Single-Cell Transcriptomics Links Loss of Human Pancreatic ß-Cell Identity to ER Stress

The maintenance of pancreatic islet architecture is crucial for proper ß-cell function. We previously reported that disruption of human islet integrity could result in altered ß-cell identity. Here we combine ß-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of ß-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and ß-cell phenotype, and strengthen the concept of altered ß-cell identity as a mechanism underlying the loss of functional ß-cell mass. Overall design: Islets were dispersed into single cells by incubation with 0.025% trypsin (Gibco) supplemented with 10 mg/mL DNase (Pulmozyme, Genentech, San Francisco, CA, USA) for 6–7 min and filtered through a 70 µm cell-strainer. For ß-cell lineage tracing, the two lentiviral vectors pTrip-RIP405Cre-ERT2-?U3 (RIP-CreERT2) and pTrip–loxP-NEO-STOP-loxP-eGFP-?U3 (CMVstopGFP) were used, kindly provided by P. Ravassard. Dispersed cells were transduced overnight with a 1:1 mixture of the two lentiviruses in regular CMRL medium containing 8 µg/mL polybrene. In the morning the medium was refreshed, and 4-hydroxy-tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) was added to a final concentration of 1 µmol/L in the evening to activate RIP-CreERT2 and induce GFP expression in ß-cells specifically. After overnight incubation, the medium was refreshed and all cells were seeded onto 2% w/v agarose microwell chips containing 2865 microwells/chip with a diameter of 200 µm/microwell, resulting in cellular aggregates of ~1000 islet cells.