Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

In vitro generation of mouse polarized embryo-like structures from embryonic and trophoblast stem cells


Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and arrangement of tissues of the early embryo. Here, we describe a protocol to generate the first embryo-like structures by directing the assembly of mouse ESCs and extra-embryonic trophoblast stem cells (TSCs) in a 3D extracellular matrix (ECM) into structures we call ‘polarized embryo-like structures’. By establishing the medium and culture conditions needed to support the growth of both stem cell types simultaneously, embryonic architecture is generated within 4 d of co-culture. This protocol can be performed by those proficient in standard ESC culture techniques and can be used in developmental studies to investigate the interactions between embryonic and extra-embryonic tissues during mammalian development.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Generation of polarized embryo-like system structures  from embryonic and extra-embryonic stem cells to form ETS embryos.
Fig. 2: Molecular markers indicating the lineage identity of the cell types in the embryonic and extra-embryonic compartments of polarized embryo-like structures (ETS embryos).


  1. 1.

    Boroviak, T., Loos, R., Bertone, P., Smith, A. & Nichols, J. The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification. Nat. Cell Biol. 16, 516–28 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Ying, Q.-L. & Smith, A. G. Defined conditions for neural commitment and differentiation. Methods Enzymol. 365, 327–341 (2003).

    CAS  Article  Google Scholar 

  3. 3.

    Murry, C. E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008).

    CAS  Article  Google Scholar 

  4. 4.

    Irion, S., Nostro, M. C., Kattman, S. J. & Keller, G. M. Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications. Cold Spring Harb. Symp. Quant. Biol. 73, 101–110 (2008).

    CAS  Article  Google Scholar 

  5. 5.

    Hattori, N. Cerebral organoids model human brain development and microcephaly. Mov. Disord. 29, 185–185 (2014).

    Article  Google Scholar 

  6. 6.

    Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–6 (2011).

    CAS  Article  Google Scholar 

  7. 7.

    Xia, Y. et al. The generation of kidney organoids by differentiation of human pluripotent cells to ureteric bud progenitor-like cells. Nat. Protoc. 9, 2693–704 (2014).

    Article  Google Scholar 

  8. 8.

    Takasato, M. et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat. Cell Biol. 16, 118–26 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Takasato, M., Er, P. X., Chiu, H. S. & Little, M. H. Generation of kidney organoids from human pluripotent stem cells. Nat. Protoc. 11, 1681–1692 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Meinhardt, A. et al. 3D reconstitution of the patterned neural tube from embryonic stem cells. Stem Cell Rep. 3, 1–13 (2014).

    Article  Google Scholar 

  11. 11.

    ten Berge, D. et al. Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3, 508–18 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    van den Brink, S. C. et al. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development 141, 4231–42 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Bedzhov, I. & Zernicka-Goetz, M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell 156, 1032–44 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Arnold, S. J. & Robertson, E. J. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat. Rev. Mol. Cell Biol. 10, 91–103 (2009).

    CAS  Article  Google Scholar 

  16. 16.

    Tanaka, S., Kunath, T., Hadjantonakis, A., Nagy, A. & Rossant, J. Promotion of trophoblast stem cell proliferation by FGF4. Science 282, 2072–2075 (1998).

    CAS  Article  Google Scholar 

  17. 17.

    Harrison, S. E., Sozen, B., Christodoulou, N., Kyprianou, C. & Zernicka-goetz, M. Assembly of embryonic and extra-embryonic stem cells to mimic embryogenesis in vitro. Science (2017).

    Article  CAS  Google Scholar 

  18. 18.

    Wilkinson, D. G., Bhatt, S. & Herrmann, B. G. Expression of the mouse T gene and its role in mesoderm formation. Nature 343, 657–659 (1990).

    CAS  Article  Google Scholar 

  19. 19.

    Scialdone, A. et al. Resolving early mesoderm diversification through single-cell expression profiling. Nature 535, 289–293 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    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).

    CAS  Article  Google Scholar 

  21. 21.

