Children's Hospital Boston




		Evan Y. Snyder, MD/PhD Assistant Professor of Neurology Harvard Medical School/ Children's Hospital Department of Neurology (Neuroscience) *MRRC Project(s)* R01-NS33852 (Pending) Grafting Human Neural Stem Cells Human Neural Stem Cells in a Primate Model of Parkinsonism The use of neural stem cells (NSCs) for the study of brain development and plasticity and for gene therapy and neural repair is the major interest of this laboratory. Exploring the hypothesis that the NSC provides the cellular basis for much of the plasticity present in the mammalian nervous system, we seek to understand the processes by which murine and human NSCs make their commitment and differentiation "decisions" during development, degeneration, and regeneration. Additionally, in related work we seek to determine whether the transplantation of multipotent NSCs might serve as vehicles for CNS gene therapy and repair in various mouse and primate models of neurodegeneration. We propose that exploiting some of the inherent biologic properties of NSCs may provide novel strategies for redressing CNS dysfunction (the emerging field of "restorative neurobiology/ neurology"). Many of the early facets of this work were begun as part of a New Program Development Project of this MRRC. Research Description Highlights of Major Accomplishments Demonstration that neural stem cells (NSC) transplanted into the CNS integrate, migrate and differentiate into multiple cell types according to their local environment. Discovery that transplanted NSCs can be used effectively for gene transfer in models of single-gene neurodegenerative disease, for cell replacement in models of inherited and acquired disease affecting one or more specific cell types, and for reconstitution of complex cytoarchitecture in models of dysmorphogenetic disorders. Demonstration that NSCs expressing specific genes exhibit a tropism for infiltrating brain tumors and may be uniquely effective as a novel platform for gene therapy against brain tumors. Major Results Our laboratory pursues two overlapping and complementary areas of research. The first is to understand the processes of commitment, differentiation, and plasticity in the mammalian nervous system during development, degeneration, and regeneration. The second area is to use such cells for therapeutic purposes. The pursuit of one goal typically nurtures the other. 1. Basic neural stem cell (NSC) biology 2. Transplantation of neural stem cells (NSCs) for gene therapy and repair 1. Basic neural stem cell (NSC) biology The first area of inquiry is the basic biology of neural cells with "stem-like" properties. We were among the first investigators to recognize that cells with this degree of plasticity and multipotency exist in the CNS (and do so far beyond the traditional "windows" of "development"); that such cells may represent the cellular basis for a great deal of plasticity "programmed" into the developing and "post-developmental" CNS; that therapeutic advantage might be realized by the harnessing of this plasticity; and that such cells can be extracted from the CNS, expanded, propagated and manipulated in vitro and yet reintegrate seamlessly following reimplantation. In vivo, following transplantation into germinal zones throughout the neuraxis and across developmental stages (from embryo to adult), we demonstrated that true NSC clones (regardless of their region of origin) can integrate non-disruptively into host CNS and PNS in an anatomically-appropriate manner, differentiating into integral cytoarchitectural components of multiple cell types (both neuronal and glial) in a manner appropriate to their site of engraftment, accurately reflecting/reporting the prevalent developmental milieu, responding to the same spatial and temporal cues as endogenous host progenitors of the respective region with which they have intermingled. Thus, donor-derived neurons receive appropriate synapses; the blood-brain