Human stem cell-derived oligodendrocytes: From humanized animal models to cell therapy in myelin diseases.

Oligodendrocytes are main targets in demyelinating and dysmyelinating diseases of the central nervous system (CNS), but are also involved in accidental, neurodegenerative and psychiatric disorders. The underlying pathology of these diseases is not fully understood and treatments are still lacking. The recent discovery of the induced pluripotent stem cell (iPSC) technology has open the possibility to address the biology of human oligodendroglial cells both in the dish and in vivo via engraftment in animal models, and paves the way for the development of treatment for myelin disorders. In this review, we make a short overview of the different sources human oligodendroglial cells, and animal models available for pre-clinical cell therapy. We discuss the anatomical and functional benefit of grafted iPSC-progenitors over their brain counterparts, their use in disease modeling and the missing gaps that still prevent to study their biology in the most integrated way, and to translate iPSC-stem cell based therapy to the clinic.

High Dose Pharmaceutical Grade Biotin (MD1003) Accelerates Differentiation of Murine and Grafted Human Oligodendrocyte Progenitor Cells In Vivo.

Accumulating evidences suggest a strong correlation between metabolic changes and neurodegeneration in CNS demyelinating diseases such as multiple sclerosis (MS). Biotin, an essential cofactor for five carboxylases, is expressed by oligodendrocytes and involved in fatty acid synthesis and energy production. The metabolic effect of biotin or high-dose-biotin (MD1003) has been reported on rodent oligodendrocytes in vitro, and in neurodegenerative or demyelinating animal models. However, clinical studies, showed mild or no beneficial effect of MD1003 in amyotrophic lateral sclerosis (ALS) or MS. Here, we took advantage of a mouse model of myelin deficiency to study the effects of MD1003 on the behavior of murine and grafted human oligodendrocytes in vivo. We show that MD1003 increases the number and the differentiation potential of endogenous murine oligodendroglia over time. Moreover, the levels of MD1003 are increased in the plasma and brain of pups born to treated mothers, indicating that MD1003 can pass through the mother's milk. The histological analysis of the grafted animals shows that MD1003 increased proliferation and accelerates differentiation of human oligodendroglia, but without enhancing their myelination potential. These findings provide important insights into the role of MD1003 on murine and human oligodendrocyte maturation/myelination that may explain the mitigated outcome of ALS/MS clinical trials.

Beneficial contribution of induced pluripotent stem cell-progeny to Connexin 47 dynamics during demyelination-remyelination.

Oligodendrocytes are extensively coupled to astrocytes, a phenomenon ensuring glial homeostasis and maintenance of central nervous system myelin. Molecular disruption of this communication occurs in demyelinating diseases such as multiple sclerosis. Less is known about the vulnerability and reconstruction of the panglial network during adult demyelination-remyelination. Here, we took advantage of lysolcithin-induced demyelination to investigate the expression dynamics of the oligodendrocyte specific connexin 47 (Cx47) and to some extent that of astrocyte Cx43, and whether this dynamic could be modulated by grafted induced pluripotent stem cell (iPSC)-neural progeny. Our data show that disruption of Cx43-Cx47 mediated hetero-cellular gap-junction intercellular communication following demyelination is larger in size than demyelination. Loss of Cx47 expression is timely rescued during remyelination and accelerated by the grafted neural precursors. Moreover, mouse and human iPSC-derived oligodendrocytes express Cx47, which co-labels with astrocyte Cx43, indicating their integration into the panglial network. These data suggest that in rodents, full lesion repair following transplantation occurs by panglial reconstruction in addition to remyelination. Targeting panglial elements by cell therapy or pharmacological compounds may help accelerating or stabilizing re/myelination in myelin disorders.

Stem cell derived oligodendrocytes to study myelin diseases.

