Influence of monensin on ganglioside anabolism and neurite stability in cultured chick neurons

We have examined the effects of monensin, a monovalent cationophore that disrupts exo- and endocytosis of membrane vesicles and diminishes Golgi anabolic function, on the incorporation of [3H]-galactose into glycosphingolipids in neurited primary cultures of chick embryo central nervous system neurons. A linear rate of incorporation into all ganglioside species from extracellular-labeled galactose was observed. Specific activity of anabolic labeling was markedly lower in GT1b and GQ1b than in the other major gangliosides of the embryonic neuron (GM3, GD3; GM2, GD2; GM1, GD1a, GD1b). With 100 nM monensin in the extracellular medium, the rate of labeling of GT1b diminished markedly to 20% of control; GD1a, GD1b, and GD2, to 35%; GQ1B to 48%; GD3 to 60%. Vigorous incorporation of label into GM3 was entirely undiminished by monensin. From these findings, it is suggested that ganglioside biosynthesis is compartmentalized in the cytodifferentiating embryonic neuron, with GM3 entirely, and GD3 and GQ1b partially, an extra-Golgi product. Extensive loss of neurites that occurred after several hours of exposure of the neurons to monensin could not be correlated directly with decreased ganglioside anabolism.     

Metabolism and function of gangliosides in developing neurons

Previous experiments have shown that the addition of a mixture of gangliosides to the growth medium induced morphological changes in primary neuronal cultures, producing especially a trophic effect and a sprouting of neurites (neuritogenesis). The study reported here examined the changes of some biochemical parameters that paralleled the morphological modifications of cultured neurons from chick brain hemispheres treated with gangliosides. Neurons cultured from 3 to 7 days in the presence of various concentrations of a purified mixture or of single-species of gangliosides (GM1, GD1a, GT1b) revealed that these glycolipids were easily incorporated into the cells as a function of their exogenous concentrations. Incubation of neurons with N-acetyl-D-[U-14C]mannosamine showed a final labeling of all endogenous cellular and exogenous incorporated gangliosides; however, the radioactivity recovered decreased as a function of the number of sialic acid units of the exogenously added gangliosides. The treatment of neuronal cells from 3 to 7 days in culture with a mixture of 10(-8) M and 10(-5) M gangliosides led to the following observations on some neurochemical parameters: no effect on the influx of choline and dopamine; no effect on the spontaneous choline efflux, whereas the K+-provoked one is abolished; decrease of the spontaneous and K+-stimulated release of dopamine; no effect on the spontaneous release of GABA for 10(-8) M gangliosides but an increase of both spontaneous and K+-provoked release for 10(-5) M gangliosides. The data suggest that the possible insertion of gangliosides into the neuronal membranes may imply structural modifications that may influence enzymatic activities, neurotransmitter transport, and finally, some nerve cell mechanisms.     

Ganglioside potentiation of NGF-independent conditioned medium enhancement of neuritic outgrowth from spinal cord and ciliary ganglia explants

Culture medium conditioned (CM) by embryonic chick skeletal muscle or RN22 Schwannoma cells enhanced dramatically the neuritic development of chick embryonic spinal cord slices explanted onto a collagen substratum. The addition of a mixture of bovine brain gangliosides (BBG) or the monosialoganglioside GM1 to this medium potentiated the nerve growth factor (NGF)-independent CM-mediated neuritogenesis. A 3-4 fold increase in spinal cord outgrowth was due to increased neurite number, length and branching. The ability of the gangliosides to potentiate the positive neuritogenic action of CM was not limited solely to spinal cord cultures since similar results were obtained in parallel studies employing organized cultures of embryonic chick ciliary ganglia. These studies demonstrate the ability of gangliosides to enhance the trophic action of factor(s) present in CM. They suggest further that gangliosides may play a modulatory role in the development of the nervous system.     

