GDC-0994 Although inhibition of p MAPK signaling prevented

Although inhibition of p38 MAPK signaling prevented spontaneous differentiation of huSCs ex vivo, this effect was reversible. After 2 weeks of expansion in the presence of p38i, the drug was chased for a period of 5 days, during which we observed a significant increase in MYOG expression (Figure S4A). After the 5-day chase, we also observed terminal differentiation of expanded GDC-0994 into multinucleate MyHC-expressing myotubes (Figure S4B). These data indicate that the inhibition of huSC differentiation by p38i is reversible and that cells expanded with p38i retain the ability to terminally differentiate. To test directly the self-renewal and tissue regenerative potential of expanded huSCs in vivo, we analyzed cell engraftment upon xenotransplantation into immunodeficient mice. As adult stem cells, huSCs are expected to yield progeny that either regenerate muscle tissue or replenish the pool of self-renewing stem cells. In transplantation studies, we compared the p38i-treated activated huSCs to two control cell populations as follows: (1) freshly isolated, uncultured quiescent huSCs; and (2) activated huSCs grown in the absence of p38i. We first tested huSC regenerative potential by analyzing the expression of human-specific nuclear lamin A/C (LMNA) and human-specific ITGB1 following transplantation of huSCs into tibialis anterior muscles. Expression of human/mouse laminin also was used to visualize gross muscle morphology. We observed robust engraftment of human LMNA-labeled nuclei that correlated with the expression of human ITGB1 in muscle fibers (Figures 4A and 4B; Figure S4C). The p38i-treated activated huSCs engrafted to form human ITGB1-expressing chimeric muscle fibers with approximately 4-fold and 11-fold greater efficiency than freshly isolated quiescent huSCs and control activated huSCs, respectively (221 ± 20 chimeric fibers per muscle cross-section from p38i-treated activated huSCs versus 49 ± 14 from quiescent huSCs and 21 ± 13 from untreated activated huSCs; p < 0.001 in separate comparisons of each control condition to the p38i-treated condition) (Figure 4A). Furthermore, human-mouse chimeric muscle fibers formed by the engraftment of p38i-expanded huSCs expressed human dystrophin (DMD) (Figure S4D). We also analyzed expanded huSC engraftment by studying fusion of huSCs engineered ex vivo by adenoviral infection to express a GFP transgene, giving rise to chimeric GFP-expressing muscle fibers in transplanted NSG mice (Figures S4E and S4F). These analyses demonstrate as a proof-of-concept that the prospectively isolated and expanded cells are genetically manipulable and capable of expressing donor-derived DMD in chimeric fibers. To study the potential of transplanted huSCs to manifest a defining stem cell property by undergoing self-renewal to yield PAX7-expressing huSCs in vivo, we first examined the expression of PAX7 in nuclei of unfused human cells in recipient muscles. In this analysis, we observed PAX7-expressing nuclei of human origin that resided beneath the basal lamina (Figure 4B; Figures S4G and S4H). To compare quantitatively the potential of freshly isolated quiescent huSCs, control activated huSCs, and p38i-treated activated huSCs to engraft as mononuclear stem cells, we analyzed the abundance of unfused, donor human cells by flow cytometry. We initially confirmed that unfused donor cells expressed PAX7 as evidence that they had formed renewed huSCs (Figures S4I and S4J). Comparable to the analysis of fusion as an index of donor cell engraftment, p38i-treated activated huSCs had approximately 3.6-fold and 7.6-fold greater potential for engraftment as mononuclear SCs than quiescent huSCs and control activated huSCs, respectively (Figure 4C). Histological quantification of the number of human-derived, PAX7-expressing nuclei in cross-sections of transplanted muscle indicated a similar enhancement of engraftment by p38i-treated activated huSCs versus both quiescent huSCs and untreated activated huSCs. We, therefore, conclude that huSCs expanded in culture via p38 inhibition are capable of engrafting both as constituents of multinucleate muscle fibers and as self-renewed mononuclear huSCs.