Production of neurons, astrocytes and oligodendrocytes from mammalian CNS stem cells

The isolation and expansion of precursor cells in a serum-free culture system allows for the systematic characterization of their properties and the intrinsic and extrinsic signals that regulate their function. The discovery of neural stem cells in the adult mouse brain was made possible by the creation of a novel culture system subsequently termed the neurosphere assay. Therein, the dissociated adult mouse periventricular area was plated in the presence of epidermal growth factor, but in the absence of adhesive substrates, which resulted in the generation of spheres of proliferating cells that detached from the plate bottom and remained suspended in the media. Since its inception, the neurosphere culture system has been widely used in the neural precursor cell field and has been extensively adapted for the isolation and expansion of corneal, cardiac, skin, prostate, mammary and brain tumor stem cells. The original neurosphere culture protocol, which takes approximately 10 d to complete, is described here in detail.     

Isolation of astrocytes,neurons, and synaptosomes of rat brain cortex

A simplified method was developed for the bulk separation of neuronal perikarya and astroglial celis from adult rat brain without the involvement of density gradients. Activities of various enzymes involved in glutamate metabolism were estimated and compared with those of synaptosomes. The activities of glutamate dehydrogenase and aspartate aminotransferase were higher in synaptosomes than in neuronal perikarya or glia. Glutamine synthetase was distributed in all the three fractions while glutaminase activity was higher in astrocytes than in synaptosomes and was not detectable in neuronal perikarya. The significance of these results in relation to metabolic compartmentation was discussed.     

Proteome analysis of primary neurons and astrocytes from rat cerebellum

Neurons and astrocytes are predominant cell types in brain and have distinguished morphological and functional features. Although several proteomics studies were carried out on the brain, work on individual brain cells is limited. Generating individual proteomes of neurons and astrocytes, however, is mandatory to assign protein expression to cell types rather than to tissues. We aimed to provide maps of rat primary neurons and astrocytes using two-dimensional gel electrophoresis with subsequent in-gel digestion, followed by MALDI-TOF/TOF. 428 protein spots corresponding to 226 individual proteins in neurons and 406 protein spots representing 228 proteins in astrocytes were unambiguously identified. Proteome data include proteins from several cascades differentially expressed in neurons and astrocytes, and specific expressional patterns of antioxidant, signaling, chaperone, cytoskeleton, nucleic acid binding, proteasomal, and metabolic proteins are demonstrated. We herein present a reference database of primary rat primary neuron and astrocyte proteomes and provide an analytical tool for these structures. The concomitant expressional patterns of several protein classes are given and potential neuronal and astrocytic marker candidates are presented.     

Enzyme levels in cultured astrocytes,oligodendrocytes and Schwann cells,and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.Special issue dedicated to Dr. Lewis Sokoloff.     

Postnatal changes in the localization of prostaglandin D synthetase from neurons to oligodendrocytes in the rat brain

Prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) in the brain of 2- and 8-week-old rats was biochemically indistinguishable as judged by Mr (26,000), pI values (4.35-4.75), Km for prostaglandin H2 (approximately 20 microM), and requirement of sulfhydryl compounds. Furthermore, the enzyme was immunologically the same at both ages, as judged by immunotitration, Ouchterlony immunodiffusion, and immunoblotting analyses using a polyclonal and two monoclonal antibodies specific for the enzyme. However, the cellular localization of the enzyme was markedly different between the two ages, as examined by immunoperoxidase staining with the specific antibodies. The immunoreactivity was found in many neurons in 2-week-old rats but was not detected in most neurons in 8-week-old rats except for multipolar neurons in layers I-II of the neocortex and several clusters of dendrites in the pyriform cortex. In the adult rat, the immunoreactivity was mainly localized in oligodendrocytes. By immunoelectron microscopy, the immunoreactive deposits were seen in rough-surfaced endoplasmic reticulum and outer nuclear membrane of the glial cells.     

Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes

The RII-B isoform of the regulatory subunit (R) of cAMP-dependent protein kinase II is abundantly and selectively expressed in cerebral cortex (Erlichman, J., Sarkar, D., Fleischer, N., and Rubin, C. S. (1980) J. Biol. Chem. 255, 8179-8184). In contrast to the cytosolic RII-H isoform from heart and other non-neural tissues, a substantial fraction of cerebral cortex RII-B is tightly associated with cell organelles. In order to study the cellular basis for the localization and abundance of RII-B in this complex and heterogeneous tissue, rat cerebral cortex was fractionated into highly purified populations of neurons, astrocytes, and oligodendrocytes. In neurons and astrocytes more than 80% of the total cAMP-binding activity is contributed by RII subunits, whereas the myelin-producing oligodendrocytes contain nearly equal proportions of RI (from protein kinase I) and RII. Approximately 70% of RII and RI subunits are associated with the particulate fraction in each of the three types of brain cells. The nature of the RII isoforms expressed in the cytosolic and particulate fractions of the purified brain cells was established by performing Western immunoblot and indirect immunoprecipitation analyses with selective and sensitive polyclonal antibodies directed against RII-B. Astrocytes and neurons exhibit high levels of RII-B, whereas oligodendrocytes contain the RII-H isoform. Thus, the expression of RII isoforms is not uniform among brain cells that are anatomically and developmentally related. Rather, it appears that RII-B and RII-H are expressed in a cell-specific fashion within cerebral cortex and this might reflect an RII-mediated adaptation of protein kinase II to the specialized metabolic and functional roles of neurons, astrocytes, and oligodendrocytes.     

Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes,oligodendrocytes, microglial cells and neurones

Multidrug resistance proteins (Mrps) are ATP-driven export pumps that mediate the export of organic anions from cells. So far only little information is available on expression and physiological functions of Mrps in brain. The expression of mRNAs of six Mrp paralogs in rat brain, as well as in rat cultures enriched for neurones, astrocytes, oligodendrocytes and microglial cells, was studied by qualitative and semiquantitative RT-PCR analysis. In adult rat brain as well as in neural cell cultures the mRNAs coding for Mrp1, Mrp3, Mrp4 and Mrp5 were detected. Semiquantitative analysis revealed that the mRNAs coding for Mrp1 and Mrp5 were more abundant in the four cell culture types than mRNAs of the other Mrps. mRNAs coding for Mrp3 and Mrp4 were found at significant levels in cultured astrocytes and microglial cells, whereas cultures of neurones and oligodendrocytes contained only marginal quantities of these mRNAs. Putative physiological functions of Mrps in brain cells are discussed.     

Response of membrane potential and intracellular pH to hypercapnia in neurons and astrocytes from rat retrotrapezoid nucleus

We compared the response to hypercapnia (10%) in neurons and astrocytes among a distinct area of the retrotrapezoid nucleus (RTN), the mediocaudal RTN (mcRTN), and more intermediate and rostral RTN areas (irRTN) in medullary brain slices from neonatal rats. Hypercapnic acidosis (HA) caused pH(o) to decline from 7.45 to 7.15 and a maintained intracellular acidification of 0.15 +/- 0.02 pH unit in 90% of neurons from both areas (n = 16). HA excited 44% of mcRTN (7/16) and 38% of irRTN neurons (6/16), increasing firing rate by 167 +/- 75% (chemosensitivity index, CI, 256 +/- 72%) and 310 +/- 93% (CI 292 +/- 50%), respectively. These responses did not vary throughout neonatal development. We compared the responses of mcRTN neurons to HA (decreased pH(i) and pH(o)) and isohydric hypercapnia (IH; decreased pH(i) with constant pH(o)). Neurons excited by HA (firing rate increased 156 +/- 46%; n = 5) were similarly excited by IH (firing rate increased 167 +/- 38%; n = 5). In astrocytes from both RTN areas, HA caused a maintained intracellular acidification of 0.17 +/- 0.02 pH unit (n = 6) and a depolarization of 5 +/- 1 mV (n = 12). In summary, many neurons (42%) from the RTN are highly responsive (CI 248%) to HA; this may reflect both synaptically driven and intrinsic mechanisms of CO(2) sensitivity. Changes of pH(i) are more significant than changes of pH(o) in chemosensory signaling in RTN neurons. Finally, the lack of pH(i) regulation in response to HA suggests that astrocytes do not enhance extracellular acidification during hypercapnia in the RTN.     

