Astrocyte‐specific activation of TNFR2 promotes oligodendrocyte maturation by secretion of leukemia inhibitory factor

Tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2 have pleiotropic effects in neurodegenerative disorders. For example, while TNFR1 mediates neurodegenerative effects in multiple sclerosis, TNFR2 is protective and contributes to remyelination. The exact mode of TNFR2 action, however, is poorly understood. Here, we show that TNFR2‐mediated activation of the PI3K‐PKB/Akt pathway in primary astrocytes increased the expression of neuroprotective genes, including that encoding the neurotrophic cytokine leukemia inhibitory factor (LIF). To investigate whether intercellular signaling between TNFR2‐stimulated astrocytes and oligodendrocytes plays a role in oligodendrocyte maturation, we established an astrocyte–oligodendrocyte coculture model, composed of primary astrocytes from huTNFR2‐transgenic (tgE1335) mice and oligodendrocyte progenitor cells (OPCs) from wild‐type mice, capable of differentiating into mature myelinating oligodendrocytes. In this model, selective stimulation of human TNFR2 on astrocytes, promoted differentiation of cocultured OPCs to myelin basic protein‐positive mature oligodendrocytes. Addition of LIF neutralizing antibodies inhibited oligodendrocyte differentiation, indicating a crucial role of TNFR2‐induced astrocyte derived LIF for oligodendrocyte maturation. GLIA 2014;62:272–283     

Regulation of oligodendrocyte precursor maintenance by chondroitin sulphate glycosaminoglycans

Chondroitin sulfate proteoglycans (CSPGs) have been proven to inhibit morphological maturation of oligodendrocytes as well as their myelination capabilities. Yet, it remained unclear, whether CSPGs and/or their respective chondroitin sulfate glycosaminoglycan (CS‐GAG) side chains also regulate the oligodendrocyte lineage progression. Here, we initially show that CS‐GAGs detected by the monoclonal antibody 473HD are expressed by primary rat NG2‐positive oligodendrocyte precursor cells (OPCs) and O4‐positive immature oligodendrocytes. CS‐GAGs become down‐regulated with ongoing oligodendrocyte differentiation. Enzymatic removal of the CS‐GAG chains by the bacterial enzyme Chondroitinase ABC (ChABC) promoted spontaneous differentiation of proliferating rat OPCs toward O4‐positive immature oligodendrocytes. Upon forced differentiation, the enzymatic removal of the CS‐GAGs accelerated oligodendrocyte differentiation toward both MBP‐positive and membrane forming oligodendrocytes. These processes were attenuated on enriched CSPG fractions, mainly consisting of Phosphacan/RPTPβ/ζ and to less extent of Brevican and NG2. To qualify CS‐GAGs as universal regulators of oligodendrocyte biology, we finally tested the effect of CS‐GAG removal on OPCs from different sources such as mouse cortical oligospheres, mouse spinal cord neurospheres, and most importantly human‐induced pluripotent stem cell‐derived radial glia‐like neural precursor cells. For all culture systems used, we observed a similar inhibitory effect of CS‐GAGs on oligodendrocyte differentiation. In conclusion, this study clearly suggests an important fundamental principle for complex CS‐GAGs to regulate the oligodendrocyte lineage progression. Moreover, the use of ChABC in order to promote oligodendrocyte differentiation toward myelin gene expressing cells might be an applicable therapeutic option to enhance white matter repair. GLIA 2016;64:270–286     

Low‐density lipoprotein receptor‐related protein 1 is a novel modulator of radial glia stem cell proliferation,survival, and differentiation

