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• W2996475925 abstract "•Cell fate under extended potential conditions bifurcate to form EPI and PE-like lineages•EPSCs and TSCs self-organize into EPS-blastoids, resembling the late-stage blastocysts•EPS-blastoids undertake pre- to post-implantation transition in vitro•EPS-blastoids initiate implantation in vivo Mammalian blastocysts comprise three distinct cell lineages essential for development beyond implantation: the pluripotent epiblast, which generates the future embryo, and surrounding it the extra-embryonic primitive endoderm and the trophectoderm tissues. Embryonic stem cells can reintegrate into embryogenesis but contribute primarily to epiblast lineages. Here, we show that mouse embryonic stem cells cultured under extended pluripotent conditions (EPSCs) can be partnered with trophoblast stem cells to self-organize into blastocyst-like structures with all three embryonic and extra-embryonic lineages. Morphogenetic and transcriptome profiling analyses reveal that these blastocyst-like structures show distinct embryonic-abembryonic axes and primitive endoderm differentiation and can initiate the transition from the pre- to post-implantation egg cylinder morphology in vitro. Mammalian blastocysts comprise three distinct cell lineages essential for development beyond implantation: the pluripotent epiblast, which generates the future embryo, and surrounding it the extra-embryonic primitive endoderm and the trophectoderm tissues. Embryonic stem cells can reintegrate into embryogenesis but contribute primarily to epiblast lineages. Here, we show that mouse embryonic stem cells cultured under extended pluripotent conditions (EPSCs) can be partnered with trophoblast stem cells to self-organize into blastocyst-like structures with all three embryonic and extra-embryonic lineages. Morphogenetic and transcriptome profiling analyses reveal that these blastocyst-like structures show distinct embryonic-abembryonic axes and primitive endoderm differentiation and can initiate the transition from the pre- to post-implantation egg cylinder morphology in vitro. Mammalian development begins in the fertilized egg with potential to form all embryonic and extra-embryonic lineages. As development progresses, embryo cells lose potency to diversify into specialized cell types. In the mouse embryo, the earliest cell fate decisions are completed by the time of blastocyst formation, just before implantation. The blastocyst is defined by the cavity that emerges three and half days after fertilization (E3.5) and initially comprises two cell populations: the outer extra-embryonic trophectoderm (TE) and the pluripotent inner cell mass (ICM), positioned to one side of the cavity. The ICM gives rise to the epiblast (EPI), the source of fetal cell lineages, and the extra-embryonic primitive endoderm (PE) (Rossant and Tam, 2009Rossant J. Tam P.P. Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse.Development. 2009; 136: 701-713Crossref PubMed Scopus (449) Google Scholar). Following implantation, the blastocyst morphologically transforms into the so-called egg cylinder. This remodelling first requires specification of parietal endoderm (PaE), PE-derived cells that migrate from the PE epithelium along the mural TE encompassing the blastocyst cavity by E4.75 (Carnegie and Cabaca, 1991Carnegie J.A. Cabaca O. The influence of extracellular matrix components on the proliferation and migration of inner cell mass-derived parietal endodermal cells.Biol. Reprod. 1991; 45: 572-580Crossref PubMed Scopus (13) Google Scholar, Hagan, 1982Hagan J.H. Looking to the future.J. Mo. Dent. Assoc. 1982; 62: 10-15PubMed Google Scholar, Salamat et al., 1995Salamat M. Miosge N. Herken R. Development of Reichert's membrane in the early mouse embryo.Anat. Embryol. (Berl). 