Principles of early human development and germ cell program from conserved model systems

Journal name:
Nature
Volume:
546,
Pages:
416–420
Date published:
DOI:
doi:10.1038/nature22812
Received
Accepted
Published online

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during weeks 2–3 of early post-implantation development1. Using in vitro models of hPGC induction2, 3, 4, recent studies have suggested that there are marked mechanistic differences in the specification of human and mouse PGCs5. This may be due in part to the divergence in their pluripotency networks and early post-implantation development6, 7, 8. As early human embryos are not accessible for direct study, we considered alternatives including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs originate from the posterior pre-primitive-streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. We use this model together with human and monkey in vitro models simulating peri-gastrulation development to show the conserved principles of epiblast development for competency for primordial germ cell fate. This process is followed by initiation of the epigenetic program9, 10, 11 and regulated by a balanced SOX17BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models provides synthetic insights into early human development.

At a glance

Figures

  1. Specification of PGCs in gastrulating porcine embryos.
    Figure 1: Specification of PGCs in gastrulating porcine embryos.

    Serial sections with immunostainings. a, Bilaminar disc embryo (~E9.5–E10). Arrowhead marks the epiblast/trophectoderm boundary. Scale bar, 20 μm. b, Pre-primitive-streak embryo (pre-PS; ~E11). Scale bar, 10 μm. c, Early primitive streak embryo (early-PS; ~E11.5–E12) with SOX17 and BLIMP1 expression. Close-up (dashed lines) shows four SOX17+BLIMP1 cells (arrows). Dashed lines highlight SOX17+BLIMP+ cells. The hypoblast is SOX17+BLIMP1+. Scale bar, 10 μm. d, Primitive streak embryo (PS; ~E12) with a pPGC cluster showing SOX17 and NANOG expression. Four SOX17+ cells without NANOG in the most anterior pPGC cluster (arrows in middle image). The rightmost image (arrows) shows five SOX17+BLIMP1 cells. Arrowheads show anterior primitive streak with SOX17+ definitive endoderm cells. Dashed lines highlight SOX17+BLIMP+ cells. Scale bar, 10 μm. Inset shows the whole embryo. e, Late primitive streak embryo (late-PS; ~E12.5–E13.5) with a pPGC cluster (arrow) showing NANOG, SOX17, TFAP2C, BLIMP1, T and Sda/GM2 expression. Arrowheads, early migratory pPGCs. Scale bar, 25 μm. A–P, anterior–posterior axis. f, Quantification of EdU incorporation in pPGCs and somatic cells. Numbers denote analysed cells (error bars, mean ± s.e.m.). g, Sagittal section of E14.5 embryo immunostained for OCT4 and 5hmC, and the pPGC cluster (white square on the right). Arrows, migratory PGCs. Scale bar, 20 μm. h, Quantification of 5hmC in analysed cells (boxes, mean and interquartile ranges; whiskers, maximum and minimum; Mann–Whitney U-test, *P < 0.01). i, Immunostaining for UHRF1 in E14 embryos. Dashed line delimits the pPGC cluster. Scale bar, 20 μm.

  2. Competence for pPGC specification.
    Figure 2: Competence for pPGC specification.

    a, Representation of porcine epiblasts and signalling for pPGC induction. b, Top-down views of epiblasts. Scale bar, 0.25 mm. c, Serial sections of a pre-PS embryo showing pSMAD1/5/8 and T. Close-up (dashed lines) of posterior epiblast cells (arrows) with nuclear pSMAD but no T. Scale bar, 20 μm. d, Schematic for 64 h epiblast culture. e, Epiblasts prior to culture. Scale bar, 0.5 mm. f, Epiblasts after 64 h culture. g, Scatter plot for triple-positive pPGCs staining (median; Mann–Whitney U-test, P < 0.05). h, Triple-immunostaining of epiblasts. Scale bar, 20 μm. WNTi, WNT inhibitor; BMPi, BMP inhibitor.

  3. Simulation of human peri-gastrulation and hPGC competency.
    Figure 3: Simulation of human peri-gastrulation and hPGC competency.

    a, Schematics showing progressive gain and loss of competency for hPGCs. b, Induction of hPGCs (percentage of NANOS3–tdTomato+ alkaline phosphatase (AP)+ cells) in day 4–5 embryoids. c, Induction of definitive endoderm (DE; percentage of CXCR4+ cells) during 12–24 h of mesendoderm differentiation. d, Immunostaining of hPGCs induced from pre-ME (12 h) and of definitive endoderm induced from mesendoderm (24 h). Dashed lines, SOX17+OCT4+ hPGCs. Scale bar, 50 μm. e, Gene expression (reverse-transcription with quantitative PCR (RT–qPCR)) changes during mesendoderm induction. f, Induction of hPGCs (percentage of NANOS3–tdTomato+AP+ cells) from pre-ME (12 h) or mesendoderm (24 h). Pre-ME are competent for hPGCs in response to cytokines or SOX17, but mesendoderm is not. g, Immunostaining of day 4 embryoids from pre-ME (12 h) or mesendoderm (24 h). SOX17 in pre-ME induced OCT4+BLIMP1+ hPGCs (dashed line), but in mesendoderm, it induced definitive endoderm (BLIMP1+FOXA2+) cells. Scale bar, 50 μm.

