Human mesenchymal stem cell transplantation extends survival, improves motor
performance and decreases neuroin
flammation in mouse model of amyotrophic
lateral sclerosis
A. Vercelli
a,b,⁎
, O.M. Mereuta
a,b, D. Garbossa
a,b, G. Muraca
a,b, K. Mareschi
b, D. Rustichelli
b, I. Ferrero
b,
L. Mazzini
c, E. Madon
b, F. Fagioli
ba
Department of Anatomy, Pharmacology and Forensic Medicine, National Institute of Neuroscience, Italy
b
Department of Pediatrics, Regina Margherita Children's Hospital, University of Turin, Italy
c
Neurologic Clinic, Ospedale Maggiore, Novara, Italy
a b s t r a c t
a r t i c l e i n f o
Article history: Received 5 July 2007 Revised 5 May 2008 Accepted 22 May 2008 Available online 4 June 2008 Keywords: SOD1 Motoneuron Microglia Astrocyte Cell therapy NeuroprotectionAmyotrophic lateral sclerosis (ALS) is a lethal disease affecting motoneurons. In familial ALS, patients bear mutations in the superoxide dismutase gene (SOD1). We transplanted human bone marrow mesenchymal stem cells (hMSCs) into the lumbar spinal cord of asymptomatic SOD1G93Amice, an experimental model of
ALS. hMSCs were found in the spinal cord 10 weeks after, sometimes close to motoneurons and were rarely GFAP- or MAP2-positive. In females, where progression is slower than in males, astrogliosis and microglial activation were reduced and motoneuron counts with the optical fractionator were higher following transplantation. Motor tests (Rotarod, Paw Grip Endurance, neurological examination) were significantly improved in transplanted males. Therefore hMSCs are a good candidate for ALS cell therapy: they can survive and migrate after transplantation in the lumbar spinal cord, where they prevent astrogliosis and microglial activation and delay ALS-related decrease in the number of motoneurons, thus resulting in amelioration of the motor performance.
© 2008 Elsevier Inc. All rights reserved.
Introduction
Amyotrophic lateral sclerosis (ALS), Lou Gehrig's disease, is a devastating degenerative disease involving motoneurons, affecting around 2 people per 100,000 (Govoni et al., 2003). Motoneuron degeneration leads to weakness, muscle atrophy, fasciculations, spasticity (Rowland, 1998). Motoneuron degeneration arises from a complex cascade involving crosstalk among motoneurons, glia and muscles and evolving through the action of converging toxic mechanisms (Strong, 2003). 5–10% cases are familial, and mutations in any of several genes have been found in patients (Wang et al., 2007). One of these mutations involves the gene for Cu/Zn superoxide dismutase 1 (SOD1,Rosen et al., 1993). Expression of ALS-associated human SOD1 mutations (e.g. SOD1G93A) in mice produces a dom-inantly inherited syndrome with clinical and histopathological aspects of ALS (Doble and Kennel, 2000; Selverstone Valentine et al., 2005).
Treatments that interfere with a specific event in the neurotoxic cascade produce a modest increase in rodent lifespan, even though multi-intervention approaches have recently been shown to be more
effective (Carri et al., 2006). Trophic factors such as insulin-like growth factor (IGF-1, Nagano et al., 2005) and vascular endothelial growth factor (VEGF,Storkebaum et al., 2005; Wang et al., 2007) can delay the progression of the disease in ALS animal models. Stem cell transplanta-tion is an attractive strategy for neurological diseases as an alternative or in addition to pharmacological treatments (Mazzini et al., 2003, 2004; Silani et al. 2004; Svendsen and Langston, 2004; Corti et al., 2007). Stem cells could replace or protect motoneurons by releasing neurotrophic factors. Wild-type cells are able to protect the motoneurons carrying the mutation, whereas mutant protein-expressing cells can induce pathol-ogy in wild type motoneurons (Clement et al., 2003). Human neural stem cell grafts ameliorate motoneuron disease in SOD1 transgenic rats (Xu et al., 2006). Moreover, whole bone marrow transplantation has been shown to delay the disease onset and to increase lifespan in SOD1G93Amice, and to participate in striated muscle regeneration (Corti et al., 2004a, 2004b, 2007). This strongly suggests that the environment of the dying neuron is crucial to its ability to function and survive.
