Osteogenic differentiation kit

The differentiation capacities of MSCs into adipogenic, osteogenic, or chondrogenic lineages were performed using following kits: the MesenCult Adipogenic Differentiation kit (Stemcell, Canada), Osteogenic Differentiation kit (Gibco), or Chondrogenic Differentiation kit (Gibco) according to the manufacturer’s procedures as we previously reported [9 (link), 11 (link)]. In brief, MSCs were seeded into 12-well plates at a density of 5 × 10⁴ cells per well and cultured until they were approximately 90–100% confluent. Then, the medium was replaced by corresponding differentiation medium and refreshed every 4 days. Oil red O staining, alizarin red S staining, and Alcian blue staining were performed to measure adipogenesis, osteogenesis, and chondrogenesis after cultured for 2 weeks, respectively. Finally, the stained cells were observed under a microscope. In addition, total RNA was extracted when the corresponding differentiation assays were completed.

To evaluate pASCs differentiation, cells were cultivated to passage 3 and maintained for 7 and 21 days, respectively, in adipogenic and osteogenic differentiation kit solutions (Gibco, Thermo Fisher Scientific) following the manufacturer's instructions. adipogenic differentiation was confirmed for positive staining by Oil Red O (Sigma-Aldrich, USA). The medium was removed after 7 days, and the wells were washed with PBS. Cells were fixed with 1% formalin for 15 min. Wells were washed again with 60% isopropanol and Oil Red O (Sigma-Aldrich) was added for 10 min. The wells were washed and visualized under a microscope to detect lipid vacuoles. For osteogenic differentiation, the medium was removed after 21 days, and the wells were washed with PBS. Cells were fixed with 4% formaldehyde for 30 min and washed twice with distilled water. Alizarin Red S 2% (pH 4.2) (Sigma-Aldrich) was added and maintained for 3 min for staining. Then, the wells were again washed with distilled water and taken for microscope viewing.