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

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.     

Cell motility of neural stem cells is reduced after SPIO‐labeling,which is mitigated after exocytosis

MRI is used for tracking of superparamagnetic iron oxide (SPIO)‐labeled neural stem cells. Studies have shown that long‐term MR tracking of rapidly dividing cells underestimates their migration distance. Time‐lapse microscopy of random cellular motility and cell division was performed to evaluate the effects of SPIO‐labeling on neural stem cell migration. Labeled cells divided symmetrically and exhibited no changes in cell viability, proliferation, or apoptosis. However, SPIO‐labeling resulted in decreased motility of neural stem cells as compared with unlabeled controls. When SPIO‐labeled neural stem cells and human induced pluripotent stem cells were transplanted into mouse brain, rapid exocytosis of SPIO by live cells was observed as early as 48 h postengraftment, with SPIO‐depleted cells showing the farthest migration distance. As label dilution is negligible at this early time point, we conclude that MRI underestimation of cell migration can also occur as a result of reduced cell motility, which appears to be mitigated following SPIO exocytosis. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

Relaxation effects of ferucarbotran‐labeled mesenchymal stem cells at 1.5T and 3T: Discrimination of viable from lysed cells

Human mesenchymal stem cells (hMSCs) were labeled with Ferucarbotran by simple incubation and cultured for up to 14 d. Iron content was determined by spectrometry and the intracellular localization of the contrast agent uptake was studied by electron and confocal microscopy. At various time points after labeling, ranging from 1 to 14 d, samples with viable or lysed labeled hMSCs, as well as nonlabeled controls, underwent MRI. Spin‐echo (SE) and gradient‐echo (GE) sequences with multiple TRs and TEs were used at 1.5T and 3T on a clinical scanner. Spectrometry showed an initial iron oxide uptake of 7.08 pg per cell. Microscopy studies revealed lysosomal compartmentalization. Contrast agent effects of hMSCs were persistent for up to 14 d after labeling. A marked difference in the T₂ effect of compartmentalized iron oxides compared to free iron oxides was found on T₂‐weighted sequences, but not on T‐weighted sequences. The observed differences may be explained by the loss of compartmentalization of iron oxide particles, the uniformity of distribution, and the subsequent increase in dephasing of protons on SE images. These results show that viable cells with compartmentalized iron oxides may—in principle—be distinguished from lysed cells or released iron oxides. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells: comparison of ferumoxides, monocrystalline iron oxide, cross-linked iron oxide (CLIO)-NH2 and tat-CLIO.

OBJECTIVE: We wanted to compare the human neural stem cell (hNSC) labeling efficacy of different superparamagnetic iron oxide nanoparticles (SPIONs), namely, ferumoxides, monocrystalline iron oxide (MION), cross-linked iron oxide (CLIO)-NH(2) and tat-CLIO. MATERIALS AND METHODS: The hNSCs (5 x 10(5) HB1F3 cells/ml) were incubated for 24 hr in cell culture media that contained 25 microg/ml of ferumoxides, MION or CLIO-NH(2), and with or without poly-L-lysine (PLL) and tat-CLIO. The cellular iron uptake was analyzed qualitatively with using a light microscope and this was quantified via atomic absorption spectrophotometry. The visibility of the labeled cells was assessed with MR imaging. RESULTS: The incorporation of SPIONs into the hNSCs did not affect the cellular proliferations and viabilities. The hNSCs labeled with tat-CLIO showed the longest retention, up to 72 hr, and they contained 2.15+/-0.3 pg iron/cell, which are 59 fold, 430 fold and six fold more incorporated iron than that of the hNSCs labeled with ferumoxides, MION or CLIO-NH(2), respectively. However, when PLL was added, the incorporation of ferumoxides, MION or CLIO-NH(2) into the hNSCs was comparable to that of tat-CLIO. CONCLUSION: For MR imaging, hNSCs can be efficiently labeled with tat-CLIO alone or with a combination of ferumoxides, MION, CLIO-NH(2) and the transfection agent PLL.     

Low‐intensity pulsed ultrasound increases cellular uptake of superparamagnetic iron oxide nanomaterial: Results from human osteosarcoma cell line U2OS

Purpose:

To determine whether low‐intensity pulsed ultrasound (LIPUS) is able to facilitate the uptake of a superparamagnetic iron oxide (SPIO) nanomaterial by cells that do not express high endocytosis capacity.

