Self-organization of human embryonic stem cells on micropatterns

Here, the authors developed a reproducible in vitro protocols that allow the study of spatial organization associated with this developmental transition. They use 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. The protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO), human laminin-521 as extracellular matrix coating, and either conditioned or chemically defined medium (mTeSRSR). 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 LN521 coating allows for a simpler coating protocol with robust results. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation.