PattersonTalk_Goldman nl SC rev04

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Unformatted text preview: REVIEW © 2005 Nature Publishing Group Stem and progenitor cell–based therapy of the human central nervous system Steve Goldman Multipotent neural stem cells, capable of giving rise to both neurons and glia, line the cerebral ventricles of all adult animals, including humans. In addition, distinct populations of nominally glial progenitor cells, which also have the capacity to generate several cell types, are dispersed throughout the subcortical white matter and cortex. A number of approaches have evolved for using neural progenitor cells in cell therapy. Four strategies are especially attractive for clinical translation: first, transplantation of oligodendrocyte progenitor cells as a means of treating the disorders of myelin; second, transplantation of phenotypically restricted neuronal progenitor cells to treat diseases of discrete loss of a single neuronal phenotype, such as Parkinson disease; third, implantation of mixed progenitor pools to treat diseases characterized by the loss of several discrete phenotypes, such as spinal cord injury; and fourth, mobilization of endogenous neural progenitor cells to restore neurons lost as a result of neurodegenerative diseases, in particular Huntington disease. Together, these may present the most compelling strategies and near-term disease targets for cell-based neurological therapy. Diseases of the brain and spinal cord represent especially daunting challenges for cell-based strategies of repair, given the multiplicity of cell types in the adult central nervous system (CNS) and the precision with which they must interact in both space and time. Nonetheless, stem cell–based strategies of neural repair have garnered much attention over the past decade. Studies of lower species have informed us of the role that newly added neurons may have in the natural histories of adult neural networks1,2, and the identification and mapping of persistent neural stem and progenitor cells in the human CNS have highlighted the potential for structural neuroplasticity in adulthood3–6. At the same time, these studies have served to emphasize the inadequacy of the adult human brain’s response to disease and injury, which is typified by a lack of clinically significant regeneration in all but the youngest brains. Against this backdrop, we have witnessed an explosion in our understanding of stem cell biology. Pluripotent embryonic stem (ES) cells, neural tissue–derived stem cells and phenotype-specified progenitor cells have all been investigated for their ability to generate neurons and glia, and the molecular mechanisms by which they do so. This review focuses on the potential utility of neural stem and progenitor cells as substrates for structural repair of the brain and the spinal cord. It emphasizes those clinical targets for which stem and progenitor cell therapy might be the most appropriate and imminent, given what we know and don’t know circa 2005. The review does not cover disorders, such as stroke and Alzheimer disease, which have been reviewed elsewhere7,8, and for which substantial gaps in our understanding of disease mechanisms may prevent near-term clinical translation. Rather, I focus Division of Cell and Gene Therapy, Departments of Neurology and Neurosurgery, 601 Elmwood Ave., Box 645, University of Rochester Medical Center, Rochester, New York 14642, USA. Correspondence should be addressed to S.G.([email protected]). Published online 7 July 2005; doi:10.1038/nbt1119 here on those disorders for which our understanding of both the disease process and our intended cellular vector may be sufficient to anticipate therapeutic success once the two are appropriately paired. Stem and progenitor cells of the adult human nervous system Neural stem cells and more restricted neuronal and glial progenitor cells are dispersed widely throughout the adult vertebrate brain1,2,9,10. Long after fetal development, multipotent neural stem cells continue to line the forebrain ventricles11,12, and committed neuronal progenitor cells also remain within the ventricular wall, throughout its extensions to the olfactory bulb and the hippocampus13,14. In addition, a larger pool of glial progenitors also pervades both the ventricular zone and tissue parenchyma15. Although initially construed as astroglial and oligodendrocytic precursors, progenitor cells of the adult brain parenchyma also retain multilineage competence, and can become robustly neurogenic in culture16,17. As such, these may best be viewed as multipotential progenitor cells, restricted to generate glia by virtue of the adult tissue environment18–20. Each of these progenitor cell phenotypes persists in adult humans as well3–6,17,19, 21–25. Together, these different classes of progenitors, along with the ventricular zone neural stem cells from which they coderive, constitute the major known categories of neural precursor cells in the adult human CNS26,27 (see Fig. 1). Cell genesis in the adult CNS. Both the neuronal progenitor cells of the subependyma and hippocampal dentate, and the glial progenitors of the adult white matter, may be considered transit amplifying progenitor cells. As initially defined in the skin and gastrointestinal mucosae, transit amplifying cells comprise the phenotypically biased, still-mitotic progeny of uncommitted stem cells28–31. As stem cell progeny depart these localized niches of stem cell expansion, their daughters may commit to more restricted lineages, still mitotic but subject to senescence, that comprise the transit-amplifying pools. By this definition, the neuronal progenitor 862 VOLUME 23 NUMBER 7 JULY 2005 N ATURE BIOTECHNOLOGY REVIEW Ventricle 'C' cell Neuronal progenitors Neural stem cell Subependyma Migrating neurons 'A' cell NES TUBA1 ELAV4(Hu) 'B' cell © 2005 Nature Publishing Group Hippocampus Dentate neuron NES MSI1 SOX2 SGZ NES TUBA1 ELAV4(Hu) 'D' cell Dentate progenitors MSI1 White matter GFAP CD44 MSI1 WMPC A2B5 PDGFRA OLIG2 Katie Ris CNP OLIG2 O4 Figure 1 Stem and progenitor cells of the adult human brain. This cartoon illustrates the basic categories of progenitor cells in the adult brain and their lineal relationships, as well as markers and combinations thereof that permit their enrichment. The human temporal lobe is schematized here; it includes periventricular neural stem cells (red), which generate at least three populations of potentially neurogenic transit amplifying progenitors of both neuronal and glial lineages (blue). These include the neuronal progenitor cells of the ventricular subependyma, those of the subgranular zone of the dentate