Multi-lineage Differentiation Potential of hDIAS Cells

To determine the multi-lineage differentiation potential of cells from each tissue type, hDIAS cells from each donor were grown in established adipogenic, osteogenic, and chondrogenic culture conditions [10 (link)]. For adipogenic and osteogenic differentiation, 1.5x10⁴ cells from each donor were plated in each well of a 24-well plate. Adipogenic differentiation medium consisted of DMEM with high glucose/GlutaMAX™-I, 5% FBS, 1% P/S/F, 1% NEAA, 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine (Sigma-Aldrich), and 0.2 mM indomethacin (Sigma-Aldrich). Osteogenic differentiation medium consisted of DMEM with high glucose/GlutaMAX™-I, 10% FBS, 1% P/S/F, 1% NEAA, 100 nM dexamethasone, 10 mM β-glycerophosphate (Sigma-Aldrich), 250 mM ascorbate-2-phosphate, and 50 ng/mL bone morphogenetic protein-2 (BMP-2) (Peprotech). For chondrogenic differentiation, 2.5x10⁵ cells from each donor were added to a round bottom polystyrene 96-well plate (Costar 3799, Corning, NY) 0.2 mL of CHG, supplemented with 50 ng/mL BMP-2, and 10 ng/mL transforming growth factor beta-1 (TGF-β1) (Peprotech), and centrifuged at 500 xg for 5 minutes to form cell pellets [30 (link)]. All groups were then maintained at 37°C and 5% CO₂ for 4 weeks, with media exchanged every other day.