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JOURNAL OF HEMATOTHERAPY & STEM RESEARCH 12:671–679 (2003) ©Mary Ann Liebert, Inc.

State-of-the-Art Review

Adult Neural Stem Cells and Repair of the Adult Central

EYLEEN LAY KEOW GOH, DENGKE MA, GUO-LI MING, and HONGJUN SONG

ABSTRACT

Neural stem cells are present not only in the developing nervous systems, but also in the adult cen- tral nervous system of mammals, including humans. The mature central nervous system has been traditionally regarded as an unfavorable environment for the regeneration of damaged axons of ma- ture and the generation of new neurons. In the adult central nervous system, however, newly generated neurons from adult neural stem cells in specific regions exhibit a striking ability to migrate, send out long axonal and dendritic projections, integrate into pre-existing neuronal cir- cuits, and contribute to normal brain functions. Adult stem cells with potential neural capacity re- cently have been isolated from various neural and nonneural sources. Rapid advances in the biology have raised exciting possibilities of replacing damaged or lost neurons by activation of endogenous neural stem cells and/or transplantation of -expanded stem cells and/or their neuronal progeny. Before the full potential of adult stem cells can be realized for regenerative med- icine, we need to identify the sources of stem cells, to understand mechanisms regulating their pro- liferation, fate specification, and, most importantly in the case of neuronal lineages, to characterize their functional properties. Equally important, we need to understand the neural development pro- cesses in the normal and diseased adult central nervous system environment, which is quite differ- ent from the embryonic central nervous system, where neural development has been traditionally investigated. Here we will review some recent progress of adult research that is ap- plicable to developmental neurobiology and also has potential implications in clinical neuroscience.

INTRODUCTION ture from primordial germ cells of an early ) are pluripotent, capable of giving rise to every of an TEMCELLSARETHEESSENTIALBUILDING BLOCKS of organism, except the trophoblasts of the (4). So- Smulticellular organisms (1–3). They have two defin- matic stem cells and germ-line stem cells are produced ing properties—they can self-renew to produce more stem later in development from pluripotent stem cells. Hema- cells and they can differentiate to generate specialized cell topoietic stem cells are probably the best characterized so- types. Stem cells come in different varieties, depending matic stem cells and are able to generate all the cell types on when and where they are produced during develop- of the blood and the (1–3). Stem cells are ment and how versatile they are (1– 3). Totipotent stem also responsible for generation of other somatic systems cells can give rise to a full organism once implanted in during development, such as the central nervous system the uterus of a living animal. Both embryonic stem (ES) (CNS) (5–9). Neural stem cells (NSCs) in the central ner- cells (produced in culture from the epiblast cells of a blas- vous system (CNS) have trilineage ability, capable of gen- tocyst) and embryonic germ (EG) cells (produced in cul- erating neurons, oligodendrocytes, and astrocytes (5– 9).

Institute for Cell Engineering, Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

