Myogenic differentiation of hAFSCs in vitro. (A) Viability assay for cells cultured in three different myogenic induction media for 14 days. Cell viability in CM was 2.7- (P < 0.0001) and 1.58- (P < 0.0001) fold higher than that of cells cultured in medium containing 5-azaC or TGF-β respectively. (B) Real-time PCR analysis of expression of myogenic lineage markers in three different media at Days 3 and 7. The expression of early myogenic differentiation markers (PAX7 and MYOD) was dominant at Day 3 and the expression of the mid to late myogenic marker (DYSTROPHIN) became dominant at Day 7 (**P < 0.01; *P < 0.05). (C) Double staining using primary myogenic antibodies and DAPI through ICC analysis (200×) at Day 7. Cells were strongly positive for MYOD and DESMIN. Ctrl(+), positive control C2C12 cells; Ctrl(-), negative control with human fibroblasts.