Abstract 3494: Electromechanically Functional Cardiac Tissue Constructs Engineered From Embryonic Stem Cells
We have previously developed a novel cardiac tissue engineering technique to culture highly dense, aligned, and interconnected neonatal rat myocytes in cell-laden hydrogels with elongated pores. Here we report the use of mouse embryonic stem-cell (mESC) derived cardiac progenitors in combination with this technique to generate highly functional 3D tissue constructs. We created D3 mESCs clones encoding puromycin resistance controlled by an α-MHC promoter and selected pure cardiac progenitors by adding 5μg/ml puromycin to differentiating embryoid bodies. The selection and culture procedures were optimized to yield highly differentiated monolayers of electrically coupled cardiomyocytes (fig A⇓). Using optical mapping of membrane potentials, we measured conduction velocities of 22.0 +/− 3 cm/s (fig B⇓), significantly higher than those previously reported for cardiomyocytes derived from mouse or human ESCs. Under these conditions, pure α-MHC progenitors cultured in fibrin gels were viable, but round, unconnected, and quiescent. Addition of 20 – 60% neonatal rat cardiac fibroblasts (CFs) produced cardiomyocyte spreading, alignment, and synchronous beating within 6 days of culture (fig C⇓). For different percents of added CFs, 2 week old tissue constructs conducted action potentials at velocities of 7.8 –19 cm/s, with optimum conduction obtained for 40% CFs and 6×106 progenitors/ml (fig D⇓). These constructs generated contractile forces of up to 486μN and physiological force-length relationships (fig E⇓). In conclusion, we demonstrate that the formation of highly functional, mESC-derived cardiac tissue constructs requires the presence of supporting non-myocytes.