Systemic treatment with adipose-derived mesenchymal stem cells ameliorates clinical and pathological features in the amyotrophic lateral sclerosis murine model

Accepted Manuscript Systemic treatment with adipose-derived mesenchymal stem cells ameliorates clinical and pathological features in the amyotrophic lateral sclerosis murine model Silvia Marconi, Marta Bonaconsa, Ilaria Scambi, Giovanna Maddalena Squintani, Wang Rui, Ermanna Turano, Daniela Ungaro, Stefania D’Agostino, Francesca Barbieri, Stefano Angiari, Alessia Farinazzo, Gabriela Constantin, Ubaldo Del Carro, Bruno Bonetti, Raffaella Mariotti PII: DOI: Reference:

S0306-4522(13)00454-5 http://dx.doi.org/10.1016/j.neuroscience.2013.05.034 NSC 14628

To appear in:

Neuroscience

Accepted Date:

19 May 2013

Please cite this article as: S. Marconi, M. Bonaconsa, I. Scambi, G.M. Squintani, W. Rui, E. Turano, D. Ungaro, S. D’Agostino, F. Barbieri, S. Angiari, A. Farinazzo, G. Constantin, U.D. Carro, B. Bonetti, R. Mariotti, Systemic treatment with adipose-derived mesenchymal stem cells ameliorates clinical and pathological features in the amyotrophic lateral sclerosis murine model, Neuroscience (2013), doi: http://dx.doi.org/10.1016/j.neuroscience. 2013.05.034

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Systemic treatment with adipose-derived mesenchymal stem cells ameliorates clinical and pathological features in the amyotrophic lateral sclerosis murine model

Silvia Marconi, PhD1, Marta Bonaconsa, PhD1, Ilaria Scambi, PhD1, Giovanna Maddalena Squintani, MD2, Wang Rui, MD1, Ermanna Turano, PhD1, Daniela Ungaro, MD3, Stefania D’Agostino, PhD1, Francesca Barbieri, MD1, Stefano Angiari, PhD4, Alessia Farinazzo, PhD1, Gabriela Constantin, MD4, Ubaldo Del Carro, MD3, Bruno Bonetti, MD, PhD1, Raffaella Mariotti, PhD1.

1

Department of Neurological Sciences (DSNNMM), University of Verona, Verona, Italy.

2

University Hospital of Verona, Verona, Italy.

3

4

San Raffaele Hospital, Neurophysiology Unit, Milan, Italy.

Department of Pathology and Diagnostics, University of Verona, Verona, Italy.

Running Head: Mesenchymal stem cells ameliorate murine ALS

Keywords: amyotrophic lateral sclerosis, motorneuron disease, neurotrophins, GDNF, neuroprotection.

Address for correspondence: Dr. Raffaella Mariotti, Department of Neurological Sciences (DSNNMM) Section of Anatomy and Histology Faculty of Medicine Strada Le Grazie 8, 37134 Verona, Italy; e-mail: [email protected]

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Abstract

Therapeutic strategies for the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are actually minimally effective on patients’ survival and quality of life. Although stem cell therapy has raised great expectations, information on the involved molecular mechanisms is still limited. Here we assessed the efficacy of the systemic administration of adipose-derived mesenchymal stem cells (ASC), a previously untested stem cell population, in superoxide-dismutase1 (SOD1)-mutant transgenic mice, the animal model of familial ALS. The administration of ASC to SOD1-mutant mice at the clinical onset significantly delayed motor deterioration for 4-6 weeks, as shown by clinical and neurophysiological tests. Neuropathological examination of ASC-treated SOD1-mutant mice at day 100 (i. e. the time of their best motor performance) revealed a higher number of lumbar motorneurons than in PBS-treated SOD1-mutant mice and a restricted number of undifferentiated GFP-labeled ASC in the spinal cord. By examining in spinal cord tissue factors that may prolong neuronal survival, we found a significant up-regulation of levels of glial-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) after ASC treatment. Considering that ASC produce bFGF but not GDNF, these findings indicate that ASC may promote neuroprotection either directly and/or by modulating the secretome of local glial cells toward a neuroprotective phenotype. Such neuroprotection resulted in a strong and long-lasting effect on motor performance and encourages the use of ASC in human pathologies, in which current therapies are not able to maintain a satisfying neurological functional status.

