Research to enhance potentials of mesenchymal calls by introducing Nanog gene and its application to regenerative medicine of bone and cartilage

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In order to apply regenerative medicine to clinical settings of oral surgery, we introduced Nanog gene, a pluripotency sustaining factor in embryonic stem cells (ES cells), into mesenchymal cells and attempted to promote their self-renewal and differentiation potentials. The purpose of the present study was to enhance the differentiation potential of mesenchymal stem cells by introducing Nanog gene and to realize the efficient formation of bone or cartilage. First of all, Nanog gene was transduced into some established cell lines and its function was analyzed. When Nanog expression was examined at gene and protein level by PCR and Western blotting, it was almost undetectable in mice cell lines, C3H10T1/2, MC3T3-E1 and ATDC5. Then, we investigated the cell differentiation of above-mentioned cells into which Nanog cDNA was introduced by viral vectors in a temporal or constitutive manner. The results suggested that Nanog may control the osteogenic differentiation. For gene silencing experiments using Nanog RNAi, virus-mediated siRNA delivery was conducted to verify its effect on cell differentiation and proliferation. Furthermore, the cells were cloned to investigate the silencing effects of Nanog. Finally, we examined the molecular mechanisms of osteogenic differentiation in the cells under constitutive expression of Nanog. Western blotting analysis revealed phosphorylation of Smad1/5/8, which are downstream effectors in BMP signaling, was sustained in response to the introduction of Nanog gene. As to phosphorilation of STAT3, which is important for self-renewal of ES cells, it was shown by western blotting to decrease in Nanog-transduced cells. Taken together, Nanog was suggested to have important functions in controlling osteogenic/chondrogenic differentiation as well as in ES cells.