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An alternative pluripotent state confers interspecies chimaeric competency

Abstract

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

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Figure 1: The effects of culture parameters on epiblast explants.
Figure 2: Characterization of rsEpiSCs.
Figure 3: Global transcriptomic and epigenomic analysis.
Figure 4: In vivo relevance of rsEpiSCs.
Figure 5: Primate region specific PSCs.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray, RNA-seq, ChIP-seq and MethylC-seq data have been deposited in the Gene Expression Omnibus under accession number GSE60605.

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Acknowledgements

We would like to thank S. Mitalipov and J. Thomson for providing rhesus ESCs and iPSCs, respectively, F. Gage for providing chimpanzee iPSCs, K. Zhang for assistance with cell line derivation, M. Ku of the H. A. and Mary K. Chapman Charitable Foundations Genomic Sequencing Core for performing RNA-seq and mouse ChIP-seq experiments, M. Chang of the Integrative Genomic and Bioinformatics Core for bioinformatics analysis, W. T. Berggren and the staff of the Salk STEM Core for preparation of custom-mTeSR1 base medium and supply of validated stem culture materials, G. Pao and K. Hasegawa for discussions, Y. Dayn from transgenic core facility and J. Luo for blastocyst injections, Y. Tsunekawa for providing the mutant eGFP human ESCs reporter line, E. O’Connor and K. Marquez of Human Embryonic Stem Cell Core Facility of Sanford Consortium for Regenerative Medicine for FACS sorting, R. H. Benitez, A. Goebl, R. D. Soligalia for assistance with genome editing, M. F. Pera for critical reading of the manuscript, and M. Schwarz, and P. Schwarz for administrative help. M.L. and K.S. are supported by a California Institute for Regenerative Medicine Training Grant. We thank J. L. Mendoza for his support on this project. This work was funded in part by UCAM (mouse studies). J.R.E. is an Investigator of the Howard Hughes Medical Institute. P.G. was supported by Fundacion Pedro Guillen. Work in the laboratory of J.C.I.B. was supported by G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust and The Moxie Foundation.

Author information

Authors and Affiliations

Authors

Contributions

J.W., D.O. and J.C.I.B. conceived the study. J.W. and D.O. derived mESC, EpiSC and rsEpiSC lines. J.W., D.O. and C.R.E. designed and performed in vivo embryo grafting experiments. J.W., D.O., M.L., K.S., L.M., Z.L., T.H. and P.R. designed and performed all in vitro experiments; J.M.C., J.L. and P.G. helped project design and discussions and performed microarray experiments. I.T., Y.T. performed bisulfite sequencing experiments; M.K. performed teratoma studies; C.L., Y.H., Z.Z., J.R.N. and J.E. performed whole-genome bisulfite sequencing experiments and analysed data. T.D. and B.R. performed ChIP-seq experiments. C.B. and M.L. performed bioinformatics analysis. A.S. analysed global metabolic profiling data; E.A. and N.K. provided technical support. J.W., D.O., M.L. and J.C.I.B. prepared the manuscript.

Corresponding author

Correspondence to Juan Carlos Izpisua Belmonte.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The effects of culture parameters on epiblast explants.

Isolated epiblasts from E5.75 embryos were plated onto mitotically inactivated MEFs in the following media. a, In EpiSC derivation medium containing 20% KnockOut serum replacement (KSR), 20 ng ml−1 Activin-A and 12 ng ml−1 FGF2. Day 3 outgrowth was stained with pluripotency markers OCT4 and SSEA-1. b, In N2B27 media supplemented with indicated growth factors and small molecules. After 4 days, outgrowths of plated epiblasts were stained for OCT4 and SSEA-1. c, Percentages of SSEA-1+/OCT4+ cells in day 4 epiblast outgrowths in N2B27KSR+F/A and N2B27F/R1 culture conditions. A simple randomization method was applied to randomly pick the microscope fields of views for counting the number of SSEA-1+/OCT4+ cells and total cell numbers. PC, phase contrast. For examining different culture parameter effects, all isolated E5.75 epiblasts were pooled together and randomly allocated to each condition.

