An Evolutionarily Conserved Region in the Second lntron of the Human Nestin Gene Directs Gene Exmession to CNS Progenitor Cells and to Early Neural Ciest Cells

Central nervous system (CNS) progenitor cells transiently proliferate in the embryonic neural tube and give rise to neurons and glial cells. A characteristic feature of the CNS progenitor cells is expression of the intermediate filament nestin and it was previously shown that the rat nestin second intron functions as an enhancer, directing gene expression to CNS progenitor cells. In this report we characterize the nestin enhancer in further detail. Cloning and sequence analysis of the rat and human nestin second introns revealed local domains of high sequence similarity in the 3' portion of the introns. Transgenic mice were generated with the most conserved 714 bp in the 3' portion of the intron, or with the complete, 1852 bp, human second intron, coupled to the reporter gene lacZ. The two constructs gave a very similar nestin-like expression pattern, indicating that the important control elements reside in the 714 bp element. Expression was observed starting in embryonic day (E)7.5 neural plate, and at E10.5 CNS progenitor cells throughout the neural tube expressed lacZ. At E12.5, lacZ expression was more restricted and confined to proliferating regions in the neural tube. An interesting difference, compared to the rat nestin second intron, was that the human intron at E10.5 mediated lacZ expression also in early migrating neural crest cells, which is a site of endogenous nestin expression. In conclusion, these data show that a relatively short, evolutionarily conserved region is sufficient to control gene expression in CNS progenitor cells, but that the same region differs between rodents and primates in its capacity to control expression in neural crest cells.     

Isolation and characterization of neural stem/progenitor cells from post-stroke cerebral cortex in mice

The CNS has the potential to marshal strong reparative mechanisms, including activation of endogenous neurogenesis, after a brain injury such as stroke. However, the response of neural stem/progenitor cells to stroke is poorly understood. Recently, neural stem/progenitor cells have been identified in the cerebral cortex, as well as previously recognized regions such as the subventricular or subgranular zones of the hippocampus, suggesting that a contribution of cortex-derived neural stem/progenitor cells may repair ischemic lesions of the cerebral cortex. In the present study, using a highly reproducible murine model of cortical infarction, we have found nestin-positive cells in the post-stroke cerebral cortex, but not in the non-ischemic cortex. Cells obtained from the ischemic core of the post-stroke cerebral cortex formed neurosphere-like cell clusters expressing nestin; such cells had the capacity for self-renewal and differentiated into electrophysiologically functional neurons, astrocytes and myelin-producing oligodendrocytes. Nestin-positive cells from the stroke-affected cortex migrated into the peri-infarct area and differentiated into glial cells in vivo . Although we could not detect differentiation of nestin-positive cells into neurons in vivo , our current observations indicate that endogenous neural stem/progenitors with the potential to become neurons can develop within post-stroke cerebral cortex.     

Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage

Proliferating populations of undifferentiated neural stem cells were isolated from the embryonic day 14 rat cerebral cortex or the adult rat subventricular zone. These cells were pluripotent through multiple passages, retaining the ability to differentiate in vitro into neurons, astrocytes, and oligodendrocytes. Two weeks to 2 months after engraftment of undifferentiated, BrdU-labeled stem cells into the normal adult spinal cord, large numbers of surviving cells were seen. The majority of the cells differentiated with astrocytic phenotype, although some oligodendrocytes and undifferentiated, nestin-positive cells were detected; NeuN-positive neurons were not seen. Labeled cells were also engrafted into the contused adult rat spinal cord (moderate NYU Impactor injury), either into the lesion cavity or into the white or gray matter both rostral and caudal to the injury epicenter. Up to 2 months postgrafting, the majority of cells either differentiated into GFAP-positive astrocytes or remained nestin positive. No BrdU-positive neurons or oligodendrocytes were observed. These results show robust survival of engrafted stem cells, but a differentiated phenotype restricted to glial lineages. We suggest that in vitro induction prior to transplantation will be necessary for these cells to differentiate into neurons or large numbers of oligodendrocytes.     

