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Self-organization of the human embryo in the absence of maternal tissues

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Abstract

Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here, we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages, and morphogenetic rearrangements leading to generation of a bilaminar disc, formation of a pro-amniotic cavity within the embryonic lineage, appearance of the prospective yolk sac, and trophoblast differentiation. Using human embryos and human pluripotent stem cells, we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together, our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous, highlighting the remarkable and unanticipated self-organizing properties of human embryos.

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Figure 1: Establishment of an in vitro system to study human implantation and early post-implantation morphogenesis.
Figure 2: Preservation of the pluripotent lineage in human embryos cultured through implantation stages in vitro.
Figure 3: Analysis of embryonic and extra-embryonic lineages in human embryos cultured through implantation stages in vitro.
Figure 4: Remodelling of the epiblast in human embryos cultured through implantation stages.
Figure 5: Self-organization of hESCs in response to ECM signalling.
Figure 6: Self-organization of hiPSCs in response to ECM signalling.

Change history

  • 17 May 2016

    In the version of this Technical Report originally published online, in Fig. 1d (which presents pilot in vitro culture experiments) it erroneously stated that 20% HCS was used in the IVC2 medium; it should have stated that 30% KSR was used. This error arose by a miscommunication between the postdoctoral fellow who contributed to the pilot in vitro culture experiments and prepared the media, and the postdoctoral fellow who performed the experiments. The composition of the media has been verified based on the laboratory notebooks that describe the media preparation. This error has been corrected in the labels and caption of Fig. 1d, in the description of the results in the main text, and in the Methods section, in all versions of the Technical Report.

References

  1. Edwards, R. G., Bavister, B. D. & Steptoe, P. C. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature 221, 632–635 (1969).

    CAS  Article  Google Scholar 

  2. Edwards, R. G., Steptoe, P. C. & Purdy, J. M. Fertilization and cleavage in vitro of preovulator human oocytes. Nature 227, 1307–1309 (1970).

    CAS  Article  Google Scholar 

  3. Koot, Y. E., Teklenburg, G., Salker, M. S., Brosens, J. J. & Macklon, N. S. Molecular aspects of implantation failure. Biochim. Biophys. Acta 1822, 1943–1950 (2012).

    CAS  Article  Google Scholar 

  4. Enders, A. C., Schlafke, S. & Hendrickx, A. G. Differentiation of the embryonic disc, amnion, and yolk sac in the rhesus monkey. Am. J. Anat. 177, 161–185 (1986).

    CAS  Article  Google Scholar 

  5. Pera, M. F. & Trounson, A. O. Human embryonic stem cells: prospects for development. Development 131, 5515–5525 (2004).

    CAS  Article  Google Scholar 

  6. Weimar, C. H., Post Uiterweer, E. D., Teklenburg, G., Heijnen, C. J. & Macklon, N. S. In-vitro model systems for the study of human embryo-endometrium interactions. Reprod. Biomed. Online 27, 461–476 (2013).

    CAS  Article  Google Scholar 

  7. 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  Google Scholar 

  8. Bedzhov, I. & Zernicka-Goetz, M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell 156, 1032–1044 (2014).

    CAS  Article  Google Scholar 

  9. Hertig, A. T., Rock, J. & Adams, E. C. A description of 34 human ova within the first 17 days of development. Am. J. Anat. 98, 435–493 (1956).

    CAS  Article  Google Scholar 

  10. Hur, Y. S. et al. Effect of artificial shrinkage on clinical outcome in fresh blastocyst transfer cycles. Clin. Exp. Reprod. Med. 38, 87–92 (2011).

    Article  Google Scholar 

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

    Article  Google Scholar 

  12. Pera, M. F. et al. What if stem cells turn into embryos in a dish? Nat. Methods 12, 917–919 (2015).

    CAS  Article  Google Scholar 

  13. Gardner, D. K. The impact of physiological oxygen during culture, and vitrification for cryopreservation, on the outcome of extended culture in human IVF. Reprod. Biomed. Online 32, 137–141 (2015).

    Article  Google Scholar 

  14. Covello, K. L. et al. HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev. 20, 557–570 (2006).

    CAS  Article  Google Scholar 

  15. Ezashi, T., Das, P. & Roberts, R. M. Low O2 tensions and the prevention of differentiation of hES cells. Proc. Natl Acad. Sci. USA 102, 4783–4788 (2005).

    CAS  Article  Google Scholar 

  16. Rivera-Perez, J. A., Jones, V. & Tam, P. P. Culture of whole mouse embryos at early postimplantation to organogenesis stages: developmental staging and methods. Methods Enzymol. 476, 185–203 (2010).

    CAS  Article  Google Scholar 

  17. Bedzhov, I., Graham, S. J., Leung, C. Y. & Zernicka-Goetz, M. Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo. Phil. Trans. R. Soc. B 369 (2014).

