGICAL DISEASE ........................... 74 Allen L. Ho, Sassan Keshavarzi and Michael L. Levy Abstract............................................................................................................................... 74 Exploiting NSCs for Therapeutic Transplantation ......................................................... 74            .................................................... 76 In Vivo Imgaging of Transplanted NSCs ......................................................................... 86 Conclusion .......................................................................................................................... 89

8. BIOLOGICAL HORIZONS FOR TARGETING BRAIN MALIGNANCY ...................................................................................... 93 Samuel A. Hughes, Pragathi Achanta, Allen L. Ho, Vincent J. Duenas and Alfredo Quiñones Hinojosa Abstract............................................................................................................................... 93 Introduction ........................................................................................................................ 93 Neural Stem Cells............................................................................................................... 94 Exogenous and Endogenous NSCs Respond to Gliomas ................................................ 95 Mechanisms for NSC Homing to Gliomas ....................................................................... 95 Exploiting NSCs as Vehicles for Delivering Toxic Payloads........................................... 96 Conclusion .......................................................................................................................... 98

9. STEM CELLS IN THE TREATMENT OF STROKE .............................. 105 Klaudia Urbaniak Hunter, Chester Yarbrough and Joseph Ciacci Abstract............................................................................................................................. 105 Introduction ...................................................................................................................... 105 Stem Cell Therapy ........................................................................................................... 106 Stem Cell Biology In Vitro............................................................................................ 106 Stem Cell Biology and Animal Models ........................................................................... 108 Cellular Reconstitution of Stroke Lesions ..................................................................... 110 Stroke Treatment Via Enhanced Trophic Factor Delivery............................................111 The Potential of Cord Blood ............................................................................................111 Conclusion ........................................................................................................................ 113

10. GENE- AND CELL-BASED APPROACHES FOR NEURODEGENERATIVE DISEASE .......................................117 Klaudia Urbaniak Hunter, Chester Yarbrough and Joseph Ciacci Abstract............................................................................................................................. 117 Introduction ...................................................................................................................... 117 Cellular and Molecular Pathophysiology of Alzheimer’s Disease ............................... 118 Cellular and Molecular Pathophysiology of Parkinson’s Disease ............................... 119

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Cellular and Molecular Pathophysiology of Huntington’s Disease ............................. 121 Stem Cell Therapy for Neurodegenerative Diseases ..................................................... 121 Immunotherapy and Alzheimer’s Disease ..................................................................... 122 Gene-Based Approaches to Therapy .............................................................................. 125 Conclusion ........................................................................................................................ 126

INDEX................................................................................................................ 131

ACKNOWLEDGEMENTS Many thanks to Cynthia Conomos and Erin O’Brien for their invaluable contributions in bringing this text to fruition.

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Chapter 1

In Vivo Imaging of Cellular Transplants Justin Chan, Jayant P. Menon, Rohit Mahajan and Rahul Jandial*

Abstract

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e will talk about the techniques of in vivo imaging currently used in today’s research and biomedical field, giving a general view of how each technique works and examples of practical applications of each technique. We will cover fluorescent (BL/CL), PET, SPECT and quantum dot imaging. Afterwards, we will cover how in vivo imaging is used in a biomedical sense; more specifically we will see how researchers studying cancer and neurodegenerative disease employ in vivo imaging.

Introduction Imaging has always been an important part of medical science and research. It has always been important to study samples under microscope with high resolution and contrast. Unfortunately, because of the nature of microscope imaging, the only way to image cells and tissues of animals and humans has been to study the sample ex vivo, outside of the living body in an artificial bath, or from a dead sample. The body has several dynamic functions that clearly can not be studied with static, post mortem samples. This is where in vivo imaging comes in. In vivo imaging allows researchers to study the movement and nature of target cells, tissue, molecules, etc. within a living organism. In vivo imaging requires a tag in order to make a contrast of the target tissue from the background and a method of imaging and recording the reporter tag. Though in vivo imaging is a simple idea it consists of vastly different routes; tags can consist of gene mutations leading to special protein synthesis, to a literal attachment onto a cell, to use of metal oxides to create a magnetic contrast. The possibilities are endless.

Fluorescent Imaging In vivo cellular imaging allows researchers to study cell motility in a living organism, as opposed to studying artificial samples or placing dead tissue in an artificial environment. Previously, one had to resort to time-lapse static images from animal models or analyze dead tissue to track and research dynamic events. However now with the use of such noninvasive techniques such as fluorescence, magnetic resonance imaging, positron emission and the ability to capture in vivo trackings in picture and in video, there are now several better ways to study cell motility. In vivo imaging of cellular motility has an infinite amount of uses, from imaging cell in animal models to better understand its physiology to cell-based therapy of disease. Fluorescent imaging uses reporter technologies, in which the cell or molecule of interest is tagged in vivo and then studied based on its presence. There are two types of fluorescent imaging: direct and indirect. Direct fluorescence imaging involves an actual engineered probe which tags target cells by targeting a specific receptor or enzyme. Active direct probes are characterized by the fact that *Corresponding Author: Rahul Jandial—Division of Neurosurgery, City of Hope Comprehensive Cancer Center & Beckman Research Institute Duarte, California, USA Email: [email protected]

Frontiers in Brain Repair, edited by Rahul Jandial. ©2010 Landes Bioscience and Springer Science+Business Media.

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Figure 1. New biotechnological tools. A) Reporter gene technology applied to the development of a bioluminescence and chemiluminescence (BL/CL) whole-cell biosensor. An analyte or a stimulus specifically activates a regulatory gene sequence that controls the reporter gene expression and the synthesis of the reporter protein, which is then measured by BL/CL techniques. B) Dual-reporter system. The signal from the control protein, which is constitutively expressed, is used as an internal reference tocorrect the analyte/stimulus-dependent signal to reduce experimental variability; alternatively, two reporter genes can be used to detect two analytes simultaneously or sequentially in the same system. C) Gene fusion of sequences of a reporter protein and a binding protein gene to obtain a BL/CL bifunctional fusion protein. D) Fusion proteins containing a suitable binding protein (e.g., MagA protein) enable bacterial magnetic particles displaying specific proteins on their surface to be obtained.2 Used with permission from Roda A et al. Trends Biotechnol 2006; 22(6):295-303. © 2006 Elsevier.

they fluoresce even if they are not attached to a cell or target. Thus they tend to cause background fluorescence, making for noisy imaging. Activatable direct probes have fluorescence that is activated by the target enzyme and thus improve contrast between target cell and background noise compared to active probes (Fig. 1).1 Indirect fluorescence imaging introduces and transcribes a reporter gene into the cell of interest, which then makes fluorescent proteins. If one is trying to study the movement of the proteins that the cell makes, one can encode its gene to tag the protein studied with fluorescence without changing the protein function.1 Commonly used tags include green fluorescent proteins (GFP) which are isolated from jellyfish Aequorea Victoria and red fluorescent proteins and can even be made into multiple reporter tags that flash in tandem or in sequence (called a dual-reporter system), or code for different enzymes within the same cell (BL/CL bifunctional molecule).2 In this way, fluorescent proteins can be used to visualize physical processes never visualized before. Imaging of cellular motility can be used in the study of neurons in the nervous system and tracking individual neurons in live samples of brain tissue during brain development.3 There are several types of fluorescent dyes that can be used. In the specific study of neuron cells, the dye Dil was widely used but because it was excited at a wavelength at around 500 nm, tissues absorbed light at this wavelength and suffered phototoxicity. However, since there are a plethora of dyes in the market for use, one can use a different dye that excites at a different wavelength, such as Cy5 and Cy7, that excite at 600 nm instead. In this study, researchers were able to use fluorescent dyes to image neural cells undergoing mitosis and moving in bursts within proliferative zones of the brain, showing a dynamism that

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could not have been otherwise seen without this cellular imaging technology. The team also visualized the actual growth and extension of an axon, something that was made possible only by the in vivo tracking.3 In vivo microscopy is used to visualize fluorescencently tagged cells in tissues from hosts. For any imaging of reporter cells, the higher the resolution and therefore sensitivity and accurateness of the imaging technique the better, so strides have been made to improve microscopy in these fields. Intravital confocal, two-photon and multiphoton microscopy are types of in vivo microscopy that have yielded high resolution imaging of reporter cells, imaging from 0.5 to 3 microns.1 These methods can be used to dynamically track fluorescent reporting tags, such as diffusion of tagged cells, or binding or activation of these cells to enzymes, against time. In a confocal fluorescence microscope a spatial pinhole is used to get rid of background lighting in order to increase contrast between the sample and background. Point illumination is used in an optically conjugate plane in front of the microscope in order to get rid of background noise in different focal planes. Only light from a single focal plane can be detected, getting rid of noise and also allowing one to create not only a 2D but also a 3D view of the target tissue. A multiphoton fluorescence microscope uses a long wavelength light to excite reporter tags (in this case flurophores) within the sample. The fluorophore absorbs the energy from two photons hitting the fluorophore at the same time; then an electron is excited to a higher energy state and decays, releasing energy imaged as light. Two-photon fluorescence microscope is a variant of multiphoton. Recently, in vivo microscopy can even be adapted into flexible fiber probes in order to get to places within the body and directly image those areas that older microscopy techniques were unable to image. In this method, light is passed through several optical fibers and bundled into a flexible probe, which is then inserted into the body to take images of target tissues. Planar imaging is the most common method to record fluorescence deeper within the tissues. The two types of planar imaging used are called epi-illumination and transillumination. Epi-illumination, also called fluorescence reflectance imaging (FRI), is when light is shone onto a tissue and captured on the same side of the tissue. The light emitted from the tissue can either be from surface flurophores or deeper within the tissue, owing to the diffusive nature of photons in this technique. Epi-illumination can not visualize depth of tissues or nonlinear dependencies of signals from neighboring tissues or deeper tissues.1 Transillumination shines light through the tissue instead of reflecting off the tissue. Light source is placed on one side of the tissue and detector on the other, picking up shadows or emitted fluorescence made by the tissue. Transillumination is far less used in comparison to epi-illumination, though it allows one to see the entire depth of the tissue unlike epi-illumination (Fig. 2). Though fluorescence and bioluminescence only has a limited level of resolution compared to other types of imaging, it provides good biochemical and physiological information. It is also comparatively cheap and fast and presents no radiation hazard to the model used.4

Quantum Dots Currently a new type of fluorescence is becoming very important for in vivo imaging. A quantum dot is a semiconductor whose excitons are confined, thereby creating different properties, in this case light. Quantum dots are said to be better than previously mentioned organic fluorescent dyes because they are much, much brighter than fluorescent dyes and also are much more stable and therefore undergo far less photodestruction. Because the CdSe layer of quantum dots is protected by a layer of ZnS, they are able to last much longer than organic dyes can. How are quantum dots taken up by cells in the first place? For cellular labeling, it is difficult to get a relatively large quantum dot molecule through the lipid bilayer of the cellular membrane and into the cell itself. Techniques used to insert QD into cells include endocytosis, direct injection of QD in miniscule amounts, electroporation and mediated uptake.5 In electroporation, an electric charge is used move through the membrane. Mediated uptake involves the encapsulation of quantum dots in reagents; the resulting vesicles are more easily passed through the lipid bilayer.

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Figure 2. Examples of transillumination imaging. (Top row) Imaging of an MMTV/neu transgenic mouse that exhibits multifocal spontaneous mammary tumorigenesis injected with a cathepsin-sensitive probe. A) Epi-illumination image of the animal obtained at the excitation wavelength; the arrow indicates the position of the tumor, in this case appearing dark owing to increased vascularity; B) transillumination image at the excitation wavelength; C) transillumination imaging at the emission wavelength (fluorescence) and; D) corrected image where the fluorescence transillumination image is divided by the image at the excitation wavelength. This correction operation improves the contrast and suppresses nonspecific signals. (Bottom row) Postmortem imaging of a fluorescence tube inserted in the center of the animal through the esophagus. E) Epi-illumination image of the animal obtained at the excitation wavelength, the position of the tube is indicated with the dotted line box and the red arrow. The actual tube is not visible in this photograph. F) Transillumination image at the excitation wavelength, G) transillumination image at the emission wavelength (fluorescence) and, H) corrected image where the fluorescence transillumination image is divided by the image at the excitation wavelength.1 Used with permission from Ntziachristos V. Annu Rev Biomed 2006; 8:1-33. A color version of this image is available at www.landesbioscience.com/curie.

There are a few drawbacks to quantum dots that are currently being worked on. One main issue with quantum dots is that they require an external illumination source in order to get excited which creates a lot of background noise. Scientists have come up with self-illuminating quantum dots in order to get rid of the need for external illumination. These self-illuminating quantum dots use bioluminescence energy transfer, which takes chemical energy and turns it into photon energy, causing fluorescence.6 This is achieved by coupling the quantum dots to a mutant of bioluminescent protein Renilla reniformis luciferase.5 This luciferase emits its own light when it binds to the quantum dot, which in turn excites the bound quantum dots. The light emitted by both the quantum dot and the luciferase are engineered to be of similar wavelength and therefore overlap and create a very bright fluorescence without the need for external illumination.7 Quantum dots can also be imaged with infared excitation, which not only lessens problems with autofluourescence in the background but also allows researchers to image more deeply into the tissue of interest. This can be done in vivo as well with no problem; excitation with infared will even cause quantum dots to continue fluorescing up to four months.8

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A major hindrance to future in vivo clinical application is that quantum dots currently are mostly synthesized with heavy metal nanocrystals for example cadmium and selenium (CdSe) these are known toxins and have very long half lives in biologic tissue. For example, Cadmium poisoning causes irreversible kidney damage.

PET and SPECT Positron emission topography (PET) and single photon emission topography (SPECT) are another way to image cells. PET uses radioactive substances to examine the metabolic activity of various body structures. Metabolic and physiological activities of target tissues can be imaged through capture of gamma rays that are emitted when the excited radioactive substances decay and release energy. Both are used to track movement of cellular implants tagged with a PET-specific reporter gene and can also give information as to how long the cell is able to live in the body and what it does in the body. A main advantage of PET and SPECT is the extreme sensitivity of these imaging techniques. Their sensitivity is down to the nanomolar, allowing researchers to image samples even at extremely low concentrations. In addition this allows for specialized systems of small animal model imaging with a larger resolution than is available in other imaging techniques. The sensitivity of PET and SPECT allows one to track cell implants through several mechanisms in the body.9-16 Radionuclide imaging generally uses three types of reporter genes: receptors, transporters and enzymes. Receptor-based reporters bind a radioactive tracer onto a gene-encoded protein. Enzymatic-based reporters rely on the production of enzymes from reporter genes which allows metabolites of radioactive tracers to be made. An example of a receptor-based reporter gene is a system which uses the dopamine D2 receptor (D2R) which is tagged with fluroethylspiperone (FESP) to show quantitatively the amount of D2R expression in vivo in animal models. This system is one of few in vivo cellular imaging systems that can be used for imaging cellular movement in the brain because PET and SPECT can cross the blood brain barrier easily. The main enzyme-based reporter system used in PET is the HSV1-tk reporter system. HSV1-tk is called a “suicide” gene because it phosphorylates substrates, causing an accumulation of diphosphates and triphosphates in the cell which will kill the cell in high concentrations.17-20 This allows HSV1-tk to be used as a negative selection marker in PET imaging to get rid of TK-expressing cells. Because PET imaging allows imaging of very small amounts of reporter, TK toxicity can be minimized and substrates labeled with a positron or single-photon emitting radioisotope can detect HSV1-tk expression. This tracking of expression can be used to report the location and level of expression.21 Though PET and SPECT imaging allows for higher resolution imaging, it is still considered low compared to the resolution that is achieved by MRI. In addition, this type of tracing is slow compared to fluorescence and is also hazardous to the model used. However, if the sample being imaged is in extremely low concentration, the high sensitivity of PET and SPECT gives it the upper hand in these cases.4

MRI Another method used for in vivo imaging, especially in cell therapy, is called magnetic resonance imaging, or MRI. Because MRI uses a special type of imaging, it requires a different kind of reporter to image. In general, MRI incorporates iron oxide nanoparticles, ultra small paramagnetic oxides (USPIO), or superparamagnetic oxides (SPIO) into the cell as contrast agents for MRI through a few methods.22 For example, when one is trying to study blood-borne molecules, one can simply inject the particles into the blood stream and it will get picked up by blood-borne cells of interest. A specific example of this would be using MRI to monitor macrophages after a stroke; using Prussian blue stain, researchers were able to determine that macrophages had picked up the iron after 6 days.22

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One can also inject the iron oxide nanoparticles into a specific tissue area of interest, if one wants to study the movement of specific cells instead of all the cells in the blood stream. In this way, only cells in the neighboring region of the site of injection are integrated with the iron oxide particles. This can be used as the labeled group of cells move, as they leave a trail of cells as they move through the body. This method can also be imaged more specifically by picking out different motilities of different specific cells versus background cells, allowing for selective in situ labeling.22 In vitro labeling of a cell culture can also be used for in vivo cell study. Various in vitro techniques such as endocytosis are used to get the iron oxide particles into the cells of interest. Then the cells are reimplanted into the host animal model; the movement of these cells from the implantation site across the body of the host can be tracked using the USPIO reporter now in the cells. This can be used to estimate the migration of cells, such as stem cells, across an organ. Though MRIs have the same function as fluorescent imaging techniques, it works in a vastly different way; there is no light being emitted and captured here. As stated before, MRI uses tiny metallic oxides such as iron oxide in order to create a contrast between target cells and background. This allows a very high soft tissue contrast, making it useful for neurological, cardiovascular, skeletal and oncological imaging. MRI works instead by using magnetic fields and aligning hydrogen atoms in the body. Radio waves change the alignment of certain hydrogen atoms causing the proton of the atom to spin out of alignment. When the proton snaps back into alignment, it releases its own rotating magnetic field. Finally, since different tissues in the body take different amounts of time to realign after being hit by radio wave, one can discern different tissues in the body and construct an image. Magnetic resonance spectroscopy also plays a key role in this type of imaging. MRS uses several different magnetic resonances that excite different tissues of the body. Using this data, one can measure levels of different metabolites in the body, allowing one to obtain biochemical information about the tissue of interest. Aforementioned reporters such as USPIO and SPIO are called contrast agents which will delineate areas of interest. They will add extra brightness or darkness to the specific area of interest and can thus be injected into the body in several different ways. For example, contrast agents can be injected intravenously if one is studying the blood stream, or eaten if one is studying bowel movement. MRI is a well-liked imaging procedure because it allows for extremely high levels of resolution. In addition, it presents no radiation hazard to the model used. However, it gives less biochemical and physiological data than PET or fluorescent imaging and requires a higher amount of supervision and experience in its usage.4

In Vivo Imaging and Tumors In vivo cellular imaging has helped find answers in battles against cancer, neurodegenerative disease and any other diseases that require the tracking of cell therapy. Noninvasive in vivo imaging of tumor response to various gene therapies and vaccines has helped in developing a cure for cancer. Because cells involved in the body’s immune system move very rapidly throughout the entire body and tissues in relation to their activation via disease signals, it is important that researchers have a tool with which to tag these cells and track their movement to better understand it, as it is insufficient to study such movement ex vivo in sample tissues (Fig. 3).4 For various types of cancer, the main methods of imaging used are magnetic resonance, positron emission topography and optical imaging. With these methods, scientists can better track cell movement by studying inflammation and infection of tissues and detecting the success of tissue implantation by monitoring the amount of immune cells start accumulating around the new tissue in response to it. The two main types of cancer immunotherapy right now involve insertion of antibodies, or enhancing the body’s immune system via T-cell activation. Thus, imaging can be used to monitor cell therapies used in cancer models against a tumor.23,24 There are several strategies to image tumor immunology, depending on whether one uses direct or indirect imaging.

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Figure 3. Schematic representation of the direct and indirect imaging strategy. A) Direct labeling procedures comprise the imaging of cells labeled ex vivo and reinfused in a subject under examination. Before the migration of a sample population can be visualized it needs to be harvested (1), labeled ex vivo with a radio labeled molecule, paramagnetic particles or fluorescent probes (2) and reinfused (3). Only after reinfusion of the labeled population can their distribution and behavior in the subject bevisualized by imaging strategies (4). Direct labeling is based on the ability of a cell to retain the label. This technique is adequate for tagging terminally differentiated cells such as dendritic cells and macrophages. B) This approach does not enable long-term monitoring of cell viability and proliferation in the body because the label is lost ordiluted owing to apoptosis or mitosis, respectively. C) Indirect imaging usually entails the use of transgenes, where expression of the reporter gene is exploited to monitorgenetically modified cells with a transgene-specific probe. Immunocompetent stem and/or progenitor cells are harvested (1) and transduced ex vivo (2) with an exogenousreporter gene encoding a reporter product. Only after reinfusion of the transduced population (3) and administration of the reporter probe (4) can their distribution and behavior in the subject be visualized by imaging strategies (5). Transcription and translation of the reporter gene leads to the production of a reporter product, which can trap a reporter probe within the cell. Indirect labeling procedures comprise stable genetic modification of the cells to monitor their fate and the fate of their progeny in vivo over time, following repeated in vivo probe administrations. Different probes can be used to tag the reporter product (such as radionuclide-labeled molecules, paramagnetic contrast agents or fluorescent molecules). This approach is fundamental for imaging proliferating cell populations during their migration, activation and division. D) Transduction of immune and/or stem cells with a reporter gene means that the entire cell progeny can trap the reporter probe, permitting their imaging without loss of signaling potential owing to mitosis. Used with permission from Lucignani G et al. Trends Biotechnol 2006; 24(9):410-418. © 2006 Elsevier.

Positron emission tomography (PET) uses localized radiotracers bound to specific enzymes or proteins; the most common ones used are D2R dopamine receptors or HSV1-tk. When using indirect imaging, HSV1-tk is the reporter gene used and 9-(4-(18F)-fluoro-3-hydroxymethylbutyl) guanine (FHBG) 24 or iodine labeled 2v-fluoro-2v-deoxy-1-beta-D-arabinofuranosyl-5-iodourac il (FIAU) can be used as HSV1-tk’s reporter probe.25,26 In a particular study of tumor cells using PET, researchers were able to visualize localization and accumulation of HSV1-tk-tagged activated T-lymphocytes towards tumors in mice models. Direct imaging of tumor immunology uses (64Cu) PTSM or (18F)FDG to tag cells.27,28

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MRI can be used for similar imaging. By using SPIO as reporter genes, one can track movement of injected T-lymphocytes within a tumor itself. MRI represents a type of direct imaging and uses cross linked iron oxides (CLIO-HD) to label cells. MRI can be used to track T-cell distribution and behavior towards target T-cells in 3D. In one specific study, researchers targeted a HER-2/ neu tumor with an antigen tagged with modified natural killer cells. Using MRI, they were able to determine that modified NK cells were able to target the tumor, whereas nonmodified ones did not, paving a way towards NK-mediated therapy.29 Optical imaging is based on simple fluorescence and bioluminescence and is used in tumor immunotherapy as a type of indirect imaging. Resultant emissions from BLI may not be enough for sufficient detection; MRI and especially PET imaging techniques are more sensitive. However, new fluorescent proteins shift emitted light wavelengths closer to infrared wavelengths, making their emissions more easily detectable. Optical imaging has been used to track details of T-cell anti-tumor immunotherapy and cell behavior after bone marrow transplantation in real time. For example, the study of implanted dendritic cells in vivo after bone marrow transplantation determined using BLI that transplanted expanded dendritic cells are able to move around quickly in vivo and have long life spans as well.30 In another study, researchers were able to tag NK-modified T-cells and track the kinetics of their ability to find and attack tumor cells.31 In multimodal imaging, more than one type of imaging can be used to track cells. By using a fusion of two reporter genes, such as the fusion HSV1-tk-GFP fusion protein, one can use both PET and BLI imaging to provide a repetitive noninvasive imaging technique to a specific cell. For example, using the fusion HSV1-tk-GFP fusion protein, one can track both the activation of a T-cell when it is introduced to anti-CD3 and anti-CD28 antibodies via HSV1-tk emission, while at the same time one can track T-cell movement via GFP emission.4

In Vivo Imaging and Neurological Disease In vivo imaging is particularly useful to the study of neurological disease for a few reasons. In vivo imaging allows researchers to track cell change in real time in response to disease or experience. Because of the vast variety of dynamics between cells in the brain, it is important to be able to image in real time. For example, neurons are relatively stable compared to constantly dynamic and changing microglial cells and changes in these dynamics must be made clear in degenerative diseases, which make neurons less stable, or autoimmune disease, which effect the dynamism of microglial cells.32-35 Such real time imaging can also allow researchers to observe the sequence in which a disease occurs. For example, in an immune attack, axonal damage, removal of myelin sheath and inflammation all occur but in different orders; using real time imaging, one can see in vivo which step causes what. Real time imaging also allows researchers to visualize exactly how certain neurological therapies cure diseases on a cellular basis (Fig. 4).36 The two main methods of in vivo imaging used for the nervous system is optical imaging (like multiphoton microscopy) and transgenic technology. While multiphoton microscopy has allowed scientists to image live animals with little phototoxicity and background noise. Transgenic technology has played a major role in nervous system imaging. Transgenic mice have been shown to express fluorescent proteins in their neurons. Fluorescent proteins are much easier to use than dyes and allow for much more specific tagging. This led to even more specific labeling of dendrites, axons and interneurons. In addition, this allows sections of the nervous system and all of its projection tracts to be labeled. Finally, one can image synapic vesicles, vesicles recycling and mitochondria using fluorescent proteins in the nervous system.36 Intrinsic optical imaging can be used to monitor activity of neurons. This technique relies on changes in light absorption based on changes in neuronal activity. How are these different methods applied in real world imaging? Imaging is used heavily in studying Parkinson’s and Huntington’s disease. Parkinson’s disease is the world’s second most common neurodegenerative disorder. The main symptoms of PD are akinesia, rigidity, tremor and

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Figure 4. Advantages of in vivo imaging in neurological disease models. A) In vivo imaging reveals the dynamics of cells in the nervous system. Static views of fixed tissue (left and right) cannot reveal that in the healthy brain dendritic branches are relatively stable, whereas microglial cells are highly dynamic. B) Intravital microscopy allows the identification of damage and assessment of repair in disease models. Trauma (flash) leads to axon transection and compensatory growth. When the nerve tissue is fixed and examined after trauma, a mixture of damaged and intact axons is often seen (right). In vivo imaging can clearly differentiate whether single fibres have regenerated (middle top, growth cone with arrow) or rather escaped damage (middle bottom). C) In vivo observations can give insight into disease pathogenesis. In neuroinflammatory lesions, demyelinated axons often degenerate, but the cause of this degeneration is unknown. Inflammatory and glial cells could create a neurotoxic environment (middle top); alternatively, immune cells could directly attack the denuded axons (middle bottom). D) Direct visualization of pathogenic structures before and after treatment can provide information about therapeutic mechanisms. Alzheimer’s plaques can be visualized using vital dyes (violet). Whereas static views (left and right) can show that a therapy slows progression (on the right: plaque size without therapy (outlined in red) is bigger than with therapy (outlined in green)), imaging plaques over time can reveal how such an improvement is achieved, that is, whether growth of pre-existing plaques is slowed (middle top), or new seeding of plaques is prevented (middle bottom). Used with permission from Misgeld T et al. Nat Rev Neurosci 2006; 7(6):449-463. © Nature Publishing Group. A color version of this image is available at www.landesbioscience.com/curie.

postural gait. PD occurs asymmetrically throughout the body and in increasingly greater strengths as the disease progresses. Studies have shown that one main cause for PD is loss of dopamine in the nervous system in the striatum, where dopamine is released. Dopaminergic neurons are affected and degenerated due to a decrease of neuron innervation from the striatum.37 Huntington’s disease is a genetic, inherited and autosomal dominant trait due to an abnormal repeat of CAG triplets on the short arm of chromosome 4.38 Its symptoms include involuntary movement and neuronal degeneration around the striatum. The link between the huntingtin gene and neurodegeneration are largely unknown.

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PET has been used in PD studies to image the dopaminergic system. By imaging levels of dopamine transporter binding, one can study dopamine terminal degeneration, which explains the disease’s symptoms of lowered motor and cognitive performance.39 In addition, dopamine release in cortical areas during task and psychological studies can be imaged as well using in vivo PET techniques.40 PET is also used to track in vivo progression of both PD and HD. Using repeated imagings of the nervous system, researchers can quantify rate of loss of DA synthesis in PD patients and loss of D2 receptors in HD patients. Most importantly, however, PET is used to track the effectiveness of therapeutic methods towards PD and HD. For example, using transplantation of fetal tissue into diseased brains, researchers were able to image restoration of dopamine storage and reinnervation of striatal neurons in the striatum of PD patients.41 In addition, by tagging hexokinase activity, researchers have been able to study metabolic effects of fetal tissue grafting onto the striatum in HD patients. These studies showed improvement in striatal function after grafting.42 In trauma cases where the CNS suffers axotomy, or axonal transection, researchers have used fluorescent imaging to study axonal regeneration. Using Thy1-XFP mice, individual axons were labeled fluorescently and repeatedly imaged before and after axotomy, showing that the axons grew spontaneously but without direction, revealing a form of axonal degeneration after axotomy. On the other hand researchers have imaged zebrafish larvae regenerating axons towards their normal spinal targets after an increase in cyclic AMP, possibly paving a way towards regenerating axons in mammals.43 46 In vivo imaging can also be used in neurovascular diseases, such as ischaemeic stroke. By injecting blood vessels with a fluorescent protein, researchers can use multiphoton microscopy to image and measure the flow.47 By imaging blood flow during vascular occlusion, one can see reversal of blood flow and the effect this has on the brain. Repeated imaging of the nervous system during an ischaemeic stroke showed that strokes caused rapid spine loss and other dendritic changes.48 Inflammatory diseases, such as multiple sclerosis, involve the blood brain barrier; tracking the movement of immune cells past the blood brain barrier has allowed researchers to come up with therapeutic interventions. Labeling immune cells with fluorescent or genetic markers has helped researchers clarify the molecular interactions that occur during the capture and adhesion of immune cells to the CNS.49

Conclusion Like any new technological advance, in vivo imaging has its drawbacks. Along with expense and unavailability, some present phototoxicity to their patients, while others work to reduce phototoxicity at the expense of resolution or sensitivity. Future technologies in all areas of in vivo imaging work towards finding a better balance, as well as continually miniaturizing probes and imaging devices in order to better image different organs and tissues, to better image smaller and smaller amounts of sample and to better manipulate probes applied to tissues. For example, “nanoscopic” imaging strives to delve into the molecular realm of imaging, also requiring much less volume needed for excitation.50 Fluorescence imaging advances have helped small animal imaging. A new technology called nonionizing radiation allows researchers to image several different targets at the same time, increasing uses of fluorescence for biomedical studies.1 Researchers are continually finding new ways to use imaging for neurodegenerative disease and for cancer treatment. Other than multimodality discussed earlier, new probes are being developed which can divulge information about behaviour of different cell populations.4 And another new technique, called “biophotonic whole-body imaging,” uses a wide-detecting photon emission range in order to not only longitudinally image an animal throughout a course of treatment, but also screen several different animals at the same time.51 Furthermore, Optical Coherence Tomography uses interferometric techniques, much like how ultrasound uses the intereference patterns of reflected waves to image, to achieve millimeter deep tissue imaging with submicrometer resolution. And finally, Functional Photoacoustic Imaging (fPAM) where pulsed lasers excite biological tissues to release ultrasonic waves (photoacoustic waves) that are picked up by a focused ultrasound transducer. The magnitude

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of these waves is proportional to the local tissue characteristics and so divulge phyisiologic properties of tissues several millimeters away from the transducer with micrometer resolution.. In vivo imaging depths of 3mm have been detected in live animals. Deoxyhemoglobin and melanin are the most optically dense molecules in mammalian tissues and so fPAM is an excellent method to image subcutaneous microvasculature. fPAM can image oxygen consumption with high fidelity in tissues and could potentially image brain functions based on local hemoglobin oxygen saturation.52 Overall, despite how useful in vivo imaging has been to both research and biomedical advances, it is clear that the technology still has great potential in the future; it simply depends on where researchers and doctors want to take the technology next.

References 1. Ntziachristos V. Fluorescence molecular imaging. Annu Rev Biomed Eng 2006; 8:1-33. 2. Roda A, Pasini P, Mirasoli M et  al. Biotechnological applications of bioluminescence and chemiluminescence. Trends Biotechnol 2004; 22(6):295-303. 3. Fishell G, Blazeski R, Godement P et al. Optical microscopy. 3. Tracking fluorescently labeled neurons in developing brain. FASEB J 1995; 9(5):324-34. 4. Lucignani G, Ottobrini L, Martelli C et al. Molecular imaging of cell-mediated cancer immunotherapy. Trends Biotechnol 2006; 24(9):410-8. 5. Medintz I, Uyeda H, Goldman E et al. Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 2005; 4(6):435-46. 6. Xu Y, Piston D, Johnson C. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 1999; 96(1):151-6. 7. Frangioni J. Self-illuminating quantum dots light the way. Nat Biotechnol 2006; 24(3):326-8. 8. Ballou B, Lagerholm B, Ernst L et al. Noninvasive imaging of quantum dots in mice. Bioconjug Chem 2004; 15(1):79-86. 9. Chatziioannou A, Cherry S, Shao Y et  al. Performance evaluation of microPET: a high-resolution lutetium oxyorthosilicate PET scanner for animal imaging. J Nucl Med 1999; 40(7):1164-75. 10. Surti S, Karp J, Perkins A et  al. Imaging performance of A-PET: a small animal PET camera. IEEE Trans Med Imaging 2005; 24(7):844-52. 11. Acton P, Kung H. Small animal imaging with high resolution single photon emission tomography. Nucl Med Biol 2003; 30(8):889-95. 12. Green M, Seidel J, Vaquero J et  al. High resolution PET, SPECT and projection imaging in small animals. Comput Med Imaging Graph 2001; 25(2):79-86. 13. Ishizu K, Mukai T, Yonekura Y et al. Ultra-high resolution SPECT system using four pinhole collimators for small animal studies. J Nucl Med 1995; 36(12):2282-7. 14. Weber D, Ivanovic M. Pinhole SPECT: ultra-high resolution imaging for small animal studies. J Nucl Med 1995; 36(12):2287-9. 15. Yang Y, Tai Y, Siegel S et al. Optimization and performance evaluation of the microPET II scanner for in vivo small-animal imaging. Phys Med Biol 2004; 49(12):2527-45. 16. Beekman F, van der Have F, Vastenhouw B et al. U-SPECT-I: a novel system for submillimeter-resolution tomography with radiolabeled molecules in mice. J Nucl Med 2005; 46(7):1194-200. 17. Eck S, Alavi J, Alavi A et al. Treatment of advanced CNS malignancies with the recombinant adenovirus H5.010RSVTK: a phase I trial. Hum Gene Ther 1996; 7(12):1465-82. 18. Shand N, Weber F, Mariani L et al. A phase 1-2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir. GLI328 European-Canadian Study Group. Hum Gene Ther 1999; 10(14):2325-35. 19. Alauddin M, Shahinian A, Gordon E et  al. Preclinical evaluation of the penciclovir analog 9-(4-((18) F)fluoro-3-hydroxymethylbutyl)guanine for in vivo measurement of suicide gene expression with PET. J Nucl Med 2001; 42(11):1682-90. 20. Germano I, Fable J, Gultekin S et al. Adenovirus/herpes simplex-thymidine kinase/ganciclovir complex: preliminary results of a phase I trial in patients with recurrent malignant gliomas. J Neurooncol 2003; 65(3):279-89. 21. Gambhir S, Barrio J, Wu L et  al. Imaging of adenoviral-directed herpes simplex virus type 1 thymidine kinase reporter gene expression in mice with radiolabeled ganciclovir. J Nucl Med 1998; 39(11):2003-11. 22. Hoehn M, Wiedermann D, Justicia C et  al. Cell tracking using magnetic resonance imaging. J Physiol 2007; 584(Pt 1):25-30. 23. Wu A, Senter P. Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol 2005; 23(9):1137-46.

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24. Kenanova V, Wu A. Tailoring antibodies for radionuclide delivery. Expert Opin Drug Deliv 2006; 3(1):53-70. 25. Dubey P, Su H, Adonai N et  al. Quantitative imaging of the T-cell antitumor response by positron-emission tomography. Proc Natl Acad Sci USA 2003; 100(3):1232-7. 26. Koehne G, Doubrovin M, Doubrovina E et al. Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes. Nat Biotechnol 2003; 21(4):405-13. 27. Adonai N, Nguyen K, Walsh J et  al. Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc Natl Acad Sci USA 2002; 99(5):3030-5. 28. Botti C, Negri D, Seregni E et  al. Comparison of three different methods for radiolabelling human activated T-lymphocytes. Eur J Nucl Med 1997; 24(5):497-504. 29. Daldrup-Link H, Meier R, Rudelius M et al. In vivo tracking of genetically engineered, anti-HER2/neu directed natural killer cells to HER2/neu positive mammary tumors with magnetic resonance imaging. Eur Radiol 2005; 15(1):4-13. 30. Schimmelpfennig C, Schulz S, Arber CJ et al. Ex vivo expanded dendritic cells home to T-cell zones of lymphoid organs and survive in vivo after allogeneic bone marrow transplantation. Am J Pathol 2005; 167(5):1321-31. 31. Edinger M, Cao Y, Verneris M et  al. Revealing lymphoma growth and the efficacy of immune cell therapies using in vivo bioluminescence imaging. Blood 2003; 101(2):640-8. 32. Trachtenberg J, Chen B, Knott G et  al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 2002; 420(6917):788-94. 33. Grutzendler J, Kasthuri N, Gan W. Long-term dendritic spine stability in the adult cortex. Nature 2002; 420(6917):812-6. 34. Davalos D, Grutzendler J, Yang G et  al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 2005; 8(6):752-8. 35. Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 2005; 308(5726):1314-8. 36. Misgeld T, Kerschensteiner M. In vivo imaging of the diseased nervous system. Nat Rev Neurosci 2006; 7(6):449-63. 37. Kirik D, Breysse N, Björklund T et al. Imaging in cell-based therapy for neurodegenerative diseases. Eur J Nucl Med Mol Imaging 2005; 32(Suppl 2):S417-34. 38. Gusella J, Wexler N, Conneally P et al. A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 1983; 306(5940):234-8. 39. Brooks D. PET studies on the function of dopamine in health and Parkinson’s disease. Ann N Y Acad Sci 2003; 991:22-35. 40. Koepp M, Gunn R, Lawrence A et al. Evidence for striatal dopamine release during a video game. Nature 1998; 393(6682):266-8. 41. Freed C, Greene P, Breeze R et al. Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N Engl J Med 2001; 344(10):710-9. 42. Gaura V, Bachoud-Lévi A, Ribeiro M et  al. Striatal neural grafting improves cortical metabolism in Huntington’s disease patients. Brain 2004; 127(Pt 1):65-72. 43. Holländer H, Mehraein P. (On the mechanics of myelin sphere formation in Wallerian degeneration. Intravital microscopic studies of single degenerating motor fibers of the frog). Z Zellforsch Mikrosk Anat 1966; 72(2):276-80. 44. Williams P, Hall S. Prolonged in vivo observations of normal peripheral nerve fibres and their acute reactions to crush and deliberate trauma. J Anat 1971; 108(Pt 3):397-408. 45. Pan Y, Misgeld T, Lichtman J et al. Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo. J Neurosci 2003; 23(36):11479-88. 46. Bhatt D, Otto S, Depoister B et  al. Cyclic AMP-induced repair of zebrafish spinal circuits. Science 2004; 305(5681):254-8. 47. Zhang S, Boyd J, Delaney K et  al. Rapid reversible changes in dendritic spine structure in vivo gated by the degree of ischemia. J Neurosci 2005; 25(22):5333-8. 48. Iadecola C. Neurovascular regulation in the normal brain and in Alzheimer’s disease. Nat Rev Neurosci 2004; 5(5):347-60. 49. Miller D, Khan O, Sheremata W et al. A controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med 2003; 348(1):15-23. 50. Hell S. Toward fluorescence nanoscopy. Nat Biotechnol 2003; 21(11):1347-55. 51. Ntziachristos V, Ripoll J, Wang L et  al. Looking and listening to light: the evolution of whole-body photonic imaging. Nat Biotechnol 2005; 23(3):313-20. 52. Hao FZ, Konstantin M, George S, Lihong VW. Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging. Nature Biotechnology 2006; 24(7):848-51

Chapter 2

Culture and Manipulation of Neural Stem Cells Jennifer Katz,* Bryan Keenan and Evan Y. Snyder

Abstract

D

espite advances in the treatment of cancer, the prognosis of patient diagnosed with metastatis cancer to the brain remains poor. The role of neural stem cells as a viable tool in the treatment of metastatic cancer to the brain alone or in conjuction with current therapeutic modalities is promising. Both murine and human neural stem cells (NSCs) have been shown to migrate through the central nervous system (CNS) and infiltrate tumors and other pathological disease states of the brain. Genetic modification of NSCs to produce cytotoxic or immunomodulatory agents in the vicinity of a primary tumor and/or satellite lesion or has proven instrumental to the reduction of tumor bulk in murine models. Although the use of stem cells proves to be a volatile social topic, scientists have discovered that NSCs are present in the adult brain and continue to propagate and differentiate. These cells may be isolated and cultured to produce clonal NSC lines that are capable of self renewal and differentiation when introduced into the CNS. In this chapter, we describe protocols currently used in our lab for the successful maintenance of NSCs in vitro advancing the role of neural stem cells in the treatment of brain tumors.