Localization of nestin in amygdaloid kindled rat: an immunoelectron microscopic study

BACKGROUND: Nestin is a class VI intermediate filament protein, expressed during early embryonic development in mammals. Postnatally, nestin and its mRNA are down-regulated and gradually disappear. Recently, nestin expression has been detected in the adult nervous system, and it has been suggested that this protein may be related to neurogenesis, although, its role in the mechanism of neurogenesis is not known. METHODS: The present study examined the localization of nestin in CNS tissue of the amygdaloid kindled rat by light and electron microscopy. RESULTS: Kindled animals showed nestin expression mainly in the piriform cortex and the perirhinal cortex. By light microscopy, nestin was shown to be expressed in astrocytes, neurons, and endothelial cells. Electron microscopy demonstrated nestin expression in endothelial cells, astrocytic perivascular end feet, the rare pericyte, neurons and oligodendrocytes. CONCLUSION: We conclude that epilepsy causes widespread nestin expression in many cell types in the CNS, including non-neural cells.     

Adult Nestin-expressing Subependymal Cells Differentiate to Astrocytes in Response to Brain Injury

The adult brain contains a small population of central nervous system (CNS) cells in the subependyma which, like embryonic CNS progenitor cells, express the intermediate filament nestin. In this report, the differentiation capacity in vivo of these cells was analysed following a standardized trauma. Before the trauma, the subependymal cells expressed nestin but not the astrocytic and neuronal differentiation markers glial fibrillary acidic protein (GFAP) and neurofilament respectively. In response to injury, the majority of the subependymal cells coexpressed nestin and GFAP, but never nestin and neurofilament. Furthermore, cells coexpressing nestin and GFAP were found progressively further away from the subependyma and closer to the lesion at later time points after the injury, indicating that these cells migrate towards the lesion. Nestin was in addition re-expressed in reactive astrocytes near the lesion and in non-reactive astrocytes very far from the lesion throughout the ipsilateral cortex. In conclusion, our data indicate that the nestin-positive subependymal cells are an in vivo source for the generation of new astrocytes but not neurons after injury, and that nestin re-expression in astrocytes following traumatic stimuli can be used as a sensitive marker for astroglial activation.     

Down-regulation of GAP-43 During Oligodendrocyte Development and Lack of Expression by Astrocytes In Vivo: Implications for Macroglial Differentiation

The discovery of molecular markers which are selectively expressed during the development of specific classes of rat central nervous system macroglia has greatly advanced our understanding of how these cells are related. In particular, it has been shown in tissue culture that oligodendrocytes and some astrocytes (type-2) may be derived from a common progenitor cell (O-2A progenitor). However, the existence of type-2 astrocytes in vivo has yet to be unequivocally established. Recently, it has been reported that the neural-specific growth-associated protein-43 (GAP-43, otherwise known as B-50, F1, pp46 and neuromodulin) may be expressed by cells of the O-2A lineage in vitro. We set out to examine the cellular specificity of GAP-43 in O-2A progenitors and their descendants in vitro and in vivo. Using a polyclonal antiserum against a GAP-43 fusion protein we have shown the presence of immunoreactive GAP-43 in the membranes of bipotential O-2A glial progenitor cells and type-2 astrocytes by Western blotting and immunocytochemistry of cells in culture. In contrast to previous studies, double labelling with mature oligodendrocyte markers showed that GAP-43 is down-regulated during oligodendrocyte differentiation in vitro. Immunohistochemical staining of sections of developing rat brain demonstrated the same developmental regulation of GAP-43, suggesting that oligodendrocytes only express GAP-43 at immature stages. In addition, normal and reactive astrocytes in tissue sections were not labelled with GAP-43.     

Differential expression of Musashi1 and nestin in the adult rat hippocampus after ischemia

Both nestin and the neural RNA-binding protein Musashi1 (Msi1) are expressed in neural stem cells in the subventricular zone. Neurogenesis in the hippocampus has received much attention, so we evaluated the expression of Msi1 and nestin in the adult rat hippocampus after transient forebrain ischemia. Both Msi1 and nestin were induced in the reactive astrocytes after ischemia, especially in the CA1 region, until 35 days after ischemia. Induction of both molecules suggested that reactive astrocytes might have immature characteristics. In the subgranular zone (SGZ) of the hippocampal dentate gyrus, Msi1-positive cells formed clusters after ischemia. These cells were labeled by bromodeoxyuridine (BrdU) but did not express glial fibrillary acidic protein. In contrast, very few nestin-positive cells were labeled by BrdU. Our results suggest that neuronal progenitor cells in the SGZ expressed Msi1 but not nestin.     