  4. Induction of hPGCs by SOX17–BLIMP1 and combined representation of hPGC and pPGC specification.
    Figure 4: Induction of hPGCs by SOX17–BLIMP1 and combined representation of hPGC and pPGC specification.

    a, Induction of hPGCs by transcription factors in comp-hPSCs; SOX17 and BLIMP1 induce hPGCs. b, Day 1–4 embryoids with NANOS3–tdTomato reporter respond to ectopic SOX17 and BLIMP1 with or without cytokines. c, Induction of hPGCs (percentage of NANOS3–tdTomato+AP+ cells) shown in b. d, t-SNE analysis of RNA-seq data. e, Heat map of representative gene expression includes previous data4. f, Day 4 embryoids induced by cytokines. Dashed line indicates positive signals. Arrowheads, OCT4+BLIMP1+ hPGCs expressing FOXA2. Scale bar, 50 μm. g, SOX17 and BLIMP1 gene dosage is critical during hPGC specification: BLIMP1 represses SOX17-induced endodermal genes. h, Representation of human germ cell origin and program based on in vitro simulations from hPSCs and in vivo origin of pPGCs in porcine embryos.

  5. Expression of key germ cell genes in early pPGCs.
    Extended Data Fig. 1: Expression of key germ cell genes in early pPGCs.

    a, Representation of mouse, pig and human embryos before gastrulation. b, Sections of early-PS and primitive-streak-stage embryo showing SOX17, BLIMP1, NANOG and OCT4. Yellow dashed insets show cells at high magnification and white dashed lines mark SOX17+ and/or BLIMP1 cells. Scale bar, 20 μm. c, Primitive-streak-stage embryo (~E12) with a cluster of pPGCs (arrow) with multiple combinations of PGC gene expression (SOX17, BLIMP1, NANOG, TFAP2C, OCT-4, Sda/GM2 and mesoderm gene, T). Arrowheads at the anterior streak point to primitive endoderm (SOX17+BLIMP1+ and NANOG cells. Scale bar, 20 μm. d, Late-PS (~E12.5–E13.5) embryo with a pPGC cluster (arrow) showing NANOG, SOX17 (split colour image of e), BLIMP1, and T expression. Arrowheads mark early migratory PGCs in the primitive endoderm. Scale bar, 25 μm. e, E14 embryo stained for SOX17, BLIMP1 and TFAP2C. Yellow dashed insets show cells at high magnification and white dashed lines mark SOX17+BLIMP1+ and TFAP2C cells. Scale bar, 20 μm. f, Immunostaining for PRDM14 co-stained with Sda/GM2 and SOX17 in E14 (pPGC cluster) embryos and E26 gonads. Arrows point to pPGCs in the gonad. Scale bar, 20 μm.

  6. Proliferation and development of early pPGCs.
    Extended Data Fig. 2: Proliferation and development of early pPGCs.

    a, OCT4 RNA in situ hybridization identifies the pPGC cluster (arrow) in the posterior end of E13.5–E15.5 embryos. Insets show whole embryos. b, Whole-mount OCT4 immunohistochemistry of a porcine embryo. Dashed square marks the area shown at higher magnification on the top right. Arrow points to the pPGC cluster. Bottom right: cross section of the embryo (line in the wholemount image) shows migratory pPGCs (red cells). c, Number of pPGCs at different stages as indicated. d, Immunostaining of EdU-labelled embryos at the indicated stages with different antibody combinations identifying the PGCs. The pPGC cluster is highlighted with dashed white line. Arrows show SOX17+EdU+ and SOX17+BLIMP1+EdU+ cells. Scale bar, 20 μm. e, Immunostained migratory pPGCs (arrows); inset show cells at higher magnification. f, Immunostained gonadal pPGCs. Inset shows SOX2+ neural tissue.

  7. Epigenetic reprogramming in pre- and early migratory pPGCs, and key germ cell markers in migratory pPGC and cultured porcine epiblast.
    Extended Data Fig. 3: Epigenetic reprogramming in pre- and early migratory pPGCs, and key germ cell markers in migratory pPGC and cultured porcine epiblast.

    a, A cluster of pPGCs (dashed line) at E13 stained for TET1 and OCT4. Scale bar, 20 μm. b, Serial sections of E14 embryos immunostained for different epigenetic markers combined with BLIMP1, NANOG and SOX17. Dashed lines highlight pPGC clusters. Scale bar, 20 μm. c, Quantification of 5-methylcytosine and H3K9me2 in embryos of different stages. Numbers of cells analysed are indicated (boxes, mean and interquartile ranges; whiskers, maximum and minimum; *P < 0.01; Mann–Whitney U-test). d, Serial sections of E16 embryos showing migratory pPGCs (arrows) immunostained for the indicated epigenetic marks. Scale bar, 20 μm. e, Triple immunostaining of epiblasts cultured under different conditions. Scale bar, 10 μm.

