Endocrine Abstracts

Congenital Hyperinsulinism (CHI) is primarily a β-cell disorder with an incomplete pathogenesis. The purpose of this study was to generate in vitro models of the disease for the purposes of investigating the relationship between gene defect and β-cell development and function. We obtained post-operative resections of pancreatic tissue from four patients with hyperinsulinism. The tissue was collagenase treated and maintained in cell culture conditions. Cell lines developed spontaneously from each patient and were expanded and cryopreserved for subsequent studies. PCR, immunohistochemistry and intracellular Ca²⁺ microfluorimetry techniques were used to examine the molecular and physiological properties of these cell lines. Genotyping reveled that all of the patients carried mutations in the SUR1 gene, ABCC8. In each of the cell lines – designated Nes139 (V1430fs), Nes140 (A4V/IVS38-1G>T), Nes143 (R620C/N), Nes144 (T172fs/ c.1818-?_1923+?del) islet and pancreatic endocrine progenitor cell selective markers have been identified and their expression stable over several passages and continuous cell culture. These include; Nkx2.2, NeuroD1, Gata6, Maf B, Pax6, Sox4, FoxA2, Arx, Hlbx9, Pbx1, Gata4, Hnf1B, Hnf1A, Hnf4A, Pdx1 and Sox9. Each of the cell lines failed to express Ngn3, Brn4, Pax4, Islet1, IAPP and insulin. Functional studies were undertaken with a number of agonists to raise cytosolic Ca²⁺ level and assess cell viability in the undifferentiated state, n=6 experiments in each case. Whilst ATP (0.1 mM) and histamine (0.1 mM) readily raised intracellular Ca²⁺, each of the cell lines failed to consistently respond Acetylcholine (0.1 mM), depolarising concentrations of KCl (40 mM) and glucose (15 mM). Collectively, the data show that the CHI-derived cell lines display an appropriate molecular and physiological phenotype for populations of proliferating pancreatic progenitor cells. Our results suggest they represent cells of the secondary transition phase of pancreatic development. Future studies will look towards the inductive potential of these cells to produce mature insulin-secreting β-cells.