Abstract
BackgroundHuman embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration.MethodsWe developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines (H7, RUES2, or ESI-17) were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation.ResultsUse of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint.ConclusionSimultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation.
Highlights
Human embryonic stem cell-derived cardiomyocytes show tremendous promise for cardiac regeneration, but the successful development of Human embryonic stem cells (hESCs)-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts
We report here our efforts to overcome the above limitations using a novel approach that involves the simultaneous imaging of GCaMP3, the aforementioned genetically encoded calcium-sensitive fluorescent protein that functions as a graft-autonomous reporter of graft activation, and di-2-ANEPEQ, a water-soluble voltage dye that labels both host and graft tissue
These observations support our earlier conclusion that graft-derived RH237-derived Optical action potential (oAP) from such hearts are factitious, and that these signals instead arise from subendocardial host tissue located beneath the graft that shine through to the epicardial surface [13]
Summary
Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While there are other remaining challenges related to scalability, graft cell immune rejection, and tumor formation, this phenomenon of graft-related arrhythmias has emerged as arguably the greatest barrier to the successful development of hESCCM-based cardiac therapies. To address this issue, the field must develop new, more powerful tools to investigate the electrophysiological properties of hESC-CMengrafted hearts and to test strategies to improve their electrical stability
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