Activation of Synthetic Notch Gene Networks Via Material-Conjugated Synthetic Ligands

Synthetic notch (synNotch) receptors have emerged as powerful tools in controlling cell differentiation and morphogenesis¹. SynNotch user-defined networks have great potential for robustly controlling cell behavior by enabling environmental ligands to directly (no signaling intermediates) induce target gene expression. This approach can improve the multiplexed and spatial control of cell phenotype for the development of more complex 3D multicellular tissues, improving the relevancy of tissue models and therapeutics. However, synNotch activation has relied on cell-cell contact with sender cells that present membrane-tethered ligands. Cell-presented ligand systems require an additional cell engineering step and remain challenging to control in 3D environments. To enable the control over synNotch gene networks in engineered 3D tissues, the development of novel tools that can both present ligands and retain the capacity to activate mechanosensitive synNotch receptors are required.

To this aim, we have demonstrated that the covalent immobilization of synthetic ligands to materials can efficiently activate synNotch gene networks. Green fluorescent protein (GFP) was conjugated to microparticles and hydrogel polymers by carbodiimide or click chemistry linkers, respectively. Murine fibroblasts were engineered to express GFP-responsive synNotch receptors that drive the expression of reporter protein, mCherry. SynNotch engineered cells were cocultured with GFP-conjugated microparticles or encapsulated within GFP-conjugated gelatin methacryloyl hydrogels, and the extent of synNotch activation was quantified based on the percent and intensity of mCherry-expressing cells. We have found that material-conjugated ligands can induce synNotch gene activation similar to ligand-presenting sender cells. Modular conjugation approaches and the capacity for multi-ligand presentation can be used as a platform to spatially control target gene activation in 3D hydrogels. In future work, we will characterize the effect of material properties on SynNotch activation and use these tools to control multi-fate differentiation in 3D engineered tissues.

1. Morsut L, et al. Cell. 2016;164(4):780-91.