Erythropoietin promotes oligodendrogenesis and myelin repair following lysolecithin-induced injury in spinal cord slice culture

Here, we sought to delineate the effect of EPO on the remyelination processes using an in vitro model of demyelination. We report that lysolecithin-induced demyelination elevated EPO receptor (EpoR) expression in oligodendrocyte progenitor cells (OPCs), facilitating the beneficial effect of EPO on the formation of oligodendrocytes (oligodendrogenesis). In the absence of EPO, the resultant remyelination was insufficient, possibly due to a limiting number of oligodendrocytes rather than their progenitors, which proliferate in response to lysolecithin-induced injury. By EPO treatment, lysolecithin-induced proliferation of OPCs was accelerated and the number of myelinating oligodendrocytes and myelin recovery was increased. EPO also enhanced the differentiation of neural progenitor cells expressing EpoR at high level toward the oligodendrocyte-lineage cells through activation of cyclin E and Janus kinase 2 pathways. Induction of myelin-forming oligodendrocytes by high dose of EPO implies that EPO might be the key factor influencing the final differentiation of OPCs. Taken together, our data suggest that EPO treatment could be an effective way to enhance remyelination by promoting oligodendrogenesis in association with elevated EpoR expression in spinal cord slice culture after lysolecithin-induced demyelination.     

LINGO-1 and its role in CNS repair

LINGO-1 is selectively expressed in the CNS on both oligodendrocyte precursor cells (OPCs) and neurons. Its expression is developmentally regulated in the normal CNS, as well as up-regulated in human or rat models of neuropathologies. LINGO-1 functions as a negative regulator of oligodendrocyte differentiation and myelination, neuronal survival and axonal regeneration. Across diverse animal CNS disease models, targeted LINGO-1 inhibition was found to promote neuron and oligodendrocyte survival, axon regeneration, oligodendrocyte differentiation, remyelination and improved functional recovery. The targeted inhibition of LINGO-1 therefore presents a novel therapeutic approach for the treatment of neurological diseases.     

Erythropoietin (EPO) Increases Myelin Gene Expression in CG4 Oligodendrocyte Cells through the Classical EPO Receptor

Erythropoietin (EPO) has protective effects in neurodegenerative and neuroinflammatory diseases, including in animal models of multiple sclerosis, where EPO decreases disease severity. EPO also promotes neurogenesis and is protective in models of toxic demyelination. In this study, we asked whether EPO could promote neurorepair by also inducing remyelination. In addition, we investigated whether the effect of EPO could be mediated by the classical erythropoietic EPO receptor (EPOR), since it is still questioned if EPOR is functional in nonhematopoietic cells. Using CG4 cells, a line of rat oligodendrocyte precursor cells, we found that EPO increases the expression of myelin genes (myelin oligodendrocyte glycoprotein [MOG] and myelin basic protein [MBP]). EPO had no effect in wild-type CG4 cells, which do not express EPOR, whereas it increased MOG and MBP expression in cells engineered to overexpress EPOR (CG4-EPOR). This was reflected in a marked increase in MOG protein levels, as detected by Western blot. In these cells, EPO induced by 10-fold the early growth response gene 2 (Egr2), which is required for peripheral myelination. However, Egr2 silencing with a siRNA did not reverse the effect of EPO, indicating that EPO acts through other pathways. In conclusion, EPO induces the expression of myelin genes in oligodendrocytes and this effect requires the presence of EPOR. This study demonstrates that EPOR can mediate neuroreparative effects.     

The early expression of VAChT and VIP in mouse sympathetic ganglia is not induced by cytokines acting through LIFRbeta or CNTFRalpha

Sympathetic ganglia consist of noradrenergic and cholinergic neurons. The cholinergic marker protein vesicular acetylcholine transporter (VAChT) and the neuropeptide vasoactive intestinal peptide (VIP), co-expressed in mature cholinergic sympathetic neurons, are first detectable during embryonic development of rat sympathetic ganglia. However, the subpopulation of cholinergic sympathetic neurons which innervates sweat glands in mammalian footpads starts to express VAChT and VIP during the first postnatal weeks, under the influence of sweat gland-derived signals. In vitro evidence suggests that the sweat gland-derived cholinergic differentiation factor belongs to a group of neuropoietic cytokines, including LIF, CNTF and CT-1, that act through a LIFRbeta-containing cytokine receptor. To investigate whether the embryonic expression of cholinergic properties is elicited by a related cytokine, the expression of VAChT and VIP was analyzed in stellate ganglia of mice deficient for the cytokine receptor subunits LIFRbeta or CNTFRalpha. The density of VAChT- and VIP-immunoreactive cells in stellate ganglia of new-born animals was not different in LIFRbeta(-/-) and CNTFRalpha(-/-) ganglia as compared to ganglia from wild-type mice. These results demonstrate that the early, embryonic expression of VAChT and VIP is not induced by cytokines acting through LIFRbeta- or CNTFRalpha-containing receptors.     

Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control.     

