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- MATERIALS AND METHODS
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. Magn Reson Med, 2011. © 2010 Wiley-Liss, Inc.
The central nervous system (CNS) is the most complex of all systems in the human body, and it has extremely limited regeneration capabilities. Tissue repair of CNS damage is a difficult task, with two possible paradigms—manipulation of the endogenous progenitor cells or transplantation of exogenous cells. Although there have been some reports on mobilizing endogenous progenitors (1, 2), the efficacy of this approach is limited. Alternatively, transplantation of neural precursors or stem cells introduces a large amount of exogenous cells with the potential to migrate, differentiate, and integrate into neural circuitries (3). In order for cell-based therapy to be applied clinically, techniques are required that can evaluate the migration and function of cells in vivo, help to guide treatment, and maximize the therapeutic effect.
There are currently several noninvasive imaging techniques available with which one can monitor cellular and biological processes, including noninvasive in vivo cellular imaging of neurografted stem cells in real time. For transplanted cells, a major goal is to monitor cells within the living organism and in their native environment, in particular to track the cell injection, migration, and viability, and to assess their ability to provide effective treatment. MR imaging of stem cells is a particularly useful noninvasive imaging modality, as it is clinically translatable, does not expose patients to radiation, allows whole body imaging, and has a high resolution. Indeed, MR imaging of magnetically labeled stem cells has been now been applied in several clinical cell therapy trials in patients (4).
Current strategies for in vivo tracking of transplanted cells by magnetic resonance imaging (MRI) depend on labeling of cells with an MR contrast agent (5). Superparamagnetic iron oxide (SPIO) nanoparticles have previously been used clinically as cell tracking T2(*) contrast agents which, through T2 relaxation effects, induce hypointensities on MR images. Unfortunately, Feridex® (Berlex Laboratories), the SPIO contrast agent approved for clinical use in liver imaging, is no longer manufactured and available commercially. Therefore, these experiments were performed using a similar SPIO—Molday ION Rhodamine B (BioPAL, Worcester, MA), which is tagged to a fluorescent marker and is detectable by fluorescence imaging. One advantage of SPIO particles over other agents is that they are biocompatible and biodegradable through the normal biochemical pathways for iron metabolism (6). However, there are several potential pitfalls with long-term MR tracking using SPIO contrast agents. Dilution of the iron oxide label is a major limitation, as the MR signal is lost over time due to cellular proliferation, especially in the case of rapidly dividing cells. In addition, the contrast agent could possibly be transferred to surrounding endogenous cells (e.g., macrophages of microglia) after death of transplanted cells (7). If endogenous cells take up the SPIO nanoparticles, it could be difficult to interpret the imaging data, which may then give inaccurate information on the transplanted cells. Another limitation of MRI cell tracking using SPIO labeling is that this technique does not offer information about the survival or function of cells. Such information can be provided by the use of reporter gene-based imaging techniques. Bioluminescence imaging (BLI) is a well-established reporter gene-based imaging modality that has been successfully used for monitoring cell survival in small animals (5, 8, 9). Although BLI is not clinically translatable, the technique is useful for following the survival of cells in animal models until a robust, clinically applicable MR-reporter gene (10–12) becomes available.
Several recent studies have been conducted on the reliability of long-term tracking of transplanted, magnetically labeled cells and the persistence of the contrast agent in various organs. Baligand et al. xenografted SPIO-labeled myoblasts into the mouse leg and showed that the SPIO label still visible 3 months after transplantation even though cells were only viable for less than 1 week (13). Therefore, evaluating the presence of viable cells during long-term studies can be difficult. Similar results were reported for cells transplanted into heart tissue, indicating that MRI of SPIO-labeled cells do not reliably track long-term engrafted cells. MR signal from labeled cardiomyoblasts persisted significantly longer after cells died, as determined by BLI (14). Terrovitis also found that MRI overestimates ferumoxide-labeled stem cell survival following transplantation in the heart (15). Every tissue will likely interact with the contrast agent differently; therefore, it is important to carefully study these interactions and the potential long-term retention of released SPIO in local tissue. Studies designed to investigate the long-term fate of iron following intracerebral transplantation of magnetically labeled stem cells, to our knowledge, have not yet been reported.
