human Monkey Chimera
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HumanMonkeyChimera.pdf
Article
Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo
Graphical abstract
Highlights
d Generation of human-monkey chimeric embryos ex vivo with
hEPSCs
d hEPSCs differentiated into hypoblast and epiblast lineages
d scRNA-seq analyses revealed developmental trajectories of
human and monkey cells
d The approach may allow for enhancing chimerism between
evolutionarily distant species
Tan et al., 2021, Cell 184, 2020–2032 April 15, 2021 ª 2021 Elsevier Inc. https://doi.org/10.1016/j.cell.2021.03.020
Authors
Tao Tan, Jun Wu, Chenyang Si, ...,
Weizhi Ji, Yuyu Niu,
Juan Carlos Izpisua Belmonte
Correspondence [email protected] (T.T.), [email protected] (J.W.), [email protected] (W.J.), [email protected] (Y.N.), [email protected] (J.C.I.B.)
In brief
Human cells, in the form of extended
pluripotent stem cells, have the ability to
contribute to both embryonic and extra-
embryonic lineages in ex-vivo-cultured
monkey embryos.
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Article
Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo Tao Tan,1,4,* Jun Wu,2,4,5,* Chenyang Si,1,4 Shaoxing Dai,1,4 Youyue Zhang,1,4 Nianqin Sun,1 E Zhang,1 Honglian Shao,1
Wei Si,1 Pengpeng Yang,1 Hong Wang,1 Zhenzhen Chen,1 Ran Zhu,1 Yu Kang,1 Reyna Hernandez-Benitez,2
Llanos Martinez Martinez,3 Estrella Nuñez Delicado,3 W. Travis Berggren,2 May Schwarz,2 Zongyong Ai,1 Tianqing Li,1
Concepcion Rodriguez Esteban,2 Weizhi Ji,1,* Yuyu Niu,1,* and Juan Carlos Izpisua Belmonte2,6,* 1State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China 2Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA 3Universidad Católica San Antonio de Murcia (UCAM), Campus de los Jerónimos, No 135 12, Guadalupe 30107, Spain 4These authors contributed equally 5Present address: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 6Lead contact
*Correspondence: [email protected] (T.T.), [email protected] (J.W.), [email protected] (W.J.), [email protected] (Y.N.), [email protected] (J. C.I.B.)
https://doi.org/10.1016/j.cell.2021.03.020
SUMMARY
Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages in- side monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These re- sults may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.
INTRODUCTION
Pluripotent stem cells (PSCs) are capable of indefinite self-
renewal in culture and generating all adult cell types (De Los An-
geles et al., 2015; Hackett and Surani, 2014; Rossant and Tam,
2017; Wu and Izpisua Belmonte, 2016). PSCs have recently been
harnessed for interspecies organogenesis via blastocyst
complementation, a technique that holds potential to provide
large quantities of in-vivo-generated human cells, tissues, and
organs for regenerative medicine applications, including organ
transplantation (Suchy and Nakauchi, 2018; Wu et al., 2016).
One of the requirements for successful interspecies blastocyst
complementation with human PSCs (hPSCs) is their ability to
contribute to chimera formation. The chimeric competency of
hPSCs has been systematically tested in several animal species
(Wu et al., 2016), but despite sustained efforts from different lab-
oratories, the general consensus is that hPSCs do not consis-
tently and robustly contribute to chimera formation when the
host animal has a high evolutionary distance from humans
(e.g., mice and pigs; Wu et al., 2016, 2017). This is the case
2020 Cell 184, 2020–2032, April 15, 2021 ª 2021 Elsevier Inc.
even when human cell apoptosis is inhibited (Das et al., 2020;
Huang et al., 2018; Wang et al., 2018). Xenogeneic barriers be-
tween hPSCs and evolutionarily distant host animal species
have been suggested to account for limited chimerism (Wu
et al., 2016, 2017), though the use of hPSCs for chimera studies
in host species evolutionarily close to humans remains unex-
plored to date.
Cultured PSCs reflect the continuum of pluripotency that is
seen in vivo, and different cell culture formulations result in
distinct pluripotency states in vitro (Morgani et al., 2017; Smith,
2017; Weinberger et al., 2016). hPSCs in different pluripotency
states exhibit distinct transcriptional, epigenetic, and metabolic
features. They also differ in their chimeric potential when intro-
duced into animal embryos (De Los Angeles, 2019; Harvey
et al., 2019; Zhang et al., 2018). Recently, we and others identi-
fied human extended PSCs (hEPSCs) that demonstrated
improved chimeric capability in mouse conceptuses (Gao
et al., 2019; Yang et al., 2017b). To date, however, the chimeric
competency of hEPSCs has not been determined in other
species.
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Leveraging a recently developed culture system that enables
cynomolgus monkey (monkey for short) embryos to develop
up to 20 days ex vivo (Ma et al., 2019; Niu et al., 2019), we per-
formed microinjection of hEPSCs into monkey blastocysts and
examined their contribution to cultured monkey embryos at
different time points (Figure 1A). We found that hEPSCs could
integrate into the inner cell masses (ICMs) of late monkey blasto-
cysts and contributed to both embryonic and extra-embryonic
lineages in peri- and early post-implantation stages during pro-
longed embryo culture. We also determined the differentiation
trajectory of hEPSCs within cultured monkey embryos by sin-
gle-cell RNA sequencing (scRNA-seq) analysis.
RESULTS
Generation of human-monkey chimeric blastocysts in vitro
To determine the chimera competency of hPSCs in a closely
related non-human primate species, we used a well-character-
ized hEPSC line generated by cellular reprogramming, iPS1-
EPSCs, which demonstrated improved chimerism in embryonic
day 10.5 (E10.5) mouse conceptuses over other reported hPSCs
(Yang et al., 2017b). Consistent with the previous report, iPS1-
EPSCs exhibited a dome-shaped colony morphology and ex-
pressed the core pluripotency transcription factors OCT4,
NANOG, and SOX2 (Figure S1A). To generate human-monkey
chimeric embryos, early blastocysts from cynomolgus monkeys
(6 days post-fertilization [d.p.f.6]) were injected with 25 iPS1-
EPSCs labeled with tdTomato (TD). Injected embryos were first
cultured to the late blastocyst stage (d.p.f.7) for analysis. The
proliferation of hEPSCs within monkey blastocysts was evident
(Video S1). In total, TD+ iPS1-EPSCs were detected in all
d.p.f.7 monkey blastocysts (100%, n = 132) (Figure S1B).
Chimeric contribution of hEPSCs to peri- and post- implantation monkey embryos We next took advantage of a recently established prolonged em-
bryo culture system that supports ex vivo primate (human and
monkey) embryogenesis to the gastrulation stage (Deglincerti
et al., 2016; Ma et al., 2019; Niu et al., 2019; Shahbazi et al.,
2016; Xiang et al., 2020; Zhou et al., 2019). In this embryo culture
system, the zona pellucida is removed and the denuded blasto-
cysts are allowed to attach to the culture dish for further develop-
ment. We used this system to trace the fate of hEPSCs in
monkey embryos at peri- and post-implantation stages. Similar
to noninjected controls (92.31%, n = 104) (Niu et al., 2019),
most embryos injected with hEPSCs attached at approximately
d.p.f.10 (92.79%, n = 111). After attachment, the injected em-
bryos continued to grow, and an embryonic disc became visible
at approximately d.p.f.11 as seen with controls (Figure 1B). TD+
human cells were found in the embryonic disc of more than half
of the injected embryos at d.p.f. 9, but this ratio progressively
declined at approximately one-third by d.p.f.13 (Figure 1C). To
determine whether introduced hEPSCs may affect embryo
development, we evaluated and compared the developmental
status of injected and control embryos (Niu et al., 2019). We
found that the developmental ratios of injected embryos were
slightly lower than that of controls (Figure 1D). Furthermore,
similar to controls, the developmental ratios of injected embryos
dropped sharply at approximately d.p.f.15, which may reflect the
limitation of the 2D attachment embryo culture system
(Figure 1D).
To study the developmental potential of hEPSCs in monkey
embryos, we performed immunofluorescence (IF) studies using
antibodies specific for several embryonic and extra-embryonic
lineages. Analyses were performed between d.p.f.9 and
d.p.f.19. At the peri-implantation stage (d.p.f.9), on average,
10.2 ± 7.2 (n = 9) iPS1-EPSCs were found incorporated into
the ICM (the number of TD+OCT4+ cells found within or close
to NANOG+ or OCT4+ monkey cells) (Figure 2A). Although these
cells expressed OCT4, only a few of them expressed NANOG
(Figure S1C). Supporting their epiblast (EPI)-like identity, we
did not observe GATA6 (a hypoblast [HYP] marker) expression
in these cells (Figure S1C). In addition, TD+GATA4+ HYP-like
cells were also detected (Figure 2B). In contrast to the results re-
ported in mice (Yang et al., 2017b), only a few iPS1-EPSCs were
detected in the trophectoderm (TE) layer of monkey blastocysts
and expressed TE (TE or trophoblast) marker genes (e.g.,
TFAP2C and CK7) (Figure 2C).
At d.p.f.11, TD+OCT4+cells were also detected in the EPI layer
of monkey embryos, and these cells rarely expressed T+ (also
known as Brachyury), a marker of gastrulation. By contrast, T+
monkey cells were detected at the dorsal amnion of the embryos
(Figure 2D). In addition, TD+ human cells expressing a HYP
marker, platelet-derived growth factor receptor-alpha
(PDGFRa), were found intermingled within monkey HYP cells
(Figure 2E). OCT4+ human cells expressing COL6A1, a marker
of extra-embryonic mesenchyme cells (EXMCs), were found
outside of the EPI layer, suggesting ongoing differentiation of
hEPSCs toward EXMCs (Figure S1D). At d.p.f.13, hEPSCs
were detected beneath the EPI layer and expressed an endo-
derm marker, SOX17, suggesting that they have initiated gastru-
lation (Figure 2F). Interestingly, we found that from d.p.f.13
onward human cells tended to group together and segregate
from the monkey EPI layer. These human cells appeared to un-
dergo differentiation into gastrulating cells as evidenced by the
gained expression of OTX2 (Martyn et al., 2018; Vincent et al.,
2003) while maintaining OCT4 expression (Figure S1E). Overall,
we found that hEPSCs exhibited reasonable contribution to the
EPI (with the highest contribution of 7.08% observed at
d.p.f.15), relatively lower contribution to the HYP (with the high-
est contribution of 4.96% observed at d.p.f.19), and limited
contribution to the TE in peri- and post-implantation monkey em-
bryos (Figure 2G).
Transcriptional landscape of human-monkey chimeric embryos To further delineate the developmental trajectory of human-
monkey chimeric embryos, scRNA-seq analysis was carried
out to profile the transcriptomes of human and monkey cells at
different developmental stages. Following embryo dissociation,
single human (TD+) and monkey (TD�) cells were manually collected using fluorescence microscopy and subjected to
scRNA-seq. In total, we sequenced 227 human and 302 monkey
cells isolated from chimeric embryos at different time points dur-
ing ex vivo culture (d.p.f.9–d.p.f.17; Table S2). TD expression and
Cell 184, 2020–2032, April 15, 2021 2021
Figure 1. Generation and developmental capability of human-monkey ex-vivo chimeras
(A) Schematic of the generation and analyses of chimeric embryos derived from blastocyst injection of hEPSCs (created with BioRender.com). hEPSCs, human
extended pluripotent stem cells; EPI, epiblast; HYP, hypoblast; TE, trophectoderm; EXMC, extra-embryonic mesenchyme cell; GAS, gastrulating cell; IF,
immunofluorescence.
(B) Representative bright-field images of hEPSC-injected monkey embryos cultured in vitro until d.p.f.19 (n = 111 embryos for d.p.f.9; n = 91 embryos for d.p.f.11;
n = 60 embryos for d.p.f.13; n = 38 embryos for d.p.f.15; n = 12 embryos for d.p.f.17 and n = 3 embryos for d.p.f.19). Scale bar, 100 mm. Yellow dotted lines indicate
ICM (d.p.f.9) or embryonic disc (d.p.f.11 to d.p.f.19).
(C) Histogram showing the percentages of host monkey embryos containing human cells within ICM or embryonic disc (yellow dotted lines in B).
(D) Dynamics of developmental ratios of chimeric (n = 126, d.p.f.8) and non-chimeric monkey embryos (n = 104, data from Niu et al., 2019). Embryos without clear
embryonic disc structure and/or appear dead were excluded from the analysis.
See also Figure S1 and Video S1.
