WS16.01Elucidating the pathogenesis of cystic fibrosis-related diabetes with induced pluripotent stem cells

Abstract

Objectives

Diabetes is the most common comorbidity of Cystic Fibrosis (CF), affecting nearly 50% of adult CF patients and severely worsening CF related disease mortality. While the root cause of CF-related diabetes (CFRD) is a mutation in the CFTR gene, the molecular mechanisms leading to CFRD are unclear, largely due to a lack of models that faithfully recapitulate human disease. The overall objectives of this study are to establish induced pluripotent stem cell (iPSC) derived islets (SC-islets) as a novel model for CFRD disease study and determine the consequence of the CFTR mutation in islet endocrine cell types.

Methods

iPSCs with G542X/G542X or F508del/F508del CFTR mutations (CF-iPSCs) were differentiated to SC-islets alongside wild-type (WT) H1 stem cells. SC-islet differentiation efficiency and hormone secretion were quantified with intracellular flow cytometry and enzyme linked immunosorbent assays (ELISAs).

Results

G542X-, F508del-, and H1-SCs differentiate to NKX6.1+/INS+ beta cells with 39±3%, 39±3%, and 42±2% efficiency, and GCG+/INS- alpha cells with 19±2%, 19±1%, and 35±4% efficiency respectively. G542X-, F508del-, and H1-beta cells are glucose responsive in vitro and secrete insulin at 1.8±0.4, 1.6±0.3, and 1.8±0.4 mIU/ml/10³ cells in high (20 mM) glucose conditions. G542X-, F508del-, and H1-alpha cells secrete 0.060±0.003, 0.84±0.24, and 0.70±0.12 pmol/L/10³ cells at low (2 mM) glucose conditions.

Conclusion

Our results demonstrate that CF-iPSCs differentiate to alpha and beta cells, and that there is no significant difference in insulin secretion for CF SC-islets and H1 SC-islets in vitro. Notably, our results show that G542X-islets secrete significantly lower glucagon at low glucose conditions compared to F508del- and H1-islets. Overall, these studies suggest a role for CFTR in alpha cell function and establish SC-islets as a novel and valuable model for studying CFRD.