Magnetic reporter genes for MRI-based stem cell tracking

Introduction: Over the past decades, several labelling techniques have been used in an attempt to track stem cells using magnetic resonance imaging (MRI). However, very few of these were able to definitely determine the precise location of stem cells within a living organism and monitor throughout a long term period, without loss or diffusion of the signal. A novel MRI cell tracking method described in 2005 proposed that reporter genes that could effectively increase the iron content of a target cell would allow a stronger contrast when imaged via MR. Being a fundamental part of the iron metabolism, transferrin receptor 1 (TfR 1) and ferritin heavy chain 1 (Fth 1) were naturally suggested to have the potential to increase the iron load of cells when overexpressed. More recently, there has been some interest in the reporter gene MagA, which is a known iron transporter found in magnetotactic bacteria. Aim: To evaluate the suitability of using TfR 1, Fth 1 and MagA as potential magnetic reporter genes for MRI-based cell tracking. Methods: Several cell and stem cell lines were transduced with a 2nd generation HIV-based lentiviral system containing one or more magnetic reporters. Viral transduction resulted in genome incorporation of bicistronic construct(s) with TfR 1 gene alongside a gene encoding a green fluorescent reporter (GFP) and/or Fth 1 and MagA gene alongside a red fluorescent reporter (RFP). This allowed for identification and monitoring of positive cells with complementing imaging modalities: MRI and fluorescence based methods. Transgenes were evaluated for integration stability over passages and their influence on iron homeostasis was assessed; also, integration and/or overexpression were confirmed at the mRNA and protein level. Finally, the influence of magnetic reporters on intracellular iron retention and MRI contrast capacity was tested both in vitro and in a model organism, the chick embryo. Results: After analysing all three potential magnetic reporters, TfR 1 was found to be the most promising, as its overexpression induced an adjustment of iron homeostasis in Chinese hamster ovary K1 cells, leading to higher intracellular iron accumulation relative to controls. The same adjustment was found in mouse mesenchymal stem cells (mMSC), but only when TfR 1 was overexpressed in conjunction with Fth 1, also leading to an increase in iron retention capacity. However, a limitation was found when overexpressing Fth 1 in mMSC, as permanent iron supplementation was needed in order to keep these cells viable. In contrast with previous studies, MagA gene integration posed some restrictions in certain cell lines studied. The results presented here show that while some cell types are able to stably maintain MagA expression over several passages, others fail to survive and die shortly after transduction, suggesting that a potential toxic effect may be originating from MagA gene integration. From the surviving cells, two were compared side by side and contradictory results were obtained, demonstrating that MagA would only be a suitable magnetic reporter for some cell types. Conclusion: The results obtained with this project are of relevance for reporter gene-based MRI cell tracking as they show that no single magnetic reporter is capable of generating detectable MRI contrast for a global cohort of cell lines. On the contrary, overexpression of endogenous genes or integration of foreign genes should be performed with caution and analysed on a case by case basis. Finally, for some cell type and magnetic reporter gene combinations, this study suggests that MRI could be a promising method for the longitudinal monitoring of engrafted cells, especially when the cells have been cultured in media supplemented with low concentrations of iron