Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative β-catenin

Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
BMC Biology (Impact Factor: 7.98). 10/2009; 7(1):67. DOI: 10.1186/1741-7007-7-67
Source: PubMed


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.
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 beta-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative beta-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.
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 beta-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.

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    • "Furthermore, the first generation of differentiation protocols also relied on media supplemented with undefined serum, which led to variable differentiation efficiencies from one serum lot to the next. As a result, skeletal myocytes typically represented less than 10% of the total differentiated cells [18] [19]. "
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    ABSTRACT: 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 2 weeks 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.
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    • "It is worth noting that bexarotene has efficacies comparable to RA at converting the P19 stem cells into muscle lineage [46]. While RA may enhance skeletal myogenesis by expanding the progenitor population [53], bexarotene appears to affect germ layer fate determinations, and more specifically, promote mesoderm differentiation [46]. "
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    ABSTRACT: Pluripotent stem cells are able to differentiate into many types of cell lineages in response to differentiation cues. However, a pure population of lineage-specific cells is desirable for any potential clinical application. Therefore, induction of the pluripotent stem cells with lineage-specific regulatory signals, or small molecule inducers, is a prerequisite for effectively directing lineage specification for cell-based therapeutics. In this article, we provide in-depth analysis of recent research findings on small molecule inducers of the skeletal muscle lineage. We also provide perspectives on how different signaling pathways and chromatin dynamics converge to direct the differentiation of skeletal myocytes.
    Full-text · Article · Oct 2013 · Cell and Bioscience
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    • "As shown in figure 2B and C, DMSO induced about 8% of cardiac and 6% of skeletal myocytes in the P19 cells, respectively, as quantified by quantitative immunofluorescence microscopy, consistent with previous studies. Cotreatment of the EBs with RA enhanced the generation of skeletal myocytes, while attenuating cardiac differentiation (Fig. 2B and C), as previously reported32. Bexarotene enhances the specification of muscle lineage in a concentration dependent manner and the efficacies of bexarotene on the differentiation of P19 cells into skeletal myocytes is comparable to RA26. "
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    ABSTRACT: Cell-based therapies using pluripotent stem cells hold great promise as regenerative approaches to treat many types of diseases. Nevertheless many challenges remain and, perhaps foremost, is the issue of how to direct and enhance the specification and differentiation of a desired cell type for potential therapeutics. We have examined the molecular basis for the inverse correlation of cardiac and skeletal myogenesis in small molecule-enhanced stem cell differentiation. Our study shows that activation of premyogenic factor Pax3 coincides with inhibiting gene expression of early cardiac factor GATA4. Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1. Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification. Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.
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