Stem Cells and Regenerative Medicine: Patents and Clinical Trials (Stem Cells - Laboratory and Clinical Research Series)

STEM CELLS - LABORATORY AND CLINICAL RESEARCH SERIES

STEM CELLS AND REGENERATIVE MEDICINE. VOLUME 5. PATENTS AND CLINICAL TRIALS

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STEM CELLS - LABORATORY AND CLINICAL RESEARCH SERIES Focus on Stem Cell Research Erik V. Greer (Editor) 2004. ISBN: 1-59454-043-8 Trends in Stem Cell Research Erik V. Greer (Editor) 2005. ISBN: 1-59454-315-1 New Developments in Stem Cell Research Erik V. Greer (Editor) 2006. ISBN: 1-59454-847-1 Neural Stem Cell Research Erik V. Greer (Editor) 2006. ISBN: 1-59454-846-3 Stem Cell Therapy Erik V. Greer (Editor) 2006. ISBN: 1-59454-848-X Embryonic Stem Cell Research Erik V. Greer (Editor) 2006. ISBN: 1-59454-849-8 Frontiers in Stem Cell Research Julia M. Spanning (Editor) 2006. ISBN: 1-60021-294-8 Stem Cells and Cancer Devon W. Parsons (Editor) 2007. ISBN: 1-60021-517-3 Hematopoietic Stem Cell Transplantation Research Advances Karl B. Neumann (Editor) 2008. ISBN: 978-1-60456-042-8 Stem Cell Applications in Diseases Mikkel L. Sorensen (Editor) 2008. ISBN: 978-1-60456-241-5

Stem Cell Applications in Diseases Mikkel L. Sorensen (Editor) 2008. ISBN: 978-1-60876-925-4 (Online Book) Leading-Edge Stem Cell Research Prasad S. Koka (Editor) 2008. ISBN: 978-1-60456-268-2 Stem Cell Research Progress Prasad S. Koka (Editor) 2008. ISBN: 978-1-60456-308-5 Stem Cell Research Progress Prasad S. Koka (Editor) 2008. ISBN: 978-1-60876-924-7 (Online Book) Progress in Stem Cell Applications Allen V. Faraday and Jonathon T. Dyer (Editors) 2008. ISBN: 978-1-60456-316-0 Developments in Stem Cell Research Prasad S. Koka (Editor) 2008. ISBN: 978-1-60456-341-2 Developments in Stem Cell Research Prasad S. Koka (Editor) 2008. ISBN: 978-1-60741-213-7 (Online Book) Stem Cells and Regenerative Medicine. Volume 1. Adult Neurogenesis and Neural Stem Cells Philippe Taupin 2008. ISBN: 978-1-60456-472-3 Stem Cells and Regenerative Medicine. Volume 2. Embryonic and Adult Stem Cells Philippe Taupin 2008. ISBN: 978-1-60456-473-0 Stem Cells and Regenerative Medicine. Volume 3. Pharmacology and Therapy Philippe Taupin 2008. ISBN: 978-1-60456-474-7

Gut Stem Cells: Multipotent, Clonogenic and the Origin of Gastrointestinal Cancer Shigeki Bamba and William R. Otto 2008. ISBN: 978-1-60456-968-1 Stem Cell Transplantation, Tissue Engineering and Cancer Applications Bernard N. Kennedy (Editor) 2008. ISBN: 978-1-60692-107-4 Stem Cells and Regenerative Medicine. Volume 4. Neurological Diseases and Cellular Therapy Philippe Taupin 2010. ISBN: 978-1-60741-783-5 Stem Cells Philippe Taupin 2009. ISBN: 978-1-60692-214-9 Stem Cell Plasticity Suraksha Agrawal, Piyush Tripathi and Sita Naik 2009. ISBN: 978-1-60741-473-5 Neural Stem Cells and Cellular Therapy Philippe Taupin 2010. ISBN: 978-1-60876-017-6 Stem Cells and Regenerative Medicine. Volume 5. Patents and Clinical Trials Philippe Taupin 2010. ISBN: 978-1-60741-782-8

STEM CELLS - LABORATORY AND CLINICAL RESEARCH SERIES

STEM CELLS AND REGENERATIVE MEDICINE. VOLUME 5. PATENTS AND CLINICAL TRIALS

PHILIPPE TAUPIN

Nova Biomedical Books New York

Copyright © 2010 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, 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 in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Taupin, Philippe. Stem cells and regenerative medicine / Philippe Taupin. p. ; cm. Includes bibliographical references and index. ISBN : 978-1-61728-666-7 (E-Book) 1. Stem cells. 2. Regeneration (Biology) I. Title. [DNLM: 1. Stem Cells. 2. Regenerative Medicine. QU 325 T227s 2008] QH588.S83.T38 2008 616'.02774--dc22 2008004342

Published by Nova Science Publishers, Inc.  New York

Contents Preface

ix

Introduction:

Therapeutic Neuronal Stem Cells: Patents at the Forefront

1

Chapter I

Adult Neurogenesis and Pharmacology in Alzheimer’s Disease and Depression

9

Adult Neurogenesis in the Pathogenesis of Alzheimer’s Disease

17

Chapter III

Therapeutic Potential of Adult Neural Stem Cells

21

Chapter IV

Human Neural Progenitor and Stem Cells

33

Chapter V

Cryopreservation of Early Postmitotic Neuronal Cells in Culture

41

Chapter VI

Adult Neural Stem Cells for the Treatment of Neuroinflammation

49

Adult Periodontal-Derived Neural Progenitor and Stem Cells

57

Magnetic Resonance Imaging for Monitoring Neurogenesis in the Adult Hippocampus

65

Apigenin and Related Compounds Stimulate Adult Neurogenesis

75

Chapter X

Nootropic Agents Stimulate Neurogenesis

83

Chapter XI

Fourteen Compounds and their Derivatives for the Treatment of Diseases and Injuries Characterized by Reduced Neurogenesis and Neurodegeneration

89

Brevetoxin Derivative Compounds for Stimulating Neuronal Growth

99

Chapter II

Chapter VII Chapter VIII Chapter IX

Chapter XII

viii

Contents

Chapter XIII

OTI-010 Osiris Therapeutics/JCR Pharmaceuticals

107

Chapter XIV

ADA-Transduced Hematopoietic Stem Cell Therapy for ADA-SCID

127

Index

145

Preface Stem Cells and Regenerative Medicine Volume 5 - Patents and Clinical Trials aims at providing an overview and in depth analysis of recent developments in stem cell research and therapy. It is composed of recently published review articles that went through peer-review process. Stem cells are the building blocks of the body. They can develop into any of the cells that make up our bodies. 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, cerebral strokes, and traumatic brain and spinal cord injuries. Stem cell research is therefore as important for our understanding the physio- and pathology of the body, as for development and therapy, including for the nervous system. Volume V provides an overview and in depth analysis of recent developments in the front of patent applications filed and clinical trials initiated in the field of stem cells, in the aim to bring stem cell research to therapy. Introduction - Background: Neural stem cells (NSCs) hold the promise to cure a broad range of neurological diseases and injuries, particularly neurodegenerative diseases and spinal cord injuries. The recent confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS opens new opportunities and avenues for cellular therapy. Objectives: To provide an overview of the current patent situation related to NSCs and to highlight the limitations and challenges of bringing NSC research to therapy. Method: Reviewing the early studies and patents in NSC research. Conclusion: NSCs are in clinical trials for Batten and Parkinson’s diseases. However, clinical development and other limitations will make it difficult for pharmaceutical/biotech companies to break even with these early patents. Chapter I - Background: Neural stem cells (NSCs) hold the promise to cure a broad range of neurological diseases and injuries, particularly neurodegenerative diseases and spinal cord injuries. The recent confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS opens new opportunities and avenues for cellular therapy. Objectives: To provide an overview of the current patent situation related to NSCs and to highlight the limitations and challenges of bringing NSC research to therapy. Method: Reviewing the early

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studies and patents in NSC research. Conclusion: NSCs are in clinical trials for Batten and Parkinson’s diseases. However, clinical development and other limitations will make it difficult for pharmaceutical/biotech companies to break even with these early patents. Chapter III - 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 NSCs reside in the adult CNS, new treatments for neurological diseases and injuries are being considered. Particularly, the transplantation of adult-derived neural progenitor and stem cells to restore brain functions. In 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. Chapter IV - Background: The application is in the field of neural stem cells (NSCs) and cellular therapy. Objective: It aims at establishing conditions for the isolation and propagation of neural progenitor and stem cells from human fetal tissue, with high rate of growth and high yields of differentiation into the neuronal, astroglial and oligodendroglial pathways. Methods: Neural progenitor and stem cells were isolated from fetal forebrain tissue and propagated as neurospheres, in defined medium in the presence of leukemia inhibitory factor (LIF). Three protocols were designed to differentiate human fetal neural progenitor and stem cells into their progenies. Results: The application claims the generation of human fetal neural progenitor and stem cells with a doubling rate between 5-10 days. It claims the differentiation of the neural progenitor and stem cells in vitro, into neurons, astrocytes and oligodendrocytes with high yields, e.g., 20 to 35% for neuronal cells. Conclusion: The establishment of human neural progenitor and stem cells with high rate of growth and high yields of differentiation provides a source of cells for therapy, particularly for the treatment of neurodegenerative diseases, like Alzheimer’s and Parkinson’s diseases. Chapter V - Background: The application is in the field of embryonic stem cells (ESCs) and the cryopreservation of neuronal cells in culture. Objective: It aims at establishing conditions for the cryopreservation of cultured neuronal cells readily usable, upon thawing. That is cryopreserved neuronal cells with a high rate of survivability and with a potential to differentiate into mature neuronal cells rapidly, after thawing. Methods: Neuronal precursor cells and neuronal cells were generated in vitro, from cultures of human ESCs (hESCs). Neuronal cells were frozen and cryopreserved at a stage when they begin to express neuronal class III β-tubulin. Results/conclusion: The application claims that neuronal cells elicit a rate of survivability greater than 50% and neurite outgrowth within 10 to 14 hours, after thawing. The cryopreservation of post-mitotic neuronal or neural-like cells provides a source of nerve cells readily usable for research and therapy. Chapter VI - Background: The application is in the field of stem cells and their therapeutic application for the treatment of inflammation. Objective: It aims at characterizing the potential of adult-derived neural progenitor and stem cells for the treatment of inflammation of the central nervous system (CNS). Methods: Neural progenitor and stem cells were isolated and expanded from the subventricular zone (SVZ) of adult mice (aNSCs).

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They were administered intravenously in an animal model of multiple sclerosis (MS). Results: Mice transplanted either at the disease onset or at the onset of the first relapse show clinical signs of improvements. Adult NSCs exert their therapeutic activity by reducing neuroinflammation. Conclusion: The application claims the use of aNSCs and multipotent somatic stem cells for the treatment of inflammation, associated with neurological diseases, disorders and injuries particularly, and for inducing tolerance to the immune central and/or peripheral system. Chapter VII - The application is in the field of neural stem cells (NSCs) and cellular therapy. It aims to identify and characterize neural progenitor and stem cells from adult periodontal tissue. Neural progenitor and stem cells were isolated and characterized from periodontal tissue originating from biopsies of adult patients. Adult human periodontalderived neural progenitor and stem cells can be induced to differentiate into neuronal and glial cells, osteoblasts and cells of the periodontium. They survive and integrate when transplanted into organotypic hippocampal slice cultures. The application claims the use of periodontal neural progenitor and stem cells for cellular therapy, particularly for the treatment of the periodontal diseases and neurodegenerative diseases and neurological injuries. Periodontal tissue can be harvested with minimal invasive procedures from the patient himself, providing a promising source of tissue for NSC-based therapy and autologous transplantation. Chapter VIII - Background: The application is in the field of bioimaging and adult neurogenesis. Objective: It aims at correlating the volume of cerebral blood (CBV) in the dentate gyrus (DG) of the human hippocampus, determined by magnetic resonance imaging (MRI), with neurogenesis in the brain of adult rodents. Methods: Adult mice were submitted to voluntary exercise or administration of fluoxetine or valproic acid (VPA). The CBV of DG was determined by MRI and neurogenesis was quantified by immunohistofluorescence. The CBV in human subjects selected and grouped according to their fitness activity was determined by MRI in the DG. Results: A selective increase in the CBV of the DG is observed in rodents housed in activity cages or administered with fluoxetine and VPA. A selective increase in the CBV of the DG is also observed in exercising humans. The selective increase of the CBV in the human DG correlates with the selective increase of the CBV in the DG and neurogenesis induced by exercise or fluoxetine and VPA in rodents. Conclusion: This indicates that neurogenesis is increased in the DG of exercising humans. The application claims the imaging of the DG of patients by MRI as a paradigm to monitor neurogenesis and identify treatments involving stimulation of neurogenesis. Chapter IX - The application is in the field of adult neurogenesis and its therapeutic potential. It aims to characterize the activity of apigenin and related compounds on adult neurogenesis in vivo and in vitro. Apigenin and related compounds are derivatives used in food products. They were administered intraperitoneally and orally in adult rodents and assessed for their activity in promoting the generation of neuronal cells and learning and memory performance. They were also tested on adult rat hippocampal-derived neural progenitor and stem cells to assess their neurogenic property. Apigenin and related compounds stimulate adult neurogenesis in vivo and in vitro, by promoting neuronal differentiation. Apigenin promotes learning and memory performance in the Morris water task. The application claims the use of apigenin and related compounds for stimulating adult

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neurogenesis and for the treatment of neurological diseases, disorders and injuries, by stimulating the generation of neuronal cells in the adult brain. Chapter X - The application is in the field of adult neurogenesis, neural stem cells and cellular therapy. It aims to characterize the activity of nootropic agents on adult neurogenesis in vitro. Nootropic agents are substances improving cognitive and mental abilities. AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) and nootropic agents were assessed for the potential to differentiate human neural progenitor and stem cells into neuronal cells in vitro. They were also tested for their behavioural activity on the novel object recognition task. AMPA, piracetam, FK-960 and SGS-111 induce and stimulate neuronal differentiation of human-derived neural progenitor and stem cells. SGS-111 increases the number of visits to the novel object. The neurogenic activity of piracetam and SGS-111 is mediated through AMPA receptor. The neurogenic activity of SGS-111 may contribute and play a role in its nootropic activity. These results suggest that nootropic agents may elicit some of their effects through their neurogenic activity. The application claims the use of nootropic agents for their neurogenic activity and for the treatment of neurological diseases, disorders and injuries, by stimulating or increasing the generation of neuronal cells in the adult brain. Chapter XI - The application is in the field of adult neurogenesis and neuronal degeneration and regeneration. It aims to characterize the activity of 15 compounds and their derivatives on adult neurogenesis, apoptosis, necrosis and neuron dysfunction in vitro. The activity of the compounds and their derivatives was tested on cultures of adult human-derived neural progenitor and stem cells, in presence of staurosporine, peptide β-amyloid and hydrogen peroxide, and was assessed by immunocytology. The compounds and their derivatives stimulate the differentiation of adult-derived neural progenitor and stem cells into the neuronal pathway, and inhibit staurosporine-induced apoptosis, peptide β-amyloidinduced necrosis and hydrogen peroxide-induced neuron dysfunction. The application claims the use of the 15 compounds and their derivatives for the treatment of diseases and injuries particularly characterized by reduced neurogenesis and neuronal loss, including Alzheimer’s disease (AD), depression, cerebral strokes and traumatic brain and spinal cord injuries. Chapter XII - The application is in the field of neuronal growth and the activity of brevetoxins and their derivatives. It aims to characterize the activity of four generic formulae of brevetoxin derivatives - formulae I, II, III, and IV - and their compounds on the growth of neurites. The activity of the brevetoxin derivative compounds was tested on primary cultures of neocortical neurons. It was assayed in the presence and absence of antagonists of receptors and second-messenger signaling pathways, particularly NMDA receptors and the calmodulindependent protein-kinase kinase pathway. Brevetoxin derivatives stimulate neurite growth, particularly the growth of minor processes from which the axons form, on neurons in primary cultures. The activity is mediated by voltage-gated sodium channels and the N-methyl-D2+

pathway. The application claims the use of the four aspartate-mediated intracellular Ca generic formulae of brevetoxin derivatives (I, II, III, and IV) and their compounds for enhancing neuronal growth and for the treatment of neurodegenerative diseases and neurological disorders and injuries, such as Alzheimer’s disease, amyotrophic lateral sclerosis, cerebral strokes, traumatic brain, and spinal cord injuries. Chapter XIII - Osiris Therapeutics is developing the donor-derived mesenchymal stem cell (MSC) therapy OTI-010, which repopulates the bone marrow stroma and thus supports

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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 XIV - 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.

Introduction: Therapeutic Neuronal Stem Cells: Patents at the Forefront* Abstract Background: Neural stem cells (NSCs) hold the promise to cure a broad range of neurological diseases and injuries, particularly neurodegenerative diseases and spinal cord injuries. The recent confirmation that neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS opens new opportunities and avenues for cellular therapy. Objectives: To provide an overview of the current patent situation related to NSCs and to highlight the limitations and challenges of bringing NSC research to therapy. Method: Reviewing the early studies and patents in NSC research. Conclusion: NSCs are in clinical trials for Batten and Parkinson’s diseases. However, clinical development and other limitations will make it difficult for pharmaceutical/biotech companies to break even with these early patents.

1. Background Neural stem cells (NSC) are the self-renewing multipotent cells that generate the main phenotypes of the nervous system, neurons, astrocytes and oligodendrocytes [1]. They hold the potential to treat and cure a broad range of neurological diseases and injuries, ranging from neurodegenerative diseases, like Alzheimer and Parkinson’s diseases, to retinal diseases, cerebral strokes and spinal-cord injuries. Neurogenesis in the adult mammalian brain occurs primarily in two regions, the dentate gyrus (DG) of the hippocampus and subventricular zone (SVZ), in various species including human [2,3]. It is hypothesized that newborn neuronal cells in the adult brain originate from residual stem cells. In support of this contention, neural progenitor and stem cells have been isolated and characterized in vitro, from various regions of the adult CNS [4]. The confirmation that neurogenesis occurs in the adult mammalian brain and that NSCs reside in the adult mammalian CNS has tremendous consequences for our understanding of developmental biology and therapy. The adult brain *

Declaration of interest; The author states no conflict of interest and has received no payment in preparation of this manuscript.

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has the potential to, and may be amenable to, repair. Therapeutic interventions may involve the stimulation of endogenous neural progenitor or stem cells, and the transplantation of adult-derived neural progenitor and stem cells.

2. Neural Stem Cells, Therapeutic Potential and Patents The advent of adult neurogenesis and NSC research has been associated with the filing and granting of patents applications. The criteria for selection of the patents cited was to identify some of the early patents of the field of NSC research, on which several pharmaceutical and biotech companies have been building and which have resulted in clinical trials. Patent WO/1993/001275, assigned to S Weiss and BA Reynolds, and first disclosed in 1993, lays the ground for protection of the intellectual property, for the preparation and use of neural progenitor and stem cells in vitro, ex vivo and in vivo [5]. With other patents, including patents WO/1995/013364, WO/1996/015224 and WO/1996/015226, it claims the use of neural progenitor and stem cells, and their progeny, isolated as neurospheres of various ages and from various species, from either normal or diseased CNS tissue, as a model to study neural development, function, to screen the effect of biological agents and to develop novel therapies [6-8]. These patents are based on the published work by Reynolds and Weiss [9], reporting the isolation and characterization of the neural progenitor and stem cells from the fetal and adult mammalian tissues [4,10]. In 2001, Palmer and collaborators reported the isolation and characterization of neural progenitor cells from human postmortem tissues [11]. The inventors filed for a patent application, patent WO/2002/036749, to claim their work; the culture and expansion of stem cells from postmortem tissues for therapy, and methodologies to isolate and propagate stem cells from biopsies and postmortem tissues [12]. Patents have been filed for methods to improve culture conditions, like promoting survival, proliferation and differentiation of neural progenitor and stem cells in vitro. Patent WO/2000/050572 covers a method for the in vitro proliferation of NSC cultures, using a growth factor and collagenase [13]. Collagenase is used as a cell-dissociating agent; its use improves cell isolation, viability and proliferation. Patent WO/2000/033791 relates to the isolation, purification and use of the co-factor of fibroblast growth factor-2 (FGF-2), glycosylated cystatin C (CCg) [14]. FGF-2 requires CCg for its mitogenic activity on selfrenewing multipotent NSCs in vitro, from single cells, to stimulate neurogenesis in vivo, and to promote the growth and expansion of neural progenitor and stem cells, isolated and cultured for human postmortem tissues [11,15]. These factors and conditions may contribute to promote the use of NSCs for therapy. Fetal and adult stem cells are multipotents; they generate lineage-specific cell types restricted to the tissues from which they are derived. Embryonic stem cells are pluripotents; they generate cells of the three germ layers – ectoderm, mesoderm and endoderm. Neural progenitor and stem cells, isolated from the adult brain and cultured in vitro, have been reported to give rise to lineages other than neuronal, in vitro and ex vivo, particularly blood cells [16]. Stem cells isolated from adult tissues other than the brain, like the skin, blood and bone marrow, have been reported to give rise to lineages other than the one from which they

Introduction: Therapeutic Neuronal Stem Cells: Patents at the Forefront

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are derived, particularly neuronal lineages [17-19]. It is proposed that adult stem cells would have a broader potential than previously thought [20]. This broader potential would have tremendous possibilities for cellular therapy, particularly in the CNS, as neuronal phenotypes could be generated from tissues other from than the nervous system. Of particular interest would be the generation of nerve cells from skin tissues, potentially allowing autologous transplantation and limiting potential damage to the CNS associated with the isolation of cells. Patent applications for the broader potential of adult stem cells, particularly adult NSCs to generate haematopoietic cells and non-haematopoietic lineages generated from haematopoietic stem cells, have been filed WO/1999/016863 and WO/2001/071016 [21,22]. However, the broader potential of stem cells is the source of debates and controversies, as it could originate from cell fusion, transdifferentiation or transformation, all of which could have adverse effects on their therapeutic potentials. Alternatively, lineage non-specific adult stem cells could be generated by de-differentiation or reprogramming, or by the introduction of developmental genes to generate stem or progenitor cells of a tailored phenotype [23,24]. Adult-derived neural progenitor and stem cells can be genetically modified [25] to express tropic factors or neurotransmitter-synthesizing enzymes. Patent applications on methods to genetically engineered neural progenitor and stem cells in vitro have also been submitted. These include various processes for genetically modifying neural progenitor and stem cells, including with reporter genes like the Escherichia coli β-galactosidase or green fluorescent protein, and the isolation of neural progenitor and stem cells from transgenic animals. Patent US-05,750,376 covers the in vitro growth and proliferation of genetically modified NSCs [26]. Adult neural progenitor and stem cells have been genetically engineered to express acid sphingomyelinase reverse lysosomal storage pathology in animal models of Niemann-Pick’s disease [27], a strategy subsequently filed under patent WO/2006/074387 [28]. Hence, NSCs offer a tremendous potential not only for the treatment of neurodegenerative diseases and injuries but also for the treatment of genetic diseases of the CNS.

3. Expert Opinion The advent of adult neurogenesis and NSC research offer tremendous opportunities for cellular therapy in the CNS. Neural progenitor and stem cells can be isolated either from patients themselves - allowing autologous transplantation - or from postmortem tissues, providing unlimited sources of material for transplantation. Neural progenitor and stem cells are currently in, or are being considered for, clinical trials for the treatment of Batten and Parkinson’s diseases [29]. Batten disease (BD) is a rare and fatal genetic disorder that begins in childhood. In BD, various genes responsible for the breakdown of lipofuscins in the nerve cells are missing or faulty. It is proposed that grafted stem cells would migrate to the areas of neurodegeneration and compensate for the missing enzymes. Fetal-derived neural progenitor and stem cells are being utilized in the clinical trials for BD, whereas autologous transplantation is considered for the clinical trials for Parkinson’s disease [30,31]. Although stem cell therapy is promising, there are limitations, pitfalls and risks to consider and monitor, particularly with the use of neural progenitor and stem cells. Among

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these is the fact that NSCs are still elusive; they have yet to be unequivocally identified and characterized [32,33]. Current protocols devised to isolate and culture self-renewing multipotent NSCs in vitro yield heterogeneous populations of neural progenitor and stem cells [34]. Some authors have used strategies - for example the combination of the use of antibodies against membranous molecular markers and fluorescence-activated cell sorting specifically to isolate and purify subpopulations of cells characterized as self-renewing multipotent NSCs in vitro. Patents WO/2000/047762, WO/2004/020597 and US-7,381,561 disclose methods for identifying, isolating and enriching neural stem/progenitor cells, and for using antibodies to membranous markers, like CD49, CD133 CD15 as well as proprietary monoclonal antibodies [35-37]. However, these populations of cells expand as heterogeneous populations in vitro, limiting their therapeutic potential. To circumvent this, it is proposed to predifferentiate in vitro neural progenitor and stem cells toward the neuronal, astroglial or oligodendroglial lineages prior to transplantation. One of the limitations of such a strategy would concern in particular predifferentiated neuronal cells that might not integrate with the neuronal network upon transplantation. Among other limitations, pitfalls and risks to be considered are the controversies and debates over the broader potential of adult stem cells and the risks of tumour formation and inflammation that are associated with the transplantation of neural progenitor and stem cells, particularly if these neural progenitor and stem cells originate from donors whose compatibility may not be matched to those of the patient. There are also risks with autologous transplantation, as the isolation of brain tissues represents an invasive surgery, with risks to the patients [4]. The scientific and technical challenges that need to be overcome also have significant consequences for the pharmaceutical/biotech companies engaged in this field of research and development. Due to the lengthy and multiple processes involved in bringing NSC research to therapy, the 20-year protection of patents may not be sufficient for a pharmaceutical/biotech company to bring a product to therapy within a time-frame that allows it to benefit from the protection of its intellectual property. This is a particular concern for patented discoveries relating to seminal studies conducted in an academic environment, university or private research institute, where the process of filing patent applications is, in most cases, dictated by the publication process of the scientific manuscripts relating these inventions. Nonetheless, several patents filed originally by universities or private research institute, and referenced in this manuscript, have originated start-ups, giving them a research niche and value for raising funds. Successful pharmaceutical/biotech companies have built-up their portfolios of intellectual property from these ‘early’ patents, giving other protections to their discoveries and securing more time in their endeavour to bring their technology to therapy. In all, adult-derived neural progenitor and stem cells represent a tremendous opportunity for cellular and gene therapy. But, their potential to restore brain function remains to be validated. In this respect, limitations, pitfalls and risks need to be assessed and resolved. Among these, NSCs need to be identified unequivocally and characterized, neural progenitor and stem cells need to be maintained as homogenous populations in culture, and their broader potentials need to be fully understood and characterized. Their tumorigenic and inflammatory potentials must be monitored. Gene-based therapy of NSCs may also bolster cellular therapy, broadening their therapeutic potential to genetic diseases. Beside its therapeutic potential,

Introduction: Therapeutic Neuronal Stem Cells: Patents at the Forefront

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NSC biology has the potential to better understand and unravel the mechanisms underlying neurological diseases and disorders, particularly by isolating and characterizing neural progenitor and stem cells from patients, leading to new discoveries and clinical applications.

Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Therapeutic neuronal stem cells: patents at the forefront. Expert Opinion on Therapeutic Patents 18(10):1107-10. Copyright 2008, Informa UK Ltd.

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McKay R. Stem cells in the central nervous system. Science 1997; 276: 66-71 Eriksson PS, Perfilieva E, Bjork-Eriksson T, et al. Neurogenesis in the adult human hippocampus. Nat Med 1998; 4: 1313-7, •• Tissue samples originate from patients treated with bromodeoxyuridine (BrdU), a marker of DNA synthesis, as part of their treatments. Immunohistology for BrdU reveals the presence of newborn neuronal cells, born during the treatment, with BrdU, in the adult DG. [3] Curtis MA, Kam M, Nannmark U, et al. Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Science 2007; 315: 1243-9 [4] Taupin P. Neural progenitor and stem cells in the adult central nervous system. Ann Acad Med Singapore 2006; 35: 814-7 [5] Weiss S, Reynolds B. Novel growth factor-responsive progenitor cells which can be proliferated in vitro. WO/1993/001275; 1993 [6] Weiss S, Reynolds BA, Van Der Kooy D, et al. In situ modification and manipulation of stem cells of the central nervous system. WO/1995/013364; 1995 [7] Weiss S, Reynolds B. In vitro induction of dopaminergic cells. WO/1996/015224; 1996 [8] Weiss S, Reynolds B. Regulation of neural stem cell proliferation. WO/1996/015226; 1996 [9] Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992; 255: 1707-10 •• Neural progenitor and stem cells are isolated from the striatal area containing the SVZ, one of the main neurogenic regions of the adult mammalian brain. Neural progenitor and stem cells are propagated and cultured, as neurospheres, in defined medium, in the presence of high concentration of trophic factor, epidermal growth factor. [10] Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 1992; 12: 4565-74 [11] Palmer TD, Schwartz PH, Taupin P, et al. Cell culture. Progenitor cells from human brain after death. Nature 2001 ; 411: 42-3, • Post-mortem neural progenitor and stem

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Philippe Taupin cells were isolated and cultured in vitro, in defined medium, in the presence of a cocktail of trophic factors and CCg. Palmer TD, Gage FH, Schwartz PH, Taupin PJ. Postmortem stem cells. WO/2002/036749; 2002 Uchida N. Use of collagenase in the preparation of neural stem cell cultures. WO/2000/050572; 2000 Gage FH, Taupin PJ, Ray. Co-factors for trophic factors, and methods of use thereof. JWO/2000/033791; 2000 Taupin P, Ray J, Fischer WH, et al. FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. Neuron 2000; 28: 385-97 Bjornson CR, Rietze RL, Reynolds BA, et al. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999; 283: 534-7 Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000; 290: 1775 -9 Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000; 290: 1779-82 Toma JG, Akhavan M, Fernandes KJ, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001; 3: 778-84 D’Amour KA, Gage FH. Are somatic stem cells pluripotent or lineage-restricted? Nat Med 2002; 8: 213 -4 Bjornson CR, Rietze RL, Reynolds BA, Vescovi AL. Generation of hematopoietic cells from multipotent neural stem cells. WO/1999/016863; 1999 Lagasse E, Weissman IL. Pluripotential stem cells. WO/2001/071016; 2001 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126: 663-76 Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318: 1917-20 Gage FH, Coates PW, Palmer TD, et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci USA 1995; 92: 11879 -83 Weiss S, Reynolds B, Hammang JP, Baetge EE. In vitro growth and proliferation of genetically modifi ed multipotent neural stem cells and their progeny. US-05,750,376; 1998 Shihabuddin LS, Numan S, Huff MR, et al. Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology. J Neurosci 2004; 24: 10642-51 Uchida N, Jacobs Y, Tamaki S. Methods for the treatment of lysosomal storage disorders. WO/2006/074387; 2006 Taupin P. HuCNS-SC (StemCells). Curr Opin Mol Ther 2006; 8: 156-63 StemCells, Inc. website. Available from: http://www.stemcellsinc.com NeuroGeneration website. Available from: http://www.neurogeneration.com Seaberg RM, van der Kooy D. Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J Neurosci 2002; 22: 1784 -93

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[33] Ray J, Gage FH. Differential properties of adult rat and mouse brain-derived neural stem/progenitor cells. Mol Cell Neurosci 2006; 31: 560-73 [34] Reynolds BA, Rietze RL. Neural stem cells and neurospheres-re-evaluating the relationship. Nat Methods 2005; 2: 333-6 [35] Buck DW, Uchida N, Weissman I. Enriched central nervous system cell populations. WO/2000/047762; 2000 [36] Uchida N, Capela A. Enriched central nervous system stem cells and progenitor cell populations, and methods for identifying, isolating and enriching for such populations. WO/2004/020597; 2004 [37] Uchida N, Capela A. Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations. US-7,381,561; 2008

Chapter I

Adult Neurogenesis and Pharmacology in Alzheimer’s Disease and Depression Abstract The confirmation that neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) has significantly contributed to modify our understanding of the functioning of the nervous system, and hold the promise to treat a broad range of neurological diseases and injuries. Adult neurogenesis is modulated in neurological diseases and disorders, like in Alzheimer’s disease (AD) and depression, and by drugs used to treat these diseases and disorders. This suggests that adult neurogenesis is affected in the pathologies of the nervous system, and mediates the activities of drugs, used to treat neurological diseases and disorders. Hence, adult neurogenesis is as important for our understanding of the etiology and pathogenesis of neurological diseases and disorders, as for their pharmacology. Research on the pharmacology of adult neurogenesis may lead to new treatments and drug design.

Introduction The advent of adult neurogenesis and NSC research has opened a new era in brain research and therapy. The confirmation that adult neurogenesis occurs in the adult brain and NSCs reside in the adult CNS, in mammals including in human, suggests that adult newborn neuronal cells contribute to the functioning of the nervous system and that the adult brain may be amenable to repair [1,2]. Cellular therapy may involve the stimulation of endogenous neural progenitor or stem cells, or the transplantation of neural progenitor and stem cells, to repair degenerated or damaged pathways [3]. Research is also actively engaged in characterizing the contribution and involvement of adult neurogenesis to the pathologies of the nervous systems, particularly neurological diseases and disorders. Over the past years, significant studies have linked adult neurogenesis to neurological diseases and disorders, particularly AD and depression, and their pharmacological treatments. Hence, new drugs and strategies may be designed and developed

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to treat neurological diseases and disorders; the stimulation of endogenous neurogenesis by drugs and the mediation of drugs activity by newborn neuronal cells, to promote functional recovery.

