The promotion of human mesenchymal stem cell proliferation by superparamagnetic iron oxide nanoparticles
Introduction
Stem cell therapy is a rapidly evolving area of research in regenerative medicine. Being able to monitor the fate of transplanted cells in vivo is crucial for developing successful cell therapies. Furthermore, an effective, noninvasive, nontoxic method is required for ideal cell tracking. Recently, magnetic resonance imaging (MRI) has afforded the superb spatial resolution and repeated noninvasive imaging of magnetically labeled cells and has been the most attractive modality for cellular imaging [1], [2]. For MRI detection, however, the cells need to be internally labeled with contrast agents. Of the two main classes of contrast agents, superparamagnetic iron oxide (SPIO) nanoparticles and gadolinium-based chelates, most approaches are with the usage of the former class as cellular MRI probe. SPIO nanoparticles are commercially available and already FDA-approved for use in humans [3], [4], but not yet in intracellular labeling. To achieve sufficiently intracellular uptake of SPIO nanoparticles for efficient MRI, most studies use transfection agents [3], [4], [5], [6]; most of these agents, however, are cationic and toxic [7], [8].
Regardless of the toxicity of transfection agents, the potential effects of SPIO nanoparticles on stem cells remain vitally important. Although it is believed that SPIO nanoparticles are inert, biocompatible nanomaterials capable of being eventually metabolized, the term “biocompatible” can be misleading due to a lack of criteria for evaluating the toxic effects of nanomaterials. Furthermore, the cytotoxicity of SPIO nanoparticles remains an unresolved issue [9], [10], [11], [12], [13], [14], [15], [16]. A recent study showed that Resovist (Ferucarbotran) without using a transfection agent was nontoxic and more efficiently taken up by human mesenchymal stem cells (hMSCs) than Feridex (Ferumoxides) [17]. Therefore, Ferucarbotran would be an ideal candidate for studying the precise action of SPIO nanoparticles on cellular responses.
In view of the present biocompatibility definition of nanoparticles, most studies have focused on whether cytotoxicity was observed [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. In this study, however, we examined the cellular effects of Ferucarbotran labeling in different views on hMSCs to explore the biosafety of SPIO nanoparticles for cellular MRI. The recent finding that magnetic nanoparticles possess intrinsic peroxidase-like activity [18] and the important role of H2O2 in cell growth led us to investigate whether SPIO nanoparticles can affect cell growth by decreasing intracellular H2O2.
In addition, after the internalization into cells, SPIO nanoparticles could be transferred to lysosomes, in which degradation of SPIO nanoparticles may occur and free iron (Fe) could be released into the cytoplasm [19], [20]. It is well known that Fe plays an important role in cell cycle progression, cell proliferation and apoptosis. Therefore, we also examined the effects of Ferucarbotran labeling on the cell cycle proceeding and the expression of several proteins critical for cell cycle progression.
Section snippets
Culture of hMSCs from bone marrow
Human mesenchymal stem cells (hMSCs) were isolated from bone marrow of normal donors. The bone marrow aspirate was added to low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 25 U/ml heparin in 1:1 ratio, fractionated by Ficoll-Paque density gradient centrifugation. The hMSC-enriched low-density fraction was collected, rinsed with DMEM, and plated in T25 flasks at 5 × 107 nucleated cells per flask in 5 ml regular growth medium consisting of low-glucose DMEM supplemented with
Cellular MRI of Ferucarbotran
We first examined the labeling efficiency of Ferucarbotran by a clinical 1.5-T MRI system. After that, 1.2 × 105 hMSCs were incubated with Ferucarbotran (3 and 10 μg/ml) for 1 h; under T2 weighted image mode (T2WI), the MRI images of centrifuged cell pellets in test tubes placed in a water bath were easily detected (Fig. 1A). In addition, a series of diluted Ferucarbotran-labeled hMSCs were processed for MRI to investigate the limit of imaging; the MR images of hMSCs labeled with 10 μg/ml
Conclusions
This is the first report that intracellular SPIO nanoparticles can promote cell growth of hMSCs by diminishing intracellular H2O2 and affect protein regulators of the cell cycle (Fig. 5). In contrast to the majority of studies, the present study shows that the labeling of SPIO nanoparticles is advantageous to cell growth, which leaves a matter for future consideration: whether this growth promotion is also good for stem cells themselves. Given our findings, a more comprehensive survey of
Acknowledgements
This work was supported by the grants from National Health Research Institutes (NHRI) (NM-097-PP-02 and NM-097-PP-09), Taiwan and from the National Science Council of Taiwan (96-2628-B-400-001-MY3 and 97-2314-B-002-115-MY2).
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Both authors contributed equally to this work.