Pluripotent stem cell‐derived radial glia‐like cells as stable intermediate for efficient generation of human oligodendrocytes

Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor‐mediated expansion with pre‐exposure to the differentiation‐inducing agent retinoic acid and subsequent immunoisolation of CD133‐positive cells. This protocol yields an adherent and self‐renewing population of hindbrain/spinal cord radial glia (RG)‐like neural precursor cells (RGL‐NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL‐NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate‐determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL‐NPCs efficiently convert into NG2‐positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL‐NPCs expedite the generation of PSC‐derived oligodendrocytes with O4‐, 4860‐, and myelin basic protein (MBP)‐positive cells that already appear within 7 weeks following growth factor withdrawal‐induced differentiation. Thus, RGL‐NPCs may serve as robust tool for time‐efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. GLIA 2015;63:2152–2167     

Protection of oligodendrocyte precursor cells by low doses of HSP90 inhibitors in cell culture

Oligodendrocyte precursor cells (OPCs) become myelin-forming after their differentiation into post-mitotic oligodendrocytes. OPCs are extremely efficient at myelin repair and contribute to remyelination. However, remyelination fails in multiple sclerosis (MS), which suggest that the OPCs are ineffective in this disorder. We have studied previously the expression of heat shock protein 90 (HSP90) in OPCs and have reported autoantibodies against HSP90 in MS patients, which recognize the antigen on the OPC surface. The present study investigated a protective effect of HSP90 inhibitors observed in cultured OPCs. Radicicol and 17-allylamino-17-demethoxygeldanamycin (17-AAG) at non-cytotoxic doses targeted cell-surface HSP90 in OPCs. Thus, 0.01 nM 17-AAG or 10 nM radicicol competed with the anti-HSP90 antibodies for binding to cell-surface HSP90. These low doses of HSP90 inhibitors prevented HSP90-antibody-induced OPC death and protected the oligodendrocyte population against antibody attack. Adult oligodendrocytes were protected by these low doses of HSP90 inhibitors in a similar fashion to perinatal cells. The present results show that, despite OPCs being very sensitive to HSP90 inhibitors, low and non-cytotoxic doses of 17-AAG and radicicol protect oligodendrocytes from anti-HSP90 antibody attack. They may have therapeutic potential for MS patients that have anti-HSP90 autoantibodies and provide a novel strategy for therapeutic intervention with HSP90 inhibitors.     

Schwann cell-like differentiation by adult oligodendrocyte precursor cells following engraftment into the demyelinated spinal cord is BMP-dependent

The development of remyelinating strategies designed to enhance recruitment and differentiation of endogenous precursor cells available to a site of demyelination in the adult spinal cord will require a fundamental understanding of the potential for adult spinal cord precursor cells to remyelinate as well as an insight into epigenetic cues that regulate their mobilization and differentiation. The ability of embryonic and postnatal neural precursor cell transplants to remyelinate the adult central nervous system is well documented, while no transplantation studies to date have examined the remyelinating potential of adult spinal-cord-derived oligodendrocyte precursor cells (adult OPCs). In the present study, we demonstrate that, when transplanted subacutely into spinal ethidium bromide/X-irradiated (EB-X) lesions, adult OPCs display a limited capacity for oligodendrocyte remyelination. Interestingly, the glia-free environment of EB lesions promotes engrafted adult OPCs to differentiate primarily into cells with immunophenotypic and ultrastructural characteristics of myelinating Schwann cells (SCs). Astrocytes modulate this potential, as evidenced by the demonstration that SC-like differentiation is blocked when adult OPCs are co-transplanted with astrocytes. We further show that inhibition of bone morphogenetic protein (BMP) signaling through noggin overexpression by engrafted adult OPCs is sufficient to block SC-like differentiation within EB-X lesions. Present data suggest that the macroglial-free environment of acute EB lesions in the ventrolateral funiculus is inhibitory to adult spinal cord-derived OPC differentiation into remyelinating oligodendrocytes, while the presence of BMPs and absence of noggin promotes SC-like differentiation, thereby unmasking a surprising lineage fate for these cells.     