1995; 192: 275-281Crossref PubMed Scopus (35) Google Scholar). The initially amorphous EPI then transforms into a polarized cup-shaped epithelium and the polar TE, adjacent to the EPI, develops into flanking extra-embryonic ectoderm (ExE) (Bedzhov and Zernicka-Goetz, 2014Bedzhov I. Zernicka-Goetz M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation.Cell. 2014; 156: 1032-1044Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). By E5.5, both tissues become covered by visceral endoderm (VE), a second PE-derived tissue. Cultures of mouse embryonic stem cells (ESCs) and extra-embryonic trophoblast stem cells (TSCs) have been derived from the pre-implantation blastocyst and are transcriptionally equivalent to the EPI and TE lineages of the pre-implantation embryo (Evans and Kaufman, 1981Evans M.J. Kaufman M.H. Establishment in culture of pluripotential cells from mouse embryos.Nature. 1981; 292: 154-156Crossref PubMed Scopus (6429) Google Scholar, Tanaka et al., 1998Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Promotion of trophoblast stem cell proliferation by FGF4.Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1078) Google Scholar). Extra-embryonic endoderm (XEN) stem cells, also derived from blastocysts, show exclusive contribution to endoderm lineages in chimeric embryos (Kunath et al., 2005Kunath T. Arnaud D. Uy G.D. Okamoto I. Chureau C. Yamanaka Y. Heard E. Gardner R.L. Avner P. Rossant J. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.Development. 2005; 132: 1649-1661Crossref PubMed Scopus (294) Google Scholar). Although ESCs are derived from ICM, they lose ability to contribute to extra-embryonic tissues under conventional conditions (Ying et al., 2008Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523Crossref PubMed Scopus (2534) Google Scholar). However, recently protocols were established to generate pluripotent stem cells with both embryonic and extra-embryonic potential (Gao et al., 2019Gao X. Nowak-Imialek M. Chen X. Chen D. Herrmann D. Ruan D. Chen A.C.H. Eckersley-Maslin M.A. Ahmad S. Lee Y.L. et al.Establishment of porcine and human expanded potential stem cells.Nat. Cell Biol. 2019; 21: 687-699Crossref PubMed Scopus (161) Google Scholar, Yang et al., 2017aYang J. Ryan D.J. Wang W. Tsang J.C. Lan G. Masaki H. Gao X. Antunes L. Yu Y. Zhu Z. et al.Establishment of mouse expanded potential stem cells.Nature. 2017; 550: 393-397Crossref PubMed Scopus (148) Google Scholar, Yang et al., 2017bYang Y. Liu B. Xu J. Wang J. Wu J. Shi C. Xu Y. Dong J. Wang C. Lai W. et al.Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency.Cell. 2017; 169: 243-257.e25Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). The bi-potency of such extended pluripotent stem cells (EPSCs) was indicated by their contribution to both embryonic and extra-embryonic parts of chimeric conceptuses (Yang et al., 2017aYang J. Ryan D.J. Wang W. Tsang J.C. Lan G. Masaki H. Gao X. Antunes L. Yu Y. Zhu Z. et al.Establishment of mouse expanded potential stem cells.Nature. 2017; 550: 393-397Crossref PubMed Scopus (148) Google Scholar, Yang et al., 2017bYang Y. Liu B. Xu J. Wang J. Wu J. Shi C. Xu Y. Dong J. Wang C. Lai W. et al.Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency.Cell. 2017; 169: 243-257.e25Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Recent studies have shown that stem cells can self-assemble in vitro to generate embryo-like structures, offering new opportunities for understanding embryogenesis (Beccari et al., 2018Beccari L. Moris N. Girgin M. Turner D.A. Baillie-Johnson P. Cossy A.C. Lutolf M.P. Duboule D. Arias A.M. Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids.Nature. 2018; 562: 272-276Crossref PubMed Scopus (211) Google Scholar, Harrison et al., 2017Harrison S.E. Sozen B. Christodoulou N. Kyprianou C. Zernicka-Goetz M. Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.Science. 2017; 356: eaal1810Crossref PubMed Scopus (244) Google Scholar, Rivron et al., 2018Rivron N.C. Frias-Aldeguer J. Vrij E.J. Boisset J.C. Korving J. Vivié J. Truckenmüller R.K. van Oudenaarden A. van Blitterswijk C.A. Geijsen N. Blastocyst-like structures generated solely from stem cells.Nature. 2018; 557: 106-111Crossref PubMed Scopus (242) Google Scholar, Sozen et al., 2018Sozen B. Amadei G. Cox A. Wang R. Na E. Czukiewska S. Chappell L. Voet T. Michel G. Jing N. et al.Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.Nat. Cell Biol. 2018; 20: 979-989Crossref PubMed Scopus (160) Google Scholar, Zheng et al., 2019Zheng Y. Xue X. Yue S. Sicong W. Esfahani S.N. Li Z. Muncie J.M. Lakins J.N. Weaver V.M. Gumucio D.L. Fu J. Controlled modelling of human epiblast and amnion development using stem cells..Nature. 2019; 573: 421-425Crossref PubMed Scopus (198) Google Scholar, Warmflash et al., 2014Warmflash A. Sorre B. Etoc F. Siggia E.D. Brivanlou A.H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells..Nature Methods. 2014; 11: 847-854Crossref PubMed Google Scholar). We previously showed that mouse ESCs and TSCs can organize into bicompartmental embryo-like structures (ET-embryos) that resemble egg cylinders and recapitulate spatiotemporal patterns of mesodermal and primordial germ-cell gene expression (Harrison et al., 2017Harrison S.E. Sozen B. Christodoulou N. Kyprianou C. Zernicka-Goetz M. Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.Science. 2017; 356: eaal1810Crossref PubMed Scopus (244) Google Scholar). The additional incorporation of XEN cells generates ETX-embryos that undertake further anterior specification and early gastrulation (Sozen et al., 2018Sozen B. Amadei G. Cox A. Wang R. Na E. Czukiewska S. Chappell L. Voet T. Michel G. Jing N. et al.Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.Nat. Cell Biol. 2018; 20: 979-989Crossref PubMed Scopus (160) Google Scholar). However, these structures recapitulate early post-implantation development, circumventing the unique pre-implantation blastocyst stage. Recent studies described generation of blastocyst-like structures (termed blastoids or blastocyst-like cysts; iBCs) from stem cells (Kime et al., 2019Kime C. Kiyonari H. Ohtsuka S. Kohbayashi E. Asahi M. Yamanaka S. Takahashi M. Tomoda K. Induced 2C expression and implantation-competent blastocyst-like cysts from primed pluripotent stem cells.Stem Cell Rep. 2019; 13: 485-498Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Rivron et al., 2018Rivron N.C. Frias-Aldeguer J. Vrij E.J. Boisset J.C. Korving J. Vivié J. Truckenmüller R.K. van Oudenaarden A. van Blitterswijk C.A. Geijsen N. Blastocyst-like structures generated solely from stem cells.Nature. 2018; 557: 106-111Crossref PubMed Scopus (242) Google Scholar). It is reported that these structures developed EPI-like and TE-like lineages with a small proportion specifying PE-like cells, but their subsequent development was arrested (Kime et al., 2019Kime C. Kiyonari H. Ohtsuka S. Kohbayashi E. Asahi M. Yamanaka S. Takahashi M. Tomoda K. Induced 2C expression and implantation-competent blastocyst-like cysts from primed pluripotent stem cells.Stem Cell Rep. 2019; 13: 485-498Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Rivron et al., 2018Rivron N.C. Frias-Aldeguer J. Vrij E.J. Boisset J.C. Korving J. Vivié J. Truckenmüller R.K. van Oudenaarden A. van Blitterswijk C.A. Geijsen N. Blastocyst-like structures generated solely from stem cells.Nature. 2018; 557: 106-111Crossref PubMed Scopus (242) Google Scholar). Here, we tested the hypothesis that full specification of the PE-lineage is required to overcome this limited development by devising an approach to establish all three blastocyst lineages in blastocyst-like structures, and, testing their subsequent potential. The ability to generate embryo-like structures from cultured stem cells brings new potential for understanding mammalian development. We hypothesized that utilizing extended potential (EP) culture conditions to promote formation of both embryonic and extra-embryonic lineages (Yang et al., 2017bYang Y. Liu B. Xu J. Wang J. Wu J. Shi C. Xu Y. Dong J. Wang C. Lai W. et al.Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency.Cell. 2017; 169: 243-257.e25Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) could improve developmental potential of blastoids. With this aim, we combined mouse EPSCs and TSCs in a multi-inverted-pyramid microwell-based system using sequential changes of differing media. We optimized the first culture medium (LCDM; STAR Methods) to capture the bi-potency of EPSCs for embryonic and extra-embryonic lineages (Figures 1A and S1A; STAR Methods). Upon formation of compacted amorphous EPSC aggregates, we added TSCs to microwells and changed the medium to one previously described (Kubaczka et al., 2014Kubaczka C. Senner C. Araúzo-Bravo M.J. Sharma N. Kuckenberg P. Becker A. Zimmer A. Brüstle O. Peitz M. Hemberger M. et al.Derivation and maintenance of murine trophoblast stem cells under defined conditions.Stem Cell Rep. 2014; 2: 232-242Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Rivron et al., 2018Rivron N.C. Frias-Aldeguer J. Vrij E.J. Boisset J.C. Korving J. Vivié J. Truckenmüller R.K. van Oudenaarden A. van Blitterswijk C.A. Geijsen N. Blastocyst-like structures generated solely from stem cells.Nature. 2018; 557: 106-111Crossref PubMed Scopus (242) Google Scholar). As natural blastocyst development occurs under low oxygen tension, we switched from normoxic (20% O2) to hypoxic (5% O2) culture conditions upon addition of TSCs (Figures 1A and S1A; STAR Methods). This increased cystic structure formation from 2.5% to 15.17% (Figures S1A–S1C, n = 400). After 72-96 h of hypoxic culture, a cohesive TSC epithelium surrounded an enlarged cavity and an internal acentric EPSC compartment (Figures 1A, 1B, and S1A–S1C). Under these EP conditions, 61.2% (n = 142/233) of EPSC-derived structures developed a PE-like epithelium flanking the cavity (Figures 1C and S1D). By comparison, only 15.9% (n = 19/123) of conventional ESC-derived blastoids specified PE-like cells (Figure 1D). Under these conditions, we were unable to detect any contribution of EPSCs toward the TE lineage in such EPSC-derived blastocysts (Figures S1E and S1F) and could not observe synthetic blastocyst formation when EPSCs were cultured in the absence of TSCs (Figure S1G). The morphology, size, cell numbers, and lineage ratios in EPSC-derived blastocysts indicated a range of mid- to late blastocyst stages (Figures 1E–1G) having an average of 10.35 PE-like cells per structure (Figure S1H). We term these structures extended potential blastoids (EPS-blastoids). The TE and PE cells lining natural mammalian blastocyst cavities exhibit apicobasal polarity that is key for TE fate determination and for PE cells to sort into an epithelial layer (Eckert et al., 2004Eckert J.J. McCallum A. Mears A. Rumsby M.G. Cameron I.T. Fleming T.P. PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo.Reproduction. 2004; 127: 653-667Crossref PubMed Scopus (44) Google Scholar, Saiz et al., 2013Saiz N. Grabarek J.B. Sabherwal N. Papalopulu N. Plusa B. Atypical protein kinase C couples cell sorting with primitive endoderm maturation in the mouse blastocyst.Development. 2013; 140: 4311-4322Crossref PubMed Scopus (50) Google Scholar). Such polarity was evident from the apical positioning of atypical protein kinase C (aPKC) in both the TSC-layer and PE-like cells of EPS-blastoids (Figure 1H). Moreover, the distribution of actin and adherens junctions necessary to establish a barrier and accumulate fluid in the blastocoel (Sheth et al., 1997Sheth B. Fesenko I. Collins J.E. Moran B. Wild A.E. Anderson J.M. Fleming T.P. Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 alpha+ isoform.Development. 1997; 124: 2027-2037PubMed Google Scholar) was established correctly in EPS-blastoids (Figures 1I and 1J). Thus, we concluded that TSCs and EPSCs in the EPS-blastoids self-organize to resemble natural blastocysts. As specification of PE would be critical for subsequent blastoid development, we examined formation of apico-basally polarized epithelium, an important feature of PE maturation. We found that the PE-like cells in EPS-blastoids not only expressed apical aPKC (Figure 1H), but also produced Podocalyxin (Figure 2A), an anti-adhesive sialomucin later secreted into the apical lumen (Bedzhov and Zernicka-Goetz, 2014Bedzhov I. Zernicka-Goetz M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation.Cell. 2014; 156: 1032-1044Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, Meder et al., 2005Meder D. Shevchenko A. Simons K. Füllekrug J. Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells.J. Cell Biol. 2005; 168: 303-313Crossref PubMed Scopus (151) Google Scholar). Moreover, the PE-like cells also secreted the basement membrane protein, Laminin (Laurie et al., 1982Laurie G.W. Leblond C.P. Martin G.R. Localization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin to the basal lamina of basement membranes.J. Cell Biol. 1982; 95: 340-344Crossref PubMed Scopus (346) Google Scholar) at their basal domains (Figure 2B). These results indicate that maturation of PE-like cells resembles the formation of bona-fide PE epithelium. We next sought to further compare how culture conditions influence stem cell pluripotency. Conventional self-renewal culture conditions, in the presence of GSK3B and MEK inhibitors and the cytokine leukemia inhibitory factor (called 2iLif), reduce intercellular heterogeneity maintaining ESCs in a naive pluripotent state (Ying et al., 2008Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523Crossref PubMed Scopus (2534) Google Scholar). Upon 2i withdrawal, ESCs change from naive to primed pluripotency, followed by the onset of differentiation (Shahbazi et al., 2017Shahbazi M.N. Scialdone A. Skorupska N. Weberling A. Recher G. Zhu M. Jedrusik A. Devito L.G. Noli L. Macaulay I.C. et al.Pluripotent state transitions coordinate morphogenesis in mouse and human embryos.Nature. 2017; 552: 239-243Crossref PubMed Scopus (129) Google Scholar, Ying et al., 2008Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523Crossref PubMed Scopus (2534) Google Scholar). To test the efficacy of stem cell-types and culture conditions in specifying PE-like cells, we compared aggregates generated, using ESCs or EPSCs, after 2i withdrawal or under EP conditions (Figure 2C). Only 13.31% of ESC aggregates expressed PE markers, PDGFRa and Sox17, 24 h after 2i withdrawal, rising to 18.18% after 48 h (Figures 2C and 2D). The efficiency of PE-like cell formation by ESCs could be increased to 24.77% after 24 h and 38.10% after 48 h by culturing them in EP conditions, suggesting partial conversion to an EP state (Figures 2C and 2D). By contrast, 60.19% EPSC aggregates specified PE-like cells after 24 h, rising to 65.14% after 48 h under their EP conditions (Figures 2C and 2D). The efficiency of PE-like cell formation by EPSCs decreased to 18.79% after 24 h or 24.71% after 48 h culture in Lif conditions, suggesting exit from the EP state (Figures 2C and 2D). These results led us to conclude that the ability to generate PE-like epithelium depends upon the potency of the stem cells influenced by culture conditions. To analyze the developmental potential of ESCs and EPSCs, we dissociated ES- or EPS-blastoids after 96 h of culture for single-cell transcriptome analysis (Figure S2A; see STAR Methods). Single cell clustering revealed three distinct cell populations representing the three blastocyst lineages within both blastoid groups (Figures S2A and S2B) but the abundance of cells able to specify an endoderm-like transcriptome was 1.7-fold higher in EPS-blastoids than ES-blastoids (6.4% versus 10.6%; Figure S2C). To identify the differences between the EPS-blastoids and E4.5 blastocysts, we first identified the differentially expressed genes (DEGs) between each lineage in pairwise combinations (Figure S2D), yielding 86 DEGs for the TE lineage, 63 DEGs for the Epi lineage and 41 DEGs for the PE lineage. To further show the general underlying differences, a Gene Ontology analysis (see STAR Methods) across all lineages was performed and identified an enrichment in functional terms linked to apoptotic process in the EPS-blastoid and an enrichment in methylation and metabolic processes in the E4.5 blastocysts (Figures S2D and S2E). A phylogenetic tree analysis and clusters in the UMAP dimensional reduction plots further illustrated close proximity between the EPS-blastoids and E4.5 blastocysts (Figures S2F and S2G). To determine whether endoderm identity in our synthetic platform captured the pre-implantation PE or post-implantation definitive endoderm (DE) lineage identity, we compared our endoderm transcriptomes with published dataset from early blastocyst to midgestation (E3.5–E8.75) stages (Nowotschin et al., 2019Nowotschin S. Setty M. Kuo Y.Y. Liu V. Garg V. Sharma R. Simon C.S. Saiz N. Gardner R. Boutet S.C. et al.The emergent landscape of the mouse gut endoderm at single-cell resolution.Nature. 