Long-term MR cell tracking of neural stem cells grafted in immunocompetent versus immunodeficient mice reveals distinct differences in contrast between live and dead cells

Abstract

Neural stem cell (NSC)-based therapy is actively being pursued in preclinical and clinical disease models. Magnetic resonance imaging (MRI) cell tracking promises to optimize current cell transplantation paradigms, however, it is limited by dilution of contrast agent during cellular proliferation, transfer of label from dying cells to surrounding endogenous host cells, and/or biodegradation of the label. Here, we evaluated the applicability of magnetic resonance imaging for long-term tracking of transplanted neural stem cells labeled with superparamagnetic iron oxide and transfected with the bioluminescence reporter gene luciferase. Mouse neural stem cells were transplanted into immunodeficient, graft-accepting Rag2 mice or immunocompetent, graft-rejecting Balb/c mice. Hypointense voxel signals and bioluminescence were monitored over a period of 93 days. Unexpectedly, in mice that rejected the cells, the hypointense MR signal persisted throughout the entire time-course, whereas in the nonrejecting mice, the contrast cleared at a faster rate. In immunocompetent, graft-rejecting Balb/c mice, infiltrating leukocytes, and microglia were found surrounding dead cells and internalizing superparamagnetic iron oxide clusters. The present results indicate that live cell proliferation and associated label dilution may dominate contrast clearance as compared with cell death and subsequent transfer and retention of superparamagnetic iron oxide within phagocytes and brain interstitium. Thus, interpretation of signal changes during long-term MR cell tracking is complex and requires caution.

FIG. 1

MRI black pixel analysis. A Z-projection of minimum intensity was created and regions of interest were manually drawn for the ipsilateral hemisphere (red) and contralateral hemisphere (yellow). Pixel intensity histograms were created for each region of interest (top—contralateral, bottom—ipsilateral) and the minimum pixel value for the contralateral hemisphere served as the baseline threshold (blue line). The threshold was applied to the hemisphere with cell injection and the total number of pixels below this baseline threshold (red box) was counted as the number of black pixels due to the iron oxide.

FIG. 2

Monitoring survival and rejection of transplanted C17.2 cells. a: Bioluminescence imaging of C17.2 cells transplanted into graft accepting Rag2 and graft rejecting Balb/c mice. Mice were imaged weekly from day 2 to day 92. b: Quantification of BLI signal normalized to the signal on day 2 for each mouse. c: Anti-β-galactosidase staining (green) for transplanted C17.2 cells 16 days after transplantation. Grafted cells could be detected in Rag2 mice but not in Balb/c mice, except for one mouse that did not reject.

FIG. 3

Histopathological assessment of immunological/inflammatory response within the graft site 16 days after transplantation. a and b: Anti-CD45 staining for infiltrating leukocytes (green), iron oxide (red), and Hoechst dye (blue) in Balb/c (a) and Rag2 (b) mice. c and d: Anti-Iba1 staining for microglia (green) and iron oxide (red) in Balb/c (c) and Rag2 (d) mice.

FIG. 4

Serial in vivo MRI. a and b: T₂-weighted MR images of Balb/c (a) and Rag2 (b) mice at days 2, 11, 30, 50, 72, and 93 after transplantation. In all mice, hypointensities were detectable within the corpus callosum the day after transplantation and persisted for up to 93 days. c and d: T₂*-weighted MR images of Balb/c (c) and Rag2 (d) mice at days 2, 11, 30, 50, 72, and 93.

FIG. 5

MRI black pixel quantification for T₂-weighted (a) and T₂*-weighted (b) MR images for Balb/c (n = 3) and Rag2 (n = 5) mice on day 2, 11, 30, 50, 72, and 93 (*Significant, P < 0.05; **Significant, P < 0.01).

FIG. 6

3D Reconstruction of postmortem high-resolution MRI of a Balb/c mouse brain at 95 days after transplantation (about 80 days after complete rejection of the graft).

FIG. 7

Histological detection of iron oxide label dilution. Anti-β-galactosidase staining (green) for C17.2 cells is positive in Rag2 mice at days 16, 57, and 95 (a–c) and negative in Balb/c mice (d–f). The amount of iron oxide nanoparticles (red) gradually decreased over time in both mouse strains but was detected at all time points. Scale bar 200 μm for (a–f).

FIG. 8

Histological detection of iron oxide label and host tissue response at day 16 after grafting. Staining for anti-CD45 pan-leukocyte antigen (green), iron oxide (red), and nuclei (blue) in Balb/c mice (a–c) and Rag2 mice (d–f) demonstrates large deposits of SPIO contrast agent (asterisks) that are surrounded by CD45⁺ immune cells in Balb/c mice (arrows) very little CD45 positivity can be detected in Rag2 animals. The tissue space occupied by these deposits is anucleated, indicating that these are extracellular deposits. Anti-Iba1 staining (g, green) shows that some iron oxide nanoparticles colocalize within microglia (arrows). Prussian blue staining (h) shows iron oxide nanoparticles (blue) clustered together in a hyponucleated brain region. Scale bar 20 μm for (a–g), 100 μm for (h).