Oligodendroglial lineage cells express nuclear p57kip2 in multiple sclerosis lesions

Impaired remyelination in multiple sclerosis (MS) might be due to the failure of oligodendrocyte precursor cells (OPC) to differentiate into myelinating oligodendrocytes. Animal experimental data have shown that p57kip2 inhibits oligodendroglial differentiation, indicating that this factor could contribute to remyelination failure. This study investigates oligodendroglial p57kip2 expression and its association with remyelination in MS lesions. To analyze the potential association of p57kip2 expression with human oligodendroglial maturation, double immunofluorescence staining was performed on brain tissue from 30 MS patients and 20 controls. Anti‐p57kip2 antibody was combined with either anti‐Nogo‐A to label mature oligodendrocytes or anti‐Olig2 antibodies to identify immature OPCs. We evaluated MS lesions with or without remyelination, the periplaque white matter (PPWM) as well as control white matter (WM). p57kip2‐expressing cells were assessed and correlated with the extent of remyelination. Most Nogo‐A‐positive oligodendrocytes (range, 87–98%) and all Olig2^strong‐positive OPCs expressed p57kip2 in MS lesions, in the PPWM and in control WM. p57kip2 expression in oligodendrocytes and OPCs were similar in MS lesions with remyelination compared to MS lesions lacking remyelination. Interestingly, all oligodendroglial lineage cells showed nuclear p57kip2 expression only, with mature oligodendrocytes expressing p57kip2 at low or intermediate levels and OPCs featuring strong expression levels, indicating that this factor may be dynamically expressed during maturation processes. Therefore, p57kip2 appears to be widely expressed in the human oligodendroglial lineage, and potential beneficial effects on remyelination in the MS brain are not based on subcellular p57kip2 localization shifts, as suggested by previous animal experiments. GLIA 2013;61:1250–1260     

The presence of astrocytes in areas of demyelination influences remyelination following transplantation of oligodendrocyte progenitors

To date, most experiments examining the myelination potential of transplanted cells have been undertaken into either the immature nervous system or into acutely demyelinating lesions. Since these are situations where myelination or remyelination are occurring, such studies provide little information on the likely outcome of introducing myelinogenic cells into area of chronic demyelination. In an attempt to gain a greater understanding of the interaction between astrocytes and oligodendrocyte progenitors in areas of demyelination, we undertook transplantation experiments in which an identical preparation of oligodendrocyte progenitors (OPCs) was (1) transplanted directly into astrocyte-free areas of acute demyelination (3 days after induction), (2) transplanted cranial to similar areas of demyelination (20 days after induction) or (3) transplanted cranial to areas of demyelination (20 days after induction) that had been injected with astrocytes at 3 days to confront OPCs with demyelinated axons in an astrocytic environment. The acute astrocyte-free lesions were remyelinated by oligodendrocytes and Schwann cells while the delayed interaction of OPCs with demyelinating lesions resulted in only oligodendrocyte remyelination, the extent of which was reduced when the area of demyelination contained astrocytes. The results of these experiments illustrate that the introduction of OPCs into an astrocyte-free area of demyelination soon after its induction favours Schwann cell differentiation while the presence of established astrocytes in an area of demyelination has an inhibitory effect on the extent of oligodendrocyte remyelination achieved by OPCs.     

