CLONING AND STEM CELLS Volume 9, Number 1, 2007 © Mary Ann Liebert, Inc. DOI: 10.1089/clo.2006.0E16 Molecular Imaging of Embryonic Stem Cell Misbehavior and Suicide Gene Ablation FENG CAO, 1 MICHA DRUKKER, 2 SHUAN LIN, 1 AHMAD Y. SHEIKH, 3 XIAOYAN XIE, 1 ZONGJIN LI, 1 ANDREW J. CONNOLLY, 2 IRVING L. WEISSMAN, 2 and JOSEPH C. WU 1,4 ABSTRACT Numerous studies have demonstrated the potential use of stem cells for the repair and re- generation of injured tissues. However, tracking transplanted stem cell fate and function in vivo remains problematic. To address these issues, murine embryonic stem (ES) cells were stably transduced with self-inactivating lentiviral vectors carrying either a triple fusion (TF) or double fusion (DF) reporter gene construct. The TF consisted of monomeric red fluores- cence protein (mrfp), firefly luciferase (Fluc), and herpes simplex virus truncated thymidine kinase (HSV-ttk) reporter genes. The DF consisted of enhanced green fluorescence protein (egfp) and Fluc reporter genes but lacked HSV-ttk. Stably transduced ES-TF or ES-DF cells were selected by fluorescence activated cell sorting based on either mrfp (TF) or egfp (DF) expression. Afterwards, cells were injected subcutaneously into the right (ES-TF cells) and left (ES-DF cells) shoulders of adult female nude mice. Cell survival was tracked noninva- sively by bioluminescence and positron emission tomography imaging of Fluc and HSV-ttk reporter genes, respectively. Imaging signals progressively increased from day 2 to day 14, consistent with ES cell survival and proliferation in vivo. However, teratoma formation oc- curred in all nude mice after 5 weeks. Administration of ganciclovir (GCV), targeting the HSV-ttk gene, resulted in selective ablation of teratomas arising from the ES-TF cells but not ES-DF cells. These data demonstrate the novel use of multimodality imaging techniques to (1) monitor transplanted ES cell survival and proliferation in vivo and (2) assess the efficacy of suicide gene therapy as a backup safety measure against teratoma formation. 107 INTRODUCTION S TEM CELL–BASED THERAPY holds great promise for the treatment of numerous diseases. Stem cells transplanted into tissues such as the pan- creas (Ramiya et al., 2000), brain (Lindvall et al., 2004), spinal cord (McDonald et al., 1999), heart (Gnecchi et al., 2005), and bone marrow (Chris- tensen et al., 2004) of rodents and primates have shown some degree of engraftment, regeneration, 1 The Department of Radiology and Bio-X Program, Stanford University School of Medicine, Stanford, California. 2 The Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, Cal- ifornia. 3 The Department of Surgery, Stanford University School of Medicine, Stanford, California. 4 The Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, Califor- nia.