Abstract 9374: Zinc Finger Nuclease Technology for Human Pluripotent Stem Cell Genomic Modification and In Vivo Tracking

Abstract

INTRODUCTION & HYPOTHESIS: Heart disease is the leading cause of death in developed nations and remains a significant public health problem both in the United States and globally. Human embryonic stem cell therapy demonstrates to be an effective means of treating ischemic heart diseases, as it possesses the capability of generating de novo tissue populations. To investigate such populations’ in vivo and in vitro behavioral characteristics as well as clinical potentials, molecular imaging proves to be a vital tool. However, thus far, genetic integration mainly relies upon the unsafe, unstable, and untranslatable method of random integration. We hypothesized that to address such barrier, we can utilize novel zinc finger nuclease (ZFN) technology to integrate reporter genes into human pluripotent stem cells for cardiac cell derivation, delivery, and molecular imaging. METHODS & RESULTS: Using ZFN technology, we integrated a triple fusion gene construct of mRFP-Fluc-HSVtk into the safe AAVS1 locus for fluorescence, bioluminescence, and positron emission tomography imaging. No discernible off-target cleavages or random integration was detected, and we achieved a high efficiency of ZFN-mediated targeted integration. We observed long-term reporter gene expression as well as pluripotency conservation; the cells retained both expression of pluripotency markers - Oct4, Tra-1-60, Sox2, Tra-1-81, Nanog and SSEA4 - as wells as ability to differentiate into 3 germ layers via in vivo teratoma formation and in vitro embryoid body formation. We then differentiated the ZFN-modified ESCs into cardiomyocytes and endothelial cells in vitro . Parallel characterization of differentiation efficiency and cardiac marker expression of ZFN-modified ESCs and blank ESCs showed no significant differences between the two. Functionally, we tracked the survival of ZFN-edited pluripotent cells in murine models, demonstrating the cardiovascular applicability of ZFN-edited cells. CONCLUSION: This study demonstrates the great potentials and applicability of ZFN transgenesis technology use in safe, stable, and effective genetic integration for the molecular imaging of human pluripotent stem cells and beyond.