gyrus, and the glial progenitor cells of the subcortical white matter. Each transit amplifying pool may then give rise to differentiated progeny appropriate to their locations, including neurons (purple), oligodendrocytes (green) and parenchymal astrocytes (orange). A–D cell stage terminology derived from10. Markers defining each stage have been reviewed previously142; figure adapted from reference 26. Astrocyte Oligodendrocyte cell of the forebrain ventricular subependyma was first proposed as a transit-amplifying cell type, on the basis of its neuronal bias during mitotic expansion, and its ability to replenish the stem cell pool under appropriate mitotic stimulation32–34. Similarly, the neuronal progenitor cells of the hippocampal dentate gyrus, which continue to divide while migrating within the subgranular zone35, may be considered transitamplifying progenitors26, as may be the glial progenitor cells of the adult white matter; like their neuronally fated counterparts, glial progenitors can divide and yield phenotypically restricted daughters, but are incapable of sustained self-renewal19. The therapeutic significance of these different classes of transit amplifiers lies in the potential of each to be targeted selectively, for mobilization and directed induction to its terminal phenotype, as required therapeutically. Gene therapeutics may be used to target specific progenitor phenotypes for mobilization in response to expressed growth and differentiation factors8, as well as to transduce both resident progenitors and their daughters to express therapeutic transgenes36. Isolation and purification of neural progenitor cells. Cell transplantation strategies for treating CNS disease require the acquisition of human neural stem and progenitor cells in both high purity and large amounts. To address this need, several groups have established lines of neural precursors, by exposing initially uncommitted cells continuously to mitogens in serum-deficient culture11,37–39. Human neural stem cell lines have similarly been established by this approach40,41 that give rise to functionally mature neurons in vitro42–44 and in vivo41,45,46. These preparations, however, were typically generated by preferential expansion over prolonged periods of time in vitro, and were thus exposed from inception not only to mitogens, but also to neuronal and glial paracrine agents in the initially mixed cultures. As a result, neural stem cells generated by such means may not reflect the lineage potential or differentiation competence of the cells from which they derived. To address this weakness of selective expansion as a means of isolating stem and progenitor cells, surface antigen–based sorting has also been used for their selective extraction47,48 (see Fig. 2). Although presently available antigenic markers for human neural stem cells are not sufficiently specific to purify these cells from human samples47, separation based upon a combination of size, lectin binding and antigenicity has been reported in rodents49,50. As an alternative strategy to surface-based selection, my group has shown that specific stem and progenitor cell types may also be isolated by progenitor cell–selective reporter gene expression51. Individual phenotypes may be identified by transducing either tissue dissociates or cultured cells with fluorescent reporters52 placed under the control of promoters for genes selectively activated in progenitor cells52. The fluorescent progenitor cells may then be isolated by fluorescence-activated cell sorting (FACS). The tubulin α1 promoter, an early neuronal regulatory sequence53, was first used to directly extract neuronal progenitors from both the adult ventricular zone and hippocampus5,23, using tubulin α1–specified, green fluorescent protein (GFP)-based FACS51. Using this strategy, oligodendrocyte progenitors were similarly isolated from human brain tissue, based on their activation of the early oligodendrocyte promoter 2′,3′-cyclic nucleotide 3′-phosphodiesterase 2 (CNP2)17, as were neural stem cells, based on their transcription of nestin and musashi homolog 1, two genes expressed by neural stem cells23,41. Nestin enhancer–driven GFP, in particular, reliably identified neural progenitor cells in both the ventricular zone and the hippocampus5,23,41,54–56. Using the above approaches, each of the known classes of adult human neural progenitors can now be isolated to purity. Besides providing the cells for transplantation, their purification has also permitted assessment of their distinctive patterns of gene expression. It can be anticipated that the latter information may prove especially useful in identifying targets for drug discovery and thus for manipulating the fates of endogenous progenitor cells. Myelin disorders The congenital dysmyelinating and acquired demyelinating diseases of the brain are especially attractive targets for cell-based therapeutic strategies because they are caused by the loss of a single cell type, the oligodendrocyte. Oligodendrocytes are the sole source of myelin in the adult CNS, and their loss or dysfunction is at the heart of a wide variety of diseases of both children and adults. Given the relative homogeneity of the affected phenotype, the diseases of myelin are especially attractive targets for cell-based therapy of brain and spinal N ATURE BIOTECHNOLOGY VOLUME 23 NUMBER 7 JULY 2005 863 REVIEW cord disease. Fortuitously, from the standpoint of donor acquisition, glial progenitor cells very likely constitute the most abundant progenitor cell in both the adult and mid-gestation fetal brain19,57. These cells may be isolated and harvested in bulk from human brain tissue, and are readily biased to oligodendrocyte phenotype. As a result, they have been assessed in a variety of models of both demyelinating and dysmyelinating diseases. © 2005 Nature Publishing Group To assess the potential of neural cell–based treatment for these diseases, our group63–65 has transplanted sorted human oligodendrocyte progenitor cells (OPCs) of both fetal and adult origin into newborn shiverer mice, a demyelinated mouse deficient in myelin basic protein. The donor OPCs dispersed widely throughout the shiverer forebrain white matter, and developed as astrocytes and myelinating oligodendrocytes in a context-dependent fashion65 (see Fig. 3). These human tissue–derived donor OPCs migrated as widely as did immortalized Pediatric leukodystrophies and cerebral palsies. Children suffer from mouse neural stem cells, which had been previously reported by Snyder a variety of diseases of myelin failure or loss, including the hereditary and colleagues66 to be similarly capable of context-dependent