| 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 beta-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 beta-like cell phenotype in
Nes160 cells.
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