*Purpose: Pancreatic beta cells dedifferentiate upon exposure to metabolic and inflammatory stress characterized by loss of beta-cell identity genes, expression of beta-cell disallowed genes, and loss of function. Here we sought to elucidate cell signaling pathways regulating genes during adaptive stress responses to identify cellular targets for restoring and maintaining islet cell function during islet isolation procedures and transplantation.

*Methods: Human and mouse islets were isolated form donor pancreases and treated with high glucose (16.7 mM) and cytokine cocktail (IL-1β, TNF-α, and IFN-γ) for 2h and 24h. Transgenic mice harboring INS1^CRE/ERT2:NFATc2^f/f were used for beta-cell knock out (BKO) islets. Gene expression was analyzed by RNA-Seq and QPCR. Western blot analyses used anti-RFX6 and MCT-1 antibodies. Promoter analyses were performed by promoter-reporter and ChIP assay. Ca²⁺ fluorometric imaging quantified intracellular Ca²⁺ changes. ELISA measured glucose-stimulated insulin secretion functional assays on islets.

*Results: Both human and mouse islets exposed to acute (2h) metabolic and inflammatory stress showed enhanced upregulation of several beta-cell differentiation genes. In contrast, long-term exposure of islets (>24h) to stress resulted in downregulation of beta-cell differentiation genes and induction of more than twenty disallowed beta-cell genes. Transcriptional changes of several of these key genes including RFX6 and MCT-1 during extended stress exposure were due to downstream ER stress, Ca²⁺ impairment, and loss of CN/NFATc2 signaling. Ca²⁺ and CN/NFATc2 signaling were restored in beta cells by small molecule ISX9, a differentiation inducer. Transgenic BKO islets showed spontaneous beta-cell dedifferentiation, dysregulated insulin secretion, and could not be restored by ISX9.

*Conclusions: CN/NFATc2 regulate genes that maintain beta cells in a differentiated state during islet cell stress and are lost upon sustained ER stress and overstimulated intracellular Ca²⁺. Reactivating Ca²⁺/CN/NFAT signaling with ISX9 induces redifferentiation of stressed islets and restores them to a functional state. The study suggests that CN/NFATc2 can be targeted to restore and maintain potency of islets during inflammatory stresses imposed throughout procedures of islet transplantation.

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