Pluripotent stem cells as a model system for the investigation of cardiovascular development and disease

Abstract

Cardiovascular diseases account for 32% of total worldwide mortality, according to the World Health Organization (WHO), thus underscoring the need for innovative therapeutic approaches. The pluripotency characteristic of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, with their ability to differentiate into various cell types, makes them valuable tools in cardiovascular research. Therefore, the central aspect of this work was to use pluripotent cells to explore the early steps of cardiovascular development, as these are challenging to investigate directly in vivo due to limited accessibility. In summary, several ECM proteins were shown to play a role in cardiovascular development. First, a notable reduction in the formation of vessel-like structures within ILK-deficient (-/-) EBs was observed. ILK deficiency affects tyrosine kinase receptor-dependent signalling pathways that are important for vital processes such as proliferation, migration and apoptosis by inhibiting intracellular Ca²? elevation. ILK plays a key role in shaping endothelial cell (EC) development and function by modulating caveolae positioning and maintaining the structural integrity of actin and tubulin subcellular networks. Disruption of laminin gamma1, an extracellular matrix (ECM) molecule involved in severe skeletal muscle pathologies and cardiac disorders such as muscular dystrophy and cardiac hypertrophy, results in altered basement membrane formation, changes in beta1 integrin distribution and collagen VI deposits in 3D cardiomyocyte models. These ECM deposits create isolated pacemaker regions, providing valuable insights into disrupted electrical signal propagation and arrhythmia.<br /> The discovery of iPS cells enables the generation of cell lines with gene defects directly from patients with cardiovascular diseases. In LQT syndromes, a gene mutation in a specific ion channel establishes a clear link between the genotype and the consequent disease phenotype. Therefore, a mouse model of LQTS3 with the prevalent human delta KPQ mutation in the cardiac sodium channel was used to investigate a monogenetic disorder, which leads to prolonged cardiac APD due to delayed repolarization, extended QT interval, and ventricular arrhythmia. The study aimed to determine whether disease-specific cardiomyocytes, derived from iPS cells obtained from mouse fibroblasts after reprogramming and subsequent in vitro differentiation, could accurately replicate the distinct phenotype observed in mice. The experiments revealed that these disease-specific cardiomyocytes effectively mirror the unique characteristics, such as prolonged action potentials and early after-depolarizations. This finding underscores the potential utility of iPS-derived cardiomyocytes for investigating monogenetic disease mechanisms, even before reaching full terminal maturation. To optimize cardiomyocyte production, a scalable method using a lentiviral-non-clonal gene transfer with antibiotic selection for purifying iPS cell-derived cardiomyocytes was investigated. Genetically modified iPS cell-derived cardiomyocytes retain disease characteristics and are suitable for automated drug screening using planar patch clamp analysis and scalable microelectrode array technologies. Next, iPS cell-derived cardiomyocytes from patients harboring a heterozygous p.R1644H mutation demonstrated the iPS cell-derived cardiomyocytes' potential for evaluating drug responses relevant to LQTS3 treatment in humans. Drugs such as mexiletine and ranolazine were found to effectively mitigated LQTS3 features, aligning with clinical effectiveness.<br /> Finally, the application of optogenetics to ES cell-derived cardiomyocytes using LWO, a red light-sensitive opsin showed that its red-light responsiveness surpasses traditional pharmacological methods in terms of temporal efficacy in activating and deactivating the Gi intracellular signaling pathways. The specificity and efficacy of optogenetic techniques make them promising for high-throughput all-optical drug screening. This is because they minimise false positive or negative results by activating target proteins specifically, without interfering with other signalling components or limiting diffusion problems.<br /> In conclusion, pluripotent stem cells, particularly ES and iPS cells, emerge as valuable tools for studying cardiovascular development. They can also serve as reliable models for drug screening, offering insights and applications that contribute to the advancement of cardiovascular research and therapeutics. This correspondence between iPS-derived cells and clinical data suggests that they could be used to predict and validate therapeutic agents.