Brief Communication
Transplantation of embryonic stem cells into the infarcted mouse heart: formation of multiple cell types

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Abstract

Initial studies have suggested that transplantation of embryonic stem (ES) cells following myocardial infarction (MI) in animal models is beneficial; however, the mechanism of benefit is largely unknown. The present study investigated the fate of mouse ES cells transplanted post-MI to determine if the ES cells give rise to the range of major cell types present in the native myocardium. MI was produced by coronary artery ligation in C57BL/6 mice. Two different mouse ES cell lines, expressing eGFP and β-galactosidase, respectively, were tested. Post-MI intramyocardial injection of 3 × 104 ES cells was compared to injection of media alone. Histochemistry and immunofluorescence were used to track the transplanted ES cells and identify the resulting cell types. Echocardiography assessed the cardiac size and function in a blinded fashion. Two weeks post-MI, engraftment of the transplanted ES cells was demonstrated by eGFP or β-galactosidase-positive cells in the infarct region without evidence for tumor formation. Co-immunolabeling demonstrated that the transplanted ES cells had become cardiomyocytes, vascular smooth muscle, and endothelial cells. Echocardiographic analysis showed that ES cell transplantation resulted in reduced post-MI remodeling of the heart and improved cardiac function. In conclusion, transplanted mouse ES cells can regenerate infarcted myocardium in part by becoming cardiomyocytes, vascular smooth muscle, and endothelial cells that result in an improvement in cardiac structure and function. Therefore, ES cells hold promise for myocardial cellular therapy.

Introduction

In animal models of myocardial infarction (MI), transplantation of a wide variety of different cell types into the heart has been reported to improve cardiac structure and function [1], [2]. Some cell types including skeletal myoblasts and bone marrow-derived cells are being tested in initial clinical trials [3]. However, the optimal donor cell population for post-MI treatment is not known. The long-term impact of cellular transplantation likely varies substantially depending on donor cell type.

Pluripotent ES cells hold significant appeal for cell therapy because these cells have the ability to differentiate into any cell type in the body including the different cell types found in the heart. The cardiogenic capacity of ES cells has been well-defined by in vitro studies as well as in chimeric mice [4]. These cells can proliferate indefinitely in culture and could provide a potentially unlimited reservoir of cells for therapy and tissue engineering. Results in a rat MI model suggested that transplanted mouse ES cells can engraft and survive long-term [5], [6], [7]. There is evidence that transplanted ES cells post-MI can become cardiac myocytes, but whether the transplanted cells differentiate into the full spectrum of cell types optimal for myocardial repair and regeneration has not been examined. The purpose of the present work was to test the ability of mouse ES cells to regenerate the post-MI mouse heart by determining the impact of transplantation on cardiac function and if transplanted cells differentiated into the range of major cell types present in the myocardium.

Section snippets

Mouse ES cells and in vitro differentiation

Mouse ES cell lines were maintained on mitomycin C (Sigma) treated STO fibroblasts (ATCC). The cell culture medium contained 15% fetal bovine serum (Invitrogen), 0.1 mM β-mercaptoethanol, 2 mM l-glutamine, 0.1 mM MEM non-essential amino acids, and 0.1% (v/v) leukemia inhibitory factor (LIF). R1 ES cells were labeled with β-galactosidase using a gene trap insertion into the ubiquitously expressed RNF4 gene as described earlier in [8]. HM1 ES cells were transfected with eGFP (EGFPN1 vector,

Results

To track transplanted donor cells, R1 mouse ES cells expressing β-galactosidase driven by the RNF4 promoter (R1-β-gal) [8] and HM1 mouse ES cells expressing enhanced green fluorescent protein (eGFP) under the control of the CMV promoter (HM1-eGFP) were used. To verify in vitro that selected ES cell lines retained the capacity to differentiate into the major cell types present in the heart and concurrently express the desired reporter protein, EBs were formed. Both HM1-eGFP and R1-β-gal cells

Discussion

The results demonstrate that undifferentiated mouse ES cells transplanted directly into the mouse heart following surgically induced MI engraft and repair myocardium resulting in improved cardiac function relative to media-injected control mice. This finding is consistent with earlier studies transplanting mouse ES cells or derivatives into infarcted rat hearts [5], [6], [7]. The major new findings in the present work are: 1) transplanted ES cells post-MI integrate into the heart and become

Acknowledgments

We acknowledge the assistance of Thankful Sanftleben in the preparation of the manuscript, Ravi C. Ballijepalli for assistance with confocal microscopy and Sarah Lodding for culture of ES cells. Support was provided by NIH PO1 HL47053 (T.J.K. and G.E.L.), NIH R21 HL72089 (T.J.K.), AHA SDG 0430227N (D.K.S.), and WiCell Research Institute (T.J.K.).

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Present address: Cardiovascular Research Institute, University of Vermont, Colchester, VT 05446, USA.

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