Maturation and electrophysiological properties of human pluripotent stem cell-derived oligodendrocytes.
ABSTRACT: Rodent-based studies have shown that the membrane properties of oligodendrocytes play prominent roles in their physiology and shift markedly during their maturation from the oligodendrocyte precursor cell (OPC) stage. However, the conservation of these properties and maturation processes in human oligodendrocytes remains unknown, despite their dysfunction being implicated in human neurodegenerative diseases such as multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Here, we have defined the membrane properties of human oligodendrocytes derived from pluripotent stem cells as they mature from the OPC stage, and have identified strong conservation of maturation-specific physiological characteristics reported in rodent systems. We find that as human oligodendrocytes develop and express maturation markers, they exhibit a progressive decrease in voltage-gated sodium and potassium channels and a loss of tetrodotoxin-sensitive spiking activity. Concomitant with this is an increase in inwardly rectifying potassium channel activity, as well as a characteristic switch in AMPA receptor composition. All these steps mirror the developmental trajectory observed in rodent systems. Oligodendrocytes derived from mutant C9ORF72-carryng ALS patient induced pluripotent stem cells did not exhibit impairment to maturation and maintain viability with respect to control lines despite the presence of RNA foci, suggesting that maturation defects may not be a primary feature of this mutation. Thus, we have established that the development of human oligodendroglia membrane properties closely resemble those found in rodent cells and have generated a platform to enable the impact of human neurodegenerative disease-causing mutations on oligodendrocyte maturation to be studied.
Project description:Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons (MN). Importantly, MN degeneration is intimately linked to oligodendrocyte dysfunction and impaired capacity of oligodendrocyte precursor cells (OPCs) to regenerate the myelin sheath enwrapping and protecting neuronal axons. Thus, improving OPC reparative abilities represents an innovative approach to counteract MN loss. A pivotal regulator of OPC maturation is the P2Y-like G protein-coupled receptor 17 (GPR17), whose role in ALS has never been investigated. In other models of neurodegeneration, an abnormal increase of GPR17 has been invariably associated to myelin defects and its pharmacological manipulation succeeded in restoring endogenous remyelination. Here, we analyzed GPR17 alterations in the SOD1G93A ALS mouse model and assessed in vitro whether this receptor could be targeted to correct oligodendrocyte alterations. Western-blot and immunohistochemical analyses showed that GPR17 protein levels are significantly increased in spinal cord of ALS mice at pre-symptomatic stage; this alteration is exacerbated at late symptomatic phases. Concomitantly, mature oligodendrocytes degenerate and are not successfully replaced. Moreover, OPCs isolated from spinal cord of SOD1G93A mice display defective differentiation compared to control cells, which is rescued by treatment with the GPR17 antagonist montelukast. These data open novel therapeutic perspectives for ALS management.
Project description:In this study, we sought evidence for alpha-synuclein (ASYN) expression in oligodendrocytes, as a possible endogenous source of ASYN to explain its presence in glial inclusions found in multiple system atrophy (MSA) and Parkinson's disease (PD). We identified ASYN in oligodendrocyte lineage progenitors isolated from the rodent brain, in oligodendrocytes generated from embryonic stem cells, and in induced pluripotent stem cells produced from fibroblasts of a healthy individual and patients diagnosed with MSA or PD, in cultures in vitro. Notably, we observed a significant decrease in ?SYN during oligodendrocyte maturation. Additionally, we show the presence of transcripts in PDGFR?/CD140a(+) cells and SOX10(+) oligodendrocyte lineage nuclei isolated by FACS from rodent and human healthy and diseased brains, respectively. Our work identifies ASYN in oligodendrocyte lineage cells, and it offers additional in vitro cellular models that should provide significant insights of the functional implication of ASYN during oligodendrocyte development and disease.
Project description:Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation.Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo.OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases, including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions.We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions.
Project description:Identification of additional uses for existing drugs is a hot topic in drug discovery and a viable alternative to de novo drug development. HAMI3379 is known as an antagonist of the cysteinyl-leukotriene CysLT2 receptor, and was initially developed to treat cardiovascular and inflammatory disorders. In our study we identified HAMI3379 as an antagonist of the orphan G protein-coupled receptor GPR17. HAMI3379 inhibits signaling of recombinant human, rat, and mouse GPR17 across various cellular backgrounds, and of endogenous GPR17 in primary rodent oligodendrocytes. GPR17 blockade by HAMI3379 enhanced maturation of primary rat and mouse oligodendrocytes, but was without effect in oligodendrocytes from GPR17 knockout mice. In human oligodendrocytes prepared from inducible pluripotent stem cells, GPR17 is expressed and its activation impaired oligodendrocyte differentiation. HAMI3379, conversely, efficiently favored human oligodendrocyte differentiation. We propose that HAMI3379 holds promise for pharmacological exploitation of orphan GPR17 to enhance regenerative strategies for the promotion of remyelination in patients.
Project description:Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs). However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM) promotes the in vitro differentiation of OPCs under pathological conditions. Primary OPCs were prepared from neonatal rat brains, and differentiated into myelin-basic-protein expressing oligodendrocytes over time. This in vitro OPC differentiation was inhibited by prolonged chemical hypoxic stress induced by non-lethal CoCl(2) treatment. However, AM promoted the OPC differentiation under the hypoxic stress conditions, and the AM receptor antagonist AM(22-52) canceled the AM-induced OPC differentiation. In addition, AM treatment increased the phosphorylation level of Akt in OPC cultures, and correspondingly, the PI3K/Akt inhibitor LY294002 blocked the AM-induced OPC differentiation. Taken together, AM treatment rescued OPC maturation under pathological conditions via an AM-receptor-PI3K/Akt pathway. Oligodendrocytes play critical roles in white matter by forming myelin sheath. Therefore, AM signaling may be a promising therapeutic target to boost oligodendrocyte regeneration in CNS disorders.
