Stem Cells and Regenerative Medicine: Pharmacology and Therapy

STEM CELLS AND REGENERATIVE MEDICINE, VOLUME III: PHARMACOLOGY AND THERAPY No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

STEM CELLS AND REGENERATIVE MEDICINE, VOLUME III: PHARMACOLOGY AND THERAPY

PHILIPPE TAUPIN

Nova Science Publishers, Inc. New York

Copyright © 2008 by Nova Science Publishers, Inc.

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CONTENTS Preface

vii

Introduction

1

Chapter I

Neurogenesis and Alzheimer’s Disease

Chapter II

Neurogenesis and the Effect of Antidepressants

11

Chapter III

Adult Neural Stem Cells and Cellular Therapy

19

Chapter IV

Stem Cells Engineering for Cell-Based Therapy

33

Chapter V

HuCNS-SC Stem Cells

43

Chapter VI

Oti-010 Osiris Therapeutics/JCR Pharmaceuticals

59

Chapter VII

ADA-Transduced Hematopoietic Stem Cell Therapy for ADASCID

77

Therapeutic Potential of Adult Neural Stem Cells

93

Chapter VIII

5

Conclusion and Perspectives: Adult Neural Stem Cells – The Promise of the Future

105

Index

121

PREFACE The subject of this book is stem cell research and regenerative medicine. Stem cells are undifferentiated cells that have the ability to differentiate into different lineages of the body. Stem cells carry tremendous potential for the treatment of a broad range of disease and injuries. Stem cells exist in embryonic, fetal, and adult tissues, including the adult central nervous system. This book aims at, in depth, the recent developments in stem cell research and regenerative medicine. Though this book encompasses all the fields of stem cell research and regenerative medicine, it emphasizes adult neurogenesis and neural stem cell research and therapy within the context of pharmacology and therapy. Chapter I - Alzheimer’s disease (AD) is a neurodegenerative disease, characterized in the brain by amyloid plaque deposits and neurofibrillary tangles. It is the most common form of dementia among older people. There is at present no cure for AD, and current treatments consist mainly in drug therapy. Potential therapies for AD involve gene and cellular therapy. The recent confirmation that neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) provide new opportunities for cellular therapy in the CNS, particularly for AD, and to better understand brain physiopathology. Hence, researchers have aimed at characterizing neurogenesis in patients with AD. Studies show that neurogenesis is increased in these patients, and in animal models of AD. The effect on neurogenesis of drugs used to treat AD is currently being investigated, to determine if neurogenesis contributes to their therapeutic activities. Chapter II - The recent evidence that neurogenesis occurs throughout adulthood and neural stem cells (NSCs) reside in the adult central nervous system (CNS) suggests that the CNS has the potential for self-repair. Along with this potential, the function of newlygenerated neuronal cells in the adult brain remains the focus of intense research. The hippocampus of patients with depression shows signs of atrophy and neuronal loss. This suggests that adult neurogenesis may contribute to the biology of depression. The observations that antidepressants like fluoxetine increase neurogenesis in the dentate gyrus (DG), and that neurogenesis is required for the behavioral effect of antidepressants, lead to a new theory on depression and the design of new strategies and drugs for the treatment of depression. However, the role of adult neurogenesis in the etiology of depression remains the source of controversy and debate. Chapter III - Considerable effort and means have been invested to find treatments for neurological diseases and injuries, yet there is still no cure for these ailments, and new alternatives for therapy must be explored. Because they generate the main phenotypes of the

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central nervous system (CNS), neural stem cells (NSCs) hold the promise to cure a broad range of CNS diseases and injuries. With the confirmation that neurogenesis occurs in the adult brain, and the recent isolation and characterization in vitro of neural progenitor and stem cells from the adult CNS, new avenues for the treatment of neurological diseases and injuries are being considered. Cell therapeutic interventions may involve both in vivo stimulation and transplantation of neural progenitor and stem cells of the adult brain. Chapter IV - Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, heart and muscular diseases, to neurological diseases, disorders and injuries. Significant progress has been made in stem cell research over the past decade: the derivation of embryonic stem cells (ESCs) from human tissues, the development of cloning technology by somatic cell nuclear transfer (SCNT); the confirmation that neurogenesis occurs in the adult mammalian brain, and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), including that of humans. Despite these advances, it may be decades before stem cell research will translate into therapy. Stem cell research is also subject to ethical and political debate, controversy and legislation, which slow its progress. Cell engineering has proven successful in bringing genetic research to therapy. In this chapter, I will review two examples of how investigators are applying cell engineering to stem cell biology to circumvent stem cells’ ethical and political constraints, thereby bolstering stem cell research and therapy. Chapter V - HuCNS-SC, a proprietary human neural stem cell product, is being developed as a cellular therapy for the potential treatment of Batten disease, one of a group of disorders known as neural ceroid lipofuscinoses (NCL). Developer StemCells, Inc. is also investigating the therapy for spinal cord injury and other central nervous system disorders, such as demyelinating disease, stroke, and Alzheimer's disease. A phase I trial of HuCNS-SC for infantile and late-infantile NCL has been initiated, following the March 2006 US Food and Drug Administration approval of StemCells' investigational new drug application. Chapter VI - Osiris Therapeutics is developing the donor-derived mesenchymal stem cell (MSC) therapy OTI-010, which repopulates the bone marrow stroma and thus supports engraftment of hematopoietic stem cells from the same donor. This stem cell therapy, which has been awarded Orphan Drug status, is currently in development for the potential enhancement of bone marrow transplants in cancer patients, for the prevention of graftversus-host disease (GVHD), and for the treatment of Crohn's disease. Japanese licensee JCR Pharmaceuticals is investigating the therapy for the potential treatment of GVHD in patients undergoing bone marrow transplantation to treat leukemia. Phase II clinical trials in acute gastrointestinal GVHD and in adult and pediatric patients with treatment-refractory severe GVHD are currently underway. Chapter VII - San Raffaele Telethon Institute for Gene Therapy is developing an adenosine deaminase transduced hematopoietic stem cell therapy for the potential intravenous treatment of adenosine deaminase deficiency in severe combined immunocompromised individuals (ADA-SCID). Chapter VIII - The central nervous system (CNS) elicits limited capacity to recover from injury. Though considerable efforts and means have been deployed to find treatments for neurological diseases, disorders and injuries, there is still no cure for these ailments, and new alternatives for therapy must be explored. Because they generate the main phenotypes of the nervous system, neural stem cells (NSCs) hold the promise to cure a broad range of neurological diseases and injuries. With the confirmation that neurogenesis occurs in the adult

Preface

ix

brain and that NSCs reside in the adult CNS, new treatments for neurological diseases and injuries are being considered, in particular, the transplantation of adult-derived neural progenitor and stem cells to restore brain functions. This manuscript will review the recent developments in adult neurogenesis and NSCs, and patent applications filed in relation to discoveries made in this new field of research. Conclusion and Perspectives - Stem cells are self-renewing undifferentiated cells that give rise to multiple types of specialized cells of the body. In the adult, stem cells are multipotent and contribute to homeostasis of the tissues and regeneration after injury. Until recently, it was believed that the adult brain was devoid of stem cells, hence unable to make new neurons and regenerate. With the recent evidence that neurogenesis occurs in the adult brain and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), the adult brain has the potential to regenerate and may be responsive to repair. The function of NSCs in the adult CNS remains the source of intense research and debate. The promise of future study of adult NSCs lies in redefining the function and physiopathology of the CNS, as well as in the treatment of a broad range of CNS diseases and injuries.

INTRODUCTION Stem cells are the building blocks of the body. They can develop into any of the cells that make up our bodies. Every single cell of the body “stems” from this type of cell at the origin of their name. Stem cells are self-renewing undifferentiated cells that produce multiple types of specialized cells of the body [1]. Stem cells are present in embryonic, fetal and adult tissues. During development, stem cells form from the tissues. In the adult, stem cells contribute to homeostasis of the tissues and regeneration after injuries. Until recently, it was believed that the adult mammalian brain was devoid of stem cells, hence unable to make new neurons and regenerate [2]. With the recent evidence and confirmation that neurogenesis occurs in the adult brain and neural stem cells (NSCs) residing in the adult central nervous system (CNS) in mammals, including in humans, the adult brain has the potential to regenerate and may be amenable to repair [3, 4]. Embryonic stem cells (ESCs) are self-renewing pluripotent cells. They are undifferentiated cells that generate all the cell types of the body [5]. As such, they hold the potential to cure a broad range of diseases and injuries, ranging from diabetes, liver and heart diseases, to neurological diseases. In contrast, adult stem cells are multipotents; they generate lineage specific cell types restricted to the tissues in which they reside. Recent studies reveal that adult stem cells may have a broader potential than originally thought [6]. The broader potential of adult stem cells has tremendous consequences for cellular therapy. Stem cells live in specialized microenvironments or “niches” that regulate their activity [7]. The environment or niches in which stem cells reside may hold the key to the developmental potential of adult stem cells. Because of their potential, stem cells carry a lot of hope for the treatment of a broad range of diseases and injuries, spanning from cancers, diabetes, genetic diseases, graft-versus-host disease, eye, heart and liver diseases, inflammatory and autoimmune disorders, to neurological diseases and injuries, particularly neurodegenerative diseases, like Alzheimer’s and Parkinson’s diseases, and spinal cord injuries. Cancer may also be a stem cell disease [8]. Hence, stem cell research is as important for our understanding the physio- and pathology of the body, as for development and therapy, including for the CNS. Over the past decade, significant progress has been made in stem cell research; the derivation of ESCs from human blastocysts [9], the development of somatic cell nuclear transfer technology [10] and the confirmation that neurogenesis occurs in the adult

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mammalian brain, including in humans [4]. These advances have contributed to bringing stem cell research closer to therapy. However, there are scientific and technical challenges lying ahead, and decades may pass before this research translates into therapy. These three freestanding volumes of Stem Cells and Regenerative Medicine aim at providing an overview and in-depth analysis of recent developments in stem cell research and therapy. They are composed of recently published review articles, reports, commentaries and letters to Editors, relating to these developments. Each of them focuses on a specific subject of stem cell biology, spanning from basic science to clinical, pharmacological, ethical and commercial aspects of stem cell research. These manuscripts went through a peer-review process. Volume I, Adult Neurogenesis and Neural Stem Cells, provides an overview and in-depth analysis of the new field of stem cell research that is the generation of new neuronal cells and the existence of stem cells, in the adult brain of mammals. These discoveries have forced us to re-think and re-evaluate how the brain is functioning, and reveal that the adult brain has the potential for self-repair. This volume covers the basic science of adult neurogenesis and neural stem cell research, from the origin, mechanisms, function, and the therapeutic potential of adult NSCs. Volume II, Embryonic and Adult Stem Cells, provides an analysis of various types of stem cells and their therapeutic potential. It concentrates particularly on embryonic and neural stem cells. This volume covers the broader potential of adult stem cells; its biology, significance and potential for therapy. This volume also describes the potential of stem cells for autologous transplantation, the stem cell theory of carcinogenesis, particularly the existence of brain tumor stem cells, and the therapeutic potential of gene therapy for cellbased therapy. Volume III, Pharmacology and Therapy, addresses developments in basic science, translational and clinical research that are underway to bring stem cell research to therapy, particularly for the treatment of Batten’s diseases, graft-versus-host disease and adenosine deaminase deficiency. This volume covers the importance of stem cell research for the understanding of drug activities and design. It also addresses the ethical issues and constraints involved in stem cell research, and its commercial applications.

REFERENCES [1] [2] [3] [4]

[5]

Potten C.S., Loeffler, M. (1990) Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development. 110, 1001-20. Rakic P. (1985) Limits of neurogenesis in primates. Science. 227, 1054-6. Kaplan M.S. (2001) Environment complexity stimulates visual cortex neurogenesis: death of a dogma and a research career. Trends Neurosci. 24, 617-20. Eriksson P.S, Perfilieva E., Bjork-Eriksson T., Alborn A.M., Nordborg C., Peterson D.A., Gage F.H. (1998) Neurogenesis in the adult human hippocampus. Nat Med. 4, 1313-7. Wobus A.M., Boheler K.R. (2005) Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev. 85, 635-78.

Introduction [6]

3

Anderson D.J., Gage F.H., Weissman I.L. (2001) Can stem cells cross lineage boundaries? Nat Med. 7, 393-5. [7] Scadden D.T. (2006) The stem-cell niche as an entity of action. Nature. 441, 1075-9. [8] Trosko J.E., Chang C.C. (1989) Stem cell theory of carcinogenesis. Toxicol Lett. 49, 283-95. [9] Thomson J.A, Itskovitz-Eldor J., Shapiro S.S., Waknitz M.A., Swiergiel J.J., Marshall V.S., Jones J.M. (1998) Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145-7. [10] Campbell K.H., McWhir J., Ritchie W.A., Wilmut I. (1996) Sheep cloned by nuclear transfer from a cultured cell line. Nature. 380, 64-6.

Chapter I

NEUROGENESIS AND ALZHEIMER’S DISEASE ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disease, characterized in the brain by amyloid plaque deposits and neurofibrillary tangles. It is the most common form of dementia among older people. There is at present no cure for AD, and current treatments consist mainly in drug therapy. Potential therapies for AD involve gene and cellular therapy. The recent confirmation that neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) provide new opportunities for cellular therapy in the CNS, particularly for AD, and to better understand brain physiopathology. Hence, researchers have aimed at characterizing neurogenesis in patients with AD. Studies show that neurogenesis is increased in these patients, and in animal models of AD. The effect on neurogenesis of drugs used to treat AD is currently being investigated, to determine if neurogenesis contributes to their therapeutic activities.

1. INTRODUCTION The recent confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS in various species, including humans, is as important for cellular therapy in the CNS, particularly for neurodegenerative diseases like AD, as for our understanding of developmental biology (Gage, 2000; Taupin and Gage, 2002). Environmental enrichment, drugs, trophic factors, neurotransmitters, and a broad range of physiopathological conditions, including AD, modulate adult neurogenesis (Taupin, 2005). Recently, using a combination of mouse models and X-irradiation to inhibit neurogenesis, it was reported that antidepressants, like fluoxetine increase hippocampal neurogenesis, which contributes to their behavioral effects (Santarelli et al., 2003). Researchers have aimed at investigating whether neurogenesis may contribute to the therapeutic effects of drugs used to treat other neurological diseases and disorders, particularly AD (Jin et al., 2006). Alzheimer’s disease is associated with the loss of nerve cells in areas of the brain, such as the hippocampus, that are vital to memory and other mental abilities (Hardy and Selkoe, 2002; St George-Hyslop and Petit, 2005). Hence, cognitive impairments that worsen over time are major disabilities of AD. Two classes of drugs are currently used to treat patients

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with AD: acetylcholinesterase (AChE) inhibitors, such as tacrine, donepezil, galantamine and rivastigmine, and an N-methyl-D-aspartate (NMDA)-glutamate receptor antagonist, i.e., memantine (Arrieta et al., 1998; Scarpini et al., 2003, Wilkinson et al., 2004; McShane et al., 2006). These drugs produce improvements in cognitive and behavioral symptoms, but their role in the pathogenesis of AD is unknown. AChE inhibitors are thought to improve cognitive function by enhancing cholinergic neurotransmission in regions of the brain affected by AD.

2. NEUROGENESIS IN ALZHEIMER’S DISEASE Jin et al. (2004) studied neurogenesis from brain autopsies of AD patients. The expression of markers for immature neuronal cells (doublecortin, polysialylated nerve cell adhesion molecule, and neurogenic differentiation factor) increase in the subgranular zone (SGZ), granular layer of the dentate gyrus (DG), and CA1 region of hippocampal Ammon’s horn (Jin et al., 2004a). The SGZ is a layer beneath the granular layer. Newly-generated neuronal cells in the SGZ migrate to the granular layer where they differentiate into neuronal cells of the DG (Taupin and Gage, 2002). Studies also show that neurogenesis is modulated in animal models of AD. Animal models have been devised to study genes involved in AD, such as presenilin 1 (PSEN1) and amyloid-beta protein precursor (APP) (German and Eisch, 2004). PSEN1 and APP are associated with most cases of early-onset AD, a rare hereditary form of dementia (St George-Hyslop and Petit, 2005). Neurogenesis is positively regulated in the DG of transgenic mice that express the Swedish and Indiana APP mutations, a mutant form of human APP (Jin et al., 2004b), and negatively regulated in the DG and subventricular zone (SVZ) of knock-out mice for PSEN1 and APP (Feng et al., 2001; Wen et al., 2002). The DG and SVZ are the two main regions of the CNS where neurogenesis occurs in the adult (Taupin and Gage, 2002). These animal studies were performed using bromodeoxyurine (BrdU) labeling, a thymidine analog that incorporates into the DNA of dividing cells during the S-phase of the cell cycle, and is used for birthdating cells and monitoring cell proliferation (Miller and Nowakowski, 1988; Taupin and Gage, 2002). The discrepancies between the studies could be explained by the limitation of the transgenic animal models as representative of complex diseases, by the study of adult phenotypes, such as adult neurogenesis (Dodart et al., 2002). Especially in mutant or deficient mice, single genes, such as PSEN1 and APP, may not fully reproduce the features of AD associated with loss of multiple cell types. Four to 10% of nerve cells in regions, such as the hippocampus, in which degeneration occurs in AD are tetraploids (Yang et al., 2001). Nerve cells may have entered the cell cycle and undergone DNA replication, but did not complete the cell cycle. It is proposed that cell cycle re-entry and DNA duplication precedes neuronal death in degenerating regions of the CNS (Herrup et al., 2004). As BrdU incorporates the DNA of dividing cells during the Sphase of the cell cycle, BrdU labeling will not allow discriminating cell proliferation versus cell cycle re-entry and DNA duplication without cell division. The existence of aneuploid cells may account for some of the newly-generated neuronal cells observed using BrdUlabeling in experimental models of AD. Therefore, reports suggest that neurogenesis is enhanced in AD. This is yet to be confirmed in light of recent data showing the existence of tetraploid cells in regions in which degeneration occurs in AD.

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3. EFFECT ON NEUROGENESIS OF DRUGS USED TO TREAT AD Researchers have aimed to identify the effect of drugs used to treat AD on neurogenesis. The effects of tacrine, galantamine and memantine on neurogenesis were assessed in adult mice, using the BrdU-labeling paradigm (Jin et al., 2006). The three drugs increased neurogenesis in the DG and SVZ by 26–45%, except tacrine that did not alter BrdU labeling in the DG. These results showed that drugs used to treat AD increased neurogenesis in the adult brain, which may contribute to their therapeutic effects (Jin et al., 2006). Neurogenesis is enhanced in AD (Jin et al., 2004a), and drugs used to treat AD also increase neurogenesis. The function of increased neurogenesis in the AD-affected brain, and the effects of drugs used to treat AD, remains to be elucidated. Some speculations can be raised. The increased neurogenesis in AD may represent a regenerative attempt by the CNS to compensate for the loss of nerve cells. It may also represent a compensatory process to increase CNS plasticity in the diseased brain (Taupin, 2006). One can speculate that drugs used to treat AD would then attempt to amplify such processes. The mechanism of action of these drugs on neurogenesis remains to be explained as well. Lesions of the cholinergic forebrain impair hippocampal neurogenesis in adult rats, and muscarinic receptors have been identified on newly-generated neuronal cells in the SGZ and SVZ. This suggests that the cholinergic pathway promotes neurogenesis (Cooper-Kuhn et al., 2004; Mohapel et al., 2005). Since AChE inhibitors are thought to improve cognitive function by enhancing cholinergic neurotransmission in ADaffected brain regions, and tacrine and galantamine may promote neurogenesis through a similar mechanism. The reason that tacrine that does not alter BrdU labeling in the DG remains to be further investigated. NMDA receptor antagonists on one hand promote neurodegeneration (Ikonomidou et al., 1999). On the other hand, they promote neurogenesis in the adult brain (Cameron et al., 1995, 1998; Gould et al., 1997; Nacher et al., 2001, 2003). Therefore, the therapeutic effect of memantine, an NMDA-glutamate receptor antagonist, in AD may also be mediated through stimulation of neurogenesis. These hypotheses and the mechanisms of action of drugs used for the treatment of AD are yet to be investigated and confirmed.

4. STEM CELL THERAPY FOR THE TREATMENT OF AD Because AD is associated with the loss of nerve cells, cellular therapy is considered for the treatment of this disease. With the recent evidence that neurogenesis occurs in the adult brain and NSCs reside in the adult CNS, new strategies for the treatment of neurodegenerative diseases, in particular AD, are being considered and are promising. These include the transplantation of adult-derived neural progenitor and stem cells, and the stimulation of endogenous neural progenitor cells. Experimental studies reveal that adult derived-neural progenitor and stem cells engraft the host tissues (Gage et al., 1995; Shihabuddin et al., 2000), and promote functional recovery in animal models of neurodegenerative diseases such as multiple sclerosis (Pluchino et al., 2003). In AD, as in multiple sclerosis, the degeneration is widespread, therefore direct transplantation of neural progenitor and stem cells in the brain may not offer an optimum strategy for treating these diseases. Neural progenitor and stem cells migrate to diseased and injured sites in the brain

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when administered by systemic injection (Macklis et al., 1993; Pluchino et al., 2003; Fujiwara et al., 2004). Such methods of delivering neural progenitor and stem cells that are noninvasive may prove to be valuable in the treatment of AD. Neurogenesis is enhanced in the diseased brain, particularly in AD (Jin et al., 2004a). This suggests that the brain has the potential to self-repair, and that endogenous progenitor cells may be recruited to replace degenerated nerve cells and promote functional recovery. Future studies will aim at identifying factors that promote neurogeneis in AD as candidates for cellular therapy.

5. CONCLUSION Neurogenesis is enhanced in AD, and drugs used to treat AD, though acting through different mechanisms of action, increase neurogenesis. This may contribute to their therapeutic effects. Santarelli et al. (2003) using a combination of mouse models and Xirradiation (to inhibit neurogenesis) reported that antidepressants such as fluoxetine increase hippocampal neurogenesis, which alters their behavioral activities. Meshi et al. (2006), using the same approaches, reported that neurogenesis does not mediate the behavioral effects of environmental enrichment (Meshi et al., 2006). Inhibition of neurogenesis is, therefore, key in determining causal relationship between neurogenesis and a physiological or pathological function. Therefore, the consequence of inhibiting neurogenesis on the effect of drugs used for the treatment of AD in establishing a causal relationship between their therapeutic effects and the stimulation of neurogenesis is yet to be evaluated. Nonetheless, the evidence that drugs used to treat AD positively regulate neurogenesis may lead to new drug design and new strategies to treat AD. To this end, unraveling the cellular and molecular mechanisms of the actions of drugs to treat AD on neurogenesis will be a key strategy.

ACKNOWLEDGMENTS Reproduced from: Taupin P. Neurogenesis and Alzheimer's Disease. Drug Target Insights (2006) 1: 1-4, with permission from Libertas Academia.

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Cooper-Kuhn, C.M., Winkler, J., Kuhn, H.G. (2004). Decreased neurogenesis after cholinergic forebrain lesion in the adult rat. J Neurosci Res, 77, 155-65. Dodart, J.C., Mathis, C., Bales, K.R., Paul, S.M. (2002). Does my mouse have Alzheimer's disease? Genes Brain Behav, 1, 142-55. Feng, R., Rampon, C., Tang, Y.P., Shrom, D., Jin, J., Kyin, M., Sopher, B., Miller, M.W., Ware, C.B., Martin, G.M., Kim, S.H., Langdon, R.B., Sisodia, S.S., Tsien, J.Z. (2001). Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. Neuron. 32, 911-26. Erratum in: (2002) Neuron, 33, 313. Fujiwara, Y., Tanaka, N., Ishida, O., Fujimoto, Y., Murakami, T., Kajihara, H., Yasunaga, Y., Ochi, M. (2004). Intravenously injected neural progenitor cells of transgenic rats can migrate to the injured spinal cord and differentiate into neurons, astrocytes and oligodendrocytes. Neurosci Lett, 366, 287-91. Gage, F.H., Coates, P.W., Palmer, T.D., Kuhn, H.G., Fisher, L.J., Suhonen, J.O., Peterson, D.A., Suhr, S.T., Ray, J. (1995). Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci, USA, 92, 11879-83. Gage, F.H. (2000). Mammalian neural stem cells. Science, 287, 1433-8. German, D.C., Eisch, A.J. (2004). Mouse models of Alzheimer's disease: insight into treatment. Rev Neurosci, 15, 353-69. Gould, E., McEwen, B.S., Tanapat, P., Galea, L.A., Fuchs, E. (1997). Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation. J Neurosci, 17, 2492-8. Hardy, J., Selkoe, D.J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 297, 353-6. Erratum in: (2002) Science, 297, 2209. Herrup, K., Neve, R., Ackerman, S.L., Copani, A. (2004). Divide and die: cell cycle events as triggers of nerve cell death. J Neurosci, 24, 9232-9. Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vöckler, J., Dikranian, K., Tenkova, T,I., Stefovska, V., Turski, L., Olney, J.W. (1999). Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science, 283, 70-4. Jin, K., Peel, A.L., Mao, X.O., Xie, L., Cottrell, B.A., Henshall, D.C., Greenberg, D.A. (2004a). Increased hippocampal neurogenesis in Alzheimer's disease. Proc Natl Acad Sci, USA, 101, 343-7. Jin, K., Galvan, V., Xie, L., Mao, X.O., Gorostiza, O.F., Bredesen, D.E., Greenberg, D.A. (2004b). Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw,Id) mice. Proc Natl Acad Sci, USA, 101, 13363-7. Jin, K., Xie, L., Mao, X.O., Greenberg, D.A. (2006). Alzheimer's disease drugs promote neurogenesis. Brain Res, 1085, 183-8. Macklis, J.D. (1993). Transplanted neocortical neurons migrate selectively into regions of neuronal degeneration produced by chromophore-targeted laser photolysis. J Neurosci, 13, 3848-63. Meshi, D., Drew, M.R., Saxe, M., Ansorge, M.S., David, D., Santarelli, L., Malapani, C., Moore, H., Hen, R. (2006). Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment. Nat Neurosci, 9, 729-31. McShane, R., Areosa Sastre, A., Minakaran, N. (2006). Memantine for dementia. Cochrane Database Syst Rev, 2, CD003154.

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Miller, M.W., Nowakowski, R.S. (1988). Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res, 457, 44-52. Mohapel, P., Leanza, G., Kokaia, M., Lindvall, O. (2005). Forebrain acetylcholine regulates adult hippocampal neurogenesis and learning. Neurobiol Aging, 26, 939-46. Nacher, J., Rosell, D.R., Alonso-Llosa, G., McEwen, B.S. (2001). NMDA receptor antagonis treatment induces a long-lasting increase in the number of proliferating cells, PSANCAM-immunoreactive granule neurons and radial glia in the adult rat dentate gyrus. Eur J Neurosci, 13, 512-20. Nacher, J., Alonso-Llosa, G., Rosell, D.R., McEwen, B.S. (2003). NMDA receptor antagonist treatment increases the production of new neurons in the aged rat hippocampus. Neurobiol Aging, 24, 273-84. Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani, G., Dina, G., Galli, R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi, A.L., Martino, G. (2003). Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis.Nature, 422, 688-94. Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee, J., Duman, R., Arancio, O., Belzung, C., Hen, R. (2003). Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science, 301, 805-9. Scarpini, E., Scheltens, P., Feldman, H. (2003). Treatment of Alzheimer's disease: current status and new perspectives. Lancet Neurol, 2, 539-47. Shihabuddin, L.S., Horner, P.J., Ray, J., Gage, F.H. (2000). Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci, 20, 8727-35. St.George-Hyslop, P.H., Petit, A. (2005). Molecular biology and genetics of Alzheimer's disease. C R Biol, 328, 119-30. Taupin, P., Gage, F.H. (2002). Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res, 69, 745-9. Taupin, P. (2005). Consideration of adult neurogenesis from basic science to therapy. Med Sci Monit, 11, LE16-7. Taupin, P. (2006). Adult neurogenesis and neuroplasticity. Restor Neurol Neurosci, 24. 9-15. Wen, P.H., Shao, X., Shao, Z., Hof , P.R., Wisniewski, T., Kelley, K., Friedrich, V.L .Jr., Ho, L., Pasinetti, G.M., Shioi, J., Robakis, N.K., Elder, G.A. (2002). Overexpression of wild type but not an FAD mutant presenilin-1 promotes neurogenesis in the hippocampus of adult mice. Neurobiol Dis, 10, 8-19. Wilkinson, D.G., Francis, P.T., Schwam, E., Payne-Parrish, J. (2004). Cholinesterase inhibitors used in the treatment of Alzheimer's disease: the relationship between pharmacological effects and clinical efficacy. Drugs Aging, 21, 453-78. Yang, Y., Geldmacher, D.S., Herrup, K. (2001). DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci, 21, 2661-8.

Chapter II

NEUROGENESIS AND THE EFFECT OF ANTIDEPRESSANTS ABSTRACT The recent evidence that neurogenesis occurs throughout adulthood and neural stem cells (NSCs) reside in the adult central nervous system (CNS) suggests that the CNS has the potential for self-repair. Along with this potential, the function of newly-generated neuronal cells in the adult brain remains the focus of intense research. The hippocampus of patients with depression shows signs of atrophy and neuronal loss. This suggests that adult neurogenesis may contribute to the biology of depression. The observations that antidepressants like fluoxetine increase neurogenesis in the dentate gyrus (DG), and that neurogenesis is required for the behavioral effect of antidepressants, lead to a new theory on depression and the design of new strategies and drugs for the treatment of depression. However, the role of adult neurogenesis in the etiology of depression remains the source of controversy and debate.

1. INTRODUCTION Neurogenesis, the generation of new neuronal cells, occurs in the adult brain of mammals (Gage, 2000; Gross, 2000), including humans (Eriksson et al., 1998). Neurogenesis occurs primarily in two regions of the adult brain: the DG and the subventricular zone (SVZ) (Taupin and Gage, 2001). Newly-generated neuronal cells establish synaptic and functional connections with nerve cells of the pre-existing network (Stanfield and Trice, 1988; Markakis and Gage, 1999; van Praag et al., 2002). It is hypothesized that newborn neuronal cells arise from stem cells in the adult brain. NSCs are the self-renewing multipotent cells that generate the main phenotypes of the nervous system (neurons, astrocytes and oligodendrocytes) as such, they hold the potential to treat a broad range of CNS diseases and injuries (Mckay, 1997). Neural progenitor and stem cells have been isolated and characterized in vitro from the adult brain, further supporting the existence of NSCs in the adult CNS (Reynolds and Weiss, 1992; Gage et al., 1995). The existence of NSCs in the adult brain has tremendous implications for cellular therapy in the CNS, and for our understanding of developmental biology (Taupin, 2006a).

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Depression is a major public health problem that affects 12-17% of the population (Kessler et al., 1994). Various classes of drugs are currently prescribed for the treatment of depression (Wong and Licinio, 2001; Brunello et al., 2002). Among them are selective serotonin reuptake inhibitors (SSRIs), like fluoxetine; monoamine oxidase inhibitors (MAOIs), like tranylcypromine; specific norepinephrine reuptake inhibitors (SNRIs), like reboxetine; and phosphodiesterase-IV inhibitors, like rolipram; alleviate symptoms of depression. It is hypothesized that an imbalance in serotonin (5-hydroxytryptamine or 5-HT) and noradrenaline (NA) pathways may underlie the pathogenesis of depressive disorders (Hindmarch, 2001; Owens, 2004). SSRIs, like fluoxetine, may produce their therapeutic effects by increasing brain levels of 5-HT, a neurotransmitter implicated in the modulation of mood and anxiety-related disorders (Whittington et al., 2004; Ryan 2005). Among the 5-HT receptor subtypes, the 5-HT1A receptor has been prominently implicated in the modulation of mood and anxiety-related disorders (Gross et al., 2002). There is increasing evidence that the hippocampus, a structure classically involved in leaning and memory, is involved in the modulation of emotional responses, particularly depression. Clinical magnetic resonance imaging and post-mortem studies in depression patients, as well as in animal studies, reveal that chronic stress and depression result in loss of nerve cells and atrophy in the hippocampus, and that these effects can be reversed by antidepressants (Watanabe et al., 1992a, 1992b; Sheline et al., 1996; Czeh et al., 2001; Campbell et al., 2004; Videbech and Ravnkilde, 2004; Colla et al., 2006). This suggests that neurogenesis may be an underlying factor in the contribution of the hippocampus to depression. In support of this contention, glucocorticoids, stress-related hormones, induce brain atrophy (Sapolsky, 2000; McEwen, 2001) and decrease neurogenesis (Gould et al., 1991; Cameron and Gould, 1994), whereas antidepressants, like fluoxetine, promote neurogenesis (Malberg et al., 2000; Malberg and Duman, 2003). Investigators have aimed at confirming and connecting the mechanism underlying adult neurogenesis to the etiology of depression.

2. NEUROGENESIS CONTRIBUTES TO THE THERAPEUTIC EFFECTS OF ANTIDEPRESSANTS The effect of antidepressants – including fluoxetine, tranylcypromine, reboxetine and rolipram – on adult neurogenesis was assessed by means of bromodeoxyuridine (BrdU) labeling, immunohistochemistry for neuronal specific markers, and confocal microscopy (Malberg et al., 2000, 2004). BrdU is a thymidine analog that incorporates into the DNA of dividing cells and is used for birthdating cells and monitoring cell proliferation (Miller and Nowakowski, 1988; Kuhn et al., 1996; Taupin, 2006b). Chronic administration of these antidepressants increases neurogenesis in the DG, but not in the SVZ of adult rats, suggesting that hippocampal neurogenesis contributes to the therapeutic effects of antidepressants (Malberg et al., 2000, 2004). To study the functional implication of such observations, Santarelli et al. (2003) aimed at determining if an increase in neurogenesis is required for the effect of antidepressants. X-ray irradiation of the hippocampal area in adult rats causes long-term reduction in cell proliferation in the DG (Tada et al., 2000). Hippocampal x-ray irradiation, but not irradiation of other brain areas – like the SVZ or the cerebellar region – prevented the neurogenic effect

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of antidepressants, such as fluoxetine, in adult mice (Santarelli et al., 2003). The behavioral effect of the antidepressants on the novelty-suppressed feeding (NSF) test subjects was also abolished after hippocampal irradiation. The NSF test, in which animals are food-deprived, placed into a novel environment containing food, and assessed for the latency to begin eating, is devised to assess chronic antidepressant efficacy in rodents (Bodnoff et al., 1988). Furthermore, 5-HT1A receptor null mice were insensitive to the neurogenic and behavioral effects of fluoxetine. In all, these data show that SSRIs, like fluoxetine, increase hippocampal neurogenesis, which contributes to their behavioral effects (Santarelli et al., 2003).

3. A NEUROGENIC THEORY OF DEPRESSION Stress, an environmental factor, is an important causal factor in precipitating episodes of depression in humans, and potently suppresses hippocampal neurogenesis in adult monkeys (Gould et al., 1998; Malberg and Duman, 2003), probably due to increased glucocorticoid release (Gould et al., 1991; Cameron and Gould, 1994). Neurogenesis plays an important role in the biology of depression, particularly the stimulation of neurogenesis by antidepressants contributing to their behavioral effects (Malberg et al., 2000; Santarelli et al., 2003). It is proposed that stress-induced decrease of neurogenesis in the DG is an important causal factor in precipitating episodes of depression. The waning and waxing of neurogenesis in the hippocampal formation are therefore important causal factors, respectively, in the precipitation of, and recovery from, episodes of clinical depression, probably mediated by the increase in brain serotonin levels (Jacobs et al., 2000). The mechanism underlying the increased neurogenesis mediated by antidepressants remains to be identified. Studies reveal that the 5-HT, particularly 5-HT1A, receptor subtypes mediate the involvement of adult neurogenesis in depression (Banasr et al., 2004), and that fluoxetine targets a population of early progenitor cells in the DG, rather than stem-like cells in the DG (Encinas et al., 2006). The effect of antidepressants on neurogenesis may be mediated by trophic factors, like brain-derived neurotrophic factor (BDNF). On one hand, antidepressant treatments increase the level of expression of BDNF in the patents’ brain, and BDNF has an antidepressant effect (Siuciak et al., 1997; Chen et al., 2001; Saarelainen et al., 2003). On the other hand, administration of BDNF increases adult neurogenesis in the hippocampus (Scharfman et al., 2005). This suggests that the effect of antidepressants on neurogenesis may be mediated by BDNF, through its signaling pathway, particularly the mitogen-activated protein (MAP) kinase pathway (Duman et al., 2006). The MAP kinase pathway is a BDNF signaling cascade mediated by the activation of MAP kinase (MAPK) that phosphorylates and activates the extracellular signal-regulated kinase (ERK) pathway (Huang and Reichardt, 2003). A hypothesis supported by recent findings shows that exercise promotes hippocampal neurogenesis, BDNF expression, and has an antidepressant effect (van Praag et al., 1999; Eadie et al., 2005; Russo-Neustadt and Chen, 2005; Bjornebekk et al., 2005; Ernst et al., 2006). Though these studies provide compelling evidence of the role of BDNF in depression and neurogenesis, the activity of BDNF is yet to be linked to the increase of neurogenesis mediated by antidepressants. There are, however, controversies and debates over the involvement of the hippocampus and adult neurogenesis in the etiology of depression. Among them, i) a link between

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neurogenesis, loss of nerve cells, atrophy and decrease of hippocampal volume in depression subjects has not yet been demonstrated; ii) studies show that hippocampal volume remains unchanged in depressive patients (Axelson et al., 1991; Inagaki et al., 2004; Bielau et al., 2005); iii) the hippocampal formation may not be primarily involved in depressive episodes, as other areas of the brain may play a critical role in depression (Nestler et al., 2002; Ebmeier et al., 2006); iv) there are questions regarding the validity of animal models of depression as representative of the human disorder; and v) neurogenesis may be more a contributing factor to CNS plasticity rather than to specific physiological or pathological processes (Taupin, 2006c). The involvement of adult neurogenesis in depression, therefore, remains unclear (Feldmann et al., 2006). All these data involved the hippocampus, a structure traditionally involved in learning and memory, and adult neurogenesis in depression and anxiety disorders (Thomas and Peterson, 2003). Antidepressant treatments may increase neural plasticity and adult neurogenesis, especially in the hippocampus. However, the neurogenic theory of depression remains the source of debate and controversy, and requires further study (Feldmann et al., 2006). More data and evidence are needed to confirm the involvement of adult neurogenesis in depression.

4. CONCLUSION These studies show that antidepressants increase hippocampal neurogenesis, and establish a causal relationship between the stimulation of neurogenesis and the effect of antidepressants. New neuronal cells that survived and integrated into the pre-existing network survived for long periods, over two years in humans (Eriksson et al., 1998). Therefore, antidepressants may have long-term consequences on the architecture and functioning of the CNS. The function of newly-generated neuronal cells in the adult brain remains a source of intense research and debate. Though the hippocampus and neurogenesis play an important role in depression, these data remain a source of controversy, and the involvement of adult neurogenesis in the etiology of depression is yet to be characterized. Nonetheless, the evidence that stimulation of neurogenesis contributes to the effects of antidepressants may hold the key to understanding the long-term consequences of the effects of antidepressants on the physiopathology of the CNS, and lead to new drug design and new strategies to treat depressive disorders. To this end, explaining the cellular and molecular mechanisms of the action of antidepressants on neurogenesis will be a determining factor.

ACKNOWLEDGMENTS Reproduced from: Taupin P. Neurogenesis and the Effects of Antidepressants. Drug Target Insights (2006) 1: 13-7, with permission from Libertas Academia.

