Oligodendrocyte-type-2 Astrocyte Research Articles

Oligodendrocyte progenitor cells derived from mouse embryonic stem cells give rise to type-1 and type-2 astrocytes in vitro

Oligodendrocyte progenitor cells (OPCs) in primary culture can give rise to mature oligodendrocytes and type-2, but not type-1 astrocytes depending on the culture conditions. The OPCs thus are called oligodendrocyte-type-2 astrocyte (O2-A) progenitor cells. Mouse embryonic stem cells (mESCs) have been efficiently differentiated into OPCs; however, the fate plasticity of mESC-derived OPCs is not well characterized. In the present study, using GFP-Olig2 mESC line, we showed that the Olig2+/GFP+/A2B5+/NG2+ OPCs derived from GFP-Olig2 mESCs can mature into oligodendrocytes when co-cultured with mESC-derived neurons. Interestingly, when induced to astrocytic differentiation by bone morphogenetic protein-4, these mESC-derived OPCs can not only generate type-2 astrocytes, but also type-1 astrocytes. These results challenge the dogma that OPCs in culture can only generate type-2, but not type-1 astrocytes, and support the in vivo finding that during perinatal development, OPCs can give rise to a subset of type-1 astrocytes.

Abstract 282: PAI-2 promotes astrocyte differentiation from glial precursor cells via enhancing STAT3 signaling

Abstract Regulation of the generation of astrocytes and oligodendrocytes is a key process in the development of normal central nervous system (CNS) and glial tumors including oligodendrocytoma, astrocytoma and glioblastoma. Glial cell differentiation occurs in two sequentially generated glial progenitor cells, the glial-restricted precursor (GRP) cells and the oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells. The molecular mechanisms of cell generation in these precursor cells are only incompletely understood. In this study, PAI-2 (Plasminogen Activator inhibitor-2) was identified to be upregulated during glial cell differentiation. Ectopic expression of PAI-2 induced GRP cells to differentiate into astrocytes and inhibited their proliferation. In contrast, suppression of endogenous PAI-2 by small interfering RNA in GRP cells promoted cell proliferation and inhibited their ability to differentiate into astrocytes. More interestingly, we observed a higher Rb protein level and a correlated lower E2F1 transcriptional activity during glial differentiation due to increased PAI-2 protein level. In consistent with these findings, ectopic expression of PAI-2 indeed stabilized Rb protein. We also found that PAI-2 upregulated GFAP expression in glial progenitor cells, an antigenic marker for astrocytes. Astrocytogenesis is controlled by two different signaling, the IL-6, LIF or CNTF-induced STAT3 pathway and the BMP-4-induced SMAD1 pathway. Interestingly, PAI-2 significantly enhanced CNTF, LIF or IL-6-induced GFAP expression as illustrated by both reporter assay and immunostaining, but not BMP4-induced GFAP expression. Furthermore, We demonstrated that PAI-2 could enhance CNTF-induced STAT3 phosphorylation and the complex formation between STAT3 and p300. It has also been reported that Rb could enhance the transcriptional activity of several transcription factors via enhancing p300/CBP activity by releasing them from some inhibitory transcriptional repressors such as EID1 (E1A-like inhibitor of differentiation). Indeed, suppression of Rb by small interfering RNA or overexpression of EID1 abrogated PAI-2's effect on STAT3 activity. Also, suppression of endogenous EID1 by small interfering RNA could further enhance PAI-2's effect. Finally, we illustrated Glial precursor cells from PAI-2 knockout mice are less responsive to CNTF and IL-6 than wild-type cells. Based on these data, we concluded that PAI-2 promotes glial progenitor cells to differentiate into astrocytes via stabilizing Rb, which can abrogate EID's inhibitory effect, and then enhancing STAT3 transcriptional activity induced by CNTF or IL-6. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 282. doi:10.1158/1538-7445.AM2011-282

A composite likelihood approach to the analysis of longitudinal clonal data on multitype cellular systems under an age-dependent branching process.

