Understanding developmental biology requires knowledge of both the environmental factors regulating stem cell differentiation, which are increasingly being defined, and their spatial organization within a structurally heterogeneous niche, which is still largely unknown. Here we introduce spatially organized stem cell developmental models to interrogate the role of space in fate specification. Specifically, we developed Differential Environmental Spatial Patterning (δESP) to organize different microenvironments around single embryonic stem cell (ESC) colonies via sequential micropatterning. We first used δESP to decouple and understand the roles of cell organization and niche organization on ESCs deciding between self-renewal and differentiation fate choices. We then approximated in vitro an embryonic developmental step, specifically proximal–distal (PD) patterning of the mouse epiblast at pre-gastrulation, by spatially organizing two extraembryonic environments around ESCs, demonstrating that spatial organization of these three cell types is sufficient for PD patterns to form in vitro.
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