Assessment of myogenic maturation for human iPSC-derived iPAX7 myogenic progenitors in vitro and in vivo
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE244521 | GEO | 2024/04/02
REPOSITORIES: GEO
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Project description:Although skeletal muscle cells can be generated from human iPSCs, transgene-free protocols include only limited options for their purification and expansion. In this study we found that FACS-purified myogenic progenitors generated from healthy controls and Pompe disease iPSCs can be robustly expanded as much as 5 x 1011 fold. At all steps during expansion, cells could be cryopreserved or differentiated into myotubes with a high fusion index. In vitro, cells were amenable to maturation into striated and contractile myofibers. Insertion of the acid alpha glucosidase cDNA into the AAVS1 locus in iPSCs using CRISPR/cas9 prevented glycogen accumulation in myotubes generated from a patient with classic infantile Pompe disease. In vivo, the expression of human-specific nuclear and sarcolemmar antigens indicated that myogenic progenitors engraft into murine muscle to form human myofibers. This protocol is useful for modeling of skeletal muscle disorders and for using patient-derived, gene-corrected cells to develop cell-based strategies.
2018-05-03 | GSE111163 | GEO
Project description:Numerous studies have been conducted to improve the supply of myogenic stem cells for patients with muscle disease, including Duchenne muscular dystrophy. Collecting muscle samples from patients in order to obtain myoblasts or satellite cells is very invasive. Thus, a new method for obtaining myogenic stem cells is required. Here, we established stably expandable induced myogenic stem cells (iMSCs) with defined factors. The iMSCs showed high expression levels of Pax7, Myf5, and MyoD. Also, the iMSCs differentiate into myotubes which are multinucleated fiber. Additionally, the iMSCs differentiated into myotubes, which are multinucleated fibers. We confirmed its myogenic differentiation capacity in mdx mice by detecting dystrophin-positive cells in iMSCs-injected TA muscles. The iMSCs showed higher proliferation capacity than MDSCs in both in vitro and vivo. This study suggests the possibility to apply directly converted expandable myogenic stem cells to muscle disease patients.
2018-11-06 | GSE94506 | GEO
Project description:Skeletal muscle is a highly organized and regenerative tissue that maintains its homeostasis primarily by activation and differentiation of muscle stem cells. Mimicking an in vitro skeletal muscle differentiation program that contains self-renewing adult muscle stem cells and aligned myotubes has been challenging. Here, we set out to engineer a biomimetic skeletal muscle construct that can self-regenerate and produce aligned myotubes using induced myogenic progenitor cells (iMPCs), a heterogeneous culture consisting of skeletal muscle stem, progenitor and differentiated cells. Utilizing electrospinning, we fabricated polycaprolactone (PCL) substrates that enabled iMPC-differentiation into aligned myotubes by controlling PCL fiber orientation. Newly-conceived constructs contained highly organized multinucleated myotubes in conjunction with self-renewing muscle stem cells, whose differentiation capacity was augmented by Matrigel supplementation. Additionally, we demonstrate using single cell RNA sequencing (scRNA-seq) that iMPC-derived constructs faithfully recapitulate a step-wise myogenic differentiation program. Notably, when the constructs were subjected to a damaging myonecrotic agent, self-renewing muscle stem cells rapidly differentiated into aligned myotubes, akin to skeletal muscle repair in vivo. Taken together, we report on a novel in vitro system that mirrors myogenic regeneration and muscle fiber alignment, and can serve as a platform to study myogenesis, model muscular dystrophies or perform drug screens.
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Project description:Satellite cells (SC) are muscle stem cells which can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo. Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.
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Project description:The ability to recapitulate muscle differentiation in vitro has proven invaluable to investigate mechanisms of myogenesis, muscle cell function and muscle diseases. However, obtaining myoblasts from patients with neuromuscular diseases poses ethical and procedural challenges which limit investigations of molecular mechanisms of muscle pathophysiology. Alternative myogenic models have been developed, such as the derivation of myogenic cells from skin fibroblasts through activation of an endogenous myogenic program triggered by exogenous expression of murine Myod. In the context of this RNA-seq dataset, we compare the transcriptome of myo-converted human fibroblasts and isogenic in vitro differentiated myoblasts. We show that myogenic induction of fibroblasts elicits genome-wide transcriptomic changes indicative of strong myogenic commitment and differentiation, including marked upregulation of genes implicated in cell cycle exit and in several myogenic pathways. Yet, we find that myotubes are further along myogenic commitment than myo-converted fibroblasts under the conditions tested. Extension of myo-conversion to 7 days does not significantly enhance myogenic commitment, from a gene expression standpoint. This suggests that myo-converted fibroblasts and myotubes retain some cell type specificity of gene expression profiles. Although these myogenic cell types are not identical, our results nonetheless favor a view of myo-converted fibroblasts as a robust and practical model to investigate cellular and genomic properties of cells from patients with muscle pathologies.
