Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial–mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo’s architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial–mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Bedzhov, I. & Zernicka-Goetz, M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell 156, 1032–1044 (2014).
Shahbazi, M. N. et al. Pluripotent state transitions coordinate morphogenesis in mouse and human embryos. Nature 552, 239–243 (2017).
Harrison, S. E., Sozen, B., Christodoulou, N., Kyprianou, C. & Zernicka-Goetz, M. Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro. Science 356, eaal1810 (2017).
Rivera-Perez, J. A. & Magnuson, T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3. Dev. Biol. 288, 363–371(2005).
Robertson, E. J. Dose-dependent Nodal/Smad signals pattern the early mouse embryo. Semin. Cell Dev. Biol. 32, 73–79 (2014).
Wood, S. A., Allen, N. D., Rossant, J., Auerbach, A. & Nagy, A. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365, 87–89 (1993).
Bradley, A., Evans, M., Kaufman, M. H. & Robertson, E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984).
ten Berge, D. et al. Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3, 508–518 (2008).
van den Brink, S. C. et al. Symmetry breaking, germ layer specification and axial organization in aggregates of mouse embryonic stem cells. Development 141, 4231–4242 (2014).
Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847–854 (2014).
Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A. & Rossant, J. Promotion of trophoblast stem cell proliferation by FGF4. Science 282, 2072–2075 (1998).
Kimura-Yoshida, C. et al. Canonical Wnt signaling and its antagonist regulate anterior–posterior axis polarisation by guiding cell migration in mouse visceral endoderm. Dev. Cell 9, 639–650 (2005).
Yamamoto, M. et al. Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo. Nature 428, 387–392 (2004).
Tam, P. P., & Beddington, R. S. Establishment and organization of germ layers in the gastrulating mouse embryo. Ciba Found. Symp. 165, 27–42 (1992).
Viotti, M., Nowotschin, S. & Hadjantonakis, A. K. Afp::mCherry, a red fluorescent transgenic reporter of the mouse visceral endoderm. Genesis 49, 124–133 (2011).
Kunath, T. et al. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132, 1649–1661 (2005).
Takaoka, K., Yamamoto, M. & Hamada, H. Origin and role of distal visceral endoderm, a group of cells that determines anterior-posterior polarity of the mouse embryo. Nat. Cell Biol. 13, 743–752 (2011).
Papanayotou, C. et al. A novel nodal enhancer dependent on pluripotency factors and smad2/3 signaling conditions a regulatory switch during epiblast maturation. PLoS Biol. 12, e1001890 (2014).
Hoshino, H., Shioi, G. & Aizawa, S. AVE protein expression and visceral endoderm cell behavior during anterior-posterior axis formation in mouse embryos: asymmetry in OTX2 and DKK1 expression. Dev. Biol. 402, 175–191 (2015).
Williams, M., Burdsal, C., Periasamy, A., Lewandoski, M. & Sutherland, A. Mouse primitive streak forms in situ by initiation of epithelial to mesenchymal transition without migration of a cell population. Dev. Dyn. 241, 270–283 (2012).
Laurie, 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. 95, 340–344 (1982).
Sasaki, H. & Hogan, B. L. Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118, 47–59 (1993).
Lewis, S. L. & Tam, P. P. Definitive endoderm of the mouse embryo: formation, cell fates, and morphogenetic function. Dev. Dyn. 235, 2315–2329 (2006).
Burtscher, I. & Lickert, H. Foxa2 regulates polarity and epithelialization in the endoderm germ layer of the mouse embryo. Development 136, 1029–1038 (2009).
Balmer, S., Nowotschin, S. & Hadjantonakis, A. K. Notochord morphogenesis in mice: current understanding & open questions. Dev. Dyn. 245, 547–557 (2016).
Saitou, N. & Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425 (1987).
Peng, G. et al. Spatial transcriptome for the molecular annotation of lineage fates and cell identity in mid-gastrula mouse embryo. Dev. Cell 36, 681–697 (2016).
Bedzhov, I., Leung, C. Y., Bialecka, M. & Zernicka-Goetz, M. In vitro culture of mouse blastocysts beyond the implantation stages. Nat. Protoc. 9, 2732–2739 (2014).
Fehling, H. J. et al. Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217–4227 (2003).
Rhee, J. M. et al. In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice. Genesis 44, 202–218 (2006).
Sozen, B., Amadei, G., Na, E., Michel, G. & Zernicka-Goetz, M. Stem cells reconstituting gastrulating embryo-like structures in vitro. Nat. Protoc. Exch. https://doi.org/10.1038/protex.2018.072 (2018).
Piette, D., Hendrickx, M., Willems, E., Kemp, C. R. & Leyns, L. An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies. Nat. Protoc. 3, 1194–1201 (2008).
Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat. Methods 10, 1096–1098 (2013).
