Increased susceptibility of human fetal astrocytes to human T-lymphotropic virus type I in culture.

Human T-lymphotropic virus type I (HTLV-I) has been considered as an agent responsible for tropical spastic paraparesis and HTLV-I associated myelopathy. However, the pathogenesis of the diseases remains unclear. In a previous study we demonstrated that HTLV-I could infect adult human astrocytes and oligodendrocytes in vitro, although the rates of infected cells were low, at a rate of 0.1% and 0.01-0.05% respectively. Since mother-to-child transmission has been proposed as one of the major pathways for the prevalence of HTLV-I endemic, in the present study we investigated the susceptibility of human fetal astrocytes to HTLV-I in culture. After two days of co-culturing fetal brain cells with irradiated MT-2 cells (an HTLV-I-producing T-cell line), immunofluorescence staining revealed many positive astrocytes for HTLV-I p19 antigen. Multinucleated giant cells doubly immunoreactive to glial fibrillary acidic protein and HTLV-I antigen were frequently observed and showed a characteristic feature of hairy or fluffy external appearance. The percentage of infected astrocytes became as high as 19.4% at Day 21 of co-culture and then decreased. Electron microscopic examination revealed type C virus-like particles in astrocytes. These results indicate that human fetal astrocytes are more susceptible to HTLV-I infection than adult human astrocytes in tissue culture.     

Phagocytic activity of human adult astrocytes and oligodendrocytes in culture

The phagocytic activity of human glial cells was examined in primary cultures obtained from normal human brain obtained at autopsy. Highly enriched cultures of astrocytes and oligodendrocytes were established using an enzyme digestion-Percoll density gradient method. These cultures were exposed to medium containing carbon particles (indian ink) for one-24 hours. Under phase contrast and immunofluorescence microscopy, carbon particles were demonstrated in the cytoplasm and processes of both astrocytes and oligodendrocytes. Electron microscopic examination revealed carbon particles phagocytized and segregated in these cells. These findings may support a view that both astrocytes and oligodendrocytes participate in phagocytosis in certain neurological diseases.     

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.     

Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture

We have used 4 cell-type-specific markers to identify individual glial and neuronal cells in dissociated cell cultures of neonatal rat sciatic nerve, dorsal root ganglia (DRG), optic nerve, cerebellum, corpus callosum, cerebral cortex and leptomeninges. Schwann cells were identified with antibodies against rat neural antigen-1 (Ran-1), neurons with tetanus toxin, astrocytes with antibody against the glial fibrillary acidic protein (GFAP) and oligodendrocytes with antibody against galactocerebroside. All of these ligands react with cell surface molecules except for anti-GFAP antibody which binds to intracellular glial filaments. Using two-fluorochrome immunofluorescence we have studied the distribution of various glycoproteins and glycolipids on these 4 major neural cell types in short-term cultures. We have found that (1) although Ran-1 is expressed on glial and neuronal tumours, it was not found on normal astrocytes, oligodendrocytes or neurons; (2) Thy-1 was present on fibroblasts and some neurons but not on the majority of leptomeningeal cells or on oligodendrocytes or astrocytes in short-term cultures (however, it was expressed on some astrocytes in longer term cultures); (3) the 'large external transformation sensitive' (LETS) protein could be detected on fibroblasts and leptomeningeal cells but not on neurons or glial cells; (4) GM1 was present on all neurons, most oligodendrocytes and approx. 50% of other cell types; sulfatide and GM3 were only detectable on oligodendrocytes, while globoside was only found on some neurons. In addition, we were able to identify putative microglial cells by the presence of cell surface receptors for IgG and by their phagocytic activity; they did not express and of the cell-type-specific defining markers.     

Antigen expression by glial cells grown in culture

Cell type-specific markers are currently available for identifying major cell types in neural cell cultures. The markers considered specific for glial cells are numerous, but only galactocerebroside and myelin basic protein for oligodendrocytes, and glial fibrillary acidic protein for astrocytes are widely adopted by investigators for that purpose. Other surface and intracellular markers which are specific for oligodendrocytes or astrocytes in vitro are briefly described. The possibility of using different classes of gangliosides as cell type-specific markers in neural cell culture is discussed. The presence of "transitional" or "bipotential" glial cells that express both oligodendrocytic and astrocytic phenotypes in human glial cell cultures and the regulatory effect of cyclic AMP derivatives on these cells are reported. In addition, the presence of Ia antigens on the surface of a selected population of cultured human oligodendrocytes and astrocytes is described.     

Comparative biochemical, morphological, and immunocytochemical studies between C-6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres.

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8-17) of glial cells derived from aged (18-month-old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20-24 were considered "early" and passages 73-90 were considered "late." Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum-free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early-passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP- and GalC-positive cells, respectively. Late-passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (+/- serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM+transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes.     

Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM-strain): studies in vivo and in vitro

The pathogenicity and cell tropism of mouse hepatitis virus (MHV-JHM-strain) in the developing mouse (Balb/c) and rat (Wistar and Lewis) brain were analysed. Intracranial infection of Balb/c mice at postnatal day 5 induced a lethal encephalitis in all animals. Of Wistar rats infected at day 2 or 5 after birth, 30 to 70%, respectively, survived. The distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus-infected cell type throughout the central nervous system. More than 75% of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. The cell tropism of MHV-JHM was examined further in virus-infected glial cell cultures derived from brains of rats or mice. In the glial cultures derived from Wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or Lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. Infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. Infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. These phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus-induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. The present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of MHV-JHM.     

Embryonic-derived glial-restricted precursor cells (GRP cells) can differentiate into astrocytes and oligodendrocytes in vivo

We have isolated and characterized a unique glial-restricted precursor cell (GRP) from the embryonic spinal cord. Clonal analysis demonstrated that these cells are able to generate oligodendrocytes and two distinct type of astrocytes (type 1 and type 2) when exposed to appropriate signals in vitro. We now show that many aspects of these cells are retained in vivo. GRP cells are restricted to the glial lineage in vivo as they seem to be unable to generate neuronal phenotypes in an in vivo neurogenic environment. GRP cells survive and migrate in the neonatal and adult brain. Transplanted GRP cells differentiate into myelin-forming oligodendrocytes in a myelin-deficient background and also generate immature oligodendrocytes in the normal neonatal brain. In addition, GRP cells also consistently generated glial fibrillary protein-expressing cells in the neonatal and adult brain, a property not consistently expressed by other glial precursor cells like the O-2A/OPC cells. We suggest that the lineage restriction of GRP cells and their ability to generate both oligodendrocytes and astrocytes in vivo together with their embryonic character that allows for extensive in vitro expansion of the population makes the cell useful for clinical application.     

Modulation of antigenic expression in cultured adult human oligodendrocytes by derivatives of adenosine 3',5'-cyclic monophosphate

Oligodendrocytes were isolated from adult human brains obtained at autopsy by enzyme treatment - Percoll density gradient centrifugation, and grown in culture. During the first week in vitro, these cultures consisted of an enriched population (93-98%) of galactocerebroside-immunoreactive oligodendrocytes. After 2 weeks and onward, a large number of GFAP-positive astrocytes and glial cells doubly positive for galactocerebroside and GFAP markers was found among the oligodendrocytes. When these cultures were exposed to dibutyryl cyclic AMP, 8-bromocyclic AMP and RO-1724, an inhibitor of cyclic nucleotide phosphodiesterase, for 4-14 days, the majority of cells returned to express oligodendrocytic phenotype. These findings suggest the presence of heretofore unidentified "transitional" or "bipotential" glial cells in human brains that express both oligodendrocytic and astrocytic phenotypes, and the regulatory role of cyclic AMP derivatives which may induce a stable antigen expression in oligodendrocytes.     

Mesencephalic type I astrocytes mediate the survival of substantia nigra dopaminergic neurons in culture

We previously demonstrated that substantia nigra (SN) support cells selectively increase SN dopamine (DA) neuron survival in dissociated primary culture. Increased survival was elicited specifically by nigral support cells; glia from other brain regions exerted lesser effects. We now report that Type I astrocytes, the principal component of SN support cell monolayers, mediate the enhanced DA cell survival. Initially, the predominant glial subtypes in SN support cell cultures were identified. Postnatal day 1 rat SN was dissociated and cells were grown to confluence (7-9 days in vitro; DIV). Monolayers were immunostained with antibodies against glial fibrillary acidic protein (GFAP; an astrocyte-specific marker), myelin basic protein (MBP; an oligodendrocyte marker), or A2B5 (recognizes 0-2A progenitors and Type II astrocytes). The number of GFAP+ cells far exceeded MBP+ and A2B5+ cells, suggesting that astrocytes constituted the predominant subpopulation. Further, direct comparison of GFAP+ (Type I and Type II astrocytes) and A2B5+ (Type II astrocytes) cells indicated that the vast majority were Type I astrocytes. Greater than 98% of cells reacted with glial antibodies. To definitively characterize the cellular subtype that augments survival of DA neurons, glial subcultures were established. At 2 DIV, enriched populations of Type I or Type II astrocytes, or oligodendrocytes, were tested for the ability to elicit DA neuron survival. Embryonic day 16 rat SN dissociates were added and DA cell number was assessed with antibody against tyrosine hydroxylase (TH), the DA biosynthetic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