  18. Roode, M. et al. Human hypoblast formation is not dependent on FGF signalling. Dev. Biol. 361, 358–363 (2012).

    CAS  Article  Google Scholar 

  19. Teklenburg, G. et al. Cell lineage specific distribution of H3K27 trimethylation accumulation in an in vitro model for human implantation. PLoS ONE 7, e32701 (2012).

    CAS  Article  Google Scholar 

  20. Niakan, K. K. & Eggan, K. Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse. Dev. Biol. 375, 54–64 (2013).

    CAS  Article  Google Scholar 

  21. O’Leary, T. et al. Tracking the progression of the human inner cell mass during embryonic stem cell derivation. Nat. Biotechnol. 30, 278–282 (2012).

    Article  Google Scholar 

  22. Niakan, K. K., Han, J., Pedersen, R. A., Simon, C. & Pera, R. A. Human pre-implantation embryo development. Development 139, 829–841 (2012).

    CAS  Article  Google Scholar 

  23. Haigh, T., Chen, C., Jones, C. J. & Aplin, J. D. Studies of mesenchymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage. Placenta 20, 615–625 (1999).

    CAS  Article  Google Scholar 

  24. Dobreva, M. P., Pereira, P. N., Deprest, J. & Zwijsen, A. On the origin of amniotic stem cells: of mice and men. Int. J. Dev. Biol. 54, 761–777 (2010).

    CAS  Article  Google Scholar 

  25. Hill, J. P. The developmental history of the primates. Phil. Trans. R. Soc. Lond. B 221, 45–178 (1932).

    Article  Google Scholar 

  26. Luckett, W. P. The development of primordial and definitive amniotic cavities in early Rhesus monkey and human embryos. Am. J. Anat. 144, 149–167 (1975).

    CAS  Article  Google Scholar 

  27. Palis, J. & Yoder, M. C. Yolk-sac hematopoiesis: the first blood cells of mouse and man. Exp. Hematol. 29, 927–936 (2001).

    CAS  Article  Google Scholar 

  28. Taniguchi, K. et al. Lumen formation is an intrinsic property of isolated human pluripotent stem cells. Stem Cell Rep. 5, 954–962 (2015).

    CAS  Article  Google Scholar 

  29. Bryant, D. M. et al. A molecular switch for the orientation of epithelial cell polarization. Dev. Cell 31, 171–187 (2014).

    CAS  Article  Google Scholar 

  30. Bryant, D. M. & Mostov, K. E. From cells to organs: building polarized tissue. Nat. Rev. Mol. Cell Biol. 9, 887–901 (2008).

    CAS  Article  Google Scholar 

  31. Vuoristo, S., Jedrusik, A., Shahbazi, M. N. & Zernicka-Goetz, M. Culture of human embryos through implantation stages in vitro. Protoc. Exch. (2016)10.1038/protex.2016.017.

  32. Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    CAS  Article  Google Scholar 

  33. Lee, G. Y., Kenny, P. A., Lee, E. H. & Bissell, M. J. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4, 359–365 (2007).

    CAS  Article  Google Scholar 

  34. Faure, E. et al. A workflow to process 3D + time microscopy images of developing organisms and reconstruct their cell lineage. Nat. Commun. 7, 8674 (2016).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to the patients donating their embryos, colleagues in the M.Z.-G. laboratory, C. Lee (Gurdon Institute), and embryologists at the CARE, Bourn Hall (K. Elder and P. Snell) and Kings College Guy’s Hospital IVF clinics for help and discussions. We thank P. Braude, D. Glover and C. Ogilvie for insightful discussion and I. Bedzhov for help in a pilot experiment. This work was supported by the Wellcome Trust grant to M.Z.-G. Work in the K.K.N. laboratory was supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK, the Medical Research Council and the Wellcome Trust. M.N.S. was initially supported by a Ramon Areces Spanish Foundation Fellowship, and subsequently by an EMBO Postdoctoral Fellowship. S.V. was supported by a Post Doc Pool Grant from the Finnish Cultural Foundation. G.R. was supported by a Newton Fellowship.

Author information

Authors and Affiliations

Authors

Contributions

M.N.S., A.J. and S.V. carried out all experiments and data analyses. G.R. analysed microscopy data and generated 3D reconstructions. A.H. prepared illustrations and contributed experimentally. N.M.E.F. and K.K.N. helped with human embryo cultures and contributed experimentally, and L.G.D. helped with human embryo cultures. A.C., S.F., D.I., Y.K. and K.K.N. oversaw and provided human embryos for these studies. M.Z.-G. conceived the project and supervised the study. M.N.S. and M.Z.-G. wrote the manuscript with help from all of the authors.

Corresponding author

Correspondence to Magdalena Zernicka-Goetz.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 3 3D analysis of embryonic and extra-embryonic lineages in human embryos cultured through implantation stages.

