Cell Transplantation, Vol. 22, pp. 993–1010, 2013 Printed in the USA. All rights reserved. Copyright 2013 Cognizant Comm. Corp.
0963-6897/13 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368912X657468 E-ISSN 1555-3892 www.cognizantcommunication.com
Role of Matrix Metalloproteinases in Migration and Neurotrophic Properties of Nasal Olfactory Stem and Ensheathing Cells Adlane Ould-Yahoui,*† Oualid Sbai,*† Kévin Baranger,*† Anne Bernard,*† Yatma Gueye,*† Eliane Charrat,*† Benoît Clément,‡§ Didier Gigmes,‡§ Vincent Dive,¶ Stéphane D. Girard,*† François Féron,*† Michel Khrestchatisky,*† and Santiago Rivera*† *Aix-Marseille Univ, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie (NICN), UMR 7259, 13344, Marseille, France †CNRS, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie (NICN), UMR 7259, 13344, Marseille, France ‡Aix-Marseille Univ, Institut de Chimie Radicalaire, Equipe Chimie Radicalaire, Organique et Polymères de Spécialité, UMR 7273, Marseille, France §CNRS, Institut de Chimie Radicalaire, Equipe Chimie Radicalaire, Organique et Polymères de Spécialité, UMR 7273, Marseille, France ¶Département d’Ingénierie et d’Etudes des Protéines (DIEP), CEA/Saclay, Gif-sur-Yvette, France Adult olfactory ectomesenchymal stem cells (OE-MSCs) and olfactory ensheathing cells (OECs), both from the nasal olfactory lamina propria, display robust regenerative properties when transplanted into the nervous system, but the mechanisms supporting such therapeutic effects remain unknown. Matrix metalloproteinases (MMPs) are an important family of proteinases contributing to cell motility and axonal outgrowth across the extracellular matrix (ECM) in physiological and pathological conditions. In this study, we have characterized for the first time in nasal human OE-MSCs the expression profile of some MMPs currently associated with cell migration and invasiveness. We demonstrate different patterns of expression for MMP-1, MMP-2, MMP-9, and MT1-MMP upon cell migration when compared with nonmigrating cells. Our results establish a correspondence between the localization of these proteinases in the migration front with the ability of cells to migrate. Using various modulators of MMP activity, we also show that at least MMP-2, MMP-9, and MT1-MMP contribute to OE-MSC migration in an in vitro 3D test. Furthermore, we demonstrate under the same conditions of culture used for in vivo transplantation that OE-MSCs and OECs secrete neurotrophic factors that promote neurite outgrowth of cortical and dorsal root ganglia (DRG) neurons, as well as axo-dendritic differentiation of cortical neurons. These effects were abolished by the depletion of MMP-2 and MMP-9 from the culture conditioned media. Altogether, our results provide the first evidence that MMPs may contribute to the therapeutic features of OE-MSCs and OECs through the control of their motility and/or their neurotrophic properties. Our data provide new insight into the mechanisms of neuroregeneration and will contribute to optimization of cell therapy strategies. Key words: Adult olfactory stem cells; Olfactory ensheathing cells (OECs); Matrix metalloproteinases (MMPs); TIMPs; Cell therapy; Nervous system
INTRODUCTION The olfactory nasal tissue is a paradigm of continuous remodeling in the nervous system. The mucosa includes two tissues separated by a basal lamina: (i) the epithelium, which harbors the olfactory sensory neurons, and (ii) the lamina propria, which is home for the ensheathing cells that nourish and guide the growing axons during their migration towards the olfactory bulb. Both compartments contain a population of stem cells with specific characteristics: bipotent epithelial stem cells in the epithelium and multipotent mesenchymal-like stem cells in the lamina propria (28). We have recently characterized the human lamina propria
stem cells as a subtype of mesenchymal stem cells with neurogenic and osteogenic properties and named them olfactory ectomesenchymal stem cells (OE-MSCs) (6). OE-MSCs are a potential source of cells for autologous cell transplantation therapy in Parkinson’s disease (30), hearing loss (34), and lesion-induced amnesia (31). OECs are also known for their therapeutic properties. They have been successfully assessed in animal models of spinal cord injury, stroke, degenerative diseases, and peripheral nerve trauma [for review, see (43)]. Following convincing preclinical tests in rodents (26,27), clinical studies based on autologous transplants of OECs in paraplegic patients