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Figure 2. hEPSCs contribute to chimera
formation in peri- and post-implantation
monkey embryos
(A) Representative IF images showing integration
of TD-positive hEPSCs into ICM of host monkey
embryos at d.p.f.9 (n = 7). The embryos were
stained for OCT4 (green) and NANOG (gray). Scale
bar, 250 mm. Bottom, enlargements of the insert
(white dotted line) in the top panel. Arrows indicate
TD-positive hEPSCs expressing OCT4 and
NANOG. Yellow dotted line indicates ICM. Scale
bar, 50 mm.
(B) Representative IF images showing hEPSCs
differentiated into HYP-like cells within host
monkey embryos at d.p.f.9. The embryos were
stained for GATA4 (gray) and NANOG (green).
Scale bar, 250 mm. Bottom, enlargements of the
insert (white dotted line) in the top panel. Arrow
indicates a TD-positive hEPSC expressing
GATA4. Yellow dotted line indicates ICM. Scale
bar, 100 mm.
(C) Representative IF images showing integration
of TD-positive hEPSCs into TE of host monkey
embryos at d.p.f.9 (n = 3). The embryos were
stained for TFAP2C (gray) and CK7 (green). Scale
bar, 100 mm. Bottom, enlargements of the insert
(white dotted line) in the top panel. Arrow indicates
a TD-positive hEPSC expressing TFAP2C and
CK7. Scale bar, 50 mm.
(D) Representative IF images showing incorporation
of hEPSCs into EPI of host monkey embryo at
d.p.f.11 (n = 3). The embryos were stained for T
(gray) and OCT4 (green). Scale bar, 250 mm. Bot-
tom: enlargements of inserts (white dotted line) in
the top panel. Notably, hEPSCs rarely express T
(arrow), a marker of mesoderm, whereas the
expression of T is detected in monkey cells
(arrowhead). Yellow dotted line indicates EPI. Scale
bar, 25 mm.
(E) Expression of HYP marker, PDGFRa, in hEPSCs
at d.p.f.11 (n = 2). The embryos were stained for
PDGFRa (green). Scale bar, 50 mm. Bottom: en-
largements of the insert (white dotted line) in the top
panel. Arrow indicates a tdTomato-positive hEPSC
expressing PDGFRa. Yellow dotted line indicates
EPI. Scale bar, 10 mm.
(F) IF images of sections of monkey embryos at
d.p.f.13 (n = 5) staining for SOX17 (green).
Arrows indicate tdTomato-positive hEPSCs expressing SOX17. Scale bar, 50 mm. Yellow dotted line indicates EPI.
(G) Levels of chimerism of hEPSCs within EPI, HYP, and TE. EPI cells expressed only OCT4, and HYP cells expressed GATA6 and/or GATA4, whereas TE expressed
CK7 (a total of 25 embryos and 17,938 cells were analyzed). EPI, epiblast; HYP, hypoblast; TE, trophectoderm; TD, tdTomato; DAPI, 40,6-diamidino-2-phenylindole. See also Figure S1.
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the ratio of reads mapped to the human or cynomolgus monkey
genomes were used to further confirm each cell’s species of
origin (Figures S2A and S2B). After stringent filtering, 200 human
and 272 monkey cells were used for further analyses (Table S2).
On average, 9,798 genes (transcripts per million [TPM] > 0) and
27,936,953 reads were detected per cell. There was no statistical
difference in the number of genes and reads detected between
human and monkey cells (Figure S2C). For comparison, we
also included published scRNA-seq datasets containing cells
from monkey and human embryos in the analyses (Nakamura
et al., 2016; Niu et al., 2019; Xiang et al., 2020; Zhou et al.,
2019). To avoid batch-specific systematic variations of scRNA-
seq caused by integration of different datasets, we used an ‘‘an-
chors’’ method that is recommended for batch-effect removal
(Stuart et al., 2019) (Figure S2C).
We first performed t-distributed stochastic neighbor embed-
ding (t-SNE) analysis on the scRNA-seq datasets. Based on
the expression of known lineage markers, we identified four ma-
jor cell clusters that were present in all samples (both chimeric
and control embryos): EPI, HYP, TE, and EXMC (Figures 3A,
3B, and S2D). These cells also expressed lineage-specific
markers that showed conservation between humans and
Cell 184, 2020–2032, April 15, 2021 2023
A
C
D
B Figure 3. Transcriptional landscape of hu- man-monkey chimeric embryos
(A) t-SNE plot of cells from chimeric and control
non-chimeric embryos. Cells were identified as
EPI, HYP, TE, and EXMC. Cells were colored by
different species origins and datasets.
(B) Expression of lineage-specific marker genes of
EPI, HYP, and TE exhibited on t-SNE plots. A
gradient of gray, yellow, and red indicates low to
high expression.
(C) The phylogenetic tree shows the cluster of EPI,
HYP, and TE cells from chimeric embryos at
different stages (d.p.f.9, 11, 13, 15, and 17). Cells
are highlighted by species origins (human or
monkey), different stages (d.p.f.9, 11, 13, 15, and
17), and different cell types (EPI, HYP, and TE).
(D) Bar plot showing the distribution of cells from
different origins in the four lineages (EPI, EXMC,
HYP, and TE). EPI, epiblast; HYP, hypoblast; TE,
trophectoderm; EXMC, extra-embryonic mesen-
chyme cell.
See also Figure S2 and Table S2.
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monkeys identified in a previous study (Figure S2E) (Zhou et al.,
2019). The presence of these cell types in chimeric embryos sug-
gests that the development of host embryos was by and large
unaffected by the injected hEPSCs (Ma et al., 2019; Niu et al.,
2019), which is consistent with the morphological analysis
(Figures 1B and 1D). Interestingly, phylogenetic tree analysis
(based on gene expression levels) revealed that while most mon-
key cells within chimeric embryos (chimeric monkey cells for
short) segregated into distinct cell-type-specific clusters (EPI,
HYP, and TE), chimeric human HYP- and TE-like cells clustered
with EPI-like cells (Figure 3C). Thus, it seems that the chimeric
monkey cells exhibited a more faithful lineage segregation than
the introduced hEPSCs. In agreement with IF results, very few
human TE-like cells were identified in the scRNA-seq data (Fig-
ures 3A and 3D) and were therefore excluded from subsequent
analyses. Together, these results demonstrate that hEPSCs
can differentiate into several peri- and early post-implantation
cell types after being introduced into monkey early blastocysts
followed by ex vivo embryo culture.
Transcriptome dynamics of hEPSCs during human- monkey chimera development We next investigated the transcriptional kinetics of chimeric hu-
man and monkey cells. We first constructed a force-directed K-
nearest neighbor graph (SPRING) (Weinreb et al., 2018) based on
transcriptomic properties of all cells (see STAR Methods). All
cells bifurcated into three branches: EPI, HYP, and TE (Fig-
ure 4A). The correlations of gene expression patterns between
chimeric and control (human and monkey) embryos were deter-
2024 Cell 184, 2020–2032, April 15, 2021
mined (Figure 4B). Similar correlation co-
efficients were obtained when chimeric
human cells were compared to control
human (0.460) or monkey (0.459) cells
(Figure 4B, right panels). Intriguingly,
when compared to control embryos,
chimeric monkey cells exhibited higher
correlation coefficients than chimeric human cells (Figure 4B,
left panels). We next generated lineage-specific correlation
matrices. We found that chimeric human EPI-like cells were
similar to EPI cells in human embryos, whereas chimeric human
HYP- and EXMC-like cells shared the highest correlation coeffi-
cients with chimeric monkey HYP cells and EXMCs, respectively
(Figure 4C). Of note, we also found that chimeric monkey EPI
cells and EXMCs more resembled chimeric than control human
cells. Interestingly, chimeric human EPI-like cells were found to
gradually gravitate toward the chimeric monkey EPI cells, with
R2 values increasing from 0.363 (pre-implantation EPI [Pre_EPI])
to 0.464 (post-implantation late EPI [PostL_EPI]) and then to
0.693 (gastrulating [Gast] cells) (Figure 4C). Taken together,
these results demonstrate that the monkey embryonic microen-
vironment exerts influence on the transcriptional states of human
cells and vice versa.
As monkey cells exhibited transcriptomic changes in the pres-
ence of human cells, we next sought to delineate the develop-
mental dynamics of monkey cells within chimeric embryos. We
first identified differentially expressed genes (DEGs) between
chimeric and control monkey embryos. Comparisons of EPI
cells, HYP cells, and EXMCs revealed that 424, 7, and 241 genes
were downregulated, whereas 5, 2, and 13 genes were upregu-
lated, respectively, in cells from chimeric compared to control
embryos, although the expression levels of lineage marker genes
remained comparable (Figures 4D and S3A). Gene Ontology
(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
enrichment analyses identified a number of signaling pathways
that were up- and downregulated in chimeric monkey EPI cells,
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E
B
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HYP cells, and EXMCs. For example, the Hippo and transforming
growth factor b (TGF-b) signaling pathways were downregulated
in chimeric monkey EPI cells and EXMCs (Figure 4E),
respectively.
Having shown that the transcriptomic profiles of monkey EPI
cells were altered in chimeric embryos, we next investigated
whether the monkey EPI embryonic niche was also affected by
human cells. To this end, CellPhoneDB (v2.0.1) (Vento-Tormo
et al., 2018) was applied to identify potential cellular interactions
between EPI and other lineages (HYP and EXMC) in both
chimeric and control embryos (see STAR Methods). We sought
to identify interactions that were specific to chimeric or control
embryos. We found more ligand-receptor interactions in
chimeric embryos when compared to control embryos (e.g.,
117 [chimeric] versus 10 [control] specific ligand-receptor inter-
actions were detected in monkey EPI cells) (Figures 4F and S3B;
Table S3). KEGG analysis was performed to reveal specific
signaling pathways that were enriched within chimeric and
control embryos. We found several signaling pathways (e.g.,
phosphatidylinositol 3-kinase [PI3K]-Akt and mitogen-activated
protein kinase [MAPK] signaling pathways) that were strength-
ened in chimeric embryos and new signaling pathways (e.g.,
WNT signaling pathway) that were specifically enriched in
chimeric embryos (Figure 4G). Using the same method, we
also determined human and monkey cell-cell interactions within
chimeric embryos and identified distinct ligand-receptor interac-
tions in EPI cells, such as FGF5-FGFR4, NOTCH4-JAG2,
WNT2B-FZD4, WISP3-SORL1, and PLXNB2-PTN (Figures S3C
and S3D; Table S3). Taken together, our results suggest that
cell-cell interactions are reinforced within the chimeric embryos
and potentially lead to activation of additional signaling
pathways.
Chimeric human EPI-like cells display a distinct developmental trajectory EPI development is characterized by pluripotency transitions that
may exhibit different dynamics between species. As proper EPI
specification and differentiation are critical for chimera formation
and development, we examined the lineage allocation of human
EPI-like cells within the chimeric embryos and compared it with
the datasets of in-vitro-cultured human and monkey embryos
Figure 4. Developmental trajectory of human-monkey chimeric embry
(A) The differentiation trajectory of chimeric cells and control non-chimeric cells
colored by cell lineages.
(B) Overall similarity under the four comparisons (chimera-monkey versus control
versus control non-chimeric human [‘‘Chimera-Human vs. Control-Human’’], c
Control-Monkey’’], and chimera-human versus control non-chimeric monkey [‘‘C
(C) Heatmap of the correlation coefficients among different cell origins under cor
(D) Histogram showing the numbers of DEGs for different lineages (EPI, HYP, and
embryos. The red and blue colors represent up- and down-regulated genes, res
(E) GO and KEGG enrichment analyses of the DEGs in (D). Red and blue represen
respectively.
(F) The Venn diagrams showing the overlap of the ligand-receptor interactions bet
non-chimeric (Control-Monkey) monkey cells.
(G) Comparison of KEGG pathways enriched by the specific interactions betwee
cells in (F). EPI, epiblast; EXMC, extra-embryonic mesenchyme cell; HYP, hypobla
gastrulating cell; DEG, differentially expressed gene.
See also Figure S3 and Table S3.