Acetoacetate and glucose as substrates for lipid synthesis by rat brain oligodendrocytes and astrocytes in serum-free culture

We have compared glucose and acetoacetate as precursors for lipogenesis and cholesterogenesis by oligodendrocytes and astrocytes, using mixed glial cultures enriched in oligodendrocytes. In order to differentiate between metabolic processes in oligodendrocytes and those in astrocytes, the other major cell type present in the mixed culture, we carried out parallel incubations with cultures from which the oligodendrocytes had been removed by treatment with anti-galactocerebroside serum and guinea-pig complement. The following results were obtained: 1. Both oligodendrocytes and astrocytes in culture actively utilize acetoacetate as a precursor for lipogenesis and cholesterogenesis. 2. In both cell types, the incorporation of acetoacetate into fatty acids and cholesterol exceeds that of glucose by a factor of 5-10 when the precursors are present at concentrations of 1 mM and higher. 3. Glucose stimulates acetoacetate incorporation into fatty acids and cholesterol, whereas acetoacetate reduces the entry of glucose into these lipids. This suggests that glucose is necessary for NADPH generation, but that otherwise the two precursors contribute to the same acetyl-CoA pool. 4. Both with acetoacetate and with glucose as precursor, oligodendrocytes are more active in cholesterol synthesis than astrocytes. 5. Using incorporation of 3H2O as an indicator for total lipid synthesis, we estimated that acetoacetate contributes one third of the acetyl groups and glucose one twentieth when saturating concentrations of both substrates are present.     

Cytosolic and mitochondrial isoforms of NADP+-dependent isocitrate dehydrogenases are expressed in cultured rat neurons,astrocytes, oligodendrocytes and microglial cells

NADP+-dependent isocitrate dehydrogenases (ICDHs) are enzymes that reduce NADP+ to NADPH using isocitrate as electron donor. Cytosolic and mitochondrial isoforms of ICDH have been described. Little is known on the expression of ICDHs in brain cells. We have cloned the rat mitochondrial ICDH (mICDH) in order to obtain the sequence information necessary to study the expression of ICDHs in brain cells by RT-PCR. The cDNA sequence of rat mICDH was highly homologous to that of mICDH cDNAs from other species. By RT-PCR the presence of mRNAs for both the cytosolic and the mitochondrial ICDHs was demonstrated for cultured rat neurons, astrocytes, oligodendrocytes and microglia. The expression of both ICDH isoenzymes was confirmed by western blot analysis using ICDH-isoenzyme specific antibodies as well as by determination of ICDH activities in cytosolic and mitochondrial fractions of the neural cell cultures. In astroglial and microglial cultures, the total ICDH activity was almost equally distributed between cytosolic and mitochondrial fractions. In contrast, in cultures of neurons and oligodendrocytes about 75% of total ICDH activity was present in the cytosolic fractions. Putative functions of ICDHs in cytosol and mitochondria of brain cells are discussed.     

Binding constants of soluble NGF-receptors in rat oligodendrocytes and astrocytes in culture

The neuronotrophic factor NGF binds to peripheral neurons of the dorsal root ganglion and the sympathetic nervous system. NGF binds to a cell surface receptor, NGFR, on these cells and displays Kd's of 10(-9) and 10(-11)M. NGF receptors have also been reported for basal forebrain magnocellular neurons. In addition, NGF specifically binds to NGFR on Schwann cells although the biological significance of this binding is not known. Here we report that NGF binds in a saturable and specific fashion to receptors on cultured isolated populations of rat astrocytes but not to oligodendrocytes. The binding to astrocytes in culture displayed a Kd of 2.7 +/- 1.0 nM with 36,000 receptors per cell.     

Progenitors of oligodendrocytes: Limiting dilution analysis in fetal rat brain culture

In this paper, we describe the use of a combination of cell culture techniques and limiting dilution analysis to determine the number of oligodendrocyte progenitor cells and the oligodendrocyte clone size in primary dispersed cultures of 20- to 21-day-old fetal rat brain. Single-cell suspensions (1,2,3 × 10⁶ cells/ml) were plated in either microwell or 100 mm dishes. After 22 days in culture the number of differentiated oligodendrocytes was ascertained by determining the amount of myelin basic protein by radioimmunoassay. The total amount of myelin basic protein was the same in the two types of dish, indicating that proliferation and differentiation were unaffected when oligodendrocytes were grown in microwells. The fraction (F₀) of microwells containing no oligodendrocytes was determined at each cell dilution. F₀ decreased exponentially with increasing total cell concentration. The linearity of the plot of ln F₀ versus cell number indicates that the number of oligodendrocyte progenitor cells is limiting. From the equation describing the Poisson distribution of progenitor cells in microwells we calculated that, at the time of plating, primary cultures of fetal rat brain contain one oligodendrocyte progenitor cell per 1.3 × 10⁵ brain cells, or a total population of 300–500 progenitor cells per brain. The mean oligodendrocyte clone size was determined to be approximately 825 at 22 days and close to 2000 by 35 days in culture. Therefore, each progenitor cell must undergo approximately 11 divisions, on the average, during postnatal development.