chorionic plate of the placental disk ( 4 ). These
extraembryonic endoderm tissues play crucial
roles in nourishing the embryo through nutrient
provision at the maternal-fetal interface ( 5 ), es-
pecially before the establishment of placental
circulation [approximately embryonic day 10.5
(E10.5) in mice], in anterior-posterior pattern-
ing of the epiblast during gastrulation ( 6 ), and
in yolk sac hematopoiesis ( 7 ). VE cells derived
from the PrE are further suggested to contrib-
ute to formation of the fetal gut ( 8 ).
To gain further insight into how the PrE
functions during pre- and early postimplan-
tation development, it would be useful to es-
tablish stem cell lines that fully retain PrE
developmental potential beyond that pos-
sessed by XENCs, which can only contribute
to the distal region of the PrE ( 3 ). We tested for
culture conditions that enabled robust growth
of PrE-derived cells from blastocysts (Fig. 1A
and table S1) and examined the impact of se-
rum, which provides undefined differentiation
signals. As reported ( 3 ), serum-containing cul-
ture allowed efficient derivation of XENCs
from the inner cell mass (ICM). Derivation
efficiency of XENCs from the ICM was further
facilitated by addition of 3mM CHIR99021,
a GSK3 inhibitor, but was suppressed by the
mitogen-activated protein kinase (MAPK)
kinase (MEK) inhibitor PD0325901 (Fig. 1B
and fig. S1A). By contrast, serum-free medium
could not support colony formation from the
ICM by itself, but addition of 3mM CHIR99021
promoted characteristic outgrowths and sub-
sequent generation of dome-shaped colonies
(Fig. 1B and fig. S1A). This effect of CHIR99021
was inhibited by PD0325901, leading to the
induction of ESCs. These results suggested
that the CHIR99021-induced cells exhibited
a competitive period for fate decision with
ESCs and originated from PrE rather than
epiblasts.
We next investigated whether CHIR99021-
induced cells retained the functional proper-
ties of PrE by maintaining them under various
serum-free culture conditions (Fig. 1C). The
maintenance of these cells was dependent on
CHIR99021. Leukemia inhibitory factor (LIF)
did not replace CHIR99021 to maintain pro-
liferation, suggesting that these cells are dif-
ferent from ESCs. However, increasing the
concentration of CHIR99021 to 10mM accel-erated their proliferation and resulted in a
more compacted colony morphology. The im-
pact of fibroblast growth factor 4 (FGF4)
and/or platelet-derived growth factor–AA
(PDGF-AA), which are known to expand the
PrE in blastocysts ( 9 , 10 ), was also tested and
found to accelerate the growth rate of the
CHIR99021-induced cells even further, with-
out any additional morphological changes.
The CHIR99021-induced cells transformed
into XENC-like cells in serum+C3 medium but
were not maintained in ESC medium (fig. S1, B
and C). Therefore, CHIR99021-induced cells
appear to be PrE derivatives captured at an
earlier stage than were XENCs. Together with
their self-renewing property in culture, we ten-
tatively refer to CHIR99021-induced cells de-
rived and maintained in +C10F4PDGF medium
as primitive endoderm stem cells (PrESCs).
Next, we explored the developmental origin
of PrESCs by comparing the gene expression
profiles revealed through single-cell RNA-
sequencing (scRNA-seq) data from the ICM of
E3.5 and E4.5 blastocysts. To enable a direct
comparison, scRNA-seq analysis for PrESCs
was performed in parallel with ESCs, TSCs,576 4 FEBRUARY 2022•VOL 375 ISSUE 6580 science.orgSCIENCE
Fig. 3. Developmental potential of PrESCs.(A) Distribution of GFP-labeled
XENCs and PrESCs 18 hours after blastocyst injection. Scale bar, 50mm.
(B) Distribution of GFP-labeled XENCs and PrESCs in E14.5 chimeric
conceptuses. Scale bar, 1 mm. (C) Schematic representation of the PrESC
complementation assay. (D) Complementation of PrE-depleted blastocysts
by GFP-labeled PrESCs generated through 0.5 or 1mM PD0325901 treatment.
E18.5 chimeric conceptuses revealed either partial or full reconstitution of
extra-embryonic endoderm derivatives by PrESCs. Scale bar, 1 mm.RESEARCH | REPORTS