Multiple intravenous administrations of human umbilical cord blood cells benefit in a mouse model of ALS.
ABSTRACT A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25 × 10⁶ cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages.
Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10⁶ or 2.5 × 10⁶ cells from 13 weeks of age. A third, pre-symptomatic, group received 10⁶ cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10⁶ cells pre-symptomatically or 2.5 × 10⁶ cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups.
These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.
- SourceAvailable from: Jong-Deog Kim[Show abstract] [Hide abstract]
ABSTRACT: Motor neuron disease (MND) is a fatal neurodegenerative disorder caused by progressive and selective degeneration of motor neurons (MNs). Because of the versatile nature, stem cells have the potential to repair or replace the degenerated cells. In this review, we discussed stem cell based therapies including the use of embryonic stem cells (ESCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs) and genetically engineered cells to produce the neurotrophic factors for the treatment of MND. To achieve this goal, the knowledge of specificity of the cell target, homing and special markers are required.KSBB Journal. 10/2013; 28(5).
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ABSTRACT: Fetal alcohol spectrum disorder (FASD) is caused by intrauterine exposure to alcohol and can cause a full range of abnormalities to brain development, as well as long-term sequelae of cognitive, sensory and motor impairments. The incidence is estimated to be as high as 2% to 5% in children born within the US, however the prevalence is even higher in low socioeconomic populations. Despite the various mechanisms thought to explain the etiology of FASD, molecular targets of ethanol toxicity during development are not completely understood. More recent findings explore the role of GABA-A and GABA-B mechanisms, as well as cell death, cell signaling and gene expression malfunctions. Stem cell based therapies have grown exponentially over the last decade, which have lead to novel clinical interventions across many disciplines. Thus, early detailed understanding of the therapeutic potential of stem cell research has provided promising applications across a wide range of illnesses. Consequently, these potential benefits may ultimately lead to a reduced incidence and severity of this highly preventable and prevalent birth defect. It is recognized that stem cell derivations provide unique difficulties and limitations of therapeutic applications. This review will outline the current knowledge, along with the benefits and challenges of stem cell therapy for FASD.Progress in Neuro-Psychopharmacology and Biological Psychiatry 10/2014; 54:149–156. · 4.03 Impact Factor
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ABSTRACT: Amyotrophic lateral sclerosis (ALS), characterized by the progressive loss of both upper and lower motor neurons, is a fatal neurodegenerative disorder. This disease is often accompanied by a tremendous physical and emotional burden not only for the patients, but also for their families and friends as well. There is no clinically relevant treatment available for ALS. To date, only one Food and Drug Administration (FDA)-approved drug, Riluzole, licensed 18 years ago, has been proven to marginally prolong patients' survival without improving the quality of their lives. Because of the lack of an effective drug treatment and the promising outcomes from several preclinical studies, researchers have highlighted this disease as a suitable candidate for stem cell therapy. This review article highlights the finding of key preclinical studies that present a rationale for the use of different types of stem cells for the treatment of ALS, and the most recent updates on the stem cell-based ALS clinical trials around the world.Neurology India 05/2014; 62(3):239-48. · 1.08 Impact Factor
Multiple Intravenous Administrations of Human
Umbilical Cord Blood Cells Benefit in a Mouse Model of
Svitlana Garbuzova-Davis1,2,3,4*, Maria C. O. Rodrigues1,7, Santhia Mirtyl1, Shanna Turner1, Shazia
Mitha1, Jasmine Sodhi1, Subatha Suthakaran1, David J. Eve1,2, Cyndy D. Sanberg6, Nicole Kuzmin-
Nichols6, Paul R. Sanberg1,2,4,5
1Center of Excellence for Aging and Brain Repair, University of South Florida, College of Medicine, Tampa, Florida, United States of America, 2Department of
Neurosurgery and Brain Repair, University of South Florida, College of Medicine, Tampa, Florida, United States of America, 3Department of Molecular Pharmacology and
Physiology, University of South Florida, College of Medicine, Tampa, Florida, United States of America, 4Department of Pathology and Cell Biology, University of South
Florida, College of Medicine, Tampa, Florida, United States of America, 5Department of Psychiatry, University of South Florida, College of Medicine, Tampa, Florida, United
States of America, 6Saneron CCEL Therapeutics, Inc., Tampa, Florida, United States of America, 7Ribeira ˜o Preto School of Medicine, University of Sa ˜o Paulo, Sa ˜o Paulo,
Background: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that
can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (256106
cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic
G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is
impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of
multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages.
Methodology/Principal Findings: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice
received 106or 2.56106cells from 13 weeks of age. A third, pre-symptomatic, group received 106cells from 9 weeks of age.
Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various
assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in
mouse spinal cords. Results showed that mice receiving 106cells pre-symptomatically or 2.56106cells symptomatically
significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice.
Astrocytes and microglia were significantly reduced in all cell-treated groups.
Conclusions/Significance: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the
symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This
multiple cell infusion approach may promote future clinical studies.
Citation: Garbuzova-Davis S, Rodrigues MCO, Mirtyl S, Turner S, Mitha S, et al. (2012) Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells
Benefit in a Mouse Model of ALS. PLoS ONE 7(2): e31254. doi:10.1371/journal.pone.0031254
Editor: R. Lee Mosley, University of Nebraska Medical center, United States of America
Received October 6, 2011; Accepted January 4, 2012; Published February 3, 2012
Copyright: ? 2012 Garbuzova-Davis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Department of Neurosurgery and Brain Repair, at the University of South Florida (USF). MCOR, received a fellowship
from the Sa ˜o Paulo Research Foundation which supported her work at USF. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript. No additional external funding received for this study.
Competing Interests: SGD, DJE are consultants and PRS is a co-founder of Saneron CCEL Therapeutics, Inc. This does not alter the authors’ adherence to all the
PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative
disorder characterized by a loss of upper and lower motor
neurons. Symptoms include spasticity, fasciculations, muscle
weakness and atrophy, combined with progressive paralysis
ultimately leading to death, usually within three to five years of
diagnosis. The sporadic form of ALS (sALS) predominates, with
only 5–10% of cases identified as genetically linked; of those that
have a familial etiology (fALS), 20% show missense mutations in
the Cu/Zn superoxide dismutase (SOD1) gene on chromosome 21
. In sALS cases, the etiology of the disease is still undefined.
However, the clinical presentation and underlying pathology of
sALS and fALS are similar. Although numerous hypotheses about
the etiopathology of this multifactorial disease have been proposed
[2–4] including neurovascular pathology , reliable treatment to
halt disease progression and restore function remains elusive.