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Andrews, S. FastQC a Quality Control to Tool for High Throughput Sequence Data https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (2010).
Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA–Seq. Bioinformatics 25, 1105–1111 (2009).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Hong, F. et al. RankProd: a bioconductor package for detecting differentially expressed genes in meta-analysis. Bioinformatics 22, 2825–2827 (2006).
Saldanha, A. J. Java Treeview—extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).
Huang da, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
The authors thank colleagues in the M.Z.G. laboratory for insightful comments. The M.Z.G. laboratory is supported by grants from the European Research Council (669198) and the Wellcome Trust (098287/Z/12/Z). B.S. is also supported by Akdeniz University, Turkey. T.V. and L.C. are funded by Wellcome. T.V. is also funded by the University of Leuven, Belgium (PFV/10/016). The authors thank A. Weberling, M. Mole, N. Christodoulou, C. Kyprianou and J. Guo for their help, A. Hupalowska for inspiration for a model in Fig. 7f, and L. Wittler, I. Urban, A. Landsberger, C. Schick and H. Schlenger for technical support.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a. Confocal images of ETX embryos developed from fluorescence reporter cell lines at indicated time intervals: EGFP XEN cells, green; mTmG ES cells, red; wild-type TS cells. At day 2 of a typical experiment around 75% (23/31) of ETX embryos have an outer layer of XEN cells on ES cell side. Each image represents a different ETX embryo. Bar=20μm. b. Still images of time-lapse recording during development of a single ETX embryo. Lineages were pseudocoloured below to help visualization. XEN layer, green; ES cells, red; TS cells, blue. Time-lapse images captured at 45 min intervals. Representative of 3 separate time-lapse movies of 10 ETX embryos. Bar=20μm.
a. Induction of T/Brachyury in a representative ETX embryo at day 4.25 (top); localised T/Brachyury in a representative ETX embryo at day 5 (middle) and in E6.5 embryo (bottom). Yellow arrowheads indicate induction (top) or asymmetric T/Brachyury expression throughout posterior (middle and bottom). White boxes indicate zoomed inset. Purple dashed lines outline TS-derived extra-embryonic compartment or ExE for clarity. Yellow dashed line outline embryonic/extra-embryonic boundary. Non-nuclear anti-T/Brachyury VE fluorescence is non-specific staining. Representative of 43 ETX embryos, 4 experiments; 20 E6.5 embryos, 2 experiments. Bar=20μm. b. Still images from time-lapse recording of development of ETX embryo derived from T:GFP reporter ES cells. Time-lapse images captured at 45 min intervals. Representative of 3 separate time-lapse movies of 3 ETX embryos. Bar=20μm. Bottom row shows intensity gradient for T:GFP signal. L, low; H, high. c. Single planes of ET embryo (top left) and ETX embryo (top middle) 8 hours after onset of T/Brachyury expression, and E6.75 natural embryo (top right). Arrowheads indicate side of T/Brachyury expression. White dashed lines outline TS-derived extra-embryonic compartment or ExE for clarity. Middle row shows reconstruction of the embryonic compartment overlaid over DAPI; red dots indicate Oct4-positive and green dots indicate Oct4 and T/Brachyury double positive cells. Bottom panels show reconstruction of embryonic compartment alone. Representative of 4 ET embryos, 4 ETX embryos and 3 natural embryos; each from separate experiments. Bar=20μm. d. Mean ratio of T/Brachyury expressing cells to total cells in embryonic compartment. One-way ANOVA, n=4 ET embryos, n=4 ETX embryos and n=3 natural embryos. Columns are means ± SEM. Non-nuclear anti-T/Brachyury/Oct4 VE fluorescence is non-specific staining. e. Majority of EX (ES + XEN cells only) structures (92.3%) at day 4 and 5 under the same culture conditions did not express T/Brachyury; remaining (7.6%) displayed non-regionalised expression of T/Brachyury. Representative of 20 ETX embryos per time point; 3 experiments. Bar=20μm. f. Proportion of ETX embryos expressing T/Brachyury at day 5 is significantly higher than EX structures at day 5. Two-sided Student’s t-test, n=4 experiments for ETX embryos, n=3 experiments for EX structures. Each dot represents the percentage of positive or negative structures for T:GFP expression scored from each separate independent experiment. The number of structures scored in each independent experiment to calculate the percentage are reported in supplementary table 3 (101 ETX embryos and 43 EX structures scored in total). Columns are means ± SEM. g. Proportion of T/Brachyury expressing ETX embryos or EX structures with asymmetric T/Brachyury expression. Two-sided Student’s t-test. Columns are means ± SEM. n=4 experiments for ETX embryos, n=3 experiments for EX structures. The number of structures scored in each independent experiment to calculate the percentage are reported in supplementary table 3 (101 ETX embryos and 43 EX structures scored in total). h. ETX embryo at day 6. Boxed area on maximum projected images shows ROI with subset of triple positive cells for T/Brachyury, Oct4 and AP-2γ next to embryonic/extra-embryonic boundary (yellow dashed lines). Single orthogonal YZ plane and rotated X axis views provided to visualise expression of PGC markers on the boundary. 38%, 10/26 ETX embryos, 2 experiments i. RT-qPCR analysis of candidate PGC specification genes performed on T:GFP positive and negative cells from day 6 ETX embryo. Two-sided Student’s t-test, n=7 biological replicates. Columns are means ± SD. j. Day 5 ETX embryo revealing reciprocal gradients of Oct4 and T/Brachyury, both pseudocoloured with “fire” lookup table in Fiji to show expression levels. White dashed lines outline the TS-derived extra-embryonic compartment for clarity. L, low; H, high. Representative of 6 ETX embryos, 3 experiments. Bar=20μm.
