Engineering 2D Cardiac Tissues Using Biomimetic Protein Micropatterns Based on the Extracellular Matrix in the Embryonic Heart

Cardiovascular disease is the leading cause of death worldwide. Due to the extremely low natural regeneration rate of heart muscle, development of new therapeutics directed towards heart repair is challenging. A potential approach to regenerate damaged heart is offered by cardiac tissue engineering. Specifically, it aims at engineering cardiac muscle in vitro and implanting it into the site of injury so that it can be integrated into the host tissue and restore the heart’s function. To ensure the effectiveness of this technique, the engineered tissue needs to recapitulate structural and functional properties of the native myocardium. Myocardium consists of laminar sheets of uniaxially aligned cardiac muscle cells (cardiomyocytes) wrapped around the heart. Therefore, achieving high cardiomyocyte alignment in engineered muscle is crucial. In this study we aimed at stimulating cardiomyocyte alignment by mimicking their niche in the embryonic heart. We hypothesized that recapitulating the extracellular cues that guide myocardial development in the embryo can guide cardiac tissue organization in vitro. To test this hypothesis, we imaged the structure of fibronectin – the most abundant protein in embryonic heart’s extracellular matrix (ECM) – and derived a 2D pattern from it that was then microcontact printed onto a substrate to guide cell alignment. We compared chick cardiomyocyte alignment on the biomimetic pattern and line patterns that have been extensively studied in the past. Results revealed a unique cell density-dependent response of cardiomyocytes to the biomimetic pattern that allowed us to elucidate the role of cell-cell and cell-ECM interactions in cardiomyocyte alignment on fibronectin patterns by looking at the effect of local pattern features on alignment and inhibiting N-cadherin-based cell-cell junctions. Further, to engineer more clinically relevant tissues, we differentiated human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) into cardiomyocytes and seeded them onto the fibronectin patterns. Cardiac tissues produced with these cells showed significant differences compared to the chick tissues due to their immature phenotype. We showed that co-culture with cardiac fibroblasts (CFBs) as well as maturation of iPSC-derived cardiomyocytes (iPSC-CMs) increased tissue alignment, indicating the important role of both of these factors in developing novel methods to engineer functional cardiac tissues.