A recurrent statistical problem in cell biology is to draw inference about cell kinetics from observations collected at discrete time points. We investigate this problem when multiple cell clones are observed longitudinally over time. The theory of age-dependent branching processes provides an appealing framework for the quantitative analysis of such data. Likelihood inference being difficult in this context, we propose an alternative composite likelihood approach, where the estimation function is defined from the marginal or conditional distributions of the number of cells of each observable cell type. These distributions have generally no closed-form expressions but they can be approximated using simulations. We construct a bias-corrected version of the estimating function, which also offers computational advantages. Two algorithms are discussed to compute parameter estimates. Large sample properties of the estimator are presented. The performance of the proposed method in finite samples is investigated in simulation studies. An application to the analysis of the generation of oligodendrocytes from oligodendrocyte type-2 astrocyte progenitor cells cultured in vitro reveals the effect of neurothrophin-3 on these cells. Our work demonstrates also that the proposed approach outperforms the existing ones.

Late Effects of Radiation on the Central Nervous System: Role of Vascular Endothelial Damage and Glial Stem Cell Survival

Selective irradiation of the vasculature of the rat spinal cord was used in this study, which was designed specifically to address the question as to whether it is the endothelial cell or the glial progenitor cell that is the target responsible for late white matter necrosis in the CNS. Selective irradiation of the vascular endothelium was achieved by the intraperitoneal (ip) administration of a boron compound known as BSH (Na(2)B(12)H(11)SH), followed by local irradiation with thermal neutrons. The blood-brain barrier is known to exclude BSH from the CNS parenchyma. Thirty minutes after the ip injection of BSH, the boron concentration in blood was 100 microg (10)B/ g, while that in the CNS parenchyma was below the detection limit of the boron analysis system, <1 microg (10)B/g. An ex vivo clonogenic assay of the O2A (oligodendrocyte-type 2 astrocyte) glial progenitor cell survival was performed 1 week after irradiation and at various times during the latent period before white matter necrosis in the spinal cord resulted in myelopathy. One week after 4.5 Gy of thermal neutron irradiation alone (approximately one-third of the dose required to produce a 50% incidence of radiation myelopathy), the average glial progenitor cell surviving fraction was 0.03. The surviving fraction of glial progenitor cells after a thermal neutron irradiation with BSH for a comparable effect was 0.46. The high level of glial progenitor cell survival after irradiation in the presence of BSH clearly reflects the lower dose delivered to the parenchyma due to the complete exclusion of BSH by the blood-brain barrier. The intermediate response of glial progenitor cells after irradiation with thermal neutrons in the presence of a boron compound known as BPA (p-dihydroxyboryl-phenylalanine), again for a dose that represents one-third the ED(50) for radiation-induced myelopathy, reflects the differential partition of boron-10 between blood and CNS parenchyma for this compound, which crosses the blood-brain barrier, at the time of irradiation. The large differences in glial progenitor survival seen 1 week after irradiation were also maintained during the 4-5-month latent period before the development of radiation myelopathy, due to selective white matter necrosis, after irradiation with doses that would produce a high incidence of radiation myelopathy. Glial progenitor survival was similar to control values at 100 days after irradiation with a dose of thermal neutrons in the presence of BSH, significantly greater than the ED(100), shortly before the normal time of onset of myelopathy. In contrast, glial progenitor survival was less than 1% of control levels after irradiation with 15 Gy of thermal neutrons alone. This dose of thermal neutrons represents the approximate ED(90-100) for myelopathy. The response to irradiation with an equivalent dose of X rays (ED(90): 23 Gy) was intermediate between these extremes as it was to thermal neutrons in the presence of BPA at a slightly lower dose equivalent to the approximate ED(60) for radiation myelopathy. The conclusions from these studies, performed at dose levels approximately iso-effective for radiation-induced myelopathy as a consequence of white matter necrosis, were that the large differences observed in glial progenitor survival were directly related to the dose distribution in the parenchyma. These observations clearly indicate the relative importance of the dose to the vascular endothelium as the primary event leading to white matter necrosis.