Adult Neurogenesis in Alzheimer’s Disease Studies from autopsies reveal that the expression of markers of the cell cycle and immature neuronal cells, like doublecortin and polysialylated nerve cell adhesion molecule, is increased in hippocampal regions, particularly the dentate gyrus (DG), of the brains of AD patients [4]. This suggests that neurogenesis is enhanced in the hippocampus of patients with AD. In animal models of AD, neurogenesis is enhanced in the DG of transgenic mice that express the Swedish and Indiana amyloid precursor protein (APP) mutations, a mutant form of human APP [5]. It is decreased in the DG and subventricular zone (SVZ) of other animal models, like mice carrying a mutant form of presenilin-1 (PS-1) or APP [6,7]. It is decreased in the DG of platelet-derived growth factor promoter expressing amyloid precursor protein (PDAPP) transgenic mouse, a mouse model of AD with age-dependent accumulation of protein amyloid, and in transgenic mice over expressing familial AD variants of APP or PS-1 [8,9]. These studies reveal discrepancies between the modulations of neurogenesis in animal models of AD and patients with AD (Table 1). AD is a complex genetic disorder. Animal models, like PS1/APP-deficient mice, provide crucial information about the physiological function of proteins involved in the disease; they do not represent the disease [10]. In addition, the effect of genetic mutations during development may have adverse effects on adult phenotypes. As such, they may not be suitable to study phenotypes, like adult neurogenesis. Two classes of drugs are currently used to treat patients with AD; acetylcholinesterase (AChE) inhibitors, like tacrine, donepezil, galantamine and rivastigmine, and N-methyl-Daspartate (NMDA) glutamate receptor antagonists, like memantine [11-13]. These drugs produce improvements in cognitive and behavioral symptoms of AD. The activity of drugs, used to treat AD, on adult neurogenesis has been characterized. Galantamine and memantine increase neurogenesis in the DG and SVZ of adult rodents, by 26-45%, as revealed by bromodeoxyuridine (BrdU) labeling and its correlation with the labeling of neuronal versus glial markers, like βIII tubulin, doublecortin, M1 muscarinic receptor and glial fibrillary acidic protein (GFAP) (Table 1) [14]. This suggests that the activity of these drugs may be mediated through adult neurogenesis. In all, adult neurogenesis is enhanced in the brain of patients with AD and drugs used to treat AD, AChE and NMDA antagonists, modulate adult neurogenesis. These drugs may act via their pharmacological activities, on messenger signaling pathways, and/or via a neurogenic or trophic activity, to modulate neurogenesis in the adult brain. They may act directly or indirectly on newborn neuronal cells of the adult brain.

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Table 1. Adult neurogenesis and pharmacology in Alzheimer’s disease and depression. Disease/Model

Regulation

References

Autopsies

Enhanced

4

Transgenic mice Swedish and Indiana5 APP mutations Knock-out/deficient mice for PS1 and APP Transgenic mice PDAPP Mice overexpressing AD variants of APP or PS-1 Galantamine (5 mg/kg, 14 days) Memantine (5.5 mg/kg, 14 days

Enhanced

5

Decreased

6,7

Decreased Decreased

8 9

Increased Increased

10 10

Alzheimer’s disease

Depression Stress Decreased 11,12 Glucocorticoids Decreased 13 Autopsies Not altered 14 Fluoxetine (5 mg/kg, 1, 5, 14 or Increased 15 28 days) Fluoxetine (10 mg/kg/day, 5, 11 Increased 16 or 28 days) Adult neurogenesis is modulated in neurological diseases and disorders, like AD and depression, and by drugs used to treat these diseases and disorders. Enhanced neurogenesis may be a result, rather than a cause, of neurodegenerative diseases, like AD. Antidepressants, like fluoxetine, produce their activities via distinct mechanisms, some independent of adult neurogenesis.

Adult Neurogenesis in Depression Stress, an important causal factor in precipitating episodes of depression, and glucocorticoids, stress-related hormones, decrease hippocampal neurogenesis [15-17]. A post-mortem study reveals that adult neurogenesis is not altered in the hippocampus of patients with major depression [18]. Hence, the relationship between adult neurogenesis and depression remains undefined. Various classes of drugs are currently prescribed for the treatment of depression [19]. Among them, selective serotonin reuptake inhibitors (SSRIs) -like fluoxetine-, monoamine oxidase inhibitors (MAOIs), like tranylcypromine, selective norepinephrine reuptake inhibitors (SNRIs), like reboxetine, tricyclic antidepressants (TCAs), like imipramine and desipramine, and phosphodiesterase-IV inhibitors, like rolipram, alleviate symptoms of

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depression. The activity of drugs, used to treat depression, on adult neurogenesis has been characterized by BrdU labeling and its correlation with the labeling of neuronal versus glial markers, like βIII tubulin, neuronal nuclear antigen, neuron-specific enolase and GFAP. Chronic administration of antidepressants, like fluoxetine, increases neurogenesis in the DG, but not the SVZ of adult rats and nonhuman primates (Table 1) [20-22]. Agomelatine, a melatonergic agonist and serotoninergic antagonist defining a new class of antidepressant, increases adult hippocampal neurogenesis in rodents [23]. These data suggest that adult neurogenesis may contribute to the activities of drugs used to treat depression. In support to this contention, X-irradiation of the hippocampal region, but not other brain regions, like the SVZ or the cerebellar region, prevents the behavioral effect of the antidepressants, like fluoxetine, in adult mice (129SvEvTac) [24]. In all, drugs used to treat depression, particularly SSRIs, modulate adult neurogenesis that may mediate their behavioral activities. Brain-derived neurotrophic factor (BDNF) has antidepressant effects [25]. The administration of BDNF increases adult neurogenesis in the hippocampus, and the level of expression of BDNF is increased in the brains of patents subjected to antidepressant treatments [26,27]. This suggests that the activity of antidepressants on adult neurogenesis may be mediated by via neurogenic or trophic activities, and that BDNF is a candidate to for mediating the activity of anti-depressants on neurogenesis in the adult hippocampus.

Discussion Studies reviewed show that adult neurogenesis is modulated in patients and animal models of neurological diseases and disorders, particularly AD and depression, and mediates the activities of drugs used to treat these diseases and disorders. However, the significance and contribution of adult neurogenesis to these processes remain to be elucidated and further confirmed. Four to 10% of neurons in regions of degeneration, like the hippocampus, in the brain of patients with AD express cell cycle proteins, like cyclin B, or are tetraploids [28,29]. The origin and fate of these cells is yet to be determined. Data suggest that they may originate from abortive cell cycle re-entry and DNA duplication, without cell proliferation. Hence, neither immunohistochemistry for cell cycle protein, particularly cyclin B - a phase G2 marker -, nor BrdU labeling may allow discriminating cell proliferation and neurogenesis versus cell cycle re-entry and DNA duplication, without cell proliferation [30]. BrdU labeling is the paradigm the most used to study adult neurogenesis in situ, including in primates human and non-human [1,2,22]. BrdU is a thymidine analog used for birth dating and monitoring cell proliferation [31,32]. As a thymidine analog, BrdU is not a marker for cell proliferation, but a marker for DNA synthesis. Therefore studying neurogenesis with BrdU requires distinguishing cell proliferation and neurogenesis from other events involving DNA synthesis, like DNA repair, abortive cell cycle re-entry and gene duplication, without cell division. Some of the data observed by means of immunohistochemistry for cell cycle markers and BrdU labeling may then not represent adult neurogenesis, but rather labeled nerve cells that may have entered the cell cycle and underwent DNA replication, but did not

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complete the cell cycle [30]. Furthermore, recent studies reveal that the blood-brain barrier may be affected in the patients with AD [33]. In these conditions, an increase in BrdU labeling in the brain could originate from an increase in BrdU uptake rather than an increase in cell proliferation and neurogenesis [30]. Adult neurogenesis in AD must therefore be reevaluated and -examined in light of these data. Despite these limitations, these data suggest that adult neurogenesis is affected in the etiology and pathogenesis of neurological diseases and disorders, particularly neurodegenerative diseases, like AD. Several studies have reported that neurogenesis is increased in the adult brain, after experimental brain injuries [34,35]. These data support the hypothesis that increased neurogenesis in neurodegenerative diseases could result from damage or stimulation induction of neurogenesis. Hence, enhanced neurogenesis may be a result, rather than a cause, of neurodegenerative diseases, like AD. Nonetheless, the contribution of adult neurogenesis to the physiopathology of AD and depression remains to be determined. Neuronal death and synapses loss have been reported as consequences of AD. Hence, the relative contribution and involvement of enhanced neurogenesis versus other components of AD, like neuronal death, synapses loss and amyloid formation, in the pathology of AD remain to be understood. In depression, the relative importance of the neurogenic, versus the neurogenic-independent, component of depression remains to be determined [36]. Responses to these issues will lead to better understand and treat these diseases. Drugs used to treat AD and depression modulate adult neurogenesis. They may act via their pharmacological activities, on messenger signaling pathways, and/or via neurogenic or trophic activities, to modulate neurogenesis in the adult hippocampus. Particularly, the activity of anti-depressants on adult neurogenesis may be mediated by the trophic factor BDNF, through the TrkB neurotrophin receptor [37]. In addition, different states of aggregation of β-amyloid have been reported to have different effects on adult neurogenesis [38], and adult neurogeneis has been reported to mediate the behavioral effect of antidepressants like, fluoxetine [24]. Hence, pathways underlying the activity of AChE inhibitors, NMDA glutamate receptor antagonists and anti-depressants on adult neurogenesis, as well as β-amyloid, are targets for the treatments of AD and depression aiming at the neurogenic component of these diseases. Combinations of treatments will therefore be needed to treat the various components of AD and depression.

Conclusion and Perspectives Neurogenesis occurs in the adult brain and is a functional neurogenesis. However, the function and contribution of newborn neuronal cells of the adult brain in the physio- and pathology of the adult CNS remain to be established. Results show that adult neurogenesis is modulated in neurological diseases and disorders and by drugs used to treat these diseases, particularly AD and depression. Adult neurogenesis may mediate the activities of drugs used to treat AD and depression. The mechanisms underlying the activities of these drugs remain to be elucidated. These drugs may act either directly or indirectly to stimulate neurogenesis to mediate their activities. They may mediate their activity through adult neurogenesis either

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through pharmacological and/or neurogenic activities. The involvement and relative contribution of adult neurogenesis, versus other components of the diseases, and drugs to treat these diseases remain to be further evaluated and confirmed. The confirmation that neurogenesis occurs in the adult brain and NSCs reside in the adult CNS is not only important for our understanding of the functioning and physio- and pathology of the nervous system, but also for cellular and drug therapies. The elucidation of the contribution of adult neurogenesis to neurological diseases and disorder may contribute to a better understanding the etiology and mechanisms of neurological diseases and disorders, particularly AD and depression, as well as new drug design and strategies to treat these diseases and disorders.

Acknowledgments Reproduced from: Taupin P. Adult neurogenesis and pharmacology in Alzheimer's disease and depression. Journal of Neurodegeneration and Regeneration (2008) 1(1):51-5, with permission of Weston Medical Publishing, LLC.

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Eriksson PS, Perfilieva E, Bjork-Eriksson T, et al. 1998. Neurogenesis in the adult human hippocampus. Nat Med, 4:1313-7. Curtis MA, Kam M, Nannmark U, Anderson MF, Axell MZ, Wikkelso C, Holtas S, van Roon-Mom WM, Bjork-Eriksson T, Nordborg C, Frisen J, Dragunow M, Faull RL, Eriksson PS. 2007. Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Science, 315:1243-9. Cummings BJ, Uchida N, Tamaki SJ, Salazar DL, Hooshmand M, Summers R, Gage FH, Anderson AJ. 2005. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A, 102:14069-74. Jin K, Peel AL, Mao XO, et al. 2004. Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci U S A, 101:343-7. Jin K, Galvan V, Xie L, et al. 2004. Enhanced neurogenesis in Alzheimer’s disease transgenic (PDGF-APPSw,Ind) mice. Proc Natl Acad Sci U S A, 101:13363-7. Wen PH, Shao X, Shao Z, et al. 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. Zhang C, McNeil E, Dressler L, Siman R. 2007. Long-lasting impairment in hippocampal neurogenesis associated with amyloid deposition in a knock-in mouse model of familial Alzheimer's disease. Exp Neurol, 204:77-87. Donovan MH, Yazdani U, Norris RD, et al. 2006. Decreased adult hippocampal neurogenesis in the PDAPP mouse model of Alzheimer's disease. J Comp Neurol, 495:70-83.

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Verret L, Jankowsky JL, Xu GM, et al. 2007. Alzheimer's-type amyloidosis in transgenic mice impairs survival of newborn neurons derived from adult hippocampal neurogenesis. J Neurosci, 27:6771-80. Dodart JC, Mathis C, Bales KR, Paul SM. 2002. Does my mouse have Alzheimer’s disease? Genes Brain Behav, 1:142-55. Arrieta JL, Artalejo FR. 1998. Methodology, results and quality of clinical trials of tacrine in the treatment of Alzheimer's disease: a systematic review of the literature. Age Ageing, 27:161-79. Wilkinson DG, Francis PT, Schwam E, et al. 2004. Cholinesterase inhibitors used in the treatment of Alzheimer's disease: the relationship between pharmacological effects and clinical efficacy. Drugs Aging, 21:453-78. McShane R, Areosa Sastre A, Minakaran N. 2006. Memantine for dementia. Cochrane Database Syst Rev, 2:CD003154. Jin K, Xie L, Mao XO, Greenberg DA. 2006. Alzheimer's disease drugs promote neurogenesis. Brain Res, 1085:183-8. Cameron HA, Gould E. 1994. Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neurosci, 61:203-9. Gould E, Tanapat P, McEwen BS, et al. 1998. Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc Natl Acad Sci U S A, 95:3168-71. Pham K, Nacher J, Hof PR, McEwen BS. 2003. Repeated restraint stress suppresses neurogenesis and induces biphasic PSA-NCAM expression in the adult rat dentate gyrus. Eur J Neurosci, 17:879-86. Reif A, Fritzen S, Finger M, et al. 2006. Neural stem cell proliferation is decreased in schizophrenia, but not in depression. Mol Psychiatry, 11:514-22. Wong ML, Licinio J. 2001. Research and treatment approaches to depression. Nat Rev Neurosci, 2:343-51. Malberg JE, Eisch AJ, Nestler EJ, Duman RS. 2000. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci, 20:9104-10. Malberg JE, Duman RS. 2003. Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment. Neuropsychopharmacol, 28:156271. Perera TD, Coplan JD, Lisanby SH, et al. 2007. Antidepressant-induced neurogenesis in the hippocampus of adult nonhuman primates. J Neurosci, 27:4894-901. Banasr M, Soumier A, Hery M, et al. 2006. Agomelatine, a new antidepressant, induces regional changes in hippocampal neurogenesis. Biol Psychiatry, 59:1087-96. Santarelli L, Saxe M, Gross C, et al. 2003. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science, 301:805-9. Siuciak JA, Lewis DR, Wiegand SJ, et al. 1997. Antidepressant-like effect of brainderived neurotrophic factor (BDNF). Pharmacol Biochem Behav, 56:131-7. Chen B, Dowlatshahi D, MacQueen GM, et al. 2001. Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication. Biol Psychiatry, 50:260-5.

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[27] Scharfman H, Goodman J, Macleod A, et al. 2005. Increased neurogenesis and the ectopic granule cells after intrahippocampal BDNF infusion in adult rats. Exp Neurol, 192:348-56. [28] Vincent I, Rosado M, Davies P. 1996. Mitotic mechanisms in Alzheimer's disease? J Cell Biol, 132:413-25. [29] Yang Y, Geldmacher DS, Herrup K. 2001. DNA replication precedes neuronal cell death in Alzheimer’s disease. J Neurosci, 21:2661-8. [30] Taupin P. 2007. BrdU Immunohistochemistry for Studying Adult Neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Res Rev, 53:198-214. [31] Miller MW, Nowakowski RS. 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. [32] del Rio JA, Soriano E. 1989. Immunocytochemical detection of 5'-bromodeoxyuridine incorporation in the central nervous system of the mouse. Brain Res. Dev Brain Res, 49:311-7. [33] Desai BS, Monahan AJ, Carvey PM, Hendey B. 2007. Blood-brain barrier pathology in Alzheimer's and Parkinson's disease: implications for drug therapy. Cell Transplant, 16:285-99. [34] Gould E, Tanapat P. 1997. Lesion-induced proliferation of neuronal progenitors in the dentate gyrus of the adult rat. Neurosci, 80:427-36. [35] Magavi SS, Leavitt BR, Macklis JD. 2000. Induction of neurogenesis in the neocortex of adult mice. Nature, 405:951-5. [36] Holick KA, Lee DC, Hen R, Dulawa SC. 2008. Behavioral effects of chronic fluoxetine in BALB/cJ mice do not require adult hippocampal neurogenesis or the serotonin 1A receptor. Neuropsychopharmacology, 33:406-17. [37] Saarelainen T, Hendolin P, Lucas G, et al. 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. [38] López-Toledano MA, Shelanski ML. 2004. Neurogenic effect of beta-amyloid peptide in the development of neural stem cells. J Neurosci, 24:5439-44.

Chapter II

Adult Neurogenesis in the Pathogenesis of Alzheimer’s Disease Alzheimer’s disease (AD) is a progressive neurodegenerative disease, leading to severe incapacity and death. It is the most common form of dementia among elderly; it affects 30% of individuals over the age of 80. There is currently no cure for AD. Once the disease is diagnosed, the average life expectancy of patients with AD is 8.5 years. More than 26 millions of people worldwide are affected by the disease, a number expected to quadruple by 2050 [1]. Neurogenesis occurs throughout adulthood in mammals. It occurs primarily in two regions of the adult brain, the dentate gyrus of the hippocampus and the subventricular zone, in various species including in humans. It is hypothesized that newly generated neuronal cells in the adult brain originate from residual stem cells. Neural stem cells are the self-renewing multipotent cells that generate the main phenotypes of the nervous system [2]. The confirmation that adult neurogenesis occurs in the adult brain and neural stem cells reside in the adult central nervous system has tremendous implications for our understanding of development and for therapy. Newly generated neuronal cells of the adult brain may be involved in the physio-, patho- and pharmacology of the nervous system and the adult brain has the potential to self-repair [3]. Studies from autopsies and animal models indicate that neurogenesis is enhanced in adult brains with AD [4]. Enhanced neurogenesis in the AD brain would result from damaged or stimulation induction of neurogenesis, rather than being a cause of the disease. It may be involved in a regenerative attempt, to compensate for the neuronal loss. Amyloid plaques and neurofibrillary tangles are hallmarks of AD. Amyloid plaques are primarily composed of deposits of protein β-amyloid. They originate from the synthesis of mutant forms of the protein or the abnormal processing of the amyloid precursor protein (APP). Neurofibrillary tangles are composed of hyperphosphorylated Tau protein. The APP and TAU genes are located on chromosomes 21 and 17, respectively. According to the amyloid hypothesis, though subject to debates, amyloid deposits may be a cause of AD [5]. There are two forms of AD: late-onset AD diagnosed after the age of 65 and early-onset AD diagnosed at younger age. Early-onset AD is mostly inherited, caused by mutations in so-

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called familial Alzheimer genes, like the genes of APP, presenilin-1 and presenilin-2. The PSEN-1 and the PSEN-2 genes are located on chromosomes 14 and 1, respectively. Most cases of late-onset AD are sporadic. They are believed to be caused by a combination of genetic, acquired and environmental risk factors, like the presence of ApoE varepsilon 4 allele(s) (ApoE4) in the genome. The ApoE gene is located on chromosome 19. Late-onset AD accounts for most cases of AD, over 93% [6]. A broad range of cells elicit aneuploidy, particularly for chromosomes 21 and 17, in patients with AD [7, 8]. Aneuploidy for chromosome 21and 17 has been proposed as one of the contributing factors for the pathogenesis of AD; aneuploidy for chromosomes 21, on which APP gene is located, and 17, on which TAU gene is located, would increase the risk of formation of amyloid plaques and neurofibrillary tangles. The theory would apply to aneuploidy for other chromosomes, on which genes involved in AD are presents. Aneuploidy in somatic cells results from nondisjunction of chromosomes during mitosis. The nondisjunction of chromosomes, particularly of chromosomes 21 and 17, in stem cells and/or populations of cells that retain their ability to divide would be at the origin of aneuploidy in AD patients [9]. Four to 10% of neurons in regions of degeneration, like the hippocampus, express proteins of the cell cycle and some at-risk neurons are aneuploids in the brain of patients with AD [10, 11]. Nerve cells are post-mitotic cells in the adult brain. Aneuploid neurons in regions of degeneration would originate from dying neurons re-entering the cell cycle and undergoing DNA replication, but not completing the cell cycle [12, 13]. It could result in the overexpression of genes involved in AD. Hence, aneuploid cells in AD brains could originate from nondisjunction of chromosomes during mitosis or DNA duplication, without cell division. It is proposed that the genetic imbalance in aneuploid cells signifies that they are fated to die, though these cells may live in this state for months, possibly up to 1 year [14]. Aneuploidy may therefore underlie the pathogenesis and pathology of AD. The process of adult neurogenesis holds the potential to generate populations of aneuploid cells particularly in the neurogenic areas. The nondisjunction of chromosomes during cell division of newly generated neural progenitor cells of the adult brain could lead to a population of aneuploid cells that would not proceed with its developmental program or of newly generated neuronal cells that are aneuploids (Figure 1). Such aneuploidy, particularly for chromosomes 21 and 17 and particularly in the hippocampus, would contribute to the pathogenesis of AD. Hence, in AD the process of adult neurogenesis may not necessarily signify a beneficial effect, it could contribute to the pathogenesis of the disease. Cell death is a normally occurring process in the adult brain particularly in the neurogenic areas, as a significant proportion of newly generated cells in the dentate gyrus and subventricular zone are believed to undergo apoptosis rather than achieving maturity. The effect of aneulpoidy derived from newly generated neuronal cells of the adult brain may therfore be limited, though significant since occuring in the hippocampus. The confirmation that adult neurogenesis occurs in the adult brain and neural stem cells reside in the adult central nervous system signifies that the adult brain may be amenable to repair [15]. Newly generated neuronal cells of the adult brain would not only contribute to plasticity and regeneration of the nervous system, but also to the pathogenesis of neurological diseases and disorders. Future studies will at at unraveling the involvement and contribution

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of adult neurogenesis to the physio- and pathology of the nervous system, and to bring adult neural stem cells to therapy, particularly for AD.

Figure 1. Fate of newly generated neuronal cells in the adult brain of patients with AD. Neurogenesis occurs in the adult brain. The process of adult neurogenesis holds the potential to generate populations of aneuploid cells particularly in the neurogenic regions. Cell death is a normally occurring process in the neurogenic regions of the adult brain, as a significant proportion of newly generated cells are believed to undergo apoptosis rather than achieving maturity (A). Newly generated neuronal cells in the adult brain originate from a population of stem cells; neural stem cells (NSCs) (B). Aneuploid neurons could originate from newly generated neuronal cells re-entering the cell cycle and undergoing DNA replication, but not completing the cell cycle (C). The nondisjunction of chromosomes during the process of cell division of newly generated neuronal progenitor cells of the adult brain could lead to newly generated neuronal cells that are aneuploids (D) or to a population of aneuploid cells that would not proceed with its developmental program (E). Aneuploidy in newly generated neuronal progenitor and neuronal cells of the adult hippocampus, particularly for chromosomes 21 and 17, would contribute to the pathogenesis of AD.

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Acknowledgments Reproduced from: Taupin P. Adult neurogenesis in the pathogenesis of Alzheimer’s disease. Journal of Neurodegeneration and Regeneration (2009) In press, with permission of Weston Medical Publishing, LLC.

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Brookmeyer R, Johnson E, Ziegler-Graham K, et al.: Forecasting the global burden of Alzheimer’s disease. Johns Hopkins University, Dept. of Biostatistics Working Papers. January 2007, Working Paper 130. http://www.bepress.com/jhubiostat/paper130. Taupin P: Neurogenesis in the adult central nervous system. C R Biol. 2006; 329(7): 465-475. Taupin P: Adult neurogenesis and pharmacology in Alzheimer's disease and depression. Journal of Neurodegeneration and Regeneration. 2008; 1(1): 51-55. Jin K, Peel AL, Mao XO, et al.: Increased hippocampal neurogenesis in Alzheimer's disease. Proc Natl Acad Sci U S A. 2004; 101(1): 343-347. Hardy J, Selkoe DJ: The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002; 297(5580): 353-356. Erratum in: Science.2002; 297(5590): 2209. Raber J, Huang Y, Ashford JW : ApoE genotype accounts for the vast majority of AD risk and AD pathology. Neurobiol Aging. 2004; 25(5): 641-650. Migliore L, Testa A, Scarpato R, et al.: Spontaneous and induced aneuploidy in peripheral blood lymphocytes of patients with Alzheimer's disease. Hum Genet. 1997; 101(3): 299-305. Migliore L, Botto N, Scarpato R, et al.: Preferential occurrence of chromosome 21 malsegregation in peripheral blood lymphocytes of Alzheimer disease patients. Cytogenet Cell Genet. 1999; 87(1-2): 41-46. Geller LN, Potter H: Chromosome missegregation and trisomy 21 mosaicism in Alzheimer's disease. Neurobiol Dis. 1999; 6(3): 167-179. Yang Y, Geldmacher DS. Herrup K: DNA replication precedes neuronal cell death in Alzheimer’s disease. J Neurosci. (2001) 21(8): 2661-2668. Herrup K, Arendt T: Re-expression of cell cycle proteins induces neuronal cell death during Alzheimer's disease. J Alzheimers Dis. 2002; 4(3): 243-247. Yang Y, Mufson EJ, Herrup K: Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease. J Neurosci. (2003) 23(7): 2557-2563. Taupin P: BrdU Immunohistochemistry for Studying Adult Neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Research Reviews. 2007; 53(1): 198-214. Herrup K, Neve R, Ackerman SL, et al.: Divide and die: cell cycle events as triggers of nerve cell death. J Neurosci. 2004; 24(42): 9232-9239. Taupin P: Editorial. Adult neural stem cells from promise to treatment: The road ahead. Journal of Neurodegeneration and Regeneration. 2008; 1(1): 7-8.

Chapter III

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 NSCs reside in the adult CNS, new treatments for neurological diseases and injuries are being considered. Particularly, the transplantation of adult-derived neural progenitor and stem cells to restore brain functions. In 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.

Introduction Since the seminal studies by Altman and Das (1965) and Altman (1969), reporting evidences 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 human [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, *Copyright notice. 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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neurons, astrocytes and oligodendrocytes [8]. The intermediate filament protein 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 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].

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, selfrenewal over an extended period of time, generation of a large number of differentiated progeny, regeneration of the tissue following injury, and a flexibility of 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 to fulfill 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, that upon dividing, a stem cell give 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].

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

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handling could account for discrepancies between the studies [21]. We further reported that FGF-2 requires a co-factor, a glycosylated form of the protease inhibitor cystatin C (CCg), requires 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 CNS, as well as drugs and novel therapies. Neural progenitor and stem cells can be isolated and characterized in vitro from 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, species, from either normal or diseased CNS tissue, as a model to study neural development, function, 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 in vitro the CNS. 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, 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, 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, like 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. 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 on methods to genetically engineered 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 neurotransmittersynthesizing enzymes, like nerve growth factor or tyrosine hydroxylase. This extends their

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use for gene therapy for the treatment of neurodegenerative diseases, but also to mark cells with reported genes, like the E. coli ß-galactosidase or green fluorescent protein.

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 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 the source of controversies, as other studies report that subependymal, but not ependymal, cells have NSC properties [32, 35], whereas the astroglial origin for adult NSCs has received further support [36-38]. A patent application has been filed for the isolation of nonembryonic 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 controversies over whether ependymal cells represent NSCs in the adult brain may jeopardize the potential of the claims filed under this patent.

Therapeutic Potential of Adult NSCs Cellular therapy is the replacement of unhealthy or damaged cells or tissues by 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.

Cellular Therapy The confirmation that neurogenesis occurs in the adult brain and NSC 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 adult-derived 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 after 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 revealed that new neuronal cells are generated at the sites of degeneration, where they originate from the SVZ.

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They 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, like 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 in the CNS of normal and in animal models, in rodents. Adult rat hippocampal-derived neural progenitor and stem cells have been grafted into the hippocampus, where they differentiate into neuronal and glial cells [12]. In another study, adult rat spinal cord derived-neural progenitor and stem cells have been transplanted into the hippocampus and spinal cord [44]. When transplantated into the spinal cord, adult spinal cord derived-neural progenitor and stem cells differentiate only in glial cells; when transplanted into the DG, they differentiate in neuronal and glial cells. These results show 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 for 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 they yield to 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 the network. Protocols have been developed to enrich for homogeneous population of neural progenitor/stem cells, by cell sorting using in particular 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 for the treatment of neurodegenerative diseases. Systemic and injection through cerebrospinal fluid obviate the need of invasive surgical procedures, and their associated risks and secondary effects. They are seen as promising ways to deliver 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 other tissues than the brain, like the skin, blood and bone marrow, give rise to neuronal lineages [55-57]. This suggests that adult

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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 adult stem cells are lineage restricted or not remains the source of debates and controversies [64]. The broader potential of adult stem cells, and particularly NSCs, would have tremendous implications for cellular therapy. The isolation of hematopoietic phenotypes from NSCs would 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-versus-host 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. Patent application 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, without the need of surgical procedure, and its associated risks and secondary effects to the patients. 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.

Gene Therapy Adult neural progenitor and stem cells can be genetically modified [12], extending their potential use for 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 valid genetically engineered neural progenitor and stem cells as a strategy to treat neurological diseases and disorders, as filed in a patent application [30]. It highlights the potential of NSCs as a gene transfer vehicle for the treatment in lysosomal storage diseases, and other genetic disorders of the CNS.

Current and Future Developments The confirmation that neurogenesis occurs in the adult brain and 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, as well as 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 the pathways. In all, adult NSCs offer a tremendous potential for the treatment of CNS diseases and injuries, but also potentially for

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other diseases, like blood-related diseases and genetic diseases. There are however limitations, debates and controversies over the use of adult NSCs for therapy. Among them, neural progenitor and stem cells are heterogeneous in culture, and NSCs remains to be identified [67]. The broader potential of adult stem cells, and particularly NSCs, remain to be elucidated. There are also unknown 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 establish connections with the wrong target cells? What is the risk that NSCs form tumors upon grafting?

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The author should has identified the most important patents of significant interest by using an asterisk symbol before each patent reference.

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[49] Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS, Gage FH, Weissman IL. (2000) Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci. USA 97, 14720-25. [50] Wang S, Roy NS, Benraiss A, Goldman SA. (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. [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 AL, 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 CR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL. (1999) Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science. 283, 534-7. [55] Brazelton TR, Rossi FM, Keshet GI, Blau HM. (2000) From marrow to brain: expression of neuronal phenotypes in adult mice. Science. 290, 1775-9. [56] Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. (2000) Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science. 290, 1779-82. [57] Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller FD. (2001) Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol. 3, 778-84. [58] D'Amour KA, Gage FH. (2002) Are somatic stem cells pluripotent or lineagerestricted? Nat Med. 8, 213-4. [59] Morshead CM, Benveniste P, Iscove NN, van der Kooy D. (2002) Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations. Nat Medecine. 8, 268-73. [60] Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. (2002) Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 297, 2256-9. [61] Castro RF, Jackson KA, Goodell MA, Robertson CS, Liu H, Shine HD. (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 DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW. (2002) Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature. 416, 542-5. [63] Ying QL, Nichols J, Evans EP, Smith AG. (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.

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[65] (*) Bjornson CR, Rietze RL, Reynolds BA, Vescovi AL. EP1019493B1 (2005). [66] Shihabuddin LS, Numan S, Huff MR, Dodge JC, Clarke J, Macauley SL, Yang W, Taksir TV, Parsons G, Passini MA, Gage FH, Stewart GR. (2004) Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology. J Neurosci. 24, 10642-51. [67] Kornblum HI, Geschwind DH. (2001) Molecular markers in CNS stem cell research: hitting a moving target. Nat Rev Neurosci. 2, 843-6. [68] Taupin P. (2006) HuCNS-SC (StemCells). Curr Opin Mol Ther. 8, 156-63.

Chapter IV

Human Neural Progenitor and Stem Cells* Abstract Background: The application is in the field of neural stem cells (NSCs) and cellular therapy. Objective: It aims at establishing conditions for the isolation and propagation of neural progenitor and stem cells from human fetal tissue, with high rate of growth and high yields of differentiation into the neuronal, astroglial and oligodendroglial pathways. Methods: Neural progenitor and stem cells were isolated from fetal forebrain tissue and propagated as neurospheres, in defined medium in the presence of leukemia inhibitory factor (LIF). Three protocols were designed to differentiate human fetal neural progenitor and stem cells into their progenies. Results: The application claims the generation of human fetal neural progenitor and stem cells with a doubling rate between 5-10 days. It claims the differentiation of the neural progenitor and stem cells in vitro, into neurons, astrocytes and oligodendrocytes with high yields, e.g., 20 to 35% for neuronal cells. Conclusion: The establishment of human neural progenitor and stem cells with high rate of growth and high yields of differentiation provides a source of cells for therapy, particularly for the treatment of neurodegenerative diseases, like Alzheimer’s and Parkinson’s diseases.

1. Introduction Neural stem cells (NSCs) are the self-renewing multipotent cells that generate the main phenotypes of the nervous system, neuronal, astroglial and oligodendroglial [1]. Neural progenitor and stem cells have been isolated and characterised in vitro from fetal, adult and post-mortem tissues, from various mammalian species, including humans [2-6]. There are now two protocols devised to culture neural progenitor and stem cells. Neural progenitor and *Patent details: Title: Cultures of human CNS neural stem cells, Inventors: Carpenter M, Filing date: 21/09/2007, Publication date: 08/05/2008, Publication no.: US20080107633A1. Declaration of Interest: The author states no conflict of interest and has received no payment in preparation of this manuscript.

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stem cells can be cultured as neurospheres, in defined medium in the presence of high concentration of the trophic factor EGF (20 ng/ml) on uncoated tissue culture dishes [2]. Neural progenitor and stem cells can also be cultured as monolayers, in defined medium in the presence of high concentration of basic fibroblast growth factor (FGF-2, 20 ng/ml) on polyornithine and laminine coated tissue culture dishes [7]. Neural progenitor and stem cells in culture express the neurofilament marker of stem cells on the CNS, nestin [8]. On differentiation, neural progenitor and stem cells in culture give rise to cells expressing markers of the three phenotypes of the nervous system, neurons, astrocytes and oligodendrocytes [2-6,9]. Differentiation of neural progenitor and stem cells in vitro yields to relatively low rate of differentiation, particularly for the neuronal phenotype. Generally, < 10% of neural progenitor and stem cells differentiate into the neuronal cells in vitro, as assessed by immunocytology and confocal microscopy for markers of immature and mature neurons, such as class III β-tubulin isotype, doublecortin and Hu and calbindin-D28k, microtubule-associated protein-2 and neuronal nuclear antigen, respectively [10-15]. Cellular therapy is the replacement of unhealthy or damaged cells or tissues by new ones. Because of their ability to generate the main phenotypes of the nervous system, NSCs hold the potential to treat and cure a broad range of neurological diseases and injuries, particularly neurodegenerative diseases, cerebral strokes and traumatic brain and spinal cord injuries. However, there are limitations to advent of NSC biology for therapy. Among them are the following: the heterogeneity of neural progenitor and stem cells in culture, the existence of niches for neurogenesis and the low yield of differentiation of adult-derived neural progenitor and stem cells in vitro and in vivo, after transplantation [16-18]. The application aims at defining culture conditions for the isolation and propagation of neural progenitor and stem cells from human fetal tissue with high rate of growth and high yields of differentiation into their progenies in vitro and in vivo, after transplantation.

