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Establishment of porcine and human expanded potential stem cells

Nature Cell Biology volume 21, pages 687–699 (2019)Cite this article

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Abstract

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

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Fig. 1: Identification of culture conditions for porcine EPSCs.
Fig. 2: Derivation of porcine EPSCs.
Fig. 3: In vitro generation of PGCLCs from pEPSCsEmb.
Fig. 4: Establishment of human EPSCs.
Fig. 5: Molecular features of porcine and human EPSCs.
Fig. 6: The requirement of individual components in EPSCM.
Fig. 7: Trophoblast differentiation potential of hEPSCs.
Fig. 8: Derivation of trophoblast stem cell-like cells from hEPSCs.

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Article 01 December 2022

Zixuan Zhao, Xinyi Chen, … Hanry Yu

Data availability

Sequencing data are deposited into ArrayExpress and the accession numbers are E-MTAB-7252 (ChIP-Seq), E-MTAB-7253 (bulk RNA-Seq) and E-MTAB-7254 (scRNA-Seq). Re-analysed previously published data are available under the accession codes ENA PRJNA326944, ENA PRJNA153427, ENA PRJNA291062 and GSE73017. The source data for the figures and supplementary figures are in Supplementary Table 10. All other relevant data are available from the corresponding author on request.

Code availability

The software and algorithms for data analyses used in this study are all well-established from previous work and are referenced throughout the manuscript. No custom code was used in this study.

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Acknowledgements

We thank our colleagues of the Wellcome Trust Sanger Institute core facilities for their generous support (J. Bussell, Y. Hooks, N. Smerdon, B. L. Ng and J. Graham) and A. Moffett for critical comments. We thank B. Petersen for performing the surgical embryo transfers and the staff of the experimental pig facility at the Friedrich-Loeffler-Institut Mariensee for their competent and enduring assistance. We acknowledge the following funding and support: the Wellcome Trust (grant nos. 098051 and 206194) to the Sanger Institute and the University of Hong Kong internal funding (P. Liu); a Wellcome Trust Clinical PhD Fellowship for Academic Clinicians (D.J.R.); a PhD fellowship from the Portuguese Foundation for Science and Technology, FCT (grant no. SFRH/BD/84964/2012; L.A.); a Marie Sklodowska-Curie Individual Fellowship (M.A.E.-M.); the BBSRC (grant no. BB/K010867/1), Wellcome Trust (grant no. 095645/Z/11/Z), EU EpiGeneSys and BLUEPRINT (W.R.); a Chongqing Agriculture Development Grant (grant no. 17407 to L.P.G., Z.H.L. and Y.H.); REBIRTH project no. 9.1, Hannover Medical School (H.N.); Shuguang Planning of Shanghai Municipal Education Commission (grant no. 16SG14) and the National Key Research and Development Program (grant no. 2017YFA0104500; L. Lu); the China Postdoctoral Science Foundation (grant no. 2017M622795; D.C.); the Strategic Priority Research Program of CAS (grant nos. XDA16030503 and XDA16030501), the National Key Research and Development Program of China Stem Cell and Translational Research (grant no. 2017YFA0105103) and Key Research and Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (grant no. 2018GZR110104004; L.Lai); the Shenzhen Municipal Government of China (DRC-SZ [2016] 884; Z.S.); the NHMRC of Australia (Senior Principal Research Fellowship grant no. 1110751; P.P.L.T.); the GRF of Hong Kong (grant nos 17119117 and 17107915) and the National Natural Science Foundation (grant nos. 81671579 (L. Lu), 31471398 (W.S.B.Y.) and U1804281 (Y. Zhang)).

Author information

Author notes

  1. Stoyan Petkov

    Present address: German Primate Center, Platform Degenerative Diseases, Gottingen, Germany

  2. Jian Yang

    Present address: Key Laboratory of Arrhythmias, Ministry of Education, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China

  3. Heiner Niemann

    Present address: Hannover Medical School (MHH), TwinCore, Hannover, Germany

  4. These authors contributed equally: Xuefei Gao, Monika Nowak-Imialek, Xi Chen.

Authors and Affiliations

  1. School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Stem Cell and Regenerative Medicine Consortium, Pokfulam, Hong Kong

    Xuefei Gao, Degong Ruan, Jian Wu & Pentao Liu

  2. The Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK

    Xuefei Gao, Xi Chen, David Ryan, Jian Yang, Liliana Antunes, Lia S. Campos, Beiyuan Fu, Yu Yong, Fengtang Yang, Sarah A. Teichmann & Pentao Liu

