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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
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.
Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).
Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981).
Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Ying, Q. L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).
Takashima, Y. et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254–1269 (2014).
Theunissen, T. W. et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15, 471–487 (2014).
Ezashi, T., Yuan, Y. & Roberts, R. M. Pluripotent stem cells from domesticated mammals. Annu. Rev. Anim. Biosci. 4, 223–253 (2016).
Brevini, T. A. et al. Culture conditions and signalling networks promoting the establishment of cell lines from parthenogenetic and biparental pig embryos. Stem Cell Rev. 6, 484–495 (2010).
Vassiliev, I. et al. In vitro and in vivo characterization of putative porcine embryonic stem cells. Cell Reprogram. 12, 223–230 (2010).
Haraguchi, S., Kikuchi, K., Nakai, M. & Tokunaga, T. Establishment of self-renewing porcine embryonic stem cell-like cells by signal inhibition. J. Reprod. Dev. 58, 707–716 (2012).
Park, J. K. et al. Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig. PLoS One 8, e52481 (2013).
Hou, D. R. et al. Derivation of porcine embryonic stem-like cells from in vitro-produced blastocyst-stage embryos. Sci. Rep. 6, 25838 (2016).
Xue, B. et al. Porcine pluripotent stem cells derived from IVF embryos contribute to chimeric development in vivo. PLoS One 11, e0151737 (2016).
Ma, Y., Yu, T., Cai, Y. & Wang, H. Preserving self-renewal of porcine pluripotent stem cells in serum-free 3i culture condition and independent of LIF and b-FGF cytokines. Cell Death Discov. 4, 21 (2018).
Yang, J. et al. Establishment of mouse expanded potential stem cells. Nature 550, 393–397 (2017).
Yang, J., Ryan, D. J., Lan, G., Zou, X. & Liu, P. In vitro establishment of expanded-potential stem cells from mouse pre-implantation embryos or embryonic stem cells. Nat. Protoc. 14, 350–378 (2019).
Esteban, M. A. et al. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J. Biol. Chem. 284, 17634–17640 (2009).
Ezashi, T. et al. Derivation of induced pluripotent stem cells from pig somatic cells. Proc. Natl Acad. Sci. USA 106, 10993–10998 (2009).
West, F. D. et al. Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells Dev. 19, 1211–1220 (2010).
Petkov, S., Glage, S., Nowak-Imialek, M. & Niemann, H. Long-term culture of porcine induced pluripotent stem-like cells under feeder-free conditions in the presence of histone deacetylase inhibitors. Stem Cells Dev. 25, 386–394 (2016).
Lai, S. et al. Generation of knock-in pigs carrying Oct4-tdTomato reporter through CRISPR/Cas9-mediated genome engineering. PLoS One 11, e0146562 (2016).
Zhang, W. et al. Pluripotent and metabolic features of two types of porcine iPSCs derived from defined mouse and human ES cell culture conditions. PLoS One 10, e0124562 (2015).
Telugu, B. P., Ezashi, T. & Roberts, R. M. Porcine induced pluripotent stem cells analogous to naive and primed embryonic stem cells of the mouse. Int. J. Dev. Biol. 54, 1703–1711 (2010).
Du, X. et al. Barriers for deriving transgene-free pig iPS cells with episomal vectors. Stem Cells 33, 3228–3238 (2015).
Chen, H. et al. Erk signaling is indispensable for genomic stability and self-renewal of mouse embryonic stem cells. Proc. Natl Acad. Sci. USA 112, E5936–E5943 (2015).
Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011).
Irie, N. et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell 160, 253–268 (2015).
Kobayashi, T. et al. Principles of early human development and germ cell program from conserved model systems. Nature 546, 416–420 (2017).
Julaton, V. T. & Reijo Pera, R. A. NANOS3 function in human germ cell development. Hum. Mol. Genet. 20, 2238–2250 (2011).
Gkountela, S. et al. The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat. Cell Biol. 15, 113–122 (2013).
Camarasa, M. V. et al. Derivation of Man-1 and Man-2 research grade human embryonic stem cell lines. In Vitro Cell. Dev. Biol. Anim. 46, 386–394 (2010).
Ye, J. et al. High quality clinical grade human embryonic stem cell lines derived from fresh discarded embryos. Stem Cell Res. Ther. 8, 128 (2017).
International Stem Cell Initiative. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat. Biotechnol. 25, 803–816 (2007).
