Next generation sequencing of laser capture microdissected single islet cells from metabolically phenotyped living donors

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Metabolically phenotyped pancreatectomised patients, defined as living donors (LD), are a novel and reproducible source of pancreatic islets for research. As conventional enzymatic isolation of islets can alter their gene expression, we routinely obtain highest-quality samples for transcriptomics by laser capture microdissection (LCM) from snap-frozen surgical tissues in our biobank, which is the largest of this type worldwide. Following our previous NGS transcriptomics and proteomic analyses on LCM bulk islets, we propose here to profile with a non-targeted approach gene expression in LCM single islet cells from LD with normoglycemia (10), impaired glucose tolerance (10) or type 2 diabetes (10). This shall enable us to unequivocally identify the cell type origin of gene expression changes found through bulk islet NGS which correlate with progressive deterioration of hyperglycemia. Generated data will be integrated with other datasets from these LD, including clinical data, proteomics of LCM bulk and single islet cells (ongoing) as well as shotgun lipidomics of LCM islets and plasma samples. Thus, the accomplishment of this ambitious, unprecedented project, shall generate a novel atlas of gene expression in islet cell types in situ along the continuum from health to T2D and thus be a study of reference in the field.