Engineered In vitro models of post-implantation human development to elucidate mechanisms of self-organized fate specification during embryogenesis

During embryogenesis, cells in different positions of the embryo acquire different fates in a seemingly autonomous process called ‘fate-patterning’. Fundamental studies have identified important signaling molecules (morphogens) that play crucial roles in coordinating developmental fate-patterning, examples include members of the transforming growth factor beta family – like bone morphogenetic proteins (BMPs), and Nodals. However, mechanistic understanding of how these morphogens coordinate fate-patterning remains unclear. Here we aim to apply bioengineering strategies to develop an in vitro model of developmental fate-patterning and employ it to interrogate the underlying mechanisms that govern this critical process. We first developed a robust, high-throughput platform to enable geometric-confinement of adherent cell types and employed it to screen various BMP4 supplemented defined media to identify conditions that coaxed geometrically-confined human pluripotent stem cell (hPSC) colonies to undergo peri-gastrulation-associated fate-patterning. This screen resulted in identification of defined conditions that spatially segregated compartments in the differentiating hPSC colonies expressing fate markers of trophoblast-like, primitive-streak-like, endoderm-like, mesoderm-like, and ectoderm-like tissues. Using a combination of experimental and computational-modelling approaches, we identified a stepwise mechanism of reaction-diffusion and positional-information underlying the observed peri-gastrulation-like fate-patterning. Here, a BMP4-Noggin reaction-diffusion network self-organized BMP signaling gradient, and this gradient patterned peri-gastrulation-associated fates in a manner consistent with positional-information. Furthermore, we found that Nodal signaling was necessary to induce the expression of the primitive-streak compartment – the precursor of gastrulation-derived fates. Interestingly, we also observed that Nodal signaling dissected gastrulation-associated and neurulation-associated gene expression profiles in differentiating hPSC lines. Specifically, in differentiating hPSCs, upregulation of Nodal signaling was observed in cells that upregulated a gene profile associated with gastrulation whereas absence of Nodal signaling correlated with upregulation of a neurulation-associated gene profile. We hypothesized that treatment of geometrically-confined hPSC colonies with BMP4 in the absence of Nodal signaling would induce fate patterning associated with neurulation. We observed experimental results consistent with this hypothesis and identified a conserved underlying mechanism of a stepwise model of reaction-diffusion and positional-information underlying the pre-neurulation-associated patterning as well. Taken together this work provides deep insight into how morphogens regulate early developmental stages of human embryogenesis – which have been previously inaccessible for experimentation.

Keywords: Biomedical Engineering
Computational Modeling
Embryonic Development
Embryonic Stem Cells
Microfabrication
Pluripotent Stem Cells
Advisor: Zandstra, Peter W