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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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.).
The authors declare no competing financial interests.
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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