    Winnier, G., Blessing, M., Labosky, P. A. & Hogan, B. L. M. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105–2116 (1995).

    CAS  Article  Google Scholar 

  22. 22.

    Lawson, K. A. et al. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424–436 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Kreuter, J. Nanoparticles and microparticles for drug and vaccine delivery. J. Anat. 189, 503–505 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Kunath, T. et al. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132, 1649–1661 (2005).

    CAS  Article  Google Scholar 

  25. 25.

    Brown, K. et al. Extraembryonic endoderm (XEN) stem cells producefactors that activate heart formation. PLoS ONE 5, e13446 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Paca, A. et al. BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm. Dev. Biol. 361, 90–102 (2012).

    CAS  Article  Google Scholar 

  27. 27.

    Artus, J. et al. BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity. Dev. Biol. 361, 245–262 (2012).

    CAS  Article  Google Scholar 

  28. 28.

    Ying, Q. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–24 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Morris, Sa et al. Dynamics of anterior-posterior axis formation in the developing mouse embryo. Nat. Commun. 3, 673 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Fehling, H. J. et al. Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217–4227 (2003).

    CAS  Article  Google Scholar 

  32. 32.

    Payer, B. et al. Generation of stella-GFP transgenic mice: a novel tool to study germ cell development. Genesis 44, 75–83 (2006).

    CAS  Article  Google Scholar 

  33. 33.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Behringer, R., Gertsenstein, M., Nagy, K. V. & Nagy, A. Manipulating the Mouse Embryo: A Laboratory Manual 4th edn. (Cold Spring Harbor Laboratory Press, 2014).

  35. 35.

    Lee, G. Y., Kenny, Pa, Lee, E. H. & Bissell, M. J. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4, 359–65 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Preparing feeder cell layers from STO or mouse embryo fibroblast (MEF) cells: treatment with mitomycin C. CSH Protoc. (2006).

    Article  Google Scholar 

  37. 37.

    Tanaka, S. Derivation and culture of mouse trophoblast stem cells in vitro. Methods Mol. Biol. 329, 35–44 (2006).

    PubMed  Google Scholar 

  38. 38.

    Rossant, J. Culturing trophoblast stem (TS) cell lines. CSH Protoc.. (2006).

    Article  PubMed  Google Scholar 

  39. 39.

    Kubaczka, C. et al. Derivation and maintenance of murine trophoblast stem cells under defined conditions. Stem Cell Rep. 2, 232–242 (2014).

    CAS  Article  Google Scholar 

  40. 40.

    Ohinata, Y. & Tsukiyama, T. Establishment of trophoblast stem cells under defined culture conditions in mice. PLoS ONE 9, e107308 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


We are grateful to our colleagues in the M.Z.-G. group and to M. Huch for advice. We are grateful to the Wellcome Trust for the Senior Research fellowship (grant no. 098287/Z/12/Z) and for a European Research Council grant (code: 669198) awarded to M.Z.-G. to fund this work. We are also grateful for the BBSRC DTP studentship that supports S.E.H. and to the Scientific and Technological Research Council of Turkey, which supports B.S.’

Author information




S.E.H., B.S., and M.Z.-G. designed the protocol, carried out the work, analyzed the results, and prepared the manuscript.

Corresponding author

Correspondence to Magdalena Zernicka-Goetz.

Ethics declarations

Competing interests

M.Z.-G. and S.E.H. declare that they are inventors on a patent application (1615343.9) submitted by Cell Guidance Systems (in which the University of Cambridge and the Wellcome Trust are beneficiaries) that covers the method and medium composition used to generate stem cell–derived embryos. B.S. declares no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

1. Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro:

2. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation:

Supplementary information

Supplementary Text and Figures

Supplementary Table 1

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Harrison, S.E., Sozen, B. & Zernicka-Goetz, M. In vitro generation of mouse polarized embryo-like structures from embryonic and trophoblast stem cells. Nat Protoc 13, 1586–1602 (2018).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing