Mechanism of osteogenic differentiation for mesenchymal stem cells induced by BMP9 and Wnt3a in vitro

Abstract:

Objective：To analyze the molecular mechanism of osteogenic differentiation of mesenchymal stem cells（MSCs） induced by canonical Wnt3a signal pathway and bone morphogenetic protein 9（BMP9）. Methods：C3H10T1/2 cells were infected by recombinant adenovirus expressing BMP9 and Wnt3a，and then alkaline phosphatase（ALP），the early osteogenic marker，was detected by quantita－tive assay. Osteogenic differentiation marker at later stage，calcium deposition was determined by Alizarin Red S staining. The protein levels of osteocalcin（OC） and osteopontin（OPN） were detected by immunocytochemical staining. The expression levels of OC，OPN and Runx2 were analyzed by real time PCR. Results：Wnt3a enhanced the ability of BMP9-induced ALP activity（F=264.962，P=0.000；t（BMP9+Wnt3a）-BMP9=7.19，P=0.000；t（BMP9+Wnt3a）-Wnt3a=16.36，P=0.000） and increased expressions of OC（F=3 920.102，P=0.000；t（BMP9+Wnt3a）-BMP9=39.10，P=0.000；t（BMP9+Wnt3a）-Wnt3a=62.56，P=0.000） and OPN（F=1 935.824，P=0.000；t（BMP9+Wnt3a）-BMP9=40.88，P=0.000；t（BMP9+Wnt3a）-Wnt3a=56.42，P=0.000），with increase of mineral calcium deposition in vitro. Furthermore，Wnt3a increased the mRNA level of Runx2（F=3 635.980，P=0.000；t（BMP9+Wnt3a）-BMP9=79.37，P=0.000；t（BMP9+Wnt3a）-Wnt3a=86.96，P=0.000）. Conclusion：Wnt3a，possibly through Runx2，acts synergistically on BMP9 induced osteogenic differentiation of MSCs.