  3. Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Mariensee, Neustadt, Germany

    Monika Nowak-Imialek, Doris Herrmann, Stoyan Petkov & Heiner Niemann

  4. REBIRTH Centre of Excellence, Hannover Medical School, Hannover, Germany

    Monika Nowak-Imialek, Doris Herrmann, Stoyan Petkov & Heiner Niemann

  5. BGI-Shenzhen, Shenzhen, China

    Dongsheng Chen, Jixing Zhong, Jiacheng Zhu, Shengpeng Wang & Zhouchun Shang

  6. China National GeneBank, BGI-Shenzhen, Shenzhen, China

    Dongsheng Chen, Jixing Zhong, Jiacheng Zhu, Shengpeng Wang & Zhouchun Shang

  7. Key Laboratory of Regenerative Biology of Chinese Academy of Sciences, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    Degong Ruan, Xiaomin Wang, Tao Nie, Peng Li, Donghai Wu, Duanqing Pei & Liangxue Lai

  8. Department of Obstetrics and Gynaecology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong

    Andy Chun Hang Chen, Yin Lau Lee & William Shu Biu Yeung

  9. Epigenetics Programme, Babraham Institute, Babraham Research Campus, Cambridge, UK

    Melanie A. Eckersley-Maslin & Wolf Reik

  10. Aston Medical Research Institute, Aston Medical School, Aston University, Birmingham, UK

    Shakil Ahmad & Asif Ahmed

  11. Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    Toshihiro Kobayashi & Azim Surani

  12. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK

    Guocheng Lan & Xiangang Zou

  13. Center for Reproductive Medicine, Shanghai East Hospital, School of Medicine, Tong Ji University, Shanghai, China

    Song-Guo Xue

  14. Chongqing Academy of Animal Sciences and Key Laboratory of Pig Industry Sciences, Department of Agriculture, Chongqing, China

    Liangpeng Ge, Zuohua Liu & Yong Huang

  15. Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China

    Duanqing Pei

  16. Biotherapy Center, The First Affiliated Hospital of Zhengzhou University, Henan, China

    Yi Zhang

  17. Institute of Immunology, School of Medicine, Shanghai Jiaotong University, Shanghai, China

    Liming Lu

  18. Faculty of Biology Medicine and Health, University of Manchester, Manchester, UK

    Susan J. Kimber

  19. Embryology Unit, Children’s Medical Research Institute and School of Medical Sciences, Sydney Medical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia

    Patrick P. L. Tam

Authors
  1. Xuefei Gao
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  2. Monika Nowak-Imialek
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  3. Xi Chen
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  46. Heiner Niemann
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  47. Pentao Liu
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Contributions

X.G. developed the culture conditions for the pEPSCs and hEPSCs and performed most of the experiments. M.N.-I. performed the pig experiments and wrote the paper. D.H. was critically involved in all porcine experiments. S.P. provided some pig reprogramming factor genes. X.C. performed most of the informatics analyses with support from S.A.T. D.C., S.W., J. Zhong, J. Zhu, Z.S. and J.W. analysed the RNA-Seq data of hEPSCs/ESCs to trophoblasts. A.C.H.C., Y.L.L. and W.S.B.Y. performed the teratoma and TSC lesion experiments at the HKU. M.A.E.-M. and W.R. performed the EPSC DNA methylation analysis. T.K. and A.S. helped X.G. with the PGCLC analysis. S.A. and A.A. measured hormones in cells differentiated from hEPSCs. L.S.C. analysed the teratoma sections. F.Y. and B.F. karyotyped cells. D.Ruan, X.W., L.G., Z.L., Y.H., T.N., D.W., D.P., P. Li, L. Lai, G.L., D. Ryan, J.Y., L.A., Y.Y., S.-G.X., Y.Z., L. Lu and X.Z. were involved in refining the culture conditions or provided intellectual input. S.J.K. provided human M1 and M10 ESCs. P.P.L.T. provided intellectual input and edited the manuscript. H.N. conceptualized the pig experiments, wrote the paper and secured funding for the pig part of the experiments. P. Liu conceived the pEPSC culture screen concept, supervised the research and wrote paper.

Corresponding authors

Correspondence to Heiner Niemann or Pentao Liu.

