Tet家族在小鼠成纤维细胞诱导为肝干细胞的谱系重编程过程中的作用及相关机制研究

"谱系重编程"是指成熟体细胞可以通过外源转录因子的导入直接重编程为其它类型的体细胞或干（祖）细胞。体细胞基因组DNA甲基化修饰是谱系重编程首先需要突破的表观遗传分子屏障之一。研究表明Tet家族能将5mC氧化成5hmC来实现DNA的去甲基化从而在ES细胞干性维持和iPSCs形成过程中发挥重要作用，但Tet家族在谱系重编程过程中的作用尚未见报道。我们实验室2013年在Cell Stem Cell上发表了将MEF重编程为肝干细胞iHepSCs的研究成果，为深入研究Tet家族的作用提供了理想平台。本课题将在前期Tet相关实验结果的基础上，通过干扰Tet的表达，结合微量细胞转录组分析技术，研究Tet家族在外源基因诱导MEF重编程为iHepSCs过程中的作用、iHepSCs干性维持中的作用以及相关机制，以完善对Tet家族在调控细胞基因组表观特性方面的功能的认识，并提出提高谱系重编程效率和质量的新思路

The "lineage reprogramming" is referred to that differentiated somatic cells could be directly converted into other somatic cells or progenitors with defined transcription factors. Both the breaking of the epigenetic molecular barriers of the somatic cell and the establishment of the epigenetic molecular landscape of the stem cells are the essential steps for lineage reprogramming and iPSCs formation. DNA methylation pattern, as one of the molecular barriers of reprogramming, has a profound impact on genome stability, transcription and development; it is even regarded as the identity of somatic cells and stem cells. In most recent years, ten-eleven translocation (TET) family enzymes, including Tet1, Tet2 and Tet3, are evidenced as critical enzymes in regulating the DNA methylation pattern, which can oxidize 5-methylcytosine to 5-hydroxymethylcytosine, an intermediate nexus in demethylation. Many recent reports showed that Tet family enzymes play a vital role in ES maintenance and iPSCs formation. However, the function and the underlying mechanism of Tet family involving in lineage reprogramming have remained unclear. We succeeded in reprogramming MEF to induced Hepatic Stem Cells (iHepSCs) with Hnf1b and Foxa3 in 2013, as a successful model in the field of lineage reprogramming，which gives us a unique advantage to explore the basis of Tet family in lineage reprogramming. Recently we found that the expression of Tet1, Tet2 and Tet3 were activated in iHepSCs and kept high expression in vitro culture. Our recent results exhibited that knockdown of Tet1 ,Tet2 and Tet3 at the same time, resulted in a morphological abnormality of iHepSCs and a dramatic decrease of the hepatic stem cell-specific gene Dlk and Sox9. In addition, we discovered that the three Tet-shRNAs could interfere with the formation of iHepSCs clone when they were co-transfected into MEF with Hnf1b and Foxa3, which suggests the function of Tet members in lineage programming. Basing on these above and other observations, and our experience in transcriptomics and CpG methylation profiling, we design to reveal the function and mechanism of Tet family in iHepSCs maintenance and iHepSCs formation by interfering with the expression of Tet family in this proposal. The ultimate destination is to optimize the reprogramming strategy with Tet family enzymes and finally improve the efficiency of reprogramming and quality of iHepSCs.