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    • To determine whether the inhibition

    2018-10-24

    To determine whether the inhibition of p38 MAPK in activated huSCs could maintain their proliferative potential and prevent expression of genes for myogenic differentiation, we grew the nmda receptor antagonist in the presence of a small molecule p38 inhibitor (p38i; SB 203580) and analyzed both their transcriptional and functional properties. Though various p38 inhibitors have been developed, SB 203580 was selected for this study because its targeting specificity has been studied extensively (Cuenda and Cohen, 1999; Davies et al., 2000; Eyers et al., 1998; Gum et al., 1998). Given previous observations of dose-dependent cell growth inhibition by p38 inhibitors in transformed muscle progenitor cell lines (Jones et al., 2005), we first studied the effects of varying doses of p38 inhibitor on PAX7 expression and cell proliferation in activated huSCs. PAX7 expression, which is correlated with the undifferentiated muscle progenitor fate (Rocheteau et al., 2012), exhibited a dose-dependent increase as the concentration of p38i was increased from 0 to 10 μM, but did not significantly change at concentrations beyond 10 μM (Figure S3A). We next examined the effect of varying concentrations of p38i on the rate of proliferation of undifferentiated cells by analyzing huSCs cultured with or without inhibitor for only 3 days post-isolation. Although concentrations of p38i up to 10 μM showed equivalent rates of EdU incorporation, when cells were cultured with 20 μM p38i, the frequency of EdU incorporation was reduced significantly (Figure S3B). Because 10 μM p38i maximally induced expression of the stem cell marker PAX7 without significantly limiting cell proliferation, this drug concentration was used for all subsequent studies of cell fate. To test for effective inhibition of p38 activity in SC cultures treated with 10 μM p38i, we quantified the amount of p38 target protein phosphorylation in control and p38i-treated cultures. These studies revealed marked decreases in the levels of phosphorylation of two p38 substrates, phopsho-HSP27 (Ser82) (Larsen et al., 1997) and phospho-GYS1 (Ser645) (Kuma et al., 2004), in the presence of p38i (Figures S3C–S3F). Consistent with a model in which p38 activity prevents myogenic lineage progression, p38i-treated cells expressed levels of MYOG, MYH3, and MYL2, for example, that were significantly reduced in comparison to untreated cells (Figure 3B). We observed similar suppression of MYOG expression by SB 203580 and an alternative, specific p38 inhibitor, SB 239063, further supporting the conclusion that pharmacologic manipulation of p38 in huSCs limits the expression of differentiation-associated genes (Figures S3G and S3H). Cells cultured in the presence of p38i also exhibited markedly diminished expression of MYOG and myosin heavy chain (MyHC) protein compared to controls (Figures S3I and S3J). Unbiased analysis of the set of genes that exhibited decreased expression due to p38i treatment revealed enrichment for genes associated with muscle contraction (GO:0006936; p = 3.7 × 10−15) and muscle organ development (p = 2.2 × 10−5). Although p38 inhibition blocked SC differentiation, we found that the expression of MYOD1 was not decreased in treated cells (Figure S3K), consistent with our findings in murine SCs of high levels of Myod1 expression in quiescent cells (Liu et al., 2013). The bulk of p38i-treated cultured huSCs also exhibited undiminished expression of the myogenic regulatory factor MYF5 relative to untreated cells (Figure S3L). Pharmacologic inhibition of p38 MAPK signaling thus maintained cultured huSCs in an undifferentiated state ex vivo. As a first test of the ability of p38 inhibition to maintain the proliferative potential of huSCs as undifferentiated cells, we measured the proliferation index of huSCs cultured in the presence or absence of p38i for 9 days by monitoring incorporation of EdU. While only 4.4% ± 0.4% of untreated cells incorporated EdU during a 1-hr pulse, 27% ± 2% of p38i-treated cells incorporated EdU (Figure 3C). Analysis of metaphase spreads of p38i-treated huSCs revealed normal chromosome number in 100% of cells after ten passages (Figure S3M). Consistent with the observation of increased EdU incorporation in p38i-treated huSCs after 9 days in culture, expression of genes associated with DNA replication and cell division, including MKI67, NDC80, AURKB, and RFC4, was increased in p38i-treated cells relative to untreated cells in the analyses of RNA-seq data (Figure S3N). Growth curves of p38i-treated huSCs maintained in culture for 3 weeks demonstrated preservation of exponential growth kinetics and massive expansion of the treated huSC cultures in comparison to parallel untreated control cultures from the same donor (Figure S3O). Thus, blocking huSC differentiation by inhibiting p38 maintains cells in an undifferentiated state in which they continued to proliferate without evidence of genetic instability.