2026 Cell 184, 2020–2032, April 15, 2021
(Nakamura et al., 2016; Niu et al., 2019; Zhou et al., 2019). Human
EPI-like cells were identified at pre-implantation, post-implanta-
tion, and gastrulating stages, and at each stage, they expressed
distinct markers (Figures 5A and S4A–S4C). A Sankey diagram
also showed the same developmental dynamics of human EPI-
like cells (Figure S4D). We next determined the relationship be-
tween hEPSCs (Yang et al., 2017b), chimeric human EPI-like
cells, EPI cells from control human and monkey embryos (Niu
et al., 2019; Zhou et al., 2019), and human and monkey PSCs
(primed and naive) (Chan et al., 2013; Chen et al., 2015; Gafni
et al., 2013; Theunissen et al., 2014). We observed that hEPSCs
were more similar to early post-implantation EPI (PostE_EPI)
and PostL_EPI cells from human and monkey embryos, respec-
tively, as well as human and monkey naive PSCs. Chimeric hu-
man PostL_EPI-like cells showed higher correlation coefficients
to primed PSCs than naive PSCs (Figure S4E). To further investi-
gate the transcriptional kinetics of hEPSCs (Yang et al., 2017b),
chimeric humanEPI-like cells, andhost monkeyEPI cells, we per-
formed RNA velocity (La Manno et al., 2018) and Slingshot ana-
lyses (Street et al., 2018) (Figure 5B). We observed two distinct
patterns of RNA velocity vectors; chimeric human PostL_EPI-
like cells bore long vectors, and gastrulating-like cells bore short
vectors. In contrast, host monkey PostL_EPI cells lacked long
vectors, and gastrulating cells bore long vectors (Figure 5B, left
two panels). These results imply that development is delayed
for chimeric human EPI-like cells. Slingshot analysis revealed
that after injection into monkey blastocysts, hEPSCs followed
the EPSC to PostL_EPI to gastrulation developmental trajectory
(Figure 5B, right panel). To further delineate the developmental
trajectory of chimeric human EPI-like cells, we mapped all EPI-
related human and monkey reads to a consensus genome and
aligned EPI development trajectories between species using a
previously reported method (Kanton et al., 2019). In agreement
with the RNA velocity analysis, we found that chimeric human
EPI-like cells differentiated more slowly than EPI cells from host
monkey, control monkey, and human embryos (Figure 5C). These
results suggest that the specification and/or differentiation of
hEPSCs toward the EPI lineages was less efficient than embry-
onic cells.
As mentioned above, global transcriptional profiles of chimeric
human EPI-like cells more resembled monkey EPI cells within the
os
. The differentiation trajectory was reconstructed using SPRING. Cells were
non-chimeric human [‘‘Chimera-Monkey vs. Control-Human’’], chimera-human
himera-monkey versus control non-chimeric monkey [‘‘Chimera-Monkey vs.
himera-Human vs. Control-Monkey’’]). Cells are colored by cell origins.
responding lineages (EPI, EXMC, HYP, Pre_EPI, PostL_EPI, and Gast).
EXMC) in the host monkey cells compared to cells from control non-chimeric
pectively.
t enrichment p values (�log10 transformed) for up- and downregulated DEGs,
ween EPI-EPI, EPI-HYP, and EPI-EXMC in host (Chimera-Monkey) and control
n host (Chimera-Monkey) and control non-chimeric (Control-Monkey) monkey
st; Pre_EPI, pre-implantation EPI; PostL_EPI, post-implantation late EPI; Gast,
A
C
D
F
E
B
Figure 5. Developmental trajectory of chimeric human EPI-like cells within monkey embryos
(A) t-SNE plot of EPI cells at different sublineages. Cells are colored by cell origins and designated as ICM, Pre_EPI, PostE_EPI, PostL_EPI, and Gast. Cells of
‘‘Control-Human 1’’ derive from dataset ‘‘Human-1’’ (Zhou et al., 2019). Cells of ‘‘Control-Monkey 1’’ derive from dataset ‘‘Monkey-1’’ (Niu et al., 2019). Cells of
‘‘Control-Monkey 2’’ derive from dataset ‘‘Monkey-2’’ (Nakamura et al., 2016). Lower right: single cells are colored according to embryonic stages.
(B) RNA velocity analysis of chimeric human (chimera-human) and host monkey (chimera-monkey) cells, respectively (left two panels). The amplitude and di-
rection of the vector reflects a transcriptional trajectory. Slingshot analysis of chimeric human EPI-like cells and hEPSCs (third panel). The curves and arrows
indicate the potential development trajectory. Cells are colored by cell lineages.
(C) Pseudotime alignment between control and chimeric cells. Left and right panels show the pseudotime alignment of chimeric cells with control human and host
monkey cells, respectively.
(legend continued on next page)
ll
Cell 184, 2020–2032, April 15, 2021 2027
Article
ll Article
chimeric embryos than EPI cells from human embryos. To further
delineate the potential processes regulating the development of
chimeric human EPI-like cells, we generated Venn diagrams us-
ing DEGs identified between human EPI-like and monkey EPI
cells within the chimeric embryos, between EPI cells from control
human and monkey embryos, between human EPI-like cells
from chimeric embryos and EPI cells from human embryos,
and between EPI cells from chimeric and control monkey em-
bryos. These analyses revealed 315 genes uniquely shared be-
tween chimeric human EPI-like cells and EPI cells from control
monkey embryos (Figure 5D; Table S4). KEGG analysis using
these genes identified PI3K-Akt, MAPK, and Peroxisome prolif-
erator-activated receptor (PPAR) signaling pathways, which
may be involved in shifting the transcriptomes of human EPI-
like cells toward the monkey EPI cells (Figures 5E and 5F). We
also found a number of hub genes that potentially played impor-
tant roles in regulating the development of chimeric human EPI-
like cells, namely CHD2, POLR2A, and RB1 (Figures S5A and
S5B). In addition, we found that a majority of chimeric human
EPI-like cells expressed S/G2/M cell-cycle-related genes, and
the expression levels of apoptosis-related genes showed no sig-
nificant difference among human and monkey cells within
chimeric and control embryos (Figures S5C and S5E). Although
chimeric human EPI-like cells showed slower than normal differ-
entiation, GO analysis using DEGs in Pre_EPI, PostL_EPI, and
gastrulating (Gast) cells revealed developmental progression
(Figure S5D). Together, these analyses reveal signaling path-
ways and factors that likely drive distinct lineage specification
and differentiation dynamics of chimeric human EPI-like cells
within monkey embryos.
DISCUSSION
There are significant ethical considerations involved in gener-
ating and studying human-animal chimeric embryos, particularly
when non-human primates are involved. Different guidelines
exist at the state, national, and international levels, and it is
important for scientists, bioethicists, policy makers, and funding
agencies to stay engaged in keeping these guidelines up to date
with the relevant science as well as for the welfare of society. For
the studies presented here, extensive reviews of research plans
and protocols were conducted in advance. Ethical consultations
and reviews were performed both at the institutional level and via
outreach to non-affiliated bioethicists with experience in state
and national bioethics policies regarding these matters. This
thorough and detailed process helped guide our experiments,
which were focused entirely on ex vivo chimeric embryos.
Furthermore, we limited our studies to early-stage chimeric em-
bryo development.
(D) The Venn diagram shows the overlap of DEGs obtained from different comp
making chimeric human EPI-like cells similar to host monkey EPI cells) and two gen
EPI cells similar to control human EPI cells) are subsets from the DEGs between
control-monkey cells, respectively.
(E) Enriched KEGG pathways for the 315 DEGs.
(F) The dynamic changes of the 315 DEGs enriched pathways along the pseudo
E_EPI, post-implantation early EPI; PostL_EPI, post-implantation late EPI; Gast,
See also Figures S4 and S5 and Table S4.
2028 Cell 184, 2020–2032, April 15, 2021
Using hPSCs to generate human-animal chimeric embryos
provides an experimental paradigm for studying early human
development and holds great potential for diverse applications
in regenerative medicine as well as for producing human tissues
and organs for replacement therapies. To achieve interspecies
blastocyst complementation, donor PSCs must be able to
contribute to chimera formation once introduced into the host
embryo. Rat and mouse PSCs robustly contribute to chimera for-
mation when introduced into mouse and rat blastocysts, respec-
tively. To date, however, robust chimerism between species that
are more evolutionarily distant has not been achieved. Several
groups, including ours, have rigorously demonstrated that
hPSCs inefficiently contributed to chimera formation in early
mouse (E8.5–E17.5) and pig (E17–E28) embryos. Levels of
chimerism were far lower than those achieved between rat and
mouse (Bayerl et al., 2020; Das et al., 2020; Hu et al., 2020; Sal-
azar-Roa et al., 2020; Theunissen et al., 2014, 2016; Wang et al.,
2018; Wu et al., 2017; Yang et al., 2017b). This conclusion was
reached by systematically testing the chimeric competency of
human embryonic stem cells (hESCs)/induced PSCs (iPSCs)
generated under different culture conditions in different labora-
tories. Different sex and genetic backgrounds were also tested.
These results likely reflect the >90-million-year evolutionary dis-
tance between primates and rodents and between primates and
ungulates. By contrast, the mouse and rat are separated by �21 million years of evolution. Thus, more pronounced xenogeneic
barriers might exist between evolutionarily distant species dur-
ing early development. These barriers likely reflect differences
in embryonic development (Rossant, 2018; Rossant and Tam,
2018; Wu et al., 2016) that manifest at the molecular and cellular
levels as differential cell adhesion, ligand-receptor incompatibil-
ity, cell competition (Zheng et al., 2021), and differences in cell-
cycle rates and developmental timing, among others. To gain
insights into early human development and identify key molecu-
lar processes underlying xenogeneic barriers, it is thus impera-
tive to study human-animal chimeras using a host species that
is more closely related to humans.
Dynamic PSC states reflect the spectrum of pluripotency
observed in vivo (Morgani et al., 2017; Riveiro and Brickman,
2020; Wu and Izpisua Belmonte, 2015). Studies in rodents
have identified a number of pluripotency states (e.g., naive and
primed) that exhibit different molecular and functional proper-
ties, including variable abilities to contribute to chimera forma-
tion (Wu and Izpisua Belmonte, 2015). A single mouse EPSC
cultured in the LCDM condition (leukemia inhibitory factor [LIF],
the GSK3 inhibitor CHIR99021, (S)-(+)-dimethindene maleate
[DiM)], and minocycline hydrochloride [MiH]) can give rise to an
entire adult mouse via tetraploid complementation, which is
not seen with other PSCs (Yang et al., 2017b). Thus, mouse
EPSCs seem to exhibit the highest chimeric capability among
arisons. The 315 genes (left panel shows genes that are putatively involved in
es (right panel shows genes that are putatively involved in making host monkey
chimera-human and control-human cells and between chimera-monkey and
time. ICM, inner cell mass; EPI, epiblast; Pre_EPI, pre-implantation EPI; Post-
gastrulating cell; DEG, differentially expressed gene.
ll Article
all reported PSCs. LCDM-cultured hEPSCs also exhibit relatively
higher chimera competency in E10.5 mouse conceptuses (�1%) when compared with other hPSCs (Yang et al., 2017b). To maxi-
mize the possibility of a positive outcome and to minimize the
number of monkey embryos used, we chose to only focus on a
well-characterized hEPSC line.
In the current study, we combined prolonged ex vivo monkey
embryo culture with hEPSC injection to study human chimerism
in a closely related non-human primate species (Macaca fascicu-
laris). We found that hEPSCs were incorporated into the devel-
opmental program of the monkey embryo until d.p.f.19. Savatier
and colleagues recently found that human naive PSCs stalled in
the G1 phase of the cell cycle and therefore could not contribute
to chimera formation when introduced into rabbit or macaque
pre-implantation embryos (Aksoy et al., 2020), which is not
observed in the present study. One possible explanation for
this discrepancy is that PSCs cultured in the LCDM condition
may acquire features that make them more proliferative and/or
better able to survive in host monkey embryos, as they did in
mouse chimeras (Yang et al., 2017a, 2017b). Recent studies
have also shown that human and monkey PSCs residing in an in-
termediate pluripotent state exhibit improved chimera contribu-
tion over naive or naive-like PSCs in pig embryos (Fu et al., 2020;
Wu et al., 2017). These observations suggest that donor PSC
culture conditions need to be catered to different host species
to enable robust interspecies chimerism.
In the current study, we found that hEPSCs contributed to
several main lineages (EPI, HYP, and EXMC) in peri- and early
post-implantation monkey embryos. Despite transcriptomic dif-
ferences, EPI-like cells derived from hEPSCs largely followed the
specification and differentiation dynamics observed in control
monkey and human embryos ex vivo. This suggests that EPI
developmental programs are evolutionarily conserved between
humans and monkeys. Recently, the ability of mouse EPSCs
and hEPSCs to differentiate into trophoblasts has been called
into question (Guo et al., 2020; Posfai et al., 2020; Yang et al.,
2017a, 2017b). Consistent with recent observations, we found
that hEPSCs rarely contributed to TE derivatives in human-mon-
key chimeric embryos. This may be due to clonal effect (only one
hEPSC line was used in this study), species differences, and/or a
lack of TE potency in hEPSCs.