  8. Characterization of NANOS3–tdTomato reporter hPSC.
    Extended Data Fig. 4: Characterization of NANOS3–tdTomato reporter hPSC.

    a, Targeting strategy for making NANOS3–tdTomato (NT) reporter. b, Representative genotyping of targeted clones using genomic DNA. c, Conventional (conv) and PGC-competent (comp) hPSCs states are reversible; the latter is equivalent to pre-ME (12 h at mesendoderm induction). Conv-hPSCs are cultured in Essential 8 medium on vitronectin coated dishes (see Methods). Comp-hPSCs are cultured in the hPSC medium containing inhibitors (i) (GSK3i, ERKi, p38i, JNKi) on MEF (see Methods). d, NANOS3–tdTomato reporter conv-hPSCs and comp-hPSCs, and day 1–4 embryoids induced with or without cytokines. e, FACS pattern and percentage of NANOS3–tdTomato+AP+ cells shown in Extended Data Fig. 4d. f, FACS pattern and percentage of NANOS3–tdTomato+AP+ cells in multiple clones derived from Wis2 or H9 hESC lines. g, Immunostaining of day 4 embryoids induced from pre-ME (12 h) or comp-hPSCs by BMP containing cytokines. Scale bar, 50 μm. h, Comparison of sensitivity of two NANOS3 reporter cell lines. FACS patterns of day 4 embryoids induced from comp-hPSCs (harbouring NANOS3–mCherry reporter or NANOS3–tdTomato reporter) with or without cytokines. i, Comparison of hPGC induction efficiency derived from pre-ME (12 h) or comp-hPSCs. Representative images and FACS patterns are shown. j, Scatter plot (mean ± s.d.) shows percentage of NANOS3–tdTomato+AP+ cells in indicated condition (n = 6). Paired t-test was used to test for statistical significance (*P < 0.05).

  9. Characterization of pre-ME and mesendoderm induced from Conv-hPSC.
    Extended Data Fig. 5: Characterization of pre-ME and mesendoderm induced from Conv-hPSC.

    a, Immunostaining of conv-hPSCs during 12–24 h mesendoderm induction. Scale bar, 50 μm. b, Gene expression (RT–qPCR) change during mesendoderm induction. c, FACS patterns of day 4 embryoids induced from pre-ME (12 h) with cytokines. Pre-ME was induced with or without GSK3i or activin A. d, FACS patterns of day 4 embryoids induced from pre-ME (12 h) with cytokines. Pre-ME was induced with or without BMP2 or the inhibitor. e, Schematics of definitive endoderm or lateral mesoderm (LM) differentiation from mesendoderm. f, FACS patterns of day 2 definitive endoderm (percentage of CXCR4+) and day 1 lateral mesoderm (percentage of PDGFRa+) induced from 24 h of mesendoderm induction. g, Relative induction efficiency of definitive endoderm or lateral mesoderm from mesendoderm induced with or without BMP2 (definitive endoderm, n = 5; lateral mesoderm, n = 6). Paired t-test was used to test for statistical significance (*P < 0.05). h, Immunostaining of definitive endoderm and lateral mesoderm in Extended Data Fig. 5f. Scale bar, 50 μm. i, Schematic of spatial-temporal progression from conv-hPSCs to mesendoderm and the signalling.

  10. Robust induction of cmPGCs from cells during mesendoderm differentiation.
    Extended Data Fig. 6: Robust induction of cmPGCs from cells during mesendoderm differentiation.

    a, Schematic of in vitro differentiation of cmPSCs. The same system was adopted as shown for conv-hPSCs differentiation in Fig. 3. b, Bright field image of undifferentiated cmPSCs. c, Immunostaining of day 2 embryoids induced with cytokines from cells at 0 h (cmPSCs), 12 h and 24 h during mesendoderm differentiation. Dashed lines highlight SOX17+BLIMP1+TFAP2C+ cmPGCs. Scale bar, 50 μm. d, Immunostaining of day 2 pre-ME-derived embryoids (12 h) in Extended Data Fig. 6c for pluripotency markers. Notably, cmPGCs express SOX17, but not SOX2. By contrast, cmPSC colonies express SOX2 but not SOX17. e, Immunostaining of day 2 monkey definitive endoderm induced from pre-ME (12 h) and mesendoderm (24 h). Scale bar, 50 μm. f, Immunostaining of day 1 monkey lateral mesoderm induced from mesendoderm (24 h). Mesendoderm were induced with or without BMP. Notably, adding BMP during mesendoderm differentiation increased the efficiency for FOXF1+HAND1+ lateral mesoderm cells, as shown in conv-hPSCs (Extended Data Fig. 5e–i). Scale bar, 50 μm.