Introduction Despite advances for the treatment of cancer, the prognosis for patients suffering from malignant brain tumors remains poor. High grade neoplasms, such as glioblastoma multiforme, are markedly invasive and spawn widely disseminated microsatellite aggregates that have limited the efficacy of surgical and adjunctive therapies.1 Evidence suggests that the brain may harbor metastatic tumor cells which are resistant to current treatments and can later develop into symptomatic metastases.2 Because current treatment options are often associated with pain, suffering and have the potential of severe side effects, the scientific and medical communities recognize the urgency in developing a more effective therapy against cancer. Until recently, the field of NSC biology focused primarily on the development of cells for the primary purpose of regenerating diseased or injured brain thereby restoring function. Recently, it has been shown that transplanted adult NSCs migrate throughout the CNS towards tumors and ‘track’ infiltrative neoplastic cells. Coupled with their ability to engraft and differentiate in vivo this phenomenon underscores the potential of NSCs as a vehicle for gene delivery in the CNS thereby vastly broadening their role as therapeutic agents.3 Numerous laboratories have since replicated and confirmed the above characteristics of NSCs and none have found these cells to be tumorigenic in animal models. A major obstacle in the treatment of brain tumors is overcoming the difficulty of delivering therapeutic agents to specific regions of the human brain. Molecules that might otherwise be effective may be unable to cross the blood-brain barrier or cross in inadequate amounts. However, NSCs are able *Corresponding Author: Jennifer Katz—Del E. Webb Neuroscience, Aging and Stem Cell Research Center, Burnham Institute for Regenerative Madicine. Email: [email protected].

Frontiers in Brain Repair, edited by Rahul Jandial. ©2010 Landes Bioscience and Springer Science+Business Media.

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to circumvent the blood-brain-barrier and have been shown to migrate to and infiltrate experimental orthotopic primary brain tumors following intravenous administration to a murine model of glioma.3 Neural stem cells can be engineered to express and disseminate bioactive molecules of therapeutic importance in vivo.4 In addition, these multipotent cells are capable of seamlessly integrating into the cytoarchitecture of various regions throughout the host brain as neurons, astroctyes and oligodendrocytes without disrupting normal function. In fact, it has been suggested that these cells may shift their differentiation fate toward replacing deficient and/or depleted cells.5 Despite the success of neural stem cell-based therapy in animal models of neurological diseases/injuries such as Parkinson’s disease, ALS, stroke and metastatic brain tumors the clinical implementation of NSC-based gene therapy still faces significant challenges. First, one must tackle the logistical issues related to the supply and isolation of donor tissue needed to generate adequate numbers of cells through expansion in culture prior to transplantation. Second, one must address the tumorigenic potential of NSCs in vivo since reports of extensive rodent NSC culturing has been shown to lead to and propagate genetic changes that may alter cell growth and differentiation.6 Third, one must demonstrate long-term cell survival, identify triggers that direct neural stem cell phenotypic fate and mitigate the risks of immunological rejection. Last and if applicable, one must ascertain the parameters required to optimize NSC engraftment, differentiation and foreign gene expression.

Neural Stem Cells Defining and Identifying the Neural Stem Cell Neural stem cells are a subgroup of stem cells and are defined as a primordial, self-renewing and self-maintaining cell that can give rise to neurons, astrocytes and oligodendroglia of the mature nervous system.5,7 They first captured the attention of scientists once it was discovered that NSCs could be isolated, genetically manipulated and differentiated in vitro then reintroduced and reintegrated into a developing, adult or a pathologically altered central nervous system.5,8 Furthermore, NSCs exhibit many unique characteristics, one of which is their migratory capability which make them potential candidates for gene therapy in various neurological diseases. NSC migration towards pathological regions in the brain was first demonstrated by Aboody and colleagues3 in an experimental brain tumor model where NSCs were administered through intratumoral, intraparenchymal, intraventricular transplantation and intravascular injection. In all paradigms examined, NSCs were able to migrate through normal brain tissue and track individual tumor cells leaving the main tumor mass. Although the exact mechanisms underlying neural stem cell migration to sites of injury or disease is unclear it is thought that NSCs are responding to chemotactic signals (or physiological processes including inflammation, angiogenesis and reactive astrocytosis) to replace dead or dysfunctional cells in those regions.3,5,9 In addition, transplanted NSCs have been shown to integrate into abnormal brains and successfully differentiate into either neurons or glia.10-13 Upon incorporation into host tissue, NSCs may promote effective changes in the phenotype of genetically based loss-of-function models of neurodegeneration such as lysosomal storage diseases.14 Collectively, these properties make the NSC an appealing candidate for gene therapy in the CNS. Stem cells reside in ‘niches’ or specialized microenvironments that regulate their activities of self-renewal, activation and differentiation. Contrary to the once popular dogma, it is now widely accepted that neurogenesis occurs within multiple regions of the adult brain, specifically the subventricular zone (SVZ) of the lateral ventricles15 and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus.16 Common components of these niches include somatic cells, a basement membrane and the extracellular matrices. The exact components within the neurogenic niche remain to be determined but it is likely that adult neurogenesis is regulated both intrinsically and extrinsically. Both the SGZ and the SVZ are highly vascularized, indicating that factors released from blood vessels may directly affect neural stem cell regulation/proliferation.17 Furthermore, NSCs can be influenced by both local cell-cell interactions as well as by complex neural circuits derived from neurons located outside of the niche. Of note, adult NSCs bear a striking resemblance to the astrocyte on both the ultrastructural and molecular level, including

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expression of glial fibrillary acidic protein (GFAP).18,19 NSCs, however, do not appear to be functionally mature nor are they terminally differentiated despite the antigens they express. The discovery that there is a renewable source of NSCs which can be isolated and cultured in vitro from neurogenic niches in the adult CNS (from human biopsies and postmortem tissues)20,21 offers a promising option for human autologous cell-based therapies in the near future. The effect of endogenous NSCs in brain repair appears to be limited. It is unclear whether this is due to a limited number of NSCs or due to other unidentified factors that seldom allow for effective neurogenesis in the injured brain. Once the signaling pathways involved in stimulating neurogenesis in the adult brain are determined then the development of new treatments involving the activation of endogenous NSCs may be used to achieve functional improvement in individuals with neurodegenerative diseases. Furthermore, understanding the mechanisms that control NSC proliferation, differentiation, survival and migration in vivo may provide insights that improve the efficiency of cell transplantation. The clinical application of neural stem cell-based therapies still faces many challenges. Although NSCs can be isolated and propagated in culture for extended periods of time, these proliferative populations often contain different cell types. This remains a challenge because immunocytochemical markers that are sufficiently specific and sensitive to define a neural stem cell have not been fully defined. Additionally, the developmental factors that direct the differentiation of these cells into specific neural cell types are also largely unknown. There are some concerns that the cells may become tumorigenic or act in unanticipated ways and exacerbate the clinical condition. Another obstacle is how to best identify the optimal tissue source from which therapeutically relevant numbers of NSCs can be efficiently isolated. Additional research is needed to better understand how transplanted cells are directed to grow, migrate, replace injured or diseased cells, or in the case of brain tumors, attack neoplastic cells in vivo. The notion of harnessing NSCs to deliver therapeutic gene products introduces yet another concern. Ascertaining the safest and most effective genes with which to arm these cells as well as the logistics of how to administer them must still be determined. Lastly, it is necessary to show conclusively whether NSCs effectively work in human disease.

Genetic Modification of Neural Stem Cells The neural stem cell has been shown to be an effective tumor-targeting delivery system in animal models of glioblastoma multiforme3,22-24 because it is a promising candidate for genetic manipulation. Numerous studies have shown that genetically modified NSCs may be used to deliver therapeutic agents for the treatment of various pathological conditions.2,25-27 Some of the challenges which remain for stem cell-based clinical approaches are as follows: which genes or chemicals are optimal/can be delivered and how often, which route is most effective and how often to administer the cells. Major therapeutic strategies have focused on the delivery of oncolytic agents to tumors, gene transfer techniques to introduce suicidal or therapeutic gene products to tumor cells and the use of immunostimulatory therapies to induce endogenous immune responses against tumors. The concept of harnessing stem cells with bioactive molecules to specifically target tumor satellites will augment current clinical treatments. Below we will discuss three main anti-tumor stem cell-related strategies currently under investigation in our lab.

Prodrug Converting Enzyme Strategy The prodrug converting enzyme strategy utilizes genetically modified NSCs to deliver bioactive products (e.g., cytosine deaminase, carboxylesterase, thymidine kinase) which convert nontoxic prodrugs into toxic molecules by specific prodrug activating enzymes. Tumor cell death occurs following incorporation of toxic analogues into the DNA of actively dividing cells. An advantage to using a prodrug approach is the prominent ‘by-stander effect’ which can be described as a ‘ripple’ in which the death of even a small number of tumor cells results in diffusion of oncolytic factors emanating from the dead cells thus killing an even broader region of tumor cells. The ‘bystander effect’ is strongest in the cytosine deaminase/5-fluorocytosine strategy which differs from other strategies abovementioned in that it does not require cell-to-cell contact.28 Furthermore, should

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bioengineered NSCs become oncogenic themselves; the bioactive genes within these cells would serve as suicide genes and act as a built in safety mechanism. Lastly, the prodrug strategy allows for the practitioner to control the initiation, duration and termination of treatment.

Immunomodulatory Strategy The ability of tumor cells to trigger an effective immune response is limited. Tumor cells often fail to express cytokines that activate local immune responses and may actually secrete immunosuppressive molecules.29 Cytokines activate immune responses by multiple mechanisms; however, their systemic use is limited by toxicity. Furthermore, the effectiveness of circulating cytokines is reduced by rapid degradation and elimination. One way to mitigate the systemic toxicity of cytokines and simultaneously activate the body’s immune system against tumor cells is to use genetically modified NSCs to deliver immunomodulatory gene products directly into the tumor milieu. Several animal models introduced to NSCs expressing certain cytokines involved in tumor-directed cytotoxic immune activity (e.g., IL-4, IL-12, IL-23) have resulted in prolonged survival.24,30,31 Utilizing the tumor tracking capability of the NSCs combined with the tumoricidal potency of constitutively expressed cytokines is an elegant example of the versatility of NSCs in the treatment of cancer.

Direct Delivery of Cytotoxic Genes TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand), a member of the TNF superfamily, is a cytokine with the unique ability to induce cell death by activating death receptor-mediated apoptosis. TRAIL binds specifically to “death” receptors DR4 and DR5 which are expressed primarily on the surface of aberrant cells, such as tumor cells and has relatively few effects on most normal cells.32 Resistance to TRAIL-induced cell death is thought to be due to expression of either defective or ‘decoy’ cell surface receptors that fail to trigger apoptosis. While previous studies have demonstrated the sensitivity of gliomas to TRAIL33 they have limited clinical relevance due to the toxic effects of administering systemic high doses of TRAIL protein and the inability to specifically target tumor. Utilizing the innate tumor tracking capabilities NSCs couple with gene modification for the expression of TRAIL serves as yet another example of the versatility of NSCs in the treatment of metastatic and, quite possibly, primary neoplasms.

Maintaining Neural Stem Cells In Vitro Neural stem cells have great potential as therapeutic agents for a variety of neuropathologic conditions because they can be readily isolated, expanded in culture and then re-administered into a host animal with great success.1 It has yet to be determined whether it is more advantageous to harvest NSCs from the embryo, fetus, newborn, juvenile, or adult. Thus far, the neurogenic potential seems to be greater in the brain regions of younger animals. Our studies currently utilize murine clone C17.2 NSCs which were initially derived from a neonatal mouse cerebellum and maintained as a single clone.34 In order to ensure that their stem-like state persists in vitro these cells were transduced with the oncogene v-myc which allow for several fundamental advantages. For example, these cells are capable of self-renewal for prolonged passages in vitro and are proven to successfully differentiate into appropriate daughter lineages when introduced into the CNS. Once the C17.2 NSCs divide and exit the cell cycle the ‘stem’ state-promoting gene v-myc is constitutively downregulated. Long-term in vivo studies in immunosuppressed mice have shown that these cells are nontumorigenic and that they differentiate appropriately in response to intrinsic cues when implanted into different brain regions.35 Another advantage of working with C17.2 NSCs is that they are relatively easy to maintain in culture and provide a consistent and reproducible cell source for experiments over a prolonged period of time.3,34 Lastly, these cells can be modified to stably express foreign genes of therapeutic importance in vivo for delivery throughout the CNS. Transplantation studies in animal models using C17.2 NSCs have provided valuable information on the behavior of these cells in vivo and highlighted the therapeutic potential of NSCs in the realm of gene therapy. Intuitively, the use of human NSCs as drug-delivery vehicles is the next scientific leap needed to assess clinical potential of NSCs in the treatment of cancer.

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The use of stem cells in science is a highly contested and volatile subject. Numerous ethical concerns surround the use of stem cells derived from human embryos and have forced scientists to find alternative sources from which to isolate these multipotent cells. In regard to NSCs, studies have determined that neurogenesis occurs throughout adulthood and that the adult brain provides a renewable source of NSCs.16,36 These reservoirs of NSCs exhibit properties comparable to their murine counterparts. Human neural stem cells (hNSCs) have been shown to exhibit tropism towards brain pathology37 and may be optimal candidates for cell-replacement therapy for nervous system disorders. Furthermore, the ability to isolate these cells from the adult human brain raises the possibility of autologous transplantation, which circumvents certain ethical issues surrounding stem cells as well as the inherent pathogenic risks associated with exogenous cell, tissue and organ transplantation. Akin to the murine NSCs, hNSCs can also be transduced by viral vectors in vitro and express transgenes in vivo. In addition, hNSCs have been shown to differentiate into mature neuronal cells when placed into a mouse mutant deficient in certain neuronal cell types.5 Finally, Aboody and colleagues3 showed that hNSCs transplanted contralateral to the tumor site migrate throughout the normal parenchyma using a murine model of glioblastoma multiforme. The potential of hNSCs as vehicles for gene therapy is promising and increased research in this field continues to demonstrate the multi-faceted role hNSCS may play in the treatment of cancer. Below, we describe a protocol outlining a simple method of culturing and passaging hNSCs in hopes of standardizing this technique and increasing reproducibility of human stem cells. The reader is advised, however, to study references relevant to their specific area of interest, because differences exist in techniques used by other groups involved in this type of research. The hNSCs we use were derived from a cadaveric human fetal forebrain and grown primarily as adherent cultures on flasks coated with laminin (although a small percentage of cells may remain in suspension). The culture of hNSCs is increasing in practice and many groups have shown that these cells are functionally stable and exhibit the capacity to self-renew for culture periods up to two years.26,38,39 The promise of utilizing genetically modified human NSCs as vehicles for CNS tumor therapy may prove useful when considering the limitation with current therapies, including surgical resection, irradiation and chemotherapy. Yet, additional studies are necessary before we can examine the true potential of these cells in treating human patients with brain tumors.

Materials and Methods Materials Materials for the Maintenance of Neural Stem Cells (Murine) 1. Neural stem cell medium: Dulbecco’s Modified Eagle’s Medium GlutaMax (DMEM) containing 10% fetal bovine serum (FBS), 5% inactivated horse serum and 1% penicillin-streptomycin; store at 4˚C. 2. 1X Dulbecco’s Phosphate-Buffered Saline without calcium and magnesium (DPBS). 3. 0.05% trypsin/ethylenediamine tetracetic acid (EDTA); store at –20˚C. 4. Sterile DMSO. 5. Falcon tissue-culture treated dishes. 6. Cryogenic vials. Materials for the Maintenance of Neural Stem Cells (Human) 1. Human neural stem cell medium: Dulbecco’s Modified Eagle’s Medium F12 (DMEM/ F12) containing 1% N-2 Supplement, 1% penicillin-streptomycin, 8 +g/ml heparin, 20 ng/ml basic fibroblast growth factor, 10 ng/ml leukemia inhibitory factor and 20 ng/ml epidermal growth factor; store at 4˚C. 2. 1X Dulbecco’s Phosphate-Buffered Saline without calcium and magnesium (DPBS). 3. Trypsin; store at –20˚C. 4. Trypsin Inhibitor, Soybean 5. Sterile DMSO. 6. Fetal bovine serum (FBS),

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7. Falcon tissue-culture treated flasks. 8. Cryogenic vials.

Methods Maintenance of Murine Neural Stem Cells In our lab, NSC cultures are grown as an adherent monolayer and should be maintained in a standard humidified incubator (37˚C, 5% CO2). Once the lines are established they can be carried in 10 cm tissue culture treated dishes. It is important to split cell lines only once a week at no more than a 1:10 dilution. NSCs are contact-inhibited and therefore cultures can become confluent during normal growth periods. However, there are some general guidelines to consider before proceeding with transplantation or injection of NSCs. When working with cells that have been previously frozen, do not use those that have been passaged for more than 6 weeks after the initial thaw. Cultures should be no more than 90% confluent or they will begin to produce an extracellular matrix causing clumping of cells and yielding poor results. If cells do not appear as a single cell suspension then they should not be used for in vivo experiments. The proper confluence can be maintained by splitting cultures at a dilution of 1:10 72-96 hours before transplantation or injection. It is important to plate cells during the last split for immunocytochemical analysis to confirm undifferentiated state at the time of use. Thawing Neural Stem Cell Lines 1. Thaw a frozen vial by placing the vial in a water bath maintained at a temperature of 37˚C for 1-2 minutes. Do not agitate cells. 2. Remove the vial from the water bath as soon as the cells have thawed and decontaminate by wiping the vial down with 70% ethanol. 3. Pipet the contents of the vial (approximately 1-2 = 106 cells/vial) equally into one 10 cm dishes containing 8 mls of prewarmed feeding medium. 4. Rinse the vial two times with 1 ml feeding medium and transfer to the dish. 5. Gently swirl each dish to evenly distribute cells. 6. Place dishes into the incubator. 7. Change the medium once the cells have attached to the plate (approximately 8 hours). 8. Once cells have become confluent they can be trypsinized and gradually expanded as described in the next step. Maintaining Neural Stem Cell Lines 1. Remove and discard culture medium by aspiration. 2. Rinse cell layer twice with 1X PBS (5 mls/10 cm dish) to remove all traces of serum. 3. Add 2 mls of 0.05% Trypsin-EDTA solution to the dish and incubate at 37˚C for 2-5 minutes, then observe cells under an inverted microscope until cell layer is dispersed. (The serum in the medium deactivates the Trypsin-EDTA so this is a crucial step that should not be left out). The dish can be gently tapped to help the cells detach. Cells that are difficult to detach can be placed back in the incubator for an additional 1-2 minutes. 4. Add 3 mls of feeding medium and aspirate cells by gently pipetting. Be careful not to introduce air bubbles while triturating. 5. Transfer 1 ml of the total 5 mls in the 10 cm dish equally (0.5 mls/dish) to two new 10 cm dishes containing 10 mls of prewarmed feeding medium. This should be done in a drop-wise manner to ensure that each dish receives 10% of the original population. Fresh media can be added to the original dish so that the cells will reattach and proliferate as a backup should the split be unsuccessful. Preparing Neural Stem Cells for Transplantation or Injection 1. Remove and discard culture medium from dish. 2. Rinse cell layer twice with 1X PBS (5 mls/10 cm dish) to remove all traces of serum. 3. Add 2 mls of 0.05% Trypsin-EDTA solution to the dish and incubate at 37˚C for 2-5 minutes, then observe cells under an inverted microscope until cell layer is dispersed.

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4. Add 3 mls of feeding medium and aspirate cells by gently pipetting. Be careful not to introduce air bubbles while triturating. 5. Pipet cells into a 15 ml tube and centrifuge for 1 minute at 1000 = g. 6. Remove supernatant by aspiration being careful to not disturb the cellular pellet. 7. Wash the cells by resuspending them in 10 mls of 1X PBS and gently triturate to form a single cell suspension. 8. Centrifuge for 1 minute at 1000 = g. 9. Repeat steps 7 and 8. 10. After the last spin, remove the supernatant by aspiration and resuspend the pellet in 10 mls of 1X PBS and gently triturate cells. Take 10 ul of cell solution and count the number of cells. 11. Centrifuge the cell solution at 1000 = g and resuspend the pellet in the appropriate volume of 1X PBS at a concentration of 4 = 104 cells/ul. 12. Place cells on ice until ready to use. Freezing Neural Stem Cells 1. Make freezing medium (e.g., 67% feeding medium 20% FBS and filter; add 13% sterile DMSO). 2. Remove and discard culture medium from dish. 3. Rinse cell layer twice with 1X PBS (5 mls/10 cm dish) to remove all traces of serum. 4. Add 2 mls of 0.05% Trypsin-EDTA solution to the dish and incubate at 37˚C for 2-5 minutes, then observe cells under an inverted microscope until cell layer is dispersed. Cells that are difficult to detach can be placed back in the incubator for an additional 1-2 minutes. 5. Add 3 mls of freezing medium and aspirate cells by gently pipetting. Be careful not to introduce air bubbles while triturating. 6. Pipet an equal volume of cells into cryogenic vials (approximately 1-2 = 106 cells per 1 ml). 7. Slow freeze cells by first placing vials of cells at –80˚C for 24 hours. Transfer the vials to –140˚C for long term storage.

Maintenance of Human Neural Stem Cells Cells have been successfully grown in tissue culture treated 25 cm2 flasks. We use uncoated flasks; however, precoating with collagen, fibronectin, laminin or poly-l-lysine does not appear to hinder cell growth. Our cells are fed once a week and/or split 1:2 each time.

Thawing Neural Stem Cell Lines The cells are very fragile when coming out of a freeze. Many will die of differentiate during the process, however, we offer a new method of thawing which improves the yield of viable cells. 1. Thaw a frozen vial by placing the vial in a water bath maintained at a temperature of 37˚C for 1-2 minutes. Do not agitate cells. 2. Remove the vial from the water bath as soon as the cells have thawed and decontaminate by wiping the vial down with 70% ethanol. 3. Pipet the contents of the vial into a 15 ml centrifuge tube with 9 ml of prewarmed feeding medium. 4. Centrifuge the tube at 700 rpm for 1 minute. 5. Gently aspirate the mixture of freezing and feeding medium. 6. Resuspend the pellet in a mixture of 50% fresh feeding medium and 50% conditioned feeding medium. 7. Transfer cell mixture to tissue coated 25 cm2 flasks with a total of 8 ml medium. After one or two weeks in culture, the percentage of conditioned medium may be reduced to 25%. Feeding with Trypsinization and Maintaining Neural Stem Cell Lines 1. Dissociate spheres mechanically by trituration.

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2. Transfer the medium into a 15 ml centrifuge tube; centrifuge for 3 minutes at 1000 rpm. 3. Add 1 ml of 0.05% Trypsin-EDTA solution into the emptied flask, mechanically dissociate by trituration any remaining spheres and incubate at 37˚C for 3 minutes. 4. Remove the supernatant from the tube (step #2) and add 0.7 mls of 0.05% Trypsin-EDTA solution. Triturate to break up the pellet and incubate at 37˚C for 5 minutes. Note: Transfer the supernatant (conditioned medium) to a new centrifuge tube and freeze for future use. 5. Add 1.5 ml of Trypsin Inhibitor to the flask and triturate well. 6. Transfer the medium from the flask into the 15 ml centrifuge tube. Rinse the flask twice with medium to collect any residual cells. Centrifuge for 3 minutes at 1000 rpm. 7. Remove the supernatant and resuspend the pellet with 2 ml of conditioned N2 medium and transfer to the flask. Add 4 ml of fresh N2 medium to the flask. Note: If splitting the cells, resuspend the pellet with 4 ml of conditioned N2 medium and distribute equally between two flasks. Add an additional 4 ml of fresh N2 medium to each flask. 8. Add the appropriate volume of bFGF and LIF to achieve the concentrations stated in the Materials section. Preparing Neural Stem Cells for Transplantation or Injection 1. Dissociate spheres mechanically by trituration. 2. Transfer the medium into a 15 ml centrifuge tube; centrifuge for 3 minutes at 1000 rpm. 3. Add 1 ml of 0.05% Trypsin-EDTA solution into the emptied flask, mechanically dissociate by trituration any remaining spheres and incubate at 37˚C for 3 minutes. 4. Remove the supernatant from the tube (step #2) and add 0.7 mls of 0.05% Trypsin-EDTA solution. Triturate to break up the pellet and incubate at 37˚C for 5 minutes. 5. Add 1.5 ml of Trypsin Inhibitor to the flask and triturate well. 6. Transfer the medium from the flask into a 15 ml centrifuge tube. Rinse the flask twice with medium to collect any residual cells. Centrifuge for 3 minutes at 1000 rpm. 7. Remove the supernatant and resuspend the pellet with 10 ml medium. Centrifuge for 3 minutes at 1000 rpm. 8. Remove the supernatant and resuspend the pellet with a small volume of 1X PBS (i.e., 30 +l or double the volume of the cell pellet). Transfer cells to a 2 ml centrifuge tube. 9. Remove 2 +l of cell solution and place into a microcentrifuge tube containing 18 +l of 1X PBS (1:10 dilution). 10. Add 1 +l of Trypan Blue tracer to the microcentrifuge tube and triturate well. 11. Count the number of cells, making sure to factor in the 1:10 dilution. 12. Adjust the volume of 1X PBS in the 2 ml centrifuge tube as needed to achieve the desired concentration of 5 = 104 cells/ul. 13. Place the tube containing the cells on ice until ready to use. Freezing Neural Stem Cells 1. Make the freezing medium as follows: 40% N2 medium 50% FBS and filter; add 10% sterile DMSO). 2. Dislodge cells by scraping or trituration. 3. Transfer the medium containing the cells into a centrifuge tube. Centrifuge for 3 minutes at 1000 rpm. 4. Remove the supernatant and resuspend the pellet with a small volume of N2 medium (i.e., 30 +l or double the volume of the cell pellet). Mix well. Remove 2 +l of cell solution and place into a microcentrifuge tube containing 18 +l of 1X PBS (1:10 dilution). 5. Add 1 +l of Trypan Blue tracer to the microcentrifuge tube and triturate well. 6. Pipet an equal volume of cells into cryogenic vials (approximately 1-2 = 106 cells/ml). 7. Slow freeze the cells by first placing vials of cells at –80˚C for 24 hours. Transfer the vials to –140˚C for long term storage.

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Conclusion Despite advances in cancer therapy, prevention or eradication of metastatic and invasive tumors is limited by the intolerable side effects of systemic therapeutic agents. In addition, it is possible that the brain provides a shielded ‘sanctuary site’ which harbors tumor microsatellites resistant to current clinical treatments. Therefore, the concept of specifically targeting infiltrative brain tumor cells using tumor-selective cell-mediated delivery of therapeutic agents is enticing. The NSC is a promising therapeutic candidate due to the ease of maintaining viable cultures, a proven tumor-tropic behavior, cell stability after genetic modification and a propensity to engraft within the nervous system following transplantation. The application of NSCs as therapeutic agents for the treatment of various human neuropathologic conditions, including brain tumors, is feasible. However, several issues must be addressed before clinical application may be considered: the optimal source from which to acquire NSCs, the tailoring of genetic modification to best treat a wide range of tumor phenotypes and the ability to regulate the activity of the NSCs during and after treatment. We anticipate that a NSC-mediated approach to cancer therapy will serve as an adjunct to current therapies. Likely, it will have the greatest impact on eradicating residual metastatic microsatellites in patients who responded well to conventional therapy but whose likelihood of developing later metastatic disease without NSC treatment is otherwise high. We believe that the use of NSCs in the treatment of metastatic tumors to the brain and other neuropathologic diseases will improve the prognosis and quality of life in patients whose outcome was, traditionally, bleak. On a larger scale, humans posses a variety of multipotent cells from hematopoietic stem cells to gastrointestinal stem cells. Continued developments in the field of human stem cell biology may serve as stepping stones towards a cure for a myriad of human ailments including metastatic cancer to the brain.

References 1. Dunn I, Black P. The neurosurgeon as local oncologist: cellular and molecular neurosurgery in malignant glioma therapy. Neurosurgery 2003; 52(6):1411-22; discussion 22-4. 2. Chang E, Lo S. Diagnosis and management of central nervous system metastases from breast cancer. Oncologist 2003; 8(5):398-410. 3. Aboody K, Brown A, Rainov N et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci USA 2000; 97(23):12846-51. 4. Park K, Ourednik J, Ourednik V et al. Global gene and cell replacement strategies via stem cells. Gene Ther 2002; 9(10):613-24. 5. Flax J, Aurora S, Yang C et  al. Engraftable human neural stem cells respond to developmental cues, replace neurons and express foreign genes. Nat Biotechnol 1998; 16(11):1033-9. 6. Palmer T, Takahashi J, Gage F. The adult rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 1997; 8(6):389-404. 7. Gage F. Mammalian neural stem cells. Science 2000; 287(5457):1433-8. 8. McKay R. Stem cells in the central nervous system. Science 1997; 276(5309):66-71. 9. Park K, Liu S, Flax J et  al. Transplantation of neural progenitor and stem cells: developmental insights may suggest new therapies for spinal cord and other CNS dysfunction. J Neurotrauma 1999; 16(8):675-87. 10. Snyder E, Yoon C, Flax J et  al. Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proc Natl Acad Sci USA 1997; 94(21):11663-8. 11. Yandava B, Billinghurst L, Snyder E. “Global” cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain. Proc Natl Acad Sci USA 1999; 96(12):7029-34. 12. Ourednik J, Ourednik V, Lynch W et al. Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat Biotechnol 2002; 20(11):1103-10. 13. Park K, Teng Y, Snyder E. The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat Biotechnol 2002; 20(11):1111-7. 14. Snyder E, Taylor R, Wolfe J. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain. Nature 1995; 374(6520):367-70. 15. Sanai N, Tramontin A, Quiñones-Hinojosa A et  al. Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 2004; 427(6976):740-4.

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16. Eriksson P, Perfilieva E, Björk-Eriksson T et  al. Neurogenesis in the adult human hippocampus. Nat Med 1998; 4(11):1313-7. 17. Alvarez-Buylla A, Lim D. For the long run: maintaining germinal niches in the adult brain. Neuron 2004; 41(5):683-6. 18. Garcia A, Doan N, Imura T et al. GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat Neurosci 2004; 7(11):1233-41. 19. Doetsch F, Caillé I, Lim D et al. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999; 97(6):703-16. 20. Palmer T, Schwartz P, Taupin P et al. Cell culture. Progenitor cells from human brain after death. Nature 2001; 411(6833):42-3. 21. Schwartz P, Bryant P, Fuja T et  al. Isolation and characterization of neural progenitor cells from postmortem human cortex. J Neurosci Res 2003; 74(6):838-51. 22. Kim S, Cargioli T, Machluf M et al. PEX-producing human neural stem cells inhibit tumor growth in a mouse glioma model. Clin Cancer Res 2005; 11(16):5965-70. 23. Ehtesham M, Kabos P, Gutierrez M et  al. Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2002; 62(24):7170-4. 24. Ehtesham M, Kabos P, Kabosova A et  al. The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma. Cancer Res 2002; 62(20):5657-63. 25. Ourednik V, Ourednik J, Park K et al. Neural stem cells—a versatile tool for cell replacement and gene therapy in the central nervous system. Clin Genet 1999; 56(4):267-78. 26. Vescovi A, Snyder E. Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo. Brain Pathol 1999; 9(3):569-98. 27. Martinez-Serrano A, Rubio F, Navarro B et al. Human neural stem and progenitor cells: in vitro and in vivo properties and potential for gene therapy and cell replacement in the CNS. Curr Gene Ther 2001; 1(3):279-99. 28. Aghi M, Rabkin S. Viral vectors as therapeutic agents for glioblastoma. Curr Opin Mol Ther 2005; 7(5):419-30. 29. Leroy P, Slos P, Homann H et al. Cancer immunotherapy by direct in vivo transfer of immunomodulatory genes. Res Immunol 1998; 149(7-8):681-4. 30. Benedetti S, Pirola B, Pollo B et al. Gene therapy of experimental brain tumors using neural progenitor cells. Nat Med 2000; 6(4):447-50. 31. Hu J, Yuan X, Belladonna M et al. Induction of potent antitumor immunity by intratumoral injection of interleukin 23-transduced dendritic cells. Cancer Res 2006; 66(17):8887-96. 32. Ashkenazi A, Dixit V. Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 1999; 11(2):255-60. 33. Roth W, Isenmann S, Naumann U et  al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem Biophys Res Commun 1999; 265(2):479-83. 34. Snyder E, Deitcher D, Walsh C et al. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 1992; 68(1):33-51. 35. Snyder E, Park K, Flax J et  al. Potential of neural “stem-like” cells for gene therapy and repair of the degenerating central nervous system. Adv Neurol 1997; 72:121-32. 36. Zhao C, Deng W, Gage F. Mechanisms and functional implications of adult neurogenesis. Cell 2008; 132(4):645-60. 37. Jeon J, An J, Kim S et al. Migration of human neural stem cells toward an intracranial glioma. Exp Mol Med 2008; 40(1):84-91. 38. Villa A, Snyder E, Vescovi A et al. Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS. Exp Neurol 2000; 161(1):67-84. 39. Carpenter M, Cui X, Hu Z et  al. In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol 1999; 158(2):265-78.

Chapter 3

Animal Models of Neurological Disease Amol Shah, Tomas Garzon-Muvdi, Rohit Mahajan, Vincent J. Duenas and Alfredo Quiñones-Hinojosa*

Abstract

T

he use of animal models to study human pathology has proved valuable in a number of fields. Animal models of neurological disease have successfully and accurately recreated many aspects of human illness allowing for in-depth study of neuropathophysiology. These models have been the source of a plethora of information, such as the importance of certain molecular mechanisms and genetic contributions in neurological disease. Additionally, animal models have been utilized in the discovery and testing of possible therapeutic treatments. Although most neurological diseases are still not yet completely understood and reliable treatment is lacking, animal models provide a major step in the right direction.

Introduction For years researchers have turned to animal models to recreate neurological pathologies and their subsequent symptoms to understand the mechanisms behind human neurological diseases, as well as possible therapeutic strategies. Animals with short generation times and well-documented genomes that are easily manipulated have provided excellent model systems.1 Rodents are commonly utilized as models for Parkinson’s disease (PD), cerebral ischemia, Huntington’s disease (HD) and Alzheimer’s disease (AD), among others.2 Hypokinetic and hyperkinetic movement disorders have also been successfully modeled in nonhuman primates.3 However, many remain skeptical regarding the use of phylogenetically lower species to study human disease. Whereas genetic and molecular basis of disease can be evaluated in lower organisms, it would seem that pathophysiological symptoms and success of possible treatments be best tested in higher, more closely related organisms such as nonhuman primates. However, different species express different behaviors in response to similar diseases and thus animal model studies should be based on the idea of functional similarity rather than anatomical identity.2 It is this concept that allows for the use of smaller, cheaper and more easily manageable models such as rodents and flies. The following review will cover various animal models of Parkinson’s disease, global ischemia, Huntington’s disease and Alzheimer’s disease, all of which are common neurological pathologies. Future directions will also be discussed briefly.

Parkinson’s Disease Parkinson’s disease (PD) is a progressive neurodegenerative disease that produces motor skill deficits primarily. This is due to loss of dopaminergic neurons in the substantia nigra with decreased dopamine input to the basal ganglia (Fig. 1). PD is characterized histologically by development of cytoplasmic Lewy bodies, abnormal aggregates of _-synuclein and other proteins. Positive *Corresponding Author: Alfredo Quiñones-Hinojosa—Neuroscience and Cellular and Molecular Medicine, Johns Hopkins University, Department of Neurosurgery, Baltimore, Maryland, USA Email: [email protected]

Frontiers in Brain Repair, edited by Rahul Jandial. ©2010 Landes Bioscience and Springer Science+Business Media.

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Figure 1. Photomicrographs of human brain sections immunostained for the dopamine transporter (DAT). Antibodies to DAT have been used as an indirect indicator of dopamine innervations of the striatum. Sections are through the striatum and globus pallidus from a person without PD (A) and from a patient with PD (B). Note the markedly reduced DAT-immunoreactivity in the PD brain section compared to the control. From: Betarbet R, Sherer T, Greenamyre J. Animal models of Parkinson’s disease. Bioessays 2002; 24(4):308-18.

symptoms of PD include tremors and rigidity, while negative symptoms include bradykinesia and hypokinesia. Successful animal models have allowed researchers to associate motor symptoms of PD with nigral dopamine deficiencies, as well as test levodopa (L-DOPA) treatment (Table 1).4 While L-DOPA remains the most effective PD treatment to this date, prolonged treatment can lead to dyskinesia.4 More studies using appropriate animal models will be required to further understand the pathogenic basis of PD and to test possible therapeutic treatments. Desirable models should include many if not all of the following: normal levels of functioning dopaminergic nigral neurons at birth with substantial selective loss at older age, easily detectable and measurable motor deficits, development of Lewy bodies and a short disease course.5

Reserpine Model Administration of reserpine, an indole alkaloid antipsychotic drug that depletes dopamine at nerve terminals in the dopaminergic nigrostriatal pathway, was shown to cause akinesia in rabbits6 and hypokinesia in rodents.7 Treatment of this animal model with L-DOPA improves motor deficits, leading to the hypothesis that PD motor symptoms were caused by dopamine depletion.6 However, the reserpine model is far from perfect, especially considering some drug treatments have been successful in the model, but ineffective in humans with PD. Unlike PD, reserpine treatment does not cause any morphological changes in nigral dopaminergic neurons and induced changes tend to be temporary.4 Nevertheless, this model has been useful in discovering the pathophysiology of PD and testing the therapeutic effectiveness of L-DOPA and other dopamine receptor agonists.

Methamphetamine Model Methamphetamine (METH) is a psychostimulant that triggers cascading release of dopamine, serotonin and norepinephrine possibly by reversing the direction of flow through the respective neurotransmitter transporters.8 Indeed, selective dopaminergic receptor antagonists have been shown to block METH toxicity. When administered at high levels METH is a neurotoxin that

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Figure 2. Pathogenesis of neuronal dysfunction produced by neurotoxins that affect dopamine neurons. The mechanism by which neurotoxins kill dopamine neurons involve mitochondrial dysfunction and oxidative damage. 6-hydroxydopamine (6-OHDA) is taken up by the dopamine transporter and it generates free radicals. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is converted by monoamine oxidase B (MAOB) to 1-methyl-4-phenylpyridinium (MPP–). MPP– is taken up by the dopamine transporter and can then be accumulated by mitochondria, leading to complex inhibition and the generation of free radicals, or by the vesicular monoamine transporter, thus reducing toxicity. Rotenone is a direct inhibitor of complex I, which also leads to free-radical generation. MPTP and rotenone treatment increase the expression of _-synuclein and, in the latter case, this leads to the formation of Lewy bodies.

results in dopamine depletion similar to that found in the reserpine model. Also like the reserpine model, histological changes such as degeneration of nigral dopaminergic neurons in the METH model are minimal, thereby only allowing for mostly biochemical studies of acutely dopamine depleted brains.9

6-OHDA Model 6-Hydroxydopamine (6-OHDA) is a neurotoxin that can enter neurons via catecholamine reuptake transporters and can cause selective degeneration of these neurons (Fig. 2). Neuronal

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death is thought to be caused by toxicity involving generation of reactive oxygen species (ROS). Intracerebral injections of 6-OHDA directly into the dopaminergic nigrostriatal pathway of rodents and monkeys initiates neuronal degeneration within 24 hours and substantial dopamine depletion in 2 to 3 days.4 Bilateral 6-OHDA lesions in rats induce posture abnormalities, poor balance and reduced spontaneous movement—motor symptoms commonly found in PD patients. However, bilaterally lesioned models are rarely used, as they require extensive care. Model animals can be unilaterally lesioned, providing an internal control and preventing need for intensive care. Unilaterally lesioned rats and nonhuman primates develop hemiparkinsonian syndrome, experiencing PD-like motor symptoms on only one side.10-12 This asymmetry provides a simple way to test efficacy of restorative treatments, by allowing direct comparison between the affected side and the contralateral control side. The extent of dopamine depletion can be measured by recording the number of turns made by a unilaterally lesioned animal in response to a dopamine agonist such as L-DOPA.2 After agonist treatment, animals turn away from the side of the lesion, creating an asymmetric rotatory behavior that can be quantified and correlated with the degree of the lesion.4 Although the 6-OHDA lesion model serves as an excellent model for PD the animal is not affected in brain regions outside the injection or and animals do not form cytoplasmic Lewy bodies characteristic of PD).5 Additionally, this model causes acute degeneration of nigral neurons, as opposed to progressive degeneration found in PD.4 Nonetheless, the 6-OHDA model closely resembles human PD and may be used for research and development of protective and/or restorative treatments.

MPTP Model Injection of another neurotoxin, 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), causes similar PD symptoms in a number of species and fulfills most characteristics of an ideal model, making it one of the best PD models to date.13 Once administered, MPTP crosses the blood-brain barrier due to its lipophilic nature and is metabolized into its active ionic metabolite form 1-methyl 4-phenyl 2,3-dihydropyridine (MPP ) by the catecholamine degrading enzyme monoamine oxidase B (Fig. 2). Due to its high affinity for the dopamine transporter, MPP

is selectively taken up by dopaminergic neurons producing toxic effects. The toxicity of MPP

probably arises from its accumulation in neuronal mitochondria inhibiting complex I of the electron transport chain (ETC).14 Inhibition of the ETC causes buildup of ROS and reduced ATP generation.15 In primates MPTP lesions cause nearly all clinical symptoms of PD, including akinesia, tremors, rigidity and postural instability.16,17 Treatment with L-DOPA proved to be successful in this PD animal model. In addition to accurately recreating parkinsonian motor symptoms, the MPTP model is useful in examining the striatal circuitry implicated in PD pathophysiology.18 Particularly, studies have found that MPTP administration greatly increases tonic neuronal activity in the internal globus pallidus, subthalamic nucleus and substantia nigra pars reticulate.19-22 This finding has re-ignited interest in surgical procedures involving stereotactic lesions of the internal globus pallidus as treatment of PD.5 Such procedures have been successful in reducing involuntary movements and reversing motor symptoms of PD.23 Work with the MPTP animal model has shown that mitochondrial dysfunction and oxidative stress have an important role in PD pathology. PD symptoms have been linked to administration of MPTP and rotenone, both of which inhibit complex I of the ETC. Furthermore, low levels of the essential ETC cofactor Coenzyme Q10 (CoQ) were found in the platelets of PD patients, while elevated CoQ levels conferred protection from MPTP toxicity in aged mice.24 CoQ is currently being tested as a possible PD treatment in humans.5 One shortcoming of the MPTP model is lack of Lewy body formation. While Lewy body-like inclusions were observed in MPTP affected neurons of aged primates, they lacked typical features present in true Lewy bodies.25 It is entirely conceivable that chronic rather than acute administration of MPTP may lead to formation of Lewy bodies. As with the other animal models pf PD, the MPTP model is acute and nonprogressive, unlike actual PD. Still, the MPTP model remains an accurate animal model for PD.