Our scRNA-seq analyses revealed transcriptomic differences
between cells in human-monkey chimeric embryos (both human
and monkey) when compared with control human and monkey
embryos (data retrieved from a publicly accessible database)
(Nakamura et al., 2016; Niu et al., 2019; Xiang et al., 2020;
Zhou et al., 2019). This is likely because introduced human cells
impacted embryonic developmental niches within the host mon-
key embryos. To gain insights into the mechanisms underlying
these changes, we studied cell-cell interactions and examined
ligand-receptor pairing. We found that cell-cell interactions be-
tween human and monkey cells within chimeric embryos
showed some distinct features when compared to controls.
We also identified a number of signaling pathways that were
potentially involved (e.g., MAPK and PI3K-AKT). It will be inter-
esting to determine whether modulating these pathway activities
in donor hPSCs and/or cultured embryos may improve interspe-
cies chimerism in future studies.
Most recently, interspecies PSC co-cultures (e.g., human and
rhesus macaque) have been developed to study interspecies
cell competition (Zheng et al., 2021). In addition, through ad-
vances in both 2D and 3D culture systems, hPSCs have been
guided to patterned structures that model post-implantation
developmental processes, including amniotic sac formation,
gastrulation, and neurulation (Martyn et al., 2018; Shao et al.,
2017; Simunovic et al., 2019; Warmflash et al., 2014; Xue et al.,
2018; Zheng et al., 2019). While these in vitro models are valuable
in studying human pluripotent EPI cells and their derivatives, they
lack extra-embryonic tissues, including trophoblast and HYP. In
comparison, the human-monkey ex vivo model reported in this
study is less accessible than in vitro PSC models but bears the
advantage of studying human development in a conceptus
context that captures the spatiotemporal dynamic cell interac-
tions from different tissues. As research progresses, all of these
different models will be invaluable and complementary to one
another to help gain insights into early human development.
In summary, our findings provide insights into the cellular and
molecular events that occur when human and monkey cells are
mixed early during embryonic development. These results
shed light on evolutionarily convergent and divergent processes
during primate embryogenesis. Ultimately, this line of funda-
mental research will help improve human chimerism in species
more evolutionarily distant that for various reasons, including so-
cial, economic, and ethical, might be more appropriate for
regenerative medicine translational therapies.
Limitations of study Despite the fact that a relatively large number of cynomolgus
monkey embryos (132) were used in this study, due to the
inherent randomness of chimera experiments and to account
for the full range of factors that potentially affect interspecies
chimerism, an expanded analysis with a larger number of mon-
key embryos will be needed. The constraint in the number of em-
bryos we could utilize led to several limitations of the current
study: (1) we only tested chimera competency of a hEPSC line
in monkey embryos and did not study other pluripotency states
from human or other species (it should be noted that monkey
EPSCs have not been reported to date), (2) we did not test the
effect of changing the numbers of hEPSCs injected in monkey
embryos, and (3) we were not able to test different injection
stages.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Animals
B Culture of human extended pluripotent stem
(hEPS) cells
Cell 184, 2020–2032, April 15, 2021 2029
ll Article
B Animal, stem cell, and ethical approvals
d METHOD DETAILS
B Oocyte collection and in vitro fertilization
B Microinjection of human EPS cells into monkey blasto-
cysts and in vitro embryo culture
B Human-monkey chimeric embryos culture in vitro
B Single cell collection and single cell RNA-seq
(scRNA-seq)
B Immunofluorescence (IF) analysis
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Statistical analysis
B Cell origin identification, scRNA-seq data pre-process-
ing and quality control
B Cell clustering, lineage identification
B Identification of differentially expressed genes
B Pseudotime alignment and pseudotime-dependent
differentially expressed genes analysis
B GO and KEGG pathway analysis
B Single-cell transcription factor regulation networks
construction and clustering
B RNA velocity analysis
B Identification of cross-talks between human and mon-
key cells within chimeric embryos
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.cell.
2021.03.020.
ACKNOWLEDGMENTS
Special thanks to the Stanford University Research Ethics Program for helpful
discussions and feedback during the protocol review process. We would like
to thank David O’Keefe for his critical reading of this manuscript and invaluable
comments. This work was supported by grants from the National Key Research
and Development Program (2018YFA0801400 and 2016YFA0101401), the Na-
tional Natural Science Foundation of China (81760271 and 31660346), Major
Basic Research Project of Science and Technology of Yunnan (2017ZF028
and 202001BC070001), Key Projects of Basic Research Program in Yunnan
Province (2017FA010), High-level Talent Cultivation Support Plan of Yunnan
Province and Yunnan Fundamental Research Projects (2019FB050), UCAM,
and The Moxie Foundation.
AUTHOR CONTRIBUTIONS
T.T., J.W., Y.N., W.J., and J.C.I.B. designed the study and supervised all ex-
periments. C.S. and Y.K. performed samples collection work, including super-
ovulation, micromanipulation, and animal care. T.T., Y.Z., N.S., E.Z., H.S.,
W.S., H.W., Z.C., R.H.-B., L.M.M., E.N.D., W.T.B., M.S., Z.A., T.L., and
C.R.E. carried out experiments or contributed critical reagents and protocols.
T.T., J.W., S.D., P.Y., and R.Z. analyzed the data and performed statistical an-
alyses. T.T., J.W., S.D., W.J., and J.C.I.B. wrote the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
INCLUSION AND DIVERSITY
We worked to ensure sex balance in the selection of non-human subjects. One
or more of the authors of this paper self-identifies as an underrepresented
ethnic minority in science. One or more of the authors of this paper self-iden-
tifies as living with a disability. One or more of the authors of this paper
2030 Cell 184, 2020–2032, April 15, 2021
received support from a program designed to increase minority representation
in science. The author list of this paper includes contributors from the location
where the research was conducted who participated in the data collection,
design, analysis, and/or interpretation of the work.
Received: September 29, 2020
Revised: January 25, 2021
Accepted: March 9, 2021
Published: April 15, 2021
REFERENCES
Aibar, S., González-Blas, C.B., Moerman, T., Huynh-Thu, V.A., Imrichova, H.,
Hulselmans, G., Rambow, F., Marine, J.C., Geurts, P., Aerts, J., et al. (2017).
SCENIC: single-cell regulatory network inference and clustering. Nat. Methods
14, 1083–1086.
Aksoy, I., Rognard, C., Moulin, A., Marcy, G., Masfaraud, E., Wianny, F., Cor-
tay, V., Bellemin-Ménard, A., Doerflinger, N., Dirheimer, M., et al. (2020). Pri-
mate naı̈ve pluripotent stem cells stall in the G1 phase of the cell cycle and
differentiate prematurely during embryo colonization. bioRxiv,
2020.2003.2027.011890.
Bayerl, J., Ayyash, M., Shani, T., Manor, Y., Gafni, O., Kalma, Y., Aguilera-Cas-
trejon, A., Zerbib, M., Amir, H., Sheban, D., et al. (2020). Tripartite Inhibition of
SRC-WNT-PKC Signalling Consolidates Human Naı̈ve Pluripotency. bioRxiv,
2020.2005.2023.112433.
Butler, A., Hoffman, P., Smibert, P., Papalexi, E., and Satija, R. (2018). Inte-
grating single-cell transcriptomic data across different conditions, technolo-
gies, and species. Nat. Biotechnol. 36, 411–420.
Chan, Y.S., Göke, J., Ng, J.H., Lu, X., Gonzales, K.A., Tan, C.P., Tng, W.Q.,
Hong, Z.Z., Lim, Y.S., and Ng, H.H. (2013). Induction of a human pluripotent
state with distinct regulatory circuitry that resembles preimplantation epiblast.
Cell Stem Cell 13, 663–675.
Chen, Y., Niu, Y., Li, Y., Ai, Z., Kang, Y., Shi, H., Xiang, Z., Yang, Z., Tan, T., Si,
W., et al. (2015). Generation of Cynomolgus Monkey Chimeric Fetuses using
Embryonic Stem Cells. Cell Stem Cell 17, 116–124.
Das, S., Koyano-Nakagawa, N., Gafni, O., Maeng, G., Singh, B.N., Rasmus-
sen, T., Pan, X., Choi, K.D., Mickelson, D., Gong, W., et al. (2020). Generation
of human endothelium in pig embryos deficient in ETV2. Nat. Biotechnol. 38,
297–302.
De Los Angeles, A. (2019). The Pluripotency Continuum and Interspecies Chi-
meras. Curr. Protoc. Stem Cell Biol. 50, e87.
De Los Angeles, A., Ferrari, F., Xi, R., Fujiwara, Y., Benvenisty, N., Deng, H.,
Hochedlinger, K., Jaenisch, R., Lee, S., Leitch, H.G., et al. (2015). Hallmarks
of pluripotency. Nature 525, 469–478.
Deglincerti, A., Croft, G.F., Pietila, L.N., Zernicka-Goetz, M., Siggia, E.D., and
Brivanlou, A.H. (2016). Self-organization of the in vitro attached human em-
bryo. Nature 533, 251–254.
Fu, R., Yu, D., Ren, J., Li, C., Wang, J., Feng, G., Wang, X., Wan, H., Li, T.,
Wang, L., et al. (2020). Domesticated cynomolgus monkey embryonic stem
cells allow the generation of neonatal interspecies chimeric pigs. Protein Cell
11, 97–107.
Gafni, O., Weinberger, L., Mansour, A.A., Manor, Y.S., Chomsky, E., Ben-Yo-
sef, D., Kalma, Y., Viukov, S., Maza, I., Zviran, A., et al. (2013). Derivation of
novel human ground state naive pluripotent stem cells. Nature 504, 282–286.
Gao, X., Nowak-Imialek, M., Chen, X., Chen, D., Herrmann, D., Ruan, D., Chen,
A.C.H., Eckersley-Maslin, M.A., Ahmad, S., Lee, Y.L., et al. (2019). Establish-
ment of porcine and human expanded potential stem cells. Nat. Cell Biol. 21,
687–699.
Guo, G., Stirparo, G.G., Strawbridge, S., Yang, J., Clarke, J., Li, M.A., Myers,
S., Özel, B.N., Nichols, J., and Smith, A. (2020). Trophectoderm Potency is Re-
tained Exclusively in Human Naı̈ve Cells. bioRxiv, 2020.2002.2004.933812.
Hackett, J.A., and Surani, M.A. (2014). Regulatory principles of pluripotency:
from the ground state up. Cell Stem Cell 15, 416–430.
ll Article
Harvey, A., Caretti, G., Moresi, V., Renzini, A., and Adamo, S. (2019). Interplay
between Metabolites and the Epigenome in Regulating Embryonic and Adult
Stem Cell Potency and Maintenance. Stem Cell Reports 13, 573–589.
Hu, Z., Li, H., Jiang, H., Ren, Y., Yu, X., Qiu, J., Stablewski, A.B., Zhang, B.,
Buck, M.J., and Feng, J. (2020). Transient inhibition of mTOR in human plurip-
otent stem cells enables robust formation of mouse-human chimeric embryos.
Sci. Adv. 6, eaaz0298.
Huang, K., Zhu, Y., Ma, Y., Zhao, B., Fan, N., Li, Y., Song, H., Chu, S., Ouyang,
Z., Zhang, Q., et al. (2018). BMI1 enables interspecies chimerism with human
pluripotent stem cells. Nat. Commun. 9, 4649.
Kanehisa, M. (2019). Toward understanding the origin and evolution of cellular
organisms. Protein Sci. 28, 1947–1951.
Kanehisa, M., and Goto, S. (2000). KEGG: kyoto encyclopedia of genes and
genomes. Nucleic Acids Res. 28, 27–30.
Kanehisa, M., Sato, Y., Furumichi, M., Morishima, K., and Tanabe, M. (2019).
New approach for understanding genome variations in KEGG. Nucleic Acids
Res. 47 (D1), D590–D595.
Kanton, S., Boyle, M.J., He, Z., Santel, M., Weigert, A., Sanchı́s-Calleja, F.,
Guijarro, P., Sidow, L., Fleck, J.S., Han, D., et al. (2019). Organoid single-cell
genomic atlas uncovers human-specific features of brain development. Nature
574, 418–422.