Oligodendroglial pathology is central to de- and dysmyelinating, but also contributes to neurodegenerative and psychiatric diseases as well as brain injury. The understanding of oligodendroglial biology in health and disease has been significantly increased during recent years by experimental in vitro and in vivo preclinical studies as well as histological analyses of human tissue samples. However, for many of these diseases the underlying pathology is still not fully understood and treatment options are frequently lacking. This is at least partly caused by the limited access to human oligodendrocytes from patients to perform functional studies and drug screens. The induced pluripotent stem cell technology (iPSC) represents a possibility to circumvent this obstacle and paves new ways to study human disease and to develop new treatment options for so far incurable central nervous system (CNS) diseases. In this review, we summarize the differences between human and rodent oligodendrocytes, provide an overview of the different techniques to generate oligodendrocytes from human progenitor or stem cells and describe the results from studies using iPSC derived oligodendroglial lineage cells. Furthermore, we discuss future perspectives and challenges of the iPSC technology with respect to disease modeling, drug screen, and cell transplantation approaches.

Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors.

Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shiRag-/- mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.

Inflammatory demyelination induces ependymal modifications concomitant to activation of adult (SVZ) stem cell proliferation.

Ependymal cells (E1/E2) and ciliated B1cells confer a unique pinwheel architecture to the ventricular surface of the subventricular zone (SVZ), and their cilia act as sensors to ventricular changes during development and aging. While several studies showed that forebrain demyelination reactivates the SVZ triggering proliferation, ectopic migration, and oligodendrogenesis for myelin repair, the potential role of ciliated cells in this process was not investigated. Using conventional and lateral wall whole mount preparation immunohistochemistry in addition to electron microscopy in a forebrain-targeted model of experimental autoimmune encephalomyelitis (tEAE), we show an early decrease in numbers of pinwheels, B1 cells, and E2 cells. These changes were transient and simultaneous to tEAE-induced SVZ stem cell proliferation. The early drop in B1/E2 cell numbers was followed by B1/E2 cell recovery. While E1 cell division and ependymal ribbon disruption were never observed, E1 cells showed important morphological modifications reflected by their enlargement, extended cytoskeleton, and reinforced cell-cell junction complexes overtime, possibly reflecting protective mechanisms against ventricular insults. Finally, tEAE disrupted motile cilia planar cell polarity and cilia orientation in ependymal cells. Therefore, significant ventricular modifications in ciliated cells occur early in response to tEAE suggesting a role for these cells in SVZ stem cell signalling not only during development/aging but also during inflammatory demyelination. These observations may have major implications for understanding pathophysiology of and designing therapeutic approaches for inflammatory demyelinating diseases such as MS.

Early netrin-1 expression impairs central nervous system remyelination.

OBJECTIVE: Chronically demyelinated multiple sclerosis (MS) lesions are frequently characterized by scarce undifferentiated oligodendrocyte progenitor cells (OPCs), suggesting the exhaustion of a local OPC pool followed by failure of recruitment and differentiation. Stimulating prompt OPC recruitment following demyelination could improve myelin repair by providing sufficient numbers of remyelinating cells during the repair-permissive period. Understanding mechanisms that determine this process may have important therapeutic implications. We therefore investigated the role of the guidance molecule netrin-1 in OPC recruitment and central nervous system (CNS) remyelination. METHODS: Netrin-1 expression was analyzed immunohistochemically in different types of MS lesions and in the murine lysolecithin model of demyelination. The influence of netrin-1 on CNS remyelination was examined using gain and loss of function experiments. RESULTS: We show that in MS lesions, astrocytes upregulate netrin-1 expression early during demyelination and netrin-1 receptors are expressed by OPCs. In contrast, in the efficiently repairing lysolecithin model of demyelination (astrocyte-free), netrin-1 expression is absent during early phases and detected concomitant with completion of OPC recruitment. In vitro migration assays demonstrated that netrin-1 is a chemorepellent for migrating adult OPCs. In mouse lesions, antibody-mediated disruption of netrin-1 function at the peak phase of recruitment increased OPC numbers. Conversely, lentiviral-mediated induction of netrin-1 expression prior to OPC recruitment reduced the number of cells recruited and impaired remyelination. INTERPRETATION: Our findings support the conclusion that netrin-1 expression within demyelinating MS plaques blocks OPC recruitment, which with repeated demyelinating episodes contributes to permanent remyelination failure.

