Letter | Published:

Self-organization of the in vitro attached human embryo

Nature volume 533, pages 251254 (12 May 2016) | Download Citation

Abstract

Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system1,2 to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy.

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Acknowledgements

We thank the members of the Brivanlou laboratory for their advice and criticisms, in particular C. Nchako, S. Tse for technical assistance, and members of the Zernicka-Goetz laboratory for their advice on how to culture embryos through attachment. We also thank A.K. Hadjantonakis for discussions, A. Wilkerson for support, and A. Brivanlou and P. Carleton-Evans for their comments on the manuscript. This work was supported by a STARR Foundation grant (number 2013-026) and Rockefeller Private funds. Images were obtained using instrumentation in The Rockefeller University Bio-Imaging Resource Center purchased with grant funds from the Sohn Conference Foundation. The Carnegie stage images are used with permission from the Virtual Human Embryo Project (http://virtualhumanembryo.lsuhsc.edu). We give special thanks for technical advice on imaging to A. North, K. Thomas, and P. Ariel, and on image analysis and rendering to T. Tong. This work would not have been possible without the generosity of the people who consented to donate their embryos to research, to whom we are indebted.

Author information

Author notes

    • Alessia Deglincerti
    •  & Gist F. Croft

    These authors contributed equally to this work.

Affiliations

  1. Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York 10065, USA

    • Alessia Deglincerti
    • , Gist F. Croft
    • , Lauren N. Pietila
    •  & Ali H. Brivanlou
  2. Department of Physiology, Development, and Neuroscience, University of Cambridge, Physiology Building, Downing Street, Cambridge CB2 3DY, UK

    • Magdalena Zernicka-Goetz
  3. Center for Studies in Physics and Biology, The Rockefeller University, New York, New York 10065, USA

    • Eric D. Siggia

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Contributions

A.D., G.C., and L.P. performed experiments; A.D. and G.C. analysed experiments; M.Z.-G. was instrumental in teaching and transferring knowledge on the mouse technology to A.D.; E.S. provided criticism of the work and manuscript; A.H.B. conceived and designed the project, established contact with the source of the biological material, provided guidance and advice throughout the work, and interfaced with the Institutional Review Board at The Rockefeller University; all authors contributed to the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ali H. Brivanlou.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Table

    This file contains Supplementary Table 1, which shows Cell scoring by marker and cell type.

Videos

  1. 1.

    Timecourse of in vitro human embryo attachment from DPF6 to DPF14

    OCT4 (green), GATA3 (blue), GATA6-Phalloidin (red). For the 3D rendering of DPF14 only, OCT4 and GATA3 channels were thresholded using the spot-finding algorithm in Imaris and then masked to remove non-nuclear background.

  2. 2.

    Example of DPF6 embryo

    DAPI (blue), OCT4 (green), CDX2 (cyan), GATA6 (red), Phalloidin (white). For this 3D rendering animation, a DAPI mask was generated by Imaris spot finding and then used to remove nuclear GATA6 from the original GATA6-phalloidin channel (creating Phalloidin-only and GATA6-only virtual channels). The Phalloidin virtual channel was then normalized across z using default settings in Imaris. A DAPI nuclear mask was also used to isolate nuclear OCT4 signal from non-nuclear background staining. A few pieces of debris outside of the embryo were manually cropped in 3D. This embryo was imaged at 40x.

  3. 3.

    Example of DPF6 embryo

    OCT4 (green), GATA3 (blue), GATA6-Phalloidin (red)

  4. 4.

    Example of DPF8 embryo.

    OCT4 (green), GATA3 (blue), GATA6-Phalloidin (red)

  5. 5.

    Example of DPF10 embryo and cavitation

    DAPI (blue), OCT4 (green), CDX2 (cyan), GATA6-Phalloidin (red). A 3D DAPI mask was used to exclude non-nuclear CDX2 and OCT4 stainings from the raw channels. Video breakdown by seconds: 0-12, 3D render of DAPI, nuclear CDX2, nuclear OCT4, and GATA6-Phalloidin; 12-16, z-sections (2µm step) from the bottom to the top of stack; 16-35, OCT4 (unmasked channel) and GATA6-Phalloidin as z-sections from the top to the bottom of the stack; 35-45, CDX2 (unmasked channel) is added at the bottom z-plane and the slice traverses from the bottom to the top of the stack showing bright CDX2 co-staining with GATA6 and OCT4 in ysTE cells; 45-50, CDX2 (unmasked channel) was replaced with nuclear (masked) CDX2, the z-slice returns to the bottom of the stack leaving progressive overlaying sections on display; 50-55 zoom out of 3D render; 55-58 DAPI staining faded back in.

  6. 6.

    Example of amniotic and yolk sac cavities in a DPF10 embryo

    OCT4 (green), CDX2 (cyan), GATA6-Phalloidin (red). 3D reconstruction of a cropped volume containing EPI, amniotic and yolk sac cavities, and ysTE cells. An oblique, 1.5µm virtual section is imposed and the 3D rendering is removed to visualize the signal from the plane passing through the structure and back. This image was obtained at 40x.

  7. 7.

    Example of amniotic cavity in a DPF10 embryo

    OCT4 (green), CDX2 (cyan), GATA6-Phalloidin (red); Z-series fly-through reconstruction.

  8. 8.

    Example of cytotrophoblast and syncytiotrophoblast phenotypes in a DPF12 embryo

    OCT4 (green), GATA3 (blue), GATA6-Phalloidin (red).

About this article

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DOI

https://doi.org/10.1038/nature17948

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