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REFERENCES Axelson, D.A., Doraiswamy, P.M., McDonald, W.M., Boyko, O.B., Tupler, L.A., Patterson, L.J., Nemeroff, C.B., Ellinwood, E.H. Jr., Krishnan, K.R. (1993). Hypercortisolemia and hippocampal changes in depression. Psychiatry Res, 47, 163-73. Banasr, M., Hery, M., Printemps, R., Daszuta, A. (2004). Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5HT receptor subtypes in the dentate gyrus and the subventricular zone. Neuropsychopharmacology, 29, 450-60. Bielau, H., Trubner, K., Krell, D., Agelink, M.W., Bernstein, H.G., Stauch, R., Mawrin, C., Danos, P., Gerhard, L., Bogerts, B., Baumann, B. (2005). Volume deficits of subcortical nuclei in mood disorders A postmortem study. Eur Arch Psychiatry Clin Neurosci, 255,401-12. Bjornebekk, A., Mathe, A.A., Brene, S. (2005). The antidepressant effect of running is associated with increased hippocampal cell proliferation. Int J Neuropsychopharmacol, 8, 357-68. Bodnoff, S.R., Suranyi-Cadotte, B., Aitken, D.H., Quirion, R., Meaney, M.J. (1988). The effects of chronic antidepressant treatment in an animal model of anxiety. Psychopharmacology (Berl), 95, 298-302. Brezun, J.M., Daszuta, A. (1999). Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats. Neurosci, 89, 999-1002. Brunello, N., Mendlewicz, J., Kasper, S., Leonard, B., Montgomery, S., Nelson, J., Paykel, E.,Versiani, M., Racagni, G. (2002). The role of noradrenaline and selective noradrenaline reuptake inhibition in depression. Eur Neuropsychopharmacol, 12, 461-75. Cameron, H.A., Gould, E. (1994). Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neurosci, 61, 203-9. Campbell, S., Marriott, M., Nahmias, C., MacQueen, G.M. (2004). Lower hippocampal volume in patients suffering from depression: a meta-analysis. Am J Psychiatry, 161, 598-607. Chen, B., Dowlatshahi, D., MacQueen, G.M., Wang, J.F., Young, L.T. (2001). Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication. Biol Psychiatry, 50, 260-5. Colla, M., Kronenberg, G., Deuschle, M., Meichel, K., Hagen, T., Bohrer, M., Heuser, I. (2007). Hippocampal volume reduction and HPA-system activity in major depression. J Psychiatr Res, 41, 553-60. Czeh, B., Michaelis, T., Watanabe, T., Frahm, J., de Biurrun, G., van Kampen, M., Bartolomucci, A., Fuchs, E. (2001). Stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. Proc Natl Acad Sci, USA, 98, 12796-801. Duman, C.H., Schlesinger, L., Kodama, M., Russell, D.S., Duman, R.S. (2006). A Role for MAP Kinase Signaling in Behavioral Models of Depression and Antidepressant Treatment. Biol Psychiatry, 61, 661-70. Eadie, B.D., Redila, V.A., Christie, B.R. (2005). Voluntary exercise alters the cytoarchitecture of the adult dentate gyrus by increasing cellular proliferation, dendritic complexity, and spine density. J Comp Neurol, 486, 39-47.

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Ebmeier, K.P., Donaghey, C., Steele, J.D. (2006). Recent developments and current controversies in depression. Lancet, 367, 153-67. Encinas, J.M., Vaahtokari, A., Enikolopov, G. (2006). Fluoxetine targets early progenitor cells in the adult brain. Proc Natl Acad Sci, USA, 103, 8233-8. Eriksson, P.S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A.M., Nordborg, C., Peterson, D.A., Gage, F.H. (1998). Neurogenesis in the adult human hippocampus. Nat Med, 4, 1313-7. Ernst, C., Olson, A.K., Pinel, J.P., Lam, R.W., Christie, B.R. (2006). Antidepressant effects of exercise: evidence for an adult-neurogenesis hypothesis? J Psychiatry Neurosci, 31, 84-92. Feldmann, R.E. Jr., Sawa, A., Seidler, G.H. (2007). Causality of stem cell based neurogenesis and depression - to be or not to be, is that the question? J Psychiatr Res, 41, 713-23. Gage, F.H. (2000). Mammalian neural stem cells. Science, 287, 1433-8. Gage, F.H., Coates, P.W., Palmer, T.D., Kuhn, H.G., Fisher, L.J., Suhonen, J.O., Peterson, D.A., Suhr, S.T., Ray, J. (1995). Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci, USA 92, 11879-83. Gould, E., Woolley, C.S., Cameron, H.A., Daniels, D.C., McEwen, B.S. (1991). Adrenal steroids regulate postnatal development of the rat dentate gyrus: II. Effects of glucocorticoids and mineralocorticoids on cell birth. J Comp Neurol, 313, 486-93. Gould, E., Tanapat, P., McEwen, B.S., Flugge, G., Fuchs, E. (1998). Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc Natl Acad Sci, USA 95, 3168-71. Gross, C.G. (2000). Neurogenesis in the adult brain: death of a dogma. Nat Rev Neurosci, 1, 67-73. Gross, C., Zhuang, X., Stark, K., Ramboz, S., Oosting, R., Kirby, L., Santarelli, L., Beck, S., Hen, R. (2002). Serotonin1A receptor acts during development to establish normal anxiety-like behaviour in the adult. Nature, 416, 396-400. Hindmarch, I. (2001). Expanding the horizons of depression: beyond the monoamine hypothesis. Hum Psychopharmacol, 16, 203-18. Huang, E.J., Reichardt, L.F. (2003). Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem, 72, 609-42. Inagaki, M., Matsuoka, Y., Sugahara, Y., Nakano, T., Akechi, T., Fujimori, M., Imoto, S., Murakami, K., Uchitomi, Y. (2004). Hippocampal volume and first major depressive episode after cancer diagnosis in breast cancer survivors. Am J Psychiatry, 161, 2263-70. Jacobs, B.L., Praag, H., Gage, F.H. (2000). Adult brain neurogenesis and psychiatry: a novel theory of depression. Mol Psychiatry, 5, 262-9. Kessler, R.C., McGonagle, K.A., Zhao, S., Nelson, C.B., Hughes, M., Eshleman, S., Wittchen, H.U., Kendler, K.S. (1994). Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States. Results from the National Comorbidity Survey. Arch Gen Psychiatry, 51, 8-19. Kuhn, H.G., Winkler, J., Kempermann, G., Thal, L.J., Gage, F.H. (1997). Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J Neurosci, 17, 5820-9. Malberg, J.E., Eisch, A.J., Nestler, E.J., Duman, R.S. (2000). Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci, 20, 9104-10.

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Malberg, J.E., Duman, R.S. (2003). Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment. Neuropsychopharmacology, 28, 1562-71. Malberg, J.E. (2004). Implications of adult hippocampal neurogenesis in antidepressant action. J Psychiatry Neurosci, 29, 196-205. Markakis, E.A., Gage, F.H. (1999). Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol, 406, 449-60. McEwen, B.S. (2001). Plasticity of the hippocampus: adaptation to chronic stress and allostatic load. Ann N Y Acad Sci, 933, 265-77. McKay, R. (1997). Stem cells in the central nervous system. Science, 276, 66-71. Miller, M.W., Nowakowski, R.S. (1988). Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res, 457, 44-52. Nestler, E.J., Barrot, M., DiLeone, R.J., Eisch, A.J., Gold, S.J., Monteggia, L.M. (2002). Neurobiology of depression. Neuron, 34, 13-25. Owens, M.J. (2004). Selectivity of antidepressants: from the monoamine hypothesis of depression to the SSRI revolution and beyond. J Clin Psychiatry, 65, 5-10. Reynolds, B.A., Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 255, 1707-10. Ryan, N.D. (2005). Treatment of depression in children and adolescents. Lancet, 366, 933-40. Russo-Neustadt, A.A., Chen, M.J. (2005). Brain-derived neurotrophic factor and antidepressant activity. Curr Pharm Des, 11, 1495-510. Saarelainen, T., Hendolin, P., Lucas, G., Koponen, E., Sairanen, M., MacDonald, E., Agerman, K., Haapasalo, A., Nawa, H., Aloyz, R., Ernfors, P., Castren, E. (2003). Activation of the TrkB neurotrophin receptor is induced by antidepressant drugs and is required for antidepressant-induced behavioral effects. J Neurosci, 23, 349-57. Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee, J., Duman, R., Arancio, O., Belzung, C., Hen, R. (2003). Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science, 301, 805-9. Sapolsky, R.M. (2000). Glucocorticoids and hippocampal atrophy in neuropsychiatric disorders. Arch Gen Psychiatry, 57, 925-35. Scharfman, H., Goodman, J., Macleod, A., Phani, S., Antonelli, C., Croll, S. (2005). Increased neurogenesis and the ectopic granule cells after intrahippocampal BDNF infusion in adult rats. Exp Neurol, 192, 348-56. Sheline, Y.I., Wang, P.W., Gado, M.H., Csernansky, J.G., Vannier, M.W. (1996). Hippocampal atrophy in recurrent major depression. Proc Natl Acad Sci. USA 93, 390813. Siuciak, J.A., Lewis, D.R., Wiegand, S.J., Lindsay, R.M. (1997). Antidepressant-like effect of brain-derived neurotrophic factor (BDNF). Pharmacol Biochem Behav, 56, 131-7. Stanfield, B.B., Trice, J.E. (1988). Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Exp Brain Res, 72, 399-406. Tada, E., Parent, J.M., Lowenstein, D.H., Fike, J.R. (2000). X-irradiation causes a prolonged reduction in cell proliferation in the dentate gyrus of adult rats. Neurosci, 99, 33-41. Taupin, P., Gage, F.H. (2002). Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res, 69, 745-9.

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Taupin, P. (2006a). Neurogenesis in the adult central nervous system.C R Biol, 329, 465-75. Taupin, P. (2006b). BrdU immunohistochemistry for studying adult neurogenesis: Paradigms, pitfalls, limitations, and validation. Brain Res Rev, 53, 198-214. Taupin, P. (2006c). Adult neurogenesis and neuroplasticity. Restor Neurol Neurosci, 24, 915. Thomas, R.M., Peterson, D.A. (2003). A neurogenic theory of depression gains momentum. Mol Interv, 3, 441-4. Van Praag, H., Kempermann, G., Gage, F.H. (1999). Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci, 2, 266-70. Van Praag, H., Schinder, A.F., Christie, B.R., Toni, N., Palmer, T.D., Gage, F.H. (2002). Functional neurogenesis in the adult hippocampus. Nature, 415, 1030-4. Videbech, P., Ravnkilde, B. (2004). Hippocampal volume and depression: a meta-analysis of MRI studies. Am J Psychiatry, 161, 1957-66. Watanabe, Y., Gould, E., McEwen, B.S. (1992a). Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res, 588, 341-5. Watanabe, Y., Gould, E., Daniels, D.C., Cameron, H., McEwen, B.S. (1992b). Tianeptine attenuates stress-induced morphological changes in the hippocampus. Eur J Pharmacol, 222, 157-62. Whittington, C.J., Kendall, T., Fonagy, P., Cottrell, D., Cotgrove, A., Boddington, E. (2004). Selective serotonin reuptake inhibitors in childhood depression: systematic review of published versus unpublished data. Lancet. 363, 1341-5. Wong, M.L., Licinio, J. (2001). Research and treatment approaches to depression. Nat Rev Neurosci. 2, 343-51.

Chapter III

ADULT NEURAL STEM CELLS AND CELLULAR THERAPY ABSTRACT Considerable effort and means have been invested to find treatments for neurological diseases and injuries, yet there is still no cure for these ailments, and new alternatives for therapy must be explored. Because they generate the main phenotypes of the central nervous system (CNS), neural stem cells (NSCs) hold the promise to cure a broad range of CNS diseases and injuries. With the confirmation that neurogenesis occurs in the adult brain, and the recent isolation and characterization in vitro of neural progenitor and stem cells from the adult CNS, new avenues for the treatment of neurological diseases and injuries are being considered. Cell therapeutic interventions may involve both in vivo stimulation and transplantation of neural progenitor and stem cells of the adult brain.

1. INTRODUCTION Cellular therapy is the replacement of unhealthy or damaged cells or tissues with new ones. Because neurodegenerative diseases, cerebral strokes, and traumatic injuries to the CNS produce neurological deficits that result from neuronal loss, cell therapy is a prominent area of investigation for the treatment of neurological diseases and injuries. Cell types of various sources and merits have been considered for cellular therapy in the CNS. Until recently, strategies developed for cellular therapy to the CNS involved heterologous transplantation, which requires finding a tissue-compatible donor or administering drugs that suppress the immune system to prevent tissue rejection. The recent confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS gives new opportunities for cellular therapy in the CNS. The existence of NSCs in the adult CNS suggests that the CNS has the potential to self-repair. Hence, tremendous efforts are being devoted to study how endogenous neural progenitor and stem cells behave in the diseased brain, and to stimulate endogenous neurogenesis at the sites of degeneration. Neural progenitor and stem cells have also been isolated and expanded in vitro from the adult brain, allowing autologous transplantation in the adult CNS. Thus, the recent

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confirmation that neurogenesis occurs in the adult brain and NSCs reside in the adult CNS opens new opportunities for cellular therapy in the CNS.

2. ADULT NEUROGENESIS AND NEURAL STEM CELLS Neurogenesis occurs mainly in two areas of the adult mammalian brain: the dentate gyrus (DG) of the hippocampus, and the subventricular zone (SVZ), in several species, including humans [1-3]. In the DG, newly-generated neuronal cells in the subgranular zone (SGZ) migrate to the granular layer, where they differentiate into mature neuronal cells, and extend axonal projections into the CA3 area. In the SVZ, cells are generated in the anterior part of the SVZ, and migrate to the olfactory bulb (OB) through the rostro-migratory stream (RMS), where they differentiate into interneurons of the OB. Newly-generated neuronal cells in the DG and OB establish functional connections with neighboring cells [1]. Quantitative studies showed that, though a significant fraction of newly-generated neuronal cells in the DG and SVZ undergo programmed cell death [4, 5], as many as 9,000 new neuronal cells – or 0.1% of the granule cell population – are generated per day in the DG, and 65.3-76.9% of the bulbar neurons are replaced during a six-week period in young adult rodents [6, 7]. The neuronal cells born during adulthood that survive to maturity and become integrated into circuits are very stable, and can survive for an extended period of time; at least two years in humans DG [2]. Thus, they may permanently and functionally replace cells born during development. Neurogenesis may also occur, albeit at lower levels, in other areas of the brain, such as the Ammon’s horn CA1, neocortex and substantia nigra (SN) in certain species [8-10]. However, some of these reports have been contradicted by other studies, and need to be further investigated [11-14]. In the adult spinal cord, recent studies have confirmed that gliogenesis, but not neurogenesis, occurs throughout the cord [15, 16]. Experimental studies show that the rate of neurogenesis in the DG and SVZ is modulated by various physiological and pathological conditions, as well as by environmental stimuli. For example, stress, neuroinflammation, and aging decrease neurogenesis in the DG, and voluntary running increases neurogenesis in the DG whereas exposure to odor deprivation or to an environment enriched in odors decreases or increases neurogenesis in the OB, respectively [17]. This modulation of neurogenesis suggests the implication of the hippocampus and SVZ in these processes. Since newly generated neural cells in the adult brain can survive for extended period of time, the modulation of neurogenesis may produce long-term effects on the architecture and functioning of the CNS. Investigators have attempted to define the function of newly-generated neuronal cells in the adult brain. Evidence suggests that newly-generated neuronal cells in the hippocampus may be involved in learning and memory [18], such as in the formation of trace memories, a form of memory that involves the hippocampus [19]. Newly generated neuronal cells in the hippocampus may also be involved in depression [20, 21]. Stress is an important causal factor in precipitating episodes of depression, and decreases hippocampal neurogenesis [22]. It is hypothesized that the waning and waxing of neurogenesis in the hippocampal formation are important causal factors, respectively, in the precipitation of, and recovery from, episodes of clinical depression [23].

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It is hypothesized that newly-generated neuronal cells in the adult brain originate from residual NSCs. NSCs are the self-renewing, multipotent cells that generate neurons, astrocytes, and oligodendrocytes in the nervous system [24] (Figure 1). Neural progenitor and stem cells have been isolated and characterized in vitro from various areas, neurogenic and non-neurogenic, of the adult CNS, including the spinal cord, suggesting that NSCs reside throughout the adult CNS.

Figure 1. Neural stem cells. Neurogenesis occurs in the adult brain. It is hypothesized that newlygenerated neuronal cells in the adult brain originate from residual neural stem cells (NSCs). NSCs are the self-renewing, multipotent cells that generate neurons, astrocytes, and oligodendrocytes in the nervous system. Neural progenitor and stem cells have been isolated and characterized in vitro from various areas, neurogenic and non-neurogenic, of the adult CNS, suggesting that NSCs reside throughout the adult CNS. Neural progenitor cells are multipotent cells, with limited self-renewing capacity. The origin and identity of NSCs are yet to be determined.

The origin and identity of NSCs remain source of debate and controversy [1]. Though recent reports further support an astroglial origin for newly-generated neuronal cells [25-29], NSCs are yet to be unequivocally identified in the adult brain. There are currently no specific markers of adult NSCs. The intermediate neurofilament nestin, the transcription factors sox-2, oct-3/4, and the RNA binding protein Musashi 1 are markers for neural progenitor and stem

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cells, but also label population of glial cells [30-36]. Though many questions are yet to be answered, the confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS has a tremendous implication for cellular therapy: the adult CNS has the potential to repair itself.

3. ADULT NEURAL STEM CELLS AND CELLULAR THERAPY With the confirmation that neurogenesis occurs in the adult brain, and the recent isolation and characterization in vitro of neural progenitor and stem cells from the adult CNS, new avenues for the treatment of neurological diseases and injuries are being considered. Cell therapeutic interventions may involve both in vivo stimulation and transplantation of neural progenitor and stem cells.

3.1. Stimulation of Endogenous Neural Progenitor Cells Recent evidence shows that new neuronal cells are generated at sites of degeneration in the diseased brain and after CNS injuries. Curtis et al. (2003) and Tattersfield et al. (2004) reported an increase in SVZ neurogenesis, leading to the migration of neural progenitor cells and the formation of new neuronal cells in the damaged areas of the striatum in Huntington's disease (HD) patients, and in animal models of HD (quinolinic acid lesion) [37, 38]. After experimental strokes (middle cerebral artery occlusion), new neuronal cells are detected at the major sites of degeneration, such as the striatum and the cortex [39-42]. Cell tracking studies revealed that these newly-generated neuronal cells at the sites of degeneration originate from the SVZ. The newly-generated cells migrate partially through the RMS to the sites of degeneration, where they differentiate, within 5 weeks, into the phenotypes of the degenerated nerve cells [37, 38, 43, 44]. Though this regenerative process is limited – estimated at 0.2% of the degenerated nerve cells in the striatum after focal ischemia [43] – this evidence overturns the long-held dogma that the adult brain cannot renew itself from injuries [1, 24]. Several hypotheses can explain the limited capacity of the CNS to recover after injuries. The number of new neurons generated may be too low to compensate for the neuronal loss. The neuronal cells that are produced are non-functional because they do not develop into fully mature neurons, because they do not develop into the right type of neurons, or because they do not integrate into the surviving brain circuitry. The generation of new neuronal cells at the sites of injury would then represent an attempt by the CNS to repair itself, further highlighting the potential of the CNS for regeneration. The SVZ origin of these newly-generated neuronal cells suggests that conditions enhancing SVZ neurogenesis could promote regeneration and functional recovery after CNS injuries. Several molecules and factors have been reported to enhance SVZ neurogenesis in rodents. Trophic factors such as epidermal growth factor and basic fibroblast growth factor, administered by intracerebroventricular or subcutaneous injection, stimulate neurogenesis in the adult SVZ [45-47]. Exogenous substances, such as Ginko biloba extract – an herbal plant used medicinally – stimulates neurogenesis in the OB [48]. Transforming growth factor-α, when infused into the adult rat striatum, leads to migration of neuronal progenitor cells from

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the SVZ and to the infusion site [49]. Nitric oxide donor [50, 51], sildenafil (Viagra) [52], glutamate [53], statins [54], erythropoietin [55], heparin-binding epidermal growth factor-like growth factor [56], vascular endothelial growth factor [57], and epidermal growth factor [58, 59] promote neurogenesis in the SVZ and improve neurological function after experimental injuries in rodents, such as strokes. These molecules and factors are potential candidates for cellular therapy in the CNS. Alternatively, since evidence suggests that NSCs may reside in neurogenic and nonneurogenic areas, the stimulation of neural progenitor and stem cells locally may also provide a strategy for promoting regeneration of the CNS. Factors such as platelet-derived growth factor and brain-derived neurotrophic factor induce striatal neurogenesis in adult rats with 6hydroxydopamine lesions, with no indication of any newly born cells differentiating into dopaminergic neurons following growth factor treatment [60]. Though neurogenesis in the SN remains the source of controversy [12-14], such factors may prove to be beneficial for recovery from Parkinson’s disease. Future investigation will aim at identifying factors promoting neurogenesis in non-neurogenic areas. The identification of the SVZ as a source of newly-generated neuronal cells at the sites of degeneration, after injuries, presents several features that can benefit cellular therapy in the CNS. First, in the intact CNS and after injuries, a significant proportion of newly-generated neuronal progenitor cells in the SVZ undergo programmed cell death rather than achieving maturity – e.g., 80% of the new neuronal cells that are generated in the SVZ after stroke in rats die within the first weeks after the insult [4, 5, 53]. This transient increase in newlygenerated neuronal progenitor cells provides a window of opportunity the newly-generated cells could be salvaged and directed to participate in the regeneration process. Factors preventing cell death, such as caspases [61-63], would thus also be potentially beneficial for cellular therapy, alone or in combination with other conditions promoting SVZ neurogenesis, such as the administration of trophic factors and/or environmental enrichment. Second, the identification of the SVZ, along the ventricles, as the source of neural progenitor and stem cells with regenerative potential after injuries also suggests that molecules and factors could be administered either by systemic, intracerebroventricular or subcutaneous injection, or through the cerebrospinal fluid (CSF) to promote neurogenesis in the brain [45-47], in addition to the spinal cord [64], as the central canal is a presumed location of putative NSCs [16]. Such less invasive procedures would be beneficial for the treatment of the injured patients. Future studies will aim at better understanding the molecular and cellular cascades involved in adult neuronal progenitor and stem cell proliferation, migration, and fate determination in the injured brain, to promote regeneration. For example, neuroinflammation inhibits neurogenesis [65, 66], and is a component of the secondary reaction after injury. Hence, strategies promoting neurogenesis after injuries would include reducing events that negatively regulate neurogenesis, such as neuroinflammation. Thus, anti-inflammatory treatments may be considered to promote neurogenesis and functional recovery after injury.

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Figure 2. Cellular therapy. The isolation and characterization of neural progenitor and stem cells from the adult brain open new opportunities for cellular transplantation in the CNS. Neural progenitor and stem cells can be isolated from various areas of the adult CNS, and cultured in vitro. Expanded cells can be maintained for weeks in culture, frozen, and stored, allowing the long-term preservation of the neural progenitor and stem cells to be used for cellular therapy. The ability to isolate neural progenitor and stem cells from the adult brain opens the possibility to perform autologous graft by isolating the patients’ neural progenitor and stem cells, hence limiting the risk of tissue rejection and obviating the need to find a matching donor.

3.2. Adult-Derived Neural Progenitor and Stem Cells as Sources of Tissue for Transplantation Adult neural progenitor and stem cells can be isolated from human post-mortem tissues [67, 68], potentially allowing the generation of neural progenitor and stem cells from multiple sources for cellular therapy. Alternatively, neural progenitor and stem cells could be isolated from an undamaged area of the patient’s brain, expanded in vitro, and grafted back to the degenerated area(s) (Figure 2). Though such strategies would carry secondary risks associated with brain surgery, autologous transplantation would obviate the need to find a matching donor for the tissues, limiting rejection risk of the grafted tissues; or the need to administer drugs that suppress the immune system, such as cyclosporine, hence, greatly enhancing the chance for cellular integration in the patient, and recovery. Neural progenitor and stem cells

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have been isolated and characterized in vitro from various areas, neurogenic and nonneurogenic, of the adult CNS, such as the hippocampus, SVZ, striatum, septum, spinal cord, and SN [1, 12]. Whether neural progenitor and stem cells isolated from diverse brain areas are equivalent and have the same potential remain to be determined. Nonetheless, experimental studies in rodents have shown that neural progenitor and stem cells isolated from the adult CNS grafted into heterotypic areas integrate and differentiate into neuronal cells of the host brain areas within two months [69-75]. The ability of the cells to differentiate into neuronal phenotypes of the target areas in heterotypic transplantation supports autologous transplantation as a possible strategy for cellular therapy in the CNS.

4. ADULT NEURAL STEM CELLS: A MODEL OF CHOICE FOR CELLULAR THERAPY FOR CNS DISEASES AND INJURIES Cell types of various sources and merits, such as cells derived from embryos – embryonic stem cells – from fetuses, or from tissues and organs, have been considered for cellular therapy in the CNS. Because they do not carry ethical and political concerns, and allow rewiring of the CNS, adult NSCs offer of model of choice for cellular therapy. Aside from the ethical and political concerns, NSCs offer a powerful tool for cellular therapy, particularly for cellular transplantation. Cell transplantation aims mainly at delivering cells to specific sites. This is particularly suitable for the treatment of diseases and injuries in which the degeneration is limited to mainly one area, as in neurodegenerative diseases like Parkinson’s disease, and after traumatic injuries to the CNS [76, 77]. When the degeneration is widespread, as in neurodegenerative diseases like Alzheimer’s disease, HD and multiple sclerosis, such a strategy is not applicable. Neural progenitor and stem cells migrate to tumor [78], injured [7981], and diseased sites [82] when transplanted into the CNS, or when administered either by systemic injection or through the CSF. A recent study in an animal model of multiple sclerosis has reported that systemic injection of neural progenitor and stem cells may provide significant clinical benefits for these disease [82]. Thus, NSC therapy may provide a therapeutic tool for the treatment of a broad range of neurological diseases and injuries, particularly for neurodegenerative diseases. Such migratory properties of NSCs can be used as a general mode for administering neural progenitor and stem cells for cellular therapy, avoiding surgical procedures, and their associated risks and secondary effects. Indeed, systemic injection and injection through CSF are regarded as promising ways to administer NSCs for cellular therapy [83, 84]. Grafted NSCs may also promote functional recovery by promoting the survival of injured neuronal cells through the secretion of neurotrophic factors [85-89], and their interaction with the injured brain [90], further underlining the relevance of NSC transplantation for cellular therapy in the CNS.

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5. CONCLUSION The recent confirmation that neurogenesis occurs in the adult brain, and isolation and characterization in vitro of neural progenitor and stem cells from the adult CNS, open new opportunities for cellular therapy in the CNS. Adult-derived neural progenitor and stem cells circumvent the ethical and political concerns associated with their embryonic and fetal counterparts, and offer the opportunity to treat a broad range of CNS diseases and injuries, making adult NSCs a model of choice for cellular therapy in the CNS.

ACKNOWLEDGMENTS Reprinted from: Taupin P. Adult neural stem cells and cellular therapy. Journal of Stem Cells (2006) 1(1): 47-55, with permission from Nova Science Publishers, Inc.

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[57] Sun, Y., Jin, K., Xie, L., Childs, J., Mao, X.O., Logvinova, A., Greenberg, D.A. (2003). VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest, 111, 1843-51. [58] Teramoto, T., Qiu, J., Plumier, J.C., Moskowitz, M.A. (2003). EGF amplifies the replacement of parvalbumin-expressing striatal interneurons after ischemia. J Clin Invest, 111, 1125-32. [59] Nakatomi, H., Kuriu, T., Okabe, S., Yamamoto, S., Hatano, O., Kawahara, N., Tamura, A., Kirino, T., Nakafuku, M. (2002). Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell, 110, 429-41. [60] Mohapel, P., Frielingsdorf, H., Haggblad, J., Zachrisson, O., Brundin, P. (2005). Platelet-Derived Growth Factor (PDGF-BB) and Brain-Derived Neurotrophic Factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions. Neuroscience, 132, 767-76. [61] Namura, S., Zhu, J., Fink, K., Endres, M., Srinivasan, A., Tomaselli, K.J., Yuan, J., Moskowitz, M.A. (1998). Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J Neurosci, 18, 3659-68. [62] Pompeiano, M., Blaschke, A.J., Flavell, R.A., Srinivasan, A., Chun, J. (2000). Decreased apoptosis in proliferative and postmitotic regions of the Caspase 3-deficient embryonic central nervous system. J Comp Neurol, 423, 1-12. [63] Ekdahl, C.T., Mohapel, P., Elmer, E., Lindvall, O. (2001). Caspase inhibitors increase short-term survival of progenitor-cell progeny in the adult rat dentate gyrus following status epilepticus. Eur J Neurosci, 14, 937-45. [64] Martens, D.J., Seaberg, R.M., van der Kooy, D. (2002). In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord. Eur J Neurosci, 16, 1045-57. [65] Monje, M.L., Toda, H., Palmer, T.D. (2003). Inflammatory blockade restores adult hippocampal neurogenesis. Science, 302, 1760-5. [66] Ekdahl, C.T., Claasen, J.H., Bonde, S., Kokaia, Z., Lindvall, O. (2003). Inflammation is detrimental for neurogenesis in adult brain. Proc Natl Acad Sci, USA. 100, 13632-7. [67] Palmer, T.D., Schwartz, P.H., Taupin, P., Kaspar, B., Stein, S.A., Gage, F.H. (2001). Cell culture. Progenitor cells from human brain after death. Nature, 411, 42-3. [68] Schwartz, P.H., Bryant, P.J., Fuja, T.J., Su, H., O'Dowd, D.K., Klassen, H. (2003). Isolation and characterization of neural progenitor cells from post-mortem human cortex. J Neurosci Res, 74, 838-51. [69] Gage, F.H., Coates, P.W., Palmer, T.D., Kuhn, H.G., Fisher, L.J., Suhonen, J.O., Peterson, D.A., Suhr, S.T., Ray, J. (1995). Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci, USA. 92, 11879-83. [70] Suhonen, J.O., Peterson, D.A., Ray, J., Gage, F.H. (1996). Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature, 383, 624-27. [71] Takahashi, M., Palmer, T.D., Takahashi, J., Gage, F.H. (1998). Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina. Mol Cell Neurosci, 12, 340-8. [72] Shihabuddin, L.S., Horner, P.J., Ray, J., Gage, F.H. (2000). Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci, 20, 8727-35.

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[73] Young, M.J., Ray, J., Whiteley, S.J., Klassen, H., Gage, F.H. (2000). Neuronal differentiation and morphological integration of hippocampal progenitor cells transplanted to the retina of immature and mature dystrophic rats. Mol Cell Neurosci, 16, 197-205. [74] Akiyama, Y., Honmou, O., Kato, T., Uede, T., Hashi, K., Kocsis, J.D. (2001). Transplantation of clonal neural precursor cells derived from adult human brain establishes functional peripheral myelin in the rat spinal cord. Exp Neurol, 167, 27-39. [75] Vroemen, M., Aigner, L., Winkler, J., Weidner, N. (2003). Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways. Eur J Neurosci, 18, 743-51. [76] Armstrong, R.J., Tyers, P., Jain, M., Richards, A., Dunnett, S.B., Rosser, A.E., Barker, R.A. (2003). Transplantation of expanded neural precursor cells from the developing pig ventral mesencephalon in a rat model of Parkinson's disease. Exp Brain Res, 151, 204-17. [77] Lepore, A.C., Bakshi, A., Swanger, S.A., Rao, M.S., Fischer, I. (2005). Neural precursor cells can be delivered into the injured cervical spinal cord by intrathecal injection at the lumbar cord. Brain Res, 1045, 206-16. [78] Brown, A.B., Yang, W., Schmidt, N.O., Carroll, R., Leishear, K.K., Rainov, N.G., Black, P.M., Breakefield, X.O., Aboody, K.S. (2003). Intravascular delivery of neural stem cell lines to target intracranial and extracranial tumors of neural and non-neural origin. Hum Gene Ther, 14, 1777-85. [79] Macklis, J.D. (1993). Transplanted neocortical neurons migrate selectively into regions of neuronal degeneration produced by chromophore-targeted laser photolysis. J Neurosci, 13, 3848-63. [80] Veizovic, T., Beech, J.S., Stroemer, R.P., Watson, W.P., Hodges, H. (2001). Resolution of stroke deficits following contralateral grafts of conditionally immortal neuroepithelial stem cells. Stroke, 32, 1012-9. [81] Boockvar, J.A., Schouten, J., Royo, N., Millard, M., Spangler, Z., Castelbuono, D., Snyder, E., O'Rourke, D., McIntosh, T. (2005). Experimental traumatic brain injury modulates the survival, migration, and terminal phenotype of transplanted epidermal growth factor receptor-activated neural stem cells. Neurosurgery, 56, 163-71. [82] Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani, G., Dina, G., Galli, R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi, A.L., Martino, G. (2003). Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature, 422, 688-94. [83] Wu, S., Suzuki, Y., Kitada, M., Kataoka, K., Kitaura, M., Chou, H., Nishimura, Y., Ide, C. (2002). New method for transplantation of neurosphere cells into injured spinal cord through cerebrospinal fluid in rat. Neurosci Lett, 318, 81-4. [84] Fujiwara,Y., Tanaka, N., Ishida, O., Fujimoto, Y., Murakami, T., Kajihara, H., Yasunaga, Y., Ochi, M. (2004). Intravenously injected neural progenitor cells of transgenic rats can migrate to the injured spinal cord and differentiate into neurons, astrocytes and oligodendrocytes. Neurosci Lett, 366, 287-91. [85] Ourednik, J., Ourednik, V., Lynch, W.P., Schachner, M., Snyder, E.Y. (2002). Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat Biotechnol, 20, 1103-10.

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[86] Lu, P., Jones, L.L., Snyder, E.Y., Tuszynski, M.H. (2003). Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol, 181, 115-29. [87] Llado, J., Haenggeli, C., Maragakis, N.J., Snyder, E.Y., Rothstein, J.D. (2004). Neural stem cells protect against glutamate-induced excitotoxicity and promote survival of injured motor neurons through the secretion of neurotrophic factors. Mol Cell Neurosci. 27, 322-31. [88] Yan, J., Welsh, A.M., Bora, S.H., Snyder, E.Y., Koliatsos, V.E. (2004). Differentiation and tropic/trophic effects of exogenous neural precursors in the adult spinal cord. J Comp Neurol, 480, 101-14. [89] Pfeifer, K., Vroemen, M., Blesch, A., Weidner, N. (2004). Adult neural progenitor cells provide a permissive guiding substrate for corticospinal axon growth following spinal cord injury. Eur J Neurosci, 20, 1695-704. [90] Park, K.I., Teng, Y.D., Snyder, E.Y. (2002). The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat Biotechnol, 20, 1111-7.

Chapter IV

STEM CELL ENGINEERING FOR CELL-BASED THERAPY ABSTRACT Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, heart and muscular diseases, to neurological diseases, disorders and injuries. Significant progress has been made in stem cell research over the past decade: the derivation of embryonic stem cells (ESCs) from human tissues, the development of cloning technology by somatic cell nuclear transfer (SCNT); the confirmation that neurogenesis occurs in the adult mammalian brain, and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), including that of humans. Despite these advances, it may be decades before stem cell research will translate into therapy. Stem cell research is also subject to ethical and political debate, controversy and legislation, which slow its progress. Cell engineering has proven successful in bringing genetic research to therapy. In this chapter, I will review two examples of how investigators are applying cell engineering to stem cell biology to circumvent stem cells’ ethical and political constraints, thereby bolstering stem cell research and therapy.

1. INTRODUCTION Cellular therapy is the replacement of lost or dysfunctional tissues with new ones. Various cell types have been evaluated and considered for therapy. In the CNS, fetal neuronal tissue has been particularly evaluated for its merit in treating neurological diseases and injuries [1]. While numerous experimental and clinical transplantation studies showed that fetal neuronal transplants improve functional deficits in models of CNS diseases [2–5], others reported less positive outcomes [6, 7]. In addition, the rate of survival of fetal neuronal cells transplanted into the adult brain is relatively low, requiring large quantities of tissue, generally from several fetuses, for therapy. Researchers are looking at other opportunities for cellular therapy, particularly in the CNS. Stem cells are undifferentiated cells that have the ability to self-renew and differentiate into other cell types. Stem cells represent a promising model for cell-based therapy [8]. ESCs are self-renewing pluripotent cells derived from the inner cell mass (ICM) of the blastocyst.

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ESCs can generate cells derived from the three primary germ layers; ectoderm, mesoderm and endoderm. Because ESCs can give rise to the various cell types of the body – an estimated 220 cell types in humans – they carry the hope of curing a broad range of diseases and injuries [9]. Stem cells exist in fetal and adult tissues. These stem cells are multipotent; they generate cells to the tissue from which they are derived [8]. In the CNS, with the confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS, the adult brain appears to have the ability of limited self-repair [10–12]. Neural stem and progenitor cells have been isolated from adult tissues [13, 14], including tissues from biopsies and postmortem tissues [15, 16], providing a potential source of tissues for the treatment of neurological diseases and injuries. Neural stem and progenitor cells derived from human fetal tissues are currently in clinical trial for the treatment of Batten’s disease, a childhood neurodegenerative disorder [17]. Preclinical data show that grafted neural stem and progenitor cells survive in damaged brain tissues, migrate to specific sites of degeneration where they differentiate into neural lineages, and have a beneficial effect on functional recovery [17]. This emphasizes the potential of neural stem and progenitor cells for therapy. There are ethical considerations in the use of stem cells for research and therapy. ESCs have been derived from the ICM of human embryos (hECSs) [18]. hESCs are derived from leftover frozen embryos, created for in vitro fertilization, destined to be discarded. Because it involves the destruction of embryos, there is a debate over the use of hESCs for research and therapy. On one hand, there are scientific reports and evidence that ESCs, including hESCs, differentiate into various cell types of the body, such as neuronal and insulin producing cells [19], backing the scientific claim of the potential of ESCs for therapy. On the other hand, opponents of the use of hESCs for research and therapy argue that it is morally unacceptable to destroy human life. The scientific and ethical debate over the use of hESCs for research and therapy considerably slows stem cell research. It is proposed that using fetal or adult tissue-derived stem cells would circumvent the ethical issues in the use of ESCs for research and therapy. However, because fetal and adult stem cells are multipotent, they would have less potential in the treatment of diseases and injuries. Recent evidence suggests that adult tissue-derived stem cells may have broader potential; they may have the capacity to give rise to cells from lineages other than the one from which they are derived [20]. This would give adult stem cells a broader spectrum in treating diseases and injuries [21]. However, data related to the broader potential of adult tissue-derived stem cells are the source of debate and controversy, and are yet to be validated therapeutically. More recently, stem cells extracted from amniotic fluid were reported to have similar potential to ESCs, fueling the search for an alternative source of cells for therapy without controversy for clinical research and therapy [22]. Although multipotent stem cells offer an alternative to ESCs for therapy, there are ethical considerations to take into account when using fetal and adult human-derived tissue for therapy. Among them, validating the use of human tissue-derived cells, including fetal and adult stem cells, involves experimentation in which human-derived cells are transplanted into animals – with different ratios of human versus host cells – generating chimeras [23]; these experiments are subject to strict regulations and controversies [24]. Other issues, like possible non-ethical origin of tissue (e.g., absence of consent from the donor), can potentially be damaging for research involving human tissues, particularly for stem cell research [25]. There is also serious ethical consideration regarding fetal sources of stem cells (versus hESCs). While the use of hESCs is considered by some to be morally unacceptable because it involves the destruction of

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embryos, the use of fetuses is morally acceptable when it involves tissues originating from spontaneous abortions (miscarriages), with the consent of the parents, as opposed to elective abortions. In all, strict ethical and political guidelines must be followed when using human tissues for therapy and particularly for stem cell research. Transgenic modifications offer the ability to modify the cells’ genome to accomplish specific functions. It has been instrumental in the study of gene functions, and as a therapeutic tool to produce biologically active substances, such as neurotransmitter synthesizing enzymes and trophic factors [26, 27]. Stem cells have properties that make them suitable candidates that could benefit from gene therapy. Though stem cells can give rise to the diverse cells types of the body, protocols to differentiate them in a wide variety and high yield of cell types, including neuronal and insulin producing cells, are yet to be established. In the CNS, aside from the replacement of lost or degenerated nerve cells by the grafted cells, studies revealed that grafted neural stem and progenitor cells may also promote functional recovery through the secretion of trophic factors [28–30]. This shows that transgenic expression of trophic factors or expression of missing proteins is increasingly likely to play a role in cellbased therapies. Stem cells, and particularly NSCs, have inherent properties to migrate to tumors, injured and diseased sites after transplantation [31, 32]. The abilities of NSCs to be genetically modified [14] and to migrate to diseased or degenerated sites provide unique opportunities to target these areas and provide missing proteins to promote recovery. Genetically modifying stem cells has also been proposed to circumvent some of the ethical issues associated with the use of ESCs for clinical research and therapy. In this review, I will discuss recent studies involving stem cells engineered to bolster stem cell research and therapy; their potentials, limitations and controversies.

