Dissecting embryonic and extra-embryonic lineage crosstalk with stem cell co-culture

SUMMARY

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated^1–14, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-β, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs) and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.

Graphical Abstract

A unified culture condition for all three primary tissues of mouse and cynomolgus monkey blastocysts is established, facilitating stem cell co-culture experiments and uncovering intricate interactions between embryonic and extra-embryonic lineages.

INTRODUCTION

How a single-celled zygote transforms into an organism with drastically different cellular phenotypes is a fundamental question in biology. In mammals, embryogenesis is accompanied by the establishment and loss of pluripotency, a transient property enabling embryonic epiblast (EPI) cells to generate all cells in an adult organism. Pluripotent EPI cells can be maintained in vitro in a spectrum of pluripotent stem cell (PSC) states (e.g., naïve, formative, and primed) by altering culture conditions^15,16. The dynamic PSC states, with their distinct molecular and functional characteristics, offer vital in vitro models to explore early mammalian development and hold significant promise for regenerative medicine.

PSCs, despite their promise, are maintained in cell cultures that significantly deviate from in vivo conditions. A notable difference is the absence of interactions with extraembryonic cells in cultured PSCs. Consequently, PSC-only models suffer from several culture artifacts that potentially limit their potential, e.g., unsynchronized and disorganized differentiation and unfettered growth. In addition, although extraembryonic tissues’ functions are well known during gastrulation (embryo patterning)^17,18 and fetal developmental stages (nutritional, respiratory and excretory support for fetal growth)^17,19,20, how they communicate with pre-gastrulation EPI remain elusive.

To overcome these limitations, we derived early post-implantation and pre-gastrulation stem cells from embryonic and extraembryonic tissues under the same condition from both mouse and cynomolgus monkey (monkey for short) blastocysts. This condition, termed FTW, activates FGF, TGF-β/Smad, and WNT/β-Catenin signaling pathways, fostering a permissive microenvironment conducive for the self-renewal of formative-like embryonic stem cells (ESCs), extraembryonic endoderm stem cells (XENs), and trophoblast stem cells (TSCs) in both standalone cultures and co-cultures, and thereby opening the door for dissecting their direct communication.

RESULTS

A common stem cell culture for all blastocyst lineages

We previously reported de novo derivation of formative-like ESCs from mouse blastocysts in the FTW condition (FTW-mESCs)¹². To enrich the FTW-mESCs population, an MEK inhibitor (PD0325901) was transiently added to suppress the proliferation of extraembryonic cells¹². Withdrawal of PD0325901 during FTW-mESCs derivation resulted in the co-appearance of epiblast-like cells (ELCs), trophoblast-like cells (TLCs), and extraembryonic endoderm-like cells (XLCs) in the same blastocyst outgrowth and upon passaging (Figure S1A). After manual picking, separate cultivation, and further passaging in the FTW condition, stable mXENs (designated as FTW-mXENs), mTSCs (designated as FTW-mTSCs), and FTW-mESCs could be derived from a single mouse blastocyst (Figures 1A and 1B). FTW-mXENs, FTW-mTSCs and FTW-mESCs expressed extraembryonic endoderm, trophoblast, and EPI markers, respectively (Figures 1C and S1B–S1E). Next, we compared FTW-mXENs and FTW-mTSCs with mXENs and mTSCs in conventional conditions^21,22 (Figures S1F–S1I). FTW-mTSCs and FTW-mXENs expressed their respective lineage markers at comparable levels to conventional mTSCs and mXENs, respectively (Figure S1J). Although similar proliferation rates were observed for conventional mTSCs and FTW-mTSCs, FTW-mXENs exhibited a significantly shorter doubling time than conventional mXENs (Figure S1K). In addition, pathway inhibitor treatments revealed differences in signaling dependencies between conventional and FTW-cultured mESCs and mXENs (Figure S1L). Regarding conventional mTSCs and FTW-mTSCs, we found little to no differences between the two upon signaling inhibitor treatments (data not shown). After random differentiation of FTW-mXENs in vitro, several parietal endoderm (PE) and visceral endoderm (VE) related genes were upregulated²³ (Figure S1M). 3 months following subcutaneous injection into an immunodeficient NOD-SCID mouse, FTW-mXENs formed a tissue mass (designated as mXEN-tumor) (Figure S1N). Immunofluorescence (IF) analysis revealed that within the mXEN-tumor many cells expressed alpha-fetoprotein (AFP)²⁴, some cells were GATA6⁺, and others stained positive for yolk sac markers FOXA1 and/or COL6A1 (Figures S1O and S1P). We did not detect T⁺ (mesoderm) or PAX6⁺ (ectoderm) cells in the mXEN-tumor (Figure S1O). For FTW-mTSCs, after random differentiation in vitro, multinucleated cells appeared (Figure S1Q) and genes related to trophoblast differentiation were upregulated⁵ (Figure S1R). FTW-mTSCs also formed a tissue mass (designated as mTSC-tumor) containing multinucleated trophoblast giant-cell like cells two weeks after injection into a NOD-SCID mouse (Figure S1S). We previously showed that FTW-mESCs could contribute to chimera formation following blastocyst injection¹². Here, we performed blastocyst injections and determined the developmental potential of FTW-mXENs and FTW-mTSCs in vivo. We found that GFP (green fluorescent protein) and mKO (monomeric Kusabira-Orange) labeled FTW-mXENs and FTW-mTSCs could contribute to chimera formation in the yolk sac and placenta tissues of mouse conceptuses (E7.5 and E11.5), respectively (Figures 1D–1G, S1T and Table S1).

Figure 1. Derivation and characterization of embryonic and extraembryonic FTW stem cells from mouse blastocyst.

(A) Schematic of FTW embryonic and extraembryonic stem cell lines derivation from mouse blastocysts. (B) Representative bright field (BF) images showcase the colony morphologies of FTW-mXENs, FTW-mTSCs, and FTW-mESCs. Scale bar, 100 μm. (C) Representative immunofluorescence (IF) images display lineage markers for extraembryonic endoderm (GATA6 and SOX17), trophoblast (CDX2 and EOMES), and epiblast (SOX2 and OCT4) in FTW-mXENs (top), FTW-mTSCs (middle), and FTW-mESCs (bottom), respectively. Scale bar, 100 μm. (D) and (F) Representative combined BF and fluorescence images depict the chimera contribution from GFP-labeled FTW-mXENs (D) and FTW-mTSCs (F) to mouse conceptuses at E11.5 stage. Scale bar, 1 mm. (E) IF staining presents a chimeric yolk sac membrane marked for GFP, GATA6, and GATA4. Scale bar, 100 μm. (G) IF staining of a chimeric sagittal section of the placenta highlights CK8 and GFP. The various placental layers are distinguished by dotted lines. Scale bar, 100 μm. See also Figure S1 and Table S1.

In sum, we discovered a common culture condition could support de novo derivation and long-term culture of embryonic and extraembryonic stem cells from mouse blastocysts.

Transcriptome profiling of mouse FTW stem cells

We next performed bulk RNA-sequencing (RNA-seq) to examine the global transcriptional profiles of mouse FTW stem cells and compared them with published datasets from established mouse embryonic and extraembryonic stem cells^4,5,25–32. We found that FTW-mXENs, FTW-mTSCs, and FTW-mESCs expressed their respective lineage markers and clustered with stem cells derived from the same tissue of origin (Figures S2A and S2B). Based on comparison with in vivo reference datasets, we found FTW-mESCs, FTW-mTSCs, and FTW-mXENs were transcriptionally most closely related to E5.5 EPI, E5.25-E5.5 extraembryonic ectoderm (ExE)³³ and E5.5 VE³⁴, respectively (Figures S2C). We also performed single-cell RNA sequencing (scRNA-seq) analysis, which revealed distinct FTW-mESCs, FTW-mXENs, and FTW-mTSCs clusters, as well as heterogeneity within each FTW stem cell type (Figures 2A, 2B, S2D–S2F and Table S2A). Consistently, by comparing with published single-cell transcriptomes derived from E3.5-E6.5 mouse embryos³⁵, FTW-mESCs, FTW-mTSCs and FTW-mXENs also showed the highest correlation with E5.5 EPI, E5.5 TE (trophectoderm) and E5.5 PrE (primitive endoderm), respectively (Figure 2C).

Figure 2. Transcriptomic profiling of mouse FTW stem cells.

(A) Uniform manifold approximation (UMAP) visual representation captures the single cell populations within FTW-mXENs, FTW-mTSCs, and FTW-mESCs. (B) Violin plots illustrate the expression levels of FTW-mESC, FTW-mXEN, and FTW-mTSC marker genes. (C) A heatmap visualizes the Pearson correlation matrix, comparing FTW-mXENs, FTW-mTSCs, FTW-mESCs, and different mouse datasets³⁵. (D) (Top) Diagram detailing the derivation timeline for FTW-mXENs, FTW-mTSCs, and FTW-mESCs. (Bottom) UMAP visual representation integrating all scRNA-seq data, which include ICM/EPI, TE, and PrE of blastocysts; ELCs, TLCs, and XLCs from Day 8 outgrowth; and established FTW-mESCs, FTW-mTSCs and FTW-mXENs. (E) (Top) Pseudotime trajectory depicts the scRNA-seq progression of distinct cell lines in mice. (Bottom left) A heatmap showcases the pseudotime-dependent gene expression changes. Notably, C1, C4, C7 represent early-stage patterns; C2, C5, C8 represent mid-stage patterns; and C3, C6 C9 capture late-stage patterns. (Bottom Right) A kinetics plot visualizes the relative expression trends of marker genes throughout developmental pseudotime. (F) (Left) A gene-expression heatmap details the differentially expressed genes for each identified cluster. (Right) Associated Gene ontology (GO) terms for each cluster are presented. See also Figure S2 and Table S2.

To examine the temporal steps of FTW stem cell derivations, in addition to established FTW stem cell lines (passage 10), we performed scRNA-seq of blastocysts (day 0) and blastocyst outgrowths (day 8). Our analysis revealed clear segregation of ELCs, TLCs, and XLCs in day 8 blastocyst outgrowths (Figure 2D). Monocle2³⁶ pseudotime analysis revealed differentiation trajectories from ICM/EPI—>ELCs—>FTW-mESCs, PrE—>XLCs—>FTW-mXENs, and TE—>TLCs—>FTW-mTSCs, respectively (Figure 2E and Table S2B), and dynamic gene expression changes alongside the pseudotime trajectories (Figure 2E). Next, we identified stage-specific genes and enriched Gene Ontology (GO) terms. Notably, several common features across all lineages emerged in our analysis, e.g. blastocyst lineages (ICM/EPI, PrE and TE) were enriched with terms related to the cellular response to hypoxia and lipid metabolic process, etc., suggesting these cells undergo a metabolic transition from in vivo to in vitro; cells in day 8 blastocyst outgrowths (ELCs, XLCs and TLCs) shared terms related to actin dynamics and cell adhesion, etc., which implicates active cell movement and dynamic changes in cell shape; established FTW stem cells, on the other hand, are characterized by terms related to glycolysis and substrate adhesion-dependent cell spreading, etc., which is indicative of their stabilization in the FTW condition (Figure 2F).

Taken together, these transcriptomic analyses confirmed lineage identities, revealed temporal properties and derivation dynamics of FTW-mESCs, FTW-mTSCs and FTW-mXENs.

Cross-lineage FTW stem cell co-cultures

Having FTW-mESCs, FTW-mXENs and FTW-mTSCs derived and maintained in the same condition enabled us to establish co-cultures to study intercellular communications among embryonic and extraembryonic lineages in their self-renewing states (Figure 3A). To this end, we labeled FTW-mESCs with GFP and subjected them to co-culture with mKO-labeled FTW-mXENs in the presence or absence of FTW-mTSCs (unlabeled). Interestingly, after 5 days of co-culture in the FTW condition, many FTW-mESC colonies were surrounded by FTW-mXENs. These FTW-mESC colonies appeared smaller and more “domed” when compared to standalone colonies (not in contact with FTW-mXENs) and FTW-mESC colonies from separate cultures (Figure 3B and Video S1). Of note is that we didn’t observe this phenomenon in FTW-mESCs co-cultured with either FTW-mTSCs or mouse embryonic fibroblasts (Figure 3B). Consistently, we found a significant reduction of the GFP signal “Area × Intensity/Colony” in FTW-mESCs co-cultured with FTW-mXENs and FTW-mXENs/FTW-mTSCs (Figure 3C) but not with FTW-mTSCs and fibroblasts, when compared with separately cultured FTW-mESCs. We also calculated the cell density (cell number per cm²) of FTW-mESCs daily in each experimental group. On day 5, a significantly lower density of FTW-mESCs was found in FTW-ESCs/TSCs/XENs and FTW-ESCs/XENs groups than in control groups (Figures 3D and S3A). We tested the effects of different cell plating ratios and found a greater reduction in FTW-mESC densities when co-cultured with more FTW-mXENs (Figure S3B). We next studied what led to the reduced FTW-mESC numbers in co-cultures. We noted that co-cultured FTW-mESCs and FTW-mXENs still highly expressed stemness related genes (Figure S3C) and showed comparable levels of cell apoptosis to separate-cultures (Figures S3D–S3G), thereby ruling out cell differentiation and apoptosis as the cause. Interestingly, we detected significantly decreased and increased subpopulations of co-cultured FTW-mESCs in the G2/M and G1/S cell cycles, respectively, suggesting the reduced FTW-mESC number in co-cultures is due to cell cycle changes (Figure S3H). Moreover, we found that the growth phenotype depended on direct contact between FTW-mESCs and FTW-mXENs (Figures S3I, S3J, and Video S1).

Figure 3. FTW-mXEN-mediated proliferation inhibition of FTW-mESCs.

