Immunocytochemical characterisation of cell cultures grown from dissociated 1-2-day post-natal rat cerebral tissue. A developmental study

A range of cell-specific markers have been employed with immunocytochemical methods to characterise and quantitate the cell types present in mixed brain cell cultures derived from dissociated 1-2-day post-natal rat cerebral hemispheres and grown in the presence of FCS. Protoplasmic astrocytes (GFAP+, A2B5-) were the major cell type to develop in culture, a confluent monolayer forming in 5-8 days. A population of smaller round cells of oligodendrocyte-like morphology appeared on this astrocyte layer. Greater than 70% of these smaller cells were GC- and thus were not oligodendrocytes. The GC- cells were A2B5+ and, in early cultures, may therefore be progenitor glial cells. Examination of GFAP and A2B5 co-expression by these smaller cells was difficult due to the dense underlying GFAP+ astrocyte layer. In less dense areas of older cultures these smaller cells with processes were GFAP+ and A2B5+: these are Type 2, fibrous astrocytes. GC+ oligodendrocytes, comprising 5-10% of the total identified cell population, were initially distributed over the astrocyte monolayer; in older cultures (after about 8 days) GC+ cells were observed in clumps over places where NF+ cells were identifiable. Such GC+ cells mostly became MBP+. Neurones accounted for about 6% of the identifiable cells in early cultures but a lower percentage in older cultures. Minor populations of ependymal cells and macrophages were present; cells displaying fibronectin, fibroblasts, were rarely identified. Use of horse serum in place of FCS gave lower yields of GC+ cells in cultures, slowed down astrocyte development, and resulted in the formation of trunks of GFAP+ cells throughout cultures. Other sera gave lower numbers of GC+ cells.     

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.     

Chronic Suppression of Bioelectric Activity and Cell Survival in Primary Cultures of Rat Cerebral Cortex: Biochemical Observations

Chronic suppression of spontaneously occurring bioelectric activity (BEA) has been shown to increase neuronal cell death in tissue culture, but may also affect astrocytes. We investigated this process in primary cultures of rat cerebral cortex by measuring the levels of NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein) in relation to general tissue markers, including measurements for cell death and proliferation. In electrically active (control) cultures, the content of DNA, protein, and NSE became maximal between 21 and 28 days in vitro (DIV) and thereafter decreased, whereas the content of GFAP rose continuously up to 43 DIV. Chronic suppression of BEA by tetrodotoxin (TTX; from 6 DIV) decreased the content of DNA, total protein, and especially NSE. The content of GFAP was decreased in all culture series investigated, but with great temporal variations among culture series. Chronic TTX treatment (started at 6 DIV) increased the efflux of lactate dehydrogenase, a marker for cell lysis, between 12 and 21 DIV, but this efflux was mainly derived from the supporting glial cells with which the cerebral cortex cultures were cocultured. Chronic, but not acute (7 h) TTX treatment decreased total [3H]thymidine incorporation into DNA from 14 DIV; this appeared to be due to a reduced number of astrocytes. Chronic suppression of BEA with xylocaine from 6 DIV had similar effects on DNA-, protein-, and NSE-content as TTX, but led to an increased content of GFAP at 21 DIV. Chronic suppression of synaptic transmission with 10 mM Mg2+ and 0.2 mM Ca2+, starting at 6 DIV, increased the content of DNA, protein, and GFAP at 21 DIV, but NSE was still decreased. We conclude that chronic suppression of BEA in cerebral cortex cultures enhances neuronal cell death, whereas astrocytes are differentially affected, depending on the suppressing agent. As astrocytes may have a modulating effect on neuronal survival, their involvement should be regarded when studying the effects of chronic suppression of BEA on neuronal development.     

Local support cells promote survival of substantia nigra dopaminergic neurons in culture.