Symptoms

Akinesia, catalepsy

No clear parkinsonian symptoms

Unilateral: rotation after apomorphine; Bilateral: akinesia

Akinesia, rigidity, tremor in some species

Decreased locomotor activity

Model

Reserpine

Methamphetamine

6-OHDA

MPTP

Paraquat-Maneb

Acute; limited histopathological change

Generally acute; nonprogressive or reversible; inclusion bodies are rare

Not yet extensively investigated/described

Screen antioxidant therapies to protect dopamine cells Preclinical testing of therapies to improve symptoms; screen pharmacological and genetic therapies designed to protect dopamine cells Preclinical testing of therapies to improve symptoms; screen pharmacological and genetic therapies designed to protect dopamine cells Screen pharmacological and genetic therapies designed to protect dopamine cells

Dopamine-related oxidative stress Oxidative stress

‘Environmental’ toxin; oxidative stress; inhibition of mitochondrial complex I

At very high doses: loss of TH in striatum; ?loss of dopamine cells in SNc

Decreased striatal TH-immunoreactivity; -degeneration of TH-immunoreactive neurons in SNc

Decreased striatal TH-immunoreactivity; degeneration of TH-immunoreactive neurons in SNc; some loss of locus ceruleus neurons; _-synuclein aggregation

Decreased striatal TH-immuno- Multiple environmenreactivity tal toxins/pesticide exposure; ?oxidative stress

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Acute; usually unilateral (hemiparkinsonian); intrastriatal injection may produce more chronic degeneration; requires intracerebral injection

Nonspecific liberation of monoamine transmitters; hypothermia

Disadvantages

Pharmacological dop- Preclinical testing of theraamine depletion pies to improve symptoms

Pathogenic Relevance Applications

None

Histopathology

Table 1. Models of neurological disease

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Akinesia, rigidity, tremor, flexed posture, piloerection

Amphetamineinduced rotation Reduced or abnormal motor activity

Rotenone

3-Nitrotyrosine

Transgenic _-Synuclein

Symptoms

Model

Table 1. Continued

Screen pharmacological and genetic therapies designed to protect dopamine ceils

Screen antioxidant therapies to protect dopamine cells

Expensive and time-consuming; mice do not have characteristic PD pathology or phenotype

Not yet extensively investigated/described; requires intracerebral injection

Labor- and time-intensive; substantial morbidity and mortality

Chronic ‘environmen- Screen pharmacological and genetic therapies designed to tal’ toxin; chronic protect dopamine cells oxidative stress; chronic inhibition of mitochondrial complex I

Decreased striatal TH-immunoreactivity; degeneration of TH-immunoreactive neurons in SNc; some loss of locus ceruleus neurons; inclusions reminiscent of Lewy bodies

Oxidative stress Decreased striatal TH-immunoreactivity; degeneration of TH-immunoreactive neurons in SNc Known pathogenic _-Synuclein-positive intraneumutations ronal inclusions; degeneration of TH-immunoreactive neurons observed in flies; modest decrease in striatal TH-immunoreactivity in mice

Disadvantages

Pathogenic Relevance Applications

Histopathology

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Rotenone Model The development of the rotenone model has brought focus to the role of pesticides and other environmental toxins in PD pathogenesis.26,27 Rotenone, a naturally occurring pesticide in the roots of some plants, functions similarly to MPTP as a specific inhibitor of complex I of the ETC. In rats, chronic exposure to rotenone was found to cause inhibition of complex I throughout the brain.4 This distinguishes this model from the MPTP model, which specifically targets complex I of mitochondria in dopaminergic neurons. In spite of its more generalized targeting, rotenone causes specific chronic degradation of and oxidative damage in the nigrostriatal dopaminergic pathway. This indicates some particular increased vulnerability of this pathway to complex I inhibition. This finding is consistent with other studies showing decreased complex I activity in PD patients and the ability of other complex I inhibitors to instigate parkinsonian symptoms in animal models.5,28 Rotenone treated rats were found to develop hypokinesia, severe rigidity and in some cases, shaking paws similar to parkinsonian resting tremors seen in humans. Additionally, nigral neurons in these rats showed formation of cytoplasmic _-synuclein and ubiquitin inclusions resembling Lewy bodies.4 One of the advantages of this model is that it results in PD-like chronic and progressive degeneration of the nigrostriatal dopaminergic pathway, as opposed to the acute degeneration seen in other models. The major disadvantage lies in the high variability of this model. Only 12 out of 25 rats treated with standardized doses of rotenone showed evidence of lesions.18 Yet, the rotenone model’s ability to cause chronic progressive degeneration of nigral neurons and formation of Lewy body-like cytoplasmic inclusions makes it one of the best models for use in development of restorative treatments such as stem cell implantation.

Genetic Models Commonly marked by early onset, familial PD has been linked to mutations in a few genes, including one encoding for _-synuclein, a major protein component of Lewy bodies.29-32 Models involving overexpression of wild type and mutated _-synuclein in Drosophila and transgenic mice have been used with some success.33-35 Flies overexpressing either wild type or mutated _-synuclein showed a number of PD features, including cytoplasmic inclusions, decreased motor functioning and dopaminergic neuron loss.36 Neuron loss was found specifically in the dorsomedial nuclei and tended to be age-dependent. There are no clear differences between wild type and mutated _-synuclein toxicity. However, it is not yet understood whether motor deficits were due to dopaminergic neuron degeneration. Extensive knowledge of the Drosophila genome makes this model viable for discovery of genes and proteins involved in PD pathogenesis. In transgenic mice, overexpression of wild type _-synuclein led to motor deficits and formation of _-synuclein and ubiquitin inclusions.33 However, unlike the Lewy bodies typically observed in PD, these inclusions lacked fibrillar aggregates and were found in nuclei as well as cytoplasms of neurons.18 Additionally, not all affected mice showed signs of nigrostriatal dopaminergic neuron degradation.4 Still, transgenic mouse models can be successfully utilized to study the role of genetics and environmental mutagens in PD development.

Cerebral Ischemia Over the last 50 years, scientific research has yielded a multitude of agents found to be neuroprotective in numerous animal models of cerebral ischemia (Fig. 3). Unfortunately most of these have been unsuccessful in humans, making the demand for more effective and accurate models even greater. The diversity of ischemic events in humans poses a major challenge for development of animal models. Current animal models include large and small animals which can be used differently. Most methods of physiological monitoring and imaging are more easily done on larger animals. Additionally, the brains of larger animals are gyrencephalic, making them more similar in structure and function to the human brain. However, large animal models are more expensive and difficult to maintain and variability of physiological attributes and infarct size can make using them quite complicated. Small animals on the other hand are less expensive and easier to manipulate. Lines of identical transgenic mice with modified genetics can be created and be used for studies

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Figure 3. Various models of cerebral ischemia. Abbreviations: VO: vessel occlusion; CBF: cerebral blood flow; MCAO: middle cerebral artery occlusion. From: Traystman R. Animal models of focal and global cerebral ischemia. ILAR J 2003; 44(2):85-95.

involving gene and protein expression. The ability to reproduce experiments with low variability attracts many scientists to smaller animal models.37 Presently, rodents serve as the most common model for cerebral ischemia.38

Focal Cerebral Ischemia Models Focal cerebral ischemia is a result of decreased blood flow to a defined region of the brain. If there is reduced cerebral blood flow (CBF) to a number of different specific sites it is termed multifocal. Usually caused by embolic or thrombotic occlusion of a major cerebral artery, focal cerebral ischemia accounts for 80% of all strokes worldwide.38 The middle cerebral artery (MCA) is the most common site of blockage and is the target of a number of animal models. Referred to as middle cerebral artery occlusion (MCAO) models, these models employ different techniques to induce MCA lesions. They vary in permanency (permanent versus temporary) and location (distal versus proximal).39,40 One MCAO model involves injection of homologous clot fragments into extracranial arteries to cause the occlusion of intracranial arteries.37 While this results in reduced CBF and tissue damage seen in ischemic stroke patients, the location of infarction is variable and inconsistent.37 Recently injection of artificial embolic compounds such as collagen and viscous silicone has been used to produce focal ischemia.41,42 Another model involves the insertion of an intraluminal monofilament into the internal carotid artery and advancing it until MCA occlusion is accomplished. This model is advantageous as it can be used to create both permanent and temporary MCA infarctions reproducibly. Reperfusion occurs when the thread is removed and the exact occlusion produced can be varied depending on the shape, size, coating and insertion length of the filament. However, vessel rupture, insufficient MCAO and development of spontaneous hyperthermia slightly detract from the strength of this model.38 The prothrombosis model of cerebral ischemia involves the injection of a photoactive dye such as Rose Bengal and irradiation with a light beam of certain frequency. The resulting formation of singlet oxygen induces endothelial damage and platelet aggregation in affected blood vessels.43 This method can be used to produce inclusions in any specific area of interest and may be used to study potential restorative drugs. Another group of MCAO models involve direct surgical occlusion of the MCA, which requires a craniotomy to access the MCA. While there are a number of surgical models, there are four main features that outline the basis of an ideal model, which include (1) minimal injury

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to the brain, (2) minimal exposure of the brain to the environment, (3) the procedure should be relatively easy to perform and should be adaptable to different species and (4) the model must be able to ensure perfusion.38

Global Cerebral Ischemia Models Global cerebral ischemia occurs when CBF is reduced throughout the entire brain. This in turn initially leads to death of particularly susceptible neurons and if maintained, may lead to complete brain death. Perhaps the earliest and most basic model is decapitation, which does not allow for much manipulation. This model does cause global ischemia and brains from decapitated animals can be frozen or homogenized for use in biochemical studies.44-46 Another model requires the use of a neck tourniquet to constrict blood flow to the brain.47 However, confounding factors such as vagal nerve compression and venous congestion make results variable. This procedure can be done both large and small animals. Studies using this neck tourniquet technique have found that after 4-6 minutes of ischemia animals experience 24 comatose hours, with subsequent normal neurological function recovery.48 Animals experience irreversible neurological damage after 8 minutes of ischemia.49 This model has also been used in conjunction with hypotension in monkeys and cats.50 First arterial blood pressure is reduced to approximately 50 mm Hg and then the tourniquet is inflated. In monkeys this method produced hippocampal damage after 15 minutes of ischemia.51 Another model employs the use of ventricular fibrillation to imitate global cerebral ischemia due to cardiac arrest.52-54 Many researchers have combined this with cardiopulmonary resuscitation (CPR) and occasionally epinephrine injections to revive animals after certain periods of time. Given the difficulty of performing CPR on small animals, this model is usually used on larger animals and is accurate in producing complete global ischemia.50 Disadvantages of this model include the high cost of maintaining large animals and the intensive care required for the animal up to 48 hours after the ischemic event.37 Recently this model has been successfully applied to mice.55 KCl administration to stop the heart followed by mouse-specific CPR procedures after 8-10 minutes of ischemia consistently produced hippocampal and caudoputamen injury in mouse brains.56 Since the ventricular fibrillation model generates body wide ischemia, other models have also been developed to specifically occlude head and thorax cephalic arteries thereby preventing ischemia of noncerebral circulations.37 Another model known as the four vessel occlusion (4-VO) model was created by Pulsinelli and Brierly in 1979 and has become a popular approach to generate forebrain ischemia in rats.50 This method involves two steps spanning two days. On the first day, atraumatic clamps are loosely positioned around each common carotid artery and the vertebral arteries are electrocauterized by way of the C1 vertebra. Then on the second day the common carotid arteries are occluded and ischemia occurs.50 Studies using this model have found striatal neuron damage after 30 minutes of ischemia, hippocampal damage after 3-6 hours of reperfusion and neocortex damage after 1-3 hours of ischemia.57,58 The 4-VO model is successful in producing ischemia in 50-75% of rats, but unfortunately some ischemic variability exists between different rat strains. A similar older model known as the two vessel occlusion (2-VO model) involves bilateral common carotid artery occlusion and systemic hypotension to cause reversible forebrain ischemia.50 Developed by Eklof and Siesjo in 1972, this model is easier to perform and allows for reperfusion unlike the 4-VO model. However unlike the 4-VO model which can be performed on anesthetized or awake animals, the 2-VO model can only be performed on anesthetized animals. Physiologically the 2-VO model tends to selectively damage susceptible neurons, such as the CA1 pyramidal neurons in the neocortex, hippocampus and caudoputamen.59 Another group of models focus entirely on the gerbil, which has been especially useful in animal models for global cerebral ischemia due to their convenient circulatory physiology. Unlike other complex organisms, gerbils lack a posterior communicating artery which connects the carotid and vertebrobasilar artery networks,60 allowing for global ischemia to be produced by bilateral occlusion of the common carotid arteries alone. Such models have proved quite successful, completely stopping CBF in most animals.61,62

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Figure 4. Schematic of a sagittal section of the human showing major sites of neuronal loss in HD. From: Rubinsztein D. Lessons from animal models of Huntington’s disease. Trends Genet 2002; 18(4):202-9.

Huntington’s Disease Huntington’s disease (HD) is a rare autosomal dominant neurological disease caused by a CAG trinucleotide repeat expansion in exon 1 of the Huntingtin gene. The increase in CAG repeats translate into an excess number of glutamine residues near the N-terminal of the Huntingtin (Htt) protein. Mutated Htt (mHtt) protein leads to neuronal damage and death in certain parts of the brain (Fig. 4). Studies show the striatum is the location of most neuronal death, with approximately 57% loss of the caudate nucleus and 65% of the putamen.63 The medium spiny neurons in particular are the most vulnerable and show altered dendrites and spines. HD is progressive and characterized by chorea, or involuntary muscle movements. Other symptoms include loss of motor coordination, bradykinesia and dystonia. Although most HD cases are late onset (ages 35-50), juvenile onset (ages 20) does occur.63 Prevalence varies globally, with incidence of about 1 in 104 in the UK versus less than 1 in 106 in Japan and Africa.63

Mouse Models of HD Mouse models have been used to study not only mHtt function, but role of wild type HD gene and Htt as well.63 Located on the short arm of chromosome 4, the HD gene normally includes a sequence of approximately 35 uninterrupted CAG repeats and production of its gene product (Htt) is asymptomatic. HD arises when the number of CAG repeats increases to 38 or greater.64 There appears to be an inverse relationship between the number of CAG repeats and the age of onset—the more repeats the earlier the onset.63 While 40-50 CAG repeats typically result in late-onset HD, 55 CAG repeats result in juvenile onset.63 Interestingly mouse models have shown that hemizygous loss of one of two wild type HD genes does not result in any HD pathology, indicating that excessive CAG repeats confer some gain of function rather than a loss of function.65-68 Transgenic mouse models in which mice express a mutant HD gene and knock-in mouse models where one of the wild type HD genes is mutated directly support this conclusion.63 Both these models develop many typical features of HD. However, it is certain that Htt is in fact an essential protein, as Htt knockout mice show embryonic death.63,69 Thus it seems that a loss of Htt function plays at least some role in HD development. Htt may be involved in proper vesicle transport and synaptic transmission. It is thought that overexpression of Htt protects cells from

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apoptosis by inhibiting pro-caspase-9 processing.70,71 In mice, overexpression of Htt was shown to protect against cellular toxicity of mHtt in both neuronal and nonneuronal cells lines.72 One of the earliest mouse models for HD was the development of a transgenic mouse expressing exon 1 of the human HD with variable CAG repeat numbers.63 While mice expressing the normal human allele coding for approximately 18 CAG repeats developed normally, mice expressing mutant alleles reminiscent of those found in juvenile onset of HD and coding for 113-156 CAG repeats developed neurological symptoms and motor deficits similar to those found in HD.73 Additionally, nuclear and process inclusions formed by the protein encoded by the mutant allele were comparable to protein aggregates found in cortical and striatal neurons of diseased HD patients.74 The abundance of inclusions positively correlates with the number of CAG repeats as well as likelihood of cell death.75 However it remains to be discovered if neuronal inclusion formation is indeed a prerequisite to HD pathology. Studies in knock-in mice have found insoluble nuclear aggregation of irregular N-terminal Htt fragments indicating that full length Htt may be cleaved near the N-terminal to produce a toxic fragment containing the polyglutamine repeats.76 This proteolytic cleavage of Htt was in fact discovered to occur in human HD brains.77 In addition to providing great insight into HD pathophysiology, mouse models have been used to study possible therapeutic treatments. One such model developed by Yamamoto et al is the conditional mouse model.78 In this model expression of mutant Htt is controlled by a tetracycline-regulated promoter. When transcription of the mutant HD gene was active, mice developed motor deficits and neuronal inclusions, but when transcription was inhibited symptoms regressed.79 This model has provided hope for HD treatment, as it indicates that the disease may be somewhat reversible.

Other Models Other simpler animals have also been used to model HD and have been useful in identifying potential suppressor genes that ameliorate the disease. The use of P-element insertions in Drosophila has allowed creation of flies expressing excess glutamine repeats.79-82 These flies exhibit multiple HD-like symptoms, including inclusion development and neuronal death and suppressor screens in these animals depict the role of improper protein folding and clearance in HD pathophysiology. Current models also now include C. elegans.83

Alzheimer’s Disease Alzheimer’s disease (AD) is a neurodegenerative terminal disease that is the most common cause of dementia in the adult population.84 It is characterized by specific damage to regions of the brain involving memory and cognition, including the hippocampus, amygdala, neocortex and basal forebrain cholinergic system.85 Clinically this damage translates into memory deficits and progressive loss of cognitive functioning. Hallmark histological markers include tau-containing neurofibrillary tangles (NFT) and `-amyloid plaques.86 NFTs consist primarily of hyperphosphorylated isoforms of tau, a microtubule associated protein and tend to aggregate in cell bodies and dendrites of affected neurons.87-93 `-amyloid plaques are aggregates of proteins with `-pleated sheet secondary structure that build up in cerebral gray matter and around cerebral and meningeal blood vessels in AD.94 These plaques are composed of A`, a 40-43 amino acid polypeptide derivative of the amyloid precursor protein (APP).95,96 A Type I transmembrane protein, APP is found in many cell types but is particularly prevalent in neurons.86 Proteolytic cleavage of APP by `- and a-secretase yields the pathogenic neurotoxic peptides A`42 and A`43 along with the more abundant but soluble A`40.97-102 Genetic factors also seem to play a major role in the development of AD. Familial AD (FAD), which has early onset, is an autosomal dominant disorder linked to mutations in APP, presinilin-1 (PS1) and presinilin-2 (PS2).77,103 Late onset of AD is not directly genetically related, although it has been associated with certain alleles of apolipoprotein E4 (apoE).77 Animal models for AD attempt to mimic the many neuropathological changes and symptoms seen in AD, but no model has recreated all aspects of the disease. Still, they have been used successfully to identify potential susceptibility genes,

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disease modifiers and drug treatments.94 Most models today focus on the overexpression of mutant gene products associated with FAD in transgenic mice.104

APP Transgenic Mouse Models One major group of AD animal models are APP transgenic mice. These mice are engineered to express mutations similar to those found in the APP gene of human FAD patients. The first FAD-linked mutation was discovered in London in 1991 and involved an isoleucine substitution for valine at codon 717.94 This mutation, commonly referred to as V717I and others similar to it have been replicated a number of transgenic mouse models (Fig. 5).94 Introduced in 1995, the first successful model used the platelet derived growth promoter-` chain (PDGP-`) to encourage expression of a human APP (hAPP) minigene (PDAPP) encoding a V717F mutated APP.104,105 PDAPP mice were found to display many typical features of AD including `-amyloid plaques, astrocytosis and dystrophic neurites. Interestingly, plaque development occurred first in the hippocampus and then the cortex and increased with age.105 Clinical assessment of PDAPP mice showed age-dependent memory loss positively correlated with plaque formation.106 Mutant hAPP has also been expressed under control of other promoters, including murine mThy-1 and protease resistant prion protein (PrP).107-109 Work with these models depicts plaque formation at only 4 months of age when mutant hAPP is co-expressed with mutant PS1 as opposed to 12 months of age without co-expression.110 These findings indicate that mutations in PS1 and likely PS2 may lead to increase deposition of FAD-linked A`. Such mutations have been found to alter processing of APP by a-secretase to increase A`42 and A`43 secretion.111 Another mutation discovered in two Swedish families involved a double pair substitution of lysine and methionine with aspartic acid and leucine at codons 670 and 671. Known as K670D/M671L or the “Swedish mutation”, this mutation has also been expressed in transgenic mice.94 The Tg2576 line is an example of such mice which expresses the K670D/M671L mutation inserted into a hamster

Figure 5. Schematic showing APP-695, -751 and -770 isoforms (A` resides partially in the ectodomain). Note the _- and ` -secretase-cleavage sites and the positions of APP mutations linked to FAD. Cleavage at residues 40 and 42 is thought to be the result of an endoproteinase, putatively termed a-secretase. A subset of a-secretase cleavages occurs at residues 39, 41 and 43. Single letter abbreviations for the amino acid residues are as follows: A: Ala; C: Cys; D: Asp; E: Glu; F: Phe; G: Gly; H: His; I: Ile; K: Lys; L: Leu; M: Met; N: Asn; P: Pro; Q: Gln; R: Arg; S: Ser; T: Thr; V: Val; W: Trp; and Y: Tyr. Price D, Sisodia S, Borchelt D. Genetic neurodegenerative diseases: the human illness and transgenic models. From: Science 1998; 282(5391):1079-83.

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PrP cosmid vector. This model showed substantial amyloid deposition along with learning and memory impairment starting as early as 9 months of age.94 Another similar model known as the APP 23 line uses an mThy-1.2 promoter to control expression of the K670D/M671L mutation. These mice showed seven-fold overexpression of APP with plaques appearing at 6 months of age.94 APP23 mice have also been used to study the cholinergic system, whose depletion has been implicated in AD development. Findings based on APP23 lesion studies suggest that the cholinergic deficit seen in AD patients is likely due to a combination of neocortical amyloidosis and loss of cholinergic basal forebrain neurons.112 Unlike the other models listed above which show no neuronal loss, APP23 mice show a significant decrease in CA1 pyramidal neurons, making it perhaps a more accurate model for AD.113-116 Another major difference between the models relates to the type of plaque formation. Whereas transgenic mice with mutant hAPP expressed under the control of the PDGF-` promoter show diffuse plaque production, mice with mutant hAPP expressed under the control of mThy-1 or PrP promoters show more mature plaque production.105,108,109,117,118

Tau Transgenic Mouse Models While APP transgenic mouse models have successfully recreated multiple neuropathological aspects of AD, modeling of tau NFTs has proved more complex. Transgenic mice overexpressing the longest human tau isoform under control of the human hThy-1 promoter showed AD-like symptoms and somatodendritic localization and hyperphosphorylation of tau.119 Enlarged axons with neurofilament- and tau-immunoreactive spheroids were also present in the spinal cord, likely due to had high levels of transgene expression in motor neurons.94 These mice also showed signs of muscle weakness. However, no NFTs formed until mice became extremely old,120 indicating that while overexpression of tau isoforms may lead to tau hyperphosphorylation and cerebral and spinal axonopathy, it may not be responsible for formation of NFTs.121 After mutations in tau were identified in 1998, a number of transgenic mouse models overexpressing mutant tau were developed. A number of these models have successfully replicated formation of NFTs in mouse neuronal and glial cells.121-128 Transgenic mice overexpressing the tau P301L mutation showed accelerated tau filament accumulation in the brain and spinal cord along with motor and behavioral deficits.122 Crossed models between APP transgenic mice and tau transgenic mice developed amyloid plaques but showed limited neuron damage and NFT formation.121 Two triple transgenic mice models have also been developed, one expressing mutant APP, PS1 and tau and the other expressing mutant APP and PS1 but wild type tau.129,130 In the former, A` accumulation occurs in the cortex and then the hippocampus while tau filament pathology in humans starts in the hippocampus and advances to the hippocampus with aging.129 It was determined that A` accumulation developed before tangle pathology. Mutant tau transgenic mice have also been used to study the role of the prolyl isomerase Pin1.94 Pin1 activity induces the phosphoThr231-Pro tau motif to make a conformational change, promoting tau dephosphorylation and restoring normal tau function.131,132 Studies of Pin1 expression in transgenic mice with the P301S mutation indicate an inverse relationship between Pin1 expression and neuron vulnerability in AD brains.133 Additionally, Pin1 knockout mice showed progressive age-dependent motor and behavior disturbances.122,134-136 These models have been able to recreate NFT pathology seen in AD. However, there is still much room for improvement.

Conclusion Animal models have been extremely useful in the study of neurological disease. They have been used to dissect disease pathogenesis at the molecular and genetic level, as well as develop novel therapeutic strategies, including preventive and restorative drug treatments. While many models exist today, many more continue to be developed and modified in order to accurately represent and recreate human pathology. Still, given the varying nature of animal and human genetics and environment it is unlikely that one single model will ever entirely depict a human disorder. Nonetheless, animal models have provided amazing insight into disease pathology and will continue to serve as invaluable tools in the quest to treat human disease.

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Chapter 4

Stem Cell Transplantation Methods Kimberly D. Tran, Allen Ho and Rahul Jandial*

Abstract

J

ust a few short years ago, we still used to think that we were born with a finite number of irreplaceable neurons. However, in recent years, there has been increasingly persuasive evidence that suggests that neural stem cell (NSC) maintenance and differentiation continue to take place throughout the mammal’s lifetime. Studies suggest that neural stem cells not only persist to mammalian adulthood, but also play a continuous role in brain tissue repair throughout the organism’s lifespan. These preliminary results further imply that NSC transplantation strategies might have therapeutic promise in treating neurodegenerative diseases often characterized by isolated or global neuronal and glial loss. The destruction of neural circuitry in neuropathologies such as stroke, Parkinson’s disease, MS, SCI prevents signals from being sent throughout the body effectively and is devastating and necessitates a cure.NSC transplantation is among one of the foremost researched fields because it offers promising therapeutic value for regenerative therapy central nervous system (CNS) diseases. Both chemotropic and exogenous cell graft mechanisms of CNS repair are under review for their therapeutic value and it is hoped that one day, these findings will be applied to human neurodegenerative disorders. The potential applications for NSC transplantations to treat both isolated and global neurodysfunction in humans are innumerable; these prospects include inherited pediatric neurodegenerative and metabolic disorders such as lysosomal storage diseases including leukodystrophies, Sandhoff disease, hypoxic-ischemic encephalopathy and adult CNS disorders characterized by neuronal or glial cell loss such as Parkinson’s disease, multiple sclerosis, stroke and spinal cord injury.

Introduction Just a few short years ago, we still used to think that we were born with a finite number of irreplaceable neurons. However, in recent years, there has been increasingly persuasive evidence that suggests that neural stem cell (NSC) maintenance and differentiation continue to take place throughout the mammal’s lifetime. These processes are most notably observed in cases of inflammation and ischemic or traumatic brain neural injury.1 The direct control mechanism governing neurogenesis and gliogenesis is not yet fully understood; thus, the cellular and molecular signals that are suspected to govern these processes (i.e., cytokines, chemokines, metalloproteases and adhesion molecules) are all under heavy investigation.2 There are many reasons to be hopeful that these investigations might prove fruitful for therapeutic brain repair strategies. In the animal model multiple sclerosis (MS), called chronic experimental autoimmune encephalomyelitis (EAE) and characterized by brain inflammation and global demyelination, Picard-Riera3 and Brundin4 both showed that self-renewing progenitor cells, residing in the subventricular zone (SVZ) of the brain or in the subependymal layer of the spinal cord, could ultimately change their physiological fate in the presence of disease. Instead of traveling down the *Corresponding Author: Rahul Jandial—Division of Neurosurgery, City of Hope Comprehensive Cancer Center & Beckman Research Institute Duarte, California, USA Email: [email protected]

Frontiers in Brain Repair, edited by Rahul Jandial. ©2010 Landes Bioscience and Springer Science+Business Media.

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rostral migratory stream (RMS) to the olfactory bulb or to the lateral columns of the spinal columns5,6 progenitor cells instead migrated towards sites of demyelination and differentiated primarily into glial cells. The same alteration in cellular destiny was equally observed in experimental models of spinal cord injury (SCI) and ischemic stroke. Within one week of the traumatic event, endogenous neural stem cells residing near the injury sites had been found to migrate to the injured area borders and promoted neurogenesis in direct support of the neural tissue repair.1,2,7 These studies suggest that neural stem cells not only persist to mammalian adulthood, but also play a continuous role in brain tissue repair throughout the organism’s lifespan. These preliminary results further imply that NSC transplantation strategies might have therapeutic promise in treating neurodegenerative diseases often characterized by isolated or global neuronal and glial loss. The destruction of neural circuitry in neuropathologies such as stroke, Parkinson’s disease, MS, SCI prevents signals from being sent throughout the body effectively and is devastating and necessitates a cure.8 NSC transplantation is among one of the foremost researched fields because it offers promising therapeutic value for regenerative therapy central nervous system (CNS) diseases. From previous studies, NSCs have been observed to respond to CNS insults.9 However, oftentimes this endogenous NSC response is not enough to counteract the severe injury inflicted on the CNS; therefore, it is in this case that exogenous NSCs may be useful to supplement the endogenous NSC pool and aid in the preservation of existing host circuitry and also in the replacement of diseased or disordered cells. Demyelination often plays an important role in both genetic metabolic disorders (i.e., leukodystrophies) and acquired neurodegenerative processes including traumatic, infectious, asphyxial, ischemic and inflammatory insults to the CNS. While not yet translated to human clinical trials, the ability of NSCs to remyelinate diseased neurons in host organisms has already been shown by Yandava10 and colleagues with the model shiverer (shi) mouse. The shi model suffers from a congenital tremor due to a deletion mutation of its myelin basic protein Mbp gene. When NSCs were transplanted into the ventricles of neonatal shi mice, up to 40% remyelination was observed in some recipients and in several test subjects, a visible decrease in the shi mice’s characteristic tremor was observed.10 Similar results were successfully reproduced with human oligodendrocyte precursors,11 thus reconfirming NSCs’ potential value to be used to globally replace cells in the CNS. We know that transplanted NSCs are able to participate in normal brain development and to either persist as undifferentiated stem cells, or to differentiate in response to local environmental cues into neurons, astroglia and oligodendrocytes.12-14 All this would be meaningless, however, if the exogenous cells did not integrate with host cells and instead formed their own isolated neural circuitry pathways. Fortunately, this is not the case and the ability of transplanted NSCs to functionally integrate with host neural circuitry was confirmed by electrophysiological recordings in slice cultures of engrafted brains.14,15 While researchers originally looked to neural stem cells’ ability to replace dying cells as their primary mechanism of treating neuropathologies, we now know that NSCs also express neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF) and brain-derived neurotrophic factors (BDNF),16,17 which may play an even greater role in circuit repair by altering the local chemical environment. The NSC-regulated environment is thought to enhance host regenerative processes such as angiogenesis and migration, while inhibiting destructive processes such as apoptosis, scarring, inflammation, excitoxicity and oxidative stress. This mechanism of preserving existing host cells through the dispersal of chemical signals is especially important when considering disorders that affect the entire CNS and not just one isolated region. Both chemotropic and exogenous cell graft mechanisms of CNS repair are under review for their therapeutic value and it is hoped that one day, these findings will be applied to human neurodegenerative disorders. The potential applications for NSC transplantations to treat both isolated and global neurodysfunction in humans are innumerable; these prospects include inherited pediatric neurodegenerative and metabolic disorders such as lysosomal storage diseases including leukodystrophies, Sandhoff disease, hypoxic-ischemic encephalopathy and adult CNS disorders characterized by neuronal or glial cell loss such as Parkinson’s disease, multiple sclerosis, stroke and spinal cord injury.

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Factors When discussing stem cell transplantation methods, it is important to not only introduce the techniques and protocols, themselves, but also to determine the ideal combination of supporting factors which play an equally vital role in determining graft success. These factors include the ideal cell source for transplantation, the appropriate cell modifications before transplantation, the experimental subject animal, the transplantation method and route of cell administration and finally optimization parameters and measurable endpoints must be considered in order to test the efficacy and level of integration of exogenous NSCs within the host organism. These topics will be discussed in depth here.

Choosing the Ideal Cell Source There are a variety of properties that make NSCs conducive to therapeutic uses. Principally, NSCs are a homogenous, well-defined cell population able to be stored and expanded and they have been shown to self-renew through many passes without changes in morphology. NSCs can be genetically manipulated to deliver therapeutic proteins or trophic factors directly to the site of injury or can be dispersed throughout the vascular system to treat widespread neuronal damage throughout the entire body. The cells themselves are multipotent and will differentiate into neurons, astrocytes and oligodendrites in response to respective deficiencies in the host CNS. They can integrate into the developing host brain through competition and interdigitation with host cells,9 or they can selectively target and replace degenerating neural tissue in response to local neurogenic and chemotropic factors. Logistically, they are a feasible and practical solution because NSCs can easily be introduced into the host tissue and irradiation is not required prior to transplantation (as it the case in bone marrow transplants). Besides their previously described effects within the CNS, NSCs are also thought to be immunoprivileged—they lack the cytochemical markers of differentiated cells and thus do not generally trigger an immune response rejecting engrafted cells. The implications of these properties are noteworthy because NSCs theoretically have the potential to provide an unlimited source of undifferentiated cells that could be induced to differentiate into any type of cell needed by a diseased individual. The ideal source of neural stem cells has been a much debated topic. The most important properties considered when judging the utility of a potential NSC source are the cells’ plastic and self-renewing capabilities. NSCs must be able to adapt in response to the varying environmental conditions to which they are exposed in order to restore function within the host circuitry. A NSC population must also be expandable without changes in morphology in order to generate a large number of clonally related progeny of neuronal, astrocytic, oligodendrocytic lineage.8 To date, the most research has been performed with adult and embryonic neural stem cells. While other sources such as multipotential NSCs from skin,18 bone marrow stromal cells19-22 and cells from umbilical cord blood23 have been shown to demonstrate some “stem-like” properties, we will focus on the vast majority of studies which cite the use of adult and embryonic NSCs.

Adult Neural Stem Cells In humans, adult neural stem cells (aNSC) have been shown to engage in neurogenesis and gliogenesis within restricted areas of CNS throughout adulthood.8 In vitro, they are capable of expansion and develop into precursor neural cells capable of neural cell replacement and repair.24-26 They are isolated from fetal and adult human brains and can be propagated and maintained for years in stable media. In this way, they provide an important renewable stem cell source for transplantation.27,28 Their growth factor (GF)-dependent expandability, stable growth rate, self-renewal and multipotent capabilities and functional plasticity all make aNSCs a very good source of stem cells.24,26,29,30 Vescovi and colleagues26 demonstrated that plasticity could be determined by growth factors; culturing cells in media containing leukemia inhibiting factor (LIF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin (NT)-3, NT-4, sonic hedgehog (Shh) and fibroblast-derived growth factor (FGF)-9 produced a neuronal fate in 40%-60% of cells, while bone morphogenetic proteins (BMPs), CNTF and LIF media produced astrocytes.24,31,32 In experiments

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where aNSCs were transplanted intraparenchymally or intrathecally (injection into the cerebrospinal fluid around the spinal cord) into healthy rodents, aNSCs survived the transplantation into the host CNS and showed predictable patterns of tissue integration and differentiation into neuronal cells.33-35 However, in experimental models of autoimmune, chemical, or traumatic CNS demyelination, aNSCs transplanted intraparenchymally, intracerbroventricularly, or intravenously migrated selectively to damaged sites and differentiated into myelinating oligodendrocytes.8,36-39 These results underline aNSCs’ potential as a source of cell replacement in neurodegenerative disorders. Equally of note, in experimental transplantations, the introduction of aNSCs into both healthy and diseased rodents did not produce teratomas, therefore suggesting a minimal tumorigenic potential of aNSCS40 which is an important difference from embryonic stem cells (ESCs) which we will examine next.

Embryonic Stem Cells Embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst-stage embryos. Though embryo-derived neural cells have not yet been consistently used for transplantations,28,41,42 they are especially of interest because they are thought to be totipotent, meaning they can give rise to all three embryonic germ layers and therefore theoretically differentiate into all the cell types of the body. ESCs hold great promise for clinical trials; however, due to ethical reasons, the source of ESCs is highly regulated and restricted to cell lines from supernumerary embryos (in vitro fertilization) or embryos designated specifically for research.43 46 ESCs theoretically would provide an endless supply of NSCs as they have been found to be immortal under conditions with leukemia inhibitory factor (LIF)47 49 and can be induced to differentiate in vitro into most cell types.48,49 Totipotent ES cells have been used in experimental transplants,50,51 but no consistent data on ES-derived, lineage-restricted cells has yet been reported. In a murine model of brain and spinal cord demyelination (EAE), injected ES cells differentiated into glial cells and remyelinated the damaged axons; however, heterologous tissues and teratoma formation was widely observed within the transplantation. This tumorgenic disadvantage of ES cells is thought to derive from unregulated differentiation52-55 and to overcome this obstacle, current protocols have been designed to generate a high percentage of type-specific neural progenitors in vitro.48,56-58 In comparison, we cannot yet empirically determine whether aNSCs or ESCs have better therapeutic value. While aNSCs have been shown to be robust in surviving transplantation procedures and integrate easily without any tumor formation, they are limited by their multipotency and inability to expand exponentially in vitro. This drawback is an extreme disadvantage as large numbers of cells are needed for stem cell replacement strategies. ESCs, on the other hand, are pluripotent and are easily expanded in culture; however, their totipotency makes ESCs highly susceptible to teratoma formation and until this issue is resolved, ESCs cannot reliably produce safe results for humans. Human ESCs are further hampered by the question of ethics surrounding the source of ESCs and the fate of the donor embryo. While long-term studies on immunorejection drugs have not yet been produced, mice who were not on immunosuppressants were not found to have any more adverse effects than those who were on immunosuppressants, suggesting that both types of NSCs integrate well enough to escape an immune-rejection response.59 In the end, no matter what the source, researchers must be able, first and foremost, to reproduce their results. Therefore, it is highly recommendable to choose a cell line that is easily obtained, maintained and expanded to produce an abundance of cells. Lee and colleagues9 cited the NSC line C17.2 as an ideal cell line. This line is immortal and has been used in studies since the early 1990s. It is homogenous and can be expanded to large quantities while still maintaining predictable engraftment patterns. This property is especially important where consistency is needed in experiments with many subject animals.9 Additionally, C17.2 has a long successful history in transplantations with the proven ability to integrate and differentiate in vivo.10,60-62 Finally, C17.2 has been engineered to quintessentially express lacZ, providing an invaluable way to track the integration and movement of NSCs in the host, posttransplantation. Neurosphere cultures can also be used and transplanted into experimental models; however, they tend to have a high cell death rate with dissociation and are generally more heterogeneous than a

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Figure 1. Neurospheres of human embryo stem/progenitor cells (NPC) prior to dissociation and transplantation. After 14 days of preceding cultivation, progenitors of both neurons and glia cells, revealed immunohistochemically by means of antibodies to ` -tubulin and GFAP, can be observed in free-floating neurospheres. Scale bar, 50 +m. Used with permission from Alexsandrova M et al. Brain Res Dev Res 2002; 134(1-2):143-148.93

cell line (which increases likelihood of teratoma formation). They tend to lose their multipotency quickly (limiting their therapeutic value) and are in general more expensive to maintain. Like in monolayer cultures, one should choose neurosphere cells with both a proven history of stable integration patterns as well as a marker for identification post engraftment (see Fig. 1).9

Method of NSC Isolation Mouse and human aNSCs are each isolated from the ventricular zone (VZ) of the fetal telencephalon, or from the subventricular zone (SVZ), the subgranule layer, or the cortical parenchyma of the adult brain.9 The polyclonal population is then left to propagate in 10% FBS-supplemented medium up to three weeks or until a stable monolayer culture is formed.9 This step helps ensure the health of the cells. Then, the heterogeneous amalgam is subjected to a serum-free serial growth factor selection procedure which alternately tests the cells for their dual-responsiveness to both epidermal growth factor (EGF) and fibroblast growth factor (FGF). These two factors are highly specialized indicators of neural stem cell lineage; therefore, after 3-4 rounds of passage and transfer alternately between the two mediums containing each individual growth factor, we are left with a small, relatively homogenous NSC population.13 This monoclonal culture can be expanded and maintained under defined conditions in a medium containing bFGF, EGF and LIF.9 As previously described, ES cells can also be isolated from the inner cell mass of a blastocyst and then expanded. This source maximizes proliferation while minimizing the selection and senescence of cells.9

Preparing NSCs before Transplant To ensure the efficacy of the graft, murine NSC transplant cells should never be more than eight weeks old after thawing. Lee and colleagues9 found that the most successful engraftments resulted from NSCs that were transplanted in their undifferentiated, active log growth phase because they were most receptive to environmental cues at this time.9 They further found that postmitotic NSCs did not engraft nor migrate satisfactorily and did not yield consistent results.9 At the time of harvest, the culture should not exceed 90% confluency, as higher confluency produces morphological changes and aggregation which subsequently yields poor results. It should be noted, however, that 90% is subjective to the observer and thus experience and consistency on

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the part of the experimenter, herself, must be taken into account. Cells should be in a free flowing, well-dissociated, single-cell suspension as Lee and colleagues9 have observed that cells in clusters greater than 10 cell diameters do not easily disaggregate and yield unreliable or uninterpretable results.9 Cell clumps that are injected directly into the parenchyma can cause hydrocephaly due to the abnormal buildup of cerebrospinal fluid in brain ventricles, thereby increasing intracranial pressure and causing irreversible brain damage in the host cranium. Mice who will eventually develop this disorder are not identifiable until a few weeks after transplantation, so it is best to avoid transplanting cell aggregates altogether.9 Prior to transplantation, it is also very useful to label the exogenous NSCs. Labeling allows for the identification and tracing of NCSs in the rodent brain posttransplantation. In many protocols, trypan blue is included in the injection solution to provide an immediate estimation of the location and initial success of the graft and to rule out damaged mice early on.9 However, in order to have long-term indications of engrafted cell viability, other markers, such as Bromodeoxyuridine (BrdU), lipophilic dye (DiI) (Molecular Probes), green fluorescent protein (GFP), or lacZ are incorporated into the cell DNA and are thus continually expressed throughout the progeny of exogenous cells. BrdU is incorporated into the cell DNA during the S phase and is generally added to the culture medium approximately 48 hours before transplantation.9 It can later be detected using anti-BrdU fluorescent tagged antibodies and is useful in determining the number of engrafted cell divisions in vivo, post transplantation. BrdU is also commonly used to differentiate between donor and host cells, especially in the case of different species (i.e., human NSCs in primate host tissue).9 DiI is often used in preclinical models of brain cancer. Although it has a weak fluorescence in water, DiI exhibits strong fluorescence and photostability when inducted into a membrane. DiI’s advantage lies, therefore, in its ability to be very easily detected after tissue sectioning. Cell DNA can also be engineered to quintessentially express vital markers such as GFP or lacZ. LacZ encodes for beta-galactosidase (beta-gal), which can be detected in an anti-beta-gal immunocytochemistry reaction. As a final step, the ability of cell lines to engraft, migrate, differentiate and express their label should be verified before transplantation. In vitro, the prospective donor cells should express characteristic markers of undifferentiated cells such as EGF and FGF and should then differentiate upon the addition of specific induction factors into their respective cell fates.13 They should also express their label. As a functional, in vivo test, cells should be injected into the ventricles and cerebella of neonatal mice, then after 3-4 weeks, these transplanted mice should be sacrificed to verify the migratory and differential abilities of the donor cells. NSC lines should generate neurons in olfactory bulb, glia in the cortex and granule neurons in the cerebellum. Cells which pass all of these restrictive conditions have a high probability of successful engraftment and integration with the host cells and should be expanded, used immediately and frozen for future use.9

Choosing the Experimental Animal Since NSC therapy has yet to be approved for human clinical trials, experimental animals must be used. Mice are generally used for several reasons. First, as mammals, they share many homologous features with humans which will be very important in years to come when these studies are translated into clinical trials. Since the murine genome has already been sequenced, one can easily correlate the murine genes and their human homologs. Additionally, we now have engineered many murine lines expressing specific models of human disease or CNS insult such as lesions, tumors, parkinsonism and global demyelination (as observed in multiple sclerosis and many congenital metabolic disorders). Logistically, mice are small, inexpensive and easily maintained. They have quick gestations and high pregnancy rates resulting in higher and more quicker yields than in other animal models. Lastly, mice can be exploited in a manner often considered unethical in humans and more highly developed animals.