In vitro modulation of changes in ganglioside patterns of differentiating neurons in the presence of an anti-GM1 antibody

Retinal cells from 7-day-old chicken embryos were cultured in the presence of a polyclonal anti-GMI antibody, at low and high density in a “sandwich cell culture”. Cells that were about 80% neurofilament positive at all times, changed their morphology and emitted processes as controls. By examining immunocytochemical expression of gangliosides, cells cultured in the presence of the antibody maintained GD3 expression longer than controls, albeit the expression of the gangliotetraosylgangliosides (GTOG) was not considerably affected. This leads to an extension of the transient period in which differentiating cells coexpressed both types of gangliosides (GD3 and GTOG). At 3–4 days in vitro the relative synthesis of GD3 was about 30% higher and that of GD1a about 40% lower than in controls, indicating a delay in the shift of the synthesis pattern. Nevertheless, the pattern of ganglioside composition resembled at 4 days in vitro. Results indicate that the anti-GMI antibody may modulate the expression and synthesis of gangliosides without a detectable decrease in neuritogenesis. Considering that the emission of neurites occurs in coexpressing GD3 and GTOG neurons, it is suggested that neuritogenesis could be irrespective of losing the GD3 expression.     

Ethanol-induced increase of sphingosine recycling for ganglioside biosynthesis: a study performed on cerebellar granule cells in culture

The effect of ethanol on ganglioside metabolism was assessed in cultured rat cerebellar granule cells. Cells were incubated in the presence of tritiated serine or galactose, and the synthesis of radioactive gangliosides was followed. The rate of de novo biosynthesis of gangliosides labeled in the oligosaccharide moiety (deriving from tritiated galactose) was not affected by the presence of ethanol. On the contrary, the biosynthesis of gangliosides labeled in the ceramide long chain base moiety (deriving from tritiated serine), dramatically decreased in the presence of alcohol. These results suggest that the gap between the extent of the biosynthesis of lipid and polar portions observed in the presence of ethanol, is filled by an increased recycling of sphingosine produced from ganglioside degradation. This hypothesis was confirmed by pulse-chase experiments with GM1 ganglioside, tritiated in the sphingosine moiety, and following radiolabeled gangliosides deriving from its metabolic processing. In fact, the radioactivity carried by gangliosides whose labeling could derive exclusively (GD1b + GT1b) or partially (GD1a) from the recycling of catabolic radiolabeled sphingosine, dramatically increased in ethanol-treated cells during the chase period. Taken together, these results suggest that ethanol increases ceramide sphingosine recycling for ganglioside biosynthesis.     

Sigma-1 receptors potentiate epidermal growth factor signaling towards neuritogenesis in PC12 cells: potential relation to lipid raft reconstitution

We previously demonstrated that overexpression of sigma-1 receptors (sigma-1R) potentiated neurite sprouting caused by nerve growth factor in PC12 cells (Takebayashi et al. 2002 J Pharmacol Exp Ther 202:1227-1237). In this study we examined if sigma-1R may be involved in the action of epidermal growth factor (EGF). EGF is conventionally recognized as a mitogenic factor that stimulates only the proliferation of various types of cells, including PC12 cells. We found here that in sigma-1 receptor-overexpressing PC12 cells (sigma-1R OE cells), EGF markedly stimulates neuritogenesis without affecting cellular proliferation. EGF receptors (EGFR) are largely reduced in lipid rafts and are enriched in non-raft regions in sigma-1R OE cells. The enrichment of EGFR in the non-raft region is correlated with enhanced downstream signaling of EGFR including the phosphorylation of both EGFR and extracellular signal-regulated kinases (ERKs). Destruction of cholesterol-containing rafts by treating cells with methyl-beta-cyclodextrin also causes a reduction of EGFR in lipid rafts, a concomitant increase in the phosphorylation of both EGFR and ERK, and an increase in the EGF-induced neurite sprouting in wildtype cells. Furthermore, while overexpression of sigma-1R increases the level of lipid raft-associated cholesterol, the overexpression alters the levels of gangliosides in lipid rafts: GM1 and GM2 are decreased, whereas GD1a is increased. We conclude that sigma-1R cause the remodeling of lipid rafts, at least by increasing the level of lipid raft-associated cholesterol and by altering the levels of certain critical lipid raft-forming gangliosides. sigma-1R may thus play an important role in directing EGF signaling towards neuritogenesis, perhaps by shifting EGFR from the lipid raft into non-raft regions.     

Interactive effects of insulin, IGF-I and GMl ganglioside on growth of PC12 cells

PC12 cells grow in serum-free medium (SFM) if high doses of insulin (5-50 μg/ml), or combined insulin (5 μg/ml) and insulin-like growth factor-I (3 ng/ml) are added daily. Ganglioside GMl counteracts these effects in a dose-responsive fashion between 10 nM and 50 μM. This provides a serum-free system to study the interactive effects of gangliosides on growth and neuritogenesis in PC12 cells.     

