Abstract 291: <i>In Vitro</i> Matured Human Embryonic Stem Cell-derived Cardiomyocytes Form Grafts With Enhanced Structure and Improved Electromechanical Integration in Injured Hearts

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have tremendous promise for application in cardiac repair, but their immature phenotype greatly limits their translational potential. The present study was designed to two hypotheses: 1) that previously reported methods to promote the maturation of hESC-CMs by culture on soft polydimethylsiloxane (PDMS) substrates can be upscaled to the quantities required for transplantation studies; and 2) that PDMS-matured hESC-CMs will stably engraft in injured hearts and form graft myocardium with enhanced structural and functional properties. First, we cultured hESC-CMs on either PDMS or tissue culture plastic (TCP) for 20 and 40 days, then phenotyped the resultant populations. All hESC-CMs were engineered to express the fluorescent voltage-sensitive protein ASAP1 to facilitate in vitro and in vivo electrophysiological studies. Relative to their counterparts on TCP, hESC-CMs on PDMS at both time-points exhibited increased cardiac gene expression as well as a more mature structural and electrophysiological phenotype in vitro. Single-cell transcriptomics confirmed enrichment of cardiac maturation markers including gene pathways involved in cardiac contraction, extracellular matrix organization, sarcomerogenesis, and adult heart development in PDMS versus TCP cultures. Next, we transplanted day 20 or 40 TCP vs PDMS ASAP1+ hESC-CMs into injured guinea pig hearts. Recipient hearts were later analyzed by ex vivo optical voltage mapping studies and histology. While CMs from both substrates showed similar capacity for engraftment, grafts formed with PDMS-matured myocytes had more mature structural properties including enhanced alignment, sarcomere lengths and maturation marker expression. Most importantly, graft formed with PDMS-matured myocytes showed improved electrophysiological properties including better host-graft electromechanical integration and more rapid and uniform propagation. We conclude that large quantities of matured hESC-CMs can indeed be economically produced by these methods. Moreover, PDMS-matured myocytes form large intramyocardial grafts with enhanced cardiac structure and electrical function, thereby establishing that maturation prior to transplantation meaningfully improves outcomes in vivo.