Letter reSeArCH
of primitive endoderm (PE; also known as the hypoblast). The EPI,
PE and TE then give rise to the embryo proper, yolk sac and placenta,
respectively. By embryonic days 6–7 (after fertilization), the embryo
will implant into the uterus to form a gastrula, followed by organogen-
esis. Owing to the limited access to embryos early after implantation
in vivo, the lineage specification and corresponding patterns of the
transcriptome and DNA methylome that are specific to the different
lineages during human implantation are still poorly understood. In this
study, with the help of donated human embryos and a robust in vitro
culture system for post-implantation embryos and single-cell multi-
omics sequencing technologies^2 –^5 , we simultaneously analysed the
gene-expression network and lineage-specific DNA methylation pat-
terns of the human peri-implantation embryos at single-cell resolution.
First, we mimicked the implantation of human embryos as
previously reported^2 ,^3 (Fig. 1a, b, Extended Data Fig. 1a–d and
Supplementary Videos 1–5). The culture of embryos was terminated
at day 14 according to bioethical guidelines^6 ,^7. Next, we profiled 7,636
individual cells from 48 human pre/post-implantation embryos at
5 consecutive developmental stages, including the blastocyst stage
(day 6, pre-implantation) and 4 later stages (days 8, 10, 12 and day14
after implantation) (Fig. 1c and Supplementary Tables 1, 2). In total,
5,911 single cells were retained for subsequent analyses following strin-
gent filtering (Extended Data Fig. 1e–h). Unsupervised t-distributed
stochastic neighbour embedding (t-SNE) analysis revealed that all of
the cells were grouped by their developmental states (Extended Data
Fig. 1i). Furthermore, t-SNE analyses partitioned the cells into four
main clusters. The analysis of the expression of known markers and
lineage scores identified these clusters as the EPI, PE, TE and yolk-sac
trophectoderm (ysTE)^3 ,^8 ,^9 (Extended Data Figs. 1j, k, 2, Supplementary
Methods and Supplementary Table 3). As there was only a limited
number (only 39 cells in total) of ysTE cells and to avoid the poten-
tial influence of embryos that lacked lineage(s) on the outcome of the
analysis, we next focused mainly on the features of the three major
lineages in those embryos that contained all three of the major lineages
(EPI, PE and TE); 3,184 individual cells were retained in the subsequent
analyses (Fig. 1c, d and Extended Data Fig. 1g).
Next, we identified genes that were specifically expressed in the
EPI, PE and TE cells, and defined these as lineage signature genes.
In addition to the canonical lineage makers, the EPI expressed
KHDC3L, TDGF1, CXCL12 and THY1, which have previously beena EPIPE ysTE TE bACKR2TFAP2AHSD3B1DLX3SOX21CD44APOA4SOX17FLRT3FOXA2CDH2GATA4PDGFRATHY1PRDM14CXCL12SOX2NANOGTDGF1KHDC3LDPPA5EPIPE6810 12 6810 12LEFTY2
PRICKLE1
PAX6
SP7
RAMP2
PSG6
ARG1
SEMA3B
LAMB3
TAC1EDN1
INHBECNR1
PTGIS
KITLG
EGFRHTR2C
HAND1
SALL1NGF
CD44
UGT2B11PPP3CA
DGKQFMOD
IRS2RBP4
LHB
AKR1D1TAC1
DKK3
EGFRCXADREMP2
HAVCR1
INSL4CGB8
CGB7CGB3
CGB2PSG5
PSG3CGB5
CGB1PSG9
PSG2PSG4
PSG6EGFR
ITGB3PSG1
ARHGDIBLAMP3
PSG8ITGA2
DPP4PSG116810 12cde TE−1.5−1.0−0.500.51.01.5OTX2 GATA6 OCT4 DAPI MergedDayDay DayFig. 2 | Transcriptome dynamics at post-implantation stages.
a, The expression patterns of lineage signature genes. Among these genes,
several genes were recognized as classical lineage-specific maker genes,
such as NANOG for EPI, GATA4 for PE and TFAP2A for TE. A marker that
has been identified in the yolk sac of mice, APOA4, was identified as one
of the signature markers in the PE lineage^25. ACKR2 was identified as a TE
marker here, consistent with its known function for regulating placenta
development in mice. The ysTE specifically expressed CD44, which has
been reported as a critical gene for trophoblast cell invasion. On the basis
of the signatures of each lineage, the EPI signatures were enriched in Gene
Ontology (GO) terms related to embryonic morphogenesis, gastrulation and
transcriptional regulation of pluripotent stem cells. The PE signatures were
enriched in GO terms related to cell-fate specification, embryonic organ
development and epithelial cell differentiation, and the TE signatures were
enriched in response to steroid hormone, extracellular matrix organization
and vasculature development (Supplementary Table 6). Such changes among
lineages are probably to balance pluripotency maintenance/transition and
lineage specification for different developmental potentials to support the
continued development of the embryo after implantation. b, Morphological
visualization of OTX2 expression in PE cells (n = 3). Series of confocal z-
sections of the embryo stained for OCT4 (red), GATA6 (green) and OTX2
(cyan). Arrowheads, GATA6+ but OTX2− cells. Arrows, GATA6+ and
OTX2+ cells. All scale bars, 20 μm. c–e, Stage-specific gene-expression
patterns for three major lineages. There were 67 (EPI), 224 (PE) and 282
(TE) genes that showed stage-specific expression features in each lineage
(c–e; Supplementary Table 6). GO analysis showed that EPI stage-specific
genes were clearly enriched in embryonic morphogenesis genes (such
as SALL1 and HAND1) between day 6 and day 12. Additionally, PSG2, a
member of the pregnancy-specific glycoprotein (PSG) gene family, was
upregulated from day 8 to day 10 in the TE lineage, which indicates that
the embryo might be preparing for mother–fetal interactions during
implantation. The colours from blue to red represent the expression levels
from low to high.29 AUGUSt 2019 | VOL 572 | NAtUre | 661