Extended Data Figure 2 Mechanistic studies of rsEpiSC derivation.

Isolated epiblasts from E5.75 embryos were plated onto mitotically inactivated MEFs in the following media. a, In N2B27 media with indicated treatments. NT, no treatment. After 4 days, outgrowths of plated epiblasts were stained with endodermal marker FOXA2, mesodermal marker T, neuroectodermal marker SOX2 and pluripotent marker OCT4. b, In N2B27 media supplemented with either 20% FBS (top) or 20% KnockOut serum replacement (KSR; bottom). Day 4 outgrowths were fixed and stained with mesodermal marker T and pluripotency marker OCT4. Nuclei were counterstained with DAPI. Scale bar, 125 μm.

Extended Data Figure 3 Highly efficient derivation of rsEpiSCs.

a, Derivation of rsEpiSCs with different passaging methods: collagenase IV (top) and trypsin (bottom). Shown are phase-contrast images of day 4 epiblast outgrowths and cells at passage 1 (P1) and passage 10 (P10). b, Derivation efficiency is compared between EpiSCs and rsEpiSCs using isolated E5.75 epiblasts from three different mouse strains. c, Derivation of rsEpiSCs using epiblasts isolated from different developmental stages of post-implantation embryos. Typical morphologies of staged embryos at E5.25, 5.75, 6.5, 7.25 and 7.5 are shown in the left panel. Day 2 and day 4 epiblast outgrowths as well as colonies of P1 are shown. d, Real-time quantitative PCR analysis of expression of pluripotent (Oct4, Sox2 and Nanog), naive (Rex1 and Klf2) and primed (Otx2 and Fgf5) PSC marker genes in mouse ESCs and rsEpiSCs derived from different post-implantation developmental stages. Error bars indicate s.d. (n = 3, biological samples). e, Derivation of rsEpiSCs from E3.5 pre-implantation blastocysts. Zona pellucida was removed with acidic Tyrode’s solution, followed by the removal of trophectoderm by immunosurgery. Isolated inner cell mass was used for the derivation of rsEpiSCs. Arrows and arrowheads point to the intact trophectoderm and destroyed trophectoderm, respectively. f, Schematic representation of dissection of isolated E6.5 epiblasts into four pieces: anterior-proximal (AP), anterior-distal (AD), posterior-proximal (PP) and posterior-distal (PD). g, Derivation of rsEpiSCs from four regions of E6.5 epiblasts. Day 2 and day 4 epiblast outgrowths as well as colonies of passage 5 (P5) are shown. h, Real-time quantitative PCR analysis of expression of pluripotent (Oct4, Sox2 and Nanog), naive (Rex1 and Klf2) and primed (Otx2 and Fgf5) PSC marker genes in rsEpiSCs derived from whole, AP, AD, PP and PD regions of E6.5 epiblasts. Error bars indicate s.d. (n = 3, biological samples). i, Derivation of rsEpiSCs using other Wnt inhibitors: XAV939 (2.5 µM) and IWP2 (2.5 µM). Day 4 epiblast outgrowths and colonies at passage 10 (P10) were shown. j, Real-time quantitative PCR analysis of expression of pluripotent (Oct4, Sox2 and Nanog), endodermal (Sox17 and Gsc), mesodermal (Eomes and Mixl1) and neuroectodermal (Sox1 and Pax6) marker genes in rsEpiSCs derived using different Wnt inhibitors. Error bars indicate s.d. (d, h, j, n = 3, biological replicates).

Source data

Extended Data Figure 4 Characterization of rsEpiSCs.

