"Systematic Assessment of 96-well Human iPSC-Cardiomyocyte Syncytia Development and Response to Hypoxia and Environmental Stress"
April 5th, 2023, 12:30 p.m. - 2:30 p.m.
Science and Engineering Hall (SEH) Room 2990
The George Washington University
Advisor: Dr. Emilia Entcheva
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are scalable and highly relevant to human physiology in their nature. These traits make them a desirable model for high-throughput preclinical drug cardiotoxicity screening and disease modeling. Despite the potential of hiPSC-CMs, there are concerns regarding their maturity and heterogeneity. Therefore, there is a need for a straightforward method that allows hiPSC-CMs to form a functional unit that is electrophysiologically mature and adaptable for high-throughput platforms, which will enable accurate prediction of proarrhythmic risk and enable large-scale studies on cardiac ion channels.
This dissertation first introduces the syncytial growth of hiPSC-CMs as a simple approach toward a potentially more mature phenotype. Then a multimodal assessment pipeline is established and validated to characterize 96-well hiPSC-CM syncytia in terms of their oxygen consumption, electrophysiology, mRNA, and protein level properties. All-optical electrophysiology (EP) is applied to achieve sophisticated cardiac EP measurements in the 96-well hiPSC-CM syncytium, including conduction velocity and reentrant arrhythmia analysis. Post-EP studied 96-well cell samples are then collected for mRNA and protein quantifications. A state-of-art peri-cellular optical oxygen sensing technique is developed, which is compatible for integration with the pipeline assessment after long-term cell oxygen consumption monitoring during cell culture growth. Further, we design a microfluidics-based 96-well perfusion system for high-throughput tissue engineering and long-term all-optical electrophysiology. The applications of the multimodal assessment pipeline are illustrated by: 1) assessing critical cardiac genes knockdown and histone deacetylase inhibitors (HDACIs)’ effects on cardiac electrophysiology, mRNA, and protein levels, 2) integrating the pipeline assessment with the microfluidics-based perfusion system to understand the influence of acute and chronic perfusion on cells, 3) examining culture condition’s influence on hiPSC-CMs and cardiac ventricular fibroblasts’ oxygen consumption 4) quantifying electrophysiological and molecular level changes induced by cardiac hypoxia.
Ultimately, this study demonstrates that the multimodal assessment pipeline provides a holistic understanding of 96-well hiPSC-CM syncytia and their responses to pharmacological, genetic, and environmental perturbations. The methodology developed and validated in this dissertation can improve the throughput and robustness of the current drug testing paradigm and contribute to revealing mechanisms underlying observed cardiac physiology and pathology changes.