Transforming growth factor β3

For chondrogenesis, 2.5 × 10⁵ cells were transferred to a 15 ml tube (BD Falcon) and cultured in chondrogenic induction medium containing 10 ng/ml transforming growth factor-β3 (Miltenyi Biotec Japan, Tokyo, Japan) and 500 ng/ml bone morphogenetic protein 2 (BMP-2, Infuse; Medtronic, Minneapolis, MN); this medium was changed every 3–4 days. After 21 days, the cell pellets were embedded, sectioned and stained with safranin O and fast green (Wako, Tokyo, Japan).
Calcification was studied by plating 100 cells in a 60 cm² dish and culturing for 14 days in α-MEM with 10% fetal bovine serum. Adherent cells were subsequently cultured in an osteogenic induction medium containing 50 μg/ml ascorbic acid 2-phosphate (Wako), 10 nM dexamethasone (Wako), and 10 mM β-glycerophosphate (Sigma-Aldrich); this medium was changed every 3–4 days. After 14 days, calcification was assessed by alizarin red (Merck Millipore, Billerica, MA) staining.
Adipogenesis was evaluated by plating 100 cells in a 60 cm² dish and culturing for 14 days to allow colony formation. Adherent cells were cultured in an adipogenic induction medium supplemented with 100 nM dexamethasone, 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), and 50 mM indomethacin (Wako) for an additional 14 days; this medium was changed every 3–4 days. Adipocyte colonies were stained with oil red O (Muto Pure Chemicals, Tokyo, Japan).