Extracellular matrix of cultured glial cells: selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.     

Localization of hyaluronate in primary glial cell cultures derived from newborn rat brain

We have devised a technique that enables one to localize hyaluronate in cultured cells. Cells were probed with the glial hyaluronate binding protein (GHAP) which was itself then visualized by conventional indirect immunofluorescence. The hyaluronate binding properties of this protein have been established. This technique was applied to the study of hyaluronate synthesis in glial cells. These cells do not themselves produce GHAP. O-2A progenitor cells were obtained from the cerebral hemispheres of newborn rats. These cells are bipotential in that they are able to differentiate into either oligodendrocytes or type 2 astrocytes depending on the composition of the culture medium. In cultures of O-2A progenitor cells maintained in the absence of serum, in which large numbers of oligodendrocytes appeared, very little hyaluronate was produced. The galC+ cells were invariably hyaluronate negative. Cultures of the same cells, maintained in the presence of 10% FCS, contained large numbers of hyaluronate producing cells. The hyaluronate producing cells were typically small, process-bearing, and GFAP+. Some, but not all, were A2B5+ and could, therefore, be identified as type 2 (GFAP+, A2B5+) astrocytes. Type 1 (GFAP+, A2B5-) astrocytes were also active in the synthesis of hyaluronate, to the extent that they were able to coat their substrate with hyaluronate. Among cells of the O-2A lineage, then, hyaluronate production would appear to be restricted to astrocytes. This may have some bearing on the origin of hyaluronate in the extracellular matrix of CNS white matter.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte-Enriched Cultures from Rat Brain

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.     

A clonal analysis of glial lineages in neonatal forebrain development in vitro

Retrovirus-mediated gene transfer combined with triple immunostaining for astro- and oligodendroglial markers (antibodies to glial fibrillary acidic protein, GD3 ganglioside, and galactocerebroside, and the O4 antibody) was used to study clonal aspects of glial lineage in primary cultures of the neonatal rat striatum. We found two major clonal populations: astrocyte clones containing GFAP+, but GD3-, O4-, and GC- cells, and oligodendrocyte clones containing cells expressing various combinations of GD3, O4, and GC, with rare GFAP+ cells. These results indicate that astrocytes and oligodendrocytes belong to separate lineages in forebrain postnatal development.     

Problems encountered when immunocytochemistry is used for quantitative glial cell identification in autoradiographic studies of cell proliferation in the brain of the unlesioned adult mouse

We have used sections of adult mouse brain to determine whether antibodies specific for oligodendroglia (anti-carbonic anhydrase II, CA II; anti-galactocerebroside, GC; anti-myelin basic protein, MBP) and astroglia (anti-glial fibrillary acidic protein, GFAP; anti-S 100 protein) are suitable for quantitative studies of the proliferation and subsequent differentiation of these cells. Unlesioned adult mice received a single injection of ³H-thymidine (TdR) and were killed between 1 h and 70 days later. Quantitative evaluations of autoradiographs of 2-m-thick serial sections stained immunocytochemically with the antibodies mentioned above or with Richardson's method for histological control led to the following conclusions. Anti-GC and anti-MBP stained only the oligodendrocytic processes and, thus, cannot be used in well-myelinated brain areas. Anti-CA II stained only a portion of the differentiated oligodendrocytes, but no proliferating cells. Anti-S 100 protein recognized all the astrocytes, but also many (interfascicular) oligodendrocytes. Anti-GFAP stained only a few astrocytes in the unlesioned mouse: all astrocytes may become GFAP-immunopositive only after wounding the brain. Thus, in contrast to in vitro studies, immunocytochemical studies with these antibodies on sections of adult animals cannot be recommended for the quantitative analysis of cell proliferation. In addition, our results show that differentiated glial cells proliferate in adult mice. Astro- and oligodendrocytes divide with the same cell cycle parameters and mode of proliferation up to about 1 month after ³H-TdR injection. In contrast to oligodendrocytes, some astrocytes might re-enter the cycle after a few weeks of quiescence.     

A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: in vivo evidence for two distinct astrocyte lineages

We have shown previously that three antibodies--anti-galactocerebroside (GC), anti-glial fibrillary acidic protein (GFAP), and the A2B5 monoclonal antibody--can be used to help distinguish three classes of glial cells in the rat optic nerve: oligodendrocytes are GC+, GFAP-, almost all type-1 astrocytes are A2B5-, GFAP+, and almost all type-2 astrocytes are A2B5+, GFAP+. In the present study we have used these antibodies to examine the timing and sequence of the development of the three types of glial cells in vivo. We show that type-1 astrocytes first appear at embryonic Day 16 (E16), oligodendrocytes at birth (E21), and type-2 astrocytes between postnatal Days 7 and 10 (P7-10). Moreover, we demonstrate quantitatively that astrocytes in the optic nerve develop in two waves, with more than 95% of type-1 astrocytes developing before P15 and more than 95% of type-2 astrocytes developing after P15. Finally, we provide indirect evidence that type-2 astrocytes do not develop from type-1 astrocytes in vivo, supporting previous direct evidence that the two types of astrocytes develop from two serologically distinct precursor cells in vitro.     

