Brain Research 1035 (2005) 73 – 85
www.elsevier.com/locate/brainres
Research report
Axon growth and recovery of function supported by human bone marrow
stromal cells in the injured spinal cord exhibit donor variations
Birgit Neuhubera,1, B. Timothy Himesa,b,1, Jed S. Shumskya, Gianluca Galloa, Itzhak Fischera,*
a
Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, 19129 PA, USA
b
Department of Veterans Affairs Hospital, Philadelphia, 19102 PA, USA
Accepted 24 November 2004
Available online 27 January 2005
Abstract
Bone marrow stromal cells (MSC) are non-hematopoietic support cells that can be easily derived from bone marrow aspirates. Human
MSC are clinically attractive because they can be expanded to large numbers in culture and reintroduced into patients as autografts or
allografts. We grafted human MSC derived from aspirates of four different donors into a subtotal cervical hemisection in adult female rats and
found that cells integrated well into the injury site, with little migration away from the graft. Immunocytochemical analysis demonstrated
robust axonal growth through the grafts of animals treated with MSC, suggesting that MSC support axonal growth after spinal cord injury
(SCI). However, the amount of axon growth through the graft site varied considerably between groups of animals treated with different MSC
lots, suggesting that efficacy may be donor-dependent. Similarly, a battery of behavioral tests showed partial recovery in some treatment
groups but not others. Using ELISA, we found variations in secretion patterns of selected growth factors and cytokines between different
MSC lots. In a dorsal root ganglion explant culture system, we tested efficacy of conditioned medium from three donors and found that
average axon lengths increased for all groups compared to control. These results suggest that human MSC produce factors important for
mediating axon outgrowth and recovery after SCI but that MSC lots from different donors vary considerably. To qualify MSC lots for future
clinical application, such notable differences in donor or lot–lot efficacy highlight the need for establishing adequate characterization,
including the development of relevant efficacy assays.
D 2004 Elsevier B.V. All rights reserved.
Theme: Development and regeneration
Topic: Transplantation
Keywords: Axon regeneration; Therapeutic factors; Axon outgrowth; Partial hemisection
1. Introduction
Treatments that enhance axonal growth and regeneration
of damaged axons in the central nervous system (CNS) have
a potential for improving recovery following spinal cord
injury (SCI). The adult, and especially the injured CNS, is
inhibitory to axonal growth. Therefore, effective repair
strategies for SCI require the creation of a permissive
* Corresponding author. Fax: +1 215 843 9802.
E-mail address: Itzhak.Fischer@drexel.edu (I. Fischer).
1
These authors contributed equally to the paper.
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2004.11.055
environment within the injured spinal cord that protects
damaged neurons from the effects of secondary injury and
also facilitates axonal regeneration. Cell transplantation is
among the most promising therapeutic approaches for
treating SCI. Ideally, cell transplants would be readily
obtainable, easy to expand and bank, and capable of
surviving long enough to facilitate sufficient and appropriate
axonal regeneration [14].
Bone marrow stromal cells (MSC) are connective tissue
progenitor cells that are distinct from hematopoietic stem
cells [45]. While MSC can be easily expanded ex vivo from
raw bone marrow, there is no generally accepted method for
MSC isolation, propagation, and characterization. As a
74
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
result, the phenotype of culture-expanded MSC can vary
considerably when derived by different methods [44] or
from different sources [42,43].
Recent studies proposed a more extensive differentiation
potential of MSC showing phenotypic plasticity that appears
to cross the boundaries of the traditional germ layers
including cardiac cells [41], skeletal muscle [31], and neural
cells [30]. Whether this apparent plasticity represents
transdifferentiation, a pool of persistent pluripotent stem
cells, cell fusion, or artifacts of culturing remains controversial [21,25,34,53].
Because of their ability to differentiate into a variety of
cells, the ease of their isolation and expansion, and their
potential use for clinical application, efforts have increased
to better understand the biology of MSC. In the injured
CNS, MSC transplantation has been shown to improve
recovery after stroke or traumatic brain injury [8]. In animal
models of SCI, grafts of MSC have been shown to promote
remyelination [1] as well as partial recovery of function
[9,23,60]. While previous studies have suggested that MSC
can differentiate into cells with neural characteristics in vitro
[11,28,47] and in vivo [9,23,30], it is unclear whether such
differentiation contributes to recovery of function in animal
models of neurotrauma.
There is growing evidence that MSC produce a variety
of neurotrophic factors as well as chemokines and
cytokines in vitro and in vivo (for review, see [8]).
Kinnaird et al. [29] found that paracrine signaling of
MSC is an important therapeutic mechanism in the treatment of ischemia. A recent study [54] showed that MSC
secrete brain natriuretic peptide (BNP), a peptide with
diuretic and vasodilatory effects in vitro, suggesting that
MSC could facilitate recovery by reducing edema and
improving perfusion. In addition, Chen et al. [6] showed
that the secretion profile of MSC is responsive to the
environment with increased secretion of certain growth
factors (e.g., BDNF, NGF) in the injured brain. Zhong et al.