Cell therapy may be a promising treatment for ALS. Although
motor neuron replacement is possible, this treatment strategy
should take into account the multifocal motor neuron degenera-
tion and death . The roles of cell-based therapeutics might be
more practical ‘‘as modifiers of the ALS-specific microenviron-
ment’’  or serving to ‘‘detoxify the local environment around
dying motor neurons’’ , therefore providing protection for
PLoS ONE | www.plosone.org1February 2012 | Volume 7 | Issue 2 | e31254
motor neurons and retarding disease progression. Neuroinflam-
mation, comprised mainly of astrocyte and microglial activation, is
a central feature in ALS, and directly contributes to neuronal
death [9–11]. Therefore, attempting to modulate inflammation,
combined with other neuroprotective strategies in ALS, seems a
more realistic approach than neuronal replacement , thus
eliminating the need for neural cell sources.
Numerous reports demonstrate the functional multipotency of
non-neural cells such as bone marrow, peripheral blood and
umbilical cord blood cells [13–16]. Based on the recently proposed
concept of biofunctional multipotency of stem cells to mediate
systemic homeostasis, stem cell multipotency should be considered
in planning for therapeutic applications . In an ALS clinical
trial, autologous ex vivo expanded mesenchymal cells from bone
marrow were transplanted directly into the thoracic spinal cord of
patients [18,19]. While beneficial effects were described only in a
few patients, no overall changes in disease progression were noted.
A second report  confirmed the lack of changes in neurological
progression of sALS patients transplanted intravenously with
allogenic peripheral blood CD34+ hematopoietic stem cells,
however, some transplanted cells were found in motor neuron
sites of the spinal cord. Likely, the cell sources chosen, specifically
bone marrow and peripheral blood, may not have been the
Human umbilical cord blood (hUCB) cells may be preferable to
other potential cell sources [21–25]. The hUCB cells are low in
pathogenicity and are immunologically immature. Hematopoietic
progenitors from cord blood are rich in the most primitive stem
cells [26–31] and are capable of developing into cells of various
tissue lineages including neural cells [32–34]. Additionally, cord
blood lymphocytes express cytokine receptor profiles (interleukins
[IL]-2, IL-4, IL-6, IL-7, tumor necrosis factor [TNF]-a, and
interferon-c) at lower levels than adult blood cells  and
produce great amounts of the anti-inflammatory cytokine IL-10
. Moreover, umbilical cord blood cells secrete trophic factors,
which can directly support neuronal survival .
In recent years, reports have shown that hematopoietic
umbilical cord blood cells are versatile instruments for the
treatment of various disorders including neurodegenerative
diseases [21,23,24]. The mononuclear cell fraction derived from
human umbilical cord blood (MNC hUCB) has been effective in
the treatment of experimental stroke [38–40], traumatic brain
injury , spinal cord injury  and Alzheimer’s disease . It
was also shown that intravenous (iv) administration of MNC
hUCB into aged rats demonstrably improved the microenviron-
ment of the aged brain . Using the G93A SOD1 mouse model
of ALS, we have previously demonstrated  that a single
systemic iv administration of MNC hUCB cells, at the low dose of
106cells, delayed disease progression by at least 2–3 weeks and
modestly increased lifespan. More recently, we investigated the
optimal MNC hUCB cell dosage, verifying that a larger dose of
256106cells administered intravenously into pre-symptomatic
G93A mice delayed disease onset by 15% and significantly
increased lifespan by 20–25% . The effects were likely due to
enduring inhibition of various inflammatory effectors, inhibition
that promoted motor neuron survival. However, converting this
large mouse dosage into a single human equivalent dose 
would require approximately 20 units of cord blood, impractical in
a clinical setting. A more feasible approach would be delivery of
multiple smaller cell doses during disease progression, thus
providing ongoing protection for motor neurons.
The aim of this study was to determine the effect of systemic
multiple MNC hUCB cell administrations into pre-symptomatic
and symptomatic G93A SOD1 mice modeling ALS. Importantly,
this is the first time that a preclinical translational study has been
designed to address efficacy of a proposed cell treatment at the
symptomatic stage of the disease.
Of the total 108 G93A SOD1 mice used in the study, seven
mice (Group1 – one, Group 2 – two, Group 3 – three, Group 4 – one)
were excluded due to death precipitated by conditions other than
disease progression, more specifically, anesthetic complications
during cell or media administrations. The number of injections per
group was: Group 1 (Gr 1, 2.56106MNC hUCB, symptomatic) -
6.5060.27 (range 4–8), Group 2 (Gr 2, 16106MNC hUCB,
symptomatic) - 6.0060.23 (range 4–8), Group 3 (Gr 3, 16106MNC
hUCB, pre-symptomatic) - 11.6560.36 (range 9–15), and Group 4
(Gr 4, Media-injected, symptomatic) - 5.4760.23 (range 4–7).
Although the range of injection numbers was similar between Gr 1
and Gr 2, the number of mice receiving 8 injections at
symptomatic stage in Gr 1 was n=5 and in Gr 2 was n=2. The
total number of injected cells was: Gr 1 - 16.2560.676106, Gr 2 -
6.0060.236106, and Gr 3 - 11.6060.376106cells.
Before distributing mice into groups, the DCT, proportional to
the expression of mutant SOD1 gene, was determined. Results
showed similar DCT for all G93A mice and no significant
differences between groups: Gr 1 - 5.9660.07, Gr 2 - 5.9260.05,
Gr 3 - 5.9160.05, Gr 4 - 6.0560.33 cycles.
Effect of multiple administrations of MNC hUCB cells on
The MNC hUCB cells were intravenously administered weekly
into G93A mice beginning at either pre-symptomatic (9 weeks old)
or symptomatic disease stage (13 weeks old). Symptomatic mice
received one of two different cell doses. Significant increases in
survival were determined in mice receiving 16106cells at pre-
symptomatic stage (p=0.0015, x2=10.07) and 2.56106cells at
symptomatic stage (p=0.0022, x2=9.393) vs. the Media injected
group (Figure 1A). Average lifespan of MNC hUCB administered
mice was: 2.56106(Gr 1, symptomatic) - 135.0061.86 days,
16106(Gr 2, symptomatic) - 130.7161.60 days, 16106(Gr 3, pre-
symptomatic) - 135.6062.47 days compared to Media-injected
mice (125.5861.40 days). Media-injected animals survived no
longer than 19.5 weeks, whereas 30% of mice receiving 2.56106
(Gr 1, symptomatic) or 16106(Gr 3, pre-symptomatic) and 14.3%
mice administered with 16106cells (Gr 2) at symptomatic stage
survived more than 140 days and 10% of mice from Gr 3 (16106
cells, pre-symptomatic) were alive up to 160 days (Figure 1B).
