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


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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Figure 1: hESCs in the pluripotent state show prepatterning in micropatterned culture.
Figure 2: hESCs differentiated on micropatterns form self-organized spatial patterns.
Figure 3: During differentiation, hESCs undergo an epithelial-to-mesenchymal transition in a region expressing markers of the primitive streak.
Figure 4: Control of cell fate extends from the edge of the colony.
Figure 5: Self-organized signaling responses in micropatterned colonies.
Figure 6: TGF-β inhibitors are required for pattern formation.


  1. 1

    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 

  2. 2

    D'Amour, K.A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Kattman, S.J. et al. Stage-specific optimization of Activin/Nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011).

    CAS  Article  Google Scholar 

  5. 5

    Ozair, M.Z., Noggle, S., Warmflash, A., Krzyspiak, J.E. & Brivanlou, A.H. SMAD7 directly converts human embryonic stem cells to telencephalic fate by a default mechanism. Stem Cells 31, 35–47 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Ben-Haim, N. et al. The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4. Dev. Cell 11, 313–323 (2006).

    CAS  Article  Google Scholar 

  7. 7

    ten Berge, D. et al. Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3, 508–518 (2008).

    CAS  Article  Google Scholar 

  8. 8

    Drukker, M. et al. Isolation of primitive endoderm, mesoderm, vascular endothelial and trophoblast progenitors from human pluripotent stem cells. Nat. Biotechnol. 30, 531–542 (2012).

    CAS  Article  Google Scholar 

  9. 9

    Blauwkamp, T.A., Nigam, S., Ardehali, R., Weissman, I.L. & Nusse, R. Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors. Nat. Commun. 3, 1070 (2012).

    Article  Google Scholar 

  10. 10

    Peerani, R. et al. Niche-mediated control of human embryonic stem cell self-renewal and differentiation. EMBO J. 26, 4744–4755 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Bauwens, C.L. et al. Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells 26, 2300–2310 (2008).

    Article  Google Scholar 

  12. 12

    Sakai, Y., Yoshiura, Y. & Nakazawa, K. Embryoid body culture of mouse embryonic stem cells using microwell and micropatterned chips. J. Biosci. Bioeng. 111, 85–91 (2011).

    CAS  Article  Google Scholar 

  13. 13

    James, D., Levine, A.J., Besser, D. & Hemmati-Brivanlou, A. TGFβ/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132, 1273–1282 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Xu, R.-H. et al. NANOG is a direct target of TGFβ/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell 3, 196–206 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. & Brivanlou, A.H. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55–63 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Yu, P., Pan, G., Yu, J. & Thomson, J.A. FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation. Cell Stem Cell 8, 326–334 (2011).

    CAS  Article  Google Scholar 

  17. 17

    Amita, M. et al. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc. Natl. Acad. Sci. USA 110, E1212–E1221 (2013).

    CAS  Article  Google Scholar 

  18. 18

    Sudheer, S., Bhushan, R., Fauler, B., Lehrach, H. & Adjaye, J. FGF inhibition directs BMP4-mediated differentiation of human embryonic stem cells to syncytiotrophoblast. Stem Cells Dev. 21, 2987–3000 (2012).

    CAS  Article  Google Scholar 

  19. 19

    Li, Y. et al. BMP4-directed trophoblast differentiation of human embryonic stem cells is mediated through a ΔNp63+ cytotrophoblast stem cell state. Development 140, 3965–3976 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Bernardo, A.S. et al. BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 9, 144–155 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Loh, K.M. & Lim, B. A precarious balance: pluripotency factors as lineage specifiers. Cell Stem Cell 8, 363–369 (2011).

    CAS  Article  Google Scholar 

  22. 22

    Thomson, M. et al. Pluripotency factors in embryonic stem cells regulate differentiation into germ layers. Cell 145, 875–889 (2011).

    CAS  Article  Google Scholar 

  23. 23

    Teo, A.K.K. et al. Pluripotency factors regulate definitive endoderm specification through eomesodermin. Genes Dev. 25, 238–250 (2011).

    CAS  Article  Google Scholar 

  24. 24

    Wang, Z., Oron, E., Nelson, B., Razis, S. & Ivanova, N. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10, 440–454 (2012).

    CAS  Article  Google Scholar 

  25. 25

    Li, L. et al. Location of transient ectodermal progenitor potential in mouse development. Development 140, 4533–4543 (2013).

    CAS  Article  Google Scholar 

  26. 26

    Chawengsaksophak, K., de Graaff, W., Rossant, J., Deschamps, J. & Beck, F. Cdx2 is essential for axial elongation in mouse development. Proc. Natl. Acad. Sci. USA 101, 7641–7645 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Williams, M., Burdsal, C., Periasamy, A., Lewandoski, M. & Sutherland, A. Mouse primitive streak forms in situ by initiation of epithelial to mesenchymal transition without migration of a cell population. Dev. Dyn. 241, 270–283 (2012).

    Article  Google Scholar 

  28. 28

    Ng, V.Y., Ang, S.N., Chan, J.X. & Choo, A.B.H. Characterization of epithelial cell adhesion molecule as a surface marker on undifferentiated human embryonic stem cells. Stem Cells 28, 29–35 (2010).

    CAS  Article  Google Scholar 

  29. 29

    Inman, G.J. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Bachiller, D. et al. The organizer factors Chordin and Noggin are required for mouse forebrain development. Nature 403, 658–661 (2000).

    CAS  Article  Google Scholar 

  31. 31

    Perea-Gomez, A. et al. Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. Dev. Cell 3, 745–756 (2002).

    CAS  Article  Google Scholar 

  32. 32

    Behringer, R.R., Wakamiya, M., Tsang, T.E. & Tam, P.P. A flattened mouse embryo: leveling the playing field. Genesis 28, 23–30 (2000).

    CAS  Article  Google Scholar 

  33. 33

    Tam, P.P.L. & Gad, J.M. in Gastrulation: From Cells to Embryo (ed. Stern, C.D.) Ch. 16 (CSHL Press, 2004).

  34. 34

    Hwang, Y.-S. et al. Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11. Proc. Natl. Acad. Sci. USA 106, 16978–16983 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Moledina, F. et al. Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells. Proc. Natl. Acad. Sci. USA 109, 3264–3269 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

    CAS  Article  Google Scholar 

  37. 37

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

    CAS  Article  Google Scholar 

  38. 38

    Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    CAS  Article  Google Scholar 

  39. 39

    Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).

    CAS  Article  Google Scholar 

  40. 40

    Spence, J.R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).

    Article  Google Scholar 

  41. 41

    Warmflash, A. et al. Dynamics of TGF-β signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor SMAD4. Proc. Natl. Acad. Sci. USA 109, E1947–E1956 (2012).

    CAS  Article  Google Scholar 

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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.).

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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.

Corresponding authors

Correspondence to Eric D Siggia or Ali H Brivanlou.

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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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