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Choosing the Surgical Procedure When considering the surgical transplantation method, it is imperative to understand the mechanism of both disease and recovery in order to determine the best possible modality and location of NSC administration. For example, in an experiment in which umbilical cord blood was administered either intravenously or intraparenchymally in rodents who had suffered a stroke, the recipients of the IV injections showed longer lasting recovery than those who receive intralesional injections.63 The authors suggested that the cord blood cells aided in recovery by not only replacing lost cells, but also by inhibiting inflammation at the injury site.64 The transplanted cells were observed to not only migrate to the site of brain infarction, but also to the spleen where they altered the spleen function.65 These results offer important implications for diseases which involve extensive neurological damage within the CNS as well as a strong interaction with the peripheral immune system which may mitigate brain damage and aid in recovery. Therefore, to best treat a neurological or congenital metabolic disorder, one must fully understand the mechanism by which the disease attacks to best choose a route of administration. In focal CNS disorders, such as Parkinson’s disease or spinal cord injuries (SCI), intralesional administration of NSCs are perhaps best suited to treat local cell deficiencies. ES-cell-derived neuronal precursors have been shown to induce dopaminergic neuron differentiation in the substantia nigra to treat parkinsonism in rodent model brains.66,67 In SCI or experimental ischemic rodent models, intralesionally-transplanted aNSCs integrated with host tissue, differentiated preferentially into glial cells, released neurotrophic growth factors (i.e., BDNF, glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF)) and facilitated neurogenesis while inhibiting astrocytosis (the abnormal destruction of nearby neurons, generally caused by hypoglycemia or oxygen deprivation). Overall motor recovery was observed.17,68-70 In another experiment involving chemically demyelinated rat spinal cords, ES cells not only integrated and differentiated within the lesion, but in fact were found to have migrated up to 8 mm away from the lesion border, differentiating into astrocytes, oligodendrocytes and neurons.50,71,72 Introduction of stem cells directly into the brain parenchyma is most therapeutically applicable in cases of local brain insults; however, heterotopic migration or dispersal could lower therapeutic efficacy. On the other hand, multifocal CNS disorders, such as amyotrophic lateral sclerosis (ALS), MS, or Alzheimer’s disease are characterized by global inflammation and demyelination throughout the entire nervous system and are thus perhaps best treated by delivering NSCs intravenously or directly into the cerebrospinal fluid circulation to better facilitate distal cell delivery.8 NSCs travel universally throughout the vascular system and reach multiple inflamed areas in the brain and spinal cord by specific homing in on target cues. Evidence suggests that aNSCs express a variety of inflammatory molecules such as adhesion molecules, chemokines, cytokines and chemokine receptors8,73 which organize the migration and differentiation of precursors during development.73-75 These molecular signals support aNSC interactions with endothelial and ependymal cells surrounding inflamed brain tissues74,76,77 and create a chemoattraction gradient78,79 that is cited as the main mechanism through which NSCs target inflamed areas of the CNS. In an EAE model (a murine model for human brain inflammation and MS), aNSC intravenous administration resulted in the functional recovery of myelin sheaths throughout the model through the selective homing to inflamed brain and spinal cord regions via membrane expression of CD44 and (VLA)-4.8 These two molecules played a vital role in the specific homing capabilities of encephalitogenic lymphocytes in EAE; therefore, Pluchino’s results suggest that aNSCs might follow a similar molecular signal pathway in order to migrate to inflamed areas.8

Potential Routes of NSC Administration Protocols for the transplantation of NSCs in midgestational, neonatal and adult mice have been previously published in detail by Lee and colleagues9 as well as by Willing and colleagues.80 These protocols will be very briefly summarized and compared here.

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Figure 2. Transillumination of anesthetized newborn mouse pup head immediately following bilateral intracerebroventricular injection of NSCs mixed with trypan blue. A) Orientation of fiber optic light source, mouse pup and investigator. B) Close-up view of transilluminated mouse pup head showing location of injected cells mixed with trypan blue. The injectate often fills as far rostrally as the olfactory bulbs and as far caudally as the third ventricle and cistern (allowing the cells access to brainstem and high cervical spinal cord).9 A color version of this image is available at www.landesbioscience.com/curie. From: Neural stem cell transplantation in mouse brain. Curr Protoc Neurosci 2008; 3:Unit 3:10. Reprinted with permission of John Wiley and Sons, Inc.

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Neonatal There are many advantages to using newborn mice for transplantation. They are easily handled and injected as their soft, translucent skulls facilitate penetration and easy visualization of engrafted cells after transplantation. NSCs tend to have better survival rates and more extensive migration when injected into neonatal mouse brains than in the case of adult mouse brains. Additionally, no preconditioning regimens (such as irradiation) are required.9 In this procedure, newborn mouse pups are transplanted within their first three days after birth. They are cryoanesthetized briefly on wet ice for three minutes. Then, guided by transillumination of the mouse head, a free-flowing single-cell suspension of NSCs labeled with trypan blue is injected bilaterally into the lateral ventricles of the cerebrum using a drawn glass micropipette. This injection method takes about 10-20 seconds per pup and gives NSCs direct access to the subventricular zone (SVZ), which has proven to be an important source of new neurons during brain development.9 The pup is returned immediately to maternal care and is given a heating pad to help it re-achieve its body temperature within 5 minutes. The pups generally do not seem overly stressed, nor do they lose any blood or cerebrospinal fluid (CSF) during the procedure. The trypan blue labeled suspension is visible immediately after surgery under transillumination and the NSCs are incorporated into the parenchyma within the first 24 hours as they do not have to cross the blood-brain barrier. This method is highly efficient and accurate, with Lee9 maintaining that an experienced technician would be able to perform the procedure in less than 5 seconds per pup with 100% accuracy (see Fig. 2).9

Midgestational In Utero NSC transplantations to mouse embryos are performed on embryonic day 13.5 (E13.5) during active cortical neurogenesis. This early administration of corrective NSCs is thought to be more effective in preventing the irreversible CNS damage caused by many congenital and early-onset neurodegenerative diseases such as Tay-Sachs and Sandhoff disease. Like in neonatal transplantations, the fetal skulls are incompletely formed at this stage of development and do not offer any resistance to the needle. Early transplanted cells migrate and integrate very quickly and effectively with the host tissue such that very few immunorejection complications are observed. Therefore, in general, the use of immunosuppressants is not required.9 The pregnant mouse is anesthetized using continuous isoflurane inhalation, then the uterine horns are exposed and the embryos located by transillumination. Again, guided by transillumination, a drawn glass pipette is used to unilaterally inject the NSC-trypan blue suspension through the thin uterine wall and into the telecephalic vesicles of 1/3 to 1/2 of each litter. We only inject a limited number of embryos because out of the 6-10 embryos present in the uterus in some mouse strains, generally only 3-4 are actually born. We therefore also need a label to differentiate embryos that were and were not injected with donor cells. The unilateral injection technique is used to minimize trauma and still is equally as effective since at this early stage of development, the two ventricles are still very well connected and material passes freely between them.9 Each embryo should be handled for no more than 10 seconds and the entire procedure should not last longer than 30 minutes to enhance survival. Ringer’s irrigant is used during the operation and contains penicillin and streptomycin; therefore, postsurgical infections are rare and supplemental antibiotics are not generally required (see Fig. 3).9

Adult NSC transplantation into the adult rodent brain allows researchers to study the efficacy of stem cell therapy on degenerative neurological disorders in the adult CNS. When cells need to be delivered to a specific target site to treat isolated neuronal loss as is the case in murine parkinsonism, stereotactic equipment is used to guide the injection. However, in the case of globally dispersed pathology, NSCs injected intravenously (generally into the jugular, carotid, penile, or tail veins) are able to target pathologic lesions throughout the nervous system (see Fig. 4).63 NSCs can be administered to the adult mouse brain via intra cerebral, intraparenchymal, intraventricular, or retinal pathways. The mouse is anesthetized by isoflurane inhalation, then a

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Figure 3. In utero transplantation of NSCs into cerebroventricles. A) Immediately after transplantation, injected donor cells mixed with trypan blue are visualized by transillumination of the embryo. The injectate often fills as far rostrally as the olfactory bulbs and as far caudally as the third ventricle. B through E Blue Xgal donor-derived cells are distributed in semiserial coronal sections at adulthood following unilateral ventricular injection of lacZ-expressing NSCs.9 A color version of this image is available at www.landesbioscience.com/curie. From: Neural stem cell transplantation in mouse brain. Curr Protoc Neurosci 2008; 3:Unit 3:10. Reprinted with permission of John Wiley and Sons, Inc.

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Figure 4. Location of common vascular cell delivery sites: common carotid artery, jugular, femoral, penile and tail veins.80 Used with permission from Allison E et al. Routes of stem cell administration in the adult rodent. In: Weiner LP. Neural Stem Cells. Methods and Protocols, 2nd Ed. Totowa: Humana Press; 2008:383-401.

mouse stereotactic atlas is used to calculate the precise coordinates of the desired injection point. Because the skull of the adult mouse is completely fused, a burr hole must be drilled before using a stereotactically-guided Hamilton syringe to inject the cells. If injecting intravascularly, it is important to avoid introducing any air emboli into the host vascular system and in all cases, one must inject very slowly and leave the needle in place for 3 min to prevent leakage of cells outside the target region.9 When NSCs are injected bilaterally into the ventricles, they gain access to subventricular germinal zone (SVZ), periventricular regions of third and fourth ventricles (source of neural stem cells in adult neurogenesis).80 Like in the case of neonatal and embryonic transplants, NSCs integrate with host tissue in neuronal maintenance and repair. They do not have to cross the blood-brain barrier; however, in contrast to the already discussed techniques above, adult transplants generally do require immunosuppressant treatment.9 In order to increase survival, it is best to minimize operation time and if the mouse is to live for a long time, then sutures are better than tissue glue.9

Considerations during Surgery

The injected cell suspension should be 20,000 cells/+L—100,000 cells/+L. A concentration lower than this range will not effect a significant result and a higher concentration will form a viscous solution that will clog the needle and yield inconsistent data. If in the donor cell suspension, there are bubbles, clumps, aggregates, or clogging of the needle, the transplant should be aborted.9 Cells tend to settle, so the cell suspension needs to be triturated frequently; however, again, it is very important not to introduce air bubbles into the suspension. Most recently, ultrasound guidance has been used as an alternative to stereotactic guidance in neurosurgical procedures. Advantages of ultrasound-guidance include the quickness and simplicity of the technique (there is no further need to move the patient for a CT scan). No irradiation is required and the new technology provides real-time imaging. For its improvements, there are also a few disadvantages to ultrasound guidance, namely: worse resolution images for deep-seated and small lesions and bigger trepanations due to the loss in precision. Stereotactic equipment requires more time and does not offer any real-time intraoperative control, however it is more precise and only requires local anaesthesia due to the

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small trepanation size. Both guidance systems are safe and highly reliable and may be used in conjunction with one another.81

Postoperative Care After transplantation, mice need to be monitored closely. Primary concerns are body temperature, hydration and infections. Mice are generally euthanized if they show signs of undue stress or suffering as manifested by poor weight gain, abnormal skin color, lethargy, or an overall disheveled appearance.9 Mice are extremely sensitive to temperature changes and hypothermia caused by the anesthesia and the incision is best avoided by placing mice on a heating pad; however, one must guard against hyperthermia as well.80 Nesting materials are provided for the security and warmth of the mice,80 while prophylactic antibiotics and acetaminophen are provided for the first week to guard against surgical infections and fever.80 Finally, mice must be checked for dehydration, which can easily be remedied by oral delivery twice a day, the substitution of soft food, or subcutaneous saline injections.80 Prenatally and neonatally transplanted NSCs have been shown to survive for long periods of time without the need for immunosuppressant therapy. However, when NSCs are introduced into the adult mouse brain, the immunosuppressant cyclosporine (10 mg/kg i.p.) is generally recommended for the remainder of the animals’ lives.9 Interestingly, recent evidence suggests that this treatment might not be necessary as undifferentiated cells lack the cytochemical markers that would ordinarily elicit an immune response.82 Since long-term cyclosporine is known to elicit some adverse side effects (i.e., dental problems that interfere with feeding, higher risk for opportunistic infections and gastrointestinal problems that could lead to death),80 the preference against its use is high. However, cyclosporin is still widely used as there is still no conclusive data stating whether immunosuppressants are needed for long-term in vivo transplants.

Optimization The differentiation of NSCs in vivo into neurons, astroglia and oligodendrocytes is directed by developmental signals or disease-specific cell type deficiencies.14 To obtain the maximum percentage yield of neurons, it is best to implant NSCs in utero during active neurogenesis. Cells delivered intravascularly yielded the highest results and results were verified by cell type-specific immunomarkers and by electrophysiological activity (donor cells were labeled with GFP to differentiate living and dead).14 In adult brain stem cell transplantations, injections into the hippocampal regions of the mouse brain were found to yield the highest number of neurons. Transplanted NSCs did not always migrate globally, but rather selectively toward a diseased region.83 Astroglia and oligodendrocytes differentiation was maximized when NSCs were transplanted postnatally during active gliogenesis, or in transgenic mice (i.e., shiverer or twitcher) with glial cell deficiencies.84 The therapeutic efficacy of stem cell grafting is highly dependent upon the methods used to optimize graft survival and minimize graft-induced lesion. Before deciding a technique, Whittemore85 and colleagues performed a variety of comparative experiments to determine the best grafting techniques into the adult rat spinal cord; they used deformation of the spinal cord (spinal cord injury) as a primary endpoint. In this way, they were able to determine which technique produced the least graft-induced trauma. Beveled needles performed by far the best and should always be used over broken and firepolished syringes.85 Before transplanting the suspension of cells, the cell count and viability must be quantified by using a hemocytometer and trypan blue. Live cells do not take up dye, while dead cells do and consequently turn dark blue.85 In this way, the cell density of the suspension can be established and subsequent calculation as to the volume to be grafted and number of grafts to be made can be determined. Nikkah and colleagues found that multiple, smaller-volume grafts yielded better cell survival and integration86 and Whittemore quantified this volume to be generally 0.5-1.0 +L/graft site, or up to 10 +L in lesion cavity.85

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Figure 5. Functional recovery associated with cortical grafts in ischaemic rats. Ratings of posture on stationary or rotating rods, illustrating normal posture and balanced, scored 5 in a normal rat (above) or severe postural impairment, scored 3 after unilateral intracerebral ischemia.94 Used with permission from Dobrossy M et al. Nat Rev Neurosci 2001; 2(12):871-879.

Anticipated Results and Methods of Detection After transplantation, it is important to test the viability and integration of exogenous NSCs in the host neural circuitry. This validation can be done in many ways: histological inspection of cell microstructures in conjunction with immunocytochemical assessment using cell type-specific immunomarkers is used to identify the location and virility of transplanted cells, while behavioral testing of the organism analyzes functional integration. Lee and colleagues showed that oligodendrocytes could be identified using CNPase, O1, O4 and myelin basic protein; astrocytes were identified using GFAP, S-and 100`; and finally, neurons were identified using the full panel of markers `-III-tubulin, neurofilament, NeuN, microtubule-associated protein-2, Hu- D and the immature progenitors nestin, vimentin, musashi1 and NG2, A2B5.9 Ultrastructure cell type identification and observation of synaptic contacts, myelin, subcellular organelles and retrograde tracing techniques helped validate these findings. Behavioral testing is performed both actively and passively. Rodents are often observed and video taped for cage behavior throughout their life span and are also subjected to electrophysiological as well as a variety of basic behavioral tests.9 In rodents with a congenital tremor (i.e., shiverer or twicher) or ataxia (i.e., reeler, meander tail, weaver) coordination tests are used to compare the organism’s performance pre and post transplantation. Rodents are timed to see how long they take to right themselves when tipped over, how long they can maintain their balance on a balance beam (normal is 5 minutes) or on a table that tilts at an increasingly acute angle (normal is about 60 degrees, while transgenetic mice often fall at only 5-10 degrees), the mean time for the animal to fall off a rotating rod at 8 revolutions/minute and sometimes, the decrement in the tremor is measured by a decreased tail displacement, or by placing EEG electrodes on the mouse flank for 15-30 sec. All these tests help give researchers an idea of how well the transplanted NSCs have aided in functional recovery of the individual organism, which should be the primary endpoint in all of these studies (see Fig. 5).

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Conclusion There is much reason to be hopeful for a future in which neural stem cell trials heretofore only involving rodents might be translated to larger mammals and eventually humans. In rats suffering from experimental parkinsonism, a rodent model for Parkinson’s disease, researchers found that human and rat NSCs injected intrastriatally into rats followed similar migratory and dopaminergic neural cell differentiation35,87,88 as in normal development. In rats with experimental intracerebral hemorrhage or transient cerebral ischemia, an application for hemorrhaging and ischemic stroke, human aNSCs injected intravascularly were found to help functional recovery by differentiating into 10% neurons and 75% astrocytes.89,90 And finally, in adult nude mice with either established experimental intracranial and/or subcutaneous flank tumors of neural and nonneural origin, a potential implication for human brain tumors, intravascularly delivered aNSCs were found to target both intracranial and extracranial tumors91,92 with high density of NSCs found in tumor regions with a very low migration/dispersal in normal tissue.92 The implications of these preliminary trials are enormously important and offer a foreseeable very-near future in which heretofore incurable neurological disorders might be treated in humans.

References 1. Takagi Y, Nozaki K, Takahashi J et  al. Proliferation of neuronal precursor cells in the dentate gyrus is accelerated after transient forebrain ischemia in mice. Brain Res 1999; 831(1-2):283-7. 2. Namiki J, Tator C. Cell proliferation and nestin expression in the ependyma of the adult rat spinal cord after injury. J Neuropathol Exp Neurol 1999; 58(5):489-98. 3. Picard-Riera N, Decker L, Delarasse C et  al. Experimental autoimmune encephalomyelitis mobilizes neural progenitors from the subventricular zone to undergo oligodendrogenesis in adult mice. Proc Natl Acad Sci USA 2002; 99(20):13211-6. 4. Brundin L, Brismar H, Danilov A et  al. Neural stem cells: a potential source for remyelination in neuroinflammatory disease. Brain Pathol 2003; 13(3):322-8. 5. Altman J. Autoradiographic and histological studies of postnatal neurogenesis. 3. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J Comp Neurol 1969; 136(3):269-93. 6. Taupin P, Gage F. Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res 2002; 69(6):745-9. 7. Duggal N, Schmidt-Kastner R, Hakim A. Nestin expression in reactive astrocytes following focal cerebral ischemia in rats. Brain Res 1997; 768(1-2):1-9. 8. Pluchino S, Zanotti L, Deleidi M et al. Neural stem cells and their use as therapeutic tool in neurological disorders. Brain Res Brain Res Rev 2005; 48(2):211-9. 9. Lee J, McKercher S, Muller F et al. Neural stem cell transplantation in mouse brain. Curr Protoc Neurosci 2008; Chapter 3:Unit 3.10. 10. Yandava B, Billinghurst L, Snyder E. “Global” cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain. Proc Natl Acad Sci USA 1999; 96(12):7029-34. 11. Windrem M, Nunes M, Rashbaum W et  al. Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain. Nat Med 2004; 10(1):93-7. 12. Lacorazza H, Flax J, Snyder E et al. Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells. Nat Med 1996; 2(4):424-9. 13. Flax J, Aurora S, Yang C et  al. Engraftable human neural stem cells respond to developmental cues, replace neurons and express foreign genes. Nat Biotechnol 1998; 16(11):1033-9. 14. Lee J, Jeyakumar M, Gonzalez R et al. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med 2007; 13(4):439-47. 15. Auerbach J, Eiden M, McKay R. Transplanted CNS stem cells form functional synapses in vivo. Eur J Neurosci 2000; 12(5):1696-704. 16. Ahmed S, Reynolds B, Weiss S. BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J Neurosci 1995; 15(8):5765-78. 17. Lu P, Jones L, Snyder E et al. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 2003; 181(2):115-29. 18. Toma J, Akhavan M, Fernandes K et  al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001; 3(9):778-84.

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19. Eglitis M, Mezey E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci USA 1997; 94(8):4080-5. 20. Azizi S, Stokes D, Augelli B et  al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts. Proc Natl Acad Sci USA 1998; 95(7):3908-13. 21. Chen J, Li Y, Wang L et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci 2001; 189(1-2):49-57. 22. Kopen G, Prockop D, Phinney D. Marrow stromal cells migrate throughout forebrain and cerebellum and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999; 96(19):10711-6. 23. Chen J, Sanberg P, Li Y et  al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke 2001; 32(11):2682-8. 24. Galli R, Gritti A, Bonfanti L et  al. Neural stem cells: an overview. Circ Res 2003; 92(6):598-608. 25. Hallbergson A, Gnatenco C, Peterson D. Neurogenesis and brain injury: managing a renewable resource for repair. J Clin Invest 2003; 112(8):1128-33. 26. Vescovi A, Snyder E. Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo. Brain Pathol 1999; 9(3):569-98. 27. Rubio F, Bueno C, Villa A et al. Genetically perpetuated human neural stem cells engraft and differentiate into the adult mammalian brain. Mol Cell Neurosci 2000; 16(1):1-13. 28. Vescovi A, Parati E, Gritti A et al. Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 1999; 156(1):71-83. 29. Gritti A, Frölichsthal-Schoeller P, Galli R et  al. Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J Neurosci 1999; 19(9):3287-97. 30. Molofsky A, Pardal R, Iwashita T et  al. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 2003; 425(6961):962-7. 31. Caldwell M, He X, Wilkie N et  al. Growth factors regulate the survival and fate of cells derived from human neurospheres. Nat Biotechnol 2001; 19(5):475-9. 32. Kim T, Lee H, Lee Y et al. Sonic hedgehog and FGF8 collaborate to induce dopaminergic phenotypes in the Nurr1-overexpressing neural stem cell. Biochem Biophys Res Commun 2003; 305(4):1040-8. 33. Englund U, Bjorklund A, Wictorin K et al. Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry. Proc Natl Acad Sci USA 2002; 99(26):17089-94. 34. Eriksson C, Björklund A, Wictorin K. Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions. Exp Neurol 2003; 184(2):615-35. 35. Yang M, Stull N, Berk M et al. Neural stem cells spontaneously express dopaminergic traits after transplantation into the intact or 6-hydroxydopamine-lesioned rat. Exp Neurol 2002; 177(1):50-60. 36. Akiyama Y, Honmou O, Kato T et  al. Transplantation of clonal neural precursor cells derived from adult human brain establishes functional peripheral myelin in the rat spinal cord. Exp Neurol 2001; 167(1):27-39. 37. Ben-Hur T, Einstein O, Mizrachi-Kol R et al. Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis. Glia 2003; 41(1):73-80. 38. Bulte J, Ben-Hur T, Miller B et  al. MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain. Magn Reson Med 2003; 50(1):201-5. 39. Wu S, Suzuki Y, Noda T et  al. Immunohistochemical and electron microscopic study of invasion and differentiation in spinal cord lesion of neural stem cells grafted through cerebrospinal fluid in rat. J Neurosci Res 2002; 69(6):940-5. 40. Vescovi A, Gritti A, Galli R et al. Isolation and intracerebral grafting of nontransformed multipotential embryonic human CNS stem cells. J Neurotrauma 1999; 16(8):689-93. 41. Frisén J, Johansson C, Lothian C et al. Central nervous system stem cells in the embryo and adult. Cell Mol Life Sci 1998; 54(9):935-45. 42. Svendsen C, Caldwell M, Ostenfeld T. Human neural stem cells: isolation, expansion and transplantation. Brain Pathol 1999; 9(3):499-513. 43. Sims M, First N. Production of calves by transfer of nuclei from cultured inner cell mass cells. Proc Natl Acad Sci USA 1994; 91(13):6143-7. 44. Cibelli J, Grant K, Chapman K et al. Parthenogenetic stem cells in nonhuman primates. Science 2002; 295(5556):819. 45. Wagers A, Sherwood R, Christensen J et  al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002; 297(5590):2256-9.

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46. Wakayama T, Rodriguez I, Perry A et  al. Mice cloned from embryonic stem cells. Proc Natl Acad Sci USA 1999; 96(26):14984-9. 47. Smith A, Heath J, Donaldson D et  al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988; 336(6200):688-90. 48. Reubinoff B, Pera M, Fong C et  al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000; 18(4):399-404. 49. Johansson C, Momma S, Clarke D et  al. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 1999; 96(1):25-34. 50. McDonald J, Howard M. Repairing the damaged spinal cord: a summary of our early success with embryonic stem cell transplantation and remyelination. Prog Brain Res 2002; 137:299-309. 51. Rosenthal N. Prometheus’s vulture and the stem-cell promise. N Engl J Med 2003; 349(3):267-74. 52. Brüstle O, Spiro A, Karram K et al. In vitro-generated neural precursors participate in mammalian brain development. Proc Natl Acad Sci USA 1997; 94(26):14809-14. 53. Deacon T, Dinsmore J, Costantini L et al. Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation. Exp Neurol 1998; 149(1):28-41. 54. Wakitani S, Takaoka K, Hattori T et al. Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology (Oxford) 2003; 42(1):162-5. 55. Yanai J, Doetchman T, Laufer N et al. Embryonic cultures but not embryos transplanted to the mouse’s brain grow rapidly without immunosuppression. Int J Neurosci 1995; 81(1-2):21-6. 56. Morizane A, Takahashi J, Takagi Y et  al. Optimal conditions for in vivo induction of dopaminergic neurons from embryonic stem cells through stromal cell-derived inducing activity. J Neurosci Res 2002; 69(6):934-9. 57. Studer L. Stem cells with brainpower. Nat Biotechnol 2001; 19(12):1117-8. 58. Zhang S, Wernig M, Duncan I et  al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001; 19(12):1129-33. 59. Pan Y, Nastav J, Zhang H et al. Engraftment of freshly isolated or cultured human umbilical cord blood cells and the effect of cyclosporin A on the outcome. Exp Neurol 2005; 192(2):365-72. 60. Snyder E, Taylor R, Wolfe J. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain. Nature 1995; 374(6520):367-70. 61. Park K, Teng Y, Snyder E. The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat Biotechnol 2002; 20(11):1111-7. 62. Parker M, Anderson J, Corliss D et  al. Expression profile of an operationally-defined neural stem cell clone. Exp Neurol 2005; 194(2):320-32. 63. Willing A, Lixian J, Milliken M et  al. Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res 2003; 73(3):296-307. 64. Vendrame M, Gemma C, de Mesquita D et al. Anti-inflammatory effects of human cord blood cells in a rat model of stroke. Stem Cells Dev 2005; 14(5):595-604. 65. Vendrame M, Gemma C, Pennypacker K et al. Cord blood rescues stroke-induced changes in splenocyte phenotype and function. Exp Neurol 2006; 199(1):191-200. 66. Bjorklund L, Sánchez-Pernaute R, Chung S et  al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci USA 2002; 99(4):2344-9. 67. Kim J, Auerbach J, Rodríguez-Gómez J et  al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 2002; 418(6893):50-6. 68. Andsberg G, Kokaia Z, Björklund A et  al. Amelioration of ischaemia-induced neuronal death in the rat striatum by NGF-secreting neural stem cells. Eur J Neurosci 1998; 10(6):2026-36. 69. Cao Q, Zhang Y, Howard R et  al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol 2001; 167(1):48-58. 70. Teng Y, Lavik E, Qu X et al. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc Natl Acad Sci USA 2002; 99(5):3024-9. 71. Liu S, Qu Y, Stewart T et  al. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc Natl Acad Sci USA 2000; 97(11):6126-31. 72. Reynolds B, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 1992; 12(11):4565-74. 73. Tran P, Miller R. Chemokine receptors: signposts to brain development and disease. Nat Rev Neurosci 2003; 4(6):444-55. 74. Prestoz L, Relvas J, Hopkins K et  al. Association between integrin-dependent migration capacity of neural stem cells in vitro and anatomical repair following transplantation. Mol Cell Neurosci 2001; 18(5):473-84. 75. Schmid R, Anton E. Role of integrins in the development of the cerebral cortex. Cereb Cortex 2003; 13(3):219-24.

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76. Butcher E, Picker L. Lymphocyte homing and homeostasis. Science 1996; 272(5258):60-6. 77. Deckert-Schlüter M, Schlüter D, Hof H et  al. Differential expression of ICAM-1, VCAM-1 and their ligands LFA-1, Mac-1, CD43, VLA-4 and MHC class II antigens in murine Toxoplasma encephalitis: a light microscopic and ultrastructural immunohistochemical study. J Neuropathol Exp Neurol 1994; 53(5):457-68. 78. Ransohoff R. The chemokine system in neuroinflammation: an update. J Infect Dis 2002; 186(Suppl 2): S152-6. 79. Trebst C, Staugaitis S, Tucky B et  al. Chemokine receptors on infiltrating leucocytes in inflammatory pathologies of the central nervous system (CNS). Neuropathol Appl Neurobiol 2003; 29(6):584-95. 80. Alison E. Willing SG-D, Paul R. Sanberg and Samuel Saporta. Routes of stem cell administration in the adult rodent. In: Weiner LP, ed. Neural stem cells, second edition methods and protocols 2nd ed. Totowa: Humana Press, 2008:383-401. 81. Melada A, Heinrich Z, Chudy D et al. The difference between ultrasound-guided and stereotactic-guided neurosurgical procedures. Minim Invasive Neurosurg 2000; 43(3):149-52. 82. Potian J, Aviv H, Ponzio N et al. Veto-like activity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens. J Immunol 2003; 171(7):3426-34. 83. Imitola J, Raddassi K, Park K et  al. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci USA 2004; 101(52):18117-22. 84. Taylor R, Lee J, Palacino J et  al. Intrinsic resistance of neural stem cells to toxic metabolites may make them well suited for cell non-autonomous disorders: evidence from a mouse model of Krabbe leukodystrophy. J Neurochem 2006; 97(6):1585-99. 85. Whittemore S, Zhang Y, Shields C et  al. Optimizing stem cell grafting into the CNS. Methods Mol Biol 2008; 438:375-82. 86. Nikkhah G, Cunningham M, Jödicke A et  al. Improved graft survival and striatal reinnervation by microtransplantation of fetal nigral cell suspensions in the rat Parkinson model. Brain Res 1994; 633(1-2):133-43. 87. Lundberg C, Martínez-Serrano A, Cattaneo E et  al. Survival, integration and differentiation of neural stem cell lines after transplantation to the adult rat striatum. Exp Neurol 1997; 145(2 Pt 1):342-60. 88. Svendsen C, Caldwell M, Shen J et al. Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson’s disease. Exp Neurol 1997; 148(1):135-46. 89. Chu K, Kim M, Jeong S et  al. Human neural stem cells can migrate, differentiate and integrate after intravenous transplantation in adult rats with transient forebrain ischemia. Neurosci Lett 2003; 343(2):129-33. 90. Jeong S, Chu K, Jung K et al. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke 2003; 34(9):2258-63. 91. Aboody K, Brown A, Rainov N et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci USA 2000; 97(23):12846-51. 92. Brown A, Yang W, Schmidt N et al. Intravascular delivery of neural stem cell lines to target intracranial and extracranial tumors of neural and nonneural origin. Hum Gene Ther 2003; 14(18):1777-85. 93. Aleksandrova M, Saburina I, Poltavtseva R et al. Behavior of human neural progenitor cells transplanted to rat brain. Brain Res Dev Brain Res 2002; 134(1-2):143-8. 94. Döbrössy M, Dunnett S. The influence of environment and experience on neural grafts. Nat Rev Neurosci 2001; 2(12):871-9.

Chapter 5

Stem Cell Origin of Brain Tumors Dawn Waters, Ben Newman and Michael L. Levy*

Abstract

T

he biology of both normal and tumor development clearly possesses overlapping and parallel features. Oncogenes and tumor suppressors are relevant not only in tumor biology, but also in physiological developmental regulators of growth and differentiation. Conversely, genes identified as regulators of developmental biology are relevant to tumor biology. This is particularly relevant in the context of brain tumors, where recent evidence is mounting that the origin of brain tumors, specifically gliomas, may represent dysfunctional developmental neurobiology. NSCs are increasingly being investigated as the cell type that originally undergoes malignant transformation—the cell of origin—and the evidence for this is discussed.

Introduction Over a century ago, the German pathologist Julius Cohnheim described the similarities between tumors and embryonic cells and suggested that “embryonic rests” were the source of tumors.1 Later, the primitive cytoarchitecture and embryonic features of many malignant brain tumors was also described by Bailey and Cushing.2 Smyth and Stern’s observations published in 1938, that “subependymal glia may actually be the point of origin of tumors of the thalamus.”3 These earlier descriptions were further advanced in 1944 when Joseph Globus and Hartwig Kuhlenbeck called attention to the subependymal cell plate in the adult brain and described this structure as one with primitive cellular composition. Based on a study with human brain tumors, they stated that “one of the most important sources for such immature embryonal residue from which neuroectodermal tumors are likely to develop under certain still unknown conditions is the subependymal plate.”4 The subependymal plate was better characterized as a “mitotically active and well defined subependymal layer is present in mammalian brains throughout life” and Lewis et al, suggested that they could be a “possible source of different histological varieties of glioma, in particular those tumors in paraventricular situation and butterfly gliomas of the corpus callosum.”5 In the late 1960s Hopewell and Wright demonstrated increased glial tumors with perventricular implantation of carcinogens in rats.6 In the 1970s, periventricular tumors were then demonstrated to occur in the subventricular region after intraventricular inoculation with avian sarcoma viruses, with a much higher rate of tumors occurring in neonatal rats versus adult rats.7-9 A single dose of ethylnitrosurea administration to pregnant rats also induced periventricular tumors in the offspring.10 Now clear evidence exists for the “subependymal plate, as described by Gobus and Kuhlenbeck is the subventricular zone (SVZ), know to be the largest cellular region of neural stem cells in the adult mammalian brain. The NSCs have been characterized by Buyalla and provide the migratory *Corresponding Author: Michael L. Levy— Pediatric Neurosurgery, Rady Childrens Hospital. San Diego, California, USA. Email: [email protected].

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Figure 1. The cells of the subventricular zone, labeled with the astrocyte marker GFAP (shown in green), line the lateral walls of the lateral ventricles. This is the largest known region of adult neural stem cells in the human brain; it is composed of the deep subcortical white matter (A), a periventricular ribbon of astrocytes that can function as neural stem cells (B), a dense layer of astrocytic processes (C) and the ependymal lining (D). Throughout adult life, astrocytes from the subventricular zone exhibit a unique capacity for multipotency and self-renewal in vitro.Used with permission from Sanai N et al. N Engl J Med 2005; 353:811-822.11 A color version of this image is available at www.landesbioscience.com/curie.

neuoblasts for neurogeneis in the olfactory bulb in rodents (Fig. 1).11 Recently, this migratory path for SVZ neural stem cells, was also described in humans.12

Reappraising the Prevailing Theory of Tumor Genesis It has been widely accepted that cancer occurs as a consequence of genetic and epigenetic alterations in a differentiated cell. These alterations could provide a proliferative advantage and ultimately lead to uncontrolled growth and spread of the malignant cells. This theory suggests that tumors, such as gliomas, result from mutations to terminally differentiated astrocytes and oligodendrocytes that “de-differentiate” into a less differentiated pheonotype.13-15 Although the

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neoplastic transformation of fully differentiated glia is widely assumed to be the mechanism of gliomagenesis, this hypothesis has never been adequately tested.

Evidence for NSC as the Cell of Origin The neurogenenic zones within the human CNS with their resident NSCs are considered the leading candidates for transformation leading to brain tumors. Specifically mitiotically active cells (NSCs and their direct progeny, transit amplifying cells) are the cells that have the greatest probability of being the brain tumor cell of origin.

NSCs and Gliomas Share Histological and Biological Similarities Gliomas have long been described by pathologists for their remarkable cellular heterogeneity and in fact the most aggressive and malignant glioma (glioblastoma multiforme) was termed based partly on the histological diversity comprising this tumor-hence “multiforme.” A transformed NSC could provide this cellular landscape due to their mulitipotnetiallity (ability to differentiate into the cell types that constitute their respective germ line). Mixed cell gliomas exist, such as oligoastrocytomas and have both oligodendrocytes and astrocytes16 and could be independent transformation of two differentiated cells, as suggested by the dedifferenteiation theory of tumor genesis. More plausible would be the transformation of a single, bipotential progenitor cell such as a NSC or a transit amplifying cell.17 These mixed cell gliomas also exhibit loss of heterozygosity on chromosomes 1p and 19q in both the astrocytic and oligodendrocytic components,18 suggesting that in oligoastrocytomas, the astrocytes and oligodendtocytes comprising the tumor have a shared cell of origin. Further, many glioma cells are undifferentiated and lack expression of differentiated cell makers, as well as demonstrate staining with markers for nestin. Nestin expression is one hallmark feature of NSCs19,20 and has become a reliable marker of NSCs.21 Gliomas and NSCs also exhibit characteristic overlapping behavior, (Table 1)11 such as high motility, association with vasculature and white matter tracts.22-24

NSCs More Likely to Accumulate Oncogenic Mutations Accumulation of oncogenic genetic hits by cells is an infrequent stochastic event that most likely takes considerable time to result in transformation. NSCs, defined by their ability to self renew are both mitoticaly active and exist during the lifetime of the animal, allowing them to potentially accumulate the necessary multiple mutations for tumor formation. Accordingly, the cellular origin of gliomas would most likely occur from the proliferative zones in the mammalian CNS such as the SVZ contain at least two types of mitotically active cells—NSCs and transit amplifying cells.25 Although

Table 1. Characteristics intrinsic to neural stem cells and gliomas11 High motility Diversity of progeny Robust proliferative potential Association with blood vessels Association with white-matter tracts Immature expression profiles Nestin expression EGF-receptor expression PTEN expression Hedgehog pathway activity Telomerase activity Wnt pathway activity

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Figure 2. Hierarchical organization of the functional compartments in renewing tissues. A) The stem-cell compartment (purple), early transiently dividing progenitor compartment (orange), late transiently dividing progenitor compartment (red) and differentiated-cell compartment (green) are schematically described. Cells in the stem-cell and transiently dividing progenitor compartments could be the target of the oncotransformation that leads to the formation of tumour stem cells. B) The neural precursors that make up similar functional compartments in the neurogenetic regions of the adult brain and that might be the source of brain tumour stem cells. C) The structure of the subventricular zone, showing how these precursors fit and are organized in the germinal neuroepithelium of the largest neurogenetic region of the adult brain. GABA, a-aminobutyric acid; GFAP, glial fibrillary acid protein; HSA, heat-stable antigen; MAP2, microtubule-associated protein 2; NCAM1, neural cell adhesion molecule 1; PSA, polysialic acid; RMS, rostral migratory stream. Used with permission from Vescovi A et al. Nat Rev Cancer 2006; 6:425-436. © 2006 Nature Publishing Group.26 A color version of this image is available at www.landesbioscience.com/curie.

