Abstract
The stresses encountered during islet isolation and culture may have deleterious effects on beta-cell physiology. However, the biological response of human islet cells to isolation remains poorly characterized. A better understanding of the network of signaling pathways induced by islet isolation and culturing may lead to strategies aimed at improving islet graft survival and function. Laser capture microdissection (LCM) was used to extract beta-cell RNA from 1) intact pancreatic islets, 2) freshly isolated islets, 3) islets cultured for 3 days, and changes in gene expression were examined by microarray analysis. We identified a strong inflammatory response induced by islet isolation that continues during in-vitro culture manifested by upregulation of several cytokines and cytokine-receptors. The most highly upregulated gene, interleukin-8 (IL-8), was induced by 3.6-fold following islet isolation and 56-fold after 3 days in culture. Immunofluorescence studies showed that the majority of IL-8 was produced by beta-cells themselves. We also observed that several pancreas-specific transcription factors were down-regulated in cultured islets. Concordantly, several pancreatic progenitor cell-specific transcription factors like SOX4, SOX9, and ID2 were upregulated in cultured islets, suggesting progressive transformation of mature beta-cell phenotype toward an immature endocrine cell phenotype. Our findings suggest islet isolation and culture induces an inflammatory response and loss of the mature endocrine cell phenotype. A better understanding of the signals required to maintain a mature beta-cell phenotype may help improve the efficacy of islet transplantation.
Highlights
Islet transplantation is a potential treatment for type1 diabetes but is limited by insufficient transplantable beta-cell mass and functional impairment after transplantation [1]
A better understanding of the network of signaling pathways induced by islet isolation and culture may lead to better strategies aimed at preventing islet cell death
We found that most insulin-positive cells co-stained with IL-8 in freshly isolated islets
Summary
Islet transplantation is a potential treatment for type diabetes but is limited by insufficient transplantable beta-cell mass and functional impairment after transplantation [1]. Our objective is to characterize the response of human beta-cells to isolation and culture in order to better maintain islets in culture and at the graft site. Islet isolation exposes these cells to a number of stresses that can adversely affect cell survival [4]. While various strategies have been explored over the past decade to improve isolated islet cell survival, there has yet to be an approach to prevent islet cell death that has translated successfully into clinical use. A better understanding of the network of signaling pathways induced by islet isolation and culture may lead to better strategies aimed at preventing islet cell death
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