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Introduction Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by progressive loss of both cortical and spinal motorneurons (Cleveland and Rothstein, 2001; Sejvar et al., 2005); ALS prevalence is 4-6 per 100,000 people and most cases are sporadic (90%), the remaining cases being familial (FALS). The pathogenesis is still unknown but in 15-20% of FALS cases mutations of the gene encoding superoxide-dismutase (SOD)1 has been reported in patients phenotypically similar to sporadic forms of ALS. Transgenic mice over-expressing the human SOD1 gene mutation G93A exhibit a phenotype that reproduces clinical and histopathological features of human ALS. Since ALS and FALS do not differ in histopathology, SOD1-mutant (mSOD1) mice provide a good model to investigate the pathogenesis of ALS and to test therapeutic approaches (Bendotti and Carri, 2004; Kato, 2008; Turner and Talbot, 2008). Several therapeutic strategies have been attempted in ALS models, but to date there is no treatment that can cure or significantly ameliorate the quality of life of ALS patients. However, there is now accumulating evidence that adult stem cell therapy may be a promising therapeutic approach for this devastating disorder (Corti et al., 2010; Garbuzova-Davis et al., 2008; Gould and Oppenheim, 2011). In particular, the transplantation of adult stem cells has been shown to delay symptom progression and prolong lifespan in murine FALS (Corti et al., 2010; Garbuzova-Davis et al., 2008). Such beneficial effects seems not to be due to neuronal replacement, but rather to the ability of stem cells to release cytokines and growth factors, which have been demonstrated to directly support motorneuron survival (Gould and Oppenheim, 2011; Li et al., 2007; Park et al., 2009). In different experimental settings, stem cells have been shown to stimulate the migration and differentiation of endogenous neuronal and glial precursors, modulate the host immune inflammatory response and support neuronal survival (Caplan and Dennis, 2006; Constantin et al., 2009; Huang et al., 2010). In view of therapeutic treatments, it is important to identify easily accessible sources of stem cells, which can provide abundant tissue and ensure autologous transplantation.

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The majority of studies on stem cell therapeutic treatments have utilized mesenchymal stem cells (MSC) derived from bone marrow (BM-MSC) or umbilical cord. In the last years much attention has been paid to adipose-derived MSC (ASC), because adipose tissue is an easily accessible and appealing source of donor tissue for autologous cell transplantation (Lin et al., 2008; Zuk et al., 2002). Furthermore, ASC can be obtained in large quantities, since they display high proliferation kinetics and slow senescence ratio in vitro (Hipp and Atala, 2008; Mitchell et al., 2006). The comparative analysis of ASC and BM-MSC clearly showed that these two cell types do not differ regarding morphology, immune phenotype, success rate of cell isolation, colony frequency and differentiation capacity (Kern et al., 2006; Krampera et al., 2007). Concerning cell surface marker expression, ASC show a peculiar profile of adhesion molecules; in particular, ASC express CD49d (α 4 integrin) (Constantin et al., 2009; De Ugarte et al., 2003; Rüster et al., 2006), which plays a key role in leukocyte extravasation. Besides the ability to migrate into damaged tissues (Meyerrose et al., 2007), ASC have been reported to exert a modulatory activity both in vivo and in vitro on a broad range of immune cells (McIntosh et al., 2006; Puissant et al., 2005). Moreover, ASC secrete a variety of growth factors and cytokines (Constantin et al., 2009), which might positively impact neural cell survival. On this basis, in the present study we tested the efficacy of systemic injection of ASC in FALS mice, evaluating clinical, neurophysiological and histopathological parameters. We also assessed the ability of injected ASC to integrate into injured central nervous system (CNS) and their influence on the local microenvironment through the release of neurotrophic molecules.

Material and Methods

Adipose-derived mesenchymal stem cell cultures Murine ASC were obtained from 6-8 weeks old female C57Bl/6-Tg(UBC-GFP)30Scha/J mice expressing green fluorescent protein (GFP) (Harlan Laboratories, Udine, Italy). The isolation 4

of stromal-vascular fraction was carried out on 5 ml of minced samples obtained from subcutaneous abdominal and inguinal fat, as previously described (Anghileri et al., 2008). Briefly, after washing with sterile HBSS, extracellular matrix was digested at 37°C with Collagenase A (Sigma Aldrich, Milan, Italy), centrifuged at 1200 g and the pellet was suspended in NH4Cl; the stromal fraction was then collected by centrifugation and filtration. Cells were then cultured in DMEM, glucose, 20% heat-inactivated adult bovine serum, penicillin and streptomycin (EuroClone SpA, Milan, Italy). After 72 h, non-adherent cells were removed. When 70-80% adherent cells were confluent, they were trypsinized, harvested and expanded. A homogeneous cell population was normally obtained after 3 to 5 weeks of culture. All the experiments were performed using ASC at 8-15 passages. Murine ASC were recognized by immunophenotype using monoclonal antibodies specific for CD106 (VCAM-1), CD9, CD44, CD80, CD138 and Sca1. In addition, the absence of haematopoietic (CD45, CD11c and CD34) and endothelial markers (CD31) was assessed, as previously described (Krampera et al., 2007). All antibodies were purchased from Pharmigen/Becton Dickinson (Palo Alto, CA, USA). For immunophenotypic analysis, ASC were detached using trypsin/EDTA, washed with phosphate-buffered saline (PBS) and resuspended at 106 cells/ml. Cell suspension was incubated at +4° C for 10 min with 15% adult bovine serum, followed by incubation with the specific monoclonal antibody at +4°C for 30 min. At least 10,000 events were analyzed by flow cytometry (FACScalibur, Becton Dickinson) using the Cell Quest software.

