Osteogenesis

Primary MSCs at passage 3 were seeded on tissue culture 6-well plates. After reaching 80% confluency, the culture medium was replaced with a medium for differentiation from StemPro^® Adipogenesis, Osteogenesis, or Chondrogenesis Differentiation Kits (all from Gibco, Grand Island, NY). The cells were cultured according to the manufacturer’s protocol for 14 or 21 days. Then, differentiated and control undifferentiated MSCs were stained with Sudan III, Alizarin Red S, and Safranin O (all from Sigma-Aldrich, St. Louis, MO, USA), respectively. Cell images were obtained using DMi8 inverted microscope (Leica Microsystems, Wetzlar, Germany).

The iMSCs were dissociated into single cells with TrypLE™ Express and counted, and 5 × 10⁴ cells were suspended in 100 μL DPBS with 5% BSA. Then cells were incubated with BB515-conjugated CD44, Precp-Cy5.5-conjugated CD73, PE-Cy7-conjugated anti-human CD90, APC-conjugated CD105, BV421-conjugated anti-human CD34, CD45, and HLA-DR (BD Biosciences, Franklin Lakes, NJ, USA) at 4 °C for 30 min in the dark. The stained cells were washed twice in DPBS and characterized by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
The differentiation potential of iMSCs was identified via osteogenesis (Gibco, New York, NY, USA), adipogenesis (STEMCELL Technologies, Vancouver, BC, Canada), and chondrogenesis (STEMCELL Technologies, Vancouver, BC, Canada) differentiation kits, according to the manufacturer’s instructions. After being cultured with differentiation medium for 2 to 3 weeks, cells were stained with alizarin red, oil red O, or alcian blue dye (Cyagen Biosciences Inc., Guangzhou, China)) for 30 min separately, then the stained cells were analyzed using a light microscope.

The differentiation potential of adipose-tissue-derived mesenchymal stem cells was verified by differentiating into adipocyte, chondrocyte, and osteocyte lines. They were cultured in a 24-well plate, 5 × 10⁴ cells/well; after 24 h of incubation, the differentiation medium was added. The commercially available Gibco’s StemPro^® Adipogenesis (A1007001), Osteogenesis (A1007201), and Chondrogenesis (A1007101) Differentiation Kits were applied according to the manufacturer’s guidelines (Gibco, Thermo Fisher Scientific, Waltham, MA United States). After 21 days of maintenance, the cells were fixed with 4% methanol-free formaldehyde (37,308, Molar Chemicals, Hungary) for 20 min at RT. Differentiation stages of AD-MSCs were validated using different dyes. For visualization of lipid-laden particles, Nile red staining (19,123, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was utilized, and Alizarin red staining (A5533, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was applied to show the mineral deposits during Osteogenesis. Toluidine blue staining (89640-5G, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was wielded to label the chondrogenic mass.

The differentiation potential of iMSCs was identified by osteogenesis (Gibco), adipogenesis (STEMCELL Technologies), and chondrogenesis (STEMCELL Technologies) differentiation kits according to the manufacturer’s protocols.