Project description:Current approaches to derive oligodendrocytes from human pluripotent stem cells (hPSCs) need extended exposure of hPSCs to growth factors and small molecules, which limits their clinical application because of the lengthy culture time required and low generation efficiency of myelinating oligodendrocytes. Compared to extrinsic growth factors and molecules, oligodendrocyte differentiation and maturation can be more effectively modulated by regulation of the cell transcription network. In the developing central nervous system (CNS), two basic helix-loop-helix transcription factors, Olig1 and Olig2, are decisive in oligodendrocyte differentiation and maturation. Olig2 plays a critical role in the specification of oligodendrocytes and Olig1 is crucial in promoting oligodendrocyte maturation. Recently viral vectors have been used to overexpress Olig2 and Olig1 in neural stem/progenitor cells (NSCs) to induce the maturation of oligodendrocytes and enhance the remyelination activity in vivo. Because of the safety issues with viral vectors, including the insertional mutagenesis and potential tumor formation, non-viral transfection methods are preferred for clinical translation. Here we report a poly(?-amino ester) (PBAE)-based nanoparticle transfection method to deliver Olig1 and Olig2 into human fetal tissue-derived NSCs and demonstrate efficient oligodendrocyte differentiation following transgene expression of Olig1 and Olig2. This approach is potentially translatable for engineering stem cells to treat injured or diseased CNS tissues.Current protocols to derive oligodendrocytes from human pluripotent stem cells (hPSCs) require lengthy culture time with low generation efficiencies of mature oligodendrocytes. We described a new approach to enhance oligodendrocyte differentiation through nanoparticle-mediated transcription modulation. We tested an effective transfection method using cell-compatible poly (?-amino ester) (PBAE)/DNA nanoparticles as gene carrier to deliver transcription factor Olig1 and Olig2 into human fetal tissue-derived neural stem/progenitor cells, and showed efficient oligodendrocyte differentiation following transgene expression of Olig1 and Olig2. We believe that this translatable approach can be applied to many other cell-based regenerative therapies as well.
Project description:Failure of remyelination is largely responsible for sustained neurologic symptoms in multiple sclerosis (MS). MS lesions contain hyaluronan deposits that inhibit oligodendrocyte precursor cell (OPC) maturation. However, the mechanism behind this inhibition is unclear. We report here that Toll-like receptor 2 (TLR2) is expressed by oligodendrocytes and is up-regulated in MS lesions. Pathogen-derived TLR2 agonists, but not agonists for other TLRs, inhibit OPC maturation in vitro. Hyaluronan-mediated inhibition of OPC maturation requires TLR2 and MyD88, a TLR2 adaptor molecule. Ablated expression of TLR2 also enhances remyelination in a lysolecithin animal model. Hyaluronidases expressed by OPCs degrade hyaluronan to hyaluronan oligomers, a requirement for hyaluronan/TLR2 signaling. MS lesions contain both TLR2(+) oligodendrocytes and low-molecular-weight hyaluronan, consistent with their importance to remyelination in MS. We thus have defined a mechanism controlling remyelination failure in MS where hyaluronan is degraded by hyaluronidases into hyaluronan oligomers that block OPC maturation and remyelination through TLR2-MyD88 signaling.
Project description:BACKGROUND:Oligodendrocytes are a type of glial cells that synthesize the myelin sheath around the axons and are critical for the nerve conduction in the CNS. Oligodendrocyte death and defects are the leading causes of several myelin disorders such as multiple sclerosis, progressive multifocal leukoencephalopathy, periventricular leukomalacia, and several leukodystrophies. Temporal activation of the Sonic Hedgehog (SHH) pathway is critical for the generation of oligodendrocyte progenitors, and their differentiation and maturation in the brain and spinal cord during embryonic development in mammals. METHODS:Our protocol utilized adherent cultures of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESCs) with a green fluorescent protein (GFP) reporter knocked into one allele of the OLIG2 gene locus, dual SMAD inhibition, and transient partial inhibition of glioma-associated oncogene 1 (GLI1) by the small molecule GANT61 during the formation of the SOX2/PAX6-positive neural stem cells (NSCs). The SHH pathway was later restimulated by a Smoothened agonist purmorphamine to induce the generation of OLIG2 glial precursors. One hundred ninety-two individual oligodendrocyte precursor cells (OPCs) from GANT61 and control group were analyzed by single-cell RNA sequencing (RNA-Seq). RESULTS:We demonstrate here that transient and partial inhibition of the SHH pathway transcription factor GLI1 in NSCs by a small molecule inhibitor GANT61 was found to generate OPCs that were more migratory and could differentiate earlier toward myelin-producing oligodendrocytes. Single-cell transcriptomic analysis (RNA-Seq) showed that GANT61-NSC-derived oligodendrocyte precursor cells (OPCs) had differential activation of some of the genes in the cytoskeleton rearrangement pathways that are involved in OPC motility and induction of maturation. At the protein level, this was also associated with higher levels of myelin-specific genes in the GANT61 group compared to controls. GANT61-NSC-derived OPCs were functional and could generate compact myelin in vitro and in vivo after transplantation in myelin-deficient shiverer mice. CONCLUSIONS:This is a small molecule-based in vitro protocol that leads to the faster generation of functional oligodendrocytes. The development of protocols that lead to efficient and faster differentiation of oligodendrocytes from progenitors provides important advances toward the development of autologous neural stem cell-based therapies using human iPSCs.
Project description:Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein--MBP--and proteolipid protein--PLP--(respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5?kPa is the most suitable rigidity for oligodendrocyte differentiation.
Project description:Changes in intracellular [Ca(2+)](i) levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.