a, Quantitative PCR analysis of expression of pluripotent, naive and primed PSC markers in mouse ESCs, EpiSCs and rsEpiSCs. Error bars indicate s.d. (n = 3, biological replicates); t-test, **P < 0.01, *P < 0.05. b, Expression of OCT4, NANOG, SOX2, DNMT3B and SSEA-1 proteins in mouse rsEpiSCs was analysed by immunofluorescence. Mouse rsEpiSCs also displayed weak alkaline phosphatase activity and showed positive H3K27me3 foci confirming an inactivated X chromosome in female rsEpiSCs. c, Western blot analyses of OCT4, NANOG and SOX2 protein levels in mouse ESCs, EpiSCs and four different lines of rsEpiSCs. β-actin was used as loading control. For NANOG, an additional long-exposure image was shown (without ESC sample loaded). For full scan associated with b, refer to Supplementary Information. d, DNA methylation patterns of Oct4, Dppa5 and Stella promoters in mESCs, EpiSCs and rsEpiSCs. e, Representative bright-field images showing colonies stained by immunohistochemistry for OCT4 expression after being plated at clonal density (500 cells per well), and cultured for 5 days. Y27632 was added at 10 µM. f, Karyotype analysis of mouse rsEpiSCs indicates a normal diploid chromosome content. g, Flow cytometry analysis of OCT4, SOX2 and NANOG expression in mouse ESCs, EpiSCs and rsEpiSCs. h, Cell-cycle profiles of mouse ESCs and rsEpiSCs analysed by flow cytometry. i, Haematoxylin and eosin staining images of teratomas generated by rsEpiSCs show lineage differentiation towards three germ layers. j, Teratomas generated by rsEpiSCs showed tri-lineage differentiation as examined by immunofluorescence analysis using FOXA2 (endoderm), TUJ1 (neuroectoderm) and ASMA (mesoderm) antibodies. k, Teratomas generated by injecting indicated number of cells in testis of NOD/SCID male mice. EpiSCs and three different lines of rsEpiSCs were used for comparison. After one month, mice were euthanized. Teratomas were retrieved and measured in size and weight. (EpiSCs, n = 1, biological replicate, two technical replicates; rsEpiSCs, n = 3, biological replicates, two technical replicates per line; error bars, s.d.) l, Flow cytometry analysis of TUJ1, ASMA and Ep-CAM expression in teratomas generated by EpiSCs and three different lines of rsEpiSCs. m, Bright-field images of isolated non-intact and non-viable E7.25–7.5 mouse embryos before and after in vitro embryo culture.

Source data

Extended Data Figure 5 Mechanistic studies of rsEpiSCs self-renewal.

a, Phase-contrast images showing colony morphologies of rsEpiSCs after 4 days of indicated treatments. Left, N2B27 media alone; right, N2B27F/R1. b, Quantitative PCR analysis of expression of pluripotent (Oct4, Sox2 and Nanog), endodermal (Sox17 and Gsc), mesodermal (Eomes and Mixl1) and neuroectodermal (Sox1 and Pax6) markers after indicated treatments for 4 days in culture. Error bars indicate s.d. (n = 3, biological replicates). c, Schematic representation of how different signalling pathways are involved in promoting or inhibiting self-renewal of rsEpiSCs.

Extended Data Figure 6 Global transcriptomic and epigenomic analysis.