differleukodystrophies (for example, adrenoleukodystrophy, Tay-Sachs, Krabbe entiation and myelination. and Canavan diseases) and the less common congenital demyelinations Notably, profound differences were observed in the behavior of fetal(for example, Pelizaeus-Merzbacher disease and vanishing white matter and adult-derived OPCs upon perinatal xenograft65. Isolates of human disease; reviewed in refs. 58,59). Even more common pediatric afflictions, OPCs derived from adult white matter myelinated the recipient brain such as cerebral palsy, may be due largely to a perinatal loss of oligoden- much more rapidly than did fetal OPCs; adult-derived progenitors drocytes and their precursors60–62. achieved widespread myelination by just 4 weeks after graft, whereas cells derived from late second trimester fetuses took over 3 months to do so. The adult OPCs also generated oligodendrocytes more efficiently Adult human tissue than fetal glial progenitors, and ensheathed more axons per donor cell than did fetal cells. In contrast, fetal glial progenitors emigrated more widely and engrafted more efficiently, differentiating as astrocytes in grey matter regions and Ventricular zone Hippocampus dentate White matter as oligodendrocytes in white matter. (neural stem cells) (dentate progenitors) (glial progenitors) The divergent behavior of fetal- and adultderived glial progenitors suggests their respecInduce desired Dissect regionally tive potential use for different disease targets. lineage defined lineages Adult OPCs, by virtue of their oligodendroHuman ES cells Fetal brain cytic bias and rapid myelination, may be most (all phenotypes) (all neural cells) appropriate for treating diseases of acute oligodendrocytic loss, such as subcortical infarcts and postinflammatory demyelinated lesions. Promoter/ Surface In contrast, fetal progenitors may prove more enhancer-based antigen-based Plasmid or sorting sorting effective for treating the congenital leukodysviral based Enhancer Virus Transfect or trophies because their extensive emigration EGFP infect better assures uniform and widespread disperPromoter/enhancers Antibodies for selection: sal, whereas their astrocytic differentiation and MACS for selection: Tα1 tubulin, CNP2, AC-133, A2B5, invasion of grey matter may offer the correction Musashi, Nestin PSA-NCAM, NG2 of enzymatic deficits in deficient cortex. Indeed, perinatal grafts of fetal progenitor cells might prove to be a means of simultaneously myelinaFACS ting and correcting enzymatic deficiencies in the pediatric leukodystrophies67. The lysosomal – + GFP GFP Sorted Depleted Sustained storage disorders are especially attractive tarcells cells cells remainder passage & gets in this regard because wild-type lysosomal expansion cDNA clone enzymes may be released by integrated donor Isolated progenitors SV40 pA cells and taken up by deficient host cells through PCMV Culture Genomic pCMV cDNA library the mannose-6-phosphate receptor pathway68. analysis construction SPORT As a result, a relatively small number of donor glia may provide sufficient enzymatic activity Transplantation Expression to correct the underlying catalytic deficit and profiling Differentiation storage disorder of a much larger number of Immortalization host cells. That being said, little data presently puro hTERT exist with regard to the amount or proportion LTR LTR of wild-type cells required to achieve local corLineage restricted progenitors Experimental xenograft rection of enzymatic activity and substrate clearance in any storage disorder, and these Figure 2 Sources, isolation and use of defined progenitor phenotypes. This figure schematizes the values will likely need to be obtained for each methods of isolating prospectively defined neural progenitor phenotypes from a variety of human cell disease target. Similarly, effective cell doses, and tissue sources, and highlights several of the experimental purposes to which these cells may be allocated and devoted. (magnetic cell sorting (MACS); fluorescence activated cell sorting (FACS)). delivery sites and time frames will need to be Katie Ris 864 VOLUME 23 NUMBER 7 JULY 2005 N ATURE BIOTECHNOLOGY REVIEW Figure 3 Neonatal xenograft of human OPCs myelinates congenitally demyelinated forebrain. (a) Sorted human fetal OPCs, implanted into neonatal myelin-deficient shiverer mice. The cells were recruited as oligodendrocytes or astrocytes in a context-dependent manner. This photo shows the striatocallosal border of a shiverer brain, 3 months after perinatal engraftment with human fetal OPCs (indicated as human nuclear antigen (hNA), blue). Donor-derived MBP+ oligodendrocytes and myelin (red) are evident in the corpus callosum, whereas donor-derived GFAP+ (green) astrocytes predominate on the striatal side. (b) A confocal optical section of engrafted corpus callosum, stained for myelin basic protein (MBP; red), human nuclear antigen (blue), and neurofilament protein (NF, green), exhibits axonal ensheathment by myelin generated by engrafted human fetal OPCs. All MBP is derived from the human OPCs, whereas the NF+ axons are those of the mouse host. (c) Another example of human donor-derived oligodendrocytes (blue), myelinating (MBP, red) host axons (green); the horizontal lines across the field are guides for computer-assisted determination of the ratio of myelinated to unmyelinated fibers in each field, by which the myelination efficacy of each graft is defined. (d) An analogous image taken from a brain implanted with adult-derived OPCs, and sacrificed at the same time as that in (c), shows the greater extent and efficiency of myelination by adult-derived progenitors. (e) A low power image showing extensive myelination of a shiverer recipient brain, 12 weeks after neonatal implantation of human fetal OPCs. Inset; a high-power image costained for human nuclear antigen (red) confirms that all MBP+ myelin (green) is donor-derived. (f,g) Electron micrograph of an engrafted shiverer homozygote shows host axons with densely compacted, donor-derived myelin sheaths (f). The asterisk indicates the field enlarged in (g), which shows the major dense lines of mature myelin. Scale bars: a, 200 µm; b–d, 10 µm; e, 50 µm; f,g, 1 µm. Adapted from ref. 65. a b c d © 2005 Nature Publishing Group e f g established in models of congenital hypomyelination before clinical trials of progenitor-based therapy can be contemplated. Moreover, the efficiency of myelination required for significant benefit remains unclear, as functional improvement may require remyelination over much if not the entire linear extent of each recipient axon. These caveats notwithstanding, there is reason for optimism that cell-based