Complete adipogenesis differentiation medium

Naïve MSCs and eMSCs-IL10 at passage 4 were seeded into 24-well plates at a density of 8000 cells per well. For adipogenic differentiation, when the cells reached 90~100% confluence, the complete adipogenesis differentiation medium (Gibco, Grand Island, NY, USA, catalog# A10070-01) was added and changed every 3–4 days. After 11 days, the cells were stained with oil red O. Briefly, cells were fixed with 4% formaldehyde solution (Fisher scientific, Fair Lawn, NJ, USA, catalog# SF98-4) for 1 h at room temperature, and stained with freshly prepared oil red O working solution containing 3 parts of 0.5% oil red O solution (Sigma, St Louis, MO, USA, catalog# O1391) and 2 parts of DPBS (Gibco, catalog# 14190-250) for 20 min. For osteogenic differentiation, the cells at roughly 50% confluency were fed with complete osteogenesis differentiation medium (Gibco, Grand Island, NY, USA, catalog# A1007201) every 3–4 days. After 19 days of inducing differentiation, the alizarin red S staining was processed. Briefly, after fixing cells with 4% formaldehyde solution for 1 h, 1% alizarin red S solution (Sigma, St Louis, MO, catalog# A5533) was used for staining. The corresponding controls were cultured in the complete growth medium and processed with the same staining procedure. Images were captured with bright field microscopy.

For osteogenic differentiation, 5 × 10³ cells/cm² were seeded in a T75 flask, replacing the growth medium after 2 days with Complete STEMPRO Osteogenesis Differentiation Medium (Gibco). After 14 and 21 days, cells were stained with Oil Red O to stain lipids. ADSCs were differentiated toward the adipogenic lineage by seeding 1 × 10⁴ cells/cm² in a T75 flask and replacing the culture medium after 2 days with Complete Adipogenesis Differentiation Medium (Gibco). After 14 and 21 days, cells were processed for Alizarin Red S staining in order to detect the calcium deposits in the culture.