Project Summary/Abstract Patients living with Type 1 Diabetes regulate their blood glucose through daily exogenous insulin administration – the only other option towards a path to insulin independence is donor islets/whole organ transplant, a limited resource, that replaces the cohort of cells that are lost in patients due to an autoimmune attack. The success of these transplants is highly variable, with several patients requiring a second or sometimes a third infusion of donor cells. Failure of allogeneic cell/tissue transplant often results from immune activation within the host, and strong systemic immunosuppression is necessary for successful engraftment of the transplant. Numerous approaches have been undertaken to alleviate the need for systemic immunosuppression – those focused on manipulating the graft have genetically removed the expression of cell surface proteins that, when mis-matched, lead to activation of the adaptive and innate immune systems. Such cells are potentially invisible to the immune system and pose the grave problem of serving as potential reservoirs of infection. If we had a method of identifying when a graft was experiencing inflammatory stress, we could design acute anti-inflammatory therapies to protect the graft from the assault – prolonging the time it takes for the graft to get accustomed to the niche and begin functioning. More recently, stem cells have emerged as a limitless resource of cells as a surrogate to donor islets, and early clinical attempts are currently underway. Replacement with stem cell derivatives circumvents the roadblock of limited transplant material – they are still faced with immune activation due to an allo-response after transplantation. Our goal is to address these challenges using state-of-the-art technologies we have pioneered. Combining the potential of stem cell therapy with an intracellular biosensing system that provides real time feedback in response to the transplantation niche, we will leapfrog allogeneic cell transplantation into the future – such a detection system can measure viability of grafts that can be predictive of successful engraftment and help define interventions to protect cells against inflammatory stress soon after transplantation. We have engineered nanoprobes that bind and respond to changes in miRNA levels and serve as intracellular sensors for cell health. Our nanoprobes are specific, sensitive, and can be detected in vivo in animal models. Here, we test our lead inflammation-sensitive biosensor in stem cell-derived insulin producing organoids – this combination can provide unique spectral read outs, in vitro and in small animal models, that result from a specific inflammatory response triggered in the cells, de-risking our final cell therapy product that will become part of a functional cure for patients living with type 1 diabetes.