therapy of the pediatric myelin disorders, in particular for the primary demyelinations such as Pelizaeus-Merzbacher disease, vanishing white matter disease, and the spastic diplegic forms of cerebral palsy, may not be far off. Adult demyelinating diseases. In adults, oligodendrocytic loss is causal in diseases as diverse as the vascular leukoencephalopathies and multiple sclerosis. When transplanted into lysolecithin-lesioned adult rat brain, adult human OPCs were able to quickly mature as oligodendrocytes and myelinate residual denuded host axons, but with relatively low efficiency compared to the robust myelination seen in congenitally hypomyelinated brain63–65. Similarly, systemic administration of neural stem cells into mice subjected to experimental allergic encephalomyelitis resulted in some local engraftment, oligodendrocytic maturation69, although the robustness and stability of donor cell–mediated remyelination remains unclear70. These observations highlight the importance of the disease environment in influencing oligodendrocytic differentiation and myelination71. Thus, although human OPCs would seem effective agents by which to remyelinate acutely demyelinated brain tissue, the complexity of the adult disease environment may make such targets less approachable than their pediatric counterparts. At the very least, neural cell–based therapeutic strategies for adult demyelination, especially those intended to remyelinate the inflammatory lesions of multiple sclerosis, will require aggressive disease modification and immunosuppression as adjuncts to cell delivery. Parkinson disease Parkinson disease (PD) is characterized by progressive deterioration of dopaminergic neurons in the substantia nigra of the midbrain72. Because these neurons project to the caudate and putamen of the neostriatum, there is a significant decrease in neostriatal dopamine levels in PD. This has spurred the development of cell-based strategies aiming to replenish dopaminergic neurons73–75. Fetal midbrain cell–based therapy. Cell-based therapies have thus far focused on implanting fetal midbrain cells, which include those destined to become dopaminergic neurons, into the neostriatum73,74. These donor dopaminergic neurons can ameliorate experimental Parkinsonian symptoms provided a sufficient number of them survive, avoid immune rejection and make appropriate postsynaptic connections with striatal neurons75. Yet, despite promising studies in experimental models, such fetal cell grafts to adult PD patients have yielded poor results thus far, characterized by limited clinical efficacy and significant morbidity, the latter manifested as refractory, medication-independent dyskinesias76. The potential causes of this disappointment are legion; they include the relative scarcity of dopaminergic cells in the donor tissues, the heterogeneity of cell types in each donor sample, and variability among those samples, as well as the often nonuniform tissue distributions of the engrafted cells, and their lack of appropriate afferent control when placed into the striatum, among others. Yet the relative simplicity of the major pathology of PD, the loss of a unifocal and phenotypically homogeneous neuronal population, along with its manifest epidemiological importance as a major disease, have continued to spur stem cell–based strategies for its cure. Indeed, the failure of fetal nigral tissue grafts to ameliorate PD has provided impetus to research into generating more uniform and accessible populations of human dopaminergic neurons, using defined dopaminergic progenitors rather than fetal tissue as the source. To address these issues, several groups have explored the possibility of generating highly enriched populations of dopaminergic neurons from mesencephalic progenitors77,78. Tissue-derived progenitors proved capable of giving rise to functional dopaminergic neurons, in sufficient numbers and with uniformity so as to permit the functional recovery of rats rendered Parkinsonian by 6-hydroxydopamine (6-OHDA)77,78. N ATURE BIOTECHNOLOGY VOLUME 23 NUMBER 7 JULY 2005 865 REVIEW Yet preparing engraftable quantities of dopaminergic progenitors from fetal tissue has proven difficult because of the small numbers of neural stem cells that may be purified from the first trimester midbrain, and the impracticality and controversial nature of sampling large numbers of aborted fetuses for this purpose. ES cells as sources of dopaminergic neurons. As a result, several groups have begun to assess human ES cells as a potential source of dopaminergic neurons. This possibility was opened by an elegant set of developmental studies, in which fibroblast growth factor 8 (FGF8) and sonic hedgehog were implicated as tandem initiators of dopaminergic neurogenesis in vivo79. By replicating these conditions in vitro, McKay and colleagues first generated dopaminergic neurons from ES cells80 and later achieved greater degrees of dopaminergic enrichment by mimicking the oxygen tension of the developing midbrain81. When implanted into 6-OHDA– lesioned rats, in which nigrostriatal dopaminergic afferents are largely lost, these ES-derived dopaminergic cells restored functional normalcy to the dopamine-depleted animals82. Similar results were obtained using monkey ES cell–derived dopaminergic neurons allografted into 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)–lesioned adult cynomolgus monkeys, in which treatment-associated behavioral improvement was noted by 10 weeks after transplantation83. These monkeys received highly-enriched dopaminergic grafts, the result of in vitro differentiation in medium supplemented with FGF20 and conditioned by a permissive stromal cell line. Nonetheless, only 2,130 dopaminergic neurons were reported per transplanted striatum, of 300,000–600,000 injected, indicating that >99% the injected cells died within the 14 weeks before sacrifice. One might imagine even more compelling results had better graft survival been achieved. These studies notwithstanding, the use of ES cell–derived dopaminergic neurons remains limited though by some of the same caveats that limit tissue-derived progenitor implantation. First, even highly enriched ES cell–derived dopaminergic preparations are by no means pure, and may be contaminated with serotonergic and γ-aminobutyric acid (GABA)ergic neurons, as well as by glia84. The cointroduction of these other transmitter phenotypes, without further dopaminergic selection, might yield as-yet unpredictable interactions among the engrafted cells, both synaptic and presynaptic. Second, the long-term survival and phenotypic stability of engrafted primate and human ES cell–derived neurons