Two proliferative stages of the oligodendrocyte lineage (A2B5+O4- and O4+GalC-) under different mitogenic control

Cell proliferation during successive stages of oligodendrocyte development was delineated in the rat brain and optic nerve. Surface antigens, A2B5, O4, and galactocerebroside (GalC) identified three cell populations emerging in sequence; the incorporation of bromodeoxyuridine into newly synthesized DNA identified the proliferative cells. In vivo, progenitor cells with phenotypes A2B5+O4- and A2B5+O4+GalC- were both proliferative, whereas differentiated GalC+ oligodendrocytes were not. Under basal conditions of culture, the proliferation of both progenitor cell types of the optic nerve was nearly abolished. Activity was restored for A2B5+O4- precursor cells with medium conditioned by either type-1 astrocytes, meningeal cells, or cerebellar interneurons. In contrast, intermediate O4+GalC- cells (proligodendrocytes) were refractory to the astroglial and meningeal signals, but remained as responsive as their precursor cells to the neuronal stimulus. These data further characterize the O4+GalC- proligodendrocyte as a distinct developmental stage, one that specifies a changing response of the cell to environmental mitogens.     

Proligodendroblast Antigen (POA), a Developmental Antigen Expressed by A007/O4-Positive Oligodendrocyte Progenitors Prior to the Appearance of Sulfatide and Galactocerebroside

Evidence is presented for the immunological identification of a developmental antigen appearing at a critical point in the oligodendroglial lineage. Specifically, monoclonal antibody A007 recognizes cells in the oligodendrocyte lineage at two distinct stages. Analyses of purified lipid standards and lipid extracts from galactocerebroside-positive (GalC+) oligodendrocytes by enzyme-linked immunosorbent assay, lipid dot blot, and immuno-TLC demonstrated that A007 recognizes sulfatide (SUL) and seminolipid. However, neither 35SO4 incorporation into SUL nor SUL accumulation could be detected in A007-positive cells lacking galactocerebroside (i.e., A007+GalC- progenitor cells) present early in development. These data suggest that A007 also recognizes an antigen, named proligodendroblast antigen (POA), that appears during the late stage of oligodendrocyte progenitor development prior to the expression by oligodendrocytes of SUL and GalC. We have previously reported that monoclonal antibody O4 also recognizes not only SUL and seminolipid, but in addition an antigen that appears prior to the expression of SUL and galactocerebroside. In the present study all A007+ cells were also O4+ (and vice versa), and the developmental patterns of the two antibodies appeared to be identical. We conclude that (1) A007 is similar or identical to O4 with respect to its antigenic specificity, and (2) during oligodendrocyte lineage progression both antibodies react first with antigen POA on the surface of the oligodendrocyte progenitor cell prior to the expression of SUL [i.e., A007+O4+(POA+)SUL-GalC- proligodendroblasts], and only later with SUL as terminally differentiating oligodendrocytes emerge (i.e., A007+O4+SUL+GalC+ oligodendrocytes).     

Sonic hedgehog-dependent emergence of oligodendrocytes in the telencephalon: evidence for a source of oligodendrocytes in the olfactory bulb that is independent of PDGFRalpha signaling.

Most studies on the origin of oligodendrocyte lineage have been performed in the spinal cord. By contrast, molecular mechanisms that regulate the appearance of the oligodendroglial lineage in the brain have not yet attracted much attention. We provide evidence for three distinct sources of oligodendrocytes in the mouse telencephalon. In addition to two subpallial ventricular foci, the anterior entopeduncular area and the medial ganglionic eminence, the rostral telencephalon also gives rise to oligodendrocytes. We show that oligodendrocytes in the olfactory bulb are generated within the rostral pallium from ventricular progenitors characterized by the expression of PLP: We provide evidence that these Plp oligodendrocyte progenitors do not depend on signal transduction mediated by platelet-derived growth factor receptors (PDGFRs), and therefore propose that they belong to a different lineage than the PDGFRalpha-expressing progenitors. Moreover, induction of oligodendrocytes in the telencephalon is dependent on sonic hedgehog signaling, as in the spinal cord. In all these telencephalic ventricular territories, oligodendrocyte progenitors were detected at about the same developmental stage as in the spinal cord. However, both in vivo and in vitro, the differentiation into O4-positive pre-oligodendrocytes was postponed by 4-5 days in the telencephalon in comparison with the spinal cord. This delay between determination and differentiation appears to be intrinsic to telencephalic oligodendrocytes, as it was not shortened by diffusible or cell-cell contact factors present in the spinal cord.     

Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture

We have previously demonstrated that lineage negative cells (Lin^neg) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone, muscle, endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin^neg UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin, neurofilament, A2B5 and Sox2) are expressed in Lin^neg cells cultured in FGF4, SCF, Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase, GalC, Olig2 and the late-stage marker MOSP are observed, as is the Schwann cell marker PMP22. In summary, Lin^neg UCB cells, when appropriately cultured, are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation.     

The mitotic history and radiosensitivity of developing oligodendrocytes in vitro

By use of pulse-chase exposure of dissociated cells of rat fetal spinal cord or brain to [3H]thymidine (TdR) and unlabeled TdR it has been shown that oligodendroglial precursors which do not express galactocerebroside (GalC) divide first and later differentiate to express GalC. The rate of proliferation of more mature GalC+ oligodendrocytes is considerably lower than that of their GalC- precursors. It has been found that oligodendrocyte precursor cells are extremely sensitive to [3H]TdR irradiation. Exposure to as little as 0.03 microCi/ml for 24 hr proved to be harmful, particularly during a critical period before birth. This critical period corresponded to the peak of division of oligodendrocyte precursor cells.     

The erbB2 gene is required for the development of terminally differentiated spinal cord oligodendrocytes

Development of oligodendrocytes and the generation of myelin internodes within the spinal cord depends on regional signals derived from the notochord and axonally derived signals. Neuregulin 1 (NRG)-1, localized in the floor plate as well as in motor and sensory neurons, is necessary for normal oligodendrocyte development. Oligodendrocytes respond to NRGs by activating members of the erbB receptor tyrosine kinase family. Here, we show that erbB2 is not necessary for the early stages of oligodendrocyte precursor development, but is essential for proligodendroblasts to differentiate into galactosylcerebroside-positive (GalC+) oligodendrocytes. In the presence of erbB2, oligodendrocyte development is normal. In the absence of erbB2 (erbB2-/-), however, oligodendrocyte development is halted at the proligodendroblast stage with a >10-fold reduction in the number of GalC+ oligodendrocytes. ErbB2 appears to function in the transition of proligodendroblast to oligodendrocyte by transducing a terminal differentiation signal, since there is no evidence of increased oligodendrocyte death in the absence of erbB2. Furthermore, known survival signals for oligodendrocytes increase oligodendrocyte numbers in the presence of erbB2, but fail to do so in the absence of erbB2. Of the erbB2-/- oligodendrocytes that do differentiate, all fail to ensheath neurites. These data suggest that erbB2 is required for the terminal differentiation of oligodendrocytes and for development of myelin.     

Human induced pluripotent stem cells differentiation into oligodendrocyte progenitors and transplantation in a rat model of optic chiasm demyelination

Background

This study aims to differentiate human induced pluripotent stem cells (hiPSCs) into oligodendrocyte precursors and assess their recovery potential in a demyelinated optic chiasm model in rats.

Methodology/Principal Findings

We generated a cell population of oligodendrocyte progenitors from hiPSCs by using embryoid body formation in a defined medium supplemented with a combination of factors, positive selection and mechanical enrichment. Real-time polymerase chain reaction and immunofluorescence analyses showed that stage-specific markers, Olig2, Sox10, NG2, PDGFRα, O4, A2B5, GalC, and MBP were expressed following the differentiation procedure, and enrichment of the oligodendrocyte lineage. These results are comparable with the expression of stage-specific markers in human embryonic stem cell-derived oligodendrocyte lineage cells. Transplantation of hiPSC-derived oligodendrocyte progenitors into the lysolecithin-induced demyelinated optic chiasm of the rat model resulted in recovery from symptoms, and integration and differentiation into oligodendrocytes were detected by immunohistofluorescence staining against PLP and MBP, and measurements of the visual evoked potentials.

Conclusions/Significance

These results showed that oligodendrocyte progenitors generated efficiently from hiPSCs can be used in future biomedical studies once safety issues have been overcome.     