  11. Chronology of transcription factors expression during hPGC induction.
    Extended Data Fig. 7: Chronology of transcription factors expression during hPGC induction.

    a, Schematic of hPGC induction from pre-ME (12 h). b, Images of day 2 and 4 embryoids in response to BMP2 alone or BMP2 with LIF, SCF and EGF (cytokines). Notably, BMP2 alone can induce hPGC at almost the same efficiency as the full cytokines, but do not survive during extended culture, as shown previously. c, Immunostaining of embryoids induced with BMP2 alone or BMP2 with LIF, SCF and EGF showing expression of SOX17, BLIMP1 and TFAP2C. Scale bar, 50 μm. d, Proportion of SOX17+ cells indicated in Extended Data Fig. 7c. e, Immunostaining of embryoids induced with BMP2 alone or BMP2 with LIF, SCF and EGF showing expression of SOX17, BLIMP1 and NANOG. Scale bar, 50 μm. f, Proportion of SOX17+ cells in Extended Data Fig. 7e.

  12. Effect of NANOG on hPGC induction, characterization of NANOS3–tdTomato reporter hPSCs containing inducible SOX17, BLIMP1 with or without TFAP2C, and similarity between cytokine- and SOX17–BLIMP1-induced hPGCs.
    Extended Data Fig. 8: Effect of NANOG on hPGC induction, characterization of NANOS3–tdTomato reporter hPSCs containing inducible SOX17, BLIMP1 with or without TFAP2C, and similarity between cytokine- and SOX17–BLIMP1-induced hPGCs.

    a, Represents overexpression of dex-inducible NANOG transgenes in NANOS3–tdTomato reporter comp-hPSC. b, Day 4 embryoids following induction of NANOG (by dex), with or without cytokines as indicated. c, FACS patterns after induction of hPGCs; NANOS3–tdTomato+AP+ cells (%) shown in Extended Data Fig. 8b. d, Represents overexpression of dex-inducible SOX17, dox-inducible BLIMP1, Shield1(S1)-inducible TFAP2C transgenes in NANOS3–tdTomato reporter comp-hPSCs. e, Immunostaining of NANOS3–tdTomato reporter comp-hPSCs + inducible SOX17/BLIMP1/TFAP2C (iSBT) 1 day after induction of SOX17, BLIMP1 and TFAP2C by addition of dex, dox or S1. Scale bar, 50 μm. f, Immunostaining of NANOS3–tdTomato reporter comp-hPSCs + iSB 1 day after induction of SOX17 or BLIMP1 by addition of dex or dox. Scale bar, 50 μm. g, Immunostaining of day 2 embyroid induced with or without dex to induce nuclear localization of SOX17. Notably, accumulation of SOX17 signal is observed in +dex condition. h, Changes in gene expression (RT-qPCR) during hPGC induction: comp-hPSCs control (AP+ cells); NANOS3–tdTomato+ hPGCs induced by SOX17–BLIMP1 or cytokines; NANOS3–tdTomato cells in cells exposed to cytokines (error bars, mean ± s.d.). i, Unsupervised hierarchical clustering (UHC) of gene expression. j, Gene set enrichment analysis (GSEA) of 123 hPGC-specific genes (Supplementary Table 1) on the transcriptome of cytokine- and SOX17–BLIMP1-induced hPGCs. k, Heat map showing expression of epigenetic modifiers related to global DNA demethylation. Same datasets as shown in Fig. 4e were used for analysis.

  13. Response of SOX17-knockout comp-hPSC to SOX17.
    Extended Data Fig. 9: Response of SOX17-knockout comp-hPSC to SOX17.

    a, Overexpression of dex-inducible SOX17 (iS) in SOX17-knockout comp-hPSCs. b, Gene expression (RT–qPCR) on day 4 of FACS-sorted NANOS3–mCherry (NC)+alkaline phosphatase (AP)+ hPGCs by RT–qPCR. c, FACS analysis of day 2 embryoids induced from wild-type comp-hPSCs, SOX17-knockout comp-hPSCs and SOX17-knockout comp-hPSCs rescued with SOX17GR transgene (iS) (percentage of CXCR4+AP+ cells). d, FACS pattern of day 2 embyoid induced from NANOS3–tdTomato reporter comp-hPSCs showing AP+CXCR4+ cells expressing NANOS3–tdTomato. e, Represents SOX17-inducible system (iSdd). Expression of SOX17 fused with destabilized domain (DD) can be induced by doxycycline (dox); addition of Shield1 (S1) can stabilize SOX17–DD protein. f, Western blots showing SOX17 expression level in day 5 embryoids from SOX17-knockout + inducible SOX17–DD (iSdd) comp-hPSCs. Embryoids were induced with cytokines. To induce SOX17, different concentration of dox and S1 were added. As controls, NANOS3–mCherry+AP+ hPGCs and NANOS3–mCherryAP cells from wild-type comp-hPSC-derived embryoids induced with cytokines were used. Histone H3 (H3) was used for internal control. g, Immunostaining of day 4 embryoids from SOX17-knockout + iSdd comp-hPSCs. Embryoids from SOX17-knockout and wild-type comp-hPSC-induced with cytokines were used as controls. Scale bar, 50 μm. h, Quantification of immunostaining data in Extended Data Fig. 9g. The numbers of OCT4+BLIMP1+ hPGCs, FOXA2+ endodermal cells and OCT4+BLIMP1+ hPGCs expressing FOXA2 were counted from 3 different embryoids. The proportions of the 3 populations are shown. i, Expression of SOX17 and BLIMP1 (RT–qPCR) in day 4 embyoids in response to different SOX17 dosage.

