Oct4‐induced oligodendrocyte progenitor cells enhance functional recovery in spinal cord injury model

The generation of patient‐specific oligodendrocyte progenitor cells (OPCs) holds great potential as an expandable cell source for cell replacement therapy as well as drug screening in spinal cord injury or demyelinating diseases. Here, we demonstrate that induced OPCs (iOPCs) can be directly derived from adult mouse fibroblasts by Oct4‐mediated direct reprogramming, using anchorage‐independent growth to ensure high purity. Homogeneous iOPCs exhibit typical small‐bipolar morphology, maintain their self‐renewal capacity and OPC marker expression for more than 31 passages, share high similarity in the global gene expression profile to wild‐type OPCs, and give rise to mature oligodendrocytes and astrocytes in vitro and in vivo. Notably, transplanted iOPCs contribute to functional recovery in a spinal cord injury (SCI) model without tumor formation. This study provides a simple strategy to generate functional self‐renewing iOPCs and yields insights for the in‐depth study of demyelination and regenerative medicine.     

Isolation and culture of rat and mouse oligodendrocyte precursor cells

The ability to isolate oligodendroglial precursor cells (OPCs) provides a powerful means to characterize their differentiation, properties and potential for myelin repair. Although much knowledge is available for isolation of OPCs from the rat central nervous system, preparation and maintenance of mouse OPCs has been until recently a challenge owing to difficulties in obtaining a sufficient quantity of purified OPCs. Here, we describe protocols to prepare highly enriched rat OPCs and nearly homogenous mouse OPCs. The mouse method generates predominantly OPCs from cortical neural progenitor cells as clonal aggregates called "oligospheres" by taking advantage of molecular genetic tools. Isolated OPCs can be further differentiated into oligodendrocytes. Collectively, we describe simple and efficient methods for the preparation and in vitro maintenance of enriched OPCs from rats and mice. Isolation and culture of a large, homogenous population of rodent OPCs should significantly facilitate studies on OPC lineage progression and their utility in myelin repair after injury.     

Cdk2 loss accelerates precursor differentiation and remyelination in the adult central nervous system

The specific functions of intrinsic regulators of oligodendrocyte progenitor cell (OPC) division are poorly understood. Type 2 cyclin-dependent kinase (Cdk2) controls cell cycle progression of OPCs, but whether it acts during myelination and repair of demyelinating lesions remains unexplored. Here, we took advantage of a viable Cdk2(-/-) mutant mouse to investigate the function of this cell cycle regulator in OPC proliferation and differentiation in normal and pathological conditions. During central nervous system (CNS) development, Cdk2 loss does not affect OPC cell cycle, oligodendrocyte cell numbers, or myelination. However, in response to CNS demyelination, it clearly alters adult OPC renewal, cell cycle exit, and differentiation. Importantly, Cdk2 loss accelerates CNS remyelination of demyelinated axons. Thus, Cdk2 is dispensable for myelination but is important for adult OPC renewal, and could be one of the underlying mechanisms that drive adult progenitors to differentiate and thus regenerate myelin.     

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.     

A role for Noggin in the development of oligodendrocyte precursor cells

Oligodendrocyte precursor cells (OPCs) can be differentiated in culture into either oligodendrocytes or type-2 astrocytes (2As), depending on the culture conditions. Whereas oligodendrocyte development can occur in the absence of inducing signals, 2A development apparently cannot. Fetal calf serum (FCS) and bone morphogenetic proteins (BMPs) are powerful inducers of 2A development in culture, but there is no compelling evidence that OPCs develop into astrocytes in vivo. We show here that BMPs are made by glial cells in the developing rat optic nerve, raising the question of why 2As do not normally develop in the optic nerve. We demonstrate that the BMP antagonist Noggin is strongly expressed by both OPCs and type-1 astrocytes in the developing optic nerve. We also show that depletion of Noggin by a small interference RNA inhibits OPC proliferation and induces 2A differentiation in the presence of a low, non-2A-inducing concentration of FCS. By contrast, enforced expression of Noggin in OPCs blocks FCS-induced 2A differentiation. These findings suggest that BMPs in FCS are largely responsible for the 2A-inducing activity of FCS and that Noggin may normally inhibit the formation of 2As in the developing CNS.     

