Carbonic anhydrase in distinct precursors of astrocytes and oligodendrocytes in the forebrains of neonatal and young rats.

Carbonic anhydrase is present in oligodendrocytes and astrocytes in the mature rat brain. Whereas carbonic anhydrase-positive oligodendrocyte precursors had been identified during the first postnatal week, no information was available about the earliest occurrence of carbonic anhydrase in the astrocytic cell line, nor had carbonic anhydrase been detected in astrocytes in neonatal rat brains. Beginning on the first postnatal day, rat brains were double immunostained with anti-carbonic anhydrase II and respective 'markers' for immature and mature astrocytes and oligodendrocytes. During the first postnatal week there were intensely carbonic anhydrase-positive cells which were ovoid or had broad processes. On the basis of their shapes and antigen contents these were considered to be precursors of oligodendrocytes. Beginning on the first postnatal day carbonic anhydrase II was also observed in some vimentin-positive radial glia and in other vimentin-positive cells that differed in their appearance from the immature oligodendrocytes. The vimentin-positive, carbonic anhydrase-positive cells were less smooth-surfaced, and had much finer processes, than the oligodendrocyte precursors. By the third postnatal day there appeared carbonic anhydrase-positive, glial fibrillary acidic protein (GFAP)-positive cells that resembled the vimentin-positive cells. It is concluded that the latter are immature astrocytes and that carbonic anhydrase is in distinct precursors of oligodendrocytes and astrocytes as early as the first postnatal day.     

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.     

Developmental fates and migratory pathways of dividing progenitors in the postnatal rat cerebellum

To investigate the developmental fates and the migratory pathways of dividing progenitors in both the white matter (WM) and the external granule layer (EGL) in the early postnatal rat cerebellum, a replication-deficient retrovirus carrying the β-galactosidase gene (BAG) was injected into the deep cerebellar tissue or the EGL of postnatal rats to label dividing progenitors. After 1–3 days post-injection (1–3 dpi) of BAG into the deep cerebellar tissue of postnatal day 4/5 (P4/5) rats, labeled immature, unipolar cells were found mainly in the WM. From 4 to 6 dpi, similar cells appeared in the internal granule (IGL), Purkinje cell, and molecular layers, although about half of the labeled cells still resided in the WM and appeared immature. The first morphologically definable Bergmann glia, astrocytes, and oligodendrocytes were also observed. From 14 to 20 dpi, most labeled cells had developed into Bergmann glia, astrocytes, oligodendrocytes, and interneurons in their appropriate layers. When BAG injections were performed at P14, unipolar cells were initially observed, but the majority of these differentiated into myelinating oligodendrocytes in the WM and IGL by 17 dpi. Few immature cells were labeled by injections administered at P20, and these did not develop into mature glia, but into cells with lacy, fine processes, possible representing immature oligodendrocytes. In contrast, BAG-labeled progenitors of EGL produced only granule neurons. Thus, within the first 2 postnatal weeks, dividing progenitors in the WM migrate as immature cells into the cortex before differentiating into a variety of glia and interneurons. The genesis of oligodendrocytes continues through the 2nd postnatal week and largely ceases by P20. EGL cells do not produce glia, but only granule cells. © 1996 Wiley-Liss, Inc.     

Immunohistochemical studies on cellular character of microtumors induced by ethylnitrosourea in the rat brain utilizing anti-leu 7 and anti-glial fibrillary acidic protein antibodies

Summary To clarify the chronologic changes in the cellular morphology of ENU-induced rat brain tumors, microtumors in the early stage were examined ummunohistochemically in comparison with macrotumors in the advanced stage. The tumor cells composing microtumors were negative for glial fibrillary acidic protein (GFAP), a specific marker of astrocylic cells, and Leu 7, a marker of oligodendrocytes, while cells of macrotumors were positive for either GFAP or Leu 7, showing characteristics of mature glial cells. The results suggested that the small round cells in the early devolopmental stage, generally thought to resemble mature oligodendrocytes, are not differentiated oligodendrocytes or astrocytes.     

