Macrophage phagocytosis alters the MRI signal of ferumoxytol-labeled mesenchymal stromal cells in cartilage defects
Improved Approach for Chondrogenic Differentiation of Human Induced Pluripotent Stem Cells
STEM CELL REVIEWS AND REPORTS
2015; 11 (2): 242-253
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.
View details for DOI 10.1038/srep25897
View details for Web of Science ID 000375769300001
View details for PubMedID 27174199
Magnetic Resonance Imaging of Stem Cell Apoptosis in Arthritic Joints with a Caspase Activatable Contrast Agent
2015; 9 (2): 1150-1160
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.
View details for DOI 10.1007/s12015-014-9581-5
View details for Web of Science ID 000353149700004
MR Imaging of Stem Cell Transplants in Arthritic Joints.
Journal of stem cell research & therapy
2014; 4 (2): 165
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.
View details for DOI 10.1021/nn504494c
View details for Web of Science ID 000349940500013
Ferumoxytol: a new, clinically applicable label for stem cell tracking in arthritic joints with MRI
2013; 8 (12): 1969-1983
About 43 million individuals in the US currently suffer from disabilities due to arthritis. Cartilage defects are the major source of pain in the affected joints. Current treatments, whilst alleviating some of the clinical symptoms, prove insufficient to cure the underlying irreversible cartilage loss. Stem cells represent a unique source for restoration of cartilage defects. Pre-clinical and clinical trials are currently pursued to investigate the potential of various types of stem cells and stem cell derived chondrocytes to repair arthritic joints. A major challenge with all stem cell-mediated tissue regeneration approaches is death of the transplanted cells with clearance by the immune system. Our current inability to diagnose successful or unsuccessful engraftment of transplanted cells non-invasively in vivo represents a major bottleneck for the development of successful stem cell therapies. A large variety of non-invasive Magnetic Resonance (MR) imaging techniques have been developed over the last decade, which enable sensitive in vivo detection of Matrix Associated Stem Cell Implants (MASI) and early diagnosis of related complications. While initially focused on successfully harvesting cellular MR imaging approaches with easily applicable SuperParamagnetic Iron Oxide Nanoparticles (SPIO), our team began to observe details that will facilitate clinical translation. We therefore started a broader effort to define a comprehensive set of novel, clinically applicable imaging approaches for stem cell transplants in patients. We established immediately clinically applicable nanoparticle labeling techniques for tracking stem cell transplants with MR imaging; we have evaluated the long term MR signal effects of iron oxide nanoparticle labeled MASI in vivo; and we have defined distinct signal characteristics of labeled viable and apoptotic MASI. This review article will provide an overview over these efforts and discuss important implications for clinical translation.
View details for DOI 10.4172/2157-7633.1000165
View details for PubMedID 25068075
Iron Administration before Stem Cell Harvest Enables MR Imaging Tracking after Transplantation
2013; 269 (1): 186-197
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. Original submitted 28 February 2012; Revised submitted 8 November 2012.
View details for DOI 10.2217/nnm.12.198
View details for Web of Science ID 000327379600010
View details for PubMedID 23534832
Enhancing In Vivo Survival of Adipose-Derived Stromal Cells Through BcI-2 Overexpression Using a Minicircle Vector
STEM CELLS TRANSLATIONAL MEDICINE
2013; 2 (9): 690-702
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.© RSNA, 2013Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.13130858/-/DC1.
View details for DOI 10.1148/radiol.13130858
View details for Web of Science ID 000325000700021
Magnetic resonance imaging and tracking of stem cells.
Methods in molecular biology (Clifton, N.J.)
2013; 1052: 1-10
Tissue regeneration using progenitor cell-based therapy has the potential to aid in the healing of a diverse range of pathologies, ranging from short-gut syndrome to spinal cord lesions. However, there are numerous hurdles to be overcome prior to the widespread application of these cells in the clinical setting. One of the primary barriers to effective stem cell therapy is the hostile environment that progenitor cells encounter in the clinical injury wound setting. In order to promote cellular survival, stem cell differentiation, and participation in tissue regeneration, relevant cells and delivery scaffolds must be paired with strategies to prevent cell death to ensure that these cells can survive to form de novo tissue. The Bcl-2 protein is a prosurvival member of a family of proteins that regulate the mitochondrial pathway of apoptosis. Using several strategies to overexpress the Bcl-2 protein, we demonstrated a decrease in the mediators of apoptosis in vitro and in vivo. This was shown through the use of two different clinical tissue repair models. Cells overexpressing Bcl-2 not only survived within the wound environment at a statistically significantly higher rate than control cells, but also increased tissue regeneration. Finally, we used a nonintegrating minicircle technology to achieve this in a potentially clinically applicable strategy for stem cell therapy.