Body weight is not only a general indicator of mouse health, but
is also a valuable marker for detecting progression of muscle
atrophy, and was measured weekly. As expected, body weight
started to slowly decline at the symptomatic age of approximately
13–14 weeks in all G93A mouse groups. By 16 weeks of age, more
than 20% of Media mice had lost 15% of their initial body weight.
Although the mean body weight loss from initial measurement to
the day of sacrifice for all G93A mice was 17.1960.80%, treated
animals lost weight more slowly, as they survived longer than the
Media group. A Kaplan-Meier plot (Figure 2A) was constructed
based on the threshold of 15% of body weight loss, a point closely
corresponding to the end-stage of disease. Mice receiving 16106
cells at pre-symptomatic stage (Gr 3) maintained their body weight
significantly longer (p=0.0152, x2=5.894) than Media-injected
mice (Gr 4).
Cell treated mice, in Gr 1 (2.56106cells, symptomatic) and Gr 3
(16106cells, pre-symptomatic), also displayed superior perfor-
mance in other tests of functional ability. Deteriorating extension
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org 2February 2012 | Volume 7 | Issue 2 | e31254
reflex was noted in G93A mice, beginning at 13 weeks of age, with
extension progressively declining until the end-stage of disease.
However, hindlimb extension of mice from Gr 1 and Gr 3
deteriorated more slowly than Media-injected mice (Gr 4) and
mice receiving 16106cells at symptomatic stage (Gr 2). At 17
weeks of age, Gr 1 and Gr 3 mice presented, respectively, 41.5%
and 51.04% of the initial hindlimb extension scores, while mice
from Gr 2 and Gr 4 presented 31.0% and 26.47% of initial values.
The mean loss in extension scores, from the initial score until time
of sacrifice, was 89.2162.07% for all G93A mice. Similarly to the
body weight analysis, Kaplan-Meier analysis was used to compare
the number of weeks until extension reflex scores of mice from
each group dropped more than 70% (Figure 2B). The Gr 1
cells, symptomatic) and Gr 3 (16106
symptomatic) mice presented significantly delayed deterioration
of hindlimb extension compared to Media (Gr 4) mice (Gr 1 vs. Gr
4, p=0.0493, x2=3.865; Gr 3 vs. Gr 4, p=0.0069, x2=7.301). A
significant difference (p=0.0269, x2=4.895) was also detected
between Gr 3 and Gr 2 mice receiving 16106cells at the pre-
symptomatic or symptomatic stage of disease, respectively.
In the grip strength test, G93A mice started to show decreased
muscle strength at approximately 13 weeks of age, with strength
progressively declining during the course of disease. The mean loss
in grip strength, from maximum to end-stage, was 87.061.12%
for all G93A mice. Kaplan-Meier analysis showed that Gr 3 mice
administered weekly with 16106cells beginning at the pre-
symptomatic stage significantly (p=0.0358, x2=4.405) delayed
loss in muscle strength vs. Media (Gr 4) animals (Figure 2C).
Although declines in performance on the rotarod test were
observed in all mice starting at week 13, mice beginning cell
Figure 1. Effect of multiple MNC hUCB cell administrations on lifespan of G93A mice. (A) Kaplan-Meier survival curves for G93A mice
receiving 2.56106(Gr 1) or 16106(Gr 2) cells at symptomatic disease stage and 16106(Gr 3) cells pre-symptomatically. Control group was Media-
injected mice (Gr 4). Significant (
) increases in survival were determined in mice receiving 16106cells at pre-symptomatic stage (p=0.0015) and
2.56106cells at symptomatic stage (p=0.0022) vs. the Media-injected group. Survival of the mouse group receiving 16106cells pre-symptomatically
tended towards significance compared to survival of mice receiving same cell dose at symptomatic stage (p=0.0595). (B) Percentages of surviving
mice within age ranges. Media-injected animals survived no longer than 19.5 weeks, whereas 30% of mice receiving 2.56106(Gr 1, symptomatic) or
16106cells (Gr 3, pre-symptomatic) and 14.3% mice administered with 16106cells (Gr 2) at symptomatic stage survived more than 140 days and 10%
of mice from Gr 3 (16106cells, pre-symptomatic) were alive for more than 150 days.
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org3February 2012 | Volume 7 | Issue 2 | e31254
treatment at the pre-symptomatic stage (Gr 3) demonstrated longer
latency at this time. At 17 weeks of age, these mice (Gr 3)
maintained 19.8% of initial rotarod latency, while mice from Gr 1
(2.56106cells, symptomatic), Gr 2 (16106cells, symptomatic), and
Gr 4 (Media) presented 11.29%, 11.96% and 11.05%, respective-
ly. The mean loss in rotarod latency, from a maximum value of
180 seconds to end-stage value was 99.760.08%, for all G93A
mice. In the Kaplan-Meier analysis, only mice from Gr 3
performed better on the rotarod test than other cell-treated mice
and took longer to lose over 70% of maximum latency than the
Media (Gr 4) group (Figure 2D).
Analysis of disease onset in mice beginning cell treatment at the
pre-symptomatic stage (Gr 3) was performed using the Kaplan-
Meier method based on a threshold of 5% of body weight loss and
15% loss in functional tests (extension reflex, grip strength, and
rotarod tests). Results demonstrated that these mice at 10–14
weeks ofage significantlymaintained
(p=0.0355, x2=4.420) and hindlimb extension (p=0.0142,
x2=6.008) compared to media-injected animals (Gr 4). Muscle
strength (grip test) and rotarod performance did not significantly
differ between Gr 3 and Gr 4 mice.
Immunohistochemical analysis of administered MNC
hUCB cells in vivo
Administered MNC hUCB cells were identified immunohisto-
chemically by a human-specific marker (HuNu) in the spinal cord,
brain, and various abdominal organs of cell-treated mice at 17
weeks of age, 4 weeks (symptomatic) or 8 weeks (pre-symptomatic)
post-transplant. Cells were widely distributed within and outside
the CNS. In the cervical (Figure 3A) and lumbar (Figure 3B)
cervical spinal cord, HuNu positive MNC hUCB cells were found
irrespective of injected cell doses or timing of initial treatment.
However, in all cell-treated mice, more than 50% of the cells were
observed within the ventral horn gray matter, areas in the spinal
cord known to be affected by ALS. Cells were frequently observed
inside the capillary lumen, but also in the spinal cord parenchyma
(Figure 3C). Some cells established in the brain, mostly in the
cerebral cortex, olfactory bulb, and brainstem. In the liver, lungs
and kidneys, a few cells were identified (Figure 4), but in the
spleen, a high density of MNC hUCB cells was detected.
