These studies add to a growing body of

These studies add to a growing body of information related to the role of p38 MAPK signaling in skeletal muscle progenitor cells. Over a decade ago, a requirement for p38 in the regulation of rodent myoblast differentiation was defined (Cuenda and Cohen, 1999; Zetser et al., 1999). Based on these prior studies, the functions of p38 MAPK are clearly diverse and complex. p38 activation correlates with rodent SC activation, and pharmacologic inhibition of p38α/β can promote muscle progenitor cell-cycle exit (Jones et al., 2005). Intriguingly, and somewhat paradoxically, genetic absence of p38α, the dominant p38 isoform regulating the myogenic program in mice (Lovett et al., 2010), enhances proliferative expansion of the murine SC population postnatally and in response to injury, although p38α is not absolutely required for myogenic differentiation in vivo (Brien et al., 2013). That the p38α isoform is not required for differentiation may be due to redundant functions of p38β or ɣ isoforms, both of which are expressed in muscle and are capable of independently regulating muscle progenitor differentiation (Wang et al., 2008). Our studies suggest that, in humans, there is a switch that occurs in the activation of p38 as SCs are activated in culture, akin to the activation of p38 observed in mouse (Jones et al., 2005), going from an inactive unphosphorylated form in quiescent huSCs to an active phosphorylated form in cultured huSCs. Altogether, our data and previously published studies of p38 in rodent muscle progentiors support a model in which inhibition of p38 activity promotes exponential growth of progenitors by maintaining an undifferentiated fate. This model seems to be conserved between mice and humans. We further demonstrate that pharmacologic manipulation of p38 signaling can be leveraged for enhancement of both ex vivo expansion and subsequent in vivo engraftment of huSCs. In our investigation, transplantation of huSCs expanded in the presence of p38i, generated more chimeric muscle fibers, and self-renewed huSCs in comparison to freshly isolated cells. What might account for the enhanced regenerative potential of expanded huSCs? One possibility is that the activated state of expanded LY2584702 relative to quiescent cells enabled the expanded cells to proliferate more during the course of muscle regeneration. In previous studies of cultured mouse SCs, this phenomenon may not have manifested as efficient engraftment because activation coincided with differentiation. By divorcing SC activation from differentiation, p38 inhibition may have revealed an increased engraftment potential of activated relative to quiescent cells. This possibility is particularly interesting given that we observed slower activation kinetics in purified huSCs relative to what has been reported for their mouse counterparts (Rodgers et al., 2014). An alternative explanation for the enhanced regenerative potential of the expanded huSCs is that p38 inhibition induces an epigenetic change in the cultured cells that promotes their engraftment potential. This hypothesis may relate to recent observations in aforementioned studies of mouse SCs, which showed an improved function in aged mouse SCs following treatment with a p38 inhibitor (Bernet et al., 2014; Cosgrove et al., 2014). The huSCs used in our study were isolated from donors with an age that greatly exceeds that of even aged mice. It is, therefore, plausible that human adult SCs exhibit a favorable response to treatment with p38i analogous to that observed in SCs from aged mice. Although the mechanisms of this epigenetic restoration of SC function have not been elucidated, our analysis of the gene expression changes stimulated by p38 inhibition identified several intriguing candidate regulators, including ENG, CRYAB, and SERPINE1. Our current understanding of the functions of these proteins suggests a plausible role for each in the aging of huSCs. ENG encodes endoglin, a membrane glycoprotein important in TGF-β signal transduction (Cheifetz et al., 1992). Increased expression of endoglin has been associated with aging phenotypes within endothelial and myeloid lineages (Aristorena et al., 2014; Blanco and Bernabeu, 2011), and excessive TGF-β signaling has been linked to impaired self-renewal of mouse SCs in the context of aging and chronic disease (Biressi et al., 2014; Carlson et al., 2008). Alpha-crystallin B chain (CRYAB) is a small heat shock protein, decreased expression of which has been linked to improved outcomes following cardiac ischemia in aged mice (Benjamin et al., 2007). SERPINE1, also known as plasminogen activator inhibitor-1 (PAI-1), is upregulated in senescent human cells and in various tissues of progeroid mouse models, including skeletal muscle (Baker et al., 2011; Mu and Higgins, 1995). Remarkably, genetic or pharmacologic inhibition of PAI-1 has been shown to improve organ function and extend the lifespan of progeroid mice (Eren et al., 2014). Future studies will be important to define the specific role of these genes during aging and muscle regeneration as regulators of huSCs downstream of p38 MAPK signaling.