Sox17 On Mouse Embryonic Stem Cell Differentiation

Stem cell (SC), which is called"omnipotent cell"in the field of medical science, has the properties of self-renewal, self-proliferation, differentiation and high plasticity. In some certain cases, SCs can differentiate into many kinds of cells, tissues and even organs. One kind of stem cells, embryonic stem cell (ESC), originates from the inner cell mass of blastula, and its directed development is regulated by internal mechanism and microenvironment, such as lots of cytokines in the complex networks. Nowadays, many embryonic stem cell lines have been founded on the base of the identification, isolation, purification, proliferation and cultivation technology of stem cells. With the establishment of the ESC model system, we are able to explore the molecular mechanisms that regulate the embryonic development processes in vitro.The Sox (SRY-related HMG-box gene) family of HMG (high mobility group)-box transcription factors are highly conserved in vertebrates, and play important roles on the development processes. As one member of Sox family, Sox17 is demonstrated to be indispensable in the endoderm development. By the way of gene targeting, Sox17 null mutant mouse embryos are deficient of gut formation and embryonic development failure. In Xenopus laevis, Sox17 induces endoderm differentiation when ectopically expressed in naive ectoderm.However, that whether Sox17 is necessary in the endoderm formation process has been only partially understood (especially in mammals), and little is known about how it works in the endoderm development. Now it has been shown that in Xenopus Sox17 interacts withβ-catenin, an important factor of Wnt signaling pathway, to co-regulate the transcription of downstream genes. Therefore, an in-depth study on the function of Sox17 may contribute to the understanding of the mechanisms of gene regulation, tissue-organ genesis and abnormality formation in the development processes.In this trial, Sox17 is overexpressed in the mouse embryonic stem cells to find out what its roles are and how it works by the ways of morphologic observation, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Immunofluorescence assay.The results show:1.We got a high transfection efficiency (about 40% to 50%) after the ESCs were transfected by the plasmid pPyCAGIP conjugated with Sox17 in it using Lipofectamine 2000.2.Compared with the negative controls (empty vectors transfected cells), the transfection of Sox17 induced the ESCs differentiation even in the presence of Leukemia inhibitory factor (LIF): gradually diffused morphology and decreased expression of pluripotency markers Oct4 and Rex-1.3.In the Sox17 transfected ESCs, the expression of primitive endoderm markers, Gata4, Gata6, Hnf1β, as well as parietal endoderm markers, LamininB1 (LamB1), Thrombomodulin (TM), Disabled homolog2 (Dab2) and Sparc, were increased. These results indicated that the Sox17 overexpression induced mouse ESCs differentiation towards primitive endoderm.4.Embryoid bodie (EB) was formed after ESC being suspension-cultured in the medium without LIF. In the EBs of day 2 (D2), D6 and D10, the primitive endoderm markers, Gata4, Gata6, Chicken ovalbumin upstream promoter transcription factorⅡ(Coup-tfⅡ), Hnf1β, parietal endoderm markers, TM, LamB1, Dab2, Sparc and visceral endoderm markers, Alpha fetoprotein (Afp), Bone morphogenetic protein 2 (Bmp2), were increased; definitive endoderm markers Intestinal fatty acid binding protein (Ifabp), Pancreatic and duodenal homeobox 1 (Pdx-1), and mesoderm marker Actc were also increased. Interestingly, the expression level ofβ-catenin, a component of Wnt signaling pathway, was enhanced, too. All of these results indicated that Sox17 played important roles on the primitive and definitive endoderm formation.In a word, Sox17 may interact withβ-catenin to play duplex effects in the development processes: induces ESCs differentiation towards primitive endoderm in the initial stages and induces definitive endoderm formation leading to the organogenesis of endodermal derivatives (liver, pancreas etc.) in the period of EB.