Stem cell repair of central nervous system injury

Neural stem cells (NSCs) have great potential as a therapeutic tool for the repair of a number of CNS disorders. NSCs can either be isolated from embryonic and adult brain tissue or be induced from both mouse and human ES cells. These cells proliferate in vitro through many passages without losing their multipotentiality. Following engraftment into the adult CNS, NSCs differentiate mainly into glia, except in neurogenic areas. After engraftment into the injured and diseased CNS, their differentiation is further retarded. In vitro manipulation of NSC fate prior to transplantation and/or modification of the host environment may be necessary to control the terminal lineage of the transplanted cells to obtain functionally significant numbers of neurons. NSCs and a few types of glial precursors have shown the capability to differentiate into oligodendrocytes and to remyeliate the demyelinated axons in the CNS, but the functional extent of remyelination achieved by these transplants is limited. Manipulation of endogenous neural precursors may be an alternative therapy or a complimentary therapy to stem cell transplantation for neurodegenerative disease and CNS injury. However, this at present is challenging and so far has been unsuccessful. Understanding mechanisms of NSC differentiation in the context of the injured CNS will be critical to achieving these therapeutic strategies.     

Minimally manipulated oligodendrocyte precursor cells retain exclusive commitment to the oligodendrocyte lineage following transplantation into intact and injured hippocampus

Oligodendrocyte precursor cells (OPCs) are widely regarded as the best characterized cell population in the mammalian CNS and until recently were believed to be a lineage-restricted precursor terminally differentiating to postmitotic oligodendrocytes. Recent evidence has suggested that OPCs may have in vitro and in vivo neuronal potential. In this report we examine the differentiation potential of cortical OPC populations following transplantation into the neurogenic environment of the intact neonatal and adult hippocampus. Donor OPCs were minimally manipulated and not subjected to long-term ex vivo manipulation such as expansion or treatment with mitogens. Minimally manipulated OPCs did not exhibit any intrinsic neuronal potential in vitro prior to transplantation. Following transplantation of GFP-OPCs into intact neonatal and adult hippocampus, cells were able to survive and integrate for at least 14 weeks but did not exhibit neuronal differentiation. Induction of a focal neurotoxic lesion also did not result in neuronal differentiation of graft-derived OPCs. These findings show that unselected and unmanipulated populations of cortical OPCs remain as precursor cells, commit to the oligodendrocyte lineage and fail to respond to the extrinsic cues of a neurogenic or injured environment.     

New insights into remyelination failure in multiple sclerosis: implications for glial cell transplantation

This review considers aspects of remyelination that require further clarification if successful strategies are to be devised to enhance remyelination in multiple sclerosis (MS). We speculate, based on our understanding of the rate with which oligodendrocyte progenitor cells (OPCs) repopulate OPC-depleted tissue in adult rats, that OPC depletion during the demyelination process could explain why remyelination fails in MS. We show that loss of OPCs in the context of large areas of demyelination would have serious consequences for remyelination as the rates of colonization of tissue by adult OPCs would lead to a situation where the cellular events associated with demyelination become uncoupled from the interaction of OPCs with demyelinated axons. Experimental studies indicate that transplanted neonatal OPCs would be able to repopulate large areas of demyelination with much greater efficiency than endogenous OPCs. This suggests that cell transplantation will have considerable potential to achieve remyelination in situations where the endogenous repair process is failing due to concurrent death of oligodendroytes and OPCs. However, we suggest that for this approach to be effective, it will be critical that the environment is permissive for remyelination.     

Calcium receptor expression and function in oligodendrocyte commitment and lineage progression: potential impact on reduced myelin basic protein in CaR-null mice

