Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition.

Moning Liu,Guang Yang,Siqin Bao,Jia Zhang,Teng Ma,Hong Su,Zixin Wang,Chunxia Hao,Gaoping Zhao,Baojiang Wu,Tong Wang,Jitong Guo,Xihe Li,Lu Chen,Guifang Cao,Yongli Song,Lixia Zhao,Yongchun Zuo,Zhiqing Yang

doi:10.3389/fcell.2021.785055

Abstract

Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.

Highlights

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells, have unlimited self-renewal capacity and retain the ability to differentiate into most cell types of adult individuals (Navarro et al, 2019)
We found that upon DOX removal, sheep induced pluripotent stem cells (iPSCs) cultured in 2i/LIF, t2iL + Gӧ, 4i/L/A, and mouse EPSC medium (mEPSCM) were dead after 96 h, and cells cultured in 5i/ L/A were dead at 48 h after one passage, concomitant with the loss of pluripotency gene expression (Figure 2E and Supplementary Figure S2A)
IPSCs were generated by porcine fetal fibroblasts with eight DOXinducible transcription factors [porcine (p) OSKM, pNANOG, human (h) LIN28, hRARG, and hLRH1] (Gao et al, 2019)

Summary

Introduction

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have unlimited self-renewal capacity and retain the ability to differentiate into most cell types of adult individuals (Navarro et al, 2019). Since the discovery of reprogramming somatic cells into iPSCs by overexpression of pluripotency-related transcription factors in the mice and humans (Takahashi and Yamanaka, 2006; Takahashi et al, 2007; Yu et al, 2007), reprogramming technology has been wildly used to generate livestock iPSCs, including bovine (Han et al, 2011; Huang et al, 2011; Cao et al, 2012; Lin et al, 2014; Heo et al, 2015; Kawaguchi et al, 2015; Nong et al, 2015; Talluri et al, 2015; Zhao et al, 2017), sheep (Bao et al, 2011; Li. Sheep Induced Pluripotent Stem Cells et al, 2011; Liu et al, 2012; Sartori et al, 2012; German et al, 2015), goat (Ren et al, 2011; Chu et al, 2015; Sandmaier et al, 2015; Tai et al, 2015; Song et al, 2016; Chen et al, 2017), and porcine (Eccles, 2011; Montserrat et al, 2011; Liu et al, 2012; Wang et al, 2013; Zhang et al, 2015; Mao et al, 2017). Sartori et al (2012) reported their sheep iPSCs were capable of a low contribution to live-born chimeric lambs, and the expression of exogenous OCT4 could be detected in one lamb skin (n 17) and one dead lamb muscle by RT-PCR (Sartori et al, 2012)

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