Differentiation of human cell line towards a pancreatic endocrine lineage

Abstract

Islet transplantations have been successful in restoring glucose homeostasis in patients with diabetes; however, the limited number of donor organs limits the success of this treatment. The lineage reprograming of different cell sources to beta cells potentially provides an unlimited supply of insulin-producing cells for regenerative therapy for patients with diabetes. The aim of this study was to investigate the ability to transdifferentiate two cell lines into an endocrine lineage. Insulin production in pancreatic beta cells can be increased using a small molecule, 3,5-disubstituted isoxazole, N-cyclopropyl-t-(thiophen-2-yl)isoxazole-3-carboxamide (isoxazole) but its effect on other cell types has not been reported. Here, we investigated the lineage reprogramming of PANC-1 pancreatic ductal cells to insulin producing cells by isoxazole treatment. Gene expression was performed using RT-PCR and qPCR for approximately 30 genes critical to beta cell development and function. In addition, quantitative proteomic profiling was performed using LC-MS by monitoring protein abundance in isoxazole-treated PANC-1 cells compared to time-matched controls. Isoxazole treatment stimulated PANC-1 cells to aggregate into islet-like clusters and gene expression analysis revealed induction of important developmental beta cell markers including NGN3, NEUROD1 and INSULIN. In addition, beta cell surface markers were also upregulated such as CD200, GPR50, TROP-2, GLUT2 and SLC30A8. Using LC-MS a catalogue of approximately 2400 identified proteins was generated; 257 proteins were differentially expressed in isoxazole-treated cells compared to DMSO-vehicle controls at p

Date of Award	31 Dec 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Gillian Edwards (Supervisor) & Karen Cosgrove (Supervisor)