have been problematic in experimental models of PD, as exemplified above by the monkey allografts. Third, any ES cells that might have escaped in vitro differentiation have the potential for undifferentiated expansion, potentially yielding teratomas or their derivative tumors after implantation85. Thus, even though tumors have not yet been reported in primate or human ES cell transplants to the CNS83,86, one must be skeptical: no long-term survival studies have yet been reported that would allow one to adequately assess the neoplastic potential of human ES cell grafts over time. Fundamentally, the risk of uncontrolled expansion by remnant ES cells is sufficiently high that complete abolition of undifferentiated ES cells from donor grafts would certainly lend more confidence to the safe clinical use of ES cell–derived dopaminergic neurons. Promoter-based isolation strategies, based on both GFP-dependent sorting87 and antibiotic selection, have allowed the enrichment of increasingly defined phenotypes from ES cells87,88. Yet whether these approaches can be scaled up to achieve the abolition of undesired phenotypes from the large numbers of cells required for clinical transplantation remains to be seen. Over and above these caveats, once dopaminergic progenitors do indeed become available in required purity and abundance, many other issues that have plagued trials of fetal mesencephalic implants, such as the lack of afferent control of cells implanted into the caudate-putamen, their nonuniform and unpredictable dispersal, and the lack of nigrostriatal reconstruction upon alternative implantation into the substantia nigra, all remain significant impediments to early clinical translation. Spinal cord injury and disease The spinal cord has been an attractive target for cell-based therapeutic attempts, in part because of the dearth of available treatment options for spinal cord injury and trauma, but also because of the rapid pace of advance in our understanding of how to effect axonal regeneration in the injured cord89–91, without which cellular replacement alone would be of limited benefit. Cell-based treatments for spinal cord trauma. Spinal cord injury is associated with the loss of both neurons and glia. Flexion-extension injuries, contusion and anterior spinal artery ischemic events, as may occur in trauma, spinal cord stroke and iatrogenic occlusion, can all result in segmental loss of both the superficial white matter tracts and the various neuronal populations of the central grey matter. Thus, cell-based strategies for reconstituting the injured spinal cord must accommodate the need to replace multiple cell types. Neural stem cell implants into the injured spinal cord have been used under the premise that regionally appropriate phenotypes might be generated from undifferentiated cells in response to local signals competent to induce cell-type specification. Yet the extent to which such site-specific phenotypic differentiation occurs can vary with both the differentiated state of the donor cells and the developmental stage of the recipient, and with whether the target region is intact or injured. Inappropriate phenotypic differentiation can be ineffective or worse; for instance, rats transplanted with neural stem cells after thoracic weight-drop injury developed forelimb allodynia and heightened pain sensitivity that were associated with aberrant axonal sprouting after ectopic astrocytic differentiation92. In this regard, the ability of neurons derived from non-homotypic neural stem cells to appropriately follow local position-dependent cues in axonal pathfinding remains unclear. These caveats notwithstanding, Okano and colleagues93 noted that neural stem cells implanted into sites of spinal cord injury yielded behavioral improvement compared with untransplanted controls. Similarly, Olson and colleagues demonstrated that neurogenin 2–transduced neural stem cells, thereby potentiated to neuronal differentiation, were associated with significant functional benefit after contusion injury92. In both cases, improvement was associated with local neurogenesis and synaptogenesis that might have served to establish alternative pathways for local neurotransmission in the injured segments of the cord. In either case though, the benefits of neural stem cell transplantation may have depended as much on serendipitous donor-host humoral interactions as on local anatomic reconstruction94. Indeed, the lack of control that can be exercised on the differentiated fate of implanted neural stem cells, or on the connectivity patterns achieved by their neuronal daughters, is a potential source of concern with regards to their therapeutic use. As such, the very environmental malleability of neural stem cells suggests that more-restricted progenitor phenotypes might be more predictable and less prone to unwanted surprise upon host engraftment. Beside the loss of neurons and astrocytes seen in spinal cord injuries and infarcts, many cord injuries are demyelinative; the sensory tracts of the posterior columns and the descending motor tracts of the corticospinal pathways are both frequent victims of flexion-extension and contusion injuries of the cord. These surface pathways are also especially predisposed to ischemic damage following cord edema, with the draining veins on the posterolateral surfaces of the spinal cord being especially vulnerable to congestion and hypoperfusion when edematous. As a result, the transplantation of myelinogenic glial progenitors has been an especially appealing strategy for treating spinal cord injury. © 2005 Nature Publishing Group 866 VOLUME 23 NUMBER 7 JULY 2005 N ATURE BIOTECHNOLOGY REVIEW Fischer and colleagues95 demonstrated that fetal tissue–derived glialrestricted progenitors implanted into the contused rat spinal cord dispersed widely, with both astrocytic and oligodendrocytic maturation. Although neither myelination nor the net efficiency of oligodendrogliogenesis was reported in this study, it nonetheless suggested the potential utility of glial progenitor grafts for treating carefully selected spinal cord injuries, especially those with limited involvement of the posterior columns and lateral funiculi. Indeed, this approach may be considered as a potential treatment for other segmental demyelinations, such as those that occur in transverse myelitis. The practical limitations on both fetal and adult cell acquisition for human allograft have driven much research on deriving tissue-specific progenitor cells from human ES cells. Oligodendrocytes derived from human ES cells were recently reported to myelinate demyelinated foci in spinal cord contusions86. This latter