In vitro Differentiation of hDPSCs

In vitro differentiation into mesenchymal lineages (adipocytes, osteocytes, and chondrocytes) was performed using previously described protocols [21 (link),24 (link)]. Briefly, third-passage hDPSCs-cryo at 70% confluence were induced to lineage specific growth conditions for 21 days. Adipogenic medium consisted of 1 µM dexamethasone, 10 µM insulin, 100 µM indomethacin, and 500 µM isobutyl methyl xanthine (IBMX). For the confirmation of adipogenesis, differentiated cells were analyzed for the accumulation of lipid droplets by staining with Oil red O solution for 30 min. Osteogenic medium consisted of 50 µM ascorbate-2-phosphate, 10 mM glycerol-2-phosphate, and 0.1 µM dexamethasone. Osteogenesis was confirmed by staining differentiated cells with Alizarin red and von Kossa for the detection of mineralization and calcium deposition, respectively. Chondrogenesis was induced by using commercial chondrogenic medium (StemPro^® Osteocyte/Chondrocyte Differentiation Basal Medium; StemPro^® Chondrogenesis supplement, Gibco^® by life technologies, Grand Island, NY, USA) and differentiation was analyzed by Alcian blue and Safranin O staining. RT-qPCR was used to evaluate the expression level of lineage-specific marker genes.