Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: differential expression of LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes

Recent findings show that the predominant multipotent neural stem cells (NSCs) isolated from postnatal and adult mouse brain express glial fibrillary acid protein (GFAP), a protein commonly associated with astrocytes, and that primary astrocyte cultures can contain GFAP-expressing cells that act as multipotent NSCs when transferred to neurogenic conditions. The relationship of GFAP-expressing NSCs to GFAP-expressing astrocytes is unclear, but has important implications. We compared the phenotype and neurogenic potential of GFAP-expressing cells derived from different CNS regions and maintained in vitro under different conditions. Multiple labeling immunohistochemistry revealed that both primary astrocyte cultures and adherent neurogenic cultures derived from postnatal or adult periventricular tissue contained subpopulations of GFAP-expressing cells that co-expressed nestin and LeX/CD15, two molecules associated with NSCs. In contrast, GFAP-expressing cells in similar cultures prepared from adult cerebral cortex did not express detectable levels of LeX/CD15, and exhibited no neurogenic potential. Fluorescence-activated cell sorting (FACS) of both primary astrocyte cultures and adherent neurogenic cultures for LeX/CD15 showed that GFAP-expressing cells competent to act as multipotent NSCs were concentrated in the LeX-positive fraction. Using neurosphere assays and a transgenic ablation strategy, we confirmed that the predominant NSCs in primary astrocyte and adherent neurogenic cultures were GFAP-expressing cells. These findings demonstrate that GFAP-expressing cells derived from postnatal and adult forebrain are heterogeneous in both molecular phenotype and neurogenic potential in vitro, and that this heterogeneity exists before exposure to neurogenic conditions. The findings provide evidence that GFAP-expressing NSCs are phenotypically and functionally distinct from non-neurogenic astrocytes.     

In vitro neuronal and glial production and differentiation of human central neurocytoma cells

Human central neurocytoma cells were cultured and characterized immunophenotypically and electrophysiologically to clarify their developmental potential. We conducted systematic in vitro studies utilizing fresh tissues from three patients. Initially small homogenous cell clusters settled down onto the bottom of the culture flasks, and, after 2 weeks from plating, mature neuron-like cells developed from these clusters and expressed neurofilament proteins (NF: specific neuronal markers). On the other hand, approximately 80% of small round cell clusters and flat glial-like cells from which these clusters developed were positively stained for glial fibrillary acidic protein (GFAP: a specific glial marker). Furthermore these neuronal and glial cells showed distinct morphology, and dual-label, indirect immunohistochemistry for GFAP and NF-200 kD disclosed that the two antigens were not found co-localized in the same cells. In single-cell clonal analysis, neuronal, glial, and mixed neuronal and glial clones were generated. Electrophysiologically, the cells of neuronal morphology possessed sodium channels, and also L-type calcium channels in whole-cell voltage clamp. The sodium channels were of a characteristic neuronal phenotype which appears in neurons. These findings suggest that small round human central neurocytoma cells exhibit both neuronal and glial differentiations and have the properties reminiscent of precursor cells derived from subventricular matrix; thus, these cultured cells may be a potential source for investigations of human CNS neuronal and glial development and differentiation. J. Neurosci. Res. 51:526–535, 1998. © 1998 Wiley-Liss, Inc.     

GFAP-positive progenitor cells produce neurons and oligodendrocytes throughout the CNS

Once thought to merely act as scaffolds in neuronal migration, recent evidence suggests that radial glia may serve as progenitors for the majority of neurons in the CNS. Cre/loxP fate-mapping experiments were carried out using a fragment of a glial-specific promoter (glial fibrillary acidic protein; GFAP) to drive expression of Cre recombinase. We show that GFAP+ progenitor cells give rise to neurons and oligodendrocytes throughout the CNS. We find very little regional heterogeneity in the neurogenic potential of radial glia between dorsal and ventral telencephalon. Additionally, radial glia serve as precursors for subpopulations of interneurons in the ventral telencephalon. Interestingly, the human GFAP promoter but not the mouse GFAP promoter is active in oligodendrocyte progenitor cells. We also demonstrate that the most commonly used Cre reporter lines are very inefficient in detecting Cre-dependent recombination in astrocytes and describe a new Cre reporter line for assessing recombination in astrocytes.     