a, Hierarchical clustering of microarray gene expression data from ESCs, EpiSCs, rsEpiSCs and in vivo isolated E5.75 and E6.5 epiblasts. b, Two-way scatter plot of gene expression data from RNA-seq of EpiSCs and rsEpiSCs. Black lines indicate fourfold cut-off in expression level difference. Pearson correlation coefficient (r) between samples is shown at the upper right corner. c, Top five Gene Ontology (GO) terms enriched in the set of genes that are differentially expressed by at least fourfold (either up or down) between rsEpiSCs and EpiSCs. d, Average H3K27me3 signal at Polycomb target genes in rsEpiSCs (purple) and EpiSCs (green). e, Plots of DNA methylation and histone methylation (H3K4me3 and H3K27me3) signals around the transcription start sites of representative classes of genes. Examples given include pluripotent genes (Sall4, Klf4 and Lin28a), neuronal-related genes (Sox2, Gbx2 and Sox1) and cell-membrane-related genes (Cldn6, Cldn3 and Cdh1). f, Global cytosine methylation at CG sites (mCG) levels of EpiSCs and rsEpiSCs (top left). The numbers of hyper- and hypo-DMRs discovered in rsEpiSCs (top right). The numbers of promoter-associated (transcription start site ± 2.5 kb), distal (>10 kb from transcription start site) and proximal (transcription start site ± 2.5 to 10 kb) rsEpiSCs hyper- and hypo-DMRs (bottom). g, Positive correlation between the amount of gene body non-CG DNA methylation and the level of gene expression. h, Gene Ontology biological process and molecular function term enrichment for genomic regions associated with rsEpiSCs hyper-DMRs. i, PC1–PC2 plane from PCA analysis of transcriptome comparison between samples from this study (rsEpiSCs (circled with red line) and EpiSCs (circled with yellow line)) and a published data set6 (in vivo epiblast isolated from different developmental stages (CAV, cavity; PS, pre-streak; LMS, late mid-streak; LS, late streak; OB, no bud; EB, early bud; LB, late bud) and EpiSCs (circled with thick blue line)). The green arrow through the in vivo samples delineates a progressing ‘developmental axis’. j, Hierarchical clustering of rsEpiSCs (red), EpiSCs (both from this study (yellow) and ref. 6 (blue)) and epiblasts of CAV, PS, LMS, LS, OB, EB and LB stages (green) using data from all annotated probes. k, Relative expression between rsEpiSCs and in vivo late-streak-stage epiblast (ref. 6) of genes characteristic of anterior mesendoderm (AME), anterior definitive endoderm (ADE), anterior primitive streak (APrS), whole primitive streak (PrS) and posterior primitive streak (PPrS). i, Primordial germ cell induction from Blimp1–YFP mESCs and rsEpiSCs. Left, before induction, both mESCs and rsEpiSCs were found negative for YFP; successful induction was observed with mESCs, as indicated by a positive YFP signal in cell aggregates, but not with rsEpiSCs. Right, PGC induction efficiency was compared between Blimp1–YFP mESCs and rsEpiSCs. Error bars indicate s.d. (n = 3, independent experiments); t-test, **P < 0.01, *P < 0.05. CAV, cavity; PS, pre-streak; ES, early streak; MS, mid-streak; LMS, late mid-streak; LS, late streak; OB/EB, no bud/early bud; LB, late bud.

Extended Data Figure 7 Metabolic profiling of EpiSCs and rsEpiSCs.

a, Basal respiration, b, c, Maximum respiration (b) and ATP production (c) were determined by calculating the average oxygen consumption rate (OCR) for each phase in EpiSCs and rsEpiSCs. d, e, f, Glycolysis (d), glycolytic capacity (e) and reserve glycolysis (f) were determined by calculating the average extracellular acidification rate (ECAR) for each phase in EpiSCs and rsEpiSCs. (Graph Pad Prism v5). g, Representative graph showing the oxygen consumption rate in response to oligomycin, FCCP and rotenone/antimycin of EpiSCs and rsEpiSCs (n = 4). h, Heat map of differentially expressed genes for mitochondrial complex COX and enzymes involved in glycolysis and the tricarboxylic acid cycle selected from the RNA-seq data set, P < 0.05. i, j, Volcano plots of hydrophilic (metabolomics) and hydrophobic (lipidomics) metabolites show broad changes in metabolite levels between EpiSCs and rsEpiSCs. Error bars indicate s.d.; t-test, **P < 0.01, *P < 0.05 (ag, n = 6, technical replicates).

Extended Data Figure 8 F/R1-based culture supports self-renewal of human ESCs as well as iPSC generation.