2. CELL ENGINEERING TO DERIVE ESCS WITHOUT THE DESTRUCTION OF EMBRYOS SCNT is a cloning strategy, originally reported by Campbell, et al. [33], in which nuclei are isolated from a donor’s somatic cells, such as fibroblasts, and are transferred into enucleated oocytes from female donors [33]. By mechanisms yet to be uncovered, the cytoplasm of the oocytes reprograms the chromosomes of the somatic cell nucleus and the cloned cells develop into blastocysts, from which ESCs can be derived [34]. One of the landmarks of SCNT is the potential to generate isogeneic ESCs, carrying a set of chromosomes identical to that of an individual, and therefore unlikely to be rejected after transplantation into that individual [35]. In addition to its potential, human cloning is the source of scientific, ethical and political debate, controversy and legislation [36–38]. SCNT has been applied in cloning various animals, to derive ESCs from various species, but is yet to be applied successfully to derive hESCs [39]. The General Assembly of the United Nations has adopted a declaration calling on governments to ban all forms of human cloning that are “incompatible with human dignity and the protection of human life.” In the US, human cellular cloning (i.e., therapeutic cloning using SCNT) is not banned, while human being cloning (i.e., reproductive cloning) is banned. Countries like England, and more recently Australia, with the aim to promote stem cell research, allow therapeutic cloning, but not reproductive cloning.

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In an attempt to circumvent the ethical and political debate over the use SCNT for clinical research, Meissner and Jaenisch [40] used a variation of SCNT, altered nuclear transfer (ANT), to derive ESCs in mice [40]. ANT is a variation of SCNT proposed by W.B. Hurlbut [41]. In ANT, a gene crucial for trophectoderm development, the gene CDX2, is inactivated in vitro in the donor cells. CDX2 encodes the earliest-known trophectodermspecific transcription factor that is activated in the eight-cell embryos, and is essential for establishment and function of the trophectoderm. Inactivating the gene CDX2 eliminates formation of the fetal–maternal interface, but spares the ICM from which ESCs could be derived. The nucleus deficient in CDX2 is then transferred into enucleated oocytes from female donors, and submitted to the same protocols as for SCNT. Because the eggs created from nuclei deficient in CDX2 produce embryos that are unable to implant into the uterus, and do develop, ANT has been proposed as a variation of nuclear transfer to derive ESCs without the destruction of embryos. Meissner and Jaenisch genetically modified the donor cells, mouse fibroblasts, by inserting into their genome a cassette coding for RNAi cdx2 and the green fluorescent protein (GFP), flanked by two LoxP sequences [40]. The cassette was inserted in the cells’ genome using a lenti viral vector. The nuclei of genetically engineered fibroblasts, selected by means of GFP fluorescence, were transferred into enucleated oocytes, and submitted to the same protocols as for SCNT. The eggs divided and produced cloned blastocysts that were morphologically abnormal and lacked functional trophoblasts. The cloned blastocysts did not implant into the uterus, and ESCs could be derived from their ICMs. To maintain the developmental potential of the generated ESCs, the expression of CDX2 is re-established by deleting the cassette RNAi cdx2, using a plasmid coding for Crerecombinase. This study was the first to report the derivation of ESCs by ANT. There is a debate as to whether a mutant embryo is equivalent to a normal embryo. Proponents of ANT claim that because the eggs created from nuclei deficient in CDX2 produce embryos that are unable to implant into the uterus and do not undergo subsequent development, ANT represents a variation of nuclear transfer to derive ESCs without the destruction of embryos [42]. Alternatively, it has been argued that finding it acceptable to destroy a CDX2 mutant embryo, but not a normal embryo, is a “flawed proposal,” as there is no basis for concluding that the action of CDX2, or any other gene, represents a transition point at which a human embryo acquires moral status [43]. Therefore, whether ANT solves the issues of the derivation of ESCs without destruction of embryos remains a source of debate [44–47]. Moreover, in the study by Meissner and Jaenisch [40], it is not known whether cloned ESCs with an inactivated gene CDX2 have the same developmental potential as ESCs derived from donated eggs. Studies have reported that SCNT may affect the developmental potential of cloned animals and ESCs [48–50]. Meissner and Jaenisch [40] also used a lenti virus to engineer donor cells [40]. All of these factors may affect the developmental and therapeutic potential of ESCs generated by ANT. Nonetheless, this study highlights the potential of cell engineering for the advancement of research in stem cell biology.

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3. ADULT NEURAL STEM CELL ENGINEERING Contrary to a long-held belief, neurogenesis occurs in the adult mammalian brain, including in humans [10, 11]. Neurogenesis occurs primarily in two areas of the adult brain, the dentate gyrus of the hippocampus and the subventricular zone along the ventricles. It is hypothesized that newly-generated neuronal cells originate from stem cells in the adult brain [12]. NSCs are the self-renewing multipotent cells that generate the main cell types of the nervous system: neurons, astrocytes and oligodendrocytes. Neural stem and progenitor cells have been isolated and characterized in vitro from various regions of the adult CNS, including the spinal cord, supporting the existence of NSCs in the CNS [51]. The generation of new neuronal cells in the adult brain and the isolation and characterization of neural stem and progenitor cells from the adult CNS suggest that the adult brain may be amenable to repair. Cell therapy in the adult CNS could involve the stimulation of endogenous neural stem or progenitor cells or the transplantation of adult-derived neural stem and progenitor cells. Adult-derived neural stem and progenitor cells have been transplanted into animal models, and have shown functional engraftment [14, 52, 53]. More recently, grafted human-derived neural stem and progenitor cells show functional integration and promote functional recovery in an animal model of spinal cord injury, supporting the potential of neural stem and progenitor cells for therapy [54]. Adult neural stem and progenitor cells can be genetically modified by retroviral-mediated transfection, rendering them a vehicle for gene therapy [14, 52]. Adult-derived neural stem and progenitor cells genetically modified to express acid sphingomyelinase can lead to a reversal of lysosomal storage pathology when transplanted into animal models of NiemannPick’s disease [55]. Niemann-Pick’s disease is a lysosomal storage disorder in which deficiency of acid sphingomyelinase leads to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. This highlights the potential of genetically modified NSCs for the treatment of lysosomal storage diseases and other genetic diseases of the CNS, and for delivering trophic factors for the treatment of neurodegenerative diseases. The relevance of genetically modified NSCs for stem cell therapy is further highlighted by the potential of NSCs for the treatment of brain tumors. Neural stem and progenitor cells migrate to tumors and injured and diseased sites when transplanted into the CNS, either by systemic injection, or through the cerebrospinal fluid [31, 32, 56–59]. The injected cells migrate to the diseased or degenerated areas where they integrate with the host tissue. The properties of NSCs to be genetically engineered and to migrate to tumor sites have been proposed for the treatment of brain tumors. It is proposed to genetically engineer NSCs with “suicide genes,” such as genes coding for cytolytic activities or anti-tumor cytokines, to attack and destroy brain tumor cells [60, 61]. This further extends the potential of genetically modified stem cells for cancer therapy, particularly in the CNS.

4. CONCLUSION Stem cell therapy holds the promise to treat a broad range of diseases and injuries. The promise of stem cell therapy, particularly in the CNS, is in regenerating and reconstructing the original pathway to promote functional recovery, but it may be years before it emerges as

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a viable therapy. Genetically engineering cells has proven valuable in understanding gene function, and in delivering missing trophic factors or neurotransmitter-synthesizing enzymes in the CNS. The studies reviewed show that genetically engineering stem cells, particularly NSCs, may offer an opportunity to bolster stem cell research and therapy. There are, however, several limitations for the application of gene therapy-based strategies for therapy. Among them are the long-term expression of the transgenes, and the risks and limitations of using genetically-engineered cells for therapy.

ACKNOWLEDGMENTS Reproduced with permission from the Institute of Physics Publishing Ltd.: Taupin, P. Stem cell engineering for cell-based therapy. Journal of Neural Engineering (2007) 4(3): R59-63. Copyright 2007, the Institute of Physics Publishing Ltd. http://jne.iop.org.

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injured motor neurons through the secretion of neurotrophic factors. Mol Cell Neurosci, 27, 322-31. [30] Bjugstad, K.B., Redmond, D.E. Jr., Teng, Y.D., Elsworth, J.D., Roth, R.H., Blanchard, B.C., Snyder, E.Y., Sladek, J.R. Jr. (2005). Neural stem cells implanted into MPTPtreated monkeys increase the size of endogenous tyrosine hydroxylase-positive cells found in the striatum: a return to control measures. Cell Transplant, 14, 183-92. [31] Zhang, R.L., Zhang, L., Zhang, Z.G., Morris, D., Jiang, Q., Wang, L., Zhang, L.J., Chopp, M. (2003). Migration and differentiation of adult rat subventricular zone progenitor cells transplanted into the adult rat striatum. Neuroscience, 116, 373-82. [32] Burnstein, R.M., Foltynie, T., He, X., Menon, D.K., Svendsen, C.N., Caldwell, M.A. (2004). Differentiation and migration of long term expanded human neural progenitors in a partial lesion model of Parkinson's disease. Int J Biochem Cell Biol, 36, 702-13. [33] Campbell, K.H., McWhir, J., Ritchie, W.A., Wilmut, I. (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature, 380, 64-6. [34] Wakayama, T. (2006). Establishment of nuclear transfer embryonic stem cell lines from adult somatic cells by nuclear transfer and its application. Ernst Schering Res Found Workshop, 60, 111-23. [35] Lanza, R.P., Chung, H.Y., Yoo, J.J., Wettstein, P.J., Blackwell, C., Borson, N., Hofmeister, E., Schuch, G., Soker, S., Moraes, C.T., West, M.D., Atala, A. (2002). Generation of histocompatible tissues using nuclear transplantation. Nat Biotechnol, 20, 689-96. [36] Trounson, A., Pera, M. (1980). Potential benefits of cell cloning for human medicine. Reprod Fertil Dev, 10, 121-5. [37] Lanza, R.P., Cibelli, J.B., West, M.D. (1999). Human therapeutic cloning. Nat Med, 5, 975-7. [38] Jaenisch, R., Wilmut, I. (2001). Developmental biology. Don't clone humans! Science, 291, 2552. [39] Kennedy, D. (2006). Editorial retraction. Science, 311, 335. [40] Meissner, A., Jaenisch, R. (2006). Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature, 439, 212-5. [41] Hurlbut, W.B. (2005). Altered nuclear transfer. N Engl J Med, 352, 1153-4. [42] Hurlbut, W.B. (2005). Altered nuclear transfer as a morally acceptable means for the procurement of human embryonic stem cells. Perspect Biol Med, 48, 211-28. [43] Melton, D.A., Daley, G.Q., Jennings, C.G. (2004). Altered nuclear transfer in stem-cell research - a flawed proposal. N Engl J Med, 351, 2791-2. [44] Weissman, I.L. (2006). Medicine: politic stem cells. Nature, 439, 145-7. [45] Solter, D. (2005). Politically correct human embryonic stem cells? N Engl J Med, 353, 2321-3. [46] Jaenisch, R., Meissner, A. (2006). Politically correct human embryonic stem cells? N Engl J Med, 354, 1208-9. [47] Taupin, P. (2006). Derivation of embryonic stem cells. Med Sci (Paris), 22, 478-80. [48] Shiels, P.G., Kind, A.J., Campbell, K.H., Waddington, D., Wilmut, I., Colman, A., Schnieke, A.E. (1999). Analysis of telomere lengths in cloned sheep. Nature, 399, 316-7. [49] Rhind, S.M., Taylor, J.E., De Sousa, P.A., King, T.J., McGarry, M., Wilmut, I. (2003). Human cloning: can it be made safe? Nat Rev Genet, 4, 855-64.

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[50] Wakayama, S., Jakt, M.L., Suzuki, M., Araki, R., Hikichi, T., Kishigami, S., Ohta, H., Van Thuan, N., Mizutani, E., Sakaide, Y., Senda, S., Tanaka, S., Okada, M., Miyake, M., Abe, M., Nishikawa, S., Shiota, K., Wakayama, T. (2006). Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts. Stem Cells, 24, 2023-33. [51] Taupin, P. (2006). Neural Progenitor and Stem Cells in the Adult Central Nervous System. Annals Academy of Medicine Singapore, 35, 814-7. [52] Shihabuddin, L.S., Horner, P.J., Ray, J., Gage, F.H. (2000). Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci, 20, 8727-35. [53] Suhonen, J.O., Peterson, D.A., Ray, J., Gage, F.H. (1996). Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature, 383, 624-7. [54] Cummings, B.J., Uchida, N., Tamaki, S.J., Salazar, D.L., Hooshmand, M., Summers, R., Gage, F.H., Anderson, A.J. (2005). Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci, USA 102, 14069-74. [55] Shihabuddin, L.S., Numan, S., Huff, M.R., Dodge, J.C., Clarke, J., Macauley, S.L., Yang, W., Taksir, T.V., Parsons, G., Passini, M.A., Gage, F.H., Stewart, G.R. (2004). Intracerebral transplantation of adult mouse neural progenitor cells into the NiemannPick-A mouse leads to a marked decrease in lysosomal storage pathology. J Neurosci, 24, 10642-51. [56] Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., Snyder, E.Y. (2000). Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci. USA 97, 12846-51. Erratum in: (2001) Proc Natl Acad Sci, USA 98, 777. [57] Veizovic, T., Beech, J.S., Stroemer, R.P., Watson, W.P., Hodges, H. (2001). Resolution of stroke deficits following contralateral grafts of conditionally immortal neuroepithelial stem cells. Stroke, 32, 1012-9. [58] Fujiwara, Y., Tanaka, N., Ishida, O., Fujimoto, Y., Murakami, T., Kajihara, H., Yasunaga, Y., Ochi, M. (2004). Intravenously injected neural progenitor cells of transgenic rats can migrate to the injured spinal cord and differentiate into neurons, astrocytes and oligodendrocytes. Neurosci Lett, 366, 287-91. [59] Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani, G., Dina, G., Galli, R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi, A.L., Martino, G. (2003). Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature, 422, 688-94. [60] Yip, S., Aboody, K.S., Burns, M., Imitola, J., Boockvar, J.A., Allport, J., Park, K.I., Teng, Y.D., Lachyankar, M., McIntosh, T., O'Rourke, D.M., Khoury, S., Weissleder, R., Black, P.M., Weiss, W., Snyder, E.Y. (2003). Neural stem cell biology may be well suited for improving brain tumor therapies. Cancer J, 9, 189-204. [61] Shah, K., Bureau, E., Kim, D.E., Yang, K., Tang, Y., Weissleder, R., Breakefield, X.O. (2005). Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression. Ann Neurol, 57, 34-41.

Chapter V

HUCNS-SC STEM CELLS SUMMARY Originator NeuroSpheres Ltd. Licensee StemCells, Inc. Status Phase I Clinical Actions Nootropic agent Indications Central nervous system disease, lysosome storage disease, neurodegenerative disease, spinal cord injury Technology Stem-cell therapy

ABSTRACT HuCNS-SC, a proprietary human neural stem cell product, is being developed as a cellular therapy for the potential treatment of Batten disease, one of a group of disorders known as neural ceroid lipofuscinoses (NCL). Developer StemCells, Inc. is also investigating the therapy for spinal cord injury and other central nervous system disorders, such as demyelinating disease, stroke, and Alzheimer's disease. A phase I trial of HuCNS-SC for infantile and late-infantile NCL has been initiated, following the March 2006 US Food and Drug Administration approval of StemCells' investigational new drug application.

1. INTRODUCTION Cellular therapy can be summarized as the replacement of dysfunctional or degenerated tissues by new ones, and as such, carries a lot of hope for the treatment of a broad range of diseases and injuries, particularly of the central nervous system (CNS). Cell types from various sources are currently being evaluated for cellular therapy, including embryonic stem cells (ESCs). ESCs are derived from the inner cell mass of blastocytes and, because they have the potential to generate virtually all the cell types found within the body, major efforts are being devoted to bringing ESCs to the clinic. Although ESCs have been popularized as a

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potential therapeutic breakthrough, there are limitations to the use in cellular therapy, the major drawback being the risk of undifferentiated ESCs developing into tumor tissue upon grafting [647207]. Neural stem cells (NSCs) offer an alternative to ESCs. NSCs are the self-renewing, multipotent cells that generate the main phenotypes of the nervous system: neurons, astrocytes and oligodendrocytes [437208]. NSCs are present in both fetal and adult mammalian tissues, from which they can be isolated and cultured in vitro, providing a source of tissue for cellular therapy [647230]. NSCs therapy is currently being proposed for the treatment of a broad range of CNS diseases and injuries, in particular neurodegenerative diseases, spinal cord injuries, stroke, and genetic disorders that damage the brain [655176]. It is possible that in the future, therapies based on NSC could aid the reconstruction of damaged brain tissues; however, a great deal of research must be done before medicine advances to this point. Batten's disease (BD) is one of a group of disorders known as neural ceroid lipofuscinoses (NCLs). NCLs are inherited autosomal recessive neurodegenerative disorders of the nervous system that usually occur in childhood. They are caused by an abnormal buildup of substances called lipofuscins in the nerve cells throughout the brain, which leads to a progressive deterioration of brain function [647967]. BD affects the nerve cells in the brain and eyes, as well as other parts of the body, causing a progressive loss of vision, decline in physical and mental capabilities, and seizures. Early symptoms usually appear between the ages of five and ten years, and the disease is often fatal by the late teens or twenties. Lipofuscinoses are normally broken down and removed from the body, but in BD, various genes responsible for this process are faulty. An enzyme called palmitoyl-protein thioesterase 1 (PPT1) is insufficiently active in the infantile form of BD, because of a mutation in the gene encoding ceroid lipofuscinosis neural 1. PPT1 is a lysosomal protein that removes fatty acyl side chains from cysteine residues of proteins in lysosomes. Deficiency of PPT1 causes an abnormal build-up of substances in the nerve cells, and subsequently leads to a decline in nervous system function [647967]. PPT1 knockout mice have been generated as a model of BD. PPT1-/- mice present neuronal loss in the hippocampus and cerebellum, as well as an accumulation of auto-fluorescent storage material within these areas [647969]. BD and other forms of NCL are relatively rare, occurring in an estimated two to four of every 100,000 live births in the US [www.ninds.nih.gov]. There is currently no specific treatment for BD and current therapy simply alleviates the symptoms of the disease. Anticonvulsant drugs alleviate the associated seizures, and occupational therapy helps individuals compensate for the loss of vision, physical and mental abilities. Because BD involves the deterioration of neuronal cell tissue, it is a candidate for cellular therapy. StemCells, Inc. is developing a proprietary NSC product for cellular therapy, under license from NeuroSpheres Ltd., comprising well-characterized, normal human CNS stem cells (HuCNS-SCs) from brain tissue. HuCNS-SC is currently under investigation for the potential treatment of neurodegenerative disorders, particularly BD [180367], [540074]. Preclinical studies have been performed in various animal models of CNS diseases and injuries. Data from these studies has supported the therapeutic potential of HuCNS-SCs, and the therapy has recently been approved for a phase I clinical trial for the treatment of BD [629732].

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2. SYNTHESIS AND SAR In 1992, Reynolds and Weiss invented a reproducible method for growing rodent CNS stem and progenitor cells in culture as clusters of cells called neurospheres [200019••]; in 1997, the procedure was adapted for human CNS stem and progenitor cells [437374]. HuCNS-SCs were derived from human fetal (16 to 20 weeks) brain tissue. After enzymatic dissociation of the tissue into single-cell suspension, cells were isolated by fluorescenceactivated cell sorting using novel combinations of antibodies to cell-surface markers [437290]. The isolated cells were cultured in neurosphere conditions, in defined medium, with a cocktail of trophic factors containing basic fibroblast growth factor, epidermal growth factor, lymphocyte inhibitory factor, neural survival factor-1 and N-acetylcysteine. In vitro studies conducted on these cells demonstrated that CD133+ cells were capable of neurosphere initiation, self-renewal and multi-potentiality, thus making them the ideal HuCNS-SC [437290]. CD133 is a major NSC marker that is defined by its five transmembrane domains (a unique structure among known cell-surface markers), and antibodies to CD133 (such as the monoclonal antibody AC133 [647217]) have previously been used to isolate human hematopoietic cells and ESC [647217]. Sorting procedures further characterized the HuCNS-SC population as CD133+, 5E12+, CD34-, CD45- and CD24-/lo (CD133+ sorted cells). After 8 weeks, 5 to 10% of single sorted CD133+ cells developed into neurospheres, whereas the sorted cell population not expressing CD133 (CD133-, 5E12+, CD34-, CD45-and CD24-/lo), representing approximately 95% of total fetal brain tissue, failed to differentiate into neurospheres. CD133+ sorted cells were grown and expanded in culture through cell dissociation and splitting techniques. After five passages, the number of CD133+ cells increased at least 1000-fold, and cultured cells retained their ability to re-initiate neurosphere formation. When plated in differentiating medium containing two growth factors (brain-derived neurotrophic factor and glial-derived neurotrophic factor), clonally derived neurospheres from CD133+ cells differentiated into neurons and astrocytes. Together, these data demonstrated that CD133+ sorted cells represent a population of cells with stem-cell properties, and that highly enriched NSC populations could be isolated with antibodies against CD133 [437290].

3. PRECLINICAL DEVELOPMENT Initial studies by CytoTherapeutics, Inc. (later StemCells, Inc.), which led to the development of HuCNS-SC, focused on the delivery of protein neurotrophic factors into the neural system. In particular, studies focused on the transfer of human nerve growth factor (NGF) into neurodegenerative animal models [199982], [199983], [225755], [243560], [243561], [243562]. Baby hamster kidney cells were genetically modified to secrete high levels of human NGF before undergoing polymer encapsulation prior to implantation. Although the therapy showed potential for the treatment of neurological disorders, the emergence of stem cells offered far broader therapeutic scope.

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3.1. Animal NSCs The potential of stem-cell therapy was examined in vitro using murine fetal NSCs. The stem cells were epidermal growth factor (EGF)-responsive, that is, were continuously propagated in vitro in the presence of EGF. In the absence of EGF and in the presence of 1% fetal calf serum, the NSCs differentiated to form astrocytes, oligodendrocytes and neurons. After 5 to 10 days, the promoter elements glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were expressed on astrocytes and oligodendrocytes, respectively [200021], [243563]. GFAP and MBP were used to direct the expression of the Escherichia coli reporter gene ßgalactosidase in a transgenic marking system in mice. Studies monitoring expression of ß-galactosidase found the gene was highly stable, as it appeared to be expressed in virtually 100% of the appropriate cells [200021]. One of the first animal studies of NSCs showed that rodent EGF-responsive NSCs could differentiate into oligodendroglia. Myelin-deficient rats, killed 13 to 14 days post-treatment, developed patches of myelin around the dorsal columns, indicating a possible valuable source of myelinating cells [200023]. Further studies with murine NSCs looked at the potential of these cells to alleviate excitotoxic striatal lesions in rats. Murine fetal stem cells containing the human GFAP promoter element were transplanted into the striatum. Animals receiving control cells demonstrated comprehensive lesions of the striatum compared with animals receiving the GFAP stem cells. The GFAP stem cells promoted the expression of human nerve growth factor (NGF), which helped to protect against striatal lesions [243558].

3.2. HuCNS-SCs An initial study of HuCNS-SCs assessed the potential of the cells to self-renew in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted 1 to 12 months prior to analysis. HuCNS-SCs from transplanted murine brain were re-isolated by immunomagnetic selection for human-specific neural cell adhesion molecules and thymus cell antigen 1 to assess the self-renewal of the transplanted cells. Some of the cells expressed the human specific antigens AC133 or hCD24 [468477]. After transplantation, most cells were AC133-/hCD24hi, but some AC133+/hCD24-/lo antigens were observed; the sorted AC133+/hCD24-/lo human cells from one-year post-transplant re-initiated neurosphere cultures, indicating that HuCNS-SCs continue to generate more AC133+ cells as well as give rise to AC133-/hCD24hi progenitors for up to one year following transplantation [468477]. An objective comparison of engraftment data between different HuCNS-SC-derived neurosphere lines showed that, ex vivo, expanded HuCNS-SC engraft robustly in the NOD/SCID mouse brain and retain a multilineage differentiation capacity. Newborn mice were injected with three neurosphere cell lines (1.55 HuCNS-SCs per hemisphere). Stereological analysis estimated that 54 to 74 transplanted cells were distributed to the cortex of one hemisphere. Further analysis using confocal microscopy showed that HuCNS-SCs gave rise to neurons, astrocytes and oligodendrocytes in a site-specific manner [636344]. This study followed on from earlier studies that had demonstrated the ability of HuCNS-SCs to engraft, proliferate, migrate and differentiate into neurons, astrocytes and oligodendrocytes

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for at least 7 months post-transplantation into the brains of NOD/SCID mice [437290], [647222]. The survival and expression of mouse NSCs and HuCNS-SCs (10 to 50 × 106 cells per ml) were compared in non-lesioned, Parkinsonian or Huntingtonian rat hosts. Analyses were conducted over 1 week to 12 months and demonstrated cell survival of 2 to 10% (mouse) and 10 to 35% (human). The transplanted cells differentiated into both neurons and astrocytes and migrated away from the injection tract in a radial fashion, extending 0.5 to 1.5 mm mediolaterally and rostrocaudally [243557]. HuCNS-SCs were transplanted into the ischemic cortex of rats 7 days after distal middle cerebral artery occlusion (MCAO), because the lesion sizes were relatively stable by this time, allowing for specific targeting of the peri-infarct area. Rats were administered three 1.0µl deposits of suspended cells (1 × 105 cells per µl) along the anterior-posterior axis into the cortex via an infusion pump. The brains of the rats were analyzed 4 weeks later. Positive survival of cells was observed only if the cells were transplanted into non-ischemic tissue. There was a negative correlation of lesion size with cell survival, suggesting that even 7 days after MCAO the environment is unfavorable to the transplanted cells. It was suggested that inflammatory cytokines might prevent the transplanted cells integrating and surviving in the lesioned tissue, and that antiinflammatory treatments might be beneficial in assisting cellular therapy. The cells also demonstrated targeted migration, with human cells mainly differentiating to the neuronal phenotype and migrating long distances (~ 1.2 mm compared with 0.2 mm for naïve rats), predominantly toward the lesion [647228], [654203]. Remyelination by HuCNS-SCs was observed in both spinal cord injury NOD/SCID and myelin-deficient shiverer mice (defective in myelin production as a result of a mutation in MBP) [623960••], [633798], [636193], [654205]. HuCNS-SCs injected into NOD/SCID spinal cord injured mice 9 days after contusion, survived, migrated and expressed differentiation markers for neurons and oligodendrocytes. Electron microscopy analysis gave evidence consistent with synapse formation between HuCNS-SCs and mouse host neurons, although no details of presynaptic output or electrophysiological evidence were presented. However, HuCNS-SCs did not contribute to glial scar formation because of glial cell proliferation, suggesting that few engrafted human cells differentiated into astrocytes in this model. The mice received four injections bilaterally of a 250nl cell suspension, at 75,000 cells per µl. By 17 weeks posttreatment, cells had migrated away from the lesion epicenter in sagittal sections, with some transplanted cells found > 1 cm from the lesion epicenter. The expression of ß-tubulin III (expressed by neurons early in their development) was observed in approximately 26% of the implanted cells [623960••], [654204], [654205]. In shiverer mice, data indicated the presence of proliferating cells because of the high expression of the proliferation marker Ki-67+ at early time points. Only a limited number of HuCNS-SCs expressed the NSC marker CD133; however, expression of nestin (an intermediate filament protein expressed in neuroepithelial stem cells during nervous system development) was prevalent. Instead of differentiating into neurons, histological assessment indicated that HuCNS-SCs largely differentiated into oligodendrocytes through a continuous process of differentiation lasting approximately 45 days until full maturation and MBP production. By the end of the study (60 days), all grafted white matter tracts in the cerebellum, fimbria and corpus callosum demonstrated dense MBP staining. However, transplanted cells within the cortex and the hippocampus, and cells remaining in the injection core, failed to express MBP by day 60. Immunoelectromagnetic analysis, using the human

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specific monoclonal antibody SC121 showed that the oligodendrocytes derived from HuCNSSC enrobed murine axons with 10 to 16 layers of myelin [633798], [636193]. HuCNS-SCs were used as a cellular delivery vehicle for enzyme replacement in a murine model (PPT1 knockout mice) of the infantile form of BD. HuCNS-SCs were cocultured with fibroblasts taken from BD individuals and used to produce and secrete the PPT1 enzyme. After cells were transplanted into the ventricle of mice, preliminary data demonstrated a reduced build-up of pathogenic toxic waste material and a larger number of surviving neurons in transplanted transgenic mice compared with control [485519]. Follow-up data from the study at 18 weeks post-transplant demonstrated that the PPT1 enzyme was replaced by HuCNS-SCs. HuCNS-SCs had migrated in a site-specific manner throughout the mouse brain and caused a reduction (in both number and intensity) in lysosomal storage material associated with disease pathology, compared with non-transplanted, age-matched controls [570810], [654206]. Further follow-up data showed the cells to engraft and secrete enzymes for up to 6 months. The brains of treated mice expressed signs of decreased lysosomal storage material in a number of cerebral regions (p < 0.0001). HuCNS-SCs demonstrated dosedependent neuroprotection (33 and 54% of neurons being scored as normal following lowand high-dose transplantation, respectively, compared with 8% in control). Enzyme assays revealed that the high dose of HuCNS-SCs caused a significant increase in enzyme levels 160 to 190 days after transplantation, to a level that was thought to be above the assumed threshold level for symptomatic disease in humans. These data indicate that grafted HuCNSSCs survive robustly in diseased and injured brains, migrate to the site of degeneration where they differentiate into neural lineages, and have a beneficial effect on functional recovery [633798], [654204].

4. METABOLISM AND PHARMACOKINETICS No data are currently available.

5. TOXICITY There were no signs of tumor formation at one year post-injection of HuCNS-SCs (105 or 106 cells) in NOD/SCID mice [437290]. No further data are currently available on the toxicity of HuCNS-SCs in animal models.

6. CLINICAL DEVELOPMENT In January 2005, StemCells filed an investigational new drug (IND) application for a phase I clinical trial in infantile and late-infantile BD [578153], but in February 2005, the Food and Drug Administration (FDA) placed the IND on hold while it raised a number of queries with StemCells regarding the trial [582789]. This application was the first time that the FDA had been asked to review a proposed clinical trial involving the use of purified human NSCs as a potential therapeutic agent. Later in the year, StemCells submitted

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amendments to the IND, including plans for patient committal to a four-year follow-up study, and the trial was approved by the FDA in October 2005 [629732]. The open-label, phase I/II trial is planned to enroll patients in the advanced stage of infantile BD, or late-infantile disease, and will evaluate two doses of HuCNS-SCs, which will be injected directly into the brain. The primary objective of the trial is to measure the safety (adverse effects, brain magnetic resonance imaging) of HuCNS-SCs, and to evaluate the ability of HuCNS-SCs to affect disease progression (cognition, communication, behavior, motor function) and quality of life (disability, behavior, communication, general health, seizures). Patients are expected to undergo continuous immunosuppression [578153], [633798].

7. SIDE EFFECTS AND CONTRAINDICATIONS A major potential flaw in the future development and therapeutic use of HuCNS-SCs is the risk of immune rejection because the stem cells are derived from fetal donors. This risk has not been addressed in the studies reported to date relating to the use of HuCNS-SCs. Allografts require immunosuppressive treatments, such as cyclosporine, to prevent host rejection. These treatments are not very well tolerated by patients, producing side effects such as renal dysfunction, tremor and hypertension. This may well affect the potential benefits of HuCNS-SC therapy.

8. PATENT SUMMARY The culture system used by StemCells to develop HuCNS-SC therapy was first disclosed in 1993 in the patent application WO-09301275, assigned to S. Weiss and B.A. Reynolds, the founders of NeuroSpheres. Several additional patent applications have been filed by NeuroSpheres, including WO-09409119, claiming a method to remyelinate neurons using NSCs; WO-09513364, disclosing methods for inducing proliferation and/or differentiation of neural progenitor cells both in vivo and in vitro; WO-09410292, disclosing methods for proliferating and differentiating NSCs into astrocytes, oligodendrocytes or neurons; US05750376, covering the in vitro growth and proliferation of genetically modified NSCs; and WO-09615226, covering the regulation of NSC proliferation. CytoTherapeutics filed two patent applications covering the use of NSCs. WO-09911758 disclosed the isolation, characterization, proliferation, differentiation and transplantation of mammalian NSCs, and WO-00050572 covers a method for the in vitro proliferation of NSC cultures using a growth factor and a collagenase enzyme said to increase cell viability and the number of proliferative cells with time. StemCells have filed WO-2004020597 disclosing methods for identifying, isolating and enriching CNS stem and progenitor cell populations. The patent covers methods that utilize reagents, such as immunoglobulins, which bind cell-surface markers, such as CD49f, CD133 and CD15. StemCells claims to hold 42 patents in the US and 108 patents worldwide (“in 14 foreign equivalent cases”), with a further 80 applications pending as of October 2005 [www.stemcellsinc.com].

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9. CURRENT OPINION The preclinical data reported to date show that grafted HuCNS-SCs can survive in damaged brain tissue and migrate to the specific site of degeneration, where they differentiate into neural lineages, and have a beneficial effect on functional recovery. The current evidence emphasizes the favorable therapeutic potential of HuCNS-SC therapy. However, additional in vivo and preliminary clinical studies are necessary to obtain a better understanding of the mechanisms of regeneration and recovery, and to further evaluate the potential therapeutic benefit of HuCNS-SCs. Although the data shows the potential and promise of HuCNS-SC therapy for the treatment of CNS diseases and injuries, there is one major concern: the risk of tissue rejection of the fetally derived cells in allograft transplantation. Preventing tissue rejection would require either genetically matching the donor with recipient, or administering life-long immunosuppressive treatments, such as cyclosporine. On one hand, optimal donor-recipient matching would require establishing HuCNS-SC banks, representing both a technical challenge and an ethical hurdle because of the stringent regulations governing the use of fetal tissues for therapeutic research. On the other hand, the toxicity of immunosuppressive drugs is well established, thereby limiting the use of HuCNS-SCs for therapy. There is also one unknown parameter regarding the use of HuCNS-SCs for therapy that has not been addressed in preclinical studies so far. Most of the studies were performed in immunosuppressed rats or NOD/SCID mice. Therefore, the activity of cytokines and chemokines of the immune system and their potential risks on HuCNS-SC therapy have not been fully investigated. Stem cells, including neurospheres, respond to the expression and activities of cytokines and chemokines. These activities may have adverse effects on the survival, migration and differentiation of HuCNS-SCs and thus affect their potential therapeutic use. Nonetheless, a range of studies has confirmed the potential of NSC for cellular therapy in the CNS (as reviewed in [243564], [284931]). The mechanisms underlying the recovery of neural progenitor and stem cells after grafting are yet to be fully characterized. The synthesis and release of neuroprotective substances by the grafted neural progenitor and stem cells have been proposed as a likely mechanism of the functional recovery [647946], [647954], but the recent of remyelination by HuCNS-SCs in both spinal cord injury NOD/SCID mice and myelin-deficient shiverer mice demonstrated that grafted neural progenitor and stem cells can also contribute to the recovery by their integration in the CNS network [623960••]. Both of these properties may be beneficial for the treatment of the diseased and injured brain, and particularly for neurodegenerative diseases such as BD. Results also demonstrate how neural progenitor and stem cells migrate toward the site of injury and degeneration when transplanted in the CNS (administered either by systemic injection, or through the cerebrospinal fluid), making them particularly suitable for the treatment of neurodegenerative diseases where the degeneration is widespread, such as in BD, Alzheimer's disease and Huntington's disease [654686], [654690]. More specifically, the recovery of myelination within animal models suggests a possible treatment for multiple sclerosis or periventricular leukomalacia (PVL), a kind of cerebral palsy caused by errors in myelination [654691].

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Neural stem and progenitor cells can also be genetically modified [437418], [US05750376], extending their potential use for the treatment of neurological diseases caused by genetic deficiencies, but also to promote neuronal survival in neurodegenerative diseases. Genetically modified neural progenitor and stem cells have been proposed for the treatment of type A Niemann Pick disease, a lysosomal storage disorder in which deficiency of acid sphingomyelinase leads to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes [647908]. Genetically engineered neural progenitor and stem cells expressing acid sphingomyelinase have been reported to reverse lysosomal storage pathology in animal models of Niemann Pick disease [647919], confirming the potential of NSCs to serve as a gene transfer vehicle for the treatment of CNS pathology, and particularly for lysosomal storage diseases. Such a strategy may be applicable to BD, by genetically engineering neural progenitor and stem cells to express PPT1 [570810]. These observations demonstrate that there may be various alternative treatments for the diseased and injured brain using NSC therapy. With the recent confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS, new opportunities for cellular therapy in the CNS are being considered [437208], [647230]. Cellular therapeutic intervention may involve the stimulation of endogenous progenitor cells, or the transplantation of neural progenitor and stem cells derived from the adult brain. Neural progenitor and stem cells may also be isolated from biopsies and post-mortem tissues, providing multiple sources of tissues for therapy [437394•], [647233]. Such strategies carry a lot of hope for the treatment of CNS diseases and injuries, and may provide an alternative to the use of fetally derived neural progenitor and stem cells, thus lacking the ethical and political constraints associated with fetally derived tissues. There are, however, several questions that need to be addressed before NSC technology can be brought to therapy: how will the selective differentiation of NSCs toward the desired phenotype(s), particularly in an environment that may not be favorable, be controlled, directed and optimized? Will the new neuronal cells establish the right CNS connections? What are the chances that they will establish connections with the wrong target cells? A process that maintains the developmental potential of NSCs will have to be devised and validated to produce the large quantities of NSCs required for therapy. NSC therapy still needs further characterization, particularly with regard to the relationship of these cells with tumor cells, before any broader evaluations of its therapeutic use for treating neurodegenerative diseases can be started.

10. LICENSING Ciba-Geigy AG By 1995, Ciba-Geigy (now Novartis) and NeuroSpheres had entered into a collaborative research agreement for the treatment of CNS disorders with stem cells; however, no development has been reported on the project by Ciba-Geigy since then [200025].

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StemCells, Inc. In March 1994, CytoTherapeutics, Inc. (now StemCells) entered into a research and license agreement with NeuroSpheres for the development of therapeutic products involving encapsulated and non-encapsulated neural stem cells. NeuroSpheres granted CytoTherapeutics an exclusive, worldwide, royalty-bearing license to develop and sell products involving the transplantation of neural stem cells developed by NeuroSpheres [180367]. This agreement was further clarified in April 1997, whereby CytoTherapeutics obtained an exclusive patent license from NeuroSpheres in the field of neural stem-cell transplantation. NeuroSpheres had an option to buy a non-exclusive license which was not exercised. An additional agreement was signed with NeuroSpheres in October 2000, for an exclusive license to non-transplant uses of the cells. Upfront payments to NeuroSpheres of 65,000 shares of CytoTherapeutics common stock were made in October 2000, and US $50,000 in January 2001. In October 2000, CytoTherapeutics reimbursed NeuroSpheres for patent costs amounting to $341,000. Annual payments of $50,000 a year were to be made to NeuroSpheres beginning in 2004 [560410].

