Non-invasive Tracking of Stem Cell-derived Cardiomyocyte Using Magnetic Beads

Embryonic Stem (ES) cells are recognized as promising tools that are potentially used for the treatment of various injuries such as cardiovascular damages. Labeling of ES cells with superparamagnetic iron oxide (SPIONs) nanoparticles enables to monitor their fate in vivo using magnetic resonance imaging (MRI). However, prior to the application of human ES cells for heart failure therapy, it is critical to validate their clinical use in large animals such as primates. In present study, we examined the uptake efficacy of magnetic labeling of cynomolgus monkey ES cells, the toxicity effects on cell behavior, division and/or myocardial differentiation processes that have not been determined. ES cells were labeled with the superparamagnetic iron oxide nanoparticles (SPIONs;Feridex®). To increase the uptake efficacy, SPIONs were dispersed in the matrix of gelatin nanoparticles. Prussian Blue (PB) staining was employed to track the accumulation of SPIONs in the cells. Myocardial differentiation of magnetically labeled ES cells was confirmed by immunocytochemsitry, transmission electron microscopy (TEM) and western blotting. Our results have shown that iron content (i.e. SPIONs amount) in ES cells was maintained throughout cell proliferation process in comparison with the Plus/lipofectamine transfection method. Subsequently, we studied the effect of SPIONs on the formation of embryoid bodies (EBs) and myocardial differentiation of ES cell in monolayer culture. The formation of EBs was not inhibited and cardiac differentiation was supported regardless of SPIO nanoparticles used. To track magnetic ES cells in vivo for long term, 3.5x106 SPIONs-gelatin ES cellswere injected intra-myocardialy into adult cynomolgus monkey (n=4) and visualized with MRI on a 2T MR scanner. On MR images, the labeled cells have shown well-defined hypointensive areas at the injection site in the intact heart with long-term localization in vivo and were confirmed with histology finding. In conclusion, effective labeling methods encompass a balance between introducing a large amount of SPIONs into the ES cells (and the consequential cytotoxicity associated with the presence of SPIONs) and the transfection method. We have demonstrated an intracellular superparamagnetic nanoparticle labeling method and analyzed the effects of iron content (e.g. SPIONs amount) in the ES cell derived EBs, including effects on proliferation, survival rate, and distribution of the contrast agent following differentiation. The increased efficiency of gelatin nanoparticles transfection and controlled release of SPIONs throughout the cell proliferation are important to extend the window of detection for in vivo MRI imaging of implanted cells. The distribution of contrast materials within the cardiomyocyte after differentiation of ES cells opens the door for specifically targeted contrast enhancement at a molecular level and the potential to visualize ES cell growth for cardiac regeneration; a subject that requires further investigation. These experiments were the first to demonstrate the potential magnetic labeling of nonhuman primate In conclusion, effective labeling methods encompass a balance between introducing a large amount of SPIONs into the ES cells (and the consequential cytotoxicity associated with the presence of SPIONs) and the transfection method. We have demonstrated an intracellular superparamagnetic nanoparticle labeling method and analyzed the effects of iron content (e.g. SPIONs amount) in the ES cell derived EBs, including effects on proliferation, survival rate, and distribution of the contrast agent following differentiation. The increased efficiency of gelatin nanoparticles transfection and controlled release of SPIONs throughout the cell proliferation are important to extend the window of detection for in vivo MRI imaging of implanted cells. The distribution of contrast materials within the cardiomyocyte after differentiation of ES cells opens the door for specifically targeted contrast enhancement at a molecular level and the potential to visualize ES cell growth for cardiac regeneration; a subject that requires further investigation. These experiments were the first to demonstrate the potential magnetic labeling of nonhuman primate (cynomolgus monkey) ES cells to develop into cardiomyocytes in vitro and in vivo. Old World monkeys, such as the cynomolgus monkey (Macaca fascicularis), are widely used for medical research and have similar gene expression profiles to humans. Therefore, their ES cells might be used as a model for elucidating primate cardiomyocyte development.

کلیدواژه ها:

Superparamagnetic iron oxide nanoparticles ، Gelatin nanoparticles ، embryonic stem cell ، Cell tracking ، Myocardial differentiation ، MRI