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.     

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.     

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 bioluminescence for tracking cell fate and function

Tracking the fate and function of cells in vivo is paramount for the development of rational therapies for cardiac injury. Bioluminescence imaging (BLI) provides a means for monitoring physiological processes in real time, ranging from cell survival to gene expression to complex molecular processes. In mice and rats, BLI provides unmatched sensitivity because of the absence of endogenous luciferase expression in mammalian cells and the low background luminescence emanating from animals. In the field of stem cell therapy, BLI provides an unprecedented means to monitor the biology of these cells in vivo, giving researchers a greater understanding of their survival, migration, immunogenicity, and potential tumorigenicity in a living animal. In addition to longitudinal monitoring of cell survival, BLI is a useful tool for semiquantitative measurements of gene expression in vivo, allowing a better optimization of drug and gene therapies. Overall, this technology not only enables rapid, reproducible, and quantitative monitoring of physiological processes in vivo but also can measure the influences of therapeutic interventions on the outcome of cardiac injuries.     

In vivo bioluminescence imaging

In vivo bioluminescent imaging (BLI) is a versatile and sensitive tool that is based on detection of light emission from cells or tissues. Bioluminescence, the biochemical generation of light by a living organism, is a naturally occurring phenomenon. Luciferase enzymes, such as that from the North American firefly (Photinus pyralis), catalyze the oxidation of a substrate (luciferin), and photons of light are a product of the reaction. Optical imaging by bioluminescence allows a low-cost, noninvasive, and real-time analysis of disease processes at the molecular level in living organisms. Bioluminescence has been used to track tumor cells, bacterial and viral infections, gene expression, and treatment response. Bioluminescence in vivo imaging allows longitudinal monitoring of a disease course in the same animal, a desirable alternative to analyzing a number of animals at many time points during the course of the disease. We provide a brief introduction to BLI technology, specific examples of in vivo BLI studies investigating bacterial/viral pathogenesis and tumor growth in animal models, and highlight some future perspectives of BLI as a molecular imaging tool.     

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 SPECT reporter gene imaging of regulatory T cells

Regulatory T cells (Tregs) were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens, including alloantigens. In vivo imaging techniques including intravital microscopy as well as whole body imaging using bioluminescence probes have contributed to the understanding of in vivo Treg function, their mechanisms of action and target cells. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT) has been used to image various cell types in vivo. It has several advantages over the aforementioned imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation of cells over time and lastly it may offer the possibility to be translated to the clinic. This study addresses whether SPECT/CT imaging can be used to visualise the migratory pattern of Tregs in vivo. Treg lines derived from CD4(+)CD25(+)FoxP3(+) cells were retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS) and the fluorescent protein mCherry and stimulated with autologous DCs. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate ((99m)TcO(4)(-)) and exposure of these cells to radioactivity did not affect cell viability, phenotype or function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6) mice by SPECT/CT using (99m)TcO(4)(-). After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models.     

In vivo imaging enters parasitology

In vivo infection routes of parasites have remained something of a "black box", in which only snapshot views of fixed tissues are available. Clearly, there exists a strong need for imaging approaches to visualise living parasites within intact organs and animals. In vivo imaging of fluorescent Plasmodium parasites now provides us with exciting insights into the infection process, from the bite of the infected mosquito to the invasion of liver cells, and alternative approaches using luciferase-expressing parasites have been used to monitor their dissemination in mice. This rapidly developing field will go a long way towards deepening our understanding of host-parasite interactions at different levels.     