a. Maximum projections from time points indicated showing formation of basement membrane during ETX embryo development. Right-most single-plane images: magnified middle plane of ETX embryo at 108h. Bar=20μm; Representative of 6 ETX embryos for each time point. b. ETX embryo after 3, 3.5, 4 and 5 days showing progression of cavitation. White dashed lines outline the cavity; purple dashed lines outline rosette; boxes, region of magnified inset. Bar=20μm; Representative of 20 ETX embryos for each time point, 4 experiments. c. Progressive formation of the pro-amniotic cavity during ETX embryo development. White boxes indicate magnified inset showing polarised Podxl. Bar=20μm; Representative of 6 ETX embryos for each time point, 2 experiments. d. Quantification of cavities in respective ES and TS-compartments of ETX embryos at days 3, 4 and 5. n=20 ETX embryos per time point. e. ETX embryos during cavitation showing laminin break-down between embryonic and extra-embryonic compartments. Yellow boxes, region of magnified inset. Yellow or black arrowheads indicate break in laminin. Lower panels have laminin staining inverted for better contrast. Black boxes indicate region of zoomed inset. Representative of 20 ETX embryos, 2 experiments. Bar=20μm. f. Quantification of laminin break-down at different developmental time points of ETX embryo embryogenesis. n=20 ETX embryos per time point, 2 experiments.
a. Images showing the same ETX embryos presented in Fig.3a-b with intensity profiles for Nodal HBE-YFP fluorescence. Insets show intensity of YFP signal in embryonic compartment. White boxes indicate area selected for intensity measurement (right-most graphs). Orange line indicates midline of the structure. Surface plot graphs (middle) show intensity of YFP. Bar=20μm. b. Proportion of structures expressing either asymmetric or symmetric Nodal at day 4 versus day 5. n=20 structures per group, 3 experiments. c. Quantitative assessment of endogenous Nodal HBE-YFP asymmetric fluorescence intensity in representative ETX embryos at day 4 (left) and day 5 (right) presented in (a). Each dot represents a cell. Mean intensity was calculated for cells in the region left or right of the midline. Two-sided Student’s t-test, n=83 (left of the midline), n=81 (right of the midline) cells in day 4 ETX embryos; n=153 (left of the midline), n=159 (right of the midline) cells in day 5 ETX embryos. Means ± SD. d. Whole mount in situ hybridization revealing Cerberus (3 embryos in 3 experiments; 8 ETX embryos in 3 experiments), Nodal (4 embryos in 2 experiments; 10 ETX embryos in 3 experiments), T/Brachyury (3 embryos in 2 experiments; 17 ETX embryos in 3 experiments), Cripto (7 embryos in 2 experiments; 13 ETX embryos in 2 experiments), Wnt3 (9 embryos in 3 experiments; 10 ETX embryos in 3 experiments) and Bmp4 (8 embryos in 2 experiments; 14 ETX embryos in 2 experiments) transcripts in natural embryos and ETX embryos at indicated time points. Bar=50μm.
