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 SOX17–BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models provides synthetic insights into early human development.
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Gene Expression Omnibus
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We thank R. Campbell and C. Lee for help with animals and hPSCs, A. Riddell for FACS and T. Otani for advice on cmPSCs. T.K. was supported by JSPS, the Uehara and Kanae Foundations; H.Z. by CSC; D.A.C. by CONACYT. The work was funded by BBSRC grant to R.A., C.A. and M.A.S (BB/M001466/1). M.A.S is a Wellcome Investigator.
The authors declare no competing financial interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
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.
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.
Extended Data Figure 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.
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).
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.
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.
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.
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.
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.
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–mCherry−AP− 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.
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.
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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Kobayashi, T., Zhang, H., Tang, W. et al. Principles of early human development and germ cell program from conserved model systems. Nature 546, 416–420 (2017). https://doi.org/10.1038/nature22812
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