Differentiation, Survival, and Function of Embryonic Stem Cell–Derived Endothelial Cells for Ischemic Heart Disease

Author:

Li Zongjin1,Wu Jenny C.1,Sheikh Ahmad Y.1,Kraft Daniel1,Cao Feng1,Xie Xiaoyan1,Patel Manishkumar1,Gambhir Sanjiv S.1,Robbins Robert C.1,Cooke John P.1,Wu Joseph C.1

Affiliation:

1. From the Department of Radiology and Molecular Imaging Program at Stanford (Z.L., F.C., X.X., M.P., S.S.G., Jo.C.W.), the Department of Medicine, Division of Cardiology (Je.C.W., J.P.C., Jo.C.W.), the Department of Cardiothoracic Surgery (A.Y.S., R.C.R.), and the Department of Medicine, Division of Hematology (D.K.), Stanford University School of Medicine, Stanford, Calif.

Abstract

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 2 =0.98) and by in vitro enzyme assay ( R 2 =0.94). Afterward, 5×10 5 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.

Publisher

Ovid Technologies (Wolters Kluwer Health)

Subject

Physiology (medical),Cardiology and Cardiovascular Medicine

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