NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS

Glial progenitor cells of the developing CNS committed to the oligodendrocyte lineage (OPCs) express the chondroitin sulfate proteoglycan, NG2. A proportion of OPCs fail to differentiate past the stage at which they express NG2 and the lipid antigen O4 and persist in the adult CNS in a phenotypically immature form. However, the physiological function of NG2(+) cells in the adult CNS is unknown. Using antibodies against NG2 we show that NG2 is expressed by a distinct cell population in the mature CNS with the homogeneous antigenic phenotype of oligodendrocyte progenitors. The morphology of NG2(+) OPCs varies from region to region, reflecting the different structural environments, but they appear to represent a homogeneous population within any one gray or white matter region. A study of nine CNS regions showed that NG2(+) OPCs are numerous throughout the CNS and numbers in the white matter are only 1.5 times that in the gray. Whereas the ratio of OPCs to myelinating oligodendrocytes in the spinal cord gray and white matter approximates 1:4, gray matter regions of the forebrain have a 1:1 ratio, a phenomenon that will have consequences for oligodendrocyte replacement following demyelination. BrdU incorporation experiments showed that NG2(+) cells are the major dividing cell population of the adult rat CNS. Since very little apoptosis was detected and BrdU became increasingly present in oligodendrocytes after a 10-day pulse chase, with a concomitant decrease in NG2(+) BrdU incorporating cells, we suggest that the size of the oligodendrocyte population may actually increase during adult life.     

Differentiation of proliferated NG2-positive glial progenitor cells in a remyelinating lesion

Cells that express the NG2 proteoglycan (NG2+ cells) constitute a large cell population in the adult mammalian central nervous system (CNS). They give rise to mature oligodendrocytes in culture and are thus considered to be oligodendrocyte progenitor cells (OPCs). They proliferate in response to a variety of insults to the CNS, but their ability to differentiate into oligodendrocytes in vivo has not been established. We used bromodeoxyuridine (BrdU) to trace the fate of NG2+ cells that proliferated in response to a chemically induced demyelinating lesion in the adult rat spinal cord. Cells that were proliferating 24 hr after lesioning were labeled by a single injection of BrdU, and their antigenic phenotype was examined at various times up to 28 days post-lesioning (28 dpl). Initially, at 2 dpl, NG2+/BrdU+ cells were found almost exclusively at the periphery of the lesion. At 7 dpl, the number of NG2+/BrdU+ cells increased in the lesion center and decreased from the surrounding areas. The number of NG2+/BrdU+ cells inside the lesion further decreased with time, concomitant with progression of remyelination and appearance of BrdU+ mature oligodendrocytes. Double labeling with (3)H-thymidine and BrdU combined with NG2 immunohistochemistry showed that some NG2+ cells in the lesion had undergone at least two rounds of cell division. These observations strongly suggest that NG2+/BrdU+ cells that appeared in response to the demyelinating insult gave rise to mature remyelinating oligodendrocytes, providing an in vivo evidence for the differentiation of NG2+ cells into oligodendrocytes.     

Regulation of oligodendrocyte precursor maintenance by chondroitin sulphate glycosaminoglycans

Chondroitin sulfate proteoglycans (CSPGs) have been proven to inhibit morphological maturation of oligodendrocytes as well as their myelination capabilities. Yet, it remained unclear, whether CSPGs and/or their respective chondroitin sulfate glycosaminoglycan (CS‐GAG) side chains also regulate the oligodendrocyte lineage progression. Here, we initially show that CS‐GAGs detected by the monoclonal antibody 473HD are expressed by primary rat NG2‐positive oligodendrocyte precursor cells (OPCs) and O4‐positive immature oligodendrocytes. CS‐GAGs become down‐regulated with ongoing oligodendrocyte differentiation. Enzymatic removal of the CS‐GAG chains by the bacterial enzyme Chondroitinase ABC (ChABC) promoted spontaneous differentiation of proliferating rat OPCs toward O4‐positive immature oligodendrocytes. Upon forced differentiation, the enzymatic removal of the CS‐GAGs accelerated oligodendrocyte differentiation toward both MBP‐positive and membrane forming oligodendrocytes. These processes were attenuated on enriched CSPG fractions, mainly consisting of Phosphacan/RPTPβ/ζ and to less extent of Brevican and NG2. To qualify CS‐GAGs as universal regulators of oligodendrocyte biology, we finally tested the effect of CS‐GAG removal on OPCs from different sources such as mouse cortical oligospheres, mouse spinal cord neurospheres, and most importantly human‐induced pluripotent stem cell‐derived radial glia‐like neural precursor cells. For all culture systems used, we observed a similar inhibitory effect of CS‐GAGs on oligodendrocyte differentiation. In conclusion, this study clearly suggests an important fundamental principle for complex CS‐GAGs to regulate the oligodendrocyte lineage progression. Moreover, the use of ChABC in order to promote oligodendrocyte differentiation toward myelin gene expressing cells might be an applicable therapeutic option to enhance white matter repair. GLIA 2016;64:270–286     