Oligodendrocytes develop from oligodendrocyte progenitor cells (OPCs), which in turn arise from a subset of neuroepithelial precursor cells during midneurogenesis. Development of the oligodendrocyte lineage involves a plethora of cell-intrinsic and -extrinsic signals. A cell surface calcium-sensing receptor (CaR) has been shown to be functionally expressed in immature oligodendrocytes. Here, we investigated the expression and function of the CaR during oligodendrocyte development. We show that the order of CaR mRNA expression as assessed by quantitative polymerase chain reaction is mature oligodendrocyte > neuron > astrocyte. We next determined the rank order of CaR expression on inducing specification of neural stem cells to the neuronal, oligodendroglial, or astrocytic lineages and found that the relative levels of CaR mRNA expression are OPC > neuron > astrocytes. CaR mRNA expression in cells at various stages of development along the oligodendrocyte lineage revealed that its expression is robustly up-regulated during the OPC stage and remains high until the premyelinating stage, decreasing thereafter by severalfold in the mature oligodendrocyte. In OPCs, high Ca(2+) acting via the CaR promotes cellular proliferation. We further observed that high Ca(2+) stimulates the mRNA levels of myelin basic protein in preoligodendrocytes, which is also CaR mediated. Finally, myelin basic protein levels were significantly reduced in the cerebellum of CaR-null mice during development. Our results show that CaR expression is up-regulated when neural stem cells are specified to the oligodendrocyte lineage and that activation of the receptor results in OPC expansion and differentiation. We conclude that the CaR may be a novel regulator of oligodendroglial development and function.     

Haplotype matching is not an essential requirement to achieve remyelination of demyelinating CNS lesions

Transplantation of oligodendrocyte precursor cells (OPCs) results in efficient remyelination in animal models of demyelination. However, the experiments so far undertaken have not addressed the need for tissue-type matching to achieve graft-mediated remyelination. Examination of MHC expression (main determinant of allograft rejection) by OPCs showed nondetectable levels under standard culture conditions and upregulation of MHC Class I expression only upon exposure to interferon gamma. We therefore hypothesized that MHC matching of OPC grafts may not be crucial to achieve transplant-mediated remyelination. Transplant experiments performed using a nonself repairing toxin-induced demyelination model showed that, similarly to allogeneic neurons, survival of allogeneic oligodendrocyte lineage cells is influenced by donor-host haplotype combination and graft composition. Transplantation of allogeneic mixed glial cell cultures resulted in remyelination failure by 1 month postengraftment due to a rejection response targeting both myelinating oligodendrocytes and OPCs, suggesting that inflammation-induced upregulation of OPC MHC I expression results in susceptibility to cytotoxic T cell attack. In contrast, remyelination persisted for at least 2 months following transplantation of OPC-enriched cultures whose overall MHC expression level was significantly decreased. While OPC-enriched preparations elicited delayed type hypersensitivity responses in hosts sensitized to alloantigens, allografting of such preparations into a central nervous system demyelinating lesion did not result in recipient priming. We conclude that while allografted oligodendrocyte lineage cells become targets of a graft rejection response once this response has been initiated, transplantation of OPC-enriched preparations can evade priming against alloantigens and graft rejection. This finding indicates that close tissue matching may not be an essential requirement for successful transplant-mediated remyelination.     

Xeno-free culture for generation of forebrain oligodendrocyte precursor cells from human pluripotent stem cells

Oligodendrocytes (OLs) show heterogeneous properties that depend on their location in the central nervous system (CNS). In this regard, the investigation of oligodendrocyte precursor cells (OPCs) derived from human pluripotent stem cells (hPSCs) should be reconsidered, particularly in cases of brain-predominant disorders for which brain-derived OPCs are more appropriate than spinal cord-derived OPCs. Furthermore, animal-derived components are responsible for culture variability in the derivation and complicate clinical translation. In the present study, we established a xeno-free system to induce forebrain OPCs from hPSCs. We induced human forebrain neural stem cells (NSCs) on Laminin 511-E8 and directed the differentiation to the developmental pathway for forebrain OLs with SHH and FGF signaling. OPCs were characterized by the expression of OLIG2, NKX2.2, SOX10, and PDGFRA, and subsequent maturation into O4⁺ cells. In vitro characterization showed that >85% of the forebrain OPCs (O4⁺) underwent maturation into OLs (MBP⁺) 3 weeks after mitogen removal. Upon intracranial transplantation, the OPCs survived, dispersed in the corpus callosum, and matured into (GSTπ⁺) OLs in the host brains 3 months after transplantation. These findings suggest our xeno-free induction of forebrain OPCs from hPSCs could accelerate clinical translation for brain-specific disorders.     

