Human stem cell modelling for heart disease of long QT syndrome
Ge, Ning
Ge, Ning
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Identifiers
http://hdl.handle.net/10379/17179
https://doi.org/10.13025/16877
https://doi.org/10.13025/16877
Repository DOI
Publication Date
2022-04-29
Type
Thesis
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Abstract
Sudden cardiac death (SCD) in otherwise healthy young people is a public health priority. Long QT syndrome (LQTS) is the commonest life-threatening cardiac arrhythmia contributing to SCD under 40 years old with an estimated prevalence of 1:2,000. Cardiac rhythm is driven by coordinated cardiac action potential, which is triggered by the flow of Na+, Ca2+ and K+ ions in and out of cardiomyocytes. To date, 15 genes have been identified as contributing to the LQTS phenotype. Loss-of-function variants in KCNQ1 (LQT1) encoding Potassium Voltage-Gated Channel Subfamily Q Member 1 and KCNH2 (LQT2) encoding Potassium Voltage-Gated Channel Subfamily H Member 2, and gain-of-function variants in SCN5A (LQT3) encoding Sodium Voltage-Gated Channel Alpha Subunit 5 account for >90% of genotyped cases. Human cell models are limited as it is not possible to maintain adult cardiomyocytes on culture dish under conventional conditions. The induced pluripotent stem cell (iPSC) technology developed by Nobel Laureate Yamanaka offers an unprecedented opportunity for disease modelling and therapeutic interventions, as iPSCs can be differentiated into different cell types in the body including cardiomyocytes. In this PhD project we firstly have generated iPSCs using a non-integrational Sendai reprogramming method, from 4 healthy individuals and 19 LQTS patients who harbour KCNQ1, KCNH2, and SCN5A pathogenic variants, including a family with an additional variant of uncertain significance (VUS) in ANK2 gene encoding an ankyrin protein that link the integral membrane proteins to the underlying spectrin-actin cytoskeleton. All iPSC lines were characterized to confirm their pluripotency, and in vitro differentiation potential to form cells from all three germ layers. The LQTS patient-derived iPSC lines retained donor-specific variants in their genome through the process of re-programming. Thus, we have established a mini-biobank of human iPSCs for LQTS modelling and drug discovery. The iPSCs are known to have heterogeneity and different iPSC lines from the same individual may not behave entirely same. The CRISPR/Cas9 technology which won the 2020 Nobel Prize in Chemistry offers an opportunity for rapid genome-editing in the genome. To overcome the iPSC heterogeneity, to confirm the variant-specific phenotype of the cardiomyocytes and to explore gene-based therapies for LQTS, we then carried out genome-editing and introduced an SCN5A pathogenic variant (c.1231G>A) into two healthy control iPSC lines using CRISPR/Cas9 technology. We identified six independent re-engineered stem cell colonies that had successfully incorporated the SCN5A c.1231G>A variant with no detected off-target. Furthermore, these genetic engineered iPSC lines exhibited normal pluripotency characteristics. This offers isogenic iPSCs to investigate LQTS variants in parallel to patients-derived iPSC lines. We subsequently differentiated these human iPSC lines from healthy controls, LQTS patients and genetically engineered LQTS isogenic lines into beating cardiomyocytes and compared cardiomyocyte function. We confirmed that cardiac markers were highly expressed in the differentiated cells. We assessed the electrophysiological characteristics of the differentiated cardiomyocytes using multi-electrode array (MEA), and importantly, showed that the cardiomyocytes retained the characteristics of the parental origin, and recapitulated the cardiac phenotype in the healthy controls and LQTS patients. We added several drugs including Nifedipine, Verapamil, Sotalol, Ondansetron, Clarithromycin, E-4031 and Mexiletine to the iPSC-derived cardiomyocytes and showed that Nifedipine, Verapamil and Mexiletine could ameliorate the patient LQTS phenotype in a dose-dependent manner, suggesting these drugs might be of therapeutic potential for LQT2 and LQT3 patients in the clinical setting. In summary, in this PhD project, we have derived a mini-biobank of iPSCs from heathy controls and LQTS patients, which offer patient-derived stem cell models to model cardiac ion channel disease in culture dish. We have generated isogenic iPSC mutant lines and showed variant-specific effects on the MEA signals of the cardiomyocytes. The preliminary drug testing experiments show that some of the tested drugs can modulate electrophysiology of patient’s cardiomyocytes. Thus, in this body of work we have built a platform and provide a pipeline for candidate drug screening and toxicity tests, and to develop concepts of combinatorial drug, gene, and cell therapy for cardiac diseases.
Publisher
NUI Galway