Purification of replicating pancreatic β-cells for gene expression studies

Carballar, Reyes; Canyelles, Maria de Lluc; Fernández, Claudia; Martí, Yasmina; Bonnin, Sarah; Castaño, Esther; Montanya, Eduard; Téllez, Noèlia

doi:http://dx.doi.org/10.1038/s41598-017-17776-2

Purification of replicating pancreatic β-cells for gene expression studies

License

Citation

Carballar R, Canyelles ML, Fernández C, Martí Y, Bonnin S, Castaño E et al. Purification of replicating pancreatic beta-cells for gene expression studies. Sci Rep. 2017 Dec 13;7(1):17515. DOI: 10.1038/s41598-017-17776-2

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 identified by EdU incorporation and purified by flow cytometry. For β-cell separation islet cells were sorted by size, granularity and Newport Green fluorescence emission that was combined with emitted fluorescence for EdU-labelled replicating cells sorting. The purity of the resulting sorted populations was evaluated by insulin staining and EdU for β-cell identification and for replicating cells, respectively. Total RNA was isolated from purified 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% efficiency of the EdU-based cell separation. RNA integrity was similar between FACS-sorted 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.