Nitric oxide-mediated cGMP synthesis in oligodendrocytes in the developing rat brain

We investigated the nature of cGMP-synthesizing cells in the developing rat forebrain using cGMP-immunocytochemistry in combination with in vitro incubation of brain slices. When brain slices of immature rats, aged between 1 and 4 weeks, were incubated with sodium nitroprusside (SNP), a nitric oxide (NO) donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), small round cells with a few processes in and around the corpus callosum were visualized with the cGMP-antibody. The morphology and the distribution of the cGMP-positive cells were consistent with the criteria for oligodendrocytes. Furthermore, the cGMP-positive cells expressed 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and gelsolin, which are marker proteins for oligodendrocytes. Therefore, we concluded that the cGMP-positive cells were oligodendrocytes. A subpopulation of the oligodendrocyte was found to be cGMP-immunoreactive also when slices were incubated in the absence of SNP. Furthermore, incubation of the slice in the presence of L-NAME, an inhibitor of NO synthase, but in the absence of SNP abolished cGMP immunostaining. In addition, some populations of neurons and astrocytes in restricted brain areas produced cGMP in response to the incubation with SNP as previously reported, whereas both ameboid and ramified microglial cells did not respond to the treatment. Atrial natriuretic peptide, a stimulator of particulate guanylyl cyclase, enhanced cGMP synthesis in astrocytes in some brain regions but not in oligodendrocytes. These findings indicate that oligodendrocytes in the immature rat brain express soluble guanylyl cyclase. No cGMP-positive oligodendrocytes were found in the mature rat brain, suggesting that cGMP may mediate signals related to myelinogenesis in the rat brain. GLIA 19:286–297, 1997. © 1997 Wiley-Liss, Inc.     

Embryonic divergence of oligodendrocyte and astrocyte lineages in developing rat cerebrum

Oligodendrocyte and astrocyte lineages were traced in rat forebrain sections using single- and double-label immunoperoxidase and indirect immunofluorescent techniques. Antibodies were directed against antigenic markers, the expressions of which overlapped in time: GD3 ganglioside in immature neuroectodermal cells; vimentin in radial glia; glial fibrillary acidic protein (GFAP) in astrocytes; and carbonic anhydrase (CA) and galactocerebroside (GC) in oligodendrocytes. A histochemical stain for iron was also used as a marker of oligodendrocytes. Small cells of the subventricular zone (SVZ) were stained with anti-GD3 but not with the other antibodies. By 16 d of gestation (E16), the SVZ generated large, round cells and thick, process-bearing cells that were GD3+/CA+/iron+. These cells then appeared in the cingulum and, with time, increased in numbers and extended thick processes as they filled the subcortical white matter. These cells eventually lost their reactivity to anti-GD3 but became GC+/CA+ with processes extending to myelin sheaths. At E15 radial glia were stained with the anti-vimentin antibody but were negative for GFAP. At birth, only the vimentin+ radial glia midline between the 2 ventricles were GFAP+, but with time more vimentin+ cells became GFAP+. By 7 d of postnatal age all the vimentin+ cells were GFAP+ and had converged predominately on the cingulum. With time these cells condensed and took on characteristic shapes of astrocytes. The embryonic separation of the oligodendrocyte and the astrocyte lineage is supported by four pieces of evidence: (1) GD3+ cells were double labeled with anti-CA, and then went on to become GC+; (2) vimentin+ and GFAP+ cells were not also GD3+; (3) ultrastructural localization of anti-GD3 was confined to cells with characteristics consistent with developing oligodendrocytes; and (4) the shapes of GD3+, CA+, GC+, or iron+ cells did not resemble those of the vimentin+ or GFAP+ cells.     