We have chosen to allograft SPIO-labeled C17.2 neural stem cells (NSCs) in the brain of two groups of mice: immunocompetent, allograft-rejecting Balb/c mice in which all cells will be eliminated; and immunodeficient, allograft-accepting Rag2 mice, in which the cells will survive, although some degree of cell death after grafting is expected. The primary goal of this study was to establish the long-term fate, at 95 days post-transplantation, of SPIO particles and the MRI signal changes following the death of grafted cells versus the proliferation of stem cells. Cell survival or rejection was monitored and validated by reporter-gene based BLI, and the persistence or clearance of SPIOs was analyzed by T2 and T2*-weighted MRI.
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- MATERIALS AND METHODS
In this study, we demonstrated that magnetic cell labeling with SPIO results in a strong MR signal detected as hypointensities on T2- and T2*-weighted MR images. The signal persists for at least 95 days after transplantation, regardless of the survival or death of transplanted cells. Longitudinal in vivo MR imaging showed that the extent of MR hypointensity gradually reduced over time in both graft-rejecting and nonrejecting animals; however, the decrease was more pronounced and at a faster rate in surviving and proliferating stem cell grafts (in Rag2 mice) than in grafts that were immunorejected (in Balb/c mice). After 95 days, about 40% of the original number of black pixels remained in the Balb/c mice, compared with about 20% in the Rag2 mice from the T2 data. This is contradictory to previous results from Zhang et al., which reported that, when killed, SPIO-labeled neural progenitor cells were transplanted into the cisterna magna, there were no Prussian blue-positive cells histologically, implying that the iron is cleared rather than being held in endogenous cells or in the interstitium (19). In this study by Zhang et al., labeled neural progenitor cells were injected into the cerebrospinal fluid, whereas in our study, they were injected into the brain parenchyma, which could account for the difference in iron clearance from dead cells. However, viable neural progenitor cells transplanted in the parenchyma were shown to extensively migrate to the tumor, which resulted in a complete loss of MR signal from the injection site (19). They also reported a near-complete clearance of hypointense MR signals for SPIO-labeled NSCs cells that were killed with five cycles of freeze-thawing before injection, as compared with the contralateral site where the same cells were injected but not killed before transplantation (20). This differential contrast was observed at 4 weeks post-transplantation and is in stark contrast to our current results. One possible explanation is the rate of cell proliferation; C17.2 cells are immortalized and divide more rapidly than SVZ-derived progenitor cells.
We have shown that C17.2 cells, transplanted into the brain parenchyma of immunodeficient Rag2 mice, were accepted, and migrated extensively. Based on the level of BLI signal, the surviving C17.2 cells underwent many cell divisions to reach the plateau of BLI signal at day 30 implying that each cell would only have a negligible amount of iron remaining. This explains why transplanted cells that were detected further away from the injection site were usually negative for the contrast agent. Another previously proposed possibility (21) is that the C17.2 cells divide asymmetrically, and if one of the daughter cells receives the entire contrast agent load, the other daughter cell that is free of contrast becomes undetectable immediately.
In Balb/c mice, the MR signal from the iron oxide nanoparticles remains high-despite cell rejection. The death of labeled cells due to immunorejection leads to a sudden release of contrast agent and the cell debris is phagocytosed by brain phagocytes and infiltrating immune cells. The brain is considered as an immune-privileged site, and it is separated from the other organ systems, including the immune system, by the blood-brain barrier. In physiological conditions, microglia are the main immune defense of the central nervous system, surveying the local environment for potential inflammation or foreign agents and coming out of quiescence when activated (22–24). Activated CNS-specific immune cells travel across the blood-brain barrier in the case of inflammation, and in concert with activated microglia, mount an immune response. Leukocytes and microglia that immunogenically respond to the transplanted cells are programmed to undergo apoptosis in the CNS after dealing with the foreign agent (25–27). The apoptosis response became evident in the experimental autoimmune encephalomyelitis (EAE) disease model where the disease is monophasic and recovery occurs rather than the autoimmune response perpetuating itself (26). Because of the delicate nature of the CNS, the immune response is modulated to avoid rampant damage (25, 26, 28). Some immune cells are able to cross the blood-brain barrier back into the circulation, but it is believed that the main fate of activated microglia and leukocytes in the CNS following the immune response is apoptosis (25–27). Because the immune cells die and remain in the area of immune response, this may explain why the iron oxide clusters that are initially released from rejected cells remain in the CNS and are not cleared from the brain parenchyma.
