Project Summary The field of regenerative medicine focuses on addressing degenerative cell behaviors arising from issues like aging, injury, autoimmune diseases, and cancer. Two main elements in regenerative medicine are cell therapies and biomaterial scaffolds. Cell therapies have become an increasingly viable therapeutic strategy as ways to control cellular behaviors have become ever more sophisticated. However, many current technologies to engineer cellular behaviors rely on bulk delivery of soluble factors, which can lead to off-target effects on surrounding tissues. Designer biomaterials provide physicochemical cues that govern migration, proliferation, and differentiation of immune and stem cells encapsulated or in proximity to these surrogate extracellular matrices. However, the influence of biomaterials over cell behaviors is limited, as many designs rely on activation of native signaling pathways with responses that can be difficult to precisely engineer. Therefore, the overall goal of this proposal is to develop a hydrogel biomaterial formulation that can control cellular behaviors in a user- defined manner for regenerative medicine purposes. This hydrogel will interface with engineered cells to enable artificial signaling in response to bioinert ligands that activate synthetic receptors. To achieve our goal, we will use these methods: (1) Cells will be engineered with synthetic signaling receptors to allow for user-defined inputs and outputs; (2) Hydrogel formulations will be designed to capture soluble ligands for dynamic activation of engineered cells. We propose to accomplish this through three Specific Aims: (1) We will engineer a hydrogel formulation that supports cell viability and is able to capture soluble bioinert ligands for tunable and inducible reporter activation of engineered cells; (2) We will utilize this hydrogel formulation and engineered cells to attenuate inflammation in a separate population of fibroblasts through production of cytokine antagonists; (3) We will differentiate a population of multipotent cells encapsulated in our hydrogel formulation into osteogenic and chondrogenic lineages. We hypothesize that this new hydrogel platform will allow for dynamic and user-defined activation of encapsulated engineered cells in a spatially constrained and ligand-inducible manner. This research will contribute a new technology for controlling cell behaviors for therapeutic functions that can be applied to a range of disease states, such as insulin secretion for diabetes, growth factor secretion for wound healing, and differentiation into muscle fibers for tissue regeneration. Here we also propose a multidisciplinary mentoring and training plan in the areas of materials engineering, cell therapy, and synthetic biology that will provide high-level training for the applicant in preparation for a career in cell therapies.