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. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:510–521, 2015     

MRI-based Visualization of Iron-labeled CD133+ Human Endothelial Progenitor Cells

The objective of this study is to build up a kind of effective approach to multiply CD133+ endothelial progenitor cells (EPCs) and visualize cells by labeling with two FDA-approved agents based on MRI technique. CD133+ cells were separated by immunomagnetic microbeads selection and grew with serum-free medium. Seven days later, CD133+ cell production was collected and co-incubated with iron complex for 24 h for labeling. The iron-labeled cells were suspended into agarose gel and scanned by MRI for visualization. Labeled cells were also analyzed for cell viability. Iron can be effectively introduced into CD133+ EPCs plasma in culture and visualized by changing the MRI signal intensity. Iron had no influence on cell viability. CONCLUSION: Iron substance can be applied to label CD133+ cells without cytotoxicity and iron-labeled cells can be visualized by MRI image. Due to the non-invasive property and repeatability of MRI technology and this kind of method could be used for tracing in vivo stem cells in the future.     

Comparative In Vitro Study on Magnetic Iron Oxide Nanoparticles for MRI Tracking of Adipose Tissue-Derived Progenitor Cells

Magnetic resonance imaging (MRI) using measurement of the transverse relaxation time (R2*) is to be considered as a promising approach for cell tracking experiments to evaluate the fate of transplanted progenitor cells and develop successful cell therapies for tissue engineering. While the relationship between core composition of nanoparticles and their MRI properties is well studied, little is known about possible effects on progenitor cells. This in vitro study aims at comparing two magnetic iron oxide nanoparticle types, single vs. multi-core nanoparticles, regarding their physico-chemical characteristics, effects on cellular behavior of adipose tissue-derived stem cells (ASC) like differentiation and proliferation as well as their detection and quantification by means of MRI. Quantification of both nanoparticle types revealed a linear correlation between labeling concentration and R2* values. However, according to core composition, different levels of labeling concentrations were needed to achieve comparable R2* values. Cell viability was not altered for all labeling concentrations, whereas the proliferation rate increased with increasing labeling concentrations. Likewise, deposition of lipid droplets as well as matrix calcification revealed to be highly dose-dependent particularly regarding multi-core nanoparticle-labeled cells. Synthesis of cartilage matrix proteins and mRNA expression of collagen type II was also highly dependent on nanoparticle labeling. In general, the differentiation potential was decreased with increasing labeling concentrations. This in vitro study provides the proof of principle for further in vivo tracking experiments of progenitor cells using nanoparticles with different core compositions but also provides striking evidence that combined testing of biological and MRI properties is advisable as improved MRI properties of multi-core nanoparticles may result in altered cell functions.     

Superparamagnetic Iron Oxide Nanoparticles Function as a Long-Term,Multi-Modal Imaging Label for Non-Invasive Tracking of Implanted Progenitor Cells

The purpose of this study was to determine the ability of superparamagnetic iron oxide (SPIO) nanoparticles to function as a long-term tracking label for multi-modal imaging of implanted engineered tissues containing muscle-derived progenitor cells using magnetic resonance imaging (MRI) and X-ray micro-computed tomography (μCT). SPIO-labeled primary myoblasts were embedded in fibrin sealant and imaged to obtain intensity data by MRI or radio-opacity information by μCT. Each imaging modality displayed a detection gradient that matched increasing SPIO concentrations. Labeled cells were then incorporated in fibrin sealant, injected into the atrioventricular groove of rat hearts, and imaged in vivo and ex vivo for up to 1 year. Transplanted cells were identified in intact animals and isolated hearts using both imaging modalities. MRI was better able to detect minuscule amounts of SPIO nanoparticles, while μCT more precisely identified the location of heavily-labeled cells. Histological analyses confirmed that iron oxide particles were confined to viable, skeletal muscle-derived cells in the implant at the expected location based on MRI and μCT. These analyses showed no evidence of phagocytosis of labeled cells by macrophages or release of nanoparticles from transplanted cells. In conclusion, we established that SPIO nanoparticles function as a sensitive and specific long-term label for MRI and μCT, respectively. Our findings will enable investigators interested in regenerative therapies to non-invasively and serially acquire complementary, high-resolution images of transplanted cells for one year using a single label.     

