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    • It has previously been shown that the GSK

    2018-11-06

    It has previously been shown that the GSK3 inhibitor CHIR99021 (CHIR) can augment mesoderm induction (Tan et al., 2013), leading to cardiomyogenesis in ESCs (Lian et al., 2012). Recombinant proteins BMP4 and ACTIVIN-A (INHBA) have similarly been used to induce mesoderm and Entinostat from ESCs (Kattman et al., 2011; Murry and Keller, 2008). These studies implicate BMP4/INHBA or CHIR treatment as a potential method for generating skeletal muscle. Furthermore, we have shown that overexpression of WNT3A or CTNNB1 enhances the formation of premyogenic mesoderm in P19 embryonal carcinoma cells, resulting in increased myogenesis (Petropoulos and Skerjanc, 2002). The loss of CTNNB1 function via dominant-negative mutation or knockdown results in the loss of MPC formation and myogenesis, supporting the use of CHIR to induce myogenesis. The PAX3/7 population that is present in the central dermomyotome appears to represent an MPC pool that is maintained throughout embryogenesis and is responsible for almost all skeletal muscle (Buckingham, 2007; Kuang et al., 2007). FGF2 prevents expression of the myoblast commitment transcription factors MYF5, MYOD1, and MYOG—collectively known as the myogenic regulatory factors (MRF)—during satellite cell activation and thus can be utilized to enhance proliferation of PAX3/7 expressing MPCs in vitro (Fedorov et al., 1998; Hall et al., 2010). Satellite cells are more efficient in reconstituting the satellite cell niche during transplantation into muscle if they do not yet upregulate the MRFs (Kuang et al., 2007; Montarras et al., 2005). We have previously shown that N2 supplemented media can enhance the terminal differentiation of myoblasts and myocytes, which would ensure MPCs are capable of complete myogenesis in vitro (Al Madhoun et al., 2011; Ryan et al., 2012). In this report, we describe the robust skeletal myogenesis of hESCs and mESCs using CHIR to induce mesoderm, FGF2 treatment to expand the MPC population, and N2-mediated terminal differentiation. This chemically defined, serum- and transgenic-free protocol yields a nearly homogeneous myogenic population from hESCs, consisting of 43% ± 4% PAX7+ve MPCs and 47% ± 3% Myosin Heavy Chain+ve (MYH) skeletal muscle.
    Results
    Discussion Although forced expression of PAX3/7 or MYOD in hESCs have resulted in enriched myogenic populations (Albini et al., 2013; Darabi et al., 2012; Goudenege et al., 2012; Rao et al., 2012), we sought to develop a viral- and transgenic- free method of in vitro differentiation. A chemically defined protocol should be more readily adaptable to a broader number of cell lines and should allow for the examination of all of the stages of differentiation, some of which may be bypassed by transgenic overexpression (Albini et al., 2013). Our study contrasts with previously published methods of transgene-free hESC myogenesis that were dependent upon serum, showing low overall myogenic induction and/or myogenic progenitor formation (Awaya et al., 2012; Hwang et al., 2013; Ryan et al., 2012). For example, we previously obtained a 4% level of MYH+ve skeletal myocytes and 7% PAX3+ve MPCs with RA treatment (Ryan et al., 2012). Our current CHIR-directed protocol improves on the serum/EB methods by directing a larger proportion of the cultures into mesoderm, leading to enriched myogenic lineage populations. Several studies have shown that Wnt signaling modulation by CHIR can maintain ESC pluripotency (Wu et al., 2013), and also can cause differentiation into tissues of all three germ layers (Denham et al., 2012; Lian et al., 2012; Tahamtani et al., 2013). However, supporting factors were required immediately following CHIR treatment for these approaches. Cell seeding density may also determine how GSK3 inhibition can generate skeletal muscle specific mesoderm versus other cell fates. Seeding density was critical first and foremost in determining how hESCs responded to external differentiation cues in our system (R.L.C., S-P.D., and W.L.S., unpublished data). At lower seeding densities (<1 × 105 cells/ml), hESC survival was reduced even in the presence of ROCK inhibitor, suggesting that cell-cell contacts are important for the initial seeding process. Additionally, sparse populations of cells responded to BMP4/INHBA treatment with increased cell death compared to higher densities. Furthermore, at higher seeding densities (>2 × 105 cells/ml), overconfluence was observed after 48 hr, again leading to compromised cell viability. Other monolayer approaches have used GSK3 to drive cardiomyogenesis; however, these protocols induce GSK3 inhibition once cells reach confluence or with supplemental BMP4 inhibition (Cao et al., 2013; Lian et al., 2012). These protocols also emphasize the critical importance of insulin depletion after GSK3 inhibition, or the need for Wnt inhibition following mesoderm induction lest cardiomyogenesis will not occur in vitro (Kattman et al., 2011; Lian et al., 2013). This is supported by the role of Dkk-mediated Wnt inhibition in specifying embryonic mesoderm to the primary heart field (Ueno et al., 2007).