The LDL family of receptors and its member low‐density lipoprotein receptor‐related protein 1 (LRP1) have classically been associated with a modulation of lipoprotein metabolism. Current studies, however, indicate diverse functions for this receptor in various aspects of cellular activities, including cell proliferation, migration, differentiation, and survival. LRP1 is essential for normal neuronal function in the adult CNS, whereas the role of LRP1 in development remained unclear. Previously, we have observed an upregulation of LewisX (LeX) glycosylated LRP1 in the stem cells of the developing cortex and demonstrated its importance for oligodendrocyte differentiation. In the current study, we show that LeX‐glycosylated LRP1 is also expressed in the stem cell compartment of the developing spinal cord and has broader functions in the developing CNS. We have investigated the basic properties of LRP1 conditional knockout on the neural stem/progenitor cells (NSPCs) from the cortex and the spinal cord, created by means of Cre‐loxp‐mediated recombination in vitro. The functional status of LRP1‐deficient cells has been studied using proliferation, differentiation, and apoptosis assays. LRP1 deficient NSPCs from both CNS regions demonstrated altered differentiation profiles. Their differentiation capacity toward oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes and neurons was reduced. In contrast, astrocyte differentiation was promoted. Moreover, LRP1 deletion had a negative effect on NSPCs proliferation and survival. Our observations suggest that LRP1 facilitates NSPCs differentiation via interaction with apolipoprotein E (ApoE). Upon ApoE4 stimulation wild type NSPCs generated more oligodendrocytes, but LRP1 knockout cells showed no response. The effect of ApoE seems to be independent of cholesterol uptake, but is rather mediated by downstream MAPK and Akt activation. GLIA 2016 GLIA 2016;64:1363–1380     

Inflammation severely alters thyroid hormone signaling in the central nervous system during experimental allergic encephalomyelitis in rat: Direct impact on OPCs differentiation failure

Differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes is severely impaired by inflammatory cytokines and this could lead to remyelination failure in inflammatory/demyelinating diseases. Due to the role of thyroid hormone in the maturation of OPCs and developmental myelination, in this study we investigated (i) the possible occurrence of dysregulation of thyroid hormone signaling in the CNS tissue during experimental neuroinflammation; (ii) the possible impact of inflammatory cytokines on thyroid hormone signaling and OPCs differentiation in vitro. The disease model is the experimental allergic encephalomyelitis in female Dark‐Agouti rats, whereas in vitro experiments were carried out in OPCs derived from neural stem cells. The main results are the following: (i) a strong upregulation of cytokine mRNA expression level was found in the spinal cord during experimental allergic encephalomyelitis; (ii) thyroid hormone signaling in the spinal cord (thyroid hormone receptors; deiodinase; thyroid hormone membrane transporter) is substantially downregulated, due to the upregulation of the thyroid hormone inactivating enzyme deiodinase 3 and the downregulation of thyroid hormone receptors, as investigated at mRNA expression level; (iii) when exposed to inflammatory cytokines, deiodinase 3 is upregulated in OPCs as well, and OPCs differentiation is blocked; (iv) deiodinase 3 inhibition by iopanoic acid recovers OPCs differentiation in the presence on inflammatory cytokines. These data suggest that cellular hypothyroidism occurs during experimental allergic encephalomyelitis, possibly impacting on thyroid hormone‐dependent cellular processes, including maturation of OPCs into myelinating oligodendrocytes. GLIA 2016;64:1573–1589     

Olig2‐lineage cells preferentially differentiate into oligodendrocytes but their processes degenerate at the chronic demyelinating stage of proteolipid protein‐overexpressing mouse

In chronic demyelinating lesions of the central nervous system, insufficient generation of oligodendrocytes (OLs) is not due to a lack of oligodendrocyte precursor cells (OPCs), because the accumulation of OPCs and premyelinating OLs can be observed within these lesions. Here we sought to identify the basis for the failure of OLs to achieve terminal differentiation in chronic demyelinating lesions through the utilization of plp1‐overexpressing (Plp ^tg/?) mice. These mice are characterized by progressive demyelination in young adults and chronic demyelinating lesions at more mature stages. We show that neural stem cells, which are the precursors of OL‐lineage cells, are present in the Plp ^tg/? mouse brain and that their multipotentiality and ability to self‐renew are comparable to those of wild‐type adults in culture. Lineage‐tracing experiments using a transgenic mouse line, in which an inducible Cre recombinase is knocked in at the Olig2 locus, revealed that Olig2‐lineage cells preferentially differentiated into OPCs and premyelinating OLs, but not into astrocytes, in the Plp ^tg/? mouse brain. These Olig2‐lineage cells matured to express myelin basic protein but after that their processes degenerated in the chronic demyelinating lesions of the Plp ^tg/? brain. These results indicate that in chronic demyelinated lesions more OL‐lineage cells are produced as part of the repair process, but their processes degenerate after maturation. © 2012 Wiley Periodicals, Inc.     

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.     