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TACs exist only briefly and then differentiate, their total cellular compartment is significantly larger than NSCs and total number of global number of divisions during their relatively short lifespan are comparable to NSCs that exist throughout life yet are less mitotically active (Fig. 2).26

Cancer Stem Cells Approximately 150 years ago pathologists Rudolph Virchow and Julius Cohnheim suggested there were histological similarities between the developing fetus and certain cancers (such as teratocarcinomas) and that both tissues have the capacity to differentiate and proliferate. This “embryonal-rest hypothesis” is the historical version of today’s cancer stem cell hypothesis.27 As defined at the American Association for Cancer Research workshop on cancer stem cells: cancer stem cells are cells that (1) self renew and (2) resupply the tumor with the various lineages of cells of which it is comprised. Self renewal can only be defined experimentally by the ability to recapitulate the generation of a continuously growing tumor or tumor initiation cell (Fig. 3).28,29 The original work establishing the CSC model was based on hematopoietic system. Evidence for leukemia-CSCs was first reported in 1994 when Lapidot et al isolated a rare population of CD34 CD38- cells from patients with acute myeloid leukemia. Infusion of these CD34 CD3cells into severe combined immune-deficient mice resulted in leukemic blast generation; however, more differentiated cells (CD34 CD38 ) did not generate leukemia.30-32 On a molecular level, CSCs share properties with normal stem cells. They have similar markers and signaling pathways, respond to environmental cues, as well as telomerase activity, apoptosis clearance and increased membrane transporter activity.15 The first report of cells with stem-like properties in brain tumors was by Ignatova et al in 2002 where surgical specimens of glioblastoma multiforme were shown to have clonogenic neurosphere forming cells that expressed both neuronal and glial markers upon differentiation (Fig. 4).26,33 Subsequently, the Dirks group demonstrated CSC in brain tumors by intracerebral transplantation of CD133 or CD133- populations into immunodeficient mice. With as few as 100 CD133 cells from the primary tumor, a new phenocopy of the tumor could be created in the transplanted mice;

Figure 3. Overview of tumor stem cells in cancer. Cancer stem cells (tumor-initiating cells) divide asymmetrically, resulting in self-renewal of the tumor-initiating cell and production of a daughter cell known as a transient-amplifying cell (progenitor cell). The transient- amplifying cell is not thought to possess self-renewing capabilities, but instead divides indefinitely to contribute to cancer progression. From: Lee JT, Herlyn M. J Cell Physiol 2007; 213:603-609. Reprinted with permission of John Wiley and Sons, Inc.29

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Figure 4. Immunofluorescence photomicrographs of representative MC-PL clones derived from different cortical glial tumor specimens (T1–T3, T6 and T7) processed for double (A, B, C, E, F) or single (B inset, D) labeling for different neural markers. The cells positive for the neuronal marker ` -III tubulin are stained green (FITC) and the cells that expressed the astroglial marker GFAP are stained blue (AMCA). The arrowhead in F points out a cell that is not labeled for GFAP, but is immunopositive for ` - III tubulin (arrowhead in G). The short arrow in F points out a cell that is immunolabeled for GFAP, but immunonegative for ` -III tubulin (short arrow in G). The long arrow in F and G points out a cell that is labeled with both immunomarkers. Scale bars are 10 +m (F and G) and 100 +m (A–E).33 A color version of this image is available at www.landesbioscience.com/curie. From: Ignatova T, Kukekov V, Laywell E et al. Glia 2002; 39:193-206. Reprinted with permission of John Wiley and Sons, Inc.33

and unsorted or CD133- primary tumor cells were unable to cause de novo tumor generation. As part of what has come to define CSCs-self-renewal capacity- was also shown by confirming the ability of serially transplanted CD133 cells to recapitulate the original tumor.30,34 These findings established the presence of BTSCs, cells which can differentiate into the neural lineages and exhibit self renewal—as demonstrated by recapitulation of primary tumors with serial transplantation.

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This has been shown for other types of brain tumors as well.35,36 The existence of these BTSCs adds further evidence toward the NSCs origin of gliomas by confirming that different brain tumours contain transformed, undifferentiated neural precursors that respond to the same mitogens that activate adult neural stem cells.26 Second, they indicate that tumour stem-like cells possess some of the molecular features of neural stem cells. Third, BTSCs, through asymmetric division, could generate a BTSCs and a progenitor cell, the latter of which may migrate away to either form or contribute to the tumor mass.26,37

Gliomas and NSCs Have Common Regulatory Pathways Neural stem cells and progenitor cells have acitivated cellular pathways, such as promitotic genes, telomerase activity and antiapoptotic genes. This innate capacity overlaps with the mechanisms underlying tumor initiation, progression, or both. Thus, NSCs may require the least amount of mutations to become transformed. 1. EGFR expression is upregulated in primary glioblastoma multiforme and transiently dividing progenitors (Type C cells).38 2. Fibroblast growth factors (FGFs) are involved in tumor proliferation and angiogenesis39,40 and also shown to regulate NSC proliferation and cell fate.41-43 3. Notch receptors and signaling is involved in NSC renewal44,45 and related to proliferative capacity of gliomas.46 4. PTEN is a tumour suppressor with an important function in the control of proliferation of neural stem cells and progenitor cells in vivo and in vitro.47-49 PTEN is inactivated in glioblastomas50,51 and its preservation in glioblastoma multiforme is clinically favorable.38 5. The Wnt-catenin pathway regulates adult neurogenesis52,53 modulating its activity may increase glioma cell growth.54 Recent experiments have also highlighted the increased ability of progenitor cells to be transformed versus differentiated cell types. If epidermal growth factor receptor (EGFR) is transfected into transgenic Ink4a-Arf< < mouse (lacking genes for cell-cycle arrest) neural stem cells, the cells lead to glioma formation.55 This contrasted with similar manipulation of differentiated mouse astrocytes. Further, if the undifferentiated mouse astrocytes were transfected with platelet-derived growth factor ( pdgf ) transgene an converted to a less differentiated state, they showed increased oncogenicity.55-57

Mouse Models of Gliomas for Investigation of Glioma Origin Mouse models gliomas are available and offer a unique opportunity to investigate tumor origin. These models, unlike glioma models in Drosophila and C. elegans, recapitulate the human pathology in terms of such characteristic structures as pleomorphic nuclei, diffusely infiltrative margins, secondary structures of Scherer, necrosis with pseudopalisading tumor cells and microvascular proliferation. Xenograft models fail to phenocopy the classic histopathological features and are not an option for elucidating developmental mechanisms. Ultimately, these models of spontaneous tumor development and progression allows for the potential identification of novel mechanisms for tumorigenesis.58

Conclusion Brain tumor classification with current histological criteria fails to accurately categorize patients as many patients with similar grade brain tumors have higly variable clinical outcomes. Clearly, this classification is one that at a minimum needs molecular modifiers. Defining the cell of origin and confirming whether NSCs are indeed the cell of origin would improve not only glioma classifications, but also detection and treatment. The differential antigenic and molecular attributes of NSCs responsible for tumorgenesis could be exploited to target malignant cells prior tumor progression to clinical presentation. Indeed, defining cell of origin could help expand the concept of chemoprevention—targeting cells in the premorbid state.

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References 1. Rather L. The Genesis of Cancer: A Study in the History of Ideas, Baltimore: Johns Hopkins University Press, 1978. 2. Bailey P, Cushing H. A classification of the tumors of the glioma group on a histogenetic basis with a correlated study of prognosis, Philadelphia, London: J.B. Lippincott Company, 1926. 3. Smyth G, Stern, K. Tumors of the thalamus: a clinicopathological study. Brain 1938; 61. 4. Globus JH, Kuhlenbeck H. The subependymal plate (matrix) and its relationship to brain tumors of the ependymal type. J Neuropathol Exp Neurol 1944;3:1-35. 5. Lewis P. Mitotic activity in the primate subependymal layer and the genesis of gliomas. Nature 1968; 217:974-5. 6. Hopewell J, Wright E. The importance of implantation site in cerebral carcinogenesis in rats. Cancer Res 1969; 29:1927-31. 7. Copeland D, Vogel F, Bigner D. The induction of intractranial neoplasms by the inoculation of avian sarcoma virus in perinatal and adult rats. J Neuropathol Exp Neurol 1975; 34:340-58. 8. Copeland D, Bigner D. The role of the subependymal plate in avian sarcoma virus brain tumor induction: comparison of incipient tumors in neonatal and adult rats. Acta Neuropathol (Berl) 1977; 38:1-6. 9. Vick N, Lin M, Bigner D. The role of the subependymal plate in glial tumorigenesis. Acta Neuropathol (Berl) 1977; 40:63-71. 10. Koestner A, Swenberg J, Wechsler W. Transplacental production with ethylnitrosourea of neoplasms of the nervous system in Sprague-Dawley rats. Am J Pathol 1971; 63:37-56. 11. Sanai N, Alvarez-Buylla A, Berger M. Neural stem cells and the origin of gliomas. N Engl J Med 2005; 353:811-22. 12. Curtis M, Kam M, Nannmark U et  al. Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Science 2007; 315:1243-9. 13. Mabon R, Svien H, Adson A et  al. Astrocytomas of the cerebellum. Arch Neurol Psychiatry 1950; 64:74-88. 14. Doetsch F, Caillé I, Lim D et al. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999; 97:703-16. 15. Sakariassen P, Immervoll H, Chekenya M. Cancer stem cells as mediators of treatment resistance in brain tumors: status and controversies. Neoplasia 2007; 9:882-92. 16. Valtz N, Hayes T, Norregaard T et  al. An embryonic origin for medulloblastoma. New Biol 1991; 3:364-71. 17. Chekenya M, Pilkington G. NG2 precursor cells in neoplasia: functional, histogenesis and therapeutic implications for malignant brain tumours. J Neurocytol 2002; 31:507-21. 18. Kraus J, Koopmann J, Kaskel P et  al. Shared allelic losses on chromosomes 1p and 19q suggest a common origin of oligodendroglioma and oligoastrocytoma. J Neuropathol Exp Neurol 1995; 54:91-5. 19. Dahlstrand J, Collins V, Lendahl U. Expression of the class VI intermediate filament nestin in human central nervous system tumors. Cancer Res 1992; 52:5334-41. 20. Tohyama T, Lee V, Rorke L et al. Nestin expression in embryonic human neuroepithelium and in human neuroepithelial tumor cells. Lab Invest 1992; 66:303-13. 21. Lendahl U, Zimmerman L, McKay R. CNS stem cells express a new class of intermediate filament protein. Cell 1990; 60:585-95. 22. Shoshan Y, Nishiyama A, Chang A et  al. Expression of oligodendrocyte progenitor cell antigens by gliomas: implications for the histogenesis of brain tumors. Proc Natl Acad Sci USA 1999; 96:10361-6. 23. Doetsch F, Petreanu L, Caille I et al. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 2002; 36:1021-34. 24. Palmer T, Willhoite A, Gage F. Vascular niche for adult hippocampal neurogenesis. J Comp Neurol 2000; 425:479-94. 25. Seri B, García-Verdugo J, Collado-Morente L et al. Cell types, lineage and architecture of the germinal zone in the adult dentate gyrus. J Comp Neurol 2004; 478:359-78. 26. Vescovi A, Galli R, Reynolds B. Brain tumour stem cells. Nat Rev Cancer 2006; 6:425-36. 27. Huntly B, Gilliland D. Leukaemia stem cells and the evolution of cancer-stem-cell research. Nat Rev Cancer 2005; 5:311-21. 28. Clarke M, Dick J, Dirks et  al. Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006; 66:9339-44. 29. Lee J, Herlyn M. Old disease, new culprit: tumor stem cells in cancer. J Cell Physiol 2007; 213:603-9. 30. Buzzeo M, Scott E, Cogle C. The hunt for cancer-initiating cells: a history stemming from leukemia. Leukemia 2007; 21:1619-27.

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31. Lapidot T, Sirard C, Vormoor J et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367:645-8. 32. Al-Hajj M, Wicha M, Benito-Hernandez et  al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100:3983-8. 33. Ignatova T, Kukekov V, Laywell E et  al. Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia 2002; 39:193-206. 34. Singh S, Hawkins C, Clarke I et al. Identification of human brain tumour initiating cells. Nature 2004; 432:396-401. 35. Taylor M, Poppleton H, Fuller C et al. Radial glia cells are candidate stem cells of ependymoma. Cancer Cell 2005; 8:323-35. 36. Merkle F, Tramontin A, García-Verdugo J et  al. Radial glia give rise to adult neural stem cells in the subventricular zone. Proc Natl Acad Sci USA 2004; 101:17528-32. 37. Berger F, Gay E, Pelletier L et  al. Development of gliomas: potential role of asymmetrical cell division of neural stem cells. Lancet Oncol 2004; 5:511-4. 38. Mellinghoff I, Wang M, Vivanco I et  al. Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N Engl J Med 2005; 353:2012-24. 39. Joy A, Moffett J, Neary K et  al. Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells. Oncogene 1997; 14:171-83. 40. Auguste P, Gürsel D, Lemière S et  al. Inhibition of fibroblast growth factor/fibroblast growth factor receptor activity in glioma cells impedes tumor growth by both angiogenesis-dependent and -independent mechanisms. Cancer Res 2001; 61:1717-26. 41. Palmer T, Markakis E, Willhoite A et al. Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 1999; 19:8487-97. 42. Gritti A, Parati E, Cova L et  al. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci 1996; 16:1091-100. 43. Vescovi A, Reynolds B, Fraser D et  al. bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells. Neuron 1993; 11:951-66. 44. Hitoshi S, Alexson T, Tropepe V et al. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 2002; 16:846-58. 45. Shen Q, Goderie S, Jin L et  al. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science 2004; 304:1338-40. 46. Purow B, Haque R, Noel M et  al. Expression of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma cell survival and proliferation. Cancer Res 2005; 65:2353-63. 47. Baker S, McKinnon P. Tumour-suppressor function in the nervous system. Nat Rev Cancer 2004; 4:184-96. 48. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature 2005; 434:843-50. 49. Groszer M, Erickson R, Scripture-Adams D et  al. Negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo. Science 2001; 294:2186-9. 50. Wechsler-Reya R, Scott M. The developmental biology of brain tumors. Annu Rev Neurosci 2001; 24:385-428. 51. Rasheed B, Wiltshire R, Bigner S et al. Molecular pathogenesis of malignant gliomas. Curr Opin Oncol 1999; 11:162-7. 52. Chenn A, Walsh C. Increased neuronal production, enlarged forebrains and cytoarchitectural distortions in beta-catenin overexpressing transgenic mice. Cereb Cortex 2003; 13:599-606. 53. Lie D, Colamarino S, Song H et  al. Wnt signalling regulates adult hippocampal neurogenesis. Nature 2005; 437:1370-5. 54. Roth W, Wild-Bode C, Platten M et al. Secreted Frizzled-related proteins inhibit motility and promote growth of human malignant glioma cells. Oncogene 2000; 19:4210-20. 55. Bachoo R, Maher E, Ligon K et al. Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell 2002; 1:269-77. 56. Dai C, Celestino J, Okada Y et  al. PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev 2001; 15:1913-25. 57. Uhrbom L, Dai C, Celestino J et  al. Ink4a-Arf loss cooperates with KRas activation in astrocytes and neural progenitors to generate glioblastomas of various morphologies depending on activated Akt. Cancer Res 2002; 62:5551-8. 58. Ding H, Roncari L, Shannon P et  al. Astrocyte-specific expression of activated p21-ras results in malignant astrocytoma formation in a transgenic mouse model of human gliomas. Cancer Res 2001; 61:3826-36.

Chapter 6

The Tumor Microenvironment Lissa Baird and Alexey Terskikh*

Abstract

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ecent advances in stem cell and developmental neurobiology have uncovered new perspectives from which we investigate various forms of cancer. Specifically, the hypothesis that tumors are comprised of a subpopulation of malignant cells similar to stem cells is of great interest to scientists and clinicians and has been dubbed the cancer stem cell hypothesis. The region where this is most relevant is within the brain. Cancer stem cells have been isolated from brain tumors that exhibit characteristics of differentiation and proliferation normally seen only in neural stem cells. These cancer stem cells may be responsible for tumor origin, survival and proliferation. Furthermore, these cells must be considered within their immediate microenvironment when investigating mechanisms of tumorgenesis. Evidence of brain tumor stem cells will be reviewed along with the role of tumor environment as the context within which these cells should be understood.

Introduction The intersection of stem cell biology with oncology has offered new insight into the cellular and molecular mechanisms underlying both normal and abnormal development. Of particular interest to clinicians and scientists is the hypothesis that solid tumors may harbor a subpopulation of malignant cells that display stem cell like properties-the cancer stem cell (CSC). A normal stem cell can both self renew and differentiate into multiple lineages. In the central nervous system (CNS), somatic (adult) neural stem cells (NSCs) can both undergo asymmetric division to create two daughter cells—one a NSC and the other a more committed progenitor able to differentiate and proliferate but not self renew. Similarly, the putative CSC would sustain and spread the tumor, as well as populate the tumor with the various types of differentiated cells. Brain tumor stem cells (BTSCs) are the CSCs of the CNS and exhibit both self renewal and multi-lineage differentiation. This subpopulation of cells within brain tumors may be responsible for tumor survival and proliferation, as well as tumor origin. The process of carcinogenesis may be more accurately viewed as the aberrant response of the entire tissue to genetic and epigenetic stress. The cancerous cell defining the tumor and constituting the CSC subpopulation must be considered within its immediate tumor microenvironment and elucidating this role may be as important as investigating the transformed glial or neuronal cells within brain tumor. The prevailing evidence in support of BTSCs will be reviewed along with the evolving and integral role of tumor environment as the context within which BTSCs should be understood.

*Corresponding Author: Alexey Terskikh—Del E. Webb Neuroscience, Aging and Stem Cell Research Center, Burnham Institute for Regenerative Medicine. Email: [email protected].

Frontiers in Brain Repair, edited by Rahul Jandial. ©2010 Landes Bioscience and Springer Science+Business Media.

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Prevailing Theory of Tumor Initiation It has been widely accepted that cancer occurs as a consequence of genetic and epigenetic alterations in a differentiated cell. These alterations provide a proliferative advantage and ultimately lead to uncontrolled growth and spread of the malignant cells. This theory suggests that tumors, such as gliomas, result from mutations to terminally differentiated astrocytes and oligodendrocytes that “de-differentiate” into a less differentiated pheonotype.1-3

Cancer Stem Cells—Discovery and Evidence of BTSC Approximately 150 years ago pathologists Rudolph Virchow and Julius Cohnheim suggested there were histological similarities between the developing fetus and certain cancers (such as teratocarcinomas) and that both tissues have the capacity to differentiate and proliferate. This “embryonal-rest hypothesis” is the historical version of today’s cancer stem cell hypothesis.4 As defined at the American Association for Cancer Research workshop on cancer stem cells: cancer stem cells are cells that1 self renew and2 resupply the tumor with the various lineages of cells of which it is comprised. Self renewal can only be defined experimentally by the ability to recapitulate the generation of a continuously growing tumor-or tumor initiation cell (Fig. 1).5,6 The original work establishing the CSC model was based on hematopoietic system. Evidence for leukemia-CSCs was first reported in 1994 when Lapidot et al isolated a rare population of CD34 CD38- cells from patients with acute myeloid leukemia. Infusion of these CD34 CD3cells into severe combined immune-deficient mice resulted in leukemic blast generation; however, more differentiated cells (CD34 CD38 ) did not generate leukemia.7,8 This CSC model was later extended to solid organ tumors (breast) by Al-Hajj et al in 2003 report of tumor recapitulation with transplantation of as little of 100 CD44 CD24- cells from the primary tumor; whereas no de novo tumorgenesis failed to occur with transplantation of 10 = 5 or greater CD44 CD24 cells.9 On a molecular level, CSCs share properties with normal stem cells. They have similar markers and signaling pathways, respond to environmental cues, as well as telomerase activity, apoptosis clearance and increased membrane transporter activity.3

Figure 1. Overview of tumor stem cells in cancer. Cancer stem cells (tumor-initiating cells) divide asymmetrically, resulting in self-renewal of the tumor-initiating cell and production of a daughter cell known as a transient-amplifying cell (progenitor cell). The transient-amplifying cell is not thought to possess self-renewing capabilities, but instead divides indefinitely to contribute to cancer progression.6 Used with permission from Lee J et al. J Cell Physiol 2007; 213:603-609.

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The first report of cells with stem-like properties in brain tumors was by Ignatova et al in 2002 where surgical specimens of glioblastoma multiforme were shown to have clonogenic neurosphere forming cells that expressed both neuronal and glial markers upon differentiation (Fig. 2).10,11 Subsequently, the Dirks group demonstrated CSC in brain tumors by intracerebral transplantation of CD133 or CD133– populations into immunodeficient mice. With as few as 100 CD133 cells from the primary tumor, a new phenocopy of the tumor could be created in the transplanted mice. Further, unsorted or CD133– primary tumor cells were unable to cause de novo tumor generation. As part of what has come to define CSCs-self-renewal capacity- was also shown by confirming the ability of serially transplanted CD133 cells to recapitulate the original tumor.7,12 These findings established the presence of BTSCs, cells which can differentiate into the neural lineages and exhibit self renewal—as demonstrated by recapitulation of primary tumors with serial transplantation. This has been shown for other types of brain tumors as well.13,14 Bao et  al further defined the molecular mechanisms by which BTSCs may escape lethal damage of ionizing radiation and confer an advantage for revisiting clinical radiation therapy. The CD133 cells isolated from fresh tumor specimens were shown to preferentially activate DNA repair checkpoints, including phosphorylation of the checkpoint proteins Chk1 and Chk2.15,16

Figure 2. Isolation and perpetuation of brain tumour stem cells in culture. The neurosphere assay is a defined serum-free culture system that allows the isolation and propagation of CNS-derived stem cells. Adult precursors are dissociated and plated in a liquid growth medium that contains the stem-cell mitogens epidermal growth factor and/or fibroblast growth factor 2. Because of the lack of serum and the low plating density, most cells die, except those that divide in response to the stem-cell mitogens. The growth-factor-responsive cells proliferate to form floating clusters of cells that are referred to as neurospheres. These can be further dissociated into a single-cell suspension and then replated in fresh medium to produce secondary neurospheres. The process can be repeated, resulting in a geometric expansion in the number of cells that are generated at each passage. Upon mitogen removal, the progeny of the proliferating precursors can be differentiated into neurons, astrocytes and oligodendrocytes, which are the three primary cell types that are found in the adult mammalian CNS. Under these conditions, the growth-factor-responsive precursors can be expanded indefinitely with little change in their growth or differentiation characteristics. These results indicate the existence of an adult neural stem cell as these cells possess the fundamental stem-cell features of extensive self-renewal, generation of many progeny and the ability to give rise to the primary cell types of the tissue from which they were obtained.11 Used with permission from Vescovi A et al. Nat Rev Cancer 2006; 6:425-436. © 2006 Nature Publishing Group.

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Caveats to BTSC With the surge in interest in CSC and BTSC, some confusion regarding methods and definitions has arisen, risking assigning properties to a heterogeneous population of cells and leading to erroneous characterization Some concerns for the current CSC hypothesis remain (Fig. 3).17 BTSCs, shown to be tumor-initiating are not independent of their site of primary culture and ultimate site of transplantation. In fact, their ability to proliferate is significantly dependent on the tumor microenvironment as discussed below. Also, the current in vivo serial transplantation assay for self-renewing BTSCs employs immunodeficient mice and in this nonphysiologic scenario one would expect higher engraftment of transplanted tumor cells. The use of proteolytic enzymes to disassociate primary brain tumor specimens, as used in all the BTSC studies, could also change the

Figure 3. Schematic diagram to illustrate potential difficulties in sorting and identifying ‘‘cancer stem cells.’’ Top, theoretical concerns; bottom, technical concerns.17 From: Hill R. Identifying cancer stem cells in solid tumors: case not proven. Cancer Res 2006; 66:1891-5; discussion 1890. Reprinted with permission of John Wiley and Sons, Inc.

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cell surface proteins by which these BTSCs are sorted. Although it is clear that CD133– cells are not tumorigenic, it remains unclear which subpopulation of CD133 cells is the bona fide BTSC conferring tumor initiating capacity. The identification of reliable markers will allow for prospective isolation and characterization of a pure population of BTSCs (not just a population of cells containing BTSCs). Then fundamental questions about these cells can be investigated, such as the underlying signaling mechanisms for self-renewal and whether the BTSC is also the tumor cell of origin.

The Tumor Microenvironment The stem cell microenvironment is known to help maintain their quiescent state, as well as preserve their potential to proliferate and differentiate.18,19 This extends to tumors as well. The tumor microenvironment, comprised of non-epithelial stromal cells (inflammatory cells, endothelial cells and fibroblasts) is increasingly being shown to be essential for tumor growth (Fig. 4).20,21 Other solid organ tumors (such as breast and cancer) demonstrate that genetic changes occur in the stoma at the earliest stages of tumorgenesis and in murine models genetic lesions confined to the stroma are sufficient to induce epithelial tumors.22-24 In fact, this concept is an extension of the “seed and soil” hypothesis posed by Paget many years ago and now studies are underscoring the role of the tumor microenvironment as the tumor “soil.” Brain tumors and BTSCs must be considered as a dysfunctional phenomenon that occurs not only in individual cells, but also in the tissue that harbors the malignant lesion. This tissue and the microenvironment it creates is an integral part of the cancer. NSCs have been shown to lie within a vascular microenvironment and similarly Calabrese et al demonstrated that CD133 Nestin tumor

Figure 4. Model of cancer stem cells (CSCs) in solid tumors. CSCs are associated with the stromal components of the tumor, including fibroblasts and/or blood vessels, which make up the CSC ‘niche’. The niche cells secrete factors that support CSC self renewal. CSCs retain differentiation potential, giving rise to nonself renewing tumor cells that make up the bulk of the tumor. CSCs may be drug and/or radiation resistant and may express CSC-specific antigens. Current research focuses on identification of the molecular mechanisms regulating these properties of CSCs (highlighted in green), as they represent potential targets for therapy.19 Used with permission from: Ailles L et al. Curr Opin Biotechnol 2007; 18:460-466.19 A color version of this image is available at www.landesbioscience.com/curie.

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Figure 5. Targeting glioblastoma treatment to the tumor stem-cell compartment. A glioblastoma is a heterogeneous tumor initiated and maintained by a minority of CD133 stem cells, which have a high tumorigenic potential and a low proliferation rate. New therapeutic strategies include (i) forcing tumor stem cells into a more differentiated phenotype characteristic of the CD133–tumor bulk, for example with bone morphogenetic proteins (BMPs); (ii) overcoming the inherent resistance of tumor stem cells to radio- or chemotherapy using suitable inhibitors, such as inhibitors of DNA repair checkpoints; (iii) selecting therapies based on the tumor’s molecular profile; (iv) and targeting the tumor stroma, which provides the specialized milieu for tumor stem cells, angiogenesis and immune response.15Used with permission from Stupp R et al. Nat Biotechnol 2007; 25:193-194.15 © 2007 Nature Publishing Group.

cells (that contain the BTSCs) were similarly associated with the vasculature.25 Further, by increasing the number of endothelial cells and blood vessels in xenografts increased the CSC population and rate of tumor growth. Accordingly, antiangiogenic treatment decreased the CSC population and tumor growth, demonstrating that the secreted factors or cell contact with the tumor microenvironment helped maintain the functional characteristics of the CSCs.19 A CSC is not subject to the same internal and external regulation as normal stem cells and cannot be identified without considering the influential microenvironment within which it resides. Therapeutically the tumor microenvironment offers a novel approach to tumor treatment by targeting the cells of the tumor niche, as well as the potential for chemoprevention. Characterizing this niche could allow for the development of in vitro assays that recreate the in vivo environment and obviating the current time consuming dependence on serial transplantation as the standard for defining CSCs.

Conclusion The BTSC, as it becomes increasingly characterized and investigated, could become the ideal target for brain tumor treatment. One approach would be to force differentiation of BTSCs and by depleting this CSC compartment achieving decreased tumor proliferative and initiating capacity. This concept was demonstrated by Piccirillo et al, with bone morphogenic protein (physiologically mature neural progenitor cells) mediated differentiation of the CD133 BTSC and reduction in tumor initiating capacity. (Fig. 5)15,26 BTSC resistance to chemotherapy and radiation therapy could be targeted with inhibitors. As the molecular signature of these cells becomes defined, each patient’s tumor could

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be better targeted by developing patient specific therapies based on the molecular characteristics. The stromal compartment and tumor microenvironment could also be targeted to modulate and inhibit tumor growth. The potential for therapeutic progress by targeting the BTSC is considerable and realizing this potential depends on the continued molecular and cellular investigation into the characterization of these cells and the molecular mechanisms underlying their unique properties.

References 1. Mabon R, Svien H, Adson A et  al. Astrocytomas of the cerebellum. Arch Neurol Psychiatry 1950; 64(1):74-88. 2. Doetsch F, Caillé I, Lim D et al. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999; 97(6):703-16. 3. Sakariassen P, Immervoll H, Chekenya M. Cancer stem cells as mediators of treatment resistance in brain tumors: status and controversies. Neoplasia 2007; 9(11):882-92. 4. Huntly B, Gilliland D. Leukaemia stem cells and the evolution of cancer-stem-cell research. Nat Rev Cancer 2005; 5(4):311-21. 5. Clarke M, Dick J, Dirks P et al. Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006; 66(19):9339-44. 6. Lee J, Herlyn M. Old disease, new culprit: tumor stem cells in cancer. J Cell Physiol 2007; 213(3):603-9. 7. Buzzeo M, Scott E, Cogle C. The hunt for cancer-initiating cells: a history stemming from leukemia. Leukemia 2007; 21(8):1619-27. 8. Lapidot T, Sirard C, Vormoor J et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367(6464):645-8. 9. Al-Hajj M, Wicha M, Benito-Hernandez A et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100(7):3983-8. 10. Ignatova T, Kukekov V, Laywell E et  al. Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia 2002; 39(3):193-206. 11. Vescovi A, Galli R, Reynolds B. Brain tumour stem cells. Nat Rev Cancer 2006; 6(6):425-36. 12. Singh S, Hawkins C, Clarke I et al. Identification of human brain tumour initiating cells. Nature 2004; 432(7015):396-401. 13. Taylor M, Poppleton H, Fuller C et al. Radial glia cells are candidate stem cells of ependymoma. Cancer Cell 2005; 8(4):323-35. 14. Merkle F, Tramontin A, García-Verdugo J et  al. Radial glia give rise to adult neural stem cells in the subventricular zone. Proc Natl Acad Sci USA 2004; 101(50):17528-32. 15. Stupp R, Hegi M. Targeting brain-tumor stem cells. Nat Biotechnol 2007; 25(2):193-4. 16. Bao S, Wu Q, McLendon R et  al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006; 444(7120):756-60. 17. Hill R. Identifying cancer stem cells in solid tumors: case not proven. Cancer Res 2006; 15;66(4):1883-90; discussion 1895-6. 18. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells and their niche. Cell 2004; 116(6):769-78. 19. Ailles L, Weissman I. Cancer stem cells in solid tumors. Curr Opin Biotechnol 2007; 18(5):460-6. 20. Kenny P, Lee G, Bissell M. Targeting the tumor microenvironment. Front Biosci 2007; 12:3468-74. 21. Egeblad M, Littlepage L, Werb Z. The fibroblastic coconspirator in cancer progression. Cold Spring Harb Symp Quant Biol 2005; 70:383-8. 22. Ishiguro K, Yoshida T, Yagishita H et al. Epithelial and stromal genetic instability contributes to genesis of colorectal adenomas. Gut 2006; 55(5):695-702. 23. Weber F, Shen L, Fukino K et  al. Total-genome analysis of BRCA1/2-related invasive carcinomas of the breast identifies tumor stroma as potential landscaper for neoplastic initiation. Am J Hum Genet 2006; 78(6):961-72. 24. Kim B, Li C, Qiao W et  al. Smad4 signalling in T-cells is required for suppression of gastrointestinal cancer. Nature 2006; 441(7096):1015-9. 25. Calabrese C, Poppleton H, Kocak M et al. A perivascular niche for brain tumor stem cells. Cancer Cell 2007; 11(1):69-82. 26. Piccirillo S, Reynolds B, Zanetti N et al. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 2006; 444(7120):761-5.

Chapter 7

Exploitation of Genetically Modified Neural Stem Cells for Neurological Disease Allen L. Ho, Sassan Keshavarzi and Michael L. Levy*

Abstract

T

he successful treatment and potential treatment of the central nervous system (CNS) pathology remains the most challenging frontier in medical science. The clinical modalities presently available are mostly of limited efficacy and with the aging population, neurodegerative diseases and CNS neoplasms are increasingly prevalent. Neural stem cells (NSCs) have provided optimism for the horizon of therapeutic progress in treating neurological diseases. These mutipotent (able to differentiate into neurons, astrocytes and oligodendrocytes) cells can be obtained directly from the CNS or derived from of embryonic stem cells (ESCs). NSCs can be genetically manipulated in vitro to express desired transgenes for improved expandability, as well as for delivery of toxic payloads. NSCs also demonstrate the ability to engraft within the CNS, migrate to CNS pathology and in certain scenarios to reconstitute the injured or diseased nervous system.

Exploiting NSCs for Therapeutic Transplantation The discovery by several groups in the early 1990’s that rodent neural stem cells, could be cultured, propagated and re-implanted into the mammalian brain with resultant integration and transgene expression has led to considerable investigation into the therapeutic potential of NSCs.1,2 These findings have spawned interesting opportunities for NSCs as a source for CNS graft material, gene transfer, neural replacement and possible CNS repair. Furthermore, NSCs possess inherent cellular properties making them uniquely favorable for potential therapeutic aims as discussed below. After demonstrating the potential for murine NSCs to integrate into mammalian brain the question arose if human NSCs have the same properties. The meander tail (mea) mouse model (characterized by cell-autonomous failure of granule neurons to develop in the cerebellum) known to be receptive to murine NSC repopulation of neurons in the cerebellum was used to address this question. Human NSCs were engrafted into the cerebellum of the newborn mea cerebella and subsequently found to repopulate the internal granule cell layer (Fig. 1).3 This clearly demonstrated the ability of human NSCs to not only engraft but also respond to local cues for appropriate lineage determination, reaffirming the ability of genetically immortalized NSCs to differentiate and follow developmental cues in vivo after transplantation. The intact wild-type adult rat brain has also been evaluated for the ability of human NSCs to engraft and integrate. Human NSCs were transplanted into the striatum and substantia nigra of adult rats and shown to integrate into surrounding brain parenchyma as well *Corresponding Author: Michael L. Levy—Pediatric Neurosurgery, Rady Children’s Hospital, San Diego. San Diego, California, USA. Email: [email protected].

Frontiers in Brain Repair, edited by Rahul Jandial. ©2010 Landes Bioscience and Springer Science+Business Media.

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Figure 1. Transplantation of human NSCs into granule neurondeficient cerebellum. (A–G) Donor-derived cells (clone H6) identified in the mature cerebellum by anti-BrDU immunoperoxidase cytochemistry (brown nuclei) following implantation into and migration from the neonatal mea EGL. A) The internal granule cell layer (IGL and arrowheads) within the parasagittal section of the cerebellum. B) Higher magnification of the posterior lobe indicated by “b” in (A). C–G) Increasing magnifications of donor-derived cells within the IGL of a mea anterior lobe (different animal from (A,B)). (G) Normarski optics: residual host granule neurons indicated by arrowheads, representative BrDU positive donor-derived neuron indicated by the arrow. H) Colabeling with anti-BrDU (green) and (I) NeuN (red) indicated with arrows. Arrowhead indicates BrDU /NeuN- cell. J) Fluorescent in situ hybridization of cells within the IGL using a human-specific probe (red). Scale bars: (A and B): 100 +m; (F, G and J): 10 +m. Used with permission from Flax J, et al. Nat Biotechnol 1998; 16:1033-1039. 3 © 1998 Nature Publishing Group. A color version of this image is available at www.landesbioscience.com/curie.

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as migrate and widely distribute in the brain.4 Important to note, in nonneurogenic areas, grafted cells differentiated primarily along an astrocytic lineage, consistent with the understanding that neuron generation via transplanted human NSCs is most effective in during fetal development or later in neurogenic zones of mature animals.5,6 The ability of NSCs to successfully integrate will be further discussed in the subsequent section on disease based examples of genetically engineered NSCs used in murine models of CNS pathology. Along with successful integration after transplantation, it was shown that NSCs exhibit an innate migratory capacity toward areas of CNS pathology, including ischemic injury, chemical injury and neoplasia.7-11 Using an immunodeficient mouse model with inoculated brain tumors, Aboody et al (2000) demonstrated NSC migration toward and distribution within the implanted tumor. This phenomenon occurred with NSC injection near and far from the implanted tumor, as well as with tail vein injection of NSCs. Furthermore, NSCs appeared to track and follow individual tumor cells escaping from the primary inoculated tumor mass (Fig. 2). This potential for home to tumors and tumor cells was corroborated by Ehtesham et al, who reported that NSCs transduced with an adenoviral vector containing the cytokine inteleukin -12 (IL-12) gene and subsequently implanted them into the brains of tumor bearing syngeneic C57Bl/6 mice. Similar to findings by Aboody et al, the NSCs exhibited tumor homing potential, distributed within tumors and tracked micro-deposits separate from the main tumor mass.12 The mechanisms responsible for this observed NSC tumor homing capacity are currently being investigated. We have shown that the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway is one important candidate for the directed migration of NSCs.10 A recent paper suggests that the glioma-produced extracellular matrix attracts NSCs.13 Other potential mechanisms include the idea that glioma and neural stem cells may share a common developmental lineage, NSCs are attracted to tumor-associated endothelial cells and/or NSCs are attracted to inflammatory signals from tumor induced injury.14 With immortalized NSCs, it is possible to engineer clonal cell lines that will migrate and become stably integrated into targeted areas of CNS pathology. These properties render NSCs as optimal candidates for gene therapy. Using NSCs to deliver therapeutic genes requires that certain fundamental requirements of gene transfer first be met. As in the aforementioned discussion on gene transfer to NSCs, it has been established that gene transfer to NSCs in vitro is not only feasible but also a relatively efficient process. However, for successful clinical applications the following requirements should be considered. First, gene transfer must be innocuous for the recipient. Currently, NSC grafting has led to successful integration in host brains and no reports of tumorgenesis from either viral oncogene immortalization of NSCs or gene delivery by NSCs. Second, it would be desirable to deliver genes that are stably and efficiently expressed over the long term. Third, circumventing the brains blood brain barrier and host immune response would enhance the efficacy of any gene transfer modality. NSCs are not limited by the blood brain barrier and in rodents do not require systemic immune suppression prior to NSCs transplantion. Fourth, the wide range of CNS pathology necessitates that gene transfer have the capacity to be achieved both locally for some applications and globally for other applications. NSCs have been demonstrated to deliver to select target tissues (i.e., in the case of brain tumors) as well as deliver to global regions (i.e., in the case of metabolic enzyme deficiencies). With any method of gene therapy it is highly desirable that the gene expression can be regulated. NSCs can be transduced with various genes and promoters allowing for cell selection, eliminated if necessary (i.e., suicide genes) and pharmacologically controlled. Lastly, application for broad clinical application would require NSCs cellular vehicles to be generated on a large scale. Clearly, immortalized NSCs proliferate continuously in vitro and allow for predictable expansion. Specific examples of therapeutic gene delivery with NSCs are discussed in the following section.

Genetically Modified NSCs and Neurological Disease The ultimate translation of NSCs for human clinical application, first requires an understanding of the behavior and potential of NSCs in animal models of neurological disorders. Indeed NSCs have been used in the gamut of neurological disorders with promising results.

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Figure 2. NSCs implanted at various intracranial sites distant from main tumorbed migrate through normal adult tissue toward glioma cells. A and B) Same hemisphere but behind tumor (Paradigm 2). Shown here is a section through the tumor from an adult nude mouse 6 days after NSC implantation caudal to tumor. In A (as per the schematic, a coned down view of a tumor populated as pictured under low power in Figs. 2A and 3A and B), note X-Gal1 blue NSCs interspersed among dark neutral red1 tumor cells. B) High power view of NSCs in juxtaposition to islands of tumor cells. C–H) Contralateral hemisphere (Paradigm 3). C–E) As indicated on the schematic, these panels are views through the corpus callosum (‘‘c’’) whereb-gal1 NSCs (red cells, arrows) are seen migrating from their site of implantation on one side of the brain toward tumor on the other. Two representative NSCs indicated by arrows in C are viewed at higher magnification in D and E , respectively, to visualize the classic elongated morphology and leading process of a migrating neural progenitor oriented toward its target. In F, b-gal1 NSCs (red) are ‘‘homing in’’ on the GFP1 tumor (green) having migrated from the other hemisphere. In G and magnified further in H, the X-Gal1blueNSCs (arrows) have now actually entered the neutral red1 tumor (arrowheads) from the opposite hemisphere. I and J) Intraventricular (Paradigm 4). Shown here is a section through the brain tumor of an adult nude mouse 6 days following NSC injection into the contralateral cerebral ventricle. In I, as per the schematic, blue X-Gal1 NSCs are distributed within the neutral red1main tumorbed (edge delineated by arrowheads). At higher power in J, the NSCs are in juxtaposition to migrating islands of red glioblastoma cells. Fibroblast control cells never migrated from their injection site in any paradigm. All X-Gal-positivity was corroborated by anti-b-gal immunoreactivity. (Scale bar: A, 20 mm and applies to C; B, 8 mm, 14 mm in D and E, 30 mm in F and G, 15 mm in H, 20 mm in I and 15 mm in J.) Used with permission from Aboody K et al. Proc Natl Acad Aci USA 2000; 97:12846-12851.7 A color version of this image is available at www.landesbioscience.com/curie.

