In vivo tracking of human neural stem cells with 19F magnetic resonance imaging

Background

Magnetic resonance imaging (MRI) is a promising tool for monitoring stem cell-based therapy. Conventionally, cells loaded with ironoxide nanoparticles appear hypointense on MR images. However, the contrast generated by ironoxide labeled cells is neither specific due to ambiguous background nor quantitative. A strategy to overcome these drawbacks is ¹⁹F MRI of cells labeled with perfluorocarbons. We show here for the first time that human neural stem cells (NSCs), a promising candidate for clinical translation of stem cell-based therapy of the brain, can be labeled with ¹⁹F as well as detected and quantified in vitro and after brain implantation.

Methodology/Principal Findings

Human NSCs were labeled with perfluoropolyether (PFPE). Labeling efficacy was assessed with ¹⁹F MR spectroscopy, influence of the label on cell phenotypes studied by immunocytochemistry. For in vitro MRI, NSCs were suspended in gelatin at varying densities. For in vivo experiments, labeled NSCs were implanted into the striatum of mice. A decrease of cell viability was observed directly after incubation with PFPE, which re-normalized after 7 days in culture of the replated cells. No label-related changes in the numbers of Ki67, nestin, GFAP, or βIII-tubulin+ cells were detected, both in vitro and on histological sections. We found that 1,000 NSCs were needed to accumulate in one image voxel to generate significant signal-to-noise ratio in vitro. A detection limit of ∼10,000 cells was found in vivo. The location and density of human cells (hunu+) on histological sections correlated well with observations in the ¹⁹F MR images.

Conclusion/Significance

Our results show that NSCs can be efficiently labeled with ¹⁹F with little effects on viability or proliferation and differentiation capacity. We show for the first time that ¹⁹F MRI can be utilized for tracking human NSCs in brain implantation studies, which ultimately aim for restoring loss of function after acute and neurodegenerative disorders.     

Dual contrast magnetic resonance imaging tracking of iron-labeled cells in vivo

Negative-contrast magnetic resonance imaging (MRI) methods utilizing magnetic susceptibility contrast agents have become one of the most widely used approaches in cellular imaging research. However, visualizing and tracking super-paramagnetic iron oxide nanoparticle (SPIO)-labeled cells on the basis of negative-contrast can limit specificity and sensitivity. Therefore, there has been a strong motivation to explore MRI methods for cellular imaging with either positive or dual contrast (both positive and negative) for identifying labeled cells; these methods offer the potential to improve significantly the sensitivity and specificity of MRI-based cell-tracking approaches. In this review, current state-of-the-art positive- and dual-contrast MRI techniques and contrast agents are described specifically for applications involving in vivo cellular tracking and imaging.     

In vivo magnetic resonance imaging tracking of SPIO-labeled human umbilical cord mesenchymal stem cells

Human umbilical cord mesenchymal stem cells (hUC-MSCs) can be efficiently labeled by superparamagnetic iron oxide (SPIO) nanoparticles, which produces low signal intensity on magnetic resonance imaging (MRI) in vitro. This study was to evaluate the feasibility of in vivo tracking for hUC-MSCs labeled by SPIO with noninvasive MRI. SPIO was added to cultures at concentrations equivalent to 0, 7, 14, 28, and 56 μg Fe/ml (diluted with DMEM/F12) and incubated for 16 h. Prussian Blue staining was used to determinate the labeling efficiency. Rats were randomly divided into three groups, control group, hUC-MSCs group, and SPIO-labeled hUC-MSCs group. All groups were subjected to spinal cord injury (SCI) by weight drop device. Rats were examined for neurological function. In vivo MRI was used to track SPIO-labeled hUC-MSCs transplanted in rats spinal cord. Survival and migration of hUC-MSCs were also explored using immunofluorescence. Significant improvements in locomotion were observed in the hUC-MSCs groups. There was statistical significance compared with control group. In vivo MRI 1 and 3 weeks after injection showed a large reduction in signal intensity in the region transplanted with SPIO-labeled hUC-MSCs. The images from unlabeled hUC-MSCs showed a smaller reduction in signal intensity. Transplanted hUC-MSCs engrafted within the injured rats spinal cord and survived for at least 8 weeks. In conclusion, hUC-MSCs can survive and migrate in the host spinal cord after transplantation, which promote functional recovery after SCI. Noninvasive imaging of transplanted SPIO-labeled hUC-MSCs is feasible.     