We tested the effects of injecting human mesenchymal stem cells (hMSCs) into the spinal cord on the progression of ALS in SOD1G93A
mice. We previously reported that hMSCs in culture can express neural markers and neural ion channels, and produce trophic factors (Mareschi et al., 2006). MSCs show unique immunologic properties for cellular therapy: they are not immunogenic, do not stimulate alloreactivity and escape lysis by cytotoxic T-cells and natural killer ⁎ Corresponding author. Dipartimento di Anatomia, Farmacologia e Medicina Legale,
corso M. D'Azeglio 52, 10126 Torino, Italy. Fax: +39 011 2367700. E-mail address:alessandro.vercelli@unito.it(A. Vercelli). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$– see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2008.05.016
Contents lists available atScienceDirect
Neurobiology of Disease
(NK)-cells (Di Nicola et al., 2002; Le Blanc, 2003; Aggarwal and Pittenger, 2005). Furthermore,Mazzini et al. (2003, 2006)reported a good tolerance and a possible beneficial effect of injected autologous hMSCs after expansion in vitro into the surgically exposed spinal cord at T7–T9 levels in a few well-monitored ALS patients.
Materials and methods
Preparation of human mesenchymal stem cells hMSC isolation and expansion
Bone marrow (BM) was obtained by aspiration from the posterior iliac crest of healthy donors after informed consensus. BM cells were layered over a 1.073 g/ml Percoll gradient (Sigma, St. Louis, MO, USA) according to a previously reported method (Mareschi et al., 2001). Briefly, the cells in the interface were collected, washed twice in the 0.1 M phosphate-buffered saline (PBS; pH 7.4), plated in MSC medium (Cambrex Bio Science, Walkersville, MD, USA) at 8 × 105
cells/cm2in T
flasks and maintained at 37 °C in an atmosphere of 5% CO2. After 3 days the non-adherent cells were removed and replaced
with fresh culture medium. Subsequent complete medium changes were performed every 4 days. After 15 days for thefirst passage and every week for the following passages, BM cells were detached by treatment with 0.25% trypsin containing 0.01% EDTA for 10 min at 37 °C. They were plated at a density of 8000/cm2and expanded for several passages up to prevent the confluence. The cells were analysed for their viability and for their immunophenotype byflow cytometry.
Characterization of hMSCs
Theflow cytometry analysis of adherent hMSCs was performed at each passage. 200,000–500,000 cells were incubated for 20 min with anti-CD45FITC/CD14PE, CD90FITC/CD106PE, CD29FITC/CD44PE, CD166FITC/CD105PE antibodies for the identification of the immuno-phenotype and with 7-aminoactinomycin D (7-AAD; Becton Dick-inson, San Jose, CA, USA) for their viability. Labelled cells were thoroughly washed with PBS 1× (Cambrex Bio Science, Verviers, Belgium) and analysed on an Epics XL cytometer (Beckman Coulter, CA, USA) with the XL2 software program. The percentage of positive cells was determined based on the fluorescent emission of the nonspecific FITC/PE isotypic antibody controls. The cells which were negative for CD45 and CD14 and positive for the other markers were considered hMSCs.
Prelabelling of hMSCs
Cells expanded for thefirst 3–8 passages were labelled by adding to the medium 10μg/ml bisbenzimide (Sigma, St. Louis, MO, USA), which binds to DNA, 24 h before transplantation. Then the cells were detached with Trypsin/EDTA, washed and resuspended in saline solution to obtain afinal concentration of 50,000 cells/μl to be used for transplantation.
Animal model
Transgenic B6SJL-TgN(SOD1-G93A)dl1Gur/J mice overexpressing
human SOD1 carrying the Gly93 to Ala mutation (G93A) were used in this study. The breeding pairs were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and the transgenic mice were genotyped using a described polymerase chain reaction (PCR) method (Rosen et al., 1993). These mice develop initial signs of neuromuscular deficits at 34.4±3.4 weeks and die at 37.9±3.0 weeks of age. Beginning at 34 weeks of age, food and water were routinely placed in the cages in order to get easy access. All procedures involving the use of live animals were performed under the supervision of a licensed veterinarian, according to the guidelines specified by the Italian Ministry of Health (DDL 116/92).
hMSC transplantation
A series of SOD1G93Amice were used for pilot studies in order to
understand the best site of injection, the diffusion of transplanted cells into the host and their potential for differentiation into neural cells. We decided to use SOD1G93Aand not wild type mice since we wanted
to test these issues directly in pathological mice, hypothesizing that the pathological brain can stimulate and attract hMSCs.
hMSCs (105cells) were transplanted in the lumbar spinal cord of the SOD1G93Amice aged 28 weeks, before ALS symptoms became manifest.
The lumbar region was chosen for transplantation due to its early involvement in this ALS model. In a separate set of experiments hMSCs were transplanted into the cisterna magna or in the spinal subarachnoideal space, but these sites of injection were abandoned since no transplanted cells were found into the spinal cord after sacrifice at 38 weeks.
The study included SOD1G93A mice transplanted with hMSCs
(n = 25) and sham-operated transgenic mice (n = 12). We also con-sidered the sex of the mice (n = 19 males and n = 18 females), since it has been reported that the progression has a different time course in the two sexes (Suzuki et al., 2007).
