Controlled Organization of Cell Fates in Spatially Confined Stem Cell Populations

Abstract

During embryonic development, cells divide and differentiate over space and time as instructed by their environment to create complex, functionally diverse tissues. Our understanding of the underlying orchestrating rules is incomplete, but recent studies have started to reveal how to instruct cultured pluripotent cell populations to undergo similar developmental-like organizational events. Here, we induce spatially polarized cell fate organization in micropatterned colonies, previously only reported for non-adherent or locally induced cell populations. Underlying this discovery was the augmentation of our micropatterning high-throughput platform through the development of analytical frameworks to automate the quantification of heterogeneous cell responses. These developments enabled rapid hypotheses generation and testing, which enabled new insights into the underlying biology of how and when cell fate organization occurs. Specifically, we developed analytical frameworks to accurately identify colonies of cells within an image and localization of positively expressing regions within these, which enabled automatic quantification of spatial fate organization. We show that when mouse pluripotent stem cells are differentiated on micropatterns towards gastrulation-like fates, their normally symmetrical spatial organization of cell fates can be modified by changing the micropattern diameter and cell density. By differentiating cells at low to medium density on circular micropatterns of 200-300 um in diameter, we induce polarized organization of primitive streak-like and anterior epiblast-like cells, reminiscent of how these populations are localized during development. We study the emergence of this organization using live imaging and found that polarization occurs largely due to reorganization within the colony post-induction. Overall, our results show that system size, both in terms of colony geometry and cell number at the time of differentiation, is critical for polarized cell fate organization. We hypothesize that this could indicate a need for developmentally relevant system sizes in polarization of micropatterned colonies, and that it might be driven by initial heterogeneities in colony morphology or cell fate distribution, or minor fluctuation that are allowed to amplify and perturb a homogeneous state. These insights on how to control and quantify fate organization in cell populations can advance both our understanding of developmental processes and how to create complex tissues with regenerative engineering.