  14. Response of SOX17-knockout comp-hPSCs to SOX17 and BLIMP1, and changes in epigenetic modifier expression after overexpression of SOX17 and BLIMP1.
    Extended Data Fig. 10: Response of SOX17-knockout comp-hPSCs to SOX17 and BLIMP1, and changes in epigenetic modifier expression after overexpression of SOX17 and BLIMP1.

    a, Overexpression of dex-inducible SOX17 (iS) and dox-inducible BLIMP1 (iB) in SOX17-knockout comp-hPSCs. b, Immunostaining of comp-hPSC 1 day after induction of SOX17 (dex) or BLIMP1 (dox), or both, and day 2 embryoids following dex-induced SOX17 with endogenous or dox-induced BLIMP1. Scale bar, 50 μm. c, Gene expression (RT–qPCR) in day 4 embryoids following induction by dex (+SOX17), dox (+BLIMP1), or dex + dox (+SOX17 and BLIMP1). Bulk cells of embryoids induced from SOX17-knockout and wild-type comp-hPSCs with cytokines were used as controls. d, Upon specification, hPGCs become refractory to activin or Wnt signalling. Left schematic shows the experimental design. The embryoids were transferred to the medium with or without GSK3i (3 μM) or activin A (100 ng ml−1) at day 0, 1 or 2 to see the effect on PGC induction. FACS patterns (right) show the induction efficiency of hPGCs (percentage of NANOS3–tdTomato+AP+) at day 4. e, Day 4 embryoids induced by cytokines with or without activin A or GSK3i.

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Author information

  1. Present address: CEIEPAA-FMVZ-UNAM, Tequisquiapan, Queretaro 76790, Mexico.

    • David A. Contreras
  2. These authors contributed equally to this work.

    • Toshihiro Kobayashi &
    • Haixin Zhang

Affiliations

  1. Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

    • Toshihiro Kobayashi,
    • Walfred W. C. Tang,
    • Naoko Irie,
    • Anastasiya Sybirna,
    • Sabine Dietmann &
    • M. Azim Surani
  2. Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK

    • Toshihiro Kobayashi,
    • Walfred W. C. Tang,
    • Naoko Irie,
    • Anastasiya Sybirna &
    • M. Azim Surani
  3. School of Biosciences, University of Nottingham, Loughborough LE12 5RD, UK

    • Haixin Zhang,
    • Sarah Withey,
    • Doris Klisch,
    • David A. Contreras,
    • Robert Webb &
    • Ramiro Alberio
  4. Wellcome Trust Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

    • Anastasiya Sybirna &
    • Sabine Dietmann
  5. School of Veterinary Medicine and Sciences, University of Nottingham, Loughborough LE12 5RD, UK

    • Cinzia Allegrucci

Contributions

T.K. designed the experiments and performed cell culture, plasmid construction, immunofluorescence, qPCR, RNA-seq, western blots, data analysis and wrote the paper. W.W.C.T. designed experiments and analysed RNA-seq. N.I. performed preliminary work and designed experiments, and S.D. performed bioinformatics. A.S. helped with a hPSC reporter. H.Z., S.W., D.K. and C.A. designed and performed immunofluorescence and culture of pig embryos and epiblasts. D.A.C. and R.W. designed and performed in situ hybridization experiments and immunofluorescence. W.W.C.T., N.I. and S.W. made equal contributions. R.A. supervised the project, designed experiments, performed dissections and wrote the paper. M.A.S. supervised the project, designed experiments, and wrote the paper. All authors contributed to the manuscript.

