Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) offer an attractive platform for cardiovascular research. Patient-specific iPSC-CMs are very useful for studying disease development, and bear potential for disease diagnostics, prognosis evaluation and development of personalized treatment. Several monolayer-based serum-free protocols have been described for the differentiation of iPSCs into cardiomyocytes, but data on their performance are scarce. In this study, we evaluated two protocols that are based on temporal modulation of the Wnt/β-catenin pathway for iPSC-CM differentiation from four iPSC lines, including two control individuals and two patients carrying an SCN5A mutation. The SCN5A gene encodes the cardiac voltage-gated sodium channel (Nav1.5) and loss-of-function mutations can cause the cardiac arrhythmia Brugada syndrome. We performed molecular characterization of the obtained iPSC-CMs by immunostaining for cardiac specific markers and by expression analysis of selected cardiac structural and ionic channel protein-encoding genes with qPCR. We also investigated cell growth morphology, contractility and survival of the iPSC-CMs after dissociation. Finally, we performed electrophysiological characterization of the cells, focusing on the action potential (AP) and calcium transient (CT) characteristics using patch-clamping and optical imaging, respectively. Based on our comprehensive morpho-functional analysis, we concluded that both tested protocols result in a high percentage of contracting CMs. Moreover, they showed acceptable survival and cell quality after dissociation (>50% of cells with a smooth cell membrane, possible to seal during patch-clamping). Both protocols generated cells presenting with typical iPSC-CM AP and CT characteristics, although one protocol (that involves sequential addition of CHIR99021 and Wnt-C59) rendered iPSC-CMs, which were more accessible for patch-clamp and calcium transient experiments and showed an expression pattern of cardiac-specific markers more similar to this observed in human heart left ventricle samples.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are currently used following the Comprehensive in vitro Proarrhythmic Assay (CiPA) initiative and subsequent recommendations in the International Council for Harmonization (ICH) guidelines S7B and E14 Q&A, to detect drug-induced cardiotoxicity. Monocultures of hiPSC-CMs are immature compared to adult ventricular cardiomyocytes and might lack the native heterogeneous nature. We investigated whether hiPSC-CMs, treated to enhance structural maturity, are superior in detecting drug-induced changes in electrophysiology and contraction. This was achieved by comparing hiPSC-CMs cultured in 2D monolayers on the current standard (fibronectin matrix, FM), to monolayers on a coating known to promote structural maturity (CELLvo™ Matrix Plus, MM). Functional assessment of electrophysiology and contractility was made using a high-throughput screening approach involving the use of both voltage-sensitive fluorescent dyes for electrophysiology and video technology for contractility. Using 11 reference drugs, the response of the monolayer of hiPSC-CMs was comparable in the two experimental settings (FM and MM). The data showed no functionally relevant differences in electrophysiology between hiPSC-CMs in standard FM and MM, while contractility read-outs indicated an altered amplitude of contraction but not changes in time course. RNA profiling for cardiac proteins shows similarity of the RNA expression across the two forms of 2D culture, suggesting that cell-to-matrix adhesion differences may explain account for differences in contraction amplitude. The results support the view that hiPSC-CMs in both 2D monolayer FM and MM that promote structural maturity are equally effective in detecting drug-induced electrophysiological effects in functional safety studies.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Research Foundation - Flanders (FWO) Introduction SCN5A encodes the α-subunit of voltage-gated cardiac sodium channel Nav1.5. Mutations in SCN5A are identified in about 20% of patients with Brugada syndrome (BrS), an inherited cardiac arrhythmia. We have identified an SCN5A founder mutation (c.4813+3_4813+6dupGGGT), leading to a loss-of-function of Nav1.5 in 25 different families. Mutation carriers show variable expression of the phenotype: from asymptomatic to syncopes and sudden cardiac death. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) to investigate the underlying pathophysiology. Material & Methods Dermal fibroblasts of six patients with different disease severity, and two unrelated healthy control individuals were reprogrammed using a commercially available reprogramming kit. iPSC-CMs were differentiated following a published protocol. We performed several differentiation rounds and investigated expression of cardiac markers using qPCR and immunocytochemistry and electrophysiological properties using patch-clamping. Results All iPSC-CMs expressed the tested markers. We observed reduction in sodium current density in patient iPSC-CMs compared to the control cells. However, our data display variability in AP characteristics between the differentiation batches, as well as between clones generated from one donor. Conclusions We established iPSC-CM models for a unique Belgian SCN5A founder mutation. Despite the observed variability, we could detect expected differences in electrophysiological properties of patient cells compared to controls.