2. Chemistry 2.1 Proliferation Medium The proliferation medium is composed of the following six components: i) a standard culture medium, such as IMDM, RPM1, DMEM, Leibovitz’s, L-15, NCTC, F-10, F-12 and MEM, ii) a source of carbohydrate, such as glucose, iii) a buffer, such as MOPS, and HEPES, iv) a source of hormones, including insulin, transferrin, progesterone, selenium; and putrescine, v) one or more growth factors known to stimulate proliferation of neural progenitor and stem cells, such as EGF, FGF-2, PDGF and nerve growth factor (NGF); and vi) leukaemia inhibitory factor (LIF; 10 ng/ml). One example of standard culture medium is a 50/50 mixture of DMEM and F-12. The culture conditions include the following: glutamine to 2 mM, heparin, pH 7.4, 35 o 37 C, 5% CO 2. The concentrations of the standard components of the proliferation medium are indicated in Table 1.

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Table 1. Concentration of the components of the proliferation medium. Component Concentration

Final

50/50 mix of DMEM/F-12 Glucose w/v Glutamine NaHCO3 HEPES apo-human transferring human insulin putrescine selenium progesterone human EGF human FGF-2 human LIF heparin

1X 0.6% 2 mM 3 mM 5 mM 100 μg/ml 25 μg/ml 60 μM 30 nM 20 nM 20 ng/ml 20 ng/ml 10 ng/ml 2 μg/ml

LIF: Leukemia inhibitory factor.

2.2 Culture of Human Fetal Neural Progenitor and Stem Cells The tissue was kept at 4 oC in saline, before processing. Samples from human embryonic forebrain tissues were dissected out and dissociated mechanically using a standard glass homogeniser. Cells were plated in uncoated flasks and cultured for 5 - 7 days in proliferation medium. They were then harvested and centrifuged at 1,000 rpm for 2 min. The pellet was resuspended in 0.2 ml of proliferation medium. Cells were dissociated by trituration using a P200 pipetman ~ 100 times. Cells were plated and grown as suspension in 20 ml proliferation medium in uncoated T75 flasks at 75,000 - 100,000 cells/ml. Medium was replaced 3 times a week, by addition of 5 ml of proliferation medium. Cells were passaged every 6 - 21 days depending on density of cells plated and cell growth. Four lines of neural progenitor and stem cells were established from the human forebrain.

3. Biology and Action 3.1 Growth Rate The growth rate of the human forebrain-derived neural progenitor and stem cells in the presence and absence of LIF is assessed. Leukemia inhibitory factor dramatically increases the rate of cellular proliferation of human fetal neural progenitor and stem cells, with a doubling rate of between 5 and 10 days. The effect of LIF was most pronounced after ~ 60 days in vitro.

Philippe Taupin

36 3.2 Immunocytofluorescence

Immunocytofluorescence was performed on cultured human fetal neural progenitor and stem cells by applying standard procedures. A broad range of primary antibodies was used. Human nestin antibody was used as a marker to identify undifferentiated neural progenitor and stem cells. Antibodies specific for various neuronal or glial proteins were used. Antibodies to neuron specific enolase, neurofilament, tau, class III β-tubulin isotype were used to identify neuronal cells. Antibodies to glial fibrillary acidic protein (GFAP) were used to identify astrocytes and antibodies to galacto-cerebroside, O4, myelin basic protein were used to identify oligodendrocytes. The proliferating neural progenitor and stem cells that are immuno-positive for human nestin, show little GFAP staining and little class III β-tubulin isotype staining. When differentiated, most of the cells lose their nestin positive immunoreactivity.

3.3 Differentiation of Human Fetal Neural Progenitor and Stem Cells The application claims three different protocols for differentiating human fetal neural progenitor and stem cells in vitro. The differentiation medium is composed of the same basic components as the proliferation medium, with the following modifications. •

•

•

Differentiation condition # 1. Differentiation medium: culture in the absence of growth factor mitogens and of LIF, in the presence of 1% serum and growth on coated glass cover slip with polyornithine or extracellular matrix components. Under these conditions, the human fetal neural progenitor and stem cell culture is enriched in neuronal differentiated cells, as indicated by the high percentage, 20 – 37%, of class III β-tubulin isotype positive cells. The differentiation pattern is irrespective of the passage number or age of cultures. Differentiation condition # 2. Differentiation medium: absence of the growth factor mitogens and of LIF, presence of 1% serum, growth on coated glass cover slip with polyornithine or extracellular matrix components and a mixture of growth factors including 10 ng/ml plateled-derived growth factor (PDGF) A/B, 10 ng/ml ciliary neurotrophic factor (CNTF), 10 ng/ml IGF-1, 10 μM forskolin, 30 ng/ml T3, 10 ng/ml LIF and 1 ng/ml NT-3. Under these conditions, the human fetal neural progenitor and stem cell culture is highly enriched in neurons (i.e., > 35% of the differentiated cell culture) and enriched in oligodendrocytes. Differentiation condition # 3. In this protocol, human fetal neural progenitor and stem cells are cultured in proliferation medium, in presence of hFGF-2, EGF and LIF. Differentiation medium: presence of 20 ng/ml hEGF and 10 ng/ml hLIF and absence of hFGF-2. Under these conditions, the human fetal neural progenitor and stem cell culture is enriched in oligodendrocytes, as observed by the high percentage of GalC immunoreactivity. Neurons were only occasionally seen, had small processes and seemed quite immature.

Human Neural Progenitor and Stem Cells

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The invention provides novel cell lines of human neural progenitor and stem cells isolated from fetal tissue. Four cell lines were established, all of which show neural stem cell properties; the cells are self-renewing, they proliferate over long periods of time and are differentiated into the main phenotypes of the nervous system, neurons, astrocytes and oligodendrocytes. These cells have doubling rate between 5 and 10 days. Leukemia inhibitory factor substantially increases the rate of proliferation of the isolated and characterised neural progenitor and stem cells in vitro.

4. Expert Opinion Neural progenitor and stem cells isolated and propagated in vitro, from fetal human forebrain tissues, elicit a high rate of growth and high yields of differentiation into neuronal lineages. The human neural progenitor and stem cells elicit a doubling rate between 5 and 10 days and high yields of differentiation into neurons, astrocytes and oligodendrocytes in vitro; for example, 20 – 35% for neuronal cells. The fetal human-derived neural progenitor and stem cells provide a source of tissue for therapy, for the treatment of a broad range of neurological diseases and injuries. Epidermal growth factor and FGF-2 are the most used mitogens for isolating and culturing neural progenitor and stem cells [2,7]. Other trophic factors, such as PDGF, in combination with FGF-2 have been reported for the isolation and propagation of neural progenitor and stem cells from human tissues, particularly from biopsies and post-mortem tissues [7]. Trophic factors, such as FGF-2, also require factors present in conditioned medium for their mitogenic activity on self-renewing multipotent NSCs in vitro, from single cells [9]. Leukemia inhibitory factor is known as a mitogen for embryonic stem cells [19,20]. The finding that LIF acts as a potent mitogen, in presence of other trophic factors including FGF-2, further reveals the importance of this trophic factor as a developmental factor. The interaction of LIF with other trophic factors to act as a mitogen for neural progenitor and stem cells as well as its mechanism of action remains to be determined. Particularly, does LIF, alone or in combination with other trophic factors, allow to culture self-renewing multipotent NSCs in vitro, from single cells? Although the defining criteria of neural progenitor and stem cells in vitro are subject to debate, the mitogenic activity on single cells in vitro and the characterisation of self-renewal and multipotentiality, from single cells, remain valid assays for characterising the activity and potency of trophic factors on selfrenewing multipotent NSCs in vitro. Human fetal neural progenitor and stem cells are now in clinical trial for the treatment of Batten’s disease [21]. The ability to isolate and propagate neural progenitor and stem cells with high rate of growth and high yields of differentiation into neuronal lineages may be beneficial for NSC-based therapy. The isolation and establishment of neural progenitor and stem cells from human fetal tissues is the subject of ethical and political debates and controversies. With the confirmation that adult neurogenesis occurs in the adult brain and NSCs reside in the adult CNS, the adult brain and post-mortem tissues offer alternative sources of neural progenitor and stem cells for therapy [22,23]. It would be interesting to

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assess the activity of the proliferation and differentiation medium reported in this application on adult and post-mortem-derived neural progenitor and stem cells. In all, this application raises interesting opportunities to isolate and propagate human neural progenitor and stem cells with high rate of growth and high yields of differentiation into neuronal lineages. The applications from this established culture of neural progenitor and stem cells are broad, ranging from drug screening and diagnostics to genomics and genetic and cellular therapies.

Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Human neural progenitor and stem cells: US20080107633A1. Expert Opinion on Therapeutic Patents 19(3):377-80. Copyright 2009, Informa UK Ltd.

References** [1] [2]

[3] [4] [5]

[6] [7]

[8] [9]

**

Taupin P, Gage FH. Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res 2002; 69: 745-9 Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992; 255: 1707-10 •• Original report of the isolation and characterisation of neural progenitor and stem cells from the adult brain of mammals. Neural progenitor and stem cells are cultured as neurospheres, in the presence of EGF. Cattaneo E, Conti L, Gritti A, et al. Non-virally mediated gene transfer into human central nervous system precursor cells. Brain Res Mol Brain Res 1996; 42: 161-6 Roy NS, Wang S, Jiang L, et al. In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat Med 2000; 6: 271-7 Palmer TD, Schwartz PH, Taupin P, et al. Cell culture. Progenitor cells from human brain after death. Nature 2001; 411: 42-3 • First isolation and chracterisation of neural progenitor and stem cells from human post-mortem tissues and biopsies. Schwartz PH, Bryant PJ, Fuja TJ, et al. Isolation and characterization of neural progenitor cells from post-mortem human cortex. J Neurosci Res 2003; 74: 838-51 Gage FH, Coates PW, Palmer TD, et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci USA 1995; 92: 11879-83 • Neural progenitor and stem cells are cultured as monolayers, in the presence of FGF-2. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate fi lament protein. Cell 1990; 60: 585-95 Taupin P, Ray J, Fischer WH, et al. FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. Neuron 2000; 28: 385-97

Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

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[10] Bernhardt R, Matus A. Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons. J Comp Neurol 1984; 226: 203-21 [11] Sloviter RS. Calcium-binding protein (calbindin-D28k) and parvalbumin immunocytochemistry: localization in the rat hippocampus with specifi c reference to the selective vulnerability of hippocampal neurons to seizure activity. J Comp Neurol 1989; 280: 183-96 [12] Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specifi c nuclear protein in vertebrates. Development 1992; 116: 201-11 [13] Marusich MF, Furneaux HM, Henion PD, et al. Hu neuronal proteins are expressed in proliferating neurogenic cells. J Neurobiol 1994; 25: 143-55 [14] Fanarraga ML, Avila J, Zabala JC. Expression of unphosphorylated class III betatubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation. Eur J Neurosci 1999; 11: 517-27 [15] Gleeson JG, Lin PT, Flanagan LA, et al. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 1999; 23: 257-71 [16] Chin VI, Taupin P, Sanga S, et al. Microfabricated platform for studying stem cell fates. Biotechnol Bioeng 2004; 88: 399-415 [17] Ninkovic J, Götz M. Signaling in adult neurogenesis: from stem cell niche to neuronal networks. Curr Opin Neurobiol 2007; 17: 338-44 [18] Taupin P. Editorial. Adult neural stem cells from promise to treatment: the road ahead. J Neurodegeneration Regen 2008; 1: 7-8 [19] Wobus AM, Boheler KR. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev 2005; 85: 635-78 [20] Taupin P. Derivation of embryonic stem cells for cellular therapy: challenges and new strategies. Med Sci Monit 2006; 12: RA75-8 [21] Taupin P. HuCNS-SC (StemCells). Curr Opin Mol Ther 2006; 8: 156-63 [22] Taupin P. The therapeutic potential of adult neural stem cells. Curr Opin Mol Ther 2006; 8: 225-31 [23] Taupin P. Therapeutic neuronal stem cells: patents at the forefront. Expert Opin Ther Patents 2008; 18: 1107-10

Chapter V

Cryopreservation of Early Postmitotic Neuronal Cells in Culture* Abstract Background: The application is in the field of embryonic stem cells (ESCs) and the cryopreservation of neuronal cells in culture. Objective: It aims at establishing conditions for the cryopreservation of cultured neuronal cells readily usable, upon thawing. That is cryopreserved neuronal cells with a high rate of survivability and with a potential to differentiate into mature neuronal cells rapidly, after thawing. Methods: Neuronal precursor cells and neuronal cells were generated in vitro, from cultures of human ESCs (hESCs). Neuronal cells were frozen and cryopreserved at a stage when they begin to express neuronal class III β-tubulin. Results/conclusion: The application claims that neuronal cells elicit a rate of survivability greater than 50% and neurite outgrowth within 10 to 14 hours, after thawing. The cryopreservation of post-mitotic neuronal or neurallike cells provides a source of nerve cells readily usable for research and therapy.

1. Introduction Embryonic stem cells (ESCs) are self-renewing, undifferentiated cells with the ability to generate the various cell types of the body [1,2]. They have the potential to cure a broad range of diseases and injuries, from diabetes and heart diseases to neurological diseases and injuries [3]. ESCs are derived in vitro from the inner cell mass of blastocysts. They have been isolated, cultured and maintained in vitro from blastocysts of non-human and human primates [4,5]. The derivation of human ESCs (hESCs) provides an unlimited source of cells for therapy. Protocols have been established to differentiate ESCs into various lineages in vitro, *Patent Evaluation: The University of Georgia Research Foundation, Inc.: US20080274446A1, Patent details: Title: Cryopreserved human neuronal culture, Assignee: The University of Georgia Research Foundation, Inc., Inventors: Stice S, Machacek D. Filing date: 01/04/2008, Publication date: 06/11/2008, Publication no.: US20080274446A1, Declaration of interest: The author states no conflict of interest and has received no payment in preparation of this manuscript.

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particularly neuronal lineages [6,7]. In this process, ESCs differentiate sequentially from pluripotent to multipotent stem cells, leading to precursor and terminally differentiated cells, and eliciting the mature phenotypes of the tissues [8]. In the nervous system, neural stem cells (NSCs) are the self-renewing multipotents cells that generate the main phenotypes of the nervous system: neuronal, astroglial and oligodendroglial [9]. Stem cells and their progenies express sequentially markers that reflect their state of differentiation. Nestin and oct3/4 are markers of stem and progenitor cells, including ESCs and neural progenitor and stem cells [10,11]. During the course of their differentiation and maturation in the nervous system, neuronal cells express - sequentially - markers of immature neuronal cells or early postmitotic neurons, e.g., doublecortin and class III β-tubulin isotype, and markers of mature neurons, like microtubule-associated protein-2 and neuronal nuclear antigen [12-15]. These markers are used routinely to assess the state of differentiation of stem cells and neuronal cells in vitro and in vivo, using standard procedures like immunohistology and reverse transcriptase polymerase chain reaction (RT-PCR), for example. It takes a few weeks to differentiate ESCs into mature neurons in vitro; a process that involves several steps and different culture conditions [6,7]. The application aims to prepare cryopreserved cultures of postmitotic neuronal cells with a high rate of survivability and readily usable, after thawing.

2. Chemistry 2.1 Defined Derivation Medium 2.1.1 Defined Derivation Medium #1 DMEM/F12, supplemented with 15% serum and 5% knockout serum replacement, 2 mM L-glutamine, 0.1 mM minimal essential medium nonessential amino acids, 50 U/ml penicillin, 50 µg/ml streptomycin, 4 ng/ml basic fibroblast growth factor (FGF-2), and 10 ng/ml leukaemia inhibitory factor (LIF); the use of penicillin and/or streptomycin is optional. 2.1.2 Defined Medium #2 DMEM/F12-based medium supplemented with N2, 2 mM L-glutamine, pencillin/streptomycin and 4 ng/ml FGF-2; the penicillin/streptomycin is optional. 2.1.3 Defined Medium #3 96% neural basal media supplemented with 1X penicillin/streptomycin, 2 mM Lglutamine, 1X B27, 20 ng/ml FGF-2 and 10 ng/ml LIF; the penicillin/streptomycin and LIF are optional components.

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2.2 Induction Medium 2.2.1 Induction Medium Neural Basal Media supplemented with 1X penicillin/streptomycin, 2 mM L-glutamine, 1X B27, 10 ng/ml LIF.

2.3 Production of Neural Progenitor Cells from Embryonic Stem Cells hESCs are cultured as previously described [5]. Cells are incubated at 37°C in a 5% CO2 humidified incubator. ESCs are differentiated into neural progenitor cells according to protocols described previously [6-8]. Briefly, hESCs are passaged onto feeder cells and allowed to proliferate in defined derivation culture medium #1 for 5 - 10 days. The cells are then cultured in defined medium #2 for 5 – 10 days. Next, the adherent hESCs are cultured in defined medium #2, without feeder cells, for 2 – 5 days. The adherent cells are isolated and cultured in defined medium #3 on coated culture plates, with laminin (5 µg/ml) and polyornithine (20 µg/ml for polystyrene plates, 50 µg/ml for glass plates). The cells can be maintained in culture indefinitely in defined medium #3. The culture of cells typically includes greater than about 85%, typically greater than about 90% neural progenitor cells. The neural progenitor cells express nestin and do not express class III β-tubulin or do not express detectable amounts of class III β-tubulin.

2.4 Induction of Neural Progenitor Cells into Neuronal or Neural-Like Cells Cultures are grown until confluence. The medium is replaced with an induction medium without FGF-2 for 2 - 5 days. Cultures are allowed to further differentiate for 2 - 3 weeks with changes in media every 3 - 4 days. As the cells in culture differentiate into neuronal or neural-like cells, the expression of nestin decreases and the expression of class III β-tubulin increases indicating the cells are becoming more mature and less proliferative. The cells are cultured for 10 - 20 days until they all begin to express neuronal class III β-tubulin. They are then frozen for cryopreservation.

2.5 Cryopreservation Cells are dissociated from the plates enzymatically, by adding an equal volume of dispase 1 g/l to the cultures. Once dissociated from the plates, fetal bovine serum is added to the culture, to inactivate the enzymes. Cells are not dissociated as single cells but remain in multicellular clumps at this point. Cells are harversted and spun in a centrifuge at 100 g, 4 min, twice. Cells are resuspended in induction medium with DMSO 10%. The medium is added to the cells dropwise. Vials were then brought to-80 at 1 °C per minute and placed into liquid nitrogen for long-term storage.

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2.6 Thawing Cryopreserved Cells When being thawed, the cryopreserved cells are incubated in a 37°C water bath until they are completely thawed. They are transferred to a culture tube using a pipette and gently resuspended in induction medium. They are then centrifuged and the supernatant discarded. The cells are suspended in induction medium and plated onto a poly-L-ornithine and laminincoated tissue culture plates.

2.7 Cultivation The cryopreserved cells are not fully mature. They express class III β-tubulin and continue to differentiate after thawing. The cells are suspended in induction medium and plated onto a poly-L-ornithine and laminin-coated tissue-culture plates. Once the cultures are thawed, neurite outgrowth can be seen within 10 – 14 h.

3. Biology and Action The expression of nestin and class III β-tubulin is monitored using conventional techniques such as RT-PCR and immunocytochemistry. The cells are cultured for 10 – 20 days, until they all begin to express neuronal class III β-tubulin. The cells are then frozen for cryopreservation. Cells are ready for cryopreservation: i) when nestin expression decreases and class III β-tubulin expression increases; ii) when nestin expression has decreased by 10% or more in the majority of the cells compared with neural progenitor cells; or iii) when nestin is undetectable. However, the cryopreserved cells are not fully mature. They express class III β-tubulin, a marker for early postmitotic neuronal cells, and are able to continue differentiating after thawing. After the cultures are thawed, neurite outgrowths can be seen, within 10 – 14 h. Postmitotic neuronal or neural-like cells can be cryopreserved with greater than 50% survivability after thawing.

4. Expert Opinion Cultured and cryopreserved postmitotic neuronal cells, prepared from ESCs, elicit a high rate of survivability and are readily usable after thawing. Postmitotic neuronal cells differentiated from hESCs and cryopreserved, as immature neuronal cells or early postmitotic neurons when they begin to express class III β-tubulin isotype, elicit a rate of survivability > 50% and neurite outgrowth within 10 - 14 h of thawing. Cryopreserved cultures of human postmitotic neuronal or neural-like cells provide a source of nerve cells for research and therapy. Cryopreserved cultures of neuronal cells elicit usually a rate of survivability in the range of 10% or below after thawing. The low rate of survival is partly the result of cryopreservation typically destroying structural components of the neurons, such as the

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cytoskeletal components, axons and neurites. The observation that cryopreserved cultures of early postmitotic neurons, expressing class III β-tubulin isotype, have greater than 10% survivability, up to 50%, and that these cells continue their process of differentiation and maturation in vitro, after thawing, has significant consequences for research and therapy. Predifferentiated nerve cells could be prepared to be readily available for transplantation in patients and for the pharmacological industry, for drug screening. Nerve cells can be prepared in vitro from different sources of tissues, in primary cultures from fetal tissue and from established cultures of stem cells, including ESCs and NSCs [16]. One of the advantages of preparing cultures of nerve cells from stem cells is the property of these latter cells to proliferate indefinitely in vitro, providing an unlimited source of tissue. In their application, the authors use ESCs as a source of cells for differentiating neuronal cells. Early postmitotic neurons, expressing the class III β-tubulin isotype, can be prepared in vitro from fetal, adult and postmortem cultures of neural progenitor and stem cells [17-21]. One advantage of using neural progenitor and stem cells isolated and cultured from adult and postmortem tissues is that they are not subject to the ethical and political debates and controversies surrounding the use of hESCs for research and therapy [1,2]. However, neural progenitor and stem cells yield to lower rates of differentiated neuronal cells, compared with ESCs, in vitro [18,22,23]. Pluripotent stem cells have been generated from somatic cells, i.e., induced-pluripotent stem cells, and are not subject to the ethical and political debates and controversies surrounding the use of hESCs. In addition, they may be used to generate isogenic lines of stem cells that would not be rejected by the patients following their transplantation [24-26]. These cells may also be used to generate early postmitotic neurons, expressing the class III β-tubulin isotype, applying the protocols described in this application. It would be interesting to validate the results from this application with the cryopreservation of early postmitotic neurons expressing the class III β-tubulin isotype, prepared from adult and postmortem neural progenitor and stem cells and induced-pluripotent stem cells, in vitro, to provide alternative sources of tissues readily usable for research and therapy [27-29]. In all, this application raises the interesting potential of cryopreserved predifferentiated neuronal cells, with a high yield of survival and the ability to proceed with their developmental programme. This opens the use of readily usable neuronal cells for research and therapy.

Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Cryopreservation of early post-mitotic neuronal cells in culture. Expert Opinion on Therapeutic Patents 19(2): 265-8. Copyright 2009, Informa UK Ltd.

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References** [1] [2] [3] [4] [5]

[6] [7] [8] [9] [10] [11] [12]

[13] [14]

[15]

[16] [17]

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Wobus AM, Boheler KR. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev 2005; 85: 635-78 • Complete review of ESCs. Taupin P. Derivation of embryonic stem cells for cellular therapy: Challenges and new strategies. Med Sci Monit 2006; 12: RA75-8 Stocum DL, Zupanc GK. Stretching the limits: Stem cells in regeneration science. Dev Dyn 2008; 237: 3648-71 Thomson JA, Kalishman J, Golos TG, et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995; 92: 7844-8 Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145-7. Erratum in: Science 1998; 282: 1827 •• First report of derivation of hESCs that were maintained in vitro for several passages. Schmandt T, Meents E, Gossrau G, et al. High-purity lineage selection of embryonic stem cell-derived neurons. Stem Cells Dev 2005; 14: 55-64 Glaser T, Perez-Bouza A, Klein K, et al. Generation of purifi ed oligodendrocyte progenitors from embryonic stem cells. FASEB J 2005; 19: 112-4 Lang KJ, Rathjen J, Vassilieva S, et al. Differentiation of embryonic stem cells to a neural fate: a route to re-building the nervous system? J Neurosci Res 2004; 76: 184-92 Taupin P, Gage FH. Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res 2002; 69: 745-9 Lendahl U, Zimmerman LB, Mckay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990; 60: 585-95 Okuda T, Tagawa K, Qi ML, et al. Oct-3/4 repression accelerates differentiation of neural progenitor cells in vitro and in vivo. Brain Res Mol Brain Res 2004; 132: 18-30 Bernhardt R, Matus A. Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons. J Comp Neurol 1984; 226: 203-21 Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development 1992; 116: 201-11 Fanarraga ML, Avila J, Zabala JC. Expression of unphosphorylated class III betatubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation. Eur J Neurosci 1999; 11: 517-27 Francis F, Koulakoff A, Boucher D, et al. Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons. Neuron 1999; 23: 247-56 Taupin P. Adult neural stem cells: a promising candidate for regenerative therapy in the CNS. Int J Integr Biol 2008; 2: 85-94 Gage FH, Coates PW, Palmer TD, et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci USA 1995; 92: 11879-83

Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

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[18] Cattaneo E, Conti L, Gritti A, et al. Non-virally mediated gene transfer into human central nervous system precursor cells. Brain Res Mol Brain Res 1996; 42: 161-6 [19] Roy NS, Wang S, Jiang L, et al. In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat Med 2000; 6: 271-7 [20] Palmer TD, Schwartz PH, Taupin P, et al. Cell culture. Progenitor cells from human brain after death. Nature 2001; 411: 42-3 • First isolation and chracterization of neural progenitor and stem cells from human postmortem tissues and biopsies. [21] Schwartz PH, Bryant PJ, Fuja TJ, et al. Isolation and characterization of neural progenitor cells from post-mortem human cortex. J Neurosci Res 2003; 74: 838-51 [22] Taupin P, Ray J, Fischer WH, et al. FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. Neuron 2000; 28: 385-97 [23] Cultures of human CNS neural stem cells: US20080107633A1; 2008 [24] Yu J, Vodyanik MA, Smuga-otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318: 1917-20 •• i-PSCs are generated from somatic cells. They may be used to generate isogenique lines of stem cells for therapy. [25] Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defi ned factors. Cell 2007; 131: 861-72 [26] Yu J, Thomson JA. Pluripotent stem cell lines. Genes Dev 2008; 22: 1987-97 [27] Lindvall O, Kokaia Z, Martinez-Serrano A. Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat Med 2004; 10(Suppl): S42-50 [28] Taupin P. The therapeutic potential of adult neural stem cells. Curr Opin Mol Ther 2006; 8: 225-31 [29] Taupin P. Therapeutic neuronal stem cells: patents at the forefront. Expert Opin Ther Patents 2008; 18: 1107-10

Chapter VI

Adult Neural Stem Cells for the Treatment of Neuroinflammation* Abstract Background: The application is in the field of stem cells and their therapeutic application for the treatment of inflammation. Objective: It aims at characterizing the potential of adult-derived neural progenitor and stem cells for the treatment of inflammation of the central nervous system (CNS). Methods: Neural progenitor and stem cells were isolated and expanded from the subventricular zone (SVZ) of adult mice (aNSCs). They were administered intravenously in an animal model of multiple sclerosis (MS). Results: Mice transplanted either at the disease onset or at the onset of the first relapse show clinical signs of improvements. Adult NSCs exert their therapeutic activity by reducing neuroinflammation. Conclusion: The application claims the use of aNSCs and multipotent somatic stem cells for the treatment of inflammation, associated with neurological diseases, disorders and injuries particularly, and for inducing tolerance to the immune central and/or peripheral system.

1. Introduction Somatic stem cells are self-renewing cells that generate the main cell types of the body [1]. Embryonic stem cells are pluripotents. They generate cell types of the three germ layers of the embryos: the neurectoderm, mesoderm and endoderm. Embryonic stem cells are derived from the inner cell mass of blastocysts [2]. Fetal and adult stem cells are multipotents, generating the cell types from the tissue from which they originate. During development, stem cells contribute to the formation of the tissues. In the adult, stem cells

*Patent Evaluation: Fondazione Centro San Raffaele del Monte Tabor: WO2007015173. Patent details: Title: Inflammation, Assignee: Fondazione Centro San Raffaele del, Monte Tabor, Inventors: Pluchino S, Martino G., Priority data: 12/07/2005, Filling date: 12/07/2006, Publication date: 08/02/2007, Publication no: WO2007015173. Declaration of interest: The author states no conflict of interest and has received no payment in preparation of this manuscript.

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contribute to homeostasis of the tissues and regeneration after injury. Stem cells can be isolated from various adult tissues, including the bone marrow, liver and skin [3]. The adult brain used to be believed to be devoid of stem cells. Underlying this belief was the fact that the adult brain lacks the ability to self-repair, particularly in neurodegenerative diseases, like Alzheimer’s and Parkinson’s diseases, and after injury. Studies in the 1980s and 1990s reported cell proliferation and neurogenesis in two regions of the adult brain, the SVZ and the dentate gyrus of the hippocampus, in rodents [4]. In 1992, Reynolds and Weiss reported the isolation and characterization of neural progenitor and stem cells from the striatal area, including the SVZ, of adult mice brains [5]. Neural stem cells (NSCs) are the self-renewing multipotent cells that generate the main phenotypes of the nervous system: neurons, astrocytes and oligodendrocytes. Neural progenitor and stem cells have been isolated and characterized from various regions of the adult CNS, including the spinal cord, and from various species, including human post-mortem tissues [4]. Neurogenesis has been reported in the dentate gyrus and SVZ of various mammalian species, including humans [6,7]. The confirmation that adult neurogenesis occurs in the adult brain and that NSCs reside in the adult CNS, opens new opportunities for the treatment of neurological diseases and injuries, particularly neurodegenerative diseases. It is proposed to stimulate endogenous neural progenitor or stem cells or to transplant adult-derived neural progenitor and stem cells, to repair the degenerated or injured pathways [8,9]. Inflammation is a self-defensive process by which cells of the immune system and proinflammatory substances of the body protect us from infections, foreign substances and injuries. During the inflammatory response, the immune cells release a host of powerful regulatory substances, including glutamate, nitric oxide, reactive oxygen species, cytokines, chemokines, interferons (IFNs) and interleukins (ILs) [10,11]. These chemicals produce both beneficial and harmful effects to the cellular environment, creating further damage. In the CNS, inflammatory responses occur in a broad range of neurological diseases, disorders and injuries, including Alzheimer’s disease, cerebral strokes, traumatic brain and spinal cord injuries [12,13]. Chronic inflammation may also be a causative factor for neurological diseases and disorders and particularly for neurodegenerative diseases, like Alzheimer’s disease [14]. Multiple sclerosis (MS) is an autoimmune disease of the nervous system that leads to paralysis and disabilities. It is associated with loss of axons and myelin sheaths [15] and is characterized by delayed-type hypersensitivity (Th1) inflammatory responses. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS. In MS and EAE, Th1 cells and the expression of Th1-associated cytokines (IFN-γ and IL-6) and of Th1inducing cytokine (IL-12) is associated with active or relapse of the disease, whereas Th2 cells and the expression of the Th2 cytokines, IL-4, IL-10 and IL-13, are associated with remission or suppression of the disease [16]. Neural progenitor and stem cells, administered intravenously, migrate to diseased and injured sites of the brain [17]. When administered in the EAE model of MS, adult NSCs (aNSCs) migrate to the sites of inflammation in the CNS and induce functional recovery [18]. The application aims to characterize the therapeutic potential of aNSCs for the treatment of MS particularly and neuroinflammatory diseases in general. It aims to identify the cellular and molecular mechanisms underlying the therapeutic activity of transplanted aNSCs in the EAE model.

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2. Chemistry Cultures of neural progenitor and stem cells (aNSCs) were established from the SVZ of adult (6- to 8-week-old) SJL and C57BL/6 mice, based on the protocol originally described by Reynolds and Weiss (in 1992) and modified by Pluchino et al. (in 2003) [5,18]. Briefly, the brains were isolated and the SVZ regions dissected out. During this process, the subependyma was removed from the dissected tissue. The tissue was cut into 1-mm 3 pieces and digested with trypsin, hyaluronidase and kynurenic acid. The dissociation of the tissue was completed by mechanical trituration, with fire-polished Pasteur pipettes. Cells were resuspended and cultured in DMEM/F12 medium, containing 2 mM L-glutamine, 0.6% glucose, 9.6 mg/ml putrescine, 6.3 ng/ml progesterone, 5.2 ng/ml sodium selenite, 0.025 mg/ml insulin, 0.1 mg/ml transferrin and 2 μg/ml heparin. Cells were cultured in the presence of NS-A medium (Euroclone) containing 20 ng/ml of epidermal growth factor and 10 ng/ml basic fibroblast growth factor. Adult neural progenitor and stem cells were cultured, as neuropheres, in untreated tissue culture flasks. Cells under 15 passages were used. For transplantation studies, aNSCs were labeled in vitro, with Escherichia coli -derived β-galactosidase (LacZ), using a third-generation lentiviral vector pRRLsin.PPT-hCMV, as previously described [18].

3. Biology and Action 3.1 Relapsing-Remitting Model of Experimental Autoimmune Encephalomyelitis The relapsing-remitting model of EAE (R-EAE) was used. R-EAE was induced experimentally by administration of 200 μg PLP139-151 in complete Freund’s adjuvant in SJL mice [19].

3.2 Syngeneic Transplantation β-gal-labelled aNSCs were dissociated as single cells and injected intravenously into REAE mice through the tail vein: 1 - 2.10 6 cells in 150 μl, prepared in PBS, were administered per mouse. The cells were administered at the first episode of the disease [13.1 ± 0.3 days post immunization (dpi)] or at the occurrence of first clinical relapse (30.9 ± 1.1 dpi). Clinical relapse is assessed by recording daily the body weight and clinical score (0 = healthy, 1 = limp tail, 2 = ataxia and/or paresis of hind limbs, 3 = paralysis of hind limbs and/or paresis of forelimbs, 4 = tetra paralysis, 5 = moribund or death). Clinical relapses are defined as the occurrence of 0.5 increases of the clinical score persisting for a minimum of three consecutive days [19]. Mice were followed up to three months post-transplantation, after which they were sacrificed.