The two strains of mice were chosen for this study to compare the differences of cell rejection versus cell acceptance. There is initial cell death after transplantation in both strains of mice, which may confound the interpretation of our results, but as this cell death will occur in both groups of mice, the differences in contrast over time must be derived from the biological differences between surviving versus rejected cells. In Rag2 mice, a significant amount of free iron clusters remain near the injection site, even at 95 days after transplantation. This suggests that some of the labeled, transplanted cells died upon transplantation or shortly thereafter, and therefore, the iron load remained high in the area surrounding the dead cells causing a persistence of MR signal. It has been documented that a large percentage (possibly 90%) of transplanted cells into the CNS may die shortly after transplantation (29–32). A large portion of the cell death occurs as apoptosis during the first week after transplantation (32). If the cells die during or immediately after transplantation, their entire iron load is released and remains in the CNS. Based on our results, this extracellular iron primarily accounts for the persistent, localized MR hypointensity for up to 95 days in both immunodeficient and immunocompetent mice. Administration of neuro-protective and pro-survival factors can be considered to reduce the amount of cell death following transplantation during this critical period and potentially change the proportion of MR signal originating from dead versus surviving, labeled cells (30). Pro-survival factors have been shown to enhance the function of cardiomyocytes transplanted into the rat heart by Laflamme et al. (33).
The interpretation of long-term MR cellular imaging is difficult, as we have shown in this study. The MR hypointensity can originate from particles within microglia or from deposits persisting in the interstitium after the immune rejection of transplanted C17.2 cells. Deposits of iron oxide in the brain continue to produce high MR signal long after transplanted cells are dead. Therefore, MRI of magnetically labeled cells alone is currently unable to distinguish transplanted labeled cells from free iron oxide released upon cell death or iron taken up by endogenous cells. The recent studies by Baligand et al. in leg muscle labeled cell transplants (13) as well as Chen et al. and Terrovitis et al. in intracardiac transplants (14, 34) are in agreement with our findings in the brain. Previous studies involving SPIOs in the brain and spinal cord have shown that SPIO-labeled cells could be used to track transplanted cells in the brain (3) and spinal cord (7); however, the limitations of long-term tracking were also recognized.
Unquestionably, the strength of MR tracking of labeled cells is short-term imaging and real-time MR-guided delivery of cellular therapeutics (4, 35, 36). For the brain, this will be of particular importance for intra-arterial injections in stroke treatment (37). This will be especially important for human translational studies, where needle positioning and verification of accurate delivery of cells into target organs is critical (4, 38). Long-term stem cell tracking in the clinic would benefit from a permanent method of tagging cells that cannot be taken up by endogenous immune cells, diluted, or held in the interstitium. A reliable, cell-specific reporter would lead to more accurate long-term MR tracking of cells. One solution to this problem may be the development of an MR reporter gene based on the expression of an existing or artificial protein that will have MR signal only when the transplanted cells are viable and functioning. Although there is significant effort to develop such a reporter system (10, 11), a robust MR reporter gene system with sensitivity sufficient for stem cell tracking is currently not available.
In conclusion, MRI of magnetically labeled cells should be undertaken cautiously, and preferably with an additional imaging method that would provide data on the viability of grafted cells. The significant persistence of SPIO-hypointensities in MRI, long after transplanted stem cells are dead, and the fact that current cell transplantation procedures are typically associated with substantial cell death following transplantation indicates that this technique cannot reliably report long-term on stem cell engraftment in the brain. Thus, MRI cell tracking with SPIO particles should only be considered for long-term use if there is a certainty that the majority of transplanted cells remain viable throughout the study. However, MRI cell tracking still remains the method of choice for visualizing the injection and location of cells shortly after transplantation.