Isolation and Osteogenic Differentiation of BM-MSCs

Femurs and tibiae were removed from the mice and the BM cells were flushed out. The washed BM cell suspensions were seeded in culture flasks (BD Falcon, Bedford, MA) in DMEM F12 medium (Thermo Fisher Scientific, Bremen, Germany) supplemented with 10% FBS and 5% horse serum (Invitrogen). After two days, the non-adherent cells were removed, and the adherent cells were further propagated for characterization of the BM-MSC phenotypes (as previously described and demonstrated in^{13 (link)}). Osteogenic differentiation was induced using DMEM supplemented with 10% FCS, 10 mM β-glycerophosphate (Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma) and 0.01 μM hydrocortisone (Sigma-Aldrich). The cultures were incubated for 21 days and the medium was changed every 3 days.