[62] demonstrated that neural cell death in response to
oxygen-glucose deprivation was reduced in hippocampal
slices co-cultured with MSC, suggesting a neuroprotective
effect possibly mediated by diffusible factors released by
MSC. Thus, the cells may create a permissive environment
for axon outgrowth and axonal guidance mediated by their
release of trophic factors, thereby improving self-repair in
the damaged CNS.
In the present study, we investigated the efficacy of
different lots of MSC, each obtained from the bone marrow
aspirate of a different donor, by evaluating their ability to
support axonal growth following engraftment in a rat model
of subtotal cervical hemisection. Functional recovery was
evaluated by an array of motor and sensory tests. In addition,
we showed variations in the secretion profiles for selected
growth factors and cytokines of MSC from different donors,
and the ability of MSC-conditioned medium to promote
axon outgrowth in an in vitro dorsal root ganglion (DRG)
culture system independent of the donor.
2. Materials and methods
2.1. Isolation and expansion of human MSC
Human MSC were isolated from bone marrow aspirates
taken from the iliac crest of four healthy adult human
volunteers under informed consent. Donors were tested for
various chronic diseases (heart, kidney or liver disease,
ulcer, cancer, diabetes, epilepsy) as well as for bacterial or
viral infections. Vital signs, hematological lab values and
donor weight were within normal range and donors were not
currently taking prescription medication. Donor age ranged
between 18 and 45 years. We took great care that harvest
and culture of MSC from different donors were performed
in a very similar manner so as to limit harvest- and culturedependent variation.
Red blood cells (RBC) were first removed from the
bone marrow aspirate by adding (1:20 v/v) ammonium
chloride buffer consisting of 155 mM ammonium
chloride, 10 mM potassium bicarbonate, and 0.1 mM
EDTA (ethylenediaminetetraacetic acid), at pH 7.2. The
resulting cell suspension was centrifuged for 10 min at
500 g to remove the RBC fraction. The mononuclear
cell pellet was then re-suspended and washed twice in
complete medium consisting of Minimal Essential
Medium-alpha (a-MEM, Invitrogen, Carlsbad, CA) supplemented with 4 mM glutamine and 10% fetal bovine
serum (FBS, Atlanta Biologicals, Norcross, GA) and
centrifuged for 10 min at 500 g. Cells were counted
by trypan blue exclusion and seeded in tissue culturetreated flasks at a density of approximately 50,000 viable
cells/cm2 and placed in a 37 8C humidified cell culture
incubator. On day 5, the non-adherent cells and spent
medium were removed from the flasks and the adherent
cells re-fed with fresh, complete medium. The adherent
cells were then cultured for an additional 5 days prior to
the first passage. Seeding density for subculture of MSC
at all subsequent passages was approximately 5000 cells/
cm2. Cells were grown to confluency before each passage
(3–4 days). Medium was exchanged every second day.
All cells used for transplantation (passage 4) were derived
from frozen stocks. Cryopreserved MSC were thawed
quickly at 37 8C and plated in a-MEM (Invitrogen)
supplemented with 20% FBS and 4 mM l-glutamine
(Invitrogen) on 100-mm plastic dishes. The same lot of
serum was used for all experiments. The serum lot chosen
allowed consistent, rapid growth and proliferation of
MSC. Twenty-four hours prior to grafting, cultures were
incubated with 2 AM PKH26 (Sigma-Aldrich, St. Louis,
MO). The following day, cells were washed several times
with HBSS and then removed from the culture plates
using 0.1% trypsin/EDTA (Cellgro, Herndon, VA). MSC
were then re-suspended in complete medium at 50,000
cells/Al and maintained on ice during transplantation
surgery. After surgery, a sample of remaining cells was
re-plated overnight to verify viability.
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
2.2. Grafting of human MSC into a subtotal cervical
hemisection
A total of 42 female Sprague–Dawley rats (225–250 g;
Taconic, Germantown, NY) were used for this study. All
procedures were approved by the institutional animal
welfare committee and conformed to NIH Guidelines for
the Care and Use of Laboratory Animals. All animals were
immune suppressed with a single daily injection of Cyclosporine A (CsA; Sandoz Pharmaceuticals Co., East
Hanover, NJ) administered subcutaneously at a dose of
1 mg/100 g/24 h starting 3 days before transplantation
procedures and continued for 2 weeks following surgery.
After this 2-week period, and for the duration of the
experiment, CsA was administered orally via drinking water
(Neoral, Sandoz, 50 Ag/ml).
Eight rats per donor (4 different donors) received a right
subtotal cervical hemisection and a transplant of MSC;
another eight rats received only gelfoam as a transplant. Ten
rats served as uninjured behavioral controls. Rats were
anesthetized by intraperitoneal (i.p.) injection of a cocktail
of acepromazine maleate (0.7 mg/kg; Fermenta Animal
Health Co., Kansas City, MO), ketamine (95 mg/kg; Fort
Dodge Animal Health, Fort Dodge, IA), and xylazine
(10 mg/kg; Bayer Co., Shawnee Mission, KS). A laminectomy was carried out at the C3–4 level to expose one spinal
cord segment. The dura over the right dorsal root entry zone
was opened with a micro-scalpel and a shallow incision was
made in the right dorsal spinal cord. A glass-pulled finetipped micro-aspiration device was used to extend the lesion
laterally and ventrally. The rostrocaudal extent of the lesion
cavity was about 2 mm. The lesion completely disrupted the
dorsal and lateral funiculus and dorsal gray matter but
spared some ventral gray matter and the ventral funiculus.
