Extended Data Fig. 8 | Development of TrB lineage. a–i, Representative
confocal staining of human embryo section. a, Staining of CK7 and F-actin. n = 5
independent embryos from three independent experiments. b, c, Cells near
the EPI–PrE bilayer had a single nucleus that expressed TEAD4 and E-cadherin.
E-cadherin displayed symmetrical distributions in the embryos (3 out of 3
embryos for each staining from two experiments). d, e, Multinucleated cells
(d1, e1 and e2) and cells with a single nucleus (d2) (3 out of 3 embryos each
staining from two experiments). Dashed lines in d and e outline the region of
interest in cells. f, g, Representative staining of CK7 and HLA-G (3 out of 3
embryos) in 12-d.p.f. (f) and 14-d.p.f. (g) human embryo. h, i, Representative
staining of TEAD4 and HLA-G (h) or TEAD4 and hCG (i) in 14-d.p.f human
embryo (3 out of 3 embryos). In f, the inset is magnification of the square. In g–i,
right panel is magnification of the square. In i, the inset is magnification of the
region indicated by a red arrow. j–l, t-SNE plot of 352 TrBs. Cells (dots) coloured
according to the original inferred lineage identity (k) and embryonic stage (l).
m, Lineage segregation path constructed by Monocle based on developmental
time (left) and cell types defined with selected markers (right). n, Heat map of
polypeptide hormone genes expressed in the six types of trophoblasts during
culture. o, Heat map indicates expression of genes specific for each cell type.
Representative genes and key GO enrichments shown. GO terms and KEGG
pathways of genes specific for different subtypes of TrBs (multiple test
corrected P < 0.01, hypergeometric tests) in the six types of TrBs from pre-
implantation stage embryos to 14-d.p.f embryos are shown in Supplementary
Table 4. Pre-CTB-expressing genes related to cell metabolism, biosynthesis
and cell differentiation, were in accord with the characteristics of trophoblast
stem cells. High expressions of NF-κB, as well as canonical and non-canonical
Wnt signalling pathway genes, indicate potential functions on CTB
development. STB-specific genes indicated hormone secretion, whereas early-
STB-expressing genes associated with cell differentiation and migration,
dependent on several signalling pathways. EVT-specific genes contributed to
regulating the immune system and angiogenesis. Scale bars, 50 μm (a–c, e–i) or
20 μm (b).