In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

A demyelinating disease induced in C57B1/6N mice by intracranial injection of a coronavirus (murine hepatitis virus strain A59) is followed by functional recovery and efficient CNS myelin repair. To study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. Using three-color immunofluorescence combined with [3H]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. We identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells with the O4 antibody. Cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of O-2A lineage cells was strikingly increased; second, the O-2A population consisted of a higher proportion of O4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the O-2A lineage showed enhanced proliferation. This proliferation was not further enhanced by adding PDGF, basic fibroblast growth factor (bFGF), or insulin-like growth factor I (IGF-I) to the defined medium. However, bFGF and IGF-I seemed to influence the fate of O-2A lineage cells in cultures of demyelinated tissue. Basic FGF decreased the percentage of cells expressing galactocerebroside. In contrast, IGF-I increased the relative proportion of oligodendrocytes. Thus, O-2A lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. These properties may be linked to the efficient remyelination achieved in this demyelinating disease.     

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.     

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.     

The proteoglycan chondroitin sulfate is present in a subpopulation of cultured astrocytes and in their precursors

We have used an antibody raised against the bovine nasal cartilage proteoglycan chondroitin sulfate (CS) digested with chondroitinase ABC (anti-CS serum) to stain cerebellar glial cells maintained in culture. In cultures grown in the presence of serum, the antibody stained a subclass of GFAP+ astrocytes which we have previously shown to selectively bind the monoclonal antibodies A2B5 and LB1. Also the direct bipotential precursors of these cells, capable of differentiating into GFAP+ astrocytes or into Gal-C+, O1+ oligodendrocytes depending on the culture conditions, were stained, but stopped to produce CS when they differentiated into 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.     

Identification of an adult-specific glial progenitor cell

We have found that glial progenitor cells isolated from the optic nerves of adult rats are fundamentally different from their counterparts in perinatal animals. In our studies on bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, we have seen that O-2Aadult progenitor cells can be distinguished from O-2Aperinatal progenitors by their morphology and antigenic phenotype, their much longer cell cycle time (65 h versus 18 h), slower rate of migration rate (4 microns h-1 versus 21 microns h-1), and their time course of differentiation into oligodendrocytes or type-2 astrocytes in vitro (less than or equal to 3 days versus greater than 5 days). At least some of the differences between O-2Aadult and O-2Aperinatal progenitor cells appear to be clearly related to the differing cellular requirements of the adult and perinatal central nervous system (CNS). The properties of the O-2Aadult progenitor cells may make these cells ideally suited for the needs of the adult CNS, where rapid exponential increases in the number of oligodendrocytes and O-2A progenitor cells would be inappropriate. However, the properties of the O-2Aadult progenitor cells are such that they may not be able to replace oligodendrocytes in sufficient numbers to repair extensive or recurrent damage in the adult brain, such as in patients suffering from the human demyelinating disease multiple sclerosis. Moreover, available information about other tissues suggests that the transition from perinatal to adult progenitor cell types may represent a developmental mechanism of general importance.     

Irradiation in vitro discriminates between different O-2A progenitor cell subpopulations in the perinatal central nervous system of rats

The effects of X irradiation on oligodendrocyte-type-2-astrocyte (O-2A) progenitor cells derived from different regions of the perinatal central nervous system (CNS) of rats were investigated in vitro. The O-2A progenitor cells can differentiate into either oligodendrocytes or type-2 astrocytes. The depletion of these cells could lead to demyelination, seen as a delayed reaction after irradiation of the CNS in vivo. To quantify cell survival, O-2A progenitor cells were grown on monolayers of type-1 astrocytes. Monolayers of type-1 astrocytes stimulate O-2A progenitor cells to divide. O-2A progenitor cells were irradiated in vitro and clonogenic cell survival was measured. The O-2A progenitor cells derived from perinatal optic nerve were quite radiosensitive in contrast to O-2A progenitor cells derived from perinatal spinal cord and perinatal corpus callosum. Furthermore, O-2A progenitor cells derived from the optic nerve formed smaller colonies, with most colonies showing early differentiation into oligodendrocytes. In contrast, more than half of the colonies derived from corpus callosum did not show any differentiation after 2 weeks in vitro and kept growing. These differences support the view that perinatal O-2A progenitor cells derived from the optic nerve are committed progenitor cells while the O-2A progenitor cells derived from the perinatal corpus callosum and the perinatal spinal cord have more stem cell properties.     