View details for DOI 10.5966/sctm.2013-0035
View details for Web of Science ID 000323801400013
MR Imaging Features of Gadofluorine-Labeled Matrix-Associated Stem Cell Implants in Cartilage Defects
2012; 7 (12)
Intravenous Ferumoxytol Allows Noninvasive MR Imaging Monitoring of Macrophage Migration into Stem Cell Transplants
2012; 264 (3): 803-811
To date, several stem cell labeling protocols have been developed, contributing to a fast growing and promising field of stem cell imaging by MRI (magnetic resonance imaging). Most of these methods utilize iron oxide nanoparticles (MION, SPIO, USPIO, VSIOP) for cell labeling, which provide negative (dark) signal effects on T2-weighted MR images. The following protocol describes stem cell labeling techniques with commercially available gadolinium chelates, which provide positive contrast on T1-weighted MR images, which can be advantageous for specific applications.
View details for DOI 10.1007/7651_2013_16
View details for PubMedID 23743862
MR imaging features of gadofluorine-labeled matrix-associated stem cell implants in cartilage defects.
2012; 7 (12)
To develop a clinically applicable imaging technique for monitoring differential migration of macrophages into viable and apoptotic matrix-associated stem cell implants (MASIs) in arthritic knee joints.With institutional animal care and use committee approval, six athymic rats were injected with intravenous ferumoxytol (0.5 mmol iron per kilogram of body weight) to preload macrophages of the reticuloendothelial system with iron oxide nanoparticles. Forty-eight hours later, all animals received MASIs of viable adipose-derived stem cells (ADSCs) in an osteochondral defect of the right femur and mitomycin-pretreated apoptotic ADSCs in an osteochondral defect of the left femur. One additional control animal each received intravenous ferumoxytol and bilateral scaffold-only implants (without cells) or bilateral MASIs without prior ferumoxytol injection. All knees were imaged with a 7.0-T magnetic resonance (MR) imaging unit with T2-weighted fast spin-echo sequences immediately after, as well as 2 and 4 weeks after, matrix-associated stem cell implantation. Signal-to-noise ratios (SNRs) of viable and apoptotic MASIs were compared by using a linear mixed-effects model. MR imaging data were correlated with histopathologic findings.All ADSC implants showed a slowly decreasing T2 signal over 4 weeks after matrix-associated stem cell implantation. SNRs decreased significantly over time for the apoptotic implants (SNRs on the day of matrix-associated stem cell implantation, 2 weeks after the procedure, and 4 weeks after the procedure were 16.9, 10.9, and 6.7, respectively; P = .0004) but not for the viable implants (SNRs on the day of matrix-associated stem cell implantation, 2 weeks after the procedure, and 4 weeks after the procedure were 17.7, 16.2, and 15.7, respectively; P = .2218). At 4 weeks after matrix-associated stem cell implantation, SNRs of apoptotic ADSCs were significantly lower than those of viable ADSCs (mean, 6.7 vs 15.7; P = .0013). This corresponded to differential migration of iron-loaded macrophages into MASIs.Iron oxide loading of macrophages in the reticuloendothelial system by means of intravenous ferumoxytol injection can be utilized to monitor differential migration of bone marrow macrophages into viable and apoptotic MASIs in a rat model.
View details for DOI 10.1148/radiol.12112393
View details for Web of Science ID 000308645500022
View details for PubMedID 22820731
Engineering stem cells for treatment of osteochondral defects
2012; 41 (1): 1-4
Somatic Differentiation and MR Imaging of Magnetically Labeled Human Embryonic Stem Cells
2012; 21 (12): 2555-2567
The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI) in pig knee specimen.Human mesenchymal stem cells (hMSCs) were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR) between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p<0.017, considering a Bonferroni correction for multiple comparisons.Collagen type II gene expression levels were not significantly different between different groups (p>0.017). However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05). GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017).hMSCs can be labeled by simple incubation with GadofluorineM-Cy. The labeled cells provide significant MR signal effects and less impaired chondrogenesis compared to Ferucarbotran-labeled hMSCs. Thus, GadoflurineM-Cy might represent an alternative MR cell marker to Ferucarbotran, which is not distributed any more in Europe or North America.