CNTF-Activated Astrocytes Release a Soluble Trophic Activity for Oligodendrocyte Progenitors

CNTF (ciliary neurotrophic factor) has been suggested to be an important survival factor for oligodendrocytes; however, this effect is inconsistently obtained and myelination appears normal in CNTF null animals. On the other hand, CNTF stimulates astrocytes to produce growth and trophic factors. Therefore, we tested the hypothesis that CNTF acts indirectly through astrocytes to promote oligodendrocyte survival. We show that CNTF-stimulated astrocytes release a trophic factor(s) that leads to more than double the number of oligodendrocyte progenitor cells (OPCs) by 48 h. The trophic activity fractionates at greater than 30 kD. By contrast, OPCs grown in CNTF supplemented chemically defined medium fared no better than cells grown without CNTF. Untreated astrocytes, and CNTF- and IL-1β -stimulated astrocytes all promoted the proliferation of OPCs to a similar extent, but only the CNTF-stimulated astrocyte conditioned media (CM) resulted in increased OPCs numbers. Cumulatively, these results confirm previous data indicating that astrocytes release potent mitogens for oligodendroglia, and demonstrate that CNTF stimulates astrocytes to release an OPC survival-promoting activity.     

Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain

Both late-gestation and adult human forebrain contain large numbers of oligodendrocyte progenitor cells (OPCs). These cells may be identified by their A2B5(+)PSA-NCAM(-) phenotype (positive for the early oligodendrocyte marker A2B5 and negative for the polysialylated neural cell adhesion molecule). We used dual-color fluorescence-activated cell sorting (FACS) to extract OPCs from 21- to 23-week-old fetal human forebrain, and A2B5 selection to extract these cells from adult white matter. When xenografted to the forebrains of newborn shiverer mice, fetal OPCs dispersed throughout the white matter and developed into oligodendrocytes and astrocytes. By 12 weeks, the host brains showed extensive myelin production, compaction and axonal myelination. Isolates of OPCs derived from adult human white matter also myelinated shiverer mouse brain, but much more rapidly than their fetal counterparts, achieving widespread and dense myelin basic protein (MBP) expression by 4 weeks after grafting. Adult OPCs generated oligodendrocytes more efficiently than fetal OPCs, and ensheathed more host axons per donor cell than fetal cells. Both fetal and adult OPC phenotypes mediated the extensive and robust myelination of congenitally dysmyelinated host brain, although their differences suggested their use for different disease targets.     

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.     

Oligodendrocyte Progenitor Cells Directly Utilize Lactate for Promoting Cell Cycling and Differentiation

Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4‐dideoxy‐1,4‐imino‐d‐arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC‐rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU‐positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate‐mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α‐cyano‐4‐hydroxy‐cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. J. Cell. Physiol. 232: 986–995, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Origin of Oligodendrocytes in the Vertebrate Optic Nerve: A Review

One of the unsolved problems in the research field of oligodendrocyte (OL) development has been the site(s) of origin of optic nerve OLs and its precursor cells (OPCs). It is generally accepted that OLs in the optic nerve are derived from the brain, and thus optic nerve OLs are immigrant cells. We previously demonstrated the brain origin of optic nerve OPCs in chick embryos. However, the site of optic nerve OPC origin has not been examined experimentally in developing rodents for the past two decades. We have recently reported that optic nerve OPCs in mice arise in the preoptic area by E12.5 and gradually migrate caudally and enter the optic nerve. These OPCs give rise to myelinating OLs in the optic nerve in the postnatal or adult stages. Surprisingly, there are species differences with respect to the origin of optic nerve OPCs between chicks and mice. Here, we summarize the site of OPC origin in the optic nerve based on our own previous and recent results, and discuss possible mechanisms underlying these species differences.     

Developing oligodendrocytes express functional GABA(B) receptors that stimulate cell proliferation and migration

GABA(B) receptors (GABA(B)Rs) are involved in early events during neuronal development. The presence of GABA(B)Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GABA(B)R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA(B1abcdf) and GABA(B2) subunits of the GABA(B)R. Using semiquantitative PCR analysis with GABA(B)R isoform-selective primers we found that the expression level of GABA(B1abd) was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA(B2) isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA(B1) and GABA(B2) subunits are under independent control during oligodendroglial development. Activation of GABA(B)Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA(B)R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA(B)Rs in oligodendrocyte development. The presence of GABA(B)Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.     

Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway

Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self‐renewal capacity from those of ES cells. All three EC cell lines maintain self‐renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self‐renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self‐renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells‐derived conditioned medium may be responsible for the self‐renewal capacity of EC and ES cells independently of LIF signaling.     

Establishment of a proteome profile and identification of molecular markers for mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) are undifferentiated cells that are required to maintain spermatogenesis throughout the reproductive life of mammals. Although SSC transplantation and culture provide a powerful tool to identify the mechanisms regulating SSC function, the precise signalling mechanisms governing SSC self‐renewal and specific surface markers for purifying SSCs remain to be clearly determined. In the present study, we established a steady SSC culture according to the method described by Shinohara's lab. Fertile progeny was produced after transplantation of cultured SSCs into infertile mouse testis, and the red fluorescence exhibited by the culture cell membranes was stably and continuously transmitted to the offspring. Next, via advanced mass spectrometry and an optimized proteomics platform, we constructed the proteome profile, with 682 proteins expressed in SSCs. Furthermore bioinformatics analysis showed that the list contained several known molecules that are regulated in SSCs. Several nucleoproteins and membrane proteins were chosen for further exploration using immunofluorescence and RT‐PCR. The results showed that SALL1, EZH2, and RCOR2 are possibly involved in the self‐renewal mechanism of SSCs. Furthermore, the results of tissue‐specific expression analysis showed that Gpat2 and Pld6 were uniquely and highly expressed in mouse testes and cultured SSCs. The cellular localization of PLD6 was further explored and the results showed it was primarily expressed in the spermatogonial membrane of mouse testes and cultured SSCs. The proteins identified in this study form the basis for further exploring the molecular mechanism of self‐renewal in SSCs and for identifying specific surface markers of SSCs.     

Heterogeneity of NG2-expressing cells in the newborn mouse cerebellum

The function and origin of NG2+ cells in the adult brain are still controversial. A large amount of data is available which strongly indicates that adult NG2-expressing cells form a heterogeneous population, constituted by oligodendrocyte precursor cells (OPCs) and a fourth novel type of glial cells named the synantocytes. Whether these two populations derive from the progressive maturation of perinatal NG2+ OPCs or are generated as separate populations is not known. We used organotypic cultures of newborn mouse cerebellum depleted, by anti-mitotic drug treatment, of their NG2+ cells with perinatal features (high proliferating rate and high oligodendrocytic differentiation ability). In these cultures, despite the lack of myelin after 14 days in vitro, numerous NG2+ cells remained. We show that these BrdU-resistant cells were able to slowly divide, as adult NG2+ cells do. Although many of these cells expressed O4, only a very small fraction of them was further engaged in oligodendrocyte lineage, as they had an extremely poor capacity to generate myelin sheaths to the Purkinje cell axons. These results support the view that at least two distinct populations of NG2+ cells coexist in the cerebellum from birth: one with the young OPC characteristics, another with adult NG2+ cell characteristics. Thus, a fraction of adult NG2+ cells do not derive from the maturation of perinatal OPCs.     

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.     

Prox1 Inhibits Proliferation and Is Required for Differentiation of the Oligodendrocyte Cell Lineage in the Mouse

Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs) in a progenitor state, but what factor may enable oligodendrocyte (OL) differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1.     

Normal timing of oligodendrocyte development depends on thyroid hormone receptor alpha 1 (TRalpha1)

The timing of oligodendrocyte development is regulated by thyroid hormone (TH) in vitro and in vivo, but it is still uncertain which TH receptors mediate this regulation. TH acts through nuclear receptors that are encoded by two genes, TRalpha and TRbeta. Here, we provide direct evidence for the involvement of the TRalpha1 receptor isoform in vivo, by showing that the number of oligodendrocytes in the postnatal day 7 (P7) and P14 optic nerve of TRalpha1-/- mice is decreased compared with normal. We demonstrate that TRalpha1 mediates the normal differentiation-promoting effect of TH on oligodendrocyte precursor cells (OPCs): unlike wild-type OPCs, postnatal TRalpha1-/- OPCs fail to stop dividing and differentiate in response to TH in culture. We also show that overexpression of TRalpha1 accelerates oligodendrocyte differentiation in culture, suggesting that the level of TRalpha1 expression is normally limiting for TH-dependent OPC differentiation. Finally, we provide evidence that the inhibitory isoforms of TRalpha are unlikely to play a part in the timing of OPC differentiation.