Qualitative evaluation of the spleens from cell-treated animals
(Figure 4, j–l) showed higher concentrations of cells in mice from
Gr 3 (16106cells, pre-symptomatic) and Gr 1 (2.56106cells,
symptomatic) than in Gr 2 (16106cells, symptomatic).
Effect of multiple administrations of MNC hUCB cells on
motor neuron survival
In both cervical and lumbar spinal cords, motor neuron counts
were directly proportional to the survival and functional
Figure 2. Evaluations of disease progression in G93A mice
through Kaplan-Meier analysis. (A) Time elapsed until animals lost
15% of their maximum body weight. Mice receiving 16106cells pre-
symptomatically (Gr 3) significantly (
Media (Gr 4) mice. A similar trend was observed in mice treated with
) maintained body weight vs.
2.56106cells (Gr 1) beginning at symptomatic disease stage. (B) Time
elapsed until hindlimb extension scores deteriorated by 70% of the
initial score. The Gr 1 and Gr 3 mice significantly (
hindlimb extension compared to Gr 4 mice. A significant difference was
also detected between Gr 3 and Gr 2 mice receiving 16106cells at pre-
symptomatic or symptomatic stage of disease, respectively. (C) Time
elapsed until muscle strength decreased by 70% from the maximum
value. Mice from Gr 3 significantly (
vs. Gr 4. Gr 1 mice tended to maintain muscle strength post-transplant.
(D) Time elapsed until rotarod latency decreased by 70% of the
maximum value. Only mice from Gr 3 performed better on the rotarod
than other cell-treated mice and tended towards significance (
taking more time to decrease latency by over 70% of the maximum
value compared to Gr 4.
) delayed decline of
) delayed muscle strength losses
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org4 February 2012 | Volume 7 | Issue 2 | e31254
improvement observed in the mice. Figure 5 shows the results of
motor neuron counts in the ventral horns of mice at 17 weeks of
age and end-stage of disease. In the cervical spinal cord
(Figure 5A), Gr 1 (2.56106cells, symptomatic) and Gr 3 (16106
cells, pre-symptomatic) mice presented significantly higher motor
neuron densities compared to Media-injected (Gr 4) (G1 vs. G4,
p,0.05; G3 vs. G4, p,0.001) and Gr 2 (16106cells, symptomatic)
mice (G1 vs. G2, p,0.01; G3 vs. G2, p,0.001) groups. Motor
neuron density in the cervical spinal cord of mice at 17 weeks of
age was: Gr 1 – 3,4986223.80, Gr 2 – 1,654692.43, Gr 3 –
5,1116230.91, Gr 4 - 1,9896129.20, and Gr 5 – 4,0876321.00
number of motor neurons/mm3. In the lumbar spinal cord, results
were similar: Gr 3 (16106cells, pre-symptomatic) and Gr 1
cells, symptomatic) mice had significantly higher
(p,0.001) motor neuron densities than the Media group (Gr 4)
at 17 weeks of age or end-stage of disease (Figure 5B). Motor
neuron density in the lumbar spinal cord of mice at 17 weeks of
Figure 3. Distribution of MNC hUCB cells in the spinal cord of
G93A mice. Administered MNC hUCB cells were identified immuno-
histochemically by a human-specific marker (HuNu) in the spinal cord of
cell-treated mice at 17 weeks of age, 4 weeks (symptomatic) or 8 weeks
(pre-symptomatic) post-transplant. In the total area of cervical (A) and
lumbar (B) cervical spinal cord, HuNu positive MNC hUCB cells were
found irrespective (p.0.05) of injected cell doses or time beginning
treatment. In all cell-treated mice, more than 50% of the observed cells
were in ventral horn gray matter. (C) Immunohistochemical staining of
MNC hUCB cells in the lumbar spinal cord. MNC hUCB cells positive for
HuNu (green, arrow) were detected in the lumbar spinal cord of mice
receiving 2.56106(a) or 16106(b) cells symptomatically or 16106cells
pre-symptomatically (c). Cells were frequently observed inside the
capillary lumen, but also in the spinal cord parenchyma. (a9), (b9), and
(c9) are merged images with DAPI. Scale bar: a–c9 is 50 mm.
Figure 4. Distribution of MNC hUCB cells in the lung, liver,
kidney and spleen of G93A mice. MNC hUCB cells immunohisto-
chemically positive for HuNu (green, arrows) were detected in the lung,
liver, kidney, and spleen of mice receiving 2.56106(a, d, g, j) or 16106
(b, e, h, k) cells symptomatically or 16106cells pre-symptomatically (c,
f, i, l). In the liver, lung and kidney, few cells were identified. In the
spleen, a high density of HuNu cells was determined in all cell-treated
mice (j–l). Scale bar: a–i is 50 mm; j–l is 200 mm.
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org 5February 2012 | Volume 7 | Issue 2 | e31254
age was: Gr 1 – 3,3046140.50, Gr 2 – 1,772686.92, Gr 3 –
4,4976208.80, Gr 4 - 1,589677.22, and Gr 5 – 4,7576444.70
number of motor neurons/mm3. In both cervical and lumbar
spinal cords, densities from Gr 2 (16106cells, symptomatic) and
Media-injected (Gr 4) mice did not differ (p.0.05) from each
other, and motor neuron densities in mice from Gr 3 and Gr 1 were
also similar (p.0.05) to controls (Gr 5) of same age. Figure 5C
demonstrates superior survival of choline acetyltransferase (ChAT)
positive motor neurons in the ventral horns of lumbar spinal cords
in cell-treated animals compared to Media mice.
Effect of multiple administrations of MNC hUCB cells on
microglia and astrocytes
At 17 weeks of age, microglial cell counts were higher in the
Media group (Gr 4) animals, while control (Gr 5) mice presented
the lowest densities. MNC hUCB cell administrations significantly
(p,0.001) decreased the number of microglia in the cervical
(Figure 6A) and lumbar (Figure 6B) ventral horns of G93A mice,
although no statistical difference was detected between the cell-
treated groups. Morphological analysis of microglial cells demon-
strated a high number of activated cells with large cell bodies and
short processes in Media-injected mice, whereas ramified microg-
lia were mostly observed in the cell-treated animals, specifically in
Gr 1 and Gr 3 (Figure 6C).
