Functional Analysis Of Tet1 In Reprogramming And Research On Integration-free HiPSC Generation

Direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs) can be achieved through the overexpression of a set of defined transcription factors. Reestablishment of the pluripotent network is crucial for a successful reprogramming, which also involves removal of the original somatic cell epigenetic landscape and establishment of a pluripotent stem cell-specific epigenetic landscape instead. Meanwhile, DNA methylation and hydroxymethylation have been proposed to play important roles in this process.In the first section of this thesis, we prove that the DNA hydroxylase Tetl can promote pluripotency genes demethylation and reactivation through 5hmC conversion, which further accelerates the establishment of the pluripotent network and improves the efficiency of iPS generation. Moreover, Tetl can substitute for certain Yamanaka factors during reprogramming and generate high quality iPSCs. These iPSCs not only express high level of pluripotency genes, but also can generate teratomas with three germ layers and chimeric mice with germline transmission ability in the study of in vivo differentiation. More importantly, we find that OT (Tetl replaces Sox2, Klf4 and c-Myc) and TSKM (Tetl replaces Oct4) iPSCs can both efficiently produce full-term all-iPSC mice through tetraploid complementation experiment. And different from the high tumorigenesis observed in traditional OSKM-iPSC mice, all the OT- and TSKM-iPSC mice can grow healthily with a normal life span and without obvious tumors. Furthermore, two new secondary induction systems mediated by OT and TSKM are established and will be valuable tools for further investigation of the mechanisms of epigenetic remodeling during reprogramming. In conclusion, our study demonstrates that Tetl and 5hmC play important roles in somatic cell reprogramming. Tetl can successfully substitute for multiply Yamanaka factors to generate high quality iPSCs.The traditional human induced pluripotent stem cells (hiPSCs) are reprogrammed by virus mediated means, which can create trans-gene integration and mutations in the human genome. In addition, drawing the somatic cells from human skin fibroblasts is much traumatic, which limits the utility of the hiPSCs in clinical applications. The secondary section of this thesis is to investigate how to generate hiPSCs with a simple, quick and safe way. Here, we describe the generation of integration-free hiPSCs by non-integrating episomal vectors from 2-5 ml human peripheral blood mononuclear cells (PBMCs) with considerable efficiency in one month. These hiPSCs are similar to human embryonic stem cells (hESCs) in cell morphology, gene expression, core pluripotency protein level and DNA methylation pattern as well as in in vitro and in vivo developmental potential. We also adapt these hiPSCs to Xeno-free culture conditions which does not influence their pluripotency. These individualized integration-free hiPSCs have multiply applications in basic biology and medical research including disease pathogenesis, drug screening as well as regenerative medicine, and may even be used in personalized treatment in the further. Our study provides an important preliminary work for the ultimate realization of the personalized hiPSCs in clinical applications.