can fully replace the endogenous PrE (Fig. 3C).
The PrE was depleted in blastocysts by treat-
ing them with PD0325901 for 48 hours (fig.
S4A) ( 13 ). Injected PrESCs efficiently recon-
stituted the PrE layer in PD0325901-treated
blastocysts and improved the frequency of
implantation of PrE-depleted blastocysts (fig.
S4B and table S5). Moreover, nine neonates
were recovered from reconstituted blastocysts
treated with 0.5 or 1mM PD0325901. In these
chimeras, PrESCs efficiently contributed to ex-
traembryonic endoderm tissues and, in four
cases, fully reconstituted these tissues (Fig.
3D, fig. S4F, and table S5). Full complemen-
tation of VE and PE by PrESCs was further
confirmed in E6.5 embryos (fig. S4E); how-
ever, no contribution of PrESCs to fetal tissues,
including the gut, was observed (n=9,allob-
tained complementation chimeras at E18.5) (Fig.
3D and fig. S4F). PrESCs therefore can function-
ally replace the endogenous PrE and thus can
be annotated as PrE stem cells for the extra-
embryonic endoderm lineage.
We further investigated the differentiation
capability of PrESCs by using a modified blas-
toid self-organization method ( 14 ), in which
ESCs, TSCs, and PrESCs were sequentially as-
sembled to form ETP (ESC/TSC/PrESC) com-
plexes (ETPs hereafter) (Fig. 4, A and B). In
this condition, respective stem cells estab-
lished uniform contacts in ETPs by day 3
(Fig. 4C). The differentiation status of the
respective stem cells was assessed by means
of scRNA-seq analysis of the respective stem
cells and day 3 and day 6 ETPs (fig. S5A). Ten
clusters were identified by means of PCA, and
clusters from different stem cells were jux-
taposed but temporally separated, suggest-
ing differentiation of respective stem cells in
ETPs. The derivatives of ESCs and TSCs ap-
propriately expressed differentiation-related
genes in day 6 ETPs (fig. S5B). Through PCA
for PrESC derivatives, PrESC descendants were
found to exist in four distinct states (fig. S5C).
A variety of PE-related or VE-related genes
were expressed in cluster 1 or cluster 8, re-
spectively (fig. S5D). PrESC can differentiate
into PE- and VE-like cells and, reciprocally,
facilitate differentiation of ESCs and TSCs in
ETPs. To evaluate to what extent these ETPs
represent normal development, day 3 ETPs
were transferred into pseudopregnant uterus,
and efficient implantation was observed, as re-
vealed by decidua development, on prospective
E7.5 embryos (23.3%, 28 of 120) (Fig. 4D). Im-
planted ETPs were elongated and associated
with yolk sac–like structures on their surface
(Fig. 4, E and F). PrESC-derived SOX17+cells
formed VE-like and PE-like sheets (Fig. 4, G and
H). Therefore, PrESCs can establish interactions
with ESCs and TSCs and generate descendants
with yolk sac–like structures in utero. ETPs, how-
ever, failed to form normal embryos (Fig. 4G).
This study presents a robust protocol for
the efficient generation of PrE stem cell lines,
which can be used to elucidate the mecha-
nismsthatunderpinPrEspecificationinvitro
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ACKNOWLEDGMENTS
Funding:This work was supported by JSPS KAKENHI under
grants 18H05366 and 19H05757 and by AMED under grant
19bk0104093h0001 to Y.O.Author contributions:Y.O. and H.K.
designed the study and wrote the manuscript. Y.O. and T.A.E.
analyzed next-generation sequencing (NGS) data. Y.O., Y.I., Y.Ka.,
A.S., T.K., M.K., Y.Ko., H.S., T.W., and O.O. performed experiments.
Competing interests:RIKEN has a patent pending (JP2019-118733)
on the method for derivation and maintenance of PrES cells. Y.O.
and H.K. are the inventors of the patent. There is no conflict of
interest relevant to this study.Data and materials availability:
RNA-seq data are available at the Gene Expression Omnibus (GEO)
website (www.ncbi.nlm.nih.gov/geo) under accession no. GSE185461.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.aay3325
Materials and Methods
Figs. S1 to S5
Tables S1 to S5
MDAR Reproducibility Checklist9 June 2019; resubmitted 16 October 2020
Accepted 21 December 2021
10.1126/science.aay3325578 4 FEBRUARY 2022•VOL 375 ISSUE 6580 science.orgSCIENCE
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