3D induced-pluripotent stem cell model of metabolic heart diseases for drug discovery and disease modelling

Cardiovascular disease is the leading cause of mortality in patients with cardiometabolic diseases such as type 2 diabetes. Elucidating and understanding the early stages of the disease is vital for seeking possible treatments. Despite numerous studies using in vitro and animal models, current preclinical models do not accurately recapitulate the adult human heart. Moreover, there are noticeable differences between rodents and humans, providing a challenge when studying the effect of cardiometabolic diseases. Therefore, there is a pressing need for an in vitro model to bridge the gap, to aid identification of disease mechanism and to provide an accurate, predictive tool for drug testing. Human induced- pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have potential as a predictive tool but are hindered by their immature phenotype.
The work presented in this thesis aims to answer several questions regarding the potential of using matured hiPSC-CM cultured in 2D and 3D cell culture (as engineered heart tissue) as an advanced in vitro model, to model cardiometabolic diseases such as type 2 diabetes and Duchenne muscular dystrophy (DMD). To improve the maturation of hiPSC-CMs, culturing the cells in 2D and 3D in a media containing oleic acid resulted in maturation of sarcomere actinin structure, electrophysiological properties, gene expression, and a metabolic switch to fatty acid oxidation, recapitulating more mature cardiomyocytes. The effect of oleic acid supplementation on the hiPSC-CM maturation was also characterised using novel synchrotron-based micro-FTIR absorption spectroscopy.
Modelling the effect of type 2 diabetes on hiPSC-CMs was performed by exposing the 2D and 3D cultured cells to a diabetic medium, which was developed by manipulating concentrations of glucose, insulin, and palmitic acid. An increase in fatty acid metabolism and a blunted insulin signalling cascade in hiPSC-CMs cultured in this media was observed, indicating that the cells had become insulin resistant. Modelling the effect of DMD was performed by using DMD patient-derived hiPSCs. Structural and metabolic maturation of hiPSC-CMs from DMD patients were found to be delayed in comparison to that of a healthy cell line. DMD hiPSC-CMs had altered functional properties such as reduced contraction speed and force. Insulin resistance was also observed in the DMD hiPSC-CMs, suggesting insulin resistance in DMD patients may be partly endogenous.
Finally, healthy, insulin resistant, and DMD hiPSC-CMs in 2D and 3D (engineered heart tissue – EHT) were treated with several drugs such as isoprenaline, isoliquiritigenin, metformin, perhexiline, and molidustat, to assess if the cell models recapitulate the human and animal model findings.
Overall, the results show that metabolically matured hiPSC-CMs cultured in 2D, or 3D generate a clinically relevant in vitro model to study the pathophysiological effects of cardiometabolic diseases. This can be a valuable predictive tool to speed up the drug discovery process and improve candidate drug success in clinical trials.

Keywords: perhexiline
maturation
in vitro disease modelling
metabolism
cardiomyocytes
heart
fatty acids
mitochondria
molidustat
Duchenne muscular dystrophy
engineered heart tissue
heart disease
drug testing
disease model
environmental factors
insulin
physiology
oleic acids
human induced-pluripotent stem cells
diabetic cardiomyopathy
Advisor: Dora, K
Heather, L
Carr, C