Cardiovascular diseases are a significant global health issue and one of the leading causes of mortality. Despite this, current treatment methods for major cardiovascular conditions still face numerous limitations and challenges. For example, in the case of heart failure, heart transplantation is often the only viable solution, and due to challenges such as difficulty in finding donor matches and the complexity of the surgical procedure, many patients are unable to benefit from this treatment. This study is part of a broader project aimed at overcoming these limitations by developing an artificial heart by repopulating decellularized porcine cardiac scaffolds with functional hiPSC-derived cardiomyocytes. The primary objective of this study is to investigate the differentiation process of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes and to evaluate the expression of relevant markers, in the obtained cells. In this study, human induced pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes using a monolayer assay. This approach, with the integration of a well-defined extracellular matrix, a specialized medium, and an optimized combination of growth factors and inhibitors, successfully regulated the differentiation process. Selected cell lineages were cultured under stringent conditions, with daily monitoring and regular media replacement to maintain the right environment to achieve complete and accurate cardiomyocyte development. The resulting differentiated cardiomyocytes were subsequently analyzed for the expression of specific markers using immunofluorescence techniques and examined with confocal microscopy to confirm their molecular and structural characteristics. The differentiated cardiomyocytes exhibited key functional characteristics and behavior, such as regular contractions. Immunofluorescence and confocal microscopy analyses confirmed the presence of essential cardiac markers, in particular Connexin 30.2 and Connexin 43, indicating that the cardiomyocytes were successfully differentiated, functional and could potentially be used to effectively repopulate decellularized scaffolds. The results of this study demonstrated that the differentiation protocols and cell culture conditions applied to human induced pluripotent stem cells (hiPSCs) were effective in generating functional cardiomyocytes. The consistent presence of essential cardiac markers, including Connexin 30.2, and Connexin 43, underscores that these cardiomyocytes, not only followed the intended differentiation pathway but also possess the functional properties and specificity required for intended cardiac applications. These findings provide an essential basis for future studies aimed at evaluating the potential use of these validated cardiomyocytes in repopulating decellularized cardiac scaffolds, contributing to the development of bioengineered cardiac structures as a potential alternative to donor heart transplants

Study on the expression of Connexin markers in hiPSC-Derived differentiated Cardiomyocytes to explore regenerative strategies for decellularized porcine cardiac scaffolds.

FAVRETTO, FEDERICO
2023/2024

Abstract

Cardiovascular diseases are a significant global health issue and one of the leading causes of mortality. Despite this, current treatment methods for major cardiovascular conditions still face numerous limitations and challenges. For example, in the case of heart failure, heart transplantation is often the only viable solution, and due to challenges such as difficulty in finding donor matches and the complexity of the surgical procedure, many patients are unable to benefit from this treatment. This study is part of a broader project aimed at overcoming these limitations by developing an artificial heart by repopulating decellularized porcine cardiac scaffolds with functional hiPSC-derived cardiomyocytes. The primary objective of this study is to investigate the differentiation process of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes and to evaluate the expression of relevant markers, in the obtained cells. In this study, human induced pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes using a monolayer assay. This approach, with the integration of a well-defined extracellular matrix, a specialized medium, and an optimized combination of growth factors and inhibitors, successfully regulated the differentiation process. Selected cell lineages were cultured under stringent conditions, with daily monitoring and regular media replacement to maintain the right environment to achieve complete and accurate cardiomyocyte development. The resulting differentiated cardiomyocytes were subsequently analyzed for the expression of specific markers using immunofluorescence techniques and examined with confocal microscopy to confirm their molecular and structural characteristics. The differentiated cardiomyocytes exhibited key functional characteristics and behavior, such as regular contractions. Immunofluorescence and confocal microscopy analyses confirmed the presence of essential cardiac markers, in particular Connexin 30.2 and Connexin 43, indicating that the cardiomyocytes were successfully differentiated, functional and could potentially be used to effectively repopulate decellularized scaffolds. The results of this study demonstrated that the differentiation protocols and cell culture conditions applied to human induced pluripotent stem cells (hiPSCs) were effective in generating functional cardiomyocytes. The consistent presence of essential cardiac markers, including Connexin 30.2, and Connexin 43, underscores that these cardiomyocytes, not only followed the intended differentiation pathway but also possess the functional properties and specificity required for intended cardiac applications. These findings provide an essential basis for future studies aimed at evaluating the potential use of these validated cardiomyocytes in repopulating decellularized cardiac scaffolds, contributing to the development of bioengineered cardiac structures as a potential alternative to donor heart transplants
2023
Study on the expression of Connexin markers in hiPSC-Derived differentiated Cardiomyocytes to explore regenerative strategies for decellularized porcine cardiac scaffolds.
Cardiac regeneration
hiPSCs
Cardiomyocytes
Differentiation
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12608/80669