Magnetic resonance and near-infrared imaging using a novel dual-modality nano-probe for dendritic cell tracking in vivo

Background aimsThe effect of cellular-based immunotherapy is highly correlated with the success of dendritic cells (DCs) homing to the draining lymph nodes (LNs) and interacting with antigen-specific CD4⁺ T cells. In this study, a novel magneto-fluorescent nano-probe was used to track the in vivo migration of DCs to the draining LNs.MethodsA dual-modality nano-probe composed of superparamagnetic iron oxide (SPIO) and near-infrared fluorescent (NIRF) dye (NIR797) was developed, and its magnetic and optical contrasting properties were characterized. DCs generated from mouse bone marrow were co-cultured with the probe at a lower concentration of 10 μg/mL. The cell phenotype and function of DCs were also investigated by fluorescence-activated cell sorting analysis and mixed leukocyte reactivity assay. Labeled DCs were injected into the footpad of C57BL/6 mice. Afterward, magnetic resonance imaging, NIRF imaging, Perls staining and CD11c immunofluorescence were used to observe the migration of the labeled DCs into draining LNs.ResultsThe synthetic SPIO-NIR797 nano-probe had a desirable superparamagnetic and near-infrared behavior. Perls staining showed perfect labeling efficiency. The cell phenotypes, including CD11c, CD80, CD86 and major histocompatibility complex class II, as well as the T-cell activation potential of the mature DCs were insignificantly affected after incubation (P > 0.05). Labeled DCs migrating into LNs could be detected by both magnetic resonance imaging and NIRF imaging simultaneously, which was further confirmed by Perls staining and immunofluorescence.ConclusionsThe novel dual-modality SPIO-NIR797 nano-probe has highly biocompatible characteristics for labeling and tracking DCs, which can be used to evaluate cancer immunotherapy in clinical applications.     

Magnetic resonance imaging for in vivo assessment of three-dimensional patellar tracking

We have developed a non-invasive measurement technique which can ultimately be used to quantify three-dimensional patellar kinematics of human subjects for a range of static positions of loaded flexion and assessed its accuracy. Knee models obtained by segmenting and reconstructing one high-resolution scan of the knee were registered to bone outlines obtained by segmenting fast, low-resolution scans of the knee in static loaded flexion. We compared patellar tracking measurements made using the new method to measurements made using Roentgen stereophotogrammetric analysis in three cadaver knee specimens loaded through a range of flexion in a test rig. The error in patellar spin and tilt measurements was less than 1.02 degrees and the error in lateral patellar shift was 0.88 mm. Sagittal plane scans provided more accurate final measurements of patellar spin and tilt, whereas axial plane scans provided more accurate measurements of lateral translation and patellar flexion. Halving the number of slices did not increase measurement error significantly, which suggests that scan times can be reduced without reducing accuracy significantly. The method is particularly useful for multiple measurements on the same subject because the high-resolution bone-models need only be created once; thus, the potential variability in coordinate axes assignment and model segmentation during subsequent measurements is removed.     

In vivo tracking of the human patella using cine phase contrast magnetic resonance imaging

Improper patellar tracking is often considered to be the cause of patellar-femoral pain. Unfortunately, our knowledge of patellar-femoral-tibial (knee) joint kinematics is severely limited due to a lack of three-dimensional, noninvasive, in vivo measurement techniques. This study presents the first large-scale, dynamic, three-dimensional, noninvasive, in vivo study of nonimpaired knee joint kinematics during volitional leg extensions. Cine-phase contrast magnetic resonance imaging was used to measure the velocity profiles of the patella, femur, and tibia in 18 unimpaired knees during leg extensions, resisted by a 34 N weight. Bone displacements were calculated through integration and then converted into three-dimensional orientation angles. We found that the patella displaced laterally, superiorly, and anteriorly as the knee extended. Further, patellar flexion lagged knee flexion, patellar tilt was variable, and patellar rotation was fairly constant throughout extension.     