Cell cycle arrest induced by ectopic expression of p27 is not sufficient to promote oligodendrocyte differentiation

Oligodendrocyte differentiation is accompanied by dramatic changes in gene expression as well as cell cycle arrest. To determine whether cell cycle arrest is sufficient to induce the changes in cell phenotype associated with differentiation, we inhibited oligodendrocyte precursor proliferation in vitro by overexpressing p27, a cyclin kinase inhibitor, using a recombinant adenovirus. Ectopic expression of p27 efficiently inhibited oligodendrocyte precursor cell division, even in the presence of exogenous mitogens, by blocking the activity of the cyclin-dependent kinase, cdk2. Although the cells had stopped dividing, they did not express galactocerebroside (GalC) or myelin basic protein (MBP), changes associated with oligodendrocyte differentiation, suggesting that they had not differentiated. After removal of exogenous mitogens, however, adenovirus-expressing oligodendrocyte precursors differentiated with a temporal profile similar to that of control, uninfected oligodendrocytes, as indicated by expression of GalC and MBP. We conclude that cell cycle arrest is not sufficient to induce differentiation of dividing oligodendrocyte precursors, and that modulation of additional, as yet unknown, signaling pathways is required for this to occur.     

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.     

Cell cycle arrest induced by ectopic expression of p27 is not sufficient to promote oligodendrocyte differentiation

Oligodendrocyte differentiation is accompanied by dramatic changes in gene expression as well as cell cycle arrest. To determine whether cell cycle arrest is sufficient to induce the changes in cell phenotype associated with differentiation, we inhibited oligodendrocyte precursor proliferation in vitro by overexpressing p27, a cyclin kinase inhibitor, using a recombinant adenovirus. Ectopic expression of p27 efficiently inhibited oligodendrocyte precursor cell division, even in the presence of exogenous mitogens, by blocking the activity of the cyclin‐dependent kinase, cdk2. Although the cells had stopped dividing, they did not express galactocerebroside (GalC) or myelin basic protein (MBP), changes associated with oligodendrocyte differentiation, suggesting that they had not differentiated. After removal of exogenous mitogens, however, adenovirus‐expressing oligodendrocyte precursors differentiated with a temporal profile similar to that of control, uninfected oligodendrocytes, as indicated by expression of GalC and MBP. We conclude that cell cycle arrest is not sufficient to induce differentiation of dividing oligodendrocyte precursors, and that modulation of additional, as yet unknown, signaling pathways is required for this to occur. J. Cell. Biochem. 76:270–279, 1999. © 1999 Wiley‐Liss, Inc.     

Contact with central nervous system myelin inhibits oligodendrocyte progenitor maturation

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated during development through the proliferation and differentiation of a distinct progenitor population. Not all oligodendrocyte progenitors generated during development differentiate, however, and large numbers of oligodendrocyte progenitors are present in the adult CNS, particularly in white matter. These "adult progenitors" can be identified through expression of the NG2 proteoglycan. Adult oligodendrocyte progenitors are thought to develop from the original pool of progenitors and in vitro are capable of differentiating into oligodendrocytes. Why these cells fail to differentiate in the intact CNS is currently unclear. Here we show that contact with CNS myelin inhibits the maturation of immature oligodendrocyte progenitors. The inhibition of oligodendrocyte progenitor maturation is a characteristic of CNS myelin that is not shared by several other membrane preparations including adult and neonatal neural membrane fractions, PNS myelin, or liver. This inhibition is concentration dependent, is reversible, and appears not to be mediated by either myelin basic protein or basic fibroblast growth factor. Myelin-induced inhibition of oligodendrocyte progenitor maturation provides a mechanism to explain the generation of a residual pool of immature oligodendrocyte progenitors in the mature CNS.     

Genetic dissection of myelin galactolipid function

The roles that the myelin galactolipids galactocerebroside (GalC) and sulfatide play in cellular differentiation, myelin formation and maintenance have been investigated for nearly 3 decades. During that time the primary approach has been to perturb lipid activity using antibodies and chemical agents in artificial systems. Recently, the isolation of the gene that encodes UDP-galactose:ceramide galactosyltransferase (CGT), the enzyme that catalyzes an essential step in the synthetic pathway of GalC and sulfatide, has enabled the generation of mice that lack myelin galactolipids. These mice display a severe tremor, hindlimb paralysis and electrophysiological defects. In addition, the CGT null mutants exhibit: 1) impaired oligodendrocyte differentiation, 2) myelin sheaths that are thin, incompletely compacted and unstable, and 3) structural abnormalities in the nodal and paranodal regions including disrupted axo-glial junctions. Collectively, these findings suggest that GalC and sulfatide are essential in myelin formation and maintenance, possibly by mediating intra- and intercellular interactions.     

p38 MAP Kinase Regulation of Oligodendrocyte Differentiation with CREB as a Potential Target

Despite a substantial understanding of the factors regulating oligodendrocyte differentiation, the signaling mechanisms involved in this process are not well-understood. This study elaborates on the findings (Bhat NR, Zhang P (1997) FASEB J 11:A925; Baron W, Metz B, Bansal R, Hoekstra D, de Vries H (2000) Mol Cell Neurosci 15:314–329) of a role for p38 MAP kinase signaling in oligodendrocyte differentiation and myelin gene expression. When proliferating oligodendrocyte progenitors were switched to a growth factor-free differentiation medium, there was a rapid activation of p38 kinase that correlated with an increased phosphorylation of CREB, a down-stream target and a factor involved in oligodendrocyte differentiation. Addition of forskolin, a known inducer of intracellular c-AMP and of oligodendrocyte differentiation, also stimulated CREB phosphorylation in a p38 kinase dependent way. Pharmacological inhibition of p38 interfered with the morphological and antigenic changes associated with differentiating oligodendrocytes as well as with the developmental and forskolin-induced expression of myelin basic protein, thereby supporting an essential role for p38 MAPK pathway in oligodendrocyte differentiation. Special issue dedicated to Anthony Campagnoni.     

The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair

Deciphering the mechanisms regulating the generation of new neurons and new oligodendrocytes, the myelinating cells of the central nervous system, is of paramount importance to address new strategies to replace endogenous damaged cells in the adult brain and foster repair in neurodegenerative diseases. Upon brain injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes (cysLTs), two families of endogenous signaling molecules, are markedly increased at the site of damage, suggesting that they may act as "danger signals" to alert responses to tissue damage and start repair. Here we show that, in brain telencephalon, GPR17, a recently deorphanized receptor for both uracil nucleotides and cysLTs (e.g., UDP-glucose and LTD(4)), is normally present on neurons and on a subset of parenchymal quiescent oligodendrocyte precursor cells. We also show that induction of brain injury using an established focal ischemia model in the rodent induces profound spatiotemporal-dependent changes of GPR17. In the lesioned area, we observed an early and transient up-regulation of GPR17 in neurons expressing the cellular stress marker heat shock protein 70. Magnetic Resonance Imaging in living mice showed that the in vivo pharmacological or biotechnological knock down of GPR17 markedly prevents brain infarct evolution, suggesting GPR17 as a mediator of neuronal death at this early ischemic stage. At later times after ischemia, GPR17 immuno-labeling appeared on microglia/macrophages infiltrating the lesioned area to indicate that GPR17 may also acts as a player in the remodeling of brain circuitries by microglia. At this later stage, parenchymal GPR17+ oligodendrocyte progenitors started proliferating in the peri-injured area, suggesting initiation of remyelination. To confirm a specific role for GPR17 in oligodendrocyte differentiation, the in vitro exposure of cortical pre-oligodendrocytes to the GPR17 endogenous ligands UDP-glucose and LTD(4) promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a "sensor" that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis.     

Novel oligodendrocyte transmembrane signaling systems

Antibodies are increasingly being used as tools to study the function of cell surface markers. Several types of responses may occur upon the selective binding of an antibody to an epitope on a receptor. Antibody binding may trigger signals that are normally transduced by endogenous ligands. Moreover, antibody binding may activate normal signals in a manner that disrupts a sequence of events that coordinates either differentiation, mitogenesis, or morphogenesis. Alternately, it is possible that binding elicits either a modified signal or no signal. This article focuses on the cascade of events that occur following specific antibody binding to myelin markers expressed by cultured murine oligodendrocytes. Binding of specific antibodies to the oligodendrocyte membrane surface markers myelin/oligodendrocyte glycoprotein (MOG), myelin/oligodendrocyte specific protein (MOSP), galactocerebroside (GalC), and sulfatide on cultured murine oligodendrocytes results in different effects with regard to phospholipid turnover, Ca²⁺ influxes, and antibody:marker distribution. The consequence of each antibody-elicited cascade of events appears to be the regulation of the cytoskeleton within the oligodendroglial membrane sheets. The antibody binding studies described in this article demonstrate that these myelin surface markers are capable of transducing signals. Since endogenous ligands for these myelin markers have yet to be identified, it is not known if these signals are normally transduced or are a modification of normally transduced signals.     