671 GOH ET AL.

While diversification of cell types is largely completed at neurons are not replaced in the adult CNS (10,11). Ac- or soon after birth, many tissues undergo self-renewal in tive is found to be restricted to two specific response to acute injury or normal usage throughout life regions in the adult CNS (6–9), namely the subgranular (1–3). Therefore, some somatic stem cells are also main- zone (SGZ) in the of the and tained throughout the whole life span for the regeneration the (SVZ). Mostly cells, but not of specific tissues, such as the epidermis, hair, small in- neurons, are generated in other regions of the intact CNS testine, and hematopoietic system (1–3). that are considered to be nonneurogenic (6–9). Cells with The adult CNS was long thought to be largely post- neurogenic potentials can, however, be derived from mitotic with very limited ability to regenerate (10– 14). these nonneurogenic regions and exhibit multipotent stem Thus, it came as a surprise when the existence of NSCs cells properties in culture (6–9). Thus, NSCs in most re- in the adult CNS was discovered (6–9). Since the initial gions of the mature CNS may have a latent neurogenic observation made over 40 years ago (15), neurogenesis, program that can be activated by the exposure to high the generation of neurons, has been found in specific re- concentrations of growth factors in culture (20). Alter- gions of the adult CNS of all mammals examined (6–9), natively, or in addition, the adult CNS environment may including humans (16). The existence of NSCs in the limit the neurogenic potentials of NSCs that have a adult CNS, the source of neurogenesis, was later sug- broadly presence in the adult CNS. gested by the isolation of multipotent NSCs from the Second, injured axons of mature neurons in the adult adult mice brain (17). Within the last decade, multipo- CNS do not spontaneously regenerate to reform connec- tent NSCs have been isolated from diverse regions of the tions (12– 14). This is largely due to the presence of adult CNS of both rodents and humans (17– 22). These growth inhibitory factors in the adult CNS environment adult NSCs can be amplified in vitro through many pas- (12–14). Many inhibitory factors have been recently iden- sages without losing their multipotentiality , capable of tified, including those associated with the CNS myelin giving rise to neurons, astrocytes, and oligodendrocytes (e.g., Nogo, myelin-associated glycoprotein, oligoden- both in culture and after transplantation to specific re- drocyte myelin glycoprotein) and the glia scar (e.g., chon- gions in vivo (7–9). In the last few years, the function- droitin sulfate proteoglycan). Many developmental re- ality of neurons derived from adult NSCs has been dem- pulsive guidance cues (e.g., semaphorins, ephrins) have onstrated both in vitro (23,24) and in vivo (25,26). Rapid also been found to be present in the lesion area and may advances in adult NSC biology have raised great expec- contribute to the lack of regeneration of axonal connec- tations that these cells can be used as potential resources tions in the adult CNS (12–14). In addition to extrinsic for neuronal replacement therapy after injury or degen- environmental factors, changes in the intrinsic properties erative neurological disease of the adult human CNS. In during neuronal maturation also contribute to the failure this review, we will focus on the recent progress in our of axon regeneration (33,34). For example, neonatal rat understanding of how functional and integrated neurons retinal ganglion cells undergo a profound and apparently are generated from adult NSCs in an apparently inhibitory irreversible loss of intrinsic axon growth ability upon re- adult CNS environment. The challenges in potential adult ceiving signal(s) from amacrine cells (33). It has also stem cell-based therapy for repairing the diseased adult been suggested that reduction of endogenous cAMP lev- CNS will also be discussed. For more details on CNS re- els during neuronal maturation leads to the loss of re- pair using various types of stem cells, several recent com- generative capacity for many axons (34). prehensive reviews may be consulted (27–31).

FUNCTIONAL NEUROGENESIS FROM MATURE CNS, AN UNFAVORABLE ENDOGENOUS NEURAL STEM ENVIRONMENT FOR NEURONAL CELLS IN THE ADULT CNS REGENERATION If the mature CNS is such an inhibitory environment Damage of the CNS, caused by injury or degenerative for neuronal regeneration, is it possible functionally to neurological diseases, such as Parkinson’s disease, Hunt- replace the lost neurons in the adult CNS? Recent land- ington’s disease, and amyotrophic lateral sclerosis, leads mark studies demonstrated that endogenous NSCs could to the severing of existing axonal connections and the generate new neurons not only in neurogenic regions in death of neurons. Both intrinsic cell properties and ex- the adult CNS, but also in nonneurogenic regions under trinsic environmental factors contribute to the lack of certain injury conditions (35,36). In addition, the adult regenerating capacity of the adult mammalian CNS CNS environment, otherwise inhibitory for regeneration (12–14,32– 34). of the existing mature neurons, is still permissive for the First, unlike blood cells, which can be continuously re- full spectrum of neuronal development of endogenous placed from hematopoietic stem cells, most of the lost adult NSCs (25,26).

