Labeling Of Mesenchymal Stem Cells Research Articles

MegaPro, a clinically translatable nanoparticle for in vivo tracking of stem cell implants in pig cartilage defects.

Rationale: Efficient labeling methods for mesenchymal stem cells (MSCs) are crucial for tracking and understanding their behavior in regenerative medicine applications, particularly in cartilage defects. MegaPro nanoparticles have emerged as a potential alternative to ferumoxytol nanoparticles for this purpose. Methods: In this study, we employed mechanoporation to develop an efficient labeling method for MSCs using MegaPro nanoparticles and compared their effectiveness with ferumoxytol nanoparticles in tracking MSCs and chondrogenic pellets. Pig MSCs were labeled with both nanoparticles using a custom-made microfluidic device, and their characteristics were analyzed using various imaging and spectroscopy techniques. The viability and differentiation capacity of labeled MSCs were also assessed. Labeled MSCs and chondrogenic pellets were implanted into pig knee joints and monitored using MRI and histological analysis. Results: MegaPro-labeled MSCs demonstrated shorter T2 relaxation times, higher iron content, and greater nanoparticle uptake compared to ferumoxytol-labeled MSCs, without significantly affecting their viability and differentiation capacity. Post-implantation, MegaPro-labeled MSCs and chondrogenic pellets displayed a strong hypointense signal on MRI with considerably shorter T2* relaxation times compared to adjacent cartilage. The hypointense signal of both MegaPro- and ferumoxytol-labeled chondrogenic pellets decreased over time. Histological evaluations showed regenerated defect areas and proteoglycan formation with no significant differences between the labeled groups. Conclusion: Our study demonstrates that mechanoporation with MegaPro nanoparticles enables efficient MSC labeling without affecting viability or differentiation. MegaPro-labeled cells show enhanced MRI tracking compared to ferumoxytol-labeled cells, emphasizing their potential in clinical stem cell therapies for cartilage defects.

Extracellular magnetic labeling of biomimetic hydrogel-induced human mesenchymal stem cell spheroids with ferumoxytol for MRI tracking.

Labeling of mesenchymal stem cells (MSCs) with superparamagnetic iron oxide nanoparticles (SPIONs) has emerged as a potential method for magnetic resonance imaging (MRI) tracking of transplanted cells in tissue repair studies and clinical trials. Labeling of MSCs using clinically approved SPIONs (ferumoxytol) requires the use of transfection reagents or magnetic field, which largely limits their clinical application. To overcome this obstacle, we established a novel and highly effective method for magnetic labeling of MSC spheroids using ferumoxytol. Unlike conventional methods, ferumoxytol labeling was done in the formation of a mechanically tunable biomimetic hydrogel-induced MSC spheroids. Moreover, the labeled MSC spheroids exhibited strong MRI T2 signals and good biosafety. Strikingly, the encapsulated ferumoxytol was localized in the extracellular matrix (ECM) of the spheroids instead of the cytoplasm, minimizing the cytotoxicity of ferumoxytol and maintaining the viability and stemness properties of biomimetic hydrogel-induced MSC spheroids. This demonstrates the potential of this method for post-transplantation MRI tracking in the clinic.

Precursor structure-determined fluorescence labeling for mesenchymal stem cells among four polyethylenimine-based carbon quantum dots

A series of polyethylenimine (PEI)-based CQDs have been synthesized via a hydrothermal method by mixing linear PEI with linear citric acid (CA with COOH groups, PEICA), linear glucose (G with OH groups, PEIG), cyclic hyaluronic acid (HA with COOH groups, PEIHA) and cyclic boron nitride (BN with OH groups, PEIBN). PEICA had the best labeling effect (100.00 ± 0.26%) and the lowest cytotoxicity (100.89 ± 18.00%) for mesenchymal stem cells (MSCs), followed by PEIG (91.83 ± 7.60%; 92.84 ± 5.56%), PEIHA (84.34 ± 7.87%; 61.27 ± 11.34%) and PEIBN (1.33 ± 0.84%; 22.72 ± 11.47%). The labeling effect of PEIHA for MSCs is lower than that of PEIG because the surface potential of PEIHA (6.58 mV) is higher than that of PEIG (0.50 mV). For PEIBN, it is likely that the precursor (BN) is less biocompatible than CA, HA and glucose. Thus, the linear acid (CA) is more appropriate to react with PEI for synthesizing CQDs with high labeling performance for MSCs. The control experimental results show that factors (such as surface potential, aromatic component, etc.) may all contribute to MSC labeling by PEICA. This work is helpful to design CQDs with high MSC labeling efficiency.

