Stempro adipogenesis

Multilineage differentiation potential of UC-MSC was assessed using StemPro Adipogenesis (A10070-01, Gibco), Osteogenesis (A10072-01, Gibco) or Chondrogenesis (A10071-01, Gibco) kits, as per manufacturer instructions. After differentiation, adipogenesis induction was assessed by Sudan Black staining. Briefly, cells were fixed with 4% paraformaldehyde solution for 45 min at room temperature, stained with a Sudan Black saturated solution in 70% ethanol for 5 min at room temperature and finally washed thoroughly with 70% ethanol. Osteogenesis induction was assessed by Alzarin Red staining. Fixed cells were stained in a 2% Alzarin Red solution (pH 4.2) for 3 min at room temperature and then washed thoroughly with distilled water. Finally, chondrogenic induction was assessed by Alcian Blue staining. Fixed cells were stained in a 1% Alcian Blue solution prepared in 0.1 N HCl for 30 min at room temperature and then washed thoroughly with distilled water. All three preparations were visualized under light microscope. Images were acquired using a Nikon Eclipse TE2000-S inverted microscope and the Eclipse Net software.

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 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.