Effects of iron oxide incorporation for long term cell tracking on MSC differentiation in vitro and in vivo

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.     

In vivo leukocyte labeling with intravenous ferumoxides/protamine sulfate complex and in vitro characterization for cellular magnetic resonance imaging

Cellular labeling with ferumoxides (Feridex IV) superparamagnetic iron oxide nanoparticles can be used to monitor cells in vivo by MRI. The objective of this study was to use histology and MRI to evaluate an in vivo, as opposed to in vitro, technique for labeling of mononuclear leukocytes as a means of tracking inflammatory processes in the brain. Long-Evans rats were intravenously injected with 20 mg/kg ferumoxides, ferumoxtran-10, or ferumoxytol with or without protamine sulfate. Leukocytes and splenocytes were evaluated by cell sorting and iron histochemistry or were implanted into the brain for MRI. Injection of ferumoxides/protamine sulfate complex IV resulted in iron labeling of leukocytes (ranging from 7.4 +/- 0.5% to 12.5 +/- 0.9% with average 9.2 +/- 0.8%) compared with ferumoxides (ranging from 3.9 +/- 0.4% to 6.3 +/- 0.5% with average 5.0 +/- 0.5%) or protamine sulfate alone (ranging from 0% to 0.9 +/- 0.7% with average 0.3 +/- 0.3%). Cell sorting analysis indicated that iron-labeled cells were enriched for cell types positive for the myelomonocytic marker (CD11b/c) and the B lymphocyte marker (CD45RA) and depleted in the T cell marker (CD3). Neither ferumoxtran-10 nor ferumoxytol with protamine sulfate labeled leukocytes. In vivo ferumoxides/protamine sulfate-loaded leukocytes and splenocytes were detected by MRI after intracerebral injection. Ferumoxides/protamine complex labeled CD45RA-positive and CD11b/c-positive leukocytes in vivo without immediate toxicity. The dose of feumoxides in this report is much higher than the approved human dose, so additional animal studies are required before this approach could be translated to the clinic. These results might provide useful information for monitoring leukocyte trafficking into the brain.     

Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation

Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP) and stem cell-derived hepatocytes (PICM-19FF). The magnetic particle is a micron-sized iron oxide particle (MPIO) that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.     

Superparamagnetic Iron Oxide Nanoparticles Labeling of Bone Marrow Stromal (Mesenchymal) Cells Does Not Affect Their “Stemness”

Superparamagnetic iron oxide nanoparticles (SPION) are increasingly used to label human bone marrow stromal cells (BMSCs, also called “mesenchymal stem cells”) to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB) staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the “stemness” of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the “gold standard” of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs.     

Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.     

Effect of Labeling with Iron Oxide Particles or Nanodiamonds on the Functionality of Adipose-Derived Mesenchymal Stem Cells

Stem cells are increasingly the focus of translational research as well as having emerging roles in human cellular therapy. To support these uses there is a need for improved methods for in vivo cell localization and tracking. In this study, we examined the effects of cell labeling on the in vitro functionality of human adipose-derived mesenchymal stem cells. Our results provide a basis for future in vivo studies investigating implanted cell fate and longevity. In particular, we investigated the effects of two different particles: micron-sized (∼0.9 µm) fluorescently labeled (Dragon Green) superparamagnetic iron oxide particles (M-SPIO particles); and, carboxylated nanodiamonds of ∼0.25 µm in size. The effects of labeling on the functionality of adipose-derived MSCs were assessed by in vitro morphology, osteogenic and adipogenic differentiation potential, CD marker expression, cytokine secretion profiling and quantitative proteomics of the intra-cellular proteome. The differentiation and CD marker assays for stem-like functionality were not altered upon label incorporation and no secreted or intra-cellular protein changes indicative of stress or toxicity were detected. These in vitro results indicate that the M-SPIO particles and nanodiamonds investigated in this study are biocompatible with MSCs and therefore would be suitable labels for cell localization and tracking in vivo.     