After hemostasis was achieved, a piece of gelfoam that had
been immersed in growth medium alone or in growth
medium containing a suspension of MSC (5 105) was
implanted into the cavity, followed by injection of another
5–10 Al of cells (5 104/Al) suspended in growth medium
onto the gelfoam using a 10-Al Hamilton syringe attached to
a 27-gauge needle. Presoaking the gelfoam permits expansion of the matrix before it is implanted, thereby preventing
potential additional injury. The dura was closed with
interrupted 10-O silk sutures, and muscle and skin were
closed in layers. Within 10 min of the spinal cord lesion, all
rats received a bolus injection of methylprednisolone
(30 mg/kg; Pharmacia and Upjohn Company, Kalamazoo,
MI) through the tail vein. A second bolus injection of the
same dose was given 2 h later. After surgery, animals were
kept on heating pads and closely observed until awake, and
then returned to their home cages.
2.3. Tissue preparation
Of the eight rats that had received cells from donors 1, 2,
3, or 4, two were chosen at random and perfused 2 weeks
75
following surgery. Of the remaining rats, four died during
the course of the experiment (one rat each with cells from
donors 1, 2, and 3, and one with a gelfoam graft) and were
not included in the analysis. Therefore, five rats that had
received MSC from donor 1, five with MSC from donor 2,
five with MSC from donor 3, six with MSC from donor 4,
and seven with the gelfoam transplants were analyzed after
11 weeks. At the end of the experiment, animals were
anesthetized with an i.p. injection of sodium pentobarbital
(100 mg/kg, Abbott Laboratories, North Chicago, IL) and
perfused transcardially with 200 ml of normal saline
solution followed by 500 ml of ice cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brain and spinal
cord were dissected out and immersed overnight in 0.1 M
phosphate buffer (PB) at 4 8C followed by cryoprotection in
30% sucrose for 3–5 days. The lesion site was identified,
blocked, embedded in OCT compound (Fisher Scientific,
Pittsburgh, PA) and kept at 80 8C. Using a cryostat,
sagittal sections (20 Am) of the lesion site were obtained.
Sections were mounted onto gelatin-coated slides and
processed for histological or immunocytochemical staining
using standard protocols from our laboratory.
2.4. Histology and quantification
Every fifth section through the lesion site was stained
to demonstrate myelin sheaths and counterstained with
cresyl violet acetate [22]. Selected sections were immunolabeled with one of three antibodies: a human-specific
mitochondria antibody (MAB1273, Chemicon International, Temecula, CA) was used to identify the MSC;
an antibody that recognizes phosphorylated epitopes of
neurofilament (RT-97; Developmental Studies Hybridoma
Bank, University of Iowa, Iowa City, IA), and an
antibody that recognizes axonal growth associated proteins (GAP-43 [5], kindly provided by Dr. Larry
Benowitz) were used to identify host axons that had
grown into the graft. The human mitochondria and RT-97
antibodies were used at a dilution of 1:100. GAP-43 was
used at a dilution of 1:2500. All primary antibodies were
diluted in 0.1M PBS, pH 7.4 containing 2% goat serum
(GS-PBS). Controls omitting the primary antibody were
routinely included. Sections were incubated at room
temperature overnight in a humidified chamber. After
several rinses in PBS, secondary antibodies, diluted 1:200
in GS-PBS, were applied (FITC-conjugated goat antimouse IgG, Texas Red-conjugated goat anti-mouse or
goat anti-rabbit IgG; Jackson Immunoresearch Labs, Inc,
West Grove, PA). Following several washes in PBS,
sections were coverslipped using Vectashield (Vector
Laboratories, Burlingame, CA).
A second series of every fifth section was stained using
the RT-97 antibody for quantitative evaluation of axon
growth. The staining procedure used was as described
above except that a biotinylated goat anti-mouse IgG
(Jackson) was used as the secondary antibody. Since
76
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
endogenous peroxidase levels are very low in perfused
CNS tissue and only very low background staining was
observed in negative controls (no primary antibody),
blocking of endogenous peroxidase was not necessary.
After several washes in PBS, staining was visualized using
a Vector ABC kit and Sigma Fast DAB tablets according to
manufacturer’s instructions. The sections were then dehydrated and coverslipped using DPX (Sigma-Aldrich).
Between 10 and 15 sections were analyzed for each
animal. Images were acquired using a Leica DMRBE
Microscope with an attached Sensys model 1401 CCD
camera/LCD filter system (Biovision Technologies, Exton,
PA) using IPLABS software (Scanalytics, Inc., Fairfax, VA)
running on an Apple Power Macintosh G4 computer.
Images were captured at 100-Am intervals through the
graft. NIH image (version 1.62) was used to outline and
measure the area of the graft and the area stained for RT-97
within the graft in each section. A summation of the
measured area of graft, the area within the graft stained for
RT-97, and the area fraction stained for RT-97 was
calculated for each animal.