Under chloral hydrate anesthesia (250 mg/kg body weight of a 0.3% solution in saline), the lumbar spinal cord was exposed through a laminectomy 2 cm under the intersection of the line connecting the inferior angles of the scapulae with the vertebral column. 2μl of hMSC suspension (transplanted animals) or saline (sham-operated animals) was gently injected into the L1–L2 neuromers, using a glass micropipette (outer diameter = 30μm) connected to a syringe body by a silicon tube.
This surgical procedure was set up before transplantation in adult mice by injecting at different levels the fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, 5% dissolved in dimethylformamide; Molecular Probes, Eugene, OR, USA), and observing the location of the injection site in the spinal cord at thefluorescent microscope after sacrifice (data not shown).
We decided to sacrifice the mice at the age of 38 weeks, the mean age of death in transgenic mice. Animals were killed by an overdose of anesthetics, perfused through the ascending aorta with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3). Since several mice died before this age, those which were found dying were killed immediately, whereas those found dead were dissected and immersionfixed.
Tissue analysis Tissue processing
The lumbar spinal cord (between the end of T13 and the beginning of L4, including the lumbar enlargement) was dissected and isolated, immersed in the samefixative for 1 h, cryoprotected in 30% sucrose solution in PB overnight and frozen in cryostat medium (Bio-Optica, Milan, Italy). The tissue was cut in coronal 50μm-thick serial sections. They were mounted onto 1% gelatine-coated slides, which were stored at−20 °C until they were reacted for histology or immunofluorescence labelling.
Quantification of alpha motoneurons
Quantification was performed only in mice which survived 38 weeks and which showed surviving hMSCs into the lumbar spinal cord. One series of serial lumbar sections (one every 600μm) was stained with cresyl violet. The nucleoli of the neurons in the ventral horns of the spinal cord were counted at 40×, in a total number of 12 sections/mouse. Only neurons with an area≥200 μm2
, classified as alpha motoneurons, were counted (Ciavarro et al., 2003). A total estimated number of alpha motoneurons was obtained with a stereological technique, the Optical Fractionator (West et al., 1991), by using a computer-assisted microscope and the StereoInvestigator software (MicroBrightField, Williston, VT, USA). Cells were counted on
the computer screen using an Optronics MicroFire digital camera mounted on a Nikon Eclipse E600 microscope.
Briefly, the Optical Fractionator is a stereological method for obtaining estimates of the total number of neurons. It is a combination of a three-dimensional probe for counting neuronal nuclei, the Optical Dissector, and a systematic uniform sampling scheme, the Fractio-nator. To determine the number of alpha motoneurons, we used a modified version of the fractionator principle. The rationale for this modification lies in the fact that the alpha motoneurons are not evenly distributed in the ventral horn of the spinal cord. When an automated stereology system, such as StereoInvestigator, is used, this leads to a high number of counting frames, most of which do not contain alpha motoneurons. Only the cells in the uppermost focal plane were excluded to avoid oversampling, but the alpha motoneurons were otherwise counted exhaustively in the ventral horn of T13–L4 lumbar segment. The total number of alpha motoneurons was then estimated by multiplying the resulting counts by 12, because only every twelve section had been used for counting. The numbers obtained in our study are thus absolute numbers and are independent of the volume of the spinal cord. The guard zones were of 5μm and the scan grid size was 240 × 160μm, the optical dissector=mean section thickness, with a Gundersen coefficient error ranging from 0.04 to 0.1.
Immunofluorescence labelling to analyse reactive astrogliosis
A series of sections (one every 600μm) from 10 transplanted and 5 sham-operated females and from 8 transplanted males was incubated for 20 min at room temperature (r.t.) with PBS containing 0.3% Triton X-100 (Sigma, St. Louis, MO, USA) to increase membrane permeability. After rinsing in PBS, sections were incubated in PBS-0.1% Triton X-100 with 10% normal donkey serum (NDS; Sigma, St. Louis, MO, USA) for 30 min at r.t. to prevent non specific binding (blocking solution). Sections were incubated overnight at 4 °C in PBS-0.1% Triton X-100 with 50% blocking solution and 1:500 rabbit anti-glial fibrillary acid protein (GFAP; DAKO, Denmark). After rinsing in PBS, the primary antibodies were detected using Cy3-conjugated donkey anti-rabbit IgG (H + L) 1:400 in PBS-0.1% Triton X-100 with 2% NDS for 1 h at r.t. Controls were performed by eliminating the primary antibody. The slides were washed, mounted in PBS-glycerol (1:1) and observed with a Nikon Eclipse E800 epifluorescence microscope under appropriate filter sets. GFAP immu-noreactivity was analysed in one section every 1200μm to evaluate reactive astrogliosis. The ventral horns were photographed using a Nikon Coolpix 995 digital camera at 40×, obtaining 24 photos for each animal. The percentage of the total area which was GFAP-positive was quantified using the Scion Image for Windows (freeware version of NIH image, Scion Corporation, Frederick, MD, USA).