Animals A mouse line carrying the human mutant Sod1 gene driven by the human SOD1 promoter (strain designation: B6SJL-TgN[SOD1-G93A]1Gur, G1H) (mSOD1) and wild-type (WT) (B6SJL) mice obtained from Jackson Laboratories (Bar Harbor, ME, USA) were used. The animals were kept under controlled environmental parameters and veterinarian assistance. All experiments were carried out with the authorization of the Committee for Research on Laboratory Animals of the 5

University of Verona and the Italian Ministry of Health, following the NIH Guide for the Use and Care of Laboratory animals, in accordance with the European Communities Council Directives (86/609/EEC), minimizing the number of animals used and avoiding their sufference. The transgenic progeny, derived from the colony established in our animal facility, was identified by polymerase chain reaction specific for human Sod1 with G93A mutation, as outlined by Jackson Laboratories and was genetically comparable (Henriques et al., 2010). A total of 58 mSOD1 and 9 WT mice were used ad hoc in different experimental paradigms; animals were intravenously (i.v.) injected through the tail vein with GFP+ ASC cells (n: 29) and PBS (29), at the onset of clinical signs (day 76-77). Experimental groups were randomized by sexes and littermates to avoid differences in disease severity. The total amount of GFP+ASC cells (2x106) was subdivided in two injections and suspended in 0.5 ml of sterile PBS without Ca2+ and Mg2+, while control animals received 0.5 ml of vehicle (PBS). The time of onset of disease of animals was evaluated starting at 50 days. All animals were assessed weekly by a blinded observer for body weight, and behavioural performances, including hindlimb extension reflex, paw grip endurance (PGE) to asses grip strength (Gurney et al., 1994) and rotarod test (Miana-Mena et al., 2005; Weydt et al., 2003) to evaluate motor coordination. In particular, mice were evaluated for signs of motor deficit according to the following scale: 4: normal; 3: hindlimb tremors when suspended by the tail; 2: gait abnormalities; 1: dragging of at least one hindlimb; 0: inability to right in 30 seconds. The PGE test was performed by placing mice on a metal grid which was then quickly turned over. The latency until the mouse lets go of the grip with both hindlimbs was timed. Each mouse was given up to two attempts to hold on to the inverted grid for an arbitrary cut-off time of 120 s. For the rotarod test, we evaluated the time spent by the animal on the rotating crod (Ugo Basile, Varese, Italy). Each animal was placed on a rotarod apparatus, the speed of rotation was gradually increased and then maintained costant at 16 rpm, and the mouse ability to remain on the rotating rod was recorded. A cut-off point of 180 s with 2 trials/session, 3 min apart, was used. PGE and rotarod tests were performed twice weekly. Presence 6

of tremors, lack of extension reflex, or failure in any motor tests in 3 consecutive sessions indicated onset symptoms. Time of clinical onset in our colony is 75 ± 3 days and duration of disease is 55 ± 5 days. Animals were considered at the end-stage when unable to right in 30s after being pushed on their side.

Neurophysiological assessment Mice were anaesthetized with tribromoethanole (Sigma; 0.02 ml/g), immobilized on a rigid table to prevent movement artefacts and placed under a heating lamp to maintain body temperature above 32°C. Neurophysiological mesuraments were performed through Cadwell EMG apparatus (Ates Device, Verona, Italy). Peripheral conduction studies were performed stimulating the sciatic nerve with steel monopolar needles (27 gauge) distally at the ankle and proximally at the greater ischiatic notch. Compound motor action potential (cMAP) was obtained with monopolar needles with the active electrode in the flexor muscles of the hindlimb and the reference electrode inserted subcutaneously between the first and second digit; cMAP amplitude and latency (distal and proximal), conduction velocity and F wave latency were calculated. The electromyographic signal was filtered with 10-2k Hz band-pass, with a sweep velocity of 10 msec and amplitude gain of 2mV. Motor evoked potentials (MEP) were obtained with the same recording apparatus; cortical MEP (cMEP) were obtained stimulating the motor cortex with monopolar needles with the cathode in the midline of the interaural line through the scalp and the anode placed 10-15 mm lateral to cathode. Current was delivered with a single shock 1.5X the resting motor threshold intensity (defined as the lowest current that allows the recording of half of responses of amplitude greater than 50 μV from 6 consecutive stimulations in resting muscles). Spinal MEP was obtained inserting the stimulating needle electrodes into the lumbar cord close to the emergence of sciatic nerve roots. Cortical and spinal MEP amplitude (cMEP and sMEP respectively), MEP/MAP ratio (to minimize variation of MEP potentials related to needle recordings), central and peripheral conduction time 7

(also calculated with the formula F latency + M latency –1/2) were considered. The electromyographic signal was filtered with 10-2k Hz band-pass, with a sweep velocity of 20 msec and amplitude gain of 500μV. In a first set of experiments, we compared the neurophysiological parameters of WT (n: 6 animals) and mSOD1 mice treated with PBS (mSOD1-PBS) at days 50, 70, 90 and 110 (6 animals at day 50, 9 at day 70, 90, 110) in order to investigate the natural course of neurophysiological disease and the correlation with clinical scores. Since neurophysiological parameters of mSOD1 mice were significantly different from WT mice at day 70, we studied the neurophysiological parameters of mSOD1 mice treated i.v. with ASC (mSOD1-ASC; 9 mice at day 70 and 90, 6 at day 110) and mSOD1-PBS mice at day 70, 90 and 110 (12 mSOD1 mice per treatment and time point) in order to verify the therapeutic effect of ASC.