Gliogenesis in rat spinal cord: evidence for origin of astrocytes and oligodendrocytes from radial precursors

We have examined glial cell lineages during rat spinal cord development by using a variety of antibodies that react with immature and mature glia. Radial glia in embryonic cord bound 1) A2B5, an antibody that reacts with a glial precursor cell population in optic nerve; 2) AbR24, which is directed against GD3 ganglioside and binds to immature neuroectodermal cells and to developing oligodendrocytes in forebrain and cerebellum; and 3) an antibody to the intermediate filament, vimentin. With time, two different populations emerged, both of which seemed to be derivatives of radial cells. One cell type expressed the astrocyte intermediate filament, GFAP, in addition to vimentin. GFAP-containing cells eventually took on the forms of astrocytes in gray and white matter. The other type expressed carbonic anhydrase, an enzyme characteristic of oligodendrocytes and enriched in myelin. Carbonic anhydrase-positive cells eventually developed into small cells with oligodendrocyte morphology. Our observations suggest a common lineage for astrocytes and oligodendrocytes from radial cells during spinal cord gliogenesis.     

Minigemistocytes and oligodendrocytic cells in mixed oligoastrocytomas: Immunohistochemical and Ultrastructural studies

In five mixed oligoastrocytomas, minigemistocytes and fibrous astrocytes showed similar immunoreactions to glial fibrillary acidic protein (GFAP), vimentin (VM) and cytokeratin. Some oligodendrocytic cells also exhibited immunoreactivities to these intermediate filaments. Intercellular myelin basic protein immunoreactive products were not associated with any tumor cell. Four recurrent tumors exhibiting GFAP and VM immunoreactions were found to consist of anaplastic astrocytes and irregular round cells with scanty cytoplasm rather than typical oligodendrocytic cells and minigemistocytes. Ultrastructurally, the three types of tumor cells showed cytoplasmic organelles in common, appearing light in electron density and exhibiting few characteristics of mature or immature oligodendrocytes. The number of intermediate filaments was the only significant ultrastructural difference among them; moderate to many in fibrous astrocytes and minigemistocytes, a few to moderate in some oligodendrocytic cells, and few in other oligodendrocytic cells. Immunohistochemically and ultrastructurally, minigemistocytes, GFAP-positive oligodendrocytic cells, and possibly GFAP-negative oligodendrocytic cells appeared to belong to an astrocytic lineage rather than transitional cells between oligodendrocytes and astrocytes.     

Phenotypic characterization of neural stem cells from human fetal spinal cord: synergistic effect of LIF and BMP4 to generate astrocytes

If cell based therapy for spinal cord injury is to become a reality, greater insights into the biology of human derived spinal cord stem cells are a prerequisite. Significant species differences and regional specification of stem cells necessitates determining the effects of growth factors on human spinal cord stem cells. Fetal spinal cords were dissociated and expanded as neurospheres in medium with bone morphogenetic protein 4 (BMP4), leukemia inhibitory factor (LIF) or BMP4 and LIF. First-generation neurospheres comprised a heterogeneous population of neural cell types and after plating emergent cells included neurons, oligodendrocytes and GFAP(+) cells which coexpressed stem cells markers and those of the neuronal lineage and were thus identified as GFAP(+) neural precursor cells (NPC). When plated, neurospheres maintained in BMP4 demonstrated a reduced proportion of emergent oligodendrocytes from 13 to 4%, whereas LIF had no statistically significant effect on cell type distribution. Combining BMP4 and LIF reduced the proportion of oligodendrocytes to 3% and that of neurons from 37 to 16% while increasing the proportion of GFAP(+) NPC from 45 to 79%. After 10 passages in control media aggregates gave rise to multiple neural phenotypes and only continued passage of neurospheres in the presence of BMP4 and LIF resulted in unipotent aggregates giving rise to only astrocytes. These results provide a means of obtaining pure populations of human spinal-cord derived astrocytes, which could be utilized for further studies of cell replacement strategies or in vitro evaluation of therapeutics.     

Postnatal cellular contributions of the hippocampus subventricular zone to the dentate gyrus, corpus callosum, fimbria, and cerebral cortex

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP(+) of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP(+) cells were observed migrating into the DG. Subsets of these EGFP(+) cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP(+) cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP(+) cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP(+) cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP(+)/RIP(+) oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis.     

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.     