2.5. Behavioral testing
A battery of behavioral tests was administered to all rats
8 weeks post injury and transplantation to assess functional
recovery. Tests were scored by trained observers who were
unaware of experimental conditions with inter-rater reliability greater than 95%.
2.5.1. BBB test
Hindlimb motor function was assessed in an open field
(5 2 ft) using a modification of the BBB locomotor
rating scale [3]. Because these rats had received unilateral
subtotal cervical hemisections, only the hindlimb on the
affected side was evaluated. Rats were observed for 2 min
and scored from 0 (no observable movements) to 21
(normal locomotion). Similarly injured rats have shown an
initial deficit followed by almost complete recovery using
this assessment [27].
2.5.2. Limb preference (cylinder) test
Forelimb exploration was assessed in a Plexiglas cylinder
(17.8 cm diameter 35.5 cm height) for 3 min. When placed
in the cylinder, animals spontaneously rear and contact the
walls with their forepaws. The number of forelimb contacts
(left, right, and both) with the cylinder walls were counted
and expressed as a percentage of total placements. Percentages of forelimb contacts with the right forelimb and with
both forelimbs were added to reflect the full usage of the
affected limb. Initial baseline performance showed no bias in
forelimb preference [35,49,51]. This test has been used
previously to demonstrate a deficit followed by partial
recovery of forelimb function after an acute subtotal cervical
hemisection and transplantation of genetically modified
fibroblasts in adult rats [35,49].
2.5.3. Postural measures
Postural adjustments and rears were also analyzed during
a 3-min recorded observation period in the cylinder [51].
Postural adjustments were defined as shifts in hindlimb
placement while the rats were rearing and preparing to
contact the walls with their forelimbs. The number of right
and left hindlimb steps was counted as the animal adjusted
its position while rearing. Data are expressed as the ratio of
hindlimb postural adjustments per rear on the affected side
over the total.
2.5.4. Grid test
Paw placement of each limb on a grid bar was assessed
as animals walked on a plastic-coated wire mesh grid
(36 cm length 38 cm width 30 cm height, with 3 2
cm openings) for 2 min. Steps where the paw gripped the
grid bar and supported the animal’s weight were counted as
correct. The number of correct paw placements was
expressed as a percentage of the total steps. Percentages
of correct paw placements were calculated for each limb. In
a variation of this test, deficits from an acute thoracic dorsal
hemisection were ameliorated by grafts of fibroblasts
secreting NT-3 [15]. Full recovery of performance of the
contralateral side was observed after a complete cervical
hemisection, with less recovery in the ipsilateral forelimb
than hindlimb [51].
2.5.5. Thermal sensitivity (heat) test
Latency for limb withdrawal in response to plantar paw
contact with a heat stimulus was measured (adapted from
[12,20,61]). Animals were habituated for 30 min in elevated
Plexiglas cages (Ugo Basile, Italy) with a moveable radiant
heat source (25–29 8C) underneath. If no paw withdrawal
occurred after 30 s, the heat stimulus was removed to
prevent tissue damage, and the animal was assigned the
maximal withdrawal latency of 30 s. Five trials were run for
each paw with a 15-min interval between each trial to
prevent sensitization. The last 4 trials were averaged to
provide the mean latency of withdrawal. This test has been
shown to reveal a reduced latency to thermal stimuli
(indicating hypersensitivity or thermal allodynia) in rats
with acute midthoracic hemisections [10,16–18] followed
by partial recovery of function in the right forepaw and left
hindpaw after a complete cervical hemisection with delayed
transplantation of genetically modified fibroblasts [51]. We
have interpreted this pattern of recovery as a result of
sprouting associated with the dorsal roots at the level of the
injury (right forepaw) with contributions from the spinothalamic tract or its descending modulation representing the
left hindpaw; therefore, we collected withdrawal data from
these two limbs.
2.6. Collection of conditioned media
Cells were quick thawed at 37 8C and added to T75
flasks each containing 15 ml of complete medium consisting
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
of a-MEM supplemented with 10% FBS and 4 mM lglutamine. Flasks were then placed in a 37 8C humidified
cell culture incubator overnight. The following day, cultures
were re-fed with fresh medium and returned to the
incubator. On day 3, conditioned medium was collected
and centrifuged at 500 g for 5 min to remove cell debris.
Cells were recovered from the flasks using 0.5% trypsin
and counted. Aliquots of conditioned media were frozen at
80 8C for short-term storage.
2.7. ELISA
Aliquots of conditioned media were thawed and warmed
to room temperature for quantitative sandwich immunoassay. All ELISA Kits (IL-6, MCP-1, VEGF, SCF, SDF,
BDNF) were purchased from R&D Systems (Minneapolis,
MN) and used as per manufacturer’s instructions. Microplates were read on a SpectraMax Plus plate reader
(Molecular Devices Corp., Sunnyvale, CA) using SoftMax
Pro software (Molecular Devices). Cytokine levels were
then extrapolated from a standard curve and normalized to
pg/million cells/day.