Activated Notch1 maintains the phenotype of radial glial cells and promotes their adhesion to laminin by upregulating nidogen

Radial glia are neural stem cells that exist only transiently during central nervous system (CNS) development, where they serve as scaffolds for neuronal migration. Their instability makes them difficult to study, and therefore we have isolated stabilized radial glial clones from E14.5 cortical progenitors (e.g., L2.3) after expression of v-myc. Activated Notch1 intracellular region (actNotch1) promotes radial glia in the embryonic mouse forebrain (Gaiano et al., (2000), and when it was introduced into E14.5 cortical progenitors or radial glial clone L2.3, the cells exhibited enhanced radial morphology and increased expression of the radial glial marker BLBP. A representative clone of L2.3 cells expressing actNotch1 called NL2.3-4 migrated more extensively than L2.3 cells in culture and in white matter of the adult rat spinal cord. Microarray and RT-PCR comparisons of mRNAs expressed in these closely related clones showed extensive similarities, but differed significantly for certain mRNAs including several cell adhesion molecules. Cell adhesion assays demonstrated significantly enhanced adhesion to laminin of NL2.3-4 by comparison to L2.3 cells. The laminin binding protein nidogen was the most highly induced adhesion molecule in NL2.3-4, and immunological analyses indicated that radial glia synthesize and secrete nidogen. Adhesion of NL2.3-4 cells to laminin was inhibited by anti-nidogen antibodies and required the nidogen binding region in laminin, indicating that nidogen promotes cell adhesion to laminin. The combined results indicate that persistent expression of activated Notch1 maintains the phenotype of radial glial cells, inhibits their differentiation, and promotes their adhesion and migration on a laminin/nidogen complex.     

Experimental transmission of human subacute spongiform encephalopathy to small rodents

Summary The distribution of glial fibrillary acidic protein (GFAP) in the central nervous system (CNS) lesions of tuberous sclerosis (TS) was examined using antiserum against GFAP and the peroxidase antiperoxidase method of Sternberger. In cortical tubers there were islands of gemistocytic astrocytes staining intensely for GFAP and occasional giant cells having some cytoplasmic staining. The majority of the cortical giant cells had no GFAP. The islands were separated by areas devoid of astrocytes with perikaryal staining. A faintly staining fibrous network was found between these islands. The majority of cells in the subependymal nodules stained. The retinal phakoma stained but not as intensely as the subependymal nodules. There was no staining whatsoever in the giant cell subependymal tumors. Absence of GFAP staining in the subependymal giant cell tumors makes their classification as astrocytomas less certain.     

Shared antigenic determinants between human hemopoietic cells and nervous tissues and tumors

Summary A panel of nine monoclonal antibodies raised against human hemopoietic cells was used for immunohistological labeling of frozen sections of human nervous tissues and tumors. Three antibodies showed a remarkably consistent labeling pattern when tested on 18 samples of normal or reactive tissue, on 34 neurogenic and 17 non-neurogenic tumors in an indirect immunofluorescence technique.VIM C6, an antibody recognizing cells of the granulocyte series, showed surface labeling of normal and reactive glial cells and of all types of glioma regardless of the grade of malignancy. VIT 13, an antibody recognizing activated T-cells labeled the processes of normal, reactive, and neoplastic glia in a manner very similar to but not identical with glial fibrillary acidic protein (GFAP). VIB C5, an antibody recognizing B cells and granulocytes, showed surface labeling restricted to malignant cells (malignant gliomas and primitive neuroectodermal tumors) and fetal brain, thus recognizing, within the nervous system, an oncofetal antigen. Due to this operational specificity within the nervous system, some of the antibodies described here might have a role as diagnostic markers for CNS tumors.This study confirms and expands previous data that sharing of antigenic determinants by hemopoietic cells and nervous tissue or neurogenic tumors is common. However, the significance of such cross-recognition is still obscure. It is tempting to speculate that cross-reacting auto-antibodies might contribute to tissue damage in some immune-mediated neurologic discases (myasthenia gravis, multiple sclerosis, CNS involvement in systemic lupus erythematosus) or to impairment of immunoregulation in multiple sclerosis or glioma patients. Furthermore, sharing of surface determinants might be responsible for the dual tissue tropism of some viruses, including the lymphotrophic virus (HTLV) in the encephalopathy of the acquired immune deficiency syndrome (AIDS).     

