MicroRNA-26b inhibits oligodendrocyte precursor cell differentiation by targeting adrenomedullin in spinal cord injury

Oligodendrocyte precursor cells (OPCs) serve as a reservoir of newborn oligodendrocytes (OLs) in pathological and homeostatic conditions. After spinal cord injury (SCI), OPCs are activated to generate myelinating OLs, contributing to remyelination and functional recovery; however, the underlying molecular mechanisms remain unclear. Here, microRNA-26b (miR-26b) expression in the spinal cord tissues of SCI rats was examined by real-time polymerase chain reaction analysis. The influences of miR-26b on locomotor recovery following SCI were assessed utilizing Basso, Beattie, and Bresnahan (BBB) scores. The effects of miR-26b on OPC differentiation were explored using immunofluorescence and western blot analyses in vitro and in vivo. The potential targets that are modulated by miR-26b were identified by bioinformatics, luciferase reporter assays, and western blot analyses. The effects of adrenomedullin (ADM) on OPC differentiation were explored in vitro using immunofluorescence and western blot analyses. We demonstrated that miR-26b was significantly downregulated after SCI. BBB scores showed that miR-26b exacerbated the locomotor function deficits induced by SCI. In vitro, miR-26b inhibited the differentiation of primary rat OPCs. In vivo, miR-26b suppressed OPC differentiation in SCI rats. Bioinformatics analyses and experimental detection revealed that miR-26b directly targeted ADM in OPCs. In addition, knockdown of ADM suppressed the differentiation of primary rat OPCs. Our study provides evidence that ADM may mediate miR-26b-inhibited OPC differentiation in SCI.     

Impact of Simulated Microgravity on Oligodendrocyte Development: Implications for Central Nervous System Repair

We have recently established a culture system to study the impact of simulated microgravity on oligodendrocyte progenitor cells (OPCs) development. We subjected mouse and human OPCs to a short exposure of simulated microgravity produced by a 3D-Clinostat robot. Our results demonstrate that rodent and human OPCs display enhanced and sustained proliferation when exposed to simulated microgravity as assessed by several parameters, including a decrease in the cell cycle time. Additionally, OPC migration was examined in vitro using time-lapse imaging of cultured OPCs. Our results indicated that OPCs migrate to a greater extent after stimulated microgravity than in normal conditions, and this enhanced motility was associated with OPC morphological changes. The lack of normal gravity resulted in a significant increase in the migration speed of mouse and human OPCs and we found that the average leading process in migrating bipolar OPCs was significantly longer in microgravity treated cells than in controls, demonstrating that during OPC migration the lack of gravity promotes leading process extension, an essential step in the process of OPC migration. Finally, we tested the effect of simulated microgravity on OPC differentiation. Our data showed that the expression of mature oligodendrocyte markers was significantly delayed in microgravity treated OPCs. Under conditions where OPCs were allowed to progress in the lineage, simulated microgravity decreased the proportion of cells that expressed mature markers, such as CC1 and MBP, with a concomitant increased number of cells that retained immature oligodendrocyte markers such as Sox2 and NG2. Development of methodologies aimed at enhancing the number of OPCs and their ability to progress on the oligodendrocyte lineage is of great value for treatment of demyelinating disorders. To our knowledge, this is the first report on the gravitational modulation of oligodendrocyte intrinsic plasticity to increase their progenies.     

Oscillatory expression of Ascl1 in oligodendrogenesis

The proneural gene Ascl1 promotes formation of both neurons and oligodendrocytes from neural stem cells (NSCs), but it remains to be analyzed how its different functions are coordinated. It was previously shown that Ascl1 enhances proliferation of NSCs when its expression oscillates but induces differentiation into transit-amplifying precursor cells and neurons when its expression is up-regulated and sustained. By time-lapse imaging and immunohistological analyses, we found that Ascl1 expression oscillated in proliferating oligodendrocyte precursor cells (OPCs) at lower levels than in transit-amplifying precursor cells and was repressed when OPCs differentiated into mature oligodendrocytes. Induction of sustained overexpression of Ascl1 reduced oligodendrocyte differentiation and promoted neuronal differentiation. These results suggest that oscillatory expression of Ascl1 plays an important role in proliferating OPCs during oligodendrocyte formation.     

CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells

Experimental animals with myelin disorders can be treated by transplanting oligodendrocyte progenitor cells (OPCs) into the affected brain or spinal cord. OPCs have been isolated by their expression of gangliosides recognized by mAb A2B5, but this marker also identifies lineage-restricted astrocytes and immature neurons. To establish a more efficient means of isolating myelinogenic OPCs, we sorted fetal human forebrain cells for CD140a, an epitope of platelet derived growth factor receptor (PDGFR)α, which is differentially expressed by OPCs. CD140a(+) cells were isolated as mitotic bipotential progenitors that initially expressed neither mature neuronal nor astrocytic phenotypic markers, yet could be instructed to either oligodendrocyte or astrocyte fate in vitro. Transplanted CD140a(+) cells were highly migratory and robustly myelinated the hypomyelinated shiverer mouse brain more rapidly and efficiently than did A2B5(+)cells. Microarray analysis of CD140a(+) cells revealed overexpression of the oligodendroglial marker CD9, suggesting that CD9(+)/CD140a(+) cells may constitute an even more highly enriched population of myelinogenic progenitor cells.     

Specification and maintenance of oligodendrocyte precursor cells from neural progenitor cells: involvement of microRNA-7a

The generation of myelinating cells from multipotential neural stem cells in the CNS requires the initiation of specific gene expression programs in oligodendrocytes (OLs). We reasoned that microRNAs (miRNAs) could play an important role in this process by regulating genes crucial for OL development. Here we identified miR-7a as one of the highly enriched miRNAs in oligodendrocyte precursor cells (OPCs), overexpression of which in either neural progenitor cells (NPCs) or embryonic mouse cortex promoted the generation of OL lineage cells. Blocking the function of miR-7a in differentiating NPCs led to a reduction in OL number and an expansion of neuronal populations simultaneously. We also found that overexpression of this miRNA in purified OPC cultures promoted cell proliferation and inhibited further maturation. In addition, miR-7a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including Pax6 and NeuroD4, or proOL genes involved in oligodendrocyte maturation. These results suggest that miRNA pathway is essential in determining cell fate commitment for OLs and thus providing a new strategy for modulating this process in OL loss diseases.     

Thymosin β4 Up-regulation of MicroRNA-146a Promotes Oligodendrocyte Differentiation and Suppression of the Toll-like Proinflammatory Pathway

Thymosin β4 (Tβ4), a G-actin-sequestering peptide, improves neurological outcome in rat models of neurological injury. Tissue inflammation results from neurological injury, and regulation of the inflammatory response is vital for neurological recovery. The innate immune response system, which includes the Toll-like receptor (TLR) proinflammatory signaling pathway, regulates tissue injury. We hypothesized that Tβ4 regulates the TLR proinflammatory signaling pathway. Because oligodendrogenesis plays an important role in neurological recovery, we employed an in vitro primary rat embryonic cell model of oligodendrocyte progenitor cells (OPCs) and a mouse N20.1 OPC cell line to measure the effects of Tβ4 on the TLR pathway. Cells were grown in the presence of Tβ4, ranging from 25 to 100 ng/ml (RegeneRx Biopharmaceuticals Inc., Rockville, MD), for 4 days. Quantitative real-time PCR data demonstrated that Tβ4 treatment increased expression of microRNA-146a (miR-146a), a negative regulator the TLR signaling pathway, in these two cell models. Western blot analysis showed that Tβ4 treatment suppressed expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two proinflammatory cytokines of the TLR signaling pathway. Transfection of miR-146a into both primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tβ4 demonstrated an amplification of myelin basic protein (MBP) expression and differentiation of OPC into mature MBP-expressing oligodendrocytes. Transfection of anti-miR-146a nucleotides reversed the inhibitory effect of Tβ4 on IRAK1 and TRAF6 and decreased expression of MBP. These data suggest that Tβ4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a.     

