Humoral and contact interactions in astroglia/stem cell co-cultures in the course of glia-induced neurogenesis

Astroglial cells support or restrict the migration and differentiation of neural stem cells depending on the developmental stage of the progenitors and the physiological state of the astrocytes. In the present study, we show that astroglial cells instruct noncommitted, immortalized neuroectodermal stem cells to adopt a neuronal fate, while they fail to induce neuronal differentiation of embryonic stem cells under similar culture conditions. Astrocytes induce neuron formation by neuroectodermal progenitors both through direct cell-to-cell contacts and via short-range acting humoral factors. Neuron formation takes place inside compact stem cell assemblies formed 30- 60 h after the onset of glial induction. Statistical analyses of time-lapse microscopic recordings show that direct contacts with astrocytes hinder the migration of neuroectodermal progenitors, while astroglia-derived humoral factors increase their motility. In non-contact co-cultures with astrocytes, altered adhesiveness prevents the separation of frequently colliding neural stem cells. By contrast, in contact co-cultures with astrocytes, the restricted migration on glial surfaces keeps the cell progenies together, resulting in the formation of clonally proliferating stem cell aggregates. The data indicate that in vitro maintained parenchymal astrocytes (1) secrete factors, which initiate neuronal differentiation of neuroectodermal stem cells; and (2) provide a cellular microenvironment where stem cell/stem cell interactions can develop and the sorting out of the future neurons can proceed. In contrast to noncommitted progenitors, postmitotic neuronal precursors leave the stem cell clusters, indicating that astroglial cells selectively support the migration of maturing neurons as well as the elongation of neurites.     

Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks

Human astrocytes differ dramatically in cell morphology and gene expression from murine astrocytes. The latter are well known to be of major importance in the formation of neuronal networks by promoting synapse maturation. However, whether human astrocyte lineage cells have a similar role in network formation has not been firmly established. Here, we investigated the impact of human astrocyte lineage cells on the functional maturation of neural networks that were derived from human induced pluripotent stem cells (hiPSCs). Initial in vitro differentiation of hiPSC-derived neural progenitor cells and immature neurons (glia+ cultures) resulted in spontaneously active neural networks as indicated by synchronous neuronal Ca²⁺ transients. Depleting proliferating neural progenitors from these cultures by short-term antimitotic treatment resulted in strongly astrocyte lineage cell-depleted neuronal networks (glia− cultures). Strikingly, in contrast to glia+ cultures, glia− cultures did not exhibit spontaneous network activity. Detailed analysis of the morphological and electrophysiological properties of neurons by patch clamp recordings revealed reduced dendritic arborization in glia− cultures. In addition, a reduced action potential frequency upon current injection in pyramidal-like neurons was observed, whereas the electrical excitability of multipolar neurons was unaltered. Furthermore, we found a reduced dendritic density of PSD95-positive excitatory synapses, and more immature properties of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) miniature excitatory postsynaptic currents (mEPSCs) in glia− cultures, suggesting that the maturation of glutamatergic synapses depends on the presence of hiPSC-derived astrocyte lineage cells. Intriguingly, addition of the astrocyte-derived synapse maturation inducer cholesterol increased the dendritic density of PSD95-positive excitatory synapses in glia− cultures.     

Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation

Stem cells that can give rise to neurons, astroglia, and oligodendroglia have been found in the developing and adult central nervous system (CNS) of rodents. Yet, their existence within the human brain has not been documented, and the isolation and characterization of multipotent embryonic human neural stem cells have proven difficult to accomplish. We show that the developing human CNS embodies multipotent precursors that differ from their murine counterpart in that they require simultaneous, synergistic stimulation by both epidermal and fibroblast growth factor-2 to exhibit critical stem cell characteristics. Clonal analysis demonstrates that human C NS stem cells are multipotent and differentiate spontaneously into neurons, astrocytes, and oligodendrocytes when growth factors are removed. Subcloning and population analysis show their extensive self-renewal capacity and functional stability, their ability to maintain a steady growth profile, their multipotency, and a constant potential for neuronal differentiation for more than 2 years. The neurons generated by human stem cells over this period of time are electrophysiologically active. These cells are also cryopreservable. Finally, we demonstrate that the neuronal and glial progeny of long-term cultured human CNS stem cells can effectively survive transplantation into the lesioned striatum of adult rats. Tumor formation is not observed, even in immunodeficient hosts. Hence, as a consequence of their inherent biology, human CNS stem cells can establish stable, transplantable cell lines by epigenetic stimulation. These lines represent a renewable source of neurons and glia and may significantly facilitate research on human neurogenesis and the development of clinical neural transplantation.     

Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro

Neural stem cells reside in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells (NSPCs) isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Recent studies have shown that endogenous or grafted NSPCs are activated after an injury and migrate toward lesioned areas. In these areas, reactive astrocytes are present and secrete numerous molecules and growth factors; however, it is not currently known whether reactive astrocytes can influence the lineage selection of NSPCs. We investigated whether reactive astrocytes could affect the differentiation, proliferation, and survival of adult NSPCs by modelling astrogliosis in vitro, using mechanical lesion of primary astrocytes. Initially, it was found that conditioned medium from lesioned astrocytes induced astrocytic differentiation of NSPCs without affecting neuronal or oligodendrocytic differentiation. In addition, NSPCs in coculture with lesioned astrocytes also displayed increased astrocytic differentiation and some of these NSPC-derived astrocytes participated in glial scar formation in vitro. When proliferation and survival of NSPCs were analyzed, no differential effects were observed between lesioned and nonlesioned astrocytes. To investigate the molecular mechanisms of the astrocyte-inducing activity, the expression of two potent inducers of astroglial differentiation, ciliary neurotrophic factor and leukemia inhibitory factor, was analyzed by Western blot and shown to be up-regulated in conditioned medium from lesioned astrocytes. These results demonstrate that lesioned astrocytes can induce astroglial differentiation of NSPCs and provide a mechanism for astroglial differentiation of these cells following brain injury.     

Stem cell therapy for central nerve system injuries： glial cells hold the key

Mammalian adult central nerve system （CNS） injuries are devastating because of the intrinsic difficulties for effective neuronal regeneration. The greatest problem to be overcome for CNS recovery is the poor regeneration of neurons and myelin-forming cells, oligodendrocytes. Endogenous neural progenitors and transplanted exogenous neuronal stem cells can be the source for neuronal regeneration. However, because of the harsh local microenvironment, they usually have very low efficacy for functional neural regeneration which cannot compensate for the loss of neurons and oligodendrocytes. Glial cells （including astrocytes, microglia, oligodendrocytes and NG2 glia） are the majority of cells in CNS that provide support and protection for neurons. Inside the local microenvironment, glial cells largely influence local and transplanted neural stem cells survival and fates. This review critically analyzes current finding of the roles of glial cells in CNS regeneration, and highlights strategies for regulating glial cells＇ behavior to create a permissive microenvironment for neuronal stem cells.     

Endogenous and exogenous ciliary neurotrophic factor enhances forebrain neurogenesis in adult mice