Materials and Methods:

The human osteosarcoma cell line U2OS and a silica‐coated SPIO functionalized peripherally with amines groups (overall diameter 8 nm) were used in this study. Adherent U2OS cells were labeled with SPIO by incubating with culture media containing the SPIO at 4.5 μg[Fe]/mL. LIPUS with the same parameters as those used in clinical application to accelerate bone fracture healing (1.5 MHz, duty cycle 1:4, spatial‐average temporal‐average intensity 30 mW/cm²) was applied to the cells at the beginning of the labeling process for 0, 0.5, 1, or 3 hours. The total incubation time with SPIO was 12 hours. SPIO labeling efficiency was evaluated with Prussian blue staining and a blueness measurement method, and magnetic resonance imaging (MRI) of cell pellets via measuring areas of SPIO‐induced signal void.

Results:

Both Prussian blue staining and in vitro MRI demonstrated that LIPUS application increased the SPIO nanomaterial labeling efficiency for U2OS cells in an exposure‐duration‐dependent manner.

Conclusion:

This study is a “proof of concept” that LIPUS can facilitate the cellular take‐up of SPIO nanomaterial. J. Magn. Reson. Imaging 2010;31:1508–1513. © 2010 Wiley‐Liss, Inc.     

Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging

PURPOSE: To evaluate the effect of using the ferumoxides-poly-l-lysine (PLL) complex for magnetic cell labeling on the long-term viability, function, metabolism, and iron utilization of mammalian cells. MATERIALS AND METHODS: PLL was incubated with ferumoxides for 60 minutes, incompletely coating the superparamagnetic iron oxide (SPIO) through electrostatic interactions. Cells were coincubated overnight with the ferumoxides-PLL complex, and iron uptake, cell viability, apoptosis indexes, and reactive oxygen species formation were evaluated. The disappearance or the life span of the detectable iron nanoparticles in cells was also evaluated. The iron concentrations in the media also were assessed at different time points. Data were expressed as the mean +/- 1 SD, and one-way analysis of variance and the unpaired Student t test were used to test for significant differences. RESULTS: Intracytoplasmic nanoparticles were stained with Prussian blue when the ferumoxides-PLL complex had magnetically labeled the human mesenchymal stem and HeLa cells. The long-term viability, growth rate, and apoptotic indexes of the labeled cells were unaffected by the endosomal incorporation of SPIO, as compared with these characteristics of the nonlabeled cells. In nondividing human mesenchymal stem cells, endosomal iron nanoparticles could be detected after 7 weeks; however, in rapidly dividing cells, intracellular iron had disappeared by five to eight divisions. A nonsignificant transient increase in reactive oxygen species production was seen in the human mesenchymal stem and HeLa cell lines. Labeled human mesenchymal stem cells did not differentiate to other lineage. A significant increase in iron concentration was observed in both the human mesenchymal stem and HeLa cell media at day 7. CONCLUSION: Magnetic cellular labeling with the ferumoxides-PLL complex had no short- or long-term toxic effects on tumor or stem cells.     

In vivo serial evaluation of superparamagnetic iron‐oxide labeled stem cells by off‐resonance positive contrast

MRI is emerging as a diagnostic modality to track iron‐oxide‐labeled stem cells. This study investigates whether an off‐resonance (OR) pulse sequence designed to generate positive contrast at 1.5T can assess the location, quantity, and viability of delivered stem cells in vivo. Using mouse embryonic stem cell transfected with luciferase reporter gene (luc‐mESC), multimodality validation of OR signal was conducted to determine whether engraftment parameters of superparamagnetic iron‐oxide labeled luc‐mESC (SPIO‐luc‐mESC) could be determined after cell transplantation into the mouse hindlimb. A significant increase in signal‐ and contrast‐to‐noise of the SPIO‐luc‐mESC was achieved with the OR technique when compared to a gradient recalled echo (GRE) sequence. A significant correlation between the quantity of SPIO‐luc‐mESC and OR signal was observed immediately after transplantation (R² = 0.74, P < 0.05). The assessment of transplanted cell viability by bioluminescence imaging (BLI) showed a significant increase of luciferase activities by day 16, while the MRI signal showed no difference. No significant correlation between BLI and MRI signals of cell viability was observed. In conclusion, using an OR sequence the precise localization and quantitation of SPIO‐labeled stem cells in both space and time were possible. However, the OR sequence did not allow evaluation of cell viability. Magn Reson Med 60:1269–1275, 2008. © 2008 Wiley‐Liss, Inc.     