Received January 24, 2012; final acceptance May 10, 2012. Online prepub date: October 3, 2012. Address correspondence to Dr. Santiago Rivera, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie (NICN), CNRS - Aix-Marseille Univ., Faculté de Médecine Secteur Nord, CS80011, 51 Bd Pierre Dramard, 13344 Marseille Cedex 15, France. Tel: +33 4 91 69 87 71; Fax: +33 4 91 25 89 70; E-mail:
[email protected]
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were performed (9,25). Although these findings highlight the clinical potential of olfactory nasal olfactory cells, the underlying molecular mechanisms, notably those supporting migration of OE-MSCs and the neurotrophic effects of both OE-MSCs and OECs remain largely unknown. Matrix metalloproteinases (MMPs) form a multigenic family of Zn2+-dependent endopeptidases that belong to the superfamily of metzincins. MMPs can be roughly classified into secreted and membrane-type MMPs (MTMMPs) (29). The four secreted tissue inhibitors of MMPs (TIMP-1 to TIMP-4) control MMP activity via the interaction of their N-terminal domain with the catalytic domain of the enzymes. In addition to the inhibition of MMPs, the TIMPs display pleiotropic effects in a wide range of cell types (42). MMPs regulate the pericellular environment in physiological and pathological conditions through the proteolytic processing of extracellular matrix (ECM) and signaling proteins (e.g., cytokines, chemokines, cell adhesion molecules, membrane receptors, etc.) (8). For instance, MMPs may mobilize growth and angiogenic factors entrapped in the ECM via proteolytic-mediated release (4) and also catalyse the conversion of inactive forms of these factors into their biologically active forms (23). For these reasons, MMPs are pivotal for fundamental cell functions, including cell migration in physiological and pathological conditions. Some MMPs such as MMP-1, -2, -9 or MT1-MMP have been extensively studied as promoters of tumor cell invasion and metastasis, but also as mediators of postlesion tissue repair [for review (7,14,22,37,38)]. In the nervous system, increasing experimental evidence identifies MMPs as regulators of neural cell motility in physiological or pathological tissue remodeling (37). For instance, the early postnatal migration of cerebellar granule cell precursors in mice parallels changes in MMP-9 expression, and the genetic- or antibody-mediated inhibition of the enzyme results in a delay in cerebellar external granular layer migration (44). Likewise, the inhibition of furin, the enzyme that catalyzes the activation of MTMMPs, inhibits the physiological migration of neuroblasts along the rostral migratory stream (RMS), suggesting the implication of MT-MMPs in this process (3). In the postischemic brain, neuroblasts that migrate from the subventricular zone (SVZ) into the injured striatum exhibit upregulated levels of MMP-9 and broad spectrum inhibitors of MMPs interfere with this migration (24). MMPs also influence the motility of differentiated neural cells; we and others have shown that MMP-2 promotes migration of cultured resting astrocytes, possibly via interaction with b1 integrin and the actin cytoskeleton (32), whereas MMP-9 contributes to astrocyte motility during glial scar formation (20). More recently, we have also shown that MMP-2 and MMP-9 support motility of cultured OECs from the nasal mucosa (18). From the above,
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it follows that cells, including grafted cells, could use MMPs to surmount natural barriers and boost neural plasticity and regeneration. Accordingly, we have characterized at the subcellular level the expression profile and distribution of MMPs potentially involved in OE-MSCs migration, and we investigated their role in cell motility. Moreover, based on recent data implicating MMPs in neurite outgrowth and axo-dendritic differentiation of cortical neurons (16,33) and neurogenesis (46), we also investigated the possible trophic effects of MMPs secreted by OE-MSCs and OECs on cortical and dorsal root ganglia (DRG) neurons. Our data demonstrate the role of MMPs in all these phenomena and suggest their contribution to the neuro-regenerative properties of grafted nasal olfactory cells. MATERIALS AND METHODS All the procedures used to generate cell cultures in the present work from human beings or animals were approved by the Ethics Committee of the Medical Faculty of Marseilles and conform to National