Recent studies suggest that brain neurons require extracellular signals for continued survival during maturity as well as development. However, factors underlying the survival of specific populations of central neurons remain to be defined. To examine the regulation of neuronal survival, we have studied the substantia nigra (SN) dopaminergic (DA) system, in dissociated cell culture. DA neuron number was monitored immunocytochemically with antibody to tyrosine hydroxylase (TH), the DA biosynthetic enzyme. Initially, mixed cultures were grown at low, medium, and high densities in serum-containing media. After 7 days, the number of neuron-specific enolase (NSE)-positive cells, a measure of total neuron number, was proportional to cell plating density. In contrast, high density culture elicited a marked, disproportionate increase in TH-immunopositive cells, suggesting that high density conditions selectively enhanced the DA subpopulation. To define the role of cellular interactions in the selective increase in DA cells, virtually pure neuron cultures were compared to support cell-neuron cocultures, in fully defined medium. In support cell-neuron cocultures, SN support cells evoked a four-fold increase in TH cells, while NSE number did not differ from controls. Moreover, local support cells elicited a greater increase in TH cell number than support cells derived from other brain regions. To determine whether increased TH cell number reflected enhanced survival, or possibly expression of TH by new populations, we monitored the time course of this effect. TH cell number remained constant after 3 days in cocultures, while declining fourfold in controls. In parallel studies, support cells were added to SN dissociates at zero time or after 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two types of Na(+)-currents in cultured rat optic nerve astrocytes: changes with time in culture and with age of culture derivation.

Na+ channel expression was studied in cultures of rat optic nerve astrocytes using whole-cell voltage-clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h infinity curves. Stellate, GFAP+/A2B5+ astrocytes showed currents with h infinity curve midpoints close to -65 mV, similar to Na+ currents in most neurons. In contrast, flat fibroblast-like GFAP+/A2B5- astrocytes showed Na+ currents with h infinity midpoints around -85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5+ astrocytes. Interestingly, Na+ current expression was maintained in A2B5+ astrocytes but began to decrease in A2B5- astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP+/A2B5-. These cells did not display measurable Na+ currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5- astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na+ channels; in P0-derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.     

Bipotential glial precursor cells of the optic nerve express the NG2 proteoglycan

We have studied the expression of the NG2 chondroitin sulfate proteoglycan on bipotential glial precursor cells in cultures of postnatal rat optic nerve. Purified populations of these precursor cells were prepared by panning dissociated optic nerve cells on dishes coated with monoclonal A2B5 antibody. Using immunofluorescence double staining, we found that NG2 was present on almost 95% of the purified A2B5+ precursor cells. The NG2 core protein from optic nerve cells was identified by immune precipitation and PAGE and was found to be identical to the 300,000 Da NG2 core protein from a clonal rat cell line B49. Over a culture period of 5 d in medium containing 10% fetal calf serum, more than 80% of the NG2+ precursor cells acquired the glial fibrillary acidic protein (GFAP), an astrocyte-specific marker. Under these conditions, fewer than 10% of the NG2+ cells expressed galactocerebroside (GC), an oligodendrocyte-specific marker. These GFAP+GC- type II astrocytes continued to express the NG2 antigen for up to 10 d in culture. During a 5 d culture period in hormonally supplemented, serum-free medium, fewer than 15% of the NG2+ cells expressed GFAP, while up to 40% expressed GC. The NG2 antigen continued to be expressed for only a short period of time by these GFAP-GC+ oligodendrocytes, so that mature oligodendrocytes in the cultures became NG2-. These results support our previous suggestion that the NG2 antigen is found on a class of neural cells that can differentiate along more than one pathway.     

Infection of rat brain primary cell cultures with an avirulent A7 strain of Semliki Forest virus

The ability of A7 Semliki Forest Virus (SFV) to infect primary brain cell cultures has been examined using cultures prepared from 1-2-day neonatal rat cerebral hemispheres. These cultures, characterised immunocytochemically using cell-specified markers, contain mainly GFAP+ protoplasmic astrocytes and smaller multiprocessed A2B5+ cells, probably fibrous astrocytes. 10% of the cells are GC+ oligodendrocytes and some neurones are also present. These cultures support virus growth and a cytopathic effect was observed. Using double labelling techniques with the cell-specific markers and anti-SFV antibody A7 has been shown to readily infect cells which carry either the A2B5+ antigen or galactocerebroside marker. Protoplasmic astrocytes (GFAP+/A2B5-) are not readily infected under the conditions used. The protein labelling studies using [35S]methionine show that host cell protein synthesis is not completely shut off and continues in the astrocyte protein region. These results suggest that cells derived from a common A2B5+, GFAP-, GC- progenitor glial cell, i.e. GC+ oligodendrocytes and A2B5+/GFAP+ fibrous astrocytes, are more readily infected than other brain cell types including the protoplasmic astrocytes.     