Evaluating the biocompatibility and biological performance of FePt-decorated nano-epoxy nanoparticles in mesenchymal stem cells.

Nanoparticles have wide potential applications in biolabeling, bioimaging, and cell tracking. Development of dual functional nanoparticles increases the versatility. We combined the fluorescent property of nano-epoxy (N-Epo) and the magnetic characteristic of FePt to fabricate the FePt-decorated N-Epo (N-Epo-FePt). The size in diameter of N-Epo-FePt (177.38 ± 39.25 nm) was bigger than N-Epo (2.28 ± 1.01 nm), both could be absorbed into mesenchymal stem cells (MSCs) via clathrin-mediated endocytosis and have multiple fluorescent properties (blue, green, and red). N-Epo-FePt prevented N-Epo-induced platelet activation, CD68+-macrophage differentiation in blood, and intracellular ROS generation in MSCs. The induction of apoptosis and the inhibitory effects of N-Epo-FePt on cell migration, MMP-9 activity, and secretion of SDF-1α were less than that of N-Epo in MSCs. N-Epo-FePt was more biocompatible without altering biological performance than N-Epo in MSCs. These results suggest that N-Epo-FePt nanoparticle can be used for fluorescence labeling of MSCs and is potential to apply to bioimaging and cell tracking of MSCs in vivo by magnetic resonance imaging or computed tomography.

Toxicity and Functional Impairment in Human Adipose Tissue-Derived Stromal Cells (hASCs) Following Long-Term Exposure to Very Small Iron Oxide Particles (VSOPs).

Magnetic nanoparticles (NPs), such as very small iron oxide NPs (VSOPs) can be used for targeted drug delivery, cancer treatment or tissue engineering. Another important field of application is the labelling of mesenchymal stem cells to allow in vivo tracking and visualization of transplanted cells using magnetic resonance imaging (MRI). For these NPs, however, various toxic effects, as well as functional impairment of the exposed cells, are described. The present study evaluates the influence of VSOPs on the multilineage differentiation ability and cytokine secretion of human adipose tissue derived stromal cells (hASCs) after long-term exposure. Human ASCs were labelled with VSOPs, and the efficacy of the labelling was documented over 4 weeks in vitro cultivation of the labelled cells. Unlabelled hASCs served as negative controls. Four weeks after labelling, adipogenic and osteogenic differentiation was histologically evaluated and quantified by polymerase chain reaction (PCR). Changes in gene expression of IL-6, IL-8, VEGF and caspase 3 were determined over 4 weeks. Four weeks after the labelling procedure, labelled and unlabelled hASCs did not differ in the gene expression of IL-6, IL-8, VEGF and caspase 3. Furthermore, the labelling procedure had no influence on the multidifferentiation ability of hASC. The percentage of labelled cells decreased during in vitro expansion over 4 weeks. Labelling with VSOPs and long-term intracellular disposition probably have no influence on the physiological functions of hASCs. This could be important for the future in vivo use of iron oxide NPs.