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Competing interests

Patent applications have been filed relating to the data presented here on behalf of the Sanger Institute and the University of Hong Kong.

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Integrated supplementary information

Supplementary Fig. 1 Culture condition screening for porcine EPSC.

a–g. The relative expression levels of endogenous OCT4 and NANOG in the survived cells after 6 days of culture in different basal media supplemented with inhibitors and cytokines combinations: a. M15 medium without Dox; b. N2B27 basal medium without Dox; c. 20% KOSR medium without Dox; d. AlbumMax II basal medium without Dox; e. N2B27 basal medium with Dox; f. Four individual basal media with Dox (M15: 411-431; N2B27: 432-453; KOSR: 454-475; AlbumMax II: 476-497); g. N2B27 basal medium without Dox. 2i: GSK3i and MEKi; t2i: GSK3i, MEKi and PKCi (Takashima, Y., et al. 2014 Cell); 4i: GSK3i, MEKi, JNKi and p38i (Irie, N., et al 2015 Cell); 5i: GSK3i, MEKi, ROCKi, BRAFi and SRCi (Theunissen, T. W., et al. 2014 Cell Stem Cell); mEPSCM: GSK3i, MEKi, JNKi, XAV939, SRCi and p38i (Yang J., et al. 2017 Nature); Details of the inhibitor combinations are presented in Supplementary Table 1. Relative expression levels were normalized to GAPDH. h. Images showing the toxicity of MEKi, PKCi and p38i to the porcine iPSCs in M15 plus Dox. i. Endogenous pluripotency gene expression in porcine iPSCs in the absence of Dox in pEPSCM (#517 minimal condition, Supplementary Fig. 2h). Gene expression was compared to that of porcine blastocysts. n = 3 independent experiments. Data are mean ± s.d. j. Detection of expression of the exogenous reprogramming factors by RT-PCR showed no detectable leaky expression in about half of the iPSC lines. For h and j, the experiments were repeated independently three times with similar results. Statistical source data are presented in Supplementary Table 1 and Supplementary Table 10. Scale bar: 100 μm.

Supplementary Fig. 2 Establishment of porcine EPSCs by reprogramming PFFs or from pre-implantation embryos.

a. Schematic diagram of reprogramming PFFs to establish EPSC lines in pEPSCM. b. Two WT pEPSCiPS lines (#10 and #11) were examined for expression of endogenous pluripotency genes and the exogenous reprogramming factors. n = 3 independent experiments. Data are mean ± s.d. c. Day-10 outgrowth from a porcine early blastocyst in pEPSCM supplemented with a ROCK inhibitor. The outgrowths were picked at day 10–12 for dissociation and re-plating to establish stable lines. d. Representative images of the pEPSCEmb (Line K3) established from pig in vivo derived embryos. Experiments were performed at least three times. e. pEPSCEmb (Line K3) retained a normal karyotype after 25 passages (10/10 metaphase spreads examined were normal). Two additional lines examined also maintained the normal karyotype after more than 25 passages. f, g. pEPSCs were cultured under seven previously reported porcine ESCs conditions for 7 days, and cell morphology and gene expression were examined. f. Immunofluorescence staining for OCT4 expression. g. RT-qPCR detection of OCT4 and NANOG in pEPSCs. n = 3 independent experiments. Data are mean ± s.d. h. Active Oct4 distal enhancer in porcine EPSCEmb and EPSCiPS. The mouse Oct4 distal and proximal enhancer constructs were used in the luciferase assay. n = 4 independent experiments. Data are mean ± s.d. i. Genome-editing in pEPSCsEmb: Knocking-in the H2B–mCherry expressing cassette into pig ROSA26 locus by Crispr/Cas9. 5 out 20 colonies picked for genotyping were correctly targeted and retained a normal karyotype. j. Bright-field and fluorescence images of the pEPSCEmb colonies with the H2B–mCherry correctly targeted to the ROSA26 locus. k. in vitro differentiation of pEPSCEmb to cells of the germ layers and the trophectoderm lineage (KRT7+). Relative expression levels were normalized to GAPDH. For c, f and i–k, the experiments were repeated independently three times with similar results. Statistical source data are provided in Supplementary Table 10. Scale bars: 100 μm.