Glial and Neuronal Protein Tyrosine Phosphatase Alpha (PTPα) Regulate Oligodendrocyte Differentiation and Myelination

CNS myelination defects occur in mice deficient in receptor-like protein tyrosine phosphatase alpha (PTPα). Here, we investigated the role of PTPα in oligodendrocyte differentiation and myelination using cells and tissues from wild-type (WT) and PTPα knockout (KO) mice. PTPα promoted the timely differentiation of neural stem cell-derived oligodendrocyte progenitor cells (OPCs). Compared to WT OPCs, KO OPC cultures had more NG2+ progenitors, fewer myelin basic protein (MBP)+ oligodendrocytes, and reduced morphological complexity. In longer co-cultures with WT neurons, more KO than WT OPCs remained NG2+ and while equivalent MBP+ populations of WT and KO cells formed, the reduced area occupied by the MBP+ KO cells suggested that their morphological maturation was impeded. These defects were associated with reduced myelin formation in KO OPC/WT neuron co-cultures. Myelin formation was also impaired when WT OPCs were co-cultured with KO neurons, revealing a novel role for neuronal PTPα in myelination. Canonical Wnt/β-catenin signaling is an important regulator of OPC differentiation and myelination. Wnt signaling activity was not dysregulated in OPCs lacking PTPα, but suppression of Wnt signaling by the small molecule XAV939 remediated defects in KO oligodendrocyte differentiation and enhanced myelin formation by KO oligodendrocytes. However, the myelin segments that formed were significantly shorter than those produced by WT oligodendrocytes, raising the possibility of a role for glial PTPα in myelin extension distinct from its pro-differentiating actions. Altogether, this study reveals PTPα as a molecular coordinator of oligodendroglial and neuronal signals that controls multiple aspects of oligodendrocyte development and myelination.     

Increase of oligodendrocyte progenitor cells after spinal cord injury

The reaction of oligodendrocyte progenitor cells (OPCs) after spinal cord injury (SCI) is poorly understood. In this study, we examined oligodendroglial reactions after contusion SCI in adult rats by immunohistochemistry. OPCs were identified by staining with monoclonal antibodies (mAbs) A2B5 and O4. Each of the A2B5-, O4-positive OPCs and galactocerebroside-positive oligodendrocytes dramatically increased in the lesion of the dorsal posterior funiculus. Bromodeoxyuridine (BrdU) incorporation studies showed that most O4-positive cells in the lesion were labeled with BrdU, suggesting that these OPCs were proliferative. In contrast, the expression of myelin basic protein was decreased in the lesion compared with controls that received laminectomy only. From the injured cord, OPCs were isolated by immunopanning with mAb A2B5. We observed an increased number of OPCs from the injured spinal cords compared with those isolated from controls and unoperated animals. After several days in culture, the OPCs from the lesion expressed galactocerebroside. These results suggest that OPCs are induced and can differentiate following SCI in the adult rat.     