Intermediate glial cells and reactive astrocytes revisited. A study in organotypic tissue culture

The existence of cells sharing features of oligodendrocytes and astrocytes has been repeatedly proposed. We have studied this problem ultrastructurally in organotypic tissue culture together with light-microscopic immunocytochemistry for the astrocyte marker glial fibrillary acidic protein (GFAP) and for 2 oligodendrocyte markers, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG). Large numbers of GFAP+ cells (astrocytes) were seen, invariably giving rise to a wealth of tapering processes. In contrast, oligodendrocytes were found far less frequently either immunocytochemically or ultrastructurally, and showed smooth contours and scarcity of processes. Ultrastructurally, the cells corresponding to the intermediate glial cells in the literature which were far more numerous in culture than MBP-stained cells, were identified as reactive astrocytes by their numbers, location and morphological similarity with the GFAP-stained cells. Other characteristics were the presence of bundles of intermediate filaments and the covering of the plasmalemma adjacent to the collagen substrate by a basal lamina, in spite of the content of microtubules and the density of the cells. It was possible to demonstrate the difference between the wrapping of axons by astrocytic digitiform processes, and true myelination by processes identifiable as oligodendrocytes. We conclude that in this model the astrocytic and oligodendrocytic cell lines appear separate from the time of initial differentiation; in other systems such as dissociated cell culture, this may not be so. The cells with 'intermediate' features are in fact a reactive form of astrocyte.     

Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: role of mesencephalic glia.

Neuronal differentiation is influenced by extracellular factors; however, only a few such factors have been identified for central neurons. To address this issue, we have screened media conditioned (CM) by several glial cell lines for neurotrophic effects on dopaminergic neurons in dissociated cell cultures of the E14.5 rat mesencephalon grown in serum-free conditions. To establish culture conditions under which dopaminergic cell survival depends on the exogenous support from neurotrophic factors, cell suspensions were seeded at varying densities and the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons was determined. This number was maximal at plating densities greater than 175,000 cells/cm2 and was 10-fold lower at the plating density of 80,000 cells/cm2. Cell density had only a minimal effect on [3H]dopamine uptake per TH-IR neuron. Treatment of cultures plated at 80,000 cells/cm2 with CM derived from the glial cell line, B49, the neural retina glial cell line, R33, and the Schwannoma cell line JS1, increased the number of surviving TH-IR neurons 160-330%. These effects were dose dependent and heat sensitive. All CM stimulated neurite elongation of TH-IR neurons, while only the B49-CM increased [3H]dopamine uptake. The neurotrophic effects of these media were not confined to dopaminergic neurons but increased overall neuronal density in culture by 50-100%. Moreover, all three CM were mitogenic for mesencephalic glia as demonstrated by glial fibrillary acidic protein (GFAP)-immunocytochemistry in combination with [3H]thymidine-autoradiography. By contrast, medium conditioned by the pheochromocytoma cell line, PC12, did not increase the number of astrocytes or promote the survival of dopaminergic neurons. Inhibition of glial proliferation reduced the neurotrophic effects of the B49-, R33-, and JS1-CM by 40-80%. These observations suggest that the glial cell lines B49, R33, and JS1 secrete factors that promote the survival of dopaminergic neurons and induce proliferation of glial precursors. The partial decrease of the survival-promoting effects of these CM on dopaminergic neurons in glial-free mesencephalic cultures further suggests that the observed neurotrophic effects result from the combined action of cell line-derived substances directly on neurons and indirectly via effects on mesencephalic astrocytes or astrocyte precursors.     

Uptake of [3H]GABA by oligodendrocytes in dissociated brain cell culture: a combined autoradiographic and immunocytochemical study

Uptake of [3H]GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography. Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers. In 12 days in vitro (DIV) cultures [3H]GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes. This was confirmed by complement-dependent antibody-mediated cytotoxicity against galactocerebroside-positive oligodendrocytes. There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes. Heavily labeled astrocytes were only occasionally observed. Oligodendrocytes accumulated [3H]GABA more rapidly than astrocytes but slower than neurons. Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes. The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by beta-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake. Oligodendrocytes did not accumulate [3H]beta-alanine, which labeled only astrocytes. Oligodendroglial [3H]GABA uptake was Na+-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA. The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.     