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Neurodegenerative Disorders The loss of dopamine producing neurons of the substantia nigra in Parkinson’s disease has been targeted for therapy in rodent and primate models. Conceptually, dopamine producing neurons and cells expressing neurotrophic factors could be delivered. Each of these has been attempted in animals with varying results. In early studies using cell transplantation of astrocytes and/or fibroblasts expressing the transgene for tyrosine hydroxylase (TH, rate limiting enzyme of dopamine synthesis), the greatest obstacle encountered was the early downregulation of the TH transgene within 1-2 weeks; thereby limiting its efficacy to provide continual production of dopamine.15 Longer TH transgene expression was reported with the use of rat NSCs in a hemiparkinsonian rat model. This was accomplished with both expression plasmids and retroviral vectors. Some behavioral improvements were observed and TH expression was maintained up to 4 months.16 The success of TH transgene expression is markedly more potent when the necessary factors and coenzymes for dopamine synthesis are present. In an experiment with 17.2 NSCs transduced with both TH gene and GTP cylohydrolase I (GTPCH1) gene, the level o L-DOPA released was 760 fold higher than when the GTPCHI gene was not cotransduced.17 Another approach used in Parkinsonian rat models, has been to provide neurotrophins for neuroprotection via NSCs to minimize nigral neuron loss as well as, replace neurons. Akerud et al transplanted glial cell line-derived neurotropic factor (GDNF) transfected C17.2 NSCs into the striatum of mice that subsequently received 6-hydroxy-dopamine (6-OHDA), a chemical that creates striatal cell death (Fig. 3). In this 6-OHDA Parkinson mouse model, the cell death in the striatum was significantly less with C17.2-GDNF cell transplants (21% cell death) versus transplants in the control group (63% cell death). Furthermore, these transplanted cells differentiated into astrocytes, neurons and oligodendrocytes in the striatum.18 The experiments were subsequently performed with persephin (a neurotrophin in the GDNF family) expressing C17.2 NSCs and again significant reduction in striatal cell death was observed.19 The ultimate modality for treating Parkinson’s disease would be to provide cellular therapy to allow for functional integration and potentially reverse neurological degeneration. This remains a major challenge, yet one study investigated the use of embryonic stem cells differentiated into dopaminergic neurons for transplantation into rat models of Parkinson’s disease. Using mouse embryonic stem cells, a highly enriched population of midbrain neural stem cells was derived and found to generate electrophysiologically active dopamine neurons that improved behavioral deficits in parkinsonian rats.20 Huntington’s disease is a progressive autosomal dominant disorder leading to striatal neuron degeneration. Earlier studies with NSCs explored the use of neuroprotective agents and found reduced striatal degeneration in a rat model of Huntington’s disease after grafting of NSCs expressing nerve growth factor (NGF).21 More recently, beneficial effects were seen after transplanting human NSCs into the striatum of quinolinic acid (QA) lesioned rats. These human neurospheres were pretreated with cytokine ciliary neurotrophic factor (CNTF), which enhances differentiation and after transplantation into the lesioned rats led to a 22% greater striatal volume versus controls.22 Furthermore, successful migration of NSCs to the striatum in QA lesioned rats has been shown, not only from intraventricular injection of NSCs, but also from injection of NSCs into the mouse tail vein.23 Amyotrophic lateral sclerosis (ALS) leads to progressive degeneration and loss of motor neurons in the cerebral cortex, brain stem and spinal cord. Currently, there is no causal treatment available for ALS. Therapy with NSCs would need to replace motor neurons through a wide area of the central nervous system. In animal models, overexpression of GDNF and VEGF has yielded some benefits. The challenge with these large growth factors is penetration through the blood brain barrier and has most often been achieved with viral vectors. Recently, NSCs have been successfully used to deliver GDNF. Using the superoxide dismutase 1 (SOD1) mouse model of ALS, human NSCs expressing GDNF were transplanted into the spinal cord of these rats and cellular integration into both gray and white matter and upregulation of cholinergic markers was observed without adverse behavioral effects.24 Using an elegant approach, Kaspar et al (2003) showed that adeno-associated virus injected into the muscle is retrogradely transported thereby delivering insulin-like growth factor 1 (IGF-1) to motorneurons. This strategy increased life span and delayed disease progression in a mouse model of ALS.25

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Figure 3. GDNF-c17.2 grafts protected substantia nigra dopaminergic neurons in a 6-OHDA model of Parkinson’s disease. A) Schematic drawing of the positions at which the cells were grafted and the 6-OHDA injection was performed. The contralateral side was left intact. The grafting was performed at day 0, the 6-OHDA injection at day 16 and perfusion at day 30. Apomorphine- and amphetamine induced circling behavior were studied at days 28 and 29, respectively. B) Quantification of the number of substantia nigra TH-positive neurons in the indicated experimental conditions. Values represent the mean SEM (n  5–7) of the number of TH-positive cells counted in serial sections through the substantia nigra. *p  0.0001 versus lesioned substantia nigra grafted or not with the MT-c17.2 cell line as determined by one-way ANOVA (significant effect of treatment, p  0.0001; F(3,32)  101.1). C–F) TH immunohistochemistry showed that grafting of the MT-c17.2 cells did not prevent the loss of dopamine neurons (compare D and E with C) Instead, GDNF-c17.2 cells (F) prevented the loss of dopamine neurons in the substantia nigra. Used with permission from Akerud P et al. J Neurosci 2001; 21:8108-8118.18

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Alzheimer’s disease poses a great therapeutic challenge due to global degeneration of neurons in the hippocampus, cortex, amygdala and other parts of the brain. Current strategies focus on using neurotrophic factors, specifically NGF and a Phase-I trial of ex vivo NGF delivery with genetically modified autologous fibroblasts has demonstrated robust growth response in one patient.26 Martinez-Serrano et al were able to demonstrate improved memory tasks at old age and less cholinergic atrophy in rats with intracerebral transplantation (into the medial septum and nucleus basalis of Meynert) of NSCs overexpressing NGF.27 NSCs can ameliorate memory dysfunction and in a mouse model with progressive neuronal loss primarily within the hippocampus, leading to specific impairments in memory. NSCs transplanted into the brain after neuronal ablation survive, migrate, differentiate and, most significantly, improved memory.28

Brain Tumors The challenge to the successful treatment of brain tumors arises from the immense difficulty of attacking invading cells within the brain, as well as the delivery of chemotherapeutic modalities past the blood brain barrier to tumors and tumors cells selectively. In 2000, several groups demonstrated the unique ability of NSCs to home to tumors, even when transplanted at various sites outside of the tumor itself.7,29 This homing ability has been exploited to deliver therapeutics in various tumors models with remarkable efficacy and promise for potential human clinical transplantation. Aboody et al employed a model where nude mice were inoculated with glioma cells and subsequently transplanted with human and murine NSCs at various locations (intratumoral, contralateral hemisphere, intraventricular and tail vein) with clear demonstration of NSCs migrating to the tumor and distributing within the tumor. Interestingly, some NSCs appeared to track single cells invading brain parenchyma outside of the tumor mass (Fig. 4). Subsequently, NSCs were transfected with a gene for cytosine deaminase Figure 4, viewed on following page. NSCs migrate extensively throughout a brain tumor mass in vivo and ‘‘trail’’ advancing tumor cells. Paradigm 1 is illustrated schematically. Section of brain under low A) and high B) power from an adult rat killed 48 h after NSC injection into an established D74 glioma, processed with X-Gal to detect blue staining b-gal-producing NSCs and counterstained with neutral red to show dark red tumor cells; arrowheads demarcate approximate edges of the tumor mass where it interfaces with normal tissue. Donor X-Gal1 blue NSCs (arrows) can be seen extensively distributed throughout the mass, interspersed among the red tumor cells. C) Tumor, 10 days after NSC injection, illustrating that, although NSCs (arrows) have infiltrated the mass, they largely stop at the junction between tumor and normal tissue (arrowheads) except where a tumor cell (dark red, elongated) is entering normal tissue; then NSCs appear to ‘‘follow’’ the invading tumor cell into surrounding tissue (upper right arrow). This phenomenon becomes more dramatic when examining NSC behavior in a more virulent and aggressively invasive tumor, the CNS-1 glioblastoma in the adult nude mouse, pictured in D. This section illustrates extensive migration and distribution of blue NSCs (arrows) throughout the infiltrating glioblastoma up to and along the infiltrating tumor edge (red arrowheads) and into surrounding tissue in juxtaposition to many dark red1 tumor cells invading normal tissue. The ‘‘tracking’’ of individual glioblastoma cells is examined in greater detail in E–L, where CNS-1 cells have been labeled ex vivo by transduction with GFP cDNA. E and F) Sister sections showing a low power view of transgene-expressing NSCs distributed throughout the main tumor mass to the tumor edge (outlined by arrowheads). Sections were either costained with X-Gal (NSCs, blue) and neutral red (tumor cells, dark red and elongated) (E) or processed for double immunofluorescence using an anti-b-gal antibody (NSCs, red) and an FITC-conjugated anti-GFP antibody (glioblastoma cells, green) (F). Low (G) and high (H) power views of tumor edge (arrowheads) with blue NSCs (blue arrow) in immediate proximity to and intermixed with an invading tumor ‘‘island’’ (dark red spindle-shaped cells) (red arrow). I and J) Low and high power views, respectively (boxed area in I is magnified in J), of a blue NSC in direct juxtaposition to a single migrating neutral red1, spindle-shaped tumor cell (arrow), the NSC ‘‘riding’’ the glioma cell in ‘‘piggy-back’’ fashion. K and L) Low and high power views, respectively, under fluorescence microscopy, of single migrating GFP1 tumor cells (green) in juxtaposition to b-gal 1 NSCs(red). Region indicated by white arrow in K and magnified in L illustrates NSCs apposed to tumor cells migrating away from the main tumor bed. (Scale bars: A, 40 mm, 30 mm in B; C, 30 mm, 25 mm in D; E, 90 mm, 100 mm in F; H, 15 mm, 60 mm in G; J, 30 mm, 60 mm in I, 70 mm in K, 35 mm in L.) Used with permission from Aboody K et al. Proc Natl Acad Sci USA 2000; 97:12846-12851.7 A color version of this image is available at www.landesbioscience.com/curie.

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Figure 4. Figure legend viewed on previous page.

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(CD), a prodrug-converting enzyme that converts 5-FC to 5-FU and transplanted some distance from the tumor. This technique let to approximately 80% reduction in tumor burden.7 NSCs have also been used to deliver interleukins with ability to extend survival in rat tumor models. Ehtesham et al transfected murine NSCs with the gene for pro-inflammatory cytokine interleukin-12 (IL-12) and demonstrated stable expression and found increased survival with intratumoral transplantation in C57/Bl6 mice.12 Herpes simplex virus1-thymidine kinase (HSVtk) has also been shown to be effective at treating gliomas when expressed by NSCs carrying the HSVtk gene. Murine NSCs expressing HSCtk were transplanted intratumoral (rat glioma cell line C6) into mice and rats both during tumor inoculation and after tumor inoculation and then treated with ganciclovir. Interestingly, there was a 70% increase in survival with NSC-HSVtk transplantation after tumor inoculation and 100% survival with cotransplantation of tumor and NSC-HSVtk.30 The ability of NSCs to express therapeutic genes has also been demonstrated using human NSC and human glioma cell lines. Kim et al employed PEX a naturally occurring fragment of human metalloproteinase-2, which acts as an inhibitor of glioma and endothelial cell proliferation, migration and angiogenesis to target human glioma cells (Gli26). Using the HB1.F3 cell line (immortalized human NSCs) transduced with a vector containing the PEX gene and subsequently transplanted intra-tumoral into the mice. The mice were evaluated for tumor size with magnetic resonance imaging and at the termination of the studies by histological analysis including tumor volume, microvessel density, proliferation and apoptosis rate. Histological analysis showed 90% reduction of tumor volume and decrease in angiogenesis (Figs. 5 and 6).31 Investigation into the mechanisms leading to NSC tropism for brain tumors is focused on the contribution of the extracellular matrix (ECM). Tumor-ECM derived from six glioblastoma cell lines was found to be highly permissive for NSC migration and laminin was the most permissive substrate for human NSC migration and tenascin-C the strongest inducer of a directed human NSC migration suggesting that the ECM of malignant gliomas is a modulator of NSC migration.13

Spinal Cord Injury The destruction of long axonal tracts in spinal cord injury occurs from acute injury with mechanical forces and exacerbation by secondary inflammatory damage, both leading to neuronal death. Currently, there are different conceptual approaches for developing novel therapeutic strategies for SCI.32 It is well-known that myelin-producing oligodendrocytes express specific molecules like NOGO that inhibit axonal regrowth after injury. Blocking or removal of these molecules has been an attractive hypothesis in the recent years. Other strategies would include bridging the spinal cord lesion, reducing scar tissue, providing nerve growth factors and repairing damaged myelin.32 For some of these paradigms, NSCs with their inherent and multifaceted properties would be ideal candidates. Indeed, transplantation of NSCs following spinal cord injury led to increased functional recovery.33-35 However, a recent report has shown that transplantation of undifferentiated NSCs induced allodynia while their directed differention and increased oligodendroglial differentiation by neurogenin-2 overexpression not only improved functional outcome, but also alleviated allodynia.36 This suggests that effective genetic manipulation of NSCs is crucial for the use of NSCs in traumatic spinal cord injury. Accordingly, potential therapy would vary depending on the time frame after injury, with minimizing inflammation the primary concern during the time of injury and regeneration the major goal when injury is well established. Axonal damage in spinal cord injury was also shown to lessen with NSCs genetically modified by recombinant retrovirus to express neurotrophin-3 (NT-3). With transplantation into injured spinal cords, the murine NSCs producing NT-3 were able to increase the growth of cholinergic motor axons and interestingly NSCs without NT-3 showed some improvement in axon growth versus fibroblast controls (Blesch et al, 2002).37 Human NSCs have been successful in the regeneration of axons in spinal cord injured mice.38 Furthermore, evaluation of NSCs in primates has occurred. Human fetal spinal cord tissue was cultured for NSCs and subsequently implanted into spinal cord injured primates. Eight weeks after transplantation, all animals were sacrificed and histological analysis revealed that not only did the human NSCs survive, but they also differentiated into neurons, astrocytes and oligodendrocytes. The bar grip

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Figure 5. Tumor volumes as determined by MRI at 7 days after therapeutic intratumoral inoculation of human neural stem cells and histologic analysis at 14 days after inoculation of human neural stem cells (n  18). A) representative images from MRI (Gd-T1 weighted images). B) representative photographs from histology (H and E staining, original magnification, x1). C) tumor volumes were estimated from MRI; columns, mean; bars, SE (P  0.03, PBS versus HB1. F3-PEX; P  0.03, HB1.F3 versus HB1.F3- EX, Mann-Whitney U test). D) tumor volumes were estimated from histologic analysis; columns, mean; bars, SE (P  0.03, PBS versus HB1.F3-PEX; P  0.03, HB1.F3 versus HB1.F3-PEX, Mann-Whitney U test).31 Used with permission from Kim S et al. Clin Cancer Res 2005; 11:5965-5970.

power and the spontaneous motor activity of the transplanted animals were higher than those of sham-operated control animals.39

Stroke Ischemic brain injury following blood vessel occlusion leads to immediate cell death and creates a surrounding region (penumbra) that is highly susceptible to infarction. Cellular therapy would aim to minimize secondary cell death and ultimately repair neuronal loss. Neuroprotective agents were the first to be explored in the management of ischemic brain injury, specifically NGF. Rats were made transiently ischemic and NSCs expressing NGF were transplanted into the rat striatum, leading to a reduction in striatal cell death versus controls 48 hours after middle cerebral artery occlusion. In this study the NSC-NGF transplants were made prior to ischemic insult.40 The

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Figure 6. Treatment effects assessed by immunohistochemistry (n  18). Primary antibodies included anti-CD31 for blood vessels, anti-Ki67 nuclear antigen for proliferating cells and anti cleaved caspase-3 for the detection of apoptosis. Sections were counterstained with H and E (original magnification, =200). Columns, mean microvessel count, proliferation index and apoptotic index as percentages of the control; bars, SE (microvessel count and proliferation index, P  0.03, PBS versus HB1.F3-PEX; P  0.03, HB1.F3 versus HB1.F3-PEX, Mann-Whitney U test). Used with permission from Kim S et al. Cancer Res 2005; 11:5965-5970.31

physiologic response of the brain in the setting of ischemia or stroke was explored in rats with the evaluation of the SVZ and hippocampus after induced ischemia. Transient middle cerebral artery occlusion in adult rats led to a marked increase of cell proliferation in the subventricular zone and 31-fold increase of the number of BrdU-NeuN-labeled neurons in the ipsilateral striatum of the stroke versus the contralateral striatum or control.41 Similarly, Nakatomi et al (2002) showed that endogenous progenitors in response to ischemia migrate into the hippocampus and generate new CA1 pyramidal neurons.42 Whether NSCs can be implanted and survive post-ischemia has been evaluated in adult rats. Murine NSCs were transplanted intraventricularly in rats 48 hours after middle cerebral artery occlusion and through histological and radiological analysis were found to survive and migrate to the ischemic brain areas with.43 More recently, human NSCs were shown to survive and integrate 7 days after ischemic insult in rats. Neurospheres were transplanted into the ischemic cortex of rats and 4 weeks after transplantation the NSCs migrated to the ischemic zone whereas in naïve rats minimal or no migration was seen. Furthermore, the majority of the migrating cells were of neuronal phenotype, possibly fate determined by the microenvironment (Fig. 7).44

Enzyme Delivery in Lysosomal Storage Diseases The earliest studies demonstrating the ability of NSCs to widely incorporate within the CNS were performed on the developing rodent brain, particularly in models with single gene deficiency. In these disease models cell engraftment without reconstitution of normal circuitry would be sufficient to correct the pathology. Snyder et al used a model of mucopolysaccharidosis Type II (MPS VII, Sly disease), a lysosomal storage disorder caused by an inherited deficiency of `-glucuronidase (GUSB) gene (a secreted enzyme involved in the degradation of glycosaminoglycans) to evaluate NSCs as a therapeutic modality. The NSCs were modified with a retrovirus coding for human

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Figure 7. Migration of transplanted human cells. A) Schematic showing extensive migration of human cells (small circles) toward the lesion in dMCAo brains compared to very little migration away from the graft (gray oval) in naive brains. B) 4 wk post transplant, sections were labeled with the human specific antibody SC121, to identify human cells and visualized with diaminobenzidine (DAB) (brown). Low magnification images depict the cells migrating long distances primarily toward the lesion in dMCAo brains. Cells transplanted into naive rats migrated little and equally in all directions. Shown are higher-magnification images of dMCAo (C and D) and naive (E and F) brains. Arrowhead indicates lesion. (Scale bar  200 micrometers.) Used with permission Kelly S et al. Proc Natl Acad Sci USA 2004; 101:11839-11844.44 A color version of this image is available at www.landesbioscience.com/curie.

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and murine GUSB and transplanted into the ventricle of newborn mice. They found the transplanted cells to engraft throughout the neuroaxis and cross-correct the disease by replacing the deficient enzyme.45 In fact, increased GUSB levels were detected up to eight months after grafting. Subsequently, Lacorazza et al showed that genetically engineered NSCs could make transgenic proteins available to large areas in the brain when transplanted into the fetal and newborn mice. They first demonstrated this ability in vitro with two murine NSC lines modified to express the alpha-subunit of human hemosaminidase A (Hex A) on human Tay-Sachs fibroblasts. Then they engrafted the cells in mice with the enzymatic deficiency and showed expression of the human enzyme for 3-8 weeks and an increase in Hex A activity by 14-76%.46 The strategy of neural stem cell use to cross correct enzymatic deficiency has recently been extended to Niemann-Pick disease. Niemann-Pick disease arises due to a lack in acid sphingomyelinase (ASM) leading to accumulation of sphingomyelin and cholesterol in lysosomes. Murine NSCs were modified to express ASM up to 10 times greater than NSC controls and transplanted into the ASM knockout mice. Although, the ASM levels were extremely low by immunostaining analysis, there was sufficient ASM expression and secretion to reverse distended lysosomal pathology and achieve regional clearance of cholesterol and shingomyelin. Indeed, transplanted NPCs survived, migrated and showed region-specific differentiation in the host brain up to 10 weeks after transplantation.47 Recently the most significant progress in NSC mediated treatment of lysosomal was demonstrated in a mouse model of Sandhoff disease, a lethal gangliosidosis. Intracranial transplantation of NSCs delayed disease onset, preserved motor function, reduced pathology, prolonged survival. Further, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Human NSCs (isolated directly from the CNS and derived secondarily from embryonic stem cells) were also shown to be effective (Fig. 8).48

The Aging Brain Neurogenesis in the aging brain is less robust and the microenvironment to support cellular transplantation may also be less favorable. Furthermore, the techniques for cellular transplantation are associated with higher morbidity when performed on the brains of older animals. Substantial decrease in new neurons in neurogenic brain regions has been described and the potential of stimulating endogenous NSC for therapeutic aims may be a difficult endeavor. It seems that this decrease is not a limitation of the NSCs per se, but a reflection of a changing milieu, as suggested by the ability to elevate neurogenesis in the aged hippocampus by decreasing glucocorticoid levels.49,50 The strategy to implant NSCs holds promise, yet must overcome obstacles that are unique to the aged brain. Hallbergson et al (2003) describe minimal neuronal differentiation of NSCs when transplanted into the aged brain, even in neurogenic regions, highlighting the challenges of therapeutic intervention in the aging brain.51

In Vivo Imgaging of Transplanted NSCs The potential of NSC transplantation is one that has mostly been explored with immuno-histological staining. In vivo imaging is critical, not only to further corroborate and understand the therapeutic capacity of NSCs but also to provide a preclinical evaluation of imaging techniques that could ultimately translated to use in humans. Cellular and molecular imaging aims to provide non-invasive imaging of cells and cellular processes in living animals and humans. In its expanded role, it may even provide information about biodistribution and location of cellular transplants, magnitude of gene expression and tracking of stem cell migration. Small animals provide the major substrate for performing transplant studies and in vivo imaging with bioluminescence (BLI) is an excellent modality for in vivo imaging of both NSCs, and tumors cells. BLI uses detection of photons that are emitted at specific wavelengths which are catalyzed by luciferases. Luciferase is a photoprotein that emits light when presented with its substrate luciferin along with oxygen and ATP. The emitted light is in a wavelength that is minimally obscured by tissue fluorescence thereby allowing adequate tissue penetration and even quantification based

Figure 8. Transplantation of mNSCs into the brains of Hexb–/–mice prolongs life, delays symptom onset and preserves motor function. A) At a time when untreated Hexb–/– mice were moribund, mNSC-transplanted Hexb–/– mice were asymptomatic. B) Kaplan-Meyer curves of the survival of mNSC-transplanted Hexb–/– mice (n  43) compared with untreated Hexb–/– control mice (n  57) (P  0.0001, log rank test). Data were obtained using mNSCs from clone C17.2. C) Lifespan of mouse neurosphere–transplanted Hexb–/– mice (n  16) was similarly prolonged compared to that of untreated Hexb–/– mice (n  14) (P  0.0001, log rank test). D) Motor function test of neurosphere-transplanted Hexb–/– mice compared with that of untreated Hexb–/– control mice (P  0.001, t-test). Data represent mean ( s.e.m; n  9 wild-type mice. Motor function deteriorated at 13 weeks in untreated control Hexb–/– mice (n  14) but was delayed to at least 16 weeks, with a more gradual decline between 16 and 18 weeks, in neurosphere-transplanted Hexb–/– mice (Rosa 26 mNSC-Tx Hexb–/–, n  16). E) Representative 1-mm-thick semiserial coronal sections through the forebrain of an adult Hexb–/– mouse transplantated at birth with lacZ-expressing mNSCs into the cerebral ventricles, showing widely disseminated integration of blue X-gal donor-derived cells. F) As in e but showing coronal section through cerebellum of adult Hexb–/– mouse transplantated as newborn into the external germinal layer. Similar cerebral and cerebellar distributions were obtained whether NSCs were from mouse clone C17.2 (here), ROSA mouse neurospheres, human neuroectoderm or hESCs. Used with permission from Lee J et al. Nat Med 2007; 13:439-447.48

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on intensity of light emission. Using firefly luciferase (Fluc) the macroscopic migratory capacity of NSCs have been shown in mice implanted with tumor cells. NSCs transduced with Fluc were transplanted into the brain parenchyma and ventricles of nude mice inoculated with tumor cells. Subsequently BLI was used to serially evaluate the animals, revealing NSC migration at 1 week and light emission from NSCs occurring within the tumor 3 weeks post transplantation. These results were confirmed with histological analysis (Fig. 9).52,53 Similar ability to in vivo image NSC migration with BLI was shown in a murine model of stroke.54 BLI also offers the ability to simultaneously image two or more cellular transplants in a single living animal. The luciferases from firefly and Renilla have different substrates (luciferin and coelenterazine, respectively) and photon emission and different wavelengths. An alternative to BLI for in vivo imaging is the use of magnetic resonance imaging (MRI) to image cells that have imbibed or been transfected with superparamagnetic iron oxide particles (SPIO). These particles are small enough to detect small clusters of cells with a high tesla MRI and not lead to cellular toxicity that would harm the animal model. SPIO are nanoparticles (approximately 7-10 nm in diameter) that do not have magnetic properties outside of their respective magnetic fields and consequently are safe to be imaged within MRI machines. The SPIO particles create magnetic field gradients that de-phase the neighboring proton magnetic moments allowing for MRI detection without the need or risk of a heavy iron oxide burden.55 SPIO labeling of nonphagocytic cells can be performed by combining iron oxide particles with commercially available transfection agents. The efficacy of SPIO particles has been demonstrated in rats and mice. In a rat model for brain tumors, SPIO particles were sufficient to track NSC migration toward the tumor.56 In mouse model of brain tumors, localized and disseminated NSC-SPIO transplants were identified and tracked in vivo with MR imaging up to 32 days post injection.55 The metabolism of SPIO particles seems to be via the formation of clusters within the endosomal compartments with gradual consumption in normal iron metabolism pathways. Furthermore, it appears that these nanoparticles do not interfere with neuronal differentiation, although differentiation of mesenchymal stem cells is limited in the presence of SPIO particles.57,58 Other imaging techniques such as positron emission tomography (PET) and single photon emission tomography (SPECT) are potential modalities for imaging of cells, however they rely upon radioligands which raise concerns over toxicity and difficulty of on-site synthesis. Ultimately for in vivo imaging of humans, MRI offers the most promising horizon. It uses an imaging modality that is increasingly available in most hospitals and does not rely upon genetic engineering, as is the case for bioluminescence. In fact, MR has been used to track magnetically labeled cells in melanoma patients.59 The horizon for neuroscience research with stem cells has generated significant interest, yet remains in its infancy. Indeed, our current knowledge mandates exploration into the vast possibilities of treating CNS pathology and genetically modified NSCs are leading candidates for human clinical application. To avoid misadventure, more investigation is necessary. The multipotency of NSCs can be harnessed by genetically engineering them to express genes that drive fate determination (such as dopaminergic neuron development with Nurr1) or eliminate the expression of genes that drive the generation of unwanted cell types. A better understanding of the cellular and molecular mechanisms controlling NSC differentiation into specific lineages during development and in adults will be critical prior to successful engraftment of diseased areas in humans. Further studies are needed to elucidate the molecular signals for proliferation, migration and integration with the aim of developing precise genetic modifications of NSCs to enhance and direct migration, integration and pathway reconstruction. A greater understanding of endogenous NSCs may allow the manipulation of environmental cues and milieu leading to their successful recruitment, particularly in the aging brain where loss or reduction of the requisite environmental cues seems to limit adult neurogenesis. NSCs also have the potential for real-time detection with molecular and cellular imaging, allowing direct visualization of gene expression providing further understanding of the efficacy of cell and gene therapy.

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Figure 9. NPC-FL-sTRAILs are effective in significantly reducing glioma growth in the brains of experimental animals. A–F) Mice were implanted with Gli36-RL cells mixed with either NPC-FL-sTRAILs (A, B, E) or NPC-FLs (C, D, F) into the right frontal lobe and were observed for tumor progression by Renilla luciferase (Rluc) imaging (A–D) and for neural precursor cell (NPC) presence in the tumors by firefly luciferase (Fluc) imaging (E, F). G) Rluc bioluminescence intensities of Gli36-RL tumors alone or mixed with either NPC-FLsTRAILs or NPC-FLs cells over time. Color-coded maps of the photon intensities from Gli36-RL or NSC-FL-sTRAIL cells are shown. H) X-gal staining showing the presence of beta-galactosidase–expressing NPC-FL-sTRAILs (blue) in the tumor (original magnification =10). Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression. Shah K et al. Ann Neurol 2005; 57:34-41.53 Reprnted with permission of John Wiley and Sons, Inc. A color version of this image is available at www.landesbioscience.com/curie.

Conclusion The optimal source (both accessible and safe) of NSCs for clinical transplantation needs to be determined. Although, embryonic stem cell derived NSCs my be the most versatile and plastic, autologous sources of NSCs from minimally invasive neurological surgery hold promise for stem

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cell therapy using a patient’s NSCs that are genetically modified and subsequently re-implanted. These experiments are ongoing in our laboratory. Genetic modification of immortalized NSCs has demonstrated effective and long-term gene transfer to the brain in murine models of CNS pathology. Novel techniques to achieve highly efficient and reproducible transduction of transgenes for the extension of cellular lifespan and expression of therapeutic proteins would be highly desirable prior to clinical transplants into patients. The goal, although challenging, would be to immortalize NSCs without using oncogenes, or at least the selective removal of the immortalized oncogene after transplantation. The modification of NSCs would be of greater utility if pharmacological control of gene expression with constitutive promoters could be more refined and effective suicide systems engineered could be created for grafted cells. The analysis of long-term cultured or immortalized NSCs need to occur prior to clinical transplantation, specifically, safety, stability, homogeneity and potential for malignant transformation need to be studied. Although the challenges remaining prior to clinical transplantation of NSCs for neurological disease are significant, the preclinical data is compelling; and of the various molecular and cellular modalities under investigation for this aim, NSCs offer the greatest potential.

References 1. Snyder E, Deitcher D, Walsh C et al. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 1992; 68(1):33-51. 2. Renfranz P, Cunningham M, McKay R. Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell 1991; 66(4):713-29. 3. Flax J, Aurora S, Yang C et  al. Engraftable human neural stem cells respond to developmental cues, replace neurons and express foreign genes. Nat Biotechnol 1998; 16(11):1033-9. 4. Rubio F, Bueno C, Villa A et al. Genetically perpetuated human neural stem cells engraft and differentiate into the adult mammalian brain. Mol Cell Neurosci 2000; 16(1):1-13. 5. Martinez-Serrano A, Rubio F, Navarro B et al. Human neural stem and progenitor cells: in vitro and in vivo properties and potential for gene therapy and cell replacement in the CNS. Curr Gene Ther 2001; 1(3):279-99. 6. Ourednik V, Ourednik J, Flax J et al. Segregation of human neural stem cells in the developing primate forebrain. Science 2001; 293(5536):1820-4. 7. Aboody K, Brown A, Rainov N et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci USA 2000; 97(23):12846-51. 8. Fallon J, Reid S, Kinyamu R et  al. In vivo induction of massive proliferation, directed migration and differentiation of neural cells in the adult mammalian brain. Proc Natl Acad Sci USA 2000; 97(26):14686-91. 9. Park K, Teng Y, Snyder E. The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat Biotechnol 2002; 20(11):1111-7. 10. Imitola J, Raddassi K, Park K et  al. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci USA 2004; 101(52):18117-22. 11. Glass R, Synowitz M, Kronenberg G et  al. Glioblastoma-induced attraction of endogenous neural precursor cells is associated with improved survival. J Neurosci 2005; 25(10):2637-46. 12. Ehtesham M, Kabos P, Kabosova A et  al. The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma. Cancer Res 2002; 62(20):5657-63. 13. Ziu M, Schmidt N, Cargioli T et  al. Glioma-produced extracellular matrix influences brain tumor tropism of human neural stem cells. J Neurooncol 2006; 79(2):125-33. 14. Yip S, Aboody K, Burns M et al. Neural stem cell biology may be well suited for improving brain tumor therapies. Cancer J 2003; 9(3):189-204. 15. Lundberg C, Horellou P, Mallet J et al. Generation of DOPA-producing astrocytes by retroviral transduction of the human tyrosine hydroxylase gene: in vitro characterization and in vivo effects in the rat Parkinson model. Exp Neurol 1996; 139(1):39-53. 16. Anton R, Kordower J, Maidment N et  al. Neural-targeted gene therapy for rodent and primate hemiparkinsonism. Exp Neurol 1994; 127(2):207-18. 17. Ryu M, Lee M, Ahn Y et  al. Brain transplantation of neural stem cells cotransduced with tyrosine hydroxylase and GTP cyclohydrolase 1 in Parkinsonian rats. Cell Transplant 2005; 14(4):193-202. 18. Akerud P, Canals J, Snyder E et  al. Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson’s disease. J Neurosci 2001; 21(20):8108-18.

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19. Akerud P, Holm P, Castelo-Branco G et al. Persephin-overexpressing neural stem cells regulate the function of nigral dopaminergic neurons and prevent their degeneration in a model of Parkinson’s disease. Mol Cell Neurosci 2002; 21(2):205-22. 20. Kim J, Auerbach J, Rodríguez-Gómez J et  al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 2002; 418(6893):50-6. 21. Martínez-Serrano A, Björklund A. Protection of the neostriatum against excitotoxic damage by neurotrophin-producing, genetically modified neural stem cells. J Neurosci 1996; 16(15):4604-16. 22. McBride J, Behrstock S, Chen E et  al. Human neural stem cell transplants improve motor function in a rat model of Huntington’s disease. J Comp Neurol 2004; 475(2):211-9. 23. Lee S, Park J, Lee K et al. Noninvasive method of immortalized neural stem-like cell transplantation in an experimental model of Huntington’s disease. J Neurosci Methods 2006; 152(1-2):250-4. 24. Azzouz M, Ralph G, Storkebaum E et  al. VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature 2004; 429(6990):413-7. 25. Kaspar B, Lladó J, Sherkat N et al. Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science 2003; 301(5634):839-42. 26. Tuszynski M, Thal L, Pay M et  al. A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat Med 2005; 11(5):551-5. 27. Martínez-Serrano A, Björklund A. Ex vivo nerve growth factor gene transfer to the basal forebrain in presymptomatic middle-aged rats prevents the development of cholinergic neuron atrophy and cognitive impairment during aging. Proc Natl Acad Sci USA 1998; 95(4):1858-63. 28. Yamasaki T, Blurton-Jones M, Morrissette D et  al. Neural stem cells improve memory in an inducible mouse model of neuronal loss. J Neurosci 2007; 27(44):11925-33. 29. Benedetti S, Pirola B, Pollo B et al. Gene therapy of experimental brain tumors using neural progenitor cells. Nat Med 2000; 6(4):447-50. 30. Li S, Tokuyama T, Yamamoto J et al. Bystander effect-mediated gene therapy of gliomas using genetically engineered neural stem cells. Cancer Gene Ther 2005; 12(7):600-7. 31. Kim S, Cargioli T, Machluf M et al. PEX-producing human neural stem cells inhibit tumor growth in a mouse glioma model. Clin Cancer Res 2005; 11(16):5965-70. 32. Schwab M. Repairing the injured spinal cord. Science 2002; 295(5557):1029-31. 33. Ogawa Y, Sawamoto K, Miyata T et  al. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J Neurosci Res 2002; 69(6):925-33. 34. Teng Y, Lavik E, Qu X et al. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc Natl Acad Sci USA 2002; 99(5):3024-9. 35. Lu P, Jones L, Snyder E et al. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 2003; 181(2):115-29. 36. Hofstetter C, Holmström N, Lilja J et al. Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nat Neurosci 2005; 8(3):346-53. 37. Blesch A, Lu P, Tuszynski M. Neurotrophic factors, gene therapy and neural stem cells for spinal cord repair. Brain Res Bull 2002; 57(6):833-8. 38. Cummings B, Uchida N, Tamaki S et al. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci USA 2005; 102(39):14069-74. 39. Iwanami A, Kaneko S, Nakamura M et  al. Transplantation of human neural stem cells for spinal cord injury in primates. J Neurosci Res 2005; 80(2):182-90. 40. Andsberg G, Kokaia Z, Björklund A et  al. Amelioration of ischaemia-induced neuronal death in the rat striatum by NGF-secreting neural stem cells. Eur J Neurosci 1998; 10(6):2026-36. 41. Arvidsson A, Collin T, Kirik D et  al. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 2002; 8(9):963-70. 42. Nakatomi H, Kuriu T, Okabe S et  al. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 2002; 110(4):429-41. 43. Zhang Z, Jiang Q, Zhang R et  al. Magnetic resonance imaging and neurosphere therapy of stroke in rat. Ann Neurol 2003; 53(2):259-63. 44. Kelly S, Bliss T, Shah A et al. Transplanted human fetal neural stem cells survive, migrate and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci USA 2004; 101(32):11839-44. 45. Snyder E, Taylor R, Wolfe J. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain. Nature 1995; 374(6520):367-70. 46. Lacorazza H, Flax J, Snyder E et al. Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells. Nat Med 1996; 2(4):424-9.

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47. Shihabuddin L, Numan S, Huff M et  al. Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology. J Neurosci 2004; 24(47):10642-51. 48. Lee J, Jeyakumar M, Gonzalez R et al. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med 2007; 13(4):439-47. 49. Cameron H, McKay R. Restoring production of hippocampal neurons in old age. Nat Neurosci 1999; 2(10):894-7. 50. Kempermann G, Gast D, Gage F. Neuroplasticity in old age: sustained fivefold induction of hippocampal neurogenesis by long-term environmental enrichment. Ann Neurol 2002; 52(2):135-43. 51. Hallbergson A, Gnatenco C, Peterson D. Neurogenesis and brain injury: managing a renewable resource for repair. J Clin Invest 2003; 112(8):1128-33. 52. Tang Y, Shah K, Messerli S et al. In vivo tracking of neural progenitor cell migration to glioblastomas. Hum Gene Ther 2003; 14(13):1247-54. 53. Shah K, Bureau E, Kim D et al. Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression. Ann Neurol 2005; 57(1):34-41. 54. Kim D, Schellingerhout D, Ishii K et  al. Imaging of stem cell recruitment to ischemic infarcts in a murine model. Stroke 2004; 35(4):952-7. 55. Magnitsky S, Watson D, Walton R et al. In vivo and ex vivo MRI detection of localized and disseminated neural stem cell grafts in the mouse brain. Neuroimage 2005; 26(3):744-54. 56. Zhang Z, Jiang Q, Jiang F et al. In vivo magnetic resonance imaging tracks adult neural progenitor cell targeting of brain tumor. Neuroimage 2004; 23(1):281-7. 57. Arbab A, Bashaw L, Miller B et  al. Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging. Radiology 2003; 229(3):838-46. 58. Kostura L, Kraitchman D, Mackay A et  al. Feridex labeling of mesenchymal stem cells inhibits chondrogenesis but not adipogenesis or osteogenesis. NMR Biomed 2004; 17(7):513-7. 59. de Vries I, Lesterhuis W, Barentsz J et  al. Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy. Nat Biotechnol 2005; 23(11):1407-13.

Chapter 8

Biological Horizons for Targeting Brain Malignancy Samuel A. Hughes, Pragathi Achanta, Allen L. Ho, Vincent J. Duenas and Alfredo Quiñones-Hinojosa*

Abstract

T

hough currently available clinical treatments and therapies have clearly extended the survival of patients with brain tumors, many of these advances are short lived, particularly with respect to high grade gliomas such as glioblastoma multiforme. The missing link to an efficacious treatment of high grade gliomas is a more complete understanding of the basic molecular and cellular origin of brain tumors. However, new discoveries of stem cell and developmental neurobiology have now borne the cancer stem cell hypothesis, drawing off of intriguing similarities between benign and malignant cells within the central nervous system. Investigation of cancer stem cell hypothesis and brain tumor propagation is the current frontier of stem cell and cancer biology. Neurosurgery is also watching closely this promising new area of focus. “Molecular neurosurgery”, glioma treatments involving biologics using neural stem cells to target the cancer at the level of individual migratory cell, is a rapidly evolving field. This coming progression of applied cancer stem cell research, coupled with current modalities, promises more comprehensive brain cancer interventions.

Introduction Brain tumors and in particular high grade aggressive gliomas, remain a difficult and evasive foe to our current armamentarium of therapeutic weapons.1-3 Clearly, steroids, surgery and adjuvant therapy (chemotherapy and radiation) have prolonged the longevity of patients. Yet the extension of life has been limited and in the case of glioblastoma multiforme, remains near one year after diagnosis and treatment with all available modalities.4-7 Part of the challenge arises from our lack of understanding of the basic molecular and cellular mechanisms regarding the origin of brain tumors. At the same time our increasing knowledge of stem cells and developmental biology offers potential to not only understand the origin of brain tumors, but also the opportunity to exploit that knowledge to target and better treat brain tumors. Classically, neurogenesis in the adult mammal and humans was thought not to exist.8,9 Modern methods and investigation have refuted this claim and understanding of the germinal zones and their resident neural stem cells (NSCs) has emerged.10-12 Remarkably, the behavior of NSCs a of neuroectodermal origin,13 at least in terms of migratory capacity, ability to incorporate into normal tissue, self renewal potential and molecular signature, has strong overlap with malignant cells in the central nervous system (CNS). Furthermore, the proliferative and metastatic capacity of brain tumors is thought to be maintained by the presence of a population of ‘stem-like’ cells called cancer *Corresponding Author: Alfredo Quiñones-Hinojosa—Neuroscience and Cellular and Molecular Medicine, Johns Hopkins University, Department of Neurosurgery, Baltimore, Maryland, USA. Email: [email protected]

Frontiers in Brain Repair, edited by Rahul Jandial. ©2010 Landes Bioscience and Springer Science+Business Media.