Chondrocyte gene expression is affected by very small iron oxide particles-labeling in long-term in vitro MRI tracking

Purpose:

To investigate the effect and dose response of very small iron oxide particles (VSOP) labeling of human chondrocytes for long‐term in vitro MRI tracking.

Materials and Methods:

Chondrocytes were isolated from cartilage biopsies from four patients. The cells for the dose–response study were labeled with 25, 50, or 100 μg/mL VSOP. Quantitative gene expression and cellular proliferation were compared with unlabeled controls at day 1, 3, and 7. The cells suited for MRI tracking were labeled with 50 μg/mL VSOP and embedded in alginate beads, followed by MRI (using T2‐weighted sequences) at day 0, 1, 3, 7, 14, 21, 28, and histology was performed at each time‐point.

Results:

Histology revealed that VSOP particles were intracellularly confined at all time‐points, whereas no extracellular VSOPs were observed. A mean reduction in T2‐value of 25.1 ms (±SD 3.5 ms) was found on T2‐maps. The chondrocyte‐specific genes aggrecan, collagen type 2, and sox9 were all affected by labeling, the two latter in a dose‐dependent manner. VSOPs had no effect on proliferation.

Conclusion:

VSOP labeling of chondrocytes affected gene expression but not proliferation. The labeled chondrocytes could be recognized by MRI for 4 weeks without significant changes in the T2 relaxation time. J. Magn. Reson. Imaging 2011;33:724–730. © 2011 Wiley‐Liss, Inc.     

Monitoring bone marrow‐originated mesenchymal stem cell traffic to myocardial infarction sites using magnetic resonance imaging

How stem cells promote myocardial repair in myocardial infarction (MI) is not well understood. The purpose of this study was to noninvasively monitor and quantify mesenchymal stem cells (MSC) from bone marrow to MI sites using magnetic resonance imaging (MRI). MSC were dual‐labeled with an enhanced green fluorescent protein and micrometer‐sized iron oxide particles prior to intra‐bone marrow transplantation into the tibial medullary space of C57Bl/6 mice. Micrometer‐sized iron oxide particles labeling caused signal attenuation in T₂*‐weighted MRI and thus allowed noninvasive cell tracking. Longitudinal MRI demonstrated MSC infiltration into MI sites over time. Fluorescence from both micrometer‐sized iron oxide particles and enhanced green fluorescent protein in histology validated the presence of dual‐labeled cells at MI sites. This study demonstrated that MSC traffic to MI sites can be noninvasively monitored in MRI by labeling cells with micrometer‐sized iron oxide particles. The dual‐labeled MSC at MI sites maintained their capability of proliferation and differentiation. The dual‐labeling, intra‐bone marrow transplantation, and MRI cell tracking provided a unique approach for investigating stem cells' roles in the post‐MI healing process. This technique can potentially be applied to monitor possible effects on stem cell mobilization caused by given treatment strategies. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

不同浓度菲立磁对大鼠间充质干细胞标记效率和细胞活力的影响

目的探讨不同浓度菲立磁标记大鼠骨髓间充质干细胞（MSCs）的磁标记效率及对细胞生长活力的影响,寻找最佳标记浓度。材料与方法菲立磁与多聚左旋赖氨酸（PLL）混合制备菲立磁-PLL复合物。将不同浓度的菲立磁-PLL复合物与培养基混合（铁的终浓度分别为150μg／ml、100μg／ml、50μg／ml、25μg／ml、10μg／ml和5μg／ml）,加入MSCs孵育过夜。分别于标记后1天、1周、2周、3周、4周行铁染色、铁含量测定及细胞活力检测。结果菲立磁-PLL标记组细胞铁含量显著高于单纯菲立磁标记组（P＜0．05）,25μg／ml浓度组细胞铁含量显著高于10μg／ml及其以下浓度组（P＜0．05）,但与50μg／ml及其以上浓度组差异无统计学意义（P＞0．05）。台盼蓝排除试验显示,100μg／ml及其以下浓度组细胞活力与对照组之间差异无统计学意义（P＞0．05）。结论以25μg/ml铁浓度标记干细胞不仅标记效率高,而且对细胞活力无明显影响．为菲立磁标记MSCs的最佳浓度。     

Magnetic poly(lactide‐co‐glycolide) and cellulose particles for MRI‐based cell tracking