(A) Illustration depicting the establishment of co-cultures using FTW stem cells to study cross-lineage communications. (B) Representative fluorescence and BF merged images display day-wise (days 1–5) the progression of separately cultured FTW-mESCs (green) and their co-culture with either FTW-mXENs (red) and/or FTW-mTSCs (blue arrowheads), or proliferative mouse embryonic fibroblasts. Scale bar, 100 μm. (C) A violin plot reveals the product of area and GFP intensity for individual FTW-mESC colonies on day 5, both in separate cultures and co-cultures. (D) Growth dynamics from days 1 to 5 for separately cultured FTW-mESCs and their co-cultures with FTW-mXENs (mean ± SD, day 1, n = 2, day 2–5, n = 5, biological replicates). (E) Schematic representation of teratoma formation using FTW-mESCs only and a combination of FTW-mESCs co-injected with FTW-mXENs (mESCs:mXENs = 4:1). Both conditions injected the same number (1 × 10⁶) of FTW-mESCs. (F) Images of teratomas generated from FTW-mESCs injected with (bottom) and without (top) FTW-mXENs. (G) Comparisons of lengths and widths of teratomas derived from FTW-mESCs injected with (orange) and without (green) FTW-mXENs. (H) Weights of the resulting teratomas from FTW-mESCs injected with (orange) and without (green) FTW-mXENs. (mean ± SD, n = 5, biological replicates). (I) A diagram shows the tissue dissection scheme for E6.5-6.75 mouse conceptus. (J) Representative BF images showcase ex vivo culture results of EPI+VE (VE+) and EPI (VE−) tissues isolated from E6.5-6.75 mouse conceptuses at indicated time points. Scale bar, 100 μm. (K) Total cell number for VE+ and VE− tissues after 48-hour ex vivo culture (mean ± SD, n = 10, biological replicates). N.S. not significant, ****p < 0.0001, P-values were calculated using two-tailed Student’s t-test. See also Figure S3.

Next, we studied whether the growth phenotype manifested during differentiation. We injected the same number of FTW-mESCs alone or with FTW-mXENs under the skin of NOD-SCID mice (Figure 3E). Although in both conditions teratomas containing tissues from all three germ lineages could be generated (Figure S3K), the average size and weight of teratomas generated by co-injection were smaller than FTW-mESCs alone (Figures 3F–3H). We also tested different FTW-ESC: FTW-XEN ratios and found in general the more FTW-mXENs were injected, the smaller the teratomas (Figure S3L). To study whether this growth restriction also exists between EPI and VE cells, we isolated E6.5-6.75 mouse conceptuses, removed ExE and ectoplacental cone, and used EPI with or without VE (VE+/−) for ex vivo culture (Figure 3I). Interestingly, the size and total cell number in the VE+ group were significantly smaller than those in the VE-group (Figures 3J, 3K, and S3M), suggesting the proliferation of EPI is also limited by the VE. In addition, we performed a PrE complementation assay³⁷. Briefly, E2.5 mouse morulae were transiently treated with an ERK inhibitor PD0325901 (PD) to inhibit PrE development, and subsequently, GFP-labeled FTW-mXENs were injected into the blastocysts to complement the extraembryonic endoderm lineages (Figure S3N). The results show that in PD-treated mouse embryos, following ex vivo culture, the number of EPI cells is significantly increased when compared to those in the mock-treated WT embryos. And the EPI cell number is restored to normal levels in the PD-treated embryos after blastocyst injection of FTW-mXEN cells (Figures S3O and S3P).

Collectively, we established embryonic and extra-embryonic stem cell co-cultures and identified a contact-dependent growth restriction of FTW-mESCs by FTW-mXENs, which may reflect an EPI size control mechanism by surrounding VE during mouse early post-implantation development.

Mechanistic insights

To gain mechanistic insights into the crosstalk among co-cultured FTW stem cells, we performed scRNA-seq analysis (Figure 4A). Pearson correlation analysis and UMAP co-embedding revealed transcriptional similarity between co-cultured and separately cultured cells (Figure 4B and S4A). We used the CellChat³⁸ toolkit to infer cell-cell communication. As expected, both the number and strength of cell-cell interactions increased in co-cultures when compared to separate cultures (Figures 4C and S4B). Interestingly, a significant proportion of these interactions originated from FTW-mXENs towards the other two cell types (Figures 4C and 4D). CellChat analysis also predicted several signaling pathways mediating these interactions, which included signaling through extracellular matrix (ECM) proteins such as LAMININ and COLLAGEN (Figure 4D). LAMININ and COLLAGEN are known components of the basement membrane (BM) lining the basal side of the post-implantation EPI, which are in part produced by the VE in mice³⁹. We next determined whether ECM protein(s) could phenocopy the growth restriction of co-cultured FTW-mESCs. We found supplementation of separately cultured FTW-mESCs with Matrigel (contains ~60% LAMININ and ~30% COLLAGEN IV), LAMININ, COLLAGEN IV, or a combination of LAMININ and COLLAGEN IV, but not VITRONECTIN could inhibit FTW-mESC growth in a dosage-dependent manner (Figures 4E, S4C–S4E). Interestingly, this growth inhibition effect of ECMs is more pronounced for FTW-mESCs than for naïve mESCs and primed EpiSCs (Figure S4F). Next, we performed gene knockout for functional validation. Knockout of Laminin γ1 (Lamc1) in FTW-mXENs could partially rescue the growth phenotype of co-cultured FTW-mESCs (Figures 4F, S4G, and S4H). As INTEGRIN-β1 plays an important role in ECM protein signaling and is a cell surface receptor for LAMININ-γ1^40–42, we generated integrin β1 (Itgb1) knockout FTW-mESCs. We found loss-of-function of Itgb1 could also mitigate the retarded growth of co-cultured FTW-mESCs (Figure S4I and S4J).

Figure 4. Mechanistic insights of growth inhibition of FTW-mESCs by FTW-mXENs.

(A) Diagram outlining the scRNA-seq experiments. (B) Heatmap illustrating the Pearson correlation among FTW-mESCs, FTW-mXENs, and FTW-mTSCs in separate cultures and co-cultures. The displayed numbers correspond to Pearson correlation coefficients. (C) Circle plots detailing the ratios of number (left) and strength (right) of cell-cell interactions between co-cultured and separately cultured samples. Red lines, increased interactions; blue lines, decreased interactions. (D) Heatmaps revealing outgoing (left) and incoming (right) signaling pathways in co-cultured mouse FTW stem cells. (E) Violin plot showing the product of area and GFP intensity for each FTW-mESC colony on day 5 in separate cultures and co-cultures (mESCs: mXENs = 2:1 or 1:1) and separate cultures supplemented with different ECM proteins. Matrigel_L: 0.5% (v/v), Matrigel_H: 2% (v/v), Laminin_L: 30 μg/ml, Laminin_H: 120 μg/ml, Collagen_L: 15 μg/ml, Collagen_H: 60 μg/ml, Vitronectin_L: 5 μg/ml, Vitronectin_H: 30 μg/ml. N.S., not significant. (F) Violin plot showing the area and GFP intensity product for each FTW-mESC colony across various experimental conditions. (G) A schematic summary capturing the key mechanistic takeaways from the observed proliferation inhibition of FTW-mESCs by FTW-mXENs. N.S. not significant, ****p < 0.0001. P-values were calculated using a two-tailed Student’s t-test. See also Figure S4.

In addition, we studied transcriptomic differences between separately cultured and co-cultured FTW-mESCs by bulk RNA-seq. Through comparative analysis, we identified 502 differentially expressed genes (DEGs) shared between FTW-mESCs in XENs/ESCs and XENs/TSCs/ESCs co-cultures when compared to separately cultured FTW-mESCs (Figure S4K). Interestingly, the majority of differentially expressed genes (DEGs) (492) were down-regulated genes in co-cultured FTW-mESCs. Consistent with their decreased proliferation, the down-regulated DEGs included genes related to cell proliferation and embryo size (e.g., several members of activator protein 1[AP-1] transcription factor complex such as Fos, Fosl1, Fosb, Batf among others^43–45 (Figure S4L). GO and Bioplanet 2019 analyses of the down-regulated DEGs revealed top enriched terms including extracellular matrix and structure organization, Interleukin-1 and TGF-beta regulation of extracellular matrix, and β1 integrin cell surface interactions (Figures S4M and S4N). In addition, we found many matrix metalloproteinases (MMPs) (e.g., Mmps 2, 3, 9, 10, 12 etc.) were down-regulated in co-cultured FTW-mESCs (Figure S4O). MMPs have been reported to modulate cell proliferation, migration, and morphogenesis by degrading ECM proteins⁴⁶, and inhibition of MMPs also led to reduced growth of EPI in vivo and ESCs in vitro⁴⁷, which are consistent with our findings.

In sum, these analyses helped gain insights into the crosstalk among co-cultured embryonic and extraembryonic stem cells and identified ECM signaling as one of the mechanisms mediating the reduced proliferation of FTW-mESCs by FTW-mXENs (Figure 4G).

FTW stem cells from monkey blastocysts

By using the FTW condition, we also succeeded in the derivation of stable extra-embryonic (FTW-cyXENs and FTW-cyTSCs) and embryonic (FTW-cyESCs, 20% KSR [knockout serum replacement] was needed) stem cell lines from 10 and 7 d.p.f. (days post-fertilization) monkey blastocysts, respectively (Figures 5A, 5B, and Table S3A). FTW-cyESCs could also be directly converted from naïve-like ESCs⁴⁸ through culture adaptation (Figure S5M). Once established, FTW-cyXENs, FTW-cyTSCs, and FTW-cyESCs proliferated well (Figures S5A, S5F, and S5L), maintained stable colony morphology (Figure 5B) and normal karyotypes (Figures S5B and S5G) after long-term culture, expressed hypoblast (HYP), trophoblast, and EPI related genes, respectively (Figures 5C, S5C, S5D, S5H, S5I, and S5M). Upon random differentiation, FTW-cyXENs could generate a XEN-tumor in vivo (Figure S5E), and visceral-/yolk sac endoderm- (VE/YE−) (FOXA1⁺ GATA4⁻) like cells and extra-embryonic mesenchyme cell- (EXMC-) (COL6A1⁺ GATA4⁻) like cells in vitro^49,50 (Figure 5D); FTW-cyTSCs were capable of differentiating into extravillous trophoblast (EVT)-like cells and multinucleated syncytiotrophoblast (SCT)-like cells in vitro (Figure 5E)⁷ and generating a TSC-tumor in vivo (Figures S5J and S5K); FTW-cyESCs could form teratomas that contained all three germ layers tissues (Figure S5N).

Figure 5. Derivation, characterization and transcriptomic profiling of monkey FTW stem cells.

(A) Schematic of FTW embryonic and extra-embryonic stem cell lines derivation from monkey blastocysts. (B) (Top) Representative BF images of a 10 d.p.f monkey blastocyst, day 12 outgrowth, and established FTW-cyXENs (P14). (Middle) Representative BF images of a 10 d.p.f monkey blastocyst, day 12 outgrowth, and established FTW-cyTSCs (P10). (Bottom) Representative BF images of a 7 d.p.f monkey blastocyst, day 7 outgrowth, and established FTW-cyESCs (P7). Scale bars, 50 μm. (C) Representative IF images showing the expression of monkey extra-embryonic endoderm (GATA6 and GATA4), trophoblast (GATA3 and CK7), and epiblast (OCT4 and SOX2) lineage markers in FTW-cyXENs (top), FTW-cyTSCs (middle), and FTW-cyESCs (bottom), respectively. Scale bar, 100 μm. (D) Representative IF co-staining images of COL6A1, FOXA1, and GATA4 in differentiated FTW-cyXENs at day 9. Blue, DAPI. Scale bars, 100 μm. (E) (Bottom) Representative IF co-staining images of GATA3 with the EVT maker HLA-G in EVT-like cells differentiated from FTW-cyTSCs. (Top) Representative IF co-staining images of GATA3 with the SCT makers HCG and HCGB in SCT-like cells differentiated from FTW-cyTSCs. (F) (Left) UMAP visualization of all scRNA-seq cells from monkey FTW stem cells. (Right) UMAP plot, showing the expression of representative markers in the different clusters/cell lines, FTW-cyESC (SOX2, POU5F1, NANOG), FTW-cyPS (MIXL1, LHX1, GSC); FTW-cyXEN (SOX17, GATA6), FTW-cyVE/YE (IHH, MIXL1), FTW-cyEXMC (COL6A1, HAND2), FTW-cyTSC (GNR2F2, LRP2), FTW-cyCTB (C1QBP, COMMD6), and FTW-cySTB (CGA, CRH). (G) Heatmap illustrating the Pearson correlation across various cell types with FTW cells and correlated with in vivo monkey datasets⁴⁹. The numbers represent Pearson correlation coefficients. (H) Heatmaps of TF enrichment scores comparing FTW-XENs between monkeys and mice. (I) Gene knockout of HMGA1, NFE2L1, and SALL1 in monkey FTW-cyXENs and the representative BF images. (J) qRT-PCR analysis of GATA4, PDGFRA, and NFE2L1 expression in NFE2L1 knockout FTW-cyXENs. (K) Representative IF co-staining images of GATA4 and GATA6 in wild type and NFE2L1 knockout FTW-cyXENs. Blue, DAPI. Scale bars, 100 μm. See also Figure S5 and Table S3A, S3B.

Next, we performed single cell transcriptomic profiling of monkey FTW stem cells. Based on marker gene expressions, we found that although the majority of FTW cells maintained stemness (FTW-cyESC [SOX2 and NAGOG], FTW-cyXEN [SOX17 and GATA6] and FTW-cyTSC [NR2F2 and LRP2] subclusters), small subpopulations (FTW-cyPS [MIXL1, LHX1 and GSC], FTW-cyVE/YE [IHH and MIXL1] and FTW-cyEXMC [COL6A1 and HAND2], and FTW-cyCTB [C1QBP and COMMD6] and FTW-cySTB [CGA and CRH] subclusters) expressed genes related to early differentiation (Figure 5F)^49,51,52. The lineage identity of each subcluster was further revealed by correlation analysis with monkey in vivo reference datasets⁴⁹ and gene set score analysis with human and monkey embryos datasets⁵³ (Figures 5G and S5O). In addition, by using SCENIC analysis we determined the regulon activities of each subcluster (Figure S5P).

The derivation of FTW-cyXENs offers an opportunity to study species-specific TF regulatory networks that regulate primate hypoblast cells. To this end, SCENIC analysis was applied to scRNA-seq datasets of monkey and mouse FTW-XENs to identify species-specific TF regulons (Figure 5H and Table S3B). For functional validations, we selected a few monkey-specific TFs (HMGA1, SALL1, and NFE2L1) and performed genetic perturbations in FTW-cyXENs using CRISPR/CAS9. Noticeably, all knock out clones (4/4 HMGA1, 4/4 SALL1, and 9/9 NFE2L1) showed stalled growth (Figure 5I). In addition, the expressions of HYP related genes (e.g., GATA6 and GATA4) were significantly impaired in these knockout clones (Figures 5J and 5K).

These results demonstrate the indispensable roles of HMGA1, SALL1, and NFE2L1 in FTW-cyXENs’ self-renewal and stemness.

