Differentiation of NPC-derived iPSCs into NP-like Cells

Fibroblast derived iPSCs were generated and identified by the same method. fibroblasts were obtained from osteoarthritis patient, and fibroblast derived iPSCs were characterized [36 (link)]. When we differentiate NPC derived iPSCs into NP-like cells, human fibroblast derived iPSCs were used as a control. To differentiate NPC derived iPSC into NP-like cells, iPSCs at a high density (1 × 10⁷ cells/ml) were dropped into the centre of a culture plate pre-coated with Matrigel, allowed to adhere for 1h, and then differentiation was induced with an NP differentiation medium (high glucose DMEM supplemented with 10ng/mL TGF-β1, 100 nM dexamethasone, 50 μg/mL ascorbic acid 2-phosphate, 1% ITS, 100U/mL penicillin, and 100 mg/mL streptomycin, all from Life Technologies) at 37°C in a 5% CO2 atmosphere. The differentiation media was changed twice per week and cells cultured in high glucose DMEM with 10% FBS served as a negative control (undifferentiatied cells). At Day 21, the cell pellets were then evaluated as described below.