Identification of glial cell proliferation in early multiple sclerosis lesions

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system which leads to destruction of myelin sheaths. The patterns of cell proliferation in the early course of the disease are largely unknown. The present study used immunohistochemical identification of proliferating glial cells in stereotactic brain biopsy material of eight patients with early chronic MS. Double-labelling with the proliferation marker MIB-1 detected proliferating oligodendrocytes (MOG), astrocytes (GFAP) and microglia/macrophages (Ki-M1P). The majority of proliferating cells were macrophages/microglia when compared with oligodendrocytes ( P >0.005) or astrocytes ( P >0.0005); only a minor proportion of microglia/macrophages, however, proliferated in situ . Astrocytic and oligodendroglial proliferation was sparse to absent and showed significant variations between different patients. There were statistically significant differences when comparing the amount of proliferation between lesions of different demyelinating activity: highest numbers of proliferating cells were found in early active lesions compared with demyelinated and early remyelinated lesions ( P >0.05) or the periplaque white matter ( P >0.01). MOG-positive oligodendrocytes proliferated occasionally in the early stages of lesion formation; this proliferation occurred in four cases but was independent of the stage of the disease. Since MOG is expressed by mature oligodendrocytes, and not by immature precursors, this might suggest a potential role for the proliferation of mature surviving oligodendrocytes with subsequent remyelination.     

Comparison of neural precursor cell fate in second trimester human brain and spinal cord

Neural transplantation holds promise for the treatment of traumatic brain and spinal cord injury by replacing lost cellular elements as well as repairing neural damage. Fetal human stem cells derived from central nervous system (CNS) tissue are potential transplantable sources for all cell types found in the mature human nervous system including neurons, astrocytes and oligodendroglia. Although nearly all areas of the fetal human neuraxis contain undifferentiated neural precursor cells, the phenotypic fate of the daughter cells might vary from one region to another during a specific developmental period. The purpose of this study was to compare the various cell types derived from neural precursors cultured from second trimester fetal human brain and spinal cord. To this end, brains (n = 8) and spinal cords (n = 8) of 15-24 week fetuses were dissociated and grown in culture medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (FGF) and leukemia inhibitory factor (LIF). The proliferating precursor cells from both brain and spinal cord grew as spherical masses that were plated on laminin-coated dishes after seven days in culture. During the next 5-7 days, the cells that emerged from these spheres were fixed and processed for immunocytochemistry. Brain derived spheres gave rise to cells expressing antigens specific for neurons (MAP-2ab and neuron specific-intermediate filaments), astrocytes (GFAP) and oligodendrocytes (A007). In contrast, cells that emerged from spinal cord derived spheres were only immunoreactive for GFAP. These data suggest that neuroepithelial precursor cells from different CNS regions, although similar in their responsiveness to proliferative growth factors, might differ in their ability to generate different cell types in the adult CNS.     

Selective apoptosis within the rat subependymal zone: a plausible mechanism for determining which lineages develop from neural stem cells

During development, the output of the subventricular zone (SZ) becomes increasingly restricted, yet it still harbors multipotential progenitors. The output of the SZ could be gated by selectively eliminating inappropriately specified progenitors. Using in situ end-labeling (ISEL) to identify apoptotic cells, nearly 60% of the ISEL(+) cells in the juvenile forebrain were localized to the SZ. Of these dying cells, at least 9% could be identified as neurons, 4% as astrocytes, and 12% as oligodendrocytes. The remainder were negative for the stem cell marker nestin, as well as other markers evaluated. To test the hypothesis that committed progenitors were under selective pressures, neural stem/progenitor cells were allowed to differentiate in vitro in the presence or absence of the caspase 3 inhibitor z-DEVD-fmk. DEVD increased neuronal production 10-fold over control cultures. By contrast, the development of astrocytes and oligodendrocytes was not affected. Altogether, these data support the hypothesis that selective forces within the postnatal rat forebrain control the types of precursors that emerge from the germinal matrix. Furthermore, they suggest that different mechanisms control neuronal versus glial cell numbers.     