In vivo tracking of transplanted mononuclear cells using manganese-enhanced magnetic resonance imaging (MEMRI)

Background

Transplantation of mononuclear cells (MNCs) has previously been tested as a method to induce therapeutic angiogenesis to treat limb ischemia in clinical trials. Non-invasive high resolution imaging is required to track the cells and evaluate clinical relevance after cell transplantation. The hypothesis that MRI can provide in vivo detection and long-term observation of MNCs labeled with manganese contrast-agent was investigated in ischemic rat legs.

Methods and Findings

The Mn-labeled MNCs were evaluated using 7-tesla high-field magnetic resonance imaging (MRI). Intramuscular transplanted Mn-labeled MNCs were visualized with MRI for at least 7 and up to 21 days after transplantation in the ischemic leg. The distribution of Mn-labeled MNCs was similar to that of ¹¹¹In-labeled MNCs measured with single-photon emission computed tomography (SPECT) and DiI-dyed MNCs with fluorescence microscopy. In addition, at 1–2 days after transplantation the volume of the site injected with intact Mn-labeled MNCs was significantly larger than that injected with dead MNCs, although the dead Mn-labeled MNCs were also found for approximately 2 weeks in the ischemic legs. The area covered by CD31-positive cells (as a marker of capillary endothelial cells) in the intact Mn-MNCs implanted site at 43 days was significantly larger than that at a site implanted with dead Mn-MNCs.

Conclusions

The present Mn-enhanced MRI method enabled visualization of the transplanted area with a 150–175 µm in-plane spatial resolution and allowed the migration of labeled-MNCs to be observed for long periods in the same subject. After further optimization, MRI-based Mn-enhanced cell-tracking could be a useful technique for evaluation of cell therapy both in research and clinical applications.     

Gadolinium-rhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging

A novel dual-labeled nanoparticle for use in labeling and tracking cells in vivo is described. We report the construction and characterization of these gadolinium-rhodamine nanoparticles. These particles are constructed from lipid monomers with diacetylene bonds that are sonicated and photolyzed to form polymerized nanoparticles. Cells are efficiently labeled with these nanoparticles. We have inoculated labeled tumor cells subcutaneouosly into the flanks of C3H mice and have been able to image these labeled tumor cells via MRI and optical imaging. Furthermore, the labeled tumor cells can be visualized via fluorescent microscopy after tissue biopsy. Our results suggest that these nanoparticles could be used to track cells in vivo. This basic platform can be modified with different fluorophores and targeting agents for studying metastisic cell, stem cell, and immune cell trafficking among other applications.     

Amphiphilic hyperbranched fluoropolymers as nanoscopic 19F magnetic resonance imaging agent assemblies

Three hyperbranched fluoropolymers were synthesized and their micelles were constructed as potential (19)F MRI agents. A hyperbranched star-like core was first synthesized via atom transfer radical self-condensing vinyl (co)polymerization (ATR-SCVCP) of 4-chloromethyl styrene (CMS), lauryl acrylate (LA), and 1,1,1-tris(4'-(2'-bromoisobutyryloxy)phenyl)ethane (TBBPE). The polymerization gave a small core with M n of 5.5 kDa with PDI of 1.6, which served as a macroinitiator. Trifluoroethyl methacrylate (TFEMA) and tert-butyl acrylate (tBA) in different ratios were then "grafted" from the core to give three polymers with M(n) of about 120 kDa and PDI values of about 1.6-1.8. After acidolysis of the tert-butyl ester groups, amphiphilic, hyperbranched star-like polymers with M(n) of about 100 kDa were obtained. These structures were subjected to micelle formation in aqueous solution to give micelles having TEM-measured diameters ranging from 3-8 nm and DLS-measured hydrodynamic diameters from 20-30 nm. These micelles gave a narrow, single resonance by (19)F NMR spectroscopy, with a half-width of approximately 130 Hz. The T1/T2 parameters were about 500 and 50 ms, respectively, and were not significantly affected by the composition and sizes of the micelles. (19)F MRI phantom images of these fluorinated micelles were acquired, which demonstrated that these fluorinated micelles maybe useful as novel (19)F MRI agents for a variety of biomedical studies.     