Astrocyte cell density showed a similar pattern as the microglia
in mice at 17 weeks of age. Media-injected animals (Gr 4)
presented the highest densities, while the lowest values were noted
in controls (Gr 5). Groups that received treatment with MNC
hUCB cells presented a significant (p,0.001) decrease in
astrocytic densities in the cervical (Figure 7A) and lumbar
(Figure 7B) spinal cords compared to Media, with no significant
difference between cell-treated groups. When the number of
reactive astrocytes was assessed in each mouse group, higher
proportions of these cells were observed in Media-injected mice at
17 weeks of age and end-stage disease compared to cell-treated
animals. These cells were distinguished by their morphology, as
exemplified in Figure 7C. The selective cell count once more
demonstrated that Media-injected mice presented higher values of
Figure 5. Characteristics of motor neuron survival in the spinal cord of G93A mice. Motor neuron counts were performed in the cervical
(A) and lumbar (B) ventral horns of G93A mice at 17 weeks of age and at end-stage of disease. Mice receiving 2.56106cells symptomatically (Gr 1) or
16106cells pre-symptomatically (Gr 3) had significantly higher motor neuron densities than the Media group (Gr 4) at 17 weeks of age or at end-
stage of disease. In both cervical and lumbar spinal cords, motor neuron densities between Gr 2 (16106cells, symptomatic) and Media-injected (Gr 4)
mice showed no significant differences (p.0.05). *p,0.05, **p,0.01, ***p,0.001. (C) Immunohistochemical staining of motor neurons in the lumbar
spinal cord of G93A mice at 17 weeks of age. Motor neuron staining for anti-choline acetyltransferase (anti-ChAT) antibody showed healthy motor
neurons in controls (a) although only a few neurons survived in the Media-treated animals (b). Cell-treated mice with (c) 2.56106cells
symptomatically (Gr 1) and (e) 16106cells pre-symptomatically (Gr 3) demonstrated higher motor neuron survival than (d) mice receiving 16106cells
symptomatically (Gr 2). Scale bar: a–e is 50 mm.
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org6February 2012 | Volume 7 | Issue 2 | e31254
reactive astrocytes than the cell treated animals, indicating that the
MNC hUCB cell treatment indeed effectively decreased astrocytic
In the present study, we evaluated the effects of multiple MNC
hUCB cell injections into a G93A SOD1 mouse model of ALS at
different disease stages. The major findings in our study
demonstrated that multiple MNC hUCB cell administrations into
systemic circulation of G93A mice effectively: (1) delay disease
progression; (2) improve animal survival; (3) enhance motor
neuron survival; (4) modulate gliosis, and (5) reduce activation of
microglia and astrocytes. Cell dose and the timing of initial
treatment have a clear influence upon disease progression. Since
the expression of mutant SOD1 gene (DCT) was similar for all
G93A mice and there were no appreciable expression differences
between groups, the benefits of MNC hUCB cells are noticeable.
Administrations of 16106cells initiated pre-symptomatically were
most advantageous in both delaying disease onset and increasing
lifespan whereas effective symptomatically-initiated cell infusions
required higher cell doses (2.56106) to delay disease progression
Figure 6. Characteristics of microglial cells in the spinal cords of G93A mice. Microglial densities were measured in the cervical (A) and
lumbar (B) ventral horns of G93A mice at 17 weeks of age and at end-stage of disease. Microglial densities were significantly (p,0.001) higher in
Media-injected mice (Gr 4) compared to control mice (Gr 5) of the same ages. MNC hUCB cell administrations significantly (p,0.001) decreased the
number of microglia in the spinal cord of G93A mice compared to Media mice. No significant differences were detected between the cell-treated
groups. *p,0.05, **p,0.01, ***p,0.001. (C) Immunohistochemical staining of microglia in the lumbar spinal cord at 17 weeks of age. Microglial cells
stained for anti-Iba-1 antibody were sparse in controls (a) and microgliosis was noted in Media-treated animals (b). MNC hUCB cells decreased
microglial density in mice from Gr 1 (c), Gr 2 (d), and Gr 3 (e). Morphological analysis of microglial cells determined numerous activated cells with
large cell bodies and short processes in Media-injected mice, whereas ramified microglia were mostly observed in cell-treated animals, particularly in
Gr 1 and Gr 3 mice and controls (inserts in a–e). Scale bar: a–e is 200 mm; in a–e inserts is 25 mm.
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org7 February 2012 | Volume 7 | Issue 2 | e31254
and so extend lifespan. Here, we are the first to demonstrate, from
a translational viewpoint, treatment benefits when initiated at the
symptomatic stage of disease. Thus, these results might provide a
superior basis for the development of clinical trials, since the
overwhelming proportion of ALS patients beginning treatment are
Hence, cell-based therapy for ALS seems a more realistic and
practical approach to developing neuroprotective strategies,
protecting motor neurons and retarding disease progression .
Hematopoietic stem cells might provide these benefits. Previously,
we have shown the effectiveness of human umbilical cord blood
administration in a mouse model of ALS [45,46]. Similar results
have been noted by other researchers [48,49]. Moreover, we have
also demonstrated that a high single dose of 256106MNC hUCB
cells, injected intravenously into pre-symptomatic G93A mice,
optimized survival and retarded disease progression .
However, to translate this experimental mouse therapy to the
clinic would require impractically high cell doses.
The lessened effectiveness of MNC hUCB cells injected into
symptomatic ALS mice had been expected, since motor neuron
deterioration and neuroinflammation are already quite advanced
when the initial symptoms manifest [50–53]. Since initiating
treatment after symptoms appear is the most likely clinical
scenario, high cell doses should be considered. However, smaller
doses might be effective if intervention begins at the first
appearance of disease symptoms. While ALS is a gradually
progressive disease, treatment strategy by a series of cell infusions
might be the best approach. The beneficial effect of multiple low-
dose administrations of MNC hUCB cells has already been
demonstrated in other models of neurodegenerative disorders: a
Figure 7. Characteristics of astrocytes in the spinal cord of G93A mice. Astrocyte densities were measured in the cervical (A) and lumbar (B)
ventral horns of G93A mice at 17 weeks of age and at end-stage of disease. Astrocytic densities were significantly (p,0.001) higher in Media-injected
mice (Gr 4) at 17 weeks of age and at end-stage of disease vs. controls (Gr 5) of the same ages. Significant (p,0.001) decrease in the number of
astrocytes was determined in cell-treated G93A mice compared to Media mice. No significant statistical differences were detected between the cell-
treated groups. Higher number of reactive astrocytes was observed in Media-injected mice at 17 weeks of age and at end-stage disease compared to
cell-treated animals. ***p,0.001. (C) Immunohistochemical staining of astrocytes in the lumbar spinal cord of G93A mice at 17 weeks of age. Anti-
GFAP antibody staining showed low astrocyte density in controls (a) and astrocytosis in Media-treated animals (b). Considerably decreased actrocytic
density was observed in mice from Gr 1 (c), Gr 2 (d), and Gr 3 (e). Astrocyte cell reactivity was also reduced in cell-treated mice vs. Media-injected mice
(inserts in a–e). Scale bar: a–e is 200 mm; in a–e inserts is 25 mm.