Effects of Glial Transplantation on Functional Recovery following Acute Spinal Cord Injury

Numerous efforts have been made to maximize the efficacy of treatment for spinal cord injury (SCI). Recently, oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells have been reported to remyelinate focal areas of demyelinated spinal cord in adult rats. We conducted a study to investigate the therapeutic potential of transplantation of O-2A cells in a rat model of acute SCI. SCI was induced with an NYU Impactor at T9 of rats. O-2A cells labeled with bromodeoxyuridine (BrdU) were transplanted into sites of SCI at 1 week after the induction of SCI. At 6 weeks after cell transplantation, a behavioral test showed significant functional improvement in animals that had received O-2A-cell transplants as compared to animals given cell-culture medium alone. An electrophysiological study revealed that the transplants did not improve the amplitude or latency of somatosensory evoked potentials, but a recording of motor evoked potentials showed that the latency of these potentials in the O-2A-cell-transplant group was significantly shorter than that in the group treated with cell-culture medium. Following transplantation of BrdU-labeled O-2A cells, cells positive for BrdU were detected at and near sites of SCI. Cells labeled for both BrdU and 2',3' -cyclic nucleotide-3-phosphodiesterase were also detected, showing that the transplanted O-2A cells differentiated into oligodendrocytes. By contrast, cells labeled for BrdU and glial fibrillary acidic protein, or for neuronal nuclei antigen, were not detected. Furthermore, a tract-tracing study showed that numbers of retrogradely labeled neurons increased in areas of the brain stem after O-2A-cell transplantation. The study data showed that after being transplanted into an animal with SCI, O-2A cells migrated to the area adjacent to the site of injury and differentiated into oligodendrocytes. The behavioral test and the electrophysiological and morphological studies showed that transplantation of O-2A cells may play an important role in functional recovery and the regeneration of axons after SCI.

Redox state as a central modulator of precursor cell function.

In our attempts to understand how the balance between self-renewal and differentiation is regulated in dividing precursor cells, we have discovered that intracellular redox state appears to be a critical modulator of this balance in oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells. The intracellular redox state of freshly isolated progenitor cells allows prospective isolation of cells with different self-renewal characteristics, which can be further modulated in opposite directions by prooxidants and antioxidants. Redox state is itself modulated by cell-extrinsic signaling molecules that alter the balance between self-renewal and differentiation: growth factors that promote self-renewal cause progenitors to become more reduced, while exposure to signaling molecules that promote differentiation causes progenitors to become more oxidized. Moreover, pharmacological antagonists of the redox effects of these cell-extrinsic signaling molecules antagonize their effects on self-renewal and differentiation, further suggesting that cell-extrinsic signaling molecules that modulate this balance converge on redox modulation as a critical component of their effector mechanism. A further example of the potential relevance of intracellular redox state to development processes emerges from our attempts to understand why different central nervous system (CNS) regions exhibit different temporal patterns of oligodendrocyte generation and myelinogenesis. Characterization of O-2A progenitor cells (O-2A/OPCs) isolated from different regions indicates that these developmental patterns are consistent with properties of the specific O-2A/OPCs resident in each region. Marked differences were seen in self-renewal and differentiation characteristics of O-2A/OPCs isolated from cortex, optic nerve, and optic chiasm. In conditions where optic nerve-derived O-2A/OPCs generated oligodendrocytes within 2 days, oligodendrocytes arose from chiasm-derived cells after 5 days and from cortical O-2A/OPCs after only 7-10 days. These differences, which appear to be cell intrinsic, were manifested both in reduced percentages of clones producing oligodendrocytes and in a lesser representation of oligodendrocytes in individual clones. In addition, responsiveness of optic nerve-, chiasm-, and cortex-derived O-2A/OPCs to thyroid hormone (TH) and ciliary neurotrophic factor (CNTF), well-characterized inducers of oligodendrocyte generation, was inversely related to the extent of self-renewal observed in basal division conditions. These results demonstrate hitherto unrecognized complexities among the precursor cells thought to be the immediate ancestors of oligodendrocytes and suggest that the properties of these different populations may contribute to the diverse time courses of myelination in different CNS regions. Strikingly, O-2A/OPCs isolated from cortex and analyzed immediately upon isolation were more reduced in their redox state than were optic nerve-derived cells, precisely as would be predicted from our analysis of the role of redox state in modulating the balance between self-renewal and differentiation. Chiasm-derived cells, which exhibited self-renewal properties intermediate between cortex- and optic nerve-derived cells, were more reduced than optic nerve cells but more oxidized that cortical O-2A/OPCs.

