Dataset Information

Bioluminescent reporter assay for monitoring ER stress in human beta cells.

ABSTRACT: During type 1 diabetes development, cells in the islets of Langerhans engage adaptive mechanisms in response to inflammatory signals to cope with stress, to restore cellular homeostasis, and to preserve cell function. Disruption of these mechanisms may induce the formation of a repertoire of stress-induced neoantigens, which are critical in the loss of tolerance to beta cells and the development of autoimmunity. While multiple lines of evidence argue for a critical role of the endoplasmic reticulum in these processes, the lack of tools to specifically monitor beta cell stress hampers the development of therapeutic interventions focusing on maintaining endoplasmic reticulum homeostasis. Here we designed and evaluated a stress-induced reporter in which induction of stress correlates with increased light emission. This Gaussia luciferase-based reporter system employs the unconventional cytoplasmic splicing of XBP1 to report ER stress in cells exposed to known ER-stress inducers. Linking this reporter to a human beta cell-specific promotor allows tracing ER-stress in isolated human beta cells as well as in the EndoC-?H1 cell line. This reporter system represents a valuable tool to assess ER stress in human beta cells and may aid the identification of novel therapeutics that can prevent beta cell stress in human pancreatic islets.

SUBMITTER: Kracht MJL

PROVIDER: S-EPMC6288136 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Bioluminescent reporter assay for monitoring ER stress in human beta cells.

Kracht Maria J L MJL de Koning Eelco J P EJP Hoeben Rob C RC Roep Bart O BO Zaldumbide Arnaud A

Scientific reports 20181210 1

During type 1 diabetes development, cells in the islets of Langerhans engage adaptive mechanisms in response to inflammatory signals to cope with stress, to restore cellular homeostasis, and to preserve cell function. Disruption of these mechanisms may induce the formation of a repertoire of stress-induced neoantigens, which are critical in the loss of tolerance to beta cells and the development of autoimmunity. While multiple lines of evidence argue for a critical role of the endoplasmic reticu ...[more]

PMID: 30532033

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Project description:Biodistribution and fate of transplanted stem cells via longitudinal monitoring has been successfully achieved in the last decade using optical imaging. However, sensitive longitudinal imaging of transplanted stem cells in deep tissue like the brain remains challenging not only due to low light penetration but because of other factors such as low or inferior expression levels of optical reporters in stem cells and stem cell death after transplantation. Here we describe an optimized imaging protocol for sensitive long-term monitoring of bone marrow-derived human mesenchymal stem cells (hMSCs) expressing a novel bioluminescent/near infrared fluorescent (NIRF) fusion reporter transplanted in mouse brain cortex. Lentivirus expressing the luc2-iRFP720 reporter, a fusion between luc2 codon-optimized firefly luciferase (luc2) and the gene encoding NIRF protein iRFP720, was generated to transduce hMSCs. These cells were analyzed for their fluorescent and bioluminescent emission and checked for their differentiation potential. In vivo experiments were performed by transplanting decreasing amounts of luc2-iRFP720 expressing hMSCs in mouse brain, followed by fluorescence and bioluminescence imaging (BLI) starting 1 wk after cell injection when the blood-brain barrier was restored. Bioluminescent images were acquired when signals peaked and used to compare different luc2 substrate performances, that is, D-luciferin (D-Luc; 25 ?M/kg or 943 ?M/kg) or CycLuc1 (25 ?M/kg). Results showed that luc2-iRFP720 expressing hMSCs maintained a good in vitro differentiation potential toward adipocytes, chondrocytes, and osteocytes, suggesting that lentiviral transduction did not affect cell behavior. Moreover, in vivo experiments allowed us to image as low as 1 × 105 cells using both fluorescence and BLI. The highest bioluminescent signals (?1 × 107 photons per second) were achieved 15 min after the injection of D-Luc (943 ?M/kg). This allowed us to monitor as low as 1 × 105 hMSCs for the subsequent 7 wk without a significant drop in bioluminescent signals, suggesting the sustained viability of hMSCs transplanted into the cortex.

Dataset Information

Bioluminescent reporter assay for monitoring ER stress in human beta cells.

Publications

Bioluminescent reporter assay for monitoring ER stress in human beta cells.

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