672 REPAIR OF ADULT CNS WITH ADULT NEURAL STEM CELLS

Traditionally, has been investigated of newborn interneurons in the adult mice. These new with tracers that can be inherited by dividing cells and neurons are first generated in the SVZ, and then migrate passed on to their progeny (7,37). The most commonly over a long distance through the rostra migratory stream used tracer is bromodeoxyuridine (BrdU), a thymidine ana- (RMS) to the olfactory bulb. With a series of electro- log that incorporates into the DNA during the S phase of physiological studies, they documented the functional mitosis (37). In one study (35), Magavi and colleagues maturation and integration of newborn interneurons in the found that after the induced death of a specific subset of olfactory bulb (26). neocortical neurons followed by BrdU pulsing, a small Taken together, these findings suggest that a neuronal number of neurons were labeled with BrdU with long-dis- developmental program could be recapitulated in both tance projections toward their targets in the neocortex. In neurogenic regions and nonneurogenic regions of the ma- another study (36), Nakatomi and colleagues induced isch- ture CNS under certain conditions. In addition, newborn emic brain injury to CA1 pyramidal neurons in the hip- neurons from endogenous adult NSCs are capable of mi- pocampus followed by intraventricular infusion of two grating, extending long axonal projections, and becom- growth factors, epidermal growth factor (EGF) and basic ing functionally integrated into existing local circuits in growth factor (FGF-2). They showed that neu- the adult CNS environment. rons labeled with BrdU probably originated from endoge- nous NSCs located in the periventricular regions of the hippocampus and then migrated into the hippocampus to MECHANISMS REGULATING regenerate nearly half of the normal number of neurons in FUNCTIONAL NEUROGENESIS the CA1 region. In addition, significant functional recov- FROM ENDOGENOUS ADULT ery was observed (36). Assuming that no significant NEURAL STEM CELLS amounts of BrdU could be incorporated into nondividing cells (37), these studies suggested that endogenous NSCs During the last decade, we have witnessed tremendous could be induced to differentiate into neurons in situ out- progress regarding embryonic neural development that side neurogenic regions in the adult CNS. will clearly have a significant impact on our understand- Because demonstration of BrdU labeling requires fix- ing of the repair mechanisms in the adult CNS (40). The ation and immunostaining (37), the functionality of the significant difference between the embryonic and adult neuronal progeny of adult NSCs cannot be examined. CNS environment suggests that there will be substantial Due to these technical limitations and the fact that many differences in the mechanisms controlling the neural de- neurons die soon after birth (38), it was not clear whether velopment in two different stages. Adult neurogenesis in neurons generated from adult NSCs are actually func- SGZ and SVZ can therefore serve as model systems for tional and can be used by the CNS to replace diseased or investigation of the underlying mechanisms regulating damaged cells. The functional integration of electrically the sequential steps of the neural development in the adult active new neurons in the adult CNS environment was CNS environment. Elucidating the molecular mecha- only demonstrated recently (25,26). Using a strategy nisms regulating neurogenesis from endogenous adult based on the finding that the integration of certain retro- NSCs will also provide the framework for developing viruses and sustained expression of transgenes requires neuronal replacement therapy for CNS diseases with en- (39), van Praag and colleagues were able to dogenous NSCs and/or transplantation of exogenous stem express green fluorescent protein (GFP) specifically in cells or their progeny. Here we will summarize our cur- proliferating cells and their progeny by stereotaxic in- rent understanding of functional neurogenesis from adult jection of engineered retroviruses into the dentate gyrus NSCs in the SGZ and SVZ. of the adult mice (25). This approach allows the GFP 1 neuronal progeny of dividing adult NSCs to be shown Proliferation of adult NSCs for their morphology and characterized for their physio- logical properties in acute brain slices with electrophys- Proliferation of adult NSCs in the SVZ and SGZ is iological approaches. These studies revealed that new- regulated by a variety of stimuli, including aging, stress, born granule neurons send out long axonal projections stroke, seizure, and physical activity (7– 9,38). Neuro- and elaborate complex dendrite trees. More importantly, transmitters (e.g., serotonin, NMDA, nitric oxide) and some of these newborn GFP 1 neurons can fire repetitive steroid hormones are also known to regulate the prolif- action potentials and receive functional synaptic inputs. eration of adult NSCs (7–9,38). In most cases, the iden- Further comparative analysis showed that these newborn tity of the proliferation signals received by adult NSCs neurons exhibit functional properties very similar to those remains unknown. Recent findings, however, had sug- of mature granule neurons in the dentate gyrus (25). gested that local vasculature (41) and astrocytes (42,43) Using a similar retroviral strategy (26), Carleton and might serve potential cellular sources for the signals. In colleagues recently followed the developmental process culture, astrocytes from hippocampus or SVZ promote