Cellular imaging at 1.5 T: detecting cells in neuroinflammation using active labeling with superparamagnetic iron oxide

The ability to visualize cell infiltration in experimental auto-immune encephalomyelitis (EAE), a well-known animal model for multiple sclerosis in humans, was investigated using a clinical 1.5-T magnetic resonance imaging (MRI) scanner, a custom-built, high-strength gradient coil insert, a 3-D fast imaging employing steady-state acquisition (FIESTA) imaging sequence and a superparamagnetic iron oxide (SPIO) contrast agent. An "active labeling" approach was used with SPIO administered intravenously during inflammation in EAE. Our results show that small, discrete regions of signal void corresponding to iron accumulation in EAE brain can be detected using FIESTA at 1.5 T. This work provides early evidence that cellular abnormalities that are the basis of diseases can be probed using cellular MRI and supports our earlier work which indicates that tracking of iron-labeled cells will be possible using clinical MR scanners.     

Magnetic resonance imaging monitoring dual-labeled stem cells for treatment of mouse nerve injury

Background aimsAdipose-derived stem cells (ADSCs) have shown great promise in the regenerative repair of injured peripheral nerves. Magnetic resonance imaging (MRI) has provided attractive advantages in tracking superparamagnetic iron oxide nanoparticle (SPION)-labeled cells and evaluating their fate after cell transplantation. This study investigated the feasibility of the use of MRI to noninvasively track ADSCs repair of peripheral nerve injury in vivo.MethodsGreen fluorescent protein (GFP)-expressing ADSCs were isolated, expanded, differentiated into an SC-like phenotype (GFP-dADSCs) at early passages and subsequently labeled with SPIONs. The morphological and functional properties of the GFP-dADSCs were assessed through the use of immunohistochemistry. The intracellular stability, proliferation and viability of the labeled cells were evaluated in vitro. Through the use of a microsurgical procedure, the labeled cells were then seeded into sciatic nerve conduits in C57/BL6 mice to repair a 1-cm sciatic nerve gap. A clinical 3-T MRI was performed to investigate the GFP-dADSCs in vitro and the transplanted GFP-dADSCs inside the sciatic nerve conduits in vivo.ResultsThe GFP-dADSCs were efficiently labeled with SPIONs, without affecting their viability and proliferation. The labeled cells implanted into the mice sciatic nerve conduit exhibited a significant increase in axonal regeneration compared with the empty conduit and could be detected by MRI. Fluorescent microscopic examination, histological analysis and immunohistochemistry confirmed the axon regeneration and MRI results.ConclusionsThese data will elucidate the neuroplasticity of ADSCs and provide a new protocol for in vivo tracking of stem cells that are seeded to repair injured peripheral nerves.     

Cell‐assembled nanoclusters of MSC‐targeting Gd‐DOTA‐peptide as a T2 contrast agent for MRI cell tracking

A cyclic peptide CC9 that targets cell membrane of mesenchymal stem cells (MSCs) is coupled with Gd‐DOTA to yield a Gd‐DOTA‐CC9 complex as MRI contrast agent. It is used to label human MSCs (hMSCs) via electroporation. Electroporation‐labeling of hMSCs with Gd‐DOTA‐CC9 induces cell‐assembly of Gd‐DOTA‐CC9 nanoclusters in the cytoplasm, significantly promotes cell‐labeling efficacy and intracellular retention time of the agent. In vitro MRI of labeled hMSCs exhibits significant signal reduction under T₂‐weighted MRI, which can allow long‐term tracking of labeled cell transplants in in vivo migration. The labeling strategy is safe in cytotoxicity and differentiation potential.     

