Micron-sized Iron Oxide Particles Research Articles

3D mapping of neuronal migration in the embryonic mouse brain with magnetic resonance microimaging

A prominent feature of the developing mammalian brain is the widespread migration of neural progenitor (NP) cells during embryogenesis. A striking example is provided by NP cells born in the ventral forebrain of mid-gestation stage mice, which subsequently migrate long distances to their final positions in the cortex and olfactory bulb. Previous studies have used two-dimensional histological methods, making it difficult to analyze three-dimensional (3D) migration patterns. Unlike histology, magnetic resonance microimaging (micro-MRI) is a non-destructive, quantitative and inherently 3D imaging method for analyzing mouse embryos. To allow mapping of migrating NP cells with micro-MRI, cells were labeled in situ in the medial (MGE) and lateral (LGE) ganglionic eminences, using targeted in utero ultrasound-guided injection of micron-sized particles of iron-oxide (MPIO). Ex vivo micro-MRI and histology were then performed 5–6days after injection, demonstrating that the MPIO had magnetically labeled the migrating NP populations, which enabled 3D visualization and automated segmentation of the labeled cells. This approach was used to analyze the distinct patterns of migration from the MGE and LGE, and to construct rostral–caudal migration maps from each progenitor region. Furthermore, abnormal migratory phenotypes were observed in Nkx2.1−/− embryos, most notably a significant increase in cortical neurons derived from the Nkx2.1−/− LGE. Taken together, these results demonstrate that MPIO labeling and micro-MRI provide an efficient and powerful approach for analyzing 3D cell migration patterns in the normal and mutant mouse embryonic brain.

Micron-sized iron oxide-containing particles for microRNA-targeted manipulation and MRI-based tracking of transplanted cells

Particle-based delivery systems for therapeutic manipulation and tracking of transplanted cells by magnetic resonance imaging (MRI) are commonly based on nanometer-sized superparamagnetic iron oxide particles (SPIOs). Here, we present a proof of concept for multifunctional, silica based micron-sized iron oxide-containing particles (sMPIO) that combine fluorescence imaging, MRI tracking, and on-the-spot targeting of specific microRNAs on a particle surface for therapeutic manipulation by RNA interference. Antisense locked nucleic acids (α-LNA) were covalently bound to the surface of silica-based, DAPI-integrated, micron-sized iron oxide particles (sMPIO-α-LNA). In vitro studies using primary human hepatocytes showed rapid particle uptake (4 h) that was accompanied by significant depletion of the targeted microRNA Let7g (80%), up-regulation of the target proteins Cyclin D1 and c-Myc, and specific proteome changes. sMPIO-α-LNA-labeled cells were successfully detected by fluorescence imaging and could be visualized by MRI after intrasplenic transplantation in rats. This new theranostic particle provides a promising tool for cell transplantation where cellular imaging and microRNA-based manipulation is needed. [165]

Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging.

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre-labeled neural cells, especially in three-dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC-derived multicellular NPC aggregates labeled with micron-sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70-80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post-cryopreservation. MRI analysis showed comparable detectability for the MPIO-labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO-labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation.

Intracellular labeling of mouse embryonic stem cell–derived neural progenitor aggregates with micron-sized particles of iron oxide

Background aimsPluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) represent an unlimited source for the treatment of various neurological disorders. NPCs are usually derived from PSCs through the formation of embryoid body (EB), an aggregate structure mimicking embryonic development. This study investigated the effect of labeling multicellular EB-NPC aggregates with micron-sized particles of iron oxide (MPIO) for cell tracking using magnetic resonance imaging (MRI). MethodsIntact and dissociated EB-NPC aggregates were labeled with various concentrations of MPIOs (0, 2.5, 5 and 10 μg Fe/mL). The labeled cells were analyzed by fluorescent imaging, flow cytometry and in vitro MRI for labeling efficiency and detectability. Moreover, the biological effects of intracellular MPIO on cell viability, cytotoxicity, proliferation and neural differentiation were evaluated. ResultsIntact EB-NPC aggregates showed higher cell proliferation and viability compared with the dissociated cells. Despite diffusion limitation at low MPIO concentration, higher concentration of MPIO (i.e., 10 μg Fe/mL) was able to label EB-NPC aggregates at similar efficiency to the single cells. In vitro MRI showed concentration-dependent MPIO detection in EB-NPCs over 2.0–2.6 population doublings. More important, MPIO incorporation did not affect the proliferation and neural differentiation of EB-NPCs. ConclusionsMulticellular EB-NPC aggregates can be efficiently labeled and tracked with MPIO while maintaining cell proliferation, phenotype and neural differentiation potential. This study demonstrated the feasibility of labeling EB-NPC aggregates with MPIO for cellular monitoring of in vitro cultures and in vivo transplantation.

