Osteogenic Differentiation of Human iPSCs

We previously reported an effective method for inducing osteogenesis in human iPSCs [21 (link),22 (link)]. Briefly, human iPSCs were incubated in the presence or absence of a SMO agonist or inhibitor in osteoblast differentiation medium (OBM). The OBM consisted of α-MEM (Invitrogen) supplemented with 10% FBS, 50 μg/ml L-ascorbic acid (Wako Pure Chemical Industries Ltd.), 10 mM β-glycerophosphate (Wako Pure Chemical Industries Ltd.), and 10 nM dexamethasone (Wako Pure Chemical Industries Ltd.). A combination of cytokines, referred to as FIT and comprising 25 ng/ml bFGF, 1 ng/ml TGF-β1 (Wako Pure Chemical Industries Ltd.), and 100 ng/ml IGF-1 (Wako Pure Chemical Industries Ltd.), was added on the following day (day 0). The iPSCs were cultured for an additional 10 days, with the OBM replenished every 3 days.