Carnosine-like immunoreactivity in the central nervous system of rats during postnatal development

In the nervous system of adult rodents, the aminoacylhistidine dipeptides (carnosine and/or homocarnosine) have been shown to be expressed in three main populations of cells: the mature olfactory receptor neurons, a subset of glial cells, and the neuroblasts of the rostral migratory stream. The current study analyzed the distribution of these dipeptides during postnatal development within the rat brain and spinal cord focusing on their pattern of appearance in the glial cells. Double staining methods using antibodies against carnosine and some markers specific for immature (vimentin) and mature (glial fibrillary acidic protein and Rip) glial cell types were used. Glial immunostaining for the aminoacylhistidine dipeptides appears starting from postnatal day 6 and reaches the final distribution in 3-week-old animals. The occurrence of carnosine-like immunoreactivity in astrocytes lags behind that in oligodendrocytes suggesting that, as previously demonstrated by in vitro studies, oligodendrocytes are also able to synthesize carnosine and/or homocarnosine in vivo. Furthermore, the spatiotemporal patterns observed support the hypothesis that the production of these dipeptides coincides with the final stages of glia differentiation. In addition, a strong carnosine-like immunoreactivity is transiently seen in a small population of cells localized in the hypothalamus and in the subfornical organ from birth to postnatal day 21. In these cells, carnosine-like immunoreactivity was not colocalized with any of the glial specific markers used. Moreover, no evidence for colocalization of carnosine and gonadotropin-releasing hormone (GnRH) has been observed.     

Localization of Pi class glutathione-S-transferase in the forebrains of neonatal and young rats: evidence for separation of astrocytic and oligodendrocytic lineages.

The Yp isoform (Pi class) of glutathione-S-transferase has recently been localized in oligodendrocytes in the brains of mature rats. To examine at what postnatal age Pi first appears in oligodendrocytes or precursor cells, antibodies against Pi were used to immunostain tissue sections from the forebrains of neonatal rats and young rats up to 17 days of age. In the brains of neonates Pi immunofluorescence was observed in ovoid cells in the subependymal layer, and in ovoid cells and cells bearing short, thick processes in the corpus callosum and cingulum. These cells did not immunostain for vimentin. During the first postnatal week Pi-positive cells showed positive immunostaining for ganglioside GD3, which is characteristic of oligodendrocyte precursors, and process-bearing Pi-positive cells appeared in the cingulum and at the lateral borders of the corpus callosum in increasing numbers. During the second postnatal week the cytoplasm of Pi-positive cells became more compact, and the processes thinner, and the Pi-positive cells and their processes began to immunostain for 2',3'-cyclic nucleotide-3'-phosphohydrolase, which is characteristic of immature and mature oligodendrocytes and myelin sheaths. By age 17 days Pi was observed in relatively mature oligodendrocytes. The observations suggest that Pi occurs in oligodendrocyte precursors, immature oligodendrocytes, and mature oligodendrocytes in the postnatal through 17 day old rat forebrain. In the accompanying paper (Cammer and Zhang, '92)--if references are permitted in the Abstract a different glutathione-S-transferase isoform, Yb (Mu class), was localized in cells of the astrocyte lineage, beginning in the forebrains of neonatal rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures

Exposure to environmental toxicants during vulnerable windows of brain development is suspected to raise the prevalence for neurological dysfunctions at later stages in life. Differentiation processes and changes in morphology, as well as a lack of physiological barriers, might be reasons that render a developing brain more susceptible to neurotoxicants than an adult. However, also the intrinsic capacity of cells to combat toxicant induced cellular stress might differ between the immature- and mature brain. In order to study whether this intrinsic protection capacity differs between immature and maturated brain cells we chose to study the maturation-dependent adverse effects of the known neurotoxicant Paraquat Dichloride (PQ) in 3D rat brain cell cultures. This in vitro system consists of the major brain cell types – neurons, astrocytes, oligodendrocytes and microglia – and over the time in vitro cultured cells undergo differentiation and maturation into a tissue-like organization. PQ was applied repeatedly over ten days in the sub-micromolar range, and effects were evaluated on neurons and glial cells. We observed that despite a higher PQ-uptake in mature cultures, PQ-induced adverse effects on glutamatergic-, GABAergic- and dopaminergic neurons, as assessed by gene expression and enzymatic activity, were more pronounced in immature cultures. This was associated with a stronger astrogliosis in immature- as compared to mature cultures, as well as perturbations of the glutathione-mediated defense against oxidative stress. Furthermore we observed evidence of microglial activation only in mature cultures, whereas immature cultures appeared to down-regulate markers for neuroprotective M2–microglial phenotype upon PQ-exposure. Taken together our results indicate that immature brain cell cultures have less intrinsic capacity to cope with cellular stress elicited by PQ as compared to mature cells. This may render immature brain cells more susceptible to the adverse effects of PQ.     