Neurogenesis in the adult mammalian CNS occurs in the subventricular zone (SVZ) and dentate gyrus. The receptor for ciliary neurotrophic factor (CNTF), CNTFRalpha, is expressed in the adult subventricular zone. Because the in vitro effects of CNTF on neural precursors have been varied, including proliferation and differentiation into neurons or glia, we investigated its role in vivo. Injection of CNTF in the adult C57BL/6 mice forebrain increased the number of cells labeled with ip BrdU in both neurogenic regions. In the dentate gyrus, CNTF also appeared to enhance differentiation of precursors into neurons, i.e., increased the proportion of NeuN+/BrdU+ cells from approximately 14 to approximately 29%, but did not affect differentiation into astrocytes (GFAP+) or oligodendrocytes (CNPase+). In the SVZ, CNTF increased the proportion of GFAP+/BrdU+ cells from approximately 1 to approximately 2%. CNTF enhanced the distance of migration of new neurons into the granule cell layer. Intraventricular injection of neutralizing anti-CNTF antibodies reduced the number of BrdU-labeled cells in the SVZ. These results suggest that endogenous CNTF regulates adult neurogenesis by increasing proliferation of neural stem cells and/or precursors. Alternatively, CNTF could maintain cells longer in the S-phase, resulting in increased BrdU labeling. In the neurogenic region of the SVZ, CNTFRalpha was exclusively present in GFAP-positive process-bearing cells, suggesting that CNTF affects neurogenesis indirectly via neighboring astroglia. Alternatively, these cells may be part of the neural precursor lineage. The restricted expression of CNTF within the nervous system makes it a potential selective drug target for cell replacement strategies.     

Radial glial cells as neuronal precursors: a new perspective on the correlation of morphology and lineage restriction in the developing cerebral cortex of mice

Radial glia is a ubiquitous cell type in the developing central nervous system (CNS) of vertebrates, characterized by radial processes extending through the wall of the neural tube which serve as guiding cables for migrating neurons. Radial glial cells were considered as glial precursor cells due to their astroglial traits and later transformation into astrocytes in the mammalian CNS. Accordingly, a hypothetical morphologically distinct type of precursor was attributed the role of neurogenesis. Recent evidence obtained in vitro and in vivo, however, revealed that a large subset of radial glia generates neurons. We further demonstrate here that the progeny of radial glial cells does not differ from the progeny of precursors labeled from the ventricular surface, implying that there is no obvious relation between precursor morphology and neuron-glia lineage decisions in the developing cerebral cortex of mice. Moreover, we show that many radial glial cells seem to maintain their process during cell division and discuss the implications of this observation for the orientation of cell division. These new data are then related to radial glial cells in other non-mammalian vertebrates persisting into adulthood and suggest that radial glia are not only neurogenic during development, but also in adulthood.     

神经干细胞及其在脑修复中的可能应用

成年哺乳动物脑内存在有能分化成神经元或神经胶质的神经干细胞,这种神经前体细胞一般位于脑室壁部位。从胎脑分离下来的神经前体细胞能在体外分裂并进一步分化成神经元和胶质细胞,许多包括生长因子在内的活性物质或分子结构参与这一分化过程并决定这些细胞的转归;从成年脑中也能分离出有繁殖能力的细胞,当这些存在于中枢神经系统内的干细胞（即多潜能细胞）和具有明确前景的前体细胞（神经母细胞和胶质母细胞）遇到与胚胎时期相同因子的影响时也会分裂、分化。由于在体外培养状况下难以提供神经前体细胞分化繁殖的所有条件,故将其植入发育中或成年中枢神经系统特定部位的做法是研究神经干细胞特性,特别是研究决定其分化微环境的有效办法。对神经干细胞的研究,为进一步探讨神经元的发生、迁移、分化和诊治多种神经疾患提供了新的机遇。     

Lineage analysis of quiescent regenerative stem cells in the adult brain by genetic labelling reveals spatially restricted neurogenic niches in the olfactory bulb