Type-2 astrocyte-like cells are more resistant than oligodendrocyte-like cells against non-N-methyl-D-aspartate glutamate receptor-mediated excitotoxicity.

Glutamate causes excitotoxicity via non-N-methyl-D-aspartate (NMDA) glutamate receptors (GluR) in oligodendrocytes. Because both oligodendrocytes and type 2 astrocytes are differentiated from oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, we investigated whether astrocytes are also vulnerable to non-NMDA GluR-mediated excitotoxicity. For these studies, oligodendrocyte-like cells (OLC) and type 2 astrocyte-like cells (2ALC) were derived from CG-4 cells, an immortalized rat O-2A progenitor cell line. About 50% of 2ALC were positive for glial fibrillary acidic protein and 90% were positive for A2B5, verifying that these cells have an type 2 astrocytic phenotype. A 24-hr exposure of OLC to 2 mM kainate, an activator of non-NMDA GluR, caused cell damage as shown by the release of lactate dehydrogenase. The extent of kainate-induced OLC damage was increased by cyclothiazide. In contrast, exposure of 2ALC to 2 mM kainate alone did not induce injury, though mild 2ALC injury was elicited by exposure to 2 mM kainate plus 100 microM cyclothiazide. Furthermore, we found that the kainate induced Ca(2+) uptake by 2ALC was 27.5% of that induced by kainate in OLC. Finally, both OLC and 2ALC expressed non-NMDA GluR subunit mRNAs, including GluR2, GluR3, GluR4, GluR6, GluR7, KA1, and KA2, but quantitative Western blot analysis revealed higher immunodetectable GluR2 and lower immunodetectable GluR3 and GluR4 in 2ALC than in OLC. Together, these results suggest that astrocytes are relatively resistant to non-NMDA GluR-mediated excitotoxicity because they have a higher expression of GluR2 and lower expression of GluR3 and GluR4.

Analysis of Fractal Dimension of O2A Glial Cells Differentiating in Vitro

Fractal dimension is a quantitative measure of morphological complexity. Glial cells of the oligodendrocyte-type 2 astrocyte (O2A) lineage exhibit increasing morphological complexity as they differentiate in vitro. Enriched populations of O2A progenitor cells isolated from neonatal rat cerebral hemispheres or optic nerves were allowed to differentiate in vitro, and their fractal dimensions were measured over time. The fractal dimensions of the maturing cells correlated with perceived complexity; cells with elaborate process branching had larger fractal dimensions than cells with a simpler morphology. An analysis of changes in fractal dimension revealed distinct rates of growth for both oligodendrocytes and type 2 astrocytes. The fractal dimension remained constant over a 10-fold range in optical magnification, demonstrating that cultured O2A glial cells exhibit self-similarity, a defining characteristic of fractal objects. These results illustrate that fractal dimension analysis of maturing cell populations is a useful method for quantitatively describing the process of cell differentiation.