11. DEVELOPMENT HISTORY In September 2003, StemCells was awarded a 1-year, US $0.34 million SBIR grant from the National Institute of Neurological Disease and Stroke to further work in the treatment of spinal cord injuries [504337]. One year later, the National Institutes of Health awarded two grants focusing on the use of StemCells' human NSCs. The company was awarded a Small Business Technology Transfer grant of $0.47 million for studies in Alzheimer's disease, to be conducted by the McLaughlin Research Institute, and the Reeve-Irvine Center at the University of California Irvine received a multiyear grant of $1.4 million to fund studies on human CNS stem-cell grafts in the treatment of spinal cord injuries [559872]. In August 2005, StemCells received a manufacturing license for its cell processing facility in California, allowing the company to conduct clinical trials of HuCNS-SCs [620544]. Developer

Country

Status

Indication

Date

Reference

StemCells, Inc.

US

Phase I

Lyosome storage disease

09-MAR-06

654808

StemCells, Inc.

US

Discovery

Central nervous system disease

14-JUL-95

180367

StemCells, Inc.

US

Discovery

Spinal cord injury

09-JUN-04

540074

NeuroSpheres, Ltd

Canada

Discontinued

Neuro-degenerative disease

01-APR-97

560410

Ciba-Geigy AG

Switzerland

No development reported

Central nervous system disease

25-SEP-95

–

HuCNS-SC Stem Cells

53

12. LITERATURE CLASSIFICATIONS Chemistry Study type HuCNS-SC isolation and differentiation.

Result Antibody-sorted cells expressing the stem-cell marker CD133 increased ~ 1000-fold in population after five passages. CD133 cells retained their ability to re-initiate neurosphere formation when plated in differentiating media

Reference 437290

Study type In vivo

Effect Studied Engraftment, proliferation, migration and differentiation

Model HuCNS-SCs (105 or 106 cells) were transplanted into the brains of NOD/SCID mice

Result HuCNS-SCs expressed potent engraftment, proliferation, migration and neural, differentiation, for at least 7 months posttransplantation

Reference 437290

In vivo

Survival, migration and differentiation

Three deposits of 105 HuCNS-SCs were transplanted into the ischemic cortex of rats 7 up to 1 days after MCAO

Analysis at 4 weeks showed that the lesion size was negatively correlated to cell survival. The cells migrated mm more than control towards the lesion, mainly differentiating to the neuronal phenotype

647228

Ex vivo

Locomotor recovery and cell migration

Four injections of 35 HuCNS-SCs were injected into spinal cord injured NOD/SCID mice 9 days after contusion

HuCNS-SC engraftment was associated with loco-motor recovery at 16 weeks ost-treatment. By 17 weeks post-treatment, cells had migrated days after con-tusion away from the lesion epicenter, in sagittal sections, with some transplanted cells found > 1 cm from the lesion epicenter

623960••

Ex vivo

MBP production

HuCNS-SCs were injected into myelindeficient shiverer mice

HuCNS-SCs largely differentiated into oligodendrocytes through a continuous process of differentiation lasting ~ 45 daysuntil full maturation and MBPproduction. By the end of the study (60 days), all grafted whitematter tracts in the cerebellum, fimbria and corpus callosum demonstrated dense MBP staining. The oligodendrocytes enrobed murine axons with 10 to 16 layers of myelin

636193

Ex vivo

Enzyme production

HuCNS-SCs cocultured with fibroblasts from BD individuals were injected into a mouse model (PPT1 knockout of BD

HuCNS-SCs engrafted and secrete enzymes for up to 6 months. Enzyme assays revealed that the high dosesof HuCNS-SC caused a significant increase in enzyme levels160 to 190days after transplantation. The brains of treated mice demonstrated signs of decreased lysosomal storage material and increased neuroprotection

633798

Biology

Philippe Taupin

54

13. ASSOCIATED PATENT Title Novel growth factor-responsive progenitor cells which can be proliferated (in vitro). Assignee Individual Publication WO-09301275 21-JAN-93 Priority US-1990726812 08-JUL-91 Inventors Weiss S, Reynolds BA.

ACKNOWLEDGMENTS Reproduced with permission from The Thomson Corporation and Taupin P: HuCNS-SC (StemCells). Current Opinion in Molecular Therapeutics (2006) 8(2): 156-63. Copyright 2006, The Thomson Corporation.

REFERENCES •• of outstanding interest • of special interest [180367] Annual Report 1994 – CytoTherapeutics, Inc. CytoTherapeutics, Inc. ANNUAL REPORT 1994 December 31. [199982] Transplantation of polymer-encapsulated cells genetically modified to secrete human nerve growth factor prevents the loss of degenerating cholinergic neurons in rats and nonhuman primates. Emerich, D.F., Winn, S.R., Hammang, J.P., Kordower, J.H., Frydel, B.R., Baetge, E.E. (1994). Cell Transplant, 3 3 212. [199983] Implants of polymer-encapsulated human NGF-secreting cells in the nonhuman primate: Rescue and sprouting of degenerating cholinergic basal forebrain neurons. Emerich, D.F., Winn, S.R., Harper, J., Hammang, J.P., Baetge, E.E., Kordower, J.H. (1994). J Comp Neurol, 349 (1): 148-164. [200019] Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Reynolds, B.A., Weiss, S. (1992). Science, 255 5052 1707-1710. •• This study reports the first isolation and characterization of neural progenitor and stem cells from the adult brain. Neural progenitor and stem cells were isolated from mouse striatal tissue containing the subventricular zone (a neurogeneic area of the adult brain) and cultured in vitro in the presence of epidermal growth factor. [200025] Does Ciba need a clearer strategic direction? Marketletter, 1995, September 25. [200021] Cell-specific expression of reporter genes in EGF-responsive neural stem cells derived from transgenic mice. Hammang, J.P., Duncan, I.D., Messing, A. (1994). Cell Transplantation, 3 (3): 230. [200023] EGF-responsive neural stem cells isolated from rat and mouse brain are capable of differentiating into oligodendrocytes and of forming myelin following transplantation into the myelin deficient rat. Duncan, I.D., Archer, D.R., Hammang, J.P. (1993). Abstr Soc Neurosci, 19 (1-3): Abs 689.

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[225755] Polymer-encapsulated genetically modified cells continue to secrete human nerve growth factor for over one year in rat ventricles: Behavioral and anatomical consequences. Winn, S.R., Lindner, M.D., Lee, A., Haggett, G., Francis, J.M., Emerich, D.F. (1996). Exp Neurol , 140 (2): 126-138. [243557] Progeny of long-term mouse and human CNS stem cell cultures survive transplantation and differentiate into neurons and glia. Borg, L.L., Vescovi, A.L., Blote, K., Kyle, A.L., Hettiaratchi, P., Reynolds, B.A., Mudrick-Donnon, L.A. (1996). Abstr Soc Neurosci, 22 (1-3): 47. [243558] Transplantation of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice attenuates excitotoxic striatal lesions. Carpenter, M.K., Winkler, C., Fricker, R., Wong, S.C., Greco, C., Emerich, D., Chen, E.Y., Chu, Y., Kordower, J., Messing, A., Bjorklund, A., Hammang, J.P. (1996). Abstr Soc Neurosci, 22 (1-3): 577. [243560] Long-term expression of nerve growth factor from polymer encapsulated cells in the rat CNS. Hammang, J.P., Dean, B.J., Lee, A., Emerich, D.F., Winn, S.R., Bamber, B., Palmiter, R.D., Baetge, E.E. (1993). Abstr Soc Neurosci, 19 (1-3): 657. [243561] The delivery of neurotrophic factors to the nervous system using polymer encapsulated cells. Baetge, E.E., Winn, S.R., Lee, A., Dean, B.J., Bamber, B., Palmiter, R.D., Hammang, J.P. (1993). Abstr Soc Neurosci , 19 (1-3): 657. [243562] Delivery of a putative Parkinson's factor (GDNF) into the rat CNS using a polymerencapsulated cell line. Baetge, E.E., Emerich, D.F., Winn, S.R., Lee, A., Lindner, M.D., Hammang, J.P. (1993). Mol Biol Cell , 4 Suppl 442A. [243563] EGF-responsive neural stem cells derived from MBP-lacZ transgenic mice can express the transgene in differentiating oligodendrocytes. Hammang, J.P., Wrabetz, L.G., Kamholz, J., Messing, A. (1993). Mol Biol Cell , 4 Suppl 374A. [243564] Neural stem cells for CNS transplantation. Baetge, E.E. (1993). Ann Ny Acad Sci, 695 285-291. [284931] Treatment of neurodegenerative diseases with neural cell transplantation. Dinsmore, J.H. (1998). Expert Opin Invest Drugs , 7 (4): 527-534. [437208] Mammalian neural stem cells. Gage, F.H. (2000). Science, 287 (5457): 1433-1438. [437290] Direct isolation of human central nervous system stem cells. Uchida, N., Buck, D.W., He, D., Reitsma, M.J., Masek, M., Phan, T.V., Tsukamoto, A.S., Gage, F.H., Weissman, I.L. (2000). Proc Natl Acad Sci, USA , 97 (26): 14720-14725. [437374] In vitro expansion of a multipotent population of human neural progenitor cells. Carpenter, M.K., Cui, X., Hu, Z.Y., Jackson, J., Sherman, S., Seiger, A., Wahlberg, L.U. (1999). Exp Neurol, 158 (2 ): 265-278. [437394] Cell culture. Progenitor cells from human brain after death. Palmer, T.D., Schwartz, P.H., Taupin, P., Kaspar, B., Stein, S.A., Gage, F.H. (2001). Nature , 411 (6833): 42-43. • This study reports the isolation and characterization of neural progenitor and stem cells from biopsies and post-mortem human brain tissues. Neural progenitor and stem cells were isolated and cultured in vitro in the presence of fibroblast growth factor, platelet derived growth factor, and the NSC factor CCg. [437418] Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Gage, F.H., Coates, P.W., Palmer, T.D., Kuhn, H.G., Fisher, L.J., Suhonen, J.O., Peterson, D.A., Suhr, S.T., Ray, J. (1995). Proc Natl Acad Sci, USA, 92 (25): 11879-11883.

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[468477] Reisolation of continuously regenerating human neural stem/progenitor AC133+ cells from transplanted mouse brain. Uchida, N., He, D., Buck, D., Reitsma, M., Masek, M., Phan, T., Tsukamoto, A., Weissman, I., Gage, R. (2000). Abstr Soc Neurosci, 26 (12): 209.9 [485519] StemsCells reports promising preclinical results in NCL StemCells, Inc. StemCells, Inc. PRESS RELEASE 2003 April 11. [504337] StemCells Inc receives grant to continue development of human neural stem cells as treatment for spinal cord injuries. StemCells, Inc. PRESS RELEASE 2003 September 08. [540074] StemCells, Inc. INT BIOTECHNOL CONV & EXHIB 2004 June 6-9 . [559872] NIH award grants for studies involving StemCell's neural stem cells. StemCells, Inc. PRESS RELEASE 2004 September 20. [560410] Form 10-K – StemCells, Inc. – for the fiscal year ended December 31, 2003. StemCells, Inc. FORM 10-K 2004 April 06. [570810] Stem Cells and Regenerative Medicine – SRI's Fourth Annual Meeting, Commercial Implications for the Pharmaceutical and Biotech Industries, Princeton, NJ, USA. IDDB MEETING REPORT 2004 October 18-19. [578153] StemCells files IND to begin Batten disease stem cell study. StemCells, Inc. PRESS RELEASE 2005 January 04. [582789] FDA and StemCells to discuss phase I Batten's disease trial. StemCells, Inc. PRESS RELEASE 2005 February 01. [620544] StemCells obtains license for cell processing facility. StemCells, Inc. PRESS RELEASE 2005 August 31. [623960] Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Cummings, B.J., Uchida, N., Tamaki, S.J., Salazar, D.L., Hooshmand, M., Summers, R., Gage, F.H., Anderson, A.J. (2005). Proc Natl Acad Sci, USA, 10239 14069-14074. •• The release of trophic factors by grafted neural progenitor and stem cells and the interaction of these cells with the injured brain are believed to play a major role in the recovery process after transplantation. In this study, where human neural progenitor and stem cells were injected into the spinal cord after injury in mice, the locomotor recovery disappeared following treatment with diphtheria toxin (diphtheria toxin kills only human cells, not mouse cells), suggesting that the grafted cells themselves are responsible for recovery. Administration of NSC might not simply stimulate the body to produce some healing factors, but also directly contribute to repair the damages themselves. [629732] StemCells's phase I Batten disease study gets FDA approval. StemCells, Inc. PRESS RELEASE 2005 October 20. [633798] Stem Cells and Regenerative Medicine – SRI's Fifth Annual Conference, Pittsburgh, PA, USA, 17-18 October 2005. Rippon HJ IDDB MEETING REPORT 2005 October 17-18. [636193] Temporal and sequential development of human oligodendrocytes from transplanted hcns-sc. Uchida, N., Capela, A., Cummings, B., Dohse, M., Anderson, A., Gage, F., Back, S., Tsukamoto, A., Tamaki, S. (2005). Abstr Soc Neurosci , 35 718.2. [636344] Quantification of engraftment and differentiation of human central nervous system stem cells (HCNS-SC) upon transplantation into the NODSCID mouse brain. Capela, A.,

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Dohse, M., Jacobs, Y., Reitsma, M., Tamaki, S., Tsukamoto, A., Uchida, N. (2005). Abstr Soc Neurosci, 35 Abs 827.15. [647207] Embryonic stem cells: Prospects for developmental biology and cell therapy. Wobus, A.M., Boheler, K.R. (2005). Physiol Rev , 85 (2): 635-678. [647217] AC133, a novel marker for human hematopoietic stem and progenitor cells. Yin, A.H., Miraglia, S., Zanjani, E.D., Almeida-Porada, G., Ogawa, M., Leary, A.G., Olweus, J., Kearney, J., Buck, D.W. (1997). Blood, 90 (12): 5002-5012. [647222] Engraftment of sorted/expanded human central nervous system stem cells from fetal brain. Tamaki, S., Eckert, K., He, D., Sutton, R., Doshe, M., Jain, G., Tushinski, R., Reitsma, M., Harris, B., Tsukamoto, A., Gage, F., Weissman, I., Uchida, N. (2002). J Neurosci Res, 69 (6): 976-986. [647228] Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Kelly, S., Bliss, T.M., Shah, A.K., Sun, G.H., Ma, M., Foo, W.C., Masel, J., Yenari, M.A., Weissman, I.L., Uchida, N., Palmer, T., Steinberg, G.K. (2004). Proc Natl Acad Sci, USA 101 (32): 11839-11844. [647230] Adult neurogenesis and neural stem cells of the central nervous system in mammals. Taupin, P., Gage, F.H. (2002). J Neurosci Res, 69 (6): 745749. [647233] FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. Taupin, P., Ray, J., Fischer, W.H., Suhr, S.T., Hakansson, K., Grubb, A., Gage, F.H. (2000). Neuron , 28 (2): 385-397. [647908] Niemann-Pick disease. Kolodny, E.H. (2000). Curr Opin Hematol , 7 1 48-52. [647919] Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology. Shihabuddin, L.S., Numan, S., Huff, M.R., Dodge, J.C., Clarke, J., Macauley, S.L., Yang, W., Taksir, T.V., Parsons, G., Passini, M.A., Gage, F.H., Stewart, G.R. (2004). J Neurosci, 24 (47): 10642-10651. [647946] Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Lu, P., Jones, L.L., Snyder, E.Y., Tuszynski, M.H. (2003). Exp Neurol, 181 (2): 115-129. [647954] Differentiation and tropic/trophic effects of exogenous neural precursors in the adult spinal cord. Yan, J., Welsh, A.M., Bora, S.H., Snyder, E.Y., Koliatsos, V.E. (2004). J Comp Neurol, 480 (1): 101-114. [647967] Neurodegenerative disease: The neuronal ceroid lipofuscinoses (Batten disease). Mitchison, H.M., Mole, S.E. (2001). Curr Opin Neurol, 14 (6): 795-803. [647969] Disruption of PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice. Gupta, P., Soyombo, A.A., Atashband, A., Wisniewski, K.E., Shelton, J.M., Richardson, J.A., Hammer, R.E., Hofmann, S.L. (2001). Proc Natl Acad Sci , USA, 98 (24): 13566-13571. [654203] Human fetal CNS stem cell neurospheres survive, migrate and differentiate following transplantation into ischemic rat cerebral cortex. Kelly, S., Bliss, T.M., Shah, A.K., Sun, G., Puja, K., Ma, M., Foo, W., Masel, J., Shallert, T., Yenari, M.A., Uchida, N., Palmer, T., Steinberg, G.K. (2003). Abstr Soc Neurosci, 844.9. [654204] Use of human central nervous system stem cells for neurodegenerative disease and CNS injury. Tamaki, S., Capela, A., Dohse, M., Eckert, K., He, D., Tushinski, R., Tsukamoto, A., Basu, S., Belichenko, P., Mobley, W., Cummings, B., Anderson, A., Uchida, N. (2003). Abstr Soc Neurosci , 656.4.

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[654205] Transplantation of human central nervous system stem cells into a NOD –SCID mouse model of spinal cord contusion injury. Cummings, B.J., Uchida, N., Salazar, D.L., Tamaki, S.J., Dohse, M., Tushinski, R., Tsukamoto, A.S., Anderson, A.J. (2003). Abstr Soc Neurosci, 415.3. [654206] Brain transplantation of human neural stem cells in mouse model of Batten's disease. Basu, S.B., Belichenko, P.V., Uchida, N., Tamaki, S., Eckert, K., Udani, V., Tsukamoto, A., Mobley, W.C. (2003). Abstr Soc Neurosci , 335.7. [654686] Resolution of stroke deficits following contralateral grafts of conditionally immortal neuroepithelial stem cells. Veizovic, T., Beech, J.S., Stroemer, R.P., Watson ,W.P., Hodges, H. (2001). Stroke, 32 (4): 1012-1029. [654690] Immunohistochemical and electron microscopic study of invasion and differentiation in spinal cord lesion of neural stem cells grafted through cerebrospinal fluid in rat. Wu, S., Suzuki, Y., Noda, T., Bai, H., Kitada, M., Kataoka, K., Nishimura, Y., Ide, C. (2002). J Neurosci Res, 69 (6): 940-945. [654691] Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani ,G., Dina, G., Galli, R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi, A.L., Martino, G. (2003). Nature, 422 (6933): 688-694. [654808] StemCells, Inc. receives IRB approval from Oregon Health & Science University to begin phase I clinical trial in Batten disease; first ever clinical trial using purified human neural stem cells. StemCells, Inc. PRESS RELEASE 2006 March 09. [655176] Survival and differentiation of neural progenitor cells derived from embryonic stem cells and transplanted into ischemic brain. Takagi, Y., Nishimura, M., Morizane, A., Takahashi, J., Nozaki, K., Hayashi, J., Hashimoto, N. (2005). J Neurosurg, 103 (2): 304310.

Chapter VI

OTI-010 OSIRIS THERAPEUTICS/JCR PHARMACEUTICALS ABSTRACT Osiris Therapeutics is developing the donor-derived mesenchymal stem cell (MSC) therapy OTI-010, which repopulates the bone marrow stroma and thus supports engraftment of hematopoietic stem cells from the same donor. This stem cell therapy, which has been awarded Orphan Drug status, is currently in development for the potential enhancement of bone marrow transplants in cancer patients, for the prevention of graftversus-host disease (GVHD), and for the treatment of Crohn's disease. Japanese licensee JCR Pharmaceuticals is investigating the therapy for the potential treatment of GVHD in patients undergoing bone marrow transplantation to treat leukemia. Phase II clinical trials in acute gastrointestinal GVHD and in adult and pediatric patients with treatmentrefractory severe GVHD are currently underway.

1. INTRODUCTION Graft-versus-host disease (GVHD) is the immunological damage and the associated consequences incurred by the introduction of immunologically competent cells into an immunocompromised host. The host must possess important transplant alloantigens that are lacking in the donor graft such that the host appears foreign to the grafted tissue [659758]. In GVHD, the donor's immune cells, particularly the alloreactive donor T-cells, recognize the patient's tissue as foreign and produce antibodies against them, attacking the patient's vital organs – the so-called graft-versus-host reaction. GVHD is also characterized by an increase in the secretion of pro-inflammatory cytokines (e.g., tumor necrosis factor (TNF)a, interferon (IFN), interleukin (IL)-1, IL-2 and IL-12) and the activation of dendritic cells (DC), macrophages, natural killer (NK) cells and cytotoxic T-cells that contribute to more inflammatory reactions within the patient [656535]. Patients are at risk of developing GVHD while undergoing any of the following procedures: bone marrow (BM) transplantation, peripheral blood progenitor (PBP) or hematopoietic stem cell (HSC) transplantation, transfusion of unirradiated blood products (transfusion-associated GVHD) or transplantation of solid organs containing

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immunologically competent cells (particularly organs containing lymphoid tissue), as well as maternal-fetal transfusions [662457]. GVHD is most commonly associated with BM transplants, which are performed following damage to the host's BM resulting from the administration of high-dose chemotherapy and/or radiation to treat certain types of leukemia and other cancers that invade the lymphatic system. During this treatment, drugs that suppress the host's immune system and permit the new donor BM to engraft without being destroyed by the host's immune system are also administered. BM transplants are also performed to treat non-malignant conditions, such as sickle cell anemia [659326]. The disparity of human leukocyte antigen (HLA) is one of the most important factors correlating with the incidence and severity of GVHD [659330], [659361]. Other major factors identified as predictors of GVHD include age and mismatches of minor histocompatibility antigens in HLA-matched transplants [659372], [659376], [659385], [659743]. In the majority of cases, most recipients undergo allogeneic BM transplants in which a genetically matched donor (usually a close family member or occasionally someone from outside the family) can be found. Only occasionally are autologous transplants performed in which the patient is given back his or her own marrow once it has been purged of malignant cells. Although autologous transplantation is associated with fewer serious side effects compared with allogeneic transplants, it can be less effective in treating certain kinds of cancer [662480]. GVHD is fatal in 50 to 80% of patients administered a HLA-matched BM transplant [570810]. GVHD is split into two forms: acute and chronic. GVHD is classified as acute if it occurs before day 100 post-transplant, and chronic if it persists or develops beyond day 100. The symptoms of acute GVHD consist of dermatitis, enteritis and hepatitis, whereas chronic GVHD is an autoimmune-like syndrome that leads to the impairment of multiple organs or organ systems [662462]. The Center for International Bone Marrow Transplant Registry (CIBMTR) has adopted a new severity index for grading acute GVHD after allogeneic marrow transplantation [658988]. Acute GVHD is graded in five steps (0 to IV) based on observable symptoms in the skin, liver and gastrointestinal tract. Grade 0 GVHD indicates no clinical evidence of disease and grades I to IV correlate with the degree of severity of the symptoms. Grade I GVHD represents only mild symptoms, characterized by rash over less than 50% of skin and no liver or gut involvement. Grade IV GVHD indicates severe symptoms characterized by erythroderma with bullous formation, bilirubin (> 3 mg/dl) or diarrhea (> 16 ml/kg/day) [659392]. Chronic GVHD is characterized by similar symptoms to those associated with autoimmune disease and can be classified as limited-chronic or extensive-chronic GVHD. The symptoms of limited chronic GVHD are localized skin involvement and/or hepatic dysfunction. The symptoms of extensive chronic GVHD are generalized or localized skin involvement and/or hepatic dysfunction, liver histology showing chronic aggressive hepatitis, bridging necrosis or cirrhosis, eye involvement (Schirmer test < 5 mm wetting) and involvement of any other target organ. The survival rate for acute GVHD grades 0 to I is 90%, compared with 60% at grades II to III, and only 20 to 50% at grade IV GVHD. The survival rate after onset of chronic GVHD is approximately 42%. A patient with one or more symptoms has a projected six-year survival rate of 60%; however, survivors are often left severely disabled [www.nih.gov]. In milder forms, the effects of GVHD may induce some benefit for the patient, particularly for cancer patients. While attacking the host tissues, the graft-derived lymphocytes also attack the cancer cells that may still be present after the transplantation

OTI-010 Osiris Therapeutics/JCR Pharmaceuticals

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therapy. This factor is believed to be the reason why allogeneic transplants are successful in curing certain cancers, particularly some forms of leukemia. This effect is known as the graftversus-tumor effect or graft-versus-leukemia effect [656537]. Successful strategies in treating each type of GVHD, such as more precise HLA matching, can significantly reduce the onset of disease. Selective T-cell depletion of the BM is another strategy used to decrease the risk of GVHD. However, this procedure can raise the risk of graft failure, infection and relapse. In addition, T-cell depletion can reduce the beneficial graft-versus-malignancy effect. The use of umbilical cord blood cells has also been proposed for use in hematopoietic cell transplantation. As a consequence of the immunological immaturity of the T-cells in umbilical cord blood, transplants using this source of cells have demonstrated a reduced incidence and severity of GVHD [658608], [658612]. The same is not true for the use of peripheral blood stem cells [659491]. If acute GVHD does develop after transplantation, glucocorticoids (such as methylprednisolone or prednisone) in combination with immunosuppressive drugs (such as cyclosporine) are administered. As a result of these advances in treatment, the incidence of grade II to IV acute GVHD after allogeneic-related transplants decreased from 45% in 1976 to < 30% in 2001 [www.nih.gov]. New drugs and strategies are currently being considered that can supplement already devised protocols. Among them are the development of various immunosuppressive treatments, new drugs, and monoclonal and anticytokine antibodies [659498], [659503], [659504]. Such treatments include the nucleoside analog pentostatin, which is a potent inhibitor of adenosine deaminase [659504], [659506], and denileukin diftitox, a recombinant protein composed of IL-2 fused to diphtheria toxin, which has a high affinity for IL-2receptor-positive activated T-cells [659532]. Other treatments include humanized monoclonal antibodies to TNFa (infliximab) and IL-2 (daclizumab). Mesenchymal stem cells (MSCs) are multipotent cells that contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose and marrow stroma [394517], [656539]. MSCs represent an important cellular component of the BM microenvironment [656703] and can be easily isolated from the adult BM stroma, where they represent a rare population of the cells (estimated at 0.001 to 0.01% of the nucleated cells, ~ 10-fold less abundant than HSCs) [658632], [658640]. MSCs have also been found in umbilical cord blood, but not peripheral blood [658631]. Once isolated, MSCs can be expanded in culture through many generations, producing billions of MSCs for cellular therapy [656543]. MSCs have been extensively studied in vitro, ex vivo, and in animal and human models [656703], [658632], [658640], [658995]. In vitro, MSCs have been shown to secrete hematopoietic cytokines and support hematopoietic progenitors [643706]. MSCs are not immunogenic and escape recognition by alloreactive T-cells and NK cells. Human MSCs (hMSCs) and their lineage derivatives express intermediate levels of HLA major histocompatibility complex (MHC) class I, and do not express co-stimulatory molecules B71, B7-2, CD40 or CD40 ligand [656546], [656548]. These features support the lack of immunogenic recognition of these cells within the host, making hMSCs an ideal candidate for cellular therapy, whereas mismatched allogeneic transplantation is one of the main limitations of therapy. MSCs have also demonstrated immunomodulary properties in vitro and in vivo; they are immunosuppressive and inhibit the proliferation of alloreactive T-cells [643694••], [656550], [656553], [656697], [658625]. It is because of these properties that MSC transplantation has been proposed for the treatment of GVHD, particularly in BM and HSC

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transplantation, and for the treatment of inflammatory and autoimmune disorders, such as Crohn's disease (CD) [659005•]. CD is an inflammatory disorder of the digestive tract. The disorder shares many symptoms with another inflammatory condition, ulcerative colitis, and the two are often referred to under the more generalized term “inflammatory bowel disease.” CD causes swelling in the intestines that extends deep into the lining of the affected organ, as well as pain, and can make the intestines empty frequently, resulting in diarrhea. CD is also associated with complications, such as blockage of the intestine, fistula, abscess, narrowing or obstruction of the bowel, nutritional complications, arthritis, and skin problems. The disease is characterized by active periods, known as flare-ups, followed by periods of remission, during which symptoms diminish or disappear altogether. CD can occur in people of all age groups, but it is more often diagnosed in people between the ages of 20 and 30, and seems to run in some families. The cause of CD is unknown, and there is currently no cure for the disease. Current treatments consist of drug therapy (antiinflammatories, cortocosteroids, immunosuppressive drugs, antibiotics and anti-diarrheal drugs), nutritional supplements, and surgery to control inflammation, correct nutritional deficiencies and relieve symptoms, or to correct complications, such as blockage, perforation, abscess or bleeding in the intestine. It is estimated that up to 75% of people who live with CD may require surgery at some point to treat a complication of the disease. Some treatments, such as anti-inflammatory drugs, also have side effects, including vomiting, heartburn, diarrhea, and greater susceptibility to infection [www.nih.gov]. Osiris Therapeutics, Inc. aims to take advantage of the properties of MSC to develop a donor-derived cellular therapy, known as OTI-010, for the potential peripheral blood support of bone marrow transplants in cancer patients, the reduction of GVHD, and treatment of CD [495015], [582522].

2. SYNTHESIS AND SAR As MHC compatibility is not necessary for MSC therapy, a parent or HLA-identical sibling may represent potential donors [659018••], [659019•]. Standard protocols for the isolation and culture of hMSCs have been previously reported [394517], [656543], [656553]. Osiris has developed protocols to isolate and culture hMSCs. MSCs are isolated from the BM of healthy adult donor volunteers (aged 18 to 45 years), and expanded until the number of adult stem cells has increased over 3000-fold. The resulting product, OTI-010, is then frozen and stored until the stem cells are required for therapy [327297], [327352]. One process developed by Osiris for the purification of hMSC magnetic bead-based selection used an antifibroblast antibody that gained about 100-fold enrichment of MSCs. The fibroblast-enriched cell fractions were then further purified by flow cytometry using antibodies for the markers CD45 and CD73 prior to expansion [350813]. The specific protocol used by Osiris for OTI-010 isolation and expansion was outlined in the paper by Aggarwal & Pittenger [643694]. Briefly, BM aspirates (10 ml) were combined with Dulbecco phosphate-buffered saline (DPBS, 40 ml) and centrifuged at 900 g for 10 min at 20°C. The cells were then resuspended and gently layered onto a Percoll cushion (density = 1.073 g/ml) at 1 to 3 × 108 nucleated cells/25 ml. The low-density hMSC-enriched

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mononuclear fraction was collected, washed with 25 ml DPBS and centrifuged to collect the cells. Cells were then resuspended in hMSC complete culture medium (Dulbecco-modified Eagle medium containing low glucose, 10% fetal bovine serum, antibiotic/antimycotic and glutamax), and plated at 3 × 107 cells/185 cm2. The cultures were maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2, and passaged prior to confluency [643694••]. The hMSCs expanded in culture showed positive surface staining for markers such as CD166, CD105, CD73, CD44 and CD29, but lacked markers such as CD34 and CD45, which are typical hematopoietic and endothelial markers [643688], [643702], [658214]. MSC have an adherent, fibroblastic phenotype and can be expanded in monolayer culture through many generations, producing billions of MSCs for cellular therapy [656543]. Culture quality was ensured on the basis of optimum MSCs growth with maximum retention of osteogenic, chondrogenic and adipogenic differentiation [394517], [656543]. OTI-010 is administered as an intravenous formulation, which was identified as the most efficient method of stem cell host integration by Liu, et al., who studied the effect of route of administration on hMSC-dependent engraftment of CD34 cells [656701]. In two separate experiments, 0.5 × 106 CD34 cells were infused with 2 × 104 hMSC either intramuscularly or intravenously into NOD/SCID mice. Flow cytometry analysis demonstrated two-fold fewer human CD45 cells present in the mice receiving intramuscular hMSCs. Examination of the BM of the mice showed 33% ± 4.5 of the cells to be positive for human CD45 after intravenous infusion, compared with 18% ± 2.5 following intramuscular injection [656701].

3. PRECLINICAL DEVELOPMENT Osiris has investigated MSCs isolated from various sources both in vitro and in vivo.

3.1. In Vitro Aggarwal & Pittenger studied the effects of hMSCs in modulating allogeneic immune cell responses [643694••]. When hMSCs were incubated with DC, a more than 50% decrease in TNFa secretion and > 50% increase in IL-10 secretion were observed. It is thought that this change may affect the maturation state and functional properties of the DC, leading to a skewing of the immune response to a more anti-inflammatory/tolerant phenotype. When incubated with naïve T-cells, hMSCs decreased IFN secretion by T-helper (Th)1 and NK cells and increased IL-4 secretion by Th2 cells. Studies also demonstrated that hMSCs increase the expression of IL-6 and IL-8, vascular endothelial growth factor (VEGF) and prostaglandin (PG)E2 in vitro. Each of these factors was found to increase by > 3-fold upon coculture with hPBMC for 24 hours. The observed inhibition of pro-inflammatory cytokines could help to reduce the severity and incidence of GVHD [643694••], [656697]. Another study looked at the relationship between selected markers and hMSC-mediated immunosuppression in vitro. It was found that co-culture of hMSC with anti-CD3/CD28 activated peripheral blood mononuclear cells (hPBMCs) caused inhibition of lymphocyte proliferation. The effect of hMSCs on lymphocyte proliferation was dose dependent, causing more than 50% inhibition at an approximately 1:10 to 1:25 MSC:lymphocyte ratio. Five-day

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studies showed how increasing MSCs increased levels of PGE2 and reduced levels of TNFa and tryptophan, which was related to the enzyme activity of indoleamine 2,3-dioxygenase (IDO). This correlation between PGE2 secretion and IDO enzyme activity demonstrated the ability of hMSC to inhibit lymphocyte proliferation in vitro. This study identifies molecules that are functionally related to the immunological responses of hMSCs, which may be important in the treatment of GVHD and other autoimmune diseases [656696]. Osiris reported that MSC are able to survive, differentiate and produce hematopoietic cytokines when treated for 24 h with chemotherapeutic agents, such as methotrexate (0.75 or 450 mg/kg) or cisplatin (1 mg/kg), but not doxorubicin (1 mg/kg), suggesting that MSC could be used during the treatment of certain types of cancer [433228]. Majumdar, et al. studied the role of MSCs as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro by outlining the phenotypic differences between MSCs and marrow-derived stromal cells (MDSCs). Flow cytometric analysis showed that MSCs are distinct from MDSC cultures and are a homogeneous cell population devoid of hematopoietic cells [656703].

3.2. In Vivo Fischer rat MSCs were infused intraportally into the livers of other Fischer rats (2 million cells/rat). Cells persisted for at least 28 days, being localized to the portal triad regions. In a second study, a different set of Fischer rats, either previously injected with MSCs or not, received intraportal infusions of an allogeneic ß-cell line (Rin-m, RT1g). After 2 weeks, rats that had been injected with both Fischer MSCs and Rin-m cells demonstrated an association of the different cell types with each other near portal triads. In contrast, in animals in which only Rin-m cells had been injected, there was no observation of these cells under the confocal microscope, indicating that the cells had been recognized and cleared by the immune system. These findings indicate how the suppressive properties of MSCs could be utilized to protect cell allografts from rejection after transplantation into the liver [643859]. Another study in Fischer rats demonstrated that MSCs do not elicit an immune response after transplantation in immunocompetent recipients. Syngeneic Fischer MSCs or allogeneic ACI MSCs were implanted via an osteoconductive matrix into the bilateral femoral gap of Fischer rats who were then sacrificed at 3, 6 and 12 weeks post-implantation (n = 4 per time point). Histological analysis showed there was no difference between the syngeneic or allogeneic implants. Allogeneic implants did not induce significant inflammatory cell infiltration or stimulate alloreactive T-cell responses [656702]. The ability of MSCs to induce tissue regeneration without an immune response was investigated in a goat model of meniscal injury. Donor MSCs were injected into the damaged meniscus in the knee joint on days 7, 14 and 21 (10 million MSCs per injection). Ex vivo examinations showed that MSCs suppressed T-cell alloreactivity selectively, maintaining some types of T-cell responses [656698].

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4. METABOLISM AND PHARMACOKINETICS No data are currently available.

5. TOXICITY There was no evidence of a localized inflammatory response as a result of infused MSCs in rats [643859]. No further data are currently available on the toxicity of OTI-010 in animal models.

6. CLINICAL DEVELOPMENT 6.1. GVHD In October 2004, clinical data from a phase I safety trial of OTI-010 therapy for the treatment of GVHD were reported. Infusion of hMSCs decreased GVHD in 22 to 45% of patients, suggesting that OTI-010 reduces the numbers of pro-inflammatory cytokines and Tcells while increasing levels of anti-inflammatory cytokines in these patients [570810]. In October 2005, Osiris received approval from the US Food and Drug Administration (FDA) to conduct a non-randomized, open-label, phase II clinical trial in adult and pediatric patients with treatment-refractory severe GVHD [628422]. By November 2005 the trial had begun, enrolling 30 patients and setting a planned completion date of January 2007. At the same time, a second phase II, double-blind, randomized, placebo-controlled clinical trial, expected to enroll 75 patients to assess the safety and efficacy of OTI-010 in acute gastrointestinal GVHD, was initiated by Osiris, with an expected completion date of April 2008 [www.clinicaltrials.gov], [632720].

6.2. Transplantation in Cancer Patients In June 1999, phase I clinical trials of OTI-010 were initiated in cancer patients receivingchemotherapy and HSC transplantation for the treatment of high-risk hematological malignancies (including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia and non-Hodgkin's lymphoma). The multicenter studies, conducted in seven US and European cancer centers, showed OTI-010 to be safe and efficacious, with 52% of patients (~ 40 patients) responding positively to the therapy [327297], [495015]. In October 2000, Osiris stated that it had completed enrollment in a phase I/II clinical trial of OTI-010 for autologous transplantation in breast cancer patients [386589]. However, data from the trial are yet to be published.

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6.3. Crohn's Disease In December 2005, Osiris began a phase II clinical study to assess the ability of OTI-010 to reduce inflammation and repair damaged tissue in patients with moderate to severe CD unresponsive to steroids and other immunosuppressants [639559]. In the phase II, randomized, open-label clinical study, OTI-010 will be infused into an expected 12 patients on two separate days, seven to 10 days apart [www.clinicaltrials.gov].

7. SIDE EFFECTS AND CONTRAINDICATIONS Osiris has not reported any significant side effects from trials to date, although published data is limited. Reports from other studies of intravenously administered MSCs, either after HSC transplantation or co-transplantation with HSCs, show that MSC therapy is well tolerated by patients [659018••], [659019•].

8. PATENT SUMMARY The patent application WO-09222584, by inventors A. Caplan and S.E. Haynesworth and assigned to Osiris in 1992, discloses a process for producing monoclonal antibodies that are specific to marrow-derived MSC. Osiris has filed several other patent applications relating to OTI-010, including WO-09639487 (published in 1996), which claims compositions and methods for maintaining and expanding the viability of human mesenchymal precursor cells in a serum-free environment. WO-09623058 discloses methods and preparations for enhancing BM engraftment in an individual by administering a culturally expanded MSC preparation. WO-09739104 claims a method for cryopreservation of an isolated, homogeneous population of viable hMSCs obtained from periosteum, BM, cord blood, peripheral blood, dermis, muscle, or other known sources of MSCs. WO-09901145 details processes for recovering peripheral blood containing a population of hMSCs from an individual and preserving these ex vivo. WO-09947163 discloses processes for MSCs to effectively reduce or inhibit host rejection following transplantation. Also disclosed is a method of inhibiting an immune response against a host by foreign tissue, for example GVHD, by treatment with MSCs. WO-09946366 states methods and preparations for using non-autologous MSCs for treating and regenerating connective tissue and enhancing bone marrow engraftment in an individual, and WO-2005093044 claims a method of promoting angiogenesis by MSCs in an organ or tissue other than the heart.