Kim, D., Paggi, J.M., Park, C., Bennett, C., and Salzberg, S.L. (2019). Graph-
based genome alignment and genotyping with HISAT2 and HISAT-genotype.
Nat. Biotechnol. 37, 907–915.
Kumar, S., Stecher, G., Li, M., Knyaz, C., and Tamura, K. (2018). MEGA X: Mo-
lecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol.
Evol. 35, 1547–1549.
La Manno, G., Soldatov, R., Zeisel, A., Braun, E., Hochgerner, H., Petukhov, V.,
Lidschreiber, K., Kastriti, M.E., Lönnerberg, P., Furlan, A., et al. (2018). RNA ve-
locity of single cells. Nature 560, 494–498.
Ma, H., Zhai, J., Wan, H., Jiang, X., Wang, X., Wang, L., Xiang, Y., He, X., Zhao,
Z.A., Zhao, B., et al. (2019). In vitro culture of cynomolgus monkey embryos
beyond early gastrulation. Science 366, eaax7890.
Martyn, I., Kanno, T.Y., Ruzo, A., Siggia, E.D., and Brivanlou, A.H. (2018). Self-
organization of a human organizer by combined Wnt and Nodal signalling. Na-
ture 558, 132–135.
Mishra, R., Grzybek, M., Niki, T., Hirashima, M., and Simons, K. (2010). Galec-
tin-9 trafficking regulates apical-basal polarity in Madin-Darby canine kidney
epithelial cells. Proc. Natl. Acad. Sci. USA 107, 17633–17638.
Morgani, S., Nichols, J., and Hadjantonakis, A.K. (2017). The many faces of
Pluripotency: in vitro adaptations of a continuum of in vivo states. BMC Dev.
Biol. 17, 7.
Nakamura, T., Okamoto, I., Sasaki, K., Yabuta, Y., Iwatani, C., Tsuchiya, H.,
Seita, Y., Nakamura, S., Yamamoto, T., and Saitou, M. (2016). A develop-
mental coordinate of pluripotency among mice, monkeys and humans. Nature
537, 57–62.
Niu, Y., Sun, N., Li, C., Lei, Y., Huang, Z., Wu, J., Si, C., Dai, X., Liu, C., Wei, J.,
et al. (2019). Dissecting primate early post-implantation development using
long-term in vitro embryo culture. Science 366, eaaw5754.
Pertea, M., Pertea, G.M., Antonescu, C.M., Chang, T.C., Mendell, J.T., and
Salzberg, S.L. (2015). StringTie enables improved reconstruction of a tran-
scriptome from RNA-seq reads. Nat. Biotechnol. 33, 290–295.
Picelli, S., Faridani, O.R., Björklund, A.K., Winberg, G., Sagasser, S., and
Sandberg, R. (2014). Full-length RNA-seq from single cells using Smart-
seq2. Nat. Protoc. 9, 171–181.
Posfai, E., Schell, J.P., Janiszewski, A., Rovic, I., Murray, A., Bradshaw, B.,
Pardon, T., El Bakkali, M., Talon, I., De Geest, N., et al. (2020). Defining totipo-
tency using criteria of increasing stringency. bioRxiv, 2020.2003.2002.972893.
Riveiro, A.R., and Brickman, J.M. (2020). From pluripotency to totipotency: an
experimentalist’s guide to cellular potency. Development 147, dev189845.
Rossant, J. (2018). Genetic Control of Early Cell Lineages in the Mammalian
Embryo. Annu. Rev. Genet. 52, 185–201.
Rossant, J., and Tam, P.P.L. (2017). New Insights into Early Human Develop-
ment: Lessons for Stem Cell Derivation and Differentiation. Cell Stem Cell
20, 18–28.
Rossant, J., and Tam, P.P.L. (2018). Exploring early human embryo develop-
ment. Science 360, 1075–1076.
Salazar-Roa, M., Trakala, M., Álvarez-Fernández, M., Valdés-Mora, F., Zhong,
C., Muñoz, J., Yu, Y., Peters, T.J., Graña-Castro, O., Serrano, R., et al. (2020).
Transient exposure to miR-203 enhances the differentiation capacity of estab-
lished pluripotent stem cells. EMBO J. 39, e104324.
Shahbazi, M.N., Jedrusik, A., Vuoristo, S., Recher, G., Hupalowska, A., Bolton,
V., Fogarty, N.N.M., Campbell, A., Devito, L., Ilic, D., et al. (2016). Self-organi-
zation of the human embryo in the absence of maternal tissues. Nat. Cell Biol.
18, 700–708.
Shao, Y., Taniguchi, K., Townshend, R.F., Miki, T., Gumucio, D.L., and Fu, J.
(2017). A pluripotent stem cell-based model for post-implantation human am-
niotic sac development. Nat. Commun. 8, 208.
Simunovic, M., Metzger, J.J., Etoc, F., Yoney, A., Ruzo, A., Martyn, I., Croft, G.,
You, D.S., Brivanlou, A.H., and Siggia, E.D. (2019). A 3D model of a human
epiblast reveals BMP4-driven symmetry breaking. Nat. Cell Biol. 21, 900–910.
Smith, A. (2017). Formative pluripotency: the executive phase in a develop-
mental continuum. Development 144, 365–373.
Street, K., Risso, D., Fletcher, R.B., Das, D., Ngai, J., Yosef, N., Purdom, E.,
and Dudoit, S. (2018). Slingshot: cell lineage and pseudotime inference for sin-
gle-cell transcriptomics. BMC Genomics 19, 477.
Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck, W.M.,
3rd, Hao, Y., Stoeckius, M., Smibert, P., and Satija, R. (2019). Comprehensive
Integration of Single-Cell Data. Cell 177, 1888–1902.e1821.
Suchy, F., and Nakauchi, H. (2018). Interspecies chimeras. Curr. Opin. Genet.
Dev. 52, 36–41.
Theunissen, T.W., Powell, B.E., Wang, H., Mitalipova, M., Faddah, D.A.,
Reddy, J., Fan, Z.P., Maetzel, D., Ganz, K., Shi, L., et al. (2014). Systematic
identification of culture conditions for induction and maintenance of naive hu-
man pluripotency. Cell Stem Cell 15, 471–487.
Theunissen, T.W., Friedli, M., He, Y., Planet, E., O’Neil, R.C., Markoulaki, S.,
Pontis, J., Wang, H., Iouranova, A., Imbeault, M., et al. (2016). Molecular
Criteria for Defining the Naive Human Pluripotent State. Cell Stem Cell 19,
502–515.
Trapnell, C., Cacchiarelli, D., Grimsby, J., Pokharel, P., Li, S., Morse, M., Len-
non, N.J., Livak, K.J., Mikkelsen, T.S., and Rinn, J.L. (2014). The dynamics and
regulators of cell fate decisions are revealed by pseudotemporal ordering of
single cells. Nat. Biotechnol. 32, 381–386.
Vento-Tormo, R., Efremova, M., Botting, R.A., Turco, M.Y., Vento-Tormo, M.,
Meyer, K.B., Park, J.E., Stephenson, E., Pola�nski, K., Goncalves, A., et al.
(2018). Single-cell reconstruction of the early maternal-fetal interface in hu-
mans. Nature 563, 347–353.
Vincent, S.D., Dunn, N.R., Hayashi, S., Norris, D.P., and Robertson, E.J.
(2003). Cell fate decisions within the mouse organizer are governed by graded
Nodal signals. Genes Dev. 17, 1646–1662.
Wang, X., Li, T., Cui, T., Yu, D., Liu, C., Jiang, L., Feng, G., Wang, L., Fu, R.,
Zhang, X., et al. (2018). Human embryonic stem cells contribute to embryonic
and extraembryonic lineages in mouse embryos upon inhibition of apoptosis.
Cell Res. 28, 126–129.
Warmflash, A., Sorre, B., Etoc, F., Siggia, E.D., and Brivanlou, A.H. (2014). A
method to recapitulate early embryonic spatial patterning in human embryonic
stem cells. Nat. Methods 11, 847–854.
Weinberger, L., Ayyash, M., Novershtern, N., and Hanna, J.H. (2016). Dynamic
stem cell states: naive to primed pluripotency in rodents and humans. Nat.
Rev. Mol. Cell Biol. 17, 155–169.
Weinreb, C., Wolock, S., and Klein, A.M. (2018). SPRING: a kinetic interface for
visualizing high dimensional single-cell expression data. Bioinformatics 34,
1246–1248.
Wu, J., and Izpisua Belmonte, J.C. (2015). Dynamic Pluripotent Stem Cell
States and Their Applications. Cell Stem Cell 17, 509–525.
Cell 184, 2020–2032, April 15, 2021 2031
ll Article
Wu, J., and Izpisua Belmonte, J.C. (2016). Stem Cells: A Renaissance in Hu-
man Biology Research. Cell 165, 1572–1585.
Wu, J., Greely, H.T., Jaenisch, R., Nakauchi, H., Rossant, J., and Belmonte,
J.C. (2016). Stem cells and interspecies chimaeras. Nature 540, 51–59.
Wu, J., Platero-Luengo, A., Sakurai, M., Sugawara, A., Gil, M.A., Yamauchi, T.,
Suzuki, K., Bogliotti, Y.S., Cuello, C., Morales Valencia, M., et al. (2017). Inter-
species Chimerism with Mammalian Pluripotent Stem Cells. Cell 168, 473–
486.e15.
Xiang, L., Yin, Y., Zheng, Y., Ma, Y., Li, Y., Zhao, Z., Guo, J., Ai, Z., Niu, Y.,
Duan, K., et al. (2020). A developmental landscape of 3D-cultured human
pre-gastrulation embryos. Nature 577, 537–542.
Xue, X., Sun, Y., Resto-Irizarry, A.M., Yuan, Y., Aw Yong, K.M., Zheng, Y.,
Weng, S., Shao, Y., Chai, Y., Studer, L., and Fu, J. (2018). Mechanics-guided
embryonic patterning of neuroectoderm tissue from human pluripotent stem
cells. Nat. Mater. 17, 633–641.
Yang, J., Ryan, D.J., Wang, W., Tsang, J.C., Lan, G., Masaki, H., Gao, X., An-
tunes, L., Yu, Y., Zhu, Z., et al. (2017a). Establishment of mouse expanded po-
tential stem cells. Nature 550, 393–397.
2032 Cell 184, 2020–2032, April 15, 2021
Yang, Y., Liu, B., Xu, J., Wang, J., Wu, J., Shi, C., Xu, Y., Dong, J., Wang, C.,
Lai, W., et al. (2017b). Derivation of Pluripotent Stem Cells with In Vivo Embry-
onic and Extraembryonic Potency. Cell 169, 243–257.e25.
Yu, G., Wang, L.G., Han, Y., and He, Q.Y. (2012). clusterProfiler: an R package
for comparing biological themes among gene clusters. OMICS 16, 284–287.
Zhang, J., Zhao, J., Dahan, P., Lu, V., Zhang, C., Li, H., and Teitell, M.A. (2018).
Metabolism in Pluripotent Stem Cells and Early Mammalian Development. Cell
Metab. 27, 332–338.
Zheng, Y., Xue, X., Shao, Y., Wang, S., Esfahani, S.N., Li, Z., Muncie, J.M., La-
kins, J.N., Weaver, V.M., Gumucio, D.L., and Fu, J. (2019). Controlled model-
ling of human epiblast and amnion development using stem cells. Nature 573,
421–425.
Zheng, C., Hu, Y., Sakurai, M., Pinzon-Arteaga, C.A., Li, J., Wei, Y., Okamura,
D., Ravaux, B., Barlow, H.R., Yu, L., et al. (2021). Cell competition constitutes a
barrier for interspecies chimerism. Nature. Published online January 28, 221.
https://doi.org/10.1038/s41586-021-03273-0.
Zhou, F., Wang, R., Yuan, P., Ren, Y., Mao, Y., Li, R., Lian, Y., Li, J., Wen, L.,
Yan, L., et al. (2019). Reconstituting the transcriptome and DNA methylome
landscapes of human implantation. Nature 572, 660–664.
