Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres.
ABSTRACT: Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for "disease in a dish" models of muscular physiology and dysfunction.
Project description:Skeletal myogenesis is a complex process that is finely regulated by myogenic transcription factors. Recent studies have shown that saturated fatty acids (SFA) can suppress the activation of myogenic transcription factors and impair the myogenic differentiation of progenitor cells. Despite the increasing evidence of the roles of miRNAs in myogenesis, the targets and myogenic regulatory mechanisms of miRNAs are largely unknown, particularly when myogenesis is dysregulated by SFA deposition. This study examined the implications of SFA-induced miR-183-5p on the myogenic differentiation in C2C12 myoblasts. Long-chain SFA palmitic acid (PA) drastically reduced myogenic transcription factors, such as myoblast determination protein (MyoD), myogenin (MyoG), and myocyte enhancer factor 2C (MEF2C), and inhibited FHL1 expression and myogenic differentiation of C2C12 myoblasts, accompanied by the induction of miR-183-5p. The knockdown of FHL1 by siRNA inhibited myogenic differentiation of myoblasts. Interestingly, miR-183-5p inversely regulated the expression of FHL1, a crucial regulator of skeletal myogenesis, by targeting the 3'UTR of FHL1 mRNA. Furthermore, the transfection of miR-183-5p mimic suppressed the expression of MyoD, MyoG, MEF2C, and MyHC, and impaired the differentiation and myotube formation of myoblasts. Overall, this study highlights the role of miR-183-5p in myogenic differentiation through FHL1 repression and suggests a novel miRNA-mediated mechanism for myogenesis in a background of obesity. [BMB Reports 2020; 53(11): 605-610].
Project description:Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-? (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.
Project description:Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis.
Project description:Long non-coding RNAs (lncRNAs) are important regulators of diverse biological processes. Here we report on functional identification and characterization of a novel long intergenic non-coding RNA with MyoD-regulated and skeletal muscle-restricted expression that promotes the activation of the myogenic program, and is therefore termed Linc-RAM (Linc-RNA Activator of Myogenesis). Linc-RAM is transcribed from an intergenic region of myogenic cells and its expression is upregulated during myogenesis. Notably, in vivo functional studies show that Linc-RAM knockout mice display impaired muscle regeneration due to the differentiation defect of satellite cells. Mechanistically, Linc-RAM regulates expression of myogenic genes by directly binding MyoD, which in turn promotes the assembly of the MyoD-Baf60c-Brg1 complex on the regulatory elements of target genes. Collectively, our findings reveal the functional role and molecular mechanism of a lineage-specific Linc-RAM as a regulatory lncRNA required for tissues-specific chromatin remodelling and gene expression.
Project description:The effectiveness of cell-based therapy to treat muscle disease has been hampered by difficulties in isolating, maintaining and propagating the stem cells that are needed for treatment. Here we report the isolation of muscle-derived stem cells from both young and old mice and their propagation over extended periods of time in culture as "free-floating" myospheres. Analysis of these sphere-forming cells showed that they express stem cell antigen-1 (Sca-1), beta1 integrin (CD29), Thy-1 (CD90), and CD34, but did not express CD45, CD31, or myogenic markers (Pax7, Myf5, and MyoD). We found that cells derived from myospheres and then grown adherently (MDACs) behaved similar to primary myoblasts, in that these cells expressed myogenic markers and were able to easily form multinucleated myotubes. Unlike the parental myospheres but analogous to primary myoblasts, MDACs expressed Pax7, Myf5, and MyoD, indicating that the parent myosphere cells were a more primitive type of cell. In support of this we demonstrated that myospheres were also able to differentiate into adipogenic and osteogenic cells in culture, as well as being able to contribute to injured muscle in vivo. In summary, we report that primitive adult muscle stem cells can be easily isolated and sustained in culture as myospheres.
Project description:The ability of human embryonic stem cells (hESCs) to differentiate into skeletal muscle cells is an important criterion in using them as a cell source to ameliorate skeletal muscle impairments. However, differentiation of hESCs into skeletal muscle cells still remains a challenge, often requiring introduction of transgenes. Here, we describe the use of WNT3A protein to promote in vitro myogenic commitment of hESC-derived cells and their subsequent in vivo function. Our findings show that the presence of WNT3A in culture medium significantly promotes myogenic commitment of hESC-derived progenitors expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), as evident from the expression of myogenic markers, including DES, MYOG, MYH1, and MF20. In vivo transplantation of these committed cells into cardiotoxin-injured skeletal muscles of NOD/SCID mice reveals survival and engraftment of the donor cells. The cells contributed to the regeneration of damaged muscle fibers and the satellite cell compartment. In lieu of the limited cell source for treating skeletal muscle defects, the hESC-derived PDGFRA(+) cells exhibit significant in vitro expansion while maintaining their myogenic potential. The results described in this study provide a proof-of-principle that myogenic progenitor cells with in vivo engraftment potential can be derived from hESCs without genetic manipulation.