Gangliosides in lesion-induced synaptogenesis: studies in the hippocampus of the rat brain

Changes in ganglioside composition, biosynthesis and individual distribution were studied in hippocampal regions after unilateral destruction of the entorhinal cortex. After 1 and 3 days postlesion (dpl), a decrease in ganglioside content was detected in area dentata (AD) and pyramidal cell regions CA1-CA3 (CA), both ipsilateral and contralateral to the lesion. By 5 dpl all the values had returned to control values, except in AD which showed a dramatic increase in total ganglioside content reaching a maximum at 12 dpl. By 30 dpl this area also showed control content. A significant increase in biosynthesis of gangliosides was observed at 5 and 8 dpl in the hippocampus ipsilateral to the lesion without changes in the contralateral counterpart. Individual ganglioside distribution showed a pronounced change in GM1 and GQ1b with small changes in the other major gangliosides. Significant differences were observed in the distribution of gangliosides between the two hippocampal regions studied in unoperated control animals. GD1a was more concentrated in AD, whereas GQ1b, GT1b and GD1b predominated in CA. The data presented here indicate that important modifications in ganglioside content as well as pattern occur in the deafferented hippocampus a phenomenon that could be related with the known effect of gangliosides on neuritogenesis observed in cell culture studies.     

Gangliosides of cultured astroglia

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.     

Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Lithium inhibits apoptosis of mouse neural progenitor cells

As undifferentiated precursor cells in the CNS, neural progenitor cells (NPCs) supply new neurons and glial cells to repair damage within the adult brain. Recently, NPCs were found to undergo apoptosis. In serum-free basic fibroblast growth factor-containing culture medium, primary culture cells from fetal mouse neuroepithelium expressed nestin, and thus might be regarded as NPCs. These NPCs demonstrated apoptosis under electron microscopy and TUNEL assay. Treatment of NPCs with lithium, a specific inhibitor of glycogen synthase kinase 3beta (GSK3beta), significantly suppressed apoptosis. Activity of pro-apoptotic protease caspase-3 was not detected in either lithium-untreated or -treated NPCs. These findings suggest that lithium may protect NPCs against apoptosis by inhibiting caspase-3-independent apoptotic pathways.     

Expression of gangliosides on glial and neuronal cells in normal and pathological adult human brain

Few studies have assessed the glycolipid phenotype of glial cells in the human central nervous system (CNS) in situ. We investigated by immunohistochemistry the expression and cellular distribution of a panel of gangliosides (GM1, GM2, acetyl-GM3, GD1a, GD1b, GD2, GD3, GT1b, GQ1b and the A2B5 antibody) in adult, human normal and pathological brain, namely multiple sclerosis (MS) and other neurological diseases (OND). In normal conditions, we found diffuse expression in the white matter of most gangliosides tested, with the exception of acetyl-GM3, GT1b and GQ1b. By double immunofluorescence with phenotypic markers, GM1 and GD1b were preferentially expressed on GFAP+ astrocytes, GD1a on NG2+ oligodendrocyte precursors, A2B5 immunostained both populations, while GD2 was selectively present on mature oligodendrocytes. In the gray matter, only GM1, GD2 and A2B5 were present on neuronal cells. Interestingly, those gangliosides present on astrocytes in normal conditions were preferentially expressed on NG2+ cells in chronic MS lesions and in OND. Selective expression of GT1b upon astrocytes and NG2+ cells was instead observed in MS lesions, but not in OND. The definition of the glycolipid phenotype of CNS glial cells may be useful to identify distinct biological glial subsets and provide insights on the potential autoantigenic role of gangliosides in CNS autoimmune diseases.     

Effect of colchicine on ganglioside composition of rat primary culture neurons.