observation paralleled earlier studies that reported myelination in the injured spinal cord by implanted mouse ES cells96. Neither of these studies, however, isolated glial progenitors or oligodendrocytes before transplantation, and neither followed animals for the long periods of time required to ensure the long-term survival and phenotypic stability of the engrafted cells. In particular, these ES cell–based approaches may prove limited by the potential of any persistent undifferentiated ES cells in the donor pool to yield teratomas or germinomas after implantation. As a result, stringent selection criteria will have to be applied so as to deplete donor cell populations of any undifferentiated ES cells, before human ES cell–based therapy of spinal cord injuries, or for that matter any other CNS disease target, may be safely contemplated. Until that time, the implantation of tissue-derived glial progenitor cells may prove to be the more clinically feasible option. Indeed, even using the better understood glial progenitor cells, it should be clear that cell therapy of spinal cord injury will need to be multimodal, and will need to be accompanied by treatments designed to maximize extant cell survival97, axonal extension and synaptogenesis98, as well as engraftment and differentiation. As with so many other disease targets, cell therapy of spinal cord injury will be unlikely to yield clinically significant restoration of function, without a concurrent modulation of the disease environment in such a way as to favor cell integration and axonal regeneration, while suppressing ongoing disease pathology. Cell-based treatment of motor neuron diseases. In contrast to the many cell types lost in spinal cord injury, the major degenerative diseases of the spinal cord, including amyotrophic lateral sclerosis (ALS or Lou Gehrig disease) and the spinal muscular atrophies of children, as well as the major viral infections of the spinal cord, including poliomyelitis and West Nile virus, are characterized by a selective loss of spinal motor neurons. Motor neuron disease was thus an early target of stem cell–based therapy. Yet while initial attempts at using ES cell implants to treat experimental motor neuron disease did yield some functional benefit, this was not accompanied by appreciable motor neuronal differentiation99. Indeed, this initial effort suggested the need for engrafting phenotypically restricted cells in such disease models, rather than being held hostage to the stochastic differentiation of multipotential stem cells. Fortunately, motor neurons have proven especially amenable to generation from human ES cells100, largely because of the well-studied development of motor neurons in normal ontogeny and its recapitulation in mouse ES cells101, in which sonic hedgehog and retinoic acid have proved sufficient to induce motor neurons from human embryoid bodies. As an alternative means of generating phenotypically-restricted spinal neurons, my group102 transduced human fetal spinal neuroepithelium with retroviruses overexpressing human telomerase reverse transcriptase (hTERT), the rate-limiting component of human telomerase. The resultant telomerase-immortalized cells included lines biased to generate neurons; one in particular preferentially generated spinal interneurons, but also generated a minor cohort of motor neurons, that could be isolated by FACS based on homeobox HB9 promoter–driven GFP expression. Like their human ES-derived counterparts, the motor neurons generated from these hTERT-immortalized progenitors were postmitotic, both antigenically and physiologically appropriate, and functionally integrated after engraftment102. But just as the use of ES cells as a source of donor neurons poses the potential for adventitiously introducing undifferentiated cells that might prove tumorigenic, the use of neurons derived from a telomerase-immortalized precursor line may similarly present the potential for undifferentiated expansion from inadvertently admitted precursor cells. Again, the issue of preimplantation purification will need to be addressed before either of these otherwise highly promising sources of phenotypically restricted donor cells may be used as clinical vectors. Yet despite the availability of highly enriched human motor neurons, from both human ES cells and telomerase-immortalized progenitors, and the identification of genetic tags that allow their isolation and targeting102–104, the specific replacement of motor neurons is a formidable challenge. Spinal cord neurons are the frequent victims of multilevel disease, with classic ALS involving most rostrocaudal segments of the CNS. Motor neurons generated from ES cells or hTERT-immortalized native spinal progenitors are essentially nonmigratory and would need to be delivered to sites of need; such multifocal targeting would be problematic at best. Indeed, the replacement of lost motor neurons in vivo requires not only multisegmental delivery or induction, but also the re-establishment of appropriate afferent innervation, and the longdistance extension of their axons, through often degenerating nerve roots, to specific loci in the distant musculature. Our poor understanding of the biology underlying these processes suggests that despite the recent progress described in generating and isolating motor neurons, cell-based treatment of the motor neuronopathies remains a difficult goal. Induced neurogenesis for treating neurodegenerative disorders The persistence of both neural stem cells and their committed neuronal progeny in the striatal wall of the lateral ventricle suggests their potential utility in restoring lost striatal neuronal populations. Several groups have now successfully targeted endogenous progenitor cells for directed mobilization and phenotypic induction, using both neurotrophic and chemotaxic cytokines, delivered to the brain by both protein infusion and viral vectors. Mobilizing endogenous progenitor cells. Compensatory replacement of striatal neurons from resident progenitors was identified in experimental stroke by several groups105–107, who described neuronal recruitment into the striatum after focal ischemic injury (Fig. 3). Similarly, Nakafuku and coworkers108 described compensatory replacement of hippocampal pyramidal neurons, another periventricular subcortical neuronal pool. Other groups have recently reported apparent compensatory neurogenesis in the striatum of Huntington disease patients109 and increased dentate neurogenesis in the hippocampus of Alzheimer disease patients110. Macklis and colleagues have similarly identified compensatory neuronal production in the neocortex, after targeted ablation