Engineering Programmable Material-To-Cell Pathways Via Synthetic Notch Receptors To Spatially Control Cellular Phenotypes In Multi-Cellular Constructs [snRNA-seq]

Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands. We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine. C3h/10t1/2 fibroblasts were engineered with 1) anti-GFP SynNotch activating myoD and miRFP AND anti-mCherry SynNotch activating ETV2 and BFP. These cells were then cultured/activated on patterned ibidi wells with No ligand, GFP rows, mcherry rows, or both GFP and mCherry interdigiting rows and cultured for 3 days before single nuclei RNA extraction and sequencing.