Expression of receptor tyrosine kinase RYK in developing rat central nervous system

Receptor tyrosine kinase RYK is a mammalian homologue of Drosophila Lio, which is involved in learning and memory and in axon guidance. We cloned a rat ryk gene and characterized its expression pattern in the central nervous system. Northern blot analysis of the whole brain revealed that the RYK mRNA was abundant during the period from 13 to 18 embryonic days (E13-18) and it decreased by E20. In the postnatal brain, the RYK signal was higher in postnatal one week (P1W) cerebrum and in P2W cerebellum than in later stages. In situ hybridization revealed that RYK was expressed throughout the central nervous system, mainly in the ventricular zone on E11 and E13. On E18 and E20, the remarkable level of RYK mRNA was detected in the ventricular zone as well as in the cortical plate of the forebrain. These two regions overlapped the immunoreactive areas of nestin and MAP2, a neural stem cell marker and a mature neural marker, respectively. Moreover, the double-labeling analysis showed that the same cells expressed both RYK and nestin in the ventricular zone. In the postnatal brain, RYK was predominantly expressed in neurons of various regions. These observations suggest that RYK plays a contributory role as a multifunctional molecule in the differentiation and maturation of neuronal cells in the central nervous system.     

Neurons and astrocytes influence the development of purified O-2A progenitor cells.

To investigate the possible role of neurons and astrocytes for oligodendrocyte development we prepared a pure population of precursor cells positive for the precursor marker GD3 with the help of fluorescence-activated cell sorting (FACS). Large numbers of highly purified cells were obtained from postnatal day 1 rat brainstems and cultured in different media and sera, and in conditioned media. As described in the literature for optic nerve O-2A progenitors, GD3-sorted brainstem cells cultured in medium containing 10% fetal calf serum (FCS) acquired a star-shaped morphology and differentiated into GD3- and GFAP-positive type-2 astrocytes. On the other hand, in serum-free medium, most of the cells differentiated into oligodendrocytes (O1-/galactocerebroside-positive). Sensory neuron conditioned media promoted survival and proliferation of the precursor cells. The spontaneous differentiation of progenitor cells into oligodendrocytes was retarded by the mitogen. Antibodies against platelet-derived growth factor (PDGF) completely blocked the mitotic effect and allowed spontaneous oligodendrocyte differentiation to occur. Cultured astrocytes also secreted PDGF as a mitogen. However, postnatal astrocytes also released a potent signal promoting oligodendrocyte differentiation. The type of factor(s) released depended on the age of the astrocytes, since only conditioned medium of postnatal but not of embryonic astrocytes promoted oligodendrocyte differentiation, suggesting that astrocyte maturation directly influences oligodendrocyte differentiation. Different concentrations of PDGF could not reproduce this differentiation-inducing effect. This study suggests that interactions between O-2A progenitor cells, neurons, and astrocytes could be required to regulate and complete the oligodendrocyte developmental pathway. Astrocytes, themselves possibly under neuronal influences, might regulate first the proliferation of the precursor cells, and, later in development, the differentiation into mature oligodendrocytes or type-2 astrocytes.     