Immunohistochemistry for microglial antigen (CD11b)
In order to detect microglial activation, other sections from 4 transplanted and 4 sham-operated females and from 4 transplanted males were incubated overnight at 4 °C with rat anti-mouse polyclonal antibody CD11b (Serotec Ltd, Oxford, UK) diluted 1:100 in PBS-0.1% Triton X-100 with 2% normal goat serum (NGS Sigma), and 2 h r.t. in 1:50 biotinylated anti-rat secondary antibody (Serotec Ltd) followed by avidin–biotin–peroxidase complex (ABC) solution (Vec-stain ABC Kits, Vectors Laboratories Inc., Burlingame, CA) and then with peroxidase substrate kit Vector SG (Vectors Laboratories Inc.) until staining was optimal as determined by light microscopic examination. Labelled sections were washed in PBS and mounted onto 1% gelatine-coated slides, air-dried overnight, dehydrated and coverslipped with Eukitt (O Knidler GmbH and Co., Freiburg, Germany). CD11b immunoreactivity was also quantified as above in order to evaluate microglial activation.
Immunohistochemistry
In order to study the differentiative potential of hMSCs some sections, in which bisbenzimide-labelled cells had been detected, were
immunoreacted against microtubule-associated protein 2 (MAP2, 1:10 monoclonal antibody AP18, a generous gift from Riederer BM, IBCM, University of Lausanne, Switzerland) and GFAP, in search for double (bisbenzimide and MAP2 or GFAP) labelled hMSCs. We counted the number of MAP2 or GFAP-positive MSCs over a total number of four hundred bisbenzimide-positive nuclei for each marker.
Clinical tests
In order to analyse the effect of MSC transplantation on the onset and progression of motor symptoms in this disease model, we performed four clinical tests: i) scoring of motor deficits by a trained observer, ii) weighing and iii) performance on the Rotarod task, all of which are commonly used to evaluate SOD1G93Aanimals (Weydt et al., 2003). In addition, we investigated the iv) Paw Grip Endurance (PaGE) test which has the dual advantage of measuring motor strength directly while requiring only minimal equipment (Weydt et al., 2003). Beginning at 24 weeks, the animals (sham-operated— three males and 5 females, and transplanted— 9 males and 10 females) were assessed weekly with the behavioral tests in randomized order by an observer blinded to the treatment. Thefirst 3 weeks of tests were considered as training.
The mice were evaluated for signs of motor deficit with the following 4 point scoring system: 4 points if normal (no sign of motor dysfunction); 3 points if hind limb tremors are evident when suspended by the tail; 2 points if gait abnormalities are present; 1 point for dragging of at least one hind limb; 0 points for inability to right itself within 30 s. Onset was defined retrospectively as the earliest time when the mice showed symptoms (scoreb4) for ≥2 consecutive weeks.
For the Rotarod test the time for which an animal could remain on the rotating cylinder was measured in a 7650 accelerating model of a Rotarod apparatus (Ugo Basile, Italy). Each animal was given three trials and the longest latency to fall was recorded; 180 s was chosen as the arbitrary cut-off time.
For the PaGE test each mouse was placed on the wire-lid of a conventional housing cage. The lid was gently shaken to prompt the mouse to hold onto the grid before the lid was swiftly turned upside down. The latency until the mouse lets go of the grip with at least both hind limbs was timed. Each mouse was given up to three attempts to hold on to the inverted lid for an arbitrary cut-off time of 90 s and the longest latency was recorded.
In motor tests some treated and control animals never achieved respective cut-off times even though they behaved otherwise normal. To eliminate this variability in maximal performance, all data were normalized to the maximal value achieved by each mouse during the whole period examined. Body weight was normalized to the weight at 27 weeks. The animals were considered end-stage when they reached a motor score of 0 points or lostN20% of their body weight, whichever occurredfirst.
Statistical analysis
Data were given as mean ± standard deviation or standard error of mean and assessed by Student's t-test and ANOVA in order to compare the values of motoneuron counts, reactive astrogliosis and microglial activation obtained for the sham-operated and MSCs transplanted females and males, respectively. pb0.05 was considered significant. Results
hMSC characterization
The cells isolated from bone marrow were hMSCs showing the specific features defined by the International Society for Cellular Therapy guidelines (Dominici et al., 2006). In fact, they were adherent
cells positive for CD90, CD106, CD29, CD44, CD105, CD166 markers, negative for the hemopoietic markers such as CD45 and CD14. Moreover, these cells were able to differentiate in osteoblasts, chondroblasts and adipocytes after exposure to specific conditioning media as previously shown (Mareschi et al., 2001; Ferrero et al., 2008). hMSC migration and expression of neural markers
When hMSCs were injected into the cisterna magna, or in the spinal subarachnoideal space, no bisbenzimide-positive nuclei were found within the spinal cord (Fig. 1). Some bisbenzimide-positive nuclei were found on the surface of cerebral cortex, or on the pial meninge, or, rarely, in the hippocampus. Therefore we decided to consider only mice which were injected into the spinal cord.