The subventricular zone (SVZ) of the lateral ventricles is the major neurogenic region in the adult mammalian brain, harbouring neural stem cells within defined niches. The identity of these stem cells and the factors regulating their fate are poorly understood. We have genetically mapped a population of Nestin-expressing cells during postnatal development to study their potential and fate in vivo . Taking advantage of the recombination characteristics of a nestin::CreER ^T2 allele, we followed a subpopulation of neural stem cells and traced their fate in a largely unrecombined neurogenic niche. Perinatal nestin::CreER ^T2 -expressing cells give rise to multiple glial cell types and neurons, as well as to stem cells of the adult SVZ. In the adult SVZ nestin::CreER ^T2 -expressing neural stem cells give rise to several neuronal subtypes in the olfactory bulb (OB). We addressed whether the same population of neural stem cells play a role in SVZ regeneration. Following anti-mitotic treatment to eliminate rapidly dividing progenitors, relatively quiescent nestin::CreER ^T2 -targeted cells are spared and contribute to SVZ regeneration, generating new proliferating precursors and neuroblasts. Finally, we have identified neurogenic progenitors clustered in ependymal-like niches within the rostral migratory stream (RMS) of the OB. These OB-RMS progenitors generate neuroblasts that, upon transplantation, graft, migrate and differentiate into granule and glomerular neurons. In summary, using conditional lineage tracing we have identified neonatal cells that are the source of neurogenic and regenerative neural stem cells in the adult SVZ and occupy a novel neurogenic niche in the OB.     

Retinoic acid induction of ES-cell-derived neurons: the radial glia connection

Neuronal induction by retinoic acid (RA) is commonly used in embryonic stem (ES) cell differentiation. Two recent papers show that this paradigm induces a population of neurogenic precursors with properties of radial glia. Upon differentiation, RA-treated cells give rise to a defined and developmentally restricted neuronal lineage. This role of RA in cell fate specification provides new perspectives for studying the radial glia-neuron transition and for generating homogenous populations of neurons from ES cells.     

Differential regulation of gliogenesis in the context of adult hippocampal neurogenesis in mice

In adult hippocampal neurogenesis, new neurons appear to originate from a cell with astrocytic properties expressing glial fibrillary acidic protein (GFAP). Also, new astrocytes are generated in the adult dentate gyrus. Whereas the putative astrocyte-like progenitor cells are consistently S-100beta-negative, many new astrocytes are S-100beta-positive. Thus, it is unclear whether the GFAP-positive progenitor cells are astrocytes in a general sense or rather neural progenitor cells with certain astrocytic characteristics. We therefore investigated the development of GFAP-expressing cells in the context of adult hippocampal neurogenesis. Proliferating cells could be either GFAP-positive or doublecortin-positive (DCX), but never both, indicating two independent populations of dividing cells in the glial and neuronal lineages. Two distinct populations of cells with astroglial properties were detected-one expressing GFAP, the other co-expressing GFAP and S-100beta. We never found S-100beta-cells to be in S-phase. No overlap between neuronal and glial markers was seen at any time point. Thus, astrogenesis occurred in parallel and to some degree independent of adult neurogenesis. The uninterrupted GFAP expression in this lineage, and neuronal markers in the other lineage, argue against a late common precursor for neurogenesis and gliogenesis in the adult hippocampus. Very few newly generated microglia and no new oligodendrocytes were detected. Environmental enrichment and voluntary wheel running-two experimental paradigms with robust stimulatory effects on adult hippocampal neurogenesis-affected hippocampal astrogenesis differentially: Running, but not enrichment, strongly induced net astrogenesis (GFAP/S-100beta), but also GFAP-positive S-100beta-negative cells, which thus appear to be a transiently amplifiable intermediate population within the glial lineage.     

Mature astrocytes transform into transitional radial glia within adult mouse neocortex that supports directed migration of transplanted immature neurons