In vivo MR imaging of intercellular adhesion molecule-1 expression in an animal model of multiple sclerosis.

Upregulation of intercellular adhesion molecule 1 (ICAM-1) is an early event in lesion formation in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Monitoring its expression may provide a biomarker for early disease activity and allow validation of anti-inflammatory interventions. Our objective was therefore to explore whether ICAM-1 expression can be visualized in vivo during EAE with magnetic resonance imaging (MRI) using micron-sized particles of iron oxide (MPIO), and to compare accumulation profiles of targeted and untargeted MPIO, and a gadolinium-containing agent. Targeted αICAM-1-MPIO/untargeted IgG-MPIO were injected at two model-characteristic phases of EAE (in myelin oligodendrocyte glycoprotein35-55 -immunized C57BL/6 J mice), that is, at the peak of the acute phase (14 ± 1 days post-immunization) and during the chronic phase (26 ± 1 days post-immunization), followed by T2 *-weighted MRI. Blood-brain barrier (BBB) permeability was measured using gadobutrol-enhanced MRI. Cerebellar microvessels were analyzed for ICAM-1 mRNA expression using quantitative PCR (qPCR). ICAM-1 and iron oxide presence was examined with immunohistochemistry (IHC). During EAE, ICAM-1 was expressed by brain endothelial cells, macrophages and T-cells as shown with qPCR and (fluorescent) IHC. EAE animals injected with αICAM-1-MPIO showed MRI hypointensities, particularly in the subarachnoid space. αICAM-1-MPIO presence did not differ between the phases of EAE and was not associated with BBB dysfunction. αICAM-1-MPIO were associated with endothelial cells or cells located at the luminal side of blood vessels. In conclusion, ICAM-1 expression can be visualized with in vivo molecular MRI during EAE, and provides an early tracer of disease activity.

Labeling pluripotent stem cell-derived neural progenitors with iron oxide particles for magnetic resonance imaging.

Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), large numbers of PSC-derived cell products are in demand for applications in drug screening, disease modeling, and especially cell therapy. In stem cell-based therapy, tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO, 0.86μm) for MRI analysis. The protocol described PSC expansion and differentiation into NPs, and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation.

Mapping stain distribution in pathology slides using whole slide imaging

Background: Whole slide imaging (WSI) offers a novel approach to digitize and review pathology slides, but the voluminous data generated by this technology demand new computational methods for image analysis. Materials and Methods: In this study, we report a method that recognizes stains in WSI data and uses kernel density estimator to calculate the stain density across the digitized pathology slides. The validation study was conducted using a rat model of acute cardiac allograft rejection and another rat model of heart ischemia/reperfusion injury. Immunohistochemistry (IHC) was conducted to label ED1+ macrophages in the tissue sections and the stained slides were digitized by a whole slide scanner. The whole slide images were tessellated to enable parallel processing. Pixel-wise stain classification was conducted to classify the IHC stains from those of the background and the density distribution of the identified IHC stains was then calculated by the kernel density estimator. Results: The regression analysis showed a correlation coefficient of 0.8961 between the number of IHC stains counted by our stain recognition algorithm and that by the manual counting, suggesting that our stain recognition algorithm was in good agreement with the manual counting. The density distribution of the IHC stains showed a consistent pattern with those of the cellular magnetic resonance (MR) images that detected macrophages labeled by ultrasmall superparamagnetic iron-oxide or micron-sized iron-oxide particles. Conclusions: Our method provides a new imaging modality to facilitate clinical diagnosis. It also provides a way to validate/correlate cellular MRI data used for tracking immune-cell infiltration in cardiac transplant rejection and cardiac ischemic injury.

Functionalizable Silica-Based Micron-Sized Iron Oxide Particles for Cellular Magnetic Resonance Imaging

Cellular therapies require methods for noninvasive visualization of transplanted cells. Micron-sized iron oxide particles (MPIOs) generate a strong contrast in magnetic resonance imaging (MRI) and are therefore ideally suited as an intracellular contrast agent to image cells under clinical conditions. However, MPIOs were previously not applicable for clinical use. Here, we present the development and evaluation of silica-based micron-sized iron oxide particles (sMPIOs) with a functionalizable particle surface. Particles with magnetite content of >40% were composed using the sol-gel process. The particle surfaces were covered with COOH groups. Fluorescein, poly-L-lysine (PLL), and streptavidin (SA) were covalently attached. Monodisperse sMPIOs had an average size of 1.18 µm and an iron content of about 1.0 pg Fe/particle. Particle uptake, toxicity, and imaging studies were performed using HuH7 cells and human and rat hepatocytes. sMPIOs enabled rapid cellular labeling within 4 h of incubation; PLL-modified particles had the highest uptake. In T2*-weighted 3.0 T MRI, the detection threshold in agarose was 1,000 labeled cells, whereas in T1-weighted LAVA sequences, at least 10,000 cells were necessary to induce sufficient contrast. Labeling was stable and had no adverse effects on labeled cells. Silica is a biocompatible material that has been approved for clinical use. sMPIOs could therefore be suitable for future clinical applications in cellular MRI, especially in settings that require strong cellular contrast. Moreover, the particle surface provides the opportunity to create multifunctional particles for targeted delivery and diagnostics.

Multimodal imaging of subventricular zone neural stem/progenitor cells in the cuprizone mouse model reveals increased neurogenic potential for the olfactory bulb pathway, but no contribution to remyelination of the corpus callosum

Multiple sclerosis is a devastating demyelinating disease of the central nervous system (CNS) in which endogenous remyelination, and thus recovery, often fails. Although the cuprizone mouse model allowed elucidation of many molecular factors governing remyelination, currently very little is known about the spatial origin of the oligodendrocyte progenitor cells that initiate remyelination in this model. Therefore, we here investigated in this model whether subventricular zone (SVZ) neural stem/progenitor cells (NSPCs) contribute to remyelination of the splenium following cuprizone-induced demyelination. Experimentally, from the day of in situ NSPC labeling, C57BL/6J mice were fed a 0.2% cuprizone diet during a 4-week period and then left to recover on a normal diet for 8weeks. Two in situ labeling strategies were employed: (i) NSPCs were labeled by intraventricular injection of micron-sized iron oxide particles and then followed up longitudinally by means of magnetic resonance imaging (MRI), and (ii) SVZ NSPCs were transduced with a lentiviral vector encoding the eGFP and Luciferase reporter proteins for longitudinal monitoring by means of in vivo bioluminescence imaging (BLI). In contrast to preceding suggestions, no migration of SVZ NSPC towards the demyelinated splenium was observed using both MRI and BLI, and further validated by histological analysis, thereby demonstrating that SVZ NSPCs are unable to contribute directly to remyelination of the splenium in the cuprizone model. Interestingly, using longitudinal BLI analysis and confirmed by histological analysis, an increased migration of SVZ NSPC-derived neuroblasts towards the olfactory bulb was observed following cuprizone treatment, indicative for a potential link between CNS inflammation and increased neurogenesis.

