Spatially Controlled Tissue Differentiation Using the Synthetic Receptor Synnotch

Spatial organization of multiple cell types is intrinsic to tissue function in vivo. One goal of tissue engineering is to recapitulate this cellular organization in vitro to create readily available and accurate models for drug screening, disease modeling, and regenerative medicine. However, the field struggles to recreate the organized complexity of native tissues with cellular scale precision. There have been many advancements in the fields of scaffold fabrication and synthetic biology that open new possibilities for tissue engineering. For example, microcontact printing enables researchers to pattern ligands onto a surface with micro-scale precision. A synergistic advancement in synthetic biology has been the Synthetic Notch receptor (synNotch), a modular receptor that allows control of transgene expression upon binding of prescribed ligands. Our goal is to combine these technologies by using microcontact printed ligands to spatially activate the synNotch receptor, and downstream transgenes, in cells. As a case study, we will use this approach to spatially control the differentiation of skeletal muscle cells by activating myoD in fibroblasts, laying the foundation for more accurate, multi-cell tissues in vitro. We have demonstrated that a surface presented ligand, GFP, can activate transgenes in cells via synNotch with spatial control. The activation can be patterned by micro-contact printing the ligand onto a cell adhesive coverslip. The spatial activation of the transgenes leads to spatially controlled myotube formation. Differentiated myotubes immuno-stain positive for alpha-actinin, a prominent myotube marker.