Differentiation, survival, and function of embryonic stem cell-derived endothelial cells for ischemic heart disease

BACKGROUND - Embryonic stem (ES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of ES cell-derived endothelial cells (ESC-ECs), use molecular imaging techniques to examine their survival in vivo, and determine the therapeutic efficacy of ESC-ECs for restoration of cardiac function after ischemic injury. METHODS AND RESULTS - Murine ES cells were transfected with a construct composed of a vascular endothelial cadherin promoter driving enhanced green fluorescence protein (pVE-cadherin-eGFP). Differentiation of ES cells to ECs was detected by FACS analysis using Flk-1 (early EC marker at day 4) and VE-cadherin (late EC marker at day 8). After isolation, these ESC-ECs express endothelial cell markers similar to adult mouse lung endothelial cells, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). For in vivo imaging, ES cells were transduced with an ubiquitin promoter driving firefly luciferase and monomeric red fluorescence protein (pUb-Fluc-mRFP). A robust correlation exists between Fluc signals and cell numbers by ex vivo imaging analysis (R=0.98) and by in vitro enzyme assay (R=0.94). Afterward, 5×10 ESC-ECs or PBS (as control) was injected into the hearts of mice undergoing LAD ligation (n=15 per group). Bioluminescence imaging showed longitudinal survival of transplanted ESC-ECs for ≈8 weeks. Echocardiogram demonstrated significant functional improvement in the ESC-EC group compared with control (P=0.04). Finally, postmortem analysis confirmed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. CONCLUSIONS - This is the first study to track the fate and function of transplanted ESC-ECs in the heart. With further validation, these ESC-ECs could become a valuable source of cell therapy for induction of angiogenesis in the treatment of myocardial ischemia.