Induction of differentiation of a human pancreas-derived cell line towards an endocrine cell fate

UoM administered thesis: Phd

  • Authors:
  • Helga Nayelli Palma Gutierrez

Abstract

Diabetes mellitus is the most common metabolic disorder worldwide and a major health problem. Islet transplantation has been a successful treatment for type 1 diabetes mellitus. However, the potential of transplantation-based therapy is currently limited by the shortage of transplantable material from human donors. New sources of insulin-secreting cells are being investigated, such as human embryonic stem cells, induced pluripotent stem cells or adult stem/progenitor cells. The aim of this study was to assess the differentiation potential of a novel human pancreatic progenitor cell line (Nes160). The effects of the small molecule inducer of differentiation isoxazole [3,5-disubstituted isoxazole; N-cyclopropyl-5-(thiophen-2-yl)isoxazole-3-carboxamide] on promoting cell differentiation in Nes160 cells towards a mature endocrine lineage were investigated in this study. The effects of isoxazole on Nes160 cells were evaluated by establishing a gene expression profile. Results of RT-PCR and qPCR analyses showed that treatment with isoxazole successfully induced pro-differentiation effects in Nes160 cells, by inducing/enhancing expression of transcription factors important to initiate the endocrine developmental program and to promote β-cell maturation such as PDX1, NGN3, NEUROD1, PAX4 and NKX6.1. Isoxazole treatment even promoted the formation of cluster-like aggregates in Nes160 cells. A larger study of the effects of isoxazole on Nes160 cells was conducted by performing microarray analysis on RNA samples collected from cells treated with isoxazole during two different time points and their corresponding time-matched controls. This analysis allowed the identification of 340 differentially expressed genes after two days of treatment with isoxazole and 425 after six days of treatment, compared to DMSO-vehicle controls and using a cutoff filter of ≥ 3 fold change for both, up and down-regulated genes. Functional analysis and processes related to the differentially expressed genes were determined using Ingenuity Pathway Analysis (IPA) and gene ontology classification (PANTHER) software. Results of those analyses showed that the biological processes that were altered by the effects of isoxazole corresponded to cell communication and cell development; and the pathways that were activated were related to remodelling of the actin cytoskeleton, transcriptional regulation, and cell movement. The results presented in this study show the potential of isoxazole to induce pro-differentiation effects and the plasticity of the Nes160 cells to initiate the pancreatic endocrine program. Further studies are needed to elucidate how to achieve a fully mature and functional β-like cell phenotype in Nes160 cells.

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Original languageEnglish
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Award date1 Aug 2018