Labeling stem cells with superparamagnetic iron oxide nanoparticles: analysis of the labeling efficacy by microscopy and magnetic resonance imaging

Abstract

Stem cell therapy has emerged as a potential therapeutic option for cell death-related heart diseases. Application of non-invasive cell tracking approaches is necessary to determine tissue distribution and lifetime of stem cells following their injection and will likely provide knowledge about poorly understood stem cells mechanisms of tissue repair. Magnetic resonance imaging (MRI) is a potentially excellent tool for high-resolution visualization of the fate of cells after transplantation and for evaluation of therapeutic strategies. The application of MRI for in vivo cell tracking requires contrast agents to achieve efficient cell labeling without causing any toxic cellular effects or eliciting any other side effects. For these reasons clinically approved contrast agents (e.g., ferumoxides) and incorporation facilitators (e.g., protamine) are currently the preferred materials for cell labeling and tracking. Here we describe how to use superparamagnetic iron oxide nanoparticles to label cells and to monitor cell fate in several disease models.

Fig. 1

Derivatization of SPION nanoparticles and labeling of cells. Schematic diagram representing the steps optimizing FeProt complex formation and incorporation by cells.

Fig. 2

Importance of facilitator agents for labeling cells with SPIONs. Representative images showing that cationic compounds, such as protamine, are necessary for the efficient uptake of SPIONs. Detection of SPIONs is with anti-dextran antibody. (a) Mesenchymal stem cells incubated only with protamine without ferumoxides. (b) Cells incubated with ferumoxides in the absence of facilitating agent. (c) Cells exposed to FeProt complexes using low protamine concentration (1 μg/mL), which is five times less than the recommended concentration (5 μg/mL). (d ) Mesenchymal cells incubated with FeProt complexes with 5 μg/mL protamine. The arrows are showing some of the ferumoxide-labeled cells. Cell nuclei are labeled blue with DAPI; no cells with SPIONs are detected in (a ), approximately 7 % cells contain SPIONs in (b ), and 95 % in (d). We did not quantify the percentage of labeled cells in group (c). Scale bar = 50 μm.

Fig. 3

Anti-dextran and Prussian Blue staining. Representative images of cells labeled with FeProt complexes for 4 h. (a–d) Mesenchymal stem cells immunostained for anti-dextran antibody. (a) Brightfield; (b ) anti-dextran antibody; (c) nuclei counterstaining with DAPI; and (d) merged images. (e) Labeled cells stained with Prussian Blue showing the presence of iron. Note the characteristic perinuclear distribution of SPIONs revealed by both staining methods. Scale bar = 50 μm.

Fig. 4

Detecting SPION labeled cells using MRI in vivo and in vitro. (a ) Appearance of FeProt labeled cells after trypsinization and immunolabeling for dextran. (b ) In vitro MRI of cells labeled for 4 h at different concentrations of cells/μL gelatin. (c) Representative image of in vivo MRI (transverse plane) showing hypointense (black) spots corresponding to ferumoxide-labeled cells injected in the leg muscles (white arrows). (d–f) Dextran immunocytochemistry confirming the presence of ferumoxide-labeled cells in sections of leg muscle; (d) nuclear counterstaining with DAPI; (e) dextran; (f) merged images in higher-magnification of the area indicated by the box in (d ) and (e ) showing a transplanted cell (nucleus marked with an asterisk) labeled with ferumoxide (arrowhead). Scale bar = 50 μm.