We refined our isolation procedure by sorting the CD73+CD105+ cells based on the presence or absence of CD90. There is, in fact, no consensus on the status of CD90 as a true MSC marker. Some studies have reported that cell populations with high levels of CD90 expression are multipotent MSCs, whereas others have categorized CD90 as a fibroblastic marker [33] and [34]. Care must be taken in interpreting these results since culture conditions have been shown to modulate the immunophenotypes of human stem cells in vitro [35]. However, in defined culture

media, the human skeletal muscle CD73+CD105+CD90− (or CD90−) and CD73+CD105+CD90+ (or CD90+) populations made up 11 ± 8% and 41 ± 2% of total viable cells, respectively ( Fig. 2A). We evaluated the osteogenic, adipogenic and chondrogenic differentiation potentials of the unsorted, CD90− and CD90+ Olaparib cell populations (Fig. 2B) from four independent donors (Table S1). The CD90−

cells differentiated into osteoblasts, as confirmed by the formation of alizarin-stained mineralized nodules; adipocytes, as confirmed by oil red O-stained lipid droplets; and chondrocytes as confirmed by tissue morphology and Alcian blue staining. In contrast, the unsorted cells displayed mixed differentiation potentials, with alizarin-stained mineralization similar to that obtained with the CD90− cells, but limited adipogenic and chondrogenic differentiation. check details Quantitative densitometry analyses of alizarin red staining revealed 86.4 ± 7.3% coverage for unsorted

cells compared to 95.9 ± 3.7% and 25.1 ± 28.3% for CD90− and CD90+ cells, respectively, while quantitative analyses of oil red O staining revealed 12.5 ± 9.7% coverage for Acyl CoA dehydrogenase unsorted cells compared to 95.4 ± 2.6% and 0.9 ± 1.1% for CD90− and CD90+ cells, respectively. Our results differ from those reported by Nesti et al., who showed that the CD90+-derived subpopulation is enriched in multipotent cells [16]. However, our population was isolated from untraumatized muscle and was expanded in a defined culture medium. The CD90+ cells displayed minimal differentiation towards all three lineages but expressed α-smooth muscle actin when stimulated with TGFβ, suggesting that myofibroblastic progenitors were present, as others have shown [36] and [37] (data not shown). These results indicated that the CD90− cell subpopulation contains hmrMSCs that are capable of differentiating into the lineages observed in HO. To determine whether these CD90− hmrMSCs arise from a common progenitor, we isolated clones from the CD90− population to determine their lineage commitment and differentiation potential. Nine clones derived from a single donor (Table S1) were obtained from 576 plated wells by limiting dilution (clonal efficiency of ~ 1.6%) and were assessed for their differentiation potential toward the osteogenic, adipogenic and chondrogenic lineages. Four clonal progenies (~ 44%) differentiated into all three lineages (Fig.