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.     

Lack of neurogenesis in the adult rat cerebellum after Purkinje cell degeneration and growth factor infusion

Although constitutive neurogenesis exclusively occurs in restricted regions of the adult mammalian brain, resident progenitors can be isolated from many different CNS sites, and neuronal neogeneration can be stimulated in vivo by injury or infusion of growth factors. To ask whether latent compensatory mechanisms, which may be exploited to promote repair processes, are present throughout the CNS, we examined the neurogenic potentialities of the adult rat cerebellum in normal conditions, following injury, and after infusion of growth factors. Degeneration of Purkinje cells was induced by intracerebroventricular administration of the toxin saporin, conjugated to anti-p75 antibodies. In addition, epidermal growth factor and basic fibroblast growth factor, or FGF8, were infused for 2 weeks to either intact or injured animals. In all conditions, proliferating cells were identified from bromodeoxyuridine (BrdU) incorporation. In the unmanipulated cerebellum there were rare dividing cells, mainly represented by NG2-positive presumptive oligodendrocyte precursors. Mitotic activity was strongly enhanced in cortical areas with Purkinje cell degeneration, being mostly sustained by microglia, plus minor fractions of NG2-expressing cells, astrocytes and oligodendrocytes. In contrast, growth factor infusion had a weak effect on both intact and injured cerebella. In all experimental conditions, we never found any BrdU-positive cells coexpressing distinctive markers for immature or differentiated cerebellar neurons. Therefore, although some progenitor cells reside in the adult cerebellum, the local environment, either intact or injured, does not provide efficient cues to direct their differentiation towards neuronal phenotypes. In addition, neurogenic potentialities cannot be induced or boosted by the application of growth factors which are effective in other CNS regions.     

Human neural stem cells genetically modified for brain repair in neurological disorders

Existence of multipotent neural stem cells (NSC) has been known in developing or adult mammalian CNS, including humans. NSC have the capacity to grow indefinitely and have multipotent potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes. Stable clonal lines of human NSC have recently been generated from the human fetal telencephalon using a retroviral vector encoding v‐myc. One of the NSC lines, HB1.F3, carries normal human karyotype of 46XX and has the ability to self‐renew, differentiate into cells of neuronal and glial lineages, and integrate into the damaged CNS loci upon transplantation into the brain of animal models of Parkinson disease, HD, stroke and mucopolysaccharidosis. F3 human NSC were genetically engineered to produce L‐dihydroxyphenylalanine (L‐DOPA) by double transfection with cDNA for tyrosine hydroxylase and guanosine triphosphate cylohydrolase‐1, and transplantation of these cells in the brain of Parkinson disease model rats led to L‐DOPA production and functional recovery. Proactively transplanted F3 human NSC in rat striatum, supported the survival of host striatal neurons against neuronal injury caused by 3‐nitropro‐pionic acid in rat model of HD. Intravenously introduced through the tail vein, F3 human NSC were found to migrate into ischemic lesion sites, differentiate into neurons and glial cells, and improve functional deficits in rat stroke models. These results indicate that human NSC should be an ideal vehicle for cell replacement and gene transfer therapy for patients with neurological diseases. In addition to immortalized human NSC, immortalized human bone marrow mesenchymal stem cell lines have been generated from human embryonic bone marrow tissues with retroviral vectors encording v‐myc or teromerase gene. These immortalized cell lines of human bone marrow mesenchymal stem cells differentiated into neurons/glial cells, bone, cartilage and adipose tissue when they were grown in selective inducing media. There is further need for investigation into the neurogenic potential of the human bone marrow stem cell lines and their utility in animal models of neurological diseases.     