Fractionation and enrichment of oligodendrocytes from developing human brain.

Enriched cultures of human oligodendrocytes were obtained from fetal brain specimens between 16 and 21 gestational weeks. Brain cells were separated over a Percoll density gradient and collected as two fractions with initial relative densities of approximately 1.035 g/ml and 1.102 g/ml, for fractions 1 and 2, respectively. After separation, 58.3 and 67.7% of the cells in fractions 1 and 2, respectively, were labeled by the antibody O4 that recognizes immature oligodendrocytes. A total of 15.5 and 29.4% of the cells in fractions 1 and 2, respectively, were positive for tubulin-beta(III), a marker for neurons but none of the freshly isolated cells were positive for glial fibrillary acidic protein (GFAP), a protein associated with astrocytes in the central nervous system. When the fractionated cells were cultured on poly-ornithine coated coverslips for 3 days and processed for immunocytochemistry, the percentage of O4+ oligodendrocytes decreased to less than 4% whereas GFAP+ cells increased to 1.8 and 12.4% for fractions 1 and 2 respectively. The percentage of tubulin-betaIII+ cells increased to 46 and 61% in cultures from the two Percoll fractions. This increase is probably due to the decrease in the number of oligodendrocytes. To avoid the loss of oligodendrocytes, cells were cultured as free-floating aggregates in the presence of 20 ng/ml of fibroblast growth factor-2 for 2 weeks. The resultant cultures became enriched for oligodendrocytes as demonstrated by cellular morphology and by positive O4 labeling. The method described here provides a means of obtaining enriched cultures of immature human oligodendrocytes for developmental and transplantation studies.     

Long-lasting coexpression of nestin and glial fibrillary acidic protein in primary cultures of astroglial cells with a major participation of nestin(+)/GFAP(-) cells in cell proliferation

Nestin, a currently used marker of neural stem cells, is transiently coexpressed with glial fibrillary acidic protein (GFAP) during development and is induced in reactive astrocytes following brain injury. Nestin expression has also been found in cultures of astroglial cells, but little is known about the fate and the mitotic activity of nestin-expressing cells in this in vitro model. The present study reveals a long-lasting expression of nestin in primary cultures of astroglial cells derived from the rat brain. Over 70% of the cells were nestin(+) at 12 weeks, with a large majority coexpressing the GFAP astrocytic marker. Time-course analyses supported a transition from a nestin(+)/GFAP(-) to a nestin(+)/GFAP(+) phenotype over time, which was further increased by cell cycle arrest. Interestingly, double staining with Ki67 revealed that over 90% of cycling cells were nestin(+) whereas only 28% were GFAP(+) in a population consisting of almost equivalent numbers of nestin(+) and GFAP(+) cells. These observations indicated that nestin(+)/GFAP(-) cells are actively engaged in mitotic activity, even after 2 weeks in vitro. Part of these cells might have retained properties of neural stem cells, insofar as 10% of cells in a primary culture of glial cells were able to generate neurospheres that gave rise to both neurons and astrocytes. Further studies will be necessary to characterize fully the proliferating cells in primary cultures of glial cells, but our present results reveal a major contribution of the nestin(+)/GFAP(-) cells to the increase in the number of astrocytes, even though nestin(+)/GFAP(+) cells proliferate also.