Optical Mapping of Human Embryonic Stem Cell-Derived Cardiomyocyte Graft Electrical Activity in Injured Hearts

Thesis (Ph.D.)--University of Washington, 2015Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac repair. hESC-CMs can be efficiently generated in high numbers for cardiac transplantation, have been shown to form stable grafts within animal models of cardiac injury and mediate beneficial mechanical effects on recipient hearts. However, the successful development of hESC-CM-based therapies requires improved tools to investigate their behavior after transplantation. Here, we utilized hESC-CMs that stably express the calcium-sensitive fluorescent protein GCaMP3 to visualize their activity in hearts ex vivo. By this method, we found the first direct evidence that GCaMP3+ hESC-CMs can integrate with host myocardium, although with variable success. Specifically, GCaMP3+ hESC-CMs coupled in 100% of uninjured, ~60% of recently injured, and ~38% of chronically injured guinea pig hearts. Further, activation kinetics measured by GCaMP3 fluorescence revealed grafts residing in injury scar of recipient hearts were considerably slower than grafts within healthy tissue. When we attempted to measure host-graft electrical interactions by optical mapping with conventional voltage-sensitive fluorescent dyes, we found hESC-CM grafts failed to label. Thus, to overcome this limitation we developed and validated an alternative strategy based on the simultaneous imaging of GCaMP3+ hESC-CM grafts (providing a graft autonomous reporter of activation) and optical action potentials (APs) from a water-soluble, voltage-sensitive fluorescent dye (di-2-ANEPEQ) that labels both graft and host tissue. By this approach, we found GCaMP3+ hESC-CM grafts from two hESC lines (RUES2 and H7) exhibited multiple potentially pro-arrhythmic properties, including conduction velocities