In vitro models of postimplantation 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 bone morphogenetic protein 4 (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 up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues.

47In vitro models of post-implantation human development are valuable to the fields of regenerative medicine 48 and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-49content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-50 gastrulation-like fate patterning upon BMP4 treatment of geometrically-confined colonies and observed 51 significant heterogeneity in their differentiation propensities along a gastrulation associable and 52 neuralization associable axis. This cell line associated heterogeneity was found to be attributable to 53 endogenous nodal expression, with upregulation of Nodal correlated with expression of a gastrulation-54 associated gene profile, and Nodal downregulation correlated with a neurulation-associated gene profile 55 expression. We harness this knowledge to establish a platform of pre-neurulation-like fate patterning in 56geometrically confined hPSC colonies that arises due to a stepwise activation of reaction-diffusion and 57 positional-information. Our work identifies a Nodal signalling dependent switch in peri-gastrulation versus 58pre-neurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC 59 differentiation outcomes, and suggests conserved mechanisms of self-organized fate specification in 60 differentiating epiblast and ectodermal tissues.

Although human dermis contains distinct fibroblast subpopulations, the functional heterogeneity of fibroblast lines from different donors is under-appreciated. We identified one commercially sourced fibroblast line (c64a) that failed to express α-smooth muscle actin (α-SMA), a marker linked to fibroblast contractility, even when treated with transforming growth factor-β1 (TGF-β1). Gene expression profiling identified insulin-like growth factor 1 (IGF1) as being expressed more highly, and Asporin (ASPN) and Wnt family member 4 (WNT4) expressed at lower levels, in c64a fibroblasts compared to three fibroblast lines that had been generated in-house, independent of TGF-β1 treatment. TGF-β1 increased expression of C-X-C motif chemokine ligand 1 (CXCL1) in c64a cells to a greater extent than in the other lines. The c64a gene expression profile did not correspond to any dermal fibroblast subpopulation identified by single-cell RNAseq of freshly isolated human skin cells. In skin reconstitution assays, c64a fibroblasts did not support epidermal stratification as effectively as other lines tested. In fibroblast lines generated in-house, shRNA-mediated knockdown of IGF1 increased α-SMA expression without affecting epidermal stratification. Conversely, WNT4 knockdown had no consistent effect on α-SMA expression, but increased the ability of fibroblasts to support epidermal stratification. Thus, by comparing the properties of different lines of cultured dermal fibroblasts, we have identified IGF1 and WNT4 as candidate mediators of two distinct dermal functions: myofibroblast formation and epidermal maintenance.

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