Establishment of 3D hiPSC cardiac differentiation platforms to investigate congenital heart disease

Abstract

Cardiac tissue engineering shows great potential for alleviating some of the immense burden of cardiovascular disease. The introduction of human induced pluripotent stem cells (hiPSCs) in 2007 led to a paradigm shift in cardiac tissue engineering, providing a reliable and patient-specific cell source for producing cardiomyocytes in vitro. However, standard methods for producing 3D engineered cardiac tissue often combine pre-differentiated cardiomyocytes with biomaterials, which does not fully recapitulate 3D cardiac development and prohibits the ability to study congenital heart diseases that initiate during development. This work demonstrated the ability to directly differentiate hiPSCs in 3D hydrogel microenvironments using multiple biomaterials and tissue geometries and deployed our direct differentiation platform to study a drug-induced and a genetic cause of congenital heart disease. Expanding on a previously established 3D direct differentiation platform, the first study utilized new hybrid biomaterial, gelatin methacryloyl (GelMA), to produce GelMA human engineered cardiac tissues (GEhECTs). GelMA is a tunable biomaterial that provided a soft (<1kPa) 3D microenvironment which supported cell growth and cardiac differentiation. Resulting GEhECTs displayed appropriate changes in cell morphology, gene expression, and contractility over time. The next study investigated the impact of encapsulation geometry on tissue homogeneity and functionality by introducing two new encapsulation geometries, square and rectangle, in comparison to the previously established microisland geometry. All tissue geometries displayed similar cardiac differentiation efficiencies (~ 65%) and temporal changes in gene expression. However, rectangular tissues had a significantly higher degree of homogeneity across the tissue volume and anisotropic contraction compared to microisland and square tissues. Additionally, square and rectangular tissues displayed more mature ultrastructural features including aligned Z-bands and organized myofibers. To study both drug-induced and genetic causes of congenital heart disease, two studies were performed, one involving a known teratogen, thalidomide, and another utilizing left ventricular noncompaction (LVNC) patient-derived hiPSC line. First, to determine if the established 3D direct differentiation platform was sensitive enough to detect thalidomide-induced changes in tissue functionality, 3D engineered cardiac tissues were exposed to 0, 10, and 70 µM thalidomide throughout differentiation and long-term culture. Thalidomide-treated tissues showed decrease cardiac differentiation efficiency, changes in extracellular matrix composition, and reduced electrophysiological function. Finally, to investigate a genetic congenital heart disease in vitro, a 3D direct differentiation platform was used with LVNC patient-derived hiPSCs to produce 2D LVNC cardiac monolayers and 3D LVNC cardiac microspheres. Both 2D LVNC cardiac monolayers and 3D LVNC cardiac microspheres exhibited decreased contraction velocity and calcium handling capabilities compared to control tissues. Overall, the findings from these studies improved and expanded this 3D direct cardiac differentiation platform and demonstrated its applicability in studying both drug-induced and genetic causes of congenital heart diseases. This 3D direct differentiation platform shows great potential for being used in preclinical testing and future disease modeling studies.