PROJECT SUMMARY Molecular imaging is a new discipline that makes possible the noninvasive visualization of cellular and molecular processes in living subjects. Here we will adopt the reporter gene and reporter probe imaging technique (developed initially for cancer researchers) to solve a different important problem in cardiology (i.e., understand pharmacokinetics and biodistribution of cardiac stem cell transplantation). However, the Achilles’ heel of reporter gene imaging has always been random integration into cellular chromosomes. Here we will develop 4 novel genome editing approaches (ZFN, TALEN, CRISPR, phiC31) that will enable us to safely and efficiently introduce human PET reporter gene (hmTK2) into human induced pluripotent stem cells (iPSCs). These cell types are chosen because of the recent discovery that adult somatic cells can be transformed into iPSCs that acquire both unlimited self-renewal and pluripotent differentiation (similar to human embryonic stem cells). We will perform comprehensive characterization of these genome edited lines. Upon validation, we will subsequently image in vivo fate of iPSC-CMs in both small and large animal myocardial infarction models to understand the biological effects of cell dosage, timing of delivery, and imaging sensitivity. We will correlate iPSC-CM survival by PET reporter gene imaging and cardiac function by magnetic resonance imaging. The information gathered from these studies should prove instrumental for marrying molecular imaging with clinical stem cell therapy in the future.

PROJECT NARRATIVE Coronary artery disease (CAD) is a progressive disease with high morbidity and mortality rates in the US. Following myocardial infarction (MI), the limited ability of the surviving cardiac cells to proliferate renders the damaged heart susceptible to unfavorable remodeling processes and morbid sequelae such as heart failure. In recent years, stem cell therapy has emerged as a promising candidate for treating ischemic heart disease as evident by the multiple clinical trials that have been undertaken. Thus, development of novel technologies to image cell fate and cardiac function is a high priority in both basic research and clinical translation. Our major goals for this R01 grant are to develop novel genome editing approaches for introducing PET reporter gene to human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and to improve the therapeutic strategies of stem cell therapy as guided by imaging. Importantly, the overall experience we gain here will be extremely valuable for eventual clinical translation of cardiac stem cell imaging in the future.