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Dissertação de Mestrado Investigação Biomédica (Medicina Regenerativa) 2022 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa
Myocardial infarction (MI) prompts a substantial loss of cardiomyocytes (CM) in the human heart, an organ that lacks significant endogenous regeneration capacities. Reduced force generation and increased stiffness caused by scarring and remodeling following MI compromise ventricular pumping, which can ultimately result in heart failure (HF). The development of differentiation protocols that allow the generation of billions of CM with high purity and quality from human pluripotent stem cells (hPSC) has raised the prospect of cell-based therapies that aim to replace lost CM with exogenously generated ones. However, current methods for CM differentiation generate cells that are transcriptionally, structurally, and functionally embryonic-like. The maturation defects of cardiomyocytes derived from stem cells (hPSC-CM) can limit their application for regenerative medicine. While certain approaches have been identified to stimulate hPSC-CM maturation, recreating the physiological complexity of cardiac tissue for regenerative therapies will likely require a combinatorial strategy that leverages knowledge from many fields such as regenerative medicine, stem cell bioengineering, and biofabrication. In this study, human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPC) and early-stage cardiomyocytes (hiPSC-CMi) in combination with cardiac fibroblasts (hiPSC-CF) were generated, characterized, and used to cellularize a hydrogel-based scaffold to create engineered cardiac tissues (ECT). First, medium formulation for ECT culture was developed based on monolayer studies. Once the ECT medium formulation was determined, it was tested in monolayer cocultures of hiPSC-CPC and -CF, where improved CM maturation features (i.e., gene expression and myofibril alignment) were found compared to hiPSC-CPC monocultures. Next, we proceeded to cellularization of ECT scaffolds with hiPSC-CPC, which successfully continued their differentiation towards CM with high viability, improved morphological features with longer time in culture, and increased expression of genes MYL2, CACNA1C, KCNJ2, GJA1, and MYH7/MYH6. CPC + CF ECT formed more compact and dense networks, but the contribution of CF to CM maturity needs further evaluation. Cellularization with hiPSC-CMi was also performed, resulting in CM with more mature features such as greater size, rod-like morphology, and increased MYL2, MYH7/MYH6, and KCNJ2 expression compared to ECT cellularized with hiPSC-CPC. A preliminary study using 3D aggregates of hiPSC-CM for ECT cellularization is also included. With the ultimate goal of engineering a cardiac patch for the remuscularization of the injured heart, this project intersects approaches to engineer cardiac tissue and evaluates the tissue response to the provided maturation cues.