Passage 2/3 MSCs (n = 5 matched donor–derived cultures for BML and non‐BML bone tissue digests) were induced toward osteogenesis, chondrogenesis, and adipogenesis, using standard protocols 21 . For osteogenesis and chondrogenesis, we used StemMACS OsteoDiff and ChondroDiff medium, respectively (Miltenyi Biotec); adipogenic cultures were grown in DMEM with 10% FCS, antibiotics, 10% horse serum (StemCell Technologies), 0.5 mM isobutylmethylxanthine, 60 μM indomethacin, and 0.5 μM hydrocortisone (all from Sigma).
Differentiation assessment was performed as previously described21 . Briefly, alkaline phosphatase activity was visualized on day 14 postinduction. Calcium deposits were stained using alizarin red on day 21, and total calcium produced by cultures was measured using a Calcium Detection Kit (Sentinel Diagnostics). Biochemical assessment of the glycosaminoglycans (GAGs) was performed on 3 of 4 chondrogenic pellets grown for 21 days. The remaining pellet was used for histologic analysis; 4‐μm sections were cut using a Leica CM1950 cryostat, fixed, and stained with toluidine blue. Adipogenic cultures were stained with oil red O on day 21 postinduction.
Differentiation assessment was performed as previously described