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org8 February 2012 | Volume 7 | Issue 2 | e31254
transgenic mouse model of Alzheimer’s disease  and a
knockout mouse model of Sanfilippo syndrome type B .
Moreover, a recent report  indicated that serial intracerebro-
ventrical injections of autologous cord blood-derived neural
progenitors given to a child with global ischemic brain injury
significantly improved neurological status.
In analyzing disease progression in G93A SOD1 mice, we
consider rapid motor neuron degeneration to be a consequence of
mutant SOD1 gene involvement. Therefore, we suggest that
improved mouse survival after MNC hUCB infusions results from
delayed disease progression. As expected, all groups of G93A
animals suffered similar percentages of body weight and functional
losses throughout their lives, due to the progressive nature of this
disease. The differences between the groups continued to be the
amount of time that elapsed to reach such endpoints. Body weight
measurements closely followed the functional curves across time,
since weight loss is a consequence of diminishing muscle mass and
later muscle atrophy. By examining the functional status of each
group through the Kaplan-Meier method, we were able to
evaluate animal performance over time, without the bias of
different survivals, a strategy already shown by Ohnishi et al. .
MNC hUCB cell infusions successfully improved most of the
evaluated functions. Together, these data indicate that MNC
hUCB cell treatment improves motor neuron survival, thus
extending functional capabilities and mouse lifespan.
Although motor neuron death is a terminal event in ALS,
directly associated with the clinical symptoms, disease pathogen-
esis involves multiple pathways in which neuroinflammation is a
critical participant [9–11]. Reactive astrocytosis and activated
microglia can be detected in cervical and lumbar spinal cord gray
matter of G93A mice before disease onset [51,52], progressively
increasing until the end-stage of disease [50,53]. The concept that
non-neuronal cells contribute to the disease process, known as the
‘‘non-cell autonomous nature of motor neuron death’’, is
supported by several authors [57–59]. The role of activated
microglia and reactive astrocytes as major inflammatory effectors
contributing to motor neuron damage in ALS has been identified
in various studies [9,52,60–65]. Astrocytes are directly involved in
the establishment of motor neuron death, while activated
microglia worsen local inflammation and disease progression
[66,67]. Therefore, inhibition of these inflammatory effectors in
ALS could have a protective effect upon motor neurons.
In the present study, we determined significant reductions of
astrocytosis and microglial density in the cell-treated G93A mice,
indicating a modulatory effect of the MNC hUCB cells upon the
inflammatory environment of the spinal cord. Previously, we
demonstrated that a single injection of 256106MNC hUCB cells
into pre-symptomatic mice significantly decreases pro-inflamma-
tory cytokines in the brain and spinal cord and reduces microglia
density in the cervical/lumbar spinal cord . We therefore
suggest that the effect of multiple MNC hUCB cell administrations
in decreasing neuroinflammation of the spinal cord, is neuropro-
tective and promotes motor neuron survival. Interestingly,
although motor neuron densities are clearly distinct among the
treatment groups, with better outcomes in mice receiving 2.56106
cells beginning at symptomatic stage (Gr 1) or 16106cells at pre-
symptomatically (Gr 3), such differences were not found in glial cell
densities. Concerning astrocytes, it is possible that density
evaluations are not as efficient as motor neuron counts in
detecting subtle differences between groups. Regarding the
microglia, however, the possibility remains that the cell treatment
is successful not only in decreasing density, but also in promoting a
shift from an ‘‘M1’’, inflammatory, towards an ‘‘M2’’, more
tolerant immunological profile . Since both microglia subtypes
present similar phenotypes, the anti-Iba-1 antibody applied in the
immunohistochemical evaluation would be insufficient to distin-
guish one from the other. Further studies are therefore necessary
to clarify this point.
In the context of reducing neuroinflammation by repeated
MNC hUCB cell administrations, we observed high concentra-
tions of grafted human cells in the spleen, suggesting that this
secondary lymphoid organ is acting as a reservoir of the
administrated cells [68,69]. Previously, we showed that even after
a single small (16106cells) intravenous injection of MNC hUCB
into pre-symptomatic G93A mice, a majority of cells were
identified in the white pulp of the spleen . We hypothesize
that in the spleen, the injected cells interact with the host cells and
modulate the immunological response, lowering the inflammatory
profile  and possibly protecting motor neurons in the remote
Numerous experimental studies have shown that the intrave-
nous delivery of cells is effective in the treatment of ALS
[45,46,48,71,72]. Moreover, it is considered a suitable route for
translation into clinical application, due to its low invasiveness. In
a previous study, we showed the migration of intravenously
injected MNC hUCB cells and their differentiation into neural-like
cells in the brain and spinal cord of G93A mice . In the
present study, we found that although the number of grafted
human cells identified in the spinal cord was low and did not
reflect the number of administered MNC hUCB cells, the
treatment was effective. These results suggest that various factors
secreted by the cells, rather than differentiation or cell-cell contact
mechanisms, are the main therapeutic mediators. In fact, the same
issue has already been approached in other neurological diseases
such as stroke, in which functional improvement was dispropor-
tionally higher than the number of administered cells migrating to
the site of injury . Moreover, a recent report demonstrated
that umbilical cord blood infusions improved neuromuscular
transmission in G93A mice, indicating a direct effect of the
treatment upon motor nerve function . The authors consider
that since the improvement was detected shortly after umbilical
cord blood cell administration, cell replacement was a less
probable mechanism of repair. In agreement with this idea, we
consider that although cell replacement is possible and should not
be overlooked, the results of the present study strongly support the
actions of neuroprotection, which may include immunomodula-
tion and secretion of trophic factors by the intravenously
transplanted cells. Possibly, there is also some degree of motor
neuron repair, which may be due more to endogenous pathways
than administered cell differentiation.
In conclusion, we demonstrated that multiple injections of
MNC hUCB cells are effective in improving motor neuron
survival, likely due to decreasing macro- and microgliosis, and, in
consequence, delaying disease progression and increasing lifespan
of a mouse model of ALS. Beginning the cell injections pre-
symptomatically provided the best outcome. Most important for
translational purposes was proving the effectiveness of high cell
doses initiated at the symptomatic disease stage. The present study
results might provide essential information and strong impetus for
future clinical trials.