A novel near-infrared fluorescence imaging probe for in vivo neutrophil tracking

The development and validation of a multiscopic near-infrared fluorescence (NIRF) probe, cinnamoyl-F-(D)L-F-(D)L-F-PEG-cyanine7 (cFlFlF-PEG-Cy7), that targets formyl peptide receptor on neutrophils using a mice ear inflammation model is described. Acute inflammation was induced in mice by topical application of phorbol-12-myristate-13-acetate to left ears 24 hours before probe administration. Noninvasive NIRF imaging was longitudinally performed up to 24 hours following probe injection. The in vivo neutrophil-targeting specificity of the probe was characterized by a blocking study with preadministration of excess nonfluorescent peptide cFlFlF-PEG and by an imaging study with a scrambled peptide probe cLFFFL-PEG-Cy7. NIRF imaging of mice injected with cinnamoyl-L-F-F-F-L-PEG-cyanine7 (cFlFlF-PEG-Cy7) revealed that the fluorescence intensity for inflamed left ears was approximately fourfold higher than that of control right ears at 24 hours postinjection. In comparison, the ratios acquired with the scrambled probe and from the blocking study were 1.5- and 2-fold at 24 hours postinjection, respectively. Moreover, a microscopic immunohistologic study confirmed that the NIRF signal of cFlFlF-PEG-Cy7 was associated with activated neutrophils in the inflammatory tissue. With this probe, in vivo neutrophil chemotaxis could be correlatively imaged macroscopically in live animals and microscopically at tissue and cellular levels.     

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.     

A novel near-infrared fluorescence imaging probe for in vivo neutrophil tracking

AbstractWe delivered adenovirus vector (Ad) via intravitreous injection and monitored transgene (luciferase) expression in living mice (BALB/c) at multiple time points. In vivo live imaging technology was able to assess dynamically intraocular luciferase expression in a single animal population throughout the entire experiment period. Using this information, we were able to determine the optimal time point for readministration of Ad into the eyes and to dynamically study the time course of expression of a second Ad administration. Optical imaging demonstrated the limited period of transgene expression in eyes. Significant transgene signal was also detected in livers. The repeat intraocular delivery of the adenovirus resulted in significant blunting of transgene expression in both eyes and livers compared to the initial delivery. Periocular corticosteroid (triamcinolone acetonide) injection combined with initial Ad delivery was effective to rescue luciferase expression on repeat Ad vector delivery. However, this effect was not observed when corticosteroid was combined with repeat Ad delivery. Although corticosteroid enhanced ocular transgene expression, it also increased transgene expression in liver, which has potential safety implications. This dynamic transgene expression in eyes was successfully traced and monitored via a live imaging technique.     

Functional assessment of human dendritic cells labeled for in vivo 19F magnetic resonance imaging cell tracking

Background aimsDendritic cells (DC) are increasingly being used as cellular vaccines to treat cancer and infectious diseases. While there have been some promising results in early clinical trials using DC-based vaccines, the inability to visualize non-invasively the location, migration and fate of cells once adoptively transferred into patients is often cited as a limiting factor in the advancement of these therapies. A novel perflouropolyether (PFPE) tracer agent was used to label human DC ex vivo for the purpose of tracking the cells in vivo by ¹⁹F magnetic resonance imaging (MRI). We provide an assessment of this technology and examine its impact on the health and function of the DC.MethodsMonocyte-derived DC were labeled with PFPE and then assessed. Cell viability was determined by examining cell membrane integrity and mitochondrial lipid content. Immunostaining and flow cytometry were used to measure surface antigen expression of DC maturation markers. Functional tests included bioassays for interleukin (IL)-12p70 production, T-cell stimulatory function and chemotaxis. MRI efficacy was demonstrated by inoculation of PFPE-labeled human DC into NOD-SCID mice.ResultsDC were effectively labeled with PFPE without significant impact on cell viability, phenotype or function. The PFPE-labeled DC were clearly detected in vivo by ¹⁹F MRI, with mature DC being shown to migrate selectively towards draining lymph node regions within 18 h.ConclusionsThis study is the first application of PFPE cell labeling and MRI cell tracking using human immunotherapeutic cells. These techniques may have significant potential for tracking therapeutic cells in future clinical trials.     

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.     

In vivo bioluminescence imaging of bone marrow-derived cells in brain inflammation

It has been accepted that bone marrow cells infiltrate the brain and play important roles in neuroinflammation. However, there is no good tool for the visualization of these cells in living animals. In this study, we generated mice that were transplanted with GFP- or luciferase-expressing bone marrow cells, and performed in vivo fluorescence imaging (FLI) and in vivo bioluminescence imaging (BLI) to visualize the infiltrated cells. Brain inflammation was induced by intrahippocampal injection of lipopolysaccharide (LPS). Immunohistochemical investigation demonstrated an increase in the infiltration of bone marrow cells into the hippocampus because of the LPS injection and differentiation of the infiltrated cells into microglia, but not into neurons or astrocytes. BLI, but not FLI, successfully detected an increase in signal intensity with the LPS injection, and the increase of BLI coincided with that of luciferase activity in hippocampus. BLI could quantitatively and continuously monitor bone marrow-derived cells in vivo.     

