Metabolic maturation of induced pluripotent stem cells derived cardiomyocytes

Cardiac metabolism and function are inherently linked, as substrate metabolism generates the energy needed for the heart to beat. With the development of protocols to differentiate human induced pluripotent stem cells (iPSCs) into beating cardiomyocytes (CMs), cardiac cells can now be studied in vitro. This promises valuable insights into the biology of human CMs, however it is hindered by the fact that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have a very immature phenotype and do not recapitulate the properties of adult CMs. iPSC-CMs are immature in several ways including but not limited to morphologically, mitochondrial and sarcromeric structure, electrophysiological properties as well as their metabolism. iPSC-CMs reflect more of a foetal CM metabolism relying on glucose as their primary energy substrate, while an adult CM has more of an oxidative metabolism. This may, in part, be due to the fact that the iPSC-CMs are frequently grown as a monolayer in high glucose media and do not have the need to generate large amounts of energy for contraction and are metabolising non-physiologically relevant concentrations of nutrients. This does not change in the majority of cardiac differentiation protocols. Even though metabolic state affects marker expression, cellular function and activity, the substrate composition is currently being overlooked. Several labs have tried different strategies to mature iPSC-CMs from long-term culture, applied mechanical and electrical stimulation, the use of maturation media containing chemicals or small compounds, to 3D culture. This thesis sought to investigate and address maturation of iPSC-CMs through stimulating fatty acid oxidation (FAO) using physiologically relevant methods of 3D culture in combination with PPARα activation via addition of the PPARα agonist WY-14643 and of the fatty acid, oleic acid (OA), into the culture media. By simultaneously targeting both maturation methods we have shown a degree of maturation through an increased movement towards reduced glycolysis and increased FAO in our iPSC-CMs. Like others this thesis has shown that iPSC-CMs mature slowly over time with changes in structural genes occurring during the first month of differentiation and continuing to increase up to 3 months, whereas metabolic changes took longer to develop. 3D culture on collagen-derived scaffolds and engineered heart tissue (EHT) accelerated this maturation time and increased mitochondrial oxidative metabolism while decreasing anaerobic glycolysis. This was also reflected genetically in the collagen-derived scaffolds with increased expression of a number of genes involved in regulating both FAO and reduced glycolytic gene expression. Treatment with OA + WY-14643 for 1 week enhanced FAO and oxygen consumption. Finally, EHTs treated for 1 week with both OA and WY-14643 were found to be generating 4 times the amount of ATP by oxidation than the 2D cultured iPSC-CMs and twice as much as the scaffolds. Although, further work is still required to optimise culture conditions by reducing glucose levels in the media and investigating the effect of insulin stimulation, the work in this thesis confirms that the dual approach of 3D culture and enhancing the culture media with additional OA and PPARα agonists induces a degree of maturation in iPSC-CMs towards the metabolic flexibility of the adult heart.