and European regulations (EU Directive Nu 86/609). Culture of Human OE-MSCs and BMSCs Human OE-MSCs and human bone marrow mesenchymal stem cells (BMSCs) were collected for a previous study from two males and two females with age ranging from 23 to 52 years old with ethical approval and informed consent (6), and aliquots of early passaged cells from all four patients were used for the current study. In proliferative conditions, stem cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM)/HAM’S F12 supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin (all from Gibco, Invitrogen, Carlsbad, CA, USA). Culture of Rat Olfactory Ensheathing Cells All experiments were conducted on adult (6–8 weeks old), pathogen-free Lewis female rats (Elevage Janvier, Le Genest Saint Isle, France). Rats were deeply anesthetized by intraperitoneal injection of 1 ml of Lethabarb (sodium pentobarbital 54.7%, Ceva Santé Animale, Libourne, France). All efforts were made to minimize animal suffering and reduce the number of animals used. After death, rats were decapitated. The olfactory mucosa was exposed and excised according to the recently described procedure (15). The tissue was immediately placed in 37°C DMEM/F12 with GlutaMAX™ (Gibco), washed two times, and incubated for 45 min at 37°C in 1 ml of an enzymatic solution of Dispase II (2.4 units/ml, in Puck’s solution; Roche Diagnostics, Penzbeg, Germany). A microspatula (Harvard Apparatus, Holliston, MA, USA) was used to gently separate the olfactory epithelium from the lamina propria under a dissection microscope. The lamina propria was then placed
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for 10 min at room temperature in 1 ml of Hank’s balanced salt solution (HBSS, calcium and magnesium free, Invitrogen) to inactivate Dispase II, transferred to 1 ml of collagenase I (0.25% in serum-free DMEM/F12; SigmaAldrich, St. Louis, MO, USA), and incubated for 5–10 min at 37°C/5% CO2. The lamina propria was mechanically triturated with the help of a Pasteur pipette (Dominique Dutscher, Brumath, France), at the start and at the end of the incubation period. The cell suspension was transferred to a 15-ml conical tube (Falcon, BD Biosciences, Franklin Lakes, NJ, USA), and collagenase activity was inhibited by adding 9 ml of HBSS. After centrifugation at 400 ´ g for 5 min, the supernatant was aspirated and the cell pellet was resuspended in DMEM/F12 supplemented with 10% FBS. A viable cell count was made with a Malassez cell (Dominique Dutscher), and cells from the lamina propria were plated in 25 cm² flasks and maintained for 15 days at 37°C and 5% CO2. The growth medium was replaced with fresh medium every 2 days. Cells were then split by trypsinization and replated at 1 ´ 105 cells/cm² in an equal final volume of 500 µl of serum-free DMEM/F12 supplemented with 25 ng/ml of transforming growth factor-a (TGF-a, Sigma-Aldrich) onto glass coverslips precoated with 2 µg/cm² of poly-l-lysine (Sigma-Aldrich) in culture dishes (Millipore, Bellirica MA, USA). The second passage was performed 1 week after. Primary Cultures of Neurons Primary cultures of cortical neurons were prepared from embryos of CD1 mice bred in our facilities, as previously reported (33). Pregnant females were deeply anesthetized with halothane (Nicholas Piramal Limited, London, UK). E17–18 embryos were quickly removed and put into cold HBSS containing 0.5% glucose (both from Invitrogen). Following decapitation, the cerebral cortices were dissected, pooled, and enzymatically dissociated for 10 min at 37°C in HBSS containing 0.1% trypsin (Invitrogen) and 10 μg/ml DNase I (Sigma-Aldrich). Adding HBSS containing 5% FBS stopped the enzymatic reaction. Further mechanical dissociation was carried out in HBSS containing 0.05% DNase by trituration through a Pasteur pipette. After 5 min centrifugation at 300 ´ g, the cell pellets were resuspended in the plating medium containing MEM, 0.6% glucose, 1 mM sodium pyruvate, 5 U/ml penicillin/streptomycin, and 10% FBS (all from Invitrogen). Cells were plated in 13-mm diameter wells (Millipore), onto 12-mm glass coverslips precoated with 1 mg/ml poly-d-lysine (SigmaAldrich) in borate buffer pH 8.5, and were grown at 37°C in a humidified atmosphere containing 5% CO2. After 75 min, the plating medium was aspirated and replaced by serum-free defined medium consisting of Neurobasal, with B27 supplement, 5 U/ml penicillin/streptomycin, 2.5 mM l-glutamine, and 25 nM glutamate (all from Invitrogen).
