Regulation of islet cell differentiation and function by calcineurin/NFATc2 signaling and applications for islet cell transplantation.

Diabetes is a leading cause of death globally and is a result of high blood glucose concentrations contributing to metabolic impairments and high associated risk of cardiovascular disease, nephropathy, neuropathy, stroke, blindness, and loss of limbs by amputation. Islet cell transplantation represents a promising minimally invasive procedure to reverse diabetes and prevent hypoglycemia by replacing all types of islet endocrine cells that regulate blood glucose. Challenges that limit widespread use of islet replacement therapy include immune-mediated loss of islet graft function and lack of available donor pancreas tissue for transplantation. Pancreatic β cells succumb to metabolic and inflammatory stress by loss of β-cell identity genes, induction of β-cell disallowed genes, and loss of function. In the first aim, I elucidated the role of CN/NFATc2 in islet stress response. This work identified CN/NFAT as a key therapeutic target for pro-differentiation and maintenance of β-cell function during stress. Specifically, I show that small molecule differentiation inducer isoxazole-9 (ISX9) could preserve CN/NFATc2 signaling by preventing overstimulation and exhaustion of intracellular calcium stores and subsequent β-cell dedifferentiation. ISX9 also preserved glucose-responsive insulin secreting capacity in islets and improved islet graft function in a mouse islet transplant model. In the second aim, I developed methods to expand and pro-differentiate islet cell precursors in culture and in vivo by HDAC inhibition and induction of CN/NFATc2. Specifically, I identified a subpopulation of RGS16+ islet cell precursors that could differentiate into functional islet organoids which produced both β- and α-like cells within 14 d exposure to ISX9. This was attributed in part to NFATc2-mediated expression of the early islet endocrine progenitor specification gene RFX6 which induced a cascade of downstream transcription factors required for islet cell differentiation. Lastly, I present data and explore the potential of Hedgehog-GLI (HH-GLI) signaling targets for driving islet cell precursors toward β- and α-like cell type specification. My preliminary data indicate that manipulation of HH signaling in islet cell precursors can influence β- and α-cells fate in islet organoids. These findings provide proof of principle that adult islet cell precursors can be expanded and engineered for potential use in islet cell replacement therapies.