Osteogenesis medium

Osteogenesis, adipogenesis, and chondrogenesis were examined to determine the iMSC multipotent differentiation potential. For osteogenic induction, 5 × 10⁴ iMSCs were seeded in 24-well plates until 90% confluence and replaced with osteogenesis medium (Gibco). The cells cultured in MSC medium served as control. After 21 days, the cells were fixed with 4% paraformaldehyde and stained with Alizarin Red to detect areas of mineralized calcium. For adipogenic induction, 5 × 10⁴ iMSCs were seeded in 24-well plates until complete confluence, and differentiation was induced by adipogenesis medium (Gibco). The cells cultured in MSC medium served as the control. After 21 days, the cells were fixed with 4% paraformaldehyde and stained with Oil Red O. For chondrogenic induction, 1 × 10⁶ cells were centrifuged in a 15-mL polypropylene falcon tube to obtain a pellet, and chondrogenic medium (Gibco) was gently added to the pellet. After 21 days, the pellet was fixed with 4% paraformaldehyde and embedded in optimum cutting temperature compound (OCT) (Thermo Fisher, Waltham, MA, USA). Cryosections were stained with Toluidine Blue to detect the presence of proteoglycans. All stained cells were observed under an optical microscope (Leica, Solms, Germany).

Tri-lineage differentiation capability of iMSC was examined as previously described [16 ]. Briefly, to detect osteogenesis, iMSC culture medium was switched to osteogenesis medium (Gibco) when 90% confluency was reached. After culture for 21 days, cells were fixed with 4% (w/v) paraformaldehyde (PFA) and Alizarin Red staining was used to detect mineralized calcium. To detect adipogenesis, iMSC were cultured under adipogenesis medium (Gibco) for 21 days, followed by Oil Red O staining. To detect chondrogenesis, 1 × 10⁶ cells were pelleted in a 15-mL polypropylene tube after centrifugation, and chondrogenic medium (Gibco) was gently added to the pellet. After 28 days, the pellet was fixed with 4% (w/v) PFA and embedded in optimum cutting temperature compound (OCT) (Thermo Fisher, Waltham, MA, USA). Cryosections (8 μm) were cut with freezing microtome (Leica, CM1950, Germany) and stained with Toluidine Blue to examine the presence of proteoglycans. The cells cultured in MSC culture medium were served as control. All images were captured under an optical microscope (Leica, DM6B, Germany).