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Cultures of DRGs CD1 mouse pups (postnatal day 2 or 3) were deeply anesthetized with halothane; the DRGs were dissected in DMEM and plated during 12 h in four-well plates on 12 mm poly-l-lysine/laminin-coated cover slips (SigmaAldrich) in DMEM. After 12 h, conditioned DMEM was removed and replaced by fresh or 72 h OEC-conditioned neurobasal media, containing B27 supplement, 5 U/ml penicillin/streptomycin, 2.5 mM l-glutamine, and 25 nM glutamate (all from Invitrogen), with or without gelatinases. Modulation of MMP Activity The following MMP inhibitors were added to serumfree culture media: (1) human recombinant truncated N-terminal form of TIMP-1 (N-TIMP-1; Hideaki Nagase) (19) with similar Ki values (0.2–0.4 nM) for MMP-1, MMP-2, MMP-3, and MMP-9 and 146 nM for MT1MMP; (2) MMP selective pseudophosphinic RXPO3R inhibitor (Dr. Dive) that does not inhibit adamalysins (5,33), with Ki for MMP-2, MMP-9, and MT1-MMP of 55, 41, and 91 nM, respectively; (3) MMP and adamalysin inhibitor GM6001 (Sigma-Aldrich) with Ki = 500 pM for MMP-2, Ki = 27 nM for MMP-3, Ki = 100 pM for MMP-8, and Ki = 200 pM for MMP-9; (4) selective MMP-2 inhibitor (MMP-2 inhibitor III, Calbiochem, La Jolla, CA, USA), which exhibits good selectivity for MMP-2 (Ki 12 nM) when compared with MMP-9 and MMP-3 (Ki 200 and 4500 nM, respectively); (5) MMP-9 inhibitor-I (Calbiochem) (IC50 = 5 nM); and (6) MT1-MMP neutralizing antibody (LEM-2/15; Alicia Garcia Arroyo) (13). MMP-2 and MMP-9 selective inhibitors were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at a final concentration of 0.04%. Controls were incubated with the same concentration of DMSO for synthetic inhibitors or IgG for anti-MT1-MMP. A range of concentrations based on values of IC50 provided by the manufacturer was used to determine the concentrations of selective MMP-2 and MMP-9 inhibitors used in Boyden chamber-based cell migration assay (Millipore). The lowest efficient concentration was used. The Silicone Stencil Assay for Synchronized Cell Polarization/Migration The silicone stencil system made from poly (dimethyl siloxane) (PDMS; Sigma) was set up in order to study the behavior of migrating cells. We adapted previous methods (10,36) to our specific experimental conditions. We manufactured reusable circular silicone inserts of defined diameter containing an internal rectangular stencil of 15 mm2 where the cells were plated, on fourwell tissue culture plates (Nunc, Roskilde, Denmark). Manufacture of the inserts consisted in mixing in a plastic pillbox (VWR, West Chester, PA, USA) 12 g base
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with 3 g of curing agent (Down Corning Corporation, Midland, MI, USA) to obtain a silicone elastomer containing 20% of curing agent. The mixture was degassed for 15–20 min using a vacuum speed device. After degassing, the mixture was poured into a Petri dish (BD Biosciences) and first heated for 60 min at 80°C followed by an additional crosslinking step at 80°C for 60 min. After 1 h at room temperature to obtain the polymer material, silicon chips were cut using a copper tube and customized rectangular miniblades. Sterilized supports were inserted into the wells, and the plates were incubated for 30 min at 37°C to allow the silicone inserts to attach to the plate surface. The stencil was loaded with 200 µl of culture medium containing 4 ´ 103 OE-MSCs (666 mm2). After 24 h, adherent cells formed a confluent monolayer and the stencil was removed using forceps. Nonadherent cells were removed following two washes with serum-free medium. Subsequently, 500 µl of fresh culture DMEM/HAM’S F12 supplemented with 1% ITS (insulin, transferrin, selenium; Invitrogen), 1% penicillin, and 1% streptomycin were added to the cells. Boyden Chamber-Based Cell Migration Assay Confluent OE-MSCs cultured in 25 cm2 flasks were detached