Islet transplantation has proved to be a useful treatment for Type 1 diabetes mellitus, but inadequate supplies of transplantable donor tissue have intensified the need to find a renewable source of β-cells. Human embryonic stem cells (HESC) are pluripotent and may offer a viable alternative to donor islets, but their targeted differentiation to a more specific β-cell phenotype is proving challenging. One strategy to restore β-cell mass is through activation of progenitor cells present in the pancreas. The aim of this study was to isolate and characterise progenitor cells from pancreatic tissue obtained from patients with Congenital Hyperinsulinism of Infancy (CHI). An enzymatic digestion was used to isolate islets from four patients with CHI and CHI-derived cell lines (NES139, NES140, NES140 and NES144) were subsequently derived that had the potential to proliferate in vitro. The previously-described cell line NES2Y was utlised as a control cell line for comparison. Using RT-PCR, exon array, qPCR, immunocytochemistry and Ca2+ microfluorimetry techniques this thesis examines both the molecular and physiological characteristics of these four CHI-derived cell lines to establish their potential as populations of pancreatic progenitor cells. Genotyping revealed that all of the patients carried mutations in the SUR1 gene, ABCC8. Pancreatic endocrine progenitor markers (e.g. PDX1, SOX9 and HLBX9) as well as islet precursor markers (e.g. NKX2.2, NKX6.1, NEUROD1, PAX6 and FOXA2) were identified and their expression was stable over continuous cell culture. However, each of the cell lines failed to express other markers, specifically NGN3, PAX4, and ISLET1. Cell lines developed from each patient then underwent a fibroblast to epithelial-like morphological transition. High-throughput exon array analysis revealed a significant down regulation of ACTA2, VIM and upregulation of CDH1 (q value< 0.05), a gene expression pattern associated with a mesenchymal-to-epithelial transition. Analysis at the mRNA level identified that CHI-derived cell lines expressed those channels and transporters associated with the β-cell function of glucose-stimulated insulin secretion (GSIS). Yet, when expression of all five endocrine hormones was investigated, mRNA expression was undetectable in three CHI-derived cell lines, except for the expression of insulin in NES143. Protein level assessment, however, failed to detect any expression of insulin. Functional studies examining whole cellular calcium dynamics and those underlying GSIS revealed that, whilst ATP (0.1 mM) and histamine (0.1 mM) readily raised intracellular Ca2+, each of the cell lines failed consistently to respond to tolbutamide (0.1 mM), glucose (20 mM), diazoxide (0.1 mM) and KCl (40 mM), except for NES140 which responded to applications of acetylcholine (0.1 mM). Given the display of cellular plasticity, molecular and physiological characteristics, the data show CHI-derived cell lines mimic pancreatic progenitor cell populations. More importantly, they represent islet precursor cells of the secondary transition phase of pancreatic development. Future studies should concentrate on the inductive potential of these cells to produce mature insulin-secreting β-cells.
|Date of Award||1 Aug 2013|
- The University of Manchester
|Supervisor||Karen Cosgrove (Supervisor) & Mark Dunne (Supervisor)|
- Pancreatic Progenitors
- Congenital Hyperinsulinism