Fate of developing astrocytes in the optic nerve of the myelin-deficient rat

There is considerable debate on the development of a glial cell line in the rat optic nerve, which is characterized by the specific expression of the A2B5 and HNK-1 epitopes. This cell line has been assumed to give rise to oligodendrocytes and so-called type 2 astrocytes. However, it is doubtful that the latter cell type really exists in vivo. In the present study, we have addressed this question by investigating the development of astrocytes in the myelindeficient (md) rat, which is characterized by dysmyelination and loss of oligodendrocytes. Defective oligodendrocytes were observed by the third postnatal day, well before the generation of type 2 astrocytes. Consequently, the number of type 2 astrocytes was reduced in cultures prepared from optic nerves of md rats vs. controls. This finding was not paralleled in vivo; i.e., no dying astrocytes were observed in md sections by conventional electron microscopy. However, immunoreactivity against the HNK-1 epitope was enhanced in md compared to control sections. Ultrastructurally, HNK-1 immunoreactivity was detected predominantly on the axonal surface at astroaxonal contact sites, which were found only at the nodes of Ranvier within controls but extended to the whole axonal surface in md animals. Only a minor portion of the immunoreactivity derived from glial cells, presumably from oligodendrocytes at the paranodal region in controls. Thus, the HNK-1 epitope is not a useful antigen for distinguishing astrocytes in the rat optic nerve. Accordingly, our results do not provide evidence for the existence of specialized type 2 astrocytes in vivo. In vitro, these cells are probably only oligodendrocytes that mimic some astroglial features if grown in serum-containing media. J. Comp. Neurol. 378:105–116, 1997. © 1997 Wiley-Liss, Inc.     

Bipotential glial progenitors are targets of neuronal cell line-derived growth factors

The growth-promoting activity of conditioned medium (CM) from the B104 CNS neuronal cell line was studied in glial cultures from neonatal rat brain. This CM at 33% (v/v; 8-12 micrograms protein/ml) produced large numbers of oligodendrocytes and multipolar glial progenitors after an 8 to 12-day treatment. At all times studied, cells of the oligodendrocyte/type 2 astrocyte (O-2A) lineage were increased due to CM-treatment, while type 1 astrocytes, microglia, and other cell types were not. Furthermore, we observed a large decrease in the percentage of oligodendrocytes in the O-2A lineage, suggesting a delay in differentiation of the progenitors. By 8 days in vitro (DIV), dose-dependent increases in numbers of galactocerebroside (GalC)-positive cells (oligodendrocytes) and A2B5-positive cells (immature oligodendrocytes and glial progenitors) occurred. In contrast, at 4 DIV only A2B5-positive cells were increased in a dose-dependent manner. The latter cells can differentiate primarily into oligodendrocytes or type 2 astrocytes depending on the culture conditions. Complement lysis studies confirmed that the A2B5-positive, but not the GalC-positive, population at 4 DIV was required for increases in oligodendrocytes to occur by 8 DIV. The [3H]thymidine labeling index of the A2B5-positive population also increased in response to CM in a dose-dependent manner, but the GalC-positive labeling index showed only small increases at 4 DIV and none at later times. Our results suggest that the delayed differentiation coupled with the selective stimulation of the bipotential glial progenitors produces the large increases in numbers of oligodendrocytes observed at 8-12 DIV.     

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.     

Characteristics of fish glial cells in culture: possible implications as to their lineage.

Regeneration of injured central nervous system axons is largely dependent on the response of the associated nonneuronal glial cells to injury. Glial cells of the mammalian central nervous system, unlike those of fish, are apparently not conducive to axonal regeneration. While the lineage of rat glial cells is well characterized and its role in the support or inhibition of regenerative growth is beginning to be understood, little is known about fish glial cells. Accordingly, glial cells in cultures of adult goldfish brain and of newly hatched goldfish larvae were studied in an attempt to establish their lineage. The cells were identified by means of indirect immunofluorescence, using antibodies against fish astrocytes and oligodendrocytes. The cell count in the cultures increased from a small number of cells at 24 h after plating to a large number of both astrocytes and oligodendrocytes after 1 week in culture. Both of these cell types had originated from proliferating cells, as shown by their uptake of tritiated thymidine and by the inhibition of cell proliferation by 5-fluoro-2'-deoxyuridine. Both astrocytes, i.e., glial fibrillary acidic protein-positive cells, and oligodendrocytes, i.e., 6D2-positive cells, were positively labeled also by A2B5 antibodies, which are known to label progenitors of type-2 astrocytes and oligodendrocytes in the rat optic nerve. The results suggest that A2B5 positive progenitor cells in the goldfish central nervous system, as in the rat optic nerve, might be a common progenitor of astrocytes and oligodendrocytes.     