Investigation of nanostar-labeled mesenchymal stem cells for in vivo cell tracking in osteoarthritis using optoacoustic imaging

Purpose: This project will investigate the potential of gold nanostar (NS)-labeled mesenchymal stem cells (MSCs) in the treatment of osteoarthritis (OA) using optoacoustic imaging (OAI). OAI, specifically multi-spectral optoacoustic tomography (MSOT) has excellent resolution at increasing depths and capabilities in functional imaging. A novel gold nanoparticle - the nanostar has been developed to enhance the signal response in OAI as well as to permit tracking of MSCs. Different nanostar surface coatings have been investigated for their uptake and effects on MSC characteristics: methoxy (NS-OMe) and carboxyl (NS-COOH). SPIONs (superparamagnetic iron oxide nanoparticles) were used to functionalize the NS (NS-SPIONs) and permit dual OAI and magnetic resonance imaging (MRI). Available carboxyl groups also allow for the conjugation of an antibody to NS, which will permit the tracking of MSC-derived extracellular vesicles (EVs). The aims of this project include optimization of MSC labeling and investigation of MSC phenotype post-labeling. Methods: Methods to assess uptake of NS included transmission electron microscopy (TEM), Image Stream X analysis (ISX) and MSOT. To assess MSC characteristics following NS labeling, viability, tri-lineage differentiation and surface marker expression were also assessed. In vivo imaging of NS in the knee joint was performed using MSOT. Results: NS-OMe have shown efficient cellular internalization and no discernable effects on MSC characteristics. The efficiency of cellular uptake of NS-COOH was shown to be sub-optimal. ISX has the capacity to both image and quantify NS conjugated to an antibody inside cells and confirmed these initial findings demonstrating that only 20% of MSCs contained NS-COOH conjugated to an antibody. MSC uptake of NS-SPIONs has also been investigated using MSOT and efficient uptake has been demonstrated. NS-SPIONs will be further studied to determine the optimal concentration for MSOT imaging. Finally, preliminary work has explored the capacity of MSOT to detect an intra-articular injection of NS. An enhanced signal in the knee joint (b) in comparison to baseline (no injection) (a) due to the presence of NS can be seen as indicated by the white arrows in the image below. Conclusions: Different nanostar surface coatings demonstrate variability in MSC uptake and internalization. NS-OMe demonstrated the most efficient uptake by cells while NS-COOH was least efficient. NS-SPIONs have also demonstrated uptake and internalization by MSCs but further work is required to elucidate the optimal concentration for labeling. This work will coincide with in vivo imaging of nanoparticles in the knee joint to determine the limit of detection of the MSOT and the minimum concentration of nanoparticles in cells required for detection with MSOT.

Gold Nanoparticles as a Computed Tomography Marker for Stem Cell Tracking.

Stem cell tracking is an essential prerequisite for effective stem cell therapy. Computed tomography (CT) imaging technique is an emerging quantitative tool to detect real time distribution of transplanted cells. Most of CT labels based on the high atomic number (Z) materials have concern over biocompatibility. The present book chapter describes a protocol for the use of biocompatible gold nanoparticles as a CT marker for efficient labeling of mesenchymal stem cells (MSCs) and subsequent cell tracking in rodent models.

Enhanced Homing of Mesenchymal Stem Cells Overexpressing Fibroblast Growth Factor 21 to Injury Site in a Mouse Model of Traumatic Brain Injury.

Mesenchymal stem cells (MSCs) are emerging as a potential therapeutic intervention for brain injury due to their neuroprotective effects and safe profile. However, the homing ability of MSCs to injury sites still needs to be improved. Fibroblast Growth Factor 21 (FGF21) was recently reported to enhance cells migration in different cells type. In this study, we investigated whether MSCs that overexpressing FGF21 (MSC-FGF21) could exhibit enhanced homing efficacy in brain injury. We used novel Molday IONEverGreen™ (MIEG) as cell labeling probe that enables a non-invasive, high-sensitive and real-time MRI tracking. Using a mouse model of traumatic brain injury (TBI), MIEG labeled MSCs were transplanted into the contralateral lateral ventricle followed by real-time MRI tracking. FGF21 retained MSC abilities of proliferation and morphology. MSC-FGF21 showed significantly greater migration in transwell assay compared to control MSC. MIEG labeling showed no effects on MSCs’ viability, proliferation and differentiation. Magnetic resonance imaging (MRI) revealed that FGF21 significantly enhances the homing of MSC toward injury site. Histological analysis further confirmed the MRI findings. Taken together, these results show that FGF21 overexpression and MIEG labeling of MSC enhances their homing abilities and enables non-invasive real time tracking of the transplanted cells, provides a promising approach for MSC based therapy and tracking in TBI.

