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

为了将再生医学应用于口腔外科的临床环境，我们将胚胎干细胞（ES细胞）中的多能维持因子Nanog基因引入间充质细胞，并试图促进其自我更新和分化潜能。本研究旨在通过引入Nanog基因增强间充质干细胞的分化潜能，实现骨或软骨的高效形成。首先，将Nanog基因转导到一些已建立的细胞系中，并分析其功能。通过PCR和Western blotting检测Nanog在基因和蛋白水平上的表达，在小鼠细胞系C3H10T1/2、MC3T3-E1和ATDC5中几乎检测不到Nanog的表达。然后，我们研究了Nanog cDNA通过病毒载体以时间或组成方式导入上述细胞的细胞分化。结果提示Nanog可能对成骨分化有控制作用。利用Nanog RNAi进行基因沉默实验，通过病毒介导的siRNA传递来验证其对细胞分化和增殖的影响。此外，我们克隆了这些细胞来研究Nanog的沉默作用。最后，我们研究了Nanog在组成型表达下细胞成骨分化的分子机制。Western blotting分析显示，在Nanog基因的引入下，BMP信号下游效应蛋白Smad1/5/8的磷酸化得以持续。而对于胚胎干细胞自我更新至关重要的STAT3的磷酸化，western blotting结果显示，在nanog转导的细胞中，STAT3的磷酸化水平降低。综上所述，Nanog在控制成骨/软骨分化以及胚胎干细胞中具有重要功能。

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.