Olga D. Lenkov

Ferumoxytol: a new, clinically applicable label for stem-cell tracking in arthritic joints with MRI

AIM To develop a clinically applicable MRI technique for tracking stem cells in matrix-associated stem-cell implants, using the US FDA-approved iron supplement ferumoxytol. MATERIALS & METHODS Ferumoxytol-labeling of adipose-derived stem cells (ADSCs) was optimized in vitro. A total of 11 rats with osteochondral defects of both femurs were implanted with ferumoxytol- or ferumoxides-labeled or unlabeled ADSCs, and underwent MRI up to 4 weeks post matrix-associated stem-cell implant. The signal-to-noise ratio of different matrix-associated stem-cell implant was compared with t-tests and correlated with histopathology. RESULTS An incubation concentration of 500 µg iron/ml ferumoxytol and 10 µg/ml protamine sulfate led to significant cellular iron uptake, T2 signal effects and unimpaired ADSC viability. In vivo, ferumoxytol- and ferumoxides-labeled ADSCs demonstrated significantly lower signal-to-noise ratio values compared with unlabeled controls (p < 0.01). Histopathology confirmed engraftment of labeled ADSCs, with slow dilution of the iron label over time. CONCLUSION Ferumoxytol can be used for in vivo tracking of stem cells with MRI.

Iron Administration before Stem Cell Harvest Enables MR Imaging Tracking after Transplantation

PURPOSE To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. MATERIALS AND METHODS This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. RESULTS In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. CONCLUSION Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.

Improved Approach for Chondrogenic Differentiation of Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However, current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation, which is required to generate endodermal, ectodermal, and mesodermal cell lineages. We report a new, straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs, which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1, GAG, and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats, magnetic resonance imaging studies showed gradual engraftment, and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs, which could improve cartilage regeneration outcomes in arthritic joints.

Magnetic resonance imaging of stem cell apoptosis in arthritic joints with a caspase activatable contrast agent.

About 43 million individuals in the U.S. encounter cartilage injuries due to trauma or osteoarthritis, leading to joint pain and functional disability. Matrix-associated stem cell implants (MASI) represent a promising approach for repair of cartilage defects. However, limited survival of MASI creates a significant bottleneck for successful cartilage regeneration outcomes and functional reconstitution. We report an approach for noninvasive detection of stem cell apoptosis with magnetic resonance imaging (MRI), based on a caspase-3-sensitive nanoaggregation MRI probe (C-SNAM). C-SNAM self-assembles into nanoparticles after hydrolysis by caspase-3, leading to 90% amplification of (1)H MR signal and prolonged in vivo retention. Following intra-articular injection, C-SNAM causes significant MR signal enhancement in apoptotic MASI compared to viable MASI. Our results indicate that C-SNAM functions as an imaging probe for stem cell apoptosis in MASI. This concept could be applied to a broad range of cell transplants and target sites.

Epigenetic DNA Methylation Linked to Social Dominance.

Social status hierarchies are ubiquitous in vertebrate social systems, including humans. It is well known that social rank can influence quality of life dramatically among members of social groups. For example, high-ranking individuals have greater access to resources, including food and mating prerogatives that, in turn, have a positive impact on their reproductive success and health. In contrast low ranking individuals typically have limited reproductive success and may experience lasting social and physiological costs. Ultimately, social rank and behavior are regulated by changes in gene expression. However, little is known about mechanisms that transduce social cues into transcriptional changes. Since social behavior is a dynamic process, we hypothesized that a molecular mechanism such as DNA methylation might play a role these changes. To test this hypothesis, we used an African cichlid fish, Astatotilapia burtoni, in which social rank dictates reproductive access. We show that manipulating global DNA methylation state strongly biases the outcomes of social encounters. Injecting DNA methylating and de-methylating agents in low status animals competing for status, we found that animals with chemically increased methylation states were statistically highly likely to ascend in rank. In contrast, those with inhibited methylation processes and thus lower methylation levels were statistically highly unlikely to ascend in rank. This suggests that among its many roles, DNA methylation may be linked to social status and more generally to social behavior.

Macrophage phagocytosis alters the MRI signal of ferumoxytol-labeled mesenchymal stromal cells in cartilage defects.

Human mesenchymal stem cells (hMSCs) are a promising tool for cartilage regeneration in arthritic joints. hMSC labeling with iron oxide nanoparticles enables non-invasive in vivo monitoring of transplanted cells in cartilage defects with MR imaging. Since graft failure leads to macrophage phagocytosis of apoptotic cells, we evaluated in vitro and in vivo whether nanoparticle-labeled hMSCs show distinct MR signal characteristics before and after phagocytosis by macrophages. We found that apoptotic nanoparticle-labeled hMSCs were phagocytosed by macrophages while viable nanoparticle-labeled hMSCs were not. Serial MRI scans of hMSC transplants in arthritic joints of recipient rats showed that the iron signal of apoptotic, nanoparticle-labeled hMSCs engulfed by macrophages disappeared faster compared to viable hMSCs. This corresponded to poor cartilage repair outcomes of the apoptotic hMSC transplants. Therefore, rapid decline of iron MRI signal at the transplant site can indicate cell death and predict incomplete defect repair weeks later. Currently, hMSC graft failure can be only diagnosed by lack of cartilage defect repair several months after cell transplantation. The described imaging signs can diagnose hMSC transplant failure more readily, which could enable timely re-interventions and avoid unnecessary follow up studies of lost transplants.

Detection of Stem Cell Transplant Rejection with Ferumoxytol MR Imaging: Correlation of MR Imaging Findings with Those at Intravital Microscopy.

