Research ReportMesenchymal and neural stem cells labeled with HEDP-coated SPIO nanoparticles: In vitro characterization and migration potential in rat brain☆
Introduction
Stem cells are often described as the best candidates for cell therapy studies due to their self-renewal capacity and their large differentiation potential. Among them, mesenchymal stem cells (MSC) remain easy to isolate and expand. They may exhibit immunodepressive characteristics which make them less sensitive to rejection by the host immune system (Le Blanc et al., 2003, Maitra et al., 2004, Nasef et al., 2007). Moreover, MSC allow autologous grafts to be performed in cell therapy protocols. Bone marrow MSC typically differentiate into connective tissue cell types (D'Ippolito et al., 2004, Jiang et al., 2002), but various laboratories have also reported the transdifferentiation potential of MSC into a neuronal-like phenotype (Black and Woodbury, 2001, Sanchez-Ramos et al., 2000, Trzaska et al., 2007). We previously showed that a subpopulation of human MSC, marrow-isolated adult multilineage inducible (MIAMI) cells may trandifferentiate in vitro in a neurotrophin-dependent manner into neuronal-like cells. These cells express neuronal markers and present electrophysiological characteristics similar to those observed in mature neurons (Tatard et al., 2007). Moreover, a fraction of MSC transplanted in adult rat brains may respond to microenvironmental cues and transdifferentiate into neuronal-like cells (Jendelova et al., 2004, Kopen et al., 1999, Zhao et al., 2002). Using different brain lesion models, it has been shown that implanted MSC may be involved in functional improvement, either directly or indirectly by their ability to produce various growth factors (Chen et al., 2002, Li et al., 2002, Mahmood et al., 2002, Zhang et al., 2005). In addition, a damaged environment resulting e.g. from ischemia or from the presence of a tumor is known to stimulate the migration of transplanted MSC (Jendelova et al., 2004, Mahmood et al., 2002, Sykova and Jendelova, 2007a) as well as of neuronal precursors (Aboody et al., 2000, Kokaia and Lindvall, 2003). MSC may thus be considered as potential candidates for cell therapy studies in the central nervous system.
However, the possible use of MSC for brain repair studies still requires an evaluation of their behavior, migratory dynamic and fate in vivo. Moreover, as only a fraction of the transplanted cells may respond to the stimuli of the microenvironment, highly sensitive procedures will be required in the future.
Magnetic resonance imaging (MRI) is a non-invasive tool that has demonstrated a high sensitivity for cell tracking after systemic or in situ injection of cells having incorporated magnetic tags. Indeed, using phagocytic cells, the detection of a single cell in mouse brain has been obtained with this technique (Heyn et al., 2006). This strategy spares laboratory animals and ultimately may be used for human stem cell therapy studies. Toward this end, new magnetic tracers readily and specifically taken up by stem cells need to be formulated. Superparamagnetic iron oxide (SPIO) nanoparticles stand as promising tools to label and track various cell types in vivo by MRI (Bulte et al., 2002, Modo et al., 2002, Sykova and Jendelova, 2007b). We developed SPIO nanoparticles coated with 1-hydroxyethylidene-1.1-bisphosphonic acid (HEDP) (Portet et al., 2001), which are interesting due to their possible functionalization allowing the targeting and uptake of a specific cell type. In order to be able to translate this tool into the clinic, the innocuousness and the non-interference of these nanoparticles with the response of stem cells to their microenvironment have to be demonstrated.
In this study, we sought to characterize the potential effects of labeling rat MSC (rMSC) with these HEDP-coated SPIO nanoparticles on their viability and functions in vitro. Toward this end, rMSC iron uptake was characterized in vitro by Prussian blue (PB) staining and MRI. Iron-labeled rMSC viability and ultrastructure were also studied, as well as their osteogenic and neuronal differentiation potentials. Furthermore, we assessed the migratory potential of these iron-labeled MSC in vivo in response to the brain neurogenic stimuli. Indeed, it has been shown that neural stem cells (NSC) migrated from the subventricular zone (SVZ) to the olfactory bulb (OB), via the rostral migratory stream (RMS), where they differentiate into post-mitotic interneurons (Coskun and Luskin, 2002). Therefore, we studied the ability of iron-labeled rMSC to migrate in a similar fashion than iron-labeled rat neural stem cells (rNSC), when transplanted into the SVZ of the lesioned or non-lesioned rat brain. This study was performed by bromodeoxyuridine (BrdU) immunohistochemistry (IHC) and PB staining. Finally, a double staining with PB/CD11b, specific for macrophage/microglia, was used to confirm the fate of the grafted cells and of the SPIO nanoparticles.
Section snippets
Iron uptake and cell characterization
Prussian blue staining, after incubation of the rMSC with the iron-oxide nanoparticles, demonstrated that the intensity and the percentage of labeled cells increased with the iron concentration used. Forty eight hours incubation with an amount of nanoparticles corresponding to 25 μg iron/mL resulted in ca. 10% of Prussian blue positive cells, 50 μg/mL efficiently labeled more than 90% of the cells whereas 100 μg iron/mL did not increase the percentage of positive cells compared to 50 μg iron/mL
Discussion
Potential adverse effects of iron nanoparticles is a topic currently under investigation (Omidkhoda et al., 2007), and the means to diminish this side effect still remain to be explored. Many authors described the use of facilitating agents (Arbab et al., 2003) such as lipofectamin (Frank et al., 2003), conjugation with Tat peptide (Lewin et al., 2000) or with internalizing antibodies (Bulte et al., 1999) to increase the uptake efficiency while diminishing cell toxicity. However, these methods
Experimental procedures
All animal experiments were conducted in accordance with the “Direction des Services Vétérinaires”, the “Ministère de l'Agriculture” of France and with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Acknowledgments
We would like to thank the SCIAM (Service Commun d'Imagerie et d'Analyse Microscopique, Angers, France) for electron microscopy imaging and the toxicology department of the hospital of Angers (Angers, France) for iron spectroscopic titration.
We would also like to thank the “Région Pays de la Loire” for financial support.
Finally, we are grateful to Pr JC. Pagès (INSERM ERI 19, faculté de médecine, Tours, France) for providing the GFP lentiviral vector and to Pr PC. Schiller (Department of
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Grant information: This work was supported by the “Région Pays de la Loire” and “INSERM”.