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Self-organization of human embryonic stem cells on micropatterns

Nature Protocols volume 11, pages 2223–2232 (2016)Cite this article

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Abstract

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped, submillimeter colonies. After 42 h of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO), human laminin-521 (LN-521) as extracellular matrix coating, and either conditioned or chemically defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation.

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Figure 1: Time line of the morphology of micropatterned colonies.
Figure 2: Immunostaining of micropatterns: pluripotency and differentiation.
Figure 3: Effect of colony size on cell fate.

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Acknowledgements

We thank the members of the Brivanlou, Siggia, and Warmflash laboratories for helpful discussions. This work was supported by NIH/NIGMS R01 GM101653 (A.H.B., E.S.), NIH/DHHS R01 HD080699 (A.H.B., E.S.), Cancer Prevention Research Institute of Texas (CPRIT) grant RR140073 (A.W.), NSF grant DGE-1325261 (A.Y.), and NSF grant MCB-1553228 (A.W.).

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Author notes

  1. Alessia Deglincerti, Fred Etoc, M Cecilia Guerra, Iain Martyn, Jakob Metzger, Albert Ruzo, Mijo Simunovic and Anna Yoney: These authors contributed equally to this work.

Authors and Affiliations

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

    Alessia Deglincerti, Fred Etoc, Iain Martyn, Jakob Metzger, Albert Ruzo, Mijo Simunovic, Anna Yoney & Ali H Brivanlou

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

    Fred Etoc, Iain Martyn, Jakob Metzger, Mijo Simunovic, Anna Yoney & Eric Siggia

  3. Department of Biosciences, Rice University, Houston, Texas, USA

    M Cecilia Guerra & Aryeh Warmflash

  4. Department of Bioengineering, Rice University, Houston, Texas, USA

    Aryeh Warmflash

Authors
  1. Alessia Deglincerti
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  9. Ali H Brivanlou
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Contributions

All authors contributed to the design of the experiments. A.D., F.E., M.C.G., I.M., J.M., A.R., M.S., A.Y., and A.W. performed and analyzed the experiments. All authors wrote the manuscript.

Corresponding authors

Correspondence to Ali H Brivanlou, Eric Siggia or Aryeh Warmflash.

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The authors declare no competing financial interests.

Supplementary information

BMP4 differentiation in micropatterned hESC colonies grown in mTeSR.

Immunofluorescence staining of ESI017 hESCs 48 hours after BMP4 (50 ng/ml) treatment. Shown are colonies of 1000, 800, and 500 μm seeded and grown following the alternative protocol described in Box 1. CDX2 (green); BRA (red); SOX2 (blue). Scale bars, 250 μm. ESCRO institutional regulatory board permission was obtained to perform these experiments. (PDF 1525 kb)

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Deglincerti, A., Etoc, F., Guerra, M. et al. Self-organization of human embryonic stem cells on micropatterns. Nat Protoc 11, 2223–2232 (2016). https://doi.org/10.1038/nprot.2016.131

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