Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

High-throughput optical action potential recordings in hiPSC-derived cardiomyocytes with a genetically encoded voltage indicator in the AAVS1 locus

“…This was consistent with Frontiers in Cell and Developmental Biology frontiersin.org previously described knock-in of the ArcLight voltage sensor into the AAVS1 locus, exhibiting a higher reporter expression in CMs when compared to undifferentiated hiPSCs (Sun et al, 2020). The observed APs were similar to those reported for hiPSC-CMs transduced with lentiviral vectors encoding VSFP (Kaestner et al, 2015;Chen et al, 2017;Goedel et al, 2018). Importantly, insertion of the reporter in one AAVS1 allele resulted in expression levels sufficient to detect and measure APs in hiPSC-derived CMs.…”

“…APs were imaged according to the protocol described by Goedel and colleagues, with some modifications (Goedel et al, 2018). Briefly, the glass bottom micro-dishes with the hiPSC-CMs were placed on the stage of an inverted fluorescence microscope (DMI6000B, Leica Microsystems, Wetzlar, Germany), equipped with an image splitter, the appropriate filter sets (a 480/40 nm band-pass excitation filter combined with a 500 nm long-pass dichroic mirror in the microscope, and a 568 nm long-pass dichroic mirror combined with 520/ 28 nm and 630/75 nm band-pass emission filters in the image splitter), a HCX PL APO 63X/1.4-0.6 oil immersion objective (Leica Microsystems) as well as a Zyla V sCMOS camera (Andor Technology, Belfast, United Kingdom) capable of high-speed imaging at a high sensitivity.…”

“…Using lentivirus and adeno-associated virus (AAV) constructs, VSFP-based voltage sensors have already been applied to faithfully report membrane potentials in hiPSC-CMs (Chen et al, 2017;Goedel et al, 2018;Esfandyari et al, 2022). Lentiviral vectors have the advantage of providing high expression of the transgenes, however, they might be toxic to the cells and can randomly integrate into the host genome leading to potentially undesirable consequences (Rothe et al, 2013;Schlimgen et al, 2016).…”

Recapitulating porcine cardiac development in vitro: from expanded potential stem cell to embryo culture models

“…Electrical stimulation was performed at 0.5 Hz using field stimulation electrodes as described above. The VSFP was excited at 480 nm, and the emitted GFP and RFP fluorescence signals were separated using an image splitter (OptoSplit II, Caim Research) equipped with CAIRN HQ535.50 566DCXR E570LP filters ( Chen et al, 2017 ; Goedel et al, 2018 ). The fluorescence over cells and over background regions was quantified in GFP and RFP channels using ImageJ (National Institutes of Health).…”