Abstract: Abstract: Background Mesenchymal stem cells (MSCs) are crucial for the healing of bone defects. Some studies have found that transient receptor potential vanillin channel protein 4 (TRPV4) can promote bone formation in bone cells, but its role in mesenchymal stem cells is not clear.Objective To investigate the effect of TRPV4 on osteogenic differentiation of mesenchymal stem cells (iMSCs) derived from human induced pluripotent stem cells (iPSCs).Methods iPSCs were induced into iMSCs and their phenotypes were detected by flow cytometry. The differentiation ability of osteoblastic, lipogenic and chondrogenic cells was evaluated.The expression of osteogenic genes and TRPV4 were found at various stages following osteogenic induction of iMSCs by using real-time quantitative PCR and Western blot, respectively. Alizarin red staining was used to detect the effects of TRPV4 small molecule agonist (GSK101) and inhibitor (GSK219) treatment of iMSCs on calcium salt deposition. iMSCs were cultured on different hardness substrates, and TRPV4 gene and protein expression were detected by real-time quantitative PCR and Western blot, respectively. Results The iMSCs induced by this experiment had the typical characteristics of mesenchymal stem cells. After osteogenic differentiation of iMSCs, the expressions of osteogenic mRNAs and the expressions of TRPV4 mRNA and protein were all increased.The alizarin red staining area of iMSCs treated with TRPV4 agonist GSK101 increased significantly compared with the control group, which was opposite in the TRPV4 antagonist GSK219 group. mRNA and protein expression of TRPV4 gene increased after iMSCs were cultured on a cell culture plate with a high hardness substrate.Conclusion TRPV4 plays a positive regulatory role in the osteogenic differentiation of iMSCs. The increased hardness of the culture matrix can regulate the osteogenic differentiation of iMSCs by promoting TRPV4 expression. The results of this study expand the understanding of the osteogenic differentiation process of iMSCs, and provide new ideas for regulating the osteogenic differentiation of iMSCs and promoting the effect of clinical transplantation to repair bone defects.