MSCs were identified as cell aggregates distributed close to the injection site, especially in the dorsal column and median posterior commissure of white matter. Cells were also recognized in the ventral horn of the lumbar spinal segment (degeneration area). Some of them were found at a few microns from the motoneurons (Fig. 2). This suggested that MSCs could migrate far from the injection site into the subarachnoidal space and also along the dorsoventral axis of the spinal cord. In some cases, cells migrated along the craniocaudal axis of the spinal cord for a distance of 1550μm from the injection site. Rare (b1%) GFAP (Figs. 2A–C) or (b1%) MAP2 (Figs. 2D–F) bisbenzi-mide-positive cells were found.
Motoneuron counts
Motoneurons were clearly recognized for their large size, for their intensely Nissl-stained cytoplasm, and for their prominent nucleolus. The
difference in the number of motoneurons between transplanted and sham-operated SOD1G93Amice was evident in histological sections and
was quantified. Stereological counts of motoneurons showed a significant difference (pb0.05) in their number between transplanted and sham female SOD1G93Amice (Figs. 3A and B respectively). Sham-operated
female SOD1G93A mice at 38 weeks of age had 3549±607 (mean±
standard error, 4 mice) motoneurons, whereas female SOD1G93Amice
which had received a hMSC injection at 28 weeks displayed 5458±682 (mean ±standard error, 7 mice) at 38 weeks of age. Unfortunately, no sham-operated male SOD1G93Amice survived up to 38 weeks of age, and
only three transplanted male SOD1G93A mice survived to allow cell
counts, which gave 3766±980 motoneurons. Nevertheless, counts of motoneurons in six sham-operated male SOD1G93A mice which died
between 35 and 37 weeks of age gave 1489±408 (pb0.05). Microglial activation
We investigated the effect of transplanted hMSCs on the microglial activation. The immunohistochemistry for CD11b, a characteristic marker of microglial cells, showed a reduced level of the activated microglia in the treated mice (Figs. 4A–D). This was confirmed by the quantitative analysis of the reaction in both transplanted and control mice (one-way Student's t-test pb0.01) (Fig. 4G). In fact, whereas sham-operated females had a 30.73 ± 1.99 value, treated females had a value of 19.612 ± 0.251 and treated males 22.674 ± 1.015.
Reactive astrogliosis
GFAP-positive profiles are directly proportional to reactive astro-cytes and the role that these cells may play in the pathogenesis of the
Fig. 1. Localization of transplanted hMSCs. Bisbenzimide-stained nuclei in the lumbar spinal cord (A, B) following intraparenchimal injection and on cerebral cortex (C, D) following injection in the cisterna magna. In A, injection site at small magnification. In C, transplanted hMSCs are located on the pial surface, whereas in D they enter the superficial cortical layers. Scale bars = 100 (A, B) and 50 (C, D)μm.
Fig. 2. Expression of neural markers by transplanted hMSCs. Double stained cells for bisbenzimide and GFAP (A–C) and MAP2 (D–F) in transverse sections of lumbar spinal cord. In G, H, localization of bisbenzimide-positive cells (blue) close to motoneurons (stained with MAP2 antibody, green). Scale bars = 30 (A–F), 50 (G) and 20 (H) μm.
Fig. 3. Motoneurons in the lumbar spinal cord. Transverse sections of transplanted (A) and sham-operated (B) female SOD1G93A
mice at 38 weeks of age. Motoneuron profiles are clearly reduced in number in B compared to A. Scale bar = 200μm.
Fig. 4. Quantification of microglial activation and of reactive astrogliosis. CD11b-immunohistochemistry at low (A, B) and high (C, D) magnification in hMSC transplanted (A and C) and sham-operated (B and D) SOD1G93A
mice. In E and F GFAP-immunofluorescence in hMSC transplanted (E) and sham-operated (F) SOD1G93A
mice. Scale bars = 200 (A, B) and 50 (C–F) μm. In G and H quantification of CD11b- and GFAP-positive profiles, respectively, in sham-operated females (dashed bar), hMSC transplanted females (empty bars) and hMSC transplanted males (filled bars).
disease. Reactive astrocytosis refers to the increase in number and the hypertrophy of astrocytes expressing GFAP and is known to occur in response to neural injuries. Using this quantitative parameter, we investigated if the release of neurotrophic factors by the transplanted MSCs could reduce the reactive astrogliosis (Figs. 4E, F).