AP-1 Activity during the Growth, Differentiation, and Death of O-2A Lineage Cells

Oligodendrocyte differentiation has been correlated with AP-1 activity, being low in progenitors and high in differentiated cells. In this study we have carried out a detailed temporal analysis of AP-1 activity in oligodendrocyte-type-2 astrocyte (O-2A) lineage cells. We show that low AP-1 activity in progenitor cells depended on the application of growth factors. Treatment of cells with B104-conditioned medium induced high AP-1 activity, increased process length, and improved growth. The role of AP-1 in proliferation and process extension was emphasized when progenitor cells overexpressing a c-Jun dominant-negative mutant had impaired growth and shortened processes. AP-1 DNA-binding activity during O-2A differentiation in vitro showed an initial down-regulation followed by up-regulation after 2 days. Increased AP-1 levels in oligodendrocytes were inhibited by overexpression of bcl-2, indicating that AP-1 in mature oligodendrocytes is involved in the regulation of apoptosis. Prevention of cell death by bcl-2 in oligodendrocytes was accompanied by progressive differentiation and expression of MOG and PLP.

Estimation problems associated with stochastic modeling of proliferation and differentiation of O-2A progenitor cells in vitro

Our previous research effort has resulted in a stochastic model that provides an excellent fit to our experimental data on proliferation and differentiation of oligodendrocyte type-2 astrocyte progenitor cells at the clonal level. However, methods for estimation of model parameters and their statistical properties still remain far away from complete exploration. The main technical difficulty is that no explicit analytic expression for the joint distribution of the number of progenitor cells and oligodendrocytes, and consequently for the corresponding likelihood function, is available. In the present paper, we overcome this difficulty by using computer-intensive simulation techniques for estimation of the likelihood function. Since the output of our simulation model is essentially random, stochastic optimization methods are necessary to maximize the estimated likelihood function. We use the Kiefer–Wolfowitz procedure for this purpose. Given sufficient computing resources, the proposed estimation techniques significantly extend the spectrum of problems that become approachable. In particular, these techniques can be applied to more complex branching models of multi-type cell systems with dependent evolutions of different types of cells.

Stochastic formulations of a clock model for temporally regulated generation of oligodendrocytes in vitro

It is possible to grow oligodendrocyte type-2 astrocyte (O-2A) progenitor cells in vitro in such a manner that they divide and generate oligodendrocytes with a timing which recapitulates the timing of normal development in vivo. The most widely accepted model of this process assumes a cell-intrinsic biological clock that resides in the O-2A progenitor cell and counts the number of mitotic divisions. The intrinsic clock model, originally proposed in 1985, remains to be the dominant theoretical concept for the analysis of timed differentiation in this cell lineage. In this paper, we proceed from different biologically plausible assumptions to provide a stochastic description of the clock model based on the theory of multitype age-dependent branching processes. This approach makes it possible to interpret the observed pattern of oligodendrocyte generation and its modification in the presence of thyroid hormone in terms of parameters that have a clear biological meaning.

Gliogenesis in the central nervous system.

Multipotential neuroepithelial stem cells are thought to give rise to all the differentiated cells of the central nervous system (CNS). The developmental potential of these multipotent stem cells becomes more restricted as they differentiate into progressively more committed cells and ultimately into mature neurons and glia. In studying gliogenesis, the optic nerve and spinal cord have become invaluable models and the progressive stages of differentiation are being clarified. Multiple classes of glial precursors termed glial restricted precursors (GRP), oligospheres, oligodendrocyte-type2 astrocyte (O-2A) and astrocyte precursor cells (APC) have been identified. Similar classes of precursor cells can be isolated from human neural stem cell cultures and from embryonic stem (ES) cell cultures providing a non-fetal source of such cells. In this review, we discuss gliogenesis, glial stem cells, putative relationships of these cells to each other, factors implicated in gliogenesis, and therapeutic applications of glial precursors.

