Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells

The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.     

Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging

Superparamagnetic iron oxide (SPIO) nanoparticles are being used for intracellular magnetic labeling of stem cells and other cells in order to monitor cell trafficking by magnetic resonance imaging (MRI) as part of cellular-based repair, replacement and treatment strategies. This review focuses on the various methods for magnetic labeling of stem cells and other mammalian cells and on how to translate experimental results from bench to bedside.     

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.     

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 divisional tracking of hematopoietic stem cells

Hematopoietic stem cell (HSC) division leads to self-renewal, differentiation, or death of HSCs, and adequate balance of this process results in sustained, lifelong, high-throughput hematopoiesis. Despite their contribution to hematopoietic cell production, the majority of cells within the HSC population are quiescent at any given time. Recent studies have tackled the questions of how often HSCs divide, how divisional history relates to repopulating potential, and how many HSCs contribute to hematopoiesis. Here, we summarize these recent findings on HSC turnover from different experimental systems and discuss hypothetical models for HSC cycling and maintenance in steady-state and upon hematopoietic challenge.     

Lentivirally generated eGFP-transgenic rats allow efficient cell tracking in vivo

Here we describe the efficient generation of eGFP-transgenic rats using a lentiviral approach. Analysis of the founder generation demonstrated that 46% of the offspring had stably integrated the provirus into the genome and of those 92% expressed eGFP in all blood-derived leukocytes. In contrast to their offspring, all founder rats were mosaic with regard to eGFP-expression, suggesting delayed viral transduction after injection. The expression level of eGFP in the F1 generation is influenced by and segregates with the site of proviral integration. Interestingly, a single copy of the transgene is sufficient for reliable detection by flow cytometry, irrespective of the leukocyte subtype analyzed. Adoptive transfer of purified CD4(+) T-lymphocytes from transgenic rats and subsequent reisolation from various organs further demonstrated that expression of the lentiviral transgene is maintained in a foreign host and therefore allows for efficient tracking of transferred cells. Taken together, lentivirally generated eGFP-transgenic rats are a powerful tool for various applications in immunology and presumably also many other fields.     

Efficient labeling of mesenchymal stem cells using cell permeable magnetic nanoparticles

For the purpose of successfully monitoring labeled cells, optimum labeling efficiency without any side effect is a prerequisite. Magnetic cellular imaging is a new and growing field that allows the visualization of implanted cells in vivo. Herein, superparamagnetic iron oxide (SPIO) nanoparticles were conjugated with a non-toxic protein transduction domain (PTD), identified by the authors and termed low molecular weight protamine (LMWP), to generate efficient and non-toxic cell labeling tools. The cells labeled with LMWP-SPIO presented the highest iron content compared to those labeled with naked SPIO and the complex of SPIO with poly-l-lysine, which is currently used as a transfection agent. In addition to the iron content assay, Prussian staining and confocal observation demonstrated the highest intracellular LMWP-SPIO presence, and the labeling procedure did not alter the cell differentiation capacity of mesenchymal stem cells. Taken together, cell permeable magnetic nanoparticles conjugated with LMWP can be suggested as labeling tools for efficient magnetic imaging of transplanted cells.     

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.     

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.     

Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm³ after 3 days, 1.24 ± 0.76 mm³ after 7 days, 0.33 ± 0.03 mm³ after 14 days, and 0.09 ± 0.05 mm³ after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.     

Indocyanine green labeling for optical and photoacoustic imaging of mesenchymal stem cells after in vivo transplantation

The transplantation of mesenchymal stem cells (MSCs) holds great promise for the treatment of a plethora of human diseases, but new noninvasive procedures are needed to monitor the cell fate in vivo. Already largely used in medical diagnostics, the fluorescent dye indocyanine green (ICG) is an established dye to track limited numbers of cells by optical imaging (OI), but it can also be visualized by photoacoustic imaging (PAI), which provides a higher spatial resolution than pure near infrared fluorescence imaging (NIRF). Because of its successful use in clinical and preclinical examinations, we chose ICG as PAI cell labeling agent. Optimal incubation conditions were defined for an efficient and clinically translatable MSC labeling protocol, such that no cytotoxicity or alterations of the phenotypic profile were observed, and a consistent intracellular uptake of the molecule was achieved. Suspensions of ICG‐labeled cells were both optically and optoacoustically detected in vitro, revealing a certain variability in the photoacoustic spectra acquired by varying the excitation wavelength from 680 to 970 nm. Intramuscular engraftments of ICG‐labeled MSCs were clearly visualized by both PAI and NIRF over few days after transplantation in the hindlimb of healthy mice, suggesting that the proposed technique retains a considerable potential in the field of transplantation‐focused research and therapy. Stem cells were labeled with the Food and Drug Administration (FDA)‐approved fluorescent dye ICG, and detected by both PAI and OI, enabling to monitor the cell fate safely, in dual modality, and with good sensitivity and improved spatial resolution.      

