|Ph.D Thesis||Department of Medicine|
|Supervisors:||Prof. Gepstein Lior|
|Prof. Schiller Jackie|
|Full Thesis text|
The establishment of a cardiomyocyte differentiating system from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), may contribute to the study of early cardiac development, personalized medicine and may serve as a model for genetically inherited diseases. In this study, we tested the potential contribution of these cellular models to each of these three fields.
Initially we aimed to characterize calcium-handling in developing hESCs and hiPSCs-derived cardiomyocytes (CMs). To this end whole-cell voltage-clamp and confocal calcium imaging were used. The presence of RyR-mediated store calcium release was established testing the effects of caffeine on three different age groups. Calcium store load gradually increased during in vitro maturation. This calcium store was shown to contribute to the automaticity of hESC-CMs in concert with the activation of a NCX-induced inward current accelerating membrane diastolic depolarization. In summary, our study establishes the presence of a functional SR calcium store that contributes to automaticity in hESC-CMs. Similarly, hiPSC-CMs also demonstrated the presence of a functional SR Ca2 store contributing to whole-cell [Ca2]i transients.
In the second part of the study we aimed to generate patient-specific hiPSCs as a new paradigm for modeling human disease and individualizing drug testing. The congenital long QT syndrome (LQTS) is a familial arrhythmogenic syndrome, characterized by abnormal ion channel function and sudden cardiac death. Here we report the development of a patient/disease-specific hiPSCs line from a patient with the LQTS-type-2. The generated hiPSCs were coaxed to differentiate into the cardiac lineage. Detailed whole-cell patch-clamp recordings revealed significant prolongation of the action-potential duration (APD) in the LQTS-hiPSCs-derived cardiomyocytes (the characteristic phenotype of LQTS) when compared to healthy, control cells. The cells displayed significant diminution of the Ikr current. Importantly, the LQTS-derived cells also displayed marked arrhythmogenicity, characterized by the development of early after depolarizations (EADs) and triggered arrhythmias. We then utilized the LQTS-hiPSC-derived cardiac tissue model to evaluate the potency of existing and novel pharmacological agents that may either aggravate (potassium channel blockers) or ameliorate (calcium channel blockers, KATP channel openers and late-sodium channel blockers) the disease phenotype. Our study illustrates the ability of the hiPSCs technology to model the abnormal functional phenotype of an inherited cardiac disorder and to identify potential new therapeutic agents. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care (personalized medicine), and aid in the development of therapies that may find their way to the bedside.