Macroglial cell development in embryonic rat brain: studies using monoclonal antibodies, fluorescence activated cell sorting, and cell culture

Astrocytes, ependymal cells, and oligodendrocytes have been shown to develop on the same schedule in dissociated cell cultures of early embryonic rat brain as in vivo. Subsequent studies showed that there are two major types of astrocyte (type-1 and type-2), which, in cultures of perinatal optic nerve, develop as two distinct lineages. In such cultures, type-2 astrocytes and oligodendrocytes develop from the same, bipotential, (O-2A) progenitor cells, which differentiate into type-2 astrocytes in 10% fetal calf serum (FCS) and into oligodendrocytes in less than or equal to 0.5% FCS. In light of these findings, we now have extended our studies on macroglial cell development in rat brain and show the following: (i) The first astrocytes to develop have a type-1 phenotype, while astrocytes with a type-2 phenotype do not develop until almost 2 weeks later, just as in the optic nerve. (ii) Most importantly, type-2 astrocytes, like the other macroglial cells, develop on the same schedule in cultures of early embryonic (less than or equal to E15) brain as they do in vivo. (iii) By contrast, both oligodendrocytes and type-2 astrocytes develop prematurely in cultures of E17 brain, and FCS influences this development in the same way it does in perinatal optic nerve cultures. (iv) Type-2 astrocyte precursors are labeled by the A2B5 monoclonal antibody, as shown previously for oligodendrocyte precursors in brain and for O-2A progenitor cells in optic nerve. Taken together with our previous findings, these results suggest that oligodendrocytes and type-2 astrocytes in brain develop from bipotential O-2A progenitor cells, whose choice of developmental pathway and timing of differentiation depend on mechanisms that operate independently of brain morphogenesis.     

EIF2B5 mutations compromise GFAP+ astrocyte generation in vanishing white matter leukodystrophy

Vanishing white matter disease (VWM) is a heritable leukodystrophy linked to mutations in translation initiation factor 2B (eIF2B). Although the clinical course of this disease has been relatively well described, the cellular consequences of EIF2B mutations on neural cells are unknown. Here we have established cell cultures from the brain of an individual with VWM carrying mutations in subunit 5 of eIF2B (encoded by EIF2B5). Despite the extensive demyelination apparent in this VWM patient, normal-appearing oligodendrocytes were readily generated in vitro. In contrast, few GFAP-expressing (GFAP+) astrocytes were present in primary cultures, induction of astrocytes was severely compromised, and the few astrocytes generated showed abnormal morphologies and antigenic phenotypes. Lesions in vivo also lacked GFAP+ astrocytes. RNAi targeting of EIF2B5 severely compromised the induction of GFAP+ cells from normal human glial progenitors. This raises the possibility that a deficiency in astrocyte function may contribute to the loss of white matter in VWM leukodystrophy.     

溶血磷脂酸对离体培养的大鼠胚胎神经干细胞向神经胶质细胞分化的影响

本研究用免疫细胞化学荧光双标技术观察了溶血磷脂酸（lysophosphatidic acid,LPA）对大鼠胚胎神经干细胞（neural stem cells,NSCs）分化为少突胶质细胞（galactocerebroside—positive,Gal-C阳性）和星形胶质细胞（grim fibrillary acidic protein-positive,GFAP阳性）的影响,并且用RT-PCR技术对NSCs可能表达的LPA受体进行分析。结果显示：（1）加入不同浓度（0．010．0μmol／L）LPA,第7天进行检测时,少突胶质细胞数量呈明显的剂量依赖性增加,峰值出现在1．0μmol／LLPA组,少突胶质细胞所占百分比从对照组的8．5％增加到32．6％;（2）星形胶质细胞的分化几乎不受LPA的影响,第7天时各LPA处理组星形胶质细胞百分比与对照组相比均无显著性差异;（3）RT-PCR结果显示,大鼠胚胎NSCs的LPA1和LPA3受体表达明显,而LPA3受体表达很弱。以上结果表明,较低浓度的LPA可能作为细胞外信号,通过LPA1和LPA3受体促进大鼠胚胎NSCs向少突胶质细胞分化和生成,但对星形胶质细胞的分化过程无明显影响。     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