by trypsin/EDTA, counted, and seeded into the upper chamber of transwell polyethylene terephthalate filter membranes with 8-µm diameter pores (Corning, Chorges, France) at a density of 2 ´ 104 cells/well, in a final volume of 300 µl serum-free medium containing 1% ITS. Cells were allowed to migrate through the mem brane filter for 24 h in the absence or presence of RXPO3R, N-TIMP-1, MMP-2I, MMP-9I, and LEM-2/15 in the upper and lower chambers. Cells migrating through the membrane pore and invading the underside surface of the membrane were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich). Nonmigratory cells on the upper mem brane surface were removed with a cotton swab, and nuclei were stained with 0.5 µg/ml DNA intercalant Hoechst #33 258 (Invitrogen). Filters were observed using a Nikon E800 upright microscope equipped with epifluorescence, tetrarhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4¢,6-diamidino-2-phenylindole (DAPI) filters (Nikon, Champigny-sur-Marne, France) and an Orca-ER CCD camera (Hamamatsu Photonics, Hamamatsu, Japon). For quantitative assessment, the number of stained migrating cells was counted with ImageJ software (NIH, Bethesda, MD, USA) on 12 random fields per membrane filter at 10´ magnification. Isolation of Intracellular, Cytoskeletal, and Membrane Fractions Subcellular fractions were obtained from cultured OE-MSCs using the ProteoExtract subcellular proteome
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extraction kit (Calbiochem) according to the manufacturer’s instructions. Proteins were extracted with different buffers from major subcellular compartments and on the basis of the difference in protein solubility. The first cytosolic fraction was obtained by incubating the cells with an extraction manufacturer’s buffer at 4°C for 10 min. A second fraction containing cell membranes was obtained by incubating the cells with an extraction buffer for 30 min at 4°C. The nucleus and cytoskeleton fractions were also obtained by the same procedure. The enrichment of each fraction was evaluated by probing the Western blots with specific antibodies against CD44 (Millipore, Billerica, MA, USA) for the membrane frac tion, b-actin (Sigma-Aldrich) for the cytoskeleton fraction, and histone-H3 (Sigma-Aldrich) for the nuclear fraction. The different subcellular fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot or gel zymography (see below). Gelatin and Collagen Gel Zymography Gel zymography was performed as previously described (33). Briefly, culture supernatants or subcellular fractions were collected, and protein concentrations were normalized using the Lowry method (Bio-Rad, Hercules CA, USA). Equal amounts of protein were subjected to 8.5% SDS-PAGE (Bio-Rad) containing 4.5 mg/ml of porcine gelatin (Sigma-Aldrich) or 1 mg/ml of native collagen type I (Sigma-Aldrich) in nondenaturing and nonreducing conditions. Gels were washed twice for 30 min in 2.5% Triton X-100 (Sigma) to remove SDS and incubated for 48 h in 50 mM Tris (pH 7.5), 10 mM CaCl2 (Sigma-Aldrich) at 37°C. Gels were then stained with 0.1% Coomassie blue G-250 (Sigma-Aldrich) for 3 h in 30% ethanol and distained with a solution containing 5% acetic acid (Sigma-Aldrich) until clear bands of gelatinolysis appeared on a dark background. Gels were digitized, and optical densities were assessed with the Bio 1D software (Vilber Lourmat, Marne-la-Vallée, France). Human recombinant active MMP-2 (300 pg) (Millipore, Malscheim, France) was used as a positive and normalizing control. Extraction of Gelatinases All procedures were performed at 4°C. Culture supernatants from OE-MSCs or OECs were collected after 72 h in neurobasal medium. For gelatinase extraction, 5 ml of supernatant were incubated 24 h with 5 ml of gelatinsepharose 4B (Amersham Bioscience, Buckinghamshire, UK) with constant shaking. After centrifugation, the supernatant was placed at –80°C, and the gelatin sepharose pellet was resuspended in working buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, 0.005% Brij-35; all from Sigma-Aldrich) and the solution was centrifuged