Competing financial interests

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Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Expression of key germ cell genes in early pPGCs. (802 KB)

    a, Representation of mouse, pig and human embryos before gastrulation. b, Sections of early-PS and primitive-streak-stage embryo showing SOX17, BLIMP1, NANOG and OCT4. Yellow dashed insets show cells at high magnification and white dashed lines mark SOX17+ and/or BLIMP1 cells. Scale bar, 20 μm. c, Primitive-streak-stage embryo (~E12) with a cluster of pPGCs (arrow) with multiple combinations of PGC gene expression (SOX17, BLIMP1, NANOG, TFAP2C, OCT-4, Sda/GM2 and mesoderm gene, T). Arrowheads at the anterior streak point to primitive endoderm (SOX17+BLIMP1+ and NANOG cells. Scale bar, 20 μm. d, Late-PS (~E12.5–E13.5) embryo with a pPGC cluster (arrow) showing NANOG, SOX17 (split colour image of e), BLIMP1, and T expression. Arrowheads mark early migratory PGCs in the primitive endoderm. Scale bar, 25 μm. e, E14 embryo stained for SOX17, BLIMP1 and TFAP2C. Yellow dashed insets show cells at high magnification and white dashed lines mark SOX17+BLIMP1+ and TFAP2C cells. Scale bar, 20 μm. f, Immunostaining for PRDM14 co-stained with Sda/GM2 and SOX17 in E14 (pPGC cluster) embryos and E26 gonads. Arrows point to pPGCs in the gonad. Scale bar, 20 μm.

  2. Extended Data Figure 2: Proliferation and development of early pPGCs. (977 KB)

    a, OCT4 RNA in situ hybridization identifies the pPGC cluster (arrow) in the posterior end of E13.5–E15.5 embryos. Insets show whole embryos. b, Whole-mount OCT4 immunohistochemistry of a porcine embryo. Dashed square marks the area shown at higher magnification on the top right. Arrow points to the pPGC cluster. Bottom right: cross section of the embryo (line in the wholemount image) shows migratory pPGCs (red cells). c, Number of pPGCs at different stages as indicated. d, Immunostaining of EdU-labelled embryos at the indicated stages with different antibody combinations identifying the PGCs. The pPGC cluster is highlighted with dashed white line. Arrows show SOX17+EdU+ and SOX17+BLIMP1+EdU+ cells. Scale bar, 20 μm. e, Immunostained migratory pPGCs (arrows); inset show cells at higher magnification. f, Immunostained gonadal pPGCs. Inset shows SOX2+ neural tissue.

  3. Extended Data Figure 3: Epigenetic reprogramming in pre- and early migratory pPGCs, and key germ cell markers in migratory pPGC and cultured porcine epiblast. (680 KB)

    a, A cluster of pPGCs (dashed line) at E13 stained for TET1 and OCT4. Scale bar, 20 μm. b, Serial sections of E14 embryos immunostained for different epigenetic markers combined with BLIMP1, NANOG and SOX17. Dashed lines highlight pPGC clusters. Scale bar, 20 μm. c, Quantification of 5-methylcytosine and H3K9me2 in embryos of different stages. Numbers of cells analysed are indicated (boxes, mean and interquartile ranges; whiskers, maximum and minimum; *P < 0.01; Mann–Whitney U-test). d, Serial sections of E16 embryos showing migratory pPGCs (arrows) immunostained for the indicated epigenetic marks. Scale bar, 20 μm. e, Triple immunostaining of epiblasts cultured under different conditions. Scale bar, 10 μm.

  4. Extended Data Figure 4: Characterization of NANOS3–tdTomato reporter hPSC. (816 KB)

    a, Targeting strategy for making NANOS3–tdTomato (NT) reporter. b, Representative genotyping of targeted clones using genomic DNA. c, Conventional (conv) and PGC-competent (comp) hPSCs states are reversible; the latter is equivalent to pre-ME (12 h at mesendoderm induction). Conv-hPSCs are cultured in Essential 8 medium on vitronectin coated dishes (see Methods). Comp-hPSCs are cultured in the hPSC medium containing inhibitors (i) (GSK3i, ERKi, p38i, JNKi) on MEF (see Methods). d, NANOS3–tdTomato reporter conv-hPSCs and comp-hPSCs, and day 1–4 embryoids induced with or without cytokines. e, FACS pattern and percentage of NANOS3–tdTomato+AP+ cells shown in Extended Data Fig. 4d. f, FACS pattern and percentage of NANOS3–tdTomato+AP+ cells in multiple clones derived from Wis2 or H9 hESC lines. g, Immunostaining of day 4 embryoids induced from pre-ME (12 h) or comp-hPSCs by BMP containing cytokines. Scale bar, 50 μm. h, Comparison of sensitivity of two NANOS3 reporter cell lines. FACS patterns of day 4 embryoids induced from comp-hPSCs (harbouring NANOS3–mCherry reporter or NANOS3–tdTomato reporter) with or without cytokines. i, Comparison of hPGC induction efficiency derived from pre-ME (12 h) or comp-hPSCs. Representative images and FACS patterns are shown. j, Scatter plot (mean ± s.d.) shows percentage of NANOS3–tdTomato+AP+ cells in indicated condition (n = 6). Paired t-test was used to test for statistical significance (*P < 0.05).