Nestin mRNA expression correlates with the central nervous system progenitor cell state in many,but not all,regions of developing central nervous system

Nestin is a recently discovered intermediate filament (IF) gene. Nestin expression has been extensively used as a marker for central nervous system (CNS) progenitor cells in different contexts, based on observations indicating a correlation between nestin expression and this cell type in vivo. To evaluate this correlation in more detail nestin mRNA expression in developing and adult mouse CNS was analysed by in situ hybridization. We find that nestin is expressed from embryonic day (E) 7.75 and that expression is detected in many proliferating CNS regions; at E10.5 nestin is expressed in cells of both the rostral and caudal neural tube, including the radial glial cells; at E15.5 and postnatal day (P) 0 expression is observed largely in the developing cerebellum and in the ventricular and subventricular areas of the developing telencephalon. Furthermore, the transition from a proliferating to a post-mitotic cell state is accompanied by a rapid decrease in nestin mRNA for motor neurons in the ventral spinal cord and for neurons in the marginal layer of developing telencephalon. In contrast to these data we observe two proliferating areas, the olfactory epithelium and the precursor cells of the hippocampal granule neurons, which do not express nestin at detectable levels. Thus, nestin mRNA expression correlates with many, but not all, regions of proliferating CNS progenitor cells. In addition to its temporal and spatial regulation nestin expression also appears to be regulated at the level of subcellular mRNA localization: in columnar neuroepithelial and radial glial cells nestin mRNA is predominantly localized to the pial endfeet.     

AN2/NG2 protein-expressing glial progenitor cells in the murine CNS: isolation, differentiation, and association with radial glia

During early neural development, the lineage specification of initially pluripotent progenitor cells is associated with proliferation, differentiation, and migration. Oligodendroglial progenitor cells migrate from their sites of origin to reach the axons that they will myelinate. We have described a cell-surface protein, AN2, expressed by oligodendroglial progenitor cells in vitro and showed that antibodies against AN2 inhibited the migration of cultured primary oligodendroglial progenitor cells, suggesting that the AN2 antigen plays a role in their migration. Recently, results from MALDI mass spectroscopy showed that AN2 is the mouse homologue of the rat NG2 protein. In this study, we have analyzed cells staining with AN2 antibodies during development and in the adult murine central nervous system (CNS), carried out double stainings with antibodies against NG2, and investigated the differentiation potential of cells in vitro after isolation from early postnatal brain using AN2 antibodies. AN2 and NG2 antibodies stained totally overlapping populations of cells in the CNS. AN2/NG2 expressing cells in embryonic and postnatal brain expressed the PDGF-alpha-receptor and in postnatal brain exhibited electrophysiological properties typical of glial progenitor cells. Cells isolated from early postnatal brain using AN2 monoclonal antibody developed into oligodendrocytes in low serum medium or into astrocytes in the presence of fetal calf serum. In the embryonic spinal cord, cells staining with AN2 antibodies were found closely apposed to radial glial cells, suggesting that glial precursors, like neurons, may use radial glia as scaffolds for migration.     

Characterization and distribution of a new cell surface marker of neuronal precursors

In a screen for novel cell surface markers of neuronal progenitors, we recently identified mAb 2F7 that recognizes an epitope present on both progenitor cells and postmitotic neurons, in the developing CNS and PNS. In the embryonic rat telencephalon, the mAb 2F7 epitope is expressed by migratory and postmigratory neurons in the developing cerebral cortex, as well as by presumptive neuronal progenitor cells of the ventricular zone. In the neonatal forebrain mAb 2F7 labels postmitotic neurons, including those of the developing cerebral cortex and olfactory bulb, as well as the axons of the corpus callosum. While mAb 2F7 immunoreactivity is present on only a low density of the neuronal progenitor cells situated in the anterior part of the subventricular zone, a progressively higher proportion of cells forming the rostral migratory stream express this epitope. mAb 2F7 labels the surfaces of neurons and neuronal precursors, but not mature oligodendrocytes and astrocytes in primary cultures derived from the rat neural tube. In vivo, migrating neural crest cells, motor neurons, and axonal projections associated with the spinal cord express the mAb 2F7 epitope. Immunoblot analyses reveal that the mAb 2F7 epitope resides on several high-molecular-weight, membrane-associated proteins, and is likely to be composed of N-linked carbohydrate. These findings suggest that mAb 2F7 recognizes a novel epitope that is present on progenitor cells and postmitotic, differentiating neurons in the developing mammalian nervous system.     

Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons,astrocytes, and oligodendrocytes in vitro

In the mammalian central nervous system (CNS), multipotential neural stem cells in the neuroepithelium generate the three major types of neural cells, namely, neurons, astrocytes, and oligodendrocytes. To explore the molecular mechanisms underlying proliferation and differentiation of these neural stem cells, we established a cell line named MNS-57 from the embryonic day 12 rat neuroepithelium by introducing the mycer fusion gene, in which c-myc can be conditionally activated by adding oestrogen to the culture medium. MNS-57 cells expressed nestin, vimentin, and the RC1 antigen, which are potential markers for neural stem cells. We show that under particular culture conditions, MNS-57 cells can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they are likely to originate from multipotential neural stem cells. Incubating MNS-57 cells with either oestrogen, which activates mycer, or growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated their growth, and the combination of oestrogen and bFGF (or EGF) had a synergistically stronger mitogenic effect than the single factors. Furthermore, both c-myc activation and bFGF appeared to be necessary for the differentiation of MNS-57 cells, and only when stimulated by both signals simultaneously, the cells committed to generating multiple neural cell types. Thus, the property of the cell line is unique in that its differentiation into neurons and glia can be conditionally manipulated invitro in an exogenous signal-dependent manner. We propose that the cell line described here will provide an useful in vitro model to understand genetic and environmental mechanisms that control the generation of neural cell diversity in the CNS. © 1995 Wiley-Liss, Inc.     

Olfactory bulb core is a rich source of neural progenitor and stem cells in adult rodent and human

The olfactory bulb (OB) core is an extension of the rostral migratory stream and thus is a potential source of neural progenitor and neural stem cells. We characterized in vivo and in vitro neuronal progenitor and neural stem cells in the adult OB core. In mouse and rat, bromodeoxyuridine (BrdU) labeling showed that the OB core accumulates newly replicated cells. Nestin, a neuroepithelial stem cell marker, was enriched in the OB core. BrdU-positive cells were immunolabeled for nestin and TUC4, a marker for early postmitotic neurons. The distributions of cells labeled for BrdU, TUC4, and nestin were similarly concentrated in the OB core. Nestin- and TUC4-positive cells were also found in the OB of young and aged humans. Isolated and cultured OB core cells from adult rat and mouse had the capacity to generate numerous neurospheres. Adult OB core neurospheres were cryopreserved and subsequently cultured. Single cell clonal analysis of neurospheres revealed the capacity for self-renewal and multipotency. Cultured adult OB core cells differentiated into neurons, astrocytes, and oligodendrocytes. Some neurons expressed choline acetlytransferase, substance P, and glutamic acid decarboxylase. Basic fibroblast growth factor potentiated the self-renewal of cells and beta-nerve growth factor stimulated differentiation. OB-derived neural stem cells in coculture with skeletal muscle cells were induced to become neurons expressing choline acetyltransferase and substance P and formed neuromuscular synaptic junctions on myocytes displaying acetylcholinesterase-positive motor end plates. Cocultured OB-derived neural stem cells with myoblast cells also generated nonneural cell progeny. We conclude that the adult mammalian OB core is a reservoir of neural progenitor cells and pluripotent neural stem cells.     

Transferrin gene expression and secretion by rat brain cells in vitro

We have previously shown by immunocytochemistry in rat primary glial cultures that transferrin (Tf) is an early developmental marker for oligodendrocytes. The present work addresses the issue of Tf gene expression and synthesis by neural cells in vitro. For this purpose, we used rat embryonic neuronal cultures and newborn glial cultures of astrocytes and oligodendrocytes. Cultured fibroblasts and C6 glioma cells were used as negative controls. We found that Tf mRNA is present in oligodendrocytes, astrocytes, and neurons. However, oligodendrocytes and astrocytes, but not neurons, were shown to synthesize and secrete Tf. Neither fibroblasts nor C6 glioma cells expressed detectable amounts of Tf mRNA. Tf mRNA levels in astrocyte cultures appeared to be under hormonal control since hydrocortisone markedly reduced message levels. These results show that both astrocytes and oligodendrocytes can synthesize and secrete Tf under cell culture conditions. However, epigenetic factors, such as hydrocortisone, may repress the expression of Tf in astrocytes in vivo.     

