There is an urgent need to develop novel treatments for heart failure. The clinical translation of novel therapies for cardiac disease is hindered by the limited availability of suitable models of the human heart. The use of human engineered cardiac tissues (hECTs) can serve to bridge the gap between current animal models, providing a species-specific model of human myocardium, and also overcomes limitations of the 2D culture systems. The field of cardiac tissue engineering is constantly evolving, with the proposition of novel strategies for the fabrication of a structurally and functionally mature model of human myocardium. These technologies have been favored by the optimization of methodologies for the differentiation of human induced pluripotent stem cells (hiPSC) into cardiomyocytes and non-myocyte cell types. Healthy cardiac development and function is supported by a complex network of interactions between diverse cell types, including cardiomyocytes, nonmyocytes, and the extracellular matrix. The objective of this study is to utilize a combination of cardiomyocytes (CM) and nonmyocytes with a matrix polymer mix to fabricate engineered myocardium (EngMyo) with functional and structural properties of the native human myocardium, along with a better representation of its cellular milieu, with the long-term goal of using these as in vitro models to test novel therapies that could impact cardiac function. We hypothesize that the presence of non-myocytes in the proper ratios, will result in enhanced functional and structural characteristics when compared to myocardial tissues fabricated with CM alone, mainly through the activation of signaling pathways associated with cardiomyocyte development and cell turnover. We will address this hypothesis in two aims.
In Aim 1, we will test the prediction that the contractility of cardiomyocytes grown in 3D is impacted by crosstalk with non-myocyte cells. First, we will differentiate hiPSC into CM, fibroblasts, endothelial, and smooth muscle cells, followed by characterization using flow cytometry and immunofluorescence. Then we will manipulate the type and number of cells that will be combined to fabricate engineered myocardium (EngMyo), and for each resulting EngMyo we will perform functional and structural characterization. Our central hypothesis predicts that the presence of non-myocytes in the proper ratios, will result in enhanced contractile force than EngMyo fabricated with hCM alone. We will determine which is the cell combination that provides the largest enhancement in force.
In Aim2 we will identify the molecular pathways activated by the presence of non-myocytes. We will perform RNAseq in the EngMyo fabricated with CM-only (control) and EngMyo fabricated with combination of CMs and non-cardiomyocyte cells. We will investigate which are the differentially expressed genes and from this analysis we seek to identify the signaling pathways activated by the presence of non-myocytes. These findings will be of great value to better understand the role of non- myocytes in myocardial function and provide with the formulation for the fabrication of reliable and reproducible models of human myocardium.
The significance of this proposal is driven by the potential impact of providing a reliable and reproducible model of human engineered myocardium, for the purpose of modeling disease and testing the effect on myocardial function of novel therapies. We will first test and compare the functional and structural characteristics of engineered myocardium fabricated with human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CM) (control) and engineered myocardium fabricated with CM and hiPSC derived non-myocyte cells at different ratios (experimental groups). Then we will perform RNAseq of the engineered myocardium to identify novel signaling pathways activated by non-myocytes which impact myocardial function.
