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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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.).
The authors declare no competing financial interests.
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Warmflash, A., Sorre, B., Etoc, F. et al. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat Methods 11, 847–854 (2014). https://doi.org/10.1038/nmeth.3016
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