Supplementary Fig. 3 In vivo differentiation potential of pEPSCs.

a. Participation of pEPSCs in preimplantation embryo development. H2B–mCherry-expressing donor pEPSCsiPS were injected into day 5 host pig parthenogenetic embryos, which developed to blastocysts. H2BmCherry+ donor cells were found in both the inner cell mass and the trophectoderm (arrowed). Scale bar: 50 μm. The experiments were repeated independently three times with similar results. b. Whole-mount fluorescence and bright-field images of 27-day pig conceptuses derived from preimplantation embryos injected with H2BmCherry+ pEPSCsEmb, showing mCherry+ cells in chimera #21. Scale bar: 1.0 cm. c. Chimeras were processed with half of which were fixed for immunofluorescence analysis, and the other half for FACS and DNA genotyping. To prepare cells for FACS analysis, tissues of each embryo were isolated from head (a), trunk (b), tail (c), and the placenta (d), and were dissociated to single cells and selected for donor H2BmCherry+ cells. Because of the various sizes of the dissociated cells analysed, the majority of starting cells were included in FSC/SSC gates. The boundary between positive and negative is defined according the negative control. The dissociated cells were also used for preparing genomic DNA samples for PCR analysis. d. PCR genotyping for mCherry DNA using the genomic DNA samples. mCherry DNA was detected only in the embryos that were mCherry+ by flow cytometry analysis. e. Schematic diagram of day 26–28 pig chimera conceptuses. The circles mark the tissue areas where tissue sections were taken for immunostaining and imaging shown in (f). f. Immunofluorescence analysis of cryosections of day 26–28 mCherry+ conceptuses or chimeric embryos and placentas for H2BmCherry+ cells in different tissues. The antibodies used include TUJ1 for neurons (Chimera #16); SOX17 and GATA4 for endodermal derivatives (Chimera #21); a-SMA for mesodermal derivatives (Chimera #21); PL-1 and KRT7 for trophoblasts (placenta of Chimera #6). H2BmCherry, GATA4 and SOX17 were found in the nucleus, whereas TUJ, A-SMA, KRT7 and PL-1 were not nuclear localized. Scale bars: 100 μm. For b–d and f, the experiments were repeated independently three times with similar results.

Supplementary Fig. 4 Differentiation of pEPSCs to pPGCLCs.

a. Generation of the NANOS3-H2BmCherry reporter EPSCsEmb by targeting the H2B–mCherry cassette to the NANOS3 locus. In the targeted allele, the T2A-H2B–mCherry sequence was in frame with the last coding exon of the pig NANOS3 locus with the stop codon TAA being deleted. We generated gRNA plasmids targeting specifically to the region covering the NANOS3 stop codon, and 4 out 15 colonies picked for genotyping were correctly targeted. After expansion, those targeted pEPSCs retained a normal karyotype. b. Diagram illustrating the strategy for expressing exogenous genes in pEPSCsEmb for pPGCLC specification and differentiation (see Methods for details). c. Expression of NANOG, BLIMP1 and TFAP2C individually or in combination with SOX17 in the pPGCLCs (H2BmCherry+) in EBs differentiation from NANOS3-H2BmCherry reporter EPSCsEmb. Scale bars: 100 μm. d. Quantitation of NANOS3-H2BmCherry positive cells in (c). n = 4 independent experiments. Data are mean ± s.d. P values were calculated using a two-tailed t-test. e. RT-qPCR analysis of PGC genes. RNA samples were prepared from day 3 EBs of pEPSCs that expressed transgenes individually or in combinations following the pPGCLC induction protocol in (b). Expression levels were normalized to GAPDH. n = 3 independent experiments. Data are mean ± s.d. Statistical source data are provided in Supplementary table 10.

Supplementary Fig. 5 Establishment and characterisation of human EPSCs.