of both interneurons111 and corticospinal112 neurons. Together, these studies have suggested the potential for recruiting new neurons from endogenous progenitor cells as a therapeutic strategy. Most of these examples of compensatory neurogenesis, however, have been modest, yielding quantitatively and thus clinically insignificant neuronal addition over the time periods assessed. The challenge thus becomes understanding the basis for compensatory neurogenesis, so as to accentuate its influence, while countering those mechanisms that may serve to limit its role in the damaged CNS. © 2005 Nature Publishing Group N ATURE BIOTECHNOLOGY VOLUME 23 NUMBER 7 JULY 2005 867 REVIEW Several humoral growth factors have been identified as modulating the expansion and fate of neural stem cells. Epidermal growth factor (EGF), transforming growth factor α and FGF2 are each mitogens for these cells, and each can potentiate neuronal replacement in the presence of permissive signals for neuronal differentiation113–114. Yet these agents appear to act solely as mitogens. In adult rodents, EGF stimulation led largely to gliogenesis, and FGF2 infusion increased neuronal recruitment to the olfactory bulb, but nowhere else; neurons generated under the sole influence of FGF2 did not depart their typical migratory paths to enter any other subcortical structures along their migratory route113. Several other ligands for receptor tyrosine kinases have been found to drive mitotic expansion by neural stem and progenitor cells, including vascular endothelial growth factor and stem cell factor, through the vascular endothelial growth factor receptor 2 and c-kit receptors, respectively115–117. Similarly, inhibition of nitric oxide synthase, which tonically suppresses progenitor turnover in the adult CNS, increases neuronal production in the olfactory subependyma, bulb and dentate gyrus118. In addition, a great number of agents have been found to modulate neurogenesis in the adult hippocampus, most notably the glucocorticoids119 and insulinlike growth factor 1 (IGF1)120 as physiological regulators2, as well as the serotinergic antidepressants among several described pharmacological regulators121 (Fig. 4). But none of these agents have been associated with the recruitment of new neurons into otherwise non-neurogenic regions, a key prerequisite to any effective restorative strategy. To achieve the addition of new neurons to otherwise non-neurogenic regions of the brain, my laboratory122–124 and others125 have focused on the trkB ligand brain-derived neurotrophic factor (BDNF), which can signal the differentiation and survival of new neurons from forebrain ventricular zone stem cells. On that basis, Benraiss et al.126 noted that a single intraventricular injection of adenoviral BDNF (AdBDNF) into the ventricles, which resulted in widespread ependymal production of BDNF, induced the production of new neurons from progenitor cells in the adjacent subependyma. Many of these new neurons invaded the neostriatum, a region of the brain that is critically important to the direction and coordination of movement, and which is otherwise non-neurogenic. A parallel study using BDNF protein infusion similarly demonstrated neuronal addition to the neostriatum127, as well as to other subcortical structures. In the neostriatum, the new neurons largely integrated as medium spiny neurons, which extended projections to the globus pallidus. These are the cells that are typically lost in Huntington disease128, which suggests that AdBDNF-induced cells may have been able to directly replace the very phenotype lost in the course of Huntington. Moreover, the new striatal neurons stably persisted for months after their production, even though viral BDNF expression persisted only a month, suggesting that once integrated, the newly generated cells could survive independently of periventricular BDNF overexpression126. Therapeutic implications of induced neurogenesis. As a treatment strategy, induced neurogenesis must be evaluated from the standpoint of the number of neurons that may be generated over a given time span, their relative contribution to the existing pool, their efficiency in extending axons to appropriate targets, and their survival thereafter. As a case in point, although AdBDNF induced over 150 new striatal neurons/ mm3/month (ref. 126), this represents <1% of the neostriatal neuronal pool. To maximize the number of new neurons recruited in response to BDNF, we129–131 asked if striatal neuronal addition might be enhanced by simultaneously blocking glial differentiation by the parental neural stem cells. As glial production by these cells may be mediated by the bone morphogenetic proteins (BMPs)129–131, gliogenesis could be supressed by overexpressing noggin, a potent inhibitor of the BMPs132,133. By thereby © 2005 Nature Publishing Group Figure 4 Compensatory and induced neuronal recruitment to the adult brain. This schematic illustrates the described loci of compensatory and experimenter-induced neurogenesis in the adult rat striatum and neocortex (a) and hippocampus (b). Loci of experimental compensatory neurogenesis include the neostriatum and hippocampal pyramidal layer after stroke. In patients, compensatory neurogenesis has similarly been reported in response to neurodegeneration in both Huntington disease and Alzheimer disease, in the striatum and dentate gyrus, respectively. Loci of induced neurogenesis include the neostriatum and diencephalon in response to brain-derived neurotrophic factor (BDNF), with potentiation of the striatal response with concurrent BMPsuppression via noggin, and the dentate gyrus of the hippocampus, in response to insulin-like growth factor 1 (IGF) and vascular endothelial growth factor (VEGF), as well as to nitric oxide synthase (NOS) inhibition and serotinergic agonists. Abbreviations: LV: lateral; ventricle; CC: corpus callosum; DG; dentate gyrus; CA1: hippocampal CA1 pyramidal cells; 3V: third ventricle; ; SCF: stem cell factor; SSRI: serotonin selective reuptake inhibitor. Loci of compensatory neurogenesis (blue numbers) (1) Striatal neuronal recruitment post-stroke105–107 and in Huntington’s patients109. (2) Cortical neuronal addition after targeted intracortical ablation111,112. (3) Hippocampal pyramidal neuronal addition postischemia108. (4) Dentate in Alzeimer patients110. Loci of induced neurogenesis (red numbers) (5) Striatal:overexpression of BDNF/noggin, yielding induced neurogenesis126 and suppression of gliogenesis and axonal projection to globus pallidus135. (6) Diencephalic and septal neurogenesis following high-dose BDNF infusion in normal rats127. (7) Dentate neurogenesis induced by IGF1 in hypophysectomized rats120; SSRIs (serotonergic reuptake inhibitors)121; NOS inhibition via L-NAME118, VEGF, SCF115–117. 