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) immunof luorescence 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.