Demyelinating processes in aging and stroke in the central nervous system and the prospect of treatment strategy

Demyelination occurs in response to brain injury and is observed in many neurodegenerative diseases. Myelin is synthesized from oligodendrocytes in the central nervous system, and oligodendrocyte death‐induced demyelination is one of the mechanisms involved in white matter damage after stroke and neurodegeneration. Oligodendrocyte precursor cells (OPCs) exist in the brain of normal adults, and their differentiation into mature oligodendrocytes play a central role in remyelination. Although the differentiation and maturity of OPCs drive endogenous efforts for remyelination, the failure of axons to remyelinate is still the biggest obstacle to brain repair after injury or diseases. In recent years, studies have made attempts to promote remyelination after brain injury and disease, but its cellular or molecular mechanism is not yet fully understood. In this review, we discuss recent studies examining the demyelination process and potential therapeutic strategies for remyelination in aging and stroke. Based on our current understanding of the cellular and molecular mechanisms underlying remyelination, we hypothesize that myelin and oligodendrocytes are viable therapeutic targets to mitigate brain injury and to treat demyelinating‐related neurodegeneration diseases.     

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.     

Regeneration and repair in multiple sclerosis: the view of experimental pathology

In order to devise a strategy to enhance remyelination in multiple sclerosis (MS) it is necessary to understand the cause of remyelination failure in MS. A case is made that areas of chronic demyelination arise because of concurrent loss of oligodendrocyte progenitor cells (OPCs) and oligodendrocytes and that because of the slow rate of repopulation that occurs in old individuals the recruited OPCs are not exposed to the acute inflammatory environment required to generate remyelinating oligodendrocytes. Based on this analysis the case is made that only areas of acute demyelination will be amenable to transplant-mediated remyelination. An analysis of the many cells that could be used to provide a source of remyelinating cells would indicate that structural repair of the CNS in MS would likely only be possible if neural precursors were used and the most promising route for their introduction would appear to be by intraventricular injection. Both neural precursors and mesenchymal stromal cells can be immunomodulatory and neuroprotective following intravenous injection; however, only neural precursors are likely to be able to contribute to structural repair of the damaged nervous system.     

CNTF promotes the survival and differentiation of adult spinal cord-derived oligodendrocyte precursor cells in vitro but fails to promote remyelination in vivo

Delivery of factors capable of promoting oligodendrocyte precursor cell (OPC) survival and differentiation in vivo is an important therapeutic strategy for a variety of pathologies in which demyelination is a component, including multiple sclerosis and spinal cord injury. Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine that promotes both survival and maturation of a variety of neuronal and glial cell populations, including oligodendrocytes. Present results suggest that, although CNTF has a potent survival and differentiation promoting effect in vitro on OPCs isolated from the adult spinal cord, CNTF administration in vivo is not sufficient to promote oligodendrocyte remyelination in the glial-depleted environment of unilateral ethidium bromide (EB) lesions.     

Effect of glial cells on remyelination after spinal cord injury

Remyelination plays a key role in functional recovery of axons after spinal cord injury. Glial cells are the most abundant cells in the central nervous system. When spinal cord injury occurs, many glial cells at the lesion site are immediately activated, and different cells differentially affect inflammatory reactions after injury. In this review, we aim to discuss the core role of oligodendrocyte precursor cells and crosstalk with the rest of glia and their subcategories in the remyelination process. Activated astrocytes influence prolif-eration, differentiation, and maturation of oligodendrocyte precursor cells, while activated microglia alter remyelination by regulating the inflammatory reaction after spinal cord injury. Understanding the interac-tion between oligodendrocyte precursor cells and the rest of glia is necessary when designing a therapeutic plan of remyelination after spinal cord injury.