2. Academic Validation
  3. Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Generation and characterization of stable pig pregastrulation epiblast stem cell lines

  • Cell Res. 2022 Apr;32(4):383-400. doi: 10.1038/s41422-021-00592-9.
Minglei Zhi # 1,  Jinying Zhang # 1,  Qianzi Tang # 2,  Dawei Yu # 3 4 5,  Shuai Gao # 6,  Dengfeng Gao 1,  Pengliang Liu 2,  Jianxiong Guo 7,  Tang Hai 3,  Jie Gao 1,  Suying Cao 8,  Zimo Zhao 1,  Chongyang Li 3,  Xiaogang Weng 9,  Mengnan He 2,  Tianzhi Chen 1,  Yingjie Wang 1,  Keren Long 2,  Deling Jiao 7,  Guanglei Li 10,  Jiaman Zhang 2,  Yan Liu 9,  Yu Lin 2,  Daxin Pang 11,  Qianqian Zhu 1,  Naixin Chen 1,  Jingjing Huang 1,  Xinze Chen 1,  Yixuan Yao 1,  Jingcang Yang 8,  Zicong Xie 11,  Xianya Huang 8,  Mengxin Liu 8,  Ran Zhang 1,  Qiuyan Li 1,  Yiliang Miao 12,  Jianhui Tian 6,  Xingxu Huang 10,  Hongsheng Ouyang 11,  Bofeng Liu 13,  Wei Xie 13,  Qi Zhou 3,  Hongjiang Wei 7,  Zhonghua Liu 14,  Caihong Zheng 15,  Mingzhou Li 16,  Jianyong Han 17
Affiliations

Affiliations

  • 1 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
  • 2 Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, China.
  • 3 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
  • 4 Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.
  • 5 Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China.
  • 6 Key Laboratory of Animal Genetics, College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • 7 State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, Yunnan, China.
  • 8 Animal Science and Technology College, Beijing University of Agriculture, Beijing, China.
  • 9 Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang, China.
  • 10 School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
  • 11 Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin, China.
  • 12 Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
  • 13 Center 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.
  • 14 Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang, China. [email protected].
  • 15 Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, and China National Center for Bioinformation, Beijing, China. [email protected].
  • 16 Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, China. [email protected].
  • 17 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China. [email protected].
  • # Contributed equally.
PMID: 34848870 DOI: 10.1038/s41422-021-00592-9
Abstract

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.

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