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Title:	Purification of replicating pancreatic β-cells for gene expression studies
Author:	Carballar, Reyes; Canyelles, Maria de Lluc; Fernández, Claudia; Martí, Yasmina; Bonnin, Sarah; Castaño Boldú, Esther; Montanya Mias, Eduard; Téllez i Besolí, Noèlia
Abstract:	β-cell proliferation is a rare event in adult pancreatic islets. To study the replication-related β-cell biology we designed a replicating β-cells sorting system for gene expression experiments. Replicating β-cells were identifed by EdU incorporation and purifed by fow cytometry. For β-cell separation islet cells were sorted by size, granularity and Newport Green fuorescence emission that was combined with emitted fuorescence for EdU-labelled replicating cells sorting. The purity of the resulting sorted populations was evaluated by insulin staining and EdU for β-cell identifcation and for replicating cells, respectively. Total RNA was isolated from purifed cell-sorted populations for gene expression analysis. Cell sorting of dispersed islet cells resulted in 96.2% purity for insulin positivity in the collected β-cell fraction and 100% efciency of the EdU-based cell separation. RNA integrity was similar between FACSsorted replicating and quiescent β-cells. Global transcriptome analysis of replicating vs quiescent β-cells showed the expected enrichment of categories related to cell division and DNA replication. Indeed, key genes in the spindle check-point were the most upregulated genes in replicating β-cells. This work provides a method that allows for the isolation of replicating β-cells, a very scarce population in adult pancreatic islets.
Subject(s):	-Expressió gènica -Malalties del pàncrees -Diabetis -Gene expression -Pancréas diseases -Diabetes
Rights:	cc-by (c) Carballar, Reyes et al., 2017 http://creativecommons.org/licenses/by/3.0/es
Document type:	Article Article - Published version
Published by:	Nature Publishing Group
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