Abstract
Pluripotent stem cells (PSCs) are the in vitro counterpart of the pluripotent epiblast of the mammalian embryo with the capacity to generate all cell types of the adult organism. During development, the three definitive germ layers are specified and simultaneously spatially organized. In contrast, differentiating PSCs tend to generate cell fates in a spatially disorganized manner. This has limited the in vitro study of specific cell-cell interactions and patterning mechanisms that occur in vivo. Here we describe a protocol to differentiate mouse PSCs in a spatially organized manner on micropatterned surfaces. Micropatterned chips comprise many colonies of uniform size and geometry facilitating a robust quantitative analysis of patterned fate specification. Furthermore, multiple factors may be simultaneously manipulated with temporal accuracy to probe the dynamic interactions regulating these processes. The micropattern system is scalable, providing a valuable tool to generate material for large-scale analysis and biochemical experiments that require substantial amounts of starting material, difficult to obtain from early embryos.