Fluorescent hiPSC-derived MYH6-mScarlet cardiomyocytes for real-time tracking, imaging, and cardiotoxicity assays

Abstract

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) hold great potential in the cardiovascular field for human disease modeling, drug development, and regenerative medicine. However, multiple hurdles still exist for the effective utilization of hiPSC-CMs as a human-based experimental platform that can be an alternative to the current animal models. To further expand their potential as a research tool and bridge the translational gap, we have generated a cardiac-specific hiPSC reporter line that differentiates into fluorescent CMs using CRISPR-Cas9 genome editing technology. The CMs illuminated with the mScarlet fluorescence enable their non-invasive continuous tracking and functional cellular phenotyping, offering a real-time 2D/3D imaging platform. Utilizing the reporter CMs, we developed an imaging-based cardiotoxicity screening system that can monitor distinct drug-induced structural toxicity and CM viability in real time. The reporter fluorescence enabled visualization of sarcomeric disarray and displayed a drug dose–dependent decrease in its fluorescence. The study also has demonstrated the reporter CMs as a biomaterial cytocompatibility analysis tool that can monitor dynamic cell behavior and maturity of hiPSC-CMs cultured in various biomaterial scaffolds. This versatile cardiac imaging tool that enables real time tracking and high-resolution imaging of CMs has significant potential in disease modeling, drug screening, and toxicology testing. Graphical abstract

Gene editing innovations and their applications in cardiomyopathy research

“…Patient-specific hiPSC-cardiomyocytes harboring a mutation in MYBPC3 and grown in two-dimensional and three-dimensional cultures showed upregulation of pathways associated with hypertrophy, and especially aberrant calcium signaling, compared to isogenic controls yielded by CRISPR-mediated repair ( Seeger et al, 2019 ). Besides studying genetic cardiomyopathies, CRISPR has been used to generate gene-specific reporter lines to study hiPSC-derived-cardiomyocyte-specific properties, such as sarcomere disarray, contractility and calcium handling ( Maria Cherian et al, 2023 ; Ribeiro et al, 2020 ; Sharma et al, 2018 ; Toepfer et al, 2020 ; Lin et al, 2018 ).…”