Improved Beta-Cell Differentiation and Function in Isolated Islets Established in Dynamic Culture to Reduce Hypoxia

Abstract

Background: Pancreatic islet transplantation effectively prevents severe hypoglycaemia in type 1 diabetes. Restoration of normal β-cell functional mass remains elusive and we have recently demonstrated loss of β-cell end-differentiation in clinical islet recipients. We hypothesised that reducing islet hypoxia in culture would restore β-cell differentiation and function. Methods: MIN6 pseudo-islets (PI) and primary human islets (HI) were cultured in either static (SC) or horizontal rotation (RC) culture for 7 days. Hypoxia was detected by pimonidazole (Pim) adduct formation and hypoxia-induced gene expression (qPCR). β-cell end-differentiation was assessed by UCN3 expression and IF staining (urocortin-3+ insulin+ cells/total insulin+ cells) with function assayed by glucose-stimulated insulin secretion (GSIS). Results: Hypoxia was confirmed in established PI (70±1.0% Pim+). This was reduced in rotation culture (Pim+: 15±1.5% RC vs. 80±2.5% SC; p<0.05) with reduced hypoxia-induced gene expression compared with adherent MIN6 (LDHA: 6.0±2.1 RC vs. 86.0±7.8 fold SC; MCT4: RC 2.9±0.4 vs. 54.6±0.6 fold SC; p<0.05). RC enhanced UCN3 gene expression (4.2±0.5 RC vs. 2.2±0.2 fold SC; p<0.05) and increased UCN3/insulin co-staining (80±1.2% RC vs. 30±2.4% SC; p<0.0001) with enhanced GSIS stimulation index (4.1±0.1 RC vs. 2.8±0.1 SC; p<0.0001). Hypoxia-induced expression was reduced in HI RC compared with freshly isolated islets (LDHA: 0.3±0.1 RC vs. 1.8±0.04 fold SC; MCT4: 0.08±0.01 RC vs. 1.1±0.2 fold SC; p<0.05) with increased UCN3 expression (5.9±1.6 RC vs. 3.3±0.9 fold SC; p<0.05) and enhanced GSIS stimulation index (4.1±0.9 RC vs. 3.0±1.2 SC; p<0.05). Conclusion: Hypoxia in established PI and isolated HI was reduced over 7 days in dynamic culture. This was associated with enhanced β-cell differentiation and function providing a potential approach to maximise optimal functional mass in islet transplantation. Disclosure N.A. Al-Jahdhami: None. S.J. Anderson: None. A. Aldibbiat: None. J.A. Shaw: Advisory Panel; Self; Novo Nordisk A/S.