2.8. DRG explant culture, immunostaining, and quantitation
Dorsal root ganglia (DRG) were obtained from E10 chick
embryos [26]. For each condition, three DRG were used in
two separate experiments. DRG explants were cultured in
24-well dishes coated with 100 Ag/ml poly-l-lysine (SigmaAldrich) containing 500 Al of unconditioned or MSCconditioned medium from three different donors. Cells from
donor 4 were not included in this experiment due to
technical reasons. After 48 h, DRG were fixed in 0.25%
glutaraldehyde. DRG were then immunostained with a
monoclonal anti-hIII-tubulin antibody (BabCO, Richmond,
CA) and the staining developed using a secondary biotinylated antibody (Jackson) and the Elite Vectastain ABC kit
(Vector Laboratories) in combination with a Fast Diaminobenzidine Tablet kit (Sigma-Aldrich).
An Olympus CK40 microscope with a 20 phase
objective connected to a Nikon Coolpix 990 digital camera
was used for analysis. To determine the average length of
axons extended from each DRG, the distance from the edge
of the explant to the main field of growth cones was
measured in four places around the perimeter by using a
micrometer slide. The average of the four measurements
and the standard error were calculated for each experiment
[57].
2.9. Statistical analysis
Behavioral data from each test were analyzed by a
one-way factorial ANOVA comparing all four donor
groups, gelfoam controls, and uninjured animals. When
ANOVA was significant (indicating the injured animals
did not recover to uninjured levels), we identified
77
individual donor groups that promoted recovery within
the range of behavioral performance demonstrated
between gelfoam controls and uninjured animals. Because
we are interested in identifying donors that promote
recovery, individual comparisons were made between the
selected experimental group and the gelfoam control
group by either a one-tailed Student’s t test or a Chisquare test (for frequency data). Significance levels were
set to 0.05 and it should be noted that P b 0.10 is
considered significant for one-tailed comparisons. Analysis of average axonal length was determined using a twotailed Student’s t test with significance levels set to 0.01.
Correlational analysis was performed between results
using simple linear regression. All data are presented as
mean values F SEM.
3. Results
3.1. Grafts of human MSC support axon outgrowth in a
donor aspirate-dependent manner
Two weeks after grafting, MSC were identified by
labeling with PKH26 and antibodies against human
mitochondria. The lesion cavity was filled with MSC (Figs.
1a and b) regardless of which donor was used. Most of the
grafted cells remained at the lesion site but some of them
migrated into the penumbra of surrounding host tissue. In no
instances were the grafted MSC observed more than 500 Am
from the lesion site. Grafts of MSC supported extensive
axonal growth, as evidenced by GAP43 and neurofilament
staining (Figs. 1c and d). GAP43 and neurofilament labeled
axons were most often located in the same regions of the
graft.
After 11 weeks, the graft site was devoid of MSC
indicated by the lack of staining with the hMITO
antibody. Nissl staining revealed fibroblast-like cells at
the graft site that were not stained with the hMITO
antibody suggesting that host cells migrated into the graft
site. Previous experiments using a contusion injury model
from our laboratory (unpublished results) and others [40]
indicated that oligodendrocytes infiltrate the graft, and
astrocytes surround the graft site extending processes into
the graft periphery. In addition, other previously published
studies showed that immune cells [46], oligodendrocyte
precursors [19,24] and Schwann cells [4] infiltrate the
injury site.
Staining with the RT-97 neurofilament antibody showed
numerous axons within the graft (Fig. 2a). Control animals
that received only a gelfoam graft showed very little
neurofilament staining through the lesion site (Fig. 2b).
We quantified the area occupied by neurofilament staining
as an area fraction of the total lesion area (Fig. 2c). Cells
obtained from different donors supported axon growth into
the graft at different levels. Whereas MSC from donors 1
(area fraction: 0.156 + 0.019), 2 (area fraction: 0.155 +
78
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
Fig. 1. Histological examination of MSC grafts in the injured spinal cord 2 weeks after grafting. MSC grafted into the injured spinal cord were identified by
(a) a fluorescent membrane dye and (b) an antibody specific for human mitochondria 2 weeks after grafting. The presence of axons within the graft was
demonstrated by immunostaining with antibodies against (c) GAP-43 and (d) neurofilaments (RT-97). Scale bar: 100 Am.
0.034), and 4 (area fraction: 0.114 + 0.014) promoted
significant ( P b 0.01) axon growth into the graft, axon
growth into MSC grafts from donor 3 (area fraction: 0.06 +
0.011) was not significantly different from the gelfoam
control (0.047 + 0.011) or a fibroblast graft (data not
shown). These findings demonstrate that while MSC can
promote axonal growth when grafted into the injured spinal
cord, there are significant variations between cells from
these different donors with respect to the ability to promote
axon growth into the graft.
Fig. 2. Axon growth into the graft is donor-dependent 11 weeks after grafting. Sections of (a) MSC- or (b) gelfoam-grafted spinal cord were stained with
antibodies against neurofilament (RT-97) to determine axon growth into the graft. (a) MSC grafts supported axonal growth, whereas (b) gelfoam grafts were
negative for neurofilament staining. (c) The area of neurofilament staining was measured and calculated as a fraction of the total graft size. The histogram
shows the different amounts of neurofilament staining obtained from MSC grafts of different donors. Only donors 1, 2, and 4 show significantly ( P b 0.01)
more axon growth into the graft compared to the gelfoam control, while axon growth into MSC grafts from donors 3 is not significantly different from control.
