Volume 32, No 4, Apr 2022

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 32 Issue 4, April 2022: 383-400   |  Open Access

ORIGINAL ARTICLES

Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Minglei Zhi^1,† , Jinying Zhang^1,† , Qianzi Tang^2,† , Dawei Yu^3,4,5,† , Shuai Gao^6,† , Dengfeng Gao¹ , Pengliang Liu² , Jianxiong Guo⁷ , Tang Hai³ , Jie Gao¹ , Suying Cao⁸ , Zimo Zhao¹ , Chongyang Li³ , Xiaogang Weng⁹ , Mengnan He² , Tianzhi Chen¹ , Yingjie Wang¹ , Keren Long² , Deling Jiao⁷ , Guanglei Li¹⁰ , Jiaman Zhang² , Yan Liu⁹ , Yu Lin² , Daxin Pang¹¹ , Qianqian Zhu¹ , Naixin Chen¹ , Jingjing Huang¹ , Xinze Chen¹ , Yixuan Yao¹ , Jingcang Yang⁸ , Zicong Xie¹¹ , Xianya Huang⁸ , Mengxin Liu⁸ , Ran Zhang¹ , Qiuyan Li¹ , Yiliang Miao¹² , Jianhui Tian^6,10,11 , Bofeng Liu¹³ , Wei Xie¹³ , Qi Zhou^3,16 , Hongjiang Wei^7,16 , Zhonghua Liu^9,* , Caihong Zheng^14,* , Mingzhou Li^2,* , Jianyong Han^1,*

¹State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
²Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, China
³State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
⁴Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China
⁵Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
⁶Key Laboratory of Animal Genetics, College of Animal Science and Technology, China Agricultural University, Beijing, China
⁷State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, Yunnan, China
⁸Animal Science and Technology College, Beijing University of Agriculture, Beijing, China
⁹Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang, China
¹⁰School of Life Science and Technology, ShanghaiTech University, Shanghai, China
¹¹Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin, China
¹²Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China
¹³Center for Stem Cell Biology and Regenerative Medicine, MOE Key Laboratory of Bioinformatics, THU-PKU Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China
¹⁴Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, and China National Center for Bioinformation, Beijing, China
^†These authors contributed equally: Minglei Zhi, Jinying Zhang, Qianzi Tang, Dawei Yu, Shuai Gao
¹⁶Senior authors: Qi Zhou, Hongjiang Wei
Correspondence: Zhonghua Liu(liuzhonghua@neau.edu.cn)Caihong Zheng(ch_zheng@aliyun.com)Mingzhou Li(mingzhou.li@sicau.edu.cn)Jianyong Han(hanjy@cau.edu.cn)

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

https://doi.org/10.1038/s41422-021-00592-9

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