Biodegradable, superparamagnetic microparticles and nanoparticles of poly(lactide‐co‐glycolide) (PLGA) and cellulose were designed, fabricated, and characterized for magnetic cell labeling. Monodisperse nanocrystals of magnetite were incorporated into microparticles and nanoparticles of PLGA and cellulose with high efficiency using an oil‐in‐water single emulsion technique. Superparamagnetic cores had high magnetization (72.1 emu/g). The resulting polymeric particles had smooth surface morphology and high magnetite content (43.3 wt % for PLGA and 69.6 wt % for cellulose). While PLGA and cellulose nanoparticles displayed highest r values per millimole of iron (399 sec^?1 mM^?1 for cellulose and 505 sec^?1 mM^?1 for PLGA), micron‐sized PLGA particles had a much higher r per particle than either. After incubation for a month in citrate buffer (pH 5.5), magnetic PLGA particles lost close to 50% of their initial r molar relaxivity, while magnetic cellulose particles remained intact, preserving over 85% of their initial r molar relaxivity. Lastly, mesenchymal stem cells and human breast adenocarcinoma cells were magnetically labeled using these particles with no detectable cytotoxicity. These particles are ideally suited for noninvasive cell tracking in vivo via MRI and due to their vastly different degradation properties, offer unique potential for dedicated use for either short (PLGA‐based particles) or long‐term (cellulose‐based particles) experiments. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

Imaging iron‐loaded mouse glioma tumors with bSSFP at 3 T

The poor prognosis for patients with high‐grade glioma is partly due to the invasion of tumor cells into surrounding brain tissue. The goal of the present work was to develop a mouse model of glioma that included the potential to track cell invasion using MRI by labeling GL261 cells with iron oxide contrast agents prior to intracranial injection. Two types of agents were compared with several labeling schemes to balance between labeling with sufficient iron to curb the dilution effect of cell division while avoiding overwhelming signal loss that could prevent adequate visualization of tumor boundaries. The balanced steady‐state free precession (bSSFP) pulse sequence was evaluated for its suitability for imaging glioma tumors and compared to T₂‐weighted two‐dimensional fast spin echo (FSE) and T₁‐weighted spoiled gradient recalled echo (SPGR) at 3 T in terms of signal‐to‐noise ratio and contrast‐to‐noise ratio efficiencies. Ultimately, a three‐dimensional bSSFP protocol consisting of a set of two images with complementary contrasts was developed, allowing excellent tumor visualization with minimal iron contrast when using pulse repetition time = 6 ms and α = 40°, and extremely high sensitivity to iron when using pulse repetition time = 22 ms and α = 20°. Quantitative histologic analysis validated that the strong signal loss seen in balanced steady state free precession pulse sequence images of iron‐loaded tumors correlated well with the presence of iron. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro

To evaluate the capacity of human monocytes to phagocytose various approved iron oxide based magnetic resonance (MR) contrast agents and to optimize in vitro labeling of these cells. Human monocytes were incubated with two superparamagnetic iron oxide particles (SPIO) as well as two ultrasmall SPIO (USPIO) at varying iron oxide concentrations and incubation times. Iron uptake in monocytes was proven by histology, quantified by atomic emission absorption spectrometry and depicted with T2* weighted fast field echo (FFE) MR images at 1.5 T. Additionally, induction of apoptosis in iron oxide labeled monocytes was determined by YO-PRO-1 staining. Cellular iron uptake was significantly (P<0.01) higher after incubation with SPIO compared with USPIO. For SPIO, the iron oxide uptake was significantly (P<0.01) higher after incubation with the ionic Ferucarbotran as compared with the non-ionic Ferumoxides. Efficient cell labeling was achieved after incubation with Ferucarbotran at concentrations 500 g Fe/ml and incubation times 1 h, resulting in a maximal iron oxide uptake of up to 50 pg Fe/cell without impairment of cell viability. In vitro labeling of human monocytes for MR imaging is most effectively obtained with the approved SPIO Ferucarbotran. Potential subsequent in vivo cell tracking applications comprise, e.g. specific targeting of inflammatory processes.     

In vivo MRI of embryonic stem cells in a mouse model of myocardial infarction.