Labeling of mesenchymal stromal cells with iron oxide-poly(L-lactide) nanoparticles for magnetic resonance imaging: uptake, persistence, effects on cellular function and magnetic resonance imaging properties

Background aimsMesenchymal stromal cells (MSC) are the focus of research in regenerative medicine aiming at the regulatory approval of these cells for specific indications. To cope with the regulatory requirements for somatic cell therapy, novel approaches that do not interfere with the natural behavior of the cells are necessary. In this context in vivo magnetic resonance imaging (MRI) of labeled MSC could be an appropriate tool. Cell labeling for MRI with a variety of different iron oxide preparations is frequently published. However, most publications lack a comprehensive assessment of the non-interference of the contrast agent with the functionality of the labeled MSC, which is a prerequisite for the validity of cell-tracking via MRI.MethodsWe studied the effects of iron oxide–poly(l-lactide) nanoparticles in MSC with flow cytometry, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), Prussian blue staining, CyQuant^® proliferation testing, colony-forming unit–fibroblast (CFU-F) assays, flow chamber adhesion testing, immunologic tests and differentiation tests. Furthermore iron-labeled MSC were studied by MRI in agarose phantoms and Wistar rats.ResultsIt could be demonstrated that MSC show rapid uptake of nanoparticles and long-lasting intracellular persistence in the endosomal compartment. Labeling of the MSC with these particles has no influence on viability, differentiation, clonogenicity, proliferation, adhesion, phenotype and immunosuppressive properties. They show excellent MRI properties in agarose phantoms and after subcutaneous implantation in rats over several weeks.ConclusionsThese particles qualify for studying MSC homing and trafficking via MRI.     

Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation

This tutorial describes a method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles. This method may provide basically all kinds of cells with sufficient magnetization to allow cell detection by high-resolution magnetic resonance imaging (MRI) and to enable potential magnetic manipulation. In order to efficiently exploit labeled cells, quantify the magnetic load and deliver or follow-up magnetic cells, we herein describe the main requirements that should be applied during the labeling procedure. Moreover we present some recommendations for cell detection and quantification by MRI and detail magnetic guiding on some real-case studies in vitro and in vivo.     

The application of super paramagnetic iron oxide-labeled mesenchymal stem cells in cell-based therapy

Mesenchymal stem cell (MSC)-based therapy has great potential for tissue regeneration. However, being able to monitor the in vivo behavior of implanted MSCs and understand the fate of these cells is necessary for further development of successful therapies and requires an effective, non-invasive and non-toxic technique for cell tracking. Super paramagnetic iron oxide (SPIO) is an idea label and tracer of MSCs. MRI can be used to follow SPIO-labeled MSCs and has been proposed as a gold standard for monitoring the in vivo biodistribution and migration of implanted SPIO-labeled MSCs. This review discusses the biological effects of SPIO labeling on MSCs and the therapeutic applications of local or systemic delivery of these labeled cells.     

Uptake and metabolism of a dual fluorochrome Tat-nanoparticle in HeLa cells

The ability to use magnetic nanoparticles for cell tracking, or for the delivery of nanoparticle-based therapeutic agents, requires a detailed understanding of probe metabolism and transport. Here we report on the development and metabolism of a dual fluorochrome version of our tat-CLIO nanoparticle termed Tat(FITC)-Cy3.5-CLIO. The nanoparticle features an FITC label on the tat peptide and a Cy3.5 dye directly attached to the cross-linked coating of dextran. This nanoparticle was rapidly internalized by HeLa cells, labeling 100% of cells in 45 min, with the amount of label per cell increasing linearly with time up to 3 h. Cells loaded with nanoparticles for 1 h retained 40-60% of their FITC and Cy3.5 labels over a period of 72 h in label-free media. Over a period of 144 h, or approximately 3.5 cell divisions, the T2 spin-spin relaxation time of cells was not significantly changed, indicating retention of the iron oxide among the dividing cell population. Using confocal microscopy and unfixed cells, both dyes were nuclear and perinuclear (broadly cytoplasmic) after Tat(FITC)-Cy3.5-CLIO labeling. Implications of the rapid labeling and slow excretion of the Tat(FITC)-Cy3.5-CLIO nanoparticle are discussed for cell tracking and drug delivery applications.     