Histopathology and immunohistochemistry Animals were deeply anaesthetized (pentobarbital 60 mg/kg, i.p.) and perfused transcardially at days 100 or 135 with phosphate buffer (0.1M, pH 7.4) followed by 4% paraformaldehyde in PBS. The spinal cord and the quadriceps muscles were soaked overnight for cryoprotection in 20% sucrose at 4°C and cut on a freezing microtome. Transverse sections (30 μmthick) were collected from lumbar spinal cord and quadriceps muscle; consecutive sections were stained with cresyl violet and for immunohistochemistry: goat polyclonal anti-coline acetyltransferase (ChAT) antibody (Chemicon International, Temecula, CA) to identify motorneurons; rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (Dako, Glostrup, Denmark) as marker of astrocytes; rat monoclonal anti-CD11b antibody (R&D Systems, Minneapolis, MN) to label microglia. Free-floating sections were pre-incubated in 5% appropriate normal serum and 0.2% Triton X-100 in PBS for 1 h, and then incubated overnight with primary antibodies. After repeated washing, the sections were incubated with appropriate biotinylated secondary antibody; the reaction was developed using the avidin-biotin peroxidase protocol and 38

3’diaminobenzidine as chromogen. As negative control, sections were processed as above omitting the primary antibodies as well as incubating with respective normal host serum at the same dilution of the primary antibodies. The preparations were observed under bright-field illumination. In order to evaluate the presence, distribution and differentiation of GFP+ ASC in spinal cord and muscle of mSOD1 mice, frozen sections were stained with DAPI and anti-ChAT, CD11b or GFAP (for spinal cord); the signal was then detected by appropriate secondary biotinylated antibodies and Streptavidin Texas Red (Vector, Peterborough, UK). Sections were viewed under a Leica tandem confocal scanner SP5 (Leica Microsystem, Manheim, Germany) at 40X and 63X objectives (numerical aperture 0.75), with acquisition of images at different wavelengths (DAPI 455 nm, GFP 509 nm, Texas Red 615 nm). All the counts were performed in three sets of sections (100 μm apart) for each animal. The density of positive elements/mm2 was then quantified at 40X magnification by two independent observers (SM and ET).

Cell counts and image analysis Spinal cord tissue derived from all mice of each group was subjected to quantitative analyses, which were pursued blindly of the experimental group. Motorneuron counts were performed on cresyl violet-stained sections using a Leitz microscope equipped with a JVC CCD KY-F58 digital camera and an X-Y-Z motorized stage. A 40X objective (numerical aperture 0.75), and the image analysis software Image Pro Plus 6.2 (Media Cybernetics, Silver Spring, MD) were used. Five regularly spaced spinal cord sections between the L1 and L5 segments were selected. In every section, four rectangular frames (16x120 μm) were randomly placed in the ventral horn of the spinal cord (two on the right side and two on the left side). Neurons were counted in each frame using the optical dissector method (Gundersen, 1986) and two inclusion lines, excluding cells within the uppermost focal plane. Only neurons with evident nucleoli were included in the cell counts, discarding glial cells identified as smaller cells without evident nucleoli and heavily stained nuclei. The values from five sections were computed for the summation, the mean number for the 9

region of interest per group was then computed from the average number derived from each animal. The data was expressed as mean ± S.E.M.. Densitometric evaluation of the immunostaining intensity of CD11b or GFAP-labelled cells was evaluated as previously described (Kassa et al., 2007; Kassa et al., 2009), using the image analysis software and 40X objective and constant brightfield illumination. A total of five regularly spaced sections per animal were sampled. The zero value of optical density was evaluated in tissue devoid of specific labelling according to a standardized protocol (Kassa et al., 2007).

ELISA assay for neural growth factors The production of neural growth factors was quantified both in supernatants of cultured ASC, as well as in spinal cord and quadriceps muscle homogenates of mSOD1 mice injected with ASC or PBS and age-matched healthy animals. To determine the production by ASC of vascular endothelial growth factor (VEGF), insulin-like growth factor I (IGF-I), and glial-derived neurotrophic factor (GDNF), supernatants were obtained from ASC (1x105) in growth medium for 24 h and analyzed by Quantikine® ELISA Immunoassay (R&D Systems, Minneapolis, USA), following the manufacturer’s instructions. Briefly, cells were grown in 24-well and the supernatants were harvested and centrifuged for 10 min to remove cell debris. Samples were added in 96-well pre-coated plates and incubated for 2 h at room temperature. After washing, specific polyclonal antibody followed by substrate solution was added and color development was measured at 450 nm (BioRad Microplate Reader, Life Science, Milan, Italy). The concentration of growth factors was calculated using the standard curve and expressed as pg/ml. The protein levels of VEGF, IGF-I, GDNF, brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF) were determined in homogenates of spinal cord and muscles (only GDNF) at day 100 and 135 from ASC-, PBS-treated mSOD1 mice as well as in WT littermates. Proteins were extracted from frozen tissue (3 samples for each condition) using PARIS kit (Applied Biosystem, Foster City, CA); briefly, tissue was cut into small pieces, and then homogenized in Cell 10

Disruption Buffer according to manufacture’s instructions. The concentration of protein was assessed by Bradford assay, as previously described (Rosati et al., 2009). Each sample was subjected to Quantikine® ELISA Immunoassay (R&D Systems) in accordance with the manufacturer’s instructions. The expression levels of neurotrophic factors in the spinal cord and muscles were expressed as ng/mg total protein.