View details for DOI 10.1371/journal.pone.0049971
View details for PubMedID 23251354
Vitamin D status of 6- to 7-year-old children living in Isfahan, Iran.
2010; 61 (4): 377-382
Magnetic resonance (MR) imaging of superparamagnetic iron oxide (SPIO)-labeled stem cells offers a noninvasive evaluation of stem cell engraftment in host organs. Excessive cellular iron load from SPIO labeling, however, impairs stem cell differentiation. The purpose of this study was to magnetically label human embryonic stem cells (hESCs) via a reduced exposure protocol that maintains a significant MR signal and no significant impairment to cellular pluripotency or differentiation potential. hESCs were labeled by simple incubation with Food and Drug Administration-approved ferumoxides, using concentrations of 50- 200 µg Fe/ml and incubation times of 3-24 h. The most reduced exposure labeling protocol that still provided a significant MR signal comparable to accepted labeling protocols was selected for subsequent studies. Labeled hESCs were compared to unlabeled controls for differences in pluripotency as studied by fluorescence staining for SSEA-1, SSEA-4, TRA-60, and TRA-81 and in differentiation capacity as studied by quantitative real-time PCR for hOCT4, hACTC1, hSOX1, and hAFP after differentiation into embryoid bodies (EBs). Subsequent MR and microscopy imaging were performed to evaluate for cellular iron distribution and long-term persistence of the label. An incubation concentration of 50 µg Fe/ml and incubation time of 3 h demonstrated a significantly reduced exposure protocol that yielded an intracellular iron uptake of 4.50 ± 0.27 pg, an iron content comparable to currently accepted SPIO labeling protocols. Labeled and unlabeled hESCs showed no difference in pluripotency or differentiation capacity. Ferumoxide-labeled hESCs demonstrated persistent MR contrast effects as embryoid bodies for 21 days. Electron microscopy confirmed persistent lysosomal storage of iron oxide particles in EBs up to 9 days, while additional microscopy visualization confirmed the iron distribution within single and multiple EBs. Labeling hESCs with ferumoxides by this tailored protocol reduces exposure of cells to the labeling agent while allowing for long-term visualization with MR imaging and the retention of cellular pluripotency and differentiation potential.
View details for DOI 10.3727/096368912X653156
View details for Web of Science ID 000315001400002
View details for PubMedID 22862886
Autologous Bone Marrow-Derived Mesenchymal Stem Cells Versus Autologous Chondrocyte Implantation An Observational Cohort Study
AMERICAN JOURNAL OF SPORTS MEDICINE
2010; 38 (6): 1110-1116
Vitamin D is essential for the maintenance of good health, and vitamin D deficiency has been reported from many countries, including those with a lot of sunshine. This study was conducted to evaluate the vitamin D status in healthy 6- to 7-year-old children in Isfahan, Iran.Five hundred and thirteen healthy children were enrolled. Serum PTH and 25-hydroxyvitamin D (25-OHD) were measured. Dietary vitamin D intake, duration of daily sunlight exposure, and percentage of exposed body surface area were determined. 25-OHD levels < 20 ng/mL and < 10 ng/mL were defined as mild and severe vitamin D deficiency, respectively. The ROC curve was utilized to obtain a local cut-off point of vitamin D deficiency.25-OHD was < 20 ng/mL in 3% and < 33 ng/mL (local cut-off point of vitamin D deficiency) in 26% of subjects. Duration of sunlight exposure and daily intake of vitamin D had significant effects on serum level of vitamin D.A high prevalence of vitamin D deficiency in Isfahan children was observed in this study. Improvements in duration of sunlight exposure and daily intake of vitamin D can prevent vitamin D deficiency in these children.