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3.3 In Situ Proliferation of Transplanted Anscs Three days before sacrifice, R-EAE mice were given one dose of bromodeoxyuridine (BrdU, 50 mg/kg) per day. BrdU is a thymidine analog used for birth dating and monitoring cell proliferation.

3.4 Immunocytology and Immunohistology For immunocytology, neurospheres were cultured onto glass chamber slides coated with matrigel. For immunohistology, brain tissues were embedded in paraffin or in agarose, and were processed according to standard procedures. In particular, brain sections were stained with Luxol fast blue and Bielshowwsky for detecting inflammatory inflitrates, demyelination and axonal loss [20]. The cells and tissues were processed for immunofluorescence, applying standard procedures. A broad range of primary antibodies was used, including antibodies againts BrdU, nestin, neuronal class III β-tubulin, NG2, CD45, CD31 and FasL/CD95-ligand. A range of assays and studies were conducted, both in vitro and in vivo, to assess the cellular and molecular mechanisms underlying the therapeutic activity of transplanted aNSCs in the EAE model. This includes adhesion assays, chemotaxis assays, cytokine and chemokine assays, apotosis experiments, RT-PCR, flow cytometry, FACS analysis and intravital microscopy. The results show that R-EAE mice, administered with aNSCs, elicit signs of clinical improvements and that aNSCs induce the programmed cell death of immune cells involved in pro-inflammatory response. Clinical improvements were observed whether aNSCs were administered at the disease onset or at the onset of the first relapse. Mice transplanted at disease onset recover between 30 and 60 dpi. During this period, they develop 2-fold fewer clinical relapses than sham-treated mice. Mice transplanted at disease onset maintained a low rate of relapse than sham-treated mice, up to the end of the follow-up study. Mice transplanted at the onset of the first relapse starts to recover later, but elicit a 3-fold reduction of the relapse rate between 60 and 90 dpi, when compared to sham-treated mice. R-EAE mice transplanted with aNSCs are characterized by a pattern of programmed cell death of proinflammatory Th1 cells, and not of the anti-inflammatory Th2 cells, in the inflamed perivascular areas of the CNS. Results further show that transplanted aNSCs survive the inflammatory episodes and maintain a pool of undifferentiated cells with the ability to proliferate, in the inflammed areas, up to the end of the follow-up study. This shows that aNSCs exert an anti-inflammatory activity, in the R-EAE model of MS, by reducing the proinflammatory response in the CNS. The transplanted aNSCs migrate to the inflamed areas of the CNS, where they maintain a pool of cells. aNSCs may retain the ability to protect against recurrent episodes of inflammation; they also promote endogenous myelin-producing cells to acquire a mature phenotype and replace damaged neural cells. The neuroprotective activity of aNSCs during neuroinflammation in the R-EAE model reveals a therapeutic potential for aNSCs for the treatment of inflammation and inflammatory diseases of the nervous system [21].

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4. Expert Opinion The present application claims the use of adult-derived neural progenitor and stem cells for the treatment of neuroinflammation and inflammatory diseases, particularly of the nervous system, and for inducing tolerance to the immune central and/or peripheral system. The transplanted adult-derived neural progenitor and stem cells prevent or decrease inflammation through either the induction of programmed cell death (apoptosis) of bloodborne CNS-infiltrating pro-inflammatory Th1 cells or/and through an immunomodulatory mechanism leading to immune tolerance. The transplanted stem cells maintain their ability to protect the CNS from chronic inflammatory reactions. The anti-inflammatory property of adult-derived neural progenitor and stem cells may be used for the treatment of inflammatory diseases, and also for the treatment of a broad range of neurological diseases, disorders and injuries in which neuroinflammation is involved, like Alzheimer’s disease, cerebral strokes, depression, epilepsy, Parkinson’s disease, and traumatic brain and spinal cord injuries [14,15]. It can also be used to treat other inflammatory diseases or inflammation associated with other diseases, like diabetes and rheumatoid arthritis. A strength of the present application is that the administration of the stem cells, by intravenous injection, is a noninvasive procedure to treat neuroinflammation. Stem cells reside in specialized microenvironments or ‘niches’ [22]. Such niches regulate and control the self-renewal and differentiation activities of NSCs. An angiogenic and astroglial niche for neurogenesis has been identified and characterized in the adult brain [23]. It is proposed that the perivascular areas, sites of neuroinflammation in the R-EAE mice, would create a microenvironment where neural progenitor and stem cells from the bloodstream would migrate and accumulate. These ‘atypical perivascular niches’ in the CNS would control the development and fate of the adult-derived neural progenitor and stem cells, and therefore their therapeutic potential for the treatment of neuroinflammatory responses. This includes the maintenance of a pool of undifferentiated neural progenitor and stem cells, to protect the CNS from chronic inflammatory reactions. The migration of the adult-derived neural progenitor and stem cells to the sites of inflammation in the CNS would be facilitated by the disruption of the blood–brain barrier, as a consequence of the neuroinflammation process [24]. This disruption allows cells from the haematopoietic system and from the administered neural progenitor and stem cells to leave the bloodstream and come into contact with the site of injury, to exert their biological activities. The potential of adult-derived neural progenitor and stem cells to reduce neuroinflammation broadens the therapeutic potential of adult-derived neural progenitor and stem cells to the treatment of inflammation and inflammatory diseases. It not only offers new opportunities and avenues for the treatment of inflammation and inflammatory diseases, but also neurological diseases, disorders and injuries, by attacking one of their causes. There are, however, questions that remain to be answered. Among them, additional controls might be needed to confirm the genetic labelling of the transplanted aNSCs in situ. What would be the optimal source of stem cells to use for such therapy, given that autologous and syngeneic transplantations are not likely candidates for the transplantation of adult-derived neural progenitor and stem cells? And how could such strategy applied to reduce neuroinflammation

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in allogeneic transplantations, particularly in the nervous system, to reduce the risk of tissue rejection?

Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Adult neural stem cells for the treatment of neuroinflammation. Expert Opinion on Therapeutic Patents 19(3): 373-6. Copyright 2009, Informa UK Ltd.

References** [1]

Potten CS, Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 1990; 110: 1001-20, • Review defi ning stem cells criteria. [2] Taupin P. Derivation of embryonic stem cells for cellular therapy: Challenges and new strategies. Med Sci Monit 2006; 12: RA75-78 [3] Stocum DL, Zupanc GK. Stretching the limits: Stem cells in regeneration science. Dev Dyn 2008; 237: 3648-71 [4] Taupin P, Gage FH. Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res 2002; 69: 745-9 [5] Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992; 255: 1707-10, •• Original report of the isolation and characterization of neural progenitor and stem cells from the adult brain of mammals. [6] Eriksson PS, Perfilieva E, Bjork-Eriksson T, et al. Neurogenesis in the adult human hippocampus. Nat Med 1998; 4: 1313-7, • First evidence of adult neurogenesis in the human brain. [7] Curtis MA, Kam M, Nannmark U, et al. Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Science 2007; 315: 1243-9 [8] Taupin P. The therapeutic potential of adult neural stem cells. Curr Opin Mol Ther 2006; 8: 225-31 [9] Taupin P. Therapeutic neuronal stem cells: patents at the forefront. Exp Opin Ther Patents 2008; 18: 1107-10 [10] Jander S, Schroeter M, Stoll G. Interleukin-18 expression after focal ischemia of the rat brain: association with the late-stage inflammatory response. J Cereb Blood Flow Metab 2002; 22: 62-70 [11] Ghirnikar RS, Lee YL, Eng LF. Inflammation in traumatic brain injury: role of cytokines and chemokines. Neurochem Res 1998; 23: 329-40 [12] Nencini P, Sarti C, Innocenti R, et al. Acute inflammatory events and ischemic stroke subtypes. Cerebrovasc Dis 2003; 15: 215-21

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[13] Schmidt OI, Heyde CE, Ertel W, et al. Closed head injury – an inflammatory disease? Brain Res. Brain Res Rev 2005; 48: 388-99 [14] Arnaud L, Robakis NK, Figueiredo-Pereira ME. It may take inflammation, phosphorylation and ubiquitination to ‘tangle’ in Alzheimer’s disease. Neurodegener Dis 2006; 3: 313-9 [15] Minghetti L. Role of inflammation in neurodegenerative diseases. Curr Opin Neurol 2005; 18: 315-21, • Inflammation as a causative factor of neurological diseases and disorders. [16] Owens T, Wekerle H, Antel J. Genetic models for CNS inflammation. Nat Med 2001; 7: 161-6 [17] Brown AB, Yang W, Schmidt NO, et al. intravascular delivery of neural stem cell lines to target intracranial and extracranial tumors of neural and non-neural origin. Hum Gene Ther 2003; 14: 1777-85 [18] Pluchino S, Quattrini A, Brambilla E, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 2003; 422: 688-94, •• Original report for the therapeutic potential of adult-derived neural progenitor and stem cells for the treatment of neuroinflammation. [19] McRae BL, Kennedy MK, Tan LJ, et al. Induction of active and adoptive relapsing experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope of proteolipid protein. J Neuroimmunol 1992; 38: 229-40 [20] Lindvall O, Kokaia Z, Martinez-Serrano A: Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat Med 2004; 10 (Suppl): S42-50 [21] Pluchino S, Zanotti L, Rossi B, et al. Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism. Nature 2005; 436: 266-71 [22] Mitsiadis TA, Barrandon O, Rochat A, et al. Stem cell niches in mammals. Exp Cell Res 2007; 313: 3377-85 [23] Ninkovic J, Götz M. Signaling in adult neurogenesis: from stem cell niche to neuronal networks. Curr Opin Neurobiol 2007; 17: 338-44 [24] Lossinsky AS, Shivers RR. Structural pathways for macromolecular and cellular transport across the blood-brain barrier during infl ammatory conditions. Histol Histopathol 2004; 19: 535-64

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Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

Chapter VII

Adult Periodontal-Derived Neural Progenitor and Stem Cells* Abstract The application is in the field of neural stem cells (NSCs) and cellular therapy. It aims to identify and characterize neural progenitor and stem cells from adult periodontal tissue. Neural progenitor and stem cells were isolated and characterized from periodontal tissue originating from biopsies of adult patients. Adult human periodontal-derived neural progenitor and stem cells can be induced to differentiate into neuronal and glial cells, osteoblasts and cells of the periodontium. They survive and integrate when transplanted into organotypic hippocampal slice cultures. The application claims the use of periodontal neural progenitor and stem cells for cellular therapy, particularly for the treatment of the periodontal diseases and neurodegenerative diseases and neurological injuries. Periodontal tissue can be harvested with minimal invasive procedures from the patient himself, providing a promising source of tissue for NSC-based therapy and autologous transplantation.

1. Introduction Neural stem cells (NSCs) are the self-renewing multipotent cells that generate the main phenotypes of the nervous system: neuronal, astroglial and oligodendroglial. During development, they undergo cell proliferation, differentiation, fate specification and maturation to form the neuronal network of the nervous system. In the adult of various mammalian species, including humans, neurogenesis occurs primarily in two regions of the brain: the hippocampus and subventricular zone [1]. Newly generated neuronal cells in the

*Patent Evaluation: Institut fur Molekulare Diagnostik und Innovative Therapie, WO2008031451. D Patent details: Title: A post-natal periodontal-derived neural stem cell, Assignee: Institut fur Molekulare Diagnostik und Innovative Therapie, Inventors: Kaltschmidt B, Widera D, Grimm WD, Kaltschmidt C., Filing date: 11/09/06, Publication date: 20/03/08, Publication no.: WO2008031451 Declaration of interest: The author states no conflict of interest and has received no payment for this manuscript.

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adult brain would originate from a population of residual NSCs. In support of the contention, neural progenitor and stem cells have been isolated and characterized in vitro from the adult brain [2]. The existence of NSCs in the adult CNS has important implications for our understanding of development and for cellular therapy. On the one hand, the function of newly generated neuronal cells in the adult brain remains to be fully understood. On the other hand, the adult CNS may be amenable to repair. To this end, endogenous neural progenitor and stem cells may be stimulated or adult-derived neural progenitor and stem cells may be transplanted to restore the degenerated or injured pathways. Adult-derived neural progenitor and stem cells offer the opportunity of performing autologous transplantation by isolating and propagating these cells from an undamaged area of the patient brain [3]. However, such a strategy remains unlikely as it would involve invasive surgical procedures and would carry the risk of provoking additional lesions in the brain of the patients [2]. Other sources of adult progenitor and stem cells are being considered for transplantation for the treatment of neurodegenerative diseases and neurological injuries. These include neural progenitor and stem cells isolated and propagated from post-mortem brain tissue and progenitor and stem cells isolated and propagated from tissues other than the nervous system [4-6]. Multipotent stem cells give rise to the phenotypes of the tissue in which they reside. However, it is possible that they have a broader potential [7]. Stem cells isolated from the haematopoietic system and the skin give rise to neuronal phenotypes in vitro [8-10]. Conversely, adult-derived neural progenitor and stem cells can give rise to cells of the haematopoietic system in vitro [11]. Whether adult stem cells elicit a broader potential or whether these observations involve experimental artefacts is still under debate. However, the broader potential of adult stem cells has important implications for stem cell-based therapy, particularly for the nervous system. Skin-derived progenitor and stem cells could be used as source of neuronal tissue for transplantation in the nervous system. The advantage of using a tissue such as the skin as a source of neuronal tissue is two-fold: (1) the cells could easily be isolated and propagated without the need of invasive surgical procedures; and (2) the cells could from the patient’s own skin, allowing autologous transplantation. Progenitor and stem cells have been isolated and characterized from various tissues of the tooth, including the dental pulp or dental pulp stem cells and the periodontal ligament or periodontal ligament stem cells [12-14]. The dental pulp and periodontal ligament derive from the embryonic germ layer, the mesoderm. Dental pulp stem cells and periodontal ligament stem cells express markers associated with mesenchymal stem cells, like the tendonspecific marker scleraxis [15,16]. Progenitor and stem cells expressing scleraxis lead to the formation of tendon, muscle attachments and blood vessels [17]. Periodontal tissues such as the cementum derive from the embryonic germ layer, the neurectoderm. They originate from the neural crest. Periodontal stem cells express markers associated with NSCs, like nestin and neural cell adhesion molecule [18,19]. Nestin is a marker of neural progenitor and stem cells and early neural crest cells [20-22]. It is expressed during development of the teeth in certain type of cells, particularly odontoblasts, and is up-regulated in odontoblasts surrounding the injury site in carious and injured teeth [23,24]. Neurotrophic factors, like nerve growth factor, brain-derived neurotrophic factor and glial cell-line derived neurotrophic factor, are highly expressed within the developing human teeth [25]. The existence of neural progenitor and

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stem cells in dental adjacencies, like in periodontal tissue or periodontal NSCs (pdNSCs), might be explained by the ontogeny of the human teeth. NSCs in dental tissues may be involved in the replacement of degenerated neuronal tissue of the teeth and/or of dental tissues as a result of diseases, like periodontal defects [26]. The aims of the application are to isolate and characterize neural progenitor and stem cells from adult periodontal tissue. The existence of neural progenitor and stem cells in dental adjacencies, like periodontal tissues, may provide a source of stem cells for cellular therapy for the treatment of periodontal diseases and neurological diseases and injuries.

2. Chemistry 2.1 Periodontal Tissue Periodontal tissue was obtained from seven patients (age 23 - 54) with periodontal defects. The tissues were isolated by intrasulcular incisions on the teeth adjacent to the defects, followed by the dissection of periodontal tissue using microsurgical instrumentation [27].

2.2 Cell Culture Adult human periodontal-derived neural progenitor and stem cells were isolated and cultured as neurospheres. Briefly, adult periodontal tissue was collected in ice cold Hank’s balanced salt solution (HBSS) containing 300 mg/ml D –glucose and dissociated with 1.33 mg/ml trypsin, 0.7 mg/ml hyaluronidase, 200 μ/ml DNAse and 0.2 mg/ml kynurenic acid at 37 ° C. Cells were centifuged at 212 x g and resuspended in Dulbecco’s modified Eagle’s medium (DMEM)/F12, in defined medium with B27 supplement (60 μl/ml), in the presence of epidermal growth factor (20 ng/ml) and basic fibroblast growth factor (20 ng/ml). Cells were passaged according to standard protocols, with the use of Accutase (PAA, Pasching).

2.3 Differentiation Adult periodontal-derived neural progenitor and stem cells were dissociated, plated onto poly- D-lysine/laminin-coated culture dishes and differentiated to: i) the neuronal pathway in the presence of 5 μM retinoic acid; and ii) the glial pathway in the presence of 10% fetal bovine serum. After 4 days, cell were fixed with 4% paraformaldehyde and processed for immunocytology. Adult periodontal-derived neural progenitor and stem cells were dissociated, plated on poly- D-lysine/laminin coated culture dishes and differentiated to the osteoblast pathway in NH OsteoDiff Medium (Miltenyi Biotec GmbH). After 20 days, cell were fixed with 4% paraformaldehyde and processed for immunocytology.

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2.4 Immunocytology Cells were processed for immunocytofluorescence according to standard procedures and were analysed by confocal microscopy. The following primary antibodies were used: antiA2B5, anti-CD117, anti CD133, anti-CD34, anti-class III β-tubulin isotype, anti-GAD67, anti-GFAP, anti-LeX, anti-L1, anti-MAP2, anti-Musashi, anti-Nestin, anti-NF-H, anti-NF-L, anti NF-M, anti-Notch1, anti-PSA-NCAM, anti-Scleraxis, anti-Sox2. Nuclear staining was done with SYTOX (Molecular Probes).

2.5 Organotypic Hippocampal Slice Cultures Organotypic hippocampal slice cultures were prepared from rat tissue and maintained as previously described [28]. Briefly, rat hippocampi (postnatal day 5) were cut perpendicularly to the longitudinal axis into slices 400 μm thick using a McIlwain tissue chopper (Mickle Laboratory Engineering). Slices were kept in ice-cold minimal essential medium (MEM) HBSS, pH 7.3, containing 25 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2 mM L-glutamine. Slices were transferred on top of Millipore membranes (diameter 30 mm, pore size 0.4 μm) in six-well plates containing 750 μl culture medium (50% MEM HBSS, 25% heat-inactivated horse serum, 2 mM L-glutamine, 5 mM NaHCO3) per well and incubated in a humidified chamber with 95% air/5% CO 2 at 37 ° C. Adult human periodontal-derived neural progenitor and stem cells were transfected using a Rat NSC Nucleofector Kit, with p max GFP (green fluorescent protein) (Amaxa). The transfected cells were differentiated for 24 h, in presence of retinoic acid. After dissociation, cells were dropped on the slices and cultured for 13 days. After 13 days, organotypic cultures were fixed and GFP fluorescence was analysed by confocal microscopy.

2.6 Further Studies Adult human periodontal-derived neural progenitor and stem cells were also studied and characterized using reverse transcription polymerase chain reaction and the Boyden migration assay to characterize the behaviour and migration of the cells.

3. Biology and Action The results show that adult human periodontal-derived progenitor and stem cells can be isolated and propagated, as neurospheres, in the presence of epidermal growth factor and basic fibroblast growth factor. The isolated and propagated progenitor and stem cells in vitro are immunopositive for nestin and Sox-2, markers of neural progenitor and stem cells, but not scleraxis. After differentiation with retinoic acid, adult human periodontal-derived neural progenitor and stem cells express markers of the neuronal lineages, like anti-class III βtubulin isotype, a marker of immature neuronal cells [29]. After differentiation with fetal

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bovine serum, adult human periodontal-derived neural progenitor and stem cells are immunopositive for glial acidic fibrillary protein (GFAP), a marker of astrocytes. After differentiation in NH OsteoDiff Medium, adult human periodontal-derived neural progenitor and stem cells are immunopositive for alkaline phosphatase, a marker for osteoblasts. Adult human periodontal-derived neural progenitor and stem cells survive and integrate when transplanted into organotypic rat hippocampal slice cultures. This shows that neural progenitor and stem cells can be isolated and characterized from adult periodontal tissue. Adult human periodontal-derived neural progenitor and stem cells can be induced to differentiate into neuronal and glial cells and osteoblasts and cells of the periodontium. Studies from organotypic hippocampal slice cultures reveal that adult periodontal-derived neural progenitor and stem cells might integrate into brain tissue after transplantation.

4. Expert Opinion Neural progenitor and stem cells can be isolated and characterized from adult periodontal tissue. They express marker of neural progenitor and stem cells, like nestin and Sox-2; they differentiate into lineages of the nervous system, as well as osteoblasts and cells of the periodontium; and they differ from other stem and progenitor cells isolated and characterized from the teeth that are of mesenchymal origin, particularly as they do not express scleraxis. Adult human periodontal-derived neural progenitor and stem cells offer a source of tissue for cellular therapy, for the treatment of periodontal diseases and of neurological diseases and injuries. Periodontal tissue originates from the embryonic germ layer, the neurectoderm. It derives from the neural crest. As such stem cells from isolated and characterized from adult periodontal tissues elicit and share similar characteristics with NSCs isolated and characterized from the adult CNS. Because of their potential to generate the main phenotypes of the nervous system, NSCs hold the potential to cure a broad range of neurological diseases and injuries, particularly neurodegenerative diseases, cerebral strokes and spinal cord injuries. However, one of the main problems facing NSC-based therapy is the source of the tissue from which to isolate and propagate neural progenitor and stem cells to be used for transplantation therapy. Neural precursor cells can be generated from various sources of stem cells in vitro, embryonic, fetal, adult and post-mortem stem cells [30,31]. The use of embryonic human tissue for therapy is the source of ethical and political debates and controversies. The use of fetal, adult and post-mortem human neural tissue is mostly limited to heterologous transplantation, thereby requiring matching the donor and recipient histocompatibility or lifelong use of immunosuppressants. Neural progenitor and stem cells could be isolated and propagated from an undamaged area of the patient brain, for autologous transplantation. However, such a strategy remains unlikely, as it would involve invasive surgical procedures and would carry the risk of provoking additional lesions in patients’ brain. Hence, peripheral sources of neural progenitor and stem cells that are easily accessible with minimal invasive surgical procedures, like pdNSCs, offer alternative sources of stem cells for therapy for the treatment of neurological diseases and injuries that could be used for

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autologous transplantation by isolating and propagating the cells from the patients’ own tissue. To this end, other sources of tissues have been proposed for generating neuronal cells for therapy, like the skin. However, the mechanisms underlying the broader potential of adult stem cells are still not fully understood. Because they derive from the neural crest, originating from the neurectoderm, pdNSCs may provide a more suitable source of neuronal cells for therapy for the treatment of neurological diseases and injuries than other sources of stem cells of non-neural origin. In conclusion, pdNSCs offer a promising model for cellular therapy for the treatment of periodontal diseases and neurological diseases and injuries. Further experimental studies remain to be performed in vivo, to validate the potential of pdNSCs for therapy.

Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Adult periodontal-derived neural progenitor and stem cells. Expert Opinion on Therapeutic Patents 19(5): 715-9. Copyright 2009, Informa UK Ltd.

References** [1] [2] [3] [4]

[5] [6] [7] [8] [9]

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Frisén J, Johansson CB, Lothian C, et al. Central nervous system stem cells in the embryo and adult. Cell Mol Life Sci 1998; 54: 935-45 Taupin P. Neural Progenitor and Stem Cells in the Adult Central Nervous System. Ann Acad Med Singapore 2006; 35: 814-7 Taupin P. Autologous transplantation in the central nervous system. Indian J Med Res 2006; 124: 613-8 Palmer TD, Schwartz PH, Taupin P, et al. Cell culture. Progenitor cells from human brain after death. Nature 2001; 411: 42-3, • First isolation and chracterization of neural progenitor and stem cells from human post-mortem tissues and biopsies. Mayer EJ, Carter DA, Ren Y, et al. Neural progenitor cells from post-mortem adult human retina. Br J Ophthalmol 2005; 89: 102-6 Taupin P. Adult neural stem cells, neurogenic niches and cellular therapy. Stem Cell Rev 2006; 2: 213-20 Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med 2001; 7: 393-5 Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000; 290: 1779-82 Toma JG, Akhavan M, Fernandes KJ, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001; 3: 778-84, • Skin progenitor and stem cells generate neuronal phenotypes and could be used for autologous transplanation for the treatment of neurological diseases and injuries.

Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

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[10] Joannides A, Gaughwin P, Schwiening C, et al. Effi cient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells. Lancet 2004; 364: 172-8 [11] Bjornson CR, Rietze RL, Reynolds BA, et al. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999; 283: 534-7 [12] Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 2000; 97: 13625-30 [13] Shi S, Robey PG, Gronthos S. Comparison of human dental pulp and bone marrow stromal stem cells by cDNA microarray analysis. Bone 2001; 29: 532-9 [14] Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004; 364: 149-55 [15] Schweitzer R, Chyung JH, Murtaugh LC, et al. Analysis of the tendon cell fate using Scleraxis, a specifi c marker for tendons and ligaments. Development 2001; 128: 385566 [16] Shi S, Bartold PM, Miura M, et al. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res 2005; 8: 191-9 [17] Cserjesi P, Brown D, Ligon KL, et al. Scleraxis: a basic helix-loop-helix protein that prefigures skeletal formation during mouse embryogenesis. Development 1995; 121: 1099-110 [18] Terling C, Rass A, Mitsiadis TA, et al. Expression of the intermediate fi lament nestin during rodent tooth development. Int J Dev Biol 1995; 39: 947-56 [19] Obara N, Suzuki Y, Nagai Y, et al. Expression of neural cell-adhesion molecule mRNA during mouse molar tooth development. Arch Oral Biol 2002; 47: 805-13 [20] Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990; 60: 585-95 [21] Stemple DL, Anderson DJ. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell 1992; 71: 973-85 [22] Lothian C, Lendahl U. An evolutionarily conserved region in the second intron of the human nestin gene directs gene expression to CNS progenitor cells and to early neural crest cells. Eur J Neurosci 1997; 9: 452-62 [23] Hildebrand C, Fried K, Tuisku F, et al. Teeth and tooth nerves. Prog Neurobiol 1995; 45: 165-222 [24] About I, Laurent-Maquin D, Lendahl U, et al. Nestin expression in embryonic and adult human teeth under normal and pathological conditions. Am J Pathol 2000; 157: 287-95 [25] Nosrat I, Seiger A, Olson L, et al. Expression patterns of neurotrophic factor mRNAs in developing human teeth. Cell Tissue Res 2002; 310: 177-87 [26] Pierret C, Spears K, Maruniak JA, et al. Neural crest as the source of adult stem cells. Stem Cells Dev 2006; 15: 286-91, • Potential of neural crest for NSC-based therapy. [27] Gassmann G, Grimm WD. Minimal invasive regenerative und plastisch rekonstruktive Parodontalchirurgie. Dent Implantol 2006; 10: 90-7 [28] Stoppini L, Buchs PA, Muller D. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 1991; 37: 173-82

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[29] Fanarraga ML, Avila J, Zabala JC. Expression of unphosphorylated class III betatubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation. Eur J Neurosci 1999; 11: 517-27 [30] Schmandt T, Meents E, Gossrau G, et al. High-purity lineage selection of embryonic stem cell-derived neurons. Stem Cells Dev 2005; 14: 55-64 [31] Taupin P. Therapeutic neuronal stem cells: patents at the forefront. Expert Opin Ther Patents 2008; 18: 1107-10.

Chapter VIII

Magnetic Resonance Imaging for Monitoring Neurogenesis in the Adult Hippocampus* Abstract Background: The application is in the field of bioimaging and adult neurogenesis. Objective: It aims at correlating the volume of cerebral blood (CBV) in the dentate gyrus (DG) of the human hippocampus, determined by magnetic resonance imaging (MRI), with neurogenesis in the brain of adult rodents. Methods: Adult mice were submitted to voluntary exercise or administration of fluoxetine or valproic acid (VPA). The CBV of DG was determined by MRI and neurogenesis was quantified by immunohistofluorescence. The CBV in human subjects selected and grouped according to their fitness activity was determined by MRI in the DG. Results: A selective increase in the CBV of the DG is observed in rodents housed in activity cages or administered with fluoxetine and VPA. A selective increase in the CBV of the DG is also observed in exercising humans. The selective increase of the CBV in the human DG correlates with the selective increase of the CBV in the DG and neurogenesis induced by exercise or fluoxetine and VPA in rodents. Conclusion: This indicates that neurogenesis is increased in the DG of exercising humans. The application claims the imaging of the DG of patients by MRI as a paradigm to monitor neurogenesis and identify treatments involving stimulation of neurogenesis.

*Patent Evaluation: The Trustee of Columbia University in the City of New York, US: WO2008020864. Patent: Title: Imaging correlates of neurogenesis with MRI, Assignee: The Trustee of Columbia University in the City of New York, US, Inventors: Small SA., Priority data: 14 November 2005, Filing date: 14 November 2006, Publication date: 21 February 2008, Publication no.: WO2008020864. Declaration of Interest Statement: The author declares no financial and competing interests with the subject matter or materials discussed in the manuscript.

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1. Introduction It is now accepted that neurogenesis occurs in the adult brain and neural stem cells reside in the adult central nervous system of mammals [1,2]. Neurogenesis occurs throughout adulthood in the dentate gyrus (DG) of the hippocampus and subventricular zone along the ventricles, in various species including humans [3,4]. In the DG, newly generated neuronal cells in the subgranular zone migrate to the granule cells layer, where they differentiate and extend axonal projections to the CA3 region, like mature granule cells [5,6]. Newly generated granule-like cells establish functional connections in the CA3 region and survive for an extended period of time [3,7]. Neurogenesis in the adult brain, particularly the hippocampus, is modulated by a broad range of environmental stimuli, physiological and pathological processes, trophic factors/cytokines and drugs [8]. Neurogenesis decreases with age and is diminished by chronic stress and depletion of serotonin, whereas an enriched environment, hippocampal-dependent learning tasks, and voluntary exercise increase neurogenesis in the adult hippocampus of rodents and non-human primates [9-15]. Neurogenesis in the adult hippocampus is involved in the formation of trace memories, a hippocampal-dependent form of learning and memory, in rodents [16]. In humans, neurogenesis is enhanced in the hippocampus of patients with Alzheimer’s disease (AD) [17]. The adult brain has therefore the potential of self-repair, and newly generated neuronal cells in the adult brain may play a critical role in physiological and pathological processes, such as learning and memory, AD and depression [18]. Chronic antidepressant treatments, such as fluoxetine treatments, valproic acid (VPA), a drug used in the treatment of long-term epilepsy, and drugs currently used to treat patients with AD, such as galantamine and memantine, increase neurogenesis in the adult hippocampus [19-21]. X-ray irradiation of the hippocampal region inhibits neurogenesis in the DG and prevents the behavioural effect of antidepressants, such as fluoxetine, in adult mice [22]. Hence, in addition to their therapeutic potential for cellular therapy, adult neurogenesis may also play a critical role in the pharmacology of neurological diseases and disorders, such as learning and memory impairments, AD, depression and epilepsy [23-25]. Alzheimer’s disease is a neurodegenerative disease associated with the loss of nerve cells in areas of the brain that are vital to memory and other mental abilities, such as the entorhinal cortex and hippocampus [26,27]. Depression is associated with reduction in the volume of the hippocampus in patients with major depression, suggesting the loss of nerve cells in the hippocampus of those patients [28-30]. Hence, there is a significant interest in identifying and characterising conditions, drugs and molecules that stimulate neurogenesis in the adult hippocampus of humans in an attempt to compensate or reverse neuronal loss, alleviate symptoms associated with learning and memory deficits, AD, depression and epilepsy, and treat these diseases and disorders. There are numerous protocols and paradigms used to study adult neurogenesis. The most used at present is the bromodeoxyuridine (BrdU)-labelling paradigm. BrdU is a thymidine analogue used for birth dating and monitoring cell proliferation [31,32]. It consists of administering BrdU and processing, staining and analysing the tissues for BrdU and other markers of interest, by immunohistology and confocal microscopy [33-36]. This paradigm is not without pitfalls and limitations and is not applicable for human studies, with the

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exception of rare cases in which patients were treated with BrdU as part of their cancer therapy and donated tissue samples for research investigations [3,4]. Other modes of investigation involve retroviral labelling and transgenic animal models and are also limited to animal studies. In humans, post-mortem immunohistology is most commonly used to investigate cell proliferation and adult neurogenesis [17]. Another technique involving birth 14

dating using C has been used successfully to investigate adult neurogenesis in humans [37]. This latter technique allows retrospective studies in post-mortem samples. MRI is a medical imaging technique for visualising the structure and function of the body. It uses a powerful magnetic field and non-ionising radiation to align the nuclear magnetisation of usually hydrogen atoms in water in the body. It provides detection with high sensitivity of the volume of cerebral blood (CBV) [38]. Agents such as gadolinium and Omniscan (gadodiamide) are used to generate image contrast to show adequately the anatomy or pathology of interest. MRI is a very sensitive, safe, non-invasive procedure that provides real time detailed images of the living body in any plane. It has sufficient spatial resolution to visualise regions and subregions of the brain, such as the hippocampus and DG, in humans and rodents [39,40]. MRI techniques have recently been developed and applied to study newly generated neuronal cells and transplanted neural progenitor and stem cells in the adult brain of rodents [41-43]. The application aims at devising an MRI paradigm to study adult neurogenesis in the hippocampus and its modulation in rodents and humans [44]. The application may be used to study adult neurogenesis in the hippocampus of humans in real time and its modulation, by various conditions and drug treatments.

2. Chemistry 2.1 Experimental Study in Rats Male F344 rats age 6 – 8 weeks (150 – 250 g) were used in this study. Rats were housed individually and divided into control and test groups. Group I or control group: animals were housed in standard laboratory cages. Group II: animals were housed in activity cages, with a running wheel linked to a computer monitoring the wheel’s use. Groups III and IV: animals were housed in standard laboratory cages and were administered drugs known to stimulate neurogenesis in the adult hippocampus, fluoxetine and VPA, respectively. Animals received daily oral injections of 10 mg/kg fluoxetine for 28 days (Group III). Animals received 2 daily intraperiteoneal injections of 300 mg/kg VPA for 28 days. VPA was also provided in the drinking water (12 g/l) (Group IV). There was a minimum of 12 animals per group. All animals received 1 daily intraperiteoneal injection of 100 mg/kg BrdU for 7 consecutive days beginning the first day of experiment/drug treatment (day 1). All animals were analysed by MRI for determination of CBV at day 1 and day 28. All mice were imaged with an MRI protocol to estimate the CBV in the entorhinal cortex, the DG, the CA3 and CA1 regions and the subiculum [39]. At the end of the experimental study, the animals were killed. Their brains were extracted and processed for histology to study and quantify neurogenesis.