9. CURRENT OPINION MSCs offer several advantages for the treatment of GVHD and inflammatory and immune disorders. The ease of isolation, in vitro expansion, genetic stability, and ability to escape alloimmunity, make MSCs a model of choice for cellular therapy and also for gene

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therapy [643707], [658995]. MSCs also have immunosuppressive properties, making them a candidate for the treatment of GVHD, particularly after allogeneic stem cell transplantation. Co-administration of MSCs and HSCs has been proposed for the treatment of GVHD, and preliminary reports indicate that this therapy is well tolerated by patients and effectively reduces chronic and acute GVHD symptoms [659018••], [659019•]. In one study of haploidentical MSCs by a different research team to Osiris, a nine-year-old boy with acute lymphoblastic leukemia who received a transplant of blood stem cells from an HLA identical, unrelated donor after irradiation, developed grade IV acute GVHD of the gut and liver. This patient received MSC therapy (the mother being chosen as the donor), and over the following days and weeks the frequency of diarrhea fell, a decline in total bilirubin was noted, the patient resumed eating, and DNA analysis showed the presence of minimal residual disease. As a result, immunosuppressive treatment was discontinued and healing of the colon epithelium was detected. One year after transplantation, the patient was again living a normal life [659018••]. Furthermore, as the beneficial effect of GVHD in cancer treatment may be preserved by discontinuing immunosuppressive treatment, MSC therapy represents a valid candidate for the potential treatment of GVHD, although more studies are required to confirm that it is well tolerated by patients, and to optimize therapy protocols. A particularly contentious issue in need of further research is whether MSCs survive long after therapy, or deplete over time. Although ultimately the transplanted HSCs contribute to the graft chimerism in successful transplantations, monitoring of long-term integration and survival of MSCs in the recipients will provide valuable information regarding the long-term beneficial effects of MSC therapy. The immunosuppressive properties of MSCs may underlie the speed of recovery after allogeneic stem cell transplantation. It is proposed that the immunoregulatory activity of the MSCs on the host tissue would suppress the incidence and severity of GVHD. This is achieved predominantly by altering the cytokine secretion profile of the host immune cells, DCs, T-cells and NK cells, inducing a shift from a pro-inflammatory environment toward an antiinflammatory or tolerant cell environment (decreased TNFa and IFN. secretion and increased IL-10 and IL-4 secretion). The potential advantageous consequences of this change could include inflammation modulation, tolerance induction and reduction of transplantation complications, such as rejection and GVHD [643694••], [656553]. Characterization of the mechanisms underlying the immunomodulatory activity of MSCs is the subject of intense studies, and contradictory data have been reported [659005••], [659032]. Understanding such mechanisms will lead to the development of more potent, efficient and safer drug therapies for GVHD. There are, however, several limitations and contraindications with MSC therapy. One of the limitations is in the establishment of MSC cultures from donors, parents or siblings. Although MSC can be readily isolated and expanded in vitro, establishing the MSC cultures can be a limiting factor. Failure to establish such cell cultures in a timely manner, and in sufficient qualities and quantities, may have profound consequences on the therapy. Since MHC compatibility is not necessary for MSC immunosuppression, and because MSC immunomodulatory activity can also be observed in third-party donor cells [656550], MSCs derived from unrelated, MHC-unmatched, healthy, third-party donors would represent a “universal donor MSC product” with the advantage of being a readily-available product that may provide an opportunity for multiple and higher MSC doses, potentially at a reduced cost [659019•].

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There are also risks associated with the long-term expansion of stem cells in culture, such as transformation and aneuploidy. Such modifications of the intrinsic properties of the cells can potentially lead to the development of malignancy in the patient following transplantation. In the protocol developed by Osiris, MSCs are expanded until the number of adult stem cells has increased over 3000-fold [327297], [327352]. Recent studies have reported that MSCs expanded in culture have an intrinsic propensity towards spontaneous transformation after a certain number of population doublings (~ 250) [660856], [660857], [660858], [660859]. Therefore, careful consideration must be taken to ensure that the patients are not at risk of developing tumors after the stem cell treatment. The potential benefits of MSC therapy may also be limited in patients suffering from certain disorders, such as arthritis. In a preclinical model of collagen-induced arthritis, it was reported that MSC therapy did not confer any benefit in the treatment of arthritis, indicating a possible contraindication of the therapy [660860]. Although it is not yet known whether such observations would translate into treatment in humans, these data highlight the need to perform careful examinations of the inflammatory environment before considering MSC therapy. Osiris overcomes the ethical, health and practical concerns that hamper the development of other stem cell products because of its stem cell source and the great care taken to ensure safety and quality of the material. Stem cell donors are monitored for up to five years after donation to ensure their health status. This is the primary reason that Osiris has progressed into the human clinical trial phase faster than any other stem cell company [www.stemcellsinc.com]. Other strategies are currently being investigated for the treatment of GVHD after allogeneic transplantation. Among them, umbilical cord blood transplantation has been reported to restore hematopoiesis after myeloablative therapy in children and in adults [658608]. Because of the immunological immaturity of the T-cells in umbilical cord blood, transplants using this source of cells have a reduced incidence and severity of GVHD, making it a promising strategy for the treatment of leukemia or other cancers after chemotherapy or radiation [658612]. In conclusion, MSC therapy represents a valid strategy for the prophylaxis and treatment of GVHD. The immunosuppressive and immunomodulatory properties of MSCs seem to play a critical role in reducing acute and chronic GVHD. It may also be beneficial for the treatment of inflammatory and autoimmune diseases and disorders, and to prevent organtransplant rejection [659005•]. OTI-010 appears to be a promising candidate for treatment of serious diseases such as CD and the treatment of GVHD after BM transplantation in cancer patients. This belief has been backed up by the FDA, which granted OTI-010 Fast Track status for the treatment of GVHD in January 2005, making this treatment the first stem cell therapy to be granted this status by the FDA. By December 2005, the therapy had also been granted Orphan Drug status for acute GVHD, adding to support for the development of OTI010 [582522], [642237]. The results of forthcoming clinical trials are widely anticipated, and future research will be aimed at optimizing MSC therapy and developing a universal donor MSC product to combat major autoimmune disorders.

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10. LICENSING JCR Pharmaceuticals Co. Ltd. In August 2003, Osiris licensed to JCR Pharmaceuticals the exclusive Japanese rights to its universal, adult MSC technology for use in conjunction with the treatment for hematologic malignancies using hematopoietic stem cell transplants [502952]. In August 2005, this agreement was clarified as refering to GVHD in patients undergoing BM transplantation to treat leukemia. By that time, Osiris had received a research milestone payment from JCR [616286].

11. DEVELOPMENT HISTORY

Developer

Country

Status

Indication

Date

Reference

Osiris Therapeutics, Inc.

US

Phase II

Bone marrow transplantation

23-OCT-00

386589

Osiris Therapeutics, Inc.

US

Phase II

Graft-versushost disease

12-OCT-05

628422

Osiris Therapeutics, Inc.

US

Phase II

Crohns disease

08-DEC-05

639559

JCR Pharmaceuticals Co. Ltd.

Japan

Discovery

Bone marrow transplantation

27-AUG-03

502952

JCR Pharmaceuticals Co. Ltd.

Japan

Discovery

Graft-versushost disease

27-AUG-03

502952

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70

12. LITERATURE CLASSIFICATIONS Chemistry Study type Stem cell isolation and expansion

Result BM aspirates were combined with DPBS and centrifuged several times at different density gradients to collect hMSCs. Cells were then resuspended in hMSC complete culture medium and plated at 3 × 107 cells/185 cm2. The cultures were maintained at 37°C in a humidified atmosphere before being passaged prior to confluency.

Reference 643694••

Study type In vitro

Effect Studied Modulation of allogeneic immune cell responses by hMSC

Model hMSCs incubated with DCs and naïve T-cells

Result When incubated with DC a > 50% decrease in TNFα secretion and a > 50% increase in IL-10 secretion were observed. hMSC decreased IFN. Secretion by Th1 and NK cells and increased IL-4 secretion by Th2 cells when incubated with naïve T-cells

Reference 643694••

In vitro

Relationship between selected markers and hMSCmediated immunosuppression HMSC engraftment and migration

Co-culture of hMSCs with anti CD3/CD28activated hPBMCs

A correlation was observed between PGE2 secretion and IDO enzyme activity on the inhibition of lymphocyte proliferation by hMSC Results showed cells to persist for at least 28 days, being localized to the portal triad regions

656696

Immune response

Donor MSCs were MSC injected into the damaged meniscus in the knee joint of goats on days 7, 14 and 21 (10 million MSC per injection)

Suppressed T-cell alloreactivity selectively, maintaining some types of T-cell responses

656698

Biology

In vivo

Ex vivo

Fischer rat MSCs were infused intraportally into the livers of other Fischer rats (2 million cells/rat)

643859

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13. ASSOCIATED PATENT Title Enhancing bone marrow engraftment using mesenchymal stem cells (MSCs). Assignee Case Western Reserve University Publication 01-AUG-96 Priority US-1995 377771 24-JAN-94 Inventors Haynesworth SE, Caplan AI, Arnold I, Gerson SL, Lazarus HM.

ACKNOWLEDGMENTS Reproduced with permission from The Thomson Corporation and Taupin P: OTI-010 Osiris Therapeutics/JCR Pharmaceuticals. Current Opinion Investigational Drugs (2006) 7(5): 473-81. Copyright 2006, The Thomson Corporation.

REFERENCES [327297] Osiris Therapeutics initiates clinical trial using donor-derived adult stem cells Osiris Therapeutics, Inc. PRESS RELEASE 1999 June 08. [327352] Cell therapy program Osiris Therapeutics, Inc. COMPANY WORLD WIDE WEB SITE 1999 June 09. [350813] Phenotype of human mesenchymal stem cells isolated directly from bone marrow Davis-Sproul, J., McNeil, R., Simonetti, D., Craig, S., Moseley, A., Deans, R., Moorman, M. (1999). Blood, 94 10 Suppl 1 Abs 3905. [386589] Research and clinical development programs Osiris Therapeutics, Inc. Company World Wide Web Site. 2000, October 23. [394517] Multilineage potential of adult human mesenchymal stem cells Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R. (1999). Science, 284 (5411): 143-147. [433228] Effects of chemotherapeutic agents on the proliferation, differentiation, and stromal support capacity of human mesenchymal stem cells Archambault, M.P., Howard, L., Butterfield, A.M., Peter, S.J. (2001). Blood, 98 11 Suppl 2 Abs 4235. [495015] BIO 2003 – International Biotechnology Convention and Exhibition (Part VII), Washington, DC, USA, 21-25 June, 2003, Aird J Iddb Meeting Report, 2003, June 21-25. [502952] Osiris licenses stem cell technology to JCR Pharmaceuticals. Osiris Therapeutics, Inc. Press Release , 2003 August 28. [570810] Stem cells and regenerative medicine - SRI’s Fourth Annual Meeting: Commercial implications for the pharmaceutical and biotechIndustries, Princeton, NJ, USA, 18-19 October 2004 IDdb author IDDB MEETING REPORT 2004 October 18-19. [582522] FDA grants Fast Track status to Osiris’s GvHD stem cell therapy Osiris Therapeutics, Inc. PRESS RELEASE 2005 January 31. [616286] Osiris and JCR Pharmaceuticals reach milestone, expand licenseagreement: Companies to expand stem cell technology into a new market opportunity Osiris Therapeutics, Inc. PRESS RELEASE 2005 August 04.

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[628422] New stem cell treatment being evaluated for critically ill bonemarrow transplant patients; osiris launches a phase II clinical trial for severe graft vs host disease Osiris Therapeutics, Inc. PRESS RELEASE 2005 October 12. 632720 Osiris’s heart attack stem cell therapy safe in phase I Osiris Therapeutics, Inc. PRESS RELEASE 2005 November 04. [639559] Osiris starts phase II stem cell trial in Crohn’s disease Osiris Therapeutics, Inc. PRESS RELEASE 2005 December 08. [642237] Osiris’s stem cells get US Orphan status for GvHD Osiris Therapeutics, Inc. PRESS RELEASE 2005 December 20. [643688] The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Barry, F.P., Boynton, R.E., Haynesworth, S., Murphy, J.M., Zaia, J. (1999). Biochem Biophys Res Commun, 265 (1) 134-139. [643694] Human mesenchymal stem cells modulate allogeneic immune cell responses Aggarwal, S., Pittenger, M.F. BLOOD 2005 (105) 4: 1815-1822. •• In this study, the authors examined the immunomodulary functions of human MSCs by coculturing them with purified subpopulations of immune cells, and report that hMSCs alter the cytokine secretion profile of the host immune cells to induce a more antiinflammatory phenotype. The authors discuss and propose mechanisms underlying the immunomodulary functions of hMSCs in allogeneic transplantation. A shift from a proinflammatory environment toward an anti-inflammatory or tolerant cell environment would result in inflammation modulation, tolerance induction and reduction of allogeneic transplantation complications. [643702] The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells Barry, F., Boynton, R., Murphy, M., Zaia, J. (2001). Biochem Biophys Res Commun, 289 (2): 519-524. [643706] Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages Majumdar, M.K., Thiede, M.A., Haynesworth, S.E., Bruder, S.P., Gerson, S.L. (2000). J Hematother Stem Cell Res, 9 (6): 841-848. [643707] Mesenchymal stem cells as vehicles for gene delivery Mosca JD, Hendricks JK, Buyaner, D., Davis-Sproul, J., Chuang, L.C., Majumdar, M.K., Chopra, R., Barry, F., Murphy, M., Thiede, M. A., Junker, U., Rigg, R.J., Forestell, S.P., Bohnlein, E., Storb, R., Sandmaier, B.M. (2000). Clin Orthop, 379 Suppl S71-S90. [643859] Intra-portal infusion of adult mesenchymal stem cells enhances survival of allogeneic beta cells in immunocompetent rats Archambault, M.P., Campbell, S.E., Vanguri, P., McIntosh, K.R. (2002). Blood, 100 11 Abs 2404. [656535] Cytolytic pathways in haematopoietic stem-cell transplantation van den Brink, M.R., Burakoff, S.J. (2002). Nat Rev Immunol, 2 (4): 273-281. [656537] Allogeneic hematopoietic cell transplantation as consolidation immunotherapy of cancer after autologous transplantation Maris, M.B., Storb, R. (2005). Acta Haematol, 114 (4): 221-229. [656539] Mesenchymal stem cells Caplan, A.I. (1991). J Orthop Res, 9 (5): 641-650. [656543] Characterization of cells with osteogenic potential from human marrow Haynesworth, S.E., Goshima, J., Goldberg, V.M., Caplan, A.I. (1992). Bone, 13 (1): 8188.

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[656546] HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells Le Blanc, K., Tammik, C., Rosendahl, K., Zetterberg, E., Ringden, O. (2003). Exp Hematol, 31 (10): 890-896. [656548] Characterization and functionality of cell surface molecules on human mesenchymal stem cells Majumdar, M.K., Keane-Moore, M., Buyaner, D., Hardy, W.B., Moorman, M.A., McIntosh, K.R., Mosca, J.D. (2003). J Biomed Sci, 10 (2): 228-241. [656550] Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo Bartholomew, A., Sturgeon, C., Siatskas, M., Ferrer, K., McIntosh, K., Patil, S., Hardy, W., Devine, S., Ucker, D., Deans, R., Moseley, A., Hoffman, R. (2002). Exp Hematol, 30 (1): 42-48. [656553] Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex Le Blanc, K., Tammik, L., Sundberg, B., Haynesworth, S.E., Ringden, O. (2003). Scand J Immunol, 57 (1): 11-20. [656696] Characterization of MSC potential to treat GVHD using molecular markers linked to MSC-mediated immunosuppression in vitro Carter, D., Tyrell, A., Bubnic, S., Marcelino, M., Kedzierski, K., Monroy, R., Mills, R., Danilkovitch, A. (2005). Blood, 106 (11). [656697] Modulation of immune cell responses by human mesenchymal stem cells Aggarwal, S., Pittenger, M.F. (2004). Blood, 104 11 Abs 1288. [656698] Evidence for selective suppression of alloreactivity by mesenchymal stem cells Beggs, K.J., Kavalkovitch, K.W., Borneman, J.N., Murphy, M.J., Barry, F.P., Stewart, M.G., Proctor, R.L., McIntosh, K.R. (2001). Blood, 98 11 Abs 2719 [656701] Mode of delivery of human mesenchymal stem cells affects engraftment of human CD34+ cells in NOD/SCID mice Liu, L., Mbalaviele, G., Lee, K., Mosca, J., Deans, R. (2000). Blood, 96 11 Abs 3309. [656702] Allogeneic rat mesenchymal stem cells do not elicit an immune response after implantation in immunocompetent recipients Archambault, M.P., McIntosh, K.R., Duty, A., Peter, S.J. (2000). Blood, 96 11 Abs 3295. [656703] Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells Majumdar, M.K., Thiede, M.A., Mosca, J.D., Moorman, M., Gerson, S.L. (1998). J Cell Physiol, 176 1 57-66. [658214] Mesenchymal stem cells can be differentiated into endothelial cells in vitro Oswald, J., Boxberger, S., Jorgensen, B., Feldmann, S., Ehninger, G., Bornhauser, M., Werner, C. (2004). Stem Cells, 22 (3): 377-384. [658608] Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors Laughlin, M.J., Barker, J., Bambach, B., Koc, O.N., Rizzieri, D.A., Wagner, J.E., Gerson, S.L., Lazarus, H.M., Cairo, M., Stevens, C.E., Rubinstein, P., Kurtzberg, J. N. (2001). Engl J Med, 344 (24): 18151822. [658612] Unrelated donor hematopoietic cell transplantation: Marrow or umbilical cord blood? Grewal, S.S., Barker, J.N., Davies, S.M., Wagner, J.E. Blood, 2003 (101): 11 4233-4244. [658625] Mesenchymal stem cells avoid allogeneic rejection Ryan, J.M., Barry, F.P., Murphy, J.M., Mahon, B.P. (2005). J Inflamm, 2 8.

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[658631] Isolation of multipotent mesenchymal stem cells from umbilical cord blood Lee, O.K., Kuo, T.K., Chen, W.M., Lee, K.D., Hsieh, S.L., Chen, T.H. (2004). Blood, 103 (5): 1669-1675. [658632] Mesenchymal stem cells: Isolation, in vitro expansion and characterization Beyer, N.N., da Silva, M.L. (2006). Handb Exp Pharmacol, 174 249-282. [658640] Mesenchymal stem cells: Isolation and therapeutics Alhadlaq, A., Mao, J.J. (2004). Stem Cells Dev , 13 (4): 436-448. [658988] IBMTR Severity Index for grading acute graft-versus-host disease: Retrospective comparison with Glucksberg grade Rowlings, P.A., Przepiorka, D., Klein, J.P., Gale, R.P., Passweg, J.R., Henslee-Downey, P.J., Cahn, J.Y., Calderwood, S., Gratwohl ,A., Socie, G., Abecasis, M.M., Sobocinski, K.A., Zhang, M.J., Horowitz, M.M. (1997). Br J Haematol, 97 (4): 855-864. [658995] Mesenchymal stem cells and their potential as cardiac therapeutics Pittenger, M.F., Martin, B.J. (2004). Circ Res, 95 (1): 9-20. [659005] Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation Le Blanc, K., Ringden, O. (2005). Biol Blood Marrow Transplant , 11 (5): 321-334. • This paper shows that the potential of MSC therapy for the treatment of GVHD may also be beneficial for the treatment of other immune disorders, such as autoimmune diseases and to prevent organ-transplant rejection. [659018] Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells Le Blanc, K., Rasmusson, I., Sundberg, B., Gotherstrom, C., Hassan, M., Uzunel, M., Ringden, O. (2004). Lancet, 363 (9419): 1439-1441 •• Of 1000 allogeneic stem cell transplantations, 25 patients developed grade IV acute GVHD. The patient reported in this study is the only patient with such severe disease who, one year after the therapy, was living a normal life at home. The other 24 patients died a median of 2 months after transplantation. This case supports MSC therapy for prophylaxis and treatment of GVHD. [659019] Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients Lazarus, H.M., Koc, O.N., Devine, S.M., Curtin, P., Maziarz, R.T., Holland, H.K., Shpall, E.J., McCarthy, P., Atkinson, K., Cooper, B.W., Gerson, S.L., Laughlin, M.J., Loberiza, F.R. Jr., Moseley, A.B., Bacigalupo, A. (2005). Biol Blood Marrow Transplant, 11 (5): 389398 • This review discusses the need to develop a universal donor product for MSC therapy. Such product would present several advantages, such as being readily available, quality controlled, allowing the administration of higher doses of MSCs, and would reduce the cost of MSC therapy. [659032] Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms Rasmusson, I., Ringden, O., Sundberg, B., Le Blanc, K. (2005). Exp Cell Res, 305 (1): 33-41. [659326] Bone marrow transplantation for sickle cell anemia: Progress and prospects Iannone, R., Ohene-Frempong, K., Fuchs, E.J., Casella, J.F., Chen, A.R. (2005). Pediatr Blood Cancer, 44 (5): 436-440. [659330] Marrow transplantation from related donors other than HLA-identical siblings Beatty, P.G., Clift, R.A., Mickelson, E.M., Nisperos, B.B., Flournoy, N., Martin, P.J.,

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Sanders, J.E., Stewart, P., Buckner, C.D., Storb, R. et al (1986). N Engl J Med , 313 (13): 765-771. [659361] The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors Morishima, Y., Sasazuki, T., Inoko, H., Juji, T., Akaza, T., Yamamoto, K., Ishikawa, Y., Kato, S., Sao, H., Sakamaki, H., Kawa, K., Hamajima, N., Asano, S., Kodera, Y. (2002). Blood, 99 (11): 4200-4206. [659372] Risk factors for acute graft-versus-host disease in histocompatible donor bone marrow transplantation Weisdorf, D., Hakke, R., Blazar, B., Miller, W., McGlave, P., Ramsay, N., Kersey, J., Filipovich, A. (1991). Transplantation, 51 (6): 1197-1203. [659376] The role of minor histocompatibility antigens in GVHD and rejection: A minireview Goulmy, E., Voogt, P., van Els, C., de Bueger, M., van Rood, J. (1991). Bone Marrow Transplant, 7 Suppl 1 49-51. [659385] Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versushost disease after bone marrow transplantation Goulmy, E., Schipper, R., Pool, J., Blokland, E., Falkenburg, J.H., Vossen, J., Gratwohl, A., Vogelsang, G.B., van Houwelingen, H.C., van Rood, J.J. (1996). N Engl J Med, 334 (5): 281-285. [659392] Acute graft-versus-host disease: Pathophysiology, clinical manifestations, and management Couriel, D., Caldera, H., Champlin, R., Komanduri, K. (2004). Cancer, 101 (9): 1936-1946 [659491] Chronic graft-versus-host disease Horwitz, M.E., Sullivan, K.M. (2006). Blood Rev, 20 (1): 15-27 [659498] New strategies in the treatment of graft-versus-host disease Basara, N., Blau, I.W., Willenbacher, W., Kiehl, M.G., Fauser, A.A. (2000). Bone Marrow Transplant, 25 Suppl 2 S12-S15 [659503] Novel pharmacotherapeutic approaches to prevention and treatment of GVHD Jacobsohn, D.A., Vogelsang, G.B. (2002). Drugs, 62 (6): 879-889. [659504] Novel strategies for steroid-refractory acute graft-versus-host disease BolanosMeade, J., Vogelsang, G.B. (2005). Curr Opin Hematol, 12 (1): 40-44. [659506] Pentostatin for the treatment of chronic graft-versus-host disease in children Goldberg, J.D., Jacobsohn, D.A., Margolis, J., Chen, A.R., Anders, V., Phelps, M., Vogelsang, G.B. (2003). J Pediatr Hematol Oncol , 25 (7): 584-588. [659532] Safety and efficacy of denileukin diftitox in patients with steroid-refractory acute graft-versus-host disease after allogeneichematopoietic stem cell transplantation Ho, V.T., Zahrieh, D., Hochberg, E., Micale, E., Levin, J., Reynolds, C., Steckel, S., Cutler, C., Fisher, D.C., Lee, S.J., Alyea, E.P., Ritz, J., Soiffer, R.J., Antin, J.H. (2004). Blood, 104 (4): 1224-1226. [659743] Non-HLA immunogenetics in hematopoietic stem cell transplantation Dickinson, A.M., Charron, D. (2005). Curr Opin Immunol, 17 (5): 517-525. [659758] The biology of graft-versus-host reactions Billingham, R.E. Harvey Lectures : Delivered Under Auspices Harvey Soc, New York, 1966-1967 1966 (62): 21-78. [660856] Adult human mesenchymal stem cell as a target for neoplastic transformation Serakinci, N., Guldberg, P., Burns, J.S., Abdallah, B., Schrodder, H., Jensen, T., Kassem, M. (2004). Oncogene, 23 (29): 5095-5098.

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[660857] Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation Miura, M., Miura, Y., Padilla-Nash, H.M., Molinolo, A.A., Fu, B., Patel, V., Seo, B.M., Sonoyama, W., Zheng, J.J., Baker, C.C., Chen, W., Ried, T., Shi, S. (2006). Stem Cells, 24 (4): 1095-1103. [660858] Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells Burns, J.S., Abdallah, B.M., Guldberg, P., Rygaard, J., Schroder, H.D., Kassem, M.(2005). Cancer Res, 15 65 (8): 3126-3135. [660859] Spontaneous human adult stem cell transformation Rubio, D., Garcia-Castro, J., Martin, M.C., de la Fuente, R., Cigudosa, J.C., Lloyd, A.C., Bernad, A. (2005). Cancer Res, 65 (8): 3035-3039. [660860] Reversal of the immunosuppressive properties of mesenchymal stem cells by tumor necrosis factor alpha in collagen-induced arthritis Djouad, F., Fritz, V., Apparailly, F., Louis-Plence, P., Bony, C., Sany, J., Jorgensen, C., Noel, D. (2005). Arthritis Rheum, 52 (5): 1595-1603. [662462] Acute graft-versus-host disease Bolanos-Meade, J., Vogelsang, G.B. (2004). Clin Adv Hematol Oncol, 2 (10): 672-82. [662457] Transfusion-associated graft-versus-host disease: A serious residual risk of blood transfusion Higgins, M.J., Blackall, D.P. (2005). Curr Hematol Rep, 4 (6): 470-6. [662480] High-dose chemotherapy and autologous bone marrow or stem cell reconstitution for solid tumors McGuire, W.P. (1998). Curr Probl Cancer, 22 (3): 135-77.

Chapter VII

ADA-TRANSDUCED HEMATOPOIETIC STEM CELL THERAPY FOR ADA-SCID SUMMARY * Originator San Raffaele Telethon Institute for Gene Therapy * Status Phase II Clinical * Indication Combined immunodeficiency * Actions Adenosine deaminase stimulator, genetically engineered autologous cell therapy, immunomodulator, retrovirus-based gene therapy * Technologies Intravenous formulation, stem cell therapy

ABSTRACT San Raffaele Telethon Institute for Gene Therapy is developing an adenosine deaminase transduced hematopoietic stem cell therapy for the potential intravenous treatment of adenosine deaminase deficiency in severe combined immunocompromised individuals (ADA-SCID).

1. INTRODUCTION Adenosine deaminase (ADA) is a ubiquitous enzyme that is essential for the breakdown of the purine base adenosine, from both food intake and the turnover of nucleic acids. ADA hydrolyzes adenosine and deoxyadenosine into inosine and deoxyinosine, respectively, via the removal of an amino group. Deficiency of the ADA enzyme results in the build-up of deoxyadenosine and deoxyATP (adenosine triphosphate), both of which inhibit the normal maturation and survival of lymphocytes. Most importantly, these metabolites affect the ability of T-cells to differentiate into mature T-cells [656430], [666686]. ADA deficiency results in a form of severe combined immunodeficiency (SCID), known as ADA-SCID [467343].

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SCIDs are rare genetic failures in the development of the immune system that, if untreated, are fatal in the first few years of life because the body produces non-functional Bcells or is unable to produce enough B- and T-cells to resist infection [666542], [668402]. However, SCID can be successfully treated with bone marrow transplantation. In the US, approximately 40 to 100 infants are diagnosed with SCID each year, although the actual number of cases may be higher [http://www.genome.gov], [467343]. The most common form of SCID is X-linked SCID, which is reported to account for approximately 50 to 60% of all SCID cases [656430]. X-linked SCID is caused by a deficiency in the common gamma chain, a component of the interleukin (IL)-2 receptor. ADA deficiency is the most common form of autosomally inherited SCID, and accounts for approximately 10 to 20% of all SCID conditions that are diagnosed [656430], [666684]. In ADA-SCID, an accumulation of the purine-toxic metabolites leads not only to impaired lymphocyte development and function, but also to skeletal, liver, lung and neurological abnormalities, indicating that ADA-SCID is more complex than other forms of SCID [469995], [666603]. Allogeneic bone marrow (BM) transplantation and enzyme replacement therapy are currently the main treatments for ADA-SCID [666620], [666621], [666625], [666626]. Allogeneic BM transplantation is the treatment of choice if a human leukocyte antigen (HLA)-identical sibling donor is available, and can result in complete recovery for most patients [470001]. Enzyme replacement therapy is conducted using bovine ADA enzyme replacement therapy together with polyethylene glycol (PEG) [515137], [515240]. The addition of PEG to bovine ADA prevents the clearance of the enzyme from the circulation and prolongs the half-life of ADA in plasma [666662]. This treatment, known as PEG-ADA, allows for the survival and maintenance of lymphocytes. However, the high rate of mortality for BM transplantation in patients who lack a matching donor, as well as the constraints and limitations associated with enzyme replacement therapy (such as a variable degree of immune recovery, the expense of the therapy and the occurrence of neutralizing antibodies or autoimmunity), underline the need for the development of new treatments for ADA-SCID. As ADA-SCID results from the defect of only a single gene, the condition was considered to be an attractive candidate for early gene therapy trials [666664]. These trials were conducted using a patient's T-cells, and demonstrated that lymphocytes with a corrected version of the ADA-encoding gene had a survival advantage and also remained in peripheral blood (PB) for a prolonged period, suggesting that gene therapy was feasible. However, the therapeutic effect of this gene therapy was difficult to measure because of the concomitant administration of PEG-ADA in all patients. In addition, the PEG-ADA therapy may have abolished the potential selective advantage for gene-corrected cells over defected cells in the ADA-SCID patients [515240], [643374], [643375]. Despite these potential influences of PEG-ADA, investigators could not justify discontinuing PEG-ADA therapy because of the demonstrated benefits of the treatment. In 2002, the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) identified a patient for which the discontinuation of PEG-ADA provided clear benefits. The patient, who had been treated with gene-corrected PB lymphocytes (PBLs), exhibited a significant expansion of gene-corrected T-lymphocytes, which experienced a selective advantage in the absence of PEG-ADA. The patient also demonstrated improved immune function, and was able to form responses to T-cell-dependent antigens such as tetanus toxoid. This breakthrough in research indicated that gene therapy in ADA-SCID patients might be performed without

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the use of PEG-ADA therapy [515240]. However, despite the benefits of using PBLs in gene therapy, this method did not completely correct the ADA metabolic defect, and multiple PBL infusions were required [470017], [470024], [666662], [666664]. In order to achieve a life-long correction in ADA metabolism, gene therapy protocols were developed to correct hematopoietic stem cells (HSCs). These cells develop into lymphocytes and have the potential of self-renewal [467343], [666662], [666665]. The candidate HSCs that were isolated from patients were CD34+ antigen expressing cells, which were genetically modified to express the wild-type ADA gene and were then grafted back into the patients. However, the frequency of multipotent genetically modified HSCs and the levels of long-term transgene expression were variable, resulting in limited clinical success [666662], [666665]; this low success rate was blamed on a poor efficiency during the transfer of genes into HSCs [515130]. HSR-TIGET is aiming to improve current gene therapy protocols for ADA-SCID by using the approach of gene transfer into autologous HSCs combined with nonmyeloablative conditioning [515038], [515136], [607777]. Nonmyeloablative conditioning, prior to the transfer of gene-corrected HSCs, is believed to convey an advantage for the transduced cells by creating space in the BM [515130]. In 2002, Dr. Alessandro Aiuti and his collaborators reported a successful clinical trial that was performed with this approach on two ADA-SCID patients [515130]; by December 2005, the trial had been extended to six patients [668664]. In August 2005, HSR-TIGET was granted Orphan Drug status by the European Medicines Agency for autologous CD34+ cells transfected with a retroviral vector containing the ADA gene for the treatment of ADA-SCID [654032].

2. SYNTHESIS AND SAR To improve upon the efficiency of engrafting genetically corrected HSCs into ADASCID patients, HSR-TIGET developed a new protocol for gene transfer into HSCs associated with nonmyeloablative conditioning [515130]. Human CD34+ HSCs were removed from the BM of two ADA-SCID patients (4.15 × 106 and 1.08 × 106 cells/kg of body weight in patient [Pt]1 and 2, respectively) and were genetically engineered using a GIADAl retroviral vector to express ADA genes. The GIADAl retroviral vector was generated by cloning ADA complementary deoxyribonucleic acid (cDNA) into the LXSN vector (encoding the neomycin-resistance marker gene). The vector was then packaged into the amphotropic Gp+Am12 cell line, and recombinant retroviral particles were produced under optimized and clinically applicable conditions for CD34+ cells. After three rounds of gene transfer and 4 days of culture, the CD34+ cells were harvested, washed and intravenously infused back into the patients (Pt1 received 8.6 × 106 CD34+ cells/kg, containing 25% transduced colonyforming units in culture (CFU-C), and Pt2 received 0.9 × 106 CD34+ cells/kg, with 21% transduced CFU-C) [515130]. At days 3 and 2 prior to cell transplantation, both patients received nonmyeloablative conditioning with busulfan (administered intravenously to Pt1, and orally to Pt2) [515130]. As demonstrated by in vitro and in vivo assays (see [515130], [643373]), this protocol allowed for efficient transduction of HSCs, while preserving their differentiation capacity into multiple lineages, including myeloid cells, B- and T-cells and natural killer (NK) cells.

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The origination of this protocol was outlined in a study by Dando, et al. [470408], who measured the efficiency of gene transfer into CD34+ cells from mobilized PB after a single exposure to retroviral supernatant. The study determined that bulk gene transfer and endpoint titer values that were obtained for cell lines under different production conditions were not predictive of the efficacy of gene transfer into hematopoietic progenitor CD34+ cells. Rather, the duration of virus production appeared to have the greatest impact on gene transfer, with a peak gene transfer rate observed 6 hours after initiation, and a 2- to 3-fold decrease in rate occurring at longer timepoints. Neither the culture vessel that was used nor the temperature during virus production demonstrated any significant effect on gene transfer into CD34+ cells. Supernatant could also be produced under defined serum-free conditions as efficiently as under serum-containing conditions for CD34+ cell gene transfer [470408].

3. PRECLINICAL DEVELOPMENT HSR-TIGET hypothesized that the re-infusion of a cell population that was enriched in lymphoid progenitors and stem cells might favor immunoreconstitution and could contribute to the generation of a pool of long-term surviving lymphocytes to help combat ADA-SCID. To determine the optimal clinically applicable protocol for gene transfer into lymphoid progenitors from the BM of patients, studies examined the effects of different cytokines on the maintenance and gene transfer of BM CD34+ cells from ADA-SCID patients compared with healthy donors [515242], [643373]. An initial ex vivo study assessed whether the presence of increasing amounts of IL-3 (0 to 600 ng/ml) or IL-7 (0 to 200 ng/ml) in the minimum culture condition (including thrombopoietin/fms-related tyrosine kinase 3-ligand/stem cell factor combination of growth factors [T/F/S]) could increase gene transfer efficiency and proliferation of CD34+ cells. When purified BM CD34+ cells were isolated from healthy donors, a dose-dependent increase in the final yield of transduced CD34+ cells in the presence of IL-3 and, to a lesser extent, IL-7 was observed, reaching a plateau at 60 and 20 ng/ml, respectively. CD34+ cells were then removed from the BM of nine ADA-SCID patients and six healthy donors, and the effect of IL-3 and IL-7 at optimal doses on stem/progenitor cells was tested. The yields of transduced CD34+ cells, as a percentage of input, were 31 ± 35 and 24 ± 13% in ADA-SCID and normal BM, respectively. The presence of IL-3 significantly increased the proportion and final yield of CD34+ cells that expressed the transgene both in ADA-SCID and normal BM. The effect of IL-3 was more pronounced on cell yield than on gene transfer rate, suggesting that an increase in cell proliferation does not strictly correlate with an enhanced efficacy of gene transfer. In six of nine ADA-SCID patients, IL-7 improved the percentages and yields of transduced CD34+ cells, but overall the increase was not statistically significant [643373]. In in vitro studies, the use of IL-3 or IL-7 significantly improved the maintenance of Bcell progenitors from ADA-SCID BM cells. In ADA-SCID patients, the addition of IL-3 or IL-7 in a 4-day culture greatly improved the number of B-cell progenitors compared with the T/F/S combination of cytokines, resulting in a 6- and 5-fold increase, respectively, and reaching values that did not differ significantly from those of freshly isolated cells. No significant differences were observed with regard to the number of NK cell progenitors before or after culture. The effect of the different cytokine treatments was less pronounced in BM

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from healthy donors compared with BM from ADA-SCID patients. IL-3 and IL-7 allowed the efficient transduction of B- and NK cell progenitors; further tests outlined how the function of these cells had been maintained, suggesting that the in vitro culture and gene transfer did not interfere with normal cell development in vitro [643373]. To investigate whether the positive in vitro findings could be replicated in vivo, the engraftment and differentiation of CD34+ cells transduced into B- and T-lymphocytes in the SCID-hu mouse model were investigated. By 8 to 9 weeks after gene therapy, the transduced CD34+ cells had been efficiently engrafted into SCID-hu mice (~ 80% overall engraftment), giving rise to B- and T-cell progeny and demonstrating the maintenance of in vivo lymphoid reconstitution capacity [643373]. To assess if the grafted transduced HSCs retained their repopulation and differentiation properties, CD34+ cells were isolated at day +330 from the BM of an ADA-SCID patient (Pt1) who underwent the gene therapy procedure in a clinical trial (see below). In vitro, the lymphoid differentiation capacity of the CD34+ cells was maintained in a B-/NK cell differentiation assay with 4 and 9% of B- and NK cells containing transduced cells, respectively [515130]. A second study injected the CD34+ cells into the BM/thymus of SCID-hu mice to study their repopulation capacity, and analyzed them 8 weeks after. Using HLA-typing, the donor cells demonstrated engraftment ranging from 85 to 98%. Quantitative polymerase chain reaction (PCR) showed that between 0.3 to 15.2% of B-cells and 0.14 to 31.2% of T-cells were transduced, indicating that genetically corrected HSCs retained their ability to reconstitute lymphopoiesis in vitro and in vivo in a secondary transplant after infusion [515130], [643373].

4. METABOLISM AND PHARMACOKINETICS No data are currently available.

5. TOXICITY Gene transfer of mouse and human CD34+ HSCs, which were genetically modified by a retroviral virus encoding ADA cDNA and the neomycin-resistance marker gene, have been extensively characterized in mouse models without reports of toxicity. Furthermore, none of the preclinical studies for ADA-SCID have demonstrated evidence of cancer in animals treated with this same gene transfer approach [515137], [666652], [666655].