Non-invasive monitoring of the kinetic infiltration and therapeutic efficacy of nanoparticle-labeled chimeric antigen receptor T cells in glioblastoma via 7.0-Tesla magnetic resonance imaging

Background aimsChimeric antigen receptor (CAR) T-cell therapy is a promising treatment strategy in solid tumors. In vivo cell tracking techniques can help us better understand the infiltration, persistence and therapeutic efficacy of CAR T cells. In this field, magnetic resonance imaging (MRI) can achieve high-resolution images of cells by using cellular imaging probes. MRI can also provide various biological information on solid tumors.MethodsThe authors adopted the amino alcohol derivatives of glucose-coated nanoparticles, ultra-small superparamagnetic particles of iron oxide (USPIOs), to label CAR T cells for non-invasive monitoring of kinetic infiltration and persistence in glioblastoma (GBM). The specific targeting CARs included anti-human epidermal growth factor receptor variant III and IL13 receptor subunit alpha 2 CARs.ResultsWhen using an appropriate concentration, USPIO labeling exerted no negative effects on the biological characteristics and killing efficiency of CAR T cells. Increasing hypointensity signals could be detected in GBM models by susceptibility-weighted imaging MRI ranging from 3 days to 14 days following the injection of USPIO-labeled CAR T cells. In addition, nanoparticles and CAR T cells were found on consecutive histopathological sections. Moreover, diffusion and perfusion MRI revealed significantly increased water diffusion and decreased vascular permeability on day 3 after treatment, which was simultaneously accompanied by a significant decrease in tumor cell proliferation and increase in intercellular tight junction on immunostaining sections.ConclusionThese results establish an effective imaging technique that can track CAR T cells in GBM models and validate their early therapeutic effects, which may guide the evaluation of CAR T-cell therapies in solid tumors.     

Migration of iron-labeled KHYG-1 natural killer cells to subcutaneous tumors in nude mice, as detected by magnetic resonance imaging

Background aimsA novel cell line of cytotoxic natural killer (NK) cells, KHYG-1, was examined in vivo for immunotherapy against prostate cancer. The feasibility of using magnetic resonance imaging (MRI) tracking to monitor the fate of injected NK cells following intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) administration was assessed.MethodsPC-3M human prostate cancer cells were injected s.c. into the flank of nude mice (day 0). KHYG-1 NK cells were labeled with an iron oxide contrast agent and injected s.c., i.v. or i.p. on day 8. Mice were imaged by MRI on days 7, 9 and 12. Tumor sections were examined with fluorescence microscopy and immunohistologic staining for NK cells.ResultsNK cells were detected in the tumors by histology after all three administration routes. NK cells and fluorescence from the iron label were co-localized. Signal loss was seen in the areas around the tumors and between the tumor lobes in the s.c. group.ConclusionsWe are the first to label this cell line of NK cells with an iron oxide contrast agent. Accumulation of NK cells was visualized by MRI after s.c. injection but not after i.v. and i.p. injection.     

Magnetic resonance contrast and biological effects of intracellular superparamagnetic iron oxides on human mesenchymal stem cells with long-term culture and hypoxic exposure

Background aimsHuman mesenchymal stem cells (hMSCs) have gained interest for treatment of stroke injury. Using in vitro culture, the purpose of this study was to investigate the long-term detectability of hMSCs by magnetic resonance imaging (MRI) after transfection with a superparamagnetic iron oxide (SPIO) and evaluate the effects of SPIO on cellular activity, particularly under an ischemic environment.MethodshMSCs were exposed to low doses of SPIOs. After a short incubation period, cells were cultured for additional 1, 7 and 14 d to evaluate proliferation, colony formation and multilinear potential. Labeled cells were imaged and evaluated in agarose to quantify R₂ and R₂1 contrast at each time point. Cells were placed in a low-oxygen, low-serum environment and tested for cytotoxicity. In addition, labeled cells were transplanted into an ischemic stroke model and evaluated with ex vivo MRI and histology.ResultsCellular events such as proliferation and differentiation were not affected at any of the exposures tested when cultured for 14 d. The low iron exposure and short incubation time are sufficient for detectability with MRI. However, the higher iron dosage results in higher calcification and cytotoxicity under in vitro ischemic conditions. Transplantation of the hMSCs labeled with an initial exposure of 22.4 μg of Fe showed excellent retention of contrast in stroke-induced rats.ConclusionsAlthough SPIO labeling is stable for long-term MRI detection and has limited effects on the multilineage potential of hMSCs, high-dose SPIO labeling may affect hMSC survival under serum and oxygen withdrawal.