Castration attenuates myelin repair following lysolecithin induced demyelination in rat optic chiasm: an evaluation using visual evoked potential, marker genes expression and myelin staining.

Multiple sclerosis (MS) is a demyelinating disease that affects the central nervous system. MS is the most common neurological disorder in young adults with a greater incidence among females. Male gonadal hormones have a protective effect on neural system development and myelin maturation. In this study, we investigate the effect of castration on lysolecithin-induced demyelination and remyelination processes using visual evoked potentials, in addition to measuring the expressions of Olig2, MBP, Nogo-A and GFAP mRNAs as oligodendrocyte or astrocyte markers; and histological assessments by myelin-specific staining. We observed more expanded demyelination with delayed repair process in castrated rats. Expression levels of the aforementioned marker genes confirmed histological and electrophysiological observations. Our results showed a pivotal role for endogenous male hormones in the context of demyelinating insults. It may also account for the different prognosis of MS between male and female genders and provide new insights for therapeutic treatments.

One-step Reprogramming of Human Fibroblasts into Oligodendrocyte-like Cells by SOX10, OLIG2, and NKX6.2.

Limited access to human oligodendrocytes impairs better understanding of oligodendrocyte pathology in myelin diseases. Here, we describe a method to robustly convert human fibroblasts directly into oligodendrocyte-like cells (dc-hiOLs), which allows evaluation of remyelination-promoting compounds and disease modeling. Ectopic expression of SOX10, OLIG2, and NKX6.2 in human fibroblasts results in rapid generation of O4+ cells, which further differentiate into MBP+ mature oligodendrocyte-like cells within 16 days. dc-hiOLs undergo chromatin remodeling to express oligodendrocyte markers, ensheath axons, and nanofibers in vitro, respond to promyelination compound treatment, and recapitulate in vitro oligodendroglial pathologies associated with Pelizaeus-Merzbacher leukodystrophy related to PLP1 mutations. Furthermore, DNA methylome analysis provides evidence that the CpG methylation pattern significantly differs between dc-hiOLs derived from fibroblasts of young and old donors, indicating the maintenance of the source cells' "age." In summary, dc-hiOLs represent a reproducible technology that could contribute to personalized medicine in the field of myelin diseases.

Vitamins E and D3 attenuate demyelination and potentiate remyelination processes of hippocampal formation of rats following local injection of ethidium bromide.

Cognitive deficits have been observed in patients with multiple sclerosis (MS) due to hippocampal insults. Antioxidant vitamins D and E are suggested for patients suffering from neurodegenerative diseases like MS, while their mechanisms of action are not well understood. Here, we have tried to study the effects of these vitamins on demyelination, cell death, and remyelination of rat hippocampus following local ethidium bromide (EB) injection. Animals received 100 mg/kg vitamin E or 5 microg/kg of vitamin D3 for 2, 7, or 28 days. The extent of demyelination, myelin staining intensity, and expression of myelin basic protein and caspase-3 were investigated using histological and immunoblotting verification. Administration of EB alone caused demyelination, cell death, and afterward an endogenous repair. Vitamins E and D3 reduced the EB-induced damage and increased the endogenous remyelination of hippocampus. Although the anti-apoptotic effect of these vitamins and protection against demyelination were predictable based on their antioxidant effect, our results indicated the positive effect of vitamins E and D3 on process of remyelination by endogenous progenitor cells and supported their possible therapeutic effects in the context of demyelinating diseases like MS.

Multiple sclerosis iPS-derived oligodendroglia conserve their properties to functionally interact with axons and glia in vivo.

Remyelination failure in multiple sclerosis (MS) is associated with a migration/differentiation block of oligodendroglia. The reason for this block is highly debated. It could result from disease-related extrinsic or intrinsic regulators in oligodendroglial biology. To avoid confounding immune-mediated extrinsic effect, we used an immune-deficient mouse model to compare induced pluripotent stem cell-derived oligodendroglia from MS and healthy donors following engraftment in the developing CNS. We show that the MS-progeny behaves and differentiates into oligodendrocytes to the same extent as controls. They generate equal amounts of myelin, with bona fide nodes of Ranvier, and promote equal restoration of their host slow conduction. MS-progeny expressed oligodendrocyte- and astrocyte-specific connexins and established functional connections with donor and host glia. Thus, MS oligodendroglia, regardless of major immune manipulators, are intrinsically capable of myelination and making functional axo-glia/glia-glia connections, reinforcing the view that the MS oligodendrocyte differentiation block is not from major intrinsic oligodendroglial deficits.

