3 isobutyl 1 methylxanthine

Human ADSCs (10⁵ cells/well) at passages 3–5 were sowed in 0.1% gelatin-coated six-well plates (Cyagen Bioscience, China) and were permitted to achieve 80–90% confluency. Adipogenic differentiation was prompted by growing cells in a medium containing 0.5 μmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 0.1 mmol/L rosiglitazone, and 100 IU insulin for 2 weeks (Cyagen Bioscience). Osteogenic differentiation was accomplished by growing cells in a medium containing 0.1 μmol/L dexamethasone, 50 μmol/L ascorbic acid, and 10 mmol/L β-glycerophosphate for 3 weeks (Cyagen Bioscience). The medium was changed every 3 days. At the end of differentiation, 4% paraformaldehyde in PBS was used to fix the cells for 15 min at room temperature. Next, the cells were stained using Oil Red O and Alizarin Red S, following the supplier's protocol, in order to evaluate adipogenic and osteogenic differentiations, respectively. The number of specifically stained ADSCs was calculated using a light microscope (Olympus, Japan).

Osteogenic differentiation was induced by culturing BMSCs in osteogenic medium supplemented with 10%FBS, 0.1 μmol/L dexamethasone, 10 μmol/L β-glycerophosphate, 10 μmol/L glutamine and 50 μg/mL ascorbate (Cyagen Biosciences, Guangzhou, China). Adipogenic differentiation was induced by culturing the BMSCs in adipogenic medium supplemented with 10%FBS, 1 μmol/L dexamethasone, 100 μg/mL 3-isobutyl-1-methylxanthine, 2 μg/L insulin, 1 μmol/L rosiglitazone and 10 μmol/L glutamine (Cyagen).