and XENCs. The homogeneity of PrESCs and
their similarity to other cell lines were exam-
ined through principal components analysis
(PCA). PrESCs were found to represent a ho-
mogenous population that is distinct from
ESCs, TSCs, and XENCs (Fig. 2A and table S2).
Gene expression profiles of ESCs, PrESCs, and
XENCs were further compared with those of
individual cells in the ICM of E3.5 and E4.5
blastocysts ( 11 ) by means of agglomerative
clustering analysis (fig. S2A). Sixteen clusters
were identified; the E4.5 ICM could be seg-
regated into putative epiblasts and PrE through
differential expression of genes from clusters
1 to 5, which were barely expressed in the
E3.5 ICM. Previously known PrE marker genes,
such asGata4andGata6, were enriched in
cluster 1, which began to express these genes
from E4.5. By contrast, pluripotent marker
genes were enriched in clusters 6 and 10,
which were expressed as early as E3.5. In cell
lines, cluster 1 genes were expressed by PrESCs
and XENCs but not by ESCs. Reciprocally, Clus-
ter 6 genes were more abundantly expressed
in ESCs than in PrESCs and XENCs. Taken
together, global clustering analysis revealed
that PrESCs were closely related to putative
PrE in the E4.5 ICM and to XENCs. To further
investigate their similarity, we examined the
expression of representative PrE marker genes,
selected mainly from cluster 1, and pluripotent
marker genes from clusters 6 and 10 (Fig. 2B).
The putative PrE expressed not only PrE mark-
ers but also some pluripotent markers such
asPou5f1[encoding octamer-binding tran-
scription factor 4 (OCT4)] andCdh1(encoding
E-cadherin). Whereas PrESCs expressed both
PrE markers and certain pluripotent markers
expressed in the putative PrE, XENCs failed to
express these pluripotent markers (table S3).
The differences between PrESCs and XENCs
were confirmed with conventional RNA-seq
analysis (Fig. 2C and table S4). Coexpression
of the PrE marker (GATA6) and pluripotent
markers (OCT4 and E-cadherin) in PrESCs was
further validated with immunofluorescence
analysis (Fig. 2D). Therefore, PrESCs retain
the molecular properties of founder PrE, which
appear around E4.5. Imprinted X-chromosome
inactivation was consistently established in
PrESCs as well as in XENCs (fig. S2, B and
C, and table S4) ( 3 , 12 ). We further examinedthe PrE origin of PrESCs by investigating
derivation efficacy of PrESCs from ICM de-
pleted of PrE by PD0325901, which dropped to
50% of the untreated ICM (fig. S2, D and E).
GATA6+OCT4+monolayer cells that were
spread out from the ICM explants after 7 days
culture in +C10F4HPDGF were also reduced
upon PrE depletion (fig. S2F). These observa-
tions also support the PrE origin of PrESCs.
Next, we investigated the differentiation ca-
pability of PrESCs by injecting 15 green fluores-
cent protein (GFP)–labeled PrESCs or XENCs
into blastocysts and examining their distribu-
tion after 18 hours. PrESCs were efficiently in-
tegrated into the PrE layer, whereas XENCs
were randomly localized in the blastocoel
(Fig. 3A and fig. S3A). In chimeric conceptuses
at later stages, PrESCs contributed to both VE
and PE, whereas XENCs were in only a small
portion of the PE (Fig. 3B and fig. S3, B to F).
To confirm the potential of PrESCs to con-
tribute to both VE and PE, individual PrESCs
were isolated and propagated. Each clone re-
tained both VE and PE potentials (fig. S3, G
and H). Next, we used a blastocyst comple-
mentation assay to examine whether PrESCsSCIENCEscience.org 4 FEBRUARY 2022•VOL 375 ISSUE 6580 577
Fig. 4. Differentiation potential of PrESCs in ETPs.(A) Schematic representation
of ETP generation. (B) ETP morphology at day 3 and day 6. Scale bar, 200mm.
(C) Higher magnification view of a day 3 ETP. TSC-derivatives are demarcated by
CDX2 expression. Scale bar, 100mm. (D) Decidual reaction induced by ETPs on
prospective E7.5. (E) The ETP descendant on prospective E7.5. (Left) Vasculature is
surrounding the ETP descendant. (Right) Merged view of PrESC-derivatives (green) in
the ETP-descendant. The outline of ETP descendant is indicated by the dashed line.
Scale bar, 500mm. (F) Histological comparison between (left) the E7.5 embryo
and (right) the ETP descendant on prospective E7.5. The yolk sac cavityÐlike
structures are indicated with arrowheads. Scale bar, 500mm. (GandH) Confocal
images of (left) the E7.5 embryo and (right) the ETP descendant. The boxed areas in
(G) are enlarged in (H). Scale bars, (G) 500mm; (H) 100mm.RESEARCH | REPORTS