  5. Extended Data Figure 5: Characterization of pre-ME and mesendoderm induced from Conv-hPSC. (819 KB)

    a, Immunostaining of conv-hPSCs during 12–24 h mesendoderm induction. Scale bar, 50 μm. b, Gene expression (RT–qPCR) change during mesendoderm induction. c, FACS patterns of day 4 embryoids induced from pre-ME (12 h) with cytokines. Pre-ME was induced with or without GSK3i or activin A. d, FACS patterns of day 4 embryoids induced from pre-ME (12 h) with cytokines. Pre-ME was induced with or without BMP2 or the inhibitor. e, Schematics of definitive endoderm or lateral mesoderm (LM) differentiation from mesendoderm. f, FACS patterns of day 2 definitive endoderm (percentage of CXCR4+) and day 1 lateral mesoderm (percentage of PDGFRa+) induced from 24 h of mesendoderm induction. g, Relative induction efficiency of definitive endoderm or lateral mesoderm from mesendoderm induced with or without BMP2 (definitive endoderm, n = 5; lateral mesoderm, n = 6). Paired t-test was used to test for statistical significance (*P < 0.05). h, Immunostaining of definitive endoderm and lateral mesoderm in Extended Data Fig. 5f. Scale bar, 50 μm. i, Schematic of spatial-temporal progression from conv-hPSCs to mesendoderm and the signalling.

  6. Extended Data Figure 6: Robust induction of cmPGCs from cells during mesendoderm differentiation. (1,029 KB)

    a, Schematic of in vitro differentiation of cmPSCs. The same system was adopted as shown for conv-hPSCs differentiation in Fig. 3. b, Bright field image of undifferentiated cmPSCs. c, Immunostaining of day 2 embryoids induced with cytokines from cells at 0 h (cmPSCs), 12 h and 24 h during mesendoderm differentiation. Dashed lines highlight SOX17+BLIMP1+TFAP2C+ cmPGCs. Scale bar, 50 μm. d, Immunostaining of day 2 pre-ME-derived embryoids (12 h) in Extended Data Fig. 6c for pluripotency markers. Notably, cmPGCs express SOX17, but not SOX2. By contrast, cmPSC colonies express SOX2 but not SOX17. e, Immunostaining of day 2 monkey definitive endoderm induced from pre-ME (12 h) and mesendoderm (24 h). Scale bar, 50 μm. f, Immunostaining of day 1 monkey lateral mesoderm induced from mesendoderm (24 h). Mesendoderm were induced with or without BMP. Notably, adding BMP during mesendoderm differentiation increased the efficiency for FOXF1+HAND1+ lateral mesoderm cells, as shown in conv-hPSCs (Extended Data Fig. 5e–i). Scale bar, 50 μm.

  7. Extended Data Figure 7: Chronology of transcription factors expression during hPGC induction. (975 KB)

    a, Schematic of hPGC induction from pre-ME (12 h). b, Images of day 2 and 4 embryoids in response to BMP2 alone or BMP2 with LIF, SCF and EGF (cytokines). Notably, BMP2 alone can induce hPGC at almost the same efficiency as the full cytokines, but do not survive during extended culture, as shown previously. c, Immunostaining of embryoids induced with BMP2 alone or BMP2 with LIF, SCF and EGF showing expression of SOX17, BLIMP1 and TFAP2C. Scale bar, 50 μm. d, Proportion of SOX17+ cells indicated in Extended Data Fig. 7c. e, Immunostaining of embryoids induced with BMP2 alone or BMP2 with LIF, SCF and EGF showing expression of SOX17, BLIMP1 and NANOG. Scale bar, 50 μm. f, Proportion of SOX17+ cells in Extended Data Fig. 7e.

  8. Extended Data Figure 8: Effect of NANOG on hPGC induction, characterization of NANOS3–tdTomato reporter hPSCs containing inducible SOX17, BLIMP1 with or without TFAP2C, and similarity between cytokine- and SOX17–BLIMP1-induced hPGCs. (763 KB)