We observed a significant reduction of the GFAP reactivity in the transplanted females compared with the control ones (one-way Student's t-test pb0.01). Because of the post-mortem degradation, it was not possible to analyse the reactive astrocytosis for the sham-operated males which died before 38 weeks of age. Anyway, we report a higher percentage of reactive astrocytosis for the transplanted males (2.52 ± 0.79) compared to the transplanted females (1.8 ± 0.25) and also a reduction of GFAP expression for the transplanted males compared to sham-operated females (3.3 ± 0.25). The statistical analysis includ-ing the three groups of animals resulted significant (one-way Student's t-test pb0.01) (Fig. 4H).
Behavioral analysis
We evaluated the effects of the transplanted hMSCs on the progression of behavioral deficits. The statistical analysis considered the results obtained beginning with the 27th week of age. All data were normalized to the maximal value achieved by each mouse during the whole period examined since some treated and control animals never achieved respective cut-off times (180 s for Rotarod test and 90 s for PaGE test). Body weight was normalized to the weight at 27 weeks. Thefirst clinical signs of motor neuron disease in SOD1G93Amice
werefine hind limb tremors (Figs. 5A and B). The treated males began to display hind limb tremors (motor score = 3 points) around 32– 35 weeks of age and achieved a motor score of 1 or 2 points at the sacrifice date (Fig. 5A). The control males (Fig. 5A) showed gait abnormalities (motor score = 2 points) starting already from 30 to 32 weeks. Two of them died spontaneously before 38 weeks of age Fig. 5. Behavioral tests. Results obtained with neurologic (A, B), Rotarod (C, D) and PaGE (E, F) tests on male (A, C, E) and female (B, D, F) SOD1G93Amice, from 27 to 38 weeks of age.
Dashed line corresponds to sham-operated mice, whereas continuous line corresponds to hMSC transplanted mice. Bars are standard deviations. Data are expressed as motor score for neurologic test, and as percentage of the performance at 27 weeks of age for Rotarod and PaGE tests.
and the other one reached a motor score of 0 points at week 37. Almost all of the transplanted females registered a motor score of 4 points until 38 weeks of age, whereas the sham-operated females showed signs of motor dysfunction (motor score = 2 or 3 points) at the 37th week of age (Fig. 5B). Regarding the weight loss characteristic for the disease progression, no difference was observed in treated mice compared to control ones (data not shown).
In the Rotarod task (Fig. 5D), the transplanted females sustained their maximal performance level, whereas the performance of the sham-operated females began to decline starting with the 35th week. However, this difference was not statistically significant. The results were similar for the PaGE test (Fig. 5F). No survival improvement was described for treated females. On the contrary, the Rotarod performance curve showed a different time course for treated and control males. Around week 28 the performance of the sham-operated males began to decline slowly and this difference became significant at 32 weeks of age. The control males failed the test after week 35 (Fig. 5C).
In the PaGE task, starting with the 34th week the transplanted males showed a reduced performance of 60–70% whereas the control ones failed the test (Fig. 5E). Treated males also displayed an improved survival: 40% survived to 38 weeks, whereas all sham-operated males died before that age.
Discussion
Our study provides evidence that mesenchymal stem cells are a good candidate for cell therapy in ALS: i) they can survive and migrate after transplantation in the lumbar spinal cord of presymptomatic SOD1G93A mice, where they ii) prevent astrogliosis and microglial
activation and,finally, iii) delay ALS-related decrease in the number of motoneurons, thus resulting in iv) an amelioration of the motor performance.
Methodological issues
Since hMSC injections in the cisterna magna did not obtain hMSCs migration into the spinal cord, we injected hMSCs intraparenchymally into the lumbar spinal cord. Labelling with bisbenzimide allowed their localization, even several months after injection. We never observed macrophages or glial cells with bisbenzimide-positive inclusions, nor large bisbenzimide-positive motoneurons, as it could be hypothesized in case of fusion with host progenitors or mature cells (Alvarez-Dolado et al., 2003; Kozorovitskiy and Gould, 2003; Horvath et al., 2006). Diffusion and long term survival of transplanted hMSCs
hMSCs display a limited proliferation in vivo: we never found dividing bisbenzimide-positive nuclei. Moreover, we observed a limited diffusion of hMSCs, in agreement with previous reports in which MSCs engraftment was less efficient in the adult than in the neonatal central nervous system, since they express neural adhesion proteins and receptors which regulate neural cell migration into the brain (Phinney et al., 2006). hMSCs transplanted into the lumbar spinal cord survive for long periods (more than 10 weeks), without immunosuppression (in agreement withLiu et al., 2006b), and diffuse 1–2 mm from the injection site. Several bisbenzimide-positive nuclei were found close to motoneurons, where hMSCs can deliver trophic and immunomodulatory factors. Allotransplantation seems to improve MSC survival without immunosuppression (Xu et al., 2006). Differentiation of hMSCs into neural cells
hMSCs under specific culture conditions can express neural markers normally expressed at various stages of neural development, such as GFAP, Nestin, Tuj-1, Tyrosine Hydroxylase and MAP2 (
Wood-bury et al., 2000; Black and WoodWood-bury, 2001; Deng et al., 2001; Kohyama et al., 2001; Minguell et al., 2001; Kim et al., 2002; Tondreau et al., 2004; Mareschi et al., 2006). At RT-PCR, undifferentiated hMSCs expressed mRNA for MAP-2, neuron-specific enolase and neurofila-ment-M (Mareschi et al., 2006), Nestin andβIII tubulin (Tondreau et al., 2004). Moreover, electrophysiology on differentiated hMSCs found K+channels usually expressed in cerebral cortex (Mareschi et al., 2006). Our experiments in vivo, in agreement with others (Brazelton et al., 2000; Sanchez-Ramos et al., 2000; Munoz-Elias et al., 2004; Bonilla et al., 2005; Xu et al., 2006), seem to support a neural differentiation of hMSCs, as shown by GFAP and MAP2 immunohistochemistry.
