Abstract 11261: Human Pluripotent Stem Cell Derived Cardiomyocytes (hPS-CM) and High Content Analysis as a Reliable High Throughput Screening Method for Drug Cardiac Toxicity
Unexpected cardiotoxicity underlies high rates of attrition during drug development. In the last decade of the 20th century, eight non-cardiovascular drugs were withdrawn from clinical use because they prolonged the cardiac QT interval. It is thought that there is over-reliance on the use of animals and materials derived from animals in preclinical assays to predict the cardiotoxic effect of new drugs in humans. Here, we describe a combination of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and the high content analysis confocal microscopy as a potential solution for this major setback in drug development.
Initially human skin fibroblasts were reprogrammed into human induced pluripotent stem cells (hiPSCs) by lentiviral transduction and utilization of the following combination of transcription factors: Oct3/4, Sox2, c-Myc and Klf4. The human pluripotent stem cell phenotype of the generated hIPSCs was confirmed by RT-PCR analysis, alkaline phosphatase staining, protein array analysis and western blotting of pluripotency markers. Cardiac differentiation of hiPSCs to hiPSC-CM was achieved using embryonic bodies’ mass culture method. Emerged beating colonies were micro-dissected and the cardiac cell phenotype was confirmed by immunofluorescence and RT-PCR analysis of cardiac markers i.e. αMHC, cTNT, NKX2.5 and connexin 43. Seventeen known cardiotoxic compounds, as well as controls, were applied to the cells in 384 well formats at a dose of 10μM for 48 hours. Then hiPSC-CM underwent high content analysis confocal microscopy to simultaneously evaluate the cell mitochondrial transmembrane potential (using TMRM dye) and plasma membrane permeability (using TOTO-3 dye). All known cardiotoxins showed a significant decrease in mitochondrial transmembrane potential ranging from 74% to 95% and an increase in plasma membrane permeability ranging from 67-327 fold in comparison to the control. These results showed 100% prediction rate of cardiotoxicity of the known cardiotoxins.
In conclusion, combining the above described two state of the art technologies 1) hiPSC-CM and 2) high content analysis confocal microscopy, we were able to provide a reliable high throughput method for cardiotoxicity testing.
- © 2013 by American Heart Association, Inc.