Protein kinase C-ε is a developmentally regulated,neuronal isoform in the chick embryo central nervous system

Protein kinase C (PKC) is expressed as many isoforms and in high quantities in the central nervous system (CNS), which suggests an important role for this enzyme in neuronal development and function. We used specific antibodies to investigate the expression of the known PKC isoforms in extracts from chick major CNS areas during embryogenesis, from day 3 (E3) of incubation to day 1 post-hatching (P1). PKC-ε was the predominant isoform and was expressed from E6 onward in all brain regions, except retina (E12 and on). PKC-α/β and -ζ isoforms were expressed at lower levels prior to PKC-ε expression and throughout embryogenesis. No other isoforms were detected in neural tissue preparations. We then used neural culture systems derived from the chick CNS to study the expression of PKC isoforms in neuroblasts, cortical neurons, and cortical glial cells. Western blotting and immunostaining of neuroblastenriched cultures, derived from E3 CNS, showed only the Ca²⁺-dependent PKC-α/β to be present. Studies on neuronal cultures derived from E6 cerebral hemispheres revealed only the Ca²⁺-independent PKC-ε to be expressed in neurons, as predicted by the developmental studies on tissue homogenates. PKC-ε immunoreactivity was seen intracellularly in differentiating neurons, regardless of their neurotransmitter phenotypes, and it correlated well with the level of neuronal activity. Furthermore, PKC-α/β immunoreactivity was verified on glia cells, as the glial lineage emerges in E15 cortical cultures. These data suggest that PKC-ε expression is associated with the final neuroblast division in neurons, and the correlation of PKC isoform expression and neural cell lineage is discussed. © 1993 Wiley-Liss, Inc.     

Heterogeneity of astrocytes in human optic nerve head

Previous studies demonstrated regional differences in the synthesis of extracellular matrix by astrocytes during optic nerve head (ONH) maturation and in glaucomatous optic neuro pathy, suggesting heterogeneity of astrocytes. To characterize different types of glial cells in human fetal and adult ONH, we used a variety of neural cell markers such as HNK-1/N-CAM, A2B5, galactocerebroside (GalC), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP). Cryostat or paraffin sections were prepared from fetal (16–25 weeks) and mature (8 months to 75 years old) ONH and processed for standard single/double immunocytochemistry. Two subpopulations of type 1 astrocytes were present in the mature prelaminar and laminar regions. Glial celia expressing only GFAP were identified as type 1A astrocytes at the edges of the cribriform plates. Cells forming the glial columns and lining the cribriform plates expressed both GFAP and fINK-1/N-CAM and were identified as type lB astrocytes. In the myelinated nerve, type 1A astrocytes form the glial limiting membrane. Cells labeled with GFAP and A2B5 were identified as type 2 astrocytes, and GFAP-negative cells labeled with GaIC, MBP, and HNK-1/N-CAM were identified as oligodendrocytes. In fetal ONH, all glial cells expressed HNK-1/N-CAM. In older fetal ONH, some glial cells also expressed GFAP. No type 2 astrocytes or oligodendrocytes were present in the fetal ONH. In conclusion, at least two subpopulations of type 1 astrocytes exist in human ONH: Type 1A astrocytes may serve as structural support for a type lB astrocytes, which retain the developmental neural marker HNK-1/N-CAM, may have a more complex function by interfacing between blood vessels and other connective tissue surfaces. These findings demonstrate the heterogeneity of astrocytes in the human ONH and suggest differential regional responses to changes in their microenvironment. © 1995 Wiley-Liss Inc.     

Glial fibrillary acidic protein-expressing cells in the neurogenic regions in normal and injured adult brains

In the adult brain, neurogenic stem cells are prevalent in the subventricular zone (SVZ) of the lateral ventricle wall and the subgranular zone (SGZ) in the dentate gyrus. Cells that have structural and molecular characteristics of astrocytes function as neurogenic stem cells in these regions, in which these cells also participate in the creation of the microenvironment that stimulates neurogenesis. In the present paper, we review the phenotypic properties, subpopulations, and proliferation of glial fibrillary acidic protein (GFAP)-expressing cells in these two neurogenic regions and their responses to different brain injuries. Cells fulfilling the criteria for astrocytes, i.e., expressing GFAP, in the SVZ and SGZ respond differently to brain injuries or neurogenic stimuli. The importance of guidance by astrocytes of newly formed neuronal cells is emphasized. The assessment of GFAP-expressing cells in the neurogenic regions is of great importance for understanding the mechanism underlying the response of neural stem cells to brain injury.     