神经干细胞生存因子在大鼠脊髓损伤前后的表达变化

目的观察大鼠脊髓损伤后干细胞来源的神经干细胞生存因子（SDNSF）mRNA在大鼠正常和损伤脊髓的农达变化,以及SDNSF的表达与Ⅵ类中间丝蛋白的表达之间的关系。方法按改良的Allen重物打击法制备大鼠脊髓损伤模型,采用RT—PCR、原位杂交方法,观察SDNSF mRNA在大鼠脊髓中的表达位置及在损伤脊髓中的表达变化。应用免疫组化的方法,显示脊髓中nestin的表达。结果RT-PCR检测SDNSF mRNA在正常大鼠脊髓中的表达,损伤后4天SDNSF的mRNA表达上升,损伤8天剑达高峰,此后SDNSF的mRNA表达逐渐减少,到16天恢复到正常水平;脊髓切片原位杂交结果发现SDNSF的mRNA阳性细胞主要分布十脊髓灰质细胞中,可能足神经元细胞,结果表明正常脊髓可表达SDNSF;脊髓损伤后8犬,原位杂交硅示SDNSF阳性细胞明显增多。同时与此切片相邻层面的切片免疫组化证实nestin阳性细胞增殖、变大、向周围发出突起,但这些阳性细胞在分布上与SDNSF无关。结论（1）SDNSF在脊髓中表达于灰质,脊髓损伤后SDNSF的mRNA表达随时间发生变化。（2）随着脊髓损伤的修复,nestin阳性细胞增殖,但是这些细胞并不表达SDNSF。     

Alterations in cellular phenotypes differentiating from embryonic rat brain neurosphere cultures by immunoselection of neuronal progenitors

The neurosphere culture system is widely used to expand neural stem/progenitor cells in vitro and to provide a source of cells for transplantation approaches to CNS disorders. This study describes the populations of neurones, astrocytes and oligodendrocytes which differentiated from embryonic day (E) 14 rat cortical and striatal tissue grown as neurosphere cultures over three passages. The percentages of cells that adopted neuronal phenotypes decreased with passage, astrocytic percentages increased and oligodendrocytic percentages remained constant. In the second part of this study, immunomagnetic separation was used to positively select neuronal progenitor cells from E14 rat cortical and striatal tissue using an antibody, 2F7, which recognises an epitope on the cell surface of pre- and post-mitotic neurones. These immunomagnetically selected cells were grown as neurosphere cultures over three passages and gave rise to significantly different percentages of neurones, astrocytes and oligodendrocytes than those found in the baseline study. In particular, the percentage of neurones arising from the second and third passages was significantly higher following immunoselection. This indicates that neuronal progenitor cells can be isolated using immunomagnetic separation and then expanded using the neurosphere culture system, to generate enriched populations of neurones that can be used in CNS repair.     

O-2A glial progenitors from mature brain respond to CNS neuronal cell line-derived growth factors

During development, myelin-forming oligodendrocytes and type 2 astrocytes are believed to arise from bipotential (O-2A) glial progenitors. Previously we found that conditioned medium (CM) from the B104 rat CNS neuronal cell line promotes growth of neonatal rat O-2A progenitors in serum-free culture conditions with subsequent increases in differentiated progeny. We now report that O-2A progenitors are present in mature rat brains and that this CM promotes the growth, motility, and bipolar morphology of these cells from 30- and 65-day-old rat brains, as shown by quantitative studies using double immunostaining and [3H]thymidine-autoradiography. In addition, the growth-promoting action of B104 CM is not neutralized by antibodies to platelet-derived growth factor, a proposed progenitor mitogen. Subsequent to the proliferation of these O-2A progenitors, increases in oligodendrocytes and type 2 astrocytes occur. These data suggest a novel therapeutic strategy for some demyelinating diseases, e.g., multiple sclerosis, where there is a deficit in oligodendrocytes. Although it has been proposed by others that mature brain O-2A progenitors are less proliferative and thereby incapable of adequately replenishing lost oligodendrocytes in these diseases, we present in vitro evidence for continued response of mature brain O-2A progenitors to this neuronal cell line-derived mitogen.     

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.