In vivo MR imaging of magnetically labeled human embryonic stem cells

INTRODUCTION: Human embryonic stem cells (hES) have emerged as a potentially new therapeutic approach for treatment of heart and other diseases applying the concept of regenerative medicine. A method for in vivo visualization and tracking of transplanted hES would increase our understanding of in vivo hES behavior in both experimental and clinical settings. The aim of this study was to evaluate the feasibility of magnetic labeling and visualization of hES with magnetic resonance imaging (MRI). METHODS: hES were established and expanded according to standard procedures. After expansion, the cells were cultured under feeder free conditions and magnetically labeled by addition of dextran-coated Ferrum-oxide particles (Endorem) to the medium. Accumulation of small particles of iron-oxide (SPIO) in hES was assessed by Prussian blue staining and electron microscopy. For in vitro MRI, the labeled and unlabeled hES were examined in cell solution and after transplantation into explanted mouse heart ( approximately 100,000 cells) on a Bruker Avance DMX 500 vertical magnet at 11.75 T. A multi-slice, multi spin-echo T(2)-weighted images were obtained. For in vivo imaging, the experiments were performed on male Sprague-Dawley using Bruker Biospec 2.35 T magnet. The hES were directly injected ( approximately 500,000 cells) after surgical procedure (thoracotomy) into anterior left ventricular (LV) wall. Multi-slice T(2)-weighted gradient echo images were obtained using cardiac gating. RESULTS: hES appeared to be unaffected by magnetic labeling and maintained their ability to proliferate and differentiate. No additive agent for membrane permeabilisation was needed for facilitation of intracellular SPIO accumulation. Prussian blue and electron microscopy have revealed numerous iron particles in the cytoplasm of hES. On T(2)-weighted images, the labeled cells have shown well-defined hyopintense areas at the site of injection in anterior LV wall both in vitro and in vivo. CONCLUSIONS: It is feasible to magnetically label and visualize hES both in vitro and in vivo. MR visualization of magnetically labeled hES may be a valuable tool for in vivo tracking of hES.     

Labelling of human adipose-derived stem cells for non-invasive in vivo cell tracking

Human adipose-derived stem cells (ASC) can be expanded in an undifferentiated state or differentiated along the osteogenic, chondrogenic, adipogenic, myogenic, endothelial and neurogenic lineage. To test their in vivo and in situ regenerative potential, their fate needs to be traced after application in suitable defect models. Non-invasive imaging systems allow for real time tracking of labelled cells in the living animal. We have evaluated a bioluminescence cell tracking approach to visualise ASC labelled with luciferase in the living animal. Two procedures have been tested to efficiently label human stem cells with a reporter gene (luciferase, green fluorescent protein), namely lipofection with Lipofectamine 2000 and electroporation with a Nucleofector device. With both lipofection and nucleofection protocols, we have reached transfection efficiencies up to 60%. Reporter gene expression was detectable for 3 weeks in vitro and did not interfere with the phenotype and the stem cell properties of the cells. By means of a highly sensitive CCD camera, we were able to achieve real time imaging of cell fate for at least 20 days after application (intravenous, intramuscular, intraperitoneal, subcutaneous) in nude mice. Moreover, we were able to influence cell mobility by choosing different modes of application such as enclosure in fibrin matrix. The optical imaging system with transient transfection is an elegant cell-tracking concept to follow survival and fate of human stem cells in small animals.     

19F nuclear magnetic resonance studies of free calcium in heart cells.

