Hmsc chondrogenic differentiation bulletkit medium

All cells were cultured at 37°C in a humidified atmosphere with 5% CO₂. SW1353 cells were obtained from the American Type Culture Collection, and TC28a2 cells were purchased from Sigma-Aldrich. SW1353 cells, TC28a2 cells, and human primary cells were cultured in DMEM with 10% CS supplemented with 1% PSG. BMSCs were purchased from Lonza and were cultured in human Mesenchymal Stem Cell (hMSC) Growth BulletKit Medium (MSCGM) (Lonza). In pellet culture of BMSCs, hMSC Chondrogenic Differentiation BulletKit Medium (Lonza) was used. Human IL-1β (PeproTech) and TGF-β3 (PeproTech) were used to treat cells. The compounds used in screenings and validation experiments were provided by Calibr (a Division of Scripps Research Institute). Mocetinostat for other experiments was obtained from LC Laboratories, and SR-18292 was purchased from Selleck Chemicals. Stocks of the compounds were diluted in DMSO. Cell viability was measured with Countess II FL Automated Cell Counter (Thermo Fisher Scientific) using Trypan Blue stain 0.4% (Thermo Fisher Scientific).

Mesenchymal lineage differentiation assays were carried out as described in Muinos-López et al. 2016 [33 (link)]. Briefly, SF-derived cells were assessed between passages 2 and 5 to confirm their osteogenic, adipogenic, and chondrogenic capacity. For osteogenic and adipogenic differentiation, 8000 cells/cm² were seeded in 12-well plates. Adipogenic differentiation was induced using DMEM supplemented with 10% FBS, 1 μM Dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 50 μM Indomethacin for 21 days. For the osteogenic differentiation, cells were cultured in DMEM supplemented with 10% FBS, 50 μg/mL L-(+)-ascorbic acid, 10 mM β-glycerol phosphate, and 10 nM Dexamethasone for 21 days. For chondrogenic differentiation, 2.5E5 cells were spun-down at 600 ×g for 10 minutes in polystyrene 15 mL conical tubes and incubated with hMSC Chondrogenic Differentiation BulletKit™ Medium (Lonza). Differentiations were analyzed at 28 days. In all differentiation assays, a negative control was included where the cells were maintained with expansion medium (DMEM containing 10% FBS) without induction factors. In all differentiation assays, medium was changed every 2-3 days.