Sonoporation-based labeling of mesenchymal stem cells with polymeric MRI contrast agents for live-cell tracking

Cell transplantation has the potential to improve repair of injured tissue function; however, it is not clear how transplanted cells participate in functional recovery. We have recently succeeded in tracking millions of transplanted living cells using a polymeric MRI contrast agent, which was delivered into monolayer-cultured cells through electroporation. However, when cells were labeled using a conventional electroporation method, only cells localized around the electrode were labeled. To improve the percentage of labeled cells and to be able to start with fewer cells, we focused on a homogeneous cell labeling system. In this study, we optimized the sonoporation of a suspension culture with microbubbles for labeling and MR tracking of mesenchymal stem cells (MSCs). When water was used as the transmission medium between the acoustic probe and cell suspension, microbubbles gently collapsed with minimal cell damage. Under this condition, the number of labeled MSCs was 25%, which is 3.3-fold greater than the number of MSCs labeled using the previous system, and the cell viability was maintained at approximately 80%. The MRI signal could be clearly observed for 2.0 × 106 MSCs. These results suggest that sonoporation can efficiently introduce the polymeric contrast agent into MSCs. We optimized the sonoporation of a suspension culture with microbubbles for labeling and MR tracking of mesenchymal stem cells (MSCs). When water was used as the transmission medium between the acoustic probe and cell suspension, microbubbles gently collapsed with minimal cell damage. Under this condition, the number of labeled MSCs was 25%, which is 3.3-fold greater than the number of MSCs labeled using the conventional electroporation system, and the cell viability was maintained at approximately 80%. The MRI signal could be clearly observed for 2.0 × 106 MSCs.

Noninvasive Tracking and Regenerative Capabilities of Transplanted Human Umbilical Cord-Derived Mesenchymal Stem Cells Labeled with I-III-IV Semiconducting Nanocrystals in Liver-Injured Living Mice.

Acute liver injury is a critical syndrome ascribed to prevalent death of hepatocytes and imperatively requires liver transplantation. Such a methodology is certainly hampered due to the deficit of healthy donors. In this regard, stem cell-based regenerative therapies are attractive in repairing injured tissues and organs for medical applications. However, it is crucial to understand the migration, engraftment, and regeneration capabilities of transplanted stem cells in the living animal models. For the first time, we demonstrate rapid labeling of umbilical cord-derived mesenchymal stem cells (MSCs) with near-infrared (NIR)-fluorescent CuInS2-ZnS nanocrystals (CIZS-NCs) to develop innovative nanobioconjugates (MSCs-CIZS-NBCs) that exhibit 98% labeling efficiency. Before nanobioconjugate synthesis, the pristine CIZS-NCs were prepared via a two-step, hot-injection, rapid and low-cost domestic-microwave-refluxing (MW-R) method within 6 min. The as-synthesized CIZS-NCs display high photoluminescence quantum yield (∼88%) and long-lived lifetime (23.4 μs). In contrast to unlabeled MSCs, the MSCs-CIZS nanobioconjugates show excellent biocompatibility without affecting the stemness, as confirmed by cell viability, immunophenotyping (CD44+, CD105+, CD90+), multi-lineage-specific gene expressions, and differentiation into adipocytes, osteocytes, and chondrocytes. The in vivo fluorescence tracking analyses revealed that the MSCs-CIZS-NBCs after tail-vein injection were initially trapped in the lungs and gradually engrafted in the injured liver within 2 h. The regeneration potential of MSCs-CIZS-NBCs was confirmed via renewal of the portal tract composed of portal veins, bile ducts, and hepatic arteries around the hepatocytes. Consequently, no apparent inflammations, necrosis, or apoptosis was observed in the acetaminophen (APAP)-induced liver-injured BALB/c mice model over 3 days after transplantation, as corroborated using laser-scanning confocal microscopy and histopathological and hematological analyses. Hence, our innovative NIR-fluorescent MSCs-CIZS-NBCs offer an off-the-self technology for noninvasive tracking of transplanted MSCs in an acute-liver-injured animal model for future image-guided cell-therapies.

