Human mesenchymal stem cell transplantation extends survival, improves motor
performance and decreases neuroinflammation in mouse model of amyotrophic
A. Vercellia,b,⁎, O.M. Mereutaa,b, D. Garbossaa,b, G. Muracaa,b, K. Mareschib, D. Rustichellib, I. Ferrerob,
L. Mazzinic, E. Madonb, F. Fagiolib
aDepartment of Anatomy, Pharmacology and Forensic Medicine, National Institute of Neuroscience, Italy
bDepartment of Pediatrics, Regina Margherita Children's Hospital, University of Turin, Italy
cNeurologic Clinic, Ospedale Maggiore, Novara, Italy
a b s t r a c t a r t i c l e i n f o
Received 5 July 2007
Revised 5 May 2008
Accepted 22 May 2008
Available online 4 June 2008
Amyotrophic 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.
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 anyof several geneshavebeen found inpatients (Wanget 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 inheritedsyndromewithclinical andhistopathologicalaspects
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-
in addition to pharmacological treatments (Mazzini et al., 2003, 2004;
cells could replace or protect motoneurons by releasing neurotrophic
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 instriatedmuscleregeneration (Corti
et al., 2004a,2004b, 2007). This stronglysuggests 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
Neurobiology of Disease 31 (2008) 395–405
⁎ Corresponding author. Dipartimento di Anatomia, Farmacologia e Medicina Legale,
corso M. D'Azeglio 52, 10126 Torino, Italy. Fax: +39 011 2367700.
E-mail address: email@example.com (A. Vercelli).
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi
(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 the first 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 by flow
Characterization of hMSCs
The flow 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
Prelabelling of hMSCs
Cells expanded for the first 3–8 passages were labelled by adding
to the medium 10 μg/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 a final concentration of 50,000 cells/μl to be used for
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).
A series of SOD1G93Amice were used for pilot studies in order to
understandthebest site of injection,thediffusion 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
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 SOD1G93Amice 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).
Underchloral hydrateanesthesia(250 mg/kg body weightof 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. 2 μl of hMSC
suspension (transplanted animals) or saline (sham-operated animals)
was gently injected into the L1–L2 neuromers, using a glass
micropipette (outer diameter=30 μm) 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 the fluorescent 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 byan 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 immersion fixed.
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 same fixative 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 50 μm-thick serial sections.
Theywere mounted onto 1% gelatine-coatedslides, which were stored
at −20 °C until they were reacted forhistologyor immunofluorescence
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 600 μm) 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
A. Vercelli et al. / Neurobiology of Disease 31 (2008) 395–405
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
obtainingestimates 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 5 μm and the scan grid size
was 240×160 μm, 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 600 μm) 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
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
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 1200 μm 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.
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.
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. The first 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 (score b4) for ≥2
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 tothe weight at
27 weeks. The animals were considered end-stage when they reached
a motor score of 0 points or lost N20% of their body weight, whichever
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.
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
A. Vercelli et al. / Neurobiology of Disease 31 (2008) 395–405
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 1550 μm from the injection site.
Rare (b1%) GFAP (Figs. 2A–C) or (b1%) MAP2 (Figs. 2D–F) bisbenzi-
mide-positive cells were found.
Motoneurons were clearly recognized for their large size, for their
difference in the number of motoneurons between transplanted and
sham-operated SOD1G93Amice was evident in histological sections and
difference (pb0.05) in their number between transplanted and sham
female SOD1G93Amice (Figs. 3A and B respectively). Sham-operated
female SOD1G93Amice 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 SOD1G93Amice survived to allow cell
counts, which gave 3766±980 motoneurons. Nevertheless, counts of
motoneurons in six sham-operated male SOD1G93Amice which died
between 35 and 37 weeks of age gave 1489±408 (pb0.05).
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.
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.
A. Vercelli et al. / Neurobiology of Disease 31 (2008) 395–405
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 SOD1G93Amice at 38 weeks of age. Motoneuron profiles are
clearly reduced in number in B compared to A. Scale bar=200 μm.
A. Vercelli et al. / Neurobiology of Disease 31 (2008) 395–405
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) SOD1G93Amice. In E and F GFAP-immunofluorescence in hMSC transplanted (E) and sham-operated (F) SOD1G93Amice. 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).
A. Vercelli et al. / Neurobiology of Disease 31 (2008) 395–405
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 tothe 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).
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
the whole period examined since some treated and control animals
neverachieved respectivecut-off times (180 s for Rotarodtest and90 s
for PaGE test). Body weight was normalized to the weight at 27 weeks.
The first clinical signs of motor neuron disease in SOD1G93Amice
were fine 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.
A. Vercelli et al. / Neurobiology of Disease 31 (2008) 395–405
andtheotheronereacheda motor score of0 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.
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
SOD1G93Amice, 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
Since hMSC injections in the cisterna magna did not obtain hMSCs
migration into the spinal cord, we injected hMSCs intraparenchymally
intothe 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 fusionwith 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 with Liu 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 Woodbury, 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 and βIII 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
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
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).
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 groupedthe experimental animalsaccording totheir
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
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, sinceastrocytesstimulated bymediatorsreleased
from microglia down-regulate the expression of neurotrophic factors
A. Vercelli et al. / Neurobiology of Disease 31 (2008) 395–405
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
SOD1G93Amice, 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 in Christou 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 and β-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 SOD1G93Afemales 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 SOD1G93Amales. 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 in Christou et al., 2007; Hedlund et al.,
Effects of MSC transplantation on motor behavior
From the 5th month of age SOD1G93Amice 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.
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 andimmunomodulatorymolecules. MSC therapycould
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;
Hedlundet al., 2007), MSCscan be collected fromthe 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
receptorsand signaling cascades.On the otherhand,we neededtotest
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.
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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