barrier remains intact where donor-derived astroglia put foot processes onto cerebral vasculature; donor-derived oligodendroglia express myelin basic protein (MBP) and myelinate neuronal processes. Transplanted rodents (and more recently, primates) exhibit no indications of neurologic dysfunction. Thus, the structures that receive contributions from donor cells develop normally. The NSC was the first solid organ stem cell "discovered". We have entertained the hypothesis that stem cells not only exist across organ systems but hold fundamental biological principles in common. We have shown, in collaboration with Dr. Lou Kunkel from this MRRC that stem cells can be isolated from the brain by assaying for cell cycle properties (via fluorescence-activated cell sorter analysis in the MRRC Cell Sorter Core) that similarly isolate stem-like cells from blood, muscle, endothelium and mesenchyme. Because NSCs share many properties with stem cells from other organ systems, the strategies by which NSCs may be used therapeutically for the CNS may be extrapolated to the reconstitution of other solid organ systems via the isolation of their respective stem cells. *2. Transplantation of neural stem cells (NSCs) for gene therapy and repair* In our second major area of investigation, we have found that the transplantation of exogenous NSCs is an effective tool for gene therapy and repair of the CNS. From work pursued in various rodent (and more recently monkey) models of cerebral and spinal dysfunction and neurodegeneration, we have accumulated substantial evidence that NSCs may be used for gene transfer (e.g., for enzymes and neurotrophins) in models of neurogenetic metabolic diseases (e.g., the mucopolysaccharidoses, gangliosidoses, and leukodystrophies) as well as in acquired trauma-induced disorders (e.g., hypoxia-ischemia, spinal contusion); to replace dysfunctional and maldeveloped neural cell types (e.g., neurons, oligodendrocytes) and structures (e.g., myelin) and other molecules (e.g. extracellular matrix) in models of inherited diseases (e.g., ataxia-telangiectasia and other cerebellar degenerative processes, amyotrophic lateral sclerosis, spinal muscular atrophy, dysmyelinating conditions) and of acquired cellular neurodegeneration (e.g., adult stroke, perinatal asphyxial injury, parkinsonism, head trauma, spinal transection/contusion, targeted neuronal apoptosis); and to rescue abnormal cytoarchitecture in various mouse models of abnormal migration and lamination (e.g., the reeler mutation). This approach has also been directed to brain tumors, a pathology whose migratory/infiltrative nature has made it elusive to all extant surgical, radiation, pharmacological and genetic approaches but which, we hypothesized, could be attacked by a successful gene therapeutic vehicle -- the NSC -- which is itself inherently migratory. We have now succeeded in isolating, propagating, and transplanting clones of human NSCs (hNSCs) that appear to follow developmentally appropriate programs in vivo (in rodents and in non-human primates). They, too, seem to express foreign genes and replace degenerating or underdeveloped neurons. As the first published clones of engraftable hNSCs (and, indeed, the first reported solid organ stem cells of human origin), these cells are being assessed for their effectiveness as gene therapy and cell replacement vehicles in preclinical situations in rodents and old world monkeys with the recognition that these actual clones may have the potential for direct human application. These studies in animal models of neurodegeneration and injury might promote rapid advancement towards novel molecular and/or cellular replacement clinical therapies for some developmental, degenerative, and acquired human neurological dysfunctions. Publications Lacorazza HD, Flax JD, Snyder EY, Jendoubi M. Expression of human b-hexosaminidase a-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells. Nature Med 1996; 4:424-429. Whittemore