On the other hand, the morphological changes and the increased immunoreactivity for neural markers following chemical induction might be consequent to cellular toxicity and related cytoskeletal changes (Lu et al., 2004; Neuhuber et al., 2004; Bertani et al., 2005, Kim et al., 2006). Massive MSC death has been described with transfer of donor labels into host macrophages, glial cells and neurons (Coyne et al., 2006). In addition, BMCs do not differentiate into astrocytes (Wehner et al., 2003). Therefore, neural differentiation has been questioned (Castro et al., 2002; Vallières and Sawchenko, 2003).
MSCs are undifferentiated cells which express at very low levels markers for different cell lines and can be induced to increase the expression of some markers (Minguell et al., 2005; Blondheim et al., 2006), which could also explain our results in vivo.
We never found CD11b-positive bisbenzimide-positive cells, therefore we tend to exclude a hMSC differentiation into microglia, as supported by the high selectivity of the process of cell isolation and characterization.
For the above reasons, we support the idea that MSC transplanta-tion more than replacing lost neurons increases neuron survival and prevents astrogliosis and microglia activation (see below) (Lepore and Maragakis, 2007; Nayak et al., 2007).
Gender issues
ALS is more frequent and rapidly progressive in males than in females (Mahoney et al., 2004; Suzuki et al., 2007). Estrogen represents a neuroprotectant in both the adult and the aging brain, both in vitro and in vivo (Suzuki et al., 2006).
Therefore, we grouped the experimental animals according to their sex, and found significant differences in survival, motor performance and histological parameters. In sham-operated groups the males died long before females and following transplantation had consistently fewer motoneurons than age-matched females, and a higher density of CD11b- of GFAP-positive profiles. This led to important decrease in motor performance in males. On the other hand, in male mice, the disease often brought the animals to death before the age we decided to examine their histology. In females the progression of the disease was slower, such that histological changes were present in the absence of motor deficits. Therefore, we studied the effect of hMSC transplantation on motor performance and on survival in males, and that on histological parameters in females. Interestingly, we found a higher percentage of the reactive astrogliosis for the transplanted males compared to the transplanted females and a reduction of GFAP expression for the transplanted males compared to sham-operated females.
Effect of hMSCs on the neuroinflammation
Neuroinflammation is a major event in ALS (Obal et al., 2001; McGeer and McGeer, 2002). Neuroinflammation is sustained by the interaction of microglia, neurons and macroglia (astrocytes and oligodendrocytes): astrocytes are both the target and the source of neuroinflammation, since astrocytes stimulated by mediators released from microglia down-regulate the expression of neurotrophic factors
and release additional inflammatory mediators, which in turn further activate microglia (Weydt and Moller, 2005; Borchelt, 2006). Reactive astrocytosis is present in the presymptomatic stage and gradually increases to end-stage disease (Feeney et al., 2001). Microglia participates in the pathogenesis of neurological disorders (Weydt and Moller, 2005). Experimental evidence from transgenic animals implicates microglia in the pathogenesis of ALS. Several studies demonstrated that the expression of proinflammatory mediators is an early event in murine ALS, even preceding the development of clinical signs. Nitric oxide, a gaseous neurotransmitters involved in both neuroprotection and excitotoxicity, produced in both glia and neurons, is upregulated in the spinal cord of SOD1G93Amice (Phul et al., 2000; Urushitami and Shimohama, 2001).
We observed reactive astrogliosis and microglial activation in the SOD1G93A mice, and that hMSC transplantation modulates
neuroin-flammation by reducing microglial activation and astrocytosis as shown by quantification in transplanted SOD1G93Acompared to
sham-operated mice. The immunosuppressive role of hMSCs, in addition to the neuroprotective one, is confirmed by their efficacy in treating multiple sclerosis (Uccelli et al., 2006). A role in preventing astrogliosis and microglial activation has been hypothesized also for neural stem cells (reviewed inChristou et al., 2007).