Cerebral cortex demyelination and oligodendrocyte precursor response to experimental autoimmune encephalomyelitis

Experimentally induced autoimmune encephalomyelitis (EAE) in mice provides an animal model that shares many features with human demyelinating diseases such as multiple sclerosis (MS). To what extent the cerebral cortex is affected by the process of demyelination and how the corollary response of the oligodendrocyte lineage is explicated are still not completely known aspects of EAE. By performing a detailed in situ analysis of expression of myelin and oligodendrocyte markers we have identified areas of subpial demyelination in the cerebral cortex of animals with conventionally induced EAE conditions. On EAE-affected cerebral cortices, the distribution and relative abundance of cells of the oligodendrocyte lineage were assessed and compared with control mouse brains. The analysis demonstrated that A2B5(+) glial restricted progenitors (GRPs) and NG2(+)/PDGFR-α(+) oligodendrocyte precursor cells (OPCs) were increased in number during "early" disease, 20 days post MOG immunization, whereas in the "late" disease, 39 days post-immunization, they were strongly diminished, and there was an accompanying reduction in NG2(+)/O4(+) pre-oligodendrocytes and GST-π mature oligodendrocytes. These results, together with the observed steady-state amount of NG2(-)/O4(+) pre-myelinating oligodendrocytes, suggested that oligodendroglial precursors attempted to compensate for the progressive loss of myelin, although these cells appeared to fail to complete the last step of their differentiation program. Our findings confirm that this chronic model of EAE reproduces the features of neocortex pathology in progressive MS and suggest that, despite the proliferative response of the oligodendroglial precursors, the failure to accomplish final differentiation may be a key contributing factor to the impaired remyelination that characterizes these demyelinating conditions.     

Alterations in the oligodendrocyte lineage, myelin, and white matter in adult mice lacking the chemokine receptor CXCR2

Oligodendrocyte precursor cell (OPC) proliferation and migration are critical for the development of myelin in the central nervous system (CNS). Previous studies showed that localized expression of the chemokine CXCL1 signals through the receptor CXCR2 to inhibit the migration and enhance the proliferation of spinal cord OPCs during development. Here, we report structural and functional alterations in the adult CNS of Cxcr2-/- mice. In Cxcr2-/- adult mice, we observed regional alterations in the density of oligodendrocyte lineage cells in Cxcr2-/- adult mice, with decreases in the cortex and anterior commissure but increases in the corpus callosum and spinal cord. An increase in the density and arborization of spinal cord NG2 positive cells was also observed in Cxcr2-/- adult mice. Compared with wild-type (WT) littermates, Cxcr2-/- mice exhibited a significant decrease in spinal cord white matter area, reduced thickness of myelin sheaths, and a slowing in the rate of central conduction of spinally elicited evoked potentials without significant changes in axonal caliber or number. Biochemical analyses showed decreased levels of myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP). In vitro studies showed reduced numbers of differentiated oligodendrocytes in Cxcr2-/- spinal cord cultures. Together, these findings indicate that the chemokine receptor CXCR2 is important for the development and maintenance of the oligodendrocyte lineage, myelination, and white matter in the vertebrate CNS.     

Progesterone attenuates astro- and microgliosis and enhances oligodendrocyte differentiation following spinal cord injury

Reactive gliosis, demyelination and proliferation of NG2+ oligodendrocyte precursor cells (OPC) are common responses to spinal cord injury (SCI). We previously reported that short-term progesterone treatment stimulates OPC proliferation whereas chronic treatment enhances OPC differentiation after SCI. Presently, we further studied the proliferation/differentiation of glial cells involved in inflammation and remyelination in male rats with SCI subjected to acute (3 days) or chronic (21 days) progesterone administration. Rats received several pulses of bromodeoyuridine (BrdU) 48 and 72 h post-SCI, and sacrificed 3 or 21 days post-SCI. Double colocalization of BrdU and specific cell markers showed that 3 days of SCI induced a strong proliferation of S100β+ astrocytes, OX-42+ microglia/macrophages and NG2+ cells. At this stage, the intense GFAP+ astrogliosis was BrdU negative. Twenty one days of SCI enhanced maturation of S100β+ cells into GFAP+ astrocytes, but decreased the number of CC1+ oligodendrocytes. Progesterone treatment inhibited astrocyte and microglia /macrophage proliferation and activation in the 3-day SCI group, and inhibited activation in the 21-day SCI group. BrdU/NG2 double labeled cells were increased by progesterone at 3 days, indicating a proliferation stimulus, but decreased them at 21 days. However, progesterone-enhancement of CC1+/BrdU+ oligodendrocyte density, suggest differentiation of OPC into mature oligondendrocytes. We conclude that progesterone effects after SCI involves: a) inhibition of astrocyte proliferation and activation; b) anti-inflammatory effects by preventing microglial activation and proliferation, and c) early proliferation of NG2+ progenitors and late remyelination. Thus, progesterone behaves as a glioactive factor favoring remyelination and inhibiting reactive gliosis.     