2023-08-21 | GSE236118 | GEO
Project description:Satellite cells (SCs), the skeletal muscle stem cells, display functional heterogeneity linked to their regenerative potential. Dramatic changes in their behaviour are also associated to muscle wasting diseases. Therefore, understanding the mechanisms controlling satellite cell heterogeneous behaviour during muscle regeneration and diseases has become increasingly important for regenerative medicine. Here, we show that miR-106bis expressed in Myf5- quiescent SCs and downregulated during SC activation and muscle regeneration. In vitro and in vivo studies to show that miR106b inhibition enhances the regenerative capability of murine and human SCs by increasing myogenic precursors. miR106b is increased in dystrophic mice and intramuscular injection of antimiR enhances muscle regeneration accompanied by significant functional recovery. miR-106b is also elevated in human dystrophic SCs and its inhibition improves intrinsic proliferative defects, increasing their myogenic differentiation potential. Therefore, this study identifies miR-106b as compelling candidate to consider for potential clinical studies.
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Project description:The ability to recapitulate muscle differentiation in vitro has proven invaluable to investigate mechanisms of myogenesis, muscle cell function and muscle diseases. However, obtaining myoblasts from patients with neuromuscular diseases poses ethical and procedural challenges which limit investigations of molecular mechanisms of muscle pathophysiology. Alternative myogenic models have been developed, such as the derivation of myogenic cells from skin fibroblasts through activation of an endogenous myogenic program triggered by exogenous expression of murine Myod. In the context of this ChIP-seq dataset, we compared the transcriptome and lamina-associated domains (LADs) as genome organizers in myo-converted human fibroblasts and in isogenic myotubes differentiated from myoblasts. We show that myogenic induction of fibroblasts elicits genome-wide transcriptomic changes indicative of myogenic commitment and differentiation. Yet, myotubes are further along myogenic commitment than myo-converted fibroblasts under the conditions tested. LADs with typical LAD properties (low gene density, containing mostly repressed genes, and H3K9me3-rich) are shared between the two cell types, but each cell type also displays nearly 700 unique LADs. Strikingly, myotube-specific LADs are smaller, more gene-rich and less heterochromatic than shared LADs or LADs unique to myo-converted fibroblasts. Thus, myo-converted fibroblasts and myotubes retain some cell type specificity of genome organization at this level. Although these myogenic cell types are not identical, our results favor a view of myo-converted fibroblasts as a robust and practical model to investigate cellular and genomic properties of cells from patients with muscle pathologies.
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Project description:We generated the human ES lines, in which the genome-wide reduction of H3K27me3 can be induced by the ectopic expression of catalytic domain of histone demethylase JMJD3 with doxycycline treatment (JMJD3c-hESCs). The overexpression of JMJD3c enhances MYOD1-mediated myogenic differentiation of hESCs. We compared the gene expression patterns of the generated myogenic cells with those of human skeletal myotubes.
2016-10-18 | GSE80106 | GEO
Project description:Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 µM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the upregulation of muscle genes at the myoblast stage while at later stages nearly 50 % of the 5AC-treated myotubes displayed a mature, well-defined sarcomere organization as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 µM ryanodine and 100 µM nifedipine, suggesting that action potential-induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity. C2C12 cells were plated at 1500 cells/cm2 (day -1), cultured for 1 day in DMEM 10%NCS, then treated (d0) with or without 10uM 5-azacytidine (5AC) for an additional 3 days. RNA was extracted on day 3 and hybridized to GeneChip Mouse Genome 430 2.0 array (Affymetrix).
2012-01-01 | E-GEOD-30192 | biostudies-arrayexpress
Project description:In this study, Affymetrix MG_U74Av2 and MG_U74Cv2 GeneChips were used to analyze gene expression over a 12 day time course in C2C12 myoblasts induced to differentiate in vitro. Triplicate cultures were studied during cell proliferation (days -2 and -1), at cell cycle withdrawal (day 0) and during myogenic fusion and maturation of multinucleated myotubes (days 2, 4, 6, 8, 10). The manuscript associated to this work is Tomczak et al., FASEB J., 2003 Dec 19. Additionally, the supplementary data can be found on our homepage at http://www.chb-genomics.org/beggslab/ Keywords = C2C12 myoblasts Keywords = myogenesis Keywords: time-course
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