Analysis of Beta-Cell Gene Expression Reveals Inflammatory Signaling and Evidence of Dedifferentiation following Human Islet Isolation and Culture

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.

C4b-binding Protein Protects β-Cells from Islet Amyloid Polypeptide-induced Cytotoxicity

“…Further, mRNA levels for C4BPA were highly up-regulated after 72 h of incubation of islets in vitro. Because C4BP is an acute phase protein, this finding is in agreement with observation that isolated islets show pro-inflammatory gene expression signature, which increases with time (28). In addition to being directly cytotoxic, IAPP induces inflammasome-dependent inflammation in the islet (29), and it is therefore likely that IAPP can therefore indirectly cause up-regulation of C4BP expression in the islet.…”

GABA Promotes Human β-Cell Proliferation and Modulates Glucose Homeostasis

“…Human islets were isolated as previously described (14). Pancreata from deceased nondiabetic adult human donors were retrieved after consent was obtained by Transplant Québec (Montreal, Canada).…”

Islet inflammation in type 2 diabetes

“…The procedure of islet preparation, based on enzymatic digestion and density gradient purification, can per se induce the expression of inflammatory mediators [34]. In addition, the length of the culture period from isolation to molecular assessments may play a role [35], and, finally, the endocrine cell heterogeneity of the islets precludes the clear determination of the properties of the beta cells [36]. A possible solution to some of these problems is the use of beta cells from frozen pancreas samples, as obtained by laser capture microdissection [37,38].…”