Development of High Content Imaging Methods for Cell Death Detection in Human Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) are being investigated as a new source of cardiac cells for drug safety assessment. We developed a novel scalable high content microscopy-based method for the detection of cell death in hPSC-CM that can serve for future predictive in vitro cardio-toxicological screens. Using rat neonatal ventricular cardiomyocytes (RVNC) or hPSC-CM, assays for nuclear remodelling, mitochondrial status, apoptosis and necrosis were designed using a combination of fluorescent dyes and antibodies on an automated microscopy platform. This allowed the observation of a chelerythrine-induced concentration-dependent apoptosis to necrosis switch and time-dependent progression of early apoptotic cells towards a necrotic-like phenotype. Susceptibility of hPSC-CM to chelerythrine-stimulated apoptosis varied with time after differentiation, but at most time points, hPSC-CM were more resistant than RVNC. This simple and scalable humanized high-content assay generates accurate cardiotoxicity profiles that can serve as a base for further assessment of cardioprotective strategies and drug safety.Electronic supplementary materialThe online version of this article (doi:10.1007/s12265-012-9396-1) contains supplementary material, which is available to authorized users.

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“…For assessment of cardiotoxicity, Mioulane and co-workers [162] triggered cell stress with the cell-permeable protein kinase C inhibitor, chelerythrine. Coupling staining cells with potentiometric dye (TMRM; tetramethylrhodamine methyl ester), caspase-3 or BOBO-1 with Cellomics Arrayscan imaging assessed mitochondrial function, apoptosis and cell death, respectively.…”

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“…In addition to validating the efficacy of drugs such as Ro 31-8220, the hiPSC-CM system will be useful in evaluating potential cardiotoxicity, which is well established issue associated with kinase inhibitors [166]. Thus, Mioulane and co-workers [167] triggered cell stress with the cell-permeable protein kinase C inhibitor, chelerythrine. Combining stains for TMRM (a potentiometric dye; tetramethylrhodamine methyl ester), caspase-3 or BOBO-1 with Cellomics Arrayscan imaging, they assessed mitochondrial function, apoptosis and cell death, respectively and demonstrated that hPSC-CMs are less susceptible to chelerythine-stimulated apoptosis compared to rat neonatal ventricular CMs.…”

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“…For example, Sirenko et al (242) have used HCI platforms to perform CalceinAM, Hoechst, and MitoTracker imaging in hiPSC-CMs to evaluate the cardiotoxicity of 131 drugs affecting Na ϩ , K ϩ , and Ca 2ϩ channels, as well as adreno-, dopamine, and histamine receptors. Mioulane et al (193) also used the Thermo Scientific Arrayscan imaging platform to study cellular stress induced by chelerythrine, a cell-permeable protein kinase C inhibitor, by performing combined TMRM, caspase-3, and BOBO-1 staining to measure mitochondrial content, apoptosis, and cell death, respectively. More detailed high content assessment of hiPSC-CM mitochondrial function has been performed by measuring glycolysis and oxidative phosphorylation (OXPHOS) using the Seahorse Extracellular Flux Analyzer (63,289).…”