GT3 and its O-acetylated derivative are the principal A2B5-reactive gangliosides in cultured O2A lineage cells and are down-regulated along with O-acetyl GD3 during differentiation to oligodendrocytes

Among the developmentally regulated antigens expressed on the surface of bipotential glial precursors isolated from neonatal rat brain are the gangliosides recognized by the monoclonal antibody A2B5. Immunostaining of thin layer chromatograms showed that oligodendrocyte-type 2 astrocyte (O2A) progenitors in culture express two ganglioside antigens that react strongly with the A2B5 antibody. Both ganglioside antigens are down-regulated as the cells differentiate to oligodendrocytes, corresponding to the disappearance of cell surface immunostaining by A2B5 in mature oligodendrocytes. By contrast, both gangliosides continue to be expressed when the cells differentiate to type-2 astrocytes. These two A2B5-reactive gangliosides in O2A lineage cells were identified as GT3 and O-acetyl GT3 by using the monoclonal antibody 18B8 that recognizes GT3 and an influenza C virus esterase that specifically removes O-acetyl moieties from sialic acids. Thin-layer chromatographic immunostaining with the JONES monoclonal antibody demonstrated that the progenitor cells in culture also express O-acetyl GD3, which is similarly down-regulated in oligodendrocytes, but retained in type-2 astrocytes. The 18B8 and JONES antibodies also immunostained the surface of O2A progenitors. Therefore, expression of GT3 and O-acetylation of GT3 and GD3 occur during the proliferative and migratory stages of glial cell development. Published 1999 Wiley-Liss, Inc.

Cells expressing the NG2 antigen contact nodes of Ranvier in adult CNS white matter

The NG2 antibody, which recognises an integral membrane chondroitin sulphate, labels a significant population of cells in adult CNS white matter tracts of the rat optic nerve and anterior medullary velum (AMV). Adult NG2+ cells are highly complex with multiple branching processes and we show by EM immunocytochemistry that they extend perinodal processes, which contact nodes of Ranvier. NG2+ cells do not react to conventional immunohistochemical markers for adult glia and so we reservedly term them NG2P cells. In vitro, NG2 labels oligodendrocyte-type-2 astrocyte (O-2A) progenitors that can give rise to oligodendrocytes or type-2 astrocytes, depending on the culture medium. Thus, it is possible that NG2P cells may be derived from the same stem cells as oligodendrocytes. Interestingly, NG2+ cells identified previously in adult CNS displayed phenotypic characteristics of O-2Aadult progenitors and it is possible that, like them, NG2P cells might retain the capacity of generating oligodendrocytes in the adult CNS. This may be an important role of NG2P cells in demyelinating diseases such as multiple sclerosis. It is significant therefore that the perinodal processes of NG2P cells contact the only sites of exposed axolemma in myelinated axons, so that NG2P cells are ideally situated to detect and respond to changes in axonal function during demyelination. A further implication of our finding is that NG2P cells may perform functions at nodes of Ranvier previously attributed to perinodal astrocytes, including the clustering and maintenance of sodium channels in the axon membrane at nodes, during development and following demyelination.

Quantitative insight into proliferation and differentiation of oligodendrocyte type 2 astrocyte progenitor cells in vitro.

As part of our attempts at understanding fundamental principles that underlie the generation of nondividing terminally differentiated progeny from dividing precursor cells, we have developed approaches to a quantitative analysis of proliferation and differentiation of oligodendrocyte type 2 astrocyte (O-2A) progenitor cells at the clonal level. Owing to extensive previous studies of clonal differentiation in this lineage, O-2A progenitor cells represent an excellent system for such an analysis. Previous studies have resulted in two competing hypotheses; one of them suggests that progenitor cell differentiation is symmetric, the other hypothesis introduces an asymmetric process of differentiation. We propose a general model that incorporates both such extreme hypotheses as special cases. Our analysis of experimental data has shown, however, that neither of these extreme cases completely explains the observed kinetics of O-2A progenitor cell proliferation and oligodendrocyte generation in vitro. Instead, our results indicate that O-2A progenitor cells become competent for differentiation after they complete a certain number of critical mitotic cycles that represent a period of symmetric development. This number varies from clone to clone and may be thought of as a random variable; its probability distribution was estimated from experimental data. Those O-2A cells that have undergone the critical divisions then may differentiate into an oligodendrocyte in each of the subsequent mitotic cycles with a certain probability, thereby exhibiting the asymmetric type of differentiation.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells transformed with c-myc and H-ras form high-grade glioma after stereotactic injection into the rat brain