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2.2 MRI Scanning in Rats MRI scans in rats were performed using a Bruker AVANCE 400WB spectrometer (Bruker NMR, Inc., Bilerica, MA, US) with an 89-mm-bore 9.4 T vertical Bruker magnet (Oxford Instruments Ltd, UK), a birdcage RF probe and a shielded gradient system up to 100 G/cm. Gadolinium (gadodiamide) sterile aqueous solution at a concentration of 287 mg/ml pH 5.5 – 7.0 is injected undiluted by means of a catheter, with an outer diameter of 0.6 mm. The catheter is placed intraperitoneally before imaging and secured with 6.0 silk suture materials.

2.3 Histology Histology for BrdU and neuronal markers was performed according to previously published protocols and procedures [9,10,14,45].

2.4 Epidemiological Study Twenty individuals, 20 – 45 years of age, non-smokers, sedentary and habitual nonexercisers, who qualify as below average fitness by the standards of the American Heart Association, were recruited for this study. Subjects were divided in two groups. Group I: subjects were submitted to exercise with moderate intensity training on a selection of aerobic activities, such as cycling on a stationary ergometer, running on a treadmill, climbing on a stairmaster or using an elliptical trainer. Subjects in this group exercised at a heart rate of 55 – 65% of their maximum heart rate for 2 weeks and 65% of maximum heart rate for 10 weeks. Group II: subjects were submitted to exercise with high-intensity training on a selection of aerobic activities, such as cycling on a stationary ergometer, running on a treadmill, climbing on a stairmaster or using an elliptical trainer. Subjects in this group exercised at a heart rate of 55 – 65% of their maximum heart rate for 2 weeks, 65 – 75% of their maximum heart rate for the next 2 weeks and 75% of maximum heart rate for 8 weeks. Cardiovascular indices and respiration were monitored and recorded. All subjects were imaged with two MRI scans, the first at the start of the study and the second at the end of the study. All subjects were imaged with an MRI protocol to estimate the CBV in the four hippocampal subregions: the entorhinal cortex, the DG, the CAl region and the subiculum [38]. The inter-individual differences in physical activity were measured by determining the maximum volume of oxygen consumption, to quantify individual differences in degree of exercise [46].

2.5 MRI Protocol in Human Subjects Two sets of coronal three-dimensional Tl-weighted images were acquired, one before and the second 4 min after intravenous administration of Omniscan (0.1 mmol/kg). Images

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were acquired perpendicular to the long axis of the hippocampus and processed using analysis software packages (MEDx Sensor Systems). The hippocampus and the four hippocampal subregions, the entorhinal cortex, the DG, the CAl region and the subiculum, were identified as follows. Hippocampus: the hippocampus is identified by following the trace of the hippocampal sulcus and of the internal white matter tracts. Entorhinal cortex: the lateral and inferior boundary of the entorhinal cortex follows the collateral sulcus, the medial boundary is the medial aspect of the temporal lobe and the superior boundary is delineated by the hippocampal sulcus and the grey/white distinction between subiculum and entorhinal cortex. Dentate gyrus: the medial boundary of the DG is the medial extent of the temporal lobe, the inferior/lateral boundary is the hippocampal sulcus/white matter tracts and the superior boundary is the top of the hippocampal formation, where the alveus is typically identified. CA1 region: the medial boundary of the CA1 region is 2 – 3 pixels lateral to the end of the subiculum, approximately at the beginning of the vertical inflection of the hippocampus and of the extension of the hippocampal sulcus/white matter tracts, the inferior boundary is the white matter of the underlying parahippocampal gyrus, and the superior boundary is the top of the hippocampal formation. Subiculum: the medial boundary of the subiculum is the medial extent of the hippocampal sulcus and/or the horizontal inflection of the hippocampus, the inferior boundary is the white matter of the underlying parahippocampal gyrus, the superior boundary is the hippocampal sulcus and the lateral boundary is a few pixels medial to the vertical inflection of the hippocampus.

3. Biology and Action The selective increase of the CBV in the DG is determined by subtracting the curve of the average CBV of the other hippocampal subregions from the curve of the CBV generated from the DG by MRI, in animal and human studies. These studies reveal that a selective increase in the CBV of the DG is observed in rodents housed in activity cages with a running wheel, or administered drugs known to stimulate neurogenesis in the adult hippocampus, fluoxetine and VPA. A selective increase in the CBV of the DG is also observed in exercising humans. The selective increase of the CBV in the human DG correlates with the selective increase of the CBV in the DG and neurogenesis induced by exercise or modulated with the drugs fluoxetine and VPA, in the rodent study. This indicates that neurogenesis is increased in the DG of exercising humans.

4. Expert Opinion The application provides a paradigm to indicate safely and in real time by MRI whether a condition or drug increases neurogenesis in the DG of living mammals, including humans. To this end, the CBV of the DG and other hippocampal subregions, such as the entorhinal cortex, the DG, the CAl region and the subiculum, are determined by MRI. In rodents, the selective increase of the CBV in the DG, determined by subtracting the curve of the average CBV of the other hippocampal subregions from the curve of the CBV generated from the

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DG, is correlated with increased neurogenesis in the subjects. In humans, the selective increase of the CBV in the DG is correlated with the selective increase of the CBV in the DG with control subjects and with the selective increase of the CBV in the DG and increased neurogenesis in animal studies. Based on this paradigm, the results presented indicate that neurogenesis is increased in the DG of exercising humans. This application claims the measure of the selective increase of the CBV in the DG as a paradigm to study adult neurogenesis and its modulation, by various conditions and drugs, in humans. The approach is based on the tight spatial and temporal coupling between neurogenesis and angiogenesis. Neurogenesis in the adult brain, particularly the hippocampus, occurs at sites of angiogenesis, defining the angiogenic niche for neurogenesis [47-51]. Hence, angiognesis reflects neurogenesis in the adult DG. Angiogenesis results in an increase in the CBV regionally. Measure of the CBV by MRI has been applied to monitoring angiogenesis [52]. MRI has sufficient spatial resolution to visualise subregions of the hippocampus, such as the entorhinal cortex, the DG, the CAl region and the subiculum, in humans and rodents [39]. The measure of the CBV and its variation regionally by MRI are therefore indicative of neurogenesis and its modulation in the adult DG. Other factors and parameters, such as cardiac output and synaptic activity, affect regionally the CBV in the brain. Hence, measure of the CBV and its variation regionally by MRI are not specifically associated with neurogenesis. To address this issue, the authors normalised the data by subtracting the curve of the average CBV from the other hippocampal subregions from the curve of the CBV generated from the DG. This normalisation is based on the assumption that other factors and parameters, such as cardiac output and synaptic activity, that affect regionally the CBV in the brain, particularly the hippocampus, would have similar effects in the DG and other subregions in the hippocampus [53]. In total, a paradigm has been devised and developed to study adult neurogenesis and its modulation in real time and in live subjects by MRI. This paradigm is based on the tight spatial and temporal coupling between neurogenesis and angiogenesis. It is very useful for studying adult neurogenesis and its modulation in human subjects, as there is basically no alternative at this time, except post-mortem studies. It allows safe, live and real-time studies of adult neurogenesis in animals and humans, an extra benefit of the strategy developed. It may therefore become a very powerful technique to identify and characterise conditions, drugs and molecules that stimulate neurogenesis in the adult hippocampus of humans, in an attempt to compensate or reverse neuronal loss, alleviate symptoms associated with learning and memory deficits, AD and depression, and treat these diseases and disorders [54]. However, there are two main limitations and pitfalls in the application of this paradigm. First, it is indicative of neurogenesis and its modulation. Second, it is based on the assumption that factors and parameters other than adult neurogenesis that affect regionally the CBV in the hippocampus would have similar effects in the DG and other subregions in the hippocampus. These limitations and pitfalls must be assessed carefully and addressed when analysing and discussing the data from these studies. The application claims that the increase of the CBV in the DG, relative to other subregions of the hippocampus, such as the CA1 region, is correlated with neurogenesis in the DG. It claims the use of the imaging of the DG of patients by MRI to monitor neurogenesis and to identify and validate treatments involving stimulation of neurogenesis in the hippocampus.

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Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Magnetic resonance imaging for monitoring neurogenesis in the adult hippocampus. Expert Opinion on Medical Diagnostics 3(2):211-6. Copyright 2009, Informa UK Ltd.

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[48] Louissaint A Jr, Rao S, Leventhal C, et al. Coordinated interaction of neurogenesis and angiogenesis in the adult songbird brain. Neuron 2002; 34: 945-60 [49] Taupin P. Adult neural stem cells, neurogenic niches and cellular therapy. Stem Cell Rev 2006; 2: 213-20 [50] Ninkovic J, Götz M. Signaling in adult neurogenesis: from stem cell niche to neuronal networks. Curr Opin Neurobiol 2007; 17: 338-44 [51] Jordan JD, Ma DK, Ming GL, et al. Cellular niches for endogenous neural stem cells in the adult brain. CNS Neurol Disord Drug Targets 2007; 6: 336-41 [52] Dunn JF, Roche MA, Springett R, et al. Monitoring angiogenesis in brain using steadystate quantification of DeltaR2 with MION infusion. Magn Reson Med 2004; 51: 55-61 [53] Delp MD, Armstrong RB, Godfrey DA, et al. Exercise increases blood fl ow to locomotor, vestibular, cardiorespiratory and visual regions of the brain in miniature swine. J Physiol 2001; 533: 849-59 [54] Taupin P. Therapeutic neuronal stem cells: patents at the forefront. Expert Opin Ther Patents 2008; 18: 1107-10

Chapter IX

Apigenin and Related Compounds Stimulate Adult Neurogenesis* Abstract The application is in the field of adult neurogenesis and its therapeutic potential. It aims to characterize the activity of apigenin and related compounds on adult neurogenesis in vivo and in vitro. Apigenin and related compounds are derivatives used in food products. They were administered intraperitoneally and orally in adult rodents and assessed for their activity in promoting the generation of neuronal cells and learning and memory performance. They were also tested on adult rat hippocampal-derived neural progenitor and stem cells to assess their neurogenic property. Apigenin and related compounds stimulate adult neurogenesis in vivo and in vitro, by promoting neuronal differentiation. Apigenin promotes learning and memory performance in the Morris water task. The application claims the use of apigenin and related compounds for stimulating adult neurogenesis and for the treatment of neurological diseases, disorders and injuries, by stimulating the generation of neuronal cells in the adult brain.

1. Introduction The hippocampus is a region of the brain located in the temporal lobe and belonging to the limbic system. It is involved in numerous physiological and pathological processes, like learning and memory - episodic and spatial memory, general declarative memory, forming new memories, anterograde and retrograde amnesia, storing and processing spatial information - Alzheimer’s disease (AD), depression, and epilepsy. It is also the site of

*

Patent Evaluation: Mars, Inc., the Salk Institute for Biological Studies: WO2008147483. De Patent details: Title: Neurogenic compounds, Assignee: Mars Incorporated, the Salk Institute, for Biological Studies, Inventors: Hammerstone JF Jr., Kelm MA, Gage FH, van Praag H, Priority data: 14/02/07, Filing date: 13/02/08, Publication date: 04/12/08, Publication no.: WO2008147483. Declaration of interest: The author states no conflict of interest and has received no payment for this manuscript.

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particular forms of plasticity, like long-term potentiation, and a region of the brain where neurogenesis occurs throughout adulthood [1]. Neurogenesis occurs primarily in two regions of the adult mammalian brain, the subventricular zone along the ventricles and the dentate gyrus (DG) of the hippocampus, in various species including humans [2]. In the hippocampus, newly generated neuronal cells in the subgranular zone migrate to the granule cell layer and extend axonal projection to the CA3 region, like mature granule cells [3,4]. Newly generated granule-like cells establish functional connections with neighbouring and target cells, and survive for extended period of time [5,6]. It is hypothesized that newborn neuronal cells in the adult brain originate from stem cells. In support of this contention, neural progenitor and stem cells have been isolated and characterized in vitro, from the adult brain [7,8]. Adult neurogenesis is modulated by a broad range of environmental stimuli, physiological and pathological processes, trophic factors/cytokines, and drugs [9]. For example, it decreases in the hippocampus with aging and stress and is enhanced in the hippocampus of mice submitted to an enriched environment, like voluntarily running in a wheel [10,11]. It is enhanced in the hippocampus of patients with AD [12]. The generation of neuronal cells and existence of neural stem cells (NSCs) in the adult central nervous system reveal that it has the capacity of self-repair [13]. It is proposed to stimulate endogenous neural progenitor and stem cells or transplant adult-derived neural progenitor and stem cells to restore and repair the degenerated or injured pathways. The potential of adult neurogenesis for regeneration is particularly promising for diseases and disorders associated with loss of nerve cells in the hippocampus, where neurogenesis can be stimulated, like AD and depression. To this aim, identifying and characterizing drugs and molecules stimulating hippocampal neurogenesis offers new perspectives and opportunities for therapy. Apigenin and related compounds are used in food supplement and have been shown to attenuate neuronal degeneration in the adult brain [14]. The application aims at characterizing the activity of apigenin and related compounds on adult NSCs in vivo and in vitro.

2. Chemistry 2.1 Compounds and Derivatives The application claims six compounds and their derivatives (Figure 1). Compounds I, II, III, IV, V, and VI and their derivatives, where R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen, hydroxyl, -OCH3, O-CH2-CH3, O-gallate, and Osugar, where the sugar is selected from glucose, maltose, galactose, and fructose. Compounds may be glycosylated, sulfated, and/or methylated. They may be polymerized, as dimers bonded via an 8 → 3′ bond. Examples of compounds I include apigenin, luteolin, baicalein, amentoflavone, kaempferol, chrysin, chryseriol, diosmetin, isorhamnetin, acacetin, apigenin7-O-glucuronide, apigenin-7-O-glucoside, luteolin-7-O-glucoside, apigenin-4′-O-glucuronide and apigenin 4′-O-sulfate-7-O-glucuronide. Examples of compounds II include geraldol, quercetin, fisetin hydrate, quercetin-3-O-glucuronide, quercetin-3,4′-diglucoside, quercetin4′-O-glucoside, quercetin-3-glucoside, quercetin-3-sulfate, quercetin glucoside sulfate, and

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3′-methylquercetin 3-O-glucuronide. Examples of compounds III include genistein, genistein7-sulfate, genistein-7-Oglucuronide, genistein-4′-O-glucuronide. An example of compound IV is resveratrol; an example of compound V is piperlongumine. Examples of compounds VI include (-)-epicatechin gallate and (-)-epigallocatechin gallate.

Figure 1. Structural backbones of apigenin and related compounds. The application claims six compounds and their derivatives: compounds I (A), II (B), III (C), IV (D), V (E), and VI (F) and their derivatives. Examples of compounds I include apigenin, luteolin, baicalein, and amentoflavone. Examples of compounds II include geraldol and quercetin. An example of compound III is genistein. An example of compound IV is resveratrol. An example of compound V is piperlongumine. An example of compound VI is (-)-epicatechin gallate.

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78 2.2 Synthesis

Compounds I to VI and their derivatives may be obtained by isolation and purification from natural sources, as previously described [15,16]. For examples, apigenin may be obtained from chamomile and parsley. Quercetin may be obtained from onion. Luteolin may be obtained from broccoli and artichokes. Baicalein may be obtained from Chinese medicinal herbs. Compounds I to VI and their derivatives may also be synthesized chemically.

3. Biology and Action 3.1 Animal Studies Mice (7 weeks old) were used in this study. 3.1.1 Intraperitoneal Administration of Apigenin Mice were divided into four groups. Group I: mice administered with 0.9% sodium chloride (NaCl). Group II: mice administered with 0.9% NaCl-Tween 80. Group III: mice administered with 12.5 mg/kg apigenin. Group IV: mice administered with 25 mg/kg apigenin. Apigenin was dissolved 0.9% NaCl-Tween 80. Mice were administered intraperitoneal (i.p.) injections of apigenin or vehicles daily for 42 days. Mice were administered i.p. injections of 5-bromo-2-deoxyuridine (BrdU, 50 mg/kg) daily for the first 8 days of the experiment. A total of 64 mice were used in this study (16 per group). Within each test group, eight mice were randomly selected to receive voluntary running exercise for 2 hours daily for 42 days [11]. Mice were submitted to the Morris water maze task on day 30 of the experiment, to assess the effect of apigenin on learning and memory [17]. At day 42, the animals were sacrificed and processed for histology for studying and quantifying neurogenesis. 3.1.2 Oral Administration of Apigenin and Epicatechin Mice were divided into eight groups. Group I: i.p. injection of 0.9% NaCl-Tween 80. Group II: i.p. injection of 25 mg/kg apigenin. Group III: i.p. injection of 20 mg/kg epicatechin. Apigenin and epicatechin are dissolved 0.9% NaCl-Tween 80. Group IV: oral intake of control food pellets. Group V: oral intake of food pellets containing apigenin 250 ppm. Group VI: oral intake of food pellets containing apigenin 500 ppm. Group VII: oral intake of food pellets containing epicatechin 250 ppm. Group VIII: oral intake of food pellets containing epicatechin 500 ppm. Mice were administered i.p. injections of apigenin, epicatechin, or vehicle daily for 10 days. Mice had access to food pellets containing apigenin or epicatechin ad libitum for the 10 days of the experiment. Mice were administered i.p. injections of BrdU (50 mg/kg) daily for the first 8 days, of the experiment. A total of 40 mice were used in this study (five per group). At day 10, the animals were sacrificed and processed for histology for studying and quantifying neurogenesis.

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3.2 Immunohistology Mice were perfused with 4% paraformaldehyde and their brains taken out. The brains were processed for histology following standard procedures. Brain sections were stained for BrdU and markers of mature neurons and glial cells, neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP), respectively, by immunofluorescence [18]. BrdU and NeuN-positive and BrdU and GFAP-postive cells were quantified in the DG.

3.3 Cell Culture Adult rat hippocampal-derived neural progenitor and stem cells were cultured in defined medium in the presence of basic fibroblast growth factor, as described previously [8]. Cells were cultured in the following conditions in defined medium in the absence of basic fibroblast growth factor: N2, dimethyl sulfoxide (DMSO), retinoic acid and forskolin, 5 μM apigenin, 10 μM acacetin, 05 and 1 μM luteolin (98%), 2 μM baicalein, 2 μM amentoflavone, 5 μM genistein (99%+), 10 μM kaempferol, 10 μM taxifolin, 10 μM geraldol, 10 μM chrysoeriol, 20 μM resveratrol, 5 μM epicatechin gallate, 5 μM epigallocatechin gallate and 5 μM piperlongumine (97%). Drugs, retinoic acid, and forskolin are dissolved in DMSO. Retinoic acid and forskolin are agents know to differentiate adult-derived neural progenitor and stem cells in the neuronal pathway [19]. Neuronal differentiation was assessed by NeuroD luciferase reporter assay (Promega), Western blot, and immunocytochemistry, for markers of immature neurons, astrocytes, and oligodendrocytes, like class III β-tubulin isotype, GFAP, and RIP, respectively [20]. Results show that that mice administered 25 mg/kg apigenin (i.p.) have a higher number of new neuronal cells in the DG than mice of control groups. Mice that received 25 mg/kg apigenin (i.p.) selected to receive voluntary running exercise have a significant higher number of new neuronal cells in the DG than mice of all other groups. Mice administered 25 mg/kg, but not 12.5 mg/kg, apigenin (i.p.) perform significantly better in the Morris water maze task than the mice selected to receive voluntary running exercise without drug treatment and the control groups. Mice administered with 25 and 12.5 mg/kg apigenin (i.p.) selected to receive voluntary running exercise perform significantly better in the Morris water maze task than the mice selected to receive voluntary running exercise without drug treatment and the control groups. Apigenin, but not epicatechin, increases cell proliferation in the DG whether administered intraperitoneally or orally. Apigenin and other compounds tested in vitro up-regulate luciferase activity in the NeuroD luciferase reporter assay and promote the differentiation of adult-derived neural progenitor and stem cells in the neuronal pathway in vitro, in the absence of basic fibroblast growth factor. This shows that apigenin increases neurogenesis in the hippocampus of adult mice. It increases learning and memory performances, with or without exercise, and exercise provides an additional benefit at low doses of apigenin. Apigenin may elicit its activity on adult neurogenesis through the induction and stimulation of neuronal differentiation.

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4. Expert Opinion Apigenin and related compounds are derivatives used in food products. They have been shown to attenuate neuronal degeneration in the adult brain. These compounds stimulate neurogenesis in the adult brain and learning and memory performances. These activities may be applied for the use of apigenin and related compounds to promote the generation of neuronal cells in patients with diseases, disorders and injuries and to promote learning and memory performances. AD and depression are associated with loss of nerve cells particularly in the hippocampus. AD is the most common form of dementia among elderly. It is a neurodegenerative disease characterized by loss of nerve cells in areas of the brain that are vital to memory and other mental abilities, like the entorhinal cortex, hippocampus and neocortex [21,22]. Depression is associated with a reduction in the volume of the hippocampus, suggesting loss of nerve cells in the hippocampus of patients with depressive disorders [23-25]. Drugs and conditions stimulating hippocampal neurogenesis, like apigenin and related compounds, may offer an opportunity to compensate or reverse some of the deficits associated with loss of nerve cells in these diseases. This application raises the opportunity to food-derived products to stimulate neurogenesis that may be beneficial for pathological conditions and impairments, like depression and learning and memory deficits. However, certain issues need to be addressed, among them, the validity of the use of BrdU to assess neurogenesis [26-30] and the mechanisms underlying the activity of these compounds on adult neurogenesis [31]. This application broadens the use of apigenin and related compounds for brain regeneration and repair [14].

Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Apigenin and related compounds stimulate adult neurogenesis. Expert Opinion on Therapeutic Patents 19(4):523-7. Copyright 2009, Informa UK Ltd.

References** [1] [2] [3]

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Taupin P. The hippocampus - Neurotransmission and Plasticity in the nervous system. Nova Science Publishers, 2008 Taupin P, Gage FH. Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res 2002; 69: 745-9 Stanfi eld BB, Trice JE. Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Exp Brain Res 1988; 72: 399-406

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Cameron HA, Woolley CS, McEwen BS, et al. Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neurosci 1993; 56: 337-44 van Praag H, Schinder AF, Christie BR, et al. Functional neurogenesis in the adult hippocampus. Nature 2002; 415: 1030-4, •• First evidence of physiological activity of newly generated neuronal cells in the adult brain. Newly generated neuronal cells were infected with retrovirus carrying green fluorescent protein and recorded electrophysiologically. Toni N, Laplagne DA, Zhao C, et al. Neurons born in the adult dentate gyrus form functional synapses with target cells. Nat Neurosci 2008; 11: 901-7 Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992; 255: 1707-10, •• Original report of the isolation and characterization of neural progenitor and stem cells from the adult brain of mammals. Gage FH, Coates PW, Palmer TD, et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci USA 1995; 92: 11879-83 Taupin P. Adult neurogenesis in the mammalian central nervous system: functionality and potential clinical interest. Med Sci Monit 2005; 11: RA247-52 Kuhn HG, Dickinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 1996; 16: 2027-33 van Praag H, Kempermann G, Gage FH. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 1999; 2: 266-70 Jin K, Peel AL, Mao XO, et al. Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci USA 2004; 101: 343-7 Taupin P. Therapeutic neuronal stem cells: patents at the forefront. Expert Opin Ther Patents 2008; 18: 1107-10 Suk K, Lee H, Kang SS, et al. Flavonoid baicalein attenuates activation-induced cell death of brain microglia. J Pharmacol Exp Ther 2003; 305: 638-45 Avallone R, Zanoli P, Puia G, et al. Pharmacological profi le of apigenin, a flavonoid isolated from Matricaria chamomilla. Biochem Pharmacol 2000; 59: 1387-94 Miean KH, Mohamed S. Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. J Agric Food Chem 2001; 49: 3106-12 Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984; 11: 47-60 Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specifi c nuclear protein in vertebrates. Development 1992; 116: 201-11 Taupin P, Ray J, Fischer WH, et al. FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. Neuron 2000; 28: 385-97 Fanarraga ML, Avila J, Zabala JC. Expression of unphosphorylated class III betatubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation. Eur J Neurosci 1999; 11: 517-27 Burns A, Byrne EJ, Maurer K. Alzheimer’s disease. Lancet 2002; 360: 163-5

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[22] Taupin P. Adult neurogenesis, neural stem cells and Alzheimer’s disease: developments, limitations, problems and promises. Curr Alzheimer Res 2008 . In press [23] Campbell S, Marriott M, Nahmias C, et al. Lower hippocampal volume in patients suffering from depression: a meta-analysis. Am J Psychiatry 2004; 161: 598-607 [24] Bielau H, Trübner K, Krell D, et al. Volume defi cits of subcortical nuclei in mood disorders. A postmortem study. Eur Arch Psychiatry Clin Neurosci 2005; 255: 401-12 [25] Taupin P. Neurogenesis and the Effects of Antidepressants. Drug Target Insights 2006; 1: 13-17 [26] Nowakowski RS, Hayes NL. New neurons: extraordinary evidence or extraordinary conclusion? Science 2000; 288: 771, • BrdU is a marker of DNA synthesis, not cell proliferation and neurogenesis. It labels cell undergoing cell proliferation, but also DNA repair, cell-cycle reentry, and DNA duplication without cell division, leading to polyploidy. [27] Gould E, Gross CG. Neurogenesis in adult mammals: some progress and problems. J Neurosci 2002; 22: 619-23 [28] Rakic P. Adult neurogenesis in mammals: an identity crisis. J Neurosci 2002; 22: 614-8 [29] Taupin P. BrdU Immunohistochemistry for Studying Adult Neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Res Rev 2007; 53: 198-214 [30] Taupin P. Protocols for Studying Adult Neurogenesis: insights and recent developments. Regen Med 2007; 2: 51-62 [31] Taupin P. Adult neurogenesis pharmacology in neurological diseases and disorders. Expert Rev Neurother 2008; 8: 311-20

Chapter X

Nootropic Agents Stimulate Neurogenesis* Abstract The application is in the field of adult neurogenesis, neural stem cells and cellular therapy. It aims to characterize the activity of nootropic agents on adult neurogenesis in vitro. Nootropic agents are substances improving cognitive and mental abilities. AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) and nootropic agents were assessed for the potential to differentiate human neural progenitor and stem cells into neuronal cells in vitro. They were also tested for their behavioural activity on the novel object recognition task. AMPA, piracetam, FK-960 and SGS-111 induce and stimulate neuronal differentiation of human-derived neural progenitor and stem cells. SGS-111 increases the number of visits to the novel object. The neurogenic activity of piracetam and SGS-111 is mediated through AMPA receptor. The neurogenic activity of SGS-111 may contribute and play a role in its nootropic activity. These results suggest that nootropic agents may elicit some of their effects through their neurogenic activity. The application claims the use of nootropic agents for their neurogenic activity and for the treatment of neurological diseases, disorders and injuries, by stimulating or increasing the generation of neuronal cells in the adult brain.

1. Introduction Neurogenesis occurs throughout adulthood in mammals [1,2]. It occurs primarily in two regions of the adult brain, the hippocampus and subventricular zone, in various species including humans [3,4]. Newly generated neuronal cells in the dentate gyrus and olfactory bulb establish functional connections with neighbouring and target cells and survive for *

Patent Evaluation: Brain Cells, Inc.: WO2007104035. Patent details: Title: Modulation of neurogenesis by nootropic, agents, Assignee: Brain Cells, Inc., Inventors: Barlow C, Carter TA, Morse A, Treuner K, Lorrain KI, Gitnick D, Pires JC., Priority data: 21/06/2006, Filing date: 08/03/2007, Publication date: 13/09/2007, Publication no.: WO2007104035. Declaration of interest: The author states no conflict of interest and has received no payment for this manuscript.

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extended period of time [5-7]. Adult neurogenesis is modulated by a broad range of environmental stimuli, physiological and pathological processes, trophic factors/cytokines and drugs. Adrenalectomy, enriched environment, voluntary exercise, hippocampal dependent learning and antidepressants stimulate neurogenesis in the adult hippocampus, whereas adrenal hormones, stress, age and drugs of abuse negatively influence neurogenesis [8]. Newly generated neuronal cells of the adult hippocampus are involved in a broad range of physiological and pathological processes, particularly learning and memory, Alzheimer’s disease, depression and schizophrenia [9-12]. It is hypothesized that new neuronal cells in the adult brain originate from a population of residual stem cells. Neural stem cells (NSCs) are the self-renewing multipotent cells that generate the main phenotypes of the nervous system. In support of this contention, neural progenitor and stem cells have been isolated and characterized from various regions of the adult CNS, including the striatal area containing the subventricular zone, the hippocampus and the spinal cord [13-15]. The existence of NSCs in the adult brain suggest that it may be amenable to repair [16,17]. To this aim, drugs and compounds that promote and stimulate adult neurogenesis in vitro and in vivo are candidates for regenerative medicine, for the treatment of neurological diseases and injuries. The hippocampus is a region of the brain located in the temporal lobe. It belongs to the limbic system. The hippocampus play a central role in learning and memory, particularly in episodic and spatial memory, general declarative memory, forming new memories, anterograde and retrograde amnesia, storing and processing spatial information. It is also the site of particular forms of plasticity such as long-term potentiation, a model of memory [18,19]. Nootropic agents are substances thought to enhance cognitive function and/or mental activity, among them piracetam, FK-960 [N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate] and SGS-111. Piracetam is a pyrrolidone derivative. It improves concentration and enhances memory. FK-960 is an antidementia piperazine derivative. SGS-111 is an analogue of piracetam [20-22]. The application aims to characterize the activity of the glutamate agonist, AMPA (αamino-3-hydroxyl-5-methyl-4-isoxazole-propionate), and nootropic agents, like piracetam (Nootropil or 2-oxo-l-pyrrolidineacetamide), FK-960 and SGS-111 (Figure 1), on adult neurogenesis in vitro. It claims the use of nootropic agents to stimulate or increase adult neurogenesis and for the treatment of neurological diseases and injuries, by stimulating or increasing neurogenesis in the adult brain.

Figure 1. Structure of the nootropic agents FK-960 and SGS-111. The application claims the nootropic agents, FK960 (A) and SGS-111 (B) , to stimulate or increase adult neurogenesis and for the treatment of neurological diseases and injuries, by stimulating or increasing neurogenesis in the adult brain.

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2. Chemistry 2.1 Cell Culture Human neural progenitor and stem cells (hNSCs) were isolated and cultured as a monolayer, in defined medium in presence of trophic factors.

2.2 Differentiation Various concentrations of drugs and compounds were tested for their differentiating potential on human-derived neural progenitor and stem cells, in the absence of trophic factors. Among them, AMPA, an agonist of the AMPA ionotropic glutamate receptor, PEPA an allosteric potentiator of AMPA receptor, the nootropic agents piracetam, FK-960 and SGS-111, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione) an antagonist of the AMPA receptor. Differentiation was assessed and quantified by immunocytochemistry.

2.3 Immunocytochemistry Immunocytochemistry was performed according to standard protocols. The cells were stained with primary antibodies against class III β-tubulin isotype, a marker of immature neurons [23].

2.4 Novel Object Recognition Task Male F344 rats were administered intraperitoneally various drugs and compounds, once daily for 7 days. Animals were tested after 7 days of drug administration on the novel object recognition task, as previously described [24].

3. Biology and Action Results show that AMPA induces and stimulates, in a dose-dependent fashion, the differentiation of human-derived neural progenitor and stem cells into immature neuronal cells, immuno-positive for class III β-tubulin isotype. In the presence of AMPA, PEPA promotes the differentiation of human-derived neural progenitor and stem cells into immature neuronal cells that are immunopositive for class III β-tubulin isotype. Piracetam, FK-960 and SGS-111 induce and promote the differentiation of human-derived neural progenitor and stem cells into immature neuronal cells, which are immunopositive for class III β-tubulin isotype. AMPA promotes the neuronal differentiation of human-derived neural progenitor and stem cells, in presence of SGS-111. NBQX inhibits the stimulation of neuronal

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differentiation by the neurogenic agents AMPA and piracetam. Administration of SGS-111 results in a significant increase in the number of visits to the novel object when compared to the familiar object, in the novel object-recognition task. This shows that AMPA-induced neuronal differentiation in vitro is mediated through the AMPA receptors. Piracetam and SGS-111 exert some of their neurogenic effects in vitro through AMPA receptor activation. SGS-111 acts as cognitive enhancer. The neurogenic activity of SGS-111 may contribute and play a role in its nootropic activity.

4. Expert Opinion AMPA, piracetam, FK-960 and SGS-111 stimulate the differentiation of human neural progenitor and stem cells into the neuronal pathway in vitro. Piracetam and SGS-111 exert their neurogenic activity in vitro through AMPA receptor activation. These agents may be used to promote the generation of neuronal cells in the adult brain, to treat and cure neurological diseases, disorders and injuries. Nootropic agents are memory enhancer. Hippocampal-dependent learning enhances neurogenesis in the adult hippocampus, increases neurogenesis in the adult hippocampus and stimulates hippocampal dependent-learning and memory performance in rodents [25-27]. The neurogenic activity of nootropic agents on human neural progenitor and stem cells in vitro suggest that nootropic agents may exert some of their activity by stimulating adult neurogenesis in the adult hippocampus. However, this remains to be evaluated in experimental studies. Neurogenesis decreases with ageing in adult rodents [28]. Nootropic agents, like piracetam, are known to enhance memory in ageing adults. It would be of interest to assess the activity of nootropic agents in vivo in ageing rodents, in adult neurogenesis and in hippocampal-dependent learning and memory performance, like the Morris water maze task [29]. This application claims the use of nootropic agents to stimulate or increase adult neurogenesis and the use of nootropic agents, alone or in combination with other substances, for the treatment of neurological diseases and injuries, by stimulating or increasing neurogenesis in the adult brain. Further experimental studies are required to characterize the neurogenic potential of nootropic agents in vivo.

Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Nootropic agents stimulate neurogenesis. Expert Opinion on Therapeutic Patents 19(5):727-30. Copyright 2009, Informa UK Ltd.