Statistics Between-group (ASC vs. PBS) and within-group (ASC) comparisons over time were performed using a generalized linear model for the clinical (PGE and Rotarod) and neurophysiological variables (cMAP, cMEP) as outcome and a categorical time predictor (70, 75, 80, 85, 90, 95, 100, 105, 110 and 120 days for PGE and Rotarod; 50, 70, 90, 110 day for cMAP and cMEP) for between-group comparison, with Bonferroni’s adjustment to perform multiple comparisons. For neural growth factors statistics, univariate analysis of variance and post-hoc test with Bonferroni’s correction were applied. Statistical analysis was performed using SPSS software, version 19 (IBM, Armonk, NY). All reported p-values were corrected for multiple comparisons and a corrected p-value < 0.05 was used to define statistical significance. For statistical evaluation of motorneuron counts and densitometric evaluation of glial cells, mean between-group differences of motorneuron numbers and CD11b or GFAP optical density units were evaluated in mSOD1 mice injected with ASC and PBS, respectively, at two different time points (100 and 135 days) with the Student-t test. Significance was set at p<0.05.

Results

Neurophysiological assessment of mSOD1 mice A first set of experiments was performed to evaluate neurophysiologically the disease course every 20 days starting from day 50. The neurophysiological assessment included several parameters 11

which evaluated either the peripheral (i. e. cMAP amplitude) or the central (cMEP amplitude, central conduction time-CCT) motor pathway involvement. Particular attention has been paid to the amplitude of cMAP, which reflects the number of larger and faster motorneurons and the amplitude of cMEP and CCT. No significant differences in all these parameters was found at day 50, whereas a significant reduction of distal and proximal cMAP amplitude and cMEP amplitude were recorded in mSOD1 mice compared to WT at day 70, 90 and 110 (Figure 1A, B). In addition, CCT was significantly increased in mSOD1 animals compared to WT mice at day 90 (data not shown). Noteworthy, while cMEP were recorded in WT mice at all time points, we were able to elicit cMEP in mSOD1 mice only in 80% of animals at day 70 and 90, and in 50% at day 110.

Systemic injection of ASC ameliorates the disease course in mSOD1 mice Clinical and neurophysiological effects of ASC The i.v. administration of ASC at clinical onset induced an improvement of the subsequent clinical disability of mSOD1 mice, with a significant delay of motor performance deterioration as compared to PBS-treated mSOD1 mice. The PGE test persisted significantly improved for 40 days in comparison to PBS-injected animals [F(1,20)=7.887; p=0.011; mSOD1-PBS n 12 animals, mSOD1-ASC n 12], the motor ability of ASC-treated mice being almost comparable to WT animals for 30 days after injection. The beneficial effect observed in ASC-treated animals rapidly disappeared around day 115 (Figure 2A). The significant clinical amelioration after ASC injection was demonstrated also with RotaRod test [F(1.16)=6.058; p=0.026; mSOD1-PBS n 9, mSOD1ASC n 9] (Figure 1C). No difference in lifespan was observed between ASC-treated (138.6 ± 8.1) and PBS-treated (137.2 ± 10.0) animals. In parallel with the clinical effect, we found that the administration of ASC exerted a significant better preservation of the distal cMAP amplitude at day 90 in treated mice as compared to controls (7.6 ± 4.0 vs 5.1 ± 2.1; p=0.02; mSOD1-PBS n 12; mSOD1-ASC n 9) and this tendency was maintained at day 110, although not reaching statistical significance (Figure 2B). The 12

registration of cMEP amplitude showed a trend similar to cMAP with minor reduction of amplitudes in ASC-treated mice as compared to PBS-treated group at day 90 and 110 (Figure 2C). In addition, F waves were recordable only in 70% of nerves of PBS-treated mSOD1 mice, while they were present in all ASC-treated mSOD1 mice. No differences in CCT were detected between the two groups at any time point (data not shown). Thus, the clinical improvement obtained by ASC treatment was confirmed by neurophysiology, since a significantly higher distal cMAP amplitude was recorded in ASC-treated as compared to control mice at days 90 and 110, the range of time in which ASC-treated animals sustained the best motor performance (Figure 2).

Analysis of ASC migration and expression of neural markers The analysis of the distribution of GFP+ASC after systemic injection confirmed the ability of these cells to migrate and persist into the damaged CNS, spleen and muscle tissue (n. 7 animals for each time point) as seen in other experimental models (Constantin et al., 2009): a restricted number of GFP+ cells were present both in the white and gray matter of the spinal cord and their number persisted almost unchanged at day 135 (Figure 3C). We next investigated whether GFP+ ASC migrated to parenchyma underwent neuronal and glial differentiation. As shown in Figure 3A, B, GFP+ASC were detectable in the anterior horn of the spinal cord in proximity to ChAT+ motorneurons at day 100 and 135, without evidence of neuronal trans-differentiation. Similarly, no expression of astroglial or microglial markers was observed on GFP+ ASC penetrated in the spinal cord (data not shown). In addition, the distribution analysis upholds the ability of GFP+ASC to migrate and integrate into muscle tissue, where GFP+ elements were present both at day 100 and 135 (Figure 3C). No damage, including tumor formation, was detected in the spinal cord and muscles after implantation; no evidence of binucleated cells was observed in the samples (data not shown).