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stem cells.11,14-18 It remains unclear whether brain tumors and their cancer stem cells emerge from the de-differentiation of mutated mature neural cells or emanate de novo from otherwise normal organogenic progenitors fated or predisposed to become neoplastic.19 Evidence supports both as potential etiologies, while not clearly accepting either scenario.20 The biology and management of brain tumors as well as other malignancies may be linked to a better understanding of stem cell biology.21,22 This could have dual benefits of understanding both how neoplastic stem cells emerge and how normal stem cells behave in response. The intricate and possibly overlapping potential of stem cells to create and populate the CNS in a physiological manner as well as be the source of malignant disease is the current frontier of stem and cancer biology. This investigation will be fostered by recent generation of more representative in vivo animal models that helped to accelerate the development of new diagnostic technologies and novel therapeutic agents.23,24 Clinical neurosurgery is being transformed by advances in imaging technologies, including high-resolution magnetic resonance imaging (MRI), MR spectroscopy and positron emission tomography (PET) scans, as well as diffusion and perfusion imaging which permit better localization and characterization of lesions and their relationship with normal brain architecture.25 These advances have led to notable improvements in surgical resection. Yet the horizon for intervention to treat glioma cells that have the intrinsic capability to infiltrate local structures and to migrate over great distances rests on what is now being called “molecular neurosurgery”. Fortunately, NSCs are excellent candidates based on their ability to track cancer cells, capacity to be genetically modified.13,26,27 If proven to be the cell of origin, cancer stem cells could even be targeted in a premorbid state in the mammalian brain.19

Neural Stem Cells NSCs are the most primordial and least committed cells of the nervous system. Given the lack of specific markers for NSCs, functional assays are used to determine their presence within a cell population.11 To be a neural “stem” cell, as opposed to a “progenitor” cell or “precursor” cell, a single neural stem cell must have the following functional properties: (1) “self-renewal”, i.e., the ability to produce daughter cells with identical properties. (2) “multipotency”, the ability to yield mature cells in all 3 fundamental neural lineages throughout the nervous system: neurons of all types; astrocytes of all types; and oligodendrocytes—in regional and developmental stage-appropriate manners; (3) the ability to populate a developing region and/or repopulate an ablated or degenerated region of the CNS with appropriate cell types.28 It has been demonstrated that a single neural cell with stem-like properties could be isolated and re-implanted into the brain where its progeny could integrate seamlessly, differentiate into integral members of the CNS, respond to prevalent developmental cues to yield appropriate multiple neural cell types (both neuronal and glial) and import foreign genes into the CNS.29,30 Neural cells with stem cell properties have been isolated from the embryonic, neonatal and adult nervous system and propagated in vitro by a variety of equally effective and safe means—both epigenetically and genetically.31,32 Populations of NSCs derived from embryonic stem cells via induction of differentiation in vitro forms the three embryonic germ layers.33,34 The ability of this differentiation into NSCs though has been described, it remains to be completely understood. Different in vitro protocols are employed to obtain embryoid bodies and embryonic stem cell derived NSCs, providing a nonsomatic source for NSCs.31,32 While there is a great deal of debate as to the best source for neural progenitors, it is the behavior of the NSC derived directly from the neuroectoderm which has established the ‘gold standard’ for what can and should be achievable by cells with normal stem-like attributes.35,36 Engrafted exogenous NSCs manipulated ex vivo to express a variety of transgenes can integrate locally at the site of implantation.13,26,27 NSCs can be exploited to disseminate therapeutic genes, or to yield some desired neural cell types globally throughout the CNS, if applied to the proper germinal zone.35,37 40 The tremendous migratory capability of NSCs in conjunction with

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their innate tropism for intracranial pathologies make them ideal therapeutic agents in a variety of neurological diseases.41 43

Exogenous and Endogenous NSCs Respond to Gliomas Aboody and colleagues first described the ability of exogenous NSCs to migrate toward and distribute within a transplanted glioma.13,26,27 NSCs migrated to the site of tumors not only when they were implanted near the tumor, but also when implanted in the contralateral side of the tumor or in the cerebral ventricles.13 The challenge of treating malignant gliomas lies in targeting the cancerous cells that migrate outside of the tumor bulk. These invasive cells lead to progression and recurrence even when the great majority of the tumor bulk is resected or treated.44-46 Surprisingly, NSCs could be seen tracking along individual cancerous cells suggesting a powerful tool for brain tumor treatment.13 A reduction in tumor size was demonstrated when NSCs were genetically modified to deliver chemotherapeutics, interleukins and thymidine kinase.27,47-51 Endogenous NSCs migrate to brain lesions such as ischemia52,53 and multiple sclerosis54 and gliomas.55,56 NSCs, particularly from the subventricular zone, have been shown to migrate toward gliomas in mice and evince the same gliotropism as exogenous NSCs.57 However, this potential does not seem to be sufficient and different genetic manipulations may be necessary to potentiate gliotropism by endogenous NSCs. Clearly this does lead to some of the cellular heterogeneity in the tumor microenvironment.58 Interestingly, the response of NSCs to gliomas, is less as animals get older and thought to be one factor that leads to the higher incidence of malignant gliomas in elderly patients.59 Whether it is an issue of pure numerical advantage of the rapidly dividing tumor cells overwhelming the small numbers of endogenous NSCs, or other unidentified factors remains to be determined. Or perhaps some ‘rogue’ endogenous NSCs themselves, under some circumstances, are the culprits giving rise to the tumor from the onset.60 The microenvironment around the tumor is thought to have pro-proliferative effect on NSCs expressing significant levels of Ki67.57,60 NSCs, like gliomas, also have a predilection for white matter tracts and blood vessel basement membranes.61,62 This capacity for self-renewing and invasion may represent important features shared by brain tumor and neural stem cells and leads to the speculation that aberrant neural stem cells give rise to brain tumor stem cells which create the bulk of the tumor and render it resistant to current therapeutic regimens.19,63-66 These are extremely important clinically relevant issues that might influence what type of new chemotherapeutic interventions should be devised—drugs that boost the response of endogenous NSCs to tumors, or perhaps those that eliminate some of the aberrant NSCs.42

Mechanisms for NSC Homing to Gliomas Several studies have demonstrated that NSCs home into regions of intracranial pathology, opening up an entire new field of NSC based treatment options for intracerebral lesions.67-71 Numerous factors such as chemokines and growth factors responsible for luring NSCs to intracranial pathology have been suggested previously.72,73 A variety of factors released and expressed by glioma cells, tumor stroma (comprised of adjacent reactive astrocytes, microglia and oligendendrocytes), tumor derived endothelium and the damage surrounding normal brain tissue have been shown to induce NSC gliomatropism.13,26,27 While factors such as stem cell factor (SCF) and monocyte chemoattractant protein-1 (MCP1) have been acknowledged as agents of NSC gliomatropism,74-77 other factors such as chemokines, growth factors still have to be characterized and their roles in NSC gliomatropism have yet to be defined.78 Chemokines are an intrinsic part of the normal development and function of many biological systems, especially in the CNS. Chemokine expression may be disturbed in diseased conditions,79 and they are an important point of inquiry into NSC attraction to pathology. Haematopoeitic chemokine receptor CXCR4 was expressed in NSCs in tandem with the expression of its cognate ligand SDF-1_ within regions of CNS injury and degeneration (particularly by reactive astrocytes and endothelium).80,81 This led to the suggestion that products of inflammation might, in addition

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to being inimical, might also function as a beacon for reparative cells. The homing of NSCs to affected regions of the CNS82 would be guided by migratory cues. This implies that the homing behavior of NSCs to pathology within the developed brain may simply be a recapitulation of NSC migration during CNS organogenesis.83 The role of CXCR4 and SDF-1_ in tumor growth, migration and angiogenesis reinforces the comparison between NSCs and neoplastic CNS cells. SDF-1_ is expressed on tumor derived endothelium and CXCR4 is found on a variety of tumor growths, including GBMs and medulloblastomas.80 This indicates a probable paracrine growth loop and an explanation for angiocentric growth of intracranial neoplasms on a molecular level.84,85 Rubin and colleagues (2003) have shown that small molecule antagonists of CXCR4 are successful in inhibiting the growth of GBM and medulloblastoma in experimental models.86 Since expression of SDF-1_ by the tumor-associated endothelium stimulates the migration of NSCs87,88 it would follow that this same inhibition of the SDF-1_/CXCR4 interaction actually prevents the gliomatropic migration of NSCs.89 In addition, this interaction also appears to be an integral part of the gliomatropism of circulating adult haematopoeietic progenitor cells.90 Finally, because the receptor CXCR4 is also expressed by metastatic breast cancer and ligand SDF-1_ is concurrently expressed by brain endothelium, a mechanism for the transmigration of circulating metastatic neoplastic cells through the blood brain barrier—another characteristic shared by stem and neoplastic cells13,91 can be proposed through the CXCR4/SDF-1_ interaction.92 Indeed, Liang and associates (2004) were able to prevent breast cancer metastasis by inhibiting CXCR4.93 Recently, vascular endothelial growth factor (VEGF) has also been targeted as a source of NSC gliomatropism56,94 VEGF, with other pro-angiogenic factors, is vital to maintaining the aggressive behavior of GBM.95-97 NSCs are recruited by a gradient of VEGF as a chemotactic effect mediated through the activation of VEGF receptors.98 VEGF infusions promoted the migration of transplanted NSCs from the contralateral hemisphere of the rodent brain across the corpus callosum to the site of infusion.99 Also, epidermal growth factor (EGF) and EGF receptor signaling over-expression, an important component of glioma malignancy, invasion and migration,100-102 has been linked to NSC migration.103 Greater than 50% of GBM cases have been shown to over-express EGFR and many have a constitutively active variant of the receptor called EGFRvIII.104 NSC migration towards tumor cells could be instituted by EGF concentration gradients.103 But the possibility still remains that glioma cells will continue to ‘outrun’ NSCs, branding ineffective any endogenous NSC response. Given that the extreme motility of malignant glioma cells is dependent upon specific gene sets,105,106 delineating the role of these genes and subsequent downstream signaling events is essential for the eventual development of therapeutic agents and suitably armed exogenous NSCs to compensate for and target these pathways to enhance their homing abilities.

Exploiting NSCs as Vehicles for Delivering Toxic Payloads Previously, most mechanisms of delivering a toxic payload were either through direct injection, or with the introduction of adeno-associated virus (AAV) engineered to express the anti-angiogenic protein angiostatin directly into the tumor.107 The effectiveness of viral-mediated gene delivery to brain tumors was limited by the ‘halo’ effect of only tumor cells within a limited radius of injected viral vector being eradicated.108 Tumor cells situated beyond that radius could escape to set up new satellite tumors.42 Infiltrating migratory cancer cells remained elusive and difficult to target. While studies continue to find increasing roles for NSCs in the treatment of neurological pathology, substantial work has been done by various groups exploiting the unique tropism of NSCs for gliomas in the context of genetic modification to deliver a variety of anti-glioma gene products.26,27 NSCs have the capacity to convey large amounts of genetic information (beyond the limits imposed by the relatively small genome of the viral vectors) that make NSCs a much more powerful and adaptive anti-tumor agent. Aboody and colleagues employed a model where nude mice were inoculated with glioma cells and subsequently transplanted with human and murine NSCs at various locations (intratumoral, contralateral hemisphere, intraventricular and tail vein) with clear demonstration

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Figure 1. From: Müller FJ, Snyder EY, Loring JF. Gene therapy: can neural stem cells deliver? Nat Rev Neurosci 2006; 7(1):75-84.

of NSCs migrating to and distributing within the tumor (Fig. 1).13 Subsequently, NSCs were transfected with a gene for cytosine deaminase (CD), a prodrug-converting enzyme that converts 5-FC to 5-FU and transplanted away from the tumor. This technique let to approximately 80% reduction in tumor burden.13 The CDA pro-drug system, in particular, engenders an extremely large ‘bystander effect’, killing even a small number of tumor cells and sending ‘ripples’ of oncolytic factors emanating from that epicenter of cell death to kill peripheral tumor cells.13 This could be mediated by expression of connexin-43 in untransduced glioma cells.50 Therefore, even if the CDA transgene were to be down-regulated in some NSCs, the oncolytic action of the

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population of NSCs would remain effective. In addition, CDA would serve as a suicide gene within the NSC—hence a built-in safety mechanism.51 Other pro-drug systems using thymidine kinase (TK) have been tried successfully in NSCs109 to target brain tumors. NSC-based delivery of an oncolytic adenovirus has been proven to be possible demonstrating the feasibility of NSC delivery of biologic agents.110 Herpes simplex virus1-thymidine kinase (HSVtk) has been shown to be effective at treating gliomas when expressed by NSCs carrying the HSVtk gene.110 Murine NSCs expressing HSCtk were transplanted intratumoral (rat glioma cell line C6) into mice and rats during and after tumor inoculation and then treated with ganciclovir.110 Interestingly, there was a 70% increase in survival with NSC-HSVtk transplantation after tumor inoculation and 100% survival with cotransplantation of tumor and NSC-HSVtk.51,110 Cytokines have been used successfully to treat some human malignancies via tumor toxicity or growth arrest.111,112 Similarly, immunotherapy for brain tumors via the direct instillation of cytokines, or via the use of genetically modified viral vectors has demonstrated some efficacy.113-115 Ehtesham et al transfected murine NSCs with the gene for pro-inflammatory cytokine interleukin-12 (IL-12), demonstrated stable expression and found increased survival with intratumoral transplantation in tumor bearing syngeneic mice.27 The authors also observed infiltration of the tumor by T-lymphocytes in response to regional expression of IL-12; a finding corroborated by others.116 NSCs engineered to delivery IL-4 resulted in a significant decrease of tumor size and increase in survival in a rodent model of gliosarcoma.26 Also, NSCs expressing TNF-related apoptosis inducing ligand (TRAIL) resulted in increased tumor cell apoptosis resulting in decreased tumor size.27 These studies indicate that NSCs could overcome the hurdle of achieving a high enough local concentration of therapeutic compounds by their specific homing ability. Malignant gliomas are known to produce pro-angiogenic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and the histopathological grading of malignancy includes assessment of vascular proliferation in the tumor sample.56 Accordingly, anti-angiogenic compounds are candidates for interfering with tumor growth as one mechanism to target gliomas and genetically modified NSCs could express the toxic payload of choice. The delivery of such molecules preferentially to the vascular endothelium within the tumors is further supported by the observation that NSCs have a predilection for and transmigrate through, tumor endothelium via association of their CXCR4 receptors with endothelial-expressed SDF-1_, as well as via _4-integrin.87 Most studies investigating the potential of NSCs in tumor therapy have employed murine cells. The ability to home and target gliomas has also been demonstrated with the use of human NSCs. Kim et al employed PEX (expand PEX), a naturally occurring fragment of human metalloproteinase-2, which acts as an inhibitor of glioma and endothelial cell proliferation, migration and angiogenesis, to target human glioma cells (Gli26).117 Using the HB1.F3 cell line, immortalized human NSCs were transduced by a vector with PEX and subsequently transplanted intratumoral into the mice. The mice showed a profound reduction in tumor volume and angiogenesis as evaluated by magnetic resonance imaging and histological examination.117 Thus, NSCs unique capability to migrate toward and distribute within brain tumors and their ability to cross the blood—brain barrier and finding even small micro-deposits of tumor cells make them even more suitable to the unique challenges of getting therapeutics into the CNS (Table 1). As more is learned about brain repair and regenerative medicine, stem cells remain applicable in other clinical scenarios with the treatment of malignant brain disease.

Conclusion More must be learned about developmental neurobiology, stem cell biology, molecular imaging and gene regulation for the successful clinical translation of NSCs. In the setting of malignant brain tumors, the NSCs would not need to differentiate and integrate into the neural circuitry, a much more challenging proposition. Instead, if they were proven to be safe and traceable, they would simply need to do what has been already demonstrated as their unique ability—migrate, distribute and track individual tumor cells. The ability to image and track cellular transplantsawith luciferase,

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Table 1. Strategies in neural stem cell (NSC)-mediated brain tumor therapeutics

Growth Regulatory and Immunomodulatory Tumoricidal

Viral Therapy

Pro-Drug Converting Enzyme/ Suicide Gene Therapy

Biophysical Agents Delivery of agents to vicinity of tumor, which requires sub sequent secondary activation

Induction of tumour growth arrest via interaction with NSCs or growth regulatory factors

Introduction of virus into vicinity of tumor cells causing cytolysis

Enzymatic conversion of pro-drug into toxins. Cytotoxicity is amplified by the ‘by-stander’ effect

Published IL4, 12 IL2a studiesb

TRAIL IFN- `

HSV adenovirus AAV

Cytosine deaminase HSV-TK

Future?

PF4 (platelet factor 4) TNF(73)

Reovirus Type 3 VSV

Deoxycytidine Nanoshells kinase photodynamic therapy

Initiation of enhanced anti-tumor immune response via local delivery and expression of high concentrations of cytokines

GM-CSF

Abbreviations: IL: interleukin; GM-CSF: Granulocyte-macrophage colony stimulating factor; AAV: adeno-associatd virus; HSV-TK: Herpes simplex virus thymidine kinase; VSV: Vesicular stomatitis virus; TNF: tumour necrosis factor; IFN: interferon. aTransduced MSCs, bRefer to references 13, 47, 27, 48, 49, 107, 108, 110-113, 116, 118-120, 122-139.

superparamagnetic iron oxide particles and quantum dots is evolving rapidly.118-120 Well characterized NSCs that can be genetically modified and banked would be another necessary component. Some have shown mesenchymal cells to be also potentially effective for treating brain tumors.121 In the future as we learn more about sources and methods for stem cell biology, the potential for autologous stem cell transplants may arise. Through NSCs mediated treatment of malignant gliomas, practical protocols for preparing and delivering stem cells will be devised, safety will be proven and the behavior of stem cells in a human brain will be observed, all of which will help the broader field of stem cell biology and regenerative medicine.

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122. Chiocca EA, Aghi M, Fulci G. Viral therapy for glioblastoma. Cancer J 2003; 9(3):167-79. 123. Chiocca EA, Broaddus WC, Gillies GT et  al. Neurosurgical delivery of chemotherapeutics, targeted toxins, genetic viral therapies in neuro-oncology. J Neurooncol 2004; 69(1-3):101-17. 124. Kew Y, Levin VA. Advances in gene therapy and immunotherapy for brain tumors. Curr Opin Neurol 2003; 16(6):665-70. 125. Kurihara H, Zama A, Tamura M et  al. Glioma/glioblastoma-specific adenoviral gene expression using the nestin gene regulator. Gene Ther 2000; 7(8):686-93. 126. Fueyo J, Alemany R, Gomez-Manzano C et  al. Preclinical characterization of the antiglioma activity of a tropism-enhanced adenovirus targeted to the retinoblastoma pathway. J Natl Cancer Inst 2003; 95(9):652-60. 127. Stojdl DF, Lichty BD, tenOever BR et al. VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 2003; 4:263-75. 128. Manome Y, Wen PY, Dong Y et al. Viral vector transduction of the human deoxycytidine kinase cDNA sensitizes glioma cells to the cytotoxic effects of cytosine arabinoside in vitro and in vivo. Nat Med 1996; 2(5):567-73. 129. Lynch WP, Sharpe AH, Snyder EY. Neural stem cells as engraftable packaging lines can mediate gene delivery to microglia: evidence from studying retroviral env-related neurodegeneration. J Virol 1999; 73(8):6841-51. 130. Arnhold S, Hilgers M, Lenartz D et al. Neural precursor cells as carriers for a gene therapeutical approach in tumor therapy. Cell Transplant 2003; 12(8):827-37. 131. Ehtesham M, Samoto K, Kabos P et al. Treatment of intracranial glioma with in situ interferon-gamma and necrosis factor-alpha gene transfer. Cancer Gene Ther 2002; 9(11):925-34. 132. Walczak H, Miller RE, Ariail K et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-ligand in vivo. Nat Med 1999; 5(2):157-63. 133. Kim I, Kim H, Im S et al. Induction of intracranial glioblastoma apoptosis by transplantation of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) expressing human neural stem cells (NSCs). In: Annual meeting of society of neuroscience. San Diego; 2004. 134. Lewin M, Carlesso N, Tung CH et al. Tat peptide-derivatized magnetic nanoparticles allow in recovery of progenitor cells. Nat Biotechnol 2000; 18(4):410-4. 135. Anderson SA, Glod J, Arbab AS et  al. Noninvasive MR imaging of magnetically labeled stem cells to directly identify neovasculature in a glioma model. Blood 2005; 105(1):420-5. 136. Jaiswal JK, Simon SM. Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol 2004; 14(9):497-504. 137. Gao X, Cui Y, Levenson RM et al. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 2004; 22(8):969-76. 138. Loo C, Lowery A, Halas N et al. Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano Lett 2005; 5(4):709-11. 139. Hirsch LR, Stafford RJ, Bankson JA et al. Nanoshell-mediated near-infrared thermal therapy of tumors undermagnetic resonance guidance. Proc Natl Acad Sci USA 2003; 100(23):13549-54.

Chapter 9

Stem Cells in the Treatment of Stroke Klaudia Urbaniak Hunter,* Chester Yarbrough and Joseph Ciacci

Abstract

S

troke is an often devastating insult resulting in neurological deficit lasting greater than 24 hours. In the United States, stroke is the third leading cause of death. In those who do not succumb, any outcome from total recovery over a period of weeks to months to persistent profound neurological deficits is possible. Present treatment centers on the decision to administer tissue plasminogen activator, subsequent medical stabilization and early intervention with rehabilitation and risk factor management. The advent of stem cell therapy presents an exciting new frontier for research in stroke treatment, with the potential to cause a paradigm shift from symptomatic control and secondary prevention to reconstitution of neural networks and prevention of neuronal cell death after neurologic injury.

Introduction Stroke is defined is brain ischemia causing neurologic deficit lasting greater than 24 hours. This period of ischemia causes death of neurons, astrocytes, oligodendrocytes and microglia in the vascular territory downstream of the pathology. The proximate cause of stroke can be thrombosis, embolization, or hemorrhage, though a complete discussion of the various causes will be left for review. Further, many medical conditions, such as hypertension, diabetes mellitus, atrial fibrillation and atherosclerosis and prior history of stroke increase the risk of suffering a stroke. Initial care hinges on supportive measures and the decision to administer tissue plasminogen activator (tPA or alteplase). The decision whether to administer tPA is an important one, as tPA been shown to decrease neurological sequelae following ischemic stroke, if given within 3 hours of onset.1 Despite advances in medical care, stroke remains the 3rd leading cause of death in the United States, affecting some 750,000 people each year, with an incidence of approximately 150 events per 100,000 people per year.2,3 The incidence of stroke may be even higher outside of the United States, especially in Europe and Asia.4-7 In approximately one-third of these instances, death ensues and approximately 60% of patients require care 2 weeks after the event.2,3 In first-strokes, the majority experience some form of hemiplegia.8 Of these, only about half of which improve enough over the first six months after the incident to become functionally independent.9 Rehabilitation is vital and must be started early in order to maximize possible outcome. Methods of rehabilitation have been exhaustively covered in the literature and will be left to readers for further review.10,11 Even with optimal rehabilitation, many patients experience disability from loss of neurological function. Several studies have shown conclusively that neurological recovery occurs in the first 3 to 6 months, with little improvement thereafter.9-13 However, full recovery occurs in only half of patients.11 Further, time to functional recovery is significantly influenced by severity of stroke, with less severe strokes taking shorter time for recovery.11 Thus, after approximately 6 months of optimal rehabilitation, fully half of *Corresponding Author: Klaudia Urbaniak Hunter—University of Michigan, Department of Radiation Oncology, UH B2C490,1500 E. Medical Center Dr, Ann Arbor, Michigan, USA. Email: [email protected]

Frontiers in Brain Repair, edited by Rahul Jandial. ©2010 Landes Bioscience and Springer Science+Business Media.

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stroke survivors are left with some neurological deficit secondary to the initial ischemic insult that affects that person’s ability to accomplish activities of daily living. Given the varied etiology of stroke and the contribution of numerous risk factors, a multitude of treatment strategies are possible in the recovery period, depending on the individual’s profile. Specific medical therapy, while important, will be left for review elsewhere. Whatever medical therapy is initiated, the goal is risk factor modification with subsequent reduction in likelihood that a patient will experience a 2nd stroke. Thus, the standard of care after acute stabilization is intensive, early rehabilitation and medical risk factor optimization. For those patients who remain with neurological impairment more than six months after ischemic insult, further improvement is unlikely to occur and current treatment options are limited. The advent of stem cell therapy (SCT) as a possible treatment modality offers an additional pathway for recovery. The current state of science and future directions will be discussed in detail below.

Stem Cell Therapy As discussed above, a patient showing continued neurological deficits 6 months post-ischemic insult will likely harbor those deficits for the remainder of his or her life. Thus, the possibility of cell replacement with stem cells is extremely attractive. However, SCT also introduces significant challenges and must fulfill certain criteria to realize its potential as a treatment. First, SCT would ideally reconstitute the areas affected by disease, adapting to the milieu and forming synaptic connections with appropriate neuroanatomical loci. Second, once these stem cells proliferate to meet the first goal, the cell would enter a stage of normal neural activity with only enough division to maintain neuronal function. This second tenet is key, as violation of it would inexorably lead to build-up of neural tissue and, possibly, to neoplasia and teratoma formation. Third, transplanted cells must affect a clinical improvement. Fourth, transplanted cells must fulfill the three previous criteria over a long period of time, forming a longstanding neural framework upon which “normal” neural transmission may occur. Recent authors have proposed SCT as a cell-replacement treatment for neurodegenerative disease (see Fig. 1).14-17 A small-scale model for SCT occurs in vivo after lesion formation, as neural stem cells (NSCs) from the subventricular zone migrate towards the lesion, differentiate and form new neurons in the area affected by the ischemic insult.18-21 However, survival and ability to regenerate lost synaptic connections are limited in endogenous injury repair.22 With local enrichment, the limitation on growth and proliferation after injury is lessened.23 Additionally, multiple groups have shown that these endogenous NSCs will respond to trophic factors and other molecular cues and that enriched cell culture media enhances levels of trophic factors in the brain.24-29 Interestingly, exercise has been shown to enhance trophic factor expression and neurogenesis in the central nervous system following insult.30-32 Taken together, this data suggests that stem cells have a finite ability to regenerate and reconstitute a neural lesion, but that stem cells’ continued response to molecular cues provides an interesting avenue for future consideration of endogenous stem cells in neurodegenerative disease and stroke.

Stem Cell Biology—In Vitro Stem cells were first studied with respect to hematopoiesis in the middle of the 1950’s.33 After identification of hematopoietic stem cells (HSC), further work has shown there to be multiple types of stem cells, each with its own repertoire of possible cell fates. Though a full discussion of stem cells will not be attempted here, one can consider the discussion of “stemness” and the continuum of differentiation in multiple reviews.15,34,35 To consider SCT as a viable option, a secure means of obtaining large numbers of undifferentiated cells is paramount. To this end, culture protocols for multiple stem cell lines have been elucidated. Specifically, human embryonic stem cells (ESC) differentiate into NSCs with exposure to epidermal growth factor, basic fibroblast growth factor and leukemia inhibitory growth factor; NSCs differentiate into neurons with exposure to retinoic acid (see Fig. 2).36-39 Specific culturing techniques for maintaining the undifferentiated state of the stem cells have been described, as have

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Figure 1. Application of stem cells for neurological disorders. Stem cells would be isolated and transplanted to the diseased brain and spinal cord, either directly or after predifferentiation/ genetic modification in culture to form specific types of neuron and glial cell, or cells producing neuroprotective molecules. In strategies relying on stimulation of the patient’s own repair mechanisms, endogenous stem cells would be recruited to areas of the adult brain and spinal cord affected by disease, where they would produce new neurons and glia (neurogenic and gliogenic areas along lateral ventricle and central canal are shown in hatched red). Stem cells could provide clinical benefits by neuronal replacement, remyelination and neuroprotection. Used with permission from Lindvall O et al. Nature 2006; 441(7097):1094-1096.14 A color version of this image is available at www.landesbioscience.com/curie.

methods to induce differentiation of progenitor cells into neurons specifying specific neurotransmitters. This fact is important, as when one is considering reconstituting the neurons forming a specific network, the neurons must be able to signal to the neurons which survived whatever insult is necessitating the treatment. Stem cells from multiple lines hold potential for SCT. The obvious choice is NSCs from the subventricular zone. However, access to these cells in vivo may be limited. However, the ultimate goal is producing large numbers of cells capable of differentiating into neurons and other neural cells. As there are ethical and legal restrictions on the use of human fetal cells, scientists must look elsewhere for a source unencumbered with such difficulties. A possible solution to the dilemma comes with the finding that hematopoeitic stem cells (HSC) and mesenchymal stem cells (MSC)

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Figure 2. Isolation and purification of hNSCs from the hESCs. The hESCs were grown on a mouse feeder layer (A). Primary neurospheres (B) were isolated and replated to eliminate other nonneural cells. The selectively harvested secondary neurospheres (arrow in C), left behind hollow cores in the surface area (star in D) where they attached earlier. They were perpetuated for an additional 5 passages (E). These 2u spheres were then passaged using a single cell dissociation protocol (F). Arrow in F shows an example of a focus of proliferating cells. G, H) The hNSCs were passaged every 5-7 days, as described in the Methods section. Starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase the total cell number and plotted as logarithm with base 2 in function of time (G). The cell perpetuation (G) and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. I) RT-PCR analysis showing the down regulation of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). J) Cytogenetic evaluation of the SD56 hNSCs line at passage 12 by standard G-banding was performed. Twenty metaphase cells were analyzed and showed a normal female chromosome complement (46, XX). Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial glial cell marker 3CB2 (M) in virtually all the progeny. N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division. Single (N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period. Note the symmetrical segregation of BrdU and nestin in the progeny. Bars: (A, B, C, D) 200 mm; (E, F) 100 mm; (K-M) 20 mm; (N-P) 10 mm. From: Daadi MM, et al. PLoS ONE 2008; 3(2):e1644.39

can be induced to transdifferentiate into NSCs.40 42 Thus, there are multiple avenues of producing the progenitor cell necessary for expansion of neural-fated cells both in vitro and in vivo.

Stem Cell Biology and Animal Models In recent years, several groups have been working on SCT in vivo in animal models of stroke. One group has shown human fetal NSCs injected into the brains of ischemic rats differentiate into neurons, migrate towards the ischemic lesion and improve functional outcome.43 Further,

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Figure 3. Fluorescence microscopy images revealing differentiated transplanted NPCs derived from ESCs. The GFPpositive cells demonstrated mature neuronlike morphological features (a), Note that NeuN-positive cells (b), GFAP-positive cells (c), GAD-positive cells (d), glutamate-positive cells (e), ChAT-positive cells (f), TH-positive cells (g) and serotonin-positive cells (h) were found in the graft. Original magnification 3400. Used with permission from Takagi Y et al. J Neurosurg 2005; 103(2):304-310.44

groups working with monkey and murine ESCs have shown implantation, proliferation and synaptic network formation when injected into ischemic lesions of mouse animal models (see Fig. 3).44,45 A separate group working with monkey ESCs showed that injected of ESCs after ischemia causing hemiplegia led to an improved motor outcome versus control animals.46 Finally, a group has reported that, in a rat model of stroke transplanted with allogeneic subventricular zone NSCs, enriched environment increased functional recovery.47 Incubation in retinoic acid induces ESCs to become neural progenitor cells.36-38 In light of the limited survival of ESCs after transplantation, one group transplanted ESCs overexpressing the anti-apoptotic gene Bcl-2 and found improved functional outcomes over animals transplanted with control ESCs.48 Though this finding is exciting, the use of an anti-apoptotic gene is problematic. As discussed above, one of the cardinal issues of SCT is monitoring the propensity for stem cells to proliferate and form teratomas. Instituting this molecular change decreases a cell’s ability to interrupt the cell cycle. Though not shown experimentally, introduction of this gene likely increases the risk that uncontrolled proliferation will occur. Thus, though the approach appears promising in animal models, caution should be maintained as investigators move to human trials.

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In addition to use of stem cells, some groups have experimented with immortalized human teratocarcinoma (NT2) cells and have shown that these cells differentiate into postmitotic neurons in vitro, after induction with retinoic acid and in vivo.49,50 Further, NT2 cells mature into neuronal phenotypes after transplantation into nude mice.51 Importantly, no neoplastic transformation was seen with study 1 year after transplantation.51 A subsequent experiment showed functional improvement in model animals transplanted with NT2 cells after ischemic event.52 This lab subsequently found similar results with a stable human NSC line, SD56, derived from ESCs.39 Thus, NT2 and SD56 cells fulfill the criteria for a possible limited clinical trial—differentiation into neurons, improvement of post-insult function and lack of neoplastic transformation. So, in addition to the various types of stem cells available, the immortalized neuronal precursor NT2 presents an important possible source for SCT. Some groups utilizing a different approach have shown that intravenous (IV) administration of various stem cell-enriched solutions to show that bone marrow stromal cells, culture-expanded autologous mesenchymal stem cells and human umbilical cord blood (UCB) and purified stem cells can lead to functional improvement in animal models.53-56 Additionally, one study showed greater functional recovery in animal models receiving IV human UBC versus intrastriatal human UBC.57 The previous discussion focuses on implantation of exogenous stem cells via various methodologies. The role for endogenous NSC proliferation and repair of damage is unclear. However, one group has shown in the adult rat that NSCs will proliferate, differentiate and migrate to form striatal neurons after ischemic insult.58 This finding is exciting, but further work must be done to determine whether such an event might occur after ischemic insult in humans. If confirmed, research into augmenting this NSC response to ischemia could result in an additional manner in which to effect functional improvement.

Cellular Reconstitution of Stroke Lesions The promising results gleaned from animal models logically lead towards trials in humans. Though it is known that transplanted human fetal brain tissue can elicit improvement in animal models of stroke, ethical considerations have precluded this approach as a possibility in humans.59-61 Thus, we must look towards other sources of human stem cells to advance stem cell science for therapeutic use. SCT has already been considered for other neurodegenerative diseases.62 Though these studies will not be reviewed in detail in this article, important data from trials for neurodegenerative disease have shown that human fetal cells can be transplanted into areas of neuronal cell death, differentiate into neurons and form synaptic connections.63-65 Though the mechanism of cell death in stroke is different from that in neurodegenerative disease, the process by which repair takes place is the same—migration of NSCs, proliferation and limited reestablishment of some synaptic connections. However, one specific challenge is unique to stroke—the location of the lesion is highly variable and can affect large or small areas of both gray and white matter. In most neurodegenerative diseases, the areas affected are predictable. In stroke, lesions anywhere in the CNS are possible, so neurological sequelae and the characteristics of the dead endogenous neurons and glia vary immensely from patient to patient. Thus, SCT for stroke will require the extremes of therapeutic versatility in order to offer the most possible patients a fuller functional recovery. Limited clinical trials have been performed in patients with basal ganglia lesions. After establishing the safety of the experimental approach, a group began a double-blinded clinical trial of 14 patients in which they reported improvement in the patients transplanted with NT2 cells, but conflicting outcomes data.66,67 Though not achieving statistical significance in all areas, this trial did show significant improvements in gross hand motor control over control and baseline measurements and in measurement of daily activities and in memory over baseline measurements.66

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Stroke Treatment via Enhanced Trophic Factor Delivery In patients with Parkinson’s disease and Alzheimer’s disease, research in animal models has shown that augmentation of trophic factor levels causes robust response from native progenitor cells.68,69 One group implanted fibroblasts genetically modified to express Nerve Growth Factor into the nucleus basalis of Meynert in 8 patients with early AD.70 One patient who expired from unrelated causes after implantation underwent autopsy and a robust trophic response was seen in the areas around implantation. Further, metabolic activity in the cortex of the other study subjects diffusely increased after implantation and psychometric testing suggested a decrease in the rate of neurologic decline. In animal stroke models studying intravenous and intraventricular administration of bone marrow cells, HSCs localize to the borders of ischemic zones, likely via chemotactic signals from chemokines.56,71,72 There is evidence that these cells may enhance neurological recovery via secretion of trophic factors, as HSCs have not been shown conclusively to differentiate into neurons.73-76 Thus, one possible mechanism of HSCs in providing neural protection is via trophic factor delivery. Genetic manipulation of stem cells to express greater amounts of trophic factors provides a promising avenue for future study.77 An additional possibility by which HSCs improve outcome after stroke follows data showing that intrastriatal administration of HSC-containing bone marrow stroma in animal stroke models improve cerebral blood flow, reduce the permeability of the blood-brain barrier earlier than control animals and increase angiogenesis in the ischemic zone.78,79 The data on trophic factor augmentation via SCT presents an exciting new avenue of stroke therapy. However, the data relies heavily on animal stroke models and has remains to be proven effective in human trials.

The Potential of Cord Blood Similarly to HSCs, umbilical cord blood cells (UCBC) home to the chemokines produced by ischemic neural tissues in vitro.72 Additionally, UCBCs have been shown to transdifferentiate into neural cells expressing neural markers in vitro, suggesting the possibility that these cells may be able to provide cellular substrate for neurogenesis (see Fig. 4).35,80-83 In animal models, intravenous co-administration of UCBCs and mannitol has been shown to increase neurotrophic factor levels, decrease ischemic zones and increase post-ischemic performance versus control mice.84 Interestingly, this same group found now evidence of UCBC translocation into the CNS after treatment.84 Increased neovascularization and neurogenesis has also been shown in immunocompromized mice treated with intravenous human UCBCs, suggesting that human UCBCs may work similarly to animal UCBCs used in prior studies.85 Though the mechanism is incompletely understood, UCBCs are currently thought to act peripherally to enhance ischemic recovery by augmenting neuroprotective trophic factors and helping normalize cerebral blood flow earlier than would occur naturally. Coupled with the brief discussion of UCB in prior sections, the attraction of utilizing UCBC in the treatment of stroke is easily understood. As discussed above, the use of SCT suffers from nonscientific concerns because of the ethical issues created by use of stem cells derived from several sources. There are many avenues that may mitigate ethical concerns, but use of UCBC could prove superior to others both from a moral perspective and from a scientific perspective. First, human UCBCs have been used to treat several conditions already.86,87 Second, because the lymphocytes in UCB are immature and lack cytotoxic capability, HLA matching requirements need not be as stringent in order to prevent graft-versus-host disease.88,89 Third, human UCB contains high concentrations of anti-inflammatory mediators, which can counteract the significant inflammation which occurs during and after stroke.90,91 Thus, human UCB may target stroke on multiple levels. A full discussion of the hematologic advantages of UCB is unwarranted in this article and is covered elsewhere.92

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Figure 4. A) UC-MC culture prior to the addition of growth factors. B) Cells become more stellate with a prominent nucleus after the addition of growth factors. The inset shows the prominent nuclear inclusion bodies of these cells. C) Western immunoblot shows expression of TuJ1 and GFAP in the growth factor-exposed culture and the control cell line of immortalized neuronal progenitor cells C17.2. D) Immunocytochemistry of GFAP in growth factor-exposed cells. E) The same cells showing expression of ` -tubulin III. F) Immunocytochemistry of Gal-C in growth factor-exposed cells. G) The same cells showing expression of ` -tubulin III. Scale bar  20 +m. Used with permission from Bicknese AR et al. Cell Transplant 2002; 11(3):261-264.80

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Conclusion Stroke remains a scourge that, despite recent medical advances such as tPA, causes a substantial amount of morbidity and mortality worldwide. The current state-of-art treatments largely strive to reduce ischemic damage, but do not act via several pathways such as growth factor delivery, cellular regeneration and decreasing inflammation. The advent of SCT presents an opportunity to promote recovery by each of these mechanisms, while also maintaining the current medical therapies as part of the medical community’s therapeutic armamentarium. However, SCT provides a multitude of potential avenues for therapy, as numerous as the various types of stem cells. Additionally, genetic manipulation to enhance growth factor delivery may improve neural protection and functional recovery from stroke. Finally, human UCBCs show enormous promise because of HLA-mismatch permissiveness and ease of banking therapeutic volumes of UCB. The discussion above presents SCT in an optimistic light. Challenges to the routine administration of SCT remain. Foremost among them is the lack of human data and randomized controlled trials in humans. Safety has not been established in stroke victims, though UCB and SCT have been used in many other disease states. Further, if any type of SCT proves to be safe and efficacious in stroke victims, many variables about administration must be studied to optimize clinical recovery. Despite these challenges, at least two Phase I studies are currently recruiting for SCT in stroke victims (ClinicalTrials.gov #NCT00473057 and #NCT00535197). These trials may pave the way for use of stem cells in humans and provide an additional weapon in the treatment of the 3rd leasing cause of death in the western world.

