Multilineage Differentiation Potential of MSCs

To assess the multiple differentiation potential, MSCs were cultured until sub-confluency and induced to differentiate into the adipogenic or osteogenic lineages for 7 and 21 days, respectively. After that, the gene expression analysis and staining were performed. The adipogenic medium consisted of α-MEM (Life Technologies), 20% FBS (Life Technologies), 2 mM glutamate (Life Technologies), 100 Units/mL penicillin/streptomycin (Sigma-Aldrich) and 55 µM 2-ME (Life Technologies), 10 mM L-ascorbic acid phosphate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 10 mg/mL insulin (Sigma-Aldrich), 0.5 mM hydrocortisone (Sigma-Aldrich), 6 mM indomethacin (R&D systems), 50 mM isobutylmethylxanthin (Enzo life science, Tokyo, Japan), as reported [66 (link)]. Osteogenic medium consisted of α-MEM (Life technologies), 20% FBS (Life Technologies), 2 mM glutamate (Life technologies), 100 Units/mL penicillin/streptomycin (Sigma-Aldrich) and 55 µM 2-ME (Life technologies), 10 mM L-ascorbic acid phosphate (FUJIFILM Wako Pure Chemical Corporation), 200 mM β-glycerophosphate (Sigma-Aldrich), as reported [67 ]. Mineralization (calcium deposition) and lipid droplet formation were evaluated, respectively, by staining with alizarin red-S or oil red-O, as reported [68 (link),69 (link)].