Cross-lineage monkey FTW stem cell co-cultures

Next, we investigated whether the growth restriction of FTW-ESCs by FTW-XENs was conserved in monkeys. To this end, we performed co-culture of FTW-cyESCs with FTW-cyXENs and/or FTW-cyTSCs. Consistent with mouse findings, we found the growth of FTW-cyESCs was greatly inhibited by FTW-cyXENs but marginally affected by FTW-cyTSCs or fibroblasts and was not due to apoptosis (Figures 6A, 6B, and S6A–S6C). To provide mechanistic insights into embryonic and extra-embryonic lineage crosstalk in monkeys, we performed scRNA-seq analysis of co-cultures of FTW-cyESCs, FTW-cyXENs, and FTW-cyTSCs and compared with separate cultures (Figure 6C and S6D). Consistent with mouse findings, we also observed reduced and increased subpopulations of FTW-cyESCs in G2/M and G1 cell cycles, respectively, in co-cultures when compared to separate cultures (Figure S6E). CellChat analysis predicted signaling through ECM proteins, e.g., LAMININ and COLLAGEN, mediated crosstalk between FTW-cyXENs and FTW-cyESCs (Figures 6D and S6F, and Table S4), in agreement with mouse results. To confirm the effects of ECM proteins, we supplemented Matrigel, LAMININ, or COLLAGEN IV to separately cultured FTW-cyESCs, and found that each could partially phenocopy FTW-cyXENs’ inhibitory effect on the growth of FTW-cyESCs in a dosage dependent manner (Figures 6E, S6G and S6H).

Figure 6. FTW-cyXEN-mediated proliferation inhibition of FTW-cyESCs.

(A) Representative merged fluorescence and BF images of days 1–5 separately cultured FTW-cyESCs (green) and FTW-cyESCs co-cultured with FTW-cyXENs (red arrowheads) and/or FTW-cyTSCs (blue arrowheads) or proliferative fibroblast. Scale bar, 100 μm. (B) Violin plot showing the area and GFP intensity product for each FTW-mESC colony under different conditions. (C) Heatmap showing the Pearson correlation between separately cultured and co-cultured FTW-cyESCs, FTW-cyXENs, and FTW-cyTSCs. The numbers represent Pearson correlation coefficients. (D) Heatmaps of outgoing (left) and incoming (right) signaling pathways in co-cultured monkey FTW stem cells. (E) Violin plot showing the product of area and GFP intensity for each FTW-cyESC colony on day 5 in separate cultures and co-cultures (cyESCs: cyXENs = 1:3.5 or 1:7) as well as separate cultures supplemented with different ECM proteins. Matrigel_L: 0.5% (v/v), Matrigel_H: 2% (v/v), Laminin_L: 2.5 μg/ml, Laminin_H: 10 μg/ml, Collagen_L: 15 μg/ml, Collagen_H: 60 μg/ml. *p < 0.05, ****p < 0.0001. P-values were calculated using a two-tailed Student’s t-test. See also Figure S6 and Table S4.

Taken together, these results extended our findings in mice to monkeys and revealed a conserved mechanism underlying growth inhibition of FTW-ESCs by FTW-XENs.

Cross-species comparisons of FTW-XENs

In addition to mice and monkeys, we found human FTW-XENs (FTW-hXENs) could also be generated from naïve ESCs and extended pluripotent stem cells (EPSCs) by culture adaptation (Figures 7A, 7B, S7A–S7C). FTW-hXENs expressed HYP markers, e.g., GATA4, GATA6, FOXA2, and SOX17, but not pluripotency markers (Figures 7C, S7C, and S7D). Although FTW-hXENs could be passaged multiple times (naïve ESC-derived: >10 passages; EPSC-derived: >25 passages) and maintained stable HYP marker gene expression, we found they grew noticeably slower than FTW-mXENs and FTW-cyXENs. We performed RNA-seq for FTW-hXENs and compared them with HYP cells from human embryos cultured ex vivo^53,54. Principle component analysis (PCA) revealed that on the PC1 axis FTW-hXENs aligned with human E8 HYP cells (Figure S7E). To determine the differentiation potential of FTW-hXENs, we cultured them on laminin-coated plates in basal medium supplemented with 10% fetal bovine serum (FBS) for 9 days. Through IF analyses we found both EXMC-like (COL6A1⁺ GATA6⁻) and VE/YE-like (FOXA1⁺ GATA6⁻) cells were present in the differentiation culture^49,50 (Figure S7F).

Figure 7. Derivation of human FTW-XENs and cross-species comparisons.

(A) Schematic illustrating the derivation of FTW-hXENs from human naïve ESCs or EPSCs. (B) Representative BF images of human naïve ESCs and FTW-hXENs (P11). Scale bars, 100 μm. (C) Representative IF images showing the expression of XEN cell markers (GATA6, GATA4, SOX17, and FOXA2) in FTW-hXENs. Scale bars, 100 μm. (D) Left, A VENN diagram showing the conserved and species-specific expressed genes in FTW-mXENs, FTW-cyXENs, and FTW-hXENs. Right, GO term enrichment analysis of conserved genes (n=2608) in FTW-mXENs, FTW-cyXENs and FTW-hXENs. (E) Hub-gene-network analysis predicts several core FTW-XENs regulators. (F) qRT-PCR analysis of Gata4, Gata6, Sox17, Foxa2, Laminin, and Pdgfra expression in FTW-mXENs and FTW-cyXENs after knockdown of the Src, Pdgfr and Jak2. See also Figure S7 and Table S5.

XENs from different species derived and cultured in the same FTW condition gave us a unique opportunity to examine species-conserved and\ -divergent features not influenced by different culture parameters. We next performed cross-species comparisons of mouse, monkey, and human FTW-XENs. Hierarchical clustering and correlation analysis showed that FTW-cyXENs clustered closer with FTW-hXENs than FTW-mXENs (Figure S7G). We found 2,608 genes were commonly shared among mouse, monkey, and human FTW-XENs, which were enriched in GO terms, including regulation of cellular PH, negative regulation of cell-matrix adhesion, positive regulation of vasculogenesis, among others (Figure 7D and Table S5A). In addition, we identified 2,132, 2,667, and 1,767 genes specifically expressed in human, monkey, and mouse FTW-XENs, respectively (Figure 7D). Top enriched species-specific GO terms include: 1) human: nervous system development and axon guidance; 2) monkey: mitochondria translation and negative regulation ubiquitin-protein ligase activity; 3) mouse: inflammatory response and neutrophil activation involved in immune response (Figure S7H). We also analyzed the expression patterns of both divergent and conserved TFs identified through cross-species comparisons among mouse, monkey, and human FTW-XENs (Figure S7I and Table S5B). To delineate evolutionarily conserved genes regulating the FTW-XENs state, pairwise transcriptomic comparisons between mouse, monkey, and human FTW-XENs and EPIs from corresponding developmental stages (mouse, E5.5 EPI⁵⁵; human, d.p.f. 8 and 9 EPIs^53,54; monkey, d.p.f. 8 and 9 EPIs⁵⁰) were carried out. Based on this analysis, we identified 735 DEGs (XEN vs. EPI) were shared by all three species (Figure S7J), and hub-gene-network analysis of these 735 genes predicted several core FTW-XENs regulators, including several well-known hypoblast-related genes (e.g., GATA4, GATA6, SOX17, and PDGFA), as well as genes with unknown functions in hypoblast development (e.g., SRC, JAK2, PIK3R1, and CCL2) (Figures 7E).

Next, we selected several candidate genes including SRC, PDGFRA, and JAK2 for functional validation via shRNA-based knockdown experiments in both mouse and monkey FTW-XENs (Figure S7K). We found that individual knockdown of SRC, PDGFRA, and JAK2 disturbed the expression patterns of several PrE/HYP-related genes in both monkey and mouse FTW-XENs. Interestingly, while knockdown of SRC and JAK2 significantly up-regulated the expression levels of FOXA2 and LAMC1 in both species (Figure 7F), PDGFRA knockdown resulted in an opposite change in GATA4 and GATA6 expression between mouse and monkey FTW-XENs (Figure 7F). These results reveal conserved and divergent functions of FTW-XENs regulators between rodent and primate species.

DISCUSSION

Most stem cell cultures include pathway inhibitors to suppress differentiation in favor of self-renewal, Surprisingly, by activating several developmental signaling pathways at once, we found embryonic and extraembryonic stem cells from multiple species could be derived and stably maintained in the same culture condition^12,53. This signaling “activated” self-renewing state can potentially be attained in a variety of stem cell types by striking a balance between differentiation and self-renewal via switching on a combination of signaling pathways including but not limited to FGF, TGF-β and WNT pathways used in this study.

In a previous study, Artus et al. observed a reduced number of PrE cells resulting from Pdgfra knockout led to an increase in the number of EPI cells in diapause mouse blastocysts⁵⁶. More recently, Kyprianou et al. demonstrated that perforations and remodeling of the basement membrane on the posterior side of gastrulating mouse embryos are vital for embryo growth, morphogenesis and gastrulation⁴⁷. These studies align with our observations and provide in vivo and ex vivo support that during mouse early post-implantation development, EPI proliferation is under control by the adjacent PrE/VE.

Several recent studies have relied on the establishment of stem cell derived embryo models to study the crosstalk between embryonic and extra-embryonic cells in mice^57–63 and humans^64,65. It should be noted that these studies were carried out under differentiating conditions, in which both starting embryonic and extraembryonic stem cells dynamically gave rise to various downstream cell types. In contrast, in our FTW co-cultures, FTW-ESCs, FTW-XENs, and FTW-TSCs maintained their stemness with minimal differentiation. This unique co-culture system offers an opportunity to explore the interactions between embryonic and extraembryonic cells at a defined early post-implantation developmental time point.

XENs or XEN-like cells have been derived from various species’ embryos, such as mice^3,4,22,37, pigs⁶⁶, rats^67–69, cows⁷⁰, and from human naïve or naïve-like PSCs^64,71. Leveraging the FTW culture in our study, we have successfully obtained XENs from mouse and monkey blastocysts, as well as from human naïve ESCs and EPSCs. The availability of FTW-XENs from mice, monkeys, and humans facilitates cross species comparison, revealing conserved and unique genes and pathways influencing the XEN state in the FTW culture. Through bioinformatic analyses and experimental validations, we identified primate specific TFs (e.g., HMGA1, SALL1, and NFE2L1) essential for FTW-cyXENs maintenance, and a group of species-conserved core FTW-XENs regulators, which includes both well-recognized genes and those previously unidentified in PrE/HYP development.

The ability to grow embryonic and extraembryonic stem cells in the same culture environment opens new avenues to dissect the molecular mechanisms underlying lineage crosstalk during early mammalian development. Based on FTW stem cell co-cultures, we have uncovered a conserved non-cell autonomous control of pluripotent cell proliferation by extraembryonic endoderm cells, which is in part mediated by the ECM signaling in both mice and monkeys. In a broader context, the stem cell co-culture strategy developed in this study may help provide superior starting cells for generating more robust integrated stem cell embryo models⁷¹ and developing more faithful differentiation protocols for regenerative medicine.

Limitations of the study

There are several limitations of the current study: 1) Due to varying growth rates among FTW-ESCs, FTW-XENs, and FTW-TSCs, cross-lineage stem cell co-cultures are sustainable only for a few passages before rapidly proliferating cells (FTW-XENs in mice; FTW-ESCs in monkeys) dominate. 2) Despite multiple attempts and varying culture parameters, we’ve been unsuccessful in deriving human FTW-TSCs from naïve ESCs. This suggests species-specific variances in sustaining TSCs in the FTW culture. 3) Much like human naïve extra-embryonic endoderm (nEnd) cells⁷¹, human FTW-XENs derived from naïve ESCs are difficult to maintain beyond 10–15 passages. In comparison, FTW-hXENs sourced from human EPSCs are sustainable long-term (>25 passages). This might be attributed to the genetic and epigenetic instability in naïve human ESCs, particularly the observed loss of imprinting^72,73, potentially affecting the longevity of FTW-hXENs. 4) Unlike mice and monkeys, we have not obtained FTW-hXENs directly from human blastocysts.

STAR★Methods

RESOURCE AVAILABILITY

Lead contact

Further information, inquiries and requests for all reagents and resources should be directed to and will be fulfilled by the lead contact Jun Wu (Jun2.Wu@UTSouthwestern.edu).

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 GSE241465.

• This paper does not report original code. Code that has been used for analysis is publicly available, referred to and listed in the references section.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Mice

C57BL/6 and CD-1 (ICR) female mice aged 8–10 weeks were purchased from Charles River or Envigo (Harlen). NOD-SCID (NOD.CB17-Prkdc^scid/J) female mice aged 8–10 weeks were purchased from the Jackson Lab. Mice were housed in a 12-hr light/12-hr dark cycle at 22.1–22.3°C and 33–44% humidity. All animal procedures were performed per the ethical guidelines of the University of Texas Southwestern Medical Center. The animal protocol was reviewed and approved by the UT Southwestern Institutional Animal Care and Use Committee (IACUC) [Protocol #2018-102430]. All experiments followed the 2021 Guidelines for Stem Cell Research and Clinical Translation released by the International Society for Stem Cell Research (ISSCR). Human-mouse chimeric studies were reviewed and approved by the UT Southwestern Stem Cell Oversight Committee (SCRO) [Registration #14].

Cynomolgus monkeys

All animals and experimental procedures were approved in 2021 by the ethical committee of the State Key Laboratory of Primate Biomedical Research and Institute of Primate Translational Medicine and Kunming University of Science and Technology (IPTM, KUST). The monkey procedures were performed by following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) for the ethical treatment of non-human primates. 2 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. All cynomolgus monkey embryo-related work was conducted at the State Key Laboratory of Primate Biomedical Research.