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Oct-3/4 (C-10) Santa Cruz Biotechnologies Cat#sc-5279; RRID: AB_628051
NANOG R&D systems Cat#AF1997; RRID: AB_355097
GATA-6 (D61E4) XP Rabbit mAb Cell Signaling Technology Cat#5851; RRID: AB_10705521
GATA4 Abcam Cat#Ab124265; RRID: AB_11000793
SOX17 R&D systems Cat#AF1924; RRID: AB_355060
PDGFRa Abcam Cat#Ab203491
Brachyury (H-210)/T Santa Cruz Biotechnologies Cat#sc-20109; RRID: AB_2255702
OTX2 Santa Cruz Biotechnologies Cat#sc-514195
AP-2g (6E4/4)/TFAP2C Santa Cruz Biotechnologies Cat#sc-12762
Anti-Collagen VI antibody/COL6A1 Abcam Cat#ab6588; RRID: AB_305585
Anti-Cytokeratin 7 Abcam Cat#ab68459; RRID: AB_1139824
OCT3-4 R&D systems Cat#AF1759; RRID: AB_354975
SOX2 Abcam Cat#ab137385; RRID: AB_2814892
Donkey anti goat IgG Alexa Fluor@647 Abcam Cat#ab150135; RRID: AB_2687955
Donkey anti goat IgG Alexa Fluor@488 Abcam Cat#ab150129; RRID: AB_2687506
Goat Anti-Rabbit IgG H&L (Alexa Fluor 647) Abcam Cat#ab150079
Goat anti-Rabbit IgG (H+L) Secondary
Antibody, DyLight 488
ThermoFisher Cat#35552; RRID: AB_844398
Goat anti-Mouse IgG (H+L) Cross-
Adsorbed Secondary Antibody, Alexa
Fluor 647
ThermoFisher Cat#A21235; RRID: AB_2535804
Goat anti-Mouse IgG / IgM (H+L) 488 ThermoFisher Cat#A10680; RRID: AB_2534062
Biological samples
Macaca fascicularis embryos State Key Laboratory of Primate
Biomedical Research
N/A
Chemicals, peptides, and recombinant proteins
Recombinant Human LIF Peprotech Cat#AF-300-05
CHIR 99021 Tocris Cat#4423; CAS: 252917-06-9
Y-27632 dihydrochloride Tocris Cat#1254; CAS: 129830-38-2
(S)-(+)-Dimethindene maleate Tocris Cat#1425; CAS: 136152-65-3
Minocycline, Hydrochloride Selleck Cat#S4226; CAS: 13614-98-7
IWR-1-endo Peprotech Cat#1128234; CAS: 1127442-82-3
KnockOut Serum Replacement ThermoFisher Cat#A3181502
Neurobasal Medium ThermoFisher Cat#21103-049
DMEM/F-12 ThermoFisher Cat#11320-033
N-2 Supplement ThermoFisher Cat#17502-048
B-27 Supplement, minus vitamin A ThermoFisher Cat#12587-010
GlutaMAX ThermoFisher Cat#35050-061
Non-Essential Amino Acids Solution ThermoFisher Cat#11140-050
2-Mercaptoethanol ThermoFisher Cat#21985-023
Penicilin-Streptomycin ThermoFisher Cat#15140-122
Mitomycin C from Streptomyces
caespitosus
Sigma-Aldrich Cat#M4287; CAS: 50-07-7
(Continued on next page)
Cell 184, 2020–2032.e1–e7, April 15, 2021 e1
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Trypsin-EDTA (0.05%), phenol red ThermoFisher Cat#25300-062
DMEM-Dulbecco’s Modified Eagle
Medium, High Glucose
ThermoFisher Cat#11965092
TrypLE Express Enzyme (1X), no phenol red ThermoFisher Cat#12604021
heat-inactivated FBS Corning Cat#35-076-CV
L-glutamine, 100 3 ThermoFisher Cat#25030
Insulin-Transferrin-Selenium-Ethanolamine
(ITS -X) (100X)
ThermoFisher Cat#51500-056
b-estradiol Sigma-Aldrich Cat#E8875; CAS: 50-28-2
N-acetyl- L-cysteine Sigma-Aldrich Cat#A7250; CAS: 616-91-1
Progesterone Sigma-Aldrich Cat#P0130; CAS: 57-83-0
KnockOut Serum Replacement ThermoFisher Cat#10828010
4’,6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich Cat# D9542CAS: 28718-90-3
Recombinant human follitropin alpha Merck Serono GONAL-F
Recombinant human chorionic
gonadotropin alpha
Merck Serono OVIDREL
CMRL-1066 ThermoFisher Cat#11530037
PBS Biological Industries Cat#02-020-1A
4% Paraformaldehyde (PFA) Biosharp Cat#BL539A
Sucrose meilunbio Cat# BGC005CAS:57-50-1
optimal cutting temperature compound
(Tissue-Tek OCT)
SAKURA Cat#4583
FBS Biological Industries Cat# 04-001-1A
BSA Sigma-Aldrich Cat#B2064CAS: 9048-46-8
PBS Meilunbio Cat#MA0008
Trypsin-EDTA (0.25%), phenol red Thermo Fisher Cat#25200-072
Triton X-100 Sigma-Aldrich Cat#T9284; CAS: 9002-93-1
FBS Biological Industries Cat# 04-002-1A
Critical commercial assays
Superscript II reverse transcriptase Invitrogen Cat#18064-014
KAPA HiFi HotStart ReadyMix KAPA Biosystems Cat#KK2601
Betaine Sigma-Aldrich Cat#61962; CAS: 107-43-7
Triton X-100 Sigma-Aldrich Cat#T9284; CAS: 9002-93-1
Magnesium chloride Sigma-Aldrich Cat#M8266; CAS: 7786-30-3
EB solution QIAGEN Cat#19086
dNTP mix TAKARA Cat#4030Q
Recombinant RNase inhibitor TAKARA Cat#2313A
RNase-OFF TAKARA Cat#9037
Agencourt Ampure XP beads Beckman Coulter Cat#A63881
HiSeq X Ten Reagent Kit v2.5 Illumina Cat#FC-501-2521
QuBit dsDNA HS Assay Kit (used with
QuBit 3.0)
Invitrogen Cat#Q32851
TruePrep DNA Library Prep Kit V2 for
Illumina
Vazyme Cat#TD501-503
High sensitivity DNA reagents (used with
Agilent Bioanalyzer 2100 system)
Agilent Technologies Cat#5067-4626
Deposited data
Single cell RNAseq data generated in
this study
This paper GEO: GSE155381
(Continued on next page)
ll
e2 Cell 184, 2020–2032.e1–e7, April 15, 2021
Article
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Experimental models: cell lines
iPS1-EPS Yang et al., 2017 N/A
Experimental models: organisms/strains
Macaca fascicularis State Key Laboratory of Primate
Biomedical Research
N/A
Oligonucleotides
Oligo-dT30VN/ISPCR oligo/TSO TSINGKE oligo store
(Picelli et al., 2014)
N/A
Software and algorithms
HISAT2 version 2.2.1 Kim et al., 2019 https://daehwankimlab.github.io/hisat2/
StringTie version 2.1.2 Pertea et al., 2015 https://ccb.jhu.edu/software/stringtie/
index.shtml
SPRING_dev Weinreb et al., 2018 https://github.com/AllonMKlein/
SPRING_dev
R package Seurat version 3.1.1 Butler et al., 2018 https://cran.r-project.org/web/packages/
Seurat/index.html
R package monocle version 2.12.0 Trapnell et al., 2014 https://github.com/cole-trapnell-lab/
monocle-release
R package SCENIC version 1.1.1-10 Aibar et al., 2017 https://github.com/aertslab/SCENIC
R package velocyto.R version 0.6 La Manno et al., 2018 https://github.com/velocyto-team/
velocyto.R
R package clusterProfiler version 3.16.0 Yu et al., 2012 https://www.bioconductor.org/packages//
2.10/bioc/html/clusterProfiler.html
CellPhoneDB version 2.0.1 Vento-Tormo et al., 2018 https://github.com/Teichlab/cellphonedb
R package slingshot version 1.1-2 Street et al., 2018 http://www.bioconductor.org/packages/
release/bioc/html/slingshot.html
ll Article
RESOURCE AVAILABILITY
Lead contact Further information and requests for reagents may be directed to and will be fulfilled by Lead Contact Juan Carlos Izpisua Belmonte
Materials availability This study did not generate any unique reagents.
Data and code availability All sequencing data were deposited at the NCBI Gene Expression Omnibus (GEO) under accession number GSE155381.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animals 10 healthy female cynomolgus monkeys (Macaca fascicularis), ranging in age from 5 to 8 years with body weights of 4 to 6 kg, were
selected for use in this study. All animals were housed at the State Key Laboratory of Primate Biomedical Research (LPBR). All animal
and experiment procedures were approved by the ethical committee of the LPBR and performed by following the guidelines of the
Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) for the ethical treatment of non-hu-
man primates.
Culture of human extended pluripotent stem (hEPS) cells Human EPS cells were cultured on MEFs in serum-free N2B27-LCDM medium under 20%O2 and 5%CO2 at 37�C . The 500mL LCDM medium is composed of 240 mL DMEM/F12 (ThermoFisher Scientific, 11320-033), 240 mL Neurobasal (ThermoFisher
Cell 184, 2020–2032.e1–e7, April 15, 2021 e3
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Scientific, 21103-049), 2.5 mL N2 supplement (ThermoFisher Scientific, 17502-048), 5 mL B27 supplement (ThermoFisher
Scientific, 12587-010), 1% GlutaMAX (ThermoFisher Scientific, 35050-061), 1%non-essential amino acids (ThermoFisher Scienti-
fic, 11140-050), 0.1mM b-mercaptoethanol (ThermoFisher Scientific, 21985-023), penicillin-streptomycin (ThermoFisher
Scientific, 15140-122), 5% knockout serum replacement (KSR, ThermoFisher Scientific, A3181502, optional). 10 ng/ml recombinant
human LIF (L, 10ng/ml; Peprotech, AF-300-05), CHIR99021 (C, human: 1 mM, Tocris, 4423), (S)-(+)-Dimethindene maleate (D, 2 mM;
Tocris, 1425), Minocycline hydrochloride (M, 2 mM; Selleck, S4226), IWR-1-endo (1 mM; Peprotech, 1128234) and Y-27632 (2 mM;
Tocris, 1254). Human EPS cells were passaged by treatment with TrypLE (ThermoFisher Scientific,12604021) every 3-4 days at a split
ratio of 1:10.
Animal, stem cell, and ethical approvals At the LPBR, the animal research protocol is guided by a set of National and Provincial regulations and institutional policies. LPBR is
also subject to additional standards that go beyond domestic regulatory requirements by also maintaining accreditation through the
Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.
An original animal research protocol was drafted based on the collaborative research proposal with the Salk Institute team. After
review by the executive leadership of LPBR and Institute of Primate Translational Medicine and Kunming University of Science and
Technology (IPTM, KUST), the protocol was submitted to the LPBR committee and discussed at two committee meetings in Oct.
2017. After that, the animal research protocol was redrafted based on suggestions from the committee and submitted to the Insti-
tutional Animal Care and Use Committee (IACUC) of LPBR in Dec. 2017. IACUC members suggested additional edits, which were
subsequently incorporated. In Jan. 2018, the protocol (LPBR201803001) received approval and was sent to the Salk Institute.
The protocol was updated and approved again in 2019 and renewed in 2020. The research protocol was also submitted to the Med-
ical Ethics Committee of LPBR in Oct. 2019 and received approval in Nov. 2019.
On the Salk Institute side, the original proposal was submitted to the Embryonic Stem Cell Research Oversight (ESCRO) committee
in September 2017. The protocol was further discussed with the Salk Institute President and the executive leadership team, which
recommended seeking additional external bioethical input. Members of the ESCRO review committee discussed the protocol with
two renowned external bioethicists in early January 2018. Additional external outreach was made to the California Institute of Regen-
erative Medicine (CIRM) in February 2018 for feedback and insight on protocol review. There was no CIRM or NIH funding involved in
this research.
The protocol was redrafted to incorporate requested information from the ESCRO committee, senior Salk Institute leadership, and
suggestions from the external bioethicists and CIRM official. The protocol (18-0001sc) received approval Feb. 2018, and the renewal
of this protocol was approved again in 2019, and again in early 2020.
