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Self-organization of human embryonic stem cells on micropatterns

Item Type:Article
Title:Self-organization of human embryonic stem cells on micropatterns
Creators Name:Deglincerti, A. and Etoc, F. and Guerra, M.C. and Martyn, I. and Metzger, J. and Ruzo, A. and Simunovic, M. and Yoney, A. and Brivanlou, A.H. and Siggia, E. and Warmflash, A.
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.
Keywords:Cell Differentiation, Cell Line, Gastrulation, Human Embryonic Stem Cells, Microtechnology
Source:Nature Protocols
Publisher:Nature Publishing Group
Page Range:2223-2232
Date:November 2016
Official Publication:https://doi.org/10.1038/nprot.2016.131
External Fulltext:View full text on PubMed Central
PubMed:View item in PubMed

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