a. Images of H1, H9, M1 and M10 human ESC colonies in pEPSCM or in pEPSCM without ACTIVIN A. Expression of OCT4 was detected by immunostaining. b. Normal karyotype in H1-EPSCs and M1-EPSCs after 25 passages in hEPSCM (10/10 metaphases scored were normal). c. Primary iPSC colony (top) and established cultures of iPSCs (bottom) in hEPSCM reprogrammed from human fibroblasts by Dox-inducible expression of exogenous OCT4, MYC, KLF4, SOX2, LRH1 and RARG. d. Analysis of the expression of the exogenous reprogramming factors by RT-qPCR revealed no obvious leaky expression in four established iPSC lines. e. The relative population doubling time of H1-ESCs, H1-naive ESCs (5i), H1-EPSCs and iPSC-EPSCs. n = 4 independent experiments. Data are mean ± s.d. P values were calculated using a two-tailed t-test. f. Expression of lineage markers (EOMES, GATA4, GATA6, T, SOX17 and RUNX1) in H1-ESCs, H1-naive ESCs (5i), H1-EPSCs and iPSC-EPSCs. n = 3 independent experiments. Data are mean ± s.d. P values were calculated between H1-ESCa and H1-EPSCs using a two-tailed t-test g. Immunostaining of H1-EPSCs and iPSC-EPSCs for pluripotency factors and cell surface markers. h. In vitro differentiation of H1-EPSCs to the germ layer lineages. i. The presence of cartilage (mesoderm. I), glandular epithelium (endoderm. II) and mature neural tissue (glia and neurons, ectoderm. III) in teratomas derived from hEPSCs. H&E staining. j. FACS analysis for expression of CD38 and TNAP on PGCLCs of H1-EPSCs. The induction of PGCLCs was performed on at least two human EPSC lines. Relative expression levels were normalized to GAPDH. For a, c, d and g–j, the experiments were repeated independently three times with similar results. Statistical source data are presented in Supplementary table 10. Scale bars: 100 μm.

Supplementary Fig. 6 RNAseq analysis of human and porcine EPSC transcriptome.

a, b. Expression of pluripotency and lineage genes in porcine (a) or human (b) EPSCs. c, d. Expression of trophoblast related genes in porcine (c) or human (d) EPSCs. For a-d, n = 2 independent replicates per sample. e. Global DNA methylation levels in porcine and human EPSCs. H1-5i human naive ESCs was included in the analysis. n = 3 independent experiments. Data are mean ± s.d. *p < 0.01, comparison of H1-5i human naive ESCs with H1-ESCs and H1-EPSCs. The P values were computed by two-tailed t-test and are presented in Supplementary table 10. f, g. RNAseq analysis of expression of genes encoding enzymes for DNA methylation or demethylation in porcine (f) and human (g) EPSCs. N = 2 independent replicates per sample. h. PCA of scRNAseq data of human H1-EPSCs and that of human preimplantation embryos (data from ref. 38). H1-EPSCs (lower panel, n = 96), Oocyte (n = 4), Zygote (n = 5), 2c-ell (n = 4), 4-cell (n = 4), 8-cell (n = 4), Morula (n = 4), ICM (n = 3), Early Blastocyst (n = 3), Middle Blastocyst (n = 3), Late Blastocyst (n = 3), TE (n = 3). N numbers represent the number of cells. i. Violin plots show the distribution and probability density of histone gene expression (scRNAseq) in human EPSCs (this study) and in human preimplantation embryos at various developmental stages (ref. 38). H1-EPSCs (n = 96), Oocyte (n = 3), zygote (n = 3), 2-cell (n = 6), 4-cell (n = 12), 8-cell (n = 20), morulae (n = 16), Late blastocyst (n = 30). N numbers represent the number of cells. Gene expression (TPM) was quantified by salmon and the values of log10(TPM + 1). Gene expression level in individual cells (represented by dots to show the distribution of the data) was superimposed on the violin plot.

Supplementary Fig. 7 Trophoblast differentiation of hEPSCs.

a. Generation of the CDX2-H2BVenus reporter EPSC line where the T2A-H2BVenus was in frame with the last coding exon of CDX2 gene with the TGA stop codon being deleted. The reporter EPSCs were subsequently cultured in hEPSCM, the FGF-containing ESC medium or in the 5i condition for subsequent analyses. b, c. Expression of trophoblast-related genes analysed by RT-qPCR of trophoblasts produced after 4-day BMP4 treatment (b) or by SB431542+PD173074+BMP4 sampled at several time points (c). Relative expression levels were normalized to GAPDH. *p < 0.01 compared with H1-ESCs and H1-5i-naive cells. n = 3 independent experiments. Data are mean ± s.d. The P values were computed by two-tailed t-test. d. Pearson correlation coefficient of gene expression in cells differentiated from H1-ESCs, H1-EPSCs and iPSC-EPSCs, incorporating published data (ref. 43) of primary human undifferentiated (PHTu) and differentiated trophoblasts (PHTd), and human tissues. e. C19MC miRNAs in EPSCs or ESCs treated with SB431542 for six days. JEG-3 and JAR represent extravillous trophoblasts and villous trophoblast cells, respectively. f. Expression of the same miRNAs in the BMP4 (4-day) treated EPSCs and ESCs. For both e and f, *p < 0.05 compared with H1-ESCs. n = 3 independent experiments. Data are mean ± s.d. The P values were computed by two-tailed t-test. miRNA expression levels are normalized to miR-103a. g. CRISPR/Cas9 mediated deletion of ~350bp in PARG exon 4 using two gRNAs (g1, g2) in CDX2-H2BVenus reporter hEPSCs. 6 out of 48 clones picked were bi-allelic mutants by PCR and sequencing. h. Trophoblast differentiation of PARG homozygous deletion CDX2-reporter EPSC cells (PARG-/-) under TGFβ inhibition for four days and analysed by flow cytometry. The experiments were repeated independently four times with similar results. i. The percentages of Venus+ cells as in (h). n = 4 independent experiments. Data are mean ± s.d. P values were computed by two-tailed t-test. Similar results were obtained using two independent PARG-/- hEPSC lines. j. RT-qPCR analysis of trophoblast genes in WT or PARG-/- H1-EPSCs after 6 days of SB431542 treatment. n = 3 independent experiments. Data are mean ± s.d. P values were computed by two-tailed t-test. Expression levels are normalized to GAPDH. Statistical source data are presented in Supplementary table 10.