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6. CLINICAL DEVELOPMENT Phase I/II Using the optimal protocol combination of T/F/S and IL-3 that was developed from a series of preclinical studies (outlined above), autologous HSC gene therapy for ADA-SCID was performed in combination with nonmyeloablative conditioning (2 mg/kg/day of busulfan) in two patients (aged 7 months [Pt1], and two years and 6 months [Pt2], respectively) [515130], [515136], [607777]. After a transient myelosuppression (neutrophil nadir = 0.15 × 103 cells/µl on day +17 and 0.4 × 103 cells/µl on day +19 for Pt1 and Pt2, respectively; platelet nadir = 154 × 103 cells/µl on day +31 and 23 × 103 cells/µl on day +30 for Pt1 and Pt2, respectively), hematopoiesis was demonstrated to recover as expected, based on the measurement of days to an absolute neutrophil count = 500 cells/µl (Pt1, 22 days; Pt2, 21 days). In Pt1, who had a follow-up of 14 months, the number of PBLs increased from < 100 to 2000 per µl at day +150; this level was maintained throughout the follow-up period. Increases in B- and NK cells, followed by T-cells (+90 days), were also observed, with T-cells developing normally into both CD3+/CD4+ cells and CD3+/CD8+ subsets. A dramatic increase in CD4+/CD45RA+ naïve T-cells and T-cell receptor circles (TREC) in CD3+ cells indicated a restoration of thymic activity that was comparable to age-matched controls. Gene therapy led to a normalization of proliferative responses to polyclonal stimuli and to nominal antigens (Candida, tetanus toxoid). PCR heteroduplex analysis revealed a normal heterogenous pattern on the T-cell receptor variable ß-chain region. Serum immunoglobulin (Ig)M, IgG and IgA increased to normal levels, allowing for the discontinuation of intravenous Ig (IVIg) 6 months after gene therapy [515130]. In Pt2, who experienced a follow-up of 12 months, lymphocytes increased to 400 cells/µl, with slower kinetics than those observed in Pt1. The increase occurred mainly in the T-cell subset, as indicated by a significant rise in TREC levels. Pt2 also demonstrated a normalization of both Ig levels and the proliferative responses to polyclonal stimuli. Both patients developed antigen-specific antibodies in response to vaccination [515130]. Quantitative real-time PCR revealed that several cell types, including granulocytes, erythroid, megakaryotic and lymphoid cells, contained the vector of genetically corrected cells. The frequency of genetically corrected cells was highest in the lymphoid subsets, indicating a stronger selective advantage for the differentiation of genetically corrected NK, B- and T-cells. In Pt1, the amount of transduced T-cells increased progressively and reached 70% by 11 months of follow-up, while virtually all NK cells in PB and BM were also transduced in this period; in Pt2, transduced CD3+ T-cells appeared later than in Pt1, but reached a 100% frequency by day +240. The persistent production of transduced granulocytes, megakaryocytes, monocytes and erythroid cells was observed at levels ranging from 5 to 20% in Pt1, indicating the engraftment of multipotent HSCs [515130]. Biochemical analysis indicated that gene therapy completely restored ADA enzymatic function in PBLs and BM CD19+ B-cells. Pt1 also demonstrated vector ADA expression at the mitochondrial ribonucleic acid (mRNA) level in differentiated cells. Both patients demonstrated increased ADA activity in the plasma, and in Pt2, BM ADA activity was found to increase 8-fold after therapy. This increase in activity was correlated with a decline in

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erythrocyte toxic adenine deoxyribonucleotide metabolites, which decreased to 10 and 40% of the initial pretherapy levels for Pt1 and Pt2, respectively; these levels matched those observed in patients who had a successful transplantation of allogeneic BM. The changes in metabolic pattern were also associated with a normalization of lactate dehydrogenase and liver enzymes, both of which are elevated in ADA-SCID [515130]. The differences observed between the two patients may have been caused by several factors, including the fact that Pt2 received one log lower autologous transduced CD34+ cells than Pt1, as well as the difference in age in the patients (Pt2 was older than Pt1), which can be an important factor in HSC engraftment. Another component may have been the degree of host BM ablation; the different routes of administration of busulfan in each patient may have affected its pharmacologic biodistribution [515130]. The clinical trial was later expanded to include two more ADA-SCID patients, and included treatment follow-ups at 10, 15, 29 and 35 months after therapy [515038], [516040], [643376]. The results from the most recent follow-up matched those found in the initial study of two patients. PBL counts in all patients were 2.5 × 109/l, 0.2 × 109/l, 1.3 × 109/l and 0.7 × 109/l for Pt1, Pt2, Pt3 and Pt4, respectively. The overall level of myeloid engraftment and the speed and degree of immune reconstitution correlated with both the dose of infused transduced CD34+ cells and the degree of myelosuppression [643376]. By December 2005, the clinical trial had been expanded to six children affected by early onset ADA-SCID who lacked an HLA-identical sibling. Following nonmyeloablative conditioning and in the absence of PEG-ADA, the patients were administered autologous BM CD34+ cells transduced with a retroviral vector (murine leukemia virus) encoding ADA. All patients were reported to be alive and healthy in the absence of enzyme replacement therapy. The degree of myelosuppression after conditioning ranged from mild (Pt2, Pt4 and Pt6) to short-term neutropenia (Pt1 and Pt5), or more prolonged thrombocytopenia and neutropenia (Pt3). Multilineage, stable engraftment of gene-corrected HSCs was achieved in the BM of all patients, with the highest levels in Pt1, Pt3 and Pt5 (5 to 10%). In all patients, the vectorADA+ cells gradually became the majority of T-, B- and NK lymphocytes. This change led to a progressive increase in PBL counts, the restoration of polyclonal thymopoiesis, and the normalization of proliferative responses to mitogens and antigens. Pt2, who received the lowest dose of transduced HSCs, displayed a partial immune reconstitution. Serum Ig levels improved in all patients, and the production of specific antibodies after IVIg discontinuation and antigen vaccination was observed in three patients. The sustained ADA activity in lymphocytes and red blood cells (RBCs) resulted in the correction of purine metabolism (adenine deoxyribonucleotides < 30 nmoles/ml RBCs in five patients) and the amelioration of systemic toxicity. None of the patients experienced severe infections or adverse events after gene therapy. The patients were to be monitored from one year to more than five years after the therapy [668664].

7. SIDE EFFECTS AND CONTRAINDICATIONS Throughout the observational period of the clinical trial, the patients were reported to be in good clinical condition without experiencing any severe infectious episodes [515130], [643374], [668664]. No patient experienced toxicity or required blood component

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transfusion. Before gene therapy, Pt2 had suffered from persistent respiratory infections, chronic diarrhea and scabies. At 12 months after gene therapy, Pt2 demonstrated no signs of respiratory infections or scabies, and recovered normally from two transient episodes of diarrhea [515130]. No activation of the oncogene LMO2, which has been associated with other gene therapy trials [666671], [666673], was observed [516040]. Details from the latest follow-up stated that all patients were living a “normal life” at home, experiencing “normal” growth and development, and remained free from enzyme replacement therapy [515130], [643374], [668664].

8. PATENT SUMMARY No data are currently available.

9. CURRENT OPINION Pioneering studies have demonstrated the potential of gene therapy for the treatment of inherited hematopoietic diseases [440300], and particularly for ADA-SCID [470017], [470024], [666662], [666664], [666665]. However, vector design, gene transfer protocols and inadequate engraftment and expansion of genetically engineered cells limited the success of earlier studies [206054], [657269], [657273], [668669]. Data presented here suggest that autologous HSC gene therapy combined with nonmyeloablative conditioning, in the absence of enzyme replacement therapy, restores lymphoid development and functions and corrects the metabolic defect of ADA-SCID, with the subsequent complete reversal of the clinical phenotype [515130]. The data from the clinical trial also suggest that early gene therapy intervention, optimal amounts of transduced HSCs, and adequate conditioning are all crucial factors in determining the speed and level of CD34+ cell engraftment [643376]. Under these optimal conditions, gene therapy appears to offer a viable strategy for the treatment of ADA-SCID, and provides a significant advancement over earlier gene therapy attempts for the treatment of this disease [515130]. The use of nonmyeloablative conditioning, together with the use of IL-3, are most likely responsible for the improved results observed in the latest protocol developed by HSR-TIGET. Two strategies are currently most commonly used for the treatment of ADA-SCID: allogeneic BM transplantation and enzyme replacement therapy. Allogeneic BM transplantation can be curative with an HLA-matched sibling donor, but the outcome for patients transplanted with non-HLA-matched sibling donors is generally poor [470001], [666668]. ADA enzyme replacement therapy is thus considered for patients lacking HLAmatched BM donors. PEG-ADA treatment has been reported to improve immune function and result in a good quality of life free of opportunistic infections. However, a gradual decline in mitogenic proliferative responses occurs after a few years of treatment and normal antigenic responses occur less often than expected, underlining a vital requirement for a close follow-up of patients to detect any premature declines in immune function [657287], [657292]. Hence, gene therapy by gene correction of autologous HSCs provides a new and encouraging therapeutic option for patients with ADA-SCID. A previous gene therapy

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clinical trial reported low numbers of transduced cells in patients who were not subjected to myeloablation, suggesting that under these conditions, no selective advantage of the genetically corrected progenitor cells was observed. In monkey studies, the lack of myeloablation appeared to enhance the engraftment of genetically modified cells [666665]. Therefore, autologous HSC gene therapy combined with nonmyeloablative conditioning represents an alternative to current therapies for the treatment of ADA-SCID. Gene therapy is not without risks, however. Previous studies in another form of SCID revealed that gene therapy carries some risks of tumor formation [440300]. SCID-X1 is an Xlinked inherited disorder due to gamma chain deficiency that is characterized by an early block in T- and NK lymphocyte differentiation [666542]. Two pioneering HSC gene therapy trials for SCID-X1 reported the correction of this gamma chain immune defect [384875], [666670]. However, in a three-year follow-up period, three of ten young children who were treated in these trials developed leukemia-like conditions [656430], [666671]. A genetic analysis of the patients' malignant cells indicated that the retroviral vector had inserted into, and activated, the oncogene LMO2, which is associated with adult T-cell leukemia [666671], [666673]. The activated oncogene was most likely one of the triggering events for the leukemia, together with other chromosomal abnormalities [666673]. Although scientists had always considered the possibility that gene insertion would activate oncogenes, no such event had been observed in more than a decade of animal studies, nor had it been observed in human clinical trials involving large numbers of genetically modified blood cells [666652], [666675]. The apparent high risk of developing malignant cells for SCID-X1 patients suggests that there are specific risk factors associated with gene therapy. Despite the fact that there has been no sign of LMO2 activation in the ADA-SCID patients treated by the HSRTIGET therapy [516040], it is important that the patients continue to be monitored for several years. While it is important to remain cautious and to assess the risk/benefit balance of using gene therapy as a disease treatment, the chance of developing malignant cells is relatively low. One study estimated the probability of activating an oncogene by insertional mutagenesis at 0.001 to 0.01% [657280]. This figure may be an overestimate, as it appears that only a subset of integration sites, including the LMO2 locus, are available to the integration machinery of retroviral vectors, and that integration itself may not be sufficient to induce tumorigenicity, as several events are required to transform a normal cell [657283]. While no tumor formation has been reported in animals and humans for ADA-SCID gene therapy (perhaps because ADA-SCID does not involve the gamma chain gene, which may be oncogenic when expressed by a retrovirus [666665]), caution and risk/benefit assessment must still be employed before the therapy is used more widely, and optimizing the gene therapy protocol is imperative to reducing potential risks. Therefore, more data need to be gathered to assess the safety and validity of gene therapy for the treatment of ADA-SCID. In conclusion, gene transfer into autologous HSCs, combined with nonmyeloablative conditioning, is a potentially safe, efficient and promising strategy for the treatment of ADASCID. Optimization of the protocol to be used, through minimizing the number of genetically-modified stem cells that are administered, developing new and safer vectors to limit oncogene activation, and performing further preclinical studies to enable a better assessment of potential risks, will be essential to the success of the therapy. The combination of T/F/S plus IL-3, which has been reported in preclinical studies to significantly improve the maintenance of B-cell progenitors from ADA-SCID BM cells in vitro and to allow the

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efficient transduction of B- and NK cell progenitors in vivo, is a particular protocol that warrants further clinical investigation by HSR-TIGET.

10. DEVELOPMENT HISTORY Developer San Raffaele Telethon Institute for Gene Therapy

Country Italy

Status Phase II

Indication Combined immunodeficiency

Date 12-JAN-06

Reference 644674

11. LITERATURE CLASSIFICATIONS Chemistry Study type Genetically engineered human HSC protocol

Result Human CD34+ HSCs were removed from the engineered human BM of two ADA-SCID patients and genetically engineered using a GIADAl retroviral vector to express ADA genes. Cloning ADA cDNA into the LXSN vector generated the GIADAl retroviral vector. This vector was then packaged into the amphotropic Gp+Am12 cell line, and recombinant retroviral particles were produced for CD34+ cells. After three rounds of gene transfer and 4 days of culture, the cells were harvested, washed and intravenously infused back into the patients.

Reference

Effect Studied The effects of IL-3 and IL-7 on gene transfer efficiency

Model

Result

Reference

CD34+ cells from the BM of nine ADASCID patients and six healthy donors

IL-3 significantly increased the proportion and final yield of CD34+ cells expressing the transgene in both ADA-SCID and normal BM. In six out of nine ADA-SCID patients, IL-7 improved the percentages and yield of transduced CD34+ cells, but the overall increase was not statistically significant.

643373

The effects of IL-3 and IL-7 on gene transfer efficiency

ADA-SCID BM CD34+ cells

IL-3 or IL-7 in a 4-day CD34+ cells culture greatly improved the number of B-cell progenitors compared with the T/F/S combination of cytokines, resulting in a 6- and 5fold increase, respectively, and reached values that did not differ significantly from those of freshlyisolated cells.

643373

515130

Biology Study type Ex vivo

In vitro

ADA-Transduced Hematopoietic Stem Cell Therapy for ADA-SCID In vivo

Engraftment and differentiation

SCID-hu mouse model

In vitro/ in vivo

Repopulation and differentiation

CD34+ cells isolated at day +330 from the BM of an ADA-SCID patient

87

Transduced CD34+ cell were efficiently engrafted into SCID-hu mice (~ 80% overall engraftment), giving rise to B- and T-cell progeny and demon-strating the maintenance of in vivo lymphoid reconstitution capacity. The lymphoid differentia-tion capacity of CD34+ cells was maintained, and genetically corrected HSCs retained their ability to reconstitute lymphopoiesis in a secondary transplant (SCID-hu mice) after infusion.

643373

Result In both patients, the number of PBLs, serum IgM, IgA and IgG levels, mRNA expression of the ADA vector, intracellular ADA enzymatic activity in PBLs, and erythrocyte enzyme activity indicated a reconstitution of B-cell functions, as well as an amelioration of the metabolic pattern.

Reference 515130

All patients were reported to be alive and healthy in the absence of enzyme replacement therapy. The degree of myelosuppression after conditioning ranged from mild (Pt2, Pt4 and Pt6) to short-term neutropenia (Pt1 and Pt5), or more prolonged thrombocytopenia and neutropenia (Pt3). None of the patients experienced severe infections or adverse events

668664

515130

Clinical Effect Studied Efficacy

Safety and efficacy

Model Autologous HSC gene therapy and nonmyeloablative conditioning in two conditioning in two ADASCID patients Expansion of the trial outlined above to six children affected by early onset ADA-SCID

ACKNOWLEDGMENTS Reproduced with permission from The Thomson Corporation and Taupin P: ADAtransduced hematopoietic stem cell therapy for ADA-SCID. IDrugs (2006) 9(6): 423-30. Copyright 2006, The Thomson Corporation.

REFERENCES •• of outstanding interest • of special interest [206054] Retroviral vector design for long-term expression in murine hematopoietic cells in vivo. Correll, P.H., Colilla, S., Karlsson, S. (1994). Blood, 84 (6): 1812-1822.

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[384875] Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P., Selz, F., Hue, C., Certain, S., Casanova, J.L., Bousso, P., Deist, F.L., Fischer, A. (2000). Science, 288 (5466): 669. [440300] Gene therapy: Trials and tribulations. Somia, N., Verma, I.M. (2000). Nat Rev Genet 1 (2): 91-99. [467343] Advances in gene therapy for ADA-deficient SCID. Aiuti, A. (2002). Curr Opin Mol Ther, 4 (5): 515-522. [469995] Cognitive and behavioral abnormalities in adenosine deaminasedeficient severe combined immunodeficiency. Rogers, M.H., Lwin, R., Fairbanks, L., Gerritsen, B., Gaspar, H.B. (2001). J Pediatr, 139 (1): 44-50. [470001] Hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency. Buckley, R.H,, Schiff, S.E., Schiff, R.I., Markert, L., Williams, L.W., Roberts, J.L., Myers, L.A., Ward, F.E. (1999). N Engl J Med, 340 (7): 508-516. [470017] Transfer of the ADA gene into bone marrow cells and peripheralblood lymphocytes for the treatment of patients affected by ADA-deficient SCID. Bordignon, C., Mavilio, F., Ferrari, G., Servida, P., Ugazio, A.G., Notarangelo, L.D., Gilboa, E., Rossini, S., O'Reilly, R.J., Smith, C.A. et al. (1993). Hum Gene Ther, 4 (4): 513-520. [470024] Successful peripheral T-lymphocyte-directed gene transfer for apatient with severe combined immune deficiency caused by adenosine deaminase deficiency. Onodera, M., Ariga, T., Kawamura, N., Kobayashi, I., Ohtsu, M., Yamada, M., Tame, A., Furuta, H., Okano, M., Matsumoto, S., Kotani, H. et al. (1998). Blood, 91 (1): 30-36. [470408] Optimisation of retroviral supernatant production conditions for the genetic modification of human CD34+ cells. Dando, J.S., Aiuti, A., Deola, S., Ficara, F., Bordignon, C. (2001). J Gene Med, 3 (3): 219-227. [515038] European Society of Gene Therapy – 11th Annual Conference (Part I), Edinburgh, UK, Douglas, J. (2003). Iddb Meeting Report, November 14-17. [515130] Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Aiuti, A., Slavin, S., Aker, M., Ficara, F., Deola, S., Mortellaro, A., Morecki, S., Andolfi, G., Tabucchi, A., Carlucci, F., Marinello, E. et al. (2002). Science, 296 (5577): 2410-2413. •• Definitive example of successful gene therapy in two patients suffering from ADASCID. More patients need to be enrolled in similar trials to confirm the safety, efficacy and optimization of the therapy, and also with regard to the requirement of nonmyeloablative conditioning. [515136] Correction of ADA-SCID defect without PEG-ADA therapy by stem/progenitor cell gene therapy combined with a non-myeloablative conditioning. Aiuti, A., Slavin, S., Aker, M., Ficara, F., Deola, S., Mortellaro, A., Tabucchi, A., Carlucci, F., Marinello, E., Morecki, S., Andolfi, G .et al. (2001). Blood, 98 (11): 780a-781a. [515137] Transfer of the ADA gene into human ADA-deficient T-lymphocytes reconstitutes specific immune functions. Ferrari, G., Rossini, S., Nobili, N., Maggioni, D., Garofalo, A., Giavazzi, R. (1992). Blood, 80 (5): 1120-1124. [515240] Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement. Aiuti, A., Vai, S., Mortellaro, A., Casorati, G., Ficara, F., Andolfi, G., Ferrari, G., Tabucchi, A., Carlucci, F., Ochs, H., Notarangelo, L. et al. (2002). Nat Med, 8 (5): 423-425.

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• In this letter to the editor, the authors critically review and discuss the various data of PBL gene therapy studies. The authors propose that combined gene transfer protocols with PBLs and HSCs might result in both correction of the immune defect and optimal levels of systemic detoxification. [515242] Ex vivo gene transfer into human lymphoid progenitors results in in vivo functionally mature transduced T- and B-cells. Ficara, F., Aiuti, A., Mocchetti, C., Carballido, F., Superchi, D., Deola, S., Bordignon, C., Carballido, J., Roncarolo, M. (2001). Blood, 98 (11): 422a. [516040] European Society of Gene Therapy – 11th Annual Congress (Part II), Edinburgh, UK, Griesenbach U, Alton E IDDB MEETING REPORT, 2003 November 14-17 607777 American Society of Gene Therapy – Eighth Annual Meeting, St Louis, MO, USA, VandenDriessche T IDDB MEETING REPORT, 2005 June 01-05. [643373] IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADASCID BM CD34+ cells while maintaining in vivo lymphoid potential. Ficara, F., Superchi, D.B., Hernandez, R.J., Mocchetti, C., Carballido-Perrig, N., Andolfi, G., Deola, S., Colombo, A., Bordignon, C., Carballido, J.M., Roncarolo, M.G. et al. (2004). Mol Ther, 10 (6): 1096-1108. •• The data presented are particularly important for the optimization and safety of gene therapy for HSCs. This study, conducted after the initiation of the gene therapy clinical trial, presents repopulation data of transduced CD34+ cells in mice. [643374] Gene therapy for adenosine-deaminase-deficient severe combined immunodeficiency. Aiuti, A. (2004). Best Pract Res Clin Hematol, 17 (3): 505-516. [643375] Gene therapy for adenosine deaminase deficiency. Aiuti, A., Ficara, F., Cattaneo, F., Bordignon, C., Roncarolo, M.G. (2003). Curr Opin Allergy Clin Immunol, 3 (6): 461466 [643376] Safety and efficacy of stem cell gene therapy combined with nonmyeloablative conditioning for the treatment of ADA-SCID. Aiuti, A., Cattaneo, F., Cassani, B., Andolfi, G., Ficara, F., Mirolo, M., Tabucchi, A., Carlucci, F., Gaetaniello, L., Miniero, R., Aker, M., et al. (2003). Blood, 102 (11): 154a. [644674] Development status and Orphan Drug status designation – ADA gene therapy. San Raffaele Telethon Institute for Gene Therapy. Company Communication, 2006, January 12. [654032] EU Orphan Drug designation. European Medicines Agency (EMEA). INTERNET SITE, 2005 August 26. [656430] Gene Therapy working group report. The Academy of Medical Sciences – Safer Medicines Report. INTERNET SITE, 2005 November 1-31. [657269] High-level human adenosine deaminase expression in dog skin fibroblasts is not sustained following transplantation. Ramesh, N., Lau, S., Palmer, T.D., Storb, R., Osborne, W.R. (1993). Hum Gene Ther, 4 (1): 3-7. [657273] Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector. Hantzopoulos, P.A., Sullenger, B.A., Ungers, G., Gilboa, E. (1989). Proc Natl Acad Sci, USA, 86 10 3519-3523. [657280] Side effects of retroviral gene transfer into hematopoietic stem cells. Baum, C., Dullmann, J., Li, Z., Fehse, B., Meyer, J., Williams, D.A., von Kalle, C. (2003). Blood, 101 (6): 2099-2114.

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[657283] Modelling the molecular circuitry of cancer. Hahn, W.C., Weinberg, R.A. (2002). Nat Rev Cancer, 2 (5): 331-341. [657287] T-lymphocyte ontogeny in adenosine deaminase-deficient severe combined immune deficiency after treatment with polyethylene glycol-modified adenosine deaminase. Weinberg, K., Hershfield, M.S., Bastian, J., Kohn, D., Sender, L., Parkman, R., Lenarsky, C. (1993). J Clin Invest, 2 596-602. [657292] Long-term efficacy of enzyme replacement therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). Chan, B., Wara, D., Bastian, J., Hershfield, M.S., Bohnsack, J., Azen, C.G., Parkman ,R., Weinberg, K., Kohn, D.B. (2005). Clin Immunol, 117 (2): 133-143. [666542] Primary immunodeficiency diseases: An experimental model for molecular medicine. Fischer, A. (2001). Lancet, 357 (9271): 1863-1869. [666603] Brief report: Hepatic dysfunction as a complication of adenosine deaminase deficiency. Bollinger, M.E., Arredondo-Vega, F.X., Santisteban, I., Schwarz, K., Hershfield, M.S., Lederman, H.M. (1996). N Engl J Med, 334 (21): 13671371. [666620] The application of bone marrow transplantation to the treatment of genetic diseases. Parkman, R. (1986). Science, 232 (4756): 1373-1378. [666621] Treatment of adenosine deaminase deficiency with polyethylene glycol-modified adenosine deaminase. Hershfield, M.S., Buckley, R.H., Greenberg, M.L., Melton, A.L., Schiff, R., Hatem, C., Kurtzberg, J., Markert, M.L., Kobayashi, R.H., Kobayashi, A.L. et al. (1987). N Engl J Med, 316 (10): 589-596. [666625] Adenosine deaminase deficiency with late onset of recurrent infections: Response to treatment with polyethylene glycol-modified adenosine deaminase. Levy, Y., Hershfield, M.S., Fernandez-Mejia, C., Polmar, S.H., Scudiery, D., Berger, M., Sorensen, R.U. (1988). J Pediatr, 113 (2): 312-317. [666626] The use of HLA-non-identical T-cell-depleted marrow transplants for correction of severe combined immunodeficiency disease. O'Reilly, R.J., Keever, C.A., Small, T.N., Brochstein, J. (1989). Immunodefic Rev, 1 (4): 273-309. 666652 The future of gene therapy. Cavazzana-Calvo, M., Thrasher, A., Mavilio, F. (2004). Nature, 427 (6977): 779-781. • This review discusses the 'gene-therapy-causes-cancer' risk, and analyzes the risk/benefit factors for using gene therapy. [666655] An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency. Ferrari, G., Rossini, S., Giavazzi, R., Maggioni, D., Nobili, N., Soldati, M., Ungers, G., Mavilio, F., Gilboa, E., Bordignon, C. (1991). Science, 251 (4999): 1363-1366. [666662] Gene therapy in peripheral blood lymphocytes and bone marrow for ADAimmunodeficient patients. Bordignon, C., Notarangelo, L.D., Nobili, N., Ferrari, G., Casorati, G., Panina, P., Mazzolari, E., Maggioni, D., Rossi, C., Servida, P., Ugazio, A.G. et al. (1995). Science, 270 (5235): 470-475. [666664] T-lymphocyte-directed gene therapy for ADA-SCID: Initial trial results after 4 years. Blaese, R.M., Culver, K.W., Miller, A.D., Carter, C.S., Fleisher, T., Clerici, M., Shearer, G., Chang, L., Chiang, Y., Tolstoshev, P., Greenblatt, J.J. et al. (1995). Science, 270 (5235): 475-480.

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[666665] Bone marrow gene transfer in three patients with adenosine deaminase deficiency. Hoogerbrugge, P.M., van Beusechem, V.W., Fischer, A., Debree, M., le Deist, F., Perignon, J.L., Morgan, G., Gaspar, B., Fairbanks, L.D., Skeoch, C.H., Moseley, A. et al. (1996). Gene Ther, 3 (2): 179-183. [666668] European Group for Blood and Marrow Transplantation; European Society for Immunodeficiency. Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: Report of the European experience 1968-99. Antoine, C., Muller, S., Cant, A., Cavazzana-Calvo, M., Veys, P., Vossen, J., Fasth, A., Heilmann, C., Wulffraat, N., Seger, R., Blanche, S. et al. (2003). Lancet, 361 (9357): 553-560. [666670] Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. Hacein-Bey-Abina, S., Le Deist, F., Carlier, F., Bouneaud, C., Hue, C., De Villartay, J.P., Thrasher, A.J., Wulffraat, N., Sorensen, R., Dupuis-Girod, S., Fischer, A. et al. (2002). N Engl J Med, 346 (16): 1185-1193. [666671] A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. Hacein-Bey-Abina, S., von Kalle, C., Schmidt, M., Le Deist, F., Wulffraat, N., McIntyre, E., Radford, I., Villeval, J.L., Fraser, C.C., Cavazzana-Calvo, M., Fischer, A. (2003). N Engl J Med, 348 (3): 255-256. [666673] LMO2-associated clonal T-cell proliferation in two patients after gene therapy for SCID-X1. Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R., Morillon, E., Sorensen, R. et al. (2003). Science, 2003, 302 (5644): 415-419 Erratum in: Science, 302 5645 568. •• This study reports that retrovirus vector insertion into a proto-oncogene activates proto-oncogene expression, which may trigger malignancy. [666675] Gene therapy insertional mutagenesis insights. Dave, U.P., Jenkins, N.A., Copeland, N.G. (2004). Science, 303 (5656): 333. [666684] Evaluation of ADA gene expression and transduction efficiency in ADA/SCIDpatients undergoing gene therapy. Carlucci, F., Tabucchi, A., Aiuti, A., Rosi, F., Floccari, F., Pagani, R., Marinello, E. (2004). nucleosides nucleotides nucleic acids, 12 ( 8-9): 1245-1248. [666686] Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity. Zavialov, A.V., Engstrom, A. (2005). Biochem J, 391, 51-57. [668402] Severe combined immunodeficiency – molecular pathogenesis and diagnosis.Gaspar, H.B., Gilmour, K.C., Jones, A.M. (2001). Arch Dis Child, 84 (2): 16973. [668664] Gene therapy of ADA-deficient SCID. Aiuti, A. (2005). Euroconference – Gene Cell Ther, December 01-02. [668669] Gene therapy of severe combined immunodeficiencies (SCID). Fischer, A. (2005). Euroconference – Gene Cell Ther, December 01-02.

Chapter VIII

THERAPEUTIC POTENTIAL OF ADULT NEURAL STEM CELLS ABSTRACT The central nervous system (CNS) elicits limited capacity to recover from injury. Though considerable efforts and means have been deployed to find treatments for neurological diseases, disorders and injuries, there is still no cure for these ailments, and new alternatives for therapy must be explored. Because they generate the main phenotypes of the nervous system, neural stem cells (NSCs) hold the promise to cure a broad range of neurological diseases and injuries. With the confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS, new treatments for neurological diseases and injuries are being considered, in particular, the transplantation of adult-derived neural progenitor and stem cells to restore brain functions. This manuscript we will review the recent developments in adult neurogenesis and NSCs, and patent applications filed in relation to discoveries made in this new field of research.

1. INTRODUCTION Since the seminal studies by Altman and Das (1965) and Altman (1969), reporting evidence that new neuronal cells are generated in discrete areas of the adult brain in rodents, it is now well accepted that neurogenesis occurs in the adult mammalian brain, including in humans [1-6]. Neurogenesis occurs primarily in two areas of the adult brain: the dentate gyrus (DG) of the hippocampus, and the subventricular zone (SVZ) along the ventricles [7]. In 1992, Reynolds and Weiss were the first to isolate and characterize in vitro, a population of undifferentiated cells immunoreactive for the intermediate filament protein (nestin) that are multipotents; upon differentiation, the isolated cells generate the main phenotypes of the CNS: neurons, astrocytes and oligodendrocytes [8]. Nestin is a marker of neuroepithelial and CNS stem cells in vitro and in vivo [9]. These cells were isolated from adult mouse striatal tissue, including the SVZ, and expanded as neurospheres, over a large number of passages in defined medium in the presence of epidermal growth factor (EGF, 20 ng/ml) [8]. They expanded without immortalization by insertion of an oncogene, and did not originate from

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tumorigenic tissue. These cell were termed neural progenitor cells (NPCs), as all attributes defining stem cells [10], particularly self-renewal, were not characterized. Reynolds and Weiss later isolated neurospheres from embryonic mouse striatal tissue [11]. In 1995, Gage et al. isolated and characterized in vitro a population of cells with similar properties from the adult rat hippocampus, the second neurogenic area of the adult brain. The NPCs grow as monolayer in the presence of basic fibroblast growth factor (FGF-2, 20 ng/ml) [12]. In 2001, Palmer et al. isolated and characterized in vitro a population of NPCs from the adult human post-mortem hippocampus [13].

2. NEURAL PROGENITOR AND STEM CELLS NSCs are the self-renewing multipotent cells that generate, through a transient amplifying population of cells, the main phenotypes of the nervous system: neurons, astrocytes and oligodendrocytes. Stem cells are defined by five attributes: proliferation, self-renewal over an extended period of time, generation of a large number of differentiated progeny, regeneration of the tissue following injury, and flexibility in the use of these options [10]. Progenitor cells are, as most broadly defined, any cells that do not fulfill all of the attributes of NSCs. Characterizing NSCs in vitro requires fulfilling three criteria: i) multipotentiality – the generation of the main phenotypes of the nervous system (neurons, astrocytes and oligodendrocytes, from single cells) through a transient amplifying population of cells; ii) self-renewal – upon dividing, a stem cell gives rise to a progenitor cell and a stem cell; and iii) the generation of a large number of progenies of several orders of magnitude more numerous than the starting cell population [7, 14]. Clonal assays from neurospheres and monolayers were established to characterize in vitro self-renewal and multipotentiality [15, 16].

2.1. Neural Progenitor and Stem Cells in Vitro In 1996, Gritti et al. isolated and characterized in vitro self-renewing multipotent NSCs from adult mouse striatal tissue, including the SVZ [17]. In 1997, Palmer et al. isolated and characterized self-renewing multipotent NSCs from adult rat hippocampus [18]. These reports confirmed the existence of putative NSCs in the adult SVZ and hippocampus. Recent studies have challenged the isolation and characterization of self-renewing multipotent NSCs from the adult hippocampus, claiming the hippocampus contains NPCs with limited proliferative capacity and not self-renewing multipotent NSCs [19, 20]. These latter studies performed in mice are controversial, as differences in species, culture conditions and handling could account for discrepancies among the studies [21]. We further reported that FGF-2 requires a co-factor, a glycosylated form of the protease inhibitor cystatin C (CCg), for its mitogenic activity on self-renewing multipotent NSCs in vitro, from single cells [16]. The isolation and characterization of neural progenitor and stem cells from the adult brain has tremendous potential for developmental biology and cellular therapy. Because neural progenitor and stem cells can be differentiated into the three major cell types of the nervous system, they provide a model to study the development and function of the adult mammalian

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CNS, as well as drugs and novel therapies. Neural progenitor and stem cells can be isolated and characterized in vitro from subjects of a wide range of age, including post-mortem [13, 22]. They have been isolated and characterized from several species, including human, providing an unlimited source of tissue for transplantation, to treat a broad range of neurological diseases and injuries [13, 23]. Neural progenitor and stem cells can be isolated from patients with neurological diseases or disorders, as well as from animal models of neurological diseases or disorders, thereby providing in vitro models to study these diseases. Several patent applications have been submitted and filed recently related to the preparation and use of neural progenitor and stem cells in vitro. The use of neural progenitor and stem cells, isolated as neurospheres, and their progeny from various ages and species, from either normal or diseased CNS tissue, as a model to study neural development and function, and to screen the effect of biological agents and to develop novel therapies, was filed in 2001 [24]. These include clonally-derived neural progenitor and stem cells. It is hypothesized that clonally-derived neural progenitor and stem cells would represent a model with less variability to study the CNS in vitro. It also includes the isolation and characterization of neural progenitor and stem cells from patients with neurological diseases or disorders, as well as from animal models of neurological diseases or disorders, to study these diseases [24]. The use of neural progenitor and stem cells and their progeny isolated from post-mortem tissue, as a model to study neural development, function, and to screen the effect of biological agents and to develop novel therapies, was also filed [25]. The isolation and characterization of neural progenitor and stem cells from human post-mortem tissues offer alternative sources of tissues to study neurogenesis in vitro, and for cellular therapy. Other patent applications to improve culture conditions to promote survival, proliferation, and differentiation of neural progenitor and stem cells were filed [22-28]. For example, the use of collagenase (0.5 mg/ml) to dissociate neurospheres improves cell viability and proliferation compared to other modes of dissociation, such as mechanical or trypsinization [28]. This would improve the yield of isolation, expansion and maintenance in culture of neural progenitor and stem cells, particularly critical when culturing neural progenitor and stem cells from human tissues and biopsies. A patent application for the isolation and purification of the co-factor of FGF-2, CCg, has also been filed [29]. CCg may be used as a pharmacologic drug to culture adult NSCs in vitro, particularly from human post-mortem tissues [13], and to stimulate neurogenesis in vivo [16]. Patent applications for methods to genetically engineer neural progenitor and stem cells in vitro have also been submitted, including the isolation of neural progenitor and stem cells from transgenic animals [30]. Neural progenitor and stem cells can be genetically modified to express trophic factors or to produce neurotransmitter-synthesizing enzymes, such as nerve growth factor or tyrosine hydroxylase. This extends their use for gene therapy for the treatment of neurodegenerative diseases, and for marking cells with reported genes, such as the E. coli ß-galactosidase or green fluorescent protein.

2.2. Origin of Newly-Generated Neuronal Cells in the Adult Brain It is hypothesized that newly-generated neuronal cells in the adult brain originate from stem cells [3, 7]. There are several hypotheses and theories of the cellular identity of NSCs in

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the adult brain [7]. Based on electron microscopy, cell cycle analysis, [3H]-thymidine autoradiography, and immunochemistry for BrdU and neuronal markers in situ, as well as in vitro studies, two conflicting theories of the origin of newly-generated neuronal cells in the adult brain have been proposed. One theory contends that NSCs of the adult SVZ are differentiated ependymal cells that express the intermediate filament protein, nestin [31]. The other theory identifies them as slowly dividing astrocyte-like cells, expressing GFAP and nestin in the SVZ and DG [32-34]. The ependymal origin of NSCs in the adult brain remains a source of controversy, as other studies report that subependymal, but not ependymal, cells have NSC properties [32, 35], whereas the astroglial origin of adult NSCs has received further support [36-38]. A patent application has been filed for the isolation of non-embryonic ependymal NSCs and their use [39]. The use of non-embryonic ependymal NSCs, and their progeny may be used as a model to study neural development function, to screen the effect of biological agents and to develop novel therapies. However, the controversy over whether ependymal cells represent NSCs in the adult brain may jeopardize the potential of the claims filed under this patent.

3. THERAPEUTIC POTENTIAL OF ADULT NSCS Cellular therapy is the replacement of unhealthy or damaged cells or tissues with new ones. Because neurodegenerative diseases, cerebral strokes and traumatic injuries to the CNS produce neurological deficits that result from neuronal loss, cell therapy is a major area of investigation for the treatment of neurological diseases and injuries.

3.1. Cellular Therapy The confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS opens new opportunities for cellular therapy. Cell therapy would involve the stimulation of endogenous neural progenitor or stem cells, or the transplantation of adultderived neural progenitor and stem cells to repair the degenerated pathways. Endogenous neural progenitor and stem cells may be mobilized in the adult brain to replace degenerating nerve cells in the diseased brain or following CNS injury to promote regeneration. Stimulation of endogenous neural progenitor or stem cells may be achieved through local stimulation or through stimulation of the SVZ, as studies have revealed that new neuronal cells are generated at the sites of degeneration, where they originate from the SVZ. These cells then migrate partially through the RMS to the sites of degeneration, where they replace some of the lost nerve cells [40, 41]. To this aim, intracerebroventricular administration of trophic factors, such as EGF and FGF-2, stimulates neurogenesis in the adult SVZ, and may be beneficial in promoting neurogenesis in the diseased and injured brain [42, 43]. Adult-derived neural progenitor and stem cells have been transplanted into the CNS of normal and animal models in rodents. Adult rat hippocampal-derived neural progenitor and stem cells have been grafted into the hippocampus, where they differentiated into neuronal and glial cells [12]. In another study, adult rat spinal-cord-derived neural progenitor and stem

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cells were transplanted into the hippocampus and spinal cord [44]. When transplantated into the spinal cord, adult spinal-cord-derived neural progenitor and stem cells differentiated only in glial cells; when transplanted into the DG, they differentiated into neuronal and glial cells. These results showed that adult-derived neural progenitor and stem cells potently engraft into the CNS, and the microenvironment controls their differentiation into the neuronal lineage. Cloned adult rat hippocampal-derived neural progenitor and stem cells transplanted into the adult eye adopt morphologies similar to those of neuronal and astroglial cells of the retina, but do not express mature neuronal or glial markers [45, 46]. Adult human-derived retinal progenitor and stem cells (retinalspheres) survive, migrate, integrate and differentiate into neural retinal cells, particularly photoreceptors, when transplanted into the eyes of rodents [47]. This suggests that adult hippocampal-derived neural progenitor and stem cells elicit limited capacity to differentiate into mature neuronal phenotypes of the retina, and that adult human retinal progenitor and stem cells may be valuable in the treatment of retinal diseases. One of the limitations of the current protocols established to isolate and culture neural stem cells from the adult brain is that the cells yield to the heterogeneous population of neural progenitor and stem cells [48]. This limits the potential of adult-derived NSC therapy, as cell types in different states of differentiation may be grafted, and may not have the ability to terminally differentiate, survive and integrate into the network. Protocols have been developed to enrich the homogeneous population of neural progenitor/stem cells via cell sorting using membranous markers [49-51]. However, once expanded in vitro, these cells develop as heterogeneous cultures, containing neural progenitor and stem cells. Future studies will aim at characterizing homogeneous populations of NSCs and maintaining their homogeneity in culture. Systemic injection of adult-derived neural progenitor and stem cells improves deficits in an animal model of multiple sclerosis [52]. This study reveals the potency of adult-derived neural progenitor and stem cells in the treatment of neurodegenerative diseases. Systemic injection and injection through cerebrospinal fluid obviate the need for invasive surgical procedures, along with their associated risks and secondary effects. They are seen as promising routes for delivering NSCs in the CNS for therapy, particularly for spinal cord injury [53]. Adult stem cells are multipotent; they generate lineage-specific cell types restricted to the tissues from which they are derived. Pluripotent stem cells generate cells of the three germ layers: ectoderm, mesoderm and endoderm. Isolated neural progenitor and stem cells from the adult brain give rise to lineages other than neuronal in vitro and ex vivo, particularly blood cells [54]. Conversely, adult stem cells isolated from tissues other than the brain, like the skin, blood and bone marrow, give rise to neuronal lineages [55-57]. This suggests that adult stem cells may have a broader potential than previously thought [58]. However, other studies have shown that cell fusion, transformation, or contamination was at the origin of some of the phenotypes observed [59-63]. Whether or not adult stem cells are lineage restricted remains the source of debates and controversies [64]. The broader potential of adult stem cells, particularly NSCs, has tremendous implications for cellular therapy. The isolation of hematopoietic phenotypes from NSCs could provide an alternative source of tissue to treat blood-related diseases, with several advantages over bone marrow or cultured hematopoietic stem cells – among them, reducing the risk of graft-versushost disease due to the absence of lymphoid cells in the transplant, the risk of reintroducing malignant or diseased cells following chemotherapy, or radiation therapy for the treatment of leukemias and other blood-related diseases in autologous transplantation. A patent application

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for the broader potential of adult NSCs to generate hematopoietic cells has been filed [65]. Neuronal phenotypes have also been generated from adult skin stem cells [57]. The isolation of neuronal phenotypes from adult skin stem cells would allow the generation of neuronal phenotypes in vitro for cellular therapy, eliminating the need for surgical procedure and its associated risks and secondary effects. It would also permit autologous transplantation in which skin stem cells would be isolated from the patients, expanded in vitro, differentiated into neuronal cells, and grafted into the patients’ brain to restore brain functions.

