Ferumoxides-protamine sulfate (FE-Pro) complexes are used for intracellular magnetic labeling of cells to non-invasively monitor cell trafficking by in vivo MRI. FE-Pro labeling is non-toxic to cells; however, the effects of FE-Pro labeling on cellular expression of transferrin receptor (TfR-1) and ferritin, proteins involved in iron transport and storage, has not been reported. FE-Pro-labeled human mesenchymal stem cells (MSCs), HeLa cells and primary macrophages were cultured from 1 week to 2 months and evaluated for TfR-1 and ferritin gene expression by RT-PCR and protein levels were determined using Western blots. MTT (proliferation assay) and reactive oxygen species (ROS) analysis were performed. FE-Pro labeling of HeLa and MSCs resulted in a transient decrease in TfR-1 mRNA and protein levels. In contrast, Fe-Pro labeling of primary macrophages resulted in an increase in TfR-1 mRNA but not in TfR-1 protein levels. Ferritin mRNA and protein levels increased transiently in labeled HeLa and macrophages but were sustained in MSCs. No changes in MTT and ROS analysis were noted. In conclusion, FE-Pro labeling elicited physiological changes of iron metabolism or storage, validating the safety of this procedure for cellular tracking by MRI.

Personalized treatment using stem, modified or genetically engineered, cells is becoming a reality in the field of medicine, in which allogenic or autologous cells can be used for treatment and possibly for early diagnosis of diseases. Hematopoietic, stromal and organ specific stem cells are under evaluation for cell-based therapies for cardiac, neurological, autoimmune and other disorders. Cytotoxic or genetically altered T-cells are under clinical trial for the treatment of hematopoietic or other malignant diseases. Before using stem cells in clinical trials, translational research in experimental animal models are essential, with a critical emphasis on developing noninvasive methods for tracking the temporal and spatial homing of these cells to target tissues. Moreover, it is necessary to determine the transplanted cell's engraftment efficiency and functional capability. Various in vivo imaging modalities are in use to track the movement and incorporation of administered cells. Tagging cells with reporter genes, fluorescent dyes or different contrast agents transforms them into cellular probes or imaging agents. Recent reports have shown that magnetically labeled cells can be used as cellular magnetic resonance imaging (MRI) probes, demonstrating the cell trafficking to target tissues. In this review, we will discuss the methods to transform cells into probes for in vivo imaging, along with their advantages and disadvantages as well as the future clinical applicability of cellular imaging method and corresponding imaging modality.

There is increasing interest in using exogenous labels such as bromodeoxyuridine (BrdU) STEM CELLS 2008;26:1366 -1375 Disclosure of potential conflicts of interest is found at the end of this article.

Superparamagnetic iron oxide nanoparticles (SPION) are increasingly used to label human bone marrow stromal cells (BMSCs, also called “mesenchymal stem cells”) to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB) staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the “stemness” of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the “gold standard” of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs.

Intracellular labels such as dextran coated superparamagnetic iron oxide nanoparticles (SPION), bromodeoxyuridine (BrdU) or green fluorescent protein (GFP) are frequently used to study the fate of transplanted cells by in vivo magnetic resonance imaging or fluorescent microscopy. Bystander uptake of labeled cells by resident tissue macrophages (TM) can confound the interpretation of the presence of intracellular labels especially during direct implantation of cells, which can result in more than 70% cell death. In this study we determined the percentages of TM that took up SPION, BrdU or GFP from labeled bone marrow stromal cells (BMSCs) that were placed into areas of angiogenesis and inflammation in a mouse model known as Matrigel™ plaque perfusion assay. Cells recovered from digested plaques at various time points were analyzed by fluorescence microscopy and flow cytometry. The analysis of harvested plaques revealed 5% of BrdU+, 5–10% of GFP+ and 5–15% of dextran+ macrophages. The transfer of the label was not dependent on cell dose or viability. Collectively, this study suggests that care should be taken to validate donor origin of cells using an independent marker by histology and to assess transplanted cells for TM markers prior to drawing conclusions about the in vivo behavior of transplanted cells.