Synthetic Morphogenesis of Early Embryonic Development Potentiated by Substratum-Guided Artificial Signaling

Introduction: Pluripotent stem cells (PSCs) have the unique ability to self-renew and differentiate into specialized cell types, making them an exceptionally useful tool for tissue engineering. Understanding the interplay of the molecular networks associated with stem cell differentiation can give insight into controlling cell fate decisions for the development of biomimetic tissues. One of the earliest steps in stem cell fate determination occurs during gastrulation when the epiblast organizes into the three germ layers – the ectoderm, mesoderm, and endoderm - a process orchestrated by the morphogens WNT, BMP, and NODAL. Due to technical challenges associated with studying gastrulation in vivo, 2D and 3D in vitro models that leverage PSC technology have been established to investigate the interplay between these signaling pathways and the spatial establishment of the germ layers.^1,2 Such studies rely on bulk medium supplementation of recombinant factors and chemical surrogates and have contributed greatly to our understanding of the molecular networks governing gastrulation; however, these efforts fail to faithfully recapitulate the spatially gated, cell-mediated signaling events associated with templating gastrulation. Here, we present a synthetic morphogenesis approach³ to human PSC gastrulation through artificial morphogenetic events driven by bioinert cues. By combining this platform with spatially constrained, micropatterned cell culture, we hope to recapitulate germ layer establishment to attain deeper insights into pattern formation and stem cell differentiation during early development.


Materials and Methods: Our method takes advantage of two innovations: (1) the synthetic signaling platform synthetic Notch (synNotch)⁴, which, like native Notch, responds to immobilized ligands to implement spatially constrained gene expression programs and (2) a defined substratum that not only supports both PSC maintenance and differentiation but can also convert soluble orthogonal ligands such as GFP into localized inputs that activate morphogenetic programs via synNotch signaling. We engineered four separate H9 hPSC lines to express a GFP-responsive synNotch receptor. In one line, GFP-induced synNotch activation renders overexpression of the reporter transgene mCherry, whereas the remaining three cell lines express either BMP4, WNT3A, or NODAL under synNotch control. We also engineered a novel substratum composed of defined peptides derived from vitronectin and fibronectin in addition to the GFP-capturing motif, GFP-TRAP. This customized substratum both supports PSC differentiation potential and facilitates GFP ligand-dependent synNotch activation in engineered cells. To constrain synNotch-PSCs in defined geometries to spatially gate morphogenetic events on the cellular level, rather than in bulk, we employ a deep UV protein patterning process. Briefly, a glass coverslip coated with the anti-fouling agent PLL-g-PEG is placed onto a quartz mask patterned with the desired geometry and exposed to deep UV (< 200 nm) for 3 minutes. The coverslip is then removed, and the UV-treated region is activated with EDC/NHS chemistry prior to functionalizing the patterned surface with our defined substratum. Cells are then seeded on the functionalized patterned surface and non-adherent cells are washed away after incubating leaving cells in the desired geometries.

Results, Conclusions, and Discussions: Results demonstrate that GFP ligand efficiently activates synNotch-PSCs cultured on the defined substratum both in monolayer and in micropatterned discs (Fig. 1A). Furthermore, the engineered surface showed superior ligand-dependent synNotch activation compared to a Geltrex™-coated surface pre-treated with GFP-TRAP (Fig. 1B). The novel substratum also supported synNotch-PSCs irrespective of maintenance or differentiation media used to culture cells, while also maintaining efficient synNotch activation upon GFP treatment (not shown). GFP-induced synNotch activation caused engineered PSCs to express WNT3a, BMP4, and NODAL transgenes; this gave rise to brachyury and/or kinase insert domain receptor (KDR) positive cells, indicating a transition to an early peri-gastrulation cell state (Fig. 1C). This was particularly prominent in the synNotch-WNT3a H9 hPSC cell line. When constraining synNotch-PSCs on our defined substratum to 500 µm discs, GFP-induced synNotch activation successfully induced WNT3a transgene expression, encouraging cells to differentiate into a brachyury-positive population primarily in the center of the disc. This region of high brachyury levels corresponded to diminished SOX2 levels, indicative of a transition from pluripotency towards a semi-patterned, early peri-gastrulation state (Fig. 1D).

We have successfully developed a defined substratum capable of supporting PSC maintenance and differentiation while also being able to convert soluble orthogonal ligands into localized inputs to activate morphogenetic programs via synNotch signaling in both monolayer and micropatterned discs. Furthermore, we have achieved ligand-inducible differentiation of synNotch-PSCs towards an early peri-gastrulation state in monolayer and patterned microdiscs. Current work extends these results on micropatterned surfaces to our NODAL and BMP4 synNotch-PSCs, in addition to our WNT3a synNotch-PSCs, to deploy synNotch as a platform to coordinate gastrulation and germ layer patterning.

Acknowledgements (Optional): This work is supported by NSF RECODE CBET-2033800 & NSF GRFP Grant No. 1937963.

References (Optional): ¹ Deglincerti et al. Nature Protocols (2016) ² Warmflash et al. Nat Methods (2014) ³ Brassard and Lutolf. Cell Stem Cell. (2019), ⁴ Morsut et al. Cell. (2016)