Chondrogenic 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.

Cells were cultured in osteogenic, adipogenic, or chondrogenic differentiation medium, respectively [osteogenesis differentiation kit, adipogenic differentiation kit, chondrogenic differentiation kit (Gibco, Carlsbad, CA, USA)], in 6-well plates for 2–3 weeks for HUCMSC differentiation. During this time, the medium was changed every 2–3 days. Cells were fixed with 4% formaldehyde for 30 min and stained with Alizarin Red S solution (pH 4.2) for 10 min. Osteogenic differentiation was observed under a microscopy (Nikon, Tokyo, Japan). Adipogenic differentiation was dyed using Oil Red O staining (Sigma-Aldrich, St Louis, MO, USA). Cartilage differentiation was determined using Alcian Blue staining.