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47. Hicks AU, Hewlett K, Windle V et al. Enriched environment enhances transplanted subventricular zone stem cell migration and functional recovery after stroke. Neuroscience 2007; 146(1):31-40. 48. Wei L, Cui L, Snider BJ et  al. Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia. Neurobiol Dis 2005; 19(1-2):183-93. 49. Andrews PW, Damjanov I, Simon D et  al. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro. Lab Invest 1984; 50(2):147-62. 50. Pleasure SJ, Lee VM. NTera 2 cells: a human cell line which displays characteristics expected of a human committed neuronal progenitor cell. J Neurosci Res 1993; 35(6):585-602. 51. Kleppner SR, Robinson KA, Trojanowski JQ et  al. Transplanted human neurons derived from a teratocarcinoma cell line (NTera-2) mature, integrate and survive for over 1 year in the nude mouse brain. J Comp Neurol 1995; 357(4):618-32. 52. Borlongan CV, Tajima Y, Trojanowski JQ et  al. Transplantation of cryopreserved human embryonal carcinoma-derived neurons (NT2N cells) promotes functional recovery in ischemic rats. Exp Neurol 1998; 149(2):310-21. 53. Bang OY, Lee JS, Lee PH et  al. Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol 2005; 57(6):874-82. 54. Chen J, Li Y, Wang L et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 2001; 32(4):1005-11. 55. Chen J, Sanberg PR, Li Y et  al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke 2001; 32(11):2682-8. 56. Shen LH, Li Y, Chen J et  al. Therapeutic benefit of bone marrow stromal cells administered 1 month after stroke. J Cereb Blood Flow Metab 2007; 27(1):6-13. 57. Willing AE, Lixian J, Milliken M et  al. Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res 2003; 73(3):296-307. 58. Thored P, Arvidsson A, Cacci E et  al. Persistent production of neurons from adult brain stem cells during recovery after stroke. Stem Cells 2006; 24(3):739-47. 59. Mattsson B, Sorensen JC, Zimmer J et al. Neural grafting to experimental neocortical infarcts improves behavioral outcome and reduces thalamic atrophy in rats housed in enriched but not in standard environments. Stroke 1997; 28(6):1225-31; discussion 31-2. 60. Nishino H, Borlongan CV. Restoration of function by neural transplantation in the ischemic brain. Prog Brain Res 2000; 127:461-76. 61. Riolobos AS, Heredia M, de la Fuente JA et  al. Functional recovery of skilled forelimb use in rats obliged to use the impaired limb after grafting of the frontal cortex lesion with homotopic fetal cortex. Neurobiol Learn Mem 2001; 75(3):274-92. 62. Korecka JA, Verhaagen J, Hol EM. Cell-replacement and gene-therapy strategies for Parkinson’s and Alzheimer’s disease. Regen Med 2007; 2(4):425-46. 63. Mendez I, Dagher A, Hong M et al. Simultaneous intrastriatal and intranigral fetal dopaminergic grafts in patients with Parkinson disease: a pilot study. Report of three cases. J Neurosurg 2002; 96(3):589-96. 64. Mendez I, Hong M, Smith S et al. Neural transplantation cannula and microinjector system: experimental and clinical experience. Technical note. J Neurosurg 2000; 92(3):493-9. 65. Mendez I, Sanchez-Pernaute R, Cooper O et  al. Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson’s disease. Brain 2005; 128(Pt 7):1498-510. 66. Kondziolka D, Steinberg GK, Wechsler L et al. Neurotransplantation for patients with subcortical motor stroke: a phase 2 randomized trial. J Neurosurg 2005; 103(1):38-45. 67. Kondziolka D, Wechsler L, Goldstein S et  al. Transplantation of cultured human neuronal cells for patients with stroke. Neurology 2000; 55(4):565-9. 68. Dass B, Olanow CW, Kordower JH. Gene transfer of trophic factors and stem cell grafting as treatments for Parkinson’s disease. Neurology 2006; 66(10 Suppl 4):S89-103. 69. Tuszynski MH, U HS, Alksne J et  al. Growth factor gene therapy for Alzheimer disease. Neurosurg Focus 2002; 13(5):e5. 70. Tuszynski MH, Thal L, Pay M et  al. A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat Med 2005; 11(5):551-5. 71. Hill WD, Hess DC, Martin-Studdard A et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol 2004; 63(1):84-96. 72. Newman MB, Willing AE, Manresa JJ et al. Stroke-induced migration of human umbilical cord blood cells: time course and cytokines. Stem Cells Dev 2005; 14(5):576-86. 73. Castro RF, Jackson KA, Goodell MA et al. Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science 2002; 297(5585):1299.

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74. Li Y, Chen J, Chen XG et al. Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology 2002; 59(4):514-23. 75. Roybon L, Ma Z, Asztely F et  al. Failure of transdifferentiation of adult hematopoietic stem cells into neurons. Stem Cells 2006; 24(6):1594-604. 76. Wagers AJ, Sherwood RI, Christensen JL et  al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002; 297(5590):2256-9. 77. Kurozumi K, Nakamura K, Tamiya T et  al. Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model. Mol Ther 2005; 11(1):96-104. 78. Borlongan CV, Lind JG, Dillon-Carter O et  al. Bone marrow grafts restore cerebral blood flow and blood brain barrier in stroke rats. Brain Res 2004; 1010(1-2):108-16. 79. Chen J, Zhang ZG, Li Y et al. Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats. Circ Res 2003; 92(6):692-9. 80. Bicknese AR, Goodwin HS, Quinn CO et  al. Human umbilical cord blood cells can be induced to express markers for neurons and glia. Cell Transplant 2002; 11(3):261-4. 81. Fu YS, Shih YT, Cheng YC et al. Transformation of human umbilical mesenchymal cells into neurons in vitro. J Biomed Sci 2004; 11(5):652-60. 82. Goodwin HS, Bicknese AR, Chien SN et al. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat and neural markers. Biol Blood Marrow Transplant 2001; 7(11):581-8. 83. Zhao ZM, Lu SH, Zhang QJ et al. (The preliminary study on in vitro differentiation of human umbilical cord blood cells into neural cells). Zhonghua Xue Ye Xue Za Zhi 2003; 24(9):484-7. 84. Borlongan CV, Hadman M, Sanberg CD et  al. Central nervous system entry of peripherally injected umbilical cord blood cells is not required for neuroprotection in stroke. Stroke 2004; 35(10):2385-9. 85. Taguchi A, Soma T, Tanaka H et al. Administration of CD34 cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J Clin Invest 2004; 114(3):330-8. 86. Bhattacharya N. Placental umbilical cord blood transfusion: a new method of treatment of patients with diabetes and microalbuminuria in the background of anemia. Clin Exp Obstet Gynecol 2006; 33(3):164-8. 87. Bhattacharya N, Mukherijee K, Chettri MK et  al. A study report of 174 units of placental umbilical cord whole blood transfusion in 62 patients as a rich source of fetal hemoglobin supply in different indications of blood transfusion. Clin Exp Obstet Gynecol 2001; 28(1):47-52. 88. Fasouliotis SJ, Schenker JG. Human umbilical cord blood banking and transplantation: a state of the art. Eur J Obstet Gynecol Reprod Biol 2000; 90(1):13-25. 89. Szabolcs P, Park KD, Reese M et al. Coexistent naive phenotype and higher cycling rate of cord blood T-cells as compared to adult peripheral blood. Exp Hematol 2003; 31(8):708-14. 90. Fukuda H, Masuzaki H, Ishimaru T. Interleukin-6 and interleukin-1 receptor antagonist in amniotic fluid and cord blood in patients with preterm, premature rupture of the membranes. Int J Gynaecol Obstet 2002; 77(2):123-9. 91. Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal cerebral ischemia. Surg Neurol 2006; 66(3):232-45. 92. George TJ, Sugrue MW, George SN et al. Factors associated with parameters of engraftment potential of umbilical cord blood. Transfusion 2006; 46(10):1803-12.

Chapter 10

Gene- and Cell-Based Approaches for Neurodegenerative Disease Klaudia Urbaniak Hunter,* Chester Yarbrough and Joseph Ciacci

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eurodegenerative diseases comprise an important group of chronic diseases that increase in incidence with rising age. In particular, the two most common neurodegenerative diseases are Alzheimer’s disease and Parkinson’s disease, both of which will be discussed below. A third, Huntington’s disease, occurs infrequently, but has been studied intensely. Each of these diseases shares characteristics which are also generalizeable to other neurodegenerative diseases: accumulation of proteinaceous substances that leads inexorably to selective neuronal death and decline in neural function. Treatments for these diseases have historically focused on symptomatic relief, but recent advances in molecular research have identified more specific targets. Additionally, stem cell therapy, immunotherapy and trophic-factor delivery provide avenues for neuronal protection that may alter the natural progression of these devastating illnesses. Upcoming clinical trials will evaluate treatment strategies and provide hope that translational research will decrease the onset of debilitating disability associated with neurodegenerative disease.

Introduction Neurodegenerative diseases comprise a wide-ranging category of maladies that affect a large number of patients, particularly so as patients get older. Within this larger group, there are smaller subdivisions of diseases affecting cognitive function, cerebellar function and deep motor function. The most prominent amongst these include Alzheimer’s disease (AD), Parkinson’s disease (PD) and Hungtington’s disease (HD). Though the diseases vary in their clinical presentation, prognosis and etiology, there are striking similarities as well. Neurodegenerative disorders tend to increase in incidence with age and tend to be progressive in nature. Pathologically, each disease causes accumulation of aberrant proteinaceous material, such as amyloid and leads inexorably to selective neuronal death. Though the anatomical location of the death may change depending on the disease, the outcome is generally the same—progressive, selective neuronal death leading to neurological compromise and, eventually, death. In the backdrop of this admittedly gloomy portrayal of neurodegenerative diseases, recent research identifying in vivo neurogenesis of neural stem cells (NSC),1 as well as the emergence of stem cell therapy (SCT) has allowed researchers to strive for more effective treatments than the pharmacologic therapies currently in use. SCT holds enormous promise for many disease processes. However, the unique environment of the Central Nervous System (CNS) also presents difficult challenges to physician-scientists as research transitions from the bench to the bedside. In particular, the intricacy of the CNS is unrivaled elsewhere in the human body. This is further complicated by *Corresponding Author: Klaudia Urbaniak Hunter—University of Michigan, Department of Radiation Oncology, UH B2C490,1500 E. Medical Center Dr, Ann Arbor, Michigan, USA. Email: [email protected]

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the privileged immune-status of the CNS caused by its isolation by the blood-brain barrier. In this chapter, the authors will address the cellular and molecular pathophysiology of neurodegenerative disease and offer the current science on SCT as a treatment modality for these ailments.

Cellular and Molecular Pathophysiology of Alzheimer’s Disease The CNS comprises a complicated and intricate network of millions of neurons, supported by several other cell types, including astrocytes, oligodendrocytes, microglia and ependymal cells. Though a complete discussion of neuroanatomical tracts will be left for review elsewhere, there are several principles of neuropathology that serve further discussion. As discussed above, the neurodegenerative diseases share amyloid aggregation and neuronal cell death as common features. The differences between the diseases arise from the distinct cell groups affected by the aggregation and cell death. Alzheimer’s disease (AD) is the most prevalent form of dementia in the world today, afflicting approximately 4 million people, or 60-80 percent of the cases of dementia in the United States.2-4 The disease has both familial and sporadic forms, both of which cause dysfunction in behavioral regulation, higher cognitive function and, most strikingly, short-term memory. AD presents unique challenges from a social and medical view. Because it is insidious and progressive, society’s costs, both direct and indirect, are high. Additionally, AD presents a problem in that most patients do not come to medical care until significant impairment has occurred. Thus, patients do not always benefit from early diagnosis and treatment. Finally, at this point, AD cannot be definitively diagnosed until autopsy. As clinicians expand the therapeutic arsenal for AD, a definite pathway for diagnosis pre-autopsy is of the utmost importance. On histological examination, an AD brain shows several characteristic features. First, affected neurons exhibit intracellular neurofibrillary tangles (NFT) of hyperphosporylated tau protein.5,6 Though NFTs occur commonly in AD, they are nonspecific, having been shown in a number of other neurodegenerative diseases.7 Further, NFTs are evident in neurons which show now histologic evidence of dysfunction, suggesting that the process of neuron cell death in AD may be protracted.5 Second, the extracellular space demonstrates neuritic plaques (NP) of amyloid ` protein (A`) surround by abnormal dendrites and axons.5 Further discussion of the molecular mechanisms of AD, as well as NFTs and NPs, will follow below. Molecularly, much work has gone into characterizing the genetic components that affect the accumulation and propagation of NFTs and NPs. NFTs consist of aggregations of hyperphosphorylated tau protein. Interestingly, recent research suggests that tau, rather than A`, is the primary neurotoxic mediator in AD.8,9 Moreover, levels of NFTs correlate more closely with cognitive function than do number of NPs.10,11 The primary component of NPs is A`. This was first suggested via analysis of certain familial versions of AD which are caused by either too much A`, as in Down’s syndrome, or by improper cleavage of A` precursor protein, as with presenilin 1 and 2 mutations.12-15 A proposed mechanism linking these pathologic and genetic findings suggests that accumulation of A` activates the intracellular caspase cascade, which leads to improper cleavage of tau and formation of NFTs (Fig. 1).15,16 As neuronal damage and cell death occur, a decrease in synapses transpires, which correlates strongly with objective clinical deterioration.17,18 The histologic and molecular dysfunction described above is informative and important, but does not provide anatomic descriptions of where this dysfunction occurs, an important consideration when looking forward to possible treatments. Grossly, brains of patients with AD may show diffuse cortical atrophy with selectively more serious atrophy of the hippocampus.5 However, these findings are nonspecific.19 On an anatomic level, the changes described above tend to occur first in the entorhinal cortex and hippocampus, then in the limbic system and, finally, diffusely throughout the cortex.20 The chronology of the pathology provides a convenient parallel with the observed clinical course and also sheds light on the involvement of several primarily cholinergic tracts of the brain.

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Figure 1. Caspase activation and intracellular A` colocalize with 6Tau in the AD brain. Fluorescent confocal imaging demonstrated that 6Tau (red) and active caspase-3 (green) colocalized within pretangle neurons (A) and dystrophic neurites (B) of the AD hippocampus. In contrast, mature extracellular tangles contained 6Tau without evidence of caspase activation (C). Intracellular A` (green), a known initiator of caspases, colocalizes with 6Tau (red) within AD neurons (D and E). In addition, 6Tau-immunoreactive dystrophic neuritis (red) were frequently associated with extracellular amyloid plaques (F). Scale bar: 5 +m (A), 1.5 +m (B), 6 +m (C), 3 +m (D and E), 25 +m (F). Used with permission from Rissman RA et al. J Clin Ivest 2004; 114(1):121-130.16 A color version of this image is available at www.landesbioscience.com/curie.

Cellular and Molecular Pathophysiology of Parkinson’s Disease Parkinson’s disease (PD), the 2nd most common neurodegenerative disease after AD, similarly primarily affects older patients, with increasing incidence after age 60.21-23 As with AD, PD has both familial and sporadic forms. Clinically, PD manifests with a “pill-rolling” tremor, bradykinesia and rigidity.24 Postural instability may or may not ensue. Additionally, many patients experience dementia, autonomic dysfunction and neuropsychiatric symptoms.25 These features are shared with several other neurologic diseases, the most similar to PD being Dementia with Lewy Bodies (DLB).26 The symptomatology of PD is thought to occur due to loss of dopaminergic neurons in the nigrostriatal pathway, which will be discussed in detail below. As with AD, PD presents a challenge in treatment and incurs high costs to society in the provision of care for these patients. The cellular and molecular mechanisms thought to cause PD will be detailed briefly below. Histologically, PD and DLB show intraneuronal inclusions as well as disordered cellular processes in affected cells.27-30 Further, it is now known that _-synuclein comprises the majority of protein within Lewy body aggregates in affected neurons.31,32 One group has shown convincingly that these pathological changes, which occur first in the dorsal motor nucleus of the vagus nerve, correlate with disease progression and cognitive impairment.33,34 The motor symptoms in PD have been convincingly shown to result from loss of dopaminergic neuron in the nigrostriatal pathway, first found in humans and subsequently shown in monkeys after ingestion of the selective neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).35,36 The molecular basis of these histologic findings will be discussed below.

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Figure 2. Complex I deficiency may be central to sporadic PD. Dysfunction of complex I leads to increased oxidative stress, free radical formation and reduction in adenosine triphosphate (ATP) formation. Decrements in ATP lead to membrane depolarization and contribute to excitotoxic neuronal injury and further free radical—mediated injury involving nitric oxide (NO) and peroxynitrite (ONOO-) and a feedforward cycle of increasing oxidative stress and injury. Slow and chronic complex I deficiency leads to accumulation and aggregation of _ -synuclein, which leads to dysfunction of the proteasome and contributes to cell death. Familial-associated mutations in _-synuclein bypass complex I deficiency, but they promote _ -synuclein accumulation and aggregation. Parkin appears to be a multipurpose neuroprotective agent that may allow the cell to more readily handle proteasomal impairment. Loss of parkin function may decrease the cells’ ability to deal with proteasomal dysfunction. DJ-1 may function as a chaperone and its absence may also decrease the cells’ ability to deal with proteasomal dysfunction. Used with permission from Dawson TM et al. Science 2003; 302(5646):819-822.46

The understanding of molecular pathways active in idiopathic PD and related disorders continues to evolve. Central to this understanding is the role of _-synuclein. Mutations in this molecule, which comprises a large part of the Lewy bodies in PD, have been found in multiple lineages afflicted with familial, early-onset PD.37-39 Further, excessive copies of the _-synuclein locus have been found to increase incidence and decrease age-of-onset of PD.40,41 Moreover, various groups have identified families with PD caused by mutations in various genes that function in the ubiquitination and processing of _-synuclein.42-45 At the same time, several authors have shown that mitochondrial dysfunction is apparent in PD patients, a finding consistent with MPTP’s inhibition of Complex I.46,47 Finally, one group has shown early-onset PD in patients with a mutation in DJ-1, a protein of unknown function that is active during oxidative response, suggesting a role for disordered stress responses in the etiology of PD.48 A proposed mechanism arose with the finding that systemic administration of the pesticide rotenone, a Complex I inhibitor, leads to formation of Lewy bodies and sequelae of PD in rats (Fig. 2).46,49 Thus, the complex molecular mechanisms contributing to PD include errors protein-processing, the electron transport chain and oxidative response. In the discussion above, molecular and cellular abnormalities are described in detail. However, no mention of a mechanism is made linking the specific molecular defects to the specific anatomic and pathologic defects found in PD. The common final pathway is accumulation of _-synuclein in dopaminergic neurons in the nigrostriatal pathway. A proposed mechanism posits

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that environmental exposure, linked with genetic polymorphisms at loci coding mitochondrial, cytoplasmic and nuclear proteins involved in oxidative response, as well as the local presence of dopamine and dopamine-related molecules available to form highly reactive dopamine-quinones, leading to the cytoplasmic accumulation of _-synuclein selectively in dopaminergic neurons.50-52

Cellular and Molecular Pathophysiology of Huntington’s Disease Huntington’s disease (HD) is a rare inherited disease arising from an increased number of trinucleotide repeats in a specific gene, huntingtin. Harboring 36 CAG repeats in the huntingtin gene is a marker for future development of the disease.53,54 Though HD is rare, the mechanism underlying the disease is a common one, occurring in several other autosomal dominant neurodegenerative diseases.53 As such, HD will be described as the prototypical neurodegenerative disease caused by trinucleotide repeats. HD may present at any age, but age at onset tends to vary inversely as the number of repeats a patient carries. HD also demonstrates anticipation, the characteristic of earlier onset and worsening phenotype with successive affected generations. Interestingly, anticipation is seen more often in children of fathers with HD.55 Clinically, HD patients exhibit a chorea, though other motor disturbances may be evident. Additionally, HD patients may show cognitive dysfunction, psychiatric disturbance and personality changes as the disease progresses. Pathologic examination of neuronal tissue from HD patients shows nuclear intranuclear inclusions (INI) of insoluble proteinaceous material in the striatum. The amino-terminus of huntingtin was subsequently shown to colocalize with these NIIs. 56,57 Additionally, neurons affected by aggregations of huntingtin also show dysfunction in their cellular processes.56 While the reason HD strikes the striatum preferentially is unknown, several authors have shown that atrophy of the caudate, globus pallidus externa and putamen precedes clinical symptoms of HD by several years.58,59 Further, animal models of HD and animals ectopically expressing abnormal CAG repeats show atrophy and pathology in similar locations to human HD patients.60,61 Molecular studies of HD have shown that huntingtin is a large cytoplasmic protein of unknown function expressed diffusely in the CNS, but in higher concentrations in large striatal interneurons, medium striatal spiny neurons, cortical pyramidal cells and cerebellar Purkinje cells.62-64 Though INIs are characteristic of HD, recent evidence points to nonfibrillar intermediates of the mutant huntingtin protein as the pathologic defect in HD.65 Further, several studies have shown little correlation between amount of INIs and either clinical course or cellular life span.66-70 Thus, though HD may be the result of a gain-of-function mutation caused by the excess CAG-repeats, further characterization of the pathologic mechanisms behind huntingtin aggregation remains to be pursued.

Stem Cell Therapy for Neurodegenerative Diseases The current armamentarium against AD, PD and HD includes pharmacological treatments, as well as surgical treatment for PD. However, these treatments are generally symptomatic and tend to lose efficacy with the progression of the disease.71 Until a truly disease-modifying pharmacologic intervention is possible, stem cells present the ultimate hope for long-term alleviation of symptoms. The allure of stem cell therapy is multifactorial. Central to the theoretical frameworks of disease discussed above is selective neuronal loss. This tenet of neurodegenerative disease presents, in each disease state, a primary area in which cell loss is worse than others. The areas of neuronal death also present a target for SCT. SCT presents an attractive idea, but also introduces significant challenges to current and future scientists. First, SCT would ideally reconstitute the areas affected by disease, adapting to the milieu and forming synaptic connections with appropriate neuroanatomical loci. Second, once these stem cells proliferate to meet the first goal, the cell would enter a stage of normal neural activity with only enough division to maintain neuronal function. This second tenet is key, as violation of it would inexorably lead to build-up of neural tissue and, possibly, to neoplasia and teratoma formation. Third, transplanted cells must affect a clinical improvement. Fourth, transplanted cells must fulfill the three previous criteria over a long period of time, forming a longstanding neural framework upon which “normal” neural transmission may occur.

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After assuming that the goals stated above are achievable, the choice of which type of stem cell to utilize for SCT becomes paramount. This choice must be made from a practical and scientific basis. The embryonic stem cell (ESC), while imbued with pluripotent capability and with considerable scientific potential, is difficult to obtain for therapeutic use. As multiple authors have noted, the supply of fetal cells is a limited one and is fraught with ethical issues. Adult neural progenitor cells (NPC) have been identified in the hippocampus and olfactory bulb.72 A recent group has reported success in maintaining NPCs in culture, with preserved mitotic capability.73 However, transdifferentiation of hematopoeitic stem cells (HSC) and mesenchymal stem cells (MSC) into NSCs has also been reported.74-76 Thus, both MSCs and HSCs provide a convenient substrate for obtaining NPCs of identical and thus non-immunogenic, genetic background to the host. There are additional issues related to the process of culturing stem cells and the yield of dopaminergic neurons which will be left for further review.77-79 In addition to possible replacement of diseased tissue, stem cells present a distinct opportunity as a transplantable producer of neurotrophic factors. With stable lentivirus transduction or cDNA transfection of target molecules, one group reported successful, continued stimulation of dopaminergic neurons and functional improvement in PD model animals via transplantation of GDNF-expressing NPCs.80,81 This avenue of SCT is a compelling one, as it does not require the complex reformation of synaptic communication that the approach discussed above requires. However, this approach remains to be tried in human subjects and no long-term proof that disease progression will be modified. At the outset of SCT, researchers held high hopes for improvement in PD patients. These hopes were mitigated by two double-blinded trials, which showed moderate improvements as well as dyskinetic side effects after cell therapy.82,83 However, one recent group has shown that intrastriatal and intranigral implants were viable, led to clinical improvement and had formed synaptic connections and fiber bundles at autopsy in two patients (Fig. 3).84-86 Thus, proof-of-principle for SCT in PD has been accomplished. At the same time PD research has pushed towards regenerative therapy, HD researchers have shown that animal models with striatal lesions of varying etiology show functional and cognitive improvement following striatal implantation of fetal striatal cells.87-90 Thus, SCT for patients with striatal lesions, such as HD patients, presents a fertile ground for therapeutic improvement. Similar to the attempts at SCT in PD and HD, groups have attempted to transplant cholinergic neurons into the nucleus basalis of Meynert in animal models with some functional improvement.91,92 However, no attempts in humans has yet been made. AD presents a singular challenge in that neuronal loss, while concentrated in certain places at onset, grows to become diffuse over the entire cortex. Thus, reconstructing the neural framework over the entire cortex presents a more difficult challenge than the more local transplantation currently theorized for treatment of PD and HD patients.

Immunotherapy and Alzheimer’s Disease As discussed above, neurodegenerative diseases share the common finding of proteinaceous accumulation as the disease progresses. Because aggregations in AD are extracellular, researchers have attempted immunotherapy as a method by which to change disease progression and cytotoxicity secondary to this accumulation (Fig. 4).93 In particular, recent research has shown that antibodies to a specific amino-terminus peptide sequence caused resolubilization of A` and decreased cytotoxicity of the A` aggregates.94-97 This important finding opens the door to active immunization, which has been achieved in AD animal models, leading to improvement in cognitive testing and in objective clearance of A` protein.98-100 These findings led to a double-blinded clinical trial in humans, which was abruptly stopped after a high percentage of the subjects receiving the vaccine developed symptoms of meningoencephalitis.101-104 Despite the disappointing side effects of the vaccine, analysis of the data showed a slowed cognitive decline in the subgroup of subjects receiving the vaccine who also mounted a significant antibody response to the vaccine.103 Further, subsequent analysis of subjects at autopsy showed clearing of cortical A` plaques and A`-positive

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Figure 3. Tyrosine hydroxylase (TH) immunostaining of one of the transplants located in the right putamen of patient 2 showing the dense neurite outgrowth into the host putamen (central boxed area in Fig. 3B). Within the graft (B, boxed in A), TH-positive neurites from grafted neurons were thick and scarcely branched, while around the graft (C, boxed area in A) and further away (D, area shown boxed in Fig. 3B) they formed a dense network of fine branches approaching normal innervation in some areas of the putamen. Compare the fibre density with the contralateral putamen (E), where there was no graft survival. F, G) GFAP immunostainings showing a representative transplant located in the putamen of patient 2. F-H) Astroglial density was not increased in the graft core but around the graft deposits there was a band of variable thickness (1 mm) of fibrous hypertrophic astrocytes (G, boxed in F). Further away, the astrocytic density and morphology were similar to those of normal striatum (H; illustrates a similar area from the putamen of patient 1). Microglial cells were identified by immunoreactivity against CD45 (common leucocyte antigen, CLA) and CD68 (activated microglia, not shown). I-J) Representative microphotographs of CD45 immunostaining showing a local circumscribed increase in microglial cell density around needle tracks (arrows), which was very similar for all the grafts located in the putamen in patient 1 (shown in I) and in both the midbrain and the striatal deposits in patient 2. At higher magnification (J) a few macrophages could be observed along the needle track (arrow), but most microglial cells showed a typical resting branched morphology (see detail in the inset in K) comparable to that observed in striatal regions at a distance (away) from the grafts (K). Scale bar: A, 500 mm; F, I, 400 mm; G, H, J, K, 200 mm; C–E and inset in K, 75 mm; B, 25 mm. Used with permission from Mendez I et al. 2005; 128(Pt 7):1498-1510.86

Figure 4. Mechanisms of clearance of amyloid- `. A) Microglial cells can be activated by binding of various ligands to various cell-surface innate immune receptors: CD14 binds lipopolysaccharide (LPS) and components of Protollin; Toll-like receptor 2 (TLR2) and TLR4 bind Protollin components; MHC class II molecules interact with T-cell receptors; CD40 binds CD40 ligand expressed by T-cells and astrocytes; complement receptors bind complement components such as C1q; and Fc receptors (FcRs) bind amyloid-` -specific antibodies. B) Activated microglial cells express various scavenger receptors (SRs) that mediate phagocytosis of amyloid-`, such as integrin- _`, CD36, CD47, SR-A and SR-BI, the triggering of which leads to phagocytosis of amyloid- ` fibrils and oligomers. Ligation of cell-surface heparin sulphate proteoglycans (HSPGs), insulin receptors and proteinase inhibitor (serpin)-enzyme complex receptor (SEC-R) on activated microglial cells leads to phagocytosis of soluble amyloid-`. Microglial cells can also degrade amyloid- ` by releasing amyloid- ` -degrading enzymes, such as metalloproteases, insulin-degrading enzyme and gelatinase A. Protollin, a proteosome-based adjuvant composed of purified outer membrane proteins of Neisseria meningitidis and lipopolysaccharide (GlaxoSmithKline Biologicals of North America). Used with permission from Weiner HL et al. Nat Rev Immunol 2006; 6(5):404-416.93

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microglial cells.105,106 Together, this data suggests that, once proper mechanisms by which to reduce postvaccine meningoencephalitis are found, immunotherapy may be an additional means by which physicians and scientists can treat AD patients.

Gene-Based Approaches to Therapy An additional alternative to repopulation of devastated areas of brain is augmented delivery of neurotrophic factors to areas of interest. As discussed above, implantation of cells expressing trophic factors has been posited as a possible treatment for neurodegenerative disease, as animal models of both AD and PD have shown robust responses to augmentation of trophic factor levels.107,108 A second path to this end involves transduction of cells in the area of interest with vectors encoding trophic factors, such that concentrations of trophic factors in the milieu might be augmented. One particular trophic factor, Nerve Growth Factor (NGF), has been suggested as a potential treatment. However, intraventricular administration of NGF results in multiple untoward effects, including pain, weight loss and Schwann cell migration.109 So, for possible treatment, NGF must be delivered to a discrete area of neuronal loss to minimize side effects. One group implanted fibroblasts genetically modified to express NGF into the nucleus basalis of Meynert in 8 patients with early AD.110 One patient who expired from unrelated causes after implantation underwent autopsy and a robust trophic response was seen in the areas around implantation (Fig. 5).110 Further, metabolic activity in the cortex of the other study subjects diffusely increased after implantation and psychometric testing suggested a decrease in the rate of decline.

Figure 5. Trophic response to NGF in the human brain. A,B) Nissl stain of autologous, NGF-secreting cell implant in brain of individual with Alzheimer disease 5 weeks after treatment. Graft (g) adjacent to nucleus basalis of Meynert (nbm; arrows). Inset, robust mRNA encoding NGF by in situ hybridization within graft. Scale bar in a, 247 +m; in b, 24 +m. Note proximity of graft to nbm, seen in similar perspective in c at higher magnification. C,D) Immunocytochemistry for cholinergic neurons (p75) shows graft implant on left (g) and adjacent neurons of nbm (arrows). Higher magnification (D) shows dense penetration of cholinergic axons into graft. Scale bar in c, 82 +m; in d, 11 +m. Reprinted with permission from Tuszynski MH et al. Nat Med. 2005; 11(5):551-555.110

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Another group has shown convincingly that stable transduction of endogenous NPCs in known NPC-populated areas with injected vectors is possible.111 Further, NPCs have been reported in the substantia nigra, thus presenting a convenient location for vector injection.112,113 Based on the results and theory presented above, multiple trials injecting viral vectors with various neurotrophic factors have been initiated into the striatum and subthalamic nucleus. One Phase I trial injecting glutamic acid decarboxylase, the enzyme catalyzing the commited step of dopamine synthesis, in an adeno-associateed virus vector into the subthalamic nucleus has shown promising results and is moving on to a Phase II trial.114

Conclusion The treatment of neurodegenerative diseases has long focused on symptomatic relief rather than alteration of the natural progression of disease. Thus, with progressive neuronal loss and loss of medication efficacy, patients were faced with a long-term course where the most frequent outcome was medication failure and worsening of symptoms without hope of cure. Recent scientific advances have shown promise in utilizing translational research to provide course-altering treatments. Though this article focuses on Alzheimer’s disease, Huntington’s disease and Parkinson’s disease, the scientific rationale behind the approaches discussed above also could apply to other neurodegenerative diseases. Continuing advances in SCT, immunotherapy and gene-based treatment provide exciting new avenues for targeting the molecular and pathologic bases of neurodegenerative disease. As the various strategies move forward in clinical trials, data on long-term efficacy and rate of disease progression will provide guidance for treatment of these patients, while research and innovation in molecular targeting and gene and cell delivery will continue to refine scientists’ and physicians’ ability to target the populations of neurons most at risk in certain neurodegenerative diseases. These developments are both intellectually stimulating as well as well-timed, as physicians are faced with the aging of Western society and the patient population.

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Index

Adeno-associated virus (AAV) 96, 99 Adult neural stem cell 13, 14, 43, 59, 64 Alzheimer’s disease (AD) 23, 33-35, 47, 80, 111, 117-119, 121, 122, 125, 126 Amyloid B proteins 118 Amyotrophic lateral sclerosis (ALS) 47, 78 Animal model 1, 5, 6, 13-16, 23, 24, 26, 29-35, 41, 46, 76, 78, 88, 94, 108-111, 121, 122, 125 App transgenic mouse model 34

Cerebral ischemia 23, 29-31, 54 Chemokine 41, 47, 95, 111 Chemoprevention 64, 72 Chronic experimental 41 Ciliary neurotrophic factor (CNTF) 43, 78 CNS pathology 74, 76, 88, 90 Confocal fluorescence microscope 3 Cross linked iron oxide (CLIO-HD) 8 CXCR4 95, 96, 98 Cytokine 16, 41, 47, 76, 78, 82, 98, 99 Cytosine deaminase (CD) 15, 62, 63, 69, 71, 72, 80, 82, 97, 99

B

D

A

`-Amyloid plaque 33, 34 Basic fibroblast growth factor (bFGF) 17, 20, 45, 98, 106 `-Glucuronidase gene 84, 86 Bioluminescence (BLI) 2-4, 8, 86, 88, 89 Bone morphogenic protein (BMP) 43, 72 Brain-derived neurotrophic factor (BDNF) 42, 43, 47 Brain tumor 13-15, 17, 21, 54, 58, 60, 62, 64, 67, 69-72, 76, 77, 80, 82, 88, 93-96, 98, 99 Brain tumor stem cell (BTSC) 63, 64, 67-73, 95 Bromodeoxyuridine (BrdU) 46, 84, 108 By-stander effects 15

C Ca1 pyramidal neuron 31, 35, 84 Cag repeat 32, 33, 121 Cancer stem cell 62, 67, 68, 70, 71, 93, 94 Cardiopulmonary resuscitation (CPR) 31 cDNA transfection 122 Cellular transplant 1, 86, 88 Central nervous system (CNS) 10, 13-17, 41-44, 46, 47, 49, 60, 67, 69, 74, 76, 78, 80, 84, 86, 88, 90, 93-96, 98, 106, 110, 111, 117, 118, 121 Cerebral blood flow 30, 31, 111

D2r dopamine receptor 7 Dementia with lewy bodies (DLB) 119 Demyelination 41, 42, 44, 46, 47 Dil 2 Dopaminergic neuron 9, 23-26, 29, 78, 79, 119-122

E Embryonic stem cell (ESC) 44, 74, 78, 86, 89, 94, 106, 122 Endogenous Nsc 95, 126 Enzyme delivery 84 Epidermal growth factor (EGF) 17, 45, 46, 60, 64, 69, 96, 106 Exogenous nscs 42, 43, 46, 53, 94, 95, 96 Experimental autoimmune encephalomyelitis (EAE) 41, 44, 47

F Familial AD (FAD) 33, 34 Fibroblast-derived growth factor (FGF) 43, 45, 46 Fibroblast growth factor (FGFs) 17, 43, 45, 46, 64, 69, 98, 106 Focal cerebral ischemia model 30 Four vessel occlusion 31 Functional photoacoustic imaging 10, 11

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G

L

Genetically modified neural stem cell 15, 16, 76, 88, 98 Genetic metabolic disorder 42 Genetic model 29 Genetic therapy 27, 28 Glial cell line-derived neurotrophic factor (GDNF) 42, 47, 78, 79, 122 Glial fibrillary acidic protein (GFAP) 15, 45, 53, 59, 61, 63, 109, 112, 123 Glioblastoma multiforme 13, 17, 60, 62, 64, 69, 93 Glioma 14, 16, 58-60, 64, 68, 76, 77, 80, 82, 89, 93-99 Gliomatropism 95, 96 Global cerebral ischemia model 31 Green fluorescent protein (GFP) 2, 8, 46, 52, 80

LacZ 44, 46, 50, 87 Lentivirus transduction 122 Leukemia inhibiting factor (LIF) 20, 43-45 Lipophilic dye (DiI) 46 Lysosomal storage disease 14, 41, 42, 84

H Hematopoeitic stem cell (HSC) 106, 107, 111, 122 Herpes simplex virus1-thymidine kinase (HSVtk) 82, 98 hNSC 17, 108 Homing 47, 76, 77, 80, 95, 96, 98 Human umbilical cord blood (UCB) 110, 111, 113 Huntington’s disease (HD) 8, 10, 23, 32, 33, 117, 121, 122

I Immortalized human teratocarcinoma cell (NT2) 110 Immortalized NSC 74, 76, 90 Immunomodulatory strategy 16 Inflammatory disease 10 Intranuclear inclusion (INI) 121 In vitro labeling 6 In vivo imaging 1, 3, 5, 6, 8-11, 86, 88 In vivo microscopy 3 Iron oxide nanoparticle 5, 6 Ischemia 23, 29-31, 53, 54, 84, 105, 109, 110 Ischemic injury 76 Ischemic stroke 10, 30, 42, 54, 105

M Magnetic resonance imaging (MRI) 1, 5, 6, 8, 82, 83, 88, 94, 98 Magnetic resonance (MR) spectroscopy 6, 94 Mesenchymal stem cell (MSC) 88, 107, 110, 122 Methamphetamine model 24 Middle cerebral artery occlusion (MCAO) 30, 83, 84 Monocyte chemoattractant protein-1 (MCP1) 95 Mouse model 29, 32-35, 64 Mouse models of HD 32 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP model) 25-27, 29, 119, 120 Multimodal imaging 8 Multiphoton fluorescence microscope 3 Multiple sclerosis (MS) 10, 41, 42, 46, 47 Mutated Htt (mHtt) 32, 33

N Neonatal transplantation 49 Nerve growth factor (NGF) 42, 47, 78, 80, 82, 83, 111, 125 Nestin 53, 60, 71, 108 Neural progenitor cell 72, 109, 122 Neural stem cell (NSC) 13-21, 41-54, 58-60, 62, 64, 67, 69, 71, 74-78, 80, 82-84, 8-89, 90, 93-99, 106-110, 117, 122 NSC culture 18 NSC engraftment 14 NSC homing 95 NSC isolation 45 NSC manipulation 13 NSC migration 14, 76, 82, 88, 96 NSC transplantation 41, 42, 49, 86 Neuritic plaque (NP) 118

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Index

Neurodegenerative disease 1, 6, 10, 15, 23, 34, 41, 42, 44, 49, 78, 106, 110, 117-119, 121, 122, 125, 126 Neurofibrillary tangle (NFT) 33, 35, 118 Neurogenesis 14, 15, 17, 41-43, 47, 49, 51, 52, 86, 88, 93, 106, 111, 117 Neurological disease 8, 9, 14, 23, 27, 32, 35, 74, 76, 90, 95 Neuroprotective agent 83 Neurosphere 44, 45, 69, 78, 84, 87, 108 Neurotrophic factor 42, 43, 47, 78, 80, 111, 122, 125, 126 Neurotrophin 43, 78, 82 Neurovascular disease 10 NOGO 82 Noninvasive in vivo imaging 6 Notch receptor 64

S

Oncogene 16, 58, 76, 90 Optical coherence tomography 10 Optical imaging 8

Sandhoff disease 41, 42, 49, 86 Serial transplantation 63, 69, 70, 72 Single photon emission tomography (SPECT) 1, 5, 88 Sonic hedgehog (Shh) 43 Spinal cord injury 41, 42, 47, 52, 82 Stem cell factor (SCF) 95 Stem cell microenvironment 71 Stem cell therapy (SCT) 49, 89, 105-111, 113, 117, 118, 121, 122, 126 Stem cell transplantation 41, 43, 48, 50, 52 Stereotactic guidance 51 Striatal neuron 10, 33, 110 Stroke 5, 10, 14, 30, 41, 42, 47, 54, 83, 84, 88, 105, 106, 108-111, 113 Subgranular zone (SGZ) 14 Subventricular zone (SVZ) 14, 41, 45, 49, 51, 58-61, 84, 95, 106, 107, 109 Superoxide dismutase 1 (SOD1) 78 Superparmagnetic iron oxide particle (SPIO) 5, 6, 8, 88

P

T

Parkinson’s disease (PD) 8-10, 14, 23, 24, 26, 28, 29, 41, 42, 47, 54, 78, 79, 111, 117, 119-122, 125, 126 PEX 82-84, 98 Planar imaging 3 Positron emission tomography 1, 5-8, 10, 88, 94 Prodrug converting enzyme 15 Prolyl isomerase Pin1 35 PTEN 60, 64

Tau transgenic mouse model 35 Thymidine kinase 5, 15, 82, 95, 98, 99 tPA 105, 113 Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) 16, 98, 99 Transduction 7, 80, 90, 122, 125, 126 Transgenic mouse 4, 8, 29, 32-35, 52 Transit amplifying cell 60 Transplantation 8, 10, 14-18, 20, 21, 41-53, 62, 63, 68-70, 72, 74-76, 78, 80, 82, 84, 86-90, 98, 109, 110, 122 Trophic factor delivery 111 Trophic factor 43, 106, 111, 125 Trypan blue 20, 46, 48-50, 52 Tumorgenesis 64, 67, 68, 71, 76 Tumor microenvironment 67, 70-73, 95 Tumor necrosis factor (TNF) 16, 98, 99 Tumor 4, 6-8, 13-17, 21, 44, 46, 54, 58-60, 62-64, 67-73, 76, 77, 80, 82, 83, 86, 88, 89, 93-99 Tumor suppressor 58 Tumor-targeting delivery system 15 Two vessel occlusion 31 Tyrosine hydroxylase 78

O

Q Quantum dot 1, 3-5, 99

R Renilla reniformis luciferase 4 Reserpine model 24, 25 Retinoic acid 106, 109, 110 Rostral migratory stream (RMS) 42, 61 Rotenone model 29

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U

V

Ultra small paramagnetic oxides (USPIO) 5, 6 Ultrasound guidance 51 Umbilical cord blood cell (UCBC) 111

Vascular endothelial growth factor (VEGF) 78, 96, 98