PECAM‐1‐targeted micron‐sized particles of iron oxide as MRI contrast agent for detection of vascular remodeling after cerebral ischemia

An increasing amount of studies have provided evidence for vascular remodeling, for example, angiogenesis, after cerebral ischemia, which may play a significant role in post-stroke brain plasticity and recovery. Molecular imaging can provide unique in vivo whole-brain information on alterations in the expression of specific endothelial markers. A possible target for molecular magnetic resonance imaging (MRI) of post-stroke (neo)vascularization is platelet endothelial cell adhesion molecule-1 (PECAM-1). Here we describe significantly increased PECAM-1 mRNA levels in ipsilesional brain tissue at 6 h, 24 h and 3 days after transient middle cerebral artery occlusion in mice, and elevated PECAM-1 staining throughout the lesion at 3, 7 and 21 days post-stroke. The potential of micron-sized particles of iron oxide (MPIO) conjugated with PECAM-1-targeted antibodies, that is, αPECAM-1-MPIO, to expose stroke-induced PECAM-1 upregulation with molecular MRI was assessed. In vitro studies demonstrated that PECAM-1-expressing brain endothelial cells could be effectively labeled with αPECAM-1-MPIO, giving rise to a fourfold increase in MRI relaxation rate R2. Injection of near-infrared fluorescent dye-labeled αPECAM-1 showed target specificity and dose efficiency of the antibody for detection of brain endothelial cells at 3 days post-stroke. However, in vivo molecular MRI at 3 and 7 days after stroke revealed no αPECAM-1-MPIO-based contrast enhancement, which was corroborated by the absence of αPECAM-1-MPIO in post mortem brain tissue. This indicates that this molecular MRI approach, which has been proven successful for in vivo detection of other types of cell adhesion molecules, is not invariably effective for MRI-based assessment of stroke-induced alterations in expression of cerebrovascular markers.

Accumulation of micron sized iron oxide particles in endothelin‐1 induced focal cortical ischemia in rats is independent of cell migration

Endogenous labeling of stem/ progenitor cells via intracerebroventricular injection of micron-sized particles of iron oxide (MPIOs) has become standard methodology for MRI of adult neurogenesis. While this method is well characterized in the naïve rodent brain, it has not been fully investigated in disease models. Here, we describe methodological challenges that can confound data analysis when this technique is applied to a rat model of stroke, the endothelin-1 model of focal cortical ischemia. We intended to track endogenous neuroblast migration from the subventricular zone to the stroke area using previously described methods for in vivo labeling of endogenous neuroblasts with MPIOs and following migration with high resolution MRI. MPIOs accumulation in stroke regions of endothelin-1-treated brains involves two dynamic steps: an initial rapid cell independent mechanism, followed by slower MPIOs accumulation. While the latter may in part be attributable to cell dependent delivery of the particles, the cell independent mechanism complicates the interpretation of the data. Strategies aimed at prelabeling the stem cell niche reduced cell independent MPIOs accumulation, but failed to abolish it. We conclude that MRI-based neural stem/progenitor cell tracking via direct injection of MPIOs into the lateral and third ventricles, requires significant validation in models of brain disease/trauma.

213 EXOGENOUS MICROPARTICLES OF IRON OXIDE BIND TO ACTIVATED ENDOTHELIAL CELLS BUT, UNLIKE MONOCYTES, DO NOT TRIGGER AN ENDOTHELIAL RESPONSE

Introduction Targeting particles to sites of inflammation is of considerable interest for molecular imaging and drug delivery. We and others have described micron-sized particles of iron oxide (MPIO) that can be directed using specific ligands to bind to mediators of vascular inflammation in vivo. Since leukocyte binding to these molecules can induce changes in the target cell, an outstanding question is whether the binding of imaging particles induces biologically significant changes in the endothelial cells, potentially initiating or propagating inflammation. We address this by looking for changes in human aortic endothelial cells (HAEC) following binding of contrast agent. Methods 1 µm MPIO dual-targeted to E-selectin and VCAM were prepared by labelling with specific antibodies, and cellular responses to binding events assessed. Calcium flux was monitored using the calcium indicator Fluo-4 and fluorescent microscopy for analysis. Production of reactive oxygen species was investigated using a nitroblue tetrazolium blue (NBT) assay. Real-time RT-PCR was used to assess gene expression of several endothelial markers of inflammation. Antibody staining of cell surface proteins and flow cytometry analysis monitored protein levels presented by HAEC. Results Binding of E+V-MPIO did not cause calcium flux in HAEC; normalised fluorescence intensity of Fluo-4 dropped to 0.81 (SEM ±0.02) of baseline 60 seconds post MPIO binding and to 0.64 (SEM±0.02) with vehicle only, whilst a significant increase in Fluo-4 fluorescence to 1.96 (SEM±0.48) was seen following THP-1 binding indicating endothelial calcium flux post-leukocyte binding (P Conclusions Our experiments demonstrate that whilst antibody-targeted microparticles mimic the binding capability of leukocytes to inflamed endothelium, they do not trigger the same cellular responses and do not appear to initiate or compound inflammation. These properties are desirable for targeted therapeutic and diagnostic agents.