Oligodendrogenesis from neural stem cells: Perspectives for remyelinating strategies

Mobilization of remyelinating cells spontaneously occurs in the adult brain. These cellular resources are specially active after demyelinating episodes in early phases of multiple sclerosis (MS). Indeed, oligodendrocyte precursor cells (OPCs) actively proliferate, migrate to and repopulate the lesioned areas. Ultimately, efficient remyelination is accomplished when new oligodendrocytes reinvest nude neuronal axons, restoring the normal properties of impulse conduction. As the disease progresses this fundamental process fails. Multiple causes seem to contribute to such transient decline, including the failure of OPCs to differentiate and enwrap the vulnerable neuronal axons. Regenerative medicine for MS has been mainly centered on the recruitment of endogenous self-repair mechanisms, or on transplantation approaches. The latter commonly involves grafting of neural precursor cells (NPCs) or neural stem cells (NSCs), with myelinogenic potential, in the injured areas. Both strategies require further understanding of the biology of oligodendrocyte differentiation and remyelination. Indeed, the success of transplantation largely depends on the pre-commitment of transplanted NPCs or NSCs into oligodendroglial cell type, while the endogenous differentiation of OPCs needs to be boosted in chronic stages of the disease. Thus, much effort has been focused on finding molecular targets that drive oligodendrocytes commitment and development. The present review explores several aspects of remyelination that must be considered in the design of a cell-based therapy for MS, and explores more deeply the challenge of fostering oligodendrogenesis. In this regard, we discuss herein a tool developed in our research group useful to search novel oligodendrogenic factors and to study oligodendrocyte differentiation in a time- and cost-saving manner.     

Human mesenchymal factors induce rat hippocampal‐ and human neural stem cell dependent oligodendrogenesis

The generation of new oligodendrocytes is essential for adult brain repair in diseases such as multiple sclerosis. We previously identified the multifunctional p57kip2 protein as a negative regulator of myelinating glial cell differentiation and as an intrinsic switch of glial fate decision in adult neural stem cells (aNSCs). In oligodendroglial precursor cells (OPCs), p57kip2 protein nuclear exclusion was recently found to be rate limiting for differentiation to proceed. Furthermore, stimulation with mesenchymal stem cell (MSC)‐derived factors enhanced oligodendrogenesis by yet unknown mechanisms. To elucidate this instructive interaction, we investigated to what degree MSC secreted factors are species dependent, whether hippocampal aNSCs respond equally well to such stimuli, whether apart from oligodendroglial differentiation also tissue integration and axonal wrapping can be promoted and whether the oligodendrogenic effect involved subcellular translocation of p57kip2. We found that CC1 positive oligodendrocytes within the hilus express nuclear p57kip2 protein and that MSC dependent stimulation of cultured hippocampal aNSCs was not accompanied by nuclear p57kip2 exclusion as observed for parenchymal OPCs after spontaneous differentiation. Stimulation with human MSC factors was observed to equally promote rat stem cell oligodendrogenesis, axonal wrapping and tissue integration. As forced nuclear shuttling of p57kip2 led to decreased CNPase‐ but elevated GFAP expression levels, this indicates heterogenic oligodendroglial mechanisms occurring between OPCs and aNSCs. We also show for the first time that dominant pro‐oligodendroglial factors derived from human fetal MSCs can instruct human induced pluripotent stem cell‐derived NSCs to differentiate into O4 positive oligodendrocytes.     

Neural stem/progenitor cells differentiate into oligodendrocytes,reduce inflammation,and ameliorate learning deficits after transplantation in a mouse model of traumatic brain injury

