Primary isolated FAPs were routinely cultured in FAP growth media containing DMEM Low glucose (22320022, ThermoFisher Scientific) supplemented with 20% fetal bovine serum (FBS) (10270-106, ThermoFisher Scientific), 1% Penicillin-Streptomycin (10,000 U/ml) (15140122, Thermo Fisher Scientific), and 5 ng/mL basic-FGF (PHG0266, ThermoFisher Scientific). Cells were expanded once before seeding for experiment unless otherwise noted. Full medium adipogenic differentiation was performed using white adipogenic induction medium containing DMEM high glucose (41966052, ThermoFisher Scientific) supplemented with 10% FBS, 1% Penicillin-Streptomycin, 0.5 mM IBMX (I5879, Sigma-Aldrich), 5 μM Troglitazone (T2573, Sigma-Aldrich), 0.25 μM dexamethasone (D2915, Sigma-Aldrich), and 5 ug/mL insulin (Sigma-Aldrich, I9278) during days 0–2. From days 2–7 cells were maintained in white adipogenic maintenance medium containing high glucose DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin, and 5 μg/mL insulin. Medium was changed every 48 h and collected on day 7 for imaging or RNA extraction. For low insulin adipogenic differentiation, cells were seeded at a density of 30,000 cells/well in a 96-well plate in FAP growth medium and allowed to attach overnight. Insulin was then added to the wells to a final concentration of 156 ng/mL. Medium was changed every 3 days before harvesting on day 5 for imaging or RNA extraction. For brown adipogenic differentiation, cells were induced from days 0–2 using brown adipogenic medium containing DMEM high glucose supplemented with 10% FBS, 1% Penicillin-Streptomycin, 5 ug/mL insulin, 1 nM T3 (T6397, Sigma-Aldrich), 1 μM rosiglitazone (AG-CR1-3570, Adipogen), 125 μM indomethacin (I8280, Sigma-Aldrich), 5 μM dexamethasone, and 0.5 mM IBMX. From days 2–10 cells were maintained in brown adipogenic maintenance medium containing high glucose DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin, and 5 μg/mL insulin, 1 nM T3, and 1 μM rosiglitazone. Medium was changed every 48 h and collected on day 10 for RNA extraction. Chondrogenic differentiation was performed using Mesenchymal Stem Cell Chondrogenic Differentiation Medium (with Inducers) (PromoCell Inc., c-28012) according to the manufacturer’s protocol and harvested on day 21 for RNA extraction. Osteogenic differentiation was performed using Mesenchymal Stem Cell Osteogenic Differentiation Medium (PromoCell Inc., c-28013) according to the manufacturer’s protocol and harvested on day 14 for RNA extraction. Fibrogenic differentiation was performed using fibrogenic induction medium consisting of low glucose DMEM supplemented with 5% horse serum (HS, 16050-122, ThermoFisher Scientific), 1% Penicillin-Streptomycin, 5 ng/mL TGFβ (eBioscience, 14-8342-82) for 3 days when cells were harvested for RNA extraction.
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