The effect of cryoprotection on the use of PLGA encapsulated iron oxide nanoparticles for magnetic cell labeling

Magnetic PLGA nanoparticles are a significant advancement in the quest to translate MRI-based cell tracking to the clinic. The benefits of these types of particles are that they encapsulate large amounts of iron oxide nanocrystals within an FDA-approved polymer matrix, combining the best aspects of inert micron-sized iron oxide particles, or MPIOs, and biodegradable small particles of iron oxide, or SPIOs. Practically, PLGA nanoparticle fabrication and storage requires some form of cryoprotectant to both protect the particle during freeze drying and to promote resuspension. While this is a commonly employed procedure in the fabrication of drug loaded PLGA nanoparticles, it has yet to be investigated for magnetic particles and what effect this might have on internalization of magnetic particles. As such, in this study, magnetic PLGA nanoparticles were fabricated with various concentrations of two common cryoprotectants, dextrose and sucrose, and analyzed for their ability to magnetically label cells. It was found that cryoprotection with either sugar significantly enhanced the ability to resuspend nanoparticles without aggregation. Magnetic cell labeling was impacted by sugar concentration, with higher sugar concentrations used during freeze drying more significantly reducing magnetic cell labeling than lower concentrations. These studies suggest that cryoprotection with 1% dextrose is an optimal compromise that preserves monodispersity following resuspension and high magnetic cell labeling.

MRI of ICAM-1 Upregulation After Stroke: the Importance of Choosing the Appropriate Target-Specific Particulate Contrast Agent

Magnetic resonance imaging (MRI) with targeted contrast agents provides a promising means for diagnosis and treatment monitoring after cerebrovascular injury. Our goal was to demonstrate the feasibility of this approach to detect the neuroinflammatory biomarker intercellular adhesion molecule-1 (ICAM-1) after stroke and to establish a most efficient imaging procedure. We compared two types of ICAM-1-functionalized contrast agent: T 1-shortening gadolinium chelate-containing liposomes and T2(*)-shortening micron-sized iron oxide particles (MPIO). Binding efficacy and MRI contrast effects were tested in cell cultures and a mouse stroke model. Both ICAM-1-targeted agents bound effectively to activated cerebrovascular cells in vitro, generating significant MRI contrast-enhancing effects. Direct in vivo MRI-based detection after stroke was only achieved with ICAM-1-targeted MPIO, although both contrast agents showed similar target-specific vascular accumulation. Our study demonstrates the potential of in vivo MRI of post-stroke ICAM-1 upregulation and signifies target-specific MPIO as most suitable contrast agent for molecular MRI of cerebrovascular inflammation.

Exogenous Microparticles of Iron Oxide Bind to Activated Endothelial Cells but, Unlike Monocytes, Do Not Trigger an Endothelial Response

Targeting particles to sites of inflammation is of considerable interest for applications relating to molecular imaging and drug delivery. We and others have described micron-sized particles of iron oxide (MPIO) that can be directed using specific ligands (e.g. antibodies, peptides and oligosaccharides) to bind to mediators of vascular inflammation in vivo. Since leukocyte binding to these molecules can induce changes in the target cell, an outstanding question has been whether the binding of imaging particles to these mediators induces biologically significant changes in the endothelial cells, potentially initiating or propagating inflammation. Here, we address these questions by looking for changes in endothelial cells following binding of contrast agent. Specifically, we have quantified calcium flux, rearrangement of the actin cytoskeleton, production of reactive oxygen species (ROS), apoptosis and potential secondary changes, such as changes in gene and protein expression follow binding events to primary endothelial cells in vitro.Although leukocytes induced changes to endothelial cell function, we did not see any significant changes to endothelial calcium flux, cytoskeletal organisation, production of ROS or induction of apoptosis in response to antibody-MPIO binding. Furthermore, there were no changes to gene expression monitored via real-time RT-PCR or presentation of protein on the cell surface measured using flow cytometry.Our experiments demonstrate that whilst antibody-targeted microparticles mimic the binding capability of leukocytes to inflamed endothelium, they do not trigger the same cellular responses and do not appear to initiate or compound inflammation. These properties are desirable for targeted therapeutic and diagnostic agents.

