title: Self-organization of human embryonic stem cells on micropatterns.
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.
authors.affiliation: Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.
authors.forename: Alessia
authors.identifier: http://orcid.org/0000-0003-0564-7746
authors.initials: A
authors.lastname: Deglincerti
authors.affiliation: Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.|Center for Studies in Physics and Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Fred
authors.initials: F
authors.lastname: Etoc
authors.affiliation: Department of Biosciences, Rice University, Houston, Texas, USA.
authors.forename: M Cecilia
authors.initials: MC
authors.lastname: Guerra
authors.affiliation: Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.|Center for Studies in Physics and Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Iain
authors.initials: I
authors.lastname: Martyn
authors.affiliation: Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.|Center for Studies in Physics and Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Jakob
authors.initials: J
authors.lastname: Metzger
authors.affiliation: Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.
authors.forename: Albert
authors.initials: A
authors.lastname: Ruzo
authors.affiliation: Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.|Center for Studies in Physics and Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Mijo
authors.initials: M
authors.lastname: Simunovic
authors.affiliation: Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.|Center for Studies in Physics and Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Yoney
authors.affiliation: Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, New York, USA.
authors.forename: Ali H
authors.initials: AH
authors.lastname: Brivanlou
authors.affiliation: Center for Studies in Physics and Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Siggia
authors.affiliation: Department of Biosciences, Rice University, Houston, Texas, USA.|Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: Aryeh
authors.initials: A
authors.lastname: Warmflash
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D054262:Gastrulation; D000066449:Human Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D055616:Microtechnology / Q000379:methods*