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.

Efficient Labeling of Human Mesenchymal Stem Cells Using Iron Oxide Nanoparticles.

Stem cells have been used in multiple clinical trials. Tracking these transplanted cells in vivo will provide real-time information on the fate of these cells. Iron oxide labeling is one such uncomplicated noninvasive labeling method. These transformed nanocrystals can be used for varied applications including stem-cell tracking, magnetic resonance imaging, and theranostics. Here we elucidate the protocol for iron oxide nanoparticles synthesis (IONPS) and labeling of mesenchymal stem cells which can be used for imaging and tracking cells to understand their fate in in vivo studies.

A Distinctive MRI-Based Absolute Bias Correction Protocol for the Potential Labelling and In Vivo Tracking of Stem Cells in a TBI Mice Model.

Traumatic brain injury (TBI) is a leading cause of death and disability. The condition is difficult to treat owing to its heterogeneous nature and complex biological pathways. Stem cell transplantation is an emerging self-deliverable therapeutic modality which could immensely improve the invigorating management of the problem. The synergistic interaction of the stem cells with the paracrine niche molecules at the site of injury is an end point that decides the cells' effective tissue-forming regenerative response. Thus, noninvasive monitoring and tracking of the infused stem cells is quite decisive after transplantation. Here, we have designed and validated a distinctive in vivo magnetic resonance imaging protocol to monitor the transplanted mesenchymal stem cells (MSCs) longitudinally in TBI-induced mice. We have further described the synthesis of improved transverse relaxivity contrast agent, a protocol for the efficient labelling of MSCs, preparation of a TBI model system in mice, and the imaging and tracking of the implanted stem cells at the injury site through 7T MRI. MGE-T2∗ imaging in association with relaxometry-based quantitative assessment using absolute bias correction provided a suitable mechanism to monitor and track the infused labelled stem cells at the TBI site. High transverse relaxivity negative contrast agent synthesis, MSC labelling procedure, and quantitative T2∗ time measurement normalized with absolute bias correction are the key features of this protocol. This procedure has immense application potential and could therefore be extrapolated to stem cell tracking during the treatment of various diseases.

Ru nanoparticles coated with γ-Fe2O3 promoting and monitoring the differentiation of human mesenchymal stem cells via MRI tracking.

Human mesenchymal stem cells (MSCs) can be successively passaged and can differentiate into multiple lineages. These attributes are important in tissue engineering, which has a great deal of attention in stem cell therapy. However, the effective labelling and tracking of MSCs in vivo remain major unresolved issues. Based on the use of iron oxides to label stem cells for magnetic resonance imaging (MRI), we synthesized nanoparticle (NPs) containing ruthenium (RuNPs), γ-Fe2O3@Ru (Fe2O3@Ru), and γ-Fe2O3@selenium (Fe2O3@Se) to label MSCs and promote osteogenic differentiation. Fe2O3@Ru and Fe2O3@Se could be used as T2-weighted MRI contrast agents. Fe2O3@Ru more effectively diffused in the cytoplasm and localized in the nuclei of MSCs, compared with Fe2O3@Se. RuNPs, Fe2O3@Ru, and Fe2O3@Se induced MSCs to differentiate into osteoblasts. Fe2O3@Ru, in particular, was a potent osteoinductive agent. Fe2O3@Ru also inhibited adipocytic differentiation. Promotion of the osteogenic differentiation of MSCs may be regulated by a Smad-dependent bone morphogenetic protein signaling pathway with reduced expression of CD44, CD73, and CD105. MSCs treated with Fe2O3@Ru NPs expressing osteoblast surface markers.