The central nervous system has limited capacity for regeneration after traumatic injury. Transplantation of neural stem/progenitor cells (NPCs) has been proposed as a potential therapeutic approach while insulin‐like growth factor I (IGF‐I) has neuroprotective properties following various experimental insults to the nervous system. We have previously shown that NPCs transduced with a lentiviral vector for IGF‐I overexpression have an enhanced ability to give rise to neurons in vitro but also in vivo, upon transplantation in a mouse model of temporal lobe epilepsy. Here we studied the regenerative potential of NPCs, IGF‐I‐transduced or not, in a mouse model of hippocampal mechanical injury. NPC transplantation, with or without IGF‐I transduction, rescued the injury‐induced spatial learning deficits as revealed in the Morris Water Maze. Moreover, it had beneficial effects on the host tissue by reducing astroglial activation and microglial/macrophage accumulation while enhancing generation of endogenous oligodendrocyte precursor cells. One or two months after transplantation the grafted NPCs had migrated towards the lesion site and in the neighboring myelin‐rich regions. Transplanted cells differentiated toward the oligodendroglial, but not the neuronal or astrocytic lineages, expressing the early and late oligodendrocyte markers NG2, Olig2, and CNPase. The newly generated oligodendrocytes reached maturity and formed myelin internodes. Our current and previous observations illustrate the high plasticity of transplanted NPCs which can acquire injury‐dependent phenotypes within the host CNS, supporting the fact that reciprocal interactions between transplanted cells and the host tissue are an important factor to be considered when designing prospective cell‐based therapies for CNS degenerative conditions. GLIA 2016;64:763–779     

Neural stem cells as a potential source of oligodendrocytes for myelin repair

Neural stem cells (NSCs) are considered to have widespread therapeutic possibilities on account of their ability to provide large numbers of cells whilst retaining multi-potentiality. Application to human demyelinating diseases requires improved understanding of the signalling requirements underlying the generation of oligodendrocytes from NSCs. During development, spinal cord oligodendrocyte precursors (OPCs) originate from the ventral, but not dorsal neuroepithelium due to the regulatory effects of the morphogen Sonic hedgehog (Shh). The developing human spinal cord shows comparable ventral-dorsal gradient of oligodendrocyte differentiation potential to the embryonic rodent spinal cord. In contrast expanded human neural precursors derived from both isolated ventral or dorsal cultures show a reduced capacity to generate oligodendrocytes, whereas comparable rodent cultures demonstrate a marked increase in oligodendrocyte formation by a hedgehog independent pathway. Inter-species difference in the capacity of neural precursors to generate oligodendrocytes emphasises the need for greater study of human derived stem cell populations.     

miR‐219 attenuates demyelination in cuprizone‐induced demyelinated mice by regulating monocarboxylate transporter 1

Remyelination is limited in patients with multiple sclerosis (MS) due to the difficulties in recruiting proliferating oligodendrocyte precursors (OPCs), the inhibition of OPC differentiation and/or maturation, and/or failure in the generation of the myelin sheath. In vitro studies have revealed that miR‐219 is necessary for OPC differentiation and monocarboxylate transporter 1 (MCT1) plays a vital role in oligodendrocyte maturation and myelin synthesis. Herein, we hypothesized that miR‐219 might promote oligodendrocyte differentiation and attenuate demyelination in a cuprizone (CPZ)‐induced demyelinated model by regulating the expression of MCT1. We found that CPZ‐treated mice exhibited significantly increased anxiety in the open field test. However, miR‐219 reduced anxiety as shown by an increase in the total distance, the central distance and the mean amount of time spent in the central area. miR‐219 decreased the quantity of OPCs and increased the number of oligodendrocytes and the level of myelin basic protein (MBP) and cyclic nucleotide 3′ phosphodiesterase (CNP) protein. Ultrastructural studies further confirmed that the extent of demyelination was attenuated by miR‐219 overexpression. Meanwhile, miR‐219 also greatly enhanced MCT1 expression via suppression of oligodendrocyte differentiation inhibitors, Sox6 and Hes5, treatment with the MCT1 inhibitor α‐cyano‐4‐hydroxycinnamate (4‐CIN) reduced the number of oligodendrocytes and the protein levels of MBP and CNP. Taken together, these results suggest a novel mode of action of miR‐219 via MCT1 in vivo and may provide a new potential remyelination therapeutic target.     

Olfactory sphere cells are a cell source for γ‐aminobutyric acid‐producing neurons

