A novel purification method of murine embryonic stem cell- and human-induced pluripotent stem cell-derived cardiomyocytes by simple manual dissociation

Cardiomyocytes derived from embryonic stem cells (ES-CMs) and induced pluripotent stem cells (iPS-CMs) are useful for toxicity and pharmacology screening. In the present study, we found that cardiomyocyte-rich beating cell clusters (CCs) emerged from murine embryonic stem cell (mESC)-derived beating EBs and from human-induced pluripotent stem cell (hiPSC)-derived beating EBs dissociated by gentle pipetting with a thin glass pipette. The percentage of cardiac troponin T (cTnT)-positive cells in the beating CCs obtained from mESC-derived and hiPSC-derived beating EBs was higher (81.5% and 91.6%, respectively) than in beating-undissociated EBs (13.7% and 67.1%, respectively). For mESCs, the yield of cTnT-positive cells from beating CCs was estimated to be 1.6 times higher than that of beating EBs. The bromodeoxyuridine labeling index of mouse ES-CMs and human iPS-CMs in beating CCs was 1.5- and 3.2-fold, respectively, greater than those in beating EBs. To investigate the utility of the cells in toxicity assessment, we showed that doxorubicin, a cardiotoxic drug, induced myofilament disruption in cardiomyocytes isolated by this method. This simple method enables preparation of mouse ES-CMs and human iPS-CMs with better proliferative activity than beating EBs not dissociated by pipetting, and the cardiomyocytes are useful for drug-induced myocardial toxicity testing.     

Phenotypic changes, signaling pathway, and functional correlates of GPR17-expressing neural precursor cells during oligodendrocyte differentiation

The developing and mature central nervous system contains neural precursor cells expressing the proteoglycan NG2. Some of these cells continuously differentiate to myelin-forming oligodendrocytes; knowledge of the destiny of NG2(+) precursors would benefit from the characterization of new key functional players. In this respect, the G protein-coupled membrane receptor GPR17 has recently emerged as a new timer of oligodendrogliogenesis. Here, we used purified oligodendrocyte precursor cells (OPCs) to fully define the immunophenotype of the GPR17-expressing cells during OPC differentiation, unveil its native signaling pathway, and assess the functional consequences of GPR17 activation by its putative endogenous ligands, uracil nucleotides and cysteinyl leukotrienes (cysLTs). GPR17 presence was restricted to very early differentiation stages and completely segregated from that of mature myelin. Specifically, GPR17 decorated two subsets of slowly proliferating NG2(+) OPCs: (i) morphologically immature cells expressing other early proteins like Olig2 and PDGF receptor-α, and (ii) ramified preoligodendrocytes already expressing more mature factors, like O4 and O1. Thus, GPR17 is a new marker of these transition stages. In OPCs, GPR17 activation by either uracil nucleotides or cysLTs resulted in potent inhibition of intracellular cAMP formation. This effect was counteracted by GPR17 antagonists and receptor silencing with siRNAs. Finally, uracil nucleotides promoted and GPR17 inhibition, by either antagonists or siRNAs, impaired the normal program of OPC differentiation. These data have implications for the in vivo behavior of NG2(+) OPCs and point to uracil nucleotides and cysLTs as main extrinsic local regulators of these cells under physiological conditions and during myelin repair.     

Quercetin Promotes Proliferation and Differentiation of Oligodendrocyte Precursor Cells After Oxygen/Glucose Deprivation-Induced Injury

The aim of this study was to investigate quercetin’s (Qu) ability to promote proliferation and differentiation of oligodendrocyte precursor cells (OPCs) under oxygen/glucose deprivation (OGD)-induced injury in vitro. The results showed that after OGD, OPCs survival rate was significantly increased by Qu as measured by Cell Counting Kit-8. Furthermore, Qu treatment reduced apoptosis of OPCs surveyed by Hoechst 33258 nuclear staining. Qu at 9 and 27 μM promoted the proliferation of OPCs the most by Brdu and Olig2 immunocytochemical staining after OGD 3 days. Also, Qu treatment for 8 days after OGD, the differentiation of OPCs to oligodendrocyte was detected by immunofluorescence staining showing that O4, Olig2, and myelin basic protein (MBP) positive cells were significantly increased compared to control group. Additionally, the protein levels of Olig2 and MBP of OPCs were quantified using western blot and mRNA levels of Olig2 and Inhibitor of DNA binding 2 (Id2) were measured by RT-PCR. Western blot showed a significant increase in Olig2 and MBP expression levels compared with controls after OGD and Qu treatment with a linear does–response curve from 3 to 81 μM. After treatment with Qu compared to its control group, Olig2 mRNA level was significantly up-regulated, whereas Id2 mRNA level was down-regulated. In conclusion, Qu at 3–27 μM can promote the proliferation and differentiation of OPCs after OGD injury and may regulate the activity of Olig2 and Id2.     