Histopathology of spinal cord in ASC-treated mSOD1 mice 13

Immunohistochemical analysis of glial and neuronal cells was performed on the lumbar portion of the spinal cord of ASC-treated (n 7) and PBS-treated mSOD1 mice (n 7) at day 100 (when behavioural tests and neurophysiology were significantly different) and at day 135 (shortly before the end point, when no clinical difference between treated groups was recorded). Interestingly, a significant increase in the number of motorneurons per section was observed at lumbar spinal cord level examined in ASC-treated mSOD1 mice as compared to PBS-treated ones at day 100 (Figure 4A, 4B and 4C), but not at day 135 (data not shown). We then investigated the effect of ASC on both astroglial and microglial activation at lumbar level. We noticed a tendency to down-regulation of GFAP-reactive astrocyte in ASC-treated mSOD1 animals at day 100, although the difference with PBS-treated mSOD1 animals was not significant (Figure 4D, 4E and 4F). No difference in the number of microglial cells was observed in the lumbar spinal cord of ASC and PBS-treated mice at any time points (Figure 4G, 4H and 4I).

Modulation of neurotrophic molecules in spinal cord homogenates The presence and persistence of ASC into the spinal cord of mSOD1 mice suggests that these cells may support the survival of motorneurons and modulate the glial reaction. Among the possible mechanisms through which ASC may modulate neural cells, we assessed by ELISA assay the production by ASC of a number of neurotrophins, known to influence their self-renewal as well as neuroregeneration. In a previous study (Constantin et al., 2009), we found that ASC were able to secrete in vitro consistent amounts of BDNF, bFGF and platelet-derived growth factor-AB. In the present study, the supernatants of ASC contained considerable quantities of IGF-1 (991.3 ± 63.3 pg/ml) and VEGF (1071.3 ± 86.1 pg/ml), but not of GDNF or CNTF. We then quantified by ELISA the tissue concentration of these neurotrophins in spinal cord and muscle homogenates to assess whether ASC were able to modulate their production into CNS and muscle tissues. The analysis was performed on spinal cord and muscle of animals sacrificed at day 100 (when ASC-treated mice showed best behavioral and histological results) and at day 135 as 14

well as in age-matched WT animals. We found that ASC treatment induced a significant increase of GDNF and bFGF into the spinal cord of ASC-treated animals at day 100 as compared to PBStreated mice [GDNF: F(2.4)=86.452; p=0.001; bFGF: F(2.5)=90.327; p=0.007; WT n 3, mSOD1PBS n 5, mSOD1-ASC n 5] (Figure 5A). At this time point, the tissue levels of BDNF and IGF-I were also increased in mSOD1 mice treated with ASC although the difference with PBS-treated animals was not significant. The quantification of tissue trophic molecules performed at end stage confirmed the ability of ASC to significantly modulate the local secretion of bFGF concentration in comparison to PBS-injected animals [F(2.5)=96.874; p=0.000;WT n 3, mSOD1-PBS n 5, mSOD1ASC n 5] (Figure 5B). The tissue concentrations in the spinal cord of VEGF and CNTF were below the detection limits in both ASC-and PBS-treated mSOD1 mice as well as in WT mice (data not shown).

Discussion

ASC as novel therapeutic approach to murine FALS Stem cells represent a promising therapeutic approach in the treatment of neurological disorders, including ALS. The efficacy of stem cell therapy has been verified in experimental models of ALS by using human umbilical cord blood stem cells ( Garbuzova-Davis et al., 2008; Garbuzova-Davis et al., 2012; Knippenberg et al., 2012), human neural stem cells (Xu et al., 2006), rodent and human BM-MSC (Corti et al., 2004; Vercelli et al., 2008), and human neuron-like cells (Garbuzova-Davis et al., 2006). However, the source and availability of stem cells represent a crucial issue for their clinical application and the invasiveness of isolation procedures may limit their application in humans. In this regard, much attention has been paid in recent years to the adipose tissue as a source of stem cells because of the high frequency and expandability of ASC and the peculiar adhesion molecule profile which favours their migration into damaged CNS after systemic injection (Constantin et al., 2009; De Ugarte et al., 2003). 15

In the present study, we demonstrate that the systemic injection of ASC in mSOD1 mice at the clinical onset significantly delayed the deterioration of motor performance as compared to vehicle-treated mSOD1 mice; in particular, mSOD1 animals injected with ASC were able to perform for 35-42 days PGE and Rotarod tests almost as well as WT mice. The clinical effect was confirmed by neurophysiological analysis, which showed a significantly better preservation of distal cMAP amplitude in ASC-treated mSOD1 mice at day 90 (i.e. the time of the best motor performance in comparison to PBS treatment). The histopathological analysis performed on the spinal cord further confirmed the transient beneficial effect of ASC, showing a significantly higher number of surviving motorneurons in ASC-treated mSOD1 mice compared to vehicle-treated ones at day 100, whereas such a difference was not appreciable at day 135 (shortly before death). However, the robust effect on motor behaviour rapidly disappeared 50 days after ASC injection. Such transiency correlates well with the lack of effect on the survival of mSOD1 mice. Regarding the reasons for such short-lasting effect, we believe that it is mainly dependent on the model employed rather than to a limited effect of ASC. In fact, transgene mSOD1 mice suffer from a genetic disease, where motorneurons are genetically committed to die. In line with this, a limited or absent effect on the survival has been reported also by most studies assessing the efficacy of stem cell-based therapy, particularly when a therapeutic (rather than preventive) administration was adopted (Garbuzova-Davis et al., 2012; Knippenberg et al., 2012; Uccelli et al., 2012).