868 VOLUME 23 NUMBER 7 JULY 2005 N ATURE BIOTECHNOLOGY Katie Ris REVIEW expanding the pool of progenitors potentially responsive to BDNF, adenoviral noggin (AdNoggin) strongly potentiated AdBDNF-induced striatal neurogenesis, roughly tripling the rate of striatal neuronal addition to >400/mm3/ month134,135. Thus, the concurrent inhibition of glial differentiation and promotion of neuronal differentiation by resident neural stem cells may be a feasible means of recruiting large numbers of new neurons to otherwise non-neurogenic regions of the adult forebrain. To judge the feasibility of this approach for treating neurodegenerative diseases, we have recently assessed the effect of AdBDNF injection in R6/2 mice, which are transgenic for a roughly 150-polyglutamine repeat in the huntingtin gene and have a relatively severe Huntington phenotype136. Our initial data indicate that R6/2 mice treated with AdBDNF and AdNoggin generated new medium spiny projection neurons to the globus pallidus, in amounts no different from identically treated normal rats and mice137. These findings offer promise for using neuronal induction from endogenous progenitor cells as a means of treating Huntington disease, a disorder that is otherwise inevitably and rapidly fatal. Indeed, activating endogenous neural stem cells in this manner may prove a treatment option not only for Huntington disease, but also other causes of striatal neuronal loss, such as striatonigral degeneration and lenticulostriate stroke. Parenchymal glial progenitor cells of the adult brain might also be considered appropriate targets for therapeutic intervention. As noted they are multipotential and appear to be maintained in a state of undifferentiated miotic competence in the normal adult17–20. Moreover, the sheer abundance of these cells in the adult human brain is striking: over 3% of all white matter cells can be isolated as nominal oligodendrocyte progenitor cells, and over half of these are mitotically active upon sorting17,19. To be sure, the extent to which the parenchymal progenitor cell pool is heterogeneous remains unclear. Nonetheless, the abundance of progenitors dispersed throughout the white matter parenchyma points to an accessible reservoir of cells potentially amenable to both genetic and pharmacological induction. The attractiveness of mobilizing endogenous progenitors as a therapeutic strategy may be tempered, however, by concern as to the risks of dysregulated mitogenic expansion in vivo. Resident stem and progenitor cells may be the source of primary brain tumors138,139, and past attempts at induced expansion using mitogenic growth factors, including FGF2 and EGF, have been hindered by formation of periventricular nodules113 and heterotopic gliosis140 that suggested early tumorigenesis. In part as a result, more recent studies of induced neurogenesis have focused on modulating the differentiation of resident precursor cells, rather than their proliferative expansion. Nonetheless, at a certain point we will need to know how to safely expand the progenitor pool in vivo before differentiating those cells to desired lineages; induced differentiation without some provision for sustaining expansion might be expected to deplete the targeted progenitor cell pools, with effects both unanticipated and unwelcome. Conclusions As enthusiasm has increased for the potential of stem cell–based treatment approaches, it has become all the more important to define rigorously those diseases most amenable to rational therapy. At our present level of understanding, several neurological disorders may present targets of opportunity for cell-based therapy. These include the pediatric and adult demyelinating diseases, spinal cord injury and two subcortical neurodegenerations, Huntington and Parkinson diseases. These seemingly diverse disorders are all characterized by relatively simple neuropathology and at least crudely understood pathophysiology, and each involves the loss of a very limited number of cell phenotypes. Whether treated by implantation or the induction of endogenous progenitors, such diseases of single phenotype may be the most appropriate and potentially fruitful initial targets for cell-based therapy of neurological disease. For instance, with a common platform of human glial progenitor cell implantation, several diseases of oligodendrocyte loss, as diverse as pediatric leukodystrophies and lysosomal storage diseases, cerebral palsy, and such monophasic acquired demyelinations as transverse myelitis, spinal cord injury and subcortical stroke, may all be approachable as therapeutic targets. Moreover, the technologies involved in enriching the progenitor cells appropriate for such transplants have proven adaptable to generating isolates amenable to both immortalization and gene expression analyses. This has led to the generation of lines of specific types of neuronal progenitors that may prove useful not only as cellular vectors for transplantation, but as tools for understanding their signaling pathways and growth control. By understanding the latter, we can hope to mobilize endogenous progenitors in vivo, and thus abrogate the need for transplantation in many disease settings. The ability of resident progenitor cells to regenerate striatal, hippocampal and even cortical neurons represent important initial steps in that direction, demonstrating the possibility of mobilizing endogenous progenitor cells to regenerate lost neural circuits and functions just as in lower species141. Together, these disparate but overlapping strategies to cell-based neural repair may bring the dream of regenerating adult human brain tissue that much closer to clinical reality. ACKNOWLEDGMENTS I thank M. Nedergaard, N. Roy, M. Windrem, F. Sim, A. Benraiss, S. Wang, E. Chmielnicki and S.-R. Cho for their contributions to the studies discussed, and apologize to the many authors whose relevant work I have not been able to cite in this short review. My thanks to F. Sim for expert help in illustration. Supported by the US National Institutes of Health–National Institute of Neurological Disorders and Stroke, the National Multiple Sclerosis Society, the New York Spinal Cord Injury Research Program, The A–T Children’s Project and the CNS foundation, Berlex Bioscience and Merck Research Laboratories. COMPETING INTERESTS STATEMENT The author declares competing financial interests (see the Nature Biotechnology website for details). Published online at 1. 2. 3. 4. Goldman, S. 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