Evaluating EMSC Differentiation Potential

The capacity of EMSCs to differentiate in vitro was evaluated. EMSCs were seeded at 1 × 10⁵/cm² and cultured for 3 weeks in osteogenic differentiation medium (0.01 μM 1α-25-dihydroxyvitamin-D3, 10 mM β-glycerophosphate, and 50 μM ascorbate-2) (Sigma-Aldrich, St. Louis, MO, USA), adipocyte differentiation medium (500 μM isobutyl-methylxanthine, 1 μM dexamethasone, 10 μM insulin, and 200 μM indomethacin) (Sigma-Aldrich, St. Louis, MO, USA), or chondrogenic differentiation medium (6.25 g/ml insulin, 50 μM ascorbate-2) (Sigma-Aldrich, St. Louis, MO, USA). Cells cultured in normal medium were used as the control. The medium was changed every 3 days. After 3 weeks, the cells in each group were stained with alizarin red, oil red O, and alcian blue (Sigma-Aldrich, St. Louis, MO, USA) to assess osteogenesis, adipogenesis, and chondrogenesis, respectively [25 (link)].