Skin-derived neural precursors competitively generate functional myelin in adult demyelinated mice.

Induced pluripotent stem cell-derived (iPS-derived) neural precursor cells may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases. So far, the therapeutic potential of reprogrammed cells has been evaluated in neonatal demyelinating models. However, the repair efficacy and safety of these cells has not been well addressed in the demyelinated adult CNS, which has decreased cell plasticity and scarring. Moreover, it is not clear if these induced pluripotent-derived cells have the same reparative capacity as physiologically committed CNS-derived precursors. Here, we performed a side-by-side comparison of CNS-derived and skin-derived neural precursors in culture and following engraftment in murine models of adult spinal cord demyelination. Grafted induced neural precursors exhibited a high capacity for survival, safe integration, migration, and timely differentiation into mature bona fide oligodendrocytes. Moreover, grafted skin-derived neural precursors generated compact myelin around host axons and restored nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Together, these results provide important insights into the biology of reprogrammed cells in adult demyelinating conditions and support use of these cells for regenerative biomedicine of myelin diseases that affect the adult CNS.

Nogo receptor inhibition enhances functional recovery following lysolecithin-induced demyelination in mouse optic chiasm.

BACKGROUND: Inhibitory factors have been implicated in the failure of remyelination in demyelinating diseases. Myelin associated inhibitors act through a common receptor called Nogo receptor (NgR) that plays critical inhibitory roles in CNS plasticity. Here we investigated the effects of abrogating NgR inhibition in a non-immune model of focal demyelination in adult mouse optic chiasm. METHODOLOGY/PRINCIPAL FINDINGS: A focal area of demyelination was induced in adult mouse optic chiasm by microinjection of lysolecithin. To knock down NgR levels, siRNAs against NgR were intracerebroventricularly administered via a permanent cannula over 14 days, Functional changes were monitored by electrophysiological recording of latency of visual evoked potentials (VEPs). Histological analysis was carried out 3, 7 and 14 days post demyelination lesion. To assess the effect of NgR inhibition on precursor cell repopulation, BrdU was administered to the animals prior to the demyelination induction. Inhibition of NgR significantly restored VEPs responses following optic chiasm demyelination. These findings were confirmed histologically by myelin specific staining. siNgR application resulted in a smaller lesion size compared to control. NgR inhibition significantly increased the numbers of BrdU+/Olig2+ progenitor cells in the lesioned area and in the neurogenic zone of the third ventricle. These progenitor cells (Olig2+ or GFAP+) migrated away from this area as a function of time. CONCLUSIONS/SIGNIFICANCE: Our results show that inhibition of NgR facilitate myelin repair in the demyelinated chiasm, with enhanced recruitment of proliferating cells to the lesion site. Thus, antagonizing NgR function could have therapeutic potential for demyelinating disorders such as Multiple Sclerosis.

The effect of vitamin A supplementation on stimulated T-cell proliferation with myelin oligodendrocyte glycoprotein in patients with multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease whereby myelin sheath of the central nervous system is destroyed. Vitamin A is known to play a role in the immune system. It has been recognized that some metabolites of vitamin A can be used effectively to treat experimental autoimmune encephalomyelitis (EAE). AIMS: The effect of vitamin A as retinyl palmitate on T-cell proliferation in MS patients. SETTING AND DESIGN: This study is a double blind clinical trial of two test groups over a period of 6 months. MATERIALS AND METHODS: Thirty five multiple sclerosis (MS) patients were divided into two groups. One group received 25,000 IU/day vitamin A (as retinyl palmitate) and the other group were administered a placebo. The peripheral blood mononuclear cells (PBMCs) were separated and stimulated with myelin oligodendrocyte glycoprotein (MOG) and phytohemagglutinin (PHA) before and after the trial period. BrdU calorimetric assay was performed to measure cell proliferation. STATISTICAL ANALYSIS: Analysis of covariance (ANCOVA) and paired t-test were used to analyze the data. RESULTS: Observations showed statistical significant differences in the reduction of cell proliferation in the presence of MOG and fetal calf serum (FCS) in the culture medium, between patients receiving vitamin A and the placebo (P = 0.046). Although, this difference was not significant between the two vitamin A and placebo groups in MOG treatment with human serum, a decrease was observed in the group of patients taking vitamin A supplements (P = 0.019). Phytohemagglutinin did not cause any change in cell proliferation between the two groups. CONCLUSION: The results suggest supplementation with retinyl palmitate in patients with MS reduce MOG stimulatory effects on T-cells.