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
3.2. Grafts of MSC from certain donors result in partial
recovery of function
Motor and sensorimotor behaviors were assessed with a
battery of behavioral tests at 8 weeks post injury. BBB
score, which is a measure of hindlimb locomotion in an
open field showed almost complete recovery in all groups
with no significant differences among groups (donor 1:
BBB score = 19.6 F 1.4, donor 2: BBB score = 19.6 F 1.4,
donor 3: BBB score = 21.0 F 0.0, donor 4: BBB score =
21.0 F 0.0, gelfoam control: BBB score = 18.7 F 1.5,
uninjured: BBB score = 21.0 F 0.0). This degree of
recovery is consistent with our previous results following an
acute subtotal cervical hemisection [27].
Forelimb exploration in the cylinder test showed minimal
recovery. Baseline performance typically results in 25% use
of each forelimb independently and 50% use of both
together [35,49,51]. Therefore, full recovery to baseline
performance would show 75% contacts with the affected
limb. In this study, rats only recovered 20–40% use of the
affected forelimb (Fig. 3a). Again, this degree of recovery is
consistent with our previous results following an acute
79
subtotal cervical hemisection in operated controls [35,49].
No differences were found among rats transplanted with
MSC from these different donors, even though axon
outgrowth was donor-dependent.
Postural measures (shifts in hindlimb placements while
rearing) can be collected during performance in the
cylinder test. Rats typically exhibit no limb preference
during postural adjustment; thus, full recovery would
constitute 50% use of the affected limb. Rats that had
received MSC transplants from donor 2 were able to use
their affected hindlimb to make postural adjustments
better than the other groups (Fig. 3b). Even though MSC
grafts from donor 1 supported axon outgrowth equally
well, no positive effects on the use of the affected
hindlimb was observed. 80% of rats that had received
MSC from donor 2 used their affected limb compared
with only 43% of control rats that had received gelfoam
(compared with 20% of rats that had received MSC from
donor 1, 20% that had received MSC from donor 3, and
17% that had received MSC from donor 4). No differences were observed among groups in the numbers or
types of rears.
Fig. 3. Tests for the recovery of motor and sensorimotor recovery. (a) Forelimb exploration and (b) postural adjustments with the hindlimb of the affected side
were evaluated in a cylinder test. (a) The number of forelimb contacts (left, right, and both) with the cylinder walls were counted and expressed as a percentage
of total placements. Percentages of forelimb contacts with the right forelimb and with both forelimbs were added to reflect the full usage of the affected limb.
No significant difference was observed among groups. (b) The number of right and left hindlimb steps was counted as the animal adjusted its position while
rearing. Data were expressed as the ratio of hindlimb postural adjustments per rear on the affected side over the total. Only rats that had received MSC
transplants from donor 2 used their affected hindlimb better than control (Chi square, P b 0.001). (c and d) Paw placement of fore- and hindlimb of the affected
side was analyzed on a grid bar. The number of correct paw placements was expressed as a percentage of the total steps. (c) Only rats that had received MSC
transplants from donor 4 demonstrated more correct right forelimb paw placements than gelfoam controls (Student’s t test, P b 0.05). (d) Rats fully recovered
the use of the affected hindlimb independent of the MSC donor.
80
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
Forelimb and hindlimb sensorimotor behavior associated
with lateral corticospinal tract function [15] can be
measured by evaluating the percentage of correct paw
placements while traversing a grid. In this study, rats
partially recovered the use of the affected forelimb (Fig.
3c) with only grafts from donor 4 promoting behavioral
recovery, independent of the amount of axon outgrowth
observed by neurofilament staining. Rats fully recovered the
use of the affected hindlimb (Fig. 3d) with no differences
among donor groups. As expected, the unaffected side
showed full recovery (data not shown). These results
following an acute subtotal cervical hemisection are consistent with the pattern of recovery seen following a
complete cervical hemisection where full recovery of
performance of the unaffected side, with less recovery in
the affected forelimb than hindlimb, was observed [51].
Forelimb and hindlimb sensorimotor behavior associated
with spinothalamic tract function can be measured by
withdrawal latency following a heat stimulus [51]. Following
spinal injury, rats typically show decreased withdrawal
latency that slowly recovers. In this study, rats showed
partial recovery of right forelimb responses with no difference among groups (Fig. 4a). Partial recovery of left
hindlimb response was observed in rats that had received
MSC from donor 3 (Fig. 4b), the same donor whose MSC
had elicited the least amount of axon outgrowth. This could
be explained by decreased regeneration of sensory fibers,
thereby reducing thermal allodynia, in the case of MSC
grafts from donor 3 compared to the rats that received grafts
from other donors. However, as we only measured the
staining for neurofilaments, we could not differentiate
between motor and sensory fibers. Uninjured rats show
forelimb withdrawal latencies of 7–8 s and hindlimb
withdrawal latencies of 8–9 s. By using these values, we
calculated that rats in all groups demonstrated about 60%
recovery with the right forelimb. Control rats and rats with
transplants from donors 1, 2, and 4 showed about 60%
recovery with the left hindlimb, whereas rats that had
received MSC from donor 3 showed about 75% recovery.