METHOD DETAILS

Antibodies, plasmids, chemicals, cell lines and recombinant DNA, oligonucleotides, and software. See Key Resources Table.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Human GATA-6 Affinity Purified Polyclonal Ab antibody	R&D Systems	Cat#AF1700; RRID:AB_2108901
Human SOX17 Affinity Purified Polyclonal Ab antibody	R&D Systems	Cat#AF1924; RRID: AB_355060
Anti-TBR2 / Eomes antibody	Abcam	Cat#ab23345; RRID: AB_778267
Anti-CDX-2, Clone CDX2-88 antibody	BioGenex	Cat#MU392A-UC; RRID: AB_2650531
Sox-2 (E-4) antibody	Santa Cruz Biotechnology	Cat#sc-365823; RRID: AB_10842165
Oct-3/4 (C-10) antibody	Santa Cruz Biotechnology	Cat#sc-5279; RRID: AB_628051
Anti-GATA4 antibody	Abcam	Cat#ab84593; RRID: AB_10670538
Human/Mouse alpha-Fetoprotein/AFP Antibody	R&D Systems	Cat#MAB1368; RRID: AB_357658
Pax-6 (AD2.35) antibody	Santa Cruz Biotechnology	Cat#SC-53108; RRID: AB_630089
Anti-Brachyury / Bry antibody [EPR18113]	Abcam	Cat#ab209665; RRID: AB_2750925
FOXA1 antibody	Abcam	Cat#ab55178; RRID: AB_941631
Rabbit Anti-Collagen VI Polyclonal Antibody	Abcam	Cat#ab6588; RRID: AB_305585
Cleaved Caspase-3 (Asp175) Antibody	Cell signaling technology	Cat#9661 RRID: AB_2341188
GATA-6 (D61E4) XP® Rabbit mAb	Cell signaling technology	Cat#5851; RRID: AB_10705521
Human GATA-4 Biotinylated Antibody	R&D Systems	Cat#BAF2606; RRID: AB_2263176
Human SOX17 Affinity Purified Polyclonal Ab antibody	R&D Systems	Cat# AF1924; RRID: AB_355060
FoxA2/HNF3-beta Antibody	Cell Signaling Technology	Cat#3143 RRID: AB_2104878
Anti-FOXA2 antibody	Abcam	Cat#ab60721; RRID: AB_941632
Anti-SOX2 antibody	Abcam	Cat#ab137385; RRID: AB_2814892
FOXA1 antibody	Abcam	Cat#ab55178; RRID: AB_941631
Rabbit Anti-Collagen VI Polyclonal Antibody	Abcam	Cat#ab6588; RRID: AB_305585
Oct-3/4 (C-10) antibody	Santa Cruz Biotechnology	Cat#sc-5279; RRID: AB_628051
Recombinant Anti-GATA3 antibody	Abcam	Cat#ab199428; RRID: AB_2819013
Cytokeratin 7 - Cytoskeleton Marker	Abcam	Cat#ab68459; RRID: AB_-1139824
AP-2γ Antibody (6E4/4)	Santa Cruz Biotechnology	Cat#sc-12762; RRID: AB_667770
Anti-HLA G antibody [G233]	Abcam	Cat#ab52454; RRID: AB_880554
hCG beta Polyclonal Antibody	Thermo Fisher Scientific	Cat#PA5–58598; RRID: AB_2642336
Anti-Human Chorionic Gonadotropin	DAKO	Cat#IR508; RRID: not available
Donkey anti rabbit IgG (H+L) 647	Invitrogen	Cat#A31573; RRID: AB_2536183
Donkey anti rabbit IgG (H+L) 594	Invitrogen	Cat#A21207; RRID: AB_141637
Donkey anti mouse IgG (H+L) 488	Invitrogen	Cat#A21202; RRID: AB_141607
Donkey anti mouse IgG (H+L) 594	Invitrogen	Cat#A32744; RRID: AB_2762826
Donkey anti goat IgG (H+L) 647	Invitrogen	Cat#A32849; RRID: AB_2762840
Donkey anti goat IgG (H+L) 594	Invitrogen	Cat#A11058; RRID: AB_2534105
Goat anti rabbit IgG (H+L) 594	Invitrogen	Cat#A11037; RRID: AB_2534095
Goat anti rabbit IgG (H+L) 488	Invitrogen	Cat#A11034; RRID: AB_2576217
Goat anti-Mouse IgG 1, Alexa Fluor 647	Invitrogen	Cat#A21240; RRID: AB_2535809
Goat anti-Mouse IgG 1, Alexa Fluor 488	Invitrogen	Cat#A21121; RRID: AB_2535764
Goat anti-Mouse IgG2b, Alexa Fluor 594	Invitrogen	Cat#A21145; RRID: AB_2535781
Goat anti-Mouse IgG2b, Alexa Fluor 488	Invitrogen	Cat#A21141; RRID: AB_2535778
Goat anti-Mouse IgG2a, Alexa Fluor 488	Invitrogen	Cat#A21131; RRID: AB_2535771
Goat anti-Mouse IgG2a, Alexa Fluor 594	Invitrogen	Cat#A21135; RRID: AB_2535774
Chemicals, Peptides, and Recombinant Proteins
CHIR-99021 (CT99021)	Selleckchem	Cat# S1263
Recombinant Human FGF-basic	Peprotech	Cat# 100-18B
Recombinant Human/Murine/Rat Activin A	Peprotech	Cat# 120-14E
Fetal Bovine Serum	GIBCO	Cat#10099141
KnockOut Serum Replacement	Thermo Fisher Scientific	Cat#A3181502
Neurobasal Medium	Thermo Fisher Scientific	Cat#21103-049
DMEM/F-12, HEPES	GIBCO	Cat# 11330032
N-2 Supplement (100X)	GIBCO	Cat# 17502048
B-27 Supplement (50X)	GIBCO	Cat# 17504044
Non-Essential Amino Acids Solution	GIBCO	Cat# 11140050
GlutaMAX	GIBCO	Cat# 35050061
Penicilin-Streptomycin	GIBCO	Cat# 15070063
Insulin-Transferrin-Selenium-Ethanolamine (ITS - X) (100X)	Thermo Fisher Scientific	Cat#51500-056
Trypsin-EDTA solution	Sigma	Cat# SLB20766
TrypLE Express	GIBCO	Cat# 12605010
2-Mercaptoethanol	GIBCO	Cat# 21985023
TrypLE™ Express Enzyme (1X), no phenol red	Thermo Fisher Scientific	Cat#12604021
PDGF	R&D Systems	Cat#220-BB
Recombinant Human NRG1/HRG1 Protein	R&D Systems	Cat#5859-NR-050
A83-01	Tocris	Cat#2939/50
Y-27632 dihydrochloride	Tocris	Cat#1254
Bovine Serum Albumin	Sigma	Cat# A6003
Forskolin	Tocris	Cat# 1099/50
Animal-Free Recombinant Human EGF	Peprotech	Cat# AF-100-15
Collagen IV	Corning	Cat# 354233
Vitronectin	GIBCO	Cat# A14700
Laminin	Biolmina	Cat#LN511
Matrigel Matrix hESC-qualified	Corning	Cat#354277
Heat-inactivated fetal bovine serum	Corning	Cat#35-076-CV
CMRL 1066	GIBCO	Cat#11530037
L-glutamine	Thermo Fisher Scientific	Cat#25030081
β-Estradiol	Sigma	Cat#E8875
Progesterone	Sigma	Cat#P0130
N-acetyl-L-cysteine	Sigma	Cat#A7250
KnockOut Serum Replacement	Thermo Fisher Scientific	Cat#10828028
M2 medium	Sigma	Cat#M7167
Annexin V-APC/PI	Procell	Cat# P-CA-207
Opti MEM	Thermo fisher Scientific	Cat# 31985070
Deposited Data
Mouse and Monkey FTW-ESCs | RNA-seq data sets	This paper	GEO: GSE241465 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241465)
Mouse extraembryonic endoderm stem cell | RNA-seq data sets	Zhong et al.4	GEO: GSE106158 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi)
Mouse trophoblast stem cell | RNA-seq data sets	Cui et al.5	GEO: GSE106158 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106158)
Mouse trophoblast stem cell | RNA-seq data sets	Kubaczka et al.25	GEO: GSE64339 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64339)
Mouse trophoblast stem cell | RNA-seq data sets	Wu et al.26	GEO: GSE25255 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25255)
Mouse pluripotent stem cell | RNA-seq data sets	Bao et al.27	GEO: GSE99494 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE99494)
Mouse pluripotent stem cell | RNA-seq data sets	Wu et al.28	GEO: GSE60605 (https://www.ncbi.nlm.nih.gov/proiects/geo/query/acc.cgi?acc=GSE60605)
Mouse pluripotent stem cell | RNA-seq data sets	Zhao et al.29	GEO: GSE73631 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73631)
Mouse pluripotent stem cell | RNA-seq data sets	Cruz-Molina et al.30	GEO: GSE89211 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89211)
Mouse extraembryonic endoderm stem cell | RNA-seq data sets	Anderson et al.31	GEO: GSE77783 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE77783)
Mouse pluripotent stem cell | RNA-seq data sets	Ye et al.32	GEO: GSE93238 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93238)
Mouse embryos | RNA-seq data sets	Cheng et al.33	GEO: GSE109071 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109071)
Mouse embryos | RNA-seq data sets	Mohammed et al.34	GEO: GSE100597 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE100597)
Mouse embryos | RNA-seq data sets	Nowotschin et al.35	GEO: GSE123124 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE123124)
Human pre-gastrulation embryos (In vitro) | RNA-seq data set	Xiang L et al.53	GEO: GSE136447 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136447)
Human pre-gastrulation embryos (In vitro) | RNA-seq data set	Zhou et al.54	GEO: GSE109555 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109555)
Experimental Models: Cell Lines
Mouse: FTW-mESCs	This manuscript	N/A
Mouse: FTW-mTSCs	This manuscript	N/A
Mouse: FTW-mXENs	This manuscript	N/A
Mouse: Conventinal-mTSCs-GFP	Tanaka et al.2	N/A
Mouse: Conventinal-mXENs-GFP	Kunath et al.3	N/A
Monkey: FTW-cyESCs	This manuscript	N/A
Monkey: FTW-cyTSCs	This manuscript	N/A
Monkey: FTW-cyXENs	This manuscript	N/A
Monkey: FTW-cyXENs (Blastocyst derived)	This manuscript	N/A
Human: FTW-hXENs (Naive converted)	This manuscript	N/A
Human: FTW-hXENs (EPSCs converted)	This manuscript	N/A
Experimental Models: Organisms/Strains
C57BL/6J	The Jackson Laboratory	RRID:IMSR_JAX:000664
CD1(ICR)	Charles River	RRID:IMSR_CRL: 022
NOD-SCID mice	Charles River	RRID:IMSR_CRL: 394
Cynomolgus monkey	Macaca fascicularis	N/A
Oligonucleotides
All primers used in this study were listed in the Table S6	This manuscript	N/A
Recombinant DNA
pSpCas9(BB)-2A-GFP (PX458)	Ran et al.76	pSpCas9(BB)-2A-GFP (PX458); Addgene plasmid #48138
Software and Algorithms
HISAT2 version 2.2.1	Kim et al.77	http://daehwankimlab.github.io/o/hisat2/;RRID:SCR_015530
StringTie version 2.1.4	Pertea et al.78	https://ccb.jhu.edu/software/stringtie/;RRID:SCR016323
Samtools version 1.7	Li et al.86	http://htslib.org/;RRID:SCR002105
GraphPad Prism 7	GraphPad Prism	https://www.graphpad.com/scientific-software/prism/
FeatureCount version 2.0.1	Liao et al.87	https://subread.sourceforge.net/
Python version 3.9.7	N/A	https://www.python.org/
Pyscenic version 0.11.2	Aibar et al.88	https://pyscenic.readthedocs.io/en/latest/#id12
R version 4.0.2	N/A	https://www.r-project.org/
Ggplot2 version 3.3.6	Wickham et al.89	https://ggplot2-book.org/
Seurat version 4.6.0	Hao et al.80	https://satijalab.org/seurat/
DESeq2 version 1.28.1	Love et al.90	https://genepattern.github.io/DESeq2/v1/index.html
Pheatmap version 1.0.12	Kolde91	https://www.rdocumentation.org/packages/pheatmap/versions/1.0.12/topics/pheatmap
Clusterprofiler version 3.16.0	Yu et al.81	https://guangchuangyu.github.io/software/clusterProfiler/
Cellchat version 1.4.0	Jin et al., 202138	https://github.com/sqjin/CellChat
Dplyr version 1.0.9	Hadley et al., 202292	https://cran.rproject.org/web/packages/dplyr/dplyr.pdf
Seurat-wrappers version 0.1.0	Satija lab	https://github.com/satijalab/seurat-wrappers
Cell Ranger version v4.0.0	10× Genomics	https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest
Cytoscape version 3.9.1	Shannon et al.93	https://cytoscape.org/
DoubletFinder version 2.0.3	McGinnis et al.83	https://github.com/chris-mcginnis-ucsf/DoubletFinder
Monocle2 version 2.16.0	Trapnell et al.84	http://monocle-bio.sourceforge.net/
Others
Ensembl database	NA	https://www.ensembl.org/biomart/martview/5c26977f2d02b6962344fc47dc737b71
animalTFDB database	NA	http://bioinfo.life.hust.edu.cn/AnimalTFDB/#!/download

Harvesting and culture of mouse embryos

Female C57BL/6 mice aged 8–10 weeks underwent superovulation through an intraperitoneal (IP) injection of 5 IU PMSG (Prospec). This was followed by another IP injection of 5 IU hCG (Sigma-Aldrich) 48 hours later. After mating with C57BL/6 male mice, embryos ranging from the 8-cell to morula stages were collected at E2.75 [note: identifying a vaginal plug was marked as embryonic day 0.5 (E0.5)]. The retrieval process involved flushing the oviducts and uterine horns using KSOM-HEPES. These embryos were then cultured in mKSOMaa until they reached the blastocyst stage, and the cultivation was conducted under a humidified environment with 5% (v/v) CO2 and 5% (v/v) O2 at 37 °C. Separately, CD-1 female mice aged 8 weeks and older, in their natural estrous cycles, were mated with CD-1 males. At E3.5, blastocysts were collected by flushing the uterine horns.

Derivation and culture of FTW-mXENs, FTW-mTSCs and FTW-mESCs

Embryo manipulations were performed under a dissection microscope (Nikon SMZ800N). In brief, the zona pellucidae (ZP) of E3.5 blastocysts were removed using a short exposure to acidic Tyrode’s solution (Millipore MR-004-D). Once the ZP was eliminated, the embryos were placed on MEFs in FTW medium, which consists of N2B27 basal medium enhanced with FGF2 (20 ng/mL, Peprotech), Activin-A (20 ng/mL, Peprotech), and CHIR99021 (3 μM, Selleckchem). After 6–8 days in culture, blastocyst outgrowths were passaged using TrypLE and re-seeded onto newly prepared MEFs. FTW-XEN, FTW-TSC, and FTW-ESC colonies were manually picked for further cultivation. Established mouse XEN, TSC, and ESC lines were cultured on MEF plates pre-coated with 0.1% gelatin in FTW medium. The cells were cultured at 37 °C under 5% CO₂, with daily media changes. For passaging, the cells were dissociated into single cells using TrypLE Express (GIBCO) and passaged onto new MEF-coated plates at a split ratio of 1:20 (FTW-mESCs), 1:10 (FTW-mTSCs) and 1:50 (FTW-mXEN) every 3–4 days.