The therapeutic potential of administering stem cells to promote angiogenesis and myocardial tissue regeneration after infarction has recently been demonstrated. Given the advantages of using embryonic stem cells and mouse models of myocardial infarction for furthering the development of this therapeutic approach, the purpose of this study was to determine if embryonic stem cells could be loaded with superparamagnetic iron oxide (SPIO) particles and imaged in a mouse model of myocardial infarction over time using MRI. Mouse embryonic stem cells were labeled with SPIO particles. When incubated with 11.2, 22.4, and 44.8 microg Fe/ml of SPIO particles, cells took up increasing amounts of iron oxide. Embryonic stem cells loaded with SPIO compared to unlabeled cells had similar viability and proliferation profiles for up to 14 days. Free SPIO injected into infarcted myocardium was not observable within 12 hr after injection. After injection of three 10-microl aliquots of 10(7) SPIO-loaded cells/ml into infarcted myocardium, MRI demonstrated that the mouse embryonic stem cells were observable and could be seen for at least 5 weeks after injection. These findings support the ability of MRI to test the long-term therapeutic potential of embryonic stem cells in small animals in the setting of myocardial infarction.     

Effectiveness of micron‐sized superparamagnetic iron oxide particles as markers for detection of migration of bone marrow‐derived mesenchymal stromal cells in a stroke model

Purpose:

To evaluate the feasibility of using micron‐sized superparamagnetic iron oxide particles (MPIOs) as an effective labeling agent for monitoring bone marrow‐derived mesenchymal stromal cell (BMSC) migration in the brain using magnetic resonance imaging (MRI) in a rat model of stroke and whether the accumulation of MPIO‐labeled BMSCs can be differentiated from the accumulation of free MPIO particles or hemoglobin breakdown at a site of neuronal damage.

Materials and Methods:

In this study BMSCs were labeled with iron oxide and their pattern of migration following intravenous injection in a rat stroke model was monitored using a clinical MRI system followed by standard histopathology. The migration pattern was compared between intravenous injection of BMSCs alone, BMSCs labeled with MPIOs, and MPIO particles alone.

Results:

The results demonstrated that while MRI was highly sensitive in the detection of iron oxide particle‐containing cells in areas of neuronal ischemia, the true origin of cells containing iron oxide particles remains ambiguous. Therefore, detection of iron particles may not be a suitable strategy for the detection of BMSCs in the brain in a stroke model.

Conclusion:

This study suggests that the use of MPIOs as labeling agents are insufficient to conclusively determine the localization of iron within cells in regions of neuronal ischemia and hemorrhage. J. Magn. Reson. Imaging 2013;37:1409–1418. © 2012 Wiley Periodicals, Inc.     

In vivo and ex vivo MR imaging of slowly cycling melanoma cells

Slowly cycling cells are believed to play a critical role in tumor progression and metastatic dissemination. The goal of this study was to develop a method for in vivo detection of slowly cycling cells. To distinguish these cells from more rapidly proliferating cells that constitute the vast majority of cells in tumors, we used the well‐known effect of label dilution due to division of cells with normal cycle and retention of contrast agent in slowly dividing cells. To detect slowly cycling cells, melanoma cells were labeled with iron oxide particles. After labeling, we observed dilution of contrast agent in parallel with cell proliferation in the vast majority of normally cycling cells. A small and distinct subpopulation of iron‐retaining cells was detected by flow cytometry after 20 days of in vitro proliferation. These iron‐retaining cells exhibited high expression of a biological marker of slowly cycling cells, JARID1B. After implantation of labeled cells as xenografts into immunocompromised mice, iron‐retaining cells were detected in vivo and ex vivo by magnetic resonance imaging that was confirmed by Prussian Blue staining. Magnetic resonance imaging detects not only iron retaining melanoma cells but also iron positive macrophages. Proposed method opens up opportunities to image subpopulation of melanoma cells, which is critical for continuous tumor growth. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.     

Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.

The purpose of this work was to evaluate the efficacy of labeling human mesenchymal stem cells (hMSCs) by ionic superparamagnetic iron oxide (SPIO) without a transfection agent and verifying its capability to be detected with clinical 1.5 T magnetic resonance (MR) at the single-cell level. Human hMSCs were incubated for 24 h with an ionic SPIO, Ferucarbotran. The labeling efficiency of hMSCs was determined by iron content measurement spectrophotometrically, and the influence of labeling on cell behavior was ascertained by examination of cell viability using the trypan blue exclusion method, cell proliferation analysis using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, mitochondrial membrane potential (MMP) change, differentiation capacity, and reactive oxygen species (ROS) production measured by dichlorofluorescein diacetate (DCFDA) fluorescent probe. Labeled hMSCs were scanned under 1.5 T MRI with three-dimensional (3D) and two-dimensional (2D) T(2)-weighted gradient echo (GRE) pulse sequences. Human hMSC labeling without transfection agent was efficient. The iron content in hMSCs was 23.4 pg Fe/cell. No significant change was found in viability, proliferation, MMP change, ROS production, or differentiation capacity. About 45.2% of the hMSCs could be detected using 1.5 T MRI at the single cell level with 3D GRE and four repetitions.     