Morphological differentiation of neuropeptide Y neurons in aggregate cultures of dissociated fetal cortical cells: a model system for glia-neuron paracrine interactions

The temporal changes in the morphological profiles of neuropeptide Y (NPY) neurons and their topographical relationship with glial cells (astrocytes) were characterized in aggregate cultures derived from fetal cortical tissue using immunocytochemical procedures. On day 6 of culture, structures labelled with NPY antibodies were small and uneven in size but many resembled neuronal cell bodies. On day 14, neuronal perikarya were well defined and several morphological types of NPY neurons could be distinguished most of which gave rise to beaded processes: unipolar or multipolar bitufted neurons whose processes branch in close proximity to the cell body; bipolar neurons; and multipolar neurons. On day 23, heavily punctate and asymmetrically labelled cell bodies were dispersed throughout the aggregate; neuronal processes were less conspicuous. At 14 and 23 days, cells expressing glial fibrillary acidic protein (GFAP) and neuronal specific enolase (NSE) were abundantly distributed throughout the aggregate. Using a double immunoreaction on 14-day-old aggregates revealed that GFAP + cells and their processes were in close apposition to and engulfing the NPY neurons. Thus, dissociated fetal NPY neurons undergo morphological differentiation in culture along with astrocytes (GFAP +) and other neuronal cell types (NSE +). Based on the topographical association of astrocytes and neurons, particularly NPY neurons, we propose that the aggregate culture system can serve as a model to study the role of paracrine interactions in the regulation of the expression of NPY.     

Expression of vimentin and glial fibrillary acidic protein in ethylnitrosourea-induced rat gliomas and glioma cell lines

Summary The expression of glial fibrillary acidic protein (GFAP) and vimentin was investigated immuno-histochemically in 104 experimental gliomas induced by transplancental application of ethylnitrosourea (ENU) in CDF rats. Immunoreactivity for vimentin was prominent in many astrocytic tumor cells and especially in small glioma cells forming anaplastic medulloblastoma-like foci in many tumors. The majority of tumor cells in oligodendroglial tumors were vimentin negative, except for some of the large polymorphous oligodendrogliomas which contained intermingled vimentin positive glioma cells. GFAP immunoreactivity was detectable only in a low fraction of tumor astrocytes and in a few exceptional cases some oligodendroglial tumor cells stained positive. Immunohistochemistry with antibodies against neurofilaments and cytokeratins revealed no staining in tumor cells of ENU-induced gliomas, while all oligoden-drogliomatous tumors stained positive for HNK-1. Immunocytological and immunoblot investigations of the two rat glioma cell clones RG2 and F98, which are both derived from ENU-induced gliomas, showed a prominent expression of vimentin in monolayer cultures and in syngeneic intracerebral transplantation tumors. F98 additionally demonstrated a fraction of GFAP positive cells especially in confluent cultures and in intracerebral tumors. RG2, on the other hand, exhibited virtually no GFAP immunoreactivity in culture but showed individual GFAP positive tumor cells in intracerebral tumors. Our results revealed a more precise picture of the cellular differentiation in ENU-induced rat gliomas and in two widely used glioma cell lines. They underline the heterogeneity of experimental rat gliomas which may comprise cells at different stages of differentiation towards the oligodendroglial or astroglial phenotype.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed

Proliferating single cells were isolated from various CNS regions (telencephalon, diencephalon, midbrain, cerebellum, pons and medulla, and spinal cord) of human fetal cadavers at 13 weeks of gestation and grown as neurospheres in long-term cultures. We investigated whether neural stem cells (NSCs) or progenitors within spheres have specific regional or temporal characteristics with regard to growth, differentiation, and region-specific gene expression, and whether these molecular specifications are reversible. Regardless of regional origin, all of the neurospheres were found to contain cells of different subtypes, which suggests that multipotent NSCs, progenitors or radial glial cells co-exist with restricted neuronal or glial progenitors within the neurospheres. Neurospheres from the forebrain grew faster and gave rise to significantly more neurons than did those from either the midbrain or hindbrain, and regional differences in neuronal differentiation appeared to be sustained during long-term passage of neurospheres in culture. There was also a trend towards a reduction in neuronal emergence from the respective neurospheres over time in culture, although the percentages of neurons generated from cerebellum-derived neurospheres increased dramatically. These results suggest that differences in neuronal differentiation for the various neurospheres are spatially and temporally determined. In addition, the properties of glial fibrillary acidic protein (GFAP)-, glutamate-, and gamma-aminobutyric acid (GABA)-expressing cells derived from neurospheres of the respective CNS regions appear to be regionally and temporally different. Isolated human neurospheres from different CNS compartments expressed distinctive molecular markers of regional identity and maintained these patterns of region-specific gene expression during long-term passage in vitro. To determine the potential of human neurospheres for regional fate plasticity, single spheres from the respective regions were co-cultured with embryonic day 16.5 (E16.5 d) mouse brain slices. Specific cues from the developing mouse brain tissues induced the human neurospheres to express different marker genes of regional identity and to suppress the expression of their original marker genes. Thus, even the early regional identities of human neurospheres may not be irreversible and may be altered by local inductive cues. These findings have important implications for understanding the characteristics of growth, differentiation, and molecular specification of human neurospheres derived from the developing CNS, as well as the therapeutic potential for neural repair.     

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.     

Astrocyte-astrocytoma cell line interactions in culture

Astrocytomas are the most common brain tumors arising in the CNS and account for 65% of all primary brain tumors. Astrocytes have been shown to have the highest predisposition to malignant transformation compared to any other CNS cell type. The majority of astrocytomas are histologically malignant neoplasm. Previous studies have shown that resident astrocytes are the first cell type to react to tumors and surround them. However, the role of these astrocytes in tumor formation and progression has not been determined. In the present study, we have co-cultured astrocytes with a permanent cell line S635c15 (derived from anaplastic astrocytoma) in order to understand the cellular interactions between astrocytes and astrocytoma cells. Our studies demonstrate that astrocytes in contact with the tumor cells become reactive and fibrous with an increase in glial fibrillary acidic protein (GFAP) immunoreactivity as early as 4 days in culture. By 8 days, astrocytes formed glial boundaries around the tumor cells which grew as round colonies. The astrocytic processes surrounding the tumor cells were also intensely GFAP positive. Since the behavior of these cells observed in culture is very similar to their interaction seen in vivo, this co-culture system may serve as an in vitro model for astrocyte and astrocytoma cell line interaction and aid in our understanding of the molecular and cellular mechanisms during early stages of tumor formation and cell interactions. © 1996 Wiley-Liss, Inc.     

Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody

The presence of the intermediate filament protein nestin has been the predominant marker used to describe stem and progenitor cells in the mammalian CNS. In this study, a 998-bp fragment in the 3' region of the nestin mRNA was cloned from human fetal brain cells (HFBC). The nucleotide sequence of the cloned cDNA revealed 21 differences with the previously published human nestin sequence, resulting in 17 amino acid changes. A 150-amino-acid fragment derived from the cloned nestin cDNA was coupled to glutathione S-transferase and used as an immunogen to generate a rabbit polyclonal antiserum that selectively detects human nestin. HFBC that proliferated in response to basic fibroblast growth factor incorporated 5-bromo-2'-deoxyuridine into their nuclei and immunostained for nestin, indicating nestin expression in proliferating CNS progenitor cells. In all cell cultures, nestin costained with the neuroepithelial cell marker vimentin. A small subset of nestin-stained cells (1-2%) immunostained with neuronal marker MAP-2 during the first week and after 4 weeks in culture. However, during the first week in culture, approximately 10-30% of the total cell population of HFBC stained for the glial cell marker GFAP, and nearly all coimmunostained for nestin. After 4 weeks in culture, a subset of GFAP-positive cells emerged that no longer costained with nestin. These results describe nestin expression not only in CNS progenitor cells but also in the cells which were in transition from a progenitor stage to glial differentiation. Collectively, these data suggest a differential temporal regulation of nestin expression during glial and neuronal cell differentiation.