Cell surface markers that have proved

Cell surface markers that have proved useful for the isolation of mouse PAX7+ satellite ion channel include ITGA7 (Pasut et al., 2012; Sacco et al., 2008), CXCR4 and β1 INTEGRIN (Sherwood et al., 2004), and VCAM1 (Chakkalakal et al., 2012). Our studies indicate that PAX7-expressing cells in fetal and adult human muscle coexpress ITGA7 and CD56, although neither of these markers alone is sufficient to distinguish these cells. This is consistent with ITGA7 and CD56 expression by human myogenic progenitors derived from PAX7-expressing induced pluripotent stem cells (Darabi et al., 2012). Clear expression of CXCR4 was observed in adult and fetal PAX7-expressing cells (Figure S6A), and β1-INTEGRIN was detected on 90% of hMFA cells (Figure 1B). Additional surface marker analyses, focusing particularly on those previously linked to mouse and/or human myogenic precursors (Cerletti et al., 2012; Darabi et al., 2012; Lecourt et al., 2010; Zheng et al., 2007), revealed increased expression of MCAM (CD146) and CD44 in human fetal CD34−CD56intITGA7hi cells as compared to CD34+ cells (Figure S6B). Finally, our microarray analyses suggest that PROMININ1 (CD133) and PDGFRA (CD140a; previously reported to mark adipocyte precursors; Berry and Rodeheffer, 2013) could also be useful in distinguishing human myogenic progenitors, as they are differentially expressed in fetal CD34−CD56intITGA7hi cells and CD34+ cells (Figure S6C; Table S6). MFA cells obtained from mouse and human muscle were previously shown to exhibit osteogenic activity (Glass et al., 2011; Hashimoto et al., 2008; Lecourt et al., 2010; Oishi et al., 2013). In our studies, both fetal and adult PAX7-expressing CD34−CD56intITGA7hi cells formed AR-positive calcium deposits and expressed osteogenic lineage genes under osteogenic conditions, consistent with prior reports of osteogenic differentiation potential within the pool of muscle cells containing PAX7+ progenitors (Hashimoto et al., 2008; Ozeki et al., 2006). Clonal assays revealed that the osteogenic activity of human fetal CD34−CD56intITGA7hi hMFA cells is unlikely to be attributable to contamination by other cells, as the majority of clone-sorted cells exhibited bipotent myogenic and osteogenic activity. Future studies are needed to investigate adult muscle progenitor bipotency and delineate the events that trigger possible osteogenic differentiation of human CD34−CD56intITGA7hi cells, their possible contributions to normal bone regeneration, and their relationship to other mesenchymal precursor cells. In contrast to fetal CD34−CD56intITGA7hi cells, which exhibit robust myogenic activity and lack adipogenic potential, human fetal CD34+ cells are adipogenic and lack myogenic capacity. While our studies evaluated the ability of these cells to form white adipocytes, a previous report indicates that CD34+ cells in fetal and adult human muscle contain brown adipogenic activity as well (Crisan et al., 2008). Gene expression profiling confirms profound differences in the transcriptional signatures of fetal human CD34−CD56intITGA7hi and CD34+ hMFA cells, with increased expression of muscle lineage genes in CD34−CD56intITGA7hi cells and increased expression of adipogenic genes in CD34+ cells. Given their differentiation profile, we speculate that CD34+ hMFA cells may represent the human counterparts of the fibroadipogenic precursor (FAP) population in mouse muscle (Joe et al., 2010; Uezumi et al., 2010), an important subset of nonmyogenic cells that appears to enhance muscle regenerative capacity (Joe et al., 2010). In summary, we report functionally distinct cell populations within the hMFA cell pool in fetal and adult muscle and provide a specific method for the prospective isolation of purified PAX7+ cells from human muscle. We anticipate that this technology will facilitate novel insights into human muscle homeostasis, aging, and disease. Phenotypically distinct cells with myogenic progenitor function also represent promising targets for muscle regenerative cell therapy and could conceivably be used to treat a variety of diseases, including muscular dystrophy and muscle injuries.