br Results br Discussion Recent advances enabling the prospe

Results
Discussion Recent advances enabling the prospective isolation of mouse satellite enzyme substrate have facilitated mechanistic analyses of their myogenic function. For example, the ability to clonally sort satellite cells with high purity made possible the demonstration that these cells can undergo asymmetric division (Kuang et al., 2007; Rocheteau et al., 2012) and repopulate the satellite cell niche in vivo (Cerletti et al., 2008). While findings in mouse models are often extrapolated to human biology, whether mouse and human myogenic cells exhibit fully equivalent properties may still be questioned, particularly given significant phenotypic discrepancies in several mouse models of human muscle disease (Bulfield et al., 1984). All of these issues can be addressed through the establishment of robust methods for direct purification of human muscle progenitors. Previous work by Pisani et al. demonstrated the utility of the sialomucin CD34 to enrich for myogenic cells within the CD34− subset of magnetically separated cells in adult muscle (Pisani et al., 2010b), consistent with immunohistochemical studies reporting the absence of CD34 in adult human muscle cells located in the satellite cell position (Lecourt et al., 2010). Pisani et al. also noted mixed myogenic and adipogenic activity within CD34+ adult muscle cells, which showed differential expression of CD56 (Pisani et al., 2010a). Findings from our study confirm that CD34 distinguishes myogenic and nonmyogenic cells within the nonhematopoietic, nonendothelial (CD45−CD11b−GlyA−CD31−) hMFA cell pool in both fetal and adult tissue: CD34+ cells are PAX7-negative, adipogenic cells that do not possess any myogenic activity, whereas within the CD45−CD11b−GlyA−CD31−CD34−/low subset, selection of CD56intITGA7hi cells yields a highly enriched population of PAX7-expressing, robustly myogenic progenitors. Yet, it is important to note that these FACS-based strategies pertain to cells isolated from fresh muscle only. Sorted cells may undergo marked changes in their surface marker profiles during ex vivo culture, and it is unclear if our protocols are applicable to cells that have undergone expansion/differentiation in culture. Fluorescence-activated cell-sorted fetal human CD34−CD56intITGA7hi cells engraft in mouse muscle to form new myofibers, albeit at low efficiency (Figures 5B–5F). Low-level engraftment of human cells into mouse tissue is not unexpected, as similar outcomes have been observed for other human, tissue-specific stem and progenitor cells upon transfer into immune-compromised mice (Doulatov et al., 2012; Racki et al., 2010). Unfortunately, given the relatively sparse presence of human myofibers in this system, we were unable to establish conditions to reliably detect costaining for PAX7 and human species-specific nuclear antigens on engrafted mouse muscle sections. We therefore were unable to determine if fluorescence-activated cell sorted fetal human CD34−CD56intITGA7hi cells can repopulate the PAX7-expressing satellite cell pool in vivo. We also were unable to test the in vivo engraftment potential of fluorescence-activated cell-sorted progenitors from adult human muscle, given the low yield of cells that could be obtained from adults (Figure 7E). Interestingly, enrichment of PAX7 expression and myogenic activity within the CD34− compartment in human muscle stands in contrast to immunophenotyping studies in mouse muscle, which localize myogenic activity to the CD34+ subset of mouse MFA cells (Beauchamp et al., 2000; Conboy et al., 2010; Montarras et al., 2005; Sacco et al., 2008; Sherwood et al., 2004). Species-specific differences in CD34 expression have also been noted in other somatic stem cell populations, including hematopoietic stem cells (HSCs), which are CD34+ in adult human bone marrow and CD34− in adult mouse bone marrow (Okuno et al., 2002; Osawa et al., 1996). Such differences appear to arise from the presence of species-specific upstream regulatory elements, which differentially regulate CD34 gene transcription in mouse and human cells (Okuno et al., 2002).