Related to Figs 3 and 4. Human blastocysts developing in vitro until day 11 were fixed and stained at indicated time points. (a) 3D rendering of DAPI stain overlaid with the centre of OCT4-expressing and GATA6-expressing cells. Dots are projected at the back of the volume. (b) Orthogonal views of embryos at different developmental stages stained for aPKC and OCT4. Note the presence of a small pro-amniotic cavity at day 8–9, which is enlarged as development proceeds. All scale bars, 50 μm.

Supplementary Figure 4 Confocal Z-sections of a day 7–8 human embryo.

Related to Fig. 4. Day 7–8 human embryo stained for OCT4, aPKC and F-actin. The different images correspond to representative confocal Z sections. Scale bar, 20 μm. Boxes indicate the magnified area (scale bar, 10 μm).

Supplementary Figure 5 Confocal Z-sections of a day 8–9 human embryo.

Related to Fig. 4. Day 8–9 human embryo stained for OCT4, aPKC and F-actin. The different images correspond to representative confocal Z sections. The arrow indicates the presence of a lumen. Scale bar, 20 μm. Boxes indicate the magnified area (scale bar, 10 μm).

Supplementary Figure 6 Confocal Z-sections of a day 9–10 human embryo.

Related to Fig. 4. Day 9–10 human embryo stained for OCT4, aPKC and F-actin. The different images correspond to representative confocal Z sections. The arrow indicates the presence of a lumen surrounded by radially organised OCT4-expressing epiblast cells. Scale bar, 20 μm. Boxes indicate the magnified area (scale bar, 10 μm).

Supplementary Figure 7 Confocal Z-sections of a day 10–11 human embryo.

Related to Fig. 4. Day 10–11 human embryo stained for OCT4, aPKC and F-actin. The different images correspond to representative confocal Z sections. The arrow indicates the presence of a small lumen surrounded by radially organised OCT4-expressing epiblast cells. Scale bar, 50 μm. Boxes indicate the magnified area (scale bar, 20 μm).

Supplementary Table 1 Summary of human embryos cultured in this study.
Supplementary Table 2 Average number of epiblast and hypoblast cells in human embryos at different stages of implantation development in vitro.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1035 kb)

Development of a day 5 human blastocyst in the in vitro culture system up to day 9–10.

Related to Fig. 1. A day 5 human embryo was cultured in the in vitro culture system for approximately 100 h. Bright field images were taken every 30 min to record its development. Scale bar, 100 μm. (AVI 6989 kb)

Development of a day 9 human blastocyst in the in vitro culture system up to day 12.

Related to Fig. 1. A day 9 human embryo was cultured in the in vitro culture system for approximately 72 h. Bright field images were taken every 30 min to record its development. Scale bar, 100 μm. (AVI 23240 kb)

3D reconstruction of embryonic lineages in a day 9–10 human embryo cultured in vitro.

Related to Fig. 3. Nuclei are shown in blue, OCT4 in grey and GATA6/F-actin in green. (AVI 31620 kb)

3D reconstruction of embryonic lineages in a day 10–11 human embryo cultured in vitro.

Related to Fig. 3. Nuclei are shown in blue, OCT4 in grey and GATA6/F-actin in green. (AVI 32179 kb)

3D reconstruction of the cellular and nuclear shape of representative trophectoderm cells at day 10–11.

Related to Fig. 3. Nuclei are shown in magenta and membranes in green. Note that cells in close proximity to the epiblast have a single nucleus, whereas cells in the periphery of the embryo are multinucleated. (AVI 37380 kb)

3D reconstruction of the pro-amniotic cavity at day 9–10.

Related to Fig. 4. The nuclei of OCT4-expressing epiblast cells is shown in grey and the pro-amniotic cavity in red. (AVI 36620 kb)

3D reconstruction of the cellular shape of representative OCT4-expressing epiblast cells at day 10–11.

Related to Fig. 4. Epiblast cells in close proximity to GATA6-expressing hypoblast cells are shown in green (note the columnar shape characteristic of cells within the epiblast disc). Epiblast cells in close proximity to cytotrophoblast cells are shown in magenta (note the squamous shape characteristic of amniotic cells). (AVI 40836 kb)

3D reconstruction of the prospective yolk sac at day 10–11.

Related to Fig. 4. The nuclei of OCT4-expressing epiblast cells is shown in grey and the prospective yolk sac in blue. (AVI 38440 kb)

3D reconstruction of the hypoblast derived cells and their position with respect to the prospective yolk sac at day 10–11.

Related to Fig. 4. Nuclei of OCT4-expressing epiblast cells are shown in grey, GATA6/F-actin is shown in green and the prospective yolk sac in blue. (AVI 31886 kb)

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Shahbazi, M., Jedrusik, A., Vuoristo, S. et al. Self-organization of the human embryo in the absence of maternal tissues. Nat Cell Biol 18, 700–708 (2016). https://doi.org/10.1038/ncb3347

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