Transient exposure to FGF2 enhances myelination in embryonic brain cell cocultures

The amount of myelination in vivo and in vitro depends on the number of oligodendrocyte progenitors, their differentiation, and on the neuron function. It has been shown that continuous administration of FGF2, a mitotic and neuroprotective factor, allows oligodendrocyte progenitors to proliferate, but prevents them from differentiating and myelinating. This study was designed to test the effect of transient exposure to FGF2 on myelination in an oligodendrocyte/neuron coculture system. At 2 days in vitro, cultures were treated with a single dose of 20 ng/ml FGF2. Cell proliferation was determined by BrdU uptake. The number of cells of the oligodendrocyte lineage was determined by immunocytology of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). The maturation of oligodendrocytes and myelination was followed by immunocytological analysis of MBP (myelin basic protein). Electron microscopy was used to study the ultrastructure of myelin. BrdU uptake procedure showed an increase in cell proliferation in FGF2-treated cultures after 48 h of treatment. At 15-18 days in vitro, CNPase(+) and MBP(+) cells were much more abundant in cultures treated with FGF2 than in control cultures. We observed differentiation and maturation of oligodendrocytes and a higher degree of myelination in FGF2-treated cultures compared to controls. Electron microscopy showed the presence of myelin structures in FGF2-treated cultures that did not differ morphologically from those observed in control cultures. Transient exposure of cultured brain cells to FGF2 increased myelination in vitro. Administration of FGF2 over a short period might thus enhance remyelination in demyelinating diseases in vivo.     

Dorsally-derived oligodendrocytes in the spinal cord contribute to axonal myelination during development and remyelination following focal demyelination

In the developing spinal cord, the majority of oligodendrocytes are derived from the ventral ventricular zone. Several recent studies suggested that a small number of oligodendrocyte precursor cells (OPCs) can also be generated in the dorsal spinal cord. However, it is not clear whether these dorsal oligodendrocyte precursor cells participate in myelination and remyelination. To investigate the fate and potential function of these dorsally-derived oligodendrocytes (dOLs) in the adult spinal cord, Cre-lox genetic fate mapping in transgenic mice was employed. We used the Pax3(Cre) knock-in mouse to drive Cre expression in the entire dorsal epithelium and the Rosa26-lacZ or Z/EG reporter line to trace their spatial distribution and population dynamics in the spinal cord. The dorsal OPCs generated from the Pax3-expressing domains migrate into all regions of spinal cord and subsequently undergo terminal differentiation and axonal myelination. In response to a focal demyelination injury, a large number of newly differentiated oligodendrocytes originated from dOLs, suggesting that dOLs may provide an important source of OPCs for axonal remyelination in multiple sclerosis or spinal cord injury.     

大鼠脊髓少突胶质前体细胞的培养与鉴定方法

目的 探讨大鼠脊髓少突胶质前体细胞（OPCs）的分离纯化及诱导分化方法.方法 取出生后3d内SD大鼠脊髓,采用0.125％胰蛋白酶消化获取原代混合胶质细胞,培养10 d左右在37℃恒温摇床采取180 r/min摇速振摇并差速贴壁40 min获得纯化的OPCs;取纯化后培养3d的OPCs免疫荧光鉴定细胞纯度或诱导分化.结果 采用胰蛋白酶消化法获取原代混合胶质细胞、振摇并差速贴壁法分离纯化能够得到纯度较高的OPCs,折光性强,呈双极或三极形态,免疫荧光染色显示95％细胞表达A2B5和NG2（OPCs标志物）,经诱导分化后表达O4及MBP（少突胶质细胞标志物）.结论 采用振摇差速贴壁法可从大鼠脊髓中分离纯化获得高纯度的OPCs,获得的OPCs体外生长稳定,经诱导可分化为成熟的少突胶质细胞.     