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Taupin P. Neurogenesis in the adult central nervous system. C R Biol 2006; 329: 46575 Duan X, Kang E, Liu CY, et al. Development of neural stem cell in the adult brain. Curr Opin Neurobiol 2008; 18: 108-15 Eriksson PS, Perfi lieva E, Bjork-Eriksson T, et al. Neurogenesis in the adult human hippocampus. Nat Med 1998; 4: 1313-7, • First evidence of adult neurogenesis in the human brain (hippocampus). Curtis MA, Kam M, Nannmark U, et al. Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Science 2007; 315: 1243-9, • Evidence that neurogenesis occurs in the human subventricular zone. van Praag H, Schinder AF, Christie BR, et al. Functional neurogenesis in the adult hippocampus. Nature 2002; 415: 1030-4, •• First evidence of physiological activity of newly generated neuronal cells in the adult brain. Newly generated neuronal cells were infected with retrovirus carrying green fluorescent protein and recorded electrophysiologically. Carlén M, Cassidy RM, Brismar H, et al. Functional integration of adult-born neurons. Curr Biol 2002; 12: 606-8 Carleton A, Petreanu LT, Lansford R, et al. Becoming a new neuron in the adult olfactory bulb. Nat Neurosci 2003; 6: 507-18 Taupin P. Adult neurogenesis in the mammalian central nervous system: functionality and potential clinical interest. Med Sci Monit 2005; 11: RA247-52 Shors TJ, Miesegaes G, Beylin A, et al. Neurogenesis in the adult is involved in the formation of trace memories. Nature 2001; 410: 372-6. Erratum in: Nature (2001) 414:938 Santarelli L, Saxe M, Gross C, et al. Requirement of hippocampal neurogenesis for the behavioural effects of antidepressants. Science 2003; 301: 805-9, •• X-ray irradiation of the hippocampal region inhibits neurogenesis and the behavioural activity of antidepressants in adult mice. Jin K, Peel AL, Mao XO, et al. Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci USA 2004; 101: 343-7, • The expression of markers of immature neuronal cells, like doublecortin and polysialylated nerve cell adhesion molecule, is increased in hippocampal regions, particularly the dentate gyrus, in the brain of patients with Alzheimer’s disease. Duan X, Chang JH, Ge S, et al. Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain. Cell 2007; 130: 1146-58 Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992; 255: 1707-10, •• Neural progenitor and stem cells isolated and characterized from the adult striatal area containing the subventricular zone in mice. Neural progenitor and stem cells isolated

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[24] [25] [26] [27] [28]

[29]

Philippe Taupin and propagated, as neurospheres, in defi ned medium in the presence of epidermal growth factor. Gage FH, Coates PW, Palmer TD, et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci USA 1995; 92: 11879-83, • Neural progenitor and stem cells isolated and characterized from adult hippocampus of rats. Neural progenitor and stem cells isolated and propagated, as monolayers, in defi ned medium in the presence of basic fibroblast growth factor. Taupin P. Neural Progenitor and Stem Cells in the Adult Central Nervous System. Ann Acad Med Singapore 2006; 35: 814-7 Taupin P. The therapeutic potential of adult neural stem cells. Curr Opin Mol Ther 2006; 8: 225-31 Taupin P. Adult neurogenesis pharmacology in neurological diseases and disorders. Expert Rev Neurother 2008; 8: 311-20 Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993; 361: 31-9 Neves G, Cooke SF, Bliss TV. Synaptic plasticity, memory and the hippocampus: a neural network approach to causality. Nat Rev Neurosci 2008; 9: 65-75 Matsuoka N, Aigner TG. FK960 [N-(4-acetyl-1-piperazinyl)-p-fl uorobenzamide monohydrate], a novel potential antidementia drug, improves visual recognition memory in rhesus monkeys: comparison with physostigmine. J Pharmacol Exp Ther 1997; 280: 1201-9 Lanni C, Lenzken SC, Pascale A, et al. Cognition enhancers between treating and doping the mind. Pharmacol Res 2008; 57: 196-213 Rueda N, Flórez J, Martínez-Cué C. Effects of chronic administration of SGS-111 during adulthood and during the pre- and post-natal periods on the cognitive defi cits of Ts65Dn mice, a model of Down syndrome. Behav Brain Res 2008; 188: 355-67 Fanarraga ML, Avila J, Zabala JC. Expression of unphosphorylated class III betatubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation. Eur J Neurosci 1999; 11: 517-27 Hammond RS, Tull LE, Stackman RW. On the delay-dependent involvement of the hippocampus in object recognition memory. Neurobiol Learn Mem 2004; 82: 26-34 Gould E, Beylin A, Tanapat P, et al. Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci 1999; 2: 260-5 van Praag H, Kempermann G, Gage FH. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 1999; 2: 266-70 Shors TJ, Townsend DA, Zhao M, et al. Neurogenesis may relate to some but not all types of hippocampal-dependent learning. Hippocampus 2002; 12: 578-84 Kuhn HG, Dickinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 1996; 16: 2027-33 Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984; 11: 47-60

Chapter XI

Fourteen Compounds and their Derivatives for the Treatment of Diseases and Injuries Characterized by Reduced Neurogenesis and Neurodegeneration* Abstract The application is in the field of adult neurogenesis and neuronal degeneration and regeneration. It aims to characterize the activity of 15 compounds and their derivatives on adult neurogenesis, apoptosis, necrosis and neuron dysfunction in vitro. The activity of the compounds and their derivatives was tested on cultures of adult human-derived neural progenitor and stem cells, in presence of staurosporine, peptide β-amyloid and hydrogen peroxide, and was assessed by immunocytology. The compounds and their derivatives stimulate the differentiation of adult-derived neural progenitor and stem cells into the neuronal pathway, and inhibit staurosporine-induced apoptosis, peptide βamyloid-induced necrosis and hydrogen peroxide-induced neuron dysfunction. The application claims the use of the 15 compounds and their derivatives for the treatment of diseases and injuries particularly characterized by reduced neurogenesis and neuronal loss, including Alzheimer’s disease (AD), depression, cerebral strokes and traumatic brain and spinal cord injuries.

*

Patent Evaluation: Neuronascent, Inc.: WO2007035722. Patent details: Title: Methods and compositions for Stimulating, neurogenesis and inhibiting neuronal degeneration. Assignee: Neuronascent, Inc. Inventors: Kelleher Andersson J. Priority data: 11/08/2006, Filing date: 19/09/2006, Publication date: 29/03/2007, Publication no.: WO2007035722. Declaration of interest: The author states no conflict of interest and has received no payment for this manuscript.

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1. Introduction Alzheimer’s and Parkinson’s diseases, cerebral strokes, depression, traumatic brain and spinal cord injuries and other neurological diseases disorders and injuries are characterized in the brain by neuronal loss and neurodegeneration, leading to a broad range of cognitive, behavioural and physical deficits and impairments, paralysis and death. These conditions affect more than 250 million individuals globally, with tremendous social and economical impacts on societies. In addition to the suffering, the cost for treating and caring patients afflicted with neurological diseases, disorders and injuries is enormous. Neurogenesis occurs in the adult mammalian brain primarily in two regions, the hippocampus and subventricular zone [1,2]. The characterization and identification of neural progenitor and stem cells in the adult brain signify that it may be amenable to repair, bringing new opportunities and hope for the treatment of a broad range of neurological diseases, disorders and injuries [3]. Adult neurogenesis is modulated, particularly in the hippocampus, by a broad range of environmental stimuli, physiological and pathological processes, trophic factors/cytokines and drug treatments [4]. Hence, it is not only adult neurogenesis and neural stem cells (NSCs) that offer new avenues for treating and curing neurological diseases, disorders and injuries (by either stimulating endogenous neural progenitor or stem cells or transplanting adult-derived neural progenitor and stem cells) but also conditions that stimulate neurogenesis in the adult hippocampus and that may contribute to compensate or reverse deficits in neurological diseases and disorders associated with loss of nerve cells in this region. Alzheimer’s disease (AD) is the most common form of dementia among elderly. The risk of developing AD doubles every 5 years after the age of 65. AD affects 50% of individuals of age over 85. It affects more than 26 million people worldwide, and this number is expected to quadruple by 2050 as populations age [5]. AD is a neurodegenerative disease associated with the loss of nerve cells in areas of the brain that are vital to memory and other mental abilities, like the entorhinal cortex, hippocampus and neocortex [6,7]. Depression is associated with reduction in the volume of the hippocampus in patients with major depression, suggesting the loss of nerve cells in the hippocampus of those patients [8-11]. Conditions that stimulate neurogenesis in the adult hippocampus may therefore be beneficial for the treatment of AD, and depression in particularly. In support of this contention, there is a close correlation between neurogenesis and AD and depression. Neurogenesis is enhanced in the hippocampus of patients with AD, suggesting that the adult brain may attempt a regenerative process [12]. Chronic stress and depletion of serotonin, known as causative factors of depression, inhibit neurogenesis in the adult hippocampus [13-15]. Chronic antidepressant treatment increases neurogenesis in the adult hippocampus of rodents [16]. X-ray irradiation of the hippocampal region inhibits neurogenesis in the DG and prevents the behavioural effect of antidepressants (like fluoxetine) in adult mice [17]. There is a significant interest in identifying and characterizing drugs and molecules that stimulate neurogenesis in the adult hippocampus in an attempt to alleviate symptoms of AD and depression and to treat these diseases [18,19]. Such compounds may also be potent in regenerative medicine for the treatment of a broad range of neurological diseases and injuries.

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The aim of this application is to characterize the activity of 15 compounds and their derivatives on nerve degeneration and adult neurogenesis in vitro. It claims compounds useful for stimulating neurogenesis and/or inhibiting neuronal degeneration.

2. Chemistry 2.1 Compounds and Derivatives The application claims 14 compounds and their derivatives (Figures 1-3). Compound I and its derivatives, where R1 is in each occurrence and independently selected from the group consisting of F, Cl, Br, R7 or −O−R7; where R7 is an optionally substituted 1 − 6 carbon alkyl or a 6 − 14 carbon aryl or aralkyl group; where R2 is selected from O or S; where R3 is (CH2)m, where m can be 1, 2 or 3; where R4 is selected from either an N or (CHn), where n equals 1 or 2 with the provision that when R4 is nitrogen then m in R3 should not be equal to 1; where R5 is a substituted heterocyclic aromatic group; and where R6 is H. Other derivatives of compound I are as follows: at least one R1 may be other than a hydrogen, the substituted heterocyclic aromatic group may be an optionally substituted 2-quinolrnyl, 2pyridyl, 2-or 4-pyrimidinyl or benzoxazolyl group, the aromatic heterocyclic group may be an optionally substituted 2-quinolinyl, 2-pyridyl, 2-or 4-pyrimidinyl, benzo [l,3]dioxol-5-yl or benzoxazolyl group and a compound of the formulae: 4-(3-cyano-6-ethoxyquinolin-2-yl)[l,4] diazepane-l-carboxylic acid (2-fluorophenyl)-amide; 4-(3-cyano-5,7-dimethyl-qumolin2-yl)-[1,4] diazepane-1-carbothioic acid (2- methoxy-phenyl)-amide; 4-benzooxazol-2-ylpiperidine-l-carbothioic acid (3-methoxyphenyl)-amide or pyrrolidine-l,2-dicarboxylic acid 2-benzo [l,3]dioxol-5-ylamide l-[(4-chloro-phenyl)-amide].

Figure 1. Structural backbones of compounds I, II, III and IV. The application claims the four compounds and their derivatives: compounds I (A), II (B), III (C) and IV (D) and their derivatives.

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Compound II and its derivatives, where R1 is in each occurrence and independently selected from the group consisting of F, Cl, Br, R7 or −O−R7, where R7 is an optionally substituted 1 – 6 carbon alkyl or a 6 – 14 carbon aryl or aralkyl group; where R2 is selected from O or S and where R3 is selected from alkyl, cyclic alkyl, aralkyl of 1 – 10 carbons, a substituted aromatic group or a substituted heteroaromatic group. Other derivatives of compound II are as follows: at least one R1 may be other than a hydrogen and a compound of the formulae: 3-(cyclopropanecarbonyl-amino)-9-aza-bicyclo[3.3.1]nonane-9-carboxylic acid p-tolylamide or 3-(2-methyl-benzoylamino)-9-azabicyclo[3.3.l] nonane-9- carboxylic acid ptolylamide. Compound III and its derivatives, where R1 is in each occurrence and independently selected from the group consisting of F, Cl, Br, R7 or −OR7, where R7 is an optionally substituted 1 – 6 carbon alkyl or a 6 – 14 carbon aryl or aralkyl group; where R2 is selected from O or S and where R3 is selected from substituted alkyl, cycloalkyl, aryl, aralkyl of 1 – 12 carbons, heteroaromatic group or heteroaromatic-alkyl group. Other derivatives of compound III are as follows: at least one R1 may be other than a hydrogen, a compound of the formula: l-(8-benzyl-8-aza-bicyclo[3.2.1]oct-3-yl)-3-(2-chloro-phenyl)-thiourea, at least one Rj may be other than a hydrogen and a compound of the formula: l-(8 benzyl-8azabicyclo[3.2.1]oct-3-yl)-3-(2-chloro-phenyl)-thiourea. Compound IV and its derivatives, where R1 is in each occurrence and independently selected from the group consisting of F, Cl, Br, R7 or −O−R7, where R7 is an optionally substituted 1 – 6 carbon alkyl or a 6 – 14 carbon aryl or aralkyl group, where R2 is selected from O or S, where R3 is selected from a 1 – 6 carbon alkyl or an ether of 1 – 6 carbons and where R4 is selected from a 6 – 14 carbon aryl, aralkyl, a substituted aromatic group, a substituted heteroaromatic group or a substituted heteroaromatic-alkyl group. Other derivatives of compound IV are as follows: R4 may be selected from an optionally substituted 3-quinolinylmethyl, 2-pyridyl, 2-pyridylmethyl, 2-or 4-pyrimidinyl, benzo[l,3]dioxol-5-yl or benzoxazolyl group. Compounds V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV and XV and their derivatives, where at least one of R1 to R6 include alkyl, aryl, alkenyl, alkynyl, alkylene, alkyldiyl, alkenylene, alkynylene, arylalkyl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl, halide, alkoxy, aryloxy, alkylthio, arylthio, silyl, siloxy, amino, alkylamino, dialkylamino. This includes derivatives with straight or branched chains, cyclic derivatives, substituted derivatives, heteroatom derivatives and heterocyclic derivatives. Other derivatives of compounds V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV and XV are as follows: at least one of R1 to R6 may be a heterocyclic or carbocycle derivative, derivatives of R1 to R6 include groups containing acyl, formyl, hydroxy, acyl halide, amide, amino, azido, acid, alkoxy, aryloxy, halide, carbonyl, ether, ester, thioether, thioester, nitrile, alkylthio, arylthio, sulfonic acid, thiol, alkenyl, alkynyl, nitro, imine, imide, alkyl, aryl and R1 to Rg include H, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, ethenyl, propenyl, butenyl, ethynyl, propynyl, butynyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, phenyl, tolyl, xylyl, benzyl, naphthyl, pyridinyl, furanyl, tetrahydro-1-napthyl, piperidinyl, indolyl, indolinyl, pyrrolidinyl, 2-(methoxymethyl) pyrrolidinyl, piperazinyl, quinolinyl, quinolyl, alkylated-1, 3-dioxolane, triazinyl, morpholinyl, phenyl pyrazolyl, indanyl, indonyl pyrazolyl, thiadiazolyl, rhodaninyl, thiolactonyl, dibenzofuranyl, benzothiazolyl, homopiperidinyl, thiazolyl, quinonuclidinyl, isoxazolidinonyl, alcohol, ether, thiol, thioether, tertiary amine, secondary amine, primary

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amine, ester, thioester, carboxylic acid, diol, diester, acrylic acid, acrylic ester, methionine ethyl ester, benzyl- 1 -cysteine ethyl ester, imine, aldehyde, ketone, amide or diene.

Figure 2. Structural backbones of compounds V, VI and VII. The application claims the three compounds and their derivatives: compounds V (A), VI (B) and VII (C) and their derivatives.

2.2 Synthesis Preparation of 2-(1,4)-diazepane-1-yl-6-ethoxy-quinonline-3-carbonitrile. A mixture of 2-chloro-6-ethoxyquinoline-3-carbonitrile and homopiperazine (10 equivalents) was heated at 150oC for 1 – 2 h (CHCl3: MeOH 9: 1). After cooling, the reaction was quenched with ice, extracted in to CHCl3 (x 3), washed with brine and dried (K2CO3). After removal of the solvent, the product was purified as the oxalic acid salt from acetone. Preparation of 4-(3cyano-6-ethoxy-quinolin-l-yl)-(l,4) diazepane-l-carboxylic acid (2-fluorophen-d-thioamide. HCl l-isothiocyanato-2-fluorobenzene (1.3 equivalents) was added to a solution of the secondary amine in dry acetonitrile. After stirring overnight, the solvent was removed and the product (NNT5) purified through the hydrochloride salt from acetone.

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Figure 3. Structural backbones of compounds VIII, IX, X, XI, XII, XIII, XIV and XV. The application claims the eight compounds and their derivatives: compounds VIII (A), IX (B), X (C), XI (D), XII (E), XIII (F), XIV (G) and XV (H) and their derivatives.

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3. Biology and Action 3.1 Cell Culture Human neural progenitors and stem cells were obtained from a commercial source. Cells were cultured for up to three passages, in DMEM/F12 in presence of leukaemia inhibitory factor (LIF, 10 ng/ml) [20]. For differentiation studies, cells were cultured for two passages and submitted to differentiation culture medium. Differentiation medium was changed every other day (50% volume change). Compounds I to XIV and their derivatives were prepared in 0.2% dimethyl sulfoxide (DMSO) and added 2 h after initiating differentiation and for 11 days, after which the cells were fixed and processed for immunocytology.

3.2 Immunocytology Immunocytology was performed following standard protocols and procedures. The cells were stained with primary antibodies against microtubule-associated protein-2 (Map-2), a marker of mature neurons [21]. The number of cells immunopositive for Map-2 was determined in the various culture conditions, in presence of compounds I to XIV and their derivatives and positive and negative controls.

3.3 Apoptosis Apotosis was induced on differentiated cultures of mature neurons (immunopositive for Map-2) with staurosporine (10 – 100 nM). The ability of compounds I to XV and their derivatives to inhibit staurosporine-induced apoptosis on differentiated cultures of mature neurons was assessed by adding various concentrations of the compounds. Staurosporineinduced apoptosis was assessed by measuring caspase-3 activity in the cultured cells.

3.4 Necrosis Necrosis was induced on differentiated cultures of mature neurons (immunopositive for Map-2) with peptide β-amyloid 1 – 42 (1 – 10 μM) or peptide β-amyloid 25 – 35 (10 – 75 μM). The ability of compounds I to XV and their derivatives to inhibit peptide β-amyloidinduced necrosis on differentiated cultures of mature neurons was assessed by adding various concentrations of the compounds. Peptide β-amyloid-induced necrosis was assessed by measuring lactate dehydrogenase activity in the cultured cells, as a measurement of neuron degeneration.

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3.5 Neuron Dysfunction Neuron dysfunction, a potential step leading to degeneration, was induced on differentiated cultures of mature neurons (immunopositive for Map-2) with hydrogen peroxide (1 – 100 μM). The ability of compounds I to XV and their derivatives to inhibit hydrogen peroxide-induced neuron dysfunction on differentiated cultures of mature neurons was assessed by adding various concentrations of the compounds. Hydrogen peroxideinduced neuron dysfunction was assessed by measuring the metabolic activity of the cells, with assays such as MTT or Alamarblue(R) assays, as a measurement of neuron respiratory activity. Results presented show that the following compounds: 4-(3-cyano-6-ethoxy-quinoKn-2yl)-[l,4]diazepanel-140 carboxylic acid (2-fluoro-phenyl)-amide; 4-(3-cyano-5,7-dimethylquinolin-2-yl)-[1,4]diazepane-1-carbothioic acid (2-methoxy-phenyl)-amide and 1-(2-chloro7,8-dimethylquinolin-3-yhnethyl)-1-(2-methoxy-ethyl)-3-(2-methoxyphenyl)-urea increase the number of neurons immuno-positive for Map-2 in culture by 140,110 and 132%, respectively, compared to control (vehicle treatment). Results presented show that 4-(3cyano-6-ethoxy-quinolin-2-yl)-[1,4]diazepane-1-carboxylic acid (2-fluoro-phenyl)-amide and 4-(3-cyano-5,7-dimethyl-quinolin-2-yl)-[l,4]diazepane-l-carbothioic acid (2-methoxyphenyl)-amide inhibit the degeneration on neurons in culture by 61% and 50%, respectively, compared with control. Results presented show that 4-(3-cyano-6-ethoxyquinolin-2-yl)[1,4]diazepane-1-carboxylic acid (2-fluoro-phenyl)-amide inhibits the dysfunction of neurons in culture by 34%, compared to control. Results presented show that 4-(3-cyano-6-ethoxyquinolin-2-yl)-[1,4]diazepane-1-carboxylic acid (2-fluoro-phenyl)-amide; 4-(3-cyano-5,7dimethylquinolin-2-yl)-[l,4]diazepane-l-carbothioic acid (2-methoxyphenyl)- amide and 4benzo-8-aza-bicyclo[3.2.1]oct-3-yl)-3- (2-chloro-phenyl)-thiourea inhibit the toxicity of peptide β-amyloid on neurons in culture by 47,84 and 68%, respectively, compared with control. This shows that compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV and XV, and their derivatives, stimulate the differentiation of adult-derived neural progenitor and stem cells into the neuronal pathway, inhibit staurosporine-induced apoptosis, and inhibit both peptide β-amyloid-induced necrosis and hydrogen peroxide-induced neuron dysfunction.

4. Expert Opinion Compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV and XV and their derivatives stimulate the differentiation of adult human-derived neural progenitor and stem cells into the neuronal pathway. These compounds and their derivatives also inhibit apoptosis, necrosis and neuron dysfunction of the differentiated neural progenitor and stem cells into mature neurons in vitro. These compounds and their derivatives may be used to promote the generation of neuronal cells or prevent the degeneration of nerve cells in the adult brain, in the aim to treat and cure neurological diseases, disorders and injuries.

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Because neuronal loss and neurodegeneration are common features of neurological diseases, disorders and injuries, compounds that would elicit a dual activity in terms of promoting regeneration and repair, i.e., stimulating the generation of neuronal cells and preventing the degeneration of neurons (including newly generated neuronal cells), would prove to be very potent candidates for the treatment of neurological diseases, disorders and injuries, particularly neurodegenerative diseases, cerebral strokes and traumatic brain and spinal cord injuries. The mechanisms of action of such compounds remain to be elucidated and the active domain(s) of the compounds to be determined. This is necessary not only to understand the activity of such compounds but also to synthesize even more potent candidates. The activity of compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV and XV and their derivatives must be studied and characterized in vivo, in animal models, to further demonstrate their potency, and validate them as candidates for the treatment of neurological diseases, disorders and injuries.

Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Fourteen compounds and their derivatives for the treatment of diseases and injuries characterized by reduced neurogenesis and neurodegeneration. Expert Opinion on Therapeutic Patents 19(4):541-7. Copyright 2009, Informa UK Ltd.

References** [1] [2] [3] [4] [5]

[6] [7]

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Taupin P. Neurogenesis in the adult central nervous system. C R Biol 2006;329:465-75 Duan X, Kang E, Liu CY, et al. Development of neural stem cell in the adult brain. Curr Opin Neurobiol 2008;18:108-15 Taupin P. The therapeutic potential of adult neural stem cells. Curr Opin Mol Ther 2006;8:225-31 Taupin P. Adult neurogenesis in the mammalian central nervous system: functionality and potential clinical interest. Med Sci Monit 2005;11:RA247-52 Brookmeyer R, Johnson E, Ziegler-Graham K, et al. Forecasting the global burden of Alzheimer’s disease. Johns Hopkins University Dept of Biostatistics Working Papers. January (2007). Working Paper 130. Available from: http://www.bepress.com/ jhubiostat/paper130. Burns A, Byrne EJ, Maurer K. Alzheimer’s disease. Lancet 2002;360:163-5 Taupin P. Adult neurogenesis, neural stem cells and Alzheimer’s disease: developments, limitations, problems and promises. Curr Alzheimer Res 2009. In press

Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

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[9] [10] [11] [12]

[13]

[14] [15]

[16] [17]

[18] [19] [20] [21]

Philippe Taupin Czéh B, Michaelis T, Watanabe T, et al. Stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. Proc Natl Acad Sci USA 2001;98:12796-801 Campbell S, Marriott M, Nahmias C, et al. Lower hippocampal volume in patients suffering from depression: a meta-analysis. Am J Psychiatry 2004;161:598-607 Bielau H, Trübner K, Krell D, et al. Volume deficits of subcortical nuclei in mood disorders. A postmortem study. Eur Arch Psychiatry Clin Neurosci 2005;255:401-12 Taupin P. Neurogenesis and the Effects of Antidepressants. Drug Target Insights 2006;1:13-7 Jin K, Peel AL, Mao XO, et al. 2004. Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci USA 2004;101:343-7, • The expression of markers of immature neuronal cells, like doublecortin and polysialylated nerve cell adhesion molecule, is increased in hippocampal regions, particularly the DG, in the brain of patients with AD. Gould E, Tanapat P, McEwen BS, et al. Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc Natl Acad Sci USA 1998;95:3168-71 Brezun JM, Daszuta A. Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats. Neuroscience 1999;89:999-1002 Pham K, Nacher J, Hof PR, et al. (2003) Repeated restraint stress suppresses neurogenesis and induces biphasic PSA-NCAM expression in the adult rat dentate gyrus. Eur J Neurosci 2003;17:879-86 Malberg JE, Eisch AJ, Nestler EJ, et al. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 2000;20:9104-10 Santarelli L, Saxe M, Gross C, et al. Requirement of hippocampal neurogenesis for the behavioural effects of antidepressants. Science 2003;301:805-9, •• X-ray irradiation of the hippocampal region inhibits neurogenesis and the behavioural activity of antidepressants in adult mice. Taupin P. Adult neurogenesis pharmacology in neurological diseases and disorders. Expert Rev Neurother 2008;8:311-20 Taupin P. Adult neurogenesis and pharmacology in Alzheimer’s disease and depression. J Neurodegeneration Regen 2008;1:51-5 Taupin P. Human neural progenitor and stem cells. Expert Opin Ther Patents 2009;19:1-4 Bernhardt R, Matus A. Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons. J Comp Neurol 1984;226:203-21

Chapter XII

Brevetoxin Derivative Compounds for Stimulating Neuronal Growth* Abstract The application is in the field of neuronal growth and the activity of brevetoxins and their derivatives. It aims to characterize the activity of four generic formulae of brevetoxin derivatives - formulae I, II, III, and IV - and their compounds on the growth of neurites. The activity of the brevetoxin derivative compounds was tested on primary cultures of neocortical neurons. It was assayed in the presence and absence of antagonists of receptors and second-messenger signaling pathways, particularly NMDA receptors and the calmodulin-dependent protein-kinase kinase pathway. Brevetoxin derivatives stimulate neurite growth, particularly the growth of minor processes from which the axons form, on neurons in primary cultures. The activity is mediated by voltage-gated 2+ sodium channels and the N-methyl-D-aspartate-mediated intracellular Ca pathway. The application claims the use of the four generic formulae of brevetoxin derivatives (I, II, III, and IV) and their compounds for enhancing neuronal growth and for the treatment of neurodegenerative diseases and neurological disorders and injuries, such as Alzheimer’s disease, amyotrophic lateral sclerosis, cerebral strokes, traumatic brain, and spinal cord injuries.

1. Introduction Neurological diseases, disorders, and injuries affect over 250 million individuals worldwide. This number is set to increase given the increase in life expectancy in developed countries and the fact that the prevalence of neurodegenerative diseases, such Alzheimer’s and Parkinson’s diseases, increases with age [1]. Neurological diseases, disorders, and injuries result from the dysfunction, degeneration, or injury of nerve cells and currently have no cure. Treatments consist primarily in drug and occupational therapies. *

Patent Evaluation: University of North Carolina at Wilmington: WO2008131411. Declaration of interest: The author states no conflict of interest and has received no payment in preparation of this manuscript.

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Brevetoxins, or Ptychodiscus brevis toxins (PbTx), are natural compounds produced by a species of marine dinoflagellate, Karenia brevis [2]. They were identified as the agents behind the symptoms of neurotoxicity resulting from the inhalation of Florida red tides. PbTx are a suite of nine cyclic polyethers belonging to two families, brevetoxin A and brevetoxin B (Figure 1, Table 1). The neurotoxic activity of PbTx is mediated through voltage-gated sodium channels (VGSCs). PbTx act as agonists of VGSCs on nerve cells [3]. VGSCs are transmembranous proteins responsible for the transmission of the action potential along the nerve axon [4,5]. Derivatives of brevetoxins are produced chemically, by modification of the natural compounds, to generate compounds of pharmaceutical value.

Figure 1. Structural backbones of brevetoxins. PbTx are a suite of 9 cyclic polyether belonging to two families with different structural backbones: A. brevetoxins A and B. brevetoxins B.

An early step in the development of nerve cells is the growth of neurites; one of which will further develop into the axon. Neurite growth is mediated by the entry of Ca2+ into the nerve cells. This entry comes about through the activation of NMDA receptors and VGCCs. The Ca2+ stimulates neurite growth by activating the calmodulin (CaM)-dependent protein kinase (CaMK) secondary-messenger signaling pathway and calmodulin-dependent protein-

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kinase kinase (CaMKK) [6]. Brevetoxin augments NMDA receptor signaling in murine neocortical neurons [7]. The application aims at characterizing the activity of brevetoxin derivatives on neurite growth in primary cultures of neurons. It also aims to determine the mechanism of action of brevetoxin derivatives from four generic formulae - I, II, III, and IV and their compounds on NMDA receptors and the CaMKK signaling pathway. Table 1. Structural backbones of natural brevetoxins. Backbone PbTx-1 PbTx-7 PbTx-10 PbTx-2 PbTx-3 PbTx-5 PbTx-6 PbTx-8 PbTx-9

R (branched group) A A A B B B B B B

CH2C(=CH2)CHO CH2C(=CH2)CH2OH CH2CH(CH3)CH2OH CH2C(=CH2)CHO CH2C(=CH2)CH2OH CH2C(=CH2)CHO, OAc at C37 CH2C(=CH2)CHO, epoxide at C27, C28 CH2COCH2Cl CH2CH(CH3)CH2OH

2. Chemistry The application claims derivatives of brevetoxins from four generic formulae, in which A, R, R1, R2, R3, X, and Y are straight and branched groups, including ‘alkyl’, ‘alkenyl’, ‘cycloalkyl’, ‘aryl’, ‘arylester’, ‘alkylamide’, ‘heteroaryl’, ‘heterocycle’, ‘heterocycloalkyl’, and ‘heterocyclyl’ (Figure 2). The compounds were synthesized using standard procedures, as previously described [8,9].

Figure 2 (Continued)

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Figure 2. Generic formulas of brevetoxin sderivatives. The application claims the use of the four generic formulae of brevetoxin derivative: A. Formula I; B. Formula II; C. formula III; and D. formula IV, and their compounds for enhancing neuronal growth and for the treatment of neurodegenerative diseases, disorders, and injuries. A, R, R1, R2, R3, X and Y are straight and branched groups, see text for details.

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3. Biology and Action 3.1 Primary Cell Culture of Neocortical Neurons The cultures were prepared from 16-day-old embryos of Swiss–Webster mice, using standard procedures. Briefly, the brains were isolated and neocortical tissue dissected out. The tissue was dissociated - mechanically and enzymatically - by trypsin. Neocortical neurons were cultured in minimum essential medium (MEM), supplemented with 2 mM Lglutamine, 10% fetal bovine serum, and 10% horse serum. The cells were plated on poly- Llysine-coated coverslips in 24-well plates at a density of 0.1 × 106 cells per well. After 3 h in culture, cells were exposed to the following concentrations of PbTx-2: 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1000 nM and untreated, for 3 h, 15 h, 24 h, 40 h, and 108 h. The activity of PbTx-2, at the various concentrations, and untreated, on neurite growth was assessed in the presence and absence of TTX (1 µM), MK801 (1 µM), nifedipine (1 µM), and STO-609 (2.6 µM). The activity of PbTx was determined by assaying neurite growth in the neocortical neurons in culture. Neurite growth was determined at the different time points and concentrations of the brevetoxin compounds, in the presence and absence of antagonists (TTX, MK801, nifedipine, STO-609), and was assayed by measuring the length of the the minor processes forming the axons by immunocytochemistry for protein gene product 9.5 (PGP 9.5). PGP 9.5 is expressed by the minor processes forming the axons on primary culture of embryonic neurons [10-12]. Neurite growth was quantified by measuring the length of neurites of 120 cells in culture. Processes shorter than cell diameter were excluded from the analysis. Quantification was performed by software analysis and at least in triplicate. The concentration–response for neocortical neurite growth was determined for PbTx-2, after 24 h treatment. The 50% inhibition constant (IC 50) values were determined for a small number of compounds. The results show that 30 nM PbTx-2 elicits a significant effect on neurite growth (minor processes forming the axons) after 15 h, 24 h, 40 h, and 108 h exposure, compared with untreated control. After 24 h exposure, PbTx-2 induces neurite growth with an optimal concentration of 30 nM (Table 2). After 3 h of exposure, the activity of 30 nM PbTx-2 on neurite growth is inhibited by tetrodotoxin (TTX) [(-) 265.8 – 20.86 µm, (+) 190.2 – 10.76 µm, untreated 187.5 – 15.56 µm], MK801 [(-) 166.5 – 17.89 µm, (+) 83.09 – 5.26 µm, control 101.50 – 7.51 µm], STO-609 [(-) 166.5 – 17.89 µm, (+) 82.43 – 11.17 µm], but not nifedipine [(-) 166.5 – 17.89 µm, (+) 156.60 – 12.48 µm]. TTX is an antagonist of VGSCs; MK801 is an antagonist of the NMDA receptor; STO-609 is an antagonist of the CaMKK; and nifendipine is an antagonist of VGCCs. These results show that PbTx-2 induces neurite growth on primary cell cultures of neocortical neurons at an early stage after plating. It further reveals that the activity of PbTx2, a ligand of VGSCs, is mediated by NMDA receptors but not by VGCCs; the activity of PbTx-2 on neurite growth is mediated by the CaMKK secondary-messenger pathway. The activation of VGSCs by PbTx-2 induces the entry of Na+ into the cells, which results in the depolarization that activates the NMDA receptors. This activation induces the entry Ca 2+

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into the nerve cells, which activates the CaMKK pathway and this, in turn, activates neurite growth in neocortical neurons in culture. Table 2. PbTx-2 concentration-response for neurite growth on neocortical neurons in culture. Concentration

Neurite growth after 24 h treatment

10 nM 30 nM 100 nM 300 nM Untreated

279.9+/-19.98μm 357.0+/-32.15μm 284.6+/-22.6μm 251.9+/-22.38μm 173.1+/-13.16μm

4. Expert Opinion PbTx derivatives promote the stimulation of neurite growth on neocortical neurons in culture, via the activation of the CaMKK pathway, by activating NMDA receptors. This activation promotes the growth of minor processes and the rapid growth of one minor process to form the axon. This activity could be applied to enhance neuronal growth in the treatment of neurodegenerative diseases and disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis, cerebral strokes, and traumatic brain and spinal-cord injuries. The identification of NMDA receptors and the CaMKK pathway as mediators of the activity of PbTx derivatives is interesting as it targets a population of nerve cells. This allows the design and use of drugs specific to subpopulations of nerve cells for potential therapeutic benefit. However, NMDA receptors and VGSCs are broadly expressed in the nervous system [3], so treatments with PbTx derivatives aiming at stimulating NMDA receptors might have secondary effects that have yet to be evaluated. Data on the sustainability of neurite growth, after removal of the derivatives of PbTx, are not presented. This raises the question of the potentiality of the drugs. It would also be of interest to test the activity of PbTx derivatives on neurite growth, in defined media, in the presence of trophic factors. This application broadens the scope of the use of brevetoxin derivatives for therapy. A previous patent application was filed for the use of such compounds for the treatment of cystic fibrosis, mucociliary dysfunction, and pulmonary diseases [13]. This new application raises the interesting opportunity of using derivatives of PbTx to enhance neuronal growth and for the treatment of neurodegenerative diseases, disorders, and injuries. Mediation of their activities by the NMDA receptors and the CaMKK pathway offers the potential to target specific populations of nerve cells. PbTx derivatives might very well be used for therapy for preventing or promoting the survival and growth of nerve cells in a diseased or injured nervous system. Preclinical studies will be of interest to study and validate the activity of these derivatives in vivo.

