Hossein Nejadnik

Autologous Bone Marrow–Derived Mesenchymal Stem Cells Versus Autologous Chondrocyte Implantation An Observational Cohort Study

Background: First-generation autologous chondrocyte implantation has limitations, and introducing new effective cell sources can improve cartilage repair. Purpose: 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). Study Design: Cohort study; Level of evidence, 3. Methods: 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. Results: 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. Conclusion: 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.

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.

Intravenous ferumoxytol allows noninvasive MR imaging monitoring of macrophage migration into stem cell transplants

PURPOSE 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSION 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.

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.

Enhancing In Vivo Survival of Adipose-Derived Stromal Cells Through Bcl-2 Overexpression Using a Minicircle Vector

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.

Somatic Differentiation and MR Imaging of Magnetically Labeled Human Embryonic Stem Cells

Magnetic resonance (MR) imaging of superparamagnetic iron oxide (SPIO)-labeled stem cells offers a non-invasive 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 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.

Magnetic Resonance Imaging and Tracking of Stem Cells

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.

Next-generation superparamagnetic iron oxide nanoparticles for cancer theranostics

Superparamagnetic iron oxide (SPIO) nanoparticles have been intensively studied for the development of contrast agents in MRI. First-generation SPIO nanoparticles had diagnostic capabilities only, whereas a new generation of SPIO nanoparticle has multifunctional characteristics for combined therapeutic and diagnostic applications. These theranostic nanoparticles hold great potential for image-guided cancer therapies. In particular, polymer-coated theranostic SPIO nanoparticles have enjoyed increasing attention as a result of good biocompatibility, biodegradability and versatile functionality endowed by polymeric matrices. This review provides an overview of recently developed polymer-coated multifunctional SPIO nanoparticles for cancer theranostics and discusses current challenges and future perspectives.