Concurrent isolation and characterization of oligodendrocytes, microglia and astrocytes from adult human spinal cord

A cellular preparation of highly enriched oligodendrocytes was obtained from adult human spinal cord by Percoll gradient centrifugation followed by either differential adhesion or fluorescence-activated cell sorting after immunostaining with an antibody against galactocerebroside (Ol). The adherent and O1-negative cell fractions were 96% microglia. The non-adherent and O1-positive fractions were 96% positive for the oligodendrocyte markers O4 and O1, 0–2% positive for glial fibrillary acidic protein, and were devoid of neuronal or microglial markers. If the oligodendrocyte fraction was co-cultured with purified dissociated rat dorsal root ganglion neurons, the oligodendrocytes adhered to the axons and their numbers increased over a 4 week period. However, myelin sheaths were not produced around axons in these cultures. In contrast, if the oligodendrocyte cell fraction was grown alone in culture for 3 weeks, the number of oligodendrocytes decreased and a layer of astrocytes developed underneath the oligodendrocytes. The oligodendrocytes could be eliminated from these cultures by subsequent passaging, thus producing cultures of pure astrocytes. The astrocytes accumulated both K⁺ and glutamate with kinetic properties similar to those reported for rodent astrocytes. We suggest that these astrocytes arose in part from an O4/O1-positive precursor which did not initially express glial fibrillary acidic protein. These results define a relatively simple method by which highly enriched populations of oligodendrocytes, astrocytes and microglia can be obtained from adult human spinal cord.     

Differential distribution of cellular forms of beta-amyloid precursor protein in murine glial cell cultures.

The production and localization of cell-associated forms of beta-amyloid precursor protein (APP) of Alzheimer's disease was investigated in primary cultures of mouse glial cells. In both oligodendrocytes and astrocytes, immunofluorescence staining with an antibody against the carboxy terminus of APP revealed an intense cytoplasmic immunoreactivity. Immunoblotting of the cell extracts detected differences in the composition of APP between oligodendrocytes and astrocytes, notably the abundance of 107 kDa subtype in oligodendrocytes. Differences in immunoblot patterns were also noted between two buffer-insoluble, membrane-rich subcellular fractions of the glial cells, nuclear-mitochondrial and microsomal; the 119 kDa APP was enriched in the former, whereas the 73 and 115 kDa APPs in the latter. The results suggest that each APP subspecies may play a distinct functional role in different cell types and subcellular fractions.     

CNS neuronal cell line-derived factors regulate gliogenesis in neonatal rat brain cultures

Recent studies suggest that heterotypic cell-cell interactions influence gliogenesis in the developing rat central nervous system. CNS neuron-derived factors have been hypothesized to exist, and several have been identified and partially characterized which affect the number of oligodendrocytes in vitro. In order to study further the role of neurons in gliogenesis, we have used serum-free culture conditions, the B104 CNS neuronal cell line as a source of soluble factors, and dissociated neonatal rat brain cells as a source of glial cells. We have analyzed the response of the glial cells to serum-free B104 conditioned medium using morphological, immunocytochemical, autoradiographic, and enzymatic methods. Dose-dependent increases in the number of morphologically identified oligodendrocytes occur in response to this conditioned medium. Galactocerebroside (GalC) is a specific marker for oligodendrocytes, and the A2B5 antigen marks bipotential glial progenitor cells and their progeny: immature oligodendrocytes and type 2 astrocytes. In the presence of conditioned medium, the number of cells expressing GalC and/or A2B5 antigen increases over time when measured at 4, 8, and 12 days in vitro. A significantly weaker effect is seen if serum is also present. Since the vast majority of A2B5-positive cells in conditioned medium treated cultures lack glial fibrillary acidic protein (GFA), indicative of type 2 astrocytes, they represent glial progenitors and immature oligodendrocytes. Double immunostaining combined with autoradiography suggests that the latter cell types are the target cells for the oligodendrocyte-promoting activity. In addition, the conditioned medium markedly increases 2',3' cyclic nucleotide 3'-phosphodiesterase (an oligodendrocyte marker) and to a lesser extent enhances glutamine synthetase activity (an astrocyte marker). Type 1 astrocytes are also more morphologically differentiated in this condition, and their percentage is decreased simultaneously. Conditioned medium from other donor neural cells either has no activity or is much less effective than B104 conditioned medium. The active factors are soluble, sensitive to both trypsin and 100 degrees C treatment for 20 min, and appear to be 30-100 kilodaltons by stirred cell ultrafiltration. In summary, we have identified a potent source of growth-stimulating factors that produce increased numbers of glial progenitor cells and oligodendrocytes; the same conditioned medium also appears to inhibit type 1 astrocyte proliferation.