Neuronal migration is an essential step in normal mammalian neocortical development, and the expression of defined cellular and molecular signals within the developing cortical microenvironment is likely crucial to this process. Therapy via transplanted or manipulated endogenous precursors for diseases which involve neuronal loss may depend critically on whether newly incorporated cells can actively migrate to repopulate areas of neuronal loss within the adult brain. Previous studies demonstrated that embryonic neurons and multipotent precursors transplanted into the neocortex of adult mice undergoing targeted apoptosis of pyramidal neurons migrate long distances into neuron-deficient regions, undergo directed differentiation, accept afferent synaptic input, and make appropriate long-distance projections. The experiments presented here: (1) use time-lapse digital confocal imaging of neuronal migration in living slice cultures to assess cellular mechanisms utilized by immature neurons during such long distance migration, and (2) identify changes within the host cortical astroglial population that may contribute to this migration. Prelabeled embryonic day 17 mouse neocortical neurons were transplanted into adult mouse primary somatosensory cortex undergoing targeted apoptotic degeneration of callosal projection neurons. Four to 7 days following transplantation, living slice cultures containing the region of transplanted cells were prepared and observed. Sequential time-lapse images were recorded using a video-based digital confocal microscope. Transplanted cells displayed bipolar morphologies characteristic of migrating neuroblasts and moved in a saltatory manner with mean rates of up to 14 microm/h. To investigate whether a permissive glial phenotype may provide a potential substrate for this directed form of neuronal migration, slice cultures were immunostained with the RC2 monoclonal antibody, which identifies radial glia that act as a substrate for neuronal migration during corticogenesis. RC2 does not label mature stellate astrocytes, which express glial fibrillary acidic protein (GFAP). RC2 expression was observed in glial cells closely apposed to migrating donor neurons within the slice cultures. The timing and specificity of RC2 expression was examined immunocytochemically at various times following transplantation. RC2 immunostaining within regions of neuronal degeneration was transient, with peak staining between 3 and 7 days following transplantation. Strongly RC2-immunoreactive cells that did not express GFAP were found within these regions, but not in distant cortical regions or within control brains. RC2-positive cells were identified in recipient transgenic mice which express beta-galactosidase under a glial specific promoter. Coexpression of RC2 and beta-galactosidase identified these cells as host astroglia. These results demonstrate that adult cortical astrocytes retain the capacity to reexpress an earlier developmental phenotype that may partially underlie the observed active migration of transplanted neurons and neural precursors. Further understanding of these processes could allow directed migration of transplanted or endogenous precursors toward therapeutic cellular repopulation and complex circuit reconstruction in neocortex and other CNS regions.     

Nestin-containing cells express glial fibrillary acidic protein in the proliferative regions of central nervous system of postnatal developing and adult mice

We are interested in the expression patterns of nestin, an embryonic intermediate filament that represent a neural precursor marker, in the mammalian central nervous system. With an immunohistochemical approach, distribution of nestin-containing cells and their colocalization with glial fibrillary acidic protein (GFAP) or neuronal nuclear specific protein (NeuN) were studied in adult and postnatal days 2-30 (P2-30) mice. Nestin-immunoreactivity was predominately distributed in certain proliferative regions, such as cerebral cortex, hippocampus, hypothalamus, subfornical organ, cerebellar cortex, area postrema, midline raphe glial structures, as well as ependymal and subependymal zones of the brain and spinal cord. The majority of nestin-immunoreactive cells, characterized by astroglial profiles of multiple and radial processes, showed a partial overlapping distribution with that of GFAP-immunoreactive astroglial cells. Double immunofluorescence confirmed that about 77% of these nestin-immunoreactive cells exhibited GFAP-immunoreactivity, indicating that a large percentage of nestin-expressing cells may have committed to astroglial cells. In developing mice, down-regulation of nestin expression was observed between P7 and P14. Although co-expression of nestin and NeuN occurred in cortical neurons of P2-7 mice, nestin-containing cells showing NeuN-immunoreactivity disappeared in CNS in older animals. Our results reveal the distribution pattern of nestin-containing neural precursors in the postnatal CNS and provide evidence on their differentiation fate to neurons and astrocytes, suggesting that nestin-containing glial cells may play an important role in remodeling and repairing in the postnatal and adult central nervous system.     

Platelet-derived growth factor receptor-alpha in ventricular zone cells and in developing neurons.