Developmental changes in gangliosides in the course of neurite outgrowth were examined in dissociated fetal rat cerebral neurons in culture. About a 2-fold increase in ganglioside levels was seen with the progression of neurite formation for up to 24 h in predominantly neuronal cultures. Ganglioside patterns appeared to be unchanged during the first 24 h, subsequently consisted of higher amounts of GD3 and b-series gangliosides (such as GD1b, GT1b, and GQ1b), and lower amounts of a-series gangliosides (GM1 and GD1a). Although the addition of colchicine to the cell growth medium inhibited neurite outgrowth in developing neurons, little if any differences in ganglioside patterns were found between control and colchicine-treated cells. Ganglioside levels decreased slightly in colchicine-treated cells in agreement with the decrease in cell attachment to culture dishes. Although colchicine treatment 8 h after plating caused complete retraction of formed neurites, the ganglioside level of the cells continued to increase during the following 16-hour incubation. Thus, the data suggest that ganglioside synthesis in differentiating neurons does not primarily accompany the expansion in cell surfaces due to neurite formation, and raises the possibility that a large proportion of gangliosides is retained in intracellular compartments.     

Ataxic neuropathy associated with disialosylated antibodies: description of new clinical and biochemical forms

INTRODUCTION: Polyneuropathies associated with IgM monoclonal gammopathy were recently recognized. Antibodies can react with glycoproteins such as myelin associated glycoprotein (MAG), or gangliosides containing one sialosyl epitope such as GM1 or several sialosyl epitopes (polysialyted gangliosides) including GD2, GD3, GT1b, GT1a, GQ1b. METHODS: We report on three patients presenting oculomotor dysfunction, chronic sensitive ataxic polyneuropathy, high sedimentation rate, IgM monoclonal paraprotein of unknown signification and antidisialosyl IgM antibodies and for two of them cold agglutinins. Such features have been previously described under the acronym "CANOMAD" (chronic ataxic neuropathy with ophthalmoplegia, M protein, agglutination and disialosyl antibodies). RESULTS: One of the patients presents extramembranous glomerulopathy and severe motor disability associated with this syndrome. The pathophysiology of the glomerulopathy seems to be linked with the polyneuropathy. Patients were treated either by intravenous immunoglobulin, corticosteroids or cyclophosphamid. Response to treatment differs in the three cases and there is currently no consensus. CONCLUSION: Our study demonstrates that spectrum of polyneuropathy associated with monoclonal polyneuropathy may be larger than originally described.     

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.     

Glycolipid sialosyltransferase activity in synaptosomes exhibits a product specificity for (2-8)disialosyl lactosyl ceramide (ganglioside GD3)

Intact synaptosomes prepared from 28-day-old rat brains were incubated with CMP-N-acetyl-(14C) neuraminic acid in Krebs-Henseleit buffer in an atmosphere of 95% O2: 5% CO2, at 37 degrees C. The activity of CMP-NANA:ganglioside sialosyltransferase using endogenous acceptors was 0.84 pmoles NANA transferred/mg synaptosomal protein/hr. Analysis of the distribution of labeled sialic acid revealed that GD3 ganglioside (alpha 2----8 disialosyl, alpha 2----3 galactosyl, beta 1----4 glucosyl, beta 1----1-ceramide) was the major product in the membrane carrying 32% of the total lipid bound label. Treatment of the reaction products with Clostridium neuraminidase liberated labeled sialic acid from GD3 and yielded labeled GM3, then unlabeled lactosyl ceramide. Lac-cer and GM3 are present in small amounts in synaptosomes, and GD3 represents less than 2% of the total ganglioside. Our findings indicate that the sialosyltransferase activity of synaptosomes exhibits a preferential product specificity for the small pool of synaptosomal membrane GD3 ganglioside that may be formed in situ, via sialosylation of its precursor (GM3 or lactosyl ceramide) which pre-exists in the synaptosomal plasma membrane. The second major labeled product quantitatively was GD1a whose precursor substrate, GM1, is quite abundant in the membrane, so that the conversion rate of GM1 to GD1a was low in comparison with GD3 formation. Sialosylation of other synaptosomal membrane gangliosides was negligible.     

Gangliosides and neutral glycolipids in ependymal, neuronal and primitive neuroectodermal tumors