3.2. Gene Therapy Adult neural progenitor and stem cells can be genetically modified [12], extending their potential use in the treatment of neurological diseases caused by genetic deficiencies. Adult neural progenitor and stem cells genetically engineered to express acid sphingomyelinase reverse lysosomal storage pathology in animal models of Niemann-Pick's disease [66]. This study validates genetically-engineered neural progenitor and stem in the treatment of neurological diseases and disorders, as filed in a patent application [30]. It highlights the potential of NSCs as a gene transfer vehicle in the treatment of lysosomal storage diseases, and other genetic disorders of the CNS.

4. CURRENT AND FUTURE DEVELOPMENTS The confirmation that neurogenesis occurs in the adult brain, and that NSCs reside in the adult CNS in mammals, has tremendous implications for cellular therapy and our understanding of development. The in vitro isolation and characterization of neural progenitor and stem cells from the adult CNS provide a model to study the adult mammalian CNS and a source of tissues for cellular therapy. Adult NSCs can be used in transplantation, including autologous transplantation in which the tissue would be isolated from biopsies, to treat various neurological diseases and disorders. Alternatively, endogenous neural progenitor and stem cells can be stimulated to promote regeneration of pathways. Adult NSCs offer tremendous potential in the treatment of CNS diseases and injuries, and potentially for diseases including blood-related and genetic diseases. However, limitations, debate and controversy exist regarding the use of adult NSCs for therapy. Among the limitations, neural progenitor and stem cells are heterogeneous in culture, and NSCs are yet to be identified [67]. The broader potential of adult stem cells, particularly NSCs, are yet to be elucidated. There are also unknowns regarding the potential of NSCs to restore brain function [68]: will the new neuronal cells establish the right connections? What are the potentials and risks that they could establish connections with the wrong target cells? What is the risk that NSCs will form tumors upon grafting?

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ACKNOWLEDGEMENTS Reproduced with permission from Bentham Science Publishers, Ltd.: Taupin P. Therapeutic potential of adult neural stem cells. Recent Patents on CNS Drug Discovery 2006; 1: 299-303. Copyright 2006, Bentham Science Publishers, Ltd.

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[36] Morshead, C.M., Garcia, A.D., Sofroniew, M.V., van Der Kooy, D. (2003). The ablation of glial fibrillary acidic protein-positive cells from the adult central nervous system results in the loss of forebrain neural stem cells but not retinal stem cells. Eur J Neurosci, 18, 76-84. [37] Imura, T., Kornblum, H.I., Sofroniew, M.V. (2003). The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP. J Neurosci, 23, 2824-32. [38] Garcia, A.D., Doan, N.B., Imura, T., Bush, T.G., Sofroniew, M.V. (2004). GFAPexpressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat Neurosci, 7, 1233-41. [39] Janson, A.M., Frisen, J., Johansson, C., Moma, S., Clarke, D., Zhao, M., Lendahl, U., Delfani, K. (2005). EP1090105B1 [40] Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z., Lindvall, O. (2002). Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med, 8, 96370. [41] Jin, K., Sun, Y., Xie, L., Peel, A., Mao, X.O., Batteur, S., Greenberg, D.A. (2003). Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum. Mol Cell Neurosci, 24, 171-89. [42] Craig, C.G., Tropepe, V., Morshead, C.M., Reynolds, B.A., Weiss, S., van der Kooy, D. (1996). In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain. J Neurosci, 16, 2649-58. [43] Kuhn, H.G., Winkler, J., Kempermann, G., Thal, L.J., Gage, F.H. (1997). Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J Neurosci, 17, 5820-9. [44] Shihabuddin, L.S., Horner, P.J., Ray, J., Gage, F.H. (2000). Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci, 20, 8727-35. [45] Takahashi, M., Palmer, T.D., Takahashi, J., Gage, F.H. (1998). Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina. Mol Cell Neurosci, 12, 340-8. [46] Nishida, A., Takahashi, M., Tanihara, H., Nakano, I., Takahashi, J.B., Mizoguchi, A., Ide, C., Honda, Y. (2000). Incorporation and differentiation of hippocampus-derived neural stem cells transplanted in injured adult rat retina. Invest Ophthalmol Vis Sci, 41, 4268-74. [47] Qiu, G., Seiler, M.J., Mui, C., Arai, S., Aramant, R.B., de Juan, E. Jr., Sadda, S. (2005). Photoreceptor differentiation and integration of retinal progenitor cells transplanted into transgenic rats. Exp Eye Res, 80, 515-25. [48] Suslov, O.N., Kukekov, V.G., Ignatova, T.N., Steindler, D.A. (2002). Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres. Proc Natl Acad Sc,. USA 99, 14506-11. [49] Uchida, N., Buck, D.W., He, D., Reitsma, M.J., Masek, M., Phan, T.V., Tsukamoto, A.S., Gage, F.H., Weissman, I.L. (2000). Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci, USA 97, 14720-25. [50] Wang, S., Roy, N.S., Benraiss, A., Goldman, S.A. (2000). Promoter-based isolation and fluorescence-activated sorting of mitotic neuronal progenitor cells from the adult mammalian ependymal/subependymal zone. Dev Neurosci, 22, 167-76.

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[51] Nagato, M., Heike, T., Kato, T., Yamanaka, Y., Yoshimoto, M., Shimazaki, T., Okano, H., Nakahata, T. (2005). Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers. J Neurosci Res. 80, 456-66. [52] Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani, G., Dina, G., Galli, R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi, A.L., Martino, G. (2003). Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature, 422, 688-94. [53] Bakshi, A., Hunter, C., Swanger, S., Lepore, A., Fischer, I. (2004). Minimally invasive delivery of stem cells for spinal cord injury: advantages of the lumbar puncture technique. J Neurosurg Spine, 1, 330-7. [54] Bjornson, C.R., Rietze, R.L., Reynolds, B.A., Magli, M.C., Vescovi, A.L. (1999). Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science, 283, 534-7. [55] Brazelton, T.R., Rossi, F.M., Keshet, G.I., Blau, H.M. (2000). From marrow to brain: expression of neuronal phenotypes in adult mice. Science, 290, 1775-9. [56] Mezey, E., Chandross, K.J., Harta, G., Maki, R.A., McKercher, S.R. (2000). Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science, 290, 1779-82. [57] Toma, J.G., Akhavan, M., Fernandes, K.J., Barnabe-Heider, F., Sadikot, A., Kaplan, D.R., Miller, F.D. (2001). Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol, 3, 778-84. [58] D'Amour, K.A., Gage, F.H. (2002). Are somatic stem cells pluripotent or lineagerestricted? Nat Med, 8, 213-4. [59] Morshead, C.M., Benveniste, P., Iscove, N.N., van der Kooy, D. (2002). Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations. Nat Medicine, 8, 268-73. [60] Wagers, A.J., Sherwood, R.I., Christensen, J.L., Weissman, I.L. (2002). Little evidence for developmental plasticity of adult hematopoietic stem cells. Science, 297, 2256-9. [61] Castro, R.F., Jackson, K.A., Goodell, M.A., Robertson, C.S., Liu, H., Shine, H.D. (2002). Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science, 297, 1299. [62] Terada, N., Hamazaki, T., Oka, M., Hoki, M., Mastalerz, D.M., Nakano, Y., Meyer, E.M., Morel, L., Petersen, B.E., Scott, E.W. (2002). Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature, 416, 542-5. [63] Ying, Q.L., Nichols, J., Evans, E.P., Smith, A.G. (2002). Changing potency by spontaneous fusion. Nature, 416, 545-8. [64] Mezey, E., Nagy, A., Szalayova, I., Key, S., Bratincsak, A., Baffi, J., Shahar, T. (2003). Comment on "Failure of bone marrow cells to transdifferentiate into neural cells in vivo". Science, 299, 1184. [65] (*) Bjornson, C.R., Rietze, R.L., Reynolds, B.A., Vescovi, A.L. EP1019493B1 (2005). [66] Shihabuddin, L.S., Numan, S., Huff, M.R., Dodge, J.C., Clarke, J., Macauley, S.L., Yang, W., Taksir, T.V., Parsons, G., Passini, M.A., Gage, F.H., Stewart, G.R. (2004). Intracerebral transplantation of adult mouse neural progenitor cells into the NiemannPick-A mouse leads to a marked decrease in lysosomal storage pathology. J Neurosci, 24, 10642-51.

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CONCLUSION AND PERSPECTIVES: ADULT NEURAL STEM CELLS – THE PROMISE OF THE FUTURE ABSTRACT Stem cells are self-renewing undifferentiated cells that give rise to multiple types of specialized cells of the body. In the adult, stem cells are multipotent and contribute to homeostasis of the tissues and regeneration after injury. Until recently, it was believed that the adult brain was devoid of stem cells, hence unable to make new neurons and regenerate. With the recent evidence that neurogenesis occurs in the adult brain and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), the adult brain has the potential to regenerate and may be responsive to repair. The function of NSCs in the adult CNS remains the source of intense research and debate. The promise of future study of adult NSCs lies in redefining the function and physiopathology of the CNS, as well as in the treatment of a broad range of CNS diseases and injuries.

1. INTRODUCTION Seminal studies in the 1960s by Altman and Das using [3H]-thymidine autoradiographic labeling were the first to report the generation of new neuronal cells in the adult rodent dentate gyrus (DG), cell proliferation in the ventricular zone, and migration and persisting neurogenesis in the adult olfactory bulb (OB) (Altman and Das, 1965; Altman, 1969). However, these studies had little impact because of the paucity of cells labeled and the difficulty of definitively identifying them. It was not until the 1990s, with the advent of new procedures using bromodeoxyuridine for labeling dividing cells in the CNS (BrdU) (Gratzner, 1982; Miller and Nowakowski, 1988) and retroviral labelings (Van Praag et al., 2002) that neurogenesis in the SVZ and DG became accepted (Gross, 2000; Taupin and Gage, 2002). Although significant progress has been made in recent years in the field of adult neurogenesis and NSCs (table 1), debate, controversy, and questions remain.

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2. ADULT NEUROGENESIS, FACTS AND DEBATES 2.1. Neurogenesis in the Adult Mammalian Brain Neurogenesis occurs primarily in two areas of the adult brain in mammals: the DG of the hippocampus and the subventricular zone (SVZ) in several species, including humans (Eriksson et al., 1998; Curtis et al., 2007a). In the DG, newly-generated neuronal cells in the subgranular zone (SGZ) migrate to the granular layer, where they differentiate into mature neuronal cells, and extend axonal projections into the CA3 area in rodents and primates. In the SVZ, cells are generated in the anterior part of the SVZ and migrate to the OB through the rostro-migratory stream (RMS), where they differentiate into interneurons of the OB in rodents and in non-human primates (Taupin P and Gage, FH. 2002). Newly-generated neuronal cells establish functional connections with neighboring cells (Van Praag et al., 2002; Carlen et al., 2002), particularly with GABAergic innervations in the DG, soon after their migration is completed (Wang et al., 2005). As many as 9,000 new neuronal cells – or 0.1% of the granule cell population – are generated per day in the DG of mice, and 65-75% of the bulbar neurons are replaced during a 6-week period in young adult rats (Kempermann et al., 1997; Kato et al., 2001; Cameron and McKay, 2001). Among them, a significant proportion undergoes programmed cell death rather than achieving maturity (Morshead and van der Kooy, 1992; Cameron and McKay, 2001; Gould et al., 2001). The newly-generated neuronal cells that survive to maturity may be very stable, and may permanently replace cells born during development, as adult-generated neuronal cells have been reported to survive for extended period of time (e.g., for at least two years in the human DG) (Altman and Das, 1965; Eriksson et al., 1998; Dayer et al., 2003; Kempermann et al., 2003). Neurogenesis may also occur, albeit at lower levels, in other areas of the mammalian brain, like the Ammon’s horn CA1, neocortex and substantia nigra (SN) (Gould et al., 1999; Rietze et al., 2000; Zhao et al., 2003). However, some of these reports have been contradicted by other studies (Kornack and Rakic, 2001; Lie et al., 2002; Frielingsdorf et al., 2004; Gould, 2007). Hence, the bulk of evidence suggests that there is little, if any, neurogenesis taking place in other brain regions.

2.2. Stem Cells in the Adult Brain The origin of newly-generated neuronal cells in the adult brain remains a source of controversy. One theory contends that they originate from differentiated ependymal cells in the lateral ventricle, while another contends that they originate from astrocyte-like cells in the SVZ and SGZ (Taupin and Gage, 2002). A glial origin for adult-generated neuronal cell has received recent support (Filippov et al., 2003; Garcia et al., 2004). Hence, the possibility of ependymal origins for NSCs has been has largely disproven; hence, astrocyte-like cells represent the most accepted model for the source of stem cells in the adult brain. It is postulated that newly-generated neuronal cells originate from residual stem cells in the adult brain. Stem cells are defined by five attributes: proliferation, self-renewal over an extended period of time, generation of a large number of differentiated progeny, maintenance of the homeostasis of the tissue, and regeneration of the tissue following injury (Potten and

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Loeffler, 1990). NSCs are the self-renewing, multipotent cells that generate neurons, astrocytes and oligodendrocytes of the nervous system. Neural progenitor cells are, as most broadly defined, any cells that do not fulfill all of the attributes of NSCs. Though NSCs are yet to be characterized in the adult CNS, self-renewing, multipotent NSC-like cells have been isolated and characterized in vitro from various areas of the adult CNS (neurogenic and nonneurogenic, including the spinal cord) suggesting that NSCs may reside throughout the CNS (Taupin and Gage, 2002). There are currently no specific markers of adult NSCs. Nestin, the transcription factors sox-2, oct-3/4, and the RNA-binding protein Musashi 1, are markers for neural progenitor and stem cells, but also label populations of glial cells (Lendahl et al., 1990; Sakakibara et al., 1996; Doetsch et al., 1999; Zappone et al., 2000; Kaneko et al., 2000; Komitova et al., 2004; Okuda et al., 2004), further fueling the controversy over the origin of newly-generated neuronal cells in the adult brain.

2.3. Rate and Modulation The rate of neurogenesis in the rodent DG and SVZ is modulated by various environmental stimuli, physio- and pathological conditions (Taupin, 2005). For example, environmental enrichment promotes the survival of newly-generated neuronal cells in the DG. Voluntary running stimulates the generation of newly-generated neuronal cells in the DG, but not the SVZ. Stress, neuroinflammation and aging decrease neurogenesis in the DG (Nithianantharajah and Hannan, 2006; Mora et al., 2007). In the diseased brain and after injuries to the CNS, such as strokes and traumatic brain injuries (TBIs), neurogenesis is stimulated in the neurogenic areas, and new neuronal cells are generated at the site of injury where they replace some of the degenerated nerve cells (Grote and Hannan, 2007). Cell tracking studies revealed that newly-generated neuronal cells at sites of injury originate from the SVZ. Newly generated neuronal cells migrate partially through the RMS to the degenerated areas. It is estimated that 0.2% of the degenerated nerve cells are replaced in the striatum after focal ischemia (Arvidsson et al., 2002). Hence, neurogenesis can be stimulated in the injured brain.

2.4. Limit and Pitfalls of BrdU Labeling The modulation of neurogenesis and its quantification have been the subject of debate, partly due to the use of BrdU (a thymidine analog) labeling as a method of assessment. As BrdU crosses the blood-brain barrier, it is generally administered intraperiteonally. It is suggested that activity such as exercise, in addition to the effects of various treatments and physio- and pathological conditions on cerebral flow, metabolism and permeability of the blood-brain barrier to reagents (in particular to BrdU) may affect the availability of BrdU to the brain. The variation of BrdU quantification observed in these conditions would then reflect the change in BrdU uptake by the cells, rather than the modulation of neurogenesis (Taupin, 2007).

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With regard to the quantification of neurogenesis with BrdU, one study suggests that the standard concentration used to assess neurogenesis (50-100 mg/kg body weight in rodents, intraperitoneal injection) may not label all the dividing cells (Taupin, 2007), whereas another study reports that it does (Burns and Kuan, 2005). Further systematic studies on BrdU labeling in the CNS are, therefore, needed to further define the conditions in which BrdU can be used for studying neurogenesis. The use of BrdU to study neurogenesis carries other limitations, as in labeling DNA repair, abortive cell cycle reentry and gene duplication, without cell proliferation (Taupin, 2007). Thus, other strategies are necessary to make educated conclusions with regard to adult neurogenesis when using BrdU labeling, as in the study of markers of the cell cycle and the use of retroviruses.

2.5. Mechanisms Underlying Adult Neurogenesis Most of the mechanisms underlying adult neurogenesis and NSC growth and fate determination are yet to be uncovered. It has been reported that cell death stimulates the proliferation of neural progenitor cells in the adult hippocampus (Gould and Tanapat, 1997). Other studies reveal that the mitotic rate is regulated by the number of available progenitor cells, rather than by cell death (Ekdahl et al., 2001; Jin et al., 2004). On the molecular level, epidermal growth factor and basic fibroblast growth factor were the first mitogens to be identified for neural progenitor and stem cells in vitro, and to stimulate neurogenesis in vivo (Reynolds and Weiss, 1992; Gage et al., 1995; Craig et al., 1996; Kuhn et al., 1997). However, other factors present in conditioned medium, like the glycosylated form of the protease inhibitor cystatin C (CCg), are also required for the proliferation of self-renewing, multipotent NSCs from single cells in vitro (Taupin et al., 2000) and remain to be characterized, as well as the pathways of these mitogens and cofactors.

2.6. Broader Potential of Adult Stem Cells Adult stem cells are multipotent; they generate lineage-specific cell types restricted to the tissues from which they are derived. Several studies have reported that adult-derived stem cells, particularly adult-derived neural progenitor and stem cells, may have a broader potential; i.e., they generate cell types of lineages other than their tissues of origin (Bjornson et al., 1999; Brazelton et al., 2000; Mezey et al., 2000). Although some studies have presented convincing results, phenomena such as contamination, transformation, transdifferentiation and cell fusion have been reported as possible explanations for the phenotypes observed in some studies (Anderson et al., 2001; Mezey, 2004).

2.7. Function of Newborn Neuronal Cells The function of adult neurogenesis has been the source of intense research and debate. Evidence suggests that newly-generated neuronal cells are involved in learning and memory, and depression (Gould et al., 1999; Shors et al., 2001; Jacobs et al., 2000; Santarelli et al.,

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2003). The involvement of adult neurogenesis in learning and memory has been challenged by other studies. Increased hippocampal neurogenesis has been observed without improvement in learning and memory performance in the Morris water maze test, in mice selectively bred for high levels of wheel running (Rhodes et al., 2003). Therefore, the function of newly-generated neuronal cells in the adult brain is yet to be determined. Finally, the evidence that neurogenesis occurs in the adult brain, and that NSCs reside in the adult CNSs provides new avenues for cellular therapy. Cell therapeutic intervention may involve the stimulation of endogenous or the transplantation of neural progenitor and stem cells of the adult CNS. However, adult NSCs are yet to be used for therapy. Though it is now accepted that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS, questions and controversy remain: What is the origin of newly generated neuronal cells in the adult brain? What are their molecular markers? What are the factors and mechanisms controlling NSC growth and fate specification? What is the potential of adult-derived stem cells? What are the functions of newly generated neuronal cells in the adult brain? How can we use adult NSCs therapeutically? Table 1. Adult neurogenesis and neural stem cells – key publications. Year 1965 1982 1988 1990 1992 1992 1995 1996 1997 1998 1999 1999 2001 2001 2005 2007

Event (references) Seminal studies on adult neurogenesis (Altman and Das, 1965) Monoclonal antibody against BrdU (Gratzner, 1982) BrdU – a marker to study neurogenesis (Miller and Nowakowski 1988) Identification of nestin as a marker for neural progenitor and stem cells (Lendahl et al., 1990) Post-mitotic cell death of newly-generated neuronal cells in the adult SVZ (Morshead and van der Kooy, 1992) Isolation and characterization of neural progenitor and stem cells from the Adult rodent SVZ (Reynolds and Weiss, 1992) Isolation and characterization of neural progenitor and stem cells from the adult rodent hippocampus, adult hippocampal-derived neural progenitor and stem cells grafted in the adult brain (Gage et al., 1995) Environmental enrichment promotes adult neurogenesis (Kempermann et al., 1997) Characterization of adult neurogenesis in the adult human hippocampus (Eriksson et al., 1998) Broader potential of adult-derived neural progenitor and stem cells (Bjornson et al., 1999) Glial origin for newly-generated neural stem cells in the SVZ (Doetsch et al.,1999) Post-mitotic cell death of newly-generated neuronal cells in the adult hippocampus (Cameron and McKay, 2001) Isolation and characterization of neural progenitor and stem cells from human postmortem tissues and biopsies (Palmer et al., 2001) Newly generated neuronal cells receive GABAergic excitatory input (Tozuka et al., 2005) Characterization of adult neurogenesis in the adult human SVZ (Curtis et al., 2007)

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3. THE FUTURE OF ADULT NEUROGENESIS Newly-generated neuronal cells represent a small fraction of nerve cells in the adult brain. But data presented above suggest that their relevance to CNS physio- and pathology, and cellular therapy is significant, but yet to be uncovered. The keys to understanding the importance of newly-generated neuronal cells is their relative contributions compared with the preexisting network of CNS functioning. One can postulate that such contributions will depend on the specific properties of adult-generated neuronal cells.

3.1. On the Functioning of Newly-Generated Neuronal Cells in the Adult Brain Adult newly-generated neuronal cells belong to three groups, based on their destiny. The first group consists of the newly-generated neuronal cells that undergo post-mitotic death (Morshead and van der Kooy, 1992; Cameron and McKay, 2001). The second group represents a population of newly-generated cells that neither undergo apoptosis nor differentiate to a defined fate. This latter group of cells likely contributes to renewing the stem cell niche. Niches are specialized microenvironments that regulate stem cell activity (Moore and Lemischka, 2006; Scadden, 2006). In the adult brain, neurogenic niches are maintained in restricted regions and have been identified and characterized (Alvarez-Buylla and Lim, 2004). These niches, an angiogenic and an astroglial niches, control NSC selfrenewal and differentiation (Palmer et al., 2000; Song et al., 2002). It is hypothesized that neurogenic niches underlie the properties and functions of NSCs in the adult CNS (AlvarezBuylla and Lim, 2004; Taupin, 2006; Lim et al., 2007). The third group consists of the newly generated neuronal cells that survive to maturity and integrate into the network (Altman and Das, 1965; Eriksson et al., 1998; Kempermann et al., 2003; Dayer et al., 2003). Several lines of evidence suggest that newly-generated neuronal cells have different properties and physiological functions than mature nerve cells that may underlie their specific functions. Young granule cells in the adult DG appear to exhibit robust long-term potentiation that, in contrast to mature granule cells, cannot be inhibited by GABA (Wang et al., 2000). More recently, newly-generated neuronal cells in the adult hippocampus were characterized as receiving GABAergic excitatory input (Ge et al., 2005, 2007; Tozuka et al., 2005), a function of GABA previously reported during development (Ben-Ari, 2002). Once cells have matured and integrated into the pre-existing network, they may functionally replace nerve cells born during development. Among the questions that arise from such a theory are: what are the physiological functions of the newly generated neural cells during the time they are distinct from their mature counterpart, what is the function of such cellular renewal, and why would it occur only and specifically in discrete areas of the adult brain?

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3.2. On the Functionality of Newly-Generated Neuronal Cells in the Adult Brain The increase of neurogenesis in diseases, disorders, and after injury might then serve a neuroadaptative process (Figure 1). Patients with neurological diseases such as Alzheimer's disease, epilepsy and Parkinson's disease (PD), or recovering from stroke or injury, are at greater risk for depression (Perna et al., 2003; Gilliam et al., 2004; Sawabini and Watts, 2004) and present memory impairments (Kotloski et al., 2002; Wang et al., 2004). Since learning and memory and depression are associated with hippocampal neurogenesis (Gould et al., 1999; Jacobs et al., 2000; Shors et al., 2001; Santarelli et al., 2003), the depressive episodes and learning impairments in patients suffering from neurological diseases or disorders may contribute to the regulation of neurogenesis in an additive, or cooperative manner with the disorder. Therefore, modulation of neurogenesis in the hippocampus might be an attempt by the CNS to compensate for other neuronal functions, such as depression, and learning and memory impairments, associated with the disease. The increase in neurogenesis would also be a factor contributing to the plasticity of the CNS, particularly in relation to the recovery of the CNS after injury. After cerebral strokes and TBIs, there is a striking amount of neurological recovery during the following months and years, despite often-permanent structural damage (Sbordone et al., 1995; Anderson et al., 2000). Though the mechanisms underlying such recovery are not fully understood, properties of the plasticity of the CNS, like the reorganization of the pre-existing network and axonal sprouting, have been implicated in the recovery (Ramic et al., 2006; Kolb and Gibb, 2007). Reorganization of the contra-lateral hemisphere has been noted in plasticity after brain injury (Cramer and Basting, 2000). Neurogenesis is increased bilaterally in the DG and the SVZ following cerebral strokes and TBIs. The bilateral increase in neurogenesis would contribute to plasticity-related recovery in the CNS, particularly following injury. The generation of newly-generated neuronal cells at sites of injury could represent a regenerative attempt by the CNS. In the diseased brain and after injury to the CNS, new neuronal cells are generated at the sites of degeneration, where they replace some of the lost nerves cells (Arvidsson et al., 2002). Hence, there is no functional recovery. The generation of new neuronal cells at the sites of injury could represent an attempt by the CNS to regenerate following injury. Several hypotheses can explain the lack of recovery of the CNS after injury. The number of new neurons generated may be too low to compensate for the neuronal loss – 0.2% of the degenerated nerve cells in the striatum after focal ischemia – (Arvidsson et al., 2002). The neuronal cells produced are non-functional because they do not develop into fully mature neurons, because they do not develop into the right type of neurons, or because they are incapable of integrating into the surviving brain circuitry. Gliogenesis has also been reported to occur at sites of injury (Fawcett and Asher, 1999). Therefore, neurogenesis and gliogenesis at sites of injury may participate in the healing process. The total number of neurons in the adult brain does not dramatically increase, and cell death is an established process in the adult brain (Gould et al., 2001). Newly-generated neuronal cells may contribute to homeostasis of the tissue. Neurogenic niches have been described in the adult brain, and may hold the molecular and cellular cues to this phenomenon (Alvarez-Buylla and Lim, 2004). On the physiopathological level, an explanation is yet to be given. It is worth mentioning that it has been suggested that because environmental enrichment promotes adult neurogenesis, and standard laboratory living conditions do not

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represent the physiological environment, neurogenesis may occur more broadly, at a low level, and remain undetected (Taupin, 2007), though such eventuality is yet to be proven in mammals. Indeed, it has been proposed that self-repair mechanisms may operate in the adult rodent SN (Zhao et al., 2003), the area of the CNS affected in PD. If such turn-over of dopaminergic neuronal cells were confirmed, progression of the disease would then be determined not only by the rate of degeneration of SN neurons, but also by the efficacy in the formation of new dopamine neurons. Thus, disturbances in the equilibrium of cellular homeostasis could result in neurodegenerative disease. In PD, therefore, neurogenesis might not only be a process for functional recovery, but it may also play a key role in the pathology of the disease. However, these data remain the source of controversy (Lie et al., 2002; Frielingsdorf et al., 2004), and this hypothesis is yet to be demonstrated. Although at this time, these hypotheses remain speculative, the future of adult neurogenesis and NSC research lies in our understanding of the specific role and relative contribution of newly-generated neuronal cells to the physio- and pathology of the CNS.

Figure 1. Functionality of adult neurogenesis. Adult neurogenesis occurs throughout adulthood. Hence, the physiological function(s) of adult neurogenesis is yet to be elucidated. Adult neurogenesis may be involved in the physiopathology of CNS functioning.

- Patients with neurological diseases such as Alzheimer's disease, epilepsy and Parkinson's disease, or recovering from stroke or injury, are at greater risk for depression and present memory impairment. Since learning and memory and depression are associated with hippocampal neurogenesis, the increase of neurogenesis in diseases, disorders, and after injury might then serve a neuroadaptative process. - After cerebral stroke and traumatic brain injury, there is a striking amount of neurological recovery in the following months and years, despite often-permanent structural damage. The increase in neurogenesis would also be a factor contributing to the plasticity of CNS, particularly in relation to the recovery in the CNS after injury. - In the diseased brain and after injury to the CNS, new neuronal cells are generated at the sites of degeneration, where they replace some of the lost nerves cells. The creation of newlygenerated neuronal cells at the site of injury could represent a regenerative attempt by the CNS and its participation in the healing process.

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- The total number of neurons in the adult brain does not dramatically increase, and cell death is an established process in the adult brain. Newly-generated neuronal cells may contribute to cellular homeostasis. The disequilibrium in cellular homeostasis may result in neurodegenerative diseases. The relative contribution of adult neurogenesis to these processes is yet to be elucidated. Specific properties of newly-generated neuronal cells yet to be determined would underlie the role of newly generated neuronal cells in CNS functioning.

4. THE PROMISE OF ADULT NEURAL STEM CELLS The promise of adult NSCs lies also in our ability to bring adult NSC research to therapy. Because of their potential to generate the main phenotype of the CNS, NSCs hold the potential to cure a broad range of CNS diseases and injuries. The confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS, opens new avenues for cellular therapy. Cell therapeutic intervention may involve the stimulation or grafting of neural progenitor and stem cells (Okano et al., 2007; Yamashima et al., 2007). The generation of new neuronal cells at sites of injury further highlights the potential of the CNS to repair itself. The SVZ origin of newly-generated neuronal cells suggests that the stimulation of neurogenesis in the SVZ would provide a strategy to promote functional recovery after injury (Curtis et al., 2007b). In addition, the potential to isolate neural progenitor and stem cells from non-degenerated brain areas from the patient himself would provide an autologous source of transplantable neural progenitor and stem cells, thereby obviating the need to find a matching donor for the tissues and the use of drugs that suppress the immune system, increasing the chance of successful graft and recovery. This strategy, however, would involve invasive surgery and the destruction of healthy brain tissue, a limiting factor for its clinical application. Neural progenitor and stem cells have also been isolated from human post-mortem tissues, providing an alternative source of tissues for cellular therapy (Palmer et al., 2001).

5. CONCLUSION The promise of the future of adult neurogenesis and NSC research lies in our understanding of the function and relative contribution of newly-generated neuronal cells in the adult brain, and our ability to bring adult NSCs to therapy. The molecular, cellular, and physiological characterization of adult NSCs is a prerequisite to this endeavor. Significant advances have already been made in recent decades. Because of the potential of adult neurogenesis and NSCs in redefining brain functioning, physio- and pathology, and their potential to cure a broad range of CNS diseases and injuries, the future of this field of research is tantalizing.

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ACKNOWLEDGMENTS Reproduced from: Taupin P. Adult neural stem cells: the promise of the future. Neuropsychiatric Disease and Treatment (2007) 3(6): 753-60, with permission of Dove Medical Press Ltd.

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INDEX A acetylcholine, 10 acetylcholinesterase, 6 acid, 37, 51, 98 acidic, 27, 46, 101 activation, 8, 9, 13, 59, 84, 85 acute lymphoblastic leukemia, 65, 67 acute myeloid leukemia, 65 adaptation, 17 adenine, 83 adenosine deaminase (ADA), viii, 2, 61, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 adenosine deaminase deficiency, 2, 88, 89, 90, 91 adenosine triphosphate, 77 adipogenic, 63 adipose, 61 administration, 12, 13, 23, 29, 60, 63, 67, 74, 78, 83, 96 adolescents, 17 adult(s), vii, viii, ix, 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 37, 39, 40, 41, 44, 51, 54, 55, 57, 58, 59, 61, 62, 65, 68, 69, 71, 72, 73, 76, 85, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 adult stem cells, 1, 2, 34, 62, 68, 71, 97, 98, 102 adult T-cell, 85 adult tissues, vii, 1, 34 adulthood, vii, 11, 20, 112 adverse event, 83, 87, 91 age, 48, 60, 62, 82, 83, 95, 114, 115, 116, 117, 118 agent, 43, 48 aging, 20, 107 aid, 44 allele, 75 allografts, 38, 64 alpha, 76

alternative(s), vii, viii, 19, 34, 44, 51, 85, 93, 95, 97, 113 alters, 8, 15 Alzheimer's disease (AD), vii, viii, 5, 6, 8, 9, 10, 25, 43, 50, 52, 111, 112, 116, 118 amendments, 49 amino, 77 amniotic fluid, 34 amyloid, vii, 5, 6, 9 amyloid-beta protein precursor (APP), 6, 9, 116 aneuploid, 6 aneuploidy, 68 angiogenesis, 29, 30, 66 angiogenic, 110 animal models, vii, 5, 6, 7, 14, 22, 37, 44, 45, 48, 50, 51, 65, 95, 96, 98 animals, 13, 34, 35, 36, 46, 64, 81, 85, 95 antagonist, 6, 7, 10 antagonists, 7 antibiotic(s), 62, 63 antibody, 62, 109, 116 antidepressant medication, 15 antidepressant(s), vii, 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 27, 118 antigen, 46, 79, 82, 83 anti-inflammatory, 23, 62, 63, 65, 72 anti-inflammatory drugs, 62 anti-tumor, 37 anxiety, 12, 14, 15, 16 anxiety disorder, 14 apoptosis, 30, 110 apoptotic, 9 artery, 22, 28, 47 arthritis, 62, 68, 76 assessment, 47, 85, 107 astrocyte(s), 9, 11, 17, 21, 26, 28, 31, 37, 39, 41, 44, 45, 46, 47, 49, 54, 93, 94, 96, 99, 100, 106, 107, 115, 117 astroglial, 21, 96, 97, 110 atmosphere, 63, 70 atrophy, vii, 11, 12, 14, 17, 18

Index

122 Australia, 35 autoimmune disease(s), 60, 64, 68, 74 autoimmune disorders, 1, 62, 68 autoimmunity, 78 autologous bone, 76 autosomal recessive, 44 availability, 107 axonal, 17, 20, 31, 32, 39, 57, 106, 111 axon(s), 32, 48, 53

B banks, 50 basal forebrain, 54 basal ganglia, 115 basic fibroblast growth factor, 22, 29, 45, 94, 100, 108 Batten disease, viii, 43, 56, 57, 58 B-cell(s), 78, 80, 81, 82, 85, 86, 87, 89 behavior, 49 behavioral effects, 5, 8, 9, 10, 13, 17, 27, 118 beneficial effect, 34, 48, 50, 67 benefits, 25, 40, 49, 68, 78 beta, 6, 28, 72, 118 bilirubin, 60, 67 binding, 21, 23, 28, 29, 107, 118 biology, vii, viii, 2, 5, 10, 11, 13, 33, 36, 38, 40, 41, 57, 75, 94 birth(s), 16, 44 blastocyst(s), 1, 3, 33, 35, 36, 39, 40, 41 bleeding, 62 blood, 59, 61, 66, 67, 68, 73, 76, 83, 85, 97, 98, 102, 107, 114, 117 blood transfusion, 76 blood-brain barrier, 107 body weight, 79, 108 bone, viii, 29, 59, 61, 62, 66, 71, 75, 76, 78, 88, 90, 97, 102, 117 bone marrow, viii, 29, 59, 62, 66, 71, 75, 76, 78, 88, 90, 97, 102, 117 bone marrow transplant, viii, 59, 62, 75, 78, 90 bovine, 63, 78 bowel, 62 brain, vii, viii, ix, 1, 2, 5, 6, 7, 9, 11, 12, 13, 14, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 37, 39, 41, 44, 45, 46, 48, 49, 50, 51, 54, 55, 56, 57, 58, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117 brain functioning, 113 brain functions, ix, 93, 98 brain tumor, 2, 37, 41 brain tumor stem cells, 2

breakdown, 77 breast, 16, 65 breast cancer, 16, 65 bromodeoxyuridine, 10, 12, 17, 105, 117 building blocks, 1

C California, 52 Canada, 52 cancer(s), viii, 1, 16, 37, 59, 60, 61, 62, 64, 65, 67, 68, 72, 76, 81, 90 cancer cells, 60 cancer treatment, 67 Candida, 82 candidates, 8, 23, 35 capacity, viii, 21, 22, 34, 46, 71, 79, 81, 87, 93, 94, 97 carcinogenesis, 2, 3 cartilage, 61 caspase(s), 23, 30 causal relationship, 8, 14 CD133, 45, 47, 49, 53 CD19, 82 CD28, 63, 70 CD3+, 82 CD34, 45, 63, 73, 79, 80, 81, 83, 84, 86, 87, 88, 89 CD34+, 73, 79, 80, 81, 83, 84, 86, 87, 88, 89 CD40, 61 CD44, 63 CD45, 45, 62, 63 CD8+, 82 cDNA, 79, 81, 86 cell, vii, viii, 1, 2, 3, 6, 9, 10, 12, 15, 16, 17, 18, 19, 20, 23, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 49, 52, 53, 55, 56, 57, 59, 61, 62, 63, 64, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 94, 95, 96, 97, 99, 100, 101, 102, 105, 106, 108, 109, 110, 111, 113, 114, 115, 116, 118 cell adhesion, 6, 46 cell culture, 67 cell cycle, 6, 9, 96, 108 cell death, 9, 10, 20, 23, 106, 108, 109, 111, 113 cell differentiation, 81 cell division, 6 cell fusion, 97, 102, 108 cell grafts, 31, 52 cell line(s), 3, 40, 46, 55, 64, 79, 80, 86 cell surface, 73 cell transplantation, 52, 55, 61, 72, 73, 79, 88 cellular homeostasis, 112, 113

