Article

A method to recapitulate early embryonic spatial patterning in human embryonic stem cells

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Abstract

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however, differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.

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Acknowledgements

The authors are grateful to S. Li and A. Yoney for technical assistance, C. Kirst for assistance with 3D image segmentation, and members of the A.H.B. and E.D.S. laboratories, A.-K. Hadjantonakis and S. Nowotschin for helpful discussions. Funding supporting this work was provided by The Rockefeller University, NYSTEM, US National Institutes of Health grants R01 HD32105 (to A.H.B.) and R01 GM 101653 (to A.H.B. and E.D.S.), US National Science Foundation grant PHY-0954398 (to E.D.S.) and the Human Frontier Science Program LT000851/2011-l (to B.S.).

Author information

Author notes

    • Aryeh Warmflash
    •  & Benoit Sorre

    These authors contributed equally to this work.

Affiliations

  1. Center for Studies in Physics and Biology, The Rockefeller University, New York, New York, USA.

    • Aryeh Warmflash
    • , Benoit Sorre
    • , Fred Etoc
    •  & Eric D Siggia
  2. Laboratory of Molecular Vertebrate Embryology, The Rockefeller University, New York, New York, USA.

    • Aryeh Warmflash
    • , Benoit Sorre
    • , Fred Etoc
    •  & Ali H Brivanlou

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Contributions

A.W. designed and performed experiments, performed analysis and wrote the paper. B.S. designed and performed experiments and contributed to writing the paper. F.E. performed experiments and contributed to writing the paper. E.D.S. designed experiments, performed analysis and wrote the paper. A.H.B. designed experiments and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Eric D Siggia or Ali H Brivanlou.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Table 1