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.