Koyanagi-Aoi, M. et al. Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells. Proc. Natl Acad. Sci. USA 110, 20569–20574 (2013).
Theunissen, T. W. et al. Molecular criteria for defining the naive human pluripotent state. Cell Stem Cell 19, 502–515 (2016).
Yang, Y. et al. Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell 169, 243–257 (2017).
Yan, L. et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1131–1139 (2013).
Dang, Y. et al. Tracing the expression of circular RNAs in human pre-implantation embryos. Genome Biol. 17, 130 (2016).
Blakeley, P. et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3613 (2015).
Chen, Y., Blair, K. & Smith, A. Robust self-renewal of rat embryonic stem cells requires fine-tuning of glycogen synthase kinase-3 inhibition. Stem Cell Rep. 1, 209–217 (2013).
Xu, R. H. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).
Amita, M. et al. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc. Natl Acad. Sci. USA 110, E1212–E1221 (2013).
Yabe, S. et al. Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas. Proc. Natl Acad. Sci. USA 113, E2598–E2607 (2016).
Chilosi, M. et al. Differential expression of p57kip2, a maternally imprinted cdk inhibitor, in normal human placenta and gestational trophoblastic disease. Lab. Invest. 78, 269–276 (1998).
Zhang, P., Wong, C., DePinho, R. A., Harper, J. W. & Elledge, S. J. Cooperation between the Cdk inhibitors p27(KIP1) and p57(KIP2) in the control of tissue growth and development. Genes Dev. 12, 3162–3167 (1998).
Okae, H. et al. Derivation of human trophoblast stem cells. Cell Stem Cell 22, 50–63 (2018).
Lee, C. Q. et al. What is trophoblast? A combination of criteria define human first-trimester trophoblast. Stem Cell Rep. 6, 257–272 (2016).
Hemberger, M., Udayashankar, R., Tesar, P., Moore, H. & Burton, G. J. ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta. Hum. Mol. Genet. 19, 2456–2467 (2010).
Ng, R. K. et al. Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5. Nat. Cell Biol. 10, 1280–1290 (2008).
Huang, S. M. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).
Thorsell, A. G. et al. Structural basis for potency and promiscuity in poly(ADP-ribose) polymerase (PARP) and tankyrase inhibitors. J. Med. Chem. 60, 1262–1271 (2017).
Hassa, P. O. & Hottiger, M. O. The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases. Front. Biosci. 13, 3046–3082 (2008).
Hemberger, M. et al. Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives. Dev. Biol. 257, 371–381 (2003).
Koh, D. W. et al. Failure to degrade poly(ADP-ribose) causes increased sensitivity to cytotoxicity and early embryonic lethality. Proc. Natl Acad. Sci. USA 101, 17699–17704 (2004).
Wang, W. et al. Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1. Proc. Natl Acad. Sci. USA 108, 18283–18288 (2011).
Petersen, B. et al. Development and validation of a highly efficient protocol of porcine somatic cloning using preovulatory embryo transfer in peripubertal gilts. Cloning Stem Cells 10, 355–362 (2008).
Holker, M. et al. Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos. Cloning Stem Cells 7, 35–44 (2005).
Pedregosa, F. & Varoquaux, G. Scikit-learn: machine learning in Python. J. Mach. Learn. Res. 12, 2825–2830 (2011).
Harrow, J. et al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1774 (2012).
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
Chang, C. W. et al. Identification of human housekeeping genes and tissue-selective genes by microarray meta-analysis. PloS One 6, e22859 (2011).
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Lee, T. I., Johnstone, S. E. & Young, R. A. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protoc. 1, 729–748 (2006).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Kent, W. J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).
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  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)).
Patent applications have been filed relating to the data presented here on behalf of the Sanger Institute and the University of Hong Kong.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
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.
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.
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.
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.
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.
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.
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 Figures 1-9, Supplementary Table titles/legends
Screening porcine EPSC culture conditions
Derivation of EPSCs from porcine preimplantation embryos
Summary of pEPSC microinjection
pEPSC chimera analysis #1- 7 #29
pEPSC chimera analysis #30- 8 #45
Highly differentially expressed genes (8 folds) in H1EPSCs vs. H1-ESCs and 10 preimplantation embryos
Antibodies used in this study
PCR primers used in this study
Probes in RT-qPCR analysis
Statistics source data
About this article
STEM CELLS (2019)
Cell Stem Cell (2019)
Experimental Cell Research (2019)
Stem Cells and Development (2019)
Journal of Cellular Physiology (2019)