Cell therapies treating pathological muscle atrophy or damage requires an adequate quantity of muscle progenitor cells (MPCs) not currently attainable from adult donors. Here, we generate cultures of approximately 90% skeletal myogenic cells by treating human embryonic stem cells (ESCs) with the GSK3 inhibitor CHIR99021 followed by FGF2 and N2 supplements. Gene expression analysis identified progressive expression of mesoderm, somite, dermomyotome, and myotome markers, following patterns of embryonic myogenesis. CHIR99021 enhanced transcript levels of the pan-mesoderm gene T and paraxial-mesoderm genes MSGN1 and TBX6; immunofluorescence confirmed that 91% ± 6% of cells expressed T immediately following treatment. By 7 weeks, 47% ± 3% of cells were MYH(+ve) myocytes/myotubes surrounded by a 43% ± 4% population of PAX7(+ve) MPCs, indicating 90% of cells had achieved myogenic identity without any cell sorting. Treatment of mouse ESCs with these factors resulted in similar enhancements of myogenesis. These studies establish a foundation for serum-free and chemically defined monolayer skeletal myogenesis of ESCs.

Class II histone deacetylases (HDAC4, HDAC5, HDAC7 and HDAC9) have been shown to interact with myocyte enhancer factors 2 (MEF2s) and play an important role in the repression of cardiac hypertrophy. We examined the role of HDACs during the differentiation of P19 embryonic carcinoma stem cells into cardiomyoctyes. Treatment of aggregated P19 cells with the HDAC inhibitor trichostatin A induced the entry of mesodermal cells into the cardiac muscle lineage, shown by the upregulation of transcripts Nkx2-5, MEF2C, GATA4 and cardiac α-actin. Furthermore, the overexpression of HDAC4 inhibited cardiomyogenesis, shown by the downregulation of cardiac muscle gene expression. Class II HDAC activity is inhibited through phosphorylation by Ca²⁺/calmodulin-dependent kinase (CaMK). Expression of an activated CaMKIV in P19 cells upregulated the expression of Nkx2-5, GATA4 and MEF2C, enhanced cardiac muscle development, and activated a MEF2-responsive promoter. Moreover, inhibition of CaMK signaling downregulated GATA4 expression. Finally, P19 cells constitutively expressing a dominant-negative form of MEF2C, capable of binding class II HDACs, underwent cardiomyogenesis more efficiently than control cells, implying the relief of an inhibitor. Our results suggest that HDAC activity regulates the specification of mesoderm cells into cardiomyoblasts by inhibiting the expression of GATA4 and Nkx2-5 in a stem cell model system.

Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies.

Background: Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation. Results: Treatment of P19 or mouse ES cells with low levels of RA led to an enhancement of skeletal myogenesis by upregulating the expression of the mesodermal marker, Wnt3a, the skeletal muscle progenitor factors Pax3 and Meox1, and the myogenic regulatory factors (MRFs) MyoD and myogenin. By chromatin immunoprecipitation, RA receptors (RARs) bound directly to regulatory regions in the Wnt3a, Pax3, and Meox1 genes and RA activated a β-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative β-catenin/engrailed repressor fusion protein, RA could not bypass the inhibition of skeletal myogenesis nor upregulate Meox1 or MyoD. Thus, RA functions both upstream and downstream of Wnt signalling. In contrast, it functions downstream of BMP4, as it abrogates BMP4 inhibition of myogenesis and Meox1, Pax3, and MyoD expression. Furthermore, RA downregulated BMP4 expression and upregulated the BMP4 inhibitor, Tob1. Finally, RA inhibited cardiomyogenesis but not in the presence of BMP4. Conclusion: RA can enhance skeletal myogenesis in stem cells at the muscle specification/progenitor stage by activating RARs bound directly to mesoderm and skeletal muscle progenitor genes, activating β-catenin function and inhibiting bone morphogenetic protein (BMP) signalling. Thus, a signalling pathway can function at multiple levels to positively regulate a developmental program and can function by abrogating inhibitory pathways. Finally, since RA enhances skeletal muscle progenitor formation, it will be a valuable tool for designing future stem cell therapies.

The inhibition of MyoD expression is important for obtaining muscle progenitors that can replenish the satellite cell niche during muscle repair. Progenitors could be derived from either embryonic stem cells or satellite cells. Hedgehog (Hh) signaling is important for MyoD expression during embryogenesis and adult muscle regeneration. To date, the mechanistic understanding of MyoD regulation by Hh signaling is unclear. Here, we demonstrate that the Hh effector, Gli2, regulates MyoD expression and associates with MyoD gene elements. Gain- and loss-of-function experiments in pluripotent P19 cells show that Gli2 activity is sufficient and required for efficient MyoD expression during skeletal myogenesis. Inhibition of Hh signaling reduces MyoD expression during satellite cell activation in vitro. In addition to regulating MyoD expression, Hh signaling regulates MyoD transcriptional activity, and MyoD activates Hh signaling in myogenic conversion assays. Finally, Gli2, MyoD, and MEF2C form a protein complex, which enhances MyoD activity on skeletal muscle-related promoters. We therefore link Hh signaling to the function and expression of MyoD protein during myogenesis in stem cells.

Sonic Hedgehog (Shh) is a critical signaling factor for a variety of developmental pathways during embryogenesis, including the specification of left-right asymmetry in the heart. Mice that lack Hedgehog signaling show a delay in the induction of cardiomyogenesis, as indicated by a delayed expression of Nkx2-5. To further examine a role for Shh in cardiomyogenesis, clonal populations of P19 cells that stably express Shh, termed P19(Shh) cells, were isolated. In monolayer P19(Shh) cultures the Shh pathway was functional as shown by the up-regulation of Ptc1 and Gli1 expression, but no cardiac muscle markers were activated. However, Shh expression induced cardiomyogenesis following cellular aggregation, resulting in the expression of factors expressed in cardiac muscle including GATA-4, MEF2C, and Nkx2-5. Furthermore, aggregated P19 cell lines expressing Gli2 or Meox1 also up-regulated the expression of cardiac muscle factors, leading to cardiomyogenesis. Meox1 up-regulated the expression of Gli1 and Gli2 and, thus, can modify the Shh signaling pathway. Finally, Shh, Gli2, and Meox1 all up-regulated BMP-4 expression, implying that activation of the Hedgehog pathway can regulate bone morphogenetic protein signals. Taken together, we propose a model in which Shh, functioning via Gli1/2, can specify mesodermal cells into the cardiac muscle lineage.