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A road-map for the maturation of human induced Pluripotent Stem Cell-derived Cardiomyocytes: a focus on metabolism

Student thesis: Doctoral ThesisDoctor of Philosophy

The ability of deriving cardiomyocytes (CMs) from embryonic stem cells (ESCs) and somatic cells reprogrammed to pluripotency (iPSCs) has opened new opportunities for basic and translational cardiovascular research, and allowed to greatly extend in vitro investigations to the human system. Progressive phases of hiPSC-CMs differentiation are characterised by the time-dependent acquisition of cardiac-specific features. However, one of the main caveats in the utilisation of hiPSC-CMs as a model for pathophysiological studies and cell therapy is the recognition of their quite immature phenotype. hiPSC-CMs have been often classified as “early-stage” and “late-stage”, even though the definition of these time-points is inconsistent throughout the literature. Indeed, the identification of specific markers of maturation constitutes a high priority in this field. 
In order to guarantee its contractile function, the cardiac tissue strongly depends on continuous energy production. In addition, cardiomyocytes have high versatility in substrate utilisation, thus allowing the heart to switch between a variety of carbon substrates depending on the conditions. Since metabolism and cardiac function are tightly intertwined, energetics plays an important role in cardiac pathology. In fact, conditions predisposing to HF are generally accompanied by metabolic remodelling. Therefore, the requirement for models to study cardiomyocyte metabolism is a prerequisite to address different aspects of cardiac pathophysiology. 
Cardiomyocytes undergo profound maturation of the pathways involved in energy metabolism during foetal development and early neonatal life. In previous studies hiPSC-CMs have been described to have a foetal-like metabolic phenotype6, with high rates of glycolysis and very low oxidation potential, but very little has been published about the time-dependent maturation of energy metabolism and the mechanisms underlying metabolic remodelling in this model. In this work hiPSC-CMs were produced from healthy donors and maintained in culture for up to 12 weeks with the aim of characterising the pathways involved in energy metabolism and substrate selection. By comparing gene expression and metabolic fluxes at different time-points we could identify differences and similarities between hiPSC-CMs maturation and embryonic development of heart-derived myocytes. Results indicated that hiPSC-CMs metabolism presented characteristics similar to the new-born and neonatal phenotype at 6 and 12 weeks post differentiation initiation respectively. Moreover, by analysing some regulatory elements of energy metabolism we identified putative points of intervention to induce further maturity in these cells by targeting energetics. 
The contribution of redox state in the coordination of metabolic reprogramming is a matter of growing importance and energy metabolism is both a source and a target of redox-regulation. The Nox family of NADPH oxidases is a group of enzymes dedicated to produce ROS in a highly specific and compartmentalised manner. Among them, Nox4 was shown to be upregulated in response to stress and to constitutively produce H2O2, which can act as a second messenger and be involved in many aspects of cardiomyocytes function. 
Whether Nox4 activity has detrimental or beneficial effects in cardiac pathology is dividing researchers. Our lab showed protective roles of Nox4 in cardiac overload and interestingly, reported a role for Nox4 in regulating energy metabolism in rodents. Here hiPSC-CMs were used to test the effects of Nox4 in regulating energy metabolism and substrate selection for the first time in the human system. Through the development of a lentiviral tool Nox4 expression levels were successfully manipulated, resulting in Nox4-dependent regulation of substrate preference during metabolic challenges.
Original languageEnglish
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Award date1 Nov 2019

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