Generation of reporter mouse FTW stem cells

We used pCAG-IP-mKO or pCAG-IP-eGFP to label FTW-mXENs, FTW-mTSCs, and FTW-mESCs. In brief, 1–2 μg of pCAG-IP-mKO/eGFP plasmids were transfected into 1 × 10⁶ – 2 × 10⁶ dissociated single cells using an electroporator (NEPA21, Nepa Gene) following the protocol recommended by the manufacturer. Then, 0.5–1.0 μg ml⁻¹ of puromycin (Invitrogen) was added to the culture medium 2–3 days after transfection. Drug-resistant colonies were manually picked between 7 to 14 days and further expanded clonally.

In vitro differentiation of FTW-mTSCs and FTW-mXENs

FTW-XENs were dissociated into single cells using TrypLE Express and seeded into a 6-well plate pre-coated with 5 μg/mL Laminin at a density of 2 × 10⁵ cells (mouse FTW-XENs) or 5 × 10⁵ cells (monkey and human FTW-XENs) per well in XEN differentiation medium for 9 days, with medium changes every other day. XEN differentiation medium was prepared using the following: 1:1 (v/v) mixture of DMEM/F12 and Neurobasal medium, 1X N2 supplement, 1X B27 minus insulin supplement, 1X GlutaMAX, 1X Nonessential amino acids, 0.1 mM β-mercaptoethanol, 0.5% Penicillin-Streptomycin, and 10% FBS.

Harvesting and culture of cynomolgus embryos

Cynomolgus monkey ovarian stimulation, oocyte recovery and in vitro fertilization were performed as previously described⁷⁴. Briefly, healthy female cynomolgus monkeys were subjected to follicular stimulation by intramuscular injection of 20 IU of recombinant human 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 aspiration 32–35 hours following rhCG administration. Follicular contents were placed in HEPES-buffered Tyrode’s albumin lactate pyruvate (TALP) medium containing 0.3% bovine serum albumin (BSA) (Sigma-Aldrich) at 37°C. Oocytes were stripped of cumulus cells by pipetting after a brief exposure (<1 min) to hyaluronidase (0.5 mg/mL) in TALP-HEPES to allow visual selection of nuclear maturity metaphase II (MII; first polar body present) oocytes. The mature oocytes were subjected to intracytoplasmic sperm injection (ICSI) immediately and then cultured in CMRL-1066 medium (Gibco, 11530037) containing 10% FBS 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 embryo development. The blastocysts were collected at 7 days post fertilization (d.p.f.). The zona pellucida of the blastocysts was removed by exposure to hyaluronidase from bovine testes (Sigma-Aldrich) for about 30 seconds and embryos were cultured in vitro until 10 days post fertilization.

Derivation and culture of FTW-cyXENs, FTW-cyTSCs, FTW-cyESCs

For the derivation of FTW-cyXENs and FTW-cyTSCs, embryos from cynomolgus monkeys at 10 days post-fertilization (d.p.f.) were delicately dissected using a 31-gauge syringe needle. They were transferred to wells in a 4-well dish pre-coated with 0.1% gelatin and layered with mitotically inactivated MEFs. The FTW medium was utilized for cultivation, following the same protocol as with mouse embryos. To promote the proliferation of hypoblast, 1% KSR (Thermo Fisher Scientific, A3181502) and PDGF (10 ng/mL, R&D, 220-BB) were also added during the derivation of FTW-cyXENs⁴. After 8–10 days, XEN-like and TSC-like colonies appeared. Single colonies were isolated and then dissociated by TrypLE (Thermo Fisher Scientific,12604021) for 3 minutes at 37°C and passaged into a well of a 4-well dish. Established FTW-cyXENs and FTW-cyTSCs were passaged by TrypLE every 6–7 days at a split ratio of 1:10 in FTW medium under 20% O₂ and 5% CO₂ at 37°C. To derive FTW-cyESCs⁷⁵, monkey blastocysts (7 days d.p.f.) were plated into a well of 4-well dish coated with 0.1% gelatin and MEFs in FTW cyESC medium [N2B27 basal medium supplemented with 20% KSR, FGF2 (6 ng/mL, Peprotech), Activin-A (25 ng/mL, Peprotech) and CHIR99021 (1.5 μM, Selleckchem)] under 5% O2 and 5% CO2. After 8–10 days, the ESC-like outgrowths appeared and were dissociated using TrypLE for 3 minutes at 37 °C. The established FTW-cyESCs were cultured in a 6-well plate pre-coated with 0.1% gelatin MEFs in FTW cyESC medium under 20% O₂ and 5% CO₂ at 37°C.

In vitro differentiation of FTW-cyXENs

FTW-cyXENs were dissociated into single cells using TrypLE and seeded into a 6-well plate pre-coated with 5 μg/mL Laminin at a density of 5 × 10⁵ cells per well and kept in differentiation medium for 9 days with medium change every other day. Differentiation medium was prepared as follows: 1:1 (v/v) mixture of DMEM/F12 and Neurobasal medium, 0.5X N2 supplement, 0.5X B27 supplement, 1% GlutaMAX, 1% Nonessential amino acids, 0.1 mM β-Mercaptoethanol, 1% Penicillin-Streptomycin, and 10% FBS.

In vitro differentiation of FTW-cyTSCs

FTW-cyTSCs were differentiated into ST-like and EVT-like cells as previously described for humans⁷. For the differentiation of EVT-like cells, FTW-cyTSCs were dissociated into single cells in a 6-well plate pre-coated with 1 mg/ml Collagen-IV at a density of 1 × 10⁵ cells per well and cultured with 3 mL of EVT medium (DMEM/F12 supplemented with 1% Penicillin-Streptomycin, 0.3% BSA, 4% KnockOut Serum Replacement, 0.1 mM β-Mercaptoethanol,1% ITS-X supplement, 100 ng/ml NRG1, 2.5 mM Y27632, 7.5 mM A83-01). Then, 2% Matrigel was added to the medium on the first day. On day 3, the medium was changed to EVT medium with 0.5% Matrigel but without NRG1. On day 6, the medium was replaced with EVT medium with 0.5% Matrigel but without NRG1 and KSR, and cells were cultured for an additional two days. For differentiation of ST-like cells in 2D, FTW-cyTSCs were dissociated in a 6-well plate pre-coated with 2.5 mg/ml Collagen-IV at a density of 2 × 10⁵ cells per well and cultured in 3 mL of ST (2D) medium (DMEM/F12 supplemented with 0.1 mM β-Mercaptoethanol, 1% Penicillin-Streptomycin, 4% KSR, 0.3% BSA, 1% ITS-X supplement, 2.5 mM Y27632, and 2 mM Forskolin). The medium was replaced on day 3, and the cells were analyzed on day 6. For the differentiation of ST-like cells in 3D, 2 × 10⁵ FTW-cyTSCs were seeded in a 3.5 cm Petri dish and cultured with 3 mL of ST (3D) medium (DMEM/F12 supplemented with 0.1 mM β-Mercaptoethanol, 1% Penicillin-Streptomycin, 4% KSR, 0.3% BSA, 1% ITS-X supplement, 2 mM Forskolin, 50 ng/ml EGF, and 2.5 mM Y27632). An equal amount of fresh ST (3D) medium was added on day 3. The cells were passed through a 40 μm mesh filter to remove dead cells and debris on day 6.

RT-PCR and qRT-PCR analysis

Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. Contaminating genomic DNA was removed by RNase-Free DNase Set (QIAGEN). RNA concentrations were measured on a spectrophotometer (DS-11+, DeNovix). cDNA was synthesized with iScript Reverse Transcription Supermix kit (BIO-RAD) and amplified with PrimeSTAR GXL DNA Polymerase (TaKaRa) or with SYBR Green PCR Master Mix (Thermo Fisher Scientific) on a Touch Thermal Cycler Real-Time PCR system (C1000, BIO-RAD). GAPDH was used as an internal normalization control. All primers used in this study are listed in Table S6.

Cell population doubling time analysis

The doubling time of the cell population was calculated using the online calculator (http://www.doubling-time.com/compute.php?lang=en).

Cell growth curve analysis

FTW-cyXENs, FTW-cyTSCs, and FTW-cyESCs (1 × 10⁵) were seeded into 6-well plates coated with MEFs in FTW medium, with media change every day. Cells were harvested by TrypLE Express at the indicated time points, depleted of MEFs by plating the cell suspension onto 0.5% gelatin-coated plates for 30 minutes, and growth curves were generated by manual cell counting.

Teratoma, XEN-tumor and TSC-tumor formation

For FTW-mTSC and FTW-mXEN tumors, a total of 5 × 10⁶ cells were resuspended in 100 μL of DMEM-Matrigel solution (1:1) and injected subcutaneously into 10-week-old immunodeficient NOD-SCID mice. After 2 weeks, FTW-mTSC tumors were dissected and fixed with PBS containing 4% formaldehyde. After 3 months, FTW-mXEN tumors were dissected and fixed with PBS containing 4% formaldehyde. For cyTSC tumors, a total of 1 × 10⁷ cells were resuspended in 100 μL of DMEM-Matrigel solution (1:1) and injected subcutaneously into 10-week-old immunodeficient NOD-SCID mice. After 7 days, FTW-mTSC tumors were dissected and fixed with PBS containing 4% formaldehyde. For co-injection experiments, the FTW-mESC only group was injected with 1 × 10⁶ FTW-mESCs, and the co-injection group was injected with 1 × 10⁶ FTW-mESCs and 2.5 × 10⁵ FTW-mXENs. After one month, teratomas were dissected, weighed, and then fixed. Paraffin-embedded teratomas were sliced and stained with hematoxylin and eosin. Teratomas were formed using the following cell numbers: 1 × 10⁶ FTW-mESCs only, or 1 × 10⁶ FTW-mESCs plus 1.25 × 10⁵ (8:1), 2.5 × 10⁵ (4:1), 5 × 10⁵ (2:1), 1 × 10⁶ (1:1), 2 × 10⁶ (1:2) FTW-mXENs. After one month, teratomas were dissected, weighed, and fixed.

Immunostaining

Samples were fixed in 4% PFA for 15 min, washed three times with PBS and permeabilized with 0.5% Triton X-100 in PBS for 30 min at room temperature. Cells were then blocked with blocking buffer [5% (w/v) BSA; and 0.1% (v/v) Tween 20 in PBS] for 1 h and incubated with the primary antibodies (Table S6) diluted in blocking buffer at room temperature for 2 h or 4°C overnight. After three washes with PBST (PBS plus 0.1% Tween 20), the cells were incubated with corresponding secondary antibodies (1:300 diluted, Table S6) in a blocking buffer at room temperature for 1 h. After an additional three times PBST washes, cells were counterstained with 300 nM DAPI solution at room temperature for 20 min before mounting. Samples were imaged using a fluorescence (Echo Laboratories, CA) or a confocal microscope (A1R, Nikon).

Flow cytometry

Cells were dissociated with TrypLE Express at 37 °C for 5 min. Then, the cells were fixed in 4% PFA at room temperature for 30 min and permeabilized with 0.5% (v/v) Triton X-100 at room temperature for 30 min. The cells were then incubated in the primary antibody (Table S6) solution for 30 min and then the secondary antibody solution for 30 min at room temperature. Samples stained with only secondary antibodies were used as the negative controls. The samples were washed twice with PBS containing 2% FBS between each step. Finally, the stained cells were suspended in PBS containing 2% FBS and analyzed by flow cytometry (FACScalibur system, BD).

Blastocyst injection of FTW-mXENs and FTW-mTSCs

FTW-mXEN injection into mouse blastocysts was performed as described previously¹² with slight modifications. Briefly, single cell suspensions of mouse and human FTW-mXENs were added to a 40 μL droplet of KSOM-HEPES containing the blastocysts and placed on an inverted microscope (Nikon) fitted with micromanipulators (Narishige). Individual cells were collected into a micropipette with a 15–20 μm internal diameter (ID), and a Piezo Micro Manipulator (Prime Tech) was used to create a hole in the zona pellucida and trophectoderm layer of mouse blastocysts. 10–12 (FTW-mXEN/FTW-mTSCs) cells were introduced into the blastocoel. After microinjection, the blastocysts were cultured in mKSOMaa. For mouse embryo transfer, 8–12 weeks old ICR female mice were used as surrogates and were mated with vasectomized ICR male mice to induce pseudopregnancy. Ketamine (30 mg/ml) / Xylazine (4 mg/mL) and Buprenorphine (1 mg/mL) were used in surgery for maintaining anesthesia and relieving pain. Injected blastocysts were transferred to the surrogate uterine at E2.5. 14–30 blastocysts were transferred within 20–30 min per surrogate.

Immunostaining and imaging of chimeric embryos

At E7.5 or E11.5, surrogates were euthanized, and embryos were isolated. Embryos were dissected and checked for fluorescence using Zeiss Axio Zoom.V16 fluorescence stereo zoom microscope equipped with a Plan-Neofluar Z 1.0x/0.25 (FWD 56 mm) objective and Axiocam 503 monochromatic camera. Embryos were fixed in 4% paraformaldehyde and incubated at 4°C for 30 min (E7.5 embryos) or overnight (E11.5 embryos). After overnight cryoprotection in 30% sucrose solution (Fisher), the embryos were embedded in a Polyfreeze Tissue freezing medium (Polyscience, Inc.) and frozen on dry ice. Sections (10 μm thick) of the different embryos were cut on a Leica cryostat (Leica CM1950). For immunostaining, 10 mM citrate buffer (0.05% Tween 20 based) was used for antigen retrieval. The primary antibodies used are summarized in Table S6. After washing with TBST three times, the cells were incubated with corresponding secondary antibodies in a blocking buffer at room temperature for 1 hour. Samples were counterstained with 300 nM DAPI solution at room temperature for 20 min and washed with PBST at least three times. Finally, slides were imaged using a fluorescence microscope (Echo Laboratories, CA).

Mouse FTW stem cells co-culture assay

FTW-mESCs-eGFP, FTW-mTSCs-WT, and FTW-mXENs-mKO/WT were seeded onto MEF-coated plates either cultured separately or mixed at different ratios for co-cultures. The seeding ratio and density were empirically tested and decided on the basis of cell growth rate. A starting number of FTW-mESCs-eGFP (1.5 × 10⁴ cells), FTW-mTSCs-WT (3 × 10⁴ cells, and FTW-mXENs-mKO (7.5 × 10³ cells) were decided on for most of the cell-cell co-culture assays. During co-culture experiments, cells were cultured in FTW medium on MEFs for 5 days and the following analyses were performed. For co-culture experiments with different cell ratios, FTW-mESCs-eGFP were seeded at a fixed cell number (1.5 × 10⁴ cells per well) and with varying numbers of FTW-mTSCs-WT and/or FTW-mXENs-WT.