In vivo tracking of the human patella.

The purpose of this study was to describe the dynamic, in vivo, three-dimensional tracking pattern of the patella for one normal male subject. Intracortical pins were inserted into the patella, tibia, and femur. The subject performed seated and squatting knee flexion/extension, and maximum voluntary quadriceps contractions. In addition, the vastus medialis oblique was subjected to maximal electrical stimulation. Motions of the markers attached to the intracortical pins were analyzed using an automated video system. Patellar and tibial motions were determined relative to a femoral reference system. While the tibia flexed 50 degrees from full extension (seated condition), the patella flexed 30.3 degrees, tilted laterally 10.3 degrees, and shifted laterally 8.6 mm. In general, these results show qualitative agreement with the data collected from cadaveric specimens [van Kampen and Huiskes, J. orthop. Res. 8, 372-382 (1990)]. The differences present may reflect different passive constraints to patellar motions, and different relative loading of the individual quadriceps components, in our study compared to the cadaveric study. Only small differences were found between patellar motions in the seated and squatting conditions. Differences in patellar displacements produced by (1) maximal electrical stimulation of the vastus medialis oblique, and (2) maximum voluntary quadriceps contraction, at 30 degrees knee flexion and full extension, may reflect the dominant influence of passive constraints, and the vastus lateralis, on normal patellar motions. Further in vivo study of patellar tracking seems warranted to evaluate surgical and conservative interventions for patellofemoral disorders.     

In vivo tracking of macrophage activated killer cells to sites of metastatic ovarian carcinoma

Radio-labelling of blood cells is an established technique for evaluating in vivo migration of normal cells to sites of pathology such as infection and haemorrhage. A limitation of cellular immunotherapies to induce anti-tumour responses is in part due to the uncertain ability of cellular effectors to reach their intended target. We extended the approach of cell radiolabelling to accurately examine the in vivo distribution of cellular immunotherapy with ex-vivo macrophage activated killer (MAK) cells. We describe the use of two methods of cell labelling for tracking the destination of autologous-derived macrophage activated killer (MAK) cells linked to the bi-specific antibody MDX-H210 delivered either by intravenous (i.v.) or intraperitoneal (i.p.) injection in ten patients with peritoneal relapse of epithelial ovarian carcinoma. Our results demonstrate the feasibility of generating high numbers and purity of GMP quality MAK cells, which can be radiolabelled with (18)F-FDG or (111)In-oxime. MAK cell administration produced minimal infusional toxicity and demonstrated a reproducible pattern of in vivo distribution and active in vivo tracking to sites of known tumour following 8 of 16 i.v. infusions or 4 of 6 i.p. infusions. However, the leakage of (18)F-FDG limited the ability to confidently confirm the tracking of MAK cells to tumour in all cases and improved PET labels are required. The addition of MDX-H210 bispecific antibody did not alter the distribution of cells to tumour sites, but did accelerate the clearance of i.v. administered MAK cells from the pulmonary circulation. This data demonstrates that cellular cancer immunotherapies may be successfully delivered to the sites of active tumour following either i.v. or i.p. injection in a proportion of patients with metastatic cancer. Incorporation of tracking studies in early cycles of cellular immunotherapy may allow selection of patients who demonstrate successful targeting of the immunotherapy for ongoing treatment.     

In vivo imaging of islet transplantation

Type 1 diabetes mellitus is characterized by the selective destruction of insulin-producing beta cells, which leads to a deficiency in insulin secretion and, as a result, to hyperglycemia. At present, transplantation of pancreatic islets is an emerging and promising clinical modality, which can render individuals with type 1 diabetes insulin independent without increasing the incidence of hypoglycemic events. To monitor transplantation efficiency and graft survival, reliable noninvasive imaging methods are needed. If such methods were introduced into the clinic, essential information could be obtained repeatedly and noninvasively. Here we report on the in vivo detection of transplanted human pancreatic islets using magnetic resonance imaging (MRI) that allowed noninvasive monitoring of islet grafts in diabetic mice in real time. We anticipate that the information obtained in this study would ultimately result in the ability to detect and monitor islet engraftment in humans, which would greatly aid the clinical management of this disease.