METHOD DETAILS
Oocyte collection and in vitro fertilization Ovarian stimulation, oocyte recovery and in vitro fertilization were performed as previously described (Mishra et al., 2010). In brief,
healthy female cynomolgus monkeys were subjected to follicular stimulation by intramuscular injection of 20 IU of recombinant hu-
man follitropin alpha (rhFSH, Gonal F, Merck Serono) for 8 days, then 1,000 IU recombinant human chorionic gonadotropin alpha
(rhCG, OVIDREL, Merck Serono) was injected on day 9. Cumulus-oocyte complexes were collected by laparoscopic follicular aspi-
ration 32-35 hours following rhCG administration. Follicular contents were placed in HEPES-buffered Tyrode’s albumin lactate py-
ruvate (TALP) medium containing 0.3% bovine serum albumin (BSA) at 37�C. Oocytes were stripped of cumulus cells by pipetting after a brief exposure (< 1 minute) to hyaluronidase (0.5 mg/mL) in TALP-HEPES to allow visual selection of nuclear maturity meta-
phase II (MII; first polar body present) oocytes. The mature oocytes were subjected to intracytoplasmic sperm injection (ICSI) imme-
diately and then cultured in CMRL-1066 medium (GIBCO, 11530037) containing 10% fetal bovine serum (FBS, GIBCO) at 37�C in 5% CO2. Fertilization was confirmed by the presence of the second polar body and two pronuclei. Zygotes were then cultured in the
chemically defined hamster embryo culture medium-9 (HECM-9) containing 10% fetal bovine serum at 37�C in 5% CO2 to allow em- bryo development. All chemicals were from Sigma Chemicals unless otherwise stated.
Microinjection of human EPS cells into monkey blastocysts and in vitro embryo culture Blastocysts at day 6 of post-fertilization (early stage blastocyst) were transferred into a 50 mL manipulation droplet of TH3 in the center
of a Petri dish covered with 3 mL of mineral oil. A previously established and well-characterized hEPS cell line generated by cellular
reprogramming iPS1-EPS, was used for cell microinjection (Yang et al., 2017b). Single cell suspensions of hEPS cells were placed
into a separate 10 uL droplet of culture medium next to the manipulation drop. 25 single tdTomato positive hEPS cells were aspirated
into a 15 mm inside diameter injection pipette with 30 degrees oblique mouth. The blastocyst was held with a holding pipette and the
injection pipette was moved to the manipulation droplet. Meanwhile, the zona pellucida was ablated using a single laser pulse, and
the injection pipette containing hEPS cells was immediately inserted into the hole in the blastocyst, close to the ICM. Injected blas-
tocysts were quickly transferred into the mixed media of HECM-9 and hEPS cell culture media (1:1) (Yang et al., 2017b), and cultured
for 24 hours to the well-expanded blastocyst stage.
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Human-monkey chimeric embryos culture in vitro The tdTomato positive human-monkey chimeric embryos were selected for follow-up experiments. Generally, the zona pellucida of
the human-monkey chimeric embryo was removed by exposure to hyaluronidase from bovine testes for about 30 s, and then the
embryos were seeded in 8-well m-plates (80826, Ibidi) with pre-equilibrated in vitro culture medium 1 (IVC1). After the embryos
attached to the well, half of the IVC1 medium was exchanged with in vitro culture medium 2 (IVC2). Thereafter, half of the medium
was replaced daily with fresh IVC2, and monkey embryos were harvested at the indicated day. Embryo culture was performed at
37�C in 5% CO2. The ingredients of IVC1 and IVC2 were published before (Niu et al., 2019).
IVC1: DMEM/F12 (11320-033, Thermo Fisher Scientific) supplemented with 20% (vol/vol) heat-inactivated FBS (35-076-CV,
Corning), 2mM L-glutamine (25030; Thermo Fisher Scientific), Penicillin (25units/ml)/streptomycin (25mg/ml) (15140-122; Thermo-
Fisher Scientific), 1X ITS-X (51500-056; ThermoFisher Scientific), 8nM b-estradiol, 200ng/ml progesterone and 25mM N-acetyl- L-
cysteine.
IVC2: 20% (vol/vol) heat-inactivated FBS of IVC1 was replaced with 30% (vol/vol) KnockOut Serum Replacement (KoSR,
10828010, Thermo Fisher Scientific).
Single cell collection and single cell RNA-seq (scRNA-seq) After washing with phosphate buffered saline (PBS) (MA0008, meilunbio), the embryo was cut into several pieces with a 1mL syringe.
The tdTomato positive and negative pieces were picked under fluorescence microscope (Leica), and digested into single cells with
0.1% trypsin (25200-072, GIBCO) at 37�C for 3 to 5 minutes. After neutralization with 2% FBS (04-002-1A, Biological Industries), cells were washed with cold PBS containing 0.1 to 1% BSA. Finally, individual tdTomato positive and negative cells were picked into a lysis
buffer on ice with a mouth pipette using a fluorescence microscope.
For scRNA-seq, the cDNA synthesis, amplification and library construction of single-cell were performed according to Smart-seq2
protocol (Picelli et al., 2014). Briefly, the single-cell was placed into the lysis buffer by a mouth pipette. The reverse transcription re-
action and pre-amplification were performed using SuperScript II (18064-071,Invitrogen), and KAPA HiFi HotStart Ready Mix
(KK2602, KAPA Biostems), respectively. Then 20 cycles of PCR were applied to obtain �20-140 ng of amplified cDNA. For library construction, the cDNA was fragmented by Tn5 transposase (TD502/TD501, Vazyme) mixed at 55�C for 10 minutes and followed by PCR amplification using TruePrep Amplify Enzyme (TD601, Vazyme). Then the libraries were purified and size selected with AM-
Pure XP magnetic beads (A63881, Beckman Coulter). All libraries were adapted for sequencing on an Illumina X-Ten platform
(sequenced by Annorad).
Immunofluorescence (IF) analysis The IF analysis was conducted similarly to what was previously published (Niu et al., 2019). Human-monkey chimeric embryos
cultured in vitro were harvested and fixed in 4% (w/v) paraformaldehyde (BL539A，Biosharp) in PBS for 30 minutes at room temper- ature and washed with PBS. The d.p.f.9 embryos were stained directly and the embryos from d.p.f.11 to 19 were dehydrated in su-
crose (BGC005, meilunbio) solutions each for 6 hours with increasing concentration from 15% (w/v) to 30% (w/v). After embedding in
optimal cutting temperature compound (4583, Tissue-Tek OCT, SAKURA), and frozen at �80�C, the embryos were prepared as cry- osections with 8 mm thickness on pre-treated glass slides (1A5105, CITOTEST), and air-dried for 1 hour. After being permeabilized in
PBST (PBS with 0.3% Triton X-100) (T9284, Sigma-Aldrich) for 30 minutes at room temperature, samples were blocked with 3% (w/v)
BSA and 10% (v/v) FBS (04-001-1A, Biological Industries) in PBS overnight at 4�C. Slides were then incubated with primary anti- bodies (Table S1) overnight at 4�C. After washing at least three times, fluorescence-conjugated secondary antibodies (Table S1), and 4’,6-diamidino-2-phenylindole (DAPI) were incubated with the slides in the dark at room temperature for 2 hours. Images
were taken using a Leica TCS SP8 confocal microscope.
QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analysis All values are depicted as mean ± SD. Statistical parameters including statistical analysis, statistical significance, and n value are
reported in the Figure legends and Supplementary Figure legends. Statistical analyses were performed using Prism Software (Graph-
Pad). For statistical comparison, one-way ANOVA was employed. A value of p < 0.05 was considered significant.
Cell origin identification, scRNA-seq data pre-processing and quality control First, human and monkey cells were identified by two different methodologies using clean data. One was to compare the number of
identical hits of sequencing reads from each cell by blastp with the randomly selected 10000 reads against the human or monkey
genome (Ensembl Homo_sapiens.GRCh38 and Macaca_fascicularis_5.0), respectively. Another was to compare the read counts
of sequencing reads mapped to the tdTomato sequence. After identification of cell origin, hisat2 (Kim et al., 2019) was used to
map sequencing reads to the human or monkey genome with the default parameters. The program StringTie (Pertea et al., 2015)
with default parameters was used to assemble alignments into potential transcripts and quantify the gene expression as transcripts
Cell 184, 2020–2032.e1–e7, April 15, 2021 e5
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per million mapped reads (TPM) value. The cells with mapped rate > = 30% and more than 2000 genes with TPM value > 0 were
processed for further analyses.
Cell clustering, lineage identification The Seurat package (v.3.1.1) (Butler et al., 2018) was used to perform single-cell clustering analysis and t-distributed stochastic
neighbor embedding (t-SNE) was applied to visualize the results. In order to identify and compare the cluster, the known lineages
from previous studies were used as a reference. The reference datasets include Human-1 (Zhou et al., 2019), Human-2 (Xiang
et al., 2020), monkey-1 (Niu et al., 2019) and monkey-2 (Nakamura et al., 2016). The reference dataset of Control-monkey is mon-
key-1 unless otherwise stated. The chimera data in our study were integrated into the above reference datasets following the below
steps. First, these datasets were created as Seurat object, respectively, with the order of Human-1, Human-2, monkey-1, chimera-
human and chimera-monkey. Then ‘‘FindIntegrationAnchors’’ function took these objects as input with parameters as ‘‘k.anchor = 5,
anchor.features = 2000’’ and returned an anchor object. Finally, ‘‘IntegrateData’’ function used the anchor object to integrate all data-
sets with the default parameter. The top-20 principal components (PCs) were used for clustering (with a resolution of 0.6) by Seurat.
Each cluster was defined by specific marker genes highlighted on the t-SNE graph using the ‘‘FeaturePlot’’ function and confirmed by
known lineages from previous studies.
Identification of differentially expressed genes Unique cluster-specific expressed genes were identified by running the Seurat ‘‘FindAllMarkers’’ and ‘‘FindClusters’’ functions (p <
0.01). The K-nearest neighbor algorithm SPRING (Weinreb et al., 2018) was used for trajectory analyses with the integrated data,
2000 highly variable genes (HVGs) and default parameters as input. To build the phylogenetic tree of all chimeric cells, the pairwise
euclidean distance calculated from integrated expression matrix was fed into MEGA (X 10.1) (Kumar et al., 2018) to build the NJ tree
under the phylogeny menu.
Pseudotime alignment and pseudotime-dependent differentially expressed genes analysis The Monocle2 (v2.12.0) (Trapnell et al., 2014) was used to construct the pseudotime course of all EPI cells. The 2000 HVGs and
expression data of human, monkey, and chimeric cells in the EPI lineage identified above separately was used as input for Monocle2.
Dimensionalities were reduced by the ‘‘reduceDimension’’ function with the parameter ‘‘max_components=2.’’ After projecting the
expression data into a lower-dimensional space, cells were ordered by performing the ‘‘orderCells’’ function. Finally, we used a dy-
namic time warping (DTW) algorithm to align different pseudotime courses among human, monkey, and chimeric cells as described
in the previous study (Kanton et al., 2019). After pseudotime alignment, we used an F-test-based ANOVA analysis to identify genes
with pseudotime-dependent expression patterns as described previously (Kanton et al., 2019). In brief, for each HVG, we established
a natural splined linear regression model (ns function in the R package splines) with six degrees of freedom (df), with expression levels
as the response variable and pseudotimes as the independent variable. Then an F-test was applied to compare the variation ex-
plained by the splined linear model with that of the residuals normalized by degrees of freedom. Bonferroni correction was performed
across tested genes, with a corrected P value threshold of 0.01 to identify genes with pseudotime-dependent expression. In addition,
the R package slingshot (v1.1-2) was used to infer the potential development trajectory of chimeric human EPI-like cells and hEPS
with default settings (Street et al., 2018).
GO and KEGG pathway analysis We used the functions enrichKEGG and enrichGO in clusterProfiler R package (Yu et al., 2012) to perform KEGG pathways (Kanehisa,
2019; Kanehisa and Goto, 2000; Kanehisa et al., 2019) and Gene Ontology (GO) biological processes enrichment analysis. A pathway
or process with a P value % 0.05 was considered to be significantly enriched. The enriched pathways and processes were visualized
with the ‘‘ggplot’’ function in the ggplot2 package in R.
Single-cell transcription factor regulation networks construction and clustering The SCENIC package (Aibar et al., 2017) was applied to infer and characterize the gene regulation networks (regulon) for our scRNA-
seq data of chimeric cells. Based on the manual from the website (https://github.com/aertslab/SCENIC), three main steps were
executed: 1. Co-expression networks between transcription factors and the potential target genes inference based on the embedded
GENIE3 package; 2. For each co-expression module, the cis-regulatory motif enrichment analysis was performed among all potential
target genes by RcisTarget. Each transcription factor and its direct target genes were defined as a regulon; and 3. The activity score
of each regulon in each cell was computed through AUCell packages. Here, we fed the transcriptome profile of all chimeric cells to
SCENIC to infer their AUC regulon activity. Then, chimeric cells were cluster based on their regulon activity with the ‘‘pheatmap’’ func-
tion in the pheatmap package (v1.0.10). For comparison, the above datasets Human-1 and monkey-1 were also performed using the
same analysis of regulon activity.
