Multilineage Differentiation of Mesenchymal Stem Cells

These procedures were described in detail in our previous study (9 (link)). Briefly, for adipogenic differentiation, BM-MSCs and AT-MSCs were seeded into 24-well plates (8x10⁴ cells per well), cultured for 12 h and treated with adipogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 7 days; the medium was refreshed every 3 days. Adherent cells were stained red with 60% Oil Red O (Sigma-Aldrich; Merck KGaA) for 1 min at room temperature. For osteogenic differentiation, BM-MSCs and AT-MSCs were seeded into 24-well plates (4x10⁴ cells per well), cultured for 12 h and treated with osteogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 21 days; the medium was refreshed every 3 days. Osteogenic differentiation was confirmed by 0.2% Alizarin Red staining for 5 min at room temperature. For chondrogenic differentiation, 2x10⁵ MSCs were collected in 15-ml centrifuge tubes and cultured with chondrogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 21 days; the medium was refreshed every 3 days. The chondroid pellets were sectioned (8 µm) with a freezing microtome. The slices were stained with 1% toluidine blue for 3 min at room temperature and were captured using a light microscope (Olympus Corporation) at a x50 magnification.