Correlation analysis was used to determine whether the
amount of axon growth into the graft as determined by
neurofilament staining predicted the behavioral results. No
such correlation could be found for any of the behavioral
tests.
3.3. Human MSC secrete therapeutic factors and support
axon outgrowth in vitro
Therapeutic factors secreted by MSC may be part of the
mechanism for axon outgrowth and recovery of function. To
determine if the differences described above are due to
secretion of different amounts of potential therapeutic
factors by MSC from different donors, we screened MSCconditioned medium from 4 donors for the presence of
certain growth factors (BDNF and VEGF) and cytokines
(IL-6, MCP-1, SCF and SDF-1a) using ELISA. All protein
levels were normalized to pg/ml/day/106 cells. As shown in
Fig. 5, growth factor and cytokine concentrations in the
medium differed widely between donors, even though cell
purification protocols were identical. Human MSC
expressed 335–640 pg/ml/day/106 cells of VEGF and 65–
105 pg/ml/day/106 cells of BDNF. Protein levels of
cytokines ranged from 315 to 735 pg/ml/day/106 cells for
IL-6, 60–280 pg/ml/day/106 cells for MCP-1, 1–13 pg/ml/
day/106 cells for SCF, and 370–810 pg/ml/day/106 cells for
SDF-1a.
To examine if MSC-conditioned medium containing
these factors had any effect on axon outgrowth, we
incubated chick DRG explant cultures with unconditioned
and conditioned MSC medium from 3 different donors for 2
days. Average axon lengths were measured, and mean and
standard error were plotted (Fig. 6a). DRG explants
incubated in unconditioned MSC medium had an average
axon length of 338 (F31) Am. Treatment of DRG explants
Fig. 4. Test for improvement in withdrawal latency after a heat stimulus. Latency for limb withdrawal in response to plantar paw contact with a heat
stimulus was measured. Five trials were run for (a) the right forelimb and (b) the left hindlimb with a 15-min interval between each trial to prevent
sensitization. The last 4 trials were averaged to provide the mean latency of withdrawal. (a) Rats showed partial recovery of right forelimb responses with
no difference among groups. (b) Partial recovery of left hindlimb response was observed only in rats with MSC grafts from donor 3 compared to gelfoam
controls (Student’s t test, P b 0.10).
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
81
explants treated with MSC-conditioned medium from donor
3 were not only longer but also less fasciculated than axons
from DRG explants treated with conditioned medium from
other donors. This may have been due to a different
composition of secreted factors that either stimulated
different expression of surface molecules on axons or
affected a different subtype of neurons.
4. Discussion
Fig. 5. Secretion of therapeutic factors by MSC. Secretion of trophic factors
and cytokines is donor-dependent. Medium of MSC from four different
donors was conditioned for three days and analyzed using ELISA. All cells
were normalized to pg/ml/day/million cells. Secretion patterns of cells from
different donors vary significantly.
with MSC-conditioned medium from all three donors
significantly ( P b 0.01) increased average axon length
compared to unconditioned control medium. Human MSCconditioned medium from donor 1 resulted in a 2.3-fold
increase in average axon length (835.42 F 66.93), donor 2
MSC-conditioned medium resulted in a 2.6-fold increase in
average axon length (958.33 F 32.18), and donor 3 MSCconditioned medium in a 3-fold increase in average axon
length (1116.67 F 70.30). Average axon lengths in DRG
cultures treated with medium from donors 1 and 3 differed
significantly ( P b 0.01) from each other.
We immunostained DRG explant cultures with antibodies against hIII-tubulin to verify that processes were
neuronal. Fig. 6b illustrates the increase in average axon
length in DRG explant cultures treated with unconditioned
or MSC-conditioned medium from all three donors.
Interestingly, it appeared that axons extended from DRG
In this study, we have demonstrated for the first time that
axon growth and recovery of function in response to a
human MSC graft in the injured rat spinal cord is donordependent. Examination of the secretion profile for certain
growth factors and cytokines revealed major differences
between four human MSC donors. While this secretion
profile did not seem to greatly affect axon growth in vitro,
axon outgrowth into MSC grafts in a subtotal cervical
hemisection differed significantly depending on the cell
donor. Behavioral tests also showed differences in the
degree of recovery among donors. Interestingly, there was
no direct correlation between the amount of in vivo axon
growth supported by MSC from any specific donor and
functional recovery, suggesting that additional conditions
are likely to be necessary for stable functional recovery.
In our in vitro assay, MSC-conditioned medium from all
three donors resulted in a significant increase in average axon
length; however, there was no correlation with our in vivo
results. In vitro, the effect of MSC-conditioned medium
appeared to correspond to the relative amount of BDNF
secreted by cells from different donors; however, amounts of
BDNF in the conditioned medium were lower than BDNF
concentrations typically used to support axon growth. In
addition, we cannot exclude a potential effect of other
neurotrophins, such as nerve growth factor or neurotrophin
Fig. 6. Effect of MSC-conditioned medium on DRG axon length in vitro. Average axon length of DRG explants increases after treatment with MSC-conditioned
medium. (a) DRG explant cultures were treated with unconditioned or MSC-conditioned medium for 48 h. Values represent average axon lengths F SEM (in Am)
of three DRG explants per condition obtained from two separate experiments. (b) Axon outgrowth from DRG explants treated with unconditioned medium or
MSC-conditioned medium from three donors stained with antibodies against hIII-tubulin.