The tetraspanin protein,CD9, is expressed by progenitor cells committed to oligodendrogenesis and is linked to beta1 integrin,CD81, and Tspan-2

Previous studies identified the tetraspanin protein CD9 in myelinating oligodendrocytes. The present report extends these observations by identifying CD9 in a subpopulation of oligodendrocyte progenitor cells (OPCs) and in premyelinating oligodendrocytes in rodents. NG2-positive cells expressed CD9 in a temporal and spatial pattern during development that was consistent with CD9 expression in OPCs just prior to their differentiation into premyelinating oligodendrocytes. NG2-positive cells in mature brains were CD9-negative. CD9 expression during oligodendrocyte development in vitro supported this hypothesis, as all CD9-positive cells became O4-positive when switched to oligodendrocyte differentiating media. CD9 immunoreactivity was enriched in myelinating oligodendrocytes and their processes, and the outer aspects of myelin internodes. Immunoprecipitation of CD9 from postnatal rat cerebrum coprecipitated beta1 integrin, CD81, and Tspan-2, another tetraspanin protein recently identified in oligodendrocytes. Following surface biotinylation of oligodendrocytes in vitro, biotinylated beta1 integrin was identified in a CD9 immunoprecipitate. These data support a molecular link between surface integrins and a CD9, Tspan-2 molecular web during the differentiation of oligodendrocytes. Oligodendrocyte production and myelination appears to be normal in CD9-deficient mice. These data support the hypothesis that CD9 helps form the tetraspanin web beneath the plasma membranes of progenitor cells committed to oligodendrogenesis, but that CD9 is not essential for oligodendrogenesis and myelination.     

NT-3 weakly stimulates proliferation of adult rat O1(-)O4(+) oligodendrocyte-lineage cells and increases oligodendrocyte myelination in vitro

The transplantation of fibroblasts, genetically modified to secrete neurotrophin-3 (NT-3) and/or brain-derived neurotrophic factor (BDNF), into spinal cord-injured rats increases the production of new oligodendrocytes and myelination (McTigue et al. [1998] J. Neurosci. 18:5354-5365). This experiment did not fully resolve whether the effect was exerted on oligodendrocyte precursors or on oligodendrocytes, or whether there was stimulation of both proliferation and differentiation of the oligodendrocyte lineage cells. To clarify the effects of NT-3 and BDNF, adult rat spinal cord was dissociated to produce cultures in which both oligodendrocyte precursors (O1(-)O4(+)) and oligodendrocytes (O1(+)) were present. Thymidine labeling of cells was determined in the presence and absence of added NT-3 and/or BDNF. In addition, the effect of these neurotrophins on myelination was determined by treating purified adult O1(+) oligodendrocyte/embryonic dorsal root ganglion (DRG) neuron cocultures with neurotrophins, only during the myelination period. O1(+) oligodendrocyte proliferation was not stimulated by NT-3 or BDNF; however, the proliferation of O1(-)O4(+) cells was increased in NT-3-treated cultures to a labeling index (LI: 24 hr) of 15-20%. This effect was observed at 5 but not at 10 days in vitro. In comparison, basic fibroblast growth factor (bFGF) induced the proliferation of both O1(+) oligodendrocytes (LI approximately 60%) and O1(-)O4(+) cells (LI approximately 75%). The amount of myelin formed in purified O1(+) oligodendrocyte/DRG neuron cocultures was significantly increased in NT-3-treated cultures compared to untreated cultures. These results indicate that NT-3 is weakly but transiently mitogenic for adult-derived oligodendrocyte precursors and support the suggestion that NT-3 promotes the maturation of O1(+) oligodendrocytes into myelin-forming cells.