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Acknowledgments Reproduced with permission from Informa Pharmaceutical Science: Taupin P. Brevetoxin derivative compounds for stimulating neuronal growth. Expert Opinion on Therapeutic Patents 19(2):269-74. Copyright 2009, Informa UK Ltd.

References** [1] [2]

[3] [4] [5]

[6] [7]

[8]

[9] [10] [11] [12]

[13]

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Ferri CP, Prince M, Brayne C, et al. 6. Alzheimer’s Disease International. Global prevalence of dementia: a Delphi consensus study. Lancet 2005; 366: 2112-7 Baden DG, Bourdelais AJ, Jacocks H, 7. et al. Natural and derivative brevetoxins: historical background, multiplicity, and effects. Environ Health Perspect 2005; 113: 621-5, • Short and concise review on brevetoxin derivatives. Chahine M, Chatelier A, Babich O, et al. Voltage-gated sodium channels in neurological disorders. CNS Neurol Disord Drug Targets 2008; 7: 144-58 Narahashi T. Tetrodotoxin: a brief history. Proc Jpn Acad Ser B Phys Biol Sci 2008; 84: 147-54 Lepage KT, Baden DG, Murray TF. Brevetoxin derivatives act as partial agonists at neurotoxin site 5 on the voltage-gated Na+ channel. Brain Res 2003; 959: 120-7, •• Evidence of the activity of brevetoxins as agonist of VGSCs. Wayman GA, Lee YS, Tokumitsu H, et al. Calmodulin-kinases: modulators of neuronal development and plasticity. Neuron 2008; 59: 914-31 Dravid SM, Baden DG, Murray TF. Brevetoxin augments NMDA receptor signaling in murine neocortical neurons. Brain Res 2005; 1031: 30-8, •• Evidence of the activity of brevetoxins on NMDA receptor. Trainer VL, Thomsen WJ, Catterall WA, et al. Photoaffinity labeling of the brevetoxin receptor on sodium channels in rat brain synaptosomes. Mol Pharmacol 1991; 40: 98894 Fuwa H, Sasaki M. Recent advances in the synthesis of marine polycyclic ether natural products. Curr Opin Drug Discov Devel 2007; 10: 784-806 Lesuisse C, Martin LJ. Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death. J Neurobiol 2002; 51: 9-23 Dotti CG, Sullivan CA, Banker GA. The establishment of polarity by hippocampal neurons in culture. J Neurosci 1988; 8: 1454-68 Fletcher TL, Banker GA. The establishment of polarity by hippocampal neurons: the relationship between the stage of a cell’s development in situ and its subsequent development in culture. Dev Biol 1989; 136: 446-54 University of North Carolina at Wilmington: WO2005028482; 2005

Papers of special note have been highlighted as either of interest(•) or of considerable •• interest (••) to readers.

Chapter XIII

OTI-010 Osiris Therapeutics/JCR Pharmaceuticals* Summary * Originator Osiris Therapeutics Inc. * Licensee JCR Pharmaceuticals Co Ltd * Status Phase II Clinical * Indications Bone marrow transplantation, Crohn's disease, Graft-versus-host disease * Actions Named whole cell modulator * Synonyms Allogen, JR-031, Mesenchymal stem cell therapy

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 graft-versus-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.

*

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.

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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 (eg, 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 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 where 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

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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 more mild 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 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 graftversus-leukemia effect [656537]. Successful strategies to treat 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 is 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-2-receptor-positive activated T-cells [659532]. Other treatments include humanized monoclonal antibodies to TNFa (infliximab) and IL-2 (daclizumab).

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Mesenchymal stem cells (MSC) are multipotent cells that contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose and marrow stroma [394517], [656539]. MSC 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 HSC) [658632], [658640]. MSC have also been found in umbilical cord blood, but not peripheral blood [658631]. Once isolated, MSC can be expanded in culture through many generations, producing billions of MSC for cellular therapy [656543]. MSC have been extensively studied in vitro, ex vivo and in animal and human models [656703], [658632], [658640], [658995]. In vitro, MSC have been shown to secrete hematopoietic cytokines and support hematopoietic progenitors [643706]. MSC are not immunogenic and escape recognition by alloreactive T-cells and NK cells. Human MSC (hMSC) 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 hMSC an ideal candidate for cellular therapy where mismatched allogeneic transplantation is one of the main limitations of therapy. MSC 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 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 of these treatments, such as antiinflammatory drugs also have side effects, such as vomiting, heartburn, diarrhea and greater susceptibility to infection [www.nih.gov].

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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].

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 hMSC have been previously reported [394517], [656543], [656553]. Osiris has developed protocols to isolate and culture hMSC. MSC 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 magneticbead-based selection using an anti-fibroblast antibody that gained about 100-fold enrichment of MSC. The fibroblastenriched 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 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 hMSC 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 MSC for cellular therapy [656543]. Culture quality was ensured on the basis of optimum MSC 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 2-fold fewer human CD45 cells present in the mice receiving intramuscular hMSC. 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].

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Preclinical Development Osiris has investigated MSC isolated from various sources both in vitro and in vivo.

In Vitro Aggarwal & Pittenger studied the effects of hMSC in modulating allogeneic immune cell responses [643694••]. When hMSC 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, hMSC decreased IFN. secretion by T-helper (Th)1 and NK cells and increased IL-4 secretion by Th2 cells. Studies also demonstrated that hMSC 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 h. 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 antiCD3/CD28activated peripheral blood mononuclear cells (hPBMC) caused inhibition of lymphocyte proliferation. The effect of hMSC on lymphocyte proliferation was dose dependent, causing more than 50% inhibition at an approximately 1:10 to 1:25 MSC:lymphocyte ratio. Five-day studies showed how increasing MSC induced 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 indicates molecules that are functionally related to the immunological responses of hMSC, 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 MSC as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro by outlining the phenotypic differences between MSC and marrow-derived stromal cells (MDSC). Flow cytometric analysis showed that MSC are distinct from MDSC cultures and are a homogeneous cell population devoid of hematopoietic cells [656703].

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In Vivo Fischer rat MSC 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 MSC 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 MSC and Rin-m cells demonstrated an association of the different cell types with each other near portal triads. In contrast, in animals where 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 MSC could be utilized to protect cell allografts from rejection after transplantation into the liver [643859]. Another study in Fischer rats demonstrated that MSC do not elicit an immune response after transplantation in immunocompetent recipients. Syngeneic Fischer MSC or allogeneic ACI MSC 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 MSC to induce tissue regeneration without an immune response was investigated in a goat model of meniscal injury. Donor MSC were injected into the damaged meniscus in the knee joint on days 7, 14 and 21 (10 million MSC per injection). Ex vivo examinations showed that MSC suppressed T-cell alloreactivity selectively, maintaining some types of T-cell responses [656698].

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

Clinical Development 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 hMSC decreased GVHD in 22 to 45% of patients, suggesting that OTI-010 reduces the numbers of pro-inflammatory cytokines and Tcells whilst 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

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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].

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 nonHodgkin'slymphoma). 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 have yet to be published.

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, 7 to 10 days apart [www.clinicaltrials.gov].

Metabolism and Pharmacokinetics No data are currently available.

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 MSC, either after HSC transplantation or co-transplantation with HSC, show that MSC therapy is well tolerated by patients [659018••], [659019•].

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Patent Summary The patent application WO-09222584, by inventors A Caplan and SE 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 hMSC obtained from periosteum, BM, cord blood, peripheral blood, dermis, muscle, or other known sources of MSC. WO-09901145 details processes for recovering peripheral blood containing a population of hMSC from an individual and preserving these ex vivo. WO-09947163 discloses processes for MSC 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 MSC. WO-09946366 states methods and preparations of using non-autologous MSC for treating and regenerating connective tissue and enhancing bone marrow engraftment in an individual, and WO-2005093044 claims a method of promoting angiogenesis by MSC in an organ or tissue other than the heart.

Current Opinion MSC 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 MSC a model of choice for cellular therapy and also for gene therapy [643707], [658995]. MSC also have immunosuppressive properties, making them a candidate for the treatment of GVHD, particularly after allogeneic stem cell transplantation. Coadministration of MSC and HSC 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 MSC 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 living a normal life again [659018••]. Furthermore, as the beneficial effect of GVHD on 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

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tolerated by patients, and to optimize therapy protocols. A particularly contentious issue in need of further research is whether MSC survive long after therapy, or deplete over time. Although ultimately the transplanted HSC contribute to the graft chimerism in successful transplantations, monitoring of long-term integration and survival of MSC in the recipients will provide valuable information regarding the long-term beneficial effect of the MSC therapy. The immunosuppressive properties of MSC may underlie the speed of recovery after allogeneic stem cell transplantation. It is proposed that the immunoregulatory activity of the MSC 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, DC, 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 MSC 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 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 with third-party donor cells [656550], MSC derived from unrelated, MHC-unmatched, healthy, third-party donors would represent a 'universal donor MSC product' with the advantages of being a readily available product that may provide an opportunity for multiple and higher MSC doses, potentially at a reduced cost [659019•]. 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, MSC are expanded until the number of adult stem cells has increased over 3000-fold [327297], [327352]. Recent studies have reported that MSC expanded in culture have an intrinsic propensity towards spontaneous transformation after a certain number of population doublings (~ 250) [660856], [660857], [660858], [660859]. Therefore, careful considerations 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 for the treatment of arthritis, indicating a possible contraindication of the therapy [660860]. Although it is not yet known whether

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such observations would translate into 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 donorsare monitored for up to five years after donation to ensure their health status. This is primarily the 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 MSC 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 organ-transplant rejection [659005•]. OTI-010 appears to be a promising candidate for 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 the 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 the support for the development of OTI-010 [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.

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 forhematologic 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].

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Development History. Developer

Country

Statuse

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

Literature classifications. Chemistry Study type

Result

Result

Stem cell isolation and expansion

BM aspirates were combined with DPBS and centrifuged several times at different density gradients to collect hMSC. 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.

643694••

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119

Biology Study type

Effect studied

Model

Result

Reference

In vitro

Modulation of allogeneic immune responses by hMSC

hMSC incubated with DC and naïve T-cells

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

643694••

In vitro

Relationship between selected markers and hMSCmediated immunosuppresion

Co-culture of hMSC with anti CD3/CD28 activated hPBMC

A correlation was observed between PGE2 secretion and IDO enzyme activity on the inhibition of lymphocyte proliferation of hMSC

656696

In vivo

hMSC engraftment and migration

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

Results showed cells to persist for at least 28 days being localized to the portal traid regions

643859

Ex vivo

Immune response

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

MSC suppressed T-cell alloreactivity selectively, maintaining some types of Tcell responses

656698

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Acknowledgments Reproduced, with permission from Thomson Reuters and Taupin P: OTI-010 Osiris Therapeutics/JCR Pharmaceuticals. Current Opinion in Investigational Drugs (2006) 7(5):473-81. Copyright 2006, Thomson Reuters (Scientific) Ltd.

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 BLOOD 1999 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 MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR SCIENCE 1999 284 5411 143-147 [433228] Effects of chemotherapeutic agents on the proliferation, differentiation, and stromal support capacity of human mesenchymal stem cells Archambault MP, Howard L, Butterfield AM, Peter SJ BLOOD 2001 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 license agreement: Companies to expand stem cell technology into a new market opportunity Osiris Therapeutics Inc PRESS RELEASE 2005 August 04 [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

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[642230]7 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 FP, Boynton RE, Haynesworth S, Murphy JM, Zaia J BIOCHEM BIOPHYS RES COMMUN 1999 265 1 134-139 [643694 Human mesenchymal stem cells modulate allogeneic immune cell responses Aggarwal S, Pittenger MF 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 anti-inflammatory 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 BIOCHEM BIOPHYS RES COMMUN 2001 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 MK, Thiede MA, Haynesworth SE, Bruder SP, Gerson SL J HEMATOTHER STEM CELL RES 2000 9 6 841-848 [643707] Mesenchymal stem cells as vehicles for gene delivery Mosca JD, Hendricks JK, Buyaner D, Davis-Sproul J, Chuang LC, Majumdar MK, Chopra R, Barry F, Murphy M, Thiede M A, Junker U, Rigg RJ, Forestell SP, Bohnlein E, Storb R, Sandmaier BM CLIN ORTHOP 2000 379 Suppl S71-S90 [643859] Intra-portal infusion of adult mesenchymal stem cells enhances survival of allogeneic beta cells in immunocompetent rats Archambault MP, Campbell SE, Vanguri P, McIntosh KR BLOOD 2002 100 11 Abs 2404 [656535] Cytolytic pathways in haematopoietic stem-cell transplantation van den Brink MR, Burakoff SJ NAT REV IMMUNOL 2002 2 4 273-281 [656537] Allogeneic hematopoietic cell transplantation as consolidation immunotherapy of cancer after autologous transplantation Maris MB, Storb R ACTA HAEMATOL 2005 114 4 221-229 [656539] Mesenchymal stem cells Caplan AI J ORTHOP RES 1991 9 5 641-650 [656543] Characterization of cells with osteogenic potential from human marrow Haynesworth SE, Goshima J, Goldberg VM, Caplan AI BONE 1992 13 1 81-88 [656546] HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O EXP HEMATOL 2003 31 10 890-896 [656548] Characterization and functionality of cell surface molecules on human mesenchymal stem cells Majumdar MK, Keane-Moore M, Buyaner D, Hardy WB, Moorman MA, McIntosh KR, Mosca JD J BIOMED SCI 2003 10 2 228241

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[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 EXP HEMATOL 2002 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 SE, Ringden O SCAND J IMMUNOL 2003 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 BLOOD 2005 106 11 [656697] Modulation of immune cell responses by human mesenchymal stem cells Aggarwal S, Pittenger MF BLOOD 2004 104 11 Abs 1288 [656698] Evidence for selective suppression of alloreactivity by mesenchymal stem cells Beggs KJ, Kavalkovitch KW, Borneman JN, Murphy MJ, Barry FP, Stewart MG, Proctor RL, McIntosh KR BLOOD 2001 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 BLOOD 2000 96 11 Abs 3309 [656702] Allogeneic rat mesenchymal stem cells do not elicit an immune response after implantation in immunocompetent recipients Archambault MP, McIntosh KR, Duty A, Peter SJ BLOOD 2000 96 11 Abs 3295 [656703] Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells Majumdar MK, Thiede MA, Mosca JD, Moorman M, Gerson SL J CELL PHYSIOL 1998 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 STEM CELLS 2004 22 3 377-384 [658608] Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors Laughlin MJ, Barker J, Bambach B, Koc ON, Rizzieri DA, Wagner JE, Gerson SL, Lazarus HM, Cairo M, Stevens CE, Rubinstein P, Kurtzberg J N ENGL J MED 2001 344 24 18151822 [658612] Unrelated donor hematopoietic cell transplantation: Marrow or umbilical cord blood? Grewal SS, Barker JN, Davies SM, Wagner JE BLOOD 2003 101 11 4233-4244 [658625] Mesenchymal stem cells avoid allogeneic rejection Ryan JM, Barry FP, Murphy JM, Mahon BP J INFLAMM 2005 2 8 [658631] Isolation of multipotent mesenchymal stem cells from umbilical cord blood Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH BLOOD 2004 103 5 1669-1675 [658632] Mesenchymal stem cells: Isolation, in vitro expansion and characterization Beyer NN, da Silva ML HANDB EXP PHARMACOL 2006 174 249-282 [658640] Mesenchymal stem cells: Isolation and therapeutics Alhadlaq A, Mao JJ STEM CELLS DEV 2004 13 4 436-448 [658988] IBMTR Severity Index for grading acute graft-versus-host disease: Retrospective comparison with Glucksberg grade Rowlings PA, Przepiorka D, Klein JP, Gale RP,

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Passweg JR, Henslee-Downey PJ, Cahn JY, Calderwood S, Gratwohl A, Socie G, Abecasis MM, Sobocinski KA, Zhang MJ, Horowitz MM BR J HAEMATOL 1997 97 4 855-864 [658995] Mesenchymal stem cells and their potential as cardiac therapeutics Pittenger MF, Martin BJ CIRC RES 2004 95 1 9-20. [659005] Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation Le Blanc K, Ringden O BIOL BLOOD MARROW TRANSPLANT 2005 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 LANCET 2004 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 HM, Koc ON, Devine SM, Curtin P, Maziarz RT, Holland HK, Shpall EJ, McCarthy P, Atkinson K, Cooper BW, Gerson SL, Laughlin MJ, Loberiza FR Jr, Moseley AB, Bacigalupo A BIOL BLOOD MARROW TRANSPLANT 2005 11 5 389-398, • 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 EXP CELL RES 2005 305 1 33-41 [659326] Bone marrow transplantation for sickle cell anemia: Progress and prospects Iannone R, Ohene-Frempong K, Fuchs EJ, Casella JF, Chen AR PEDIATR BLOOD CANCER 2005 44 5 436-440 [659330] Marrow transplantation from related donors other than HLA-identical siblings Beatty PG, Clift RA, Mickelson EM, Nisperos BB, Flournoy N, Martin PJ, Sanders JE, Stewart P, Buckner CD, Storb R, et al N ENGL J MED 1986 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 BLOOD 2002 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 TRANSPLANTATION 1991 51 6 1197-1203

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[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 BONE MARROW TRANSPLANT 1991 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 JH, Vossen J, Gratwohl A, Vogelsang GB, van Houwelingen HC, van Rood JJ N ENGL J MED 1996 334 5 281-285 [659392] Acute graft-versus-host disease: Pathophysiology, clinical manifestations, and management Couriel D, Caldera H, Champlin R, Komanduri K CANCER 2004 101 9 1936-1946 [659491] Chronic graft-versus-host disease Horwitz ME, Sullivan KM BLOOD REV 2006 20 1 15-27 [659498] New strategies in the treatment of graft-versus-host diseaseBasara N, Blau IW, Willenbacher W, Kiehl MG, Fauser AA BONE MARROW TRANSPLANT 2000 25 Suppl 2 S12-S15 [659503] Novel pharmacotherapeutic approaches to prevention and treatment of GVHDJacobsohn DA, Vogelsang GB DRUGS 2002 62 6 879889 [659504] Novel strategies for steroid-refractory acute graft-versus-host disease BolanosMeade J, Vogelsang GB CURR OPIN HEMATOL 2005 12 1 4044 [659506] Pentostatin for the treatment of chronic graft-versus-host disease in children Goldberg JD, Jacobsohn DA, Margolis J, Chen AR, Anders V, Phelps M, Vogelsang GB J PEDIATR HEMATOL ONCOL 2003 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 VT, Zahrieh D, Hochberg E, Micale E, Levin J, Reynolds C, Steckel S, Cutler C, Fisher DC, Lee SJ, Alyea EP, Ritz J, Soiffer RJ, Antin JH BLOOD 2004 104 4 1224-1226 [659743] Non-HLA immunogenetics in hematopoietic stem cell transplantation Dickinson AM, Charron D CURR OPIN IMMUNOL 2005 17 5 517-525 [659758] The biology of graft-versus-host reactions Billingham RE 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 JS, Abdallah B, Schrodder H, Jensen T, Kassem M ONCOGENE 2004 23 29 5095-5098 [660857] Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation Miura M, Miura Y, Padilla-Nash HM, Molinolo AA, Fu B, Patel V, Seo BM, Sonoyama W, Zheng JJ, Baker CC, Chen W, Ried T, Shi S STEM CELLS 2006 24 4 1095-1103 [660858] Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells Burns JS, Abdallah BM, Guldberg P, Rygaard J, Schroder HD, Kassem M CANCER RES 2005 15 65 8 3126-3135 [660859] Spontaneous human adult stem cell transformation Rubio D, Garcia-Castro J, Martin MC, de la Fuente R, Cigudosa JC, Lloyd AC, Bernad A CANCER RES 2005 65 8 3035-3039

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[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, LouisPlence P, Bony C, Sany J, Jorgensen C, Noel D ARTHRITIS RHEUM 2005 52 5 15951603 [662462] Acute graft-versus-host disease Bolanos-Meade J, Vogelsang GB CLIN ADV HEMATOL ONCOL 2004 2 10 672-82 [662457] Transfusion-associated graft-versus-host disease: A serious residual risk of blood transfusion Higgins MJ, Blackall DP CURR HEMATOL REP 2005 4 6 470-6 [662480] High-dose chemotherapy and autologous bone marrow or stem cell reconstitution for solid tumors McGuire WP CURR PROBL CANCER 1998 22 3 135-77

Chapter XIV

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.

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

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results in a form of severe combined immunodeficiency (SCID), known as ADA-SCID [467343]. 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 γ-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 a 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 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

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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 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]. Non-myeloablative 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].

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 non-myeloablative 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

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received non-myeloablative 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. 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 h 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].

Preclinical Development HSR-TIGET hypothesized that the re-infusion of a cell population that was enriched in lymphoid progenitors, in addition to 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].

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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 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].

Metabolism and Pharmacokinetics No data are currently available.

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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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 to those observed in Pt1. The increase occurred mainly in the Tcell 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

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to increase 8-fold after therapy. This increase in activity was correlated with a decline in 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 1 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 non-myeloablative 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, 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].

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Side Effects and Contraindications Throughout the observational period of the clinical trial, the patients have been reported to be in good clinical condition without experiencing any severe infectious episodes [515130], [643374], [668664]. No patient has experienced toxicity or has required blood component 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 had 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], had been 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].

Patent Summary No data are currently available.

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 non-myeloablative conditioning, together with the use of IL-3, are most likely responsible for the improved results observed in the latest protocol developed by HSRTIGET. 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],

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[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 that is 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 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 ADASCID. A previous gene therapy 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 non-myeloablative 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 γ-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 γ-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 HSR-TIGET 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 γ-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

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

Country Status

Indication

Date

Reference

San Raffaele Telethon Institute for Gene Therapy

Italy

Phase II Combined immunodeficiency

12-JAN-06

644674

Literature classifications. Chemistry Study type

Result

Reference

Genetically engineered human HSC protocol

Human CD34+ HSCs were removed from the 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

515130

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137

Biology Study type

Effect studied

Model

Result

Reference:

Ex vivo

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

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 ADASCID 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

In vitro

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 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 reached values that did not differ significantly from those of freshly isolated cells

643373

In vivo

Engraftment and differentiation

SCID-hu mouse model

Transduced CD34+ cells were efficiently engrafted into SCID-hu mice (~ 80% overall giving rise to B- and T-cell progeny and demonstrating the lymphoid reconstitution capacity

643373

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138

Literature classifications (Continued) In vitro / in vivo

Repopulation and differentiation

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

The lymphoid differentiation 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

515130

Clinical Effect studied

Model

Result

Reference:

Efficacy

Autologous HSC gene therapy and nonmyeloablative conditioning in two ADA-SCID patients

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 Bcell functions, as well as an amelioration of the metabolic pattern

515130

Expansion of the trial outlined above to six children affected by early onset ADASCID

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, Pt5), or more prolonged thrombocytopenia and neutropenia (Pt3). None of the patients experienced severe infections or adverse events

668664

Safety efficacy

and

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Acknowledgments Reproduced, with permission from Thomson Reuters and Taupin P: ADA-transduced hematopoietic stem cell therapy for ADA-SCID. IDrugs (2006) 9(6):423-30. Copyright 2006, Thomson Reuters (Scientific) Ltd.

References*

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*

•• of outstanding interest, • of special interest

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[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 SCIENCE 1991 251 4999 1363-1366 [666662] Gene therapy in peripheral blood lymphocytes and bone marrow for ADAimmunodeficient patients. Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, Mazzolari E, Maggioni D, Rossi C, Servida P, Ugazio AG et al SCIENCE 1995 270 5235 470-475 [666664] T-lymphocyte-directed gene therapy for ADA-SCID: Initial trial results after 4 years. Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ et al SCIENCE 1995 270 5235 475-480 [666665] Bone marrow gene transfer in three patients with adenosine deaminase deficiency. Hoogerbrugge PM, van Beusechem VW, Fischer A, Debree M, le Deist F, Perignon JL, Morgan G, Gaspar B, Fairbanks LD, Skeoch CH, Moseley A et al GENE THER 1996 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 LANCET 2003 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 JP, Thrasher AJ, Wulffraat N, Sorensen R, Dupuis-Girod S, Fischer A et al N ENGL J MED 2002 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 JL, Fraser CC, Cavazzana-Calvo M, Fischer A N ENGL J MED 2003 348 3 255256 [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 MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E, Sorensen R et al SCIENCE 2003 302 5644 415-419 Erratum in: SCIENCE 2003 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 UP, Jenkins NA, Copeland NG SCIENCE 2004 303 5656 333 [666684] Evaluation of ADA gene expression and transduction efficiency in ADA/SCID patients undergoing gene therapy. Carlucci F, Tabucchi A, Aiuti A, Rosi F, Floccari F, Pagani R, Marinello E NUCLEOSIDES NUCLEOTIDES NUCLEIC ACIDS 2004 12 89 1245-1248 [666686] Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity. Zavialov AV, Engstrom A BIOCHEM J 2005 391 51-57 [668402] Severe combined immunodeficiency – molecular pathogenesis and diagnosis. Gaspar HB, Gilmour KC, Jones AM ARCH DIS CHILD 2001 84 2 169-73

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[668664] Gene therapy of ADA-deficient SCID. Aiuti A EUROCONFERENCE – GENE CELL THER 2005 December 01-02 [668669] Gene therapy of severe combined immunodeficiencies (SCID). Fischer A EUROCONFERENCE – GENE CELL THER 2005 December 01-02

Index A acetone, 93 acetonitrile, 93 acetylcholinesterase, 10 acid, 3, 26, 51, 59, 60, 65, 66, 79, 91, 92, 93, 96 acidic, 61 acrylic acid, 93 action potential, 100 activation, 81, 86, 100, 103, 104, 108, 134, 135, 136 acute lymphoblastic leukemia, 114, 115 acute myeloid leukemia, 114 adenine, 133 adenosine, 109, 127, 139, 140, 141, 142 adenosine triphosphate, 127 adhesion, 10, 52, 58, 63, 72, 87, 98 adipose, 110 administration, 12, 25, 51, 53 adult, 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 37, 38, 39, 45, 46, 47, 49, 50, 51, 53, 54, 55, 57, 58, 59, 60, 61, 62, 63 adult stem cells, 2, 4, 6, 25, 26, 27, 30, 49, 58, 62, 63, 111, 116, 120 adult T-cell, 135 adult tissues, 2, 50 adulthood, 17, 66, 76, 83, 88 adults, 86, 117 adverse event, 133, 142 age, 10, 17, 23, 27, 36, 59, 66, 67, 68, 71, 81, 84, 88, 90, 99, 108, 110, 132, 133 agents, 2, 23, 24 aggregation, 13 aging, 76, 105 agonist, 12, 84, 85, 105

air, 60 alcohol, 92 alkaline, 61 alkaline phosphatase, 61 allele, 18, 123 allogeneic, 54 alternative, 23, 26, 37, 45, 61, 70, 135 alternatives, 21 Alzheimer’s disease, 9, 11, 14, 15, 16, 17, 20, 50, 53, 55, 66, 72, 75, 81, 82, 84, 87, 89, 90, 97, 98, 99, 104 American Heart Association, 68 amino acids, 42 amyloid, 10, 13, 14, 16, 17, 18, 20 amyloid deposits, 17 amyloid plaques, 18 amyloid precursor protein, 10, 17 amyloidosis, 15 amyotrophic lateral sclerosis, 99, 104 analog, 12, 52 anatomy, 67 aneuploid, 18, 19 aneuploidy, 18, 20, 116 angiogenesis, 70, 74, 115 angiogenic, 53 animal models, 3, 10, 12, 17, 23, 25, 26 animals, 3, 23, 67, 70, 78, 113, 131, 135 antagonist, 12 antagonists, 10, 13 antibiotic, 111 antibodies, 36 antibody, 36, 111 antidepressant, 12, 15, 16, 66, 72, 90, 98 antidepressant medication, 15 antidepressants, 11, 12, 15 antigen, 12, 34, 42, 79, 129, 132, 133 anti-inflammatory drugs, 110

Index

146

apoptosis, 18, 19, 53, 89, 95, 96 APP, 10, 11, 17, 18 arthritis, 110, 116, 125 aspartate, 10, 99 assessment, 135, 136 astrocyte, 24 astrocyte-like cells, 24 astrocytes, 1, 5, 22, 27, 28, 33, 34, 36, 37, 38, 50, 54, 61, 63, 79, 81, 87 astroglial, 4, 24, 25, 33, 42, 53, 57 ataxia, 51 autocrine, 6, 28, 38, 47 autoimmune, 50, 55 autoimmune disease, 50, 109, 112, 117, 123 autoimmunity, 128 autoradiography, 24 axonal, 39, 46, 52 axons, 45, 50, 99, 103

B barrier, 13, 16, 53, 55 basic fibroblast growth factor, 22, 28, 34, 42, 51, 59, 60 Batten disease, 3 BDNF, 12, 13, 15, 16 behavioral effects, 15, 16 beneficial effect, 18, 115 bilirubin, 109, 115 binding, 39 biopsies, 2, 23, 26, 37, 38, 47, 57, 62 birth, 12, 52, 66 bleeding, 110 blood, 2, 6, 13, 20, 25, 26, 27, 30, 53, 55, 58, 62, 63, 65, 67, 73, 74, 108, 109, 110, 115, 117, 122, 125, 134, 135 blood transfusion, 125 blood vessels, 58 blood-brain barrier, 13, 55 bloodstream, 53 body weight, 51, 129 bone marrow, 2, 6, 25, 26, 30, 50, 62, 63, 107, 108, 111, 115, 120, 123, 124, 125, 128, 139, 141, 142 bone marrow stromal stem cells, 63 bone marrow transplant, 107, 111, 123, 124, 128, 141 bovine, 43, 59, 61 bowel, 110 brain functions, 21, 26 brain injury, 54

BrdU, 5, 10, 12, 16, 20, 24, 52 breakdown, 3, 127 breast cancer, 114 brevis, 100 bromodeoxyuridine, 5, 10, 16, 52 buffer, 34

C Ca2+, 100 cancer, 67, 71, 107, 108, 109, 111, 112, 114, 115, 117, 121, 124, 131, 141 cancer cells, 109 candidates, 53, 84, 97 carbohydrate, 34 carbon, 91, 92 cardiac output, 70 cartilage, 110 catheter, 68 causality, 88 CD8+, 132 CD95, 52 cDNA, 63, 129, 131 cell adhesion, 10, 58 cell culture, 6, 36, 103, 116 cell cycle, 10, 12, 18, 19, 20, 24 cell death, 16, 20, 52, 53, 81 cell division, 12, 18, 19 cell fate, 39, 63 cell fusion, 3, 26, 30 cell growth, 35 cell line, 6, 37, 46, 47, 113, 129, 130, 136 cell lines, 37, 130 cell surface, 121 cellular therapy, 1, 3, 4, 23, 24, 26, 33, 39, 46, 54, 57, 58, 59, 61, 62 cementum, 58 central nervous system, 5, 7, 9, 16, 17, 18, 20, 21, 27, 28, 29, 30, 38, 46, 47, 49, 54, 62, 66, 71, 73, 76, 80, 81, 87, 97 cerebral cortex, 29 cerebral strokes, 1, 24, 34, 50, 53, 61 cerebrospinal fluid, 25 channels, 99, 100, 105 chemicals, 50 chemokine, 52 chemokines, 50, 54 chemotaxis, 52 chemotherapeutic agent, 112, 120 chemotherapy, 26, 108, 117, 125

Index childhood, 3 children, 117, 124, 133, 135, 138 chromosomal abnormalities, 135 chromosomal instability, 124 chromosome, 18, 20 chromosomes, 17, 18, 19 circulation, 128 cirrhosis, 109 classes, 10, 11 clinical trial, 1, 2, 3, 15, 37 clinical trials, 1, 2, 3, 15, 107, 114, 117, 135 cloning, 129 CNS, 1, 2, 3, 9, 13, 14, 21, 23, 24, 25, 26, 27, 28, 31, 33, 34, 37, 38, 46, 47, 49, 50, 52, 53, 55, 58, 61, 63, 74, 84, 105 Co, 2, 6, 28 CO2, 43, 111 Cochrane, 15 cognitive function, 84 collagen, 116, 125 collateral, 69 colon, 115 compatibility, 4, 111, 116, 123 competence, 30 complexity, 27 complications, 110, 116, 121 components, 13, 14, 34, 35, 36, 42, 44 compounds, 75, 76, 77, 79, 80, 84, 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105 concentration, 5, 34, 68, 84, 103, 104 conditioning, 129, 132, 133, 134, 135, 136, 138, 139, 140 conflict, 1, 33, 41, 49, 57, 75, 83, 89, 99 conflict of interest, 1, 33, 41, 49, 57, 75, 83, 89, 99 connective tissue, 115 consensus, 105 consolidation, 121 contamination, 26 control, 53, 67, 70, 78, 79, 96, 103, 110 control group, 67, 79 cooling, 93 correlation, 10, 12, 73, 90, 112 cortex, 27, 38, 47, 69 cortical neurons, 105 coupling, 70 Crohn’s disease, 120 cryopreservation, 41, 43, 44, 45, 115 cryopreserved, 41, 42, 44, 45