SR, Snyder EY. Physiologic relevance and functional potential of CNS-derived cell lines, Molec Neurobiol 1996; 12:13-38. Snyder EY, Wolfe JH. CNS cell transplantation: a novel therapy for storage diseases? Curr Opin Neurol 1996; 9:126-136 [on cover]. Snyder EY, Macklis JD. Multipotent neural stem-like cells may be uniquely suited for therapy of some neurodegenerative conditions. Clin Neurosci 1996; 3:310-316. Snyder EY, Fisher LJ. Gene therapy for neurologic diseases, Curr Opin Pediatr 1996; 8:558-568. Lynch WP, Snyder EY, Qualtierre L, Portis JL, Sharpe AH. Neither neurovirulent retroviral envelope protein nor viral particles are sufficient for induction of acute spongiform neurodegeneration: evidence from engineered neural progenitor-derived chimeric mouse brains. J Virol 1996; 70:8896-8907. Snyder EY, Flax JD, Yandava BD, Park KI, Liu S, Rosario CM, Aurora S. Transplantation and differentiation of neural "stem-like" cells: possible insights into development and therapeutic potential. In: Gage FH, Christen Y. eds. Research and Perspectives in Neurosciences, Isolation, Characterization, and Utilization of CNS Stem Cells. Springer-Verlag, 1997; 173-196. Snyder EY, Park KI, Liu S, Flax JD, Yandava BD, Rosario CM, Aurora S. Potential of neural stem-like cells for gene therapy and repair of the degenerating central nervous system. Adv Neurol 1997; 72:121-132. Taylor RM, Snyder EY. Widespread engraftment of neural progenitor and stem-like cells throughout newborn mouse brain, Transplantation Proc 1997; 29:845-847. Snyder EY, Senut M-C. Use of non-neuronal cells for gene delivery. Neurobiol Dis 1997; 4:69-102. Snyder EY, Yoon CH, Flax JD, Macklis JD. Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proc Natl Acad Sci USA 1997; 94:11663-11668. Plotkin MD, Snyder EY, Hebert SC, Depire E. Expression of the Na-K-2Cl co-transporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA’s excitatory role in immature CNS, J Neurobiol 1997; 33:781-795. Aboody-Guterman KS, Pechan PA, Rainov NG, Sena-Esteves M, Jacobs, A, Snyder EY, Wild P, Schraner E, Tobler K, Breakefield XO, Fraefel C. GFP as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo. NeuroReport 1997; 8:3801-3808. Rosario CM, Yandava BD, Kosaras B, Zurakowski D, Sidman RL, Snyder EY. Differentiation of engrafted multipotent neural progenitors towards replacement of missing granule neurons in meander tail cerebellum may help determine the locus of mutant gene action. Development 1997; 124:4213-4224. Billinghurst LL, Taylor RM, Snyder EY. Remyelination: Cellular and gene therapy. Sem Pediatr Neurol 1998; 5:211-228. Snyder EY. Neural stem-like cells: Developmental lessons with therapeutic potential. The Neuroscientist 1998; 4:408-425 [cover]. Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst L, Jendoubi M, Sidman RL, Wolfe JH, Kim SU, Snyder EY. Engraftable human neural stem cells respond to developmental cues, replace neurons, and expess foreign genes. Nature Biotech 1998; 16:1033-1039 [on cover]. Martinez-Serrano A, Snyder EY. Neural stem cell lines for CNS repair. In: Tuszynski M, Kordower J. eds. CNS Regeneration: Basic Science and Clinical Applications. San Diego: Academic Press, 1998; 203-250. Yandava BD, Billinghurst LL, Snyder EY. Global cell replacement is feasible via neural stem cell transplantation: evidence from the shiverer dysmyelinated mouse brain. Proc Natl Acad Sci USA 1999; 96:7029-7034. Lynch WP, Sharpe AH, Snyder EY. Neural stem cells as engraftable packaging lines can optimize viral vector-mediated gene delivery to the CNS: evidence from studying retroviral env-related neurodegeneration. J Virol 1999; 73:6841-6851. Liu Y, Himes BT, Solowska J, Moul J, Chow SY, Park KI, Tessler A, Murray M, Snyder EY, Fischer I. Intraspinal delivery of neurotrophin-3 (NT-3) using neural stem cells genetically modified by recombinant retrovirus Exper Neurol 1999; 158:9-26. Vescovi AL, Snyder EY. Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo, Brain Pathol 1999; 9:569-598. Wagner J, Akerud P, Castro D, Holm PC, Snyder EY, Perlmann, Arenas E. Type 1 astrocytes induce a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells. Nature Biotech 1999; 17:653-659 [on cover]. Park KI, Liu S, Flax JD, Nissim S, Stieg PE, Snyder EY. Transplantation of neural progenitor and stem-like cells: developmental insights may suggest new therapies for spinal cord and other CNS dysfunction. J Neurotrauma 1999; 16/8:675-687. Rubio FJ, Kokaia Z, del Arco A, Garcia-Simon MI, Snyder EY, Lindvall O, Satrustegui J, Martinez-Serrano A. BDNF gene transfer to the mammalian brain using CNS-derived neural precursors. Gene Therapy 1999; 6:1851-1866. Rowitch DH, St Jacques B, Lee SMK, Flax JD, Snyder EY, McMahon AP. Sonic hedgehog regulates proliferation and inhibits differentiation of CNS precursor cells. J Neurosci 1999; 19:8954-8965. Ourednik V, Ourednik J, Park KI, Snyder EY. Neural stem cells: a versatile tool for cell replacement and gene therapy in the CNS, Clin Genet 1999; 46:267-278. Villa A, Snyder EY, Vescovi A, Martinez-Serrano A. Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS. Exp Neurol 2000; 161:67-84 [co-PIs]. Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W, Small JE, Herrlinger U, Ourednik V, Black PMcL, Breakefield XO, Snyder EY. Neural stem cells display extensive tropism for pathology in adult brain: Evidence from intracranial gliomas. PNAS* 2000; 97:12846-12851. Poulsen DJ, Favara C, Snyder EY, Portis J, Chesebro B. Increased neurovirulence of polytropic mouse retroviruses delivered by inoculation of brain with infected neural progenitor cells. Virology 2000; in press. Sanchez-Ramos J, Song S, Dailey M, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, Freeman TB, Saporta S, Zigova T, Sanberg PR, Snyder EY. The Xgal caution in neural transplantation studies. Cell Transplantation 2000; in press. Snyder EY. Neural stem cells may be uniquely suited for repair of the degenerating CNS. In: Novartis Foundation Symposium: Neural Transplantation in Neurodegenerative Disease: Current Status and New Directions. London: Novartis Press, 2000; in press. Herrlinger U, Woiciechowski C, Aboody KS, Jacobs AH, Rainov NG, Snyder EY, Breakefield XO. Neural stem cells for delivery of replication-conditional HSV-1 vectors to intracerebral gliomas. Molec Therapy 2000; in press. Auguste KI, Nakajima K, Miyata T, Shiba R, Kosaras B, Ogawa M, Mikoshiba K, Snyder EY. Neural progenitor transplantation into reeler cerebellum complements mutant lamination and neuronal survival by Reelin-and non-Reelin-producing processes. J Neurosci 2000: in revision. Park KI, Snyder EY. Injury shifts migrational patterns and promotes establishment of new neurons in "non-neurogenic" regions of the CNS. Nature 2000: in revision. Zlomanczuk P, Mruggala M, de la Iglesia HO, Quesenberry PJ, Snyder EY, Schwartz WJ. Functional integration by stem cells: appropriate response to natural remote photic stimulation following incorporation within suprachiasmatic nucleus. Nature Neurosci 2000; in revision. Mitchell-Herpolsheimer C, Ames NK, Snyder EY, Kraemer SA, Jones MZ, Lovell KL. Xenogeneic transplantation of mouse neural stem cells into prenatal goat brain: intrinsic migratory ability and feasibility for therapy in a neurodegenerative lysosomal storage diseases in large mammals. Exper Neurol 2000; in revision. Himes BT, Liu Y, Solowska JM, Snyder EY, Fischer I, Tessler A. Transplants of cells genetically modified to express NT-3 rescue axotomized Clarke’s nucleus neurons after spinal cord hemisection in adult rats. Submitted. Doering L, Snyder EY. Expression of a cholinergic phenotype by neural stem cells derived from cerebellum when grafted to the medial septum/diagonal band complex of rodents. Submitted. See Dr. Snyder's publications via PubMed Contact Information E-mail: Evan Y. Snyder, MD/PhD Assistant Professor of Neurology Harvard Medical School/ Children's Hospital Department of Neurology (Neuroscience)