Culture substrate-associated YAP inactivation underlies chondrogenic differentiation of human induced pluripotent...

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for the creation of cartilage to treat articular cartilage damage. The molecular mechanisms that translate culture conditions to the chondrogenic differentiation of hiPSCs remain to be analyzed. To analyze the effects of culture substrates, we chondrogenically differentiated hiPSCs on Matrigel or laminin 511-E8 while holding the composition of the chondrogenic medium constant. Cartilage was formed from hiPSCs on Matrigel, but not on laminin 511-E8. On Matrigel, the hiPSCs were round and yes-associated protein (YAP) was inactive. In contrast, on laminin 511-E8, the hiPSCs were flat and YAP was active. Treating the laminin 511-E8 hiPSCs in a bioreactor caused cell aggregates, in which the cells were round and YAP was inactive. Subsequent culture of the aggregates in chondrogenic medium resulted in cartilage formation. Transient knockdown of YAP in hiPSCs around the start of chondrogenic differentiation successfully formed cartilage on laminin 511-E8, suggesting that the activation of YAP is responsible for the failure of cartilage formation from hiPSCs on laminin 511-E8. Consistently, the addition of YAP inhibitors to laminin 511-E8 hiPSCs caused partial cartilage formation. This study contributes to identifying the molecules that mediate the effects of culture substrates on the chondrogenic differentiation of hiPSCs as well as to developing clinically applicable chondrogenic differentiation methods.