Materials and Methods
All described procedures were approved by the Institutional
Animal Care and Use Committee at USF, ID #R3416, and
conducted in compliance with the Guide for the Care and Use of
Laboratory Animals. One hundred and eight transgenic male B6SJL-
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org9 February 2012 | Volume 7 | Issue 2 | e31254
TgN (SOD1-G93A) 1GUR mice (G93A; obtained from Jackson
Laboratories, Bar Harbor, MA, USA), over-expressing human
SOD1, carrying the Gly93RAla mutation, were used. Before
being assigned to the study, tail snips were obtained from each
mouse and relative human SOD1 gene expression was quantified
through standard procedure of real-time PCR , performed by
Charles River Laboratories (Troy, NY, USA). The gene copy
number was estimated through DCT, which is the difference in the
threshold cycles between the transgene (human SOD1) and a
mouse reference gene. The DCT is a direct index of the transgene
copy number .
The G93A mice were randomly assigned to receive intrave-
nously MNC hUCB cells or media weekly beginning at different
disease stages. Injections were made into the penile vein of mice
and started at pre-symptomatic (8–9 weeks of age) or symptomatic
(13 weeks of age) disease stages and continued weekly until
sacrifice. The cell dose of 16106was injected into pre- and
symptomatic mice and was chosen based on our previous study
 showing effectiveness of this single cell dose in delaying
disease onset. Repeated injections of 2.56106cells were performed
into symptomatic mice. This cell dose was chosen to deliver an
optimal dose of 256106cells, proven effective for both delaying
disease onset and increasing lifespan of G93A mice , over a
number of weeks. From a translational viewpoint, the weekly dose
of 2.56106cells injected into symptomatic mice should be roughly
equivalent to 1–2 cord blood units administered to patients each
month. The treated groups were divided as follows: Group 1 –
2.56106MNC hUCB (n=27, symptomatic), Group 2 – 16106
(n=28, symptomatic), and Group 3 - 16106
symptomatic). There were two control groups: Group 4 - G93A
Media-injected (n=25, symptomatic) and Group 5 - transgenic
mice (BL6/SJL) carrying the normal human allele for SOD1 gene
(nSOD1, n=20). All mice were maintained on a 12:12 h dark:
light cycle (lights on at 06:00 AM). Room temperature was 23uC.
Food and water were available ad libitum. Upon progression of
neurological symptoms, a highly palatable liquid nutritional
supplement (Ensure PlusH) was placed on the cage floor, ensuring
access by the animal.
Preparation of MNC hUCB cells for transplantation
Cryopreserved MNC hUCB cells (U-CORD-CELLTM, Sa-
neron CCEL Therapeutics, Inc., Tampa, FL, USA) were thawed
rapidly at 37uC then transferred slowly with a pipette into a
centrifuge tube containing 10 ml of Dulbecco’s Phosphate
Buffered Saline 16 (DPBS), pH 7.4 (Mediatech, Inc., Manassas,
VA, USA). The cells were centrifuged (400 rpm/15 min) at 12uC,
the supernatant discarded and the process repeated. After the final
wash, the viability of cells was assessed using the 0.4% trypan blue
dye exclusion method before and following transplantation.
Transplant cell concentrations were adjusted for each group:
25,000 cells/ml (2.56106cells/100 ml/injection, Group 1) and
10,000 cells/ml (16106cells/100 ml/injection, Groups 2 and 3).
Cell or media administration
The MNC hUCB cells were delivered intravenously into the
superficial dorsal penile vein of mice under anesthesia with
Isofluorane (2–5% at 2 L O2/min) using a calibrated vaporizer-
equipped induction chamber and nose cone. This route delivers
the injected cells to the inferior vena cava and then to the right
atrium for easy distribution throughout the body. Immediately
prior to the injection, the penis was withdrawn from its protective
sheath and wiped with 70% ethanol. A 31-gauge needle attached
to a 100 ml Hamilton syringe was inserted through the skin at a
30u angle directly into the vein. During this procedure, the plunger
was gently pulled back to aspirate blood back into the needle hub,
ensuring that the needle was in the vein. Without moving the
syringe, the plunger was then depressed to deliver the contents of
the syringe into the penile vein. Once the cells (16106or 2.56106)
in 100 ml of vehicle (DPBS, pH 7.4) had been delivered, the needle
was removed and pressure was applied to the vein for 30–
60 seconds to prevent bleeding. The Media mouse group received
100 ml of DPBS, the same volume administered to the cell-
transplanted mice. All animals, including controls, received
cyclosporin A (CsA, 20 mg/kg per os) during the post-transplant
period. Cell and media administrations were performed blind by
independent investigators to avoid subjective bias.
Characteristics of disease progression
The evaluation of animal disease progression has been
previously described [45,46]. All measures of disease progression
were performed blind by independent investigators to avoid
subjective bias. Body weight was assessed weekly throughout the
study. Extension reflex, rotarod, and grip strength tests were
started on week 9 and then repeated weekly thereafter. Five to six
randomly selected cell-treated, non-treated, and control mice from
each group were sacrificed at approximately 17 weeks of age,
corresponding to 8 or 4 weeks after initial treatment at respectively
pre-symptomatic or symptomatic disease stage, for immunohisto-
chemical analysis of surviving motor neurons, glial cells and
distribution of administered cells. The remaining G93A mice were
behaviorally monitored until disease symptoms had progressed to
the point of complete hindlimb paralysis, at which time the mice
can no longer feed or care for themselves. After the last behavioral
test, lifespan was determined.
The mouse was suspended by the tail and
the extension of each hindlimb was observed. If the mouse showed
normal hindlimb extension, a score of 2 was given. A score of 1
indicated partial hindlimb extension. If no extension was observed,
the score was 0.
The mouse was placed on a 3.2 cm diameter axle
rotating at a speed of 16 rpm (Omnitech Rotoscan, Omnitech
Electronics, OH, USA). The latency (seconds) that the mouse
stayed on the rotating axle during a 3 minute maximum period
Grip strength test (IDTECH-BIOSEB, France).
mouse was held by the tail and carefully placed with all 4 paws
on the grid. The animal was gently pulled by the tail and a sensor
recorded muscle strength (Newtons) with which the mice resisted
the pull. The test was performed three times during approximately
one minute and the average of the tests was recorded.
Mice (n=5–6/group) at 17 weeks of age and the remaining
mice from each group, when demonstrating an inability to move
and reach food and/or water due to hindlimb paralysis and muscle
atrophy, were sacrificed under deep chloral hydrate (10%)
anesthesia and perfused transcardially with 0.1 M phosphate
buffer (PB, pH 7.2) followed by 4% paraformaldehyde (PFA) in PB
solution. The cervical/lumbar segments of the spinal cord and
brain were removed, post-fixed in 4% PFA, and then cryopro-
tected in 20% sucrose in 0.1 M PB overnight. Coronal sections of
the spinal cord and sagittal sections of the brain at 30 mm were cut
in a cryostat, every fifth section was thaw-mounted onto slides, and
the tissue was stored at 220uC for immunohistochemical analysis.