Transplantation of Oligodendrocyte Precursor Cells Improves Locomotion Deficits in Rats with Spinal Cord Irradiation Injury

Demyelination contributes to the functional impairment of irradiation injured spinal cord. One potential therapeutic strategy involves replacing the myelin-forming cells. Here, we asked whether transplantation of Olig2⁺-GFP⁺-oligodendrocyte precursor cells (OPCs), which are derived from Olig2-GFP-mouse embryonic stem cells (mESCs), could enhance remyelination and functional recovery after spinal cord irradiation injury. We differentiated Olig2-GFP-mESCs into purified Olig2⁺-GFP⁺-OPCs and transplanted them into the rats’ cervical 4–5 dorsal spinal cord level at 4 months after irradiation injury. Eight weeks after transplantation, the Olig2⁺-GFP⁺-OPCs survived and integrated into the injured spinal cord. Immunofluorescence analysis showed that the grafted Olig2⁺-GFP⁺-OPCs primarily differentiated into adenomatous polyposis coli (APC⁺) oligodendrocytes (54.6±10.5%). The staining with luxol fast blue, hematoxylin & eosin (LFB/H&E) and electron microscopy demonstrated that the engrafted Olig2⁺-GFP⁺-OPCs attenuated the demyelination resulted from the irradiation. More importantly, the recovery of forelimb locomotor function was enhanced in animals receiving grafts of Olig2⁺-GFP⁺-OPCs. We concluded that OPC transplantation is a feasible therapy to repair the irradiated lesions in the central nervous system (CNS).     

Heterogeneity of NG2-expressing cells in the newborn mouse cerebellum

The function and origin of NG2+ cells in the adult brain are still controversial. A large amount of data is available which strongly indicates that adult NG2-expressing cells form a heterogeneous population, constituted by oligodendrocyte precursor cells (OPCs) and a fourth novel type of glial cells named the synantocytes. Whether these two populations derive from the progressive maturation of perinatal NG2+ OPCs or are generated as separate populations is not known. We used organotypic cultures of newborn mouse cerebellum depleted, by anti-mitotic drug treatment, of their NG2+ cells with perinatal features (high proliferating rate and high oligodendrocytic differentiation ability). In these cultures, despite the lack of myelin after 14 days in vitro, numerous NG2+ cells remained. We show that these BrdU-resistant cells were able to slowly divide, as adult NG2+ cells do. Although many of these cells expressed O4, only a very small fraction of them was further engaged in oligodendrocyte lineage, as they had an extremely poor capacity to generate myelin sheaths to the Purkinje cell axons. These results support the view that at least two distinct populations of NG2+ cells coexist in the cerebellum from birth: one with the young OPC characteristics, another with adult NG2+ cell characteristics. Thus, a fraction of adult NG2+ cells do not derive from the maturation of perinatal OPCs.     

Oligodendrocyte precursor cells: Reactive cells that inhibit axon growth and regeneration

Oligodendrocyte precursor cells (OPCs) are a newly recognized glial component of the adult central nervous system of unknown function. Antibodies against the NG2 chondroitin sulfate proteoglycan have been useful tools to identify these cells in intact tissue. Here we review studies that show that OPCs react to several types of experimentally induced brain injury. Injury stimulates OPCs to re-enter the cell cycle, divide, and accumulate at the site of damage. OPCs, together with microglia and astrocytes, form the glial scar. Glial scars are thought to inhibit or prevent axonal regeneration and reactive OPCs contribute to this inhibition by producing growth-inhibiting chondroitin sulfate proteoglycans, particularly NG2. In developing animals, NG2 is found in areas, such as the perinotochordal mesenchyme, that are avoided by growing motor and sensory axons. Within the developing CNS, NG2-expressing cells surround the developing optic chiasm and tract and separate it from the overlying diencephalon. Thus, NG2-expressing cells are well positioned to inhibit axonal growth from developing as well as regenerating neurons.     

