iPSC-Derived Cardiomyocytes and Luciferase Reporters: A Robust Reporting Platform for Monitoring Cardioprotection and Pathway Biology in Endogenous Human Tissue Cells
Poster Oct 09, 2015
Fiene, S., Thompson, A., Niles, A., Robers, M., Anson, B.
Pathophysiological conditions, medical interventions, and off-target toxicities can all result in cellular oxidative stress. In cardiac myocytes, prolonged and/or excessive oxidative stress can lead to cardiotoxicity: a primary cause of developmental delays, black-box warnings, and post-launch withdrawal of pharmaceuticals. Many protective responses to oxidative stress are mediated at the transcriptional level through antioxidant response elements (AREs). iCell® Cardiomyocytes (Cellular Dynamics International) are fully functional cardiomyocytes derived from human induced pluripotent stem cells (iPSCs). The ability to monitor ARE activity in this relevant tissue cell would provide an excellent tool for both interrogating and enhancing cardioprotective processes through basic experimental and screening paradigms, respectively. To that end, a novel ARE luciferase reporter construct, pGL4[luc2P/NRF2/Hygro] (ARE-luc: Promega) was transiently transfected into iCell Cardiomyocytes and functionally validated by inducing oxidative stress through application of tertiary butylhydroquinone (tBHQ) and monitoring increases in luciferase activity. Control experiments with GFP demonstrated that lipid-based methodologies could introduce the plasmid into the terminally differentiated iCell Cardiomyocytes with greater than 40% transfection efficiency with minimal optimization. Transfection of ARE-luc into iCell Cardiomyocytes and subsequent incubation with tBHQ produced increases in luciferase activity in a dose dependent manner. Similarly, luciferase-based CRE and NFAT reporters acted as a surrogate readout for GPCR modulation by generating dose dependent increases in luciferase activity upon transfection into iCell Cardiomyocytes and stimulation with isoproterenol. Together, these data demonstrate that a variety of luciferase-based reporters are easily transfected into iCell Cardiomyocytes. The combination of a nearly-limitless supply of relevant human tissue cells and a robust reporter system promises great utility for pathway analysis for both basic and applied research endeavors.
The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s), phase II metabolizing enzymes and transporter genes in response to xenobiotics, including prescription drugs.READ MORE
We found a distinct subpopulation of Tregs within BMSCs. Tregs and BMSCs in co-culture conferred neuroprotection that varied in a dose-dependent manner. Tregs minimized stem cell production of IL-6, a pro-inflammatory cytokine, and inhibited BMSC secretion of FGF-beta, a cytokine related to BMSC proliferation and differentiation. The ratio of Tregs found natively in BMSCs is optimally adapted to provide the maximum neuroprotective benefit of stem cell treatment after ischemic stroke.READ MORE