147

culture, 2, 4, 5, 22, 23, 25, 27, 33, 34, 36, 37, 38, 41, 42, 43, 44, 45, 47, 51, 59, 60, 62, 95, 96, 103, 104, 105, 110, 111, 112, 116, 118, 123, 129, 130, 131, 136, 137 culture conditions, 2, 22, 23, 34, 42, 95 curing, 90, 109 cycles, 27, 54 cycling, 68 cyclosporine, 109 cystic fibrosis, 104 cytokine, 50, 52 cytokines, 50, 54, 66, 76, 84, 90, 108, 110, 112, 113, 121, 130, 131 cytometry, 30, 52, 111

D dating, 12, 52, 66 death, 5, 13, 16, 17, 18, 19, 20, 27, 38, 47, 51, 52, 53, 62, 90, 105 debates, 3, 4, 17, 26, 27, 37, 45, 61 declarative memory, 75, 84 defects, 59 deficiency, 127, 128, 135, 139, 140, 141, 142 deficits, 25 delivery, 30, 55, 121, 122 dementia, 15, 17, 73, 80, 90, 105 demyelination, 52 dendritic cell, 108 density, 35, 103, 111, 118 dentate gyrus, 1, 6, 10, 15, 16, 17, 18, 21, 28, 29, 50 dentate gyrus (DG), 1, 10, 21 deoxyribonucleic acid, 129 depolarization, 103 deposition, 14 deposits, 17 depressants, 12, 13 depression, 9, 11, 12, 13, 14, 15, 20, 53, 66, 70, 72, 75, 76, 80, 82, 84, 89, 90, 98 derivatives, 75, 76, 77, 78, 80, 89, 91, 92, 93, 94, 95, 96, 97, 99, 101, 104, 105, 110 dermatitis, 108 dermis, 6, 30, 62, 115 desipramine, 11 detection, 16, 67, 73 developed countries, 99 diabetes, 41, 53 diarrhea, 109, 110, 115, 134 differentiated cells, 36, 42

Index

148

differentiation, 2, 3, 6, 21, 23, 25, 27, 29, 33, 34, 36, 37, 38, 39, 42, 45, 46, 53, 57, 60, 64, 72, 75, 79, 81, 83, 85, 86, 88, 89, 95, 96, 111, 112, 120, 130, 131, 132, 135, 138 diphtheria, 109 disabilities, 50 diseases, 1, 3, 5, 9, 11, 12, 13, 14, 18, 21, 23, 24, 26, 34, 37, 41, 50, 52, 53, 55, 57, 59, 61, 62 disorder, 3, 10, 14, 110 dissociation, 23, 51, 60 distribution, 39, 46, 98 division, 12, 18, 19, 82 DNA, 5, 12, 16, 18, 19, 20, 82, 115 DNA repair, 12, 82 donor, 61 donors, 4, 111, 116, 122, 123, 124, 130, 131, 134, 137 dopaminergic, 5 doping, 88 Down syndrome, 88 drinking water, 67 drug design, 9, 14 drug therapy, 16, 72, 110 drug treatment, 67, 79, 90 drug use, 66 drugs, 9, 10, 11, 12, 13, 15, 16, 23, 66, 67, 69, 70, 72, 76, 84, 85, 90, 104, 108, 109, 110 DSC, 112 duplication, 12, 18, 82 duration, 130

E E. coli, 24 EAE, 50, 51, 52, 53, 55 eating, 115 ectoderm, 2, 25 elderly, 17, 80, 90 electron, 24, 39, 46, 98 electron microscopy, 24 embryo, 62 embryogenesis, 63 embryonic stem, 37, 39, 41, 46, 54, 64 embryonic stem cells, 37, 39, 41, 46, 54 embryos, 49 encephalomyelitis, 50, 55 encoding, 128, 129, 131, 133 endoderm, 2, 25, 49 endothelial cells, 122 enolase, 36

enrollment, 114 enteritis, 108 entorhinal cortex, 66, 67, 68, 69, 70, 80, 90 environment, 4, 50, 66, 71, 76, 84, 115, 116, 117, 121 environmental stimuli, 66, 76, 84, 90 enzymatic activity, 138 enzymes, 3, 23, 43 ependymal, 24, 29, 30 ependymal cell, 24 epidermal growth factor, 5, 22, 51, 59, 60 epigenetic, 30 epigenetic alterations, 30 epilepsy, 53, 66, 75 epithelium, 115 epitope, 55 erythroid cells, 132 Escherichia coli, 3, 51 ester, 92 etiology, 9, 13, 14 examinations, 113, 117 exercise, 65, 66, 68, 69, 73, 78, 79, 84 experimental autoimmune encephalomyelitis, 55 exposure, 103, 130 extracellular matrix, 36 extracranial, 55 eyes, 25

F FAD, 14 failure, 109 familial, 10, 14, 18 family, 108, 142 FasL, 52 FDA, 113, 117, 120 fetal, 2, 33, 34, 35, 36, 37, 43, 45, 59, 61 fetal tissue, 33, 34, 37, 45 FGF-2, 2, 6, 22, 23, 25, 28, 34, 35, 37, 38, 42, 43, 47 fibroblast, 2, 29 fibroblast growth factor, 2, 22, 28, 29, 34, 42, 51, 59, 60, 79, 88 fibroblasts, 141 filament, 21, 24, 27, 63 fire, 51 fitness, 65, 68 flexibility, 22 flow, 30, 52 flow cytometry analysis, 30 fluid, 25

Index fluorescence, 4, 30, 60 fluoxetine, 11, 13, 15, 16, 65, 66, 67, 69, 90 food, 75, 76, 78, 80, 127 food intake, 127 food products, 75, 80 forebrain, 27, 28, 29, 33, 35, 37 fructose, 76 funds, 4 fusion, 3, 26, 30

G gadolinium, 67 gastrointestinal tract, 109 gene, 4, 12, 18, 24, 26, 38, 47, 63, 103, 115, 121, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 gene expression, 63, 141, 142 gene therapy, 4, 24, 115, 127, 128, 129, 131, 132, 133, 134, 135, 138, 139, 140, 141, 142 gene transfer, 26, 38, 47, 129, 130, 131, 134, 136, 139, 140, 141, 142 generation, 3, 22, 26, 33, 51, 63, 75, 76, 80, 83, 86, 96, 97, 130 genes, 3, 17, 18, 24, 129, 136 genetic disease, 3, 4, 27, 141 genetic disorders, 26 genetic mutations, 10 genome, 18, 128 genomics, 38 genotype, 20 germ layer, 2, 25, 49, 58, 61 GFP, 60 glass, 35, 36, 43, 52 glia, 63, 71, 81 glial, 10, 12, 25, 29, 36, 57, 58, 59, 61 glial cells, 25, 57, 61, 79 glial fibrillary acidic protein (GFAP), 12, 24, 29, 36, 60, 61 glucocorticoids, 11 glucose, 34, 51, 59, 76, 111 glucoside, 76 glutamate, 10, 13, 50, 84, 85 glutamate receptor antagonists, 10, 13 glutamine, 34, 42, 43, 51, 60 glycol, 128, 141 glycosylated, 2, 23 grades, 109 grading, 108, 122 grafting, 27

149

graft-versus-host disease, 26 grants, 120 granule cells, 16 green fluorescent protein, 3, 24, 60 groups, 67, 68, 78, 79, 92, 101, 102, 110 growth, 2, 3, 5, 6, 10, 22, 23, 29, 33, 34, 35, 36, 37, 38, 51, 58, 59, 60, 79, 88, 99, 100, 102, 103, 104, 105, 111, 130, 134, 142 growth factor, 2, 5, 10, 22, 23, 29, 34, 36, 37, 51, 58, 59, 60, 79, 88, 130, 142 growth factors, 34, 36 growth rate, 35 gut, 109, 115 gyrus, 50

H half-life, 128 handling, 23 harmful effects, 50 head injury, 55 healing, 115 health, 117 health status, 117 heart, 41 heart attack, 120 heart disease, 41 heart rate, 68 heartburn, 110 heat, 60 helix, 63 hematopoietic cells, 6, 26 hematopoietic stem cells, 26, 30, 107, 123, 129, 141 hepatitis, 108 heterogeneity, 29, 34, 124 heterogeneous, 4, 25, 27 hippocampal, 10, 11, 12, 14, 15, 16, 20, 25, 27, 28, 39, 57, 60, 61 hippocampus, 1, 5, 10, 11, 12, 13, 14, 15, 17, 18, 19, 21, 22, 25, 27, 28, 29, 38, 39, 47, 50, 54, 57, 65, 66, 67, 69, 70, 71, 72, 73, 75, 76, 79, 80, 81, 83, 84, 86, 87, 88, 90, 98 histocompatibility antigens, 108, 124 histological, 27 histology, 67, 78, 79, 109 HLA, 108, 109, 110, 111, 115, 121, 123, 124, 128, 131, 133, 134, 141 homeostasis, 50 homogeneity, 25 homogenous, 4

Index

150

hormones, 11, 34 horse, 60 host, 26, 50, 107, 108, 109, 110, 111, 115, 116, 118, 120, 121, 122, 123, 124, 125, 133 human, 1, 2, 5, 6, 9, 10, 12, 14, 21, 23, 25, 27, 28, 30, 33, 34, 35, 36, 37, 38, 41, 44, 46, 47, 50, 54, 55, 57, 58, 59, 60, 61, 62, 63 human brain, 5, 27, 38, 47, 54, 62, 87 human ESC, 41 human leukocyte antigen, 108, 123, 128 human subjects, 65, 70 humans, 17, 33, 50, 57 hydrogen, 67, 76, 89, 91, 92, 96 hydrogen atoms, 67 hydrogen peroxide, 89, 96 hydroxyl, 76, 83, 84 hypersensitivity, 50 hypothesis, 13, 17, 20

I ice, 59, 60 identification, 90, 104 identity, 24, 82 IGF-1, 36 IL-1, 50 IL-10, 50 IL-13, 50 IL-4, 50 IL-6, 50, 112 IL-8, 112 images, 67, 68 immune cells, 50, 52 immune disorders, 115, 123 immune function, 129, 135, 140 immune response, 112, 113, 115, 122 immune system, 50, 108, 113, 128 immunization, 51 immunochemistry, 24 immunocompromised, 108, 127 immunocytochemistry, 39, 44 immunodeficiency, 127, 128, 139, 140, 141, 142 immunofluorescence, 52 immunogenetics, 124 immunoglobulin, 132 immunohistochemistry, 12, 16, 73 immunomodulatory, 53, 55, 116, 117 immunoreactivity, 15, 36 immunosuppression, 112, 116, 122 immunosuppressive drugs, 109, 110

immunotherapy, 121 impairments, 66, 80, 90 in situ, 12, 24, 53 in vitro, 1, 2, 3, 4, 5, 6, 21, 22, 23, 24, 25, 26, 33, 34, 35, 36, 37, 41, 42, 45, 46, 51, 52, 58, 60, 61, 63, 75, 76, 79, 83, 84, 86, 89, 91, 96, 110, 112, 115, 116, 122, 130, 131, 136 in vivo, 2, 6, 22, 23, 30, 34, 42, 46, 52, 62, 63, 73, 75, 76, 84, 86, 97, 104, 110, 112, 122, 130, 131, 136, 139, 140, 142 incidence, 108, 109, 112, 116, 117 indices, 68 individual differences, 68 induction, 5, 13, 17, 43, 44, 53, 79, 116, 121 industry, 45 infants, 128 infection, 109, 110, 128 infections, 50 inflammation, 4, 49, 50, 52, 53, 55, 110, 114, 116, 121 inflammatory, 4, 50, 52, 53, 54, 55 inflammatory disease, 52, 53, 55 inflammatory response, 50, 52, 54 inflammatory responses, 50 infliximab, 109 inherited, 17 inherited disorder, 135 inhibition, 72, 103, 112 inhibitor, 23, 109 inhibitors, 10, 11, 13, 15 inhibitory, 33, 34, 35, 37, 42 initiation, 130, 140 injection, 25, 53 injections, 67, 78 injuries, 1, 3, 9, 13, 21, 23, 24, 26, 34, 37, 41, 49, 50, 53, 57, 58, 59, 61, 62, 75, 80, 83, 84, 86, 89, 90, 96, 97, 99, 102, 104 injury, 21, 22, 24, 25, 30, 50, 53, 55, 58 inner cell mass, 41, 49 insertion, 22, 135, 142 insulin, 34, 35, 51 integration, 29, 87, 111, 116, 135 intellectual property, 2, 4 interaction, 37, 74 interferon (IFN), IFN, 50, 108, 112, 116, 119 interferons, 50 interleukin-1, 54 interleukins, 50 intervention, 134 intestine, 110

Index intracranial, 55 intramuscular injection, 111 intravascular, 55 intravenous, 53 intravenously, 49, 50, 51, 111, 114, 129, 136 intravital microscopy, 52 intron, 63 invasive, 4, 25, 30, 57, 58, 61, 63 inventions, 4 inventors, 2, 115 irradiation, 12, 66, 72, 87, 90, 98, 115 ischemia, 54 ischemic, 29, 54 ischemic stroke, 54 isolation, 2, 3, 4, 22, 23, 24, 26, 30, 33, 34, 37, 38, 47, 50, 54, 62, 78, 81, 111, 115 IVIg, 132, 133

151

liver, 50, 109, 113, 115, 128, 133 liver enzymes, 133 localization, 39 locus, 135 long period, 37 luciferase, 79 lumbar, 30 lumbar puncture, 30 lymphatic system, 108 lymphocytes, 20, 109, 127, 128, 129, 130, 131, 132, 133, 139, 140, 142 lymphoid, 26, 108, 130, 131, 132, 134, 137, 138, 140 lymphoid cells, 26 lymphoid tissue, 108 lysine, 59, 103

M K kinetics, 132 knockout, 42

L L1, 60 labeling, 10, 12, 105 lactate dehydrogenase, 95, 133 laminin, 43, 44, 59 late-onset AD, 17 late-stage, 54 learning, 66, 70, 75, 78, 79, 80, 84, 86, 88 learning task, 66 lentiviral, 51 lesion, 16 lesions, 58, 61 leukaemia, 34, 42 leukemia, 33, 107, 108, 109, 114, 117, 133, 135 leukemias, 26 lice, 57, 61 licenses, 120 LIF, 33, 34, 35, 36, 37, 42, 43 life expectancy, 17, 99 life span, 73 ligament, 58, 63, 110 ligand, 52, 103, 110, 130 limbic system, 75, 84 limitations, 1, 3, 4, 13, 16, 20, 25, 27, 34 liquid nitrogen, 43

M1, 10 machinery, 135 macrophages, 108 magnet, 68 magnetic field, 67 magnetic resonance imaging, 65 maintenance, 23, 53 major depression, 11, 66, 90 major histocompatibility complex, 110, 122 malignancy, 109, 116, 123, 142 malignant, 26 maltose, 76 mammalian, 27 mammalian brain, 1, 5, 21, 28, 76, 90 mammalian tissues, 2 mammals, 9, 17, 26, 27, 38, 46, 54, 55 management, 124 manipulation, 5 market, 120 marrow, 2, 6, 25, 26, 30, 50, 62, 63, 107, 108, 110, 112, 115, 121, 122, 123, 141, 142 matrix, 36, 113 maturation, 42, 45, 57, 112, 127 measurement, 95, 96, 132 media, 42, 43, 104 median, 123 mediation, 10 membranes, 60 memory, 66, 70, 73, 75, 78, 79, 80, 84, 86, 88, 90 memory performance, 75, 79, 80, 86 mental activity, 84

152

Index

mesenchymal stem cell, 58, 63 mesenchymal stem cells, 58, 63, 107, 120, 121, 122, 123, 124, 125 mesoderm, 2, 25, 49, 58 meta-analysis, 72, 82, 98 metabolism, 73, 129, 133 metabolites, 98, 127, 128, 133 methylprednisolone, 109 MHC, 110, 111, 116 mice, 6, 10, 11, 12, 14, 15, 16, 22, 28, 30, 49, 50, 51, 52, 53, 65, 66, 67, 71, 72, 73, 76, 78, 79, 87, 88, 90, 98, 103, 111, 122, 131, 137, 138, 140 microarray, 63 microenvironment, 25, 53 microenvironments, 53 microscope, 113 microscopy, 24, 34, 52, 60, 66, 71 microtubule, 34, 39, 42, 46 migration, 16, 27, 29, 53, 60, 73 mitogen, 37 mitogenic, 2, 23, 37 mitosis, 18 mitotic, 18, 30, 41, 45 models, 3, 10, 12, 17, 23, 25, 26, 55, 67, 97, 110, 113, 131 molecular markers, 4 molecular mechanisms, 50, 52 molecular medicine, 141 molecules, 66, 70, 76, 90, 110, 112, 121 monkeys, 15 monoamine, 11 monoamine oxidase, 11 monoamine oxidase inhibitors, 11 monoclonal, 4 monoclonal antibodies, 4 monoclonal antibody, 121 monolayer, 22, 85, 111 monolayers, 22, 34, 38 mood, 72, 82, 98 mood disorder, 72, 82, 98 mortality, 128 mouse, 6, 7, 10, 14, 15, 16, 22, 28, 29, 31, 51, 63 mouse model, 10, 14 MRI, 65, 67, 68, 69, 70, 73 mRNA, 63, 132, 138 multiple sclerosis, 25, 30, 49, 55 multipotent, 1, 2, 4, 5, 6, 17, 22, 25, 27, 28, 30, 33, 37, 42, 49, 50, 55, 57, 62, 63, 72, 84, 110, 122, 129, 132 multipotent stem cells, 42

multipotents, 2, 21, 42, 49 muscarinic receptor, 10 muscle, 58 mutagenesis, 135, 142 mutant, 10, 14, 17 mutations, 10, 11, 17 myelin, 36, 50, 52 myelin basic protein, 36 myeloid cells, 130

N Na+, 103, 105 NaCl, 78 necrosis, 89, 95, 96, 109 neocortex, 16, 80, 90 nerve, 3, 10, 12, 20, 23, 24, 34, 41, 44, 45, 58, 66, 72, 76, 80, 87, 90, 91, 96, 98, 99, 100, 104 nerve cells, 3, 12, 24, 41, 44, 45 nerve growth factor, 23, 34, 58 nerves, 63 nervous system, 1, 3, 9, 14, 17, 18, 21, 22, 23, 33, 34, 37, 42, 46, 50, 52, 53, 54, 57, 58, 61, 62, 80, 84, 104 network, 4, 25, 57 NeuN, 39, 46 neural crest, 58, 61, 63 neural development, 2, 23, 24 neural network, 88 neural stem cell, 5, 6, 9, 14, 16, 17, 18, 19, 20, 21, 25, 27, 28, 29, 30, 33, 37, 38, 39, 42, 46, 47, 54, 55, 57, 62, 63 neural stem cells, 6, 9, 14, 16, 17, 18, 19, 20, 21, 25, 27, 28, 29, 30, 33, 38, 39, 42, 46, 47, 54, 57, 62, 63 neural stem/progenitor cell, 4, 7, 28 neural tissue, 61 neuroblasts, 5, 14, 54, 71, 87 neurodegeneration, 3, 90, 97 neurodegenerative, 1, 3, 11, 13, 17, 24, 25, 33, 34, 47, 50, 55, 57, 58, 61 neurodegenerative disease, 1, 3, 11, 13, 17, 24, 25, 33, 34, 50, 55, 57, 58, 61 neurodegenerative diseases, 1, 3, 11, 13, 24, 25, 33, 34, 50, 55, 57, 58, 61, 97, 99, 102, 104 neurodegenerative disorders, 47, 55 neurofibrillary tangles, 17, 18 neurofilament, 34, 36 neurogenic, 5, 6, 10, 12, 13, 14, 16, 18, 19, 22, 28, 39, 62

Index neuroinflammation, 49, 52, 53, 54, 55 neurological deficit, 24 neurological disease, 1, 5, 9, 11, 12, 13, 14, 18, 21, 23, 24, 26, 34, 37, 41, 49, 50, 53, 55, 59, 61, 62, 66, 72, 75, 82, 83, 84, 86, 88, 90, 96, 97, 98 neuronal cells, 1, 4, 5, 9, 10, 13, 17, 18, 19, 21, 24, 26, 27, 33, 34, 36, 37, 41, 42, 44, 45, 57, 60, 62, 66, 67, 72, 73, 75, 76, 79, 80, 81, 83, 84, 85, 86, 87, 96, 97, 98 neuronal death, 13 neuronal loss, 17, 24 neuronal markers, 24 neuronal stem cells, 5, 39, 47, 54, 64, 74, 81 neurons, 1, 5, 12, 15, 18, 19, 22, 27, 28, 29, 33, 34, 36, 37, 38, 39, 42, 44, 45, 46, 50, 54, 63, 64, 71, 73, 79, 81, 82, 85, 87, 95, 96, 97, 99, 101, 103, 104, 105 neuron-specific enolase, 12 neuroprotection, 55 neuroprotective, 52 neurotoxicity, 100 neurotransmitter, 3, 23 neurotrophic, 12, 15, 36, 58, 63 neutropenia, 133, 138 nitric oxide, 50 nitrogen, 43, 91 NK cells, 110, 112, 116, 119, 131, 132 NMDA, 10, 13 NMDA receptors, 99, 100, 103, 104 N-methyl-D-aspartate, 10 NMR, 68 nonane, 92 nondisjunction, 18, 19 non-human, 12, 41 non-invasive, 53 non-smokers, 68 norepinephrine, 11 normal, 2, 23, 25, 63 NSCs, 1, 2, 3, 4, 9, 14, 19, 21, 22, 23, 24, 25, 26, 33, 34, 37, 42, 45, 49, 50, 51, 53, 57, 58, 61 nuclear, 12, 34, 39, 42, 46 nucleic acid, 127 nutritional deficiencies, 110

O observations, 58, 117 obstruction, 110 olfactory bulb, 5, 14, 27, 54 oligodendrocytes, 1, 22, 33, 34, 36, 37, 50, 79

153

oncogene, 22 oncogenes, 135 optimization, 140 organ, 108, 110, 115, 117, 123 ornithine, 44 osteoblasts, 57, 61 oxygen, 50, 68, 73 oxygen consumption, 68

P PAA, 59 pain, 110 paracrine, 6, 28, 38, 47 paralysis, 50, 51, 90 parents, 116 paresis, 51 Parkinson’s disease, 1, 3, 33, 50, 53, 90, 99 particles, 129, 136 parvalbumin, 39 patents, 1, 2, 4, 5, 12, 27, 39, 47, 54, 64, 74, 81 pathogenesis, 9, 13, 18, 19, 20, 142 pathology, 3, 6, 13, 14, 16, 18, 19, 20, 26, 31, 67 pathways, 9, 13, 24, 26, 33, 50, 55, 58, 76, 121 patients, 3, 4, 5, 10, 11, 12, 17, 18, 19, 20, 23, 26, 45, 57, 58, 59 PCR, 42, 44, 52, 131, 132 PCs, 22 PDGF, 14, 34, 36, 37 penicillin, 42, 43 PEPA, 85 peptide, 16 perforation, 110 periodontal, 57, 58, 59, 60, 61, 62, 63 periodontal disease, 57, 59, 61, 62 periodontium, 57, 61 periosteum, 115 peripheral blood, 20, 108, 109, 110, 111, 112, 115, 128, 142 peripheral blood lymphocytes, 20 peripheral blood mononuclear cell, 112 permit, 26, 108 peroxide, 89, 96 PG, 63 pH, 34, 60, 68 pharmaceutical, 1, 2, 4 pharmacological, 9, 10, 13, 14, 15, 45 pharmacological treatment, 9 pharmacology, 9, 11, 14, 17, 20, 66, 72, 82, 88, 98 phenotype, 3, 30, 34, 52, 111, 112, 121, 134

154

Index

phenotypes, 1, 3, 6, 10, 17, 21, 22, 25, 26, 30, 33, 34, 37, 42, 50, 57, 58, 61, 62 phosphodiesterase, 11 phosphorylation, 55 photoreceptors, 25 physical activity, 68 physiological, 10 physiopathology, 13 placebo, 114 plants, 81 plaques, 17 plasma, 128, 132 plasticity, 18, 30, 76, 84, 88, 105 platelet, 10 polarity, 105 polyether, 100 polymerase, 42, 60, 131 polymerase chain reaction, 42, 60, 131 polyploidy, 82 polystyrene, 43 poor, 129, 134 population, 18, 19, 21, 22, 25, 28, 58, 84, 104, 110, 112, 115, 116, 130 pore, 60 portfolios, 4 positron, 73 positron emission tomography, 73 postmortem, 2, 3, 28, 45, 47 precursor cells, 38, 41, 47, 61, 115 predictors, 108 prednisone, 109 press, 20 prevention, 107, 124 primate, 46 primates, 12, 15, 41 probability, 135 probe, 68 production, 130, 132, 133, 139 progenitor cells, 2, 3, 5, 6, 18, 19, 22, 27, 28, 29, 30, 31, 38, 42, 43, 44, 46, 47, 61, 62, 63 progenitors, 6, 16, 28, 29, 46 progeny, 2, 6, 22, 23, 24 progesterone, 34, 35, 51 program, 18, 19, 120 pro-inflammatory, 50, 52, 53, 108, 112, 113, 116 pro-inflammatory response, 52 proliferation, 2, 3, 5, 6, 12, 15, 16, 22, 23, 27, 28, 34, 35, 36, 37, 38, 47, 50, 52, 57, 66, 71, 72, 73, 79, 81, 82, 88, 98, 110, 112, 120, 122, 123, 130, 142 promoter, 10

propagation, 33, 34, 37 prophylaxis, 117, 123 protection, 2, 4 protein, 10, 12, 17, 21, 24, 27, 34, 36, 38, 39, 42, 46, 55, 61, 63 proteins, 10, 12, 18, 20, 36, 39, 100 proteolipid protein, 55 protocol, 36, 51, 67, 68, 111, 116, 129, 130, 132, 134, 136 protocols, 4, 25, 33, 36, 43, 45, 59 proto-oncogene, 142 PSA, 15, 60 pulp, 58, 63 purification, 2, 23, 78, 111 putrescine, 34, 35, 51

Q quality control, 123 quality of life, 135

R radiation, 26, 67, 108, 117 radiation therapy, 26 rain, 18, 53 range, 1, 9, 18, 21, 23, 34, 36, 37, 41, 44, 50, 52, 53, 61, 66, 76, 84, 90 rash, 109 rat, 7, 15, 16, 22, 25, 28, 29, 39, 46, 54, 60, 61 rats, 12, 16, 27, 29 reactive oxygen species, 50 real time, 67, 69, 70 receptors, 86, 99, 101, 103, 104 recognition, 83, 85, 86, 88, 110 recovery, 10, 14, 30, 50, 55, 116, 128 red blood cells, 133 regenerate, 63 regeneration, 18, 22, 24, 26, 46, 50, 54, 76, 80, 89, 97, 110, 113 regional, 15 rejection, 54, 113, 115, 116, 117, 122, 123, 124 relapse, 49, 50, 51, 52 relapses, 51, 52 relationship, 7, 11, 15, 27, 105, 112 remission, 50, 110 repair, 2, 9, 12, 17, 18, 24, 50, 58, 63, 76, 80, 84, 90, 97, 114 replication, 12, 16, 18, 19, 20

Index repression, 46 research and development, 4 residual disease, 115 resistance, 129, 131 resolution, 67, 70, 73 respiration, 68 respiratory, 96, 134 retention, 111 retina, 25, 29, 62 retinal disease, 1, 25 retinal stem cells, 29 retinoic acid, 59, 60 retrograde amnesia, 75, 84 retrovirus, 81, 87, 135, 142 reverse transcriptase, 42 Reynolds, 2, 5, 6, 7, 21, 27, 28, 29, 30, 31, 38, 50, 51, 54, 63 rheumatoid arthritis, 53 ribonucleic acid, 132 risk, 18, 20, 26, 27, 54, 58, 61, 90, 108, 109, 114, 116, 125, 135, 141 risk factors, 18, 135 risks, 3, 4, 25, 26, 27 rodent, 6, 28, 63 rodents, 10, 12, 21, 25, 50, 65, 66, 67, 69, 70, 73, 75, 86, 90

S safety, 113, 114, 117, 136, 140 saline, 35 salt, 59, 93 scabies, 134 schizophrenia, 15, 84 sclerosis, 50 SCs, 41 secrete, 110 secretion, 108, 112, 116, 119, 121 seizure, 39 selective serotonin reuptake inhibitor, 11 selenium, 34, 35 self-renewal, 22, 37, 53 self-renewing, 1, 2, 4, 17, 22, 33, 37, 41, 49, 57 self-repair, 17, 50, 66, 76 sensitivity, 67 serotonin, 11, 16, 66, 71, 90, 98 serum, 36, 42, 43, 59, 60, 61, 103, 111, 115, 130, 138 severity, 108, 109, 112, 116, 117 shares, 110

155

sibling, 111, 123, 128, 133, 134 siblings, 116, 123 sickle cell, 108, 123 sickle cell anemia, 108, 123 side effects, 108, 110, 114 sign, 135 signaling, 10, 13 signaling pathway, 10, 13, 99, 100 signaling pathways, 10, 13 signs, 49, 52, 134 silk, 68 sites, 24, 50, 53 skin, 2, 6, 25, 26, 30, 50, 58, 62, 63, 109, 110, 122, 141 sodium, 51, 78, 99, 100, 105 software, 69, 103 solid tumors, 125 somatic cell, 6, 18, 45, 47, 142 somatic cells, 6, 18, 45, 47 somatic stem cells, 6, 30, 49 sorting, 4, 25, 30 spatial information, 75, 84 spatial learning, 81, 88 spatial memory, 75, 84 species, 1, 2, 17, 22, 23, 33, 50, 57, 66, 76, 83, 100 speed, 116, 133, 134 spinal cord, 1, 14, 25, 28, 29, 30, 34, 50, 53, 61, 84, 89, 90, 97, 99 spinal cord injury, 25, 30 sporadic, 18 stability, 115 stages, 20 standards, 68 stem cell culture, 36 stem cell lines, 6, 46, 47, 55 stem cell research, 31 stem cell therapy, 3 sterile, 68 steroids, 15, 114 storage, 3, 6, 26, 31, 43 strategies, 4, 9, 14, 39, 46, 54, 109, 117, 124, 134 strategy use, 109 strength, 53 stress, 11, 15, 66, 71, 72, 76, 84, 90, 98 stress-related, 11 striatum, 29 stroke, 29, 54 stroma, 107, 110 stromal cells, 112, 122 subcortical nuclei, 72, 82, 98

Index

156 subependymal zone, 28, 30 substances, 50 subventricular zone, 1, 10, 17, 18, 21, 49, 57 success rate, 129 sugar, 76 supernatant, 44 suppression, 50, 122 surgery, 4 surgical, 25, 26, 58, 61 survivability, 41, 42, 44 survival, 2, 15, 23, 29, 44, 45, 104, 109, 116, 121, 122, 127, 128, 142 survival rate, 109 survivors, 109 susceptibility, 110 sustainability, 104 suture, 68 SVZ, 1, 5, 10, 12, 21, 22, 24, 49, 50, 51 swelling, 110 symptoms, 10, 11, 66, 70, 90, 100, 108, 110, 115 synapses, 13 syndrome, 108 synthesis, 5, 12, 17, 82, 105

T tangles, 17 targets, 13, 104 tau, 36 T-cell receptor, 132 teeth, 58, 59, 61, 63 temperature, 130 temporal lobe, 69, 75, 84 tendon, 58, 63, 110 tendons, 63 tetanus, 129, 132 thawing, 41, 42, 44 therapeutics, 20, 122, 123 thrombocytopenia, 133 thrombopoietin, 130 thymidine, 12, 24, 52 thymus, 131 tides, 100 tissue, 2, 22, 23, 26, 33, 34, 35, 37, 44, 45, 49, 51, 54, 57, 58, 59, 60, 61, 63, 67, 71, 103, 108, 113, 114, 115, 116 tolerance, 49, 53 toxicity, 96, 113, 131, 133, 134 toxin, 109 training, 68

transcriptase, 42 transcription, 60 transduction, 130, 131, 136, 142 transfer, 26, 38, 47 transferrin, 34, 51 transformation, 3, 26, 116, 124 transfusion, 108, 134 transgene, 129, 130, 137 transgenic, 3, 10, 11, 14, 15, 23, 29 transgenic mice, 10, 15 transgenic mouse, 10 transmission, 100 transplant, 26, 50 transplantation, 2, 3, 4, 6, 9, 21, 23, 24, 26, 29, 31, 34, 45, 51, 53, 57, 58, 61, 62, 107, 108, 109, 110, 113, 114, 115, 116, 117, 121, 122, 123, 124, 128, 129, 133, 134, 139, 141, 142 transport, 55, 73 traumatic brain injury, 54 trial, 37, 113, 114, 117, 120, 129, 131, 133, 134, 135, 138, 140, 142 tricyclic antidepressant, 11 tricyclic antidepressants, 11 triggers, 20 trisomy, 20 trisomy 21, 20 trypsin, 51, 59, 103 tryptophan, 112 tumor, 108, 109, 125, 135 tumor necrosis factor (TNF), 108, 119, 125 tumorigenic, 4, 22 tumors, 27, 55, 116 tumour, 4 turnover, 127 tyrosine, 23, 130 tyrosine hydroxylase, 23

U ulcerative colitis, 110 umbilical cord, 109, 110, 117, 122 undifferentiated cells, 21, 41, 52 urea, 96

V validation, 16, 20, 73, 82 validity, 80, 136 values, 103, 130, 131, 137

Index variability, 23 variation, 70 vascular endothelial growth factor (VEGF), 112 vector, 51, 129, 132, 133, 134, 135, 136, 138, 139, 141, 142 vehicles, 78, 121 vein, 51 ventricles, 21 vertebrates, 39, 46, 81 vessels, 58 vomiting, 110 vulnerability, 39

W warrants, 136 water, 44 wetting, 109 white matter, 69 wild type, 14 WM, 14

X X-irradiation, 12

Y yield, 4, 23, 25, 34, 45, 130, 137

157