673 GOH ET AL. proliferation of adult NSCs (42,43). In the adult hip- (7–9). Proliferating cells that generate only glia under pocampus, hot spots of cell proliferation were found to normal conditions in broad regions of the adult CNS can be associated with vascular structures (41) and astrocytes generate neurons, either after isolation and culture in vitro (43). Furthermore, factors promoting endothelial cell pro- (7–9), or after specific types of injuries in vivo (35,36). liferation also increase neurogenesis in the adult song- Clonally derived adult NSCs from nonneurogenic regions birds (44), suggesting an important relationship between when transplanted back to the regions of their origin only these two processes. give rise to glia (51,52). They do, however, differentiate While the mitogenic factors from vascular and astro- into neurons when transplanted into the neurogenic SGZ cytic sources remain to be identified, several molecules (51,52). Recent in vitro studies have provided some in- have been shown to be effective in inducing proliferation sights into the cellular elements that may constitute a neu- of adult NSCs in vivo (7–9,45– 48). Infusion of EGF, FGF- rogenic niche in the neurogenic regions of the adult CNS. 2, or transforming growth factor- a (TGF-a) into the brain Cultured astrocytes derived from neurogenic hippocam- was found to promote proliferation of adult NSCs both un- pus actively regulate neurogenesis by promoting prolif- der normal and injury conditions (7,36,45). EGF and FGF- eration and neuronal fate specification of adult NSCs 2 have also been used, almost exclusively, as mitogens for derived from the adult hippocampus (43). Similarly, cul- NSCs derived from the adult CNS, either in adhesive or tured astrocytes from SVZ also promote the production in cultures (7–9). The mitogenic effects of of neurons from SVZ stem cells (42). In contrast, astro- FGF-2 for cultured NSCs at clonal densities appear to re- cytes derived from adult spinal cord, a non-neurogenic quire a co-factor, recently identified as the glycosylated region, do not promote neurogenesis (43). These studies form of cystatin C (46). Combined delivery of FGF-2 and raise the intriguing possibility that the ability to generate cystatin C to the dentate gyrus in adult mice stimulates neurons from adult NSCs may be in part due to the re- proliferation of NSCs in the SGZ (46). Very recently, gionally specified astrocytes. During early development, Sonic hedgehog (Shh) signaling was shown to regulate most of the astrocytes are generated after the majority of proliferation of adult NSCs (47,48). Loss of Shh signaling CNS neurons are born (9), thus are unlikely to instruct resulted in abnormalities in both the dentate gyrus and ol- neuronal differentiation of fetal NSCs. These studies sup- factory bulb (48). Pharmacological inhibition or stimula- port the notion that there will be substantial differences tion of Shh signaling in the adult brain also leads to de- between mechanisms regulating neurogenesis in the de- creased or elevated NSC proliferation in the hippocampus veloping embryonic CNS and those in the adult CNS. and SVZ, respectively (47,48). In vitro, Shh is sufficient The molecular mechanisms underlying fate specifica- to maintain the proliferation of NSCs derived from adult tion of adult NSCs are largely unknown (9). Members of rat hippocampus (47), while mice SVZ progenitors that the bone morphogenic protein (BMP) were shown to be lack Smoothened, a key downstream effector of Shh, able to instruct adult NSCs to adopt a glial fate (53). In formed significantly fewer neurospheres (48). Other fac- the neurogenic SVZ, the BMP inhibitor noggin, released tors that are not traditionally regarded as mitogens have from the ependymal cells in the lateral wall, can block also been implicated in regulating proliferation of adult the gliogenic effects of BMP (53). The instructive fac- NSCs (49). For example, Eph/ ephrin signaling has been tors for neuronal differentiation of adult NSCs, including shown to be involved in axon guidance, cell those released from hippocampal or SVZ astrocytes, re- migration, establishment of segmental boundaries, and for- main to be identified. mation of angiogenic capillary plexi (50). A 3-day infu- sion of the ectodomain of either EphB2 or ephrin-B2 into Navigation of neuronal progeny of adult NSCs the lateral ventricle not only disrupted migration of neu- roblasts, but also increased proliferation of NSCs in the One of the striking features of adult neurogenesis is SVZ of adult mice (49). the capability of newborn neurons to have long-range mi- The proliferation signals for adult NSCs activate an ar- gration and axonal outgrowth in a CNS environment oth- ray of interconnected cytoplasmic signal transduction erwise inhibitory for mature neurons and axons. For neu- pathways that eventually lead to the activation of gene rogenesis in the olfactory bulb, neuroblasts generated transcription in the nucleus. While many of these path- from SVZ stem cells first migrate tangentially through ways have been intensively investigated in other cell tubular structures formed by special astrocytes, then ra- types, specific cytoplasmic pathways involved prolifera- dially in the olfactory bulb to their final position (6). This tion of adult NSCs remain to be identified. is quite different from what occurs during embryonic de- velopment where newborn neurons are generally guided Fate specification of adult NSCs by radial glia cells (9). Several developmental guidance cues and adhesion molecules have been implicated in di- Several lines of evidence support the crucial roles of recting neuronal migration of these new neurons (7), in- local environment in fate specification of adult NSCs cluding members of the ephrin-B family, Slit, and poly-