Index cellular therapy, vii, 5, 8, 19, 20, 23, 24, 25, 26, 33, 43, 44, 98, 113 central nervous system (CNS), vii, viii, ix, 1, 5, 6, 7, 10, 11, 14, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 33, 34, 35, 37, 39, 43, 44, 45, 49, 50, 51, 52, 54, 55, 56, 57, 58, 93, 95, 96, 97, 98, 99, 100, 103, 105, 107, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118 cerebellum, 44, 47, 53 cerebral cortex, 29, 57, 101 cerebral ischemia, 28, 29, 30 cerebral palsy, 50 cerebral strokes, 19, 96, 111 cerebrospinal fluid (CSF), 23, 25, 31, 37, 50, 58, 97 cervical, 31 chemokines, 50 chemotherapeutic agent, 64, 71 chemotherapy, 60, 68, 76, 97 childhood, 18, 34, 44, 114 children, 17, 68, 75, 83, 85, 87 chimerism, 67 cholesterol, 37, 51 cholinergic neurons, 54 chondrogenic, 63 choroid, 28 chromosomal abnormalities, 85 chromosomal instability, 76 chromosomes, 35 chronic stress, 12, 17 circulation, 78 cirrhosis, 60 cisplatin, 64 classes, 5, 12 cleavage, 30 clinical, viii, 2, 8, 10, 13, 20, 25, 27, 33, 34, 35, 36, 44, 48, 50, 52, 58, 59, 60, 65, 66, 68, 71, 72, 75, 79, 81, 83, 84, 85, 86, 89, 113, 115, 118 clinical depression, 13, 20 clinical trials, viii, 8, 52, 59, 65, 68, 85 clone, 40 cloning, viii, 33, 35, 40, 79 clusters, 45 CO2, 63 coding, 36, 37 cofactors, 108 cognition, 49 cognitive, 5, 7, 38 cognitive deficits, 38 cognitive function, 6, 7 cognitive impairment, 5 collagen, 68, 76 colon, 67 combat, 68, 80

123

commercial, 2 communication, 49 compatibility, 62, 67, 75 competence, 102 complementary, 79 complexity, 2, 15, 39, 99 complications, 62, 67, 72 compositions, 66 concentrates, 2 concentration, 108 conditioning, 79, 82, 83, 84, 85, 87, 88, 89 Congress, 89 connective tissue, 66 consent, 34 consolidation, 72 constraints, viii, 2, 33, 51, 78 contamination, 97, 108 control, 40, 46, 48, 53, 62, 110 contusion, 47, 53, 58 corpus callosum, 47, 53 correlation, 47, 64, 70 cortex, 2, 22, 28, 30, 39, 46, 47, 53, 99 cortical, 116 costs, 52 covering, 49 critically ill, 72 Crohn's disease, viii, 59, 62, 72 cryopreservation, 66 cues, 111 culture, 24, 30, 39, 45, 49, 55, 61, 62, 63, 68, 70, 74, 79, 80, 86, 94, 95, 97, 98, 99, 117 culture conditions, 94, 95 curing, 34, 61 cycles, 2, 38, 99, 117 cyclosporine, 24, 49, 50, 61 cysteine, 44 cysteine residues, 44 cytoarchitecture, 15 cytokine(s), 37, 47, 50, 59, 61, 63, 64, 65, 67, 72, 80, 86 cytometry, 62, 63, 102 cytoplasm, 35 cytotoxic, 59

D death, 2, 16, 23, 26, 30, 39, 55, 99, 108, 110, 116, 117 deficiency, viii, 2, 37, 51, 77, 78, 85, 88, 89, 90, 91 deficits, 15, 29, 31, 33, 41, 58, 97, 116 degenerative disease, 52 delivery, 31, 45, 48, 55, 72, 73, 102 dementia, vii, 5, 6, 9

124 demyelinating disease, viii, 43 dendrites, 18 dendritic cell, 59 density, 15, 62, 70 dentate gyrus (DG), vii, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 20, 26, 29, 30, 37, 41, 93, 96, 97, 100, 101, 105, 106, 107, 110, 111, 114, 115, 118 deoxyribonucleic acid, 79 deoxyribonucleotides, 83 deposits, vii, 5, 47, 53 depression, vii, 11, 12, 13, 14, 15, 16, 17, 18, 20, 27, 108, 111, 112, 116, 117, 118 deprivation, 20 derivatives, 61 dermatitis, 60 dermis, 66, 102 destruction, 34, 36, 113 detection, 116 detoxification, 89 developing brain, 9 diabetes, viii, 1, 33 diabetic, 46 diarrhea, 60, 62, 67, 84 differentiated cells, 82 differentiation, 6, 9, 16, 27, 28, 29, 30, 31, 39, 40, 46, 47, 49, 50, 51, 53, 55, 56, 58, 63, 64, 71, 79, 81, 82, 85, 87, 93, 95, 97, 99, 101, 110, 115, 117, 118 digestive tract, 62 diphtheria, 56, 61 disability, 49 disease progression, 49 disequilibrium, 113 disorder, 14, 34, 37, 51, 62, 111 dissociation, 45, 95 DNA, 6, 10, 12, 67, 108, 116 DNA repair, 108 donations, 39 donors, 35, 36, 49, 62, 67, 68, 73, 74, 75, 80, 81, 84, 86 dopamine, 38, 112 dopaminergic, 23, 27, 112, 115 dopaminergic neurons, 23, 27, 115 drug design, 8, 14 drug therapy, vii, 5, 62 drugs, vii, 5, 7, 8, 9, 11, 12, 17, 19, 24, 44, 60, 61, 62, 95, 113 DSM, 16 DSM-II, 16 DSM-III, 16 duplication, 6, 108 duration, 80

Index

E eating, 13, 67 ectoderm, 34, 97 eggs, 36, 73 elective abortion, 35 electron, 58, 96 electron microscopy, 96 electrophysiological, 47, 115 embryo, 28, 36, 118 embryonic, vii, viii, 1, 2, 25, 26, 28, 30, 33, 38, 39, 40, 41, 43, 58, 94, 96, 99, 101 embryonic stem cells (ESCs), viii, 1, 25, 33, 34, 35, 36, 40, 41, 43, 44, 45, 58 emotional responses, 12 encapsulation, 45 encoding, 44, 78, 79, 81, 83 endoderm, 34, 97 endogenous, 7, 19, 29, 30, 37, 40, 51, 96, 98, 101, 109, 114, 117, 118 endothelial cells, 73 engineering, viii, 33, 36, 38, 51 England, 35 enrollment, 65 enteritis, 60 environment, 1, 13, 20, 26, 47, 51, 66, 67, 68, 72, 112, 116 environmental, 8, 9, 13, 20, 23, 107, 111, 117 environmental stimuli, 20, 107 enzymatic, 45, 82, 87 enzymatic activity, 87 enzyme(s), 35, 38, 44, 48, 49, 53, 64, 70, 77, 78, 83, 84, 87, 88, 90, 95 ependymal, 28, 96, 100, 101, 106 ependymal cell, 96, 106 epidermal growth factor, 22, 29, 31, 45, 46, 54, 93, 108 epidermal growth factor receptor, 31 epigenetic, 102 epigenetic alterations, 102 epilepsy, 111, 112, 115 epithelium, 67 epitopes, 72 equilibrium, 112 erythroid cells, 82 erythropoietin, 23, 29 Escherichia coli (E. coli), 46, 95 ethical, viii, 2, 25, 26, 33, 34, 35, 36, 39, 50, 51, 68 ethical issues, 2, 34, 35 etiology, vii, 11, 12, 13, 14 EU, 89 European, 65, 79, 88, 89, 91

Index evidence, vii, ix, 1, 7, 8, 11, 12, 13, 14, 16, 22, 23, 27, 34, 41, 47, 50, 60, 65, 81, 93, 99, 102, 105, 106, 109, 110, 114, 115 examinations, 64, 68 excitation, 118 excitotoxic, 46, 55 excitotoxicity, 32, 39 exercise, 13, 15, 16, 107 exogenous, 30, 32, 57 exposure, 20, 80 extracellular, 13 extracranial, 31 eye(s), 1, 44, 60, 97

F FAD, 10 failure, 61 familial, 118 family, 60, 91 FDA approval, 56 fertilization, 34 fetal, vii, 1, 26, 33, 34, 36, 38, 44, 45, 46, 49, 50, 57, 60, 63 fetal tissue, 34, 50 fetuses, 25, 33, 35 FGF-2, 57, 94, 95, 96, 100, 118 fibroblast growth factor, 16, 29, 55, 101, 116 fibroblasts, 35, 36, 48, 53, 89 filament, 28, 47, 93, 96, 99, 116 flare, 62 flexibility, 94 flow, 62, 102, 107 fluorescence, 36, 45, 101 fluoxetine, vii, 5, 8, 11, 12, 13, 17, 27 focusing, 52 food, 13, 77 Food and Drug Administration (FDA) , viii, 43, 48, 56, 65, 68, 71 food intake, 77 forebrain, 7, 9, 27, 28, 99, 100, 101, 114, 115 fusion, 102

G GABA, 110, 115 GABAergic, 106, 109, 110, 118 gastrointestinal, viii, 59, 60, 65 gastrointestinal tract, 60 gene, vii, 2, 5, 6, 28, 35, 36, 37, 38, 39, 44, 46, 51, 54, 67, 72, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 95, 98, 108, 118

125

gene expression, 28, 89, 91, 118 gene therapy, 2, 35, 37, 38, 39, 67, 77, 78, 79, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 95 gene transfer, 51, 79, 80, 81, 84, 85, 86, 88, 89, 91, 98 generation, 2, 11, 22, 24, 37, 80, 94, 98, 105, 106, 107, 111, 113 genetic disease, 1, 37, 98 genetic disorders, 44, 98 genetics, 10 genome, 35, 36, 78 germ layer, 34, 97 Ginkgo biloba, 29 glia, 10, 55 glial, 22, 27, 45, 46, 47, 96, 101, 106, 107, 109, 115 glial cells, 22, 96, 107 glial scar, 47, 115 gliomas, 41 glucocorticoids, 12, 16, 61 glucose, 63 glutamate, 6, 7, 23, 32, 39 glycol, 78, 90 government, 35 grades, 60 grading, 60, 74 graft-versus-host disease (GVHD), viii, 1, 2, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 73, 74, 75, 76, 97 grants, 52, 56, 71 granule cells, 17, 26, 110, 114 groups, 62, 110 growth, 16, 22, 23, 29, 30, 32, 39, 45, 49, 54, 57, 63, 80, 84, 91, 101, 108, 109, 114, 116 growth factor(s), 16, 22, 23, 29, 30, 45, 49, 54, 80, 91, 101, 114, 116 guidelines, 35 gut, 60, 67 gyrus, 115

H half-life, 78 healing, 56, 67, 111, 112 health, 49, 68 health status, 68 heart, viii, 1, 33, 66, 72 heart attack, 72 heart disease, 1 heartburn, 62 hematologic, 69, 74 hematological, 65 hematopoiesis, 68, 72, 82

Index

126 hematopoietic, viii, 45, 57, 59, 61, 63, 64, 69, 72, 73, 74, 75, 77, 79, 80, 84, 87, 89, 97, 102, 114 hematopoietic cells, 45, 64, 87, 98 hematopoietic stem cell(s) (HSC), viii, 59, 61, 65, 66, 74, 79, 82, 83, 84, 85, 86, 87, 89, 97, 102 hemisphere, 46, 111 hepatitis, 60 heterogeneity, 76, 101 heterogeneous, 97, 98 high-risk, 65 hippocampal, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 26, 27, 30, 31, 96, 99, 100, 109, 111, 112, 114, 115, 116, 117, 118 hippocampus, vii, 2, 5, 6, 10, 11, 12, 13, 14, 16, 17, 18, 20, 25, 26, 27, 30, 37, 39, 41, 44, 47, 93, 94, 96, 99, 100, 101, 106, 108, 109, 110, 111, 115, 116, 117, 118 histocompatibility antigens, 60, 75 histological, 47, 99, 114 histology, 60 homeostasis, ix, 1, 105, 106, 111, 113 homogeneity, 97 hormones, 12 host, 7, 25, 32, 34, 37, 39, 47, 49, 57, 59, 60, 61, 63, 66, 67, 69, 72, 75, 83 host tissue, 7, 37, 60, 67 human, viii, 1, 2, 3, 6, 14, 16, 24, 26, 28, 29, 30, 31, 33, 34, 35, 36, 37, 39, 40, 43, 44, 45, 46, 47, 48, 52, 54, 55, 56, 57, 58, 60, 61, 63, 66, 68, 71, 72, 73, 74, 75, 76, 78, 81, 85, 86, 88, 89, 90, 94, 95, 97, 99, 100, 101, 106, 109, 113, 115, 117 human brain, 26, 30, 31, 39, 55, 99, 117 human dignity, 35 human embryonic stem cells, 40 human leukocyte antigen (HLA), 60, 61, 62, 67, 73, 74, 75, 78, 81, 83, 84, 90 humans, viii, 1, 2, 5, 11, 13, 14, 20, 33, 34, 37, 40, 48, 68, 85, 93, 106 Huntington's disease, 22, 28, 38, 50 hypertension, 49 hypothesis, 9, 13, 16, 17, 112

I identical sibling, 62, 74, 78, 83 identification, 23 identity, 21, 95 IgG, 82, 87 IL-1, 59, 63, 67, 70 IL-6, 63 IL-8, 63 imaging, 41 immune disorders, 66, 74

immune function, 78, 84, 88 immune response, 63, 64, 66, 73 immune system, 19, 24, 50, 60, 64, 78, 113 immunochemistry, 96 immunocompromised, viii, 59, 77 immunodeficiency, 77, 86, 88, 89, 90, 91 immunodeficient, 46, 90 immunogenetics, 75 immunoglobulin(s), 49, 82 immunohistochemistry, 10, 12, 17, 18, 117 immunological, 59, 61, 64, 68 immunomodulator, 77 immunomodulatory, 67, 68 immunoreactivity, 15 immunosuppression, 49, 63, 67, 73 immunosuppressive, 49, 50, 61, 62, 67, 68, 76 immunosuppressive drugs, 50, 61, 62 immunotherapy, 72 impairments, 38, 111 implants, 64 in situ, 96 in vitro, viii, 11, 19, 21, 22, 24, 26, 28, 34, 36, 37, 39, 44, 46, 49, 54, 55, 61, 63, 64, 66, 67, 73, 74, 79, 80, 81, 85, 93, 94, 95, 96, 97, 98, 107, 108, 117 in vivo, viii, 19, 22, 28, 30, 41, 49, 50, 61, 63, 73, 79, 81, 86, 87, 89, 90, 93, 95, 102, 108, 114, 117 incidence, 60, 61, 63, 67, 68 Indiana, 6 indication, 23 induction, 29, 67, 72 infants, 78 infection, 61, 62, 78 infectious episodes, 83 inflammation, 62, 66, 67, 72 inflammatory, 1, 47, 59, 62, 64, 65, 66, 68, 72 inflammatory bowel disease, 62 inflammatory response, 65 infliximab, 61 inherited disorder, 85 inhibition, 15, 63, 70 inhibitor(s), 6, 7, 10, 12, 18, 30, 61, 94, 108, 115 inhibitory, 45 initiation, 45, 80, 89 injections, 47, 53 injury(ies), vii, viii, ix, 1, 11, 19, 22, 23, 25, 26, 33, 34, 37, 43, 44, 50, 51, 52, 56, 57, 58, 64, 93, 94, 95, 96, 98, 105, 106, 107, 111, 112, 113, 114, 115, 116, 117 inner cell mass (ICM), 33, 34, 36, 43 insertion, 85, 91, 93 insight, 9 insulin, 34, 35

Index integration, 24, 30, 31, 37, 50, 63, 67, 85, 101, 114, 115 intensity, 48 interaction, 25, 56 interface, 36 interferon (IFN), 59, 63, 67, 70 interleukin, 59, 78 interneurons, 20, 30, 106 intervention, 51, 84, 109, 113 intestine, 62 intracerebral, 39 intracranial, 31, 41 intramuscular injection, 63 intramuscularly, 63 intraperitoneal, 108 intravenous, viii, 63, 77, 82 intravenous Ig (IVIg), 82, 83 intravenously, 63, 66, 79, 86 intrinsic, 68 inventors, 66 irradiation, 12, 67 ischemia, 22, 30, 107, 111 ischemic, 29, 30, 47, 53, 57, 58, 101 ischemic brain injury, 30 isolation, viii, 19, 22, 24, 26, 37, 49, 53, 54, 55, 62, 66, 70, 94, 95, 96, 97, 98, 101 Italy, 86

127

leukemia(s), viii, 59, 60, 61, 65, 68, 69, 83, 85, 97 licenses, 71 ligament, 61 ligand, 61, 80 limitation, 6 literature, 8 liver, 1, 60, 64, 67, 78, 83 liver disease, 1 liver enzymes, 83 living conditions, 111 location, 23 locus, 85 long distance, 47 long period, 14 long-term potentiation, 110 lumbar puncture, 102 lung, 78 lying, 2, 39 lymphatic, 60 lymphatic system, 60 lymphocyte(s), 45, 60, 63, 70, 73, 74, 77, 78, 79, 80, 82, 83, 85, 88, 90 lymphoid, 60, 80, 81, 82, 84, 87, 89, 97 lymphoid tissue, 60 lymphoma, 65 lysosome(s), 37, 43, 44, 51

M J Japan, 69 Japanese, viii, 59, 69 JCR Pharmaceuticals, viii, 59, 69, 71

K Ki-67, 47 kidney, 45 kinase, 13, 15, 80 kinetics, 82 Korean, 39

L labeling, 6, 7, 12, 105, 107, 108, 114 lactate dehydrogenase, 83 laser, 9, 31 latency, 13 lead, vii, 8, 11, 14, 37, 67, 68 learning, 10, 14, 20, 27, 38, 108, 111, 112, 118 legislation, viii, 33, 35 lesions, 23, 30, 46, 55, 115

machinery, 85 macrophages, 59 magnetic, 12, 49, 62 magnetic resonance imaging, 12, 49 maintenance, 78, 80, 81, 85, 87, 95, 106 major depression, 15, 17 major histocompatibility complex (MHC), 61, 62, 67, 73 malignancy, 61, 68, 74, 91 malignant cells, 60, 85 mammalian brain, viii, 1, 2, 20, 28, 29, 33, 37, 93, 100, 106, 115 mammalian tissues, 44 management, 75, 117 manufacturing, 52 MAPK (MAP kinase), 13 market, 71 marrow, viii, 59, 60, 61, 64, 66, 69, 72, 73, 74, 75, 76, 90, 91, 102, 114 matrix, 64 maturation, 47, 53, 63, 77 measurement, 82 measures, 40 mechanical, 95

128 media, 53 median, 74 medication, 117 medicine, vii, 40, 44 memory, 5, 9, 12, 14, 20, 108, 111, 112, 116 memory performance, 109 mesencephalon, 31 mesenchymal stem cells (MSCs), viii, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76 mesoderm, 34, 97 meta-analysis, 15, 18 metabolism, 79, 83, 107 metabolites, 15, 77, 78, 83 methylprednisolone, 61 mice, 6, 7, 9, 10, 13, 26, 27, 29, 36, 41, 44, 46, 47, 48, 50, 53, 54, 55, 56, 57, 63, 73, 81, 87, 89, 94, 100, 102, 106, 109, 114, 116, 117 microenvironment(s), 1, 61, 97, 110 microscope, 64 microscopy, 12, 46, 47 middle cerebral artery occlusion (MCAO), 47, 53 migration, 10, 17, 22, 23, 26, 29, 31, 40, 41, 47, 50, 53, 70, 99, 101, 105, 106, 114, 117 migratory properties, 25 miscarriages, 35 mitochondrial, 82 mitogen, 13 mitotic, 101, 108, 109, 110 models, 6, 9, 33, 61, 95 modulation, 12, 20, 67, 72, 107, 111 molecular mechanisms, 8, 14 molecular medicine, 90 molecules, 22, 23, 46, 61, 64, 73 momentum, 18 monkeys, 13, 16, 40 monoamine oxidase inhibitors, 12 monoclonal, 45, 48, 61, 66, 72 monoclonal antibody(ies), 45, 48, 61, 66, 72 monocytes, 82 monolayer, 63, 94 mood, 12, 15 mood disorder, 15 mortality, 78 motor function, 49 motor neurons, 32, 40 mouse model, 5, 8, 53, 58, 81, 87 MRI, 18 mRNA, 82, 87 multiple sclerosis, 7, 25, 31, 41, 50, 58, 97, 102 multipotent, ix, 11, 21, 34, 37, 44, 55, 61, 74, 79, 82, 94, 97, 99, 100, 102, 105, 107, 108 multipotent stem cells, 34 multipotents, 1, 93

Index murine model, 48 muscarinic receptor, 7 muscle, 61, 66 mutagenesis, 85, 91 mutant, 6, 10, 36 mutation(s), 6, 44, 47, 118 myelin, 31, 46, 47, 48, 50, 53, 54 myelin basic protein (MBP), 46, 47, 53, 55 myelination, 50 myeloid, 65, 79, 83 myeloid cells, 79

N National Institutes of Health (NIH), 52, 56 natural, 59, 79 natural killer (NK), 59, 61, 63, 67, 70, 79, 80, 81, 82, 83, 85, 86 necrosis, 60 neocortex, 20, 26, 27, 106, 116 nerve(s), 5, 6, 7, 9, 11, 12, 14, 22, 35, 44, 45, 46, 54, 55, 95, 96, 107, 110, 111, 112 nerve cells, 5, 6, 7, 11, 12, 14, 22, 35, 44, 96, 107, 110, 111 nerve growth factor, 45, 46, 54, 55, 95 nervous system, viii, 11, 21, 37, 43, 44, 47, 52, 55, 93, 94, 107, 117 network, 11, 14, 50, 97, 110, 111, 115 neural ceroid lipofuscinoses (NCL), viii, 43, 44, 56 neural development, 95, 96 neural stem cells (NSCs), vii, viii, ix, 1, 2, 5, 7, 9, 10, 11, 16, 17, 19, 21, 23, 25, 26, 27, 28, 31, 33, 34, 35, 37, 38, 39, 41, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 118 neural stem/progenitor cell, 100 neuroblasts, 115 neurodegeneration, 7, 9 neurodegenerative disease(s), vii, 1, 5, 7, 19, 25, 37, 43, 44, 50, 51, 55, 57, 95, 96, 97, 112, 113, 115 neurodegenerative disorders, 44 neurofibrillary tangles, vii, 5 neurofilament, 21 neurogenesis, vii, viii, ix, 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 28, 29, 30, 33, 34, 37, 39, 51, 57, 93, 95, 96, 98, 99, 100, 101, 105, 106, 107, 108, 109, 111, 112, 113, 114, 115, 116, 117, 118, 119 neurogenic, 6, 12, 14, 18, 21, 23, 25, 27, 39, 94, 100, 107, 110, 116, 118 neuroinflammation, 20, 23, 107 neurological deficit, 19, 96

Index neurological disease, vii, viii, 1, 5, 19, 22, 25, 33, 34, 51, 93, 95, 96, 98, 111, 112 neurological disorder, 45 neuronal cells, vii, 2, 6, 7, 11, 14, 20, 21, 22, 23, 25, 33, 37, 51, 93, 95, 96, 98, 105, 106, 107, 108, 109, 110, 111, 112, 113 neuronal ceroid lipofuscinoses, 57 neuronal death, 6 neuronal degeneration, 9, 31 neuronal markers, 96 neuronal survival, 51 neurons, ix, 1, 9, 10, 11, 17, 18, 20, 21, 22, 26, 27, 30, 31, 37, 38, 39, 41, 44, 45, 46, 47, 48, 49, 54, 55, 93, 94, 99, 100, 101, 105, 106, 107, 111, 113, 114, 115, 116, 117, 118 neuroplasticity, 10, 18 neuroprotection, 30, 48, 53 neuroprotective, 50 neurotransmission, 6, 7 neurotransmitter(s), 5, 12, 35, 38, 95, 117 neurotrophic factors, 25, 32, 39, 40, 45, 55, 57 neutropenia, 83, 87 New York, 75 Niemann-Pick disease, 57 nitric oxide, 29 NK cells, 61, 63, 67, 70, 81, 82 NMDA receptors, 9 N-methyl-D-aspartate (NMDA), 6, 7, 8, 9, 10, 29 non-human primates, 106 non-myeloablative, 84, 87, 88 non-random, 65 noradrenaline, 12, 15 norepinephrine, 12 novelty, 13 nucleic acid, 77, 91 nucleotides, 91 nucleus(i), 35, 36 nutritional deficiencies, 62 nutritional supplements, 62

O observations, vii, 11, 12, 51, 68 obstruction, 62 occlusion, 22, 28, 47 occupational therapy, 44 older people, vii, 5 olfactory, 20, 26, 29, 30, 41, 99, 105, 114, 116 olfactory bulb (OB), 20, 22, 99, 105, 106, 114 olfactory epithelium, 29 oligodendrocytes, 9, 11, 21, 31, 37, 41, 44, 46, 47, 49, 53, 54, 55, 56, 93, 94, 107 oligodendroglia, 46

129

oncogene(s), 75, 85 oocyte(s), 35, 36, 39 optimization, 88, 89 Oregon, 58 organ, 60, 62, 66, 68, 74 Orphan Drug, viii, 59, 68, 79, 89 Osiris Therapeutics, viii, 59, 62, 69, 71, 72 osteogenic, 63, 72 oxide, 23

P pain, 62 paracrine, 57, 100, 118 parameter, 50 parents, 35, 67 Paris, 40 Parkinson’s disease, 1, 23, 25 Parkinsonism, 118 particles, 79, 86 parvalbumin, 30 patents, 49, 99 pathogenesis, 6, 12, 91 pathogenic, 48 pathology, 1, 37, 41, 48, 51, 57, 98, 102, 110, 112, 113 pathways, 12, 31, 72, 96, 98, 108 patients, vii, viii, 5, 6, 11, 12, 14, 15, 22, 23, 38, 49, 59, 60, 62, 65, 66, 67, 68, 69, 72, 74, 75, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 90, 91, 95, 98, 111 PB lymphocytes (PBL), 79, 83, 88, 89 pediatric patients, viii, 59, 65 perforation, 62 periosteum, 66 peripheral blood, 59, 61, 62, 63, 66, 78, 90 peripheral blood mononuclear cell, 63 periventricular leukomalacia (PVL), 50 permeability, 107 permit, 60, 98 pharmaceutical, 71 pharmacological, 2, 10 pharmacology, vii phenotype(s), vii, viii, 6, 11, 19, 22, 25, 31, 44, 47, 51, 53, 63, 72, 84, 93, 94, 97, 102, 108, 113, 114 phosphate, 62 phosphorylates, 13 photolysis, 9, 31 photoreceptors, 97 physiopathology, vii, ix, 5, 14, 105, 112 placebo, 65 plaque, vii, 5 plasma, 78, 82 plasmid, 36

130 plasticity, 7, 14, 102, 111, 112, 114, 116, 117 platelet, 23, 30, 55, 82 platelet-derived growth factor (PDGF), 9, 30, 55, 116 play, 14, 35, 56, 68, 112 plexus, 28 pluripotent cells, 1, 33 polyethylene, 78, 90 polymer, 45, 54, 55 polymerase chain reaction (PCR), 81, 82 poor, 79, 84 population, 12, 13, 20, 22, 45, 53, 55, 61, 64, 66, 68, 80, 93, 94, 97, 99, 106, 110 precipitation, 13, 20 preclinical, 50, 56, 68, 81, 82, 85 precursor cells, 31, 66, 118 predictors, 60 prednisone, 61 prevention, viii, 59, 75 primate(s), 2, 26, 27, 38, 54, 106, 116, 118 probability, 85 procedures, 23, 25, 45, 59, 97, 105 production, 10, 47, 53, 80, 82, 83, 88 progenitor cells, 7, 9, 13, 16, 21, 22, 23, 27, 28, 30, 31, 32, 34, 35, 37, 39, 40, 41, 45, 49, 51, 54, 55, 57, 58, 80, 85, 94, 99, 100, 101, 102, 107, 108, 115, 116, 117, 118 progenitors, 16, 28, 29, 30, 40, 41, 46, 61, 80, 85, 86, 89, 100, 101, 115, 116 program, 71 progressive, 44, 83, 116 pro-inflammatory, 59, 63, 65, 67, 72 proliferation, 6, 10, 12, 15, 17, 18, 23, 27, 28, 29, 30, 47, 49, 53, 57, 61, 63, 70, 71, 73, 74, 80, 91, 94, 95, 99, 100, 105, 106, 108, 114, 115, 116, 117, 118 promote, 7, 9, 12, 22, 23, 25, 32, 35, 37, 39, 41, 51, 56, 57, 95, 96, 98, 113 promoter, 46 property, 102 prophylaxis, 68, 74 prostaglandin, 63 protein(s), 6, 13, 21, 27, 28, 35, 36, 44, 45, 46, 47, 61, 93, 95, 96, 99, 101, 107, 116, 118 protocol(s), 35, 36, 61, 62, 67, 68, 79, 80, 82, 84, 85, 86, 89, 97 proto-oncogene, 91 PSA, 10 psychiatric disorders, 16 psychiatry, 16, 27, 116 psychosocial, 9 psychosocial stress, 9 public, 12

Index public health, 12 purification, 62, 95

Q quality control, 74 quality of life, 49, 84 quinolinic acid, 22, 28 quinones, 26

R radiation, 60, 68, 97 radiation therapy, 97 rain, 45 range, vii, viii, ix, 1, 5, 11, 19, 25, 26, 33, 34, 37, 43, 44, 50, 93, 95, 105, 113 rash, 60 rat(s), 7, 8, 9, 10, 12, 15, 16, 17, 22, 23, 26, 27, 28, 29, 30, 31, 40, 41, 46, 47, 50, 53, 54, 55, 57, 58, 64, 65, 70, 72, 73, 94, 96, 99, 100, 101, 106, 114, 115, 116, 117, 118 reagents, 49, 107 receptors, 16 recognition, 61 reconstruction, 44 recovery, 7, 10, 13, 20, 22, 23, 24, 25, 29, 31, 34, 35, 37, 38, 41, 48, 50, 53, 56, 58, 67, 78, 102, 111, 112, 113, 116, 117 red blood cells, 83 reduction, 12, 15, 17, 48, 62, 67, 72 refractory, viii, 59, 65, 75 regenerate, ix, 1, 105, 111 regeneration, ix, 1, 22, 23, 27, 50, 61, 64, 94, 96, 98, 105, 106 regenerative medicine, vii, 71 regression, 41 regulation(s), 34, 49, 50, 111 rehabilitation, 117 rejection, 19, 24, 49, 50, 64, 66, 67, 68, 73, 74, 75 relationship, 8, 10, 51, 63, 99 relevance, 25, 37, 110 remission, 62 remyelination, 50 renal dysfunction, 49 repair, ix, 1, 22, 27, 37, 56, 66, 96, 105, 113, 115, 117 replication, 6, 10, 116 repression, 28, 117 residual disease, 67 resistance, 79, 81 respiratory, 84

Index retention, 63 retina, 30, 31, 97, 101 retinal disease, 97 retrovirus(es), 77, 85, 91, 108 Reynolds, 11, 17, 29, 39, 45, 49, 54, 55, 75, 93, 99, 100, 101, 102, 108, 109, 114, 117 ribonucleic acid, 82 risk, 24, 44, 49, 50, 59, 61, 68, 76, 85, 90, 97, 98, 111, 112 risk factors, 85 RNA, 21, 28, 107, 118 RNAi, 36 rodent(s), 13, 20, 22, 25, 45, 46, 93, 96, 100, 105, 106, 107, 108, 109, 112, 118 royalty, 52

S safety, 49, 65, 68, 85, 88, 89 saline, 62 San Raffaele Telethon Institute for Gene Therapy, viii, 77, 78, 89 SAR, 45, 62, 79 saturation, 114 scabies, 84 science, 2, 10, 39 sclerosis, 7, 10, 25 search, 34 secrete, 32, 39, 45, 48, 53, 54, 55, 57, 61 secretion, 25, 32, 35, 40, 59, 63, 64, 67, 70, 72 seizures, 44, 49, 116 selective serotonin reuptake inhibitor, 12 self-renewal, 45, 46, 79, 94, 106, 110 self-repair, vii, 2, 8, 11, 19, 34, 112 septum, 25 series, 82 serotonin, 12, 13, 15, 18 serum, 46, 63, 66, 80, 87 severity, 60, 61, 63, 67, 68, 114 shares, 52, 62 sheep, 40 sibling(s), 67, 84 sickle cell anemia, 60, 74 side effects, 49, 60, 62, 66 signal transduction, 16 signaling, 13 signaling pathway, 13 sign(s), vii, 11, 48, 53, 84, 85 Singapore, 41 sites, 7, 19, 22, 23, 25, 34, 35, 37, 85, 96, 107, 111, 112, 113 skin, 60, 62, 73, 89, 97, 98, 102 solid tumors, 76

131

somatic cell, viii, 1, 33, 35, 40, 90 somatic cell nuclear transfer (SCNT), viii, 1, 33, 35, 36 somatic stem cells, 39, 102 sorting, 45, 97, 101 spatial learning, 117 specialized cells, ix, 1, 105 species, 5, 20, 35, 94, 95, 106 spectrum, 34 speed, 67, 83, 84 spinal cord, viii, 1, 9, 10, 20, 21, 23, 25, 27, 30, 31, 32, 37, 39, 41, 43, 44, 47, 50, 52, 53, 56, 57, 58, 97, 100, 101, 102, 107 spinal cord injury, viii, 31, 32, 37, 39, 43, 47, 50, 57, 97, 102 spine, 15 spontaneous abortion, 35 sprouting, 54, 111 stability, 66 statins, 23, 29 status epilepticus, 30, 115 stem cell culture, 55 stem cell lines, 3, 31, 39, 40 stem cell research, vii, viii, 1, 2, 33, 34, 35, 38, 103 stem cell therapy, viii, 37, 59, 68, 71, 72, 77, 87 stem cell transplantation, 67, 74, 75 stem cell(s), vii, viii, ix, 1, 2, 3, 7, 10, 11, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 44, 45, 46, 47, 49, 50, 51, 52, 54, 55, 56, 57, 58, 61, 62, 67, 68, 70, 71, 72, 73, 74, 76, 80, 85, 91, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 105, 106, 107, 108, 109, 113, 114, 116, 117, 118 steroid(s), 8, 15, 16, 66, 75 stock, 52 storage, 37, 41, 43, 44, 48, 51, 52, 53, 57, 98, 102 strategic, 54 strategies, vii, 7, 8, 11, 14, 19, 23, 24, 38, 51, 61, 68, 75, 84, 108, 116 strategy use, 61 stress, 12, 13, 16, 17, 18, 20, 27 striatum, 22, 25, 28, 29, 40, 46, 101, 107, 111 stroke(s), viii, 22, 23, 28, 29, 31, 41, 43, 44, 58, 101, 107, 111, 112, 114 stroma, viii, 59, 61 stromal, 29, 64, 71, 72, 73 stromal cells, 29, 64, 73 subcortical nuclei, 15 subcutaneous injection, 22, 23, 29 subgranular zone, 6, 20, 106 subventricular zone (SVZ), 6, 7, 11, 12, 15, 20, 22, 23, 25, 28, 37, 40, 54, 93, 94, 96, 105, 106, 107, 109, 111, 113

Index

132 success rate, 79 suffering, 15, 68, 88, 111 suicide, 37 supernatant, 80, 88 suppression, 70, 73 surgery, 24, 38, 62, 113 surgical, 25, 97, 98 survival, 25, 30, 31, 32, 33, 39, 45, 47, 50, 53, 60, 67, 72, 73, 77, 78, 91, 95, 101, 107, 115 survival rate, 60 survivors, 16, 60 susceptibility, 62 swelling, 62 Switzerland, 52 symptoms, 6, 12, 44, 60, 62, 67 synapse, 47 synaptic vesicles, 17 synaptogenesis, 29 syndrome, 60 synthesis, 50 systems, 60

T targets, 13, 16 T-cell(s), 59, 61, 63, 64, 65, 67, 68, 70, 77, 78, 79, 81, 82, 87, 90, 91 T-cell receptor, 82 technology, viii, 1, 33, 51, 69, 71 teens, 44 telomere, 40 temperature, 80 tendon, 61 tetanus, 78, 82 theory, vii, 2, 3, 11, 14, 16, 18, 27, 96, 106, 110, 116 therapeutic, vii, viii, 2, 5, 7, 8, 12, 19, 22, 25, 35, 36, 40, 44, 45, 48, 49, 50, 51, 52, 78, 84, 109, 113 therapeutic interventions, viii, 19, 22 therapeutics, 9, 74 therapy, vii, viii, 1, 2, 5, 7, 10, 11, 19, 22, 23, 24, 25, 26, 29, 33, 34, 35, 37, 38, 39, 41, 43, 44, 45, 46, 47, 49, 50, 51, 57, 59, 61, 62, 63, 65, 66, 67, 68, 71, 74, 77, 78, 79, 82, 83, 84, 85, 87, 88, 89, 90, 91, 93, 94, 95, 96, 97, 98, 109, 110, 113, 118 third party, 74 threshold level, 48 thrombocytopenia, 83, 87 thrombopoietin, 80 thymidine, 6, 12, 27, 96, 105, 107 thymopoiesis, 83 thymus, 46, 81 time, 5, 10, 17, 20, 41, 47, 48, 49, 64, 65, 67, 69, 82, 94, 106, 110, 112, 117

tissue, 19, 24, 32, 33, 34, 44, 45, 47, 50, 54, 59, 64, 66, 93, 94, 95, 97, 98, 106, 111, 113 T-lymphocytes, 78, 81, 88 toxic, 48, 78, 83 toxicity, 48, 50, 65, 81, 83 toxin, 56, 61 tracking, 22, 107 transcription, 21, 27, 36, 107 transcription factor(s), 21, 36, 107 transduction, 79, 81, 86, 91 transfection, 37 transformation, 68, 75, 76, 97, 108 transfusion, 59, 84 transgene, 28, 55, 79, 80, 86, 118 transgenic, 6, 9, 31, 35, 41, 46, 48, 54, 55, 95, 101, 116 transition, 36 transmembrane, 45 transplant, 39, 46, 48, 52, 59, 60, 67, 68, 72, 74, 75, 81, 87, 97 transplantation, viii, ix, 2, 7, 10, 19, 22, 24, 25, 30, 31, 33, 35, 37, 38, 40, 41, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 64, 65, 66, 67, 68, 69, 72, 74, 78, 83, 84, 89, 91, 93, 95, 96, 97, 98, 101, 102, 109 traumatic brain injury, 31, 112, 114, 118 tremor, 49 trial, viii, 34, 38, 43, 44, 48, 49, 56, 58, 65, 68, 71, 72, 79, 81, 83, 84, 85, 87, 89, 90 triggers, 9 tropism, 41 tryptophan, 64 tumor(s), 25, 31, 35, 37, 41, 44, 48, 51, 59, 61, 68, 76, 85, 98 tumor cells, 37, 51 tumor necrosis factor (TNF), 59, 70, 76 tumorigenic, 94 turnover, 77 tyrosine, 40, 80, 95 tyrosine hydroxylase, 40, 95

U UK, 88, 89 ulcerative colitis, 62 umbilical cord blood, 61, 68, 73, 74 undifferentiated, vii, ix, 1, 33, 44, 73, 93, 105 undifferentiated cells, vii, ix, 1, 33, 93, 105 United Nations, 35 United States, 16 uterus, 36

Index

V vaccination, 82, 83 validation, 18, 118 validity, 14, 85 values, 80, 86 variability, 95 variable, 78, 79, 82 variation, 36, 107 vascular, 23, 63 vascular endothelial growth factor (VEGF), 30, 63 vector, 36, 79, 82, 83, 84, 85, 86, 87, 89, 91 vehicles, 72 ventricle, 30, 48, 106 ventricular, 100, 105, 115 ventricular zone, 105 viral, 36 virus, 36, 80, 81, 83 vision, 44 visual, 2, 39, 99 vomiting, 62

133

W warrants, 86 Washington, 71 water, 109 wetting, 60 white matter, 47 wild type, 10 World Wide Web, 71

X X-irradiation, 5, 8, 17 X-ray, 12

Y yield, 35, 80, 86, 95, 97