The oligodendrocyte-type-2 astrocyte lineage (O-2A) comprises a progenitor cell that is able to differentiate into an oligodendrocyte or astrocyte in vitro. The lineage was originally identified in the neonatal rat central nervous system but evidence suggests that the equivalent O-2A lineage also exists in humans. Apart from its putative and widely studied role in glial repair, this cell type could potentially be involved in malignant glioma formation. In this study we demonstrate that a rat O-2A progenitor cell line carrying the bacterial beta-galactosidase reporter gene and transformed with the c-myc and H-ras oncogenes which has lost its differentiation capacity in vitro generates glioma-like growth after stereotactic injection into the adult rat brain. Tumour pathology was similar to human glioblastoma, suggesting that one of the pathways in the generation of human glioblastomas may be the transformation of adult O-2A progenitor cells. Parallel studies demonstrated the presence of a DNA-binding protein complex, termed APprog, in a panel of human glioma cell lines. This protein was initially identified in O-2A progenitor cells and not their differentiated progeny. These data lead us to propose that APprog could be used as an indicator of the lineage origin of gliomas.

Growth and Fate of PSA-NCAM+ Precursors of the Postnatal Brain

Oligodendrocyte-type 2 astrocyte (O-2A) lineage cells are derived from multipotential stem cells of the developing CNS. Precursors of O-2A progenitors express the polysialylated (PSA) form of the neural cell adhesion molecule (NCAM) and are detected in neonatal rat brain glial cultures. It is unclear how such PSA-NCAM+ "pre-progenitors" are related to neural stem cells and whether they still have the potential to differentiate along several neural lineages. Here we isolated PSA-NCAM+ pre-progenitor cells from glial cultures by immunopanning and found that most of these cells expressed nestin and PDGF-receptor-alpha but not O-2A antigens. PSA-NCAM+ cells synthesized transcripts for fibroblast growth factor (FGF) receptors 1, 2, and 3 and responded to FGF2 by survival and proliferation, growing into large clusters resembling neural spheres. FGF2-induced proliferation of PSA-NCAM+ pre-progenitors was significantly enhanced by thyroid hormone (T3), which on its own did not increase cell survival or mitosis. After adhesion and withdrawal of the mitogen, spheres generated mostly oligodendrocytes and astrocytes but very rarely neurons. PSA-NCAM immunopanned cells grown in epidermal growth factor (EGF) also adopted a mostly glial fate after differentiation. In contrast, PSA-NCAM-negative cells and striatal neonatal stem cells, grown in EGF or FGF2, generated the three CNS cell types. Like neural stem cells, PSA-negative cells generated more oligodendrocytes and fewer neurons when expanded in FGF2 and T3. Thus emergence of PSA-NCAM at the surface of neonatal brain precursors coincides with their restriction to a glial fate. T3 modulates these events by enhancing PSA-NCAM+ pre-progenitor growth in FGF2 and favoring an oligodendrocyte fate.

A stochastic model of brain cell differentiation in tissue culture.