a, Expression of pluripotency markers SOX2, NANOG, TRA-1-60, TRA-1-80 and SSEA-4 in human H1 rsESCs. b, Representative bright-field images showing colonies visualized by alkaline phosphatase (AP) staining after being plated at clonal density (1,000 cells per well) and cultured for 6 days. Y27632 was added at 10 µM. c, Real-time quantitative PCR analysis of expression of pluripotency marker genes (OCT4, SOX2, NANOG and LIN28) and lineage marker genes (T, NKX1-2 and WNT3) in H1 ESCs, human-foreskin-fibroblast-derived iPSCs, H1 rsESCs and human-foreskin-fibroblast-derived rs-iPSCs. Error bars indicate s.d. (n = 3, biological replicates). d, Haematoxylin and eosin staining images of teratomas generated from human H1 rsESCs show lineage differentiation towards three germ layers. e, Karyotype analysis of human H9 rsESCs indicates a normal diploid chromosome content. f, Representative bright-field images showing morphologies of putative iPSC colonies in conventional F/A-based human ESC culture and F/R1-based culture conditions (top). Alkaline phosphatase staining at day 25 post-nucleofection indicates a larger colony size in F/R1-based culture (bottom). g, Efficiency of iPSC generation in conventional F/A-based human ESC culture conditions and F/R1-based culture conditions. Error bars indicate s.d. (n = 3, independent experiments). h, Quality of human iPSC-like colonies generated in F/A-based and F/R1-based culture conditions. Partial and full alkaline-phosphatase-positive iPSC-like colonies were counted separately using the criteria shown on the right. Error bars indicate s.d. (n = 3, independent experiments). i, Phase-contrast image showing morphology of human-foreskin-fibroblast-derived rs-iPSCs. j, Graphic representation of H3K4me3 and H3K27me3 ChIP-Seq signals near the transcription start site (TSS) for Polycomb target genes in H1 ESCs and H1 rsESCs. k, Average H3K27me3 signal at Polycomb target genes in H1 rsESCs (purple) and H1 ESCs (green). i, Cell-cycle profiles of H1 ESCs and H1 rsESCs were analysed by flow cytometry. m, Flow cytometry analysis of OCT4, SOX2, NANOG and TRA-1-60 protein expression in H1 ESCs and H1 rsESCs.

Extended Data Figure 9 Genome editing in human rsESCs.

a, Schematic representation of the targeted mutagenesis approach employed in human ESCs and rsESCs by CRISPR/Cas9. b, Targeted mutagenesis efficiencies at the LRRK2 locus in human ESCs (treated with Y27632, 10 μM) and rsESCs. c, Schematic representation of the CRISPR/Cas9- or TALEN-mediated gene correction approaches in human ESCs and rsESCs containing a mutated GFP gene. d, GFP correction efficiencies in human ESCs (treated with Y27632, 10 μM) and rsESCs by CRISPR/Cas9 or TALEN. The y axis shows the gene correction efficiency, which was calculated as GFP-positive cells per 5 × 105 cells. Error bars indicate s.d. (n = 3, independent experiments).

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Extended Data Figure 10 Non-human primate rsPSCs.

a, Phase-contrast images of colony morphologies of rhesus macaque rsESCs (ORMES22 and ORMES23), rhesus macaque rsiPSCs and chimpanzee rsiPSCs. b, Immunofluorescence images of NANOG, SOX2, DNMT3b, TRA-1-60 and TRA-1-80 protein expression in ORMES23 rsESCs. ORMES23 rsESCs were also stained for alkaline phosphatase (AP) activity and OCT4 immunohistochemistry (top right). c, Haematoxylin and eosin staining images of teratomas generated by chimpanzee rsiPSCs show lineage differentiation towards three germ layers. d, Schematic representation of epiblast grafting experiment with GFP-labelled ORMES23 rsESCs (Please refer to Supplementary Fig. 1 for details). A, anterior; P, posterior; D, distal. e, Fluorescence images of grafted embryos after in vitro culture. GFP-labelled ORMES23 rsESCs were grafted to posterior, distal and anterior regions of epiblasts of isolated non-intact and non-viable E7.5 mouse embryos and cultured in vitro for 36 h before fixation and visualization by an inverted fluorescence microscope. Arrowhead indicates a cell clump failed to distribute. Dashed line indicates dispersed cells in the posterior region of grafted embryo. Blue, DAPI. Insets show higher-magnification images of GFP-labelled cells. f, Top, quantification of the extent of cell spreading of GFP-labelled ORMES23 rsESCs after being grafted to different regions of E7.5 mouse epiblasts. Bottom, incorporation efficiency of grafted GFP-labelled ORMES23 rsESCs in mouse E7.5 epiblasts. Error bars indicate s.d. (n, indicated on the graph, independent experiments); t-test, *P < 0.05.

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Wu, J., Okamura, D., Li, M. et al. An alternative pluripotent state confers interspecies chimaeric competency. Nature 521, 316–321 (2015). https://doi.org/10.1038/nature14413

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