Fluorescence Endomicroscopy Imaging of Mesenchymal Stem Cells in the Rat Lung.

Stem cell therapy has shown great promise for organ repair and regeneration. In the context of lung disease, such as radiation-induced lung damage (RILD) in cancer radiotherapy, mesenchymal stem cells (MSCs) have shown the ability to reduce damage possibly due to their immunomodulatory properties and other unknown mechanisms. However, once MSCs are transplanted into the body, little is known as to their localization or their mechanisms of action. In this work, we proposed, implemented, and validated a fluorescence endomicroscopy (FE) imaging technique that allows for the real-time detection and quantification of transplanted pre-labeled MSCs in vivo and tracking in a rat model. This protocol covers aspects related to MSCs extraction, labeling, FE imaging, and image analysis developed in a RILD rat model but applicable to other biological systems. © 2018 by John Wiley & Sons, Inc.

Effects of iron oxide contrast agent in combination with various transfection agents during mesenchymal stem cells labelling: An in vitro toxicological evaluation

The use of iron oxide nanoparticles for different biomedical applications, hold immense promise to develop negative tissue contrast in magnetic resonance imaging (MRI). Previously, we have optimized the labelling of mesenchymal stem cells (MSCs) with iron oxide nanoparticles complexed to different transfection agents like poly-l-lysine (IO-PLL) and protamine sulfate (Fe-Pro) on the basis of relaxation behaviour and its biological expressions. However, there is a distinct need to investigate the biocompatibility and biosafety concerns coupled with its cytotoxicity and genotoxicity. This study was prepared to evaluate the viability of cells, generation of ROS, changes in actin cytoskeleton, investigation of cell death, level of GSH and TAC, activities of SOD and GPx, and stability of DNA in MSCs after labelling. Results demonstrated a marginal alteration in toxicological parameters like ROS generation, cell length, actin cytoskeleton, total apoptosis and DNA damage was detected after stem cell labelling. Insignificant depletion of GSH and SOD level, and increase in GPx and TAC level in MSCs were measured after labelling with IO-PLL and Fe-Pro complexes, which later on recovered and normalized to its baseline. This MSCs labelling could provide a reference guideline for toxicological analysis and relaxometry based in vivo MRI detection.

Effect of Moldey Ion Rhodamine B on viability of Adipose and Bone marrow Derived Mesenchymal Stem cells - an In-vitro Study

The study was performed to access the effect of Moldey ion Rhodamine B on in-vitro labelled Bone marrow and adipose derived derived stem cells in sheep. Perirenal adipose tissue and bone marrow from femur were collected from 28 male sheep. Adipose derived mesenchymal stem cells were isolated from perirenal fat using enzymatic digestion method by using type II collagenase. Bone marrow derived mesenchymal stem cells were separated from femur by density gradient centrifugation method using percoll. These mesenchymal stem cells from bone marrow and adipose tissue were cultured and expanded using DulbeccoÂ’s Modified EagleÂ’s Medium with 10 per-cent of fetal bovine serum, one per-cent of antibiotic-antimycotic solution. Adipose and bone marrow derived mesenchymal stem cells from passage 4 to passage 6 were used for labelling with medium containing 25 µg of MIRB per ml. Cell viability was evaluated by Trypan blue exclusion test in both labelled and control (unlabelled) groups. There was no significant difference in the viability of bone marrow and adipose derived mesenchymal stem cells between the labelled and control groups. It was observed that in-vitro labelling of mesenchymal stem cells with MIRB at a concentration of 25 µg per ml of medium did not affect cell proliferation and viability.

