Abstract 17122: Human iPS Cell-derived 3D Engineered Cardiac Tissue Including Cardiomyocytes and Multiple Vascular Lineages for Cardiac Regeneration

Abstract

Background: Three-dimensional (3D) engineered cardiac tissues (ECTs) are a robust platform technology to investigate cardiovascular (CV) cell function and provide an excellent microenvironment for tissue implantation and cardiac repair. We generated 3D ECTs using cardiomyocytes (CMs), endothelial cells (ECs), and vascular mural cells (MCs) efficiently differentiated from human iPS cells (hiPSCs). Methods & Results: We employed 3 different monolayer culture-based differentiation protocols: 1) CM+EC protocol: mesoderm induction followed by VEGF (mes+VEGF) produced a distribution of 61.8±8.0% cTnT + -CMs, 19.4±9.1% VE-cadherin + -ECs, and 1.7±2.0% PDGFRβ + -MCs, (n=26, day15); 2) CM+MC protocol: mes+Dkk1 to induce CMs and MCs; and 3) MC protocol: exclusive induction of MCs. We collected the cells on differentiation day 15 and mixed them to generate 3 classes of ECTs composed of different CV cell population patterns: 1). CM+EC; 2). CM+MC, and 3). CM+EC+MC. We seeded the each cell mixture in a collagen/Matrigel mixture to form spontaneously beating ECTs. In vitro force measurement analysis showed that CM+EC+MC ECTs possessed the highest maximum capture rate (4.6±0.6 Hz, P<0.0001) and lowest excitation threshold (1.2±0.3 V/cm, P<0.01) indicating that incorporation of vascular cells augmented tissue maturation and function. We confirmed more preferential alignment of CMs to the ECT long axis in ECTs with MCs. Transmission electron microscopy revealed that incorporation of MCs increased CM sarcomeric structure. Incorporation of both ECs and MCs led to vasculature formation within ECTs during 14 days of in vitro culture. These results indicate that incorporation of vascular cells accelerated tissue structural maturation. Next CM+EC+MC ECTs were implanted onto infarcted athymic rat hearts. Echocardiogram revealed a significantly higher cardiac output at 4 weeks after implantation compared to that of sham-operated rats (137±23 vs 95±23 mL/min, P<0.05). Immunostaining 4 weeks after implantation showed epicardial engraftment of human cells as a regenerated myocardium vascularized both by graft and host-derived vasculature. Conclusion: HiPSC-derived ECTs including vascular cells showed novel properties relevant for clinical translation.