Project description:Members of the Hey family of transcriptional repressors are basic helix-loop-helix proteins that are thought to act downstream of Notch in diverse tissues. Although forced expression of Hey1, a target of Notch in myoblasts, is sufficient to recapitulate inhibitory effects of the pathway on differentiation, how Hey1 interferes with myogenic transcription has not been fully elucidated. We provide multiple lines of evidence that Hey1 does not target the intrinsic transcriptional activity of the skeletal muscle master regulator MyoD. Our results indicate instead that Hey1 is recruited to the promoter regions of myogenin and Mef2C, two genes whose induction is critical for myogenesis. Expression of Hey1 in C2C12 myoblasts correlates with reduced recruitment of MyoD to these promoters, arguing that Hey1 inhibits myogenesis by associating with and repressing expression of key myogenic targets.
Project description:The coordinated expression of myogenic regulatory factors, including MyoD and myogenin, orchestrates the steps of skeletal muscle development, from myoblast proliferation and cell-cycle exit, to myoblast fusion and myotubes maturation. Yet, it remains unclear how key transcription factors and epigenetic enzymes cooperate to guide myogenic differentiation. Proteins of the SMYD (SET and MYND domain-containing) methyltransferase family participate in cardiac and skeletal myogenesis during development in zebrafish, Drosophila and mice. Here, we show that the mammalian SMYD3 methyltransferase coordinates skeletal muscle differentiation in vitro. Overexpression of SMYD3 in myoblasts promoted muscle differentiation and myoblasts fusion. Conversely, silencing of endogenous SMYD3 or its pharmacological inhibition impaired muscle differentiation. Genome-wide transcriptomic analysis of murine myoblasts, with silenced or overexpressed SMYD3, revealed that SMYD3 impacts skeletal muscle differentiation by targeting the key muscle regulatory factor myogenin. The role of SMYD3 in the regulation of skeletal muscle differentiation and myotube formation, partially via the myogenin transcriptional network, highlights the importance of methyltransferases in mammalian myogenesis.
Project description:Nuclear acetyltransferases promote and deacetylases inhibit skeletal muscle-gene expression, suggesting the potential effectiveness of deacetylase inhibitors (DIs) in modulating skeletal myogenesis. Surprisingly, previous studies have indicated that DIs suppress myogenesis. The recent observations that histone deacetylases associate with the muscle-regulatory proteins MyoD and MEF2C only in undifferentiated myoblasts prompted us to evaluate the effect of DIs at distinct stages of the myogenic program. We found that exposure of established rodent and human muscle cells to distinct DIs has stage-specific effects. Exposure of undifferentiated skeletal myoblasts to DIs, followed by incubation in differentiation medium, enhanced the expression of muscle-specific reporters and increased the levels of endogenous muscle proteins, leading to a dramatic increase in the formation of multinucleated myotubes. By contrast, simultaneous exposure of muscle cells to differentiation medium and DIs inhibited the myogenic program. Likewise, embryos exposed in utero to nonteratogenic doses of DI at the early stages of somitic myogenesis (embryonic day 8.5) exhibited an increased number of somites and augmented expression of a muscle-specific transgene as well as endogenous muscle genes. The functional effects induced by DIs were mirrored by changes in the state of acetylation of histones present at a muscle-gene enhancer and of MyoD itself. These results represent the first evidence that DIs can enhance muscle differentiation and suggest the rationale for their use in manipulating adult and embryonic skeletal myogenesis.
Project description:Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. The proliferation and differentiation of muscle precursor cells require the concerted activity of myogenic regulatory factors including MyoD. In addition, chromatin modifiers mediate dynamic modifications of histone tails that are vital to reprogramming cells toward terminal differentiation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. We demonstrate that the lysine methyltransferase G9a is dynamically expressed in myoblasts and impedes differentiation in a methyltransferase activity-dependent manner. In addition to mediating histone H3 lysine-9 di-methylation (H3K9me2) on MyoD target promoters, endogenous G9a interacts with MyoD in precursor cells and directly methylates it at lysine 104 (K104) to constrain its transcriptional activity. Mutation of K104 renders MyoD refractory to inhibition by G9a and enhances its myogenic activity. Interestingly, MyoD methylation is critical for G9a-mediated inhibition of myogenesis. These findings provide evidence of an unanticipated role for methyltransferases in cellular differentiation states by direct posttranslational modification of a transcription factor.