High-throughput micro-patterning platform reveals Nodal-dependent dissection of peri-gastrulation-associated versus pre-neurulation associated fate patterning

In vitro models of post-implantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon BMP4 treatment of geometrically-confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line associated heterogeneity was found to be attributable to endogenous nodal expression, with upregulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal downregulation correlated with a neurulation-associated gene profile expression. We harness this knowledge to establish a platform of pre-neurulation-like fate patterning in geometrically confined hPSC colonies that arises due to a stepwise activation of reaction-diffusion and positional-information. Our work identifies a Nodal signalling dependent switch in peri-gastrulation versus pre-neurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of self-organized fate specification in differentiating epiblast and ectodermal tissues. We kindly thank Dr. Andras Nagy for providing the CA1 human embryonic stem cell line; Dr. Sean Palecek for providing the 7TGP(H9) line; Dr. Gordon Keller for providing the GKH9(H9), HES2, and the PDX1-GFP(MEL1) lines; and Dr. Andrew Elefanty for providing the RUNX1-GFP(HES3), and the MIXL1-GFP(HES3) lines. We thank Dr. Emanuel Nazareth for providing us with the micro-contact printing stamps. We gratefully acknowledge Dr. Alfonso Martinez Arias, Dr. James Briscoe, Dr. Teresa Rayon Alonso, Dr. Christopher Demers, and Dr. Celine Bauwens for providing insightful feedback to our manuscript. This work was funded by the Canadian Institutes for Health Research and Medicine by Design, a Canada First Research Excellence Program at the University of Toronto. MT received funding from CREATE Materials, Mimetics, and Manufacturing from the Natural Sciences and Engineering Research Council of Canada. DD is a PhD fellow of Research Foundation - Flanders (FWO). FMW gratefully acknowledges funding from the UK Medical Research Council and Wellcome Trust. PWZ is the Canada Research Chair in Stem Cell Bioengineering.