Undifferentiated embryonic stem cells cause teratoma formation in the heart

Cell transplantation offers the chance to repopulate myocardial infarcts with new muscle tissue. Although transplanted cardiomyocytes can survive and integrate into normal or injured myocardium, donor cardiomyocytes are not readily available for transplantation. Embryonic stem (ES) cells present an attractive alternative for the derivation of cardiomyocytes for therapeutic interventions. ES cells are pluripotent and capable of spontaneous differentiation into cells of all three germ lineages, including cardiomyocytes. However, spontaneous differentiation of ES cells in embryoid bodies produces only a low yield of cardiomyocytes. The objective of this study was to delineate whether the heart will provide an instructive environment that would guide differentiation of ES cells into cardiomyocytes. Mouse R1-ES cells were stably transfected with enhanced green fluorescent protein (EGFP) under the control of the cardiac-alpha-actin promoter. Expression of EGFP was used to track cardiac differentiation of ES cell grafts in vivo. Two million undifferentiated cells were injected into the normal myocardium of the left ventricle of immunodeficient nude mice. At 3 weeks after cell injection, formation of a tumor was observed in the hearts. Immunocytochemistry revealed mostly undifferentiated cells and ectodermal structures (keratin-filled ducts). At this time point, ES cell-derived cardiomyocytes could not be detected by EGFP staining. Sporadic endodermal structures (respiratory epithelium) could also be detected at three weeks. At 5 weeks the tumor could be characterized as a teratoma. It contained cells from the endoderm lineages (goblet cells, respiratory epithelium), mesoderm lineages (smooth muscle, cartilage) and ectoderm lineages (neuronal structures). A very small number of ES cell–derived cardiomyocytes was observed. The teratoma extended into the chest wall and the lung. No difference in the number of EGFP-positive cardiomyocytes could be observed in extra-cardiac versus intracardiac locations. These data suggest that undifferentiated ES cells are not guided to form new myocardium when injected into the heart. These data also indicate that care must be taken to remove undifferentiated elements from ES cell derivatives prior to transplantation, to avoid formation of intracardiac teratomas.