Cells in the early neuroepithelium differentiate and give rise to all cells in the central nervous system (CNS). The ways from a multipotent CNS stem cell to specialized neurons and glia are not fully understood. Using immunohistochemistry we found that neuroepithelial cells express the platelet-derived growth factor receptor-alpha (PDGFR-alpha) in the neural plate at embryonic day 8.5 and onwards in the neural tube. The protein was polarized to ventricular endfeet. Furthermore, PDGFR-alpha expression was localized to cells undergoing early neuronal development. We also found PDGFR-alpha expression in developing granule cells in the postnatal cerebellum, in Purkinje cells in the adult cerebellum and on processes of developing dorsal root ganglion cells. Previous reports mainly describe PDGFR-alpha expression in oligodendrocyte precursors and glial cells. We believe, in line with a few previous reports, that the PDGFR-alpha in addition marks a pool of undifferentiated cells, which are able to differentiate into neurons.     

A population of human brain parenchymal cells express markers of glial, neuronal and early neural cells and differentiate into cells of neuronal and glial lineages

Glial fibrillary acidic protein (GFAP)-positive cells derived from the neurogenic areas of the brain can be stem/progenitor cells and give rise to new neurons in vitro and in vivo. We report here that a population of GFAP-positive cells derived from fetal human brain parenchyma coexpress markers of early neural and neuronal cells, and have neural progenitor cell characteristics. We used a monolayer culture system to expend and differentiate these cells. During the initial proliferative phase, all cells expressed GFAP, nestin and low levels of betaIII-tubulin. When these cells were cultured in serum and then basic fibroblast growth factor, they generated two distinct progenies: (i) betaIII-tubulin- and nestin-positive cells and (ii) GFAP- and nestin-positive cells. These cells, when subsequently cultured in serum-free media without growth factors, ceased to proliferate and differentiated into two major neural cell classes, neurons and glia. In the cells of neuronal lineage, nestin expression was down-regulated and betaIII-tubulin expression became robust. Cells of glial lineage differentiated by down-regulating nestin expression and up-regulating GFAP expression. These data suggest that populations of parenchymal brain cells, initially expressing both glial and neuronal markers, are capable of differentiating into single neuronal and glial lineages through asymmetric regulation of gene expression in these cells, rather than acquiring markers through differentiation.     

Role of astroglial cell clones in the survival and differentiation of cerebellar embryonic neurons

To investigate the role of astrocytes in the survival and differentiation of cerebellar neurons during development, we have used astroglial cell clones, derived from 8-day postnatal cerebellar explants and which might be the in vitro equivalents of the 3 main types of cerebellar astrocytes, the Golgi epithelial cells and their Bergmann processes, the velate protoplasmic and the fibrous astrocytes (F. Alliot and B. Pessac, Brain Res., 306 (1984) 283-291). Nearly all single cells, dissociated from 15-day embryonic mouse cerebella and seeded at low density, adhered to layers of each of the cerebellar astroglial cell clones as well as to other glial lines or artificial substrates. However, the cerebellar embryonic neurons survived well only on monolayers of either the 'Golgi-Bergmann'-like or the 'velate protoplasmic'-like clones. On these layers, 60-80% of the neurons were still present after 5 days of co-culture, while only less than 5% survived on the other types of substrates. The differentiation pattern of the neurons surviving on the 'Golgi-Bergmann' and the 'velate protoplasmic' astroglial clones was studied with markers of postmitotic granule cells, the major neuronal population in adult cerebellum. The velate protoplasmic-like clone was the only one able to support the coordinate acquisition by most surviving neurons of the phenotypic characteristics of granule cells, i.e. a distinct morphology, a specific epitope binding the monoclonal antibody 7-8 D2 and immunoreactivity to glutamate. These data show a broad heterogeneity in the capacity of astroglial cell clones to support embryonic cerebellar neurons. In addition, they indicate that neuronal survival per se is not sufficient for the acquisition of a differentiated neuronal phenotype.     