Prox1 Inhibits Proliferation and Is Required for Differentiation of the Oligodendrocyte Cell Lineage in the Mouse

Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs) in a progenitor state, but what factor may enable oligodendrocyte (OL) differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1.     

Ddx20, an Olig2 binding factor,governs the survival of neural and oligodendrocyte progenitor cells via proper Mdm2 splicing and p53 suppression

Olig2 is indispensable for motoneuron and oligodendrocyte fate-specification in the pMN domain of embryonic spinal cords, and also involved in the proliferation and differentiation of several cell types in the nervous system, including neural progenitor cells (NPCs) and oligodendrocytes. However, how Olig2 controls these diverse biological processes remains unclear. Here, we demonstrated that a novel Olig2-binding protein, DEAD-box helicase 20 (Ddx20), is indispensable for the survival of NPCs and oligodendrocyte progenitor cells (OPCs). A central nervous system (CNS)-specific Ddx20 conditional knockout (cKO) demonstrated apoptosis and cell cycle arrest in NPCs and OPCs, through the potentiation of the p53 pathway in DNA damage-dependent and independent manners, including SMN complex disruption and the abnormal splicing of Mdm2 mRNA. Analyzes of Olig2 null NPCs showed that Olig2 contributed to NPC proliferation through Ddx20 protein stabilization. Our findings provide novel mechanisms underlying the Olig2-mediated proliferation of NPCs, via the Ddx20-p53 axis, in the embryonic CNS.Subject terms: Oligodendrocyte, Experimental models of disease     

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2⁺OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR⁺microglia. NG2⁺cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.     

Regulatory roles of ganglioside GQ1b in neuronal cell differentiation of mouse embryonic stem cells

Gangliosides play an important role in neuronal differentiation processes. The regulation of ganglioside levels is related to the induction of neuronal cell differentiation. In this study, the ST8Sia5 gene was transfected into mESCs and then differentiated into neuronal cells. Interestingly, ST8Sia5 gene transfected mESCs expressed GQ1b by HPTLC and immunofluorescence analysis. To investigate the effects of GQ1b over-expression in neurogenesis, neuronal cells were differentiated from GQ1b expressing mESCs in the presence of retinoic acid. In GQ1b expressing mESCs, increased EBs formation was observed. After 4 days, EBs were co-localized with GQ1b and nestin, and GFAP. Moreover, GQ1b co-localized with MAP-2 expressing cells in GQ1b expressing mESCs in 7-day-old EBs. Furthermore, GQ1b expressing mESCs increased the ERK1/2 MAP kinase pathway. These results suggest that the ST8Sia5 gene increases ganglioside GQ1b and improves neuronal differentiation via the ERK1/2 MAP kinase pathway.     

Transcript profiling and lipidomic analysis of ceramide subspecies in mouse embryonic stem cells and embryoid bodies

Ceramides (Cers) are important in embryogenesis, but no comprehensive analysis of gene expression for Cer metabolism nor the Cer amounts and subspecies has been conducted with an often used model: mouse embryonic stem cells (mESCs) versus embroid bodies (EBs). Measuring the mRNA levels by quantitative RT-PCR and the amounts of the respective metabolites by LC-ESI/MS/MS, notable differences between R1 mESCs and EBs were: EBs have higher mRNAs for CerS1 and CerS3, which synthesize C18- and C≥24-carbons dihydroceramides (DH)Cer, respectively; EBs have higher CerS2 (for C24:0- and C24:1-); and EBs have lower CerS5 + CerS6 (for C16-). In agreement with these findings, EBs have (DH)Cer with higher proportions of C18-, C24- and C26- and less C16-fatty acids, and longer (DH)Cer are also seen in monohexosylCers and sphingomyelins. EBs had higher mRNAs for fatty acyl-CoA elongases that produce C18-, C24-, and C26-fatty acyl-CoAs (Elovl3 and Elovl6), and higher amounts of these cosubstrates for CerS. Thus, these studies have found generally good agreement between genomic and metabolomic data in defining that conversion of mESCs to EBs is accompanied by a large number of changes in gene expression and subspecies distributions for both sphingolipids and fatty acyl-CoAs.     