674 REPAIR OF ADULT CNS WITH ADULT NEURAL STEM CELLS sialated glycoprotein neural cell adhesion molecule come synaptically connected soon after migration has (PSA-NCAM). For neurogenesis in the dentate gyrus, been completed (26). However, spiking activity does not limited neuronal migration occurs (7). Instead, newborn occur until the neurons are almost fully mature. This is neurons extend long axonal projections to the CA3 re- also different to developing embryonic neurons, which gion and their dendritic arborizations to the outer mo- can fire action potentials and release neurotransmitters lecular layer in opposite direction (25). It is possible that even before they are connected (55). One hypothesis for newly generated neurons do not express functional re- this unique neuronal maturation process is that the de- ceptors for factors that inhibit axon regeneration in the layed maturation of excitability can prevent the newborn adult CNS, and/or have different internal states (e.g., high cells from disrupting the function of circuitry already in levels of neuronal cAMP) that permit them to grow in an place in the adult. environment otherwise inhibitory for regenerating mature The mechanisms regulating maturation and synapse neurons. The active mechanisms responsible for such formation by adult NSCs are largely unknown. In vitro stereotypic guidance behaviors of newborn neurons are studies with adult hippocampal NSCs suggest that local unknown. Interestingly, many axon/ dendrite guidance hippocampal astrocytes play essential roles in the matu- cues involved in the guidance of developing granule neu- ration and synapse formation process (24). In the absence rons during fetal development, such as semaphorins, of astrocytes, the neuronal progeny of cultured adult maintain their expression in adulthood, thus may con- NSCs remain immature, both morphologically and func- tribute to the guidance of these newborn neurons (54). tionally. They display simple morphology, limited mem- brane excitability, inability to fire action potentials, and Maturation and integration by neuronal little functional synaptogenesis (24). In contrast, adult progeny of adult NSCs NSC derived neurons in the presence of astrocytes ac- quired physiological properties comparable to those of While the functional roles of adult neurogenesis re- mature CNS neurons (24). Similar active roles of astro- main elusive (7,38), recent studies provided convincing cytes in regulating synapse formation have been previ- evidence that newly generated neurons are able to inte- ously observed for neonatal neurons (56). grate into the existing neuronal circuits in the adult CNS (25,26). Functional studies of the maturation and inte- gration process during adult neurogenesis have also re- ADULT STEM CELL BASED vealed some unique features of neuronal development in THERAPY FOR CNS REPAIR the adult CNS that are different from that being observed during embryonic and neonatal stages (25,26). The discovery of the existence of active functional neu- In the case of neurogenesis in the dentate gyrus, about rogenesis in the adult CNS and rapid advances in stem half of new newborn cells die within 2 weeks after birth cell technology have fueled our hope to cure currently (38). Four weeks after retroviral labeling, some of the re- intractable CNS diseases by replacing damaged or lost maining neurons become electrically active and start to neurons. Many types of stem cells with neurogenic po- receive synaptic inputs, as shown by electrophysiologi- tential have been identified, including pluripotent ES cal recordings (35). Whether these newborn granule neu- and EG cells and multipotent fetal NSCs (4–9,17– 22). rons also make functional synaptic connections with their Besides ethical concerns and political restrictions, im- target neurons, thus actively involved in the information munocapability is another unique advantage of using flow, remains to be demonstrated. The complexity of adult stem cells over other cell sources. The realization their dendrites and density of the dendritic spine, the ma- that endogenous NSCs may be broadly present in the jor sites for excitatory synapses, continue to increase for adult CNS and can be induced to generate new func- at least several months (25). Thus, the course of neuronal tionally integrated neurons in situ has raised exciting pos- maturation for newborn granule neurons in the adult CNS sibilities for self-repair of the damaged CNS by activa- appears to be much more protracted than those generated tion of endogenous NSCs, even without transplantation. during embryonic stages. Many strategies in using stem cells for neuronal re- In the case of neurogenesis in the olfactory bulb, elec- placement therapy in different CNS diseases have been trophysiological recordings showed that tangentially proposed (27–31,57). Here we will focus our discussion migrating neurons express extrasynaptic GABA A and on several key steps and challenges ahead for neuronal AMPA receptors, while NMDA receptors appear later in replacement therapy using adult stem cells. radially migrating neurons (26). The sequental expres- sion of receptors for neurotransmitters is different from Source of adult stem cells what occurs during embryonic neuronal development, where expression of NMDA receptors normally precedes Unlike hematopoietic stem cells, which can be directly AMPA receptors. These newborn olfactory neurons be- isolated with cell surface markers, the precise identifica-