ASC and neurotrophin release in murine FALS The analysis of distribution of GFP ASC injected i.v. confirmed their capacity to penetrate and persist undifferentiated (i. e. do not undergo a process of neuronal trans-differentiation) into the damaged CNS until the end stage, as seen in other experimental models (Constantin et al., 2009; Marconi et al., 2012). The persistence of ASC in spinal cord of mSOD1 mice and the lack of additional benefit after repeated injection of ASC (2x106 cells at day 100; unpublished observation), indicate that, at least in this model, ASC are not able to rescue motorneurons from cell death. We 16

then performed a set of experiments to assess which mechanisms were responsible for the transient neuroprotection to motorneurons. The present and previous studies have documented that ASC can produce a number of neurotrophic factors able to sustain neural survival (Constantin et al., 2009; Marconi et al., 2012). Although we cannot exclude the possibility of a direct neurotrophic effect of ASC on motorneurons in the present study, the evidence rather suggests an effect mediated by a cross-talk of ASC with glial cells. In particular, the limited number of ASC detected in target tissues in previous (Constantin et al., 2009; Marconi et al., 2012) and in the present study argues against a direct neuroprotective effect of these stem cells. The data obtained here on the comparison of the neurotrophins produced by ASC and those found significantly modulated in the spinal cord of treated mSOD1 mice (i. e. GDNF and bFGF) further indicates that ASC may modulate the secretome of local glial cells towards a neuroprotective phenotype. It is interesting to note that albeit we did not find significant quantitative changes in the number of astrocytes and microglia, the analysis of the production of neurotrophins in situ do indicate that ASC changed the biological functions (secretome) of local glial cells, particularly astrocytes. Although bFGF may derive both from ASC and reactive astrocytes, the up-regulation of GDNF in the spinal cord of ASC-treated mice may be considered the best example of modulation by ASC present in the spinal cord on surrounding astrocytes (known to produce GDNF; Shen et al., 2010). In fact, since ASC do not produce GDNF in culture, we hypothesize that these cells release soluble factors able to influence the astrocytic secretome, which can in turn mediate and amplify the biological effect of ASC. Such amplification is needed considering a 1:150 ASC:astrocytes ratio in the mSOD1 spinal cord (Marconi et al., 2012). It is interesting to note that bFGF and GDNF upregulation is specifically associated with ASC treatment, since reactive astrocytosis was not able per se to increase the production of these neurotrophins in WT mice and PBS-treated mSOD1 mice. On the other hand, the possibility that increased levels of GDNF derive from muscle (Li et al., 2007; Suzuki et al., 2008; Wang et al., 2002) seems unlikely in our experimental setting, due to the limited contribution of muscle-derived GDNF. Noteworthy, we have recently observed a similar 17

ASC-induced up-regulation of GDNF by Schwann cells in crushed sciatic nerves (Marconi et al., 2012). These results, obtained in a different experimental model, further confirm findings observed also in experimental autoimmune encephalomyelitis (Constantin et al., 2009), in which a restricted number of ASC capable to reach target tissue probably initiates a cascade of events through the interaction with local glia, which result in neuroprotection. Previous studies have indeed suggested that stem cells may contribute to the repair of injured tissue by releasing in situ a large number of trophic factors, which can profoundly influence the local micro-environment ( Gould and Oppenheim, 2011; Caplan and Dennis, 2006; Kaspar, 2008). However, clinical trials with direct delivery of neurotrophic factors have been unsuccessful; in addition to problems with drug pharmacokinetics, toxicity, and antibody inactivation ( Park et al., 2009; Ekestern, 2004), we hypothesize that the reason may be related to the use of a single molecule. Among neurotrophic molecules potentially useful in ALS, GDNF protects motorneurons (Henderson et al., 1994; Sariola and Saarma, 2003) and the administration of GDNF through viral vectors or with genetically modified stem cells has been tested with promising results in animal models of FALS (Li et al., 2007; Suzuki et al., 2008; Wang et al., 2002). However, we believe that the beneficial effect exerted by ASC in the mSOD1 model is mediated by multiple mechanisms acting in concert. As opposed to the administration of a single molecule, the therapeutic advantage of ASC is related to their intrinsic plasticity in response to the signals from the micro-environment. Here we demonstrated the up-regulation of GDNF and bFGF, but a wider array of neurotrophins are probably responsible for the increased survival of motorneurons (Gu et al., 2010). In parallel, it is conceivable that other mechanisms are involved. In fact, MSC have been recently reported to influence several pathogenetic processes in experimental ALS (Uccelli et al., 2012), including down-regulation of the glial response and an anti-oxidative effect. In this regard, the role of astrocytes in non-cell autonomous toxic effect on motoneurons has been recently demonstrated (Foran et al., 2011): in fact, Uccelli et al. (2012) have indeed demonstrated that BM-MSC influence glutamate-mediated, astrocytic-dependent excitoxicity on motoneurons and human ASC have been 18

shown to enhance the glutamate uptake function of glutamate transporter 1 in SOD1(G93A) astrocytes (Gu et al., 2010). Thus, on the basis of the present results, the complex cross-talk of ASC with astrocytes could modulate different pathogenetic aspects: stimulate the astrocytic production of protective neurotrophins (in particular GDNF) and simultaneously reduce the harmful astrogliosis.