a. ETX embryos at day 5; white box indicates magnified field showing nuclei of T/Brachyury-positive cells changing from a plane perpendicular (asterisks) to parallel (arrowheads) to the basal membrane of the ES-derived compartment. Representative of 6 ETX embryos, 2 experiments. Bar=20μm. Bar in zoomed image=10μm. b. Still images from time-lapse movie of live ETX embryo built from CAG:GFP ES cells presented in Fig. 4d. White dashed lines outline XEN layer; purple dashed lines, embryonic/extra-embryonic boundary. White arrowhead indicates change in cell shape on the boundary of prospective posterior side. Representative of 3 separate time-lapse movies of 7 ETX embryos. c. Transverse sections from embryonic compartments of E6.75 embryo (top) (3 embryos) and day 5 ETX embryos (bottom) (3 ETX embryos). E-cadherin immunostaining reveals change in orientation of T/Brachyury-expressing mesenchymal cells (white arrowheads). 3 experiments. Bar=20μm. d. Immunostaining of E6.75 embryo (top) and day 5 ETX embryo (bottom) to reveal phosphorylated Histone 3 (H3S10-P) (magenta) – there is no increase in mitotic cells in the mesoderm, white boxes. ETX embryo presented in Fig. 4f re-stained for T/Brachyury in the same channel as GM130. Non-nuclear anti-T/Brachyury VE/XEN fluorescence is non-specific. Proportion of H3S10-P positive cells within and outside boxed region. LE, lateral epiblast; S, streak; R1, region 1; R2, region 2. n=3 E6.75 embryos; n=6 ETX embryos. Two-sided Student’s t-test. Means ± SD. Bar=20μm e. Day 5 ETX embryo and E6.75 embryo immunostained for N-cadherin (cyan) and T/Brachyury (magenta). Magnified images below show up-regulated N-cadherin in re-oriented T/Brachyury expressing cells that identify mesoderm formation (white arrowheads). Non-nuclear anti-T/Brachyury VE fluorescence is non-specific. Representative of 4 E6.75 embryos; 3 ETX embryos. Bar on the zoomed images=5μm f. XZ sectioned orthogonal views from the ES-derived embryonic compartment of ETX embryo also presented in Fig. 4h demonstrating laminin break-down on the T/Brachyury expressing side. Yellow arrows indicate break in laminin. g. Oblique section of an ETX embryo at day 5 showing break in laminin in T/Brachyury expressing posterior domain. Dashed lines outline the TS-derived extra-embryonic compartment. Representative of 3 ETX embryos, 2 experiments. h. Quantification of ETX embryos expressing T/Brachyury with and without DKK1 treatment (200ng/ml) for 24h presented as a bar chart. Contingency table used to perform statistical test. Two-sided Fisher’s exact test, total of 20 structures scored per group from n=2 separate experiments. The number of structures scored in each independent experiment is reported in Supplementary Table 3.
Foxa2 expression within the axial mesoderm region of natural (in vivo; 10), in vitro cultured embryos (IVC; 3), or ETX embryos (10). E7.0 natural (top) and day 6 ETX embryo (bottom) shown in a; embryo cultured in vitro from E5.25 for 48h (top) and day 6 ETX embryo (bottom) shown in b. White boxes indicate magnified region to show Foxa2 positive cells. White dashed lines outline the axial mesoderm region; purple dashed lines, ExE or TS-derived extra-embryonic compartment, 3 experiments. Bar=20μm. Bar in zoomed images=10μm.
Supplementary Figure 7 Transcriptional profiling of ETX embryos reveals global similarity of anterior-posterior patterning to natural embryos.
a, b. Average expression level of differentially expressed genes in side 1 (prospective anterior) and side 2 (prospective posterior) of ETX embryos. Box plot elements represent differentially expressed genes (DEGs) between ETX-Side 1 and ETX-Side 2, n=499 DEGs for side 1 shown in a, n=239 DEGs for side 2 shown in b. Middle line shows the median value. DEGs were identified from 3 biological replicates divided into side1 and side2. DEGs were identified using RankProd statistical test with P value <0.05 and fold change >1.5. c. Left: Gene expression heatmap from each side of the ETX embryos. Red, high gene expression; green, low gene expression. Right: Gene Ontology (GO) analysis on combined Side 1 samples (top box) and combined Side 2 samples (bottom box) illustrating significantly enriched terms. n=3 biological replicates, divided in side 1 and side 2. RankProd (p < 0.05, fc > 1.5) used to identify the differentially expressed genes. Functional enrichment of DEGs was performed using DAVID v6.8 (see method). d, e. Gene Ontology analysis highlighting developmental categories detected in side 1 and side 2 of ETX embryos. n=3 biological replicates, divided in side 1 and side 2. Functional enrichment of DEGs was performed using DAVID v6.8, then the P values were transformed to logarithmic space by using -log10(P value), then using R to draw the bar plot that indicates the significance of the biological processes (see method).
Supplementary Figures 1–7, Supplementary Table and Supplementary Video legends.
Antibodies used in this study.
qPCR primers used in this study.
Statistic source data.
Time-lapse recording of an ETX embryo from day 3.5 to 5.0 in culture.
Cell tracking in an ETX embryo.
About this article
Cite this article
Sozen, B., Amadei, G., Cox, A. et al. Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures. Nat Cell Biol 20, 979–989 (2018). https://doi.org/10.1038/s41556-018-0147-7
Cell Proliferation (2020)
Philosophical Transactions of the Royal Society B: Biological Sciences (2020)
National human embryo and embryoid research policies: a survey of 22 top research-intensive countries
Regenerative Medicine (2020)
Current Opinion in Biomedical Engineering (2020)
Developmental Biology (2020)