Extended Data Fig. 5: Human embryos at 14 d.p.f. initiate anterior–posterior polarity and generation of PSA in 3D-culture conditions.

This figure relates to Fig. 3. a, OCT4, GATA6 and TBXT T staining of sections from a 14-d.p.f. embryo (2 out of 2 embryos), showing that T⁺ cells originated from the EPI compartment close to the AME compartment boundary at 14 d.p.f. b, Representative OCT4, OTX2 and SOX2 staining. Arrow indicates an OTX2⁺ cell. n = 4 of 5 embryos from three independent experiments displayed consistent data. c–e, LEFTY1 (c, d; n = 3 out of 4 embryos from two independent experiments) and OTX2 (e; n = 3 out of 5 embryos from two independent experiments) immunofluorescence was only detected on the side of 14-d.p.f. embryonic disc. Arrows indicate LEFTY1⁺ or OTX2⁺ cells. Right image in d is magnification of the square in the left image. The exclusive expression of NANOG and SOX2 was not observed in EPIs (e). f, Staining of OCT4, HESX1 and GATA6 in the 12-d.p.f. embryos (2 out of 2 embryos). g, The violin plots show dynamic expression of HESX1 during EPI development. All violin plots have the same maximum width, black dot denotes the mean. h, Correlation of HESX1 and T expression of 14-d.p.f. EPIs, as determined by scRNA-seq. Each plot represents a single cell. i, Volcano plots show DEGs in HESX1⁺T⁻ (10 single cells) and HESX1⁻T⁺ (12 single cells) EPIs by scRNA-seq. DEGs were defined as those with uncorrected P < 0.01 (likelihood ratio test) and fold change of >2 or <−2, and median FPKM > 1 in one group. j, k, Staining of OCT4, FLK1 and T at 14 d.p.f. (2 out of 2 embryos from two independent experiments). Red arrows denote migrating T⁺ cells; white arrows denote FLK1⁺ extra-embryonic mesenchyme. l, The violin plots show expression dynamics of primitive streak genes over pluripotent-stem-cell development. All violins have the same maximum width, black dot denotes the mean. In total, 136 cells were included (Extended Data Fig. 9e): ICM, n = 49 cells; pre-EPI, n = 23 cells; post-EPI, n = 48 cells; PSA-EPI, n = 16 cells. *P < 0.05, ** P < 0.01, two-sided Wilcoxon rank-sum test. m–o, Absence of specific neural gene expression indicates 14-d.p.f embryos do not generate the initial nervous system, which meets the internationally recognized ethical limit for human embryo culture. m, Violin plots of dynamic expressions of neural-specific genes in EPIs over embryo culture. All violins have the same maximum width, black dot denote the mean. In total, 136 cells were included: ICM, n = 49 cells; pre-EPI, n = 23 cells; post-EPI, n = 48 cells; PSA-EPI, n = 16 cells. n, o, Representative staining of PAX6, OCT4, SOX1 and FOXA2 in human 14-d.p.f embryos (3/3 embryos). p–r, Development and cell proliferation of human embryos cultured in the Matr+10% Matr condition. Quantified data at each stage were based on five embryos from three independent experiments. Data are presented as mean ± s.d. p, Quantification of the dynamics of total cell number per embryo during culture. *P < 0.05, **P < 0.01, two-sided Student’s t-test. q, Dynamics of OCT4⁺ EPIs and GATA6⁺ PrEs per embryo over culture. r, Dynamics of CK7⁺ TrBs per embryo over culture. EPIs and PrEs maintained gradual proliferation at 8–10 d.p.f., after which their proliferation speeds accelerated. However, TrBs always maintained a rapid proliferation rate, which may be for establishing cell connections with the maternal environment. Scale bars, 25 μm.

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