View details for PubMedID 20806182
Vitamin D status of 6-to 7-year-old children living in Isfahan, Iran
2010; 61 (4): 377-381
Cavernous angioma: a clinical study of 35 cases with review of the literature
2009; 31 (8): 785-793
First-generation autologous chondrocyte implantation has limitations, and introducing new effective cell sources can improve cartilage repair.This study was conducted to compare the clinical outcomes of patients treated with first-generation autologous chondrocyte implantation to patients treated with autologous bone marrow-derived mesenchymal stem cells (BMSCs).Cohort study; Level of evidence, 3.Seventy-two matched (lesion site and age) patients underwent cartilage repair using chondrocytes (n = 36) or BMSCs (n = 36). Clinical outcomes were measured before operation and 3, 6, 9, 12, 18, and 24 months after operation using the International Cartilage Repair Society (ICRS) Cartilage Injury Evaluation Package, which included questions from the Short-Form Health Survey, International Knee Documentation Committee (IKDC) subjective knee evaluation form, Lysholm knee scale, and Tegner activity level scale.There was significant improvement in the patients' quality of life (physical and mental components of the Short Form-36 questionnaire included in the ICRS package) after cartilage repair in both groups (autologous chondrocyte implantation and BMSCs). However, there was no difference between the BMSC and the autologous chondrocyte implantation group in terms of clinical outcomes except for Physical Role Functioning, with a greater improvement over time in the BMSC group (P = .044 for interaction effect). The IKDC subjective knee evaluation (P = .861), Lysholm (P = .627), and Tegner (P = .200) scores did not show any significant difference between groups over time. However, in general, men showed significantly better improvements than women. Patients younger than 45 years of age scored significantly better than patients older than 45 years in the autologous chondrocyte implantation group, but age did not make a difference in outcomes in the BMSC group.Using BMSCs in cartilage repair is as effective as chondrocytes for articular cartilage repair. In addition, it required 1 less knee surgery, reduced costs, and minimized donor-site morbidity.
View details for DOI 10.1177/0363546509359067
View details for Web of Science ID 000278062200004
View details for PubMedID 20392971
Vitamin D deficiency among pregnant women and their newborns in Isfahan, Iran
EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES
2008; 116 (6): 352-356
Cavernous angioma is a vascular malformation which can be found in any region within the central nervous system.There are few clinical and demographic cavernous angioma studies with large sample sizes. Therefore, the present study was designed to provide further information on the clinical and demographic characteristics of cavernous angioma using a relatively large sample of Persian patients.Patients with cavernous angioma were recruited from the outpatient neurology clinics in Isfahan, Iran, from October 2003 to October 2006.In all cases, the diagnosis of cavernous angioma was based on brain magnetic resonance imaging. There were 35 patients (female/male: 17 : 18) identified with cavernous angioma. The mean age at presentation was 28.8 years. Initial manifestations included seizures in 16, headache in 11 and intracranial hemorrhage in eight patients. During follow-up, all patients experienced seizures and 19 developed headaches. Depression, vertigo, nausea, vomiting, disequilibrium, loss of consciousness and sensorimotor symptoms were also observed.Some of the findings of the present study were in accordance with previous studies. However, more of our patients with positive family history had solitary rather than multiple lesions, and more of our patients had generalized tonic-clonic seizures rather than partial seizures. Moreover, our data demonstrated that if there is a history of cavernous angioma with intracranial hemorrhage in family members, the presenting cavernous angioma patient is more prone to intracranial hemorrhage.
View details for DOI 10.1179/016164109X12445505689445
View details for Web of Science ID 000269131700003
View details for PubMedID 19723446
Effect of lime juice consumption on urine pH value
SAUDI MEDICAL JOURNAL
2006; 27 (12): 1923-1924
Vitamin D deficiency is one of the major health problems and unexpectedly has a high prevalence in sunny countries (e.g. Middle East). In this study we determined the prevalence of vitamin D deficiency in pregnant women and their newborns in Isfahan, a sunny city in Iran.In a cross-sectional study, 88 newborns born in Beheshty hospital, affiliated to Isfahan University of Medical Sciences (August-September, 2005) and their mothers were studied. Their data were collected by questionnaires and blood sampling was done to measure serum alkaline phosphatase (ALP), calcium, phosphorus, 25 (OH) vitamin D and parathormone (PTH). Vitamin D deficiency defined as levels of 25 (OH) D < 20 and < 12.5 ng/ml for mothers and newborns, respectively and local cut-offs defined as levels in which mean serum PTH started to increase.The prevalence of vitamin D deficiency according to 25 (OH) D < 20 ng/ml in mothers and < 12.5 ng/ml in newborns was 5.7% and 4.5%, respectively. According to local cut-offs (35 ng/ml for mothers and 26 ng/ml for newborns) 26.1% of mothers and 53.4% of newborns were vitamin D deficient.According to local definition, vitamin D deficiency is a health problem in pregnant women and their newborns in this sunny city.
View details for DOI 10.1055/s-2008-1042403
View details for Web of Science ID 000257245900008
View details for PubMedID 18700279