Purpose To determine whether endogenous labeling of macrophages with clinically applicable nanoparticles enables noninvasive detection of innate immune responses to stem cell transplants with magnetic resonance (MR) imaging. Materials and Methods Work with human stem cells was approved by the institutional review board and the stem cell research oversight committee, and animal experiments were approved by the administrative panel on laboratory animal care. Nine immunocompetent Sprague-Dawley rats received intravenous injection of ferumoxytol, and 18 Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice received rhodamine-conjugated ferumoxytol. Then, 48 hours later, immune-matched or mismatched stem cells were implanted into osteochondral defects of the knee joints of experimental rats and calvarial defects of Jax mice. All animals underwent serial MR imaging and intravital microscopy (IVM) up to 4 weeks after surgery. Macrophages of Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice express enhanced green fluorescent protein (GFP), which enables in vivo correlation of ferumoxytol enhancement at MR imaging with macrophage quantities at IVM. All quantitative data were compared between experimental groups by using a mixed linear model and t tests. Results Immune-mismatched stem cell implants demonstrated stronger ferumoxytol enhancement than did matched stem cell implants. At 4 weeks, T2 values of mismatched implants were significantly lower than those of matched implants in osteochondral defects of female rats (mean, 10.72 msec for human stem cells and 11.55 msec for male rat stem cells vs 15.45 msec for sex-matched rat stem cells; P = .02 and P = .04, respectively) and calvarial defects of recipient mice (mean, 21.7 msec vs 27.1 msec, respectively; P = .0444). This corresponded to increased recruitment of enhanced GFP- and rhodamine-ferumoxytol-positive macrophages into stem cell transplants, as visualized with IVM and histopathologic examination. Conclusion Endogenous labeling of macrophages with ferumoxytol enables noninvasive detection of innate immune responses to stem cell transplants with MR imaging.

Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection

Purpose: Stem cell transplants are an effective approach to repair large bone defects. However, comprehensive techniques to monitor the fate of transplanted stem cells in vivo are lacking. Such strategies would enable corrective interventions at an early stage and greatly benefit the development of more successful tissue regeneration approaches. In this study, we designed and synthesized a dual-modality imaging probe (Feru-AFC) that can simultaneously localize transplanted stem cells and diagnose immune rejection-induced apoptosis at an early stage in vivo. Methods: We used a customized caspase-3 cleavable peptide-dye conjugate to modify the surface of clinically approved ferumoxytol nanoparticles (NPs) to generate the dual-modality imaging probe with fluorescence “light-up” feature. We labeled both mouse mesenchymal stem cells (mMSCs, matched) and pig mesenchymal stem cells (pMSCs, mismatched) with the probe and transplanted the labeled cells with biocompatible scaffold at the calvarial defects in mice. We then employed intravital microscopy (IVM) and magnetic resonance imaging (MRI) to investigate the localization, engraftment, and viability of matched and mismatched stem cells, followed by histological analyses to evaluate the results obtained from in vivo studies. Results: The Feru-AFC NPs showed good cellular uptake efficiency in the presence of lipofectin without cytotoxicity to mMSCs and pMSCs. The fluorescence of Feru-AFC NPs was turned on inside apoptotic cells due to the cleavage of peptide by activated caspase-3 and subsequent release of fluorescence dye molecules. Upon transplantation at the calvarial defects in mice, the intense fluorescence from the cleaved Feru-AFC NPs in apoptotic pMSCs was observed with a concomitant decrease in the overall cell number from days 1 to 6. In contrast, the Feru-AFC NP-treated mMSCs exhibited minimum fluorescence and the cell number also remained similar. Furthermore, in vivo MRI of the Feru-AFC NP-treated mMSC and pMSCs transplants could clearly indicate the localization of matched and mismatched cells, respectively. Conclusions: We successfully developed a dual-modality imaging probe for evaluation of the localization and viability of transplanted stem cells in mouse calvarial defects. Using ferumoxytol NPs as the platform, our Feru-AFC NPs are superparamagnetic and display a fluorescence “light-up” signature upon exposure to activated caspase-3. The results show that the probe is a promising tool for long-term stem cell tracking through MRI and early diagnosis of immune rejection-induced apoptosis through longitudinal fluorescence imaging.

Ferumoxytol Can Be Used for Quantitative Magnetic Particle Imaging of Transplanted Stem Cells

PurposeTo evaluate, if clinically translatable ferumoxytol nanoparticles can be used for in vivo detection and quantification of stem cell transplants with magnetic particle imaging (MPI).ProceduresMesenchymal stem cells (MSCs) were labeled with ferumoxytol or ferucarbotran and underwent MPI, magnetic resonance imaging (MRI), Prussian blue staining, and inductively coupled plasma (ICP) spectrometry. Unlabeled, ferumoxytol, and ferucarbotran-labeled MSCs were implanted in calvarial defects of eight mice and underwent MPI, MRI, and histopathology. The iron concentration calculated according to the MPI signal intensity and T2 relaxation times of the three different groups were compared using an analysis of variance (ANOVA) with Bonferroni correction, and a p < 0.05.ResultsCompared to unlabeled controls, ferumoxytol- and ferucarbotran-labeled MSC showed significantly increased iron content, MPI signal and MRI signal. The ferumoxytol MPI signal was approximately 4× weaker compared to ferucarbotran at equimolar concentrations (p = 0.0003) and approximately 1.5× weaker for labeled cells when using optimized labeling protocols (p = 0.002). In vivo, the MPI signal of ferumoxytol-labeled MSC decreased significantly between day 1 and day 14 (p = 0.0124). This was confirmed by histopathology where we observed a decrease in Prussian blue stain of MSCs at the transplant site. The MRI signal of the same transplants did not change significantly during this observation period (p = 0.93).ConclusionFerumoxytol nanoparticles can be used for in vivo detection of stem cell transplants with MPI and provide quantitative information not attainable with MRI.