A unique 19F MRI agent for the tracking of non phagocytic cells in vivo

There is currently intense interest in new methods for understanding the fate of therapeutically-relevant cells, such as mesenchymal stem cells (MSCs). The absence of a confounding background signal and consequent unequivocal assignment makes 19F MRI one of the most attractive modalities for the tracking of injected cells in vivo. We describe here the synthesis of novel partly-fluorinated polymeric nanoparticles with small size and high fluorine content as MRI agents. The polymers, constructed from perfluoropolyether methacrylate (PFPEMA) and oligo(ethylene glycol) methacrylate (OEGMA) have favourable cell uptake profiles and excellent MRI performance. To facilitate cell studies the polymer was further conjugated with a fluorescent dye creating a dual-modal imaging agent. The efficacy of labelling of MSCs was assessed using 19F NMR, flow cytometry and confocal microscopy. The labelling efficiency of 2.6 ± 0.1 × 1012 19F atoms per cell, and viability of >90% demonstrates high uptake and good tolerance by the cells. This loading translates to a minimum 19F MRI detection sensitivity of ∼7.4 × 103 cells per voxel. Importantly, preliminary in vivo data demonstrate that labelled cells can be readily detected within a short acquisition scan period (12 minutes). Hence, these copolymers show outstanding potential for 19F MRI cellular tracking and for quantification of non-phagocytic and therapeutically-relevant cells in vivo.

Biological effects of iron oxide-protamine sulfate complex on mesenchymal stem cells and its relaxometry based labeling optimization for cellular MRI

Mesenchymal stem cells (MSCs) are frequently used as a therapeutic, but reliable imaging technique to longitudinally evaluate the engraftment of transplanted cells is inadequate. For magnetic resonance imaging (MRI), it is essential to understand the technical competence of in vitro stem cells labeling with iron oxide with regard to its relaxation behavior and significance of its biological expressions. The purpose of the study was to optimize the effective labeling of MSCs with high transverse relaxivity iron oxide contrast agent with protamine sulfate and also evaluate the biological effects (phenotype and function) of labeled MSCs. Our results demonstrated that 50:3µg/ml of Fe-Pro complex containing 10% serum at an incubation time of 6h were ideal for effective in vitro labeling. Relaxometry study demonstrated that almost an 8-fold increase in relaxation rate (R2) was observed in labeled MSCs by comparing with unlabeled. Marginal alteration in Oct4 and CD146 genes, and phenotypic CD45 expressions were detected after labeling. T2-weighted images and histological analysis confirmed the homing of transplanted cells to the site of injury. The relaxometry based optimized labeling method of MSCs could be extrapolated for cellular MRI and may be useful in stem cell tracking in various pre-clinical and clinical studies.

Magnetic Labelling of Mesenchymal Stem Cells with Iron-Doped Hydroxyapatite Nanoparticles as Tool for Cell Therapy.

Superparamagnetic nanoparticles offer several opportunities in nanomedicine and magnetic cell targeting. They are considered to be an extremely promising approach for the translation of cell-based therapies from the laboratory to clinical studies. In fact, after injection, the magnetic labeled cells could be driven by a static magnetic field and localized to the target site where they can perform their specific role. In this study, innovative iron-doped hydroxyapatite nanoparticles (FeHA NPs) were tested with mesenchymal stem cells (MSCs) as tools for cell therapy. Results showed that FeHA NPs could represent higher cell viability in'respect to commercial superparamagnetic iron oxide nanoparticles (SPION) at four different concentrations ranging from 10 μg/ml up to 200 μg/ml and would also upregulate an early marker involved in commitment and differentiation of MSCs. Moreover, FeHA NPs were uptaken without negatively affecting the cell behavior and their ultrastructure. Thus obtained magnetic cells were easily guided by application of a static magnetic field. This work demonstrates the promising opportunities of FeHA NPs in MSCs labeling due to the unique features of fast degradation and very low iron content of FeHA NPs compared to SPIONs. Likewise, due to the intrinsic properties of FeHA NPs, this approach could be simply transferred to different cell types as an effective magnetic carrier of drugs, growth factors, miRNA, etc., offering favorable prospects in nanomedicine.