The Stem Cell Factor Sox2 Is a Positive Timer of Oligodendrocyte Development in the Postnatal Murine Spinal Cord

Myelination in the central nervous system takes place predominantly during the postnatal development of humans and rodents by myelinating oligodendrocytes (OLs), which are differentiated from oligodendrocyte progenitor cells (OPCs). We recently reported that Sox2 is essential for developmental myelination in the murine brain and spinal cord. It is still controversial regarding the role of Sox2 in oligodendroglial lineage progression in the postnatal murine spinal cord. Analyses of a series of cell- and stage-specific Sox2 mutants reveal that Sox2 plays a biphasic role in regulating oligodendroglial lineage progression in the postnatal murine spinal cord. Sox2 controls the number of OPCs for subsequent differentiation through regulating their proliferation. In addition, Sox2 regulates the timing of OL differentiation and modulates the rate of oligodendrogenesis. Our experimental data prove that Sox2 is an intrinsic positive timer of oligodendroglial lineage progression and suggest that interventions affecting oligodendroglial Sox2 expression may be therapeutic for overcoming OPC differentiation arrest in dysmyelinating and demyelinating disorders.     

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。     

miR-124 promotes neural differentiation in mouse bulge stem cells by repressing Ptbp1 and Sox9

Hair follicle stem cells (HFSCs) are able to differentiate into neurons and glial cells. Distinct microRNAs (miRNAs) regulate the proliferation and differentiation of HFSCs. However, the exact role of miR-124 in the neural differentiation of HFSCs has not been elucidated. HFSCs were isolated from mouse whisker follicles. miR-9, let-7b, and miR-124, Ptbp1 , and Sox9 expression levels were detected by real-time polymerase chain reaction (RT-PCR). The influence of miR-124 transfection was evaluated using immunostaining. We demonstrated that miR-124 and let-7b expression levels were significantly increased after the neural differentiation. Sox9 and Ptbp1 were identified as the target of miR-124 in the HFSCs. During neural differentiation and miR-124 mimicking, Ptbp1 and Sox9 levels were decreased. Moreover, the miR-124 overexpression increased MAP2 (58.43 ± 11.26) and NeuN (48.34 ± 11.15) proteins expression. The results demonstrated that miR-124 may promote the differentiation of HFSCs into neuronal cells by targeting Sox9 and Ptbp1.     

Loss of protein-tyrosine phosphatase α (PTPα) increases proliferation and delays maturation of oligodendrocyte progenitor cells

Tightly controlled termination of proliferation determines when oligodendrocyte progenitor cells (OPCs) can initiate differentiation and mature into myelin-forming cells. Protein-tyrosine phosphatase α (PTPα) promotes OPC differentiation, but its role in proliferation is unknown. Here we report that loss of PTPα enhanced in vitro proliferation and survival and decreased cell cycle exit and growth factor dependence of OPCs but not neural stem/progenitor cells. PTPα(-/-) mice have more oligodendrocyte lineage cells in embryonic forebrain and delayed OPC maturation. On the molecular level, PTPα-deficient mouse OPCs and rat CG4 cells have decreased Fyn and increased Ras, Cdc42, Rac1, and Rho activities, and reduced expression of the Cdk inhibitor p27Kip1. Moreover, Fyn was required to suppress Ras and Rho and for p27Kip1 accumulation, and Rho inhibition in PTPα-deficient cells restored expression of p27Kip1. We propose that PTPα-Fyn signaling negatively regulates OPC proliferation by down-regulating Ras and Rho, leading to p27Kip1 accumulation and cell cycle exit. Thus, PTPα acts in OPCs to limit self-renewal and facilitate differentiation.     

Silencing or knocking out the Na(+)/Ca(2+) exchanger-3 (NCX3) impairs oligodendrocyte differentiation

Changes in intracellular [Ca(2+)](i) levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.     

Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.