Silica-Based Micron-Sized Iron Oxide Particles for Detection and Loco-Regional Stimulation of Transplanted Liver Cells

Silica-Based Micron-Sized Iron Oxide Particles for Detection and Loco-Regional Stimulation of Transplanted Liver Cells

Silica-Based Micron-Sized Iron Oxide Particles for Detection and Loco-Regional Stimulation of Transplanted Liver Cells

Silica-Based Micron-Sized Iron Oxide Particles for Detection and Loco-Regional Stimulation of Transplanted Liver Cells

Evaluation of Intracellular Labeling with Micron-Sized Particles of Iron Oxide (MPIOs) as a General Tool for In Vitro and in Vivo Tracking of Human Stem and Progenitor Cells

Magnetic resonance imaging (MRI)-based tracking is increasingly attracting attention as a means of better understanding stem cell dynamics in vivo. Intracellular labeling with micrometer-sized particles of iron oxide (MPIOs) provides a practical MRI-based approach due to superior detectability relative to smaller iron oxide particles. However, insufficient information is available about the general utility across cell types and the effects on cell vitality of MPIO labeling of human stem cells. We labeled six human cell types from different sources: mesenchymal stem cells derived from bone marrow (MSCs), mesenchymal stem cells derived from adipose tissue (ASCs), presumptive adult neural stem cells (ad-NSCs), fetal neural progenitor cells (f-NPCs), a glioma cell line (U87), and glioblastoma tumor stem cells (GSCs), with two different sizes of MPIOs (0.9 and 2.84 µm). Labeling and uptake efficiencies were highly variable among cell types. Several parameters of general cell function were tested in vitro. Only minor differences were found between labeled and unlabeled cells with respect to proliferation rate, mitotic duration, random motility, and capacity for differentiation to specific phenotypes. In vivo behavior was tested in chicken embryos and severe combined immunodeficient (SCID) mice. Postmortem histology showed that labeled cells survived and could integrate into various tissues. MRI-based tracking over several weeks in the SCID mice showed that labeled GSCs and f-NPCs injected into the brain exhibited translocations similar to those seen for unlabeled cells and as expected from migratory behavior described in previous studies. The results support MPIO-based cell tracking as a generally useful tool for studies of human stem cell dynamics in vivo.

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s). Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting. BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin). MRI on the other hand is a non-invasive technique which is clinically applicable and can be used to precisely locate cellular grafts with very high resolution, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies. In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

Feasibility of Fast Dynamic MRI for Noninvasive Monitoring During Ectopic Liver Cell Transplantation to the Spleen in a Porcine Model

Liver cell transplantation is a promising approach for the treatment of metabolic liver disorders. However, a method for noninvasive monitoring during liver cell transplantation is not available clinically. The aim of this study was to investigate the feasibility of fast dynamic MRI monitoring during liver cell infusion to the spleen, which is considered an ectopic implantation site for liver cell transplantation. Porcine liver cells were labeled with micron-sized iron oxide particles and infused to the spleens of pigs (n = 5) via the lineal artery. MRI was performed using a 3-T MR scanner. Initially, T1- and T2-weighted pulse sequences were tested. Thereafter, fast dynamic MRI was performed during cell infusion. MR findings were verified by immunohistological examinations. Images from static MRI (TR/TE, 2500/105.2) showed significantly lower signal intensity and signal-to-noise ratio after cell infusion compared with pretransplant images. T2-weighted fast dynamic MRI enabled visualization of signal decrease of the spleen during cell infusion. When cells were infused systemically, no signal changes in the spleen were observed. This study shows that fast dynamic MRI can enable noninvasive monitoring during liver cell transplantation to the spleen. This approach could be useful for preclinical studies and for quality control of clinical liver cell transplantation.