Establishment of mouse oligodendrocyte/type-2 astrocyte lineage cell line by transfection with origin-defective simian virus 40 DNA.

A permanent glial cell line has been established from the neonatal mouse primary mixed glial cell cultures by transfection with replication origin-defective simian virus 40 DNA. This cell line, designated OS3, has morphological similarity to type-2 astrocyte and expresses an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), when cultured in the presence of 10% calf serum (CS). OS3 cells do not express the O4 antigen, galactocerebroside (GalC) and A2B5 under this culture condition. When cultured in a medium containing 2% CS or a chemically defined medium, these cells undergo morphological transformation. Some of these cells express O4 antigen and/or GalC, and the percentage of GFAP positive cells decreases under these conditions. Thus depending on the culture conditions, the OS3 cells display either type-2 astrocyte properties or immature oligodendrocyte characteristics. Furthermore, the OS3 cells show similar responses to the various growth factors as do oligodendrocyte/type-2 astrocyte (O-2A) progenitors. Therefore, the OS3 cell line is an unique mouse bipotential permanent O-2A lineage cell line which may be useful to analyze the developmental properties of these glial cells.     

Loss of p27Kip1 function results in increased proliferative capacity of oligodendrocyte progenitors but unaltered timing of differentiation.

In many tissues, progenitor cells permanently withdraw from the cell cycle prior to commitment towards a differentiated phenotype. In the oligodendrocyte lineage a counting mechanism has been proposed, linking the number of cell divisions to growth arrest and differentiation. A direct prediction of this model is that an increase in the number of cell divisions would result in a delayed onset of differentiation. Since the cell cycle inhibitor p27Kip1 is an essential component of the machinery leading to oligodendrocyte progenitor growth arrest, we examined the temporal relationship between cell cycle withdrawal and expression of late differentiation markers in vivo, in mice carrying a targeted deletion in the p27Kip1 gene. Using bromodeoxyuridine to label proliferating cells, quaking (QKI) to identify embryonic glial progenitors, NG2 to identify neonatal oligodendrocyte progenitors, and myelin basic protein to label differentiated oligodendrocytes, we found an increased number of proliferating QKI- and NG2-positive cells in germinal zones of p27Kip1(-/-) mice at the peak of gliogenesis. However, no delay was observed in these mice in the appearance of the late differentiation marker myelin basic protein in the developing corpus callosum and cerebellum. Significantly, a decrease in cyclin E levels was observed in the brain of p27Kip1 null mice coincident with oligodendrocyte growth arrest. We conclude that two distinct modalities of growth arrest occur in the oligodendrocyte lineage: a p27Kip1-dependent mechanism of growth arrest affecting proliferation in early phases of gliogenesis, and a p27Kip1-independent event leading to withdrawal from the cell cycle and differentiation.     

Interactions between oligodendrocyte precursors control the onset of CNS myelination

The formation of CNS myelin is dependent on the differentiation of oligodendrocyte precursor cells (OPCs) and oligodendrocyte maturation. How the initiation of myelination is regulated is unclear, but it is likely to depend on the development of competence by oligodendrocytes and receptivity by target axons. Here we identify an additional level of control of oligodendrocyte maturation mediated by interactions between the different cellular components of the oligodendrocyte lineage. During development oligodendrocyte precursors mature through a series of stages defined by labeling with monoclonal antibodies A2B5 and O4. Newly differentiated oligodendrocytes begin to express galactocerebroside recognized by O1 antibodies and subsequently mature to myelin basic protein (MBP)-positive cells prior to formation of compact myelin. Using an in vitro brain slice culture system that supports robust myelination, the consequences of ablating cells at different stages of the oligodendrocyte lineage on myelination have been assayed. Elimination of all OPC lineage cells through A2B5+, O4+, and O1+ complement-mediated cell lysis resulted in a delay in development of MBP cells and myelination. Selective elimination of early OPCs (A2B5+) also unexpectedly resulted in delayed MBP expression compared to controls suggesting that early OPCs contribute to the timing of myelination onset. By contrast, elimination of differentiated (O1+) immature oligodendrocytes permanently inhibited the appearance of MBP+ cells suggesting that oligodendrocytes are critical to facilitate the maturation of OPCs. These data illuminate that the presence of intra-lineage feed-forward and feedback cues are important for timely myelination by oligodendrocytes.