675 GOH ET AL. tion of NSCs occurs only retrospectively, and we are still responses to differentiation stimuli is unknown. It also in search of effective methods for prospective isolation remains to be investigated whether different mitogens of NSCs (7–9). Nonetheless, many types of adult will change the epigenetic properties of proliferating stem cells with neural potentials have been derived adult NSCs, resulting in a different capacity in generat- (4–9,17– 22,58– 60). Some are derived from neural tis- ing neuronal subtypes. Because extensive amplification sues, such as multipotent NSCs from the adult CNS, may be required to generate enough cells for transplan- multipotent neural crest stem cells from gut and oligo- tation, stem cells need to be routinely examined for po- dendrocyte precursors or astrocytes that can be repro- tential abnormality, such as their karyotypes and growth grammed to generate neurons (58– 60). Surprisingly, factor dependency. some appear to be derived from nonneural tissues (58–60), such as blood, , or skin. In most Differentiation of stem cells into defined lineages cases, the neuronal identity is determined merely based on the expression of certain markers (e.g., Tuj1, NeuN), Numerous attempts to transplant multipotent NSCs di- rather than their functional properties (58–60). Interpre- rectly into the nonneurogenic regions of the adult CNS tation of some of the transplantation experiments are fur- failed to generate significant numbers of new neurons (7); ther complicated by the potential fusion events that have however, transplantation of neuronal lineage-restricted occurred between the transplanted cells and the host cells progenitors did generate neurons (64), suggesting that the (58–60). neuronal fate specification is the limiting step. Therefore, Because functionality is the foundation for the success understanding the neurogenic environment, by compara- of neuronal replacement therapy, it is essential to charac- tive studies with nonneurogenic conditions, will be es- terize the physiological properties of neurons derived from sential for developing strategies to activate endogenous different types of adult stem cells. For adult multipotent NSCs. It may be also beneficial to transplant neuroblasts NSCs derived from human white matter (22) and multi- or immature neurons instead of NSCs directly into the potent adult progenitor from bone marrow of mice (61), adult CNS. The molecular mechanisms regulating neu- electrophysiological studies have shown that the neuronal ronal fate specification and neuronal subtype differenti- progeny of these stem cells are electrically active, capable ation will be an area of intensive investigation in the im- of firing action potentials. For adult NSCs derived from mediate future. Understanding how these developmental hippocampus, extensive functional analysis showed that processes occur during embryonic stages will clearly fa- these cells retain the ability to give rise to electrically ac- cilitate our efforts. Protocols have already been devel- tive and functional neurons with all essential characteris- oped to differentiate ES cells effectively into different tic properties of mature CNS neurons, even after extensive neuronal subtypes (65), including dopaminergic neurons, propagation and amplification in cultures (24). Whether GABAergic neurons, and motor neurons. It is expected adult NSCs derived from nonneurogenic regions exhibit that similar strategies will also be developed to coax the similar abilities remains to be investigated. adult stem cells (61). The functionality of neuronal prog- There is undeniable evidence that stem cells derived eny for stem cells, including membrane excitability and from very early could be used to make numer- release of neurotransmitter, is an essential piece of the ous types of neural cell, including the principal projec- task, and should be rigorously examined. tion neurons, most of which are born during embryoge- nesis (58). It remains to be seen whether any kind of adult Survival and functional integration by stem cells has the potential to general neuronal subtypes newly generated neurons as diverse as ES cells. Studies with cultured adult NSCs derived from hippocampus did show some plasticity in A significant percentage of neurons, either derived generating neuronal subtypes (7,58,62). When trans- from endogenous NSCs (38) or from transplantation (66), planted to the RMS of adult rats, adult hippocampal NSCs died soon after in the adult CNS. Thus, strategies to sup- can migrate to olfactory bulb and give rise to neurons ex- port the survival of endogenous and/or transplanted cells pressing tyrosine hydroxylase, a phenotype never ob- are of apparent importance (66). Interestingly, enriched served in hippocampus (62). environment and learning appear to increase the survival of newborn neurons in the rodent hippocampus (38). Amplification of adult stem cells Adult NSCs and their progeny can be genetically modi- fied either in situ or in vitro to become the cellular sources In addition to EGF and FGF-2, some other factors, such for growth factors and neurotrophins that may promote as Shh (47) and neuregulin (63), also appear to be suffi- or support the survival of themselves and the surround- cient in maintaining the self-renewal and multipotential- ing neurons. ity of adult NSCs from rodents. Whether adult NSCs To achieve functional integration by the newborn neu- propagated in different mitogens will exhibit differential rons from either endogenous NSCs or transplantation, we

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