As a widely available noninvasive imaging modality with high resolution, exquisite tissue contrast, and superb anatomical detail, magnetic resonance imaging has found extensive applications in stem cell imaging in both pre-clinical and clinical settings [2
]. MRI of SPIO nanoparticles, which can be internalized by cells to generate strong MRI contrast in T2
- and T2
*-weighted images, has become a valuable tool for studying the in vivo
fate of transplanted stem cells and precursor cells. Cellular uptake of SPIO may be induced by fluid phase endocytosis or macropinocytosis. Internalized iron oxide particles remain transiently in the endosome/lysosome compartment. The effects of SPIO labeling on cellular expression of transferrin receptor and ferritin have been investigated in different types of cells [17
]. Ferritin gene transcripts and protein were significantly increased in HeLa cells labeled with SPIO, compared with unlabeled cells. Ferritin expression from SPIO-labeled mesenchymal stem cells (MSCs) grown to confluence demonstrated an insignificant increase in ferritin mRNA expression, compared with that in corresponding unlabeled cells. However, the ferritin protein levels were significantly increased at each time point compared with unlabeled MSC whole cell lysate. Some of the iron oxide-loaded endosomes fused with lysomes and, at a pH of 4.5 maintained with appropriate buffers, resulted in the dissolution of the iron oxide nanoparticles, which suggests that the iron from the nanoparticles can be released into the intracellular compartment and participate in cellular iron metabolism [18
]. The increased expression of ferritin in SPIO-labeled cells is a response to the intracellular iron increase that is due to the iron degraded from SPIO in cytoplasm. Although magnetic cell labeling is robust and allows the detection of a few cells, once the cells divide, there is an exponential loss of the iron oxide.
Several candidate MRI reporter genes, including one that encodes the iron storage protein ferritin, have been reported [8
]. Although a primary application for MR reporter genes is in monitoring gene therapy [8
], an alternative application is tracking stem or progenitor cells after transplantation. The results shown here suggest that endogenous expression of ferritin reporter gene can prevent the dilution of MRI signal. However, the use of ferritin MRI reporter gene to track cells is limited by several factors. First, the cellular contrast of MRI produced by endogenous iron is weak and the sensitivity of detection is low. Second, sufficient endogenous iron needs to be present, with different tissues having different iron concentrations. Third, the T2
signal attenuation induced by ferritin increases linearly with field strength, making it a more efficient T2
contrast agent at higher fields. The sensitivity of detection is lower at clinical field strengths (1.5 and 3 T).
Despite the strong appeal of a molecular MRI reporter system and SPIO for stem cell research, no attempt has been reported of combining ferritin MRI reporter with SPIO in tracking stem cells. We speculate that overexpressed ferritin protein by transfection in treated cells could sequester cytoplasmic iron degraded from SPIO, as an exogenous iron source, in addition to endogenous iron from the labile iron pool. This could enhance cellular contrast and result in a shortening of T2 and T2* of the cells in the tissue and produce a persistent decrease in signal intensity in areas containing SPIO-labeled cells.
In the present study, a plasmid of H-chain of murine ferritin was transfected into C6 cells and then labeled with SPIO. The cooperative effect between ferritin protein and SPIO was studied in vitro and in vivo. There was no obvious difference in cellular SPIO labeling between C6 rat glioma cells transfected with H-chain of ferritin and control C6 cells (). The concentration of iron in transfected C6 cells showed a minimal increase compared to parental C6 cells when incubated with a routine medium (p=0.684; ), while the concentration of iron was significantly increased in transfected C6 with Fe supplement (p=0.01; ). The results indicated that overexpressed ferritin protein worked, and the H-chain of murine ferritin could sequester more iron independently. We then tested the relationship between accumulation of iron associated with overexpressed ferritin protein and concentration of Fe supplement (0.2, 0.25, 0.5, 1.0, 1.5 mM). Our data showed that the intracellular iron concentration is not linearly associated with iron supplement concentration. In this study, 0.25 mM Fe supplement was the appropriate concentration for ferritin protein to sequester more iron. The intracellular concentration of iron in transfected and parental C6 cells showed no difference at first passage 2 days after SPIO labeling (n=3, p=0.383). This can be interpreted as meaning that only a small portion of SPIO degraded 2 days after labeling. The concentration of intracellular iron sharply decreased after second passage at day 4, and C6 cells overexpressing ferritin accumulated more iron than parental C6 cells from the second to sixth passages (). Our data indicated that the intracellular iron concentration in cells labeled with SPIO decreased rapidly during the first several passages. The concentration of iron in xenografts from transfected C6 cells was minimally increased compared to that from parental C6 cells without SPIO labeling (n=4, p=0.129). Although overexpressed ferritin in implanted C6 cells has the potential to sequester relatively large amount of iron, the concentration of endogenous iron is limited, and there is not enough iron entering ferritin protein under normal physiological condition. The concentration of iron in transfected C6 cells labeled with SPIO was significantly higher than in the control C6 cells labeled with SPIO (n=5, p=0.034). This is consistent with the results from in vivo MRI of inoculated mice. C6 cells overexpressing ferritin protein showed minimally enhanced visualization, compared with parental C6 cells 13 days after implantation (a, b; n=4, p=0.056). Ferritin-transfected C6 cells labeled with SPIO significantly reduced signal intensity in T2-weighted images for MRI at the same time point (c, d; n=5, p=0.021). Although we cannot deduce from this study the detailed mechanisms of interaction between overexpressed ferritin and iron degraded from SPIO, it is possible that more iron clustering in the transgenic cells leads to the effect on relaxivity.
It is worth noting that mammalian ferritins are composed of light and heavy polypeptide subunits. The H polypeptide has a potent ferroxidase activity and L
polypeptide plays a role in iron nucleation and protein stability [19
]. In addition, expression of mutant light chain induced iron overload in transgenic mice has been recently reported [19
]. In this study, however, we transfected only H-chain of murine ferritin into C6 cells. Our future work will include testing the effects of co-transfection of H and L
-chain of ferritin, or the effects of L
-chain transfection alone, on iron nucleation and MRI tracking. Furthermore, the current study tracked only rapidly dividing C6 cells. A topic of our future research will also be whether an increase of ferritin levels in slowly dividing or nondividing stem cells would also help retain iron to facilitate T2