Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells

Dario Gerace; Rosetta Martiniello-Wilks; Rosaline Habib; Ann Margaret Simpson

doi:10.1371/journal.pone.0220013

Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells

PLoS One. 2019 Jul 18;14(7):e0220013. doi: 10.1371/journal.pone.0220013. eCollection 2019.

Authors

Dario Gerace¹, Rosetta Martiniello-Wilks^{1

2}, Rosaline Habib¹, Ann Margaret Simpson¹

Affiliations

¹ The School of Life Sciences and the Centre for Health Technologies, University of Technology Sydney, Sydney, Australia.
² Translational Cancer Research Group, University of Technology Sydney, Sydney, Australia.

Abstract

Due to their ease of isolation, gene modification and tumor-homing properties, mesenchymal stem cells (MSCs) are an attractive cellular vehicle for the delivery of toxic suicide genes to a variety of cancers in pre-clinical models. In addition, the incorporation of suicide genes in stem cell-derived cell replacement therapies improves their safety profile by permitting graft destruction in the event of unexpected tumorigeneses or unwanted differentiation. Due to the functional requirement of ATP for the Firefly luciferase gene Luc2 to produce light, luciferase-based reporting of cytotoxicity can be engineered into potential cell therapies. Consequently, we nucleofected mammalian expression plasmids containing both the Luc2 and the yeast fusion cytosine deaminase uracil phosphoribosyltransferase (CDUPRT) genes for expression in murine MSCs to assess luciferase as a reporter of suicide gene cytotoxicity, and MSC as vehicles of suicide gene therapy. In vitro bioluminescence imaging (BLI) showed that following the addition of the non-toxic prodrug fluorocytosine (5-FC), CDUPRT-expressing MSCs displayed enhanced cytotoxicity in comparison to Luc2 reporter MSC controls. This study demonstrates the utility of luciferase as a reporter of CDUPRT-mediated cytotoxicity in murine MSC using BLI.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biomarkers
Cell Line
Gene Expression*
Genes, Reporter*
Genes, Transgenic, Suicide*
Immunophenotyping
Luciferases / genetics*
Luciferases / metabolism
Mesenchymal Stem Cells / cytology
Mesenchymal Stem Cells / metabolism*
Mice

Substances

Biomarkers
Luciferases

Grants and funding

The MSC isolation technique and luciferase-based suicide gene reporter bioassay were developed by RMW and the UTS TCRG. This work was funded by a Sydney Vital/Cancer Institute NSW/University of Sydney Seed Funding Research Grant, Prostate Cancer Foundation of Australia (PCFA) Research Project grant PR-37 and UTS Early Career Research Grant awarded to RMW. The NOD MSC research received further support from the Diabetes Australia Research Trust and Rebecca L. Cooper Medical Research Foundation grants awarded to AMS and RMW. DG was supported by an Australian Postgraduate Award and a scholarship from the Arrow Bone Marrow Transplant Foundation. RH was supported by a UTS Research Excellence PhD Scholarship and UTS Top-up scholarship from the Translational Cancer Research Network (TCRN).