Synthetic Ligand Microenvironments for Synthetic Notch Activation and Co-Differentiation

Introduction: The fabrication of more complex tissue models and therapeutics remains challenging, due to the requirement of multiple cell types, various biological molecules, and distinct microarchitecture to recapitulate native tissue function. There is a critical need to develop strategies to support simultaneous differentiation of multiple lineages within 3D extracellular matrix. Synthetic notch (synNotch) receptors have emerged as powerful tools for user-defined control over cell differentiation and morphogenesis through intracellular signaling regardless of microenvironmental factors. In this work, we develop synNotch engineered cells and new ligand-presenting modules that can activate synNotch circuits via programmed microenvironments. We further utilize these tools to spatially coordinate skeletal muscle and endothelial differentiation within the same culture.

Materials and Methods: Mouse fibroblasts (C3H10T1/2) are co-transduced with designed plasmids using lentiviruses to generate stable cell lines. Ligand-presenting sender cells were generated by genetically encoding cell-membrane ligands or fibronectin-fused ligands. Ligand-containing extracellular matrix (ECM) was generated by culturing fibronectin-GFP secreting cells for 8 days then decellularization to remove cellular components while maintaining ECM integrity. Ligand-mediated activation of synNotch circuits is evaluated via ligand presented by sender cells, immobilized to polystyrene culture surface, or conjugated to hydrogel polymers via click chemistry. For dual patterning, a capillary fluidic based approach was utilized to spatially control the distribution of GFP and mCherry ligand solutions. Activation of synthetic transgenes and the extent of differentiation are evaluated via inducible fluorescent reporters, morphological changes, and lineage-specific markers.

Results, Conclusions, and Discussions: We first show that coculture of fibronectin-GFP producing cells with engineered synNotch reporters, that express mCherry in the presence of GFP, lead to activation of synthetic circuits. Further, engineered cells seeded onto decellularized fibrinogen-GFP similarly expressed mCherry indicating that ECM-cell interactions are sufficient to activate synthetic gene networks. We also demonstrate that ligands conjugated to microparticles as well as hydrogel substrates are able to interact and activate synthetic gene networks. In 3D matrix, encapsulation of engineered cells within GFP-conjugated gelatin methacryloyl (GelMA) hydrogels leads to robust synthetic gene expression, where over 70% of cells express the mCherry reporter. By patterning GFP-conjugated or unconjugated GelMA, we demonstrate spatially controlled and sustained reporter expression for up to 7 days in 3D culture.

Following synthetic gene integration, we demonstrate that GFP and mCherry ligands can induce the expression of master transcription factors MyoD or ETV2 respectively. Engineered fibroblasts cells seeded on a pattern of GFP and mCherry lead to spatially defined muscular and endothelial di­­­fferentiation co-differentiation, indicated by positive α-Actinin staining or VEGFR2 staining after 72 hours. Interestingly, both differentiation events can occur orthogonally and are highly correlated with the initial GFP and mCherry pattern.

By incorporating synthetic biology techniques with engineered materials, this platform enables multi-fate codifferentiation within the same culture by introducing distinct cellular functions for synthetic ligands GFP and mCherry. Through 3D bioprinted patterning, we aim to generate more complex models of skeletal muscle that incorporate mature myofibers and integrated endothelial networks.