| TGF-βsuperfamily has been shown to play an important role in cell differentiation and establishment of germ layers in the early embryonic development. Recent results showed that TGF-beta superfamily participate in the regulation of the pluripotency and differentiation of the human embryonic stem cells (hESCs). The aim of this paper is to elucidate the role of TGF-β/Activin in the differentiation of hESCs when the TGF-β/Activin signaling pathway is inhibited. Nanog is an important target gene of TGF-beta/Activin signaling. Therefore, we have acquired the Nanog gene positive recombinants and analysed its expression patterns in several tissues of C57B/6J adult mice in order to generate conditional knockout mice of Nanog. Our results showed that TGF-beta/Activin signaling pathway regulates the pluripotency and differentiation of the embryonic stem cells (ESCs) in various aspects .SB-431542 inhibited the TGF-β/Activin pathway through TGF-β1 activin receptor-like kinases such as ALK4,ALK5,ALK7 during the differentiation of hESCs. In this paper, the inhibitor SB-431542 was added to hESCs cultured in CM (conditional medium) at the concentrations of 0,1,10μM respectively. The cells were collected at day 4,6,8,10,12 and RNAs were extracted. Then the RNAs was reverse-transcripted to cDNA and the gene expression patterns were analyzed by the method of real time PCR. The expression variations of related genes in other important pathway such as FGF,Wnt and BMP pathways were analyzed at the same time. Our results indicated that the hESCs lost their pluripotency and expressed the trophectoderm specific genes such as GCM1, hCGa, hCGb when TGF-β/Activin signaling is inhibited. The expression of these genes were upregulated when the concentration of SB-431542 was inceased and the time was prolonged. However, the variations of expression of endoderm-, mesoderm- and ectoderm-specific genes were not dramatic. Our results indicated that hESCs differentiate specifically into trophectoderm when TGF-β/Activin signaling is inhibited. In addition, expression patterns of the related genes involved in FGF, Wnt and BMP signaling pathway were anylized after 10-day-treatment with SB-43154 of different concentration. The expression of FGF2, FGF4, FGF8, BMP7 and Wnt3 were downregulated significantly, while the expression of BMP2 and BMP4 did not change so much as those of the above genes. This indicates that the TGF-beta/Activin signaling pathway regulates FGF, Wnt and BMP signaling pathway.In order to elucidate how the TGF-β/Activin signaling pathway regulates the pluripotency and differentiation of the embryonic stem cells (ESCs) in various aspects, conditional knockout of mice Nanog were produced, which is an important target gene of TGF-β/Activin signaling. We also constructed the Nanog targeting vector by replacing the Nanog exon2 with Loxp-FRT-Neo- FRT-exon2-Loxp. The linearized vectors were electrophorated into S6 ESCs. The G418-resistant clones were isolated after the screen with G418. Positive recombinants were mainly identified by PCR. In addition, we also analyzed the expression of Nanog in different organs in adult C57B/6J mice. Our data indicated that Nanog is not only expressed in germ cells, but also in cerebellum, kidney, lung and cerebrum.In this paper, we mainly studied the function of TGF-β/Activin signaling pathway in the differentiation of hESCs. This laid a foundation for studies on molecular mechanism of fate decisions in hESCs. Furthermore, hESCs will be served as a new experimental model for studying early development in our species. Acquisition of Nanog positive recombinants and expression analysis of it in various tissues in C57B/6J adult mouse made an essential ground for production of the Nanog conditional knockout mice and the further studies of mechanisms of multipotency and differentiation of adult stem cells. |
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