Manipulation of the embryoid body microenvironment to increase cardiomyogenesis
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Myocardial Infarction (MI) is one of the most prevalent and deadliest diseases in the United States. Since the host myocardium becomes irreversibly damaged following MI, current research is focused on identification of novel, less invasive, and more effective treatment options for patients. Cellular cardiomyopathy, in which viable cells are transplanted into the necrotic tissue, has the potential to regenerate and integrate with the host myocardium. Stem cells, specifically pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC), are ideal candidates for this procedure because they are pluripotent; however, ESCs must be predifferentiated to avoid teratoma formation in vivo. In this dissertation, our goal was improve upon current protocols to direct differentiation of ESCs into cardiomyocytes using an embryoid body (EB) model. We immobilized pro-cardiomyogenic proteins, specifically Sonic Hedgehog (SHH) and Bone Morphogenetic Protein 4 (BMP4) to paramagnetic beads and delivered them in the interior of the EB. While lineage commitment was indiscriminate, the presence of the beads alone appeared to guide differentiation into cardiomyocytes: there were significantly more contracting areas in EBs containing beads than in the presence of SHH or BMP4. To take advantage of this result, we immobilized Arginine-Glycine-Aspartic Acid (RGD) peptides to the beads and magnetized them following incorporation into the EB. Magnetically mediated strain increased the expression of mechanochemical markers, and in combination with BMP4 increased the percentage of cardiomyocytes. Finally, PEGylated fibrin gels were used to investigate the effect of seeding method and fibrinogen concentration on cardiomyocyte behavior and maturation. Cells seeded on top of compliant hydrogels had the most contracting regions compared to stiffer PEGylated fibrin gels, whereas cardiomyocytes seeded within the hydrogels could not remodel the matrix or maintain contractility. As an alternative to 3D culture, we seeded cardiomyocytes within gel layers, which maintained viability as well as contractile activity. We observed that PEGylated fibrin gels can maintain ESC-derived cardiomyocytes; however, the ratio of cardiomyocytes and non-cardiomyocytes should be optimized to maintain contractile phenotypes. Therefore, this dissertation presents novel methods to differentiate ESCs into cardiomyocytes, and subsequently promote their maturation in vitro, for the treatment of MI.