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In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium.

ABSTRACT: OBJECTIVES:The aim of the current study is to test the ability to label and detect murine embryonic stem cell-derived cardiovascular progenitor cells (ES-CPC) with cardiac magnetic resonance (CMR) using the novel contrast agent Gadofluorine M-Cy3 (GdFM-Cy3). BACKGROUND:Cell therapy shows great promise for the treatment of cardiovascular disease. An important limitation to previous clinical studies is the inability to accurately identify transplanted cells. GdFM-Cy3 is a lipophilic paramagnetic contrast agent that contains a perfluorinated side chain and an amphiphilic character that allows for micelle formation in an aqueous solution. Previous studies reported that it is easily taken up and stored within the cytosol of mesenchymal stem cells, thereby allowing for paramagnetic cell labeling. Investigators in our laboratory have recently developed techniques for the robust generation of ES-CPC. We reasoned that GdFM-Cy3 would be a promising agent for the in vivo detection of these cells after cardiac cell transplantation. METHODS:ES-CPC were labeled with GdFM-Cy3 by incubation. In vitro studies were performed to assess the impact of GdFM-Cy3 on cell function and survival. A total of 500,000 GdFM-Cy3-labeled ES-CPC or control ES-CPC were injected into the myocardium of mice with and without myocardial infarction. Mice were imaged (9.4-T) before and over a 2-week time interval after stem cell transplantation. Mice were then euthanized, and their hearts were sectioned for fluorescence microscopy. RESULTS:In vitro studies demonstrated that GdFM-Cy3 was easily transfectable, nontoxic, stayed within cells after labeling, and could be visualized using CMR and fluorescence microscopy. In vivo studies confirmed the efficacy of the agent for the detection of cells transplanted into the hearts of mice after myocardial infarction. A correspondence between CMR and histology was observed. CONCLUSIONS:The results of the current study suggest that it is possible to identify and potentially track GdFM-Cy3-labeled ES-CPC in murine infarct models via CMR.

SUBMITTER: Adler ED

PROVIDER: S-EPMC3638738 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium.

Adler Eric D ED Bystrup Anne A Briley-Saebo Karen C KC Mani Venkatesh V Young Wilson W Giovanonne Steven S Altman Perry P Kattman Steven J SJ Frank Joseph A JA Weinmann Hans J HJ Keller Gordon M GM Fayad Zahi A ZA

JACC. Cardiovascular imaging 20090901 9

<h4>Objectives</h4>The aim of the current study is to test the ability to label and detect murine embryonic stem cell-derived cardiovascular progenitor cells (ES-CPC) with cardiac magnetic resonance (CMR) using the novel contrast agent Gadofluorine M-Cy3 (GdFM-Cy3).<h4>Background</h4>Cell therapy shows great promise for the treatment of cardiovascular disease. An important limitation to previous clinical studies is the inability to accurately identify transplanted cells. GdFM-Cy3 is a lipophilic ...[more]

PMID: 19761992

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Project description:Introduction: Vascular remodeling and compromised alveolar development are hallmarks of chronic pulmonary diseases such as bronchopulmonary dysplasia (BPD). Despite advances in neonatal healthcare the number of BPD cases worldwide continues to increase. One approach to overcoming the premature arrest in lung development seen in BPD is to stimulate neonatal angiogenesis via delivery and engraftment of endothelial progenitor cells (EPCs). One such population is resident to the pulmonary microvasculature and expresses both FOXF1 and c-KIT. Previous studies have shown that c-KIT+FOXF1+ EPCs are highly sensitive to elevated levels of oxygen (hyperoxia) and are decreased in premature infants with BPD and hyperoxia-induced BPD mouse models. We hypothesize that restoring EPCs through transplantation of c-KIT+FOXF1+ EPCs derived in vitro from pluripotent embryonic stem cells (ESCs), will stimulate neonatal angiogenesis and alveolarization in mice with hyperoxia-induced lung injury. Methods: Utilizing a novel ESC line with a FOXF1:GFP reporter, we generated ESC-derived c-KIT+FOXF1+ EPCs in vitro. Using a second ESC line which contains FOXF1:GFP and tdTomato transgenes, we differentiated ESCs towards c-KIT+FOXF1+ EPCs and tracked them in vivo after injection into the neonatal circulation of hyperoxia-injured mice. After a recovery period in room air conditions, we analyzed c-KIT+FOXF1+ EPC engraftment and quantified the number of resident and circulating endothelial cells, the size of alveolar spaces, and the capillary density after EPC transplantations. Results and conclusion: Herein, we demonstrate that addition of BMP9 to the directed endothelial differentiation protocol results in very efficient generation of c-KIT+FOXF1+ EPCs from pluripotent ESCs. ESC-derived c-KIT+FOXF1+ EPCs effectively engraft into the pulmonary microvasculature of hyperoxia-injured mice, promote vascular remodeling in alveoli, increase the number of resident and circulating endothelial cells, and improve alveolarization. Altogether, these results provide a proof-of-principle that cell therapy with ESC-derived c-KIT+FOXF1+ EPCs can prevent alveolar simplification in a hyperoxia-induced BPD mouse model.

Project description:Acute myocardial infarction (MI) resulting from coronary ischemia is a major cause of disability and death worldwide. Transplantation of human embryonic stem cell (hESC)-derived cardiovascular progenitor cells (hCVPCs) promotes the healing of infarcted hearts by secreted factors. However, the hCVPC-secreted proteins contributing to cardiac repair remain largely unidentified. In this study, we investigated protective effects of neurotrophin (NT)-3 secreted from hCVPCs in hearts against myocardial ischemia/reperfusion (I/R) injury and explored the underlying mechanisms to determine the potential of using hCVPC products as a new therapeutic strategy. The implantation of hCVPCs into infarcted myocardium at the beginning of reperfusion following 1 hour of ischemia improved cardiac function and scar formation of mouse hearts. These beneficial effects were concomitant with reduced cardiomyocyte death and increased angiogenesis. Moreover, hCVPCs secreted a rich abundance of NT-3. The cardioreparative effect of hCVPCs in the I/R hearts was mimicked by human recombinant NT-3 (hNT-3) but canceled by NT-3 neutralizing antibody (NT-3-Ab). Furthermore, endogenous NT-3 was detected in mouse adult cardiomyocytes and its level was enhanced in I/R hearts. Adenovirus-mediated NT-3 knockdown exacerbated myocardial I/R injury. Mechanistically, hNT-3 and endogenous NT-3 inhibited I/R-induced cardiomyocyte apoptosis through activating the extracellular signal-regulated kinase (ERK) and reducing the Bim level, resulting in the cardioreparative effects of infarcted hearts together with their effects in the improvement of angiogenesis. These results demonstrate for the first time that NT-3 is a cardioprotective factor secreted by hCVPCs and exists in adult cardiomyocytes that reduces I/R-induced cardiomyocyte apoptosis via the ERK-Bim signaling pathway and promotes angiogenesis. As a cell product, NT-3 may represent as a noncell approach for the treatment of myocardial I/R injury.

Dataset Information

In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium.

Publications

In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium.

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