Ferumoxytol: a new, clinically applicable label for stem-cell tracking in arthritic joints with MRI

Abstract

Aim: To develop a clinically applicable MRI technique for tracking stem cells in matrix-associated stem-cell implants, using the US FDA-approved iron supplement ferumoxytol.

Materials & methods: Ferumoxytol-labeling of adipose-derived stem cells (ADSCs) was optimized in vitro. A total of 11 rats with osteochondral defects of both femurs were implanted with ferumoxytol- or ferumoxides-labeled or unlabeled ADSCs, and underwent MRI up to 4 weeks post matrix-associated stem-cell implant. The signal-to-noise ratio of different matrix-associated stem-cell implant was compared with t-tests and correlated with histopathology.

Results: An incubation concentration of 500 µg iron/ml ferumoxytol and 10 µg/ml protamine sulfate led to significant cellular iron uptake, T2 signal effects and unimpaired ADSC viability. In vivo, ferumoxytol- and ferumoxides-labeled ADSCs demonstrated significantly lower signal-to-noise ratio values compared with unlabeled controls (p < 0.01). Histopathology confirmed engraftment of labeled ADSCs, with slow dilution of the iron label over time.

Conclusion: Ferumoxytol can be used for in vivo tracking of stem cells with MRI.

Figure 1. Magnetic resonance signal effects of ferumoxytol-labeled stem cells

(A) T2-weighted magnetic resonance images and (B) T2 relaxation time maps of centrifuged stem-cell pellets, labeled with increasing concentrations of ferumoxytol (µg/ml). Labeled cell pellets show a marked negative signal (dark) compared with unlabeled controls. Endorem^® (Guerbet, Aulnay-sous-Bois, France; ferumoxides, 100 µg/ml) and ferumoxytol samples, labeled according to Thu et al. [39], shown for comparison.

Figure 2. In vitro evaluations of ferumoxytol-labeled cells

(A) Viability, (B) iron content and (C) T2 relaxation time of stem cells, labeled with increasing concentrations of ferumoxytol. Data are displayed as means and standard errors of triplicate samples per experimental group, with 0.5 × 10⁶ cells per sample.

Figure 3. Magnetic resonance signal effects of this study’s labeling technique compared with Thu et al.’s over time

(A) T2 relaxation time maps of stem-cell pellets, labeled with this study’s technique, Thu et al.’s technique [39] and control samples at days 1 and 14 post labeling along with (B) corresponding graph of mean T2 relaxation times and standard errors of triplicate samples per experimental group, showing no difference in T2 relaxation times between this study’s and Thu et al.’s technique [39] after 14 days post labeling.

Figure 4. Ferumoxytol uptake by adipose-derived stem cells

3,3´-diaminobenzidine–Prussian blue stains of (A) unlabeled control cells and (B) ferumoxytol-labeled cells. (C) Transmission electron microscopy image of an adipose-derived stem cell labeled with 500 µg/ml ferumoxytol showing iron nanoparticles in secondary lysosomes in the cytoplasm (arrows). (D) Fluorescence microscopy of unlabeled control cells and (E) FITC-conjugated ferumoxytol labeled cells. Only ferumoxytol-labeled cells demonstrate positive 3,3´-diaminobenzidine–Prussian blue staining and positive fluorescence. (F) Mean fluorescence per cell, as quantified in a Cell-IQ^® 2 imager (Chip-Man Technologies Ltd, Tempere, Finland), of triplicate samples per experimental group with standard deviations. *Indicates significant differences between unlabeled controls and FITC-conjugated ferumoxytol-labeled cells (p < 0.05). CLIO: Cross-linked iron oxide; FITC: Fluorescein isothiocyanate; RFU: Relative fluorescence unit.

Figure 5. Chondrogenic differentiation of control and labeled cell pellets

(A) Hematoxylin and eosin stain of control and (B) labeled cell pellets are shown. (C) Alcian blue stains of control and (D) labeled cells showing positive (blue) staining, suggesting uninhibited chondrogenesis.

Figure 6. Sagittal T2-weighted magnetic resonance images through representative knee joints with matrix-associated stem-cell transplants (arrows) in osteochondral defects of the distal femur

(A) Ferumoxytol-labeled cell transplants, (B) ferumoxides-labeled cell transplants and (C) unlabeled controls at week 0, 2 and 4 post-adipose-derived stem-cell implantation. Ferumoxytol- and ferumoxides-labeled cell transplants demonstrate a strong, negative signal effect in vivo, which slowly declines over time. Field of view was 2.5 × 2.5 cm.

Figure 7. Corresponding quantitative magnetic resonance signal effects of labeled and unlabeled matrix-associated stem-cell transplants in vivo

(A & C) SNRs of unlabeled cell transplants: (A) n = 6; (C) n = 4, showing unchanged signal over 4 weeks. SNRs of (B) ferumoxytol-labeled (n = 6) and (D) ferumoxides-labeled stem-cell transplants (n = 4), showing slow decline of signal (increase in SNR) over 4 weeks, suggesting slow dilution of the iron label. The SNR is significant between ferumoxytol-labeled and unlabeled cell transplants until week 2 and not significant between ferumoxytol- and ferumoxides-labeled cell transplants at all time points. Data are displayed as mean SNRs and standard errors of ferumoxytol-labeled ADSCs (n = 6) or ferumoxides-labeled ADSCs (n = 4) and corresponding unlabeled controls (n = 10) at different time points after stem-cell implantation. ADSC: Adipose-derived stem cell; SNR: Signal-to-noise ratio.

Figure 8. Histopathologic correlations of in vivo MRI data

Hematoxylin and eosin stains of (A) unlabeled controls showing engraftment of both labeled and unlabeled adipose-derived stem cells in the defect. (B) Ferumoxytol- and (C) ferumoxides-labeled matrix-associated stem-cell implant at 4 weeks after transplantation. FISH stains against rat Y chromosome showing red fluorescence localized to the nuclei of transplanted cells (counterstained by 4´,6-diamidino-2-phenylindole:blue) in all implants ([D] control, [E] ferumoxytol-labeled and [F] ferumoxides-labeled) suggesting transplanted XY cells in XX female rats.

Figure 9. Histologic correlation of iron and FISH stains in stem-cell transplants

Positive 3,3´-diaminobenzidine–Prussian blue staining (brown) is seen at 2 weeks after transplantation in (A) ferumoxytol-labeled adipose-derived stem cells but not at (B) 4-weeks post-transplant showing slow dilution of iron oxide label over time. (C) Unlabeled controls remain unstained. FISH stains against rat Y chromosome showing red fluorescence localized to the nuclei of transplanted cells (counterstained by 4´,6-diamidino-2-phenylindole:blue) at both (D) 2-week and (E) 4-week post-implant along with (F) unlabeled implants demonstrating transplanted XY cells in XX female rats.