For the differentiation co-culture experiments, FTW-mESCs-eGFP and FTW-mXENs-mKO were seeded, and the medium was switched to a differentiation medium containing DMEM/F12 supplemented with 10% fetal bovine serum (FBS) the next day. On day 5, the number of FTWmESCs-eGFP cells was counted.

Transwell co-culture assay

For transwell co-culture experiments, Millipore Transwell 0.4 μm PET hanging inserts (Millicell, MCH12H48) were used by placing them into 12-well plates. MEFs were coated on the top/bottom well. FTW-mESCs-eGFP (5,000 cells) were seeded for separate culture groups on the bottom wells, not the top insert. For co-culture groups, FTW-mESCs-eGFP (5,000 cells) and FTW-mXENs-WT (2,500 cells) were seeded on the bottom and top insert of the wells, respectively. Half of the medium was changed daily, and cell numbers were counted on day 5.

Mouse ECM protein inhibition assay

Mouse FTW-mESC-GFP cells or naive mESCs (cultured in 2iLIF and serum/LIF) and primed mEpiSCs were passaged using TrypLE and seeded in a well of 6-well plate at 1.5 × 10⁴ cells in FTW cyESC medium under 20% O₂ and 5% CO₂ at 37°C. After 4 hours, the ECM protein mixtures (Matrigel_L: 0.5% (v/v), Matrigel_H: 2% (v/v), Laminin_L: 30 μg/ml, Laminin_H: 120 μg/ml, Collagen_L: 15 μg/ml, Collagen_H: 60 μg/ml, Vitronectin_L: 5 μg/ml, Vitronectin_H: 30 μg/ml) were directly added into the culture medium every day for 5 days. Medium and ECM proteins were changed every day.

Cell cycle assay

Cell cycle analysis was performed using the fluorescence single staining PI (Propyridine Iodide). Separate culture (FTW-mESCs-GFP [5 × 10⁴]) and Co-culture (FTW-mESCs-GFP [5 × 10⁴] and FTW-mXEN-WT [1 × 10⁴]) cells were seeded on a 35 mm dish. Each group has three experimental repeats. Cells were fixed with ice colded 70% ethanol for 30 min and staining PI for 30 min for Subsequent experiments. Cells were analyzed on the BD flowcytometer and FlowJo software.

Mouse FTW stem cell inhibitor tests

FTW-mESC cells, FTW-mXEN cells, conventional mESCs and mXENs were passaged using TrypLE and seeded in a well of 6-well plate at 1.5 × 104 cells in FTW or conventinal medium under 20% O2 and 5% CO2 at 37°C. After 24 hours, the inhibitors (PD173074: 1 μM, IWR-1: 2.5 μM, LDN193189: 2 μM, EC359: 100 nM, SB431542: 2 μM) were directly added into the culture medium for 5 days. Medium with inhibitors was changed every day.

Annexin V-PI assay

Cell death was analyzed by Annexin V-APC/PI fluorescence dual staining cell apoptosis detection kit (Procell, Cat# P-CA-207). Separate culture (FTW-mESCs-GFP [5 × 10⁴]) and Co-culture (FTW-mESCs-GFP [5 × 10⁴] and FTW-mXEN-WT [1 × 10⁴]) cells were seeded on a 35mm dish. Cells were collected at day 3, day 4, and day 5 for further analysis. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the Annexin V-APC/PI fluorescence double staining cell apoptosis detection kit was used for subsequent experiments as the kit described. Cells were analyzed on the BD flowcytometer and FlowJo software.

Monkey FTW stem cells co-culture assay

FTW-cyESCs-GFP, FTW-cyTSCs-WT, and FTW-cyXENs-mKO or WT were seeded onto MEF-coated plates and mixed at different ratios for co-culture experiments. The seeding ratios and densities were empirically tested and decided on the basis of cell growth rate. These numbers were: FTW-cyESCs-GFP (5 × 10⁴ cells), FTW-cyTSCs-WT (30 × 10⁴ cells, and FTW-cyXENs-mKO/WT (35 × 10⁴ cells). Cells were cultured in MEF-coated plates in FTW cyESC medium during co-culture experiments for 5 days.

Monkey ECM protein inhibition assay

Monkey GFP positive FTW-cyESCs cells were passaged by treatment with TrypLE and 5 × 10⁴ cells were seeded onto a MEFs-coated well of 6-well plate in FTW medium under 20% O₂ and 5% CO2 at 37°C. After 4 hours, ECM mixtures were added (Matrigel: 0.5%, 2%; Laminin: 2.5μg/ml, 10μg/ml; CollagenIV:15 μg/ml, 60 μg/ml) directly added into the culture medium every day for 5 days. Medium and ECM proteins were changed every day.

Quantification of FTW-ESCs number

For all of the co-culture experiments, total FTW-ESC numbers were quantified by the following method. Cells were dissociated into single cells using TrypLE Express at 37 °C for 4 min, and the total number of live cells was counted. Next, the relative percentage of eGFP+ FTW-ESCs-eGFP was determined using an LSR II Flow Cytometer (BD Bioscience). The total ES cell number (tN) for each group in both the co-cultured and separate culture conditions was determined by multiplying total cell volume (V) with cell concentration (CC) and percentage of eGFP+ cells (P). tN = V × CC × P. Cell density (cells cm⁻²) was calculated by dividing the total cell number by the surface area.

Colony size and fluorescence intensity analysis

The following method analyzed all of the co-culture experiments involving colony size and fluorescence intensity. On day 5 of co-culture, fluorescently labeled cells were imaged with a Leica Microsystem DMi8 microscope using Leica Application Suite X software for analysis. The images were taken randomly by the software in a fixed area for each well and A Fiji pipeline was used to quantify the size and intensity of the ESC colonies. Briefly, images are preprocessed with median filters to filter out noise and small debris, binary images are then created through Otsu thresholding, and the watershed was applied to separate merged clones. The quantification was implemented with the Analyze Particles function of Fiji based on the binary images created earlier. The average ESC colony Pixels (P) for each group in co-cultures and separate cultures were determined by multiplying colony Size (A) with colony intensity (I). P = A × I. For mouse cell experiments, there were three independent biological replicates for each group and 3 images for each sample. All of the colonies from 9 images were analyzed. For monkey cell experiments, 10 images were taken randomly, and all colonies were analyzed in the 10 images.

Ex vivo culture of E6.5-E6.75 epiblasts with or without visceral endoderm

To obtain embryos, ICR females were mated with males from ICR (Charles River Laboratories) in the afternoon, and the presence of vaginal plugs was checked the next morning. The day on which a plug was found was considered to be E0.5. Both male and female mice were used between 6 to 25 weeks. All the animal experiments were performed under the ethical guidelines of the Kindai University, and animal protocols were reviewed and approved by the Kindai University Animal Care and Use Committee. The developmental stage of embryos is critical for adapting the cultivation of isolated epiblast to an STO-conditioned medium. The isolated epiblasts before the onset of gastrulation collapsed in culture, while those after the onset of gastrulation were stable for growth in an STO-conditioned medium and were applicable for further experiments. The embryos at E6.5-6.75 were surgically isolated in cold DMEM supplemented with 10% fetal calf serum (FCS, BioWest), and whole epiblasts were isolated by the mechanical removal of Reichert’s membrane, extra-embryonic ectoderm as well as visceral endoderm, depending on the cases, using fine forceps and a tungsten needle. The isolated epiblasts with or without visceral endoderm were plated into Nunclon Sphera-treated, 96-well U-shaped-bottom microplate (174925, Thermo Scientific) in STO-conditioned medium. The STO-conditioned medium was prepared as follows. Dulbecco’s Modified Eagle’s Medium (DMEM, SIGMA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% Penicillin-Streptomycin (10,000 U/mL, Gibco) was used to culture Mitomycin-C inactivated STO feeders for 2 days. After 48 hours, cultured epiblast outgrowths, including visceral endoderm in the case, were dissociated with TrypLE (Gibco, 12604013), and the total cell number was counted.

Extended ex vivo culture of mouse blastocysts

Embryos at the 2-cell stage were incubated in 30 μL droplets of prewarmed potassium simplex optimization medium (KSOM) (MR-004-D, Sigma-Aldrich) under a layer of mineral oil (10029, Vitrolife). After a day, embryos at the 8-cell stage were shifted to fresh prewarmed KSOM droplets containing 0.5/1 μM PD0325901 (TOCRIS, 4192) for a 48-hour treatment. Post-treatment, each blastocyst was injected with 8–10 FTW-mXENs (GFP+) and then placed in fresh KSOM droplets for about 3 hours to allow the shrunken blastocyst to re-expand. Following the removal of the zona pellucida using Acid Tyrode’s Solution (T1788, Sigma-Aldrich) and a rinse in M2 medium (M7167, Sigma-Aldrich), 6–8 blastocysts were transferred to a well in a μ-Slide 8-well plate (80826, Ibidi). This plate was pre-coated with 100 μL of undiluted Matrigel and kept at 37°C for 30 minutes in 250 μL of pre-equilibrated IVC1 medium (components see below). On day 1, the medium was refreshed with prewarmed IVC1. By the morning of day 2, IVC1 was substituted with 200μL of prewarmed IVC2 (component details provided below), and by evening, the embryos were fixed for immunofluorescence staining. IVC1: CMRL 1066 (11530037, Gibco) supplemented with 20% (v/v) heat-inactivated fetal bovine serum (FBS) (35-076-CV, Corning), 2 mM L-glutamine (25030081, Thermo Fisher Scientific), Penicillin (100 units/mL)/Streptomycin (100 mg/mL) (15140122, Thermo Fisher Scientific), Insulin-Transferrin-Selenium-X (ITS-X) (100 3) (51500056, Thermo Fisher Scientific), 8 nM β-Estradiol (E8875, Sigma-Aldrich), 200 ng/mL Progesterone (P0130, Sigma-Aldrich) and 25 μM N-acetyl-L-cysteine (A7250, Sigma-Aldrich), 4% (v/v) Matrigel. IVC2: CMRL 1066 supplemented with 25% (v/v) KnockOut Serum Replacement (10828028, Thermo Fisher Scientific), 5% rat serum (prepared in-house), 2 mM L-glutamine, Penicillin (100 units/mL)/Streptomycin (100 mg/mL), Insulin-Transferrin-Selenium-X (ITS-X), 4% (v/v) Matrigel.

FTW-mXENs and FTW-cyXENs knockdown assay

FTW-mXENs and TFW-cyXENs were cultured until they reached 50–60% confluency. Afterward, 100μl of lentivirus (at a titer of 1 × 10^8 TU/ml) carrying shRNA targeting either PDGFRA, JAK2, or SRC was added to each individual well. Cells were then subjected to puromycin selection 72 hours post-transfection. Subsequent to this, single cell clones were isolated for the Smart-seq2 protocol⁷⁵. Briefly, the reverse transcription reaction was conducted using SuperScript II (18064–071, Invitrogen). This was followed by cDNA amplification using the KAPA HiFi HotStart Ready Mix (product number KK2602 from KAPA Biosystems) and PCR, set to 19 cycles.The concentration of cDNA was determined by Qubit 3 (Invitrogen) and was diluted to 10 ng/μL using nuclease-free water. Next, TB Green^® Premix Ex Taq^™ II (RR820L, Takara) was used to establish a 25 μL real-time fluorescence PCR reaction (TB Green Premix Ex Taq II: 12.5 μL, upstream primer (10 μM): 0.5 μL, downstream primer (10 μM): 0.5 μL, cDNA: 10 ng, nuclease-free water: to 25 μL). Real-time PCR was performed with CFX ConnectTM Real Time System (Bio-Rad). The amount of target mRNA was determined using the ΔΔCt method with ACTB as the internal control.

FTW-mESCs and FTW-mXENs knockout assay

For this study, we designed all single guide RNAs (sgRNAs) using the online software Benchling CRISPR. The sgRNA sequences are provided in Table S6. These sgRNAs were incorporated into the pSpCas9(BB)-2A-eGFP (PX458)⁷⁶ plasmid from Addgene by ligating annealed oligonucleotides to a BbsI-digested vector. The plasmid containing the specific sgRNA was subsequently transfected into either FTW-mESCs or FTW-mXENs using the NEPA2 electroporator (NEPA2, Nepa Gene 1). 48 hours post-transfection, EGFP-positive cells were isolated via flow cytometry, and 2,000 of these cells were seeded into a well of a 6-well plate. Individual clones were then selected and cultivated. Clones with homozygous knockouts were validated through Sanger sequencing.

Monkey FTW-cyXENs knockout assay

For this study, we designed all single guide RNAs (sgRNAs) using the online software Benchling CRISPR. The sgRNA sequences are provided in Table S6. These sgRNAs were incorporated into the pSpCas9(BB)-2A-eGFP (PX458) plasmid from Addgene by ligating annealed oligonucleotides to a BbsI-digested vector. Following the manufacturer’s guidelines, we transfected 2×10⁶ FTW-cyXENs with 10μg of the plasmid containing the specific sgRNA using an electroporator. After 48 hours post-transfection, EGFP-positive cells were isolated using flow cytometry. We then seeded 500 of these cells in a 4-well plate. Individual clones were subsequently selected for further analysis. We extracted their genome to analyze knockout clones using the Discover-sc Single Cell WGA Kit (Vazyme, N603-02). In brief, we prepared the cell lysate per the instructions, and monoclonal cells were aspirated into the lysate using a pipette. Following this, random primers and Phi29 enzyme facilitated the isothermal PCR amplification of the genome (conditions: 30°C for 2 hours, followed by 65°C for 5 minutes). We then diluted the extracted DNA to a concentration of 10 ng/μL based on its concentration. We performed specific PCR amplification to verify knockout efficiency using Ex Premier^™ DNA Polymerase (Takara, RR370A). The amplified products underwent sequencing on the Applied Biosystems 3730xl platform (provided by Tsingke), and we analyzed the sequencing results using the SnapGene software (version 6.0.2).

Plasmids

pSpCas9(BB)-2A-eGFP (PX458) plasmid was purchased from Addgene (plasmid #48138). pLKO.1-puro.shRNA was purchased from Sigma-Aldrich (Cat#SHC001). pCAG-IP-mKO and pCAG-IP-eGFP plasmids were obtained from T. Hishida. HBLV-JAK2/PDGFRA/SRC shRNA-ZsGREEN-PURO plasmids were obtained from HANBIO.