The spleen, liver, kidneys and lungs were also removed, cut in a
cryostat, and stored at 220uC for future analysis of administered
Multiple Cell Injections for ALS Mice
PLoS ONE | www.plosone.org10February 2012 | Volume 7 | Issue 2 | e31254
Immunohistochemical staining of MNC hUCB cells.
identification of MNC hUCB cells in the cervical/lumbar spinal
cords, brains, and organs (lungs, liver, spleen, and kidneys), serial
tissue sections were stained with the human-specific marker
(HuNu) as we described previously . Briefly, the mouse
monoclonal antibody (HuNu,
combined with the secondary antibody, monovalent goat anti-
mouse Fab9 fragment conjugated to FITC (1:200; Jackson
ImmunoResearch, USA), and incubated at room temperature
(RT) for 2 hours. The tissue sections on the slides were pre-
incubated with 10% normal goat serum (NGS), 1% normal
human serum and 0.3% Triton 1006in PBS, for 30 min at RT
and, subsequently, incubated with the previously prepared
antibody cocktail overnight at 4uC. Next day, the slides were
thoroughly washed in PBS and coverslipped with Vectashield
containing DAPI (Vector Laboratories, USA). The tissue was then
examined under epifluorescence using an Olympus BX60
Immunohistochemical staining of motor neurons in the
Serial sections of the cervical and lumbar spinal
cord were rinsed in PBS to remove the freezing medium. The
tissue sections were pre-incubated in a blocking solution of 10%
NGS and 3% Triton 1006in PBS for 60 min at RT, followed by
overnight incubationwith rabbit
acetyltransferase primary antibody (1:200, Abcam, USA) at 4uC.
On the next day, the slides were rinsed in PBS and incubated with
biotinylated goat-anti-rabbit secondary antibody (1:300, Vector
Laboratories, USA), 2% NGS, and 0.3% Triton 1006in PBS for
60 min at RT. After several rinses in PBS, an avidin-biotin-
peroxidase enzyme complex (ABC-Elite kit, Vector, USA),
followed by 3,3-diaminobenzidine chromatogen (DAB, Pierce,
USA) were used to verify motor neurons within the spinal cords.
Tissues were then dehydrated, coverslipped with synthetic resin
mounting medium (Permount, Fisher-Scientific,
examined under a bright field microscope (Olympus 606).
microglia in the spinal cord.
incubation followed the same procedures listed in the previous
section (immunohistochemical staining of motor neurons in the spinal cord).
For astrocyte staining, the tissues were incubated overnight with
rabbit polyclonal anti-glial fibrillary acid protein primary antibody
(GFAP, 1:500, Abcam, USA) at 4uC. For microglial staining,
tissues were incubated overnight at 4uC with rabbit polyclonal
anti-ionized calcium binding adapter molecule-1 (Iba-1, 1:2000,
Wako, Japan). After primary antibody incubation, the slides were
rinsed in PBS and incubated with biotinylated goat-anti-rabbit
secondary antibody (1:500, Vector Laboratories, USA), 2% NGS,
and 0.3% Triton 1006in PBS for 60 min at RT. The tissue was
then rinsed in PBS and incubated with avidin-biotin-peroxidase
enzyme complex (ABC-Elite kit, Vector, USA), followed by 3,3-
diaminobenzidine chromogen (DAB, Pierce, USA). Slides were
then dehydrated, coverslipped with mounting medium and
examined under a bright field microscope (Olympus 606).
Motor neuron counts in the spinal cord.
for stereological estimation of the motor neuron numbers in the
ventral horn, an initial tissue section was randomly selected at one
anatomical border of the spinal cord level (cervical or lumbar) to
be examined. Thereafter, every fifth section of the spinal cord was
used. Series of sections previously stained with anti-choline
acetyltransferase (anti-ChAT) antibody were analyzed. The
1:50,Millipore, USA) was
Tissue preparation and pre-
number of motor neurons was determined using unbiased,
systematic stereological random sampling and the optical
fractionator method  using the Stereo Investigator software
package (MicroBrightfield, VT, USA). Spinal cord volume was
measured by Cavalieri volume estimates, using the Stereo
Investigator software. Outlines of the anatomical structures were
done using a 106objective and cell quantification was conducted
using a 406 objective. Identification of motor neurons for
counting was based on ChAT expression, cell size and shape.
Motor neuron density is expressed as number of cells per mm3.
Astrocyte and microglial cell counts in the spinal
Analysis of astrocyte and microglial cell density in the
cervical and lumbar spinal cords from 17 weeks old mice was
performed using a computerized image analysis program (Image-
Pro Plus, Media Cybernetics, Inc., Silver Springs, MD, USA) as
we previously described . Briefly, measurements of cervical/
lumbar ventral horn area were first performed by determining the
cross-point of a line passing the central canal perpendicular to the
midline. The area of ventral gray matter was determined below
this line in the right and left cervical/ lumbar spinal cords in
coronal sections (n=6/slide) from each mouse group at
microglial cells were counted within right and left ventral gray
matter of the cervical and lumbar spinal cords. Density of cells was
determined as cell number per mm2.
For astrocytes, the proportion of reactive versus non-reactive
cells was also determined, based on cell morphology. Reactive
astrocytes present larger cell bodies and thicker, easily visible
processes, as opposed to non-reactive cells, which have more
Data analysis and statistical methods.
as means 6 S.E.M. One-way ANOVA with Tukey’s Multiple
Comparison test was used to compare SOD1 gene expression and
percentage of decline in body weight and functional tests between
mouse groups. The same test was used to compare astrocyte,
microglia and motor neuron densities and HuNu positive cell
counts between mouse groups. The Kaplan-Meier method was
used to determine differences in survival rates between groups of
G93A mice with or without cell transplants, and is reported as a
evaluations, an endpoint corresponding to the end-stage of
disease was established, based on the mean percentage of
decline of motor function or body weight in all G93A mice. The
number of weeks elapsed for each animal to reach the established
endpoint was determined and the resulting data were then
evaluated through Kaplan-Meier method. For all analyses,
p,0.05 was considered significant.
Data are presented
We would like to acknowledge Karen Lynn Brocklehurst, RLATG, Mary
Jane Perkins, BS, RLATG, SRA, and Kenneth Hass, BA, LATG, from
USF’s Division of Comparative Medicine for their unstinting assistance in
animal care and surgical procedures.
Conceived and designed the experiments: SGD PRS NKN CDS.
Performed the experiments: MCOR DJE S. Mirtyl JS S. Mitha SS ST.
Analyzed the data: SGD MCOR S. Mirtyl JS S. Mitha SS ST.
Contributed reagents/materials/analysis tools: PRS. Wrote the paper:
Multiple Cell Injections for ALS Mice
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