2019; 569: 361-367Crossref PubMed Scopus (136) Google Scholar). Our analyses indicated that the PE-like cells from blastoids co-localized with PE cells from the E4.5 blastocyst but neither with cells from E3.5 blastocysts nor DE cells from E7.5 post-implantation embryos (Figures 2E–2G and S3A–S3C), indicating that endoderm-like cells resemble PE from the late blastocyst. Although PE identity was seemingly captured in both ES- and EPS-blastoids, we identified differences in expression levels of core transcription factors that define PE lineage specification (Figures 2H and 2I). Specifically, PE-like cells from EPS-blastoids expressed higher proportions of Gata6, Sox7, Gata4, Pdgfra, Marcks, Gpt2 than PE-like cells of ES-blastoids (Figures 2H, 2I, and S4A–S4C). Of these, the PE cell fate determinant Gata6 showed the most robustly elevated differential expression in EPS-blastoids (Figures 2J and S4C). Gata6 is at the head of a hierarchy of genes regulating PE development (Hermitte and Chazaud, 2014Hermitte S. Chazaud C. Primitive endoderm differentiation: from specification to epithelium formation.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014; 369: 20130537Crossref PubMed Scopus (27) Google Scholar) and its expression in blastoids correlated with this hierarchy (Figure 2K). These results indicate that gene expression profiles of EPS-blastoids bear the hallmarks of more successful PE generation than conventional ES-blastoids. After specification of three blastocyst lineages, the PE-epithelium generates two further derivatives, PaE and VE, which are important for blastocyst remodelling at implantation (Figure 3A). In EPS-blastoids, we observed Gata6-expressing PaE-like cells detached from the ICM and extending over TE-like cells of the cavity from which they were separated by basement membrane (Figures 3B and 3C). This resembles the reorganization of newly specified PaE cells of E4.75 blastocysts (Figures 3B–3D). Our single-cell transcriptomic analyses revealed a small proportion of EPS-blastoid cells that expressed Snail (21.95% of total), Folistatin (4.06% of total), Vimentin (4.87% of total), Grem2 (4.87% of total), Stra6 (2.44% of total), and Zeb1 (2.44% of total) (Figures 3E and 3F); genes associated with PaE specification (Lehtonen et al., 1983Lehtonen E. Lehto V.P. Paasivuo R. Virtanen I. Parietal and visceral endoderm differ in their expression of intermediate filaments.EMBO J. 1983; 2: 1023-1028Crossref PubMed Scopus (67) Google Scholar). This contrasts with ES-blastoids, which did not exhibit features of PaE formation (Figure 3G). Together, these results indicate that the EPSC-compartment can generate cells that morphologically and transcriptionally resemble the PE of the E4.5 blastocyst and that can initiate generation of PaE, which is critical for the pre- to post-implantation developmental transition. At the conclusion of pre-implantation development, an embryonic-abembryonic (em-ab.em) axis becomes established. Polar TE, which covers the ICM at the embryonic pole, displays high self-renewal capacity; elevated expression of core TSC transcription factors; and is fated to form ExE following implantation. Mural TE, which encloses the blastocoel at the abembryonic pole, differentiates as it initiates invasion during implantation (Latos et al., 2015Latos P.A. Sienerth A.R. Murray A. Senner C.E. Muto M. Ikawa M. Oxley D. Burge S. Cox B.J. Hemberger M. Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.Genes Dev. 2015; 29: 2435-2448Crossref PubMed Scopus (63) Google Scholar). We found that from around 96 h, EPS-blastoids showed a gradient of expression of the core self-renewal transcription factor, Cdx2, between the embryonic and abembryonic poles (Figures 4A–4C ), resembling the Cdx2 pattern in blastocysts at em-ab.em axis formation (Figures 4D–4F). Similarly, the reverse gradient of Tfap2c along the em-ab.em axis (Figures 4B–4F) and the expression of Krt8, a marker for differentiating TE, in the abembryonic part indicated the similarity of EPS-blastoids to blastocysts in the onset of mural TE differentiation (Figure 4G). To validate these observations, we analyzed TE transcriptomes from single cells of blastoids at 96 h sorted using Cdx2 and Gata2 as respective polar and mural TE markers (Nakamura et al., 2015Nakamura T. Yabuta Y. Okamoto I. Aramaki S. Yokobayashi S. Kurimoto K. Sekiguchi K. Nakagawa M. Yamamoto T. Saitou M. SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression.Nucleic Acids Res. 2015; 43: e60Crossref PubMed Scopus (81) Google Scholar). This analysis revealed the TSC cluster to be polarized into distinct populations having either Cdx2-high and Gata2-low or Cdx2-low and Gata2-high gene signatures (Figures 4H and 4I; see STAR Methods). Louvain clustering (Waltman and Eck, 2013Waltman L. Eck N.J.v. A smart local moving algorithm for large-scale modularity-based community detection.Eur. Phys. J. B. 2013; 86: 471Crossref Scopus (485) Google Scholar) delineated 3 distinct subpopulations within the TSC-derived epithelium (Figure 4J). Cells in cluster 1 (Figure 4J; yellow), showed expression of self-renewal and mitotic cell cycle transcription factors, including Essrb, Eomes, Utf1, Ddah1, Pou3f1, Cpne3, markers of the polar TE (Figure 4K). The opposing sub-cluster (Figure 4J; Cluster 3, purple) contained cells expressing high levels of terminal differentiation markers including Gata2, Gjb2, Peg3 and Peg10 (Figure 4K). The mid sub-cluster (Figure 4J; Cluster 2, green) showed an intermediate expression profile, with upregulation of early differentiation genes, Tfap2c, Krt7, Krt18, Serpine1, Rhox6, Rhox9 (Figure 4K). These results suggest that cells exhibit a progressive exit from stem-cell (polar) identity toward a differentiated (mural) state characterizing establishment of the em-ab.em axis in EPS-blastoids. These features are likely to reflect segregation of differential cellular identity from an initially multipotent TE lineage as during natural embryogenesis. We next tested whether EPS-blastoids would undertake cell rearrangements that characterize post-implantation morphogenesis. We found that, depending upon culture conditions, between 5% and 20% of EPS-blastoids became reorganized into elongated, egg cylinder-like structures, 83.5% of which had abutting EPI- and ExE-like compartments positioned along a proximal-distal axis and enveloped by a VE-like cell layer (Figures 5A, 5B, S5A, and S5B). This pattern of organization was similar to natural embryos and ETX embryos comprising ESCs, TSCs, XEN cells (Sozen et al., 2018Sozen B. Amadei G. Cox A. Wang R. Na E. Czukiewska S. Chappell L. Voet T. Michel G. Jing N. et al.Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.Nat. Cell Biol. 2018; 20: 979-989Crossref PubMed Scopus (160) Google Scholar; Figures 5C and 5D). In contrast, none of the ES-blastoids developed in this way; 91.7% failed to develop at all over 27 h (Figure S5C) and 8.3% formed non-polarized ESC-TSC aggregates with no distinguishable VE-layer after 48 h (Figure S5C). To analyze formation of the respective EPI and VE components of the egg cylinder, we filmed the development of EPS-blastoids on optical-grade plastic under IVC conditions. We found that PE-like cells segregated from other EPSCs and formed an epithelium by 24 h, which surrounded the cylindrical structure by 48 h (Figures S5D and S5E). The PE-derived VE-like layer of these post-implantation stage structures deposited the basement membrane, which is required for EPI cells to polarize and generate the pro-amniotic cavity (Figure 5E; Bedzhov and Zernicka-Goetz, 2014Bedzhov I. Zernicka-Goetz M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation.Cell. 2014; 156: 1032-1044Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, Christodoulou et al., 2018Christod" @default.
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• W2996475925 date "2019-12-01" @default.
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• W2996475925 title "Self-Organization of Mouse Stem Cells into an Extended Potential Blastoid" @default.
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• W2996475925 doi "https://doi.org/10.1016/j.devcel.2019.11.014" @default.
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