Bulk RNA-sequencing

RNA extraction was performed using an RNeasy Mini Kit (QIAGEN) using DNase treatment (QIAGEN). RNA was analyzed using a 2100 Bioanalyzer (Agilent Technologies). (Transcripts per Kilobase Million). RNA was extracted with Trizol Reagent (15596026, Invitrogen) for monkey cells. The RNA (~50 ng) reverse transcription reaction and amplification were performed using SuperScript II (18064-071, Invitrogen), and KAPA HiFi HotStart Ready Mix (KK2602, KAPA). The cDNA was analyzed using a 2100 Bioanalyzer (Agilent Technologies). The RNA Library was generated using TruePrep DNA Library Prep Kit V2 for Illumina (TD501, Vazyme), and then the library was adapted for sequencing on an Illumina NovaSeq 6000 platform (sequenced by Annoroad).

Pre-processing of raw RNA-seq data

All reads were mapped to the mouse (GRCm38/mm10), human (GRCh38/hg38), and rhesus macaque genome (Mmul_10/rheMac10) using hisat2⁷⁷ (version 2.2.1) with default settings. FeatureCount (version 2.0.1) was used to estimate read counts. Stringtie (version 2.1.4) was used to estimate fragments per kilobase of exon per million fragments mapped (FPKM) and transcripts per kilobase of exon model per million mapped reads (TPM) values according to a previous report⁷⁸, genes with an FPKM value ≥3 were considered as expressed.

Comparison analysis with published available datasets

The previously published datasets, including mouse^{4,5,25–30,32,34}, human^53,54,71 and monkey^49,50,79 embryonic single-cell RNA sequencing and mouse cell lines (nEND) microarray datasets³¹ were obtained from GEO repository (NCBI) and incorporated into our analysis. The expression levels of scRNA-seq data were transformed into log2(TPM + 1), and those of microarray data were transformed into log2(intensity).

Principal components analysis (PCA)

The principal component analysis (PCA) was performed using the prcomp function without scaling. The differentially expressed genes (DEGs) were defined as genes exhibiting more than twofold changes between the samples (P < 0.005) and the sum of the expression level of every gene was log2(TPM+1) > 0 with the variance > 0.

Similarities inference between cells

We selected the union of the top 2,000 genes of the highest variance for our dataset and the published dataset and calculated 20 canonical correlates (CCs) with diagonal CCA. After running CCA, the first 10 CCs were used for t-Distributed Stochastic Neighbor Embedding (t-SNE) visualization. The homology cell types were co-clustered in the same CCA cluster. Then, the correlation analysis was employed to detect the correspondence of cell subtype for our cells and the previously published embryonic cells by using an expression matrix of 200 high variable genes (HVGs) that contributed to the first 10 CCs.

Differentially expressed genes, GO and KEGG pathway analysis

DEGs across clusters were identified using the “FindAllMarkers” function in Seurat⁸⁰ (v4.6.0). function “FindAllMarkers”. Heatmaps representing the expression of marker genes for each cell cluster were generated using the pheatmap R package (v1.0.12). We used the functions enrichKEGG and enrichGO in clusterProfiler R package⁸¹ (v3.16.0) to perform KEGG pathways⁸² and Gene Ontology (GO) biological processes enrichment analysis. Pathways or processes with a P value of ≤ 0.05 were deemed significantly enriched. Visual representations of these enriched pathways and processes were created using the “ggplot2” function in the ggplot2 package (v3.3.6) in R.

Single cell sample preparation

Mouse blastocysts were harvested at E3.5, and the zona pellucida was removed using Tyler’s buffer, as previously described¹². Post-removal, blastocysts were rinsed twice in PBS/PVA. Digestion was carried out in TrypLE Express for 10 minutes at 37°C. Cell clusters were then mechanically disrupted using mouth glass pipettes, starting with a larger diameter (50 μm) and progressing to a smaller one (20 μm). The entire digestion process took approximately 40–50 minutes. For the FTW-derived day-8 outgrowth samples, blastocysts were collected and cultured on MEF-coated plates in FTW medium for 8 days. Subsequently, the outgrowth regions were isolated and disaggregated. Cells were dissociated for the established FTW cell lines using TrypLE Express for 3–5 minutes at 37°C.

scRNA-seq library preparing and sequencing

Single-cell suspensions were prepared at a concentration of 1,100 cells/μL in 0.04% bovine serum albumin (BSA)/PBS. For mouse blastocysts, the entire cell population was loaded onto 10X Chromium Single Cell G Chips. Libraries were constructed using the Chromium Single Cell 3’ Reagent Kit v3.1 (10X Genomics, Pleasanton, CA) by the manufacturer’s guidelines. In a nutshell, individual cells were captured into Gel beads in Emulsion (GEMs) within the GemCode device. This was followed by cell lysis, barcoded reverse transcription of RNA, amplification, and shearing. Subsequently, 5’ adaptors and sample indices were attached. Sequencing was executed on the Illumina NovaSeq 6000 platform.

scRNA-seq data analysis

We constructed the single-cell gene expression count matrix using Cell Ranger (v4.0.0) and then conducted subsequent analyses via the Seurat package. Each dataset underwent a filtration process to eliminate transcription noise cells, setting criteria as follows: 1) At least 200 genes expressed per cell. 2) Mitochondrial read percentage below 20%. 3) Ribosomal read percentage exceeding 5%. 4) Raw reads surpassing 3,000. 5) Specific minimum/maximum gene counts and maximum read thresholds for each dataset. Potential doublets in the single-cell RNA sequencing data were filtered out using the DoubletFinder⁸³ package (v2.0.3) in R. For the monkey samples, after stringent quality control, we obtained 17,587 single-cell transcriptomes, each having a median unique molecular identifier (UMI) of 21,024 and 5,197 genes. Cells that passed the quality control were amalgamated and normalized through Seurat’s SCT method. To bridge datasets from diverse origins, we utilized Seurat’s CCA method. The dimensional reduction was executed using UMAP, employing the top 30 principal components (PCs) from PCA. The clustering of cells was achieved with the FindClusters function in Seurat. The cell identities were annotated according to the expressed marker genes. An initial PCA was run for every cluster to explore correlations among cell clusters, followed by a correlation analysis based on the PC1s extracted from these clusters.

Cell clustering by nonlinear dimensional reduction

Initially, we corrected batch effects to mitigate the influence of varying experimental conditions across samples. We followed a sequence of standard processing steps: 1) Employed the “SelectIntegrationFeatures” function to choose 2,000 feature genes. 2) Used the “FindIntegrationAnchors” function to identify anchors between distinct samples. 3) Implemented the “IntegrateData” function for data integration. 4) Applied the “RunPCA” and “RunUMAP” functions, selecting 30 PCs for dimensionality reduction. 5) Leveraged the “DimPlot” function to visualize the outcome of the dimensional reduction on a 2D scatter plot. We then utilized the Seurat package for cell clustering analysis, considering only genes with an expression level above 5 for subsequent evaluation. Visualization was achieved using UMAP. Specifically, the “FindIntegrationAnchors” function was executed with parameters “k.anchor = 5, anchor.features = 2000” to generate an anchor object. The “IntegrateData” function then used this object to integrate all datasets using default parameters. Lastly, Seurat leveraged the top 10/30 PCs for clustering at a resolution of 1.

Pseudotime trajectory analysis

We utilized Monocle2⁸⁴ (v2.16.0) (available at http://cole-trapnell-lab.github.io/monocle-release) for pseudotime trajectory analysis. The dimensionality of the cells was reduced using the DDRTree method. Subsequently, the cells were ordered in pseudotime. The results were visualized using the plot_cell_trajectory, pheatmap, and plot_genes_in_pseudotime functions.”

Gene regulatory networks

The Single Cell Regulatory Network Inference and Clustering (SCENIC) method⁸⁵ is utilized to deduce gene regulatory networks from single-cell expression profiles and pinpoint cell states. This offers valuable insights into the mechanisms underlying cell heterogeneity. We employed the pySCENIC (v0.11.2) tool’s grn/ctx/aucell functions to discern crucial transcriptional regulation during cell development. Initially, the workflow takes the single-cell expression abundance profile matrix and applies the GRNBoost2 method, a per-target regression technique, to deduce co-expression modules. Indirect targets are subsequently pruned based on cis-regulatory motif discovery using cisTarget. After that, the activity of these regulatory factors is assessed via aucells, yielding a regulatory factor activity score (RAS) derived from the enrichment and scoring of the target genes of these regulatory factors. The Regulator Specificity Score (RSS) is computed based on Jensen-Shannon divergence to identify cluster-specific regulators. This process utilizes the “calcRSS” function in the R language and the results are visualized using ggplot2.

Gene set score

The “AddModuleScore” function within the Seurat package was employed to compute the scores for gene sets. The scores for the gene sets of FTW-cyTSC, FTW-cyCTB, and FTW-cySTB were derived using the differentially expressed genes (DEGs) from each sub-cluster identified in a previous study. These scores were subsequently visualized using ggplot2.

Cell cycle analysis based on scRNA-seq data

The “CellCycleScoring” function within the Seurat package was employed to determine each cell’s cycle phase. This is accomplished by assessing the expression of hallmark marker genes for the G2/M and S phases. For each cell, expression scores are derived for these classical G2/M and S phase marker genes. Cells that lack expression of these markers are likely in the G1 phase.

Analysis of Cell-Cell Communication

To evaluate the differences in cell-cell interactions among FTW-ESCs, FTW-TSCs, and FTW-XENs under co-culture and separate culture conditions, we utilized the CellChat package^38 (v1.4.0). Following the recommended protocol, we determined potential ligand-receptor interactions among the three cell lineages under each condition. We then contrasted the count and intensity of these interactions between the co-culture and separate culture setups.

In our approach, we input the normalized counts into CellChat and underwent routine preprocessing using functions such as “identifyOverExpressedGenes”, “identifyOverExpressedInteractions”, and “projectData”, all set with standard parameters. Subsequently, we deduced the potential ligand-receptor interactions between infected and non-infected cells, employing the functions “computeCommunProb”, “computeCommunProbPathway”, and “aggregateNet”. All procedures adopted default settings.

Annotation of homologous genes

To annotate homologous genes, we initially retrieved a list of human homologous genes from Ensembl BioMart (https://www.ensembl.org/biomart/martview/526377f4f1a12cc7650403f7c93d23bb) to use as a reference. We then examined gene sequences to identify orthologous genes across humans, monkeys, and mice. We annotated 14,818 expressed homologous genes from this process, of which 1,197 were identified as transcription factors (TFs). The list of TF genes was sourced from AnimalTFDB (http://bioinfo.life.hust.edu.cn/AnimalTFDB/#!/).

QUANTIFICATION AND STATISTICAL ANALYSIS

All quantitative data are presented as mean ± SD. Experiments were conducted a minimum of three times, with the repeat number specified as “n” in figure legends. Group differences were assessed using a two-sided Student’s t-test. p-values are indicated in the figures. Data visualization and analysis were performed using GraphPad Prism versions 7.0 and 8.0 (GraphPad Software, La Jolla, CA) and Microsoft Excel (Microsoft 365).

Supplementary Material

1

Figure S1. Derivation and characterization of mouse FTW stem cells, related to Figure 1. (A) Representative BF images of an E4.5 mouse blastocyst (top left), day 8 outgrowth (bottom left) and passage 1 (right) during derivation of FTW stem cells. Enlarged views of the boxed regions are presented in the middle panel. Scale bars, 100 μm. (B) Flow cytometry analysis illustrating the expression of GATA6 in FTW-mXENs (top) and EOMES in FTW-mTSCs (bottom). (C) qRT-PCR results showing the relative expression levels of lineage-specific markers in FTW-mESCs, FTW-mXENs, and FTW-mTSCs (mean ± SD, n = 3, biological replicates). (D) Representative IF images displaying PDGFRa and SOX17 expression in FTW-mXENs. Scale bar, 100 μm. (E) Representative IF images displaying TFAP2C expression in FTW-mTSCs. Scale bar, 100 μm. (F) to (I), Representative BF (F and H), and IF images (G and I) of conventional mXENs (F and G) and mTSCs (H and I). Scale bars, 100 μm. (J) RT-PCR analyses showing the expression of lineage markers in both conventional mXENs and mTSCs, as well as in the converted FTW-mXENs and FTW-mTSCs. GAPDH, loading control. (K) Doubling time of conventional mXENs (mXENs) (n = 5, biological replicates) and FTW-mXENs (n = 8, biological replicates) (top), and conventional mTSCs (mTSCs) (n = 5, biological replicates) and FTW-mTSCs (n = 5, biological replicates) (bottom). Box-and-whisker plots showing the median value (bar inside the box), 25th and 75th percentiles (bottom and top of the box, respectively), and minimum and maximum values (bottom and top whisker, respectively). (L) Quantification of cell numbers for mESCs and FTW-mESCs, as well as mXENs and FTW-mXENs, after 5 days of treatment with various inhibitors. PD17, PD173074, an FGF inhibitor; SB43, SB431542, a TGF-beta inhibitor; LDN19, LDN193189, a BMP inhibitor; EC359, a LIF inhibitor; IWR1, a WNT inhibitor (mean ± SD, n = 3, biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001). (M) qRT-PCR results showing the relative expression levels of several PE and VE markers in randomly differentiated FTW-mXENs at indicated time points (mean ± SD, n = 3, biological replicates). (N) Representative images showing the appearance and H&E staining of an XEN-tumor generated from FTW-mXENs. A higher magnification image of the boxed area is shown on the right. (O) Representative IF images displaying markers AFP, GATA6, PAX6, and T in sections from XEN-tumors. Blue, DAPI. Scale bars, 100 μm. (P) Representative IF images showing FOXA1 and COL6A1 expression in an XEN-tumor section. Scale bar, 100 μm. (Q) Representative BF and DAPI staining images of randomly differentiated FTW-mTSCs at day 12. Yellow dashed lines indicate multinucleated cells. Scale bar, 100 μm. (R) qRT-PCR results showing the relative expression levels of genes associated with trophoblast differentiation in randomly differentiated FTW-mTSCs at indicated time points (mean ± SD, n = 3, biological replicates). (S) Representative images showing the appearance (left) and H&E staining (right) of a TSC-tumor generated from FTW-mTSCs. A higher magnification image of the boxed area is shown in the right panel. (T) Representative BF and fluorescence images showing the chimeric contribution of mKO-labeled FTW-mXENs (top) and FTW-mTSCs (bottom) to E11.5 mouse conceptuses. Scale bars, 1 mm.

