355 General Discussion - hiPSC-CMs Disease Modelling and Future Perspectives 13 multi-omics approaches, researchers could establish purely the computational blueprints for development, differentiation, and drug response in hiPSC-CMs. From in vivo to in vitro - from organ to organoid. As discussed, hiPSC-CMs could bypass the limitation represented by the impossibility of mechanistic studies on the pathogenesis and progression of cardiomyopathies in tissues from living patients. Moreover, patient-specific hiPSC-CMs create the possibility of analyzing the molecular mechanism of cardiomyopathies in an early stage, before the cardiac remodeling or end-stage pathological levels are reached. Both 2D and 3D hiPSC-CMs have their advantages and disadvantages as discussed below. Still, the best model is one that is fit for purpose; as simple as possible, yet, as complex as necessary. The 2D patient-derived hiPSC-CMs models we used in this thesis showed multiple significant characteristics of the studied disease, including the metabolic disturbance (Chapter 8) and upregulation of UPR (Chapter 9). On the contrary, 2D cell culture techniques have proven to be ineffective for the prediction of cancer drug efficacy35 or mimicking the contractile deficit in titin patient-derived hiPSC-CMs. Instead, the titin mutant hiPSC-CMs in a 3D EHT model showed the obvious insufficient contractile force of the patient-derived hiPSC-CMs.22 Both examples have been attributed to the fact that cells grown in a 2D culture lack maturity, complex structure, and multicellular organization instead of a 3D model. A recent study by Biendarra-Tiegs et al. showed that atrial fibrillation can be modeled in day 60 old-hiPSC-CMs cultured on a micropatterned linear chip, which enhances the conduction velocity compared to the 2D planar culturing as this chip matched the conduction velocity measured in vivo human hearts and ex vivo human atrial tissue slices.36 This example nicely illustrates that, although the model is not a perfect copy of the human adult myocardium, specific traits can be used for disease modeling and ultimately for novel therapeutic screening. However, finding the right molecular phenotype to model the disease can be quite complex, especially when patients with the same mutation manifest the disease phenotype with a diverse severity. As described in Chapter 7, the variability in penetrance of the PLN-R14del cardiomyopathy, including the diversity in disease onset, varies from young symptomatic patients and elderly asymptomatic mutation carriers. Primary tissues from multiple human donors are problematic as there is not enough ex vivo primary tissue from a single donor to accommodate testing multiple concentrations of drugs and a single donor approach can not predict the outcome for the complete patient population. 2D in vitro hiPSC-CMs can overcome the single donor limitation as we can generate multiple hiPSC lines from genetic cardiopathy donors (Chapter 10). Despite the discovered metabolic disturbance (Chapter 8) and upregulation of UPR (Chapter 9) by using 2D hiPSC-CMs, we developed micro 3D tissues, called spheroids (Chapter 11) to study the morphology, PLN-R14del pathology and the cell-cell interactions (Chapter
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