Olfactory sphere cells (OSCs) are stem cells generated by culturing olfactory mucosa. Adult rat OSCs express oligodendrocyte progenitor cell (OPC) markers and differentiate into mature oligodendrocytes. Although OSCs also express nestin, a marker of neural stem cells (NSCs), it remains unclear whether adult rat OSCs are multipotent and capable of giving rise to neurons as well as oligodendrocytes. Valproic acid (VPA) is a histone deacetylase inhibitor that has the contradictory capacity to induce both differentiation of NSCs and dedifferentiation of OPCs. This study investigates a potential role for VPA in inducing either differentiation or dedifferentiation of adult rat OSCs. Treatment of OSCs with VPA induced hyperacetylation of histones and decreased cell proliferation in the absence of changes in the number of nestin‐positive cells. Furthermore, VPA promoted the genesis of γ‐aminobutyric acid (GABA)‐producing neurons identified by expression of Tuj1/GAD67/GABA while repressing oligodendrocyte production. These findings suggest that OSCs treated with VPA did not exhibit stem cell properties indicative of dedifferentiation but rather switched to a neuronal identity during their terminal differentiation. OSCs were then transplanted into the hippocampus of rats with kainic acid‐induced temporal lobe epilepsy and were systemically given VPA. Although grafted OSCs expressed Tuj1 and GAD67, these cells did not sufficiently inhibit epileptic activity. These results suggest that OSCs are a transplantable cell source for GABA‐producing neurons that can be modulated by VPA. However, further investigation is required to develop them for clinical applications. © 2015 Wiley Periodicals, Inc.     

Human oligodendrocytes in remyelination research

Studies on myelination and oligodendrocyte development are inevitably linked with demyelinating conditions such as multiple sclerosis (MS), leukodystrophies or spinal cord injury (SCI). Chronic loss of myelin, subsequently leading to neurodegeneration, is the ultimate cause of severe and permanent disability. Thus, fast restoration of myelin (remyelination) is essential for circumventing demyelination‐caused pathologies. Implantation of exogenous remyelinating cells has been considered as a potential remyelination strategy. Researchers have examined a variety of cell types endowed with myelin‐forming capacity (oligodendrocytes, Schwann cells, olfactory ensheathing cells etc.) in vitro and in vivo for their potential application as myelin restoring cell grafts. This review gives a summary of studies on the generation and testing of pure suspensions of human oligodendrocytes as a clinically relevant, efficient cellular tool for treating myelin pathology. We start with a brief overview of the current knowledge on the development of human oligodendrocytes from the late stages of embryogenesis up to the early postnatal stage. Insight in the specific extrinsic and intrinsic factors regulating normal oligodendrogenesis is crucial in order to achieve and maintain a sufficient population of engraftable functional oligodendrocytes in vitro. We discuss potential sources of human oligodendrocytes, including novel oligodendrocyte generation strategies employing induced pluripotent stem cells (iPSCs) and direct conversion technology. Finally, we provide a systematic overview of (the outcome of) experimental studies, in which human oligodendrocytes were tested for their (re)myelination capacity and efficiency. GLIA 2015;63:513–530     

Lineage tracing reveals dynamic changes in oligodendrocyte precursor cells following cuprizone‐induced demyelination

The regeneration of oligodendrocytes is a crucial step in recovery from demyelination, as surviving oligodendrocytes exhibit limited structural plasticity and rarely form additional myelin sheaths. New oligodendrocytes arise through the differentiation of platelet‐derived growth factor receptor α (PDGFRα) expressing oligodendrocyte progenitor cells (OPCs) that are widely distributed throughout the CNS. Although there has been detailed investigation of the behavior of these progenitors in white matter, recent studies suggest that disease burden in multiple sclerosis (MS) is more strongly correlated with gray matter atrophy. The timing and efficiency of remyelination in gray matter is distinct from white matter, but the dynamics of OPCs that contribute to these differences have not been defined. Here, we used in vivo genetic fate tracing to determine the behavior of OPCs in gray and white matter regions in response to cuprizone‐induced demyelination. Our studies indicate that the temporal dynamics of OPC differentiation varies significantly between white and gray matter. While OPCs rapidly repopulate the corpus callosum and mature into CC1 expressing mature oligodendrocytes, OPC differentiation in the cingulate cortex and hippocampus occurs much more slowly, resulting in a delay in remyelination relative to the corpus callosum. The protracted maturation of OPCs in gray matter may contribute to greater axonal pathology and disease burden in MS.     

Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast‐derived iPS cells using zinc‐finger nuclease technology

Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast‐derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human‐derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc‐finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte‐specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno‐associated virus integration site 1 (AAVS1) locus in disease and control‐derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP‐iPS astrocytes exhibited a well‐described functional astrocytic activity in vitro characterized by neuron‐dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP‐iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP‐iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte‐driven disease pathogenesis and therapy. GLIA 2016;64:63–75