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again. The pellet was then incubated for 30 min with elution buffer consisting of working buffer containing 10% DMSO. The efficacy of gelatinase extraction was measured by gel zymography. Western Blot Equal amounts of protein in Laemmli sample buffer containing 10% b-mercaptoethanol (Sigma-Aldrich) were boiled and separated by 8% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Amersham Biosciences Buckinghamshire, UK) in transfer buffer (25 mM Tris, 192 mM glycine, 20% ethanol; Sigma-Aldrich). Membranes were incubated overnight in blocking buffer (Roche Diagnostics, Mannheim, Germany) at 4°C and then probed with mouse anti-MMP-1 (EMD Chemicals, Gibbstown, NJ, USA), rabbit anti-MMP-2 (Millipore), rabbit anti-MMP-9 (Millipore), rabbit anti-MT1-MMP (Epitomics, Burlingame, CA, USA), rabbit anti-histone H3 (Sigma-Aldrich), rabbit anti-CD44 (Millipore), mouse anti-bIII tubulin (Sigma-Aldrich), and mouse anti-b-actin (Sigma-Aldrich). All antibodies were diluted in blocking buffer. After incubation with primary antibodies, membranes were incubated with a peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove PA, USA) (for further details, see Table 1). Finally, proteins were detected using a chemiluminescence kit (Roche Diagnostics, Meylan, France). Films were digitized using GeneTools software (Syngen, Cambridge, UK), and the optical density (OD) of the bands was assessed using ImageJ domain processing software. Immunocytochemistry OE-MSCs, cortical neurons, or DRGs were rinsed three times in phosphate-buffered salt (PBS) and fixed in 4% PFA for 20 min. For immunocytochemistry, cells were preincubated with 0.1% Triton X-100, 3% bovine serum albumin
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(BSA) (all from Sigma-Aldrich) for 60 min, followed by 90 min incubation at room temperature with mouse monoclonal anti-bIII tubulin, (Sigma-Aldrich), mouse monoclonal anti-Tau-1 (Sigma-Aldrich), rabbit anti-microtubule associated protein-2 (MAP-2; Abcam, Cambridge, UK), rabbit anti-MMP-9 (Millipore), mouse anti-MMP-1 (EMD Chemicals), rabbit anti-MMP-2 (Millipore), and rabbit anti-MT1-MMP (Epitomics). The secondary antibodies were labeled with Alexa Fluor® (Invitrogen), and all secondary antibodies were at a concentration of 1/800 (for further details see Table 1). The antibodies were diluted in PBS containing 0.1% Triton X-100, 3% BSA. Cells were rinsed three times in PBS and incubated for 1 h with Alexa Fluor® 488-594 antibodies (Invitrogen) and 0.5 µg/ml of nuclear marker Hoechst #33258. For cytoskeleton labeling (F-actin), cells were incubated after immunocytochemistry with Texas Red-X phalloidin (Sigma-Aldrich) for 1 h at room temperature, then rinsed in PBS, and mounted in fluorescence mounting medium (Dako, Glostrup, Denmark). Cells were observed under a LSM 700 confocal microscope (Zeiss Jena, Germany). Images were analyzed using the ZEN (Zeiss) and ImageJ software. In Situ Zymography on FITC-Labeled Gelatin In order to localize net gelatinolytic activity on OEMSCs, we used an in situ zymography method previously described (32,39). Briefly, OE-MSCs were grown on glass coverslips, and the medium was supplemented to a final concentration of 5 mM CaCl2 and 10 µg/ml of FITC-labeled DQTM gelatin intramolecularly quenched (EnzCheck Col lagenase kit from Invitrogen). After 1 h at 37°C in a humidified atmosphere containing 5% CO2, cells were rinsed in PBS, fixed with 4% PFA for 5 min, and processed for immunocytochemistry for MMPs and nuclear labeling, as stated above. Cell images were acquired with a LSM 700 confocal microscope and analyzed with the ZEN software.
