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  • Our limited understanding of huSC biology can be attributed

    2018-10-24

    Our limited understanding of huSC biology can be attributed in part to the difficulty of obtaining tissue samples and of isolating a pure population of huSCs (Boldrin et al., 2010). By comparison, techniques for the prospective isolation of mouse SCs have identified a well-defined myogenic SC population and have enabled extensive characterization of the molecular regulation of their quiescence, activation, and differentiation (Bosnakovski et al., 2008; Collins et al., 2005; Fukada et al., 2007; Liu et al., 2013; Montarras et al., 2005; Sherwood et al., 2004). Previous studies have used prospective isolation of mononuclear cell types with distinct surface protein expression to define subsets of myogenic and non-myogenic Pirfenidone in human muscle. These studies have found that a myogenic population resides within cells expressing CD56 (NCAM) (Bareja et al., 2014; Pisani et al., 2010a, 2010b; Zheng et al., 2007). Zheng et al. have identified both CD56+CD34− and CD56+CD34+ subsets with myogenic potential. The CD56+CD34+ subset is thought to represent a Pirfenidone myoendothelial population with the capacity to differentiate into myogenic, chondrogenic, or osteogenic lineages (Zheng et al., 2007). A similar study of myogenic potential within muscle-resident human cell populations showed that a CD56+CD34+ population of bipotent progenitors can give rise to both myogenic and adipogenic cell types in vitro (Pisani et al., 2010a). In a second study, the same authors confirmed that both CD56+CD34+ and CD56+CD34− cells have myogenic potential, but only the latter is restricted to a myogenic fate (Pisani et al., 2010b). More recently, Bareja et al. (2014) used CD34 as a negative selection marker for identification of a myogenic huSC within the CD56+ population.
    Results As the starting point of this study, we prospectively isolated huSCs from surgical specimens of skeletal muscle using fluorescence-activated cell sorting (FACS) to analyze cell-surface protein expression. To identify huSC surface markers, we sequentially screened known markers of mouse SCs and huSCs by analyzing extracellular protein expression in PAX7-expressing SCs associated with human muscle fiber explants (Figure S1A). Like mouse SCs, huSCs expressed β1-integrin (ITGB1), but did not express the endothelial marker CD31 or the hematopoietic marker CD45 (Sherwood et al., 2004). We observed that huSCs, in contrast to mouse SCs (Montarras et al., 2005; Sherwood et al., 2004), did not express detectable CD34. Furthermore, we found that the epidermal growth factor receptor (EGFR), which is expressed by mouse SCs (Golding et al., 2007) and exhibits a rapid decrease in expression early in the process of differentiation (Leroy et al., 2013), also was expressed by human fiber-associated SCs. Taken together, our studies defined huSCs as CD34−CD31−CD45− cells that can be further specified as a population that expresses ITGB1 and EGFR (Figures 1A and S1A). Using this surface protein signature, huSCs could be prospectively isolated from all muscles tested, including latissimus dorsi, serratus anterior, and rectus abdominis (Figure S1B). To analyze the purity of cells within the putative huSC population, we quantified the percentage of cells expressing PAX7 (Figure 1B), which serves as a marker of huSCs. This analysis revealed that 96% ± 2% (n = 8) of cells in the huSC population express PAX7 (Figure 1B), while no PAX7-expressing cells were observed in the remaining huSC-depleted population, which consisted of all cells expressing CD34, CD31, or CD45 (Figure S1C). These data indicate that the sorting strategy highly enriches myogenic cells. As an additional analysis of the myogenic identity of the sorted cell populations, single-cell-derived clones were analyzed for expression of myogenic genes (Figure S1D). Although 100% of cells in the putative huSC-derived population yielded clones that expressed PAX7, no PAX7-expressing clones were derived from the remaining huSC-depleted cells. The myogenic identity of purified huSCs also was evaluated by analyzing the expression of a suite of myogenic transcription factors relative to huSC-depleted cells (Figure 1C). In this analysis, cultured huSCs expressed significantly more PAX7, PAX3, MYF5, MYOD, MYOG, and MEF2C than the remaining huSC-depleted population. We also observed that a subset of activated huSC progeny fused to form multinucleate myotubes when cultured to ∼90% confluency (Figure S1E). In contrast, the huSC-depleted population of cells grown under the same conditions did not exhibit signs of myogenesis in culture (Figure S1F), further indicating that the purification strategy efficiently enriches myogenic cells.