Gene Expression and Profiling of Human Islet Cell Subtypes: A Master’s Thesis

Abstract

Background: The endocrine pancreas contains multiple cell types co-localized into clusters called the Islets of Langerhans. The predominant cell types include alpha and beta cells, which produce glucagon and insulin, respectively. The regulated release of these hormones maintains whole body glucose homeostasis, essential for normal metabolism and to prevent diabetes and complications from the disease. Given the heterogeneous nature of islet composition and absence of unique surface markers, many previous studies have focused on the whole islet. Sorting islet cells by intracellular hormone expression overcomes this limitation and provides pure populations of individual islet cell subsets, specifically alpha and beta cells. This technique provides the framework for characterizing human islet composition and will work towards identifying the genetic changes alpha and beta cells undergo during development, growth, and proliferation. Methods: Human islets obtained from cadaveric donors are dissociated into a single cell suspension, fixed, permeabilized, and labeled with antibodies specific to glucagon, insulin, and somatostatin. Individual alpha, beta, and delta cell populations are simultaneously isolated using fluorescence activated cell sorting. Candidate gene expression and microRNA profiles have been obtained for alpha and beta cell populations using a quantitative nuclease protection assay. Thus far, RNA has been extracted from whole islets and beta cells and subjected to next generation sequencing analysis. Results: The ratio of beta to alpha cells significantly increases with donor age and trends higher in female donors; BMI does not appear to significantly alter the ratio. Further, we have begun to investigate the unique gene expression profiles of alpha and beta cells versus whole islets, and have characterized the microRNA profiles of the two cell subsets. Conclusions: By establishing methods to profile multiple characteristics of alpha and beta cells, we hope to determine how gene, miRNA, and protein expression patterns change under environmental conditions that lead to beta cell failure or promote beta cell development, growth, and proliferation.