Enhanced magnetic cell labeling efficiency using –NH2 coated MPIOs

Strategies have been developed for labeling cells with micron sized iron oxide particles (MPIOs) for in vivo visualization of cells by magnetic resonance imaging. Although this approach is well established and has a variety of applications, current protocols employ long labeling times. It has been previously demonstrated that incubation of dextran coated iron oxide nanoparticles with positively charged transfection agents, such as poly‐L ‐lysine increases labeling efficiency. Therefore, we sought to ascertain whether preincubating MPIOs with various quantities of poly‐L ‐lysine would similarly enhance the rate of magnetic cell labeling. This was also tested against an NH₂ functionalized, commercially available MPIO. Indeed, we demonstrate significantly increased rate of magnetic cell labeling with MPIOs previously incubated with varying amounts of poly‐L ‐lysine, with robust intracellular labeling at 2 hours. Yet the most robust labeling was achieved with the MPIO‐NH₂. Interestingly, even for particle formulations which still had negative zeta potential, enhancement of magnetic cell labeling was achieved. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

Applicability and limitations of MR tracking of neural stem cells with asymmetric cell division and rapid turnover: the case of the shiverer dysmyelinated mouse brain.

LacZ-transfected C17.2 neural stem cells (NSCs) were labeled with the superparamagnetic iron oxide formulation Feridex prior to ICV injection in shi/shi neonates. Feridex labeling did not alter cell differentiation in vitro and in vivo. Initially, MR images obtained at 11.7T correlated closely to NSC distribution as assessed with anti-dextran and anti-beta-galactosidase double-fluorescent immunostaining. However, at 6 days postgrafting there was already a pronounced mismatch between the hypointense MR signal and the histologically determined cell distribution, with a surprisingly sharp cutoff rather than a gradual decrease of signal. Positive in vivo BrdU labeling of NSCs showed that significant cell replication occurred post-transplantation, causing rapid dilution of Feridex particles between mother and daughter cells toward undetectable levels. Neural differentiation experiments demonstrated asymmetric cell division, explaining the observed sharp cutoff. At later time points (2 weeks), the mismatch further increased by the presence of non-cell-associated Feridex particles resulting from active excretion or cell death. These results are a first demonstration of the inability of MRI to track rapidly dividing and self-renewing, asymmetrically dividing SCs. Therefore, MR cell tracking should only be applied for nonproliferating cells or short-term monitoring of highly-proliferative cells, with mitotic symmetry or asymmetry being important for determining its applicability.     

Estimating amounts of iron oxide from gradient echo images

Rat legs directly injected with superparamagnetic iron oxide (SPIO) were studied by dual‐echo, gradient‐echo imaging. The amount of iron injected was estimated using a point dipole model for the SPIO injection site. Saturation magnetization of 6:1 PEG/amino modified silane‐coated iron oxide particles with 5‐ to 6‐nm core and 20–25 hydrodynamic diameter was ～110 emu/g of iron. Estimates of the amount of iron injected made from signal void volumes surrounding SPIO centers yielded erroneous results varying with sample orientation in the scanner and echo time (TE). For example, a 10 μL, 3‐μg iron injection produced signal void volumes of 80 and 210 μL at TE of 9.8 and 25 ms, respectively, giving apparent iron contents of 6 ± 1 and 10 ± 2 μg respectively. A more effective approach uses the phase difference between two gradient recalled echo images. To estimate iron content, this approach fits the expected (3 cos²θ ? 1)/|r|³ spatial phase distribution to the observed phase differences. Extraneous phase effects made fitting phase at a single TE ineffective. With the dual echo method, 18 independent estimates were 2.48 ± 0.26 μg std, independently of sample orientation. Estimates in empty control regions were ?90 and ?140 ng. A 1‐μg injection indicated 0.5, 1.2, and 1.2 μg. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.