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Anran Fan, Kuiying Ma, Xinglan An, Yu Ding, Peipei An, Guangqi Song, Lina Tang, Sheng Zhang, Peng Zhang, Wentao Tan, Bo Tang, Xueming Zhang and Ziyi Li

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression of POU5F1 (OCT4), SOX2, KLF4, and MYC (C-MYC). siRNAs targeting to TET1 were used to transiently knockdown the expression of TET1 in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation of TET1. The effects of TET1 knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown of TET1 resulted in the downregulated expression of pluripotency-related genes, such as LEFTY2, KLF2, and SOX2, and the upregulated expression of differentiation-related genes including PITX2, HAND1, GATA6, and LEF1. However, POU5F1, M YC, KLF4, and NANOG were actually not downregulated. Further analysis showed that the methylation levels of the promoters for POU5F1 and M YC increased significantly after TET1 downregulation, whereas there were no obvious changes in the promoters of SOX2, KLF4, and NANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest that TET1 may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation.

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Jian Zhang, Linlin Hao, Qian Wei, Sheng Zhang, Hui Cheng, Yanhui Zhai, Yu Jiang, Xinglan An, Ziyi Li, Xueming Zhang and Bo Tang

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.