3 isobuthyl 1 methylxanthine

For adipogenic differentiation, hCSCs were cultivated in DMEM (Sigma Aldrich) containing 10% FCS (Sigma Aldrich) and plated at a density of 4 × 10³ cells /cm². After 48 h, 1 μM dexamethasone (Sigma Aldrich), 2 μM insulin (Sigma Aldrich), 500 μM 3-isobuthyl-1-methylxanthine (Sigma Aldrich) and 200 μM indomethacin (Sigma Aldrich) were added to the medium and cultivated for 72 h. Afterwards, the medium was switched and cells were cultivated for 4 days in DMEM containing 10% FCS and 2 μM insulin (Sigma Aldrich) to induce adipogenic differentiation. These two media were alternatingly used and changed every 4 days for 3 weeks. As undifferentiated control, cells were cultured in hCSC-medium as described above.

For adipocyte differentiation, ASC were amplified until 80% of confluency was reached and culture medium replaced by differentiation medium containing Dulbecco's modified Eagle medium/Nutrient Mixture F12 (DMEM/F12; Dominique Dutscher, France), FBS 10% (Hyclone, France), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 1 μg/ml insulin, 0.5 μM dexamethasone, 2 nM Triiodo-L-Thyronine 3 (T3), 0.5 μM 3-isobuthyl-1-methylxanthine (IBMX), 2 μM rosiglitazone, and 10 μg/ml transferrin, all from Sigma-Aldrich. After 7 days, adipocyte differentiation was measured by Oil red O staining of the neutral triglyceride and lipids. Cells were fixed in 10% formaldehyde (Carlo Erba, France) for 1 hour then washed with 60% isopropanol (Carlo Erba) twice, dried and colored by incubation for 10 minutes with 0.2% Oil red O (Sigma-Aldrich) in isopropanol. Brightfield images were taken with an optic microscope with a ×20 magnification. The expression of adipogenic genes (Pparγ, Dgat2, and Hsl) was measured by RT-qPCR.

2‐aminoethyl diphenylborinate (2‐APB), N‐methyl maleimide (NMM), NS8593, naltriben, dexamethasone (DEX), and 3‐isobuthyl‐1‐methylxanthine (IBMX) were purchased from Sigma‐Aldrich (St. Louis, MO). 2‐amino‐2‐[2‐(4‐octylphenyl)ethyl]‐1,3‐propanediol (FTY720) was purchased from Cayman Chemicals (Ann Arbor, MI). Recombinant human insulin was purchased from Wako (Osaka, Japan). Fura‐2 acetoxymethyl ester (AM) was purchased from Thermo Fisher Scientific (Waltham, MA).