    a, Represents overexpression of dex-inducible NANOG transgenes in NANOS3–tdTomato reporter comp-hPSC. b, Day 4 embryoids following induction of NANOG (by dex), with or without cytokines as indicated. c, FACS patterns after induction of hPGCs; NANOS3–tdTomato+AP+ cells (%) shown in Extended Data Fig. 8b. d, Represents overexpression of dex-inducible SOX17, dox-inducible BLIMP1, Shield1(S1)-inducible TFAP2C transgenes in NANOS3–tdTomato reporter comp-hPSCs. e, Immunostaining of NANOS3–tdTomato reporter comp-hPSCs + inducible SOX17/BLIMP1/TFAP2C (iSBT) 1 day after induction of SOX17, BLIMP1 and TFAP2C by addition of dex, dox or S1. Scale bar, 50 μm. f, Immunostaining of NANOS3–tdTomato reporter comp-hPSCs + iSB 1 day after induction of SOX17 or BLIMP1 by addition of dex or dox. Scale bar, 50 μm. g, Immunostaining of day 2 embyroid induced with or without dex to induce nuclear localization of SOX17. Notably, accumulation of SOX17 signal is observed in +dex condition. h, Changes in gene expression (RT-qPCR) during hPGC induction: comp-hPSCs control (AP+ cells); NANOS3–tdTomato+ hPGCs induced by SOX17–BLIMP1 or cytokines; NANOS3–tdTomato cells in cells exposed to cytokines (error bars, mean ± s.d.). i, Unsupervised hierarchical clustering (UHC) of gene expression. j, Gene set enrichment analysis (GSEA) of 123 hPGC-specific genes (Supplementary Table 1) on the transcriptome of cytokine- and SOX17–BLIMP1-induced hPGCs. k, Heat map showing expression of epigenetic modifiers related to global DNA demethylation. Same datasets as shown in Fig. 4e were used for analysis.

  9. Extended Data Figure 9: Response of SOX17-knockout comp-hPSC to SOX17. (818 KB)

    a, Overexpression of dex-inducible SOX17 (iS) in SOX17-knockout comp-hPSCs. b, Gene expression (RT–qPCR) on day 4 of FACS-sorted NANOS3–mCherry (NC)+alkaline phosphatase (AP)+ hPGCs by RT–qPCR. c, FACS analysis of day 2 embryoids induced from wild-type comp-hPSCs, SOX17-knockout comp-hPSCs and SOX17-knockout comp-hPSCs rescued with SOX17GR transgene (iS) (percentage of CXCR4+AP+ cells). d, FACS pattern of day 2 embyoid induced from NANOS3–tdTomato reporter comp-hPSCs showing AP+CXCR4+ cells expressing NANOS3–tdTomato. e, Represents SOX17-inducible system (iSdd). Expression of SOX17 fused with destabilized domain (DD) can be induced by doxycycline (dox); addition of Shield1 (S1) can stabilize SOX17–DD protein. f, Western blots showing SOX17 expression level in day 5 embryoids from SOX17-knockout + inducible SOX17–DD (iSdd) comp-hPSCs. Embryoids were induced with cytokines. To induce SOX17, different concentration of dox and S1 were added. As controls, NANOS3–mCherry+AP+ hPGCs and NANOS3–mCherryAP cells from wild-type comp-hPSC-derived embryoids induced with cytokines were used. Histone H3 (H3) was used for internal control. g, Immunostaining of day 4 embryoids from SOX17-knockout + iSdd comp-hPSCs. Embryoids from SOX17-knockout and wild-type comp-hPSC-induced with cytokines were used as controls. Scale bar, 50 μm. h, Quantification of immunostaining data in Extended Data Fig. 9g. The numbers of OCT4+BLIMP1+ hPGCs, FOXA2+ endodermal cells and OCT4+BLIMP1+ hPGCs expressing FOXA2 were counted from 3 different embryoids. The proportions of the 3 populations are shown. i, Expression of SOX17 and BLIMP1 (RT–qPCR) in day 4 embyoids in response to different SOX17 dosage.

  10. Extended Data Figure 10: Response of SOX17-knockout comp-hPSCs to SOX17 and BLIMP1, and changes in epigenetic modifier expression after overexpression of SOX17 and BLIMP1. (773 KB)

    a, Overexpression of dex-inducible SOX17 (iS) and dox-inducible BLIMP1 (iB) in SOX17-knockout comp-hPSCs. b, Immunostaining of comp-hPSC 1 day after induction of SOX17 (dex) or BLIMP1 (dox), or both, and day 2 embryoids following dex-induced SOX17 with endogenous or dox-induced BLIMP1. Scale bar, 50 μm. c, Gene expression (RT–qPCR) in day 4 embryoids following induction by dex (+SOX17), dox (+BLIMP1), or dex + dox (+SOX17 and BLIMP1). Bulk cells of embryoids induced from SOX17-knockout and wild-type comp-hPSCs with cytokines were used as controls. d, Upon specification, hPGCs become refractory to activin or Wnt signalling. Left schematic shows the experimental design. The embryoids were transferred to the medium with or without GSK3i (3 μM) or activin A (100 ng ml−1) at day 0, 1 or 2 to see the effect on PGC induction. FACS patterns (right) show the induction efficiency of hPGCs (percentage of NANOS3–tdTomato+AP+) at day 4. e, Day 4 embryoids induced by cytokines with or without activin A or GSK3i.

Supplementary information

PDF files

  1. Supplementary Information (5.2 MB)

    This file contains the uncropped gels and Supplementary Tables 2-3.

Excel files

  1. Supplementary Table 1 (36 KB)

    This file contains a list of hPGC specific genes used for GSEA analysis.

Additional data