18F]FDG labeling of neural stem cells for in vivo cell tracking with positron emission tomography: inhibition of tracer release by phloretin

The introduction of neural stem cells into the brain has promising therapeutic potential for the treatment of neurodegenerative diseases. To monitor the cellular replacement therapy, that is, to determine stem cell migration, survival, and differentiation, in vivo tracking methods are needed. Ideally, these tracking methods are noninvasive. Noninvasive tracking methods that have been successfully used for the visualization of blood-derived progenitor cells include magnetic resonance imaging and radionuclide imaging using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The SPECT tracer In-111-oxine is suitable for stem cell labeling, but for studies in small animals, the higher sensitivity and facile quantification that can be obtained with PET are preferred. Here the potential of 2'-[18F]fluoro-2'-deoxy-D-glucose ([18F]-FDG), a PET tracer, for tracking of neural stem cell (NSCs) trafficking toward an inflammation site was investigated. [18F]-FDG turns out to be a poor radiopharmaceutical to label NSCs owing to the low labeling efficiency and substantial release of radioactivity from these cells. Efflux of [18F]-FDG from NSCs can be effectively reduced by phloretin in vitro, but inhibition of tracer release is insufficient in vivo for accurate monitoring of stem cell trafficking.     

Locating and labeling neural stem cells in the brain

The phenomenon of adult neurogenesis has been demonstrated in most mammals including humans. At least two regions of the adult brain maintain stem cells throughout life; the subgranular zone (SGZ) of the hippocampal dentate gyrus, and the subventricular zone (SVZ) of the lateral ventricle wall. Both regions continuously produce neurons that mature and become integrated into functional networks that are involved in learning and memory and odor discrimination, respectively. Apart from these well‐studied regions neurogenesis has been reported in a number of other brain regions, such as amygdala and cortex. However, these studies have been contested and there is currently no well‐postulated function for non‐SVZ/SGZ neurogenesis. The studies of the regional localization of neurogenesis in the brain have been made possible due to several methods for detecting adult neurogenesis including; bromodeoxyuridine labeling (BrdU) together with markers of mature neurons, genetic labeling, by mouse transgenesis, or with the use of viral vectors. These techniques are already put to creative use and will be essential for the discovery of the nature of the adult neural stem cells. In this mini‐review, we will discuss the localization of neural stem/progenitor cells in the brain and their implications as well as discussing the pro's and con's of stem cell labeling techniques. J. Cell. Physiol. 226: 1–7, 2010. © 2010 Wiley‐Liss, Inc.     

Quantum dot labeling of mesenchymal stem cells

Background  

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, fat and muscle cells and are being investigated for their utility in cell-based transplantation therapy. Yet, adequate methods to track transplanted MSCs in vivo are limited, precluding functional studies. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. These nanoparticles are resistant to chemical and metabolic degradation, demonstrating long term photostability. Here, we investigate the cytotoxic effects of in vitro QD labeling on MSC proliferation and differentiation and use as a cell label in a cardiomyocyte co-culture.     

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.     

Effects of EdU labeling on mesenchymal stem cells

BackgroundThymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been used for tracking mesenchymal stem cells (MSCs). In the present study, we tested whether EdU was cytotoxic and whether it interfered with differentiation, cytokine secretion and migration of MSCs.MethodsEdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10^?8 mol/L of EdU for 48 h. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay.ResultsEdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express β-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein and to form capillary-like structures as well as the expression of vWF, eNOS and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1.DiscussionAt the recommended dosage of 10^?8 mol/L, EdU is non-toxic to ADSCs. EdU label did not interfere with differentiation, cytokine secretion or migratory response to SDF-1 by ADSCs.     

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.