Table 1. Primary Antibodies and Other Probes Used in Immunocytochemistry and Western Blot Assays Probes
Species
MMP-1 Anti-MMP-2 Anti-MMP-9 Anti-MT1-MMP Anti-Histone H3 Anti-b-Actin Anti-bIII tubulin Anti-CD44 Anti-Tau-1 Anti-MAP-2 Texas Red-X phalloidin
Mouse Rabbit Rabbit Rabbit Rabbit Mouse Mouse Rabbit Mouse Rabbit
Final Concentration (Immunocytochemistry)
Final Concentration (Western Blot)
1/100 1/250 1/250 1/200
1/500 1/250 1/250 1/500 1/10,000 1/3,000
1/1,000 1/400 1/500 1/400 1/400
MMP, matrix metalloproteinases; MT1, membrane type 1; MAP-2, microtubule-associated protein-2.
Source EMD Chemicals Millipore Millipore Epitomics Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Abcam Sigma-Aldrich
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RNA Extraction, RT-PCR, and TaqMan Quantitative RT-PCR Analysis Total RNA was prepared from cultures of OE-MSCs with the RNeasy Lipid Tissue Mini kit (Qiagen, Courta boeuf, France). Single-strand cDNA for a template was synthesized from 1 mg of total RNA using primer oligo (dT)12–18 (Invitrogen) and moloney murine leukaemia virus (MMLV) reverse transcriptase (Invitrogen) using manufacturer recommendations. qPCR experiments were carried out with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All reactions were performed using TaqMan Fast Universal PCR Master Mix and the following probes TaqMan® Gene Expression Assays according to the manufacturer’s instructions (Applied Biosystems): MMP-1 Hs00899658_m1, MMP-2 Hs01548727_m1, MMP-9 Hs00234579_m1, MT1-MMP Hs00237119_m1, TIMP-1 Hs00171558_m1, and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) Hs03929097_g1*. Twenty-five nano grams of previously prepared OE-MSC cDNA were used for each experiment. Samples were run in duplicates on the same 96-well plates and analyzed using the 7,500 Software v2.0 (Applied Biosystems). Thermal cycling con ditions started with initial denaturation at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 3 s, and annealing/extension at 60°C for 30 s. Relative expression levels were determined according to the ∆∆CT method. In the latter, gene expression level was quantified using the following formula: 2-∆∆CT, where ∆∆CT = ∆CT target gene – ∆CT reference gene (GAPDH) in the same sample (2). All values were normalized with respect to the 6 h time point. Cell Viability Assay Cell viability was assessed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma-Aldrich), which measures mitochondrial acti vity in living cells. Data were calculated as the percentage of living cells = 100 – [(treated cells OD550/tcontrol cells OD550) ´ 100]. Morphological Analyses of Cortical and DRG Neurons In each experiment with cortical neurons, fluorescence microphotographs of at least five randomly selected fields per well and three wells per experimental condition were taken. The following parameters were analyzed: (i) total number of cells, determined on the basis of nuclear Hoechst staining, and the MAP-2/Tau-1 ratio from doublelabeled samples and (ii) neurite length of all MAP-2 and Tau-1-positive cells was manually drawn using a computer mouse. The mean length of the neuritic arbor was obtained by dividing the total length of neurites in a field by the number of cells immunoreactive for MAP-2 and Tau-1. A neurite segment was defined as the distance between
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branching points or the distance between a branching point and the tip of the neurite. The axon is defined as a neurite with a robust immunostaining for Tau-1, whereas MAP-2 labels all neurites. In order to evaluate the axo-dentritic differentiation, we determined the following ratio: number of neurites with a robust Tau-1 immunostaining/number of neurites with MAP-2 immunostaining. DRG explants cultured for 3 days were fixed in 4% PFA and stained with the Tau-1 antibody. The area occupied by the axonal arbor was measured and normalized by the area of the explant using the LUCIA software (Laboratory Imaging, Prague, Czech Republic). Between five and seven explants per experimental group were analyzed in each independent experiment. Statistical Analysis Data were analyzed using analysis of the variance (ANOVA), followed by post hoc Tukey’s test. The mean values ± SEM were obtained from at least three independent experiments, unless otherwise indicated. Statistical significance was achieved at p