19F nuclear magnetic resonance is used in conjunction with 5,5'-difluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBapta), a fluorinated calcium chelator, to report steady-state intracellular free calcium levels ([Ca2+]i) in populations of resting, quiescent, isolated adult heart cells. 31P nuclear magnetic resonance shows that 5FBapta-loaded cells maintain normal intracellular high-energy phosphates, pH, and free Mg2+. The intracellular free calcium concentration of well perfused, isolated heart cells is 61 +/- 5 nM, measured with 5FBapta, which has a dissociation constant (Kd) for calcium chelation of 500 nM. A similar value is obtained with Quin-MF, another fluorinated calcium chelator with Kd and maximum calcium sensitivity at 80 nM. We find that the steady-state level of intracellular free calcium is increased by decreased extra-cellular sodium concentration, omission of extracellular magnesium, decreased extracellular pH, hyperglycemia, and upon treatment with lead acetate. Further, extracellular ATP caused a large transient increase in [Ca2+]i. Thus, while heart cells maintain a very low level of intracellular free Ca2+, acute alterations in extracellular environment can cause derangement of calcium homeostasis, resulting in measurable increases in [Ca2+]i.     

In vivo imaging platform for tracking immunotherapeutic cells

Cellular therapeutics show great promise for the treatment of disease, but few noninvasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technology that uses perfluoropolyether (PFPE) agents to track cells in vivo. Fluorine MRI selectively images only the labeled cells, and a 'conventional' (1)H image places the cells in their anatomical context. We labeled phenotypically defined dendritic cells (DCs) with PFPE ex vivo and observed efficient intracellular uptake of the PFPE with little effect on DC function. We injected labeled DCs into tissue or intravenously in mice and then tracked the cells in vivo using (19)F MRI. Although we focused on DCs, which are being developed as immunotherapeutics for cancer and autoimmune diseases, this technology should be useful for monitoring a wide range of cell types in vivo.     

A new transgene reporter for in vivo magnetic resonance imaging

We report a new platform technology for visualizing transgene expression in living subjects using magnetic resonance imaging (MRI). Using a vector, we introduced an MRI reporter, a metalloprotein from the ferritin family, into specific host tissues. The reporter is made superparamagnetic as the cell sequesters endogenous iron from the organism. In this new approach, the cells construct the MRI contrast agent in situ using genetic instructions introduced by the vector. No exogenous metal-complexed contrast agent is required, thereby simplifying intracellular delivery. We used a replication-defective adenovirus vector to deliver the ferritin transgenes. Following focal inoculation of the vector into the mouse brain, we monitored the reporter activity using in vivo time-lapse MRI. We observed robust contrast in virus-transduced neurons and glia for several weeks. This technology is adaptable to monitor transgene expression in vivo in many tissue types and has numerous biomedical applications, such as visualizing preclinical therapeutic gene delivery.     

Non-invasive in vivo magnetic resonance imaging assessment of acute aflatoxin B1 hepatotoxicity in rats

Acute aflatoxin B1 (AFB1)-induced hepatotoxicity was assessed in vivo in male Sprague-Dawley rats (150-300 g) using magnetic resonance imaging (MRI). MRI results were compared to serum enzyme levels, histology and electron microscopy. Twenty-four hours following intraperitoneal delivery of AFB1 (3 mg/kg body weight in a saline/dimethyl sulfoxide (DMSO; 0.03 ml/kg body weight) solution), regions of damage, characterised by increased proton signal intensities in T2-weighted images, were observed in the vicinity of the hepatic portal vein (HPV) and in the right medial regions of the liver. Image analysis of regions of apparent damage around the HPV and right medial regions, following 24 h of AFB1 exposure, indicated statistically significant (P<0.05) increases in proton image signal intensities, when compared to saline/DMSO-treated rats. No significant difference in proton image signal intensities were observed 1-2 h following AFB1 exposure. Twenty-four hours following AFB1 exposure, histopathological assessment was characterised by portal/central vein/artery congestion, sinusoid congestion, nuclear pyknosis and karyolysis, and hepatocyte vacuolation; electron microscopy (EM) examination indicated nuclear debris, swollen cytoplasmic compartments, vacuolation, and the disappearance of the smooth endoplasmic reticulum, and elevated levels of serum aspartate aminotransferase and alanine aminotransferase were found to be significantly different (P<0.01) than controls.     