Neutral glycolipid and ganglioside compositions were determined on 11 ependymal tumors, 12 medulloblastomas, 6 other neuronal tumors of the brain, 4 peripheral neuroblastomas, 1 cerebral primitive neuroectodermal tumor (PNET), and 1 PNET of the thoracic wall. Within the group of tumors that can demonstrate neuronal phenotypes, there was an association between the degree of neuronal differentiation usually demonstrated by these tumors and the proportions of both GD1a and 1b-pathway gangliosides. The amount of globoside also correlated with the amount of 1b pathway gangliosides. Patients with medulloblastomas whose 1b gangliosides made up over 15% of the total gangliosides survived longer that those with lower proportions of 1b gangliosides. The only gangliosides in the choroid plexus papilloma were GM3 and GD1a, but other ependymal tumors had significant amounts of GD1b and its metabolic precursors. Ependymoma and anaplastic ependymoma had similar neutral glycolipid compositions, which were different from subependymoma, which lacked ceramide monohexoside and ceramide dihexoside. These differences in glycolipid compositions suggest that there may be fundamental biological differences between these types of ependymal tumors. Ganglioside nomenclature is according to Svennerholm (Ledeen et al., 1988; Svennerholm, 1980).     

Neurons and astrocytes influence the development of purified O-2A progenitor cells.

To investigate the possible role of neurons and astrocytes for oligodendrocyte development we prepared a pure population of precursor cells positive for the precursor marker GD3 with the help of fluorescence-activated cell sorting (FACS). Large numbers of highly purified cells were obtained from postnatal day 1 rat brainstems and cultured in different media and sera, and in conditioned media. As described in the literature for optic nerve O-2A progenitors, GD3-sorted brainstem cells cultured in medium containing 10% fetal calf serum (FCS) acquired a star-shaped morphology and differentiated into GD3- and GFAP-positive type-2 astrocytes. On the other hand, in serum-free medium, most of the cells differentiated into oligodendrocytes (O1-/galactocerebroside-positive). Sensory neuron conditioned media promoted survival and proliferation of the precursor cells. The spontaneous differentiation of progenitor cells into oligodendrocytes was retarded by the mitogen. Antibodies against platelet-derived growth factor (PDGF) completely blocked the mitotic effect and allowed spontaneous oligodendrocyte differentiation to occur. Cultured astrocytes also secreted PDGF as a mitogen. However, postnatal astrocytes also released a potent signal promoting oligodendrocyte differentiation. The type of factor(s) released depended on the age of the astrocytes, since only conditioned medium of postnatal but not of embryonic astrocytes promoted oligodendrocyte differentiation, suggesting that astrocyte maturation directly influences oligodendrocyte differentiation. Different concentrations of PDGF could not reproduce this differentiation-inducing effect. This study suggests that interactions between O-2A progenitor cells, neurons, and astrocytes could be required to regulate and complete the oligodendrocyte developmental pathway. Astrocytes, themselves possibly under neuronal influences, might regulate first the proliferation of the precursor cells, and, later in development, the differentiation into mature oligodendrocytes or type-2 astrocytes.     

Epidermal growth factor binding and mitogenic activity on purified populations of cells from the central nervous system

Binding of 125I-epidermal growth factor (EGF) to purified populations of rat astrocytes, oligodendrocytes, and neurons was measured. Astrocytes bound 40,000-100,000 EGF molecules per cell, while oligodendrocytes bound only 6,000-10,000 EGF molecules per cell. In contrast, neurons had little or no capacity to bind 125I-EGF. EGF alone was able to stimulate incorporation of tritiated thymidine fivefold in purified astrocyte cultures incubated in serum-free medium. When EGF was added to the previously described chemically defined medium for astrocytes, incorporation of tritiated thymidine in purified astrocytes was equivalent to that observed in medium containing 10% fetal calf serum. Addition of EGF to this chemically defined medium also doubled the proliferative capability of the medium for cultured astrocytes. EGF was maximally effective in stimulating 3H-thymidine incorporation at concentrations between 1-10 ng/ml.     

Neural stem cells: isolation and differentiation into cholinergic neurons

This investigation aimed to isolate neural stem cells from neonatal hippocampus and induce them to differentiate into cholinergic neurons. The isolated neural stem cells were incubated in serum-free Dulbecco's modified Eagle medium/F12 medium added with 20 ng/ml basic fibroblast growth factor and B27. The cell line isolated from the hippocampal formation of neonatal rats expressed nestin and had the potency to form clones and differentiate into neurons, astrocytes and oligodendrocytes. Embryonic chick skeletal muscle extract was used to induce the differentiation of the neural stem cells into cholinergic neurons. Immunocytochemistry was used to detect the choline acetyltransferase antigen of cholinergic neurons for confirmation. The results showed that embryonic chick skeletal muscle extract could induce isolated neural stem cell to differentiate into a significantly larger number of cholinergic neurons than controls.