RNA velocity analysis RNA velocity was calculated using the Velocyto.R program (http://velocyto.org) on the basis of spliced and unspliced transcript reads
as previously reported (La Manno et al., 2018). At first, Velocyto used a BAM file to count spliced and unspliced reads, and generated
e6 Cell 184, 2020–2032.e1–e7, April 15, 2021
ll Article
the loom file. Those loom files were then loaded into R using the ‘read.loom.matrices’ function to generate count tables for splicing
and unsplicing reads. RNA velocity was estimated using a gene-relative model with k-nearest neighbor cell pooling (kCells = 10). For
other parameters, we used the standard R implementation of velocyto with default settings.
Identification of cross-talks between human and monkey cells within chimeric embryos The signaling cross-talks between human and monkey cells in different embryonic lineages were built based on the ligand-receptor
interactions using CellPhoneDB (v2.0.1) (Vento-Tormo et al., 2018). All the ligand-receptor interaction databases that integrated into
CellPhoneDB were used for our data analysis. Here we fed the transcriptome profile of all chimeric cells and their information
including cell type (human or monkey cell) and lineages information (EPI, HYP, and EXMC) to CellPhoneDB to infer potential interac-
tion between two types of cells. An adjusted P value of 0.05 from a Permutation test was used as a threshold to identify ligands/re-
ceptors specifically expressed between two types of cells. The other parameters were set as default. For comparison, the above
datasets Human-1 and monkey-1 were also used to perform the same analysis of ligand-receptor interactions.
Cell 184, 2020–2032.e1–e7, April 15, 2021 e7
Supplemental figures
Figure S1. Lineage specification of hEPSCs within host monkey embryos, related to Figures 1 and 2
(A) Representative IF images of OCT4 (green), NANOG (gray) or SOX2 (gray) in iPS1-hEPSCs. Scale bars, 50 mm.
(B) Representative epifluorescence images of the late blastocyst-stage monkey host embryos resulting from microinjection of 25 TD-positive iPS1-hEPSCs.
Scale bar, 50 mm.
(C) Representative IF images of NANOG (green) and GATA6 (gray) in d.p.f. 9 (n = 2) monkey embryos. Yellow dotted line, ICM. Arrow indicates a TD-positive
hEPSC close to NANOG positive cells. Scale bar, 50 mm.
(D) Representative IF images of OCT4 (green) and COL6A1 (gray) in d.p.f. 11 monkey embryos (n = 2). Yellow dotted line, EPI. Arrow indicates a TD-positive
hEPSC expressing COL6A1 and OCT4. Scale bar, 50 mm.
(E) Representative IF images OTX2 (gray) and OCT4 (green) in d.p.f.13 monkey embryos (n = 2) . Scale bar, 50 mm. EPI, epiblast; TD, tdTomato; DAPI,
4’,6-diamidino-2-phenylindole.
ll Article
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D
UTF1 MT1G
CYP2S1 VENTX DPPA5 L1TD1
ZSCAN10 NODAL
GAL TDGF1
KHDC3L GDF3 SPP1
NANOG DPPA4
KHDC1L NLRP7
PRDM14 FAM46B
ALPL CXCL12
SOX2 RNF125
SERINC5 FGF2
MEG3 THY1 PDPN
IFITM1 MT1X
VASH2 USP28 MRS2 PIM2
CHST2 DIAPH2
MAD2L2 UACA
TBC1D23 IFITM3 STOM
SERPINH1 TET1
CXADR LAPTM4B
TMSB4X TUBB2B POU5F1
0 25 50 75 100 Expression rate (%)
0 2 4 6
log10(expression+1)
Expression rate (%) 0 25 50 75
Chimera Human EPI (158 cells)
APOA4 FOXA2
GPX2 HNF1B
S100A16 MGST2
S100A10 COL18A1
CTSE RNASE1
SLCO2A1 RAB3B HNF4A
NID2 BMP4
RSPO3 GATA4 SOX17
PDGFRA APOA1 SPARC BAMBI
DPP4 IL6R
GPC3 ANPEP GATA6 FLRT3 ADD3 APOE
P4HA1 TLN2
AMOT VCAN
COL4A1 RAB15
ZC3HAV1 S100A13
ALDH2 CDH2
CKB GJA1 BMP2
CADM1 IDH1
MARCKS SERPINE2
CNN3 FN1
TMEM123
0 25 50 75 100 Expression rate (%)
0 1 2 3
log10(expression+1)
Expression rate (%) 0 25 50 75 100
Chimera Human HYP (3 cells)
ACKR2 XAGE3
C1orf115 PTN
PTGES AIM1L
FAM49A ADAP2
SLCO4A1 KRT7
HSD3B1 GRHL1
FYB HSD17B1
CLDN4 ABCG2
TINAGL1 SP6
PRKCH ADAM15
CYP19A1 PTPRE GATA3
DLX3 GCM1
GRAMD2 GJA5
MED12L GATA2 RHOU
PPARG MPP1
COL21A1 TFEB
RAB31 SLC7A2 MBNL3
TPD52L1 FAM110A
TEAD1 ADAMTS1 GRAMD3
SYNJ1 MBNL2
EPB41L3 TFAP2A
ZFHX3 GNG12
WLS PPME1 NR2F2
ATP6V1C2
0 25 50 75 100 Expression rate (%)
Expression rate (%) 0 25 50 75 100
0
1
2
log10(expression+1)
Chimera Human TE (11 cells)
−30
−20
−10
0
10
20
30
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tS N
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Chimera-Monkey Control-Monkey
tS N
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−20
−10
0
10
20
30
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Chimera-Monkey Control-Monkey
0
5
10
Chimera-Monkey Control-Monkey
lo g2
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0.2
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In te
gr at
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P M
Chimera-Monkey Control-Monkey
Before integration
After integration
E LAPTM4B
FLRT3
IDH1
BAMBI
P4HA1
POU5F1
CKB
TMSB4X
IFITM3
CNN3
MAD2L2
MARCKS
AGO2
RP11−97C16.1
SEC24A
MAML1
PRKX
CDV3
RNF44
HOXA9
ANXA3
MIR17HG
PMF1−BGLAP
STK25
ZWINT
WDSUB1
NUF2
DDX47
DYNC2LI1
ACTL6A
RFC1
ZNF16
lineage
m arker
lineage EPI HYP TE
marker EPI HYP TE
−3
−2
−1
0
1
2
3
(legend on next page)
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Figure S2. Identification of cell species origin and quality control, related to Figure 3
(A and B) Both the different number of identical hits by blastp (A) and tdTomato reads (B) can distinguish the cell origin. Red and green points represent cell
samples from human or monkey, respectively.
(C) The quality control and statistic analysis of gene numbers (nGene) and reads numbers (nCount), mapping rates and tdTomato reads numbers (nCounts tdTm)
for all chimeric cells (left four panels). The gene expression levels and tSNE plot of control monkey (Control-Monkey) and host monkey (Chimera-Monkey) cells
before and after integration (remove batch effect) (right two panel).
(D) The bubble charts showing the expression of the reported marker genes in chimeric human EPI-like (Chimera Human EPI), HYP-like (Chimera Human HYP),
and TE-like (Chimera Human TE) cells, respectively.
(E) Heatmap showing the expression of lineage markers conserved between humans and monkeys in chimeric human EPI-like, HYP-like, and TE-like cells. EPI,
epiblast; HYP, hypoblast; TE, trophectoderm.
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Figure S3. Comparative analysis of monkey cells between the chimeric and host embryos, related to Figure 4
(A) Boxplot (left panels) and heatmap (right panel) showing expression of marker genes for EPI, HYP, and EXMC lineages between the chimeric (Chimera-Monkey)
and control (Control-Monkey) embryos.
(B) The top 20 ligand-receptor interactions between EPI-EPI, EPI-HYP and EPI-EXMC, respectively, non-chimeric monkey (Control-Monkey) cells are used as
control to compare with chimeric cells (Chimera-Monkey).
(C) Venn diagram showing the common and specific ligand-receptor interactions between EPItoEPI, EPItoHYP, and EPItoEXMC in distinct cells.
(D) The expression levels (log2 [TPM+1]) of the representative ligand-receptors interactions between chimeric human (Chimera-Human) and host monkey
(Chimera-Monkey) EPI cells are shown. EPI, epiblast; HYP, hypoblast; EXMC, extra-embryonic mesenchyme cell. Human, control human EPI cells (Zhou et al.,
2019); Monkey, control monkey EPI cells (Niu et al., 2019).
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Figure S4. Characteristics of subgroups in the EPI lineage, related to Figure 5
(A) Heatmap of the expression levels of identified marker genes for the subgroups of ICM, Pre_EPI, PostE_EPI, PostL_EPI, and Gast.
(B) Expression of subgroup-specific marker genes exhibited on t-SNE plots. A gradient of gray, yellow, and red indicates low to high expression.
(C) The dynamic change of marker genes for different subgroups along the pseudotime.
(D) The sankey diagram showing the number and flow from different cell types to different lineages.
(E) Heatmap of the correlation coefficients among hEPSCs (Yang et al., 2017b), chimeric human EPI-like (Chimeric human) cells, and control human (Control-
Human 1) and monkey (Control-Monkey 1) EPI cells (Niu et al., 2019; Zhou et al., 2019) and human (hESC/iPSC) and monkey (CyESC) PSCs at primed (C) or naive
(N) pluriotency state (Chan et al., 2013; Chen et al., 2015; Gafni et al., 2013; Theunissen et al., 2014). ICM, inner cell mass; EPI, epiblast; Pre_EPI, pre-implantation
EPI; PostE_EPI, post-implantation early EPI; PostL_EPI, post-implantation late EPI; Gast, gastrulating cell; N, ‘naive’; C, conventional (primed).
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(legend on next page)
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Figure S5. Regulatory network analysis of chimeric EPI cells, related to Figure 5
(A) The regulon-activity heatmap for the chimeric (Chimera) EPI cells with human and monkey non-chimeric (Control-Human and Control-Monkey) embryos as
controls. The samples in each column were ordered based on lineage and species origins. These regulons are specific for chimeric EPI cells. (B) Regulatory
networks of the top 3 regulons for non-chimeric control human (Control-Human), monkey (Control-Monkey) and chimeric (Chimera) EPI cells. The gene
expression status (up, down, and no change) are colored with red, blue, and gray, respectively. The transcription factors located in the center regulate the
surrounding target genes. (C) Heatmap showing the expression levels of cell-cycle related genes in chimeric human EPI-like cells. Cell-cycle related genes were
retrieved from Seurat package (v.3.1.1). (D) Heatmap showing the expression levels of the markers identified from EPI sub-groups and the related biological
processes. (E) Heatmap showing the expression of apoptosis related genes in control human (Control-Human), control monkey (Control-Monkey), host monkey
(Chimera-Monkey) and chimeric human (Chimera-Human) cells. Apoptosis related genes were obtained from GSEA website. Pre_EPI, pre-implantation EPI;
PostL_EPI, post-implantation late EPI; Gast, gastrulating cell
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- Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo
- Introduction
- Results
- Generation of human-monkey chimeric blastocysts in vitro
- Chimeric contribution of hEPSCs to peri- and post-implantation monkey embryos
- Transcriptional landscape of human-monkey chimeric embryos
- Transcriptome dynamics of hEPSCs during human-monkey chimera development
- Chimeric human EPI-like cells display a distinct developmental trajectory
- Discussion
- Limitations of study
- Supplemental information
- Acknowledgments
- Author contributions
- Declaration of interests
- Acknowledgments
- References
- STAR★Methods
- KEY RESOURCES TABLE
- Resource availability
- Lead contact
- Materials availability
- Data and code availability
- Experimental model and subject details
- Animals
- Culture of human extended pluripotent stem (hEPS) cells
- Animal, stem cell, and ethical approvals
- Method details
- Oocyte collection and in vitro fertilization
- Microinjection of human EPS cells into monkey blastocysts and in vitro embryo culture
- Human-monkey chimeric embryos culture in vitro
- Single cell collection and single cell RNA-seq (scRNA-seq)
- Immunofluorescence (IF) analysis
- Quantification and statistical analysis
- Statistical analysis
- Cell origin identification, scRNA-seq data pre-processing and quality control
- Cell clustering, lineage identification
- Identification of differentially expressed genes
- Pseudotime alignment and pseudotime-dependent differentially expressed genes analysis
- GO and KEGG pathway analysis
- Single-cell transcription factor regulation networks construction and clustering
- RNA velocity analysis
- Identification of cross-talks between human and monkey cells within chimeric embryos