Functional magnetic resonance imaging as a tool for investigating human cortical motor function.

Non-invasive functional magnetic resonance imaging (fMRI) mapping techniques sensitive to the local changes of blood flow, blood volume, and blood oxygenation which accompany neuronal activation have been widely used over the last few years to investigate the functional organization of human cortical motor systems, and specifically of the primary motor cortex. Validation studies have demonstrated a good correspondence between quantitative and topographic aspects of data acquired by fMRI and positron emission tomography. The spatial and temporal resolution affordable by fMRI has allowed to achieve new important information on the distributed representation of hand movements in multiple functional modules, and on the intensity and spatial extent of neural activation in the contralateral and ipsilateral primary motor cortex in relation to parametric and nonparametric aspects of movement and to the degree of handedness. Neural populations with different functional characteristics have been identified in anatomically defined regions, and the temporal aspects of the activation during voluntary movement tracked in different components of the motor system. Finally, this technique has proved useful to deepen our understanding of the neural basis of motor imagery, demonstrating increased activity in the primary motor cortex during mental representation of sequential finger movements.     

Current issues in the utility of 19F nuclear magnetic resonance methodologies for the assessment of tumour hypoxia

It is now well established that uncontrolled proliferation of tumour cells together with the chaotic and poorly regulated blood supply of solid tumours result in tissue hypoxia, and that hypoxic regions of tumours are resistant to radiotherapy and chemotherapy. The development and application of non-invasive methods to rapidly determine the degree and extent of tumour hypoxia in an individual tumour would clearly enhance cancer treatment strategies. This review describes the current status of two (19)F nuclear magnetic resonance (NMR) methodologies that have been exploited to investigate tumour hypoxia, namely: (i) (19)F NMR oximetry following administration of perfluorocarbons, from which tumour p(O)(2) measurements can be made; and (ii) (19)F NMR measurements of the tumour retention of fluorinated 2-nitroimidazoles.     

The present status of cell tracking methods in animal models using magnetic resonance imaging technology

With the advance of stem cell transplantation research, in vivo cell tracking techniques have become increasingly important in recent years. Magnetic resonance imaging (MRI) may provide a unique tool for non-invasive tracking of transplanted cells. Since the initial findings on the stem cell migration by MRI several years ago, there have been numerous studies using various animal models, notably in heart or brain disease models. In order to develop more reliable and clinically applicable methodologies, multiple aspects should be taken into consideration. In this review, we will summarize the current status and future perspectives of in vivo cell tracking technologies using MRI. In particular, use of different MR contrast agents and their detection methods using MRI will be described in much detail. In addition, various cell labeling methods to increase the sensitivity of signals will be extensively discussed. We will also review several key experiments, in which MRI techniques were utilized to detect the presence and/or migration of transplanted stem cells in various animal models. Finally, we will discuss the current problems and future directions of cell tracking methods using MRI.     

Functional magnetic resonance imaging of awake behaving macaques

In recent years, more and more laboratories have developed functional Magnetic Resonance Imaging (fMRI) for awake non-human primates. This research is essential to provide a link between non-invasive hemodynamic signals recorded in the human brain and the vast body of knowledge gained from invasive electrophysiological studies in monkeys. Given that their brain structure is so closely related to that of humans and that monkeys can be trained to perform complicated behavioral tasks, results obtained with monkey fMRI and electrophysiology can be compared to fMRI results obtained in humans, and provide information crucial to a better understanding of the mechanisms by which different cortical areas perform their functions in the human brain. However, despite that the first publications on fMRI in awake behaving macaques appeared ～10 years ago (Logothetis et al. (1999) [1], Stefanacci et al. (1998) [2], Dubowitz et al. (1998) [3]), relatively few laboratories perform such experiments routinely, a sign of the significant technical difficulties that must be overcome. The higher spatial resolution required because of the animal’s smaller brain results in poorer signal-to-noise ratios than in human fMRI, which is further compounded by problems due to animal motion. Here, we discuss the specific challenges and benefits of fMRI in the awake monkey and review the methodologies and strategies for scanning behaving macaques.