Generating mESC-derived insulin-producing cell lines through an intermediate lineage-restricted progenitor line

Generating surrogate insulin-producing cells from embryonic stem cells (ESCs) through in vitro replication of successive steps during pancreatic development has been challenging . Here we describe a novel reproducible protocol to establish homogeneous and scalable insulin-producing cell lines from mouse (m) ESCs via differentiation of the previously described lineage-restricted clonal mESC-derived E-RoSH cells. Unlike their parental mESCs, E-RoSH cells expressed high levels of mesodermal and endodermal genes. Nutrient depletion in the presence of nicotinamide inhibited proliferation of E-RoSH cells and induced differentiation into heterogeneous cultures comprising vascular-like structures that produced detectable levels of insulin and C-peptide in an equimolar ratio. Limiting dilution of these cultures resulted in the isolation of eight independent insulin-producing cell lines in five experiments. All these lines were cloned and shown to be amenable to repeated cycles of freeze and thaw and to replicate for months with a doubling time of 3–4 days. Under such conditions, the cultured cells exhibited genomic, structural, biochemical, and pharmacological properties of pancreatic β cells, including storage of an equimolar ratio of insulin and C-peptide in granules and release of the contents of these organelles through a glucose-sensitive machinery. After transplantation, these cells reversed hyperglycemia in streptozotocin-treated SCID mice and did not form teratomas.     

A new role for the P2Y-like GPR17 receptor in the modulation of multipotency of oligodendrocyte precursor cells in vitro

Oligodendrocyte precursor cells (OPCs, also called NG2 cells) are scattered throughout brain parenchyma, where they function as a reservoir to replace lost or damaged oligodendrocytes, the myelin-forming cells. The hypothesis that, under some circumstances, OPCs can actually behave as multipotent cells, thus generating astrocytes and neurons as well, has arisen from some in vitro and in vivo evidence, but the molecular pathways controlling this alternative fate of OPCs are not fully understood. Their identification would open new opportunities for neuronal replace strategies, by fostering the intrinsic ability of the brain to regenerate. Here, we show that the anti-epileptic epigenetic modulator valproic acid (VPA) can promote the generation of new neurons from NG2⁺ OPCs under neurogenic protocols in vitro, through their initial de-differentiation to a stem cell-like phenotype that then evolves to “hybrid” cell population, showing OPC morphology but expressing the neuronal marker βIII-tubulin and the GPR17 receptor, a key determinant in driving OPC transition towards myelinating oligodendrocytes. Under these conditions, the pharmacological blockade of the P2Y-like receptor GPR17 by cangrelor, a drug recently approved for human use, partially mimics the effects mediated by VPA thus accelerating cells’ neurogenic conversion. These data show a co-localization between neuronal markers and GPR17 in vitro, and suggest that, besides its involvement in oligodendrogenesis, GPR17 can drive the fate of neural precursor cells by instructing precursors towards the neuronal lineage. Being a membrane receptor, GPR17 represents an ideal “druggable” target to be exploited for innovative regenerative approaches to acute and chronic brain diseases.     

Olig2/Plp-positive progenitor cells give rise to Bergmann glia in the cerebellum

NG2 (nerve/glial antigen2)-expressing cells represent the largest population of postnatal progenitors in the central nervous system and have been classified as oligodendroglial progenitor cells, but the fate and function of these cells remain incompletely characterized. Previous studies have focused on characterizing these progenitors in the postnatal and adult subventricular zone and on analyzing the cellular and physiological properties of these cells in white and gray matter regions in the forebrain. In the present study, we examine the types of neural progeny generated by NG2 progenitors in the cerebellum by employing genetic fate mapping techniques using inducible Cre–Lox systems in vivo with two different mouse lines, the Plp-Cre-ER^T2/Rosa26-EYFP and Olig2-Cre-ER^T2/Rosa26-EYFP double-transgenic mice. Our data indicate that Olig2/Plp-positive NG2 cells display multipotential properties, primarily give rise to oligodendroglia but, surprisingly, also generate Bergmann glia, which are specialized glial cells in the cerebellum. The NG2+ cells also give rise to astrocytes, but not neurons. In addition, we show that glutamate signaling is involved in distinct NG2+ cell-fate/differentiation pathways and plays a role in the normal development of Bergmann glia. We also show an increase of cerebellar oligodendroglial lineage cells in response to hypoxic–ischemic injury, but the ability of NG2+ cells to give rise to Bergmann glia and astrocytes remains unchanged. Overall, our study reveals a novel Bergmann glia fate of Olig2/Plp-positive NG2 progenitors, demonstrates the differentiation of these progenitors into various functional glial cell types, and provides significant insights into the fate and function of Olig2/Plp-positive progenitor cells in health and disease.     

Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.