PSA-NCAM distinguishes reactive astrocytes in 6-OHDA-lesioned substantia nigra from those in the striatal terminal fields

6-hydroxydopamine (6-OHDA) lesion of the substantia nigra (SN) causes the appearance of reactive astrocytes not only in the SN but also in the striatal terminal fields, as measured by increased size of the cells and their processes, as well as enhanced expression of glial fibrillary acidic protein (GFAP) and an epitope recognized by monoclonal antibody 19D1. We now demonstrate that polysialylated neural cell adhesion molecule (PSA-NCAM) is induced on reactive astrocytes, as well as on large neurons, on the ipsilateral side of the 6-OHDA-lesioned SN. Colocalization of GFAP and PSA-NCAM was confirmed for reactive astrocytes using a confocal laser scanning microscope. Negligible amounts of PSA-NCAM reactivity were detected contralaterally, although colocalization was noted on astrocytes with sparse, significantly thinner processes. In contrast to the increase of GFAP in the lesioned striatum, few striatal astrocytes expressed PSA-NCAM. In agreement with these results, PSA-NCAM was detected on cultured reactive astrocytes from SN but not reactive striatal astrocytes. Double immunohistochemistry for proliferating cell nuclear antigen (PCNA), a marker of dividing cells, and GFAP demonstrated that reactive astrocytes in lesioned SN were PCNA-positive whereas those in striatum were not. Although NG2 chondroitin sulfate proteoglycan expression also increased in the lesioned SN, NG2 was not colocalized with PSA-NCAM, was not expressed on astrocytes, and labeled only oligodendrocyte precursor cells. Our results suggest that PSA-NCAM can act as a marker for reactive astrocytes only at the site of the lesion and not in the terminal fields, probably because it is reexpressed only when astrocytes divide.     

Characterization of glial subpopulations in cultures of the ovine central nervous system

The cellular composition and in vitro development of glial cultures derived from the rat CNS has been well studied. However, less information is available on similar cultures from other species, particularly higher mammals. To study ovine glial development in vitro, cultures from 50-day fetal to adult animals were characterized with various immunocytochemical markers, which are frequently used to define neural cell subsets in rat cultures. As in rats, both A2B5+ and A2B5- astrocytes can be identified in ovine cultures. However, ovine A2B5+ and A2B5- could not be reliably differentiated by their morphology, which was more influenced by whether the cells were in serum-free or serum-containing media than by their A2B5-positive or -negative status. In addition, ovine A2B5+ astrocytes were present in cultures from early fetal brain before the development of identifiable oligodendrocytes, unlike rat type II astrocytes, which develop only after the appearance of oligodendrocytes. An A2B5+ cell, morphologically similar to the rat 02-A cell, can be found in cultures from fetal ovine cerebrum or cerebellum. A2B5+/glial fibrillary acidic protein (GFAP)- cells in cultures from 100- to 115-day ovine cerebellum appeared to differentiate into A2B5+ astrocytes in serum-containing media. However, in serum-free media, although the A2B5+ cells assumed a more "oligodendroglial-like" morphology, they did not express galactocerebroside or myelin basic protein, suggesting that these cells may not be bipotential as is the rat 02-A cell. Oligodendroglial differentiation was not induced by treatment with dibutyryl cyclic AMP or insulin-like growth factor I. Many cells in cultures from a variety of fetal ages did not label with any of the immunocytochemical markers used, suggesting the need for more cell-type-specific markers to identify neural cell subsets in higher mammals.     

Region- and sex-related differences in maturation of astrocytes in dissociated cell cultures of embryonic rat brain

Previous studies using dissociated cell cultures of fetal rat brain have revealed considerable regional diversity as well as sex steroid-independent sex differences in developmental schedules of dopaminergic neurons. Because these phenomena might be related to glial heterogeneity, cultures of dissociated male and female diencephalon, mesencephalon, and rhombencephalon of gestational day 14 rats were investigated with respect to the development of astrocytic markers. Cultures were incubated for 3-8 days in vitro (DIV) in serum-supplemented or serum-free medium. Vimentin and glial fibrillary acidic protein (GFAP) were quantified by counting of immunolabeled cells and immunoblotting. Vimentin and GFAP content rose from DIV 3 to 6 in all cultures. Regional variation of vimentin content was low, but large differences occurred in amounts of GFAP. GFAP reached high levels in rhombencephalon, especially when supplemented with serum, but remained very low or not detectable in mesencephalon. Simultaneous immunostaining for both cytoskeletal proteins revealed the presence of large numbers of vimentin single-labeled and small numbers of vimentin/GFAP double-labeled cells. Numbers of cells expressing GFAP showed similar regional variations as GFAP contents in both serum-free and serum-supplemented medium. They rose steeply from DIV 3 to 8 in rhomb- and diencephalon but not in mesencephalon. Transiently, female diencephalic cultures contained slightly more GFAP-immunoreactive cells than male cultures. The results thus demonstrate considerable regional heterogeneity of astrocytic maturation. However, neither the regional nor the sex differences show a consistent correlation with previous data on development of dopaminergic and other monoaminergic neurons in vitro. It seems likely that the dependence of neurons on glial environment for realization of an inherent developmental program varies among neuronal phenotypes.     

Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides, and growth characteristics

Two types of glial fibrillary acidic protein-positive (GFAP+) astrocytes were found in cultures of developing rat optic nerve. Type 1 astrocytes had a fibroblast-like morphology, did not bind tetanus toxin or the monoclonal antibody A2B5 (both of which bind to specific polysialogangliosides), and were stimulated to divide by an extract of bovine pituitary and by epidermal growth factor (EGF). Type 2 astrocytes had a neuron-like morphology, bound tetanus toxin and A2B5 antibody, and were not stimulated to divide by bovine pituitary extract or by EGF. Although both types of astrocytes were present in cultures of white matter, only type 1 astrocytes were found in cultures of gray matter. Astrocytes did not convert from one type to the other in culture: while many type 1 astrocytes adopted a neuron-like morphology when exposed to dibutyryl cyclic adenosine 3':5'-monophosphate, or pituitary or brain extracts, especially in serum-free medium, such morphologically altered cells did not bind tetanus toxin or A2B5 antibody. Although small numbers of tetanus toxin-binding, A2B5+, GFAP+ cells were present in suspensions of freshly dissected, neonatal optic nerves, most of the type 2 astrocytes in cultures of such optic nerves developed from tetanus toxin-binding, A2B5+, GFAP- cells, which were induced to express GFAP by the culture conditions. Since type 2 astrocytes have a neuron-like morphology and bind tetanus toxin and A2B5 antibody, these ligands cannot be used on their own as neuron-specific markers in central nervous system cultures.     

Electrophysiologically "complex" glial cells freshly isolated from the hippocampus are immunopositive for the chondroitin sulfate proteoglycan NG2

We have recently described a subgroup of isolated glial fibrillary acidic protein-positive (GFAP+) hippocampal astrocytes that predominantly express outwardly rectifying currents (which we term "ORAs" for outwardly rectifying astrocytes), which are similar to the currents already described for hippocampal GFAP- "complex glia." We now report that post-recording staining of cells that were first selected as "complex" by morphology and then confirmed by their electrophysiological characteristics were NG2+ approximately 90% of the time. Also, the morphology of freshly isolated NG2+ cells differs from that of isolated GFAP+ ORAs in having a smaller and round cell body with thinner processes, which usually are collapsed back onto the soma. Upon detailed examination, NG2+ cells were found to differ quantitatively in some electrophysiological characteristics from GFAP+ ORAs. The outward, transient K+ currents (IKa) in the NG2+ cells showed a slower decay than the IKa in ORAs, and their density decreased in NG2+ cells from older animals. The other two major cation currents, the voltage-activated Na+ and outwardly delayed rectifier K+ currents, were similar in NG2+ cells and ORAs. To further distinguish isolated complex cells from outwardly rectifying GFAP+ astrocytes, we performed immunocytochemistry for glial markers in fixed, freshly isolated rat hippocampal glia. NG2+ cells were negative for GFAP and also for the astrocytic glutamate transporters GLT-1 and GLAST. Thus the isolated hippocampal NG2+ glial cells, though having an electrophysiological phenotype similar to that of ORAs, are an immunologically and morphologically distinct glial cell population and most likely represent NG2+ cells in situ.     

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.     

Astrocytes cultured from mature brain derive from glial precursor cells

We have previously shown that enriched preparations of oligodendrocytes from either mature bovine brain or 30-d-old rat brain, when cultured in serum-free medium, yield mixed cultures of oligodendrocytes and astrocytes even though no GFAP+ cells were present after 24 hr in culture (Norton et al., 1986, 1988). To test the possibility that the astrocytes in these cultures arose from glial precursor cells, we followed the expression of ganglioside GD3, galactosylceramide (GC), glial fibrillary acidic protein (GFAP), and vimentin in the cultures. GD3 has already been shown to be a marker of immature neuroectodermal cells, which in the postnatal brain are glial progenitor cells (Goldman et al., 1984, 1986). The cultures from both species contained at 1 DIV only two populations of cells; 90-95% GC+/GD3- oligodendrocytes and 4-10% GD3+/GC- small, round cells. With time, the oligodendrocytes remained GD3-/GFAP-/vimentin-. The kinetics of antigen expression of the GD3+ cells could best be interpreted by the following sequence: (sequence; see text) We interpret these results to show that the astrocytes arose from a small population of GD3+ glial precursor cells present in the brain that were co-isolated with oligodendroglia. No evidence was obtained that these GD3+ cells could also differentiate into oligodendrocytes.