Analysis of structural and molecular events associated with adult rat optic chiasm and nerves demyelination and remyelination: possible role for 3rd ventricle proliferating cells.

Multiple sclerosis frequently affects the optic apparatus, particularly optic chiasm and nerves. Here, we have reported the structural and molecular characteristics of remyelination in the adult rat optic chiasm and nerves. Moreover, considering the proximity of optic chiasm and 3rd ventricle, we have tried to determine if proliferating cells residing in 3rd ventricle region are able to migrate in response to experimental demyelination of the optic chiasm. Following local demyelination by lysolecithin, remyelination pattern in longitude of optic chiasm and proximal nerves was investigated using myelin staining and marker genes expression. Furthermore, cell tracing was carried out using BrdU labeling of proliferating cells prior to gliotoxin injection. Morphometric analysis revealed that demyelination was considerable on days 7 and 14 and an incomplete remyelination occurred on day 28 post-lesion. Interestingly, myelin repair was more evident in the caudal part of chiasm, compared to rostral part and proximal optic nerves. Following chiasm and nerve demyelination, trains of BrdU+ cells were seen near the 3rd ventricle which subsequently moved to lesion site. Nestin was significantly up-regulated in 3rd ventricle surroundings. At the lesion site, Nogo-A gene expression was significantly decreased on days 7 and 14 post lesion, while Olig2, nestin, and GFAP expression was increased on day 7. The changes were then reversed by the time. Myelin repair in optic chiasm seems to be mediated by endogenous progenitors and stem cells. Adult 3rd ventricle proliferating cells may play a role in this context by mobilization into the demyelinated chiasm.

Visual evoked potentials and MBP gene expression imply endogenous myelin repair in adult rat optic nerve and chiasm following local lysolecithin induced demyelination.

Multiple sclerosis (MS) patients may suffer from optic disturbances. Toxin-induced demyelinations have frequently been developed to investigate the cellular and structural aspects of demyelination and remyelination processes, separately. The present study describes functional consequence of lysolecithin (LPC)-induced lesion in the adult rat optic nerves and chiasm by recording the visual evoked potentials (VEPs) from the visual cortex and its correlation with myelin basic protein (MBP) expression in lesion site. Records of VEP were obtained at 2, 7, 14 and 28 days post-injection. We observed that the VEPs generated by light stimuli progressively changed in both amplitude and latency after the lesion as well as in comparison with those generated in control animals. These observations were confirmed through measurement of mRNA expression level for MBP which is one of the important genes expressed in mature oligodendrocytes and Schwann cells. The level of MBP mRNAs in demyelinated chiasm and optic nerves decreased following lysolecithin injection with its least value on day 7, and then it increased to the control level 14 days post-lesion. However, it continued to increase even after that and reached a maximum level 28 days post lesion. Results of the present paper show that, LPC injection in the chiasm share functional and molecular alterations which are found in demyelinating disorders in both the optic nerves and chiasm and also these alterations were coming back to level of control animal on 28 days post lesion, which is typically seen in myelin repair process. The present paper provides new insights into the experimental toxin-induced models that may be useful for evaluating the functional recovery of demyelinated optic nerves and chiasm following various repairing strategies. It also seems to be useful for studying the protective or remyelinating effects of different therapies in e.g. optic apparatus which is more affected by MS.