Regulation of glial development by cystatin C

Cystatin C (CysC) is an endogenous cysteine proteases inhibitor produced by mature astrocytes in the adult brain. Previously we isolated CysC as a factor activating the glial fibrillary acidic protein (GFAP) promoter, and showed that CysC is expressed in astrocyte progenitors during development. Here we show that protease inhibitor activity increased daily in conditioned medium, and that this activity was mainly a result of CysC released from primary cultured cells. Human CysC added to the culture medium of primary brain cells increased the number of GFAP-positive and nestin-positive cells. Human CysC also increased the number of neurospheres formed from embryonic brain, and thus it increases the number of neural stem/precursor cells in a manner similar to glycosylated rat CysC. The addition of a neutralizing antibody, on the other hand, greatly decreased the number of GFAP and glutamate aspartate transporter (GLAST)-positive astrocytes. This decrease was reversed by the addition of CysC but not by another cysteine protease inhibitor. Thus, the promotion of astrocyte development by CysC appears to be independent of its protease inhibitor activity. The antibody increased the number of oligodendrocytes and their precursors. Therefore, CysC modifies glial development in addition to its activity against neural stem/precursor cells.     

Immunohistochemical localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in adult bovine cerebrum and cerebellum

We have previously shown that 2,3-cyclic nucleotide 3-phosphodiesterase (CNP; EC 3.1.4.37) in rat central nervous tissues can be immunohistochemically stained with anti-bovine CNP serum. However, the anti-bovine CNP serum prepared in our laboratory has only weak cross-reactivity with rat CNP. Sections of bovine nervous tissues were found to be stained effectively with the serum, and the localization of CNP has been revealed in greater detail. We describe here the immunohistochemical localization of CNP in adult bovine cerebrum and cerebellum. CNP stained was localized in myelin sheaths, oligodendrocytes, and the processes of oligodendrocytes; astrocytes and neurons were negative. All myelinated nerve fibers appeared to be stained with the anti-CNP serum. Perineuronal and perivascular oligodendrocytes, and oligodendrocytes extending their processes to isolated myelin fibers were stained. Interfascicular oligodendrocytes, however, did not react or reacted faintly to the anti-CNP serum; only their processes were reactive. Comparison with the stain for S-100 protein was helpful to distinguish oligodendrocytes from astrocytes particularly when both glial cells were situated together at the perineuronal and perivascular positions.Dedicated to Professor Yasuzo Tsukada.     

An inducer protein may control the timing of fate switching in a bipotential glial progenitor cell in rat optic nerve

In rat optic nerve, oligodendrocytes and type-2 astrocytes develop from a common (O-2A) progenitor cell. The first oligodendrocytes differentiate at birth, while the first type-2 astrocytes differentiate in the second postnatal week. We previously showed that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell. Here we provide evidence that the timing of type-2 astrocyte differentiation, by contrast, may depend on an inducing protein that appears late in the developing nerve. We show that extracts of 3- to 4-week-old, but not 1-week-old, rat optic nerve contain a protein (apparent Mr approximately 25,000) that induces O-2A progenitor cells in culture to express glial fibrillary acidic protein (GFAP), an astrocyte-specific marker in the rat central nervous system.     

Reconstitution of a developmental clock in vitro: a critical role for astrocytes in the timing of oligodendrocyte differentiation

The rat optic nerve contains three types of macroglial cells: type 1 astrocytes first appear at embryonic day 16 (E16), oligodendrocytes at birth (E21), and type 2 astrocytes between postnatal days 7 and 10. The oligodendrocytes and type 2 astrocytes develop from a common, bipotential O-2A progenitor cell. We show here that although O-2A progenitor cells in E17 optic nerve prematurely stop dividing and differentiate into oligodendrocytes within 2 days in culture, when cultured on a monolayer of type 1 astrocytes, they continue to proliferate; moreover, the first cells differentiate into oligodendrocytes after 4 days in vitro, which is equivalent to the time that oligodendrocytes first appear in vivo. Our findings suggest that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell that counts cell divisions that are driven by a growth factor (or factors) produced by type 1 astrocytes.     

A new “marker” protein for astrocytes

A monoclonal antibody (Mab J1-31) has been produced by using human brain homogenate as immunogen in mouse. Double-label immunofluorescence microscopy on cryostat sections of human, rabbit and rat brain, reveals staining of cells that are also stained with antiserum to glial fibrillary acidic protein (GFAP, a commonly used marker protein for astrocytes). However, there is no decrease in staining due to Mab J1-31 in sections incubated in antiserum to GFAP prior to incubation with the J1-31 ascites fluid. Immunoprecipitation of aqueous and detergent extracts of brain homogenate gives a single band at 30K by SDS PAGE followed by autoradiography. Immunoelectron microscopy shows that the J1-31 antigen is associated with the cytoskeleton. Thus, the Mab J1-31 recognizes a new protein present in GFAP positive cells (astrocytes) in the brain.