Concluding remarks Taken together, our data show that autologous and undifferentiated ASC have a clear therapeutic potential in slowing down the clinical course in the animal model of FALS and could thus represent a valuable tool for stem cell-based therapy of motorneuron degeneration. The profound changes of the local microenvironment, not only via their direct release of growth factors in situ, but also through the cross-talk with endogenous glial cells which amplify the beneficial effect of ASC, could have important implications for their future therapeutic use. The present results bring neurotrophic factors back into the spotlight and open the possibility of a novel form of treatment also for sporadic ALS. Especially striking was the effect of ASC on motor performance which remained for several weeks as high as in WT animals. These findings are encouraging for ALS patients, in whom current therapies are not able to reduce neurodegeneration.

Acknowledgments Supported in part by PRIN 2009 Grant, COMPAGNIA di San Paolo 2009 Grant, and European Research Council grant 261079- NEUROTRAFFICKING (G.C). We are grateful to Dr. Marina Bentivoglio for the critical reading of the manuscript and to Dr. Silvia Savazzi for the advice on statistical analysis support.

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Figure Legends

Figure 1: Neurophysiological assessment of mSOD1 mice and clinical effects of ASC treatment. (A) The registration of compound motor action potential (cMAP) amplitude, which reflects the number of lower motorneurons, is significantly reduced in mSOD1 mice compared to WT at day 70, 90 and 110; *p<0.001. (B) Cortical motor evoked potentials (cMEP) are significantly reduced in mSOD1 animals as compared to WT at day 70 and 90 and this correlate with progressive loss of cortical motorneurons; *p<0.05. (C) The Rotarod test persists enhanced for 5 weeks after injection in ACS-treated mSOD1 mice (black circle) in comparison to PBS-treated mSOD1 animals (white triangle; *p=0.026.

Figure 2: Motor performance deterioration is delayed after i.v. ASC-treatment. (A) The PGE test persists improved for 40 days after injection in ACS-treated mSOD1 (black circle) in comparison to PBS-treated mSOD1 animals (white square); the beneficial effect observed in ASCtreated animals rapidly disappears around day 120. *p=0.011. (B) Administration of ASC exerts a significant minor reduction of the distal cMAP amplitude in ASC-treated mice (black circle) as compared to controls (white square) and also the registration of cMEP (C) shows a similar trend with minor reduction of amplitudes in ASC-treated mSOD1 animals (black circle) in comparison to PBS-treated group (white square). *p<0.05.

Figure 3: Distribution analysis confirms the ability of GFP+ASC to home into damaged CNS. A restricted number of GFP+ cells is present both into white and gray matter parenchyma of the spinal cord, without evidence of neuronal differentiation: green-GFP+ASC are detectable in anterior horn of spinal cord close to red-ChAT+ neurons. Nuclei are stained in blue with DAPI; magnification 40x (A) and 63x (B). Scale bar: 40µm. (C) The distribution analysis upholds the 21

ability of GFP+ASC to migrate and persist into CNS both at day 100 and 135, and also to integrate into spleen and muscle tissue up to day 100.

Figure 4: Treatment with ASC increases motorneurons survival and reduces reactive astrogliosis. (A-C) A significant increase in the number of Nissl+ motorneurons per section is observed in ASC-treated mice compared to PBS-treated animals into spinal cord at day 100. (D-I) The analysis of astroglial and microglial activation at day 100 in cervical, dorsal and lumbar portion of spinal cord from ASC-treated mSOD1 mice shows a notable although not significant decrease of GFAP reactive astrocytes as compared to controls (D-F) while no difference in the number of microglial cells is observed in the spinal cord of ASC- and PBS-treated animals (G-I); magnification 20X. Scale bar: 30µm

Figure 5: Modulation the levels of neurotrophins in spinal cord tissue after systemic injection of ASC. (A) ASC-treatment induces a significant increase of GDNF and bFGF concentration into spinal cord at day 100 as compared to PBS-treated mice; tissue levels of BDNF and IGF-I are also increased in mice treated with ASC although the difference with PBS-treated animals is not significant. *p<0.005. (B) At end stage, ASC significantly modulate the local secretion only of bFGF in comparison to control animals. *p<0.001.

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Highlights: • Administration of ASC in SOD1(G93A) mice significantly delayed motor deterioration. • Neuropathological examination revealed a higher motoneurons in ASC-treated animals. • GFP+ASC were detectable in the anterior horn of spinal cord close to ChAT+ motoneurons. • We observed an increase in the number of motoneurons in ASC-treated mSOD1 mice. • We found an increase of neurotrophins into spinal cord of ASC-treated mice.

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