Stage-specific and opposing roles of BDNF, NT-3 and bFGF in differentiation of purified callosal projection neurons toward cellular repair of complex circuitry

Cellular repair of neuronal circuitry affected by neurodegenerative disease or injury may be approached in the adult neocortex via transplantation of neural precursors ("neural stem cells") or via molecular manipulation and recruitment of new neurons from endogenous precursors in situ. A major challenge for potential future approaches to neuronal replacement will be to specifically direct and control progressive differentiation, axonal projection and connectivity of neural precursors along a specific neuronal lineage. This goal will require a progressively more detailed understanding of the molecular controls over morphologic differentiation of specific neuronal lineages, including neurite outgrowth and elongation, in order to accurately permit and direct proper neuronal integration and connectivity. Here, we investigate controls over the morphologic differentiation of a specific prototypical lineage of cortical neurons: callosal projection neurons (CPN). We highly enriched CPN to an essentially pure population, and cultured them at three distinct stages of development from embryonic and postnatal mouse cortex by retrograde fluorescence labelling, followed by fluorescence-activated cell sorting. We find that specific peptide growth factors exert direct stage-specific positive and negative effects over the morphologic differentiation and process outgrowth of CPN. These effects are distinct from the effects of these growth factors on CPN survival [Catapano et al. (2001)J. Neurosci., 21, 8863-8872]. These data may be critical for the future goal of directing lineage-specific neuronal differentiation of transplanted or endogenous precursors/"stem cells" toward cellular repair of complex cortical circuitry.     

The Adult Rat Hippocampus Contains Primordial Neural Stem Cells

Adult-derived hippocampal progenitors generate neurons, astrocytes, and oligodendrocytesin vitroand following grafting into the adult brain. Although these progenitors have a considerable capacity forin vitroself renewal, it is not known if each lineage is generated by separate committed precursors or by multipotent stem cells. By genetic marking, we have followed individual cells through the process of proliferative expansion, commitment, and differentiation. All three lineages are generated by single marked cells and the relative proportions of each lineage can be strongly influenced by environmental cues. Differentiation is accompanied by a characteristic progression of lineage-specific markers and can be potentiated by retinoic acid, elevated cyclic AMP, or neurotrophic factors. The ability to genetically mark and clone normal diploid hippocampal progenitors provides the first definitive evidence that multipotent neural stem cells exist outside of the adult striatal subventricular zone and supports the hypothesis that FGF-2-responsive neural stem cells may be broadly distributed in the adult brain.     

Neuron-to-astrocyte transition: phenotypic fluidity and the formation of hybrid asterons in differentiating neurospheres

To the extent that their fate choice and differentiation processes can be understood and manipulated, neural stem cells represent a promising therapeutic tool for a variety of neuropathologies. We have previously shown that mature astrocytes possess neural stem cell attributes, and can give rise to neurons through the formation of multipotent neurosphere clones. Here we show that relatively mature neurons generated from neurospheres derived from postnatal subependymal zone or cerebellar cortex undergo a phenotypic transformation into astrocytes that coincides with the appearance of a nonfused, hybrid cell type that shares the morphology, antigenicity, and physiology of both neurons and astrocytes. We refer to this astrocyte/neuron hybrid as an "asteron," and hypothesize that it represents an intermediate step in the trans- or dedifferentiation of neurons into astrocytes. The present finding suggests that seemingly terminally differentiated neural cells may in fact represent points along a bidirectionally fluid continuum of differentiation, with intermediate points represented by "hybrid" cells coexpressing phenotypic markers of more than one lineage.     

Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse

We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1-GFP-expressing population. Thus, the Sox1-GFP reporter system is highly useful for identification, isolation and characterization of neural stem and progenitor cells, as well as for the validation of alternative means for isolating neural stem and progenitor cells. Further, transplantation experiments show that Sox1-GFP cells isolated from the foetal brain give rise to neurons and glia in vivo, and that many of the neurons display phenotypic characteristics appropriate for the developing brain region from which the Sox1-GFP precursors were derived. On the other hand, Sox1-GFP cells isolated from the adult subventricular zone or expanded neurosphere cultures gave rise almost exclusively to glial cells following transplantation. Thus, not all Sox1-GFP cells possess the same capacity for neuronal differentiation in vivo.