The timing of cell differentiation can be controlled both by cellintrinsic mechanisms and by cell-extrinsic signals. Oligodendrocyte type-2 astrocyte progenitor cells are known to be the precursor cells that give rise to oligodendrocytes. When stimulated to divide by purified cortical astrocytes or by platelet-derived growth factor, these progenitor cells generate oligodendrocytes in vitro with a timing like that observed in vivo. The most widely accepted model of this process assumes a cell-intrinsic biological clock that resides in the progenitor cell. The intrinsic clock model originally proposed in 1986 remains as the dominant theoretical concept for the analysis of timed differentiation in this cell lineage. However, the results of a recent experimental study (Ibarrola et al., Developmental Biology, vol. 180, 1-21, 1996) are most consistent with the hypothesis that the propensity of a clone of dividing O-2A progenitor cells initially to generate at least one oligodendrocyte may be regulated by cell-intrinsic mechanisms, but that environmental signals regulate the extent of further oligodendrocyte generation. We propose a stochastic model of cell differentiation in culture to accommodate the most recent experimental findings. Our model is an age-dependent branching stochastic process with two types of cells. The model makes it possible to derive analytical expressions for the expected number of progenitor cells and of oligodendrocytes as functions of time. The model parameters were estimated by fitting these functions through data on the average (sample mean) number of both types of cells per colony at different time intervals from start of experiment. Using this method we provide a biologically meaningful interpretation of the observed pattern of oligodendrocyte generation in vitro and its modification in the presence of thyroid hormone.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells express glutamine synthetase: developmental and cell type-specific regulation.

Glutamine synthetase (GS), the enzyme that catalyses glutamine synthesis from glutamate and ammonia, plays a central role in the detoxification of brain ammonia. In the central nervous system (CNS), GS also subserves additional important functions such as regulating glutamate, GABA and amino acid metabolism. Oligodendrocytes (OL) form the myelin sheath in the central nervous system (CNS) and are essential for efficient propagation of nerve impulses. In culture, OL arise from bipotential O-2A progenitor cells. These O-2A cells give rise to type-2 astrocytes in the presence of serum. GS is expressed in mature glial cells in vivo and in vitro, but it is unknown whether GS is present in glial progenitors. In addition, a comparison of the GS expression level among the various types of glial cells has never been done in vitro. The current study investigates in vitro GS expression levels in O-2A progenitors, astrocytes and OL. We demonstrate that the GS gene is expressed in O-2A progenitors and is expressed at different levels in each cultured glial cell type. GS also is stimulated during OL developmental maturation. Thus, the GS gene is expressed in O-2A cells and is regulated in a developmental and macroglial cell type-specific manner.

Properties of GABA A receptors in cultured rat oligodendrocyte progenitor cells

We have studied the properties of GABA responses in oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells derived from primary cultures of the neonatal rat brain. In whole cell voltage clamp recordings, rapid application of 1–10 mM GABA elicited current responses in >85% of the cells examined. The dose-response relationship pooled from nine progenitor cells was best fit by a logistic function of EC 50=113 μM and Hill coefficient=0.9. In contrast to the rate of current deactivation, the rate of current activation exhibited marked concentration-dependence. Pharmacologically, GABA, muscimol and ZAPA ((Z)-3[(aminiiminomethyl)thio]prop-2-enoic acid sulphate) produced responses with ligand-specific kinetics, whereas glycine and the GABA C receptor agonist CACA were without effect; bicuculline methochloride acted as a competitive antagonist. Neither the amplitude nor the kinetics of currents produced by 100 μM GABA were affected by the benzodiazepine flunitrazepam (1 μM). Similarly the benzodiazepine receptor inverse agonist DMCM (1 μM) was also without effect. GABA-activated currents reversed polarity within 2 mV of the calculated Cl − equilibrium potential. With brief agonist pulses deactivation was monoexponential, however, unlike neurones the rate of deactivation was voltage-independent. Desensitisation of responses to 10 mM GABA was bi-exponential and accelerated at depolarised membrane potentials. Increasing the amount of GABA A receptor desensitisation (by increasing the duration of the agonist exposure) consistently produced a slowing of deactivation.