Inhibition of myoblast differentiation by tumor necrosis factor alpha is mediated by c-Jun N-terminal kinase 1 and leukemia inhibitory factor

The proinflammatory cytokine, TNFalpha plays a major role in muscle wasting occurring in chronic diseases and muscular dystrophies. Among its other functions, TNFalpha perturbs muscle regeneration by preventing satellite cell differentiation. In the present study, the role of c-Jun N-terminal kinase (JNK), a mediator of TNFalpha, was investigated in differentiating myoblast cell lines. Addition of TNFalpha to C2 myoblasts induced immediate and delayed phases of JNK activity. The delayed phase is associated with myoblast proliferation. Inhibition of JNK activity prevented proliferation and restored differentiation to TNFalpha-treated myoblasts. Studies with cell lines expressing MyoD:ER chimera and lacking JNK1 or JNK2 genes indicate that JNK1 activity mediates the effects of TNFalpha on myoblast proliferation and differentiation. TNFalpha does not induce proliferation or inhibit differentiation of JNK1-null myoblasts. However, differentiation of JNK1-null myoblasts is inhibited when they are grown in conditioned medium derived from cell lines affected by TNFalpha. We investigated the induced synthesis of several candidate growth factors and cytokines following treatment with TNFalpha. Expression of IL-6 and leukemia inhibitory factor (LIF) was induced by TNFalpha in wild-type and JNK2-null myoblasts. However, LIF expression was not induced by TNFalpha in JNK1-null myoblasts. Addition of LIF to the growth medium of JNK1-null myoblasts prevented their differentiation. Moreover, LIF-neutralizing antibodies added to the medium of C2 myoblasts prevented inhibition of differentiation mediated by TNFalpha. Hence, TNFalpha promotes myoblast proliferation through JNK1 and prevents myoblast differentiation through JNK1-mediated secretion of LIF.     

Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-beta 1 (TGF-beta 1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-beta 1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.     

Therapeutic Effect of Transplanted Human Wharton’s Jelly Stem Cell-Derived Oligodendrocyte Progenitor Cells (hWJ-MSC-derived OPCs) in an Animal Model of Multiple Sclerosis

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination. We transplanted human Wharton’s jelly stem cell-derived oligodendrocyte progenitor cells (hWJ-MSC-derived OPCs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted OPCs on the functional and pathological manifestations of the disease. Transplanted hWJ-MSC-derived OPCs significantly reduced the clinical signs of EAE. Histological examinations showed that remyelination was significantly increased after transplantation. These results suggest that hWJ-MSC-derived OPCs promote the regeneration of myelin sheaths in the brain.     

Leukemia inhibitory factor contributes to hepatocyte-like differentiation of human bone marrow mesenchymal stem cells

The current majority of protocols for hepatocyte differentiation of mesenchymal stem cells (MSCs) are conducted using oncostatin M (OSM) as an inducer of hepatocyte-like maturation. As leukemia inhibitory factor (LIF) and OSM share similar signaling pathways, we examined whether LIF could play a role in the hepatocyte differentiation process. A differentiation protocol was designed using LIF as a maturation cytokine and this was compared with standard and control protocols applied to human MSCs of bone marrow origin. We observed that mesenchymal-derived hepatocyte-like cells (MDHLCs) acquired similar morphological changes when exposed to LIF or to OSM. Using protein and gene expression assays, we noticed a comparable hepatic marker expression in both differentiation conditions. Furthermore, LIF and OSM allowed the acquisition of equivalent levels of hepatocyte-like functionality as attested by evaluation of urea secretion and glycogen deposition. However, no increase in the expression of hepatocyte-like features could be observed in MDHLCs after a combined exposition to LIF and OSM. In conclusion, we demonstrated that LIF can play a similar role as OSM in the hepatocyte differentiation process of human MSCs.     

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.     

The remyelination Philosopher's Stone: stem and progenitor cell therapies for multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease that leads to oligodendrocyte loss and subsequent demyelination of the adult central nervous system (CNS). The pathology is characterized by transient phases of recovery during which remyelination can occur as a result of resident oligodendroglial precursor and stem/progenitor cell activation. However, myelin repair efficiency remains low urging the development of new therapeutical approaches that promote remyelination activities. Current MS treatments target primarily the immune system in order to reduce the relapse rate and the formation of inflammatory lesions, whereas no therapies exist in order to regenerate damaged myelin sheaths. During the last few years, several transplantation studies have been conducted with adult neural stem/progenitor cells and glial precursor cells to evaluate their potential to generate mature oligodendrocytes that can remyelinate axons. In parallel, modulation of the endogenous progenitor niche by neural and mesenchymal stem cell transplantation with the aim of promoting CNS progenitor differentiation and myelination has been studied. Here, we summarize these findings and discuss the properties and consequences of the various molecular and cell-mediated remyelination approaches. Moreover, we address age-associated intrinsic cellular changes that might influence the regenerative outcome. We also evaluate the extent to which these experimental treatments might increase the regeneration capacity of the demyelinated human CNS and hence be turned into future therapies.     

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.     

Induction of Oligodendrocyte Differentiation and In Vitro Myelination by Inhibition of Rho-Associated Kinase

In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.     

Leukemia inhibitory factor deficiency modulates the immune response and limits autoimmune demyelination: a new role for neurotrophic cytokines in neuroinflammation

The neurotrophic cytokines ciliary neurotrophic factor and leukemia inhibitory factor (LIF) play a key role in neuronal and oligodendrocyte survival and as protective factors in neuroinflammation. To further elucidate the potential of endogenous LIF in modulating neuroinflammation, we studied myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LIF knockout mice (LIF(-/-) mice). In the late phase of active myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, LIF(-/-) mice exhibited a markedly milder disease course. The inflammatory infiltrate in LIF(-/-) mice was characterized by an increase in neutrophilic granulocytes early and fewer infiltrating macrophages associated with less demyelination later in the disease. In good correlation with an effect of endogenous LIF on the immune response, we found an Ag-specific T cell-priming defect with impaired IFN-gamma production in LIF(-/-) mice. On the molecular level, the altered recruitment of inflammatory cells is associated with distinct patterns of chemokine production in LIF(-/-) mice with an increase of CXCL1 early and a decrease of CCL2, CCL3, and CXCL10 later in the disease. These data reveal that endogenous LIF is an immunologically active molecule in neuroinflammation. This establishes a link between LIF and the immune system which was not observed in the ciliary neurotrophic factor knockout mouse.