Neuroprotective effect of MSC transplantation
MSCs can be considered as trophic mediators (Caplan and Dennis, 2006) via the production of an assortment of cytokines, of the angiogenetic VEGF, of the prosurvival gene Akt1. MSCs can stimulate neural stem cells (Lou et al., 2003). MSCs can be genetically modified to produce and deliver neurotrophic factors in loco (Hamada et al., 2005; Kurozumi et al., 2005), or angiogenic factor (Liu et al., 2006a) respectively to protect neurons and favor revascularization in neurodegenerative diseases (McMahon et al., 2006). Subclones of MSCs already produce brain-derived neurotrophic factor andβ-nerve growth factor (Crigler et al., 2006).
We have shown that following hMSC transplantation the progres-sion of motoneuron cell death is consistently (35%) delayed in transplanted SOD1G93A females compared to the sham-operated. Unfortunately, SOD1G93Asham-operated males died long before the
sacrifice date to count motoneurons: nevertheless, counts of moto-neurons in these mice gave a significant difference if compared to data obtained in transplanted males. Moreover, the transplanted males displayed a motoneuron loss similar to the sham-operated females and more consistent than in the transplanted females, confirming the more severe disease course in the SOD1G93A
males. Trophic factors produced by MSCs such as VEGF (Caplan and Dennis, 2006) or BDNF (Crigler et al., 2006) can support motoneuron survival both diffusing at distance and by local interaction with motoneurons, since we have observed bisbenzimide-positive nuclei close to motoneurons, where they can exert their paracrine function (Xu et al., 2007). In addition, transplanted hMSCs can provide motoneurons with wild type SOD. Similar results were reported by other authors using neural stem cells which secrete GDNF (reviewed inChristou et al., 2007; Hedlund et al., 2007).
Effects of MSC transplantation on motor behavior
From the 5th month of age SOD1G93A mice display impaired
exploratory activity and motor coordination (Lalonde et al., 2005), followed by asymmetric hind limb weakness, spasticity and atrophy, exacerbated by high intensity endurance exercise training in males (Mahoney et al., 2004). We have shown that, in males, motor behavior is dramatically impaired from 32 weeks of age, significantly delayed by hMSC transplantation. All parameters here considered, scoring of motor deficits, performance on the Rotarod task and PaGE test were able to differentiate between treated and untreated male mice (Weydt
et al., 2003). hMSC transplantation had no effects on motor behavior in female at the age here considered, when sham-operated females also had normal motor performance.
Conclusions
Our study clearly supports stem cell therapy as a promising tool in the treatment of neurodegenerative diseases. In agreement with others, it suggests that the intraspinal transplantation is the most efficient way of delivering MSCs (similarly for hNT neurons,
Garbuzova-Davis et al., 2001). Moreover, hMSCs can be transplanted in the absence of immunosuppression, probably due to their immunomodulatory capability itself and to the use of MSCs from other species. MSCs can be considered as biologic minipumps migrating close to motoneurons affected by the disease, delivering trophic factors and immunomodulatory molecules. MSC therapy could be exploited by inserting genes coding for specific neurotrophic factors or immunomodulatory molecules. The low level of prolifera-tion represents an advantage on other stem cells, since we never observed the formation of teratomas.
In addition, whereas the use of neural stem cells requires heterologous transplantation and embryonic donors, and further information is needed on the mechanisms leading to cell differentia-tion in order to provide cell replacement (Christou et al., 2007; Hedlund et al., 2007), MSCs can be collected from the patient itself and have been used since a long time in bone marrow transplantation thus reducing concerns about their safety.
One limitation of this study is the use of human MSCs in a rodent: of course, signaling molecules, cytokines, and factors might have species-specificities either in their biochemical composition or in their receptors and signaling cascades. On the other hand, we needed to test safety of hMSCs on an animal model before performing tests in humans. In addition, the major issue in this therapeutic protocol consists in the way of administration, a rather invasive approach including laminectomy and opening of the meninges in patients which are already affected by a devastating disease. Nevertheless, clinical trials have been authorized and some patients have been intraspinally administered with hMSCs with very few minor side effects (Mazzini et al., 2006). Experimenting intravenous delivery, eventually after engineering MSCs with neural adhesion genes to exploit their extravasation and intraparenchimal migration into the nervous system, could allow a less invasive approach.
Acknowledgments
Supported by grants to F. Fagioli, E. Madon and A. Vercelli from Compagnia di San Paolo, Italian Ministry of Health and Vialli-Mauro Foundation. We are grateful to Dr. Ferdinando Rossi for the critical reading of the manuscript.
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