Characterization of a slowly proliferative cell along the oligodendrocyte differentiation pathway

A single bipotential glial progenitor cell of newborn rat optic nerve (the O-2A progenitor) characterized by its reactivity with antibodies to surface gangliosides (A2B5) and the presence of vimentin, can grow in microcultures in conditions which favor this progenitor's differentiation into oligodendrocytes. We selected at 8 days larger clones derived from such bipolar progenitors which had steadily proliferated on a layer of Type-1 astrocytes during the first week. Clonal growth and ratio of progenitor cells to oligodendrocytes was measured over the next two weeks by phase microscopy and double immunofluorescence labeling for the specific markers A2B5, GC (galactocerebroside, a surface marker for differentiated oligodendrocytes), O4 (a glycolipid marker of oligodendrocytes and some progenitors), GFAP (an astrocyte specific intermediate filament protein) and vimentin. Two types of clones were identified: type A clones (the majority), which were still slowly expanding at three weeks, and type B clones (the minority), which had stopped proliferating during the second week. In type A clones, some cells became GC positive multipolar oligodendrocytes, while other multipolar cells remained GC negative for days and expressed A2B5 and O4, but not GFAP or vimentin. Type B clones contained only GC positive, vimentin negative oligodendrocytes, which were generated during the second week and then decreased in number because of their restricted lifespan. Type B clones only developed in the presence of insulin or neurons. The number of GC negative cells in type A clones increased when insulin or neurons were deleted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

Identity, distribution, and development of polydendrocytes: NG2-expressing glial cells

Cells that express the NG2 proteoglycan (NG2+ cells) comprise a unique population of glial cells in the central nervous system. While there is no question that some NG2+ cells differentiate into oligodendrocytes during development, the persistence of numerous NG2+ cells in the mature CNS has raised questions about their identity, relation to other CNS cell types, and functions besides their progenitor role. NG2+ cells also express the alpha receptor for platelet-derived growth factor (PDGF αR), a receptor that mediates oligodendrocyte progenitor proliferation during development. Antigenically, NG2+ cells are distinct from fibrous and protoplasmic astrocytes, resting microglia, and mature oligodendrocytes. Therefore, we propose the term polydendrocytesto refer to all NG2-expressing glial cells in the CNS parenchyma. This distinguishes them from the classical glial cell types and identifies them as the fourth major glial population in the CNS. Recent observations suggest that polydendrocytes are complex cells that physically and functionally interact with other cell types in the CNS. Committed oligodendrocyte progenitor cells arise from restricted foci in the ventral ventricular zone in both spinal cord and brain. It remains to be clarified whether there are multiple sources of oligodendrocytes, and if so whether polydendrocytes (NG2+ cells) represent progenitor cells of all oligodendrocyte lineages. Proliferation of NG2+ cells during early development appears to be dependent on PDGF, but the regulatory mechanisms that govern NG2+ cell proliferation in the mature CNS remain unknown. Pulse-chase labeling with bromodeoxyuridine indicates that polydendrocytes that proliferate in the postnatal spinal cord differentiate into oligodendrocytes. Novel experimental approaches are being developed to further elucidate the functional properties and differentiation potential of polydendrocytes.     

Expression of fibronectin and laminin by different types of mouse glial cells cultured in a serum-free medium

The expression of fibronectin and laminin by cultured glial cells was studied. The glial culture from neonatal mouse cerebra maintained in a chemically defined, serum-free medium consisted of type-1 astrocytes, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, oligodendrocytes and type-2 astrocytes. Double-labelling immunofluorescent experiments performed using the mixed glial culture indicated that fibronectin and laminin are expressed in different patterns among the glial subtypes. The staining intensities with anti-fibronectin or anti-laminin antibodies decreased in the order: type-1 astrocytes, O-2A progenitor cells and type-2 astrocytes. Both molecules were deposited in a fibrillar matrix underneath type-1 astrocytes, whereas only intracytoplasmic localization of these molecules was observed with O-2A progenitor cells and type-2 astrocytes. Western blot analysis showed that glial fibronectin has a slightly higher molecular weight than mouse plasma fibronectin (230 kDa) and that glial laminin is a variant with a 220 kDa B chain present and the 400 kDa A chain missing. Using enzyme-linked immunosorbent assays (ELISA), these molecules were detected in the glial extracellular matrix at the concentration of 4 ng/10⁶ cells. A large amount of fibronectin (82 ng/10⁶ cells) was secreted into the culture medium, while secretion of laminin was not detected.     

Remyelination after chronic spinal cord injury is associated with proliferation of endogenous adult progenitor cells after systemic administration of guanosine

Axonal demyelination is a consistent pathological sequel to chronic brain and spinal cord injuries and disorders that slows or disrupts impulse conduction, causing further functional loss. Since oligodendroglial progenitors are present in the demyelinated areas, failure of remyelination may be due to lack of sufficient proliferation and differentiation of oligodendroglial progenitors. Guanosine stimulates proliferation and differentiation of many types of cells in vitro and exerts neuroprotective effects in the central nervous system (CNS). Five weeks after chronic traumatic spinal cord injury (SCI), when there is no ongoing recovery of function, intraperitoneal administration of guanosine daily for 2 weeks enhanced functional improvement correlated with the increase in myelination in the injured cord. Emphasis was placed on analysis of oligodendrocytes and NG2-positive (NG2+) cells, an endogenous cell population that may be involved in oligodendrocyte replacement. There was an increase in cell proliferation (measured by bromodeoxyuridine staining) that was attributable to an intensification in progenitor cells (NG2+ cells) associated with an increase in mature oligodendrocytes (determined by Rip+ staining). The numbers of astroglia increased at all test times after administration of guanosine whereas microglia only increased in the later stages (14 days). Injected guanosine and its breakdown product guanine accumulated in the spinal cords; there was more guanine than guanosine detected. We conclude that functional improvement and remyelination after systemic administration of guanosine is due to the effect of guanosine/guanine on the proliferation of adult progenitor cells and their maturation into myelin-forming cells. This raises the possibility that administration of guanosine may be useful in the treatment of spinal cord injury or demyelinating diseases such as multiple sclerosis where quiescent oligodendroglial progenitors exist in demyelinated plaques.     

Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury

Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages.     

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.     

Gliogenesis in rat optic nerve: astrocytes are generated in a single wave before oligodendrocytes

The time of origin for astrocytes in the rat optic nerve was investigated to determine whether this cell type is generated in two waves, a first wave which occurs before the formation of oligodendrocytes and a second wave which occurs after the peak period of oligodendrocyte formation. To answer this question, multiple injections of radioactive thymidine were administered to rats after the peak period of oligodendrocyte production in the optic nerve and the animals were sacrificed several weeks after the first injection. Thymidine-labeled cells in the optic nerve were identified with the electron microscope. Of the labeled cells, greater than 80% are oligodendrocytes, 4% are microglia, 2% are astrocytes, and the remainder are unclassifiable. The thymidine-labeled cells in the nerve were not immunostained for glial fibrillary acidic protein (GFAP), a marker characteristic of astrocytes. The number of thymidine-labeled glia generated after the second postnatal week is a small fraction of the total number of glia generated neonatally. No evidence exists for a second wave of astrocyte formation in the rat optic nerve as has been suggested in a study by Miller et al. (1985, Dev. Biol. 111, 35-41); rather, the vast majority of astrocytes are generated during the first 2 postnatal weeks and these data are in keeping with classical studies of gliogenesis. The question of whether astrocytes in the rat optic nerve arise directly from division of an undifferentiated, common progenitor cell or from a cell committed to the astrocyte lineage was addressed by combining thymidine autoradiography with GFAP immunocytochemistry. Rats were sacrificed 1 hr after an injection of thymidine and their nerves were processed for GFAP immunocytochemistry and autoradiography. During the first postnatal week, many thymidine-labeled cells are immunostained for GFAP. These observations demonstrate that cells committed to the astrocyte lineage divide neonatally and give rise to additional astrocytes.     

神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

A2B5+/GFAP+ Cells of Rat Spinal Cord Share a Similar Lipid Profile with Progenitor Cells: A Comparative Lipidomic Study

The central nervous system (CNS) harbors multiple glial fibrillary acidic protein (GFAP) expressing cell types. In addition to the most abundant cell type of the CNS, the astrocytes, various stem cells and progenitor cells also contain GFAP+ populations. Here, in order to distinguish between two types of GFAP expressing cells with or without the expression of the A2B5 antigens, we performed lipidomic analyses on A2B5+/GFAP+ and A2B5?/GFAP+ cells from rat spinal cord. First, A2B5+/GFAP? progenitors were exposed to the leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP) to induce their differentiation to A2B5+/GFAP+ cells or A2B5?/GFAP+ astrocytes, respectively. The cells were then analyzed for changes in their phospholipid, sphingolipid or acyl chain profiles by mass spectrometry and gas chromatography. Compared to A2B5+/GFAP? progenitors, A2B5?/GFAP+ astrocytes contained higher amounts of ether phospholipids (especially the species containing arachidonic acid) and sphingomyelin, which may indicate characteristics of cellular differentiation and inability for multipotency. In comparison, principal component analyses revealed that the lipid composition of A2B5+/GFAP+ cells retained many of the characteristics of A2B5+/GFAP? progenitors, but their lipid profile was different from that of A2B5?/GFAP+ astrocytes. Thus, our study demonstrated that two GFAP+ cell populations have distinct lipid profiles with the A2B5+/GFAP+ cells sharing a phospholipid profile with progenitors rather than astrocytes. The progenitor cells may require regulated low levels of lipids known to mediate signaling functions in differentiated cells, and the precursor lipid profiles may serve as one measure of the differentiation capacity of a cell population.     

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.     

Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell.

Optic nerves of neonatal rats contain a bipotential glial progenitor cell which can be induced by tissue culture conditions to differentiate into either an oligodendrocyte (the myelin-forming cell of the CNS) or a type 2 astrocyte (an astrocyte population found only in the myelinated tracts of the CNS). In our previous studies most oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells differentiated within 3 days in vitro with relatively little division of the progenitors or their differentiated progeny. We have now found that the O-2A progenitors are stimulated to divide in culture by purified populations of type 1 astrocytes, another glial cell-type found in the rat optic nerve. This cell-cell interaction appears to be mediated by a soluble factor(s) and results in the production of large numbers of both progenitor cells and oligodendrocytes. As type 1 astrocytes are the major glial cell-type in the optic nerve when oligodendrocytes first begin to be produced in large numbers in vivo, our results suggest that this astrocyte subpopulation may play an important role in expanding the oligodendrocyte population during normal development.