Alizarin red

Osteogenic differentiation was induced when the cells were at approximately 80% confluency using a commercially osteogenic medium (StemPro Osteogenesis Differentiation Kit, Life Technologies GmbH, Darmstadt, Germany). The medium was changed three times a week. After one week of differentiation culture, the cells were evaluated for osteogenic differentiation by detection of ALP expression using an ALP staining method (BCIP/NBT Color Development Substrate, Promega). After three weeks, the cells were again evaluated for osteogenic differentiation using alizarin red staining (Muto Pure Chemicals Co., Tokyo, Japan). Quantitation of alizarin red staining was performed using the absorbance at 415 nm of the solution eluted from stained cells. Adipogenic differentiation induction of the cells was initiated when they had proliferated and were overconfluent using a commercially available adipogenic differentiation medium (StemPro Adipogenic Differentiation Kit, Life Technologies GmbH). After three weeks of differentiation, the cells were stained with oil red solution (0.5% Oil-Red, Sigma-Aldrich) to evaluate the lipid content of the cells as an indicator of adipogenic capability.

ADSCs and iADSCs were seeded at 3.2 × 10⁴ cells per well in a Nunc cell-culture- treated 4-well dish (ThermoFisher Scientific, Waltham, MA, USA). After 2 days, the adipogenic differentiation was induced by incubation in a MesenCult Adipogenic Differentiation Kit (Mouse) (STEMCELL Technologies). After 4 and 7 days, cells were stained with Oil Red O (Sigma-Aldrich, St Louis, MO, USA) to determine the presence of intracellular lipid droplets, and the expression of differentiation markers after 7 days of differentiation was examined using real-time quantitative PCR (RT-qPCR). Osteogenic differentiation of ADSCs and iADSCs was induced by incubation in a MesenCult Osteogenic Stimulatory Kit (Mouse) (STEMCELL Technologies) for 11–21 days. Cells after 21 days of differentiation were then stained with Alizarin red (MUTO PURE CHEMICALS, Tokyo, Japan) to detect Ca²⁺ deposits. Cells after 11–21 days of differentiation were also examined for expression of differentiation markers via RT-qPCR. For Alizarin red staining, Nunc cell-culture-treated 4-well dishes (ThermoFisher Scientific) were coated with 0.01% type I collagen (C4243, Sigma-Aldrich).