Assessing and Enhancing the Functional and Structural Maturation of Human Pluripotent Stem-Cell Derived Cardiomyocytes with Engineered Microenvironments

Assessing and Enhancing the Contractile and Structural Maturation of Human Stem-Cell Derived Cardiomyocytes with Engineered Microenvironments Marita Lynn Rodriguez Chair of Supervisory Committee: Professor Nathan J. Sniadecki Department of Mechanical Engineering There is great potential for human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) to serve as a test bed for developmental, pharmacological, and regenerative studies. These cells can serve as therapeutic agents, which can be implanted into damage heart tissue to supplant dead cells. They can be used to assess new pharmacological treatments for heart disease. Moreover, they can be used as model systems to study the progression of developmental and pathological states of the heart. However, upon differentiation into cardiomyocytes, these cells are distinctly immature i.e. their cell size, shape, cardiac-specific markers, ploidy, nucleation, calcium handling properties, action potentials, contractility, metabolism, etc. more closely mimic that of an embryonic-stage cardiomyocyte. Therefore, in order for these cells to serve as a valid replacement or model for more developed cardiomyocytes, their structural and functional maturation must be assessed and enhanced. One of the most important functional characteristics of a cardiomyocyte is its ability to produce contractile forces. Therefore, having the ability to quantify this contraction would provide a powerful assessment tool for hPSC-CMs. Arrays of micropost have previously been employed as a means to measure the isotonic contraction of cardiomyocytes. In this work, a new micropost technique was developed in order to allow for real-time measurements of hPSC-CM contractility, to enable contractile assessment under various different culture conditions. Previous studies with immature cardiomyocytes have shown that a number of different methods are able to enhance their contractile and structural maturation. Here, hPSC-CM maturation was achieved via: i) prolonged cell culture, ii) cell alignment, iii) controlling cell-cell contact between adjacent cells, iv) altering substrate stiffness, v) electrically-stimulating the cells, and vii) treating the cells with various different biochemical agents. Assessment of hPSC-CM structural maturation was achieved by immunofluorescent analysis, while high speed imaging of micropost deflections and fluorescent calcium transients was used to quantify functional maturation. Through these studies, I found that the micropost assay is capable of accessing the contractile state of immature human cardiomyocytes, which makes it a powerful tool for developmental studies, pharmacological screening, and disease modeling applications. Furthermore, the pro-maturation environment that I developed was able to elicit cardiomyocyte maturation in the absence of any biochemical cues. Ultimately, I believe that these novel culture and analysis techniques will provide future researchers with a means to culture large populations of rapidly matured stem cell-derived cardiomyocytes, in order to effectively perform developmental, pharmacological, and therapeutic studies in a more rapid and high-throughput manner.