Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis

Abstract

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation.Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltagesensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

Designing of spider silk proteins for human induced pluripotent stem cell-based cardiac tissue engineering

“…A cornerstone of this development is the advent of human induced pluripotent stem cells (hiPSCs) and protocols for their efficient differentiation into cardiac lineages [ [12] , [13] , [14] , [15] ]. The hiPSCs can be derived from patients’ own cells and easily modified by the genome editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) to introduce or correct specific mutations or to introduce reporters for monitoring specific processes [ 16 , 17 ].…”

A cell-based platform for oxidative stress monitoring in motor neurons using genetically encoded biosensors of H2O2

“…In the present work, we established a valid approach for studying neurodegeneration in cell- based models using a combination of methods such as CRISPR/Cas9 genome editing, genetically encoded biosensors, and iPSC differentiation. Nowadays, GE biosensors are more often applied for research of different physiological and pathological processes [47,52,53] where they replaced molecular probes. In the case of redox processes, it provided information about dynamics of components of redox balance, e.g., hydrogen peroxide or GSH/GSSG ratio, in different cell compartments and tissues [23,47,54,55].…”

iPSC-Cardiomyocyte Models of Brugada Syndrome—Achievements, Challenges and Future Perspectives

“…An advantage of iPSC-CMs compared to native CMs in this case is that iPSC-lines with stable GEVI/GECI expression can be created, stored and repeatedly differentiated to cardiomyocytes. Several GECIs (GCaMP5G, R-GECO1) and one GEVI (ArcLight) have been used in control and patient-derived iPSC-CMs and shown to consistently represent the calcium transients [ 80 , 81 , 82 , 83 , 84 ] and the transmembrane APs [ 82 , 83 , 84 , 85 , 86 ]. Although these reporters offer significant advantages over the traditional dyes, a major drawback is that the transgenes integrate randomly into the genome, raising serious concerns about potential gene disruption and alteration of local and global gene expression that could adversely affect normal cellular functions.…”