2

Figure S2. Transcriptomic analyses of mouse FTW stem cells, related to Figure 2. (A) Uniform manifold approximation (UMAP) visualization integrating FTW-mXENs, FTW-mTSCs, FTW-mESCs with previously published datasets of mPSCs^{15,27,29,30,32} mTSCs^5,25,26 and mXENs^4,31. (B) Expression levels (log2 [FPKM\10+1]) of markers for epiblast, trophoblast, and extraembryonic endoderm in FTW-mESCs, FTW-mTSCs, and FTW-mXENs, as determined from RNA-seq datasets. (C) PCA plot of RNA-seq data from FTW-mESCs, FTW-mXENs, and FTW-mTSCs, alongside published datasets spanning from E3.5 to E6.5 mouse embryos^33,34. (D) UMAP representation of single cell populations from FTW-mXENs, FTW-mTSCs, and FTW-mESCs. (E) UMAP visualization illustrating the expression of representative markers in the mouse FTW stem cells: FTW-mESCs (Nanog, Pou5f1, Sox2), FTW-XENs (Gata6, Sox17, Pdgfra), and FTW-TSCs (Cdx2, Gata3, Krt7). (F) Pie chart illustrating the proportion of different marker gene expression in different cell clusters.

3

Figure S3. Mouse FTW stem cell co-cultures and proliferation restriction of FTW-mESCs by FTW-mXENs, related to Figure 3. (A) Bar chart illustrating the cell densities of FTW-mESCs on day 5 in separate cultures compared to various co-culture conditions. (B) Bar chart illustrating the cell densities of FTW-mESCs in separate cultures and co-cultures with FTW-mTSCs and FTW-mXENs at varying ratios. (C) Representative IF images showing the expression of GATA6 and SOX2 in co-cultured FTW-mXENs (mKO) and FTW-mESCs (GFP), respectively. Scale bars, 100 μm. (D) Representative IF images showing the expression of Activated Caspase 3 in separately cultured FTW-mESCs (bottom) and FTW-mESCs co-cultured with FTW-mXENs (top). Scale bars, 100 μm. (E) Bar chart illustrating the percentages of Caspase3+ cells in separately cultured FTW-mESCs (left) and FTW-ESCs co-cultured with FTW-mXENs (right). (mean ± SD, n = 3, biological replicates, N.S., not significant). (F) Flow cytometry analysis of apoptotic cells (Annexin V+ / PI+) for separately cultured (top) and co-cultured (bottom) FTW-mESCs from day 3 to day 5. (G) Quantification of the results from (F) (mean ± SD, n = 3, biological replicates, N.S., not significant). (H) Bar chart illustrating the distribution of cell cycle phases in separately cultured and co-cultured FTW-mESCs from day 3 to day 5 (mean ± SD, n = 3, biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (I) Representative merged BF and fluorescence images of FTW-mESCs and FTW-mXENs in micropatterned co-cultures from day 1 to day 4. Scale bar, 100 μm. Yellow arrowhead, a standalone FTW-mESC colony. Red arrowheads, FTW-mESC colonies adjacent to FTW-mXENs. (J) Bar chart illustrating the cell densities of FTW-mESCs on day 5 in separate cultures, contact co-cultures, and non-contact (transwell) co-cultures (mean ± SD, n = 3, biological replicates). (K) Representative H&E staining images of the teratoma generated from FTW-mESCs alone (top) and FTW-mESCs co-injected with FTW-mXENs (bottom). (L) Weights of teratoma formed by FTW-mESCs and FTW-mESCs co-injected with FTW-mXENs at different mix ratios (mean ± SD, n = 3, biological replicates). (M) Bar chart illustrating the area of each in vitro cultured E6.5-6.75 mouse epiblast with (red) or without (green) VE (mean ± SD, n = 10, biological replicates). *p < 0.01, **p < 0.001. (N) Schematic of PrE complementation experiments. (O) Representative IF images displaying GATA6 and OCT4 in WT ex vivo E5.5 mouse embryos (top), ex vivo E5.5 mouse embryos treated with 0.5 μM PD (middle), and ex vivo E5.5 mouse embryos injected with FTW-mXEN (GFP+) cells after 0.5 μM PD treatment (bottom). Scale bars, 25 μm. (P) Bar chart displaying the number of OCT4+ and GATA6+ cells in ex vivo E5.5 mouse embryos under various conditions (mean ± SD, n =9, biological replicates, N.S., not significant).

4

Figure S4. Mechanistic insights of growth inhibition of FTW-mESCs by FTW-mXENs, related to Figure 4. (A) UMAP visualization of monkey FTW stem cells from both separately-cultured and co-cultured conditions. Colors indicate the cell source, distinguishing between separately-cultured and co-cultured cells. (B) Bar graphs showing the number (left) and strength (right) of cell-cell interactions in co-cultured versus separately cultured mouse FTW stem cells. (C) Representative merged BF and fluorescence images of separately cultured and co-cultured FTW-mESCs, as well as separately cultured FTW-mESCs supplemented with different ECM proteins. Scale bar, 100 μm. (D) Bar chart illustrating the cell densities of FTW-mESCs on day 5 in both separate cultures and co-cultures, as well as in separate cultures supplemented with different ECM proteins (mean ± SD, n = 3, biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, N.S., not significant). (E) Bar chart illustrating the cell densities of FTW-mESCs on day 5 when supplemented with COLLEGEN IV, LAMININ, or a combination of COLLEGEN IV, LAMININ (mean ± SD, n = 3, biological replicates, ****p < 0.0001). (F) Bar plot illustrating the cell densities of mESCs (under 2iLIF and serum/LIF conditions), FTW-mESCs, and EpiSCs on day 5, when supplemented with COLLEGEN IV, LAMININ, or a combination of COLLEGEN IV, LAMININ (mean ± SD, n = 3, biological replicates, **p < 0.01, ***p < 0.001, N.S., not significant). (G) Representative merged BF and fluorescence images of separately-cultured and co-cultured FTW-mESCs (with WT or Lamc1^−/− FTW-mXENs [clones #4 and #6]). Scale bar, 100 μm. (H) Bar chart illustrating the cell densities of FTW-mESCs on day 5 in both separate cultures and co-cultures (with WT or Lamc1^−/− FTW-mXENs) (mean ± SD, n = 3, biological replicates). (I) Representative merged BF and fluorescence images of separately cultured and co-cultured FTW-mESCs (WT or Itgb1^−/− [clones #2 and #6]). Scale bar, 100 μm. (J) Bar chart illustrating the cell densities of FTW-mESCs (WT or Itgb1^−/−) on day 5 in both separate cultures and co-cultures (mean ± SD, n = 3, biological replicates). (K) A VENN diagram illustrating the 502 DEGs (492 down-regulated; 10 up-regulated) common between separately cultured FTW-mESCs and those co-cultured with either FTW-mXENs or both FTW-mXENs and FTW-mTSCs. (L) A heatmap showing the expression levels of AP-1 family members in separately cultured and co-cultured FTW-mESCs. (M) and (N), Enriched GO terms (M) and Bioplanet terms (N) for 492 down-regulated genes from (K). (O) MMP expression levels (FPKM value) in separately cultured FTW-mESCs, FTW-mESCs co-cultured with FTW-mXENs (top), and FTW-mESCs co-cultured with both FTW-mXENs and FTW-mTSCs (bottom).

5

Figure S5. Derivation and characterization of monkey FTW stem cells, related to Figure 5. (A) Growth curve of FTW-cyXENs (passage 26) (n=9, biological replicates). (B) Karyotype analysis of established FTW-cyXENs. (C) RT-PCR results showing the expression of several hypoblast markers in FTW-cyXENs. M, DNA ladder. N.C., non-template control. (D) Representative IF images showing the expression of SOX17, FOXA2, GATA4, GATA3, and OCT4 in FTW-cyXENs. Blue, DAPI. Scale bars, 50 μm. (E) Representative H&E staining images of an XEN-tumor generated from FTW-cyXENs (3 months post-injection). (F) Growth curve of FTW-cyTSCs (passage 16) (n=3, biological replicates). (G) Karyotype analysis of established FTW-cyTSCs. (H) RT-PCR results showing the expression of several trophoblast markers in FTW-cyTSCs. M, DNA ladder. N.C., non-template control. (I) Representative IF images showing TFAP2C expression in FTW-cyTSCs. Blue, DAPI. Scale bar, 50 μm. (J) Representative images showing the appearance and H&E staining of a TSC-tumor generated from FTW-cyTSCs (7 days post-injection). (K) Representative IF images showing the expression of EVT (HLA-G), SCT (HCG), and TSC (GATA3 and TFAP2C) marker genes in FTW-cyTSCs derived TSC-tumor sections. Blue, DAPI. Scale bars, 50 μm. (L) Growth curve of FTW-cyESCs (passage 9) (n=3, biological replicates). (M) Representative IF images showing the expression of OCT4 and SOX2 in FTW-cyESCs derived from naïve ESCs. Blue, DAPI. Scale bars, 50 μm. (N) Representative images showing the appearance and H&E staining of a teratoma generated from FTW-cyESCs (2 months post-injection). (O) Violin plots illustrating gene set signature scores for in vivo monkey Post-paTE, PreE-TE, PreL-TE⁴⁹, alongside in vitro human Early-EVTs, Early-STBs, Post-CTBs, STBs, EVTs, and Pre-CTBs⁵³ in FTW-cyTSCs. (P) Heatmaps showing the eight regulon groups (G1–8) across all FTW stem cell clusters, with representative TF regulons indicated (numbers of target genes predicted by SCENIC are provided in brackets).

6

Figure S6. Monkey FTW stem cell co-cultures and proliferation restriction of FTW-cyESCs by FTW-cyXENs, related to Figure 6. (A) Growth curves of FTW-cyESCs in both separate cultures and co-cultures with FTW-cyXENs (n=3, biological replicates). (B) Flow cytometry analysis of apoptotic cells (Annexin V+ / PI+) for separately cultured (top) and co-cultured (bottom) FTW-cyESCs from day 3 to day 5. (C) Quantification of the results from (B) (mean ± SD, n=3, biological replicates, N.S., not significant). (D) UMAP visualization of monkey FTW stem cells from both separately-cultured and co-cultured conditions. Colors indicate the cell source, distinguishing between separately-cultured and co-cultured cells. (E) Bar chart displaying the distribution of cell cycle phases in separately-cultured versus co-cultured monkey FTW stem cells. (F) Heatmaps illustrating outgoing (left) and incoming (right) signaling pathways in co-cultured monkey FTW stem cells. (G) Representative merged BF and fluorescence images of separately cultured and co-cultured FTW-cyESCs, as well as separately cultured FTW-cyESCs supplemented with different ECM proteins. Scale bar, 100 μm. (H) Bar chart displaying the cell densities of FTW-cyESCs on day 5 for both separately-cultured and co-cultured conditions, as well as separate cultures supplemented with different ECM proteins (n=3, biological replicates, ***p < 0.001, ****p < 0.0001, N.S., not significant).

7

Figure S7. Human XENs derivation and cross-species comparison, related to Figure 7. (A) Representative time-lapse IF co-staining images of SOX2 and GATA6 showing the conversion of human naïve ESCs into FTW-hXENs. Scale bars, 100 μm. (B) Representative BF (left) and merged BF and fluorescence (right) images of the EPSC-derived FTW-hXENs (SOX17-tdTomato reporter) at different passages (P3, P16 and P25). Scale bar, 100 μm. (C) IF co-staining images of hypoblast markers (GATA4, GATA6, SOX17, and FOXA2) in EPSC-derived FTW-hXENs. Scale bars, 100 μm. (D) qRT-PCR results showing the relative expression levels of lineage-specific markers in FTW-hXENs compared with FTW-hiPSCs (mean ± SD, n = 3, biological replicates). (E) PCA plot of RNA-seq data from separately cultured and co-cultured FTW-hXENs, differentiated FTW-hXEN at day 9 (FTW-hXENs-dif), and published in vivo datasets of hypoblasts from D6 to D14^53,54. (F) IF co-staining images of FTW-hXENs VE/YE-like marker (FOXA1+ GATA6-) (left) and EXMC-like marker (COL6A1+ GATA6-) (right) in differentiation cultures. Scale bars, 100 μm. (G) Heatmap showing the Pearson correlation among human, monkey, and mouse FTW-XENs. The numbers represent Pearson correlation coefficients. (H) Heatmap of differentially expressed genes in human (n=2132), monkey (n=2667), and mouse (n=1767) FTW-XENs. The color key from blue to red indicates low to high expression levels. Shown on the right are the enriched GO terms. (I) Scaled expression patterns of representative TFs in mouse, monkey, and human FTW-XENs. (J) A VENN diagram showing the conserved and species-specific DEGs (XENs vs. EPIs) among human, monkey, and mouse FTW-XENs. (K) qRT-PCR results showing the expression of SRC, PDGFR, and JAK2 in different knockdown (KD) clones in mouse (top) and monkey (bottom) FTW-XENs.

8

Supplementary video 1. Time-lapse video showing the growth of co-cultured FTW-mESCs and FTW-mXENs, related to Figure 3 and S3. The yellow arrow indicates the FTW-mESC colonies surrounded by FTW-mXENs; The white arrow indicates the standalone FTW-mESC colonies.

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Table S1. Chimera efficiency of the FTW-mXENs and FTW-mTSCs, related to Figure 1.

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Table S2. DEGs of FTW-ESCs, XENs, and TSCs subclusters (Table S2A) and expression patterns of representative genes alongside the monocle pseudotime trajectories (Table S2B), related to Figure 2.

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Table S3. Summary of cell lines derivation from monkey embryos (Table S3A) and the identified species-specific TF regulons in monkey and mouse FW-XENs (Table S3B), related to Figure 5.

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Table S4. Cell signaling pathways involved in cell-cell communications during co-culture of FTW-cyXENs and FTW-cyESCs, related to Figure 6.

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Table S5. Divergent and conserved genes and TFs identified from the cross-species comparisons of mouse, monkey and human FTW-XENs, Related to Figure 7

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Table S6. Primer and antibody used in this study, Related to the STAR Method

Highlights.

• A common culture condition for mouse and monkey embryonic and extraembryonic stem cells

• Stem cell co-cultures reveal crosstalk between embryonic and extraembryonic lineages

• Extraembryonic endoderm cells limit pluripotent cell growth via ECM signaling

• Cross-species study found common/unique factors regulating extraembryonic endoderm cells

INCLUSION AND DIVERSITY

We support inclusive, diverse, and equitable conduct of research.