Supplementary Fig. 8 Derivation and characterisation of trophoblast stem cell-like cells (pTSCs) from porcine EPSCs.

a. H3K27me3 and H3K4me3 marks at the loci encoding factors associated with placenta development in pEPSCEmb and human H1-EPSCs. b. Images of primary TSC colonies (top) formed from individual pEPSCEmb on day 7 cultured in human TSC condition, and of established pTSCs at passage 7 (bottom). Dashed lines mark the area of putative trophoblasts, which were picked for establishing stable pTSC lines. c. RT-qPCR analysis of pluripotency and trophoblast genes in four pTSC lines and their parental pEPSCEmb. n = 3 independent experiments. Data are mean ± s.d. *p < 0.01 comparison between pEPSCs to pTSCs. P values were computed by two-tailed t-test. Expression levels are normalized to GAPDH. d. Expression of trophoblast factors GATA3 and KRT7 in pTSCs detected by immunostaining. DAPI stained nuclei. e. Confocal image of immunostaining of sections of lesions formed by pTSCs in NOD-SCID mice for cells expressing SDC1 and KRT7. f. Histology of the pTSC-derived lesions. H&E staining. g. Confocal images of immunostaining of porcine blastocysts 1 to 2 days following injection of H2B–mCherry-expressing pTSCs into porcine parthenogenetic or IVF morulae and early blastocysts (50 blastocysts in two injections). Arrow indicates H2B–mCherry+ cells in the trophectoderm which expressed the porcine trophectoderm transcription factor GATA3 and CDX2. For a, b and d–g, the experiments were repeated independently three times with similar results. Statistical source data are presented in Supplementary table 10. Scale bars: 100 μm.

Supplementary Fig. 9 Unprocessed images of all gels and blots.

Unprocessed images for Fig. 6c, e. Unprocessed images for Supplementary Fig. 1j. Unprocessed images for Supplementary Fig. 2i. Unprocessed images for Supplementary Fig. 3d. Unprocessed images for Supplementary Fig. 4a. Unprocessed images for Supplementary Fig. 5d. Unprocessed images for Supplementary Fig. 7a, g.

Supplementary information

Supplementary Information

Supplementary Figures 1-9, Supplementary Table titles/legends

Reporting Summary

Supplementary Table 1

Screening porcine EPSC culture conditions

Supplementary Table 2

Derivation of EPSCs from porcine preimplantation embryos

Supplementary Table 3

Summary of pEPSC microinjection

Supplementary Table 4

pEPSC chimera analysis #1- 7 #29

Supplementary Table 5

pEPSC chimera analysis #30- 8 #45

Supplementary Table 6

Highly differentially expressed genes (8 folds) in H1EPSCs vs. H1-ESCs and 10 preimplantation embryos

Supplementary Table 7

Antibodies used in this study

Supplementary Table 8

PCR primers used in this study

Supplementary Table 9

Probes in RT-qPCR analysis

Supplementary Table 10

Statistics source data

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Gao, X., Nowak-Imialek, M., Chen, X. et al. Establishment of porcine and human expanded potential stem cells. Nat Cell Biol 21, 687–699 (2019). https://doi.org/10.1038/s41556-019-0333-2

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