82
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
3, which we did not evaluate in our ELISA screening.
Neurotrophins including BDNF are known to stimulate axon
outgrowth (for review, see [39]). Aside from secreted trophic
factors, extracellular matrix molecules produced by MSC
could also affect axon growth. These molecules were not
present in the conditioned medium used for in vitro testing. In
vivo, the presence of certain subsets of these molecules,
which may be donor-dependent, could have an inhibitory
effect on axon growth. Furthermore, factors secreted from
astrocytes, oligodendrocytes, and immune cells may suppress
the positive effects of BDNF or other neurotrophins secreted
by MSC. In addition, other factors differentially secreted by
MSC from different donors may alter host cell secretion to
varying degrees, making the environment more or less
hostile. It is also possible that factors released by the injured
tissue affect the secretion profile of transplanted MSC as has
been shown by Chen and colleagues [6], and that cells from
different donors or lots may respond differently.
In this study, we evaluated axonal growth in vivo by
neurofilament staining. This staining allows the visualization of processes but does not identify their source. It is
likely that the RT-97-positive processes observed within the
MSC graft are composed of CNS as well as dorsal root
axons. Although we did not do tracing experiments in this
study, in previous studies [35,37] we have shown that
growth of CNS axons is supported by cellular grafts
expressing neurotrophins, suggesting that at least part of
the processes labeled with neurofilament are extended from
CNS neurons.
To investigate if the amount of axonal growth in vivo
correlated with functional recovery, we used different
behavioral tests evaluating recovery of motor and sensory
axons. However, the results obtained in these tests did not
correlate with the amount of axon growth into the graft.
MSC grafts release neurotrophins that can stimulate axon
outgrowth; however, the stimulated outgrowth may not be
associated with functionally relevant tracts (e.g., improvement in forelimb exploration in the cylinder is linked to
regeneration of rubrospinal axons). Also, without activation
of functionally relevant synaptic connections (e.g., stimulation through targeted exercise), axon outgrowth may not be
directed toward the correct target. Considering the unstimulated release of neurotrophins by MSC grafts, it is not
surprising that the amount of axon outgrowth does not
correlate with behavioral recovery. Alternatively, compensatory reorganization of neural circuitry after injury may
lead to synaptic reorganization to maintain function even
without requiring sprouting or axon regeneration.
The modest recovery observed in motor and sensory tests
indicates that the purification methods and transplantation
protocols are still not optimal. We were unable to detect
MSC in the injury site at the end of the study, suggesting
that cell survival is limited. Poor graft survival could
potentially limit the growth of axons because trophic factors
would not be present for a sufficient amount of time. Also,
after the disappearance of MSC, axons may lack a
permissive matrix through which to grow. Together, this
may result in insufficient re-growth measured as functional
recovery by behavioral testing techniques. We are currently
testing improved immune suppression protocols to enhance
MSC survival. Prolonged survival of MSC in the graft may
result in improved behavioral recovery.
Previous studies have shown that transplantation of MSC
into a contusion injury in the spinal cord promotes axon
outgrowth and results in moderate recovery of function
[9,23,60]; however, although several possible mechanisms
have been proposed, the process by which MSC contribute to
this improvement remains to be elucidated. Transdifferentiation of MSC into neural cells [30] is considered a
controversial potential mechanism for recovery of function,
as cell fusion or the presence of a subpopulation of
pluripotent stem cells cannot be ruled out [21,25,34,53].
However, even if MSC were able to transdifferentiate, it is
unlikely that the small number of MSC expressing neural
markers would result in functional recovery even if the cells
were integrated in neural circuitry. Aside from transdifferentiation, other potential mechanisms for the therapeutic
effect of MSC depend on factors secreted by MSC or
presented on the MSC surface. Co-culture of MSC with
hippocampal slices results in reduced cell death after
oxygen-glucose deprivation, suggesting that MSC are able
to promote neuron survival [62]. Chen et al. [7] provided
evidence that MSC transplantation into the infarcted brain
leads to a reduction of cell death and an increase in cell
proliferation. In culture, it has been shown that MSC, cocultured with neural stem cells, preferentially induce neuronal differentiation [38,60]. Several growth factors released
by MSC are known to affect functional recovery in CNS
injury [55]. BDNF is important for the regeneration of
damaged rubrospinal tract axons [35,36,56]. VEGF reduces
apoptosis and promotes regeneration of the corticospinal
tract [13,59]. Factors such as MCP-1 and SDF-1a have been
shown to be involved in the homing of MSC to injured tissue
[2,58]. These factors may be important if less invasive
techniques for cell delivery, like intra-thecal injection, are
used. However, MSC also secrete factors, like IL-6 that may
have a negative effect on recovery by increasing neutrophil
and macrophage infiltration and inhibiting axonal growth
[32]. MSC secrete various other cytokines that may modulate
the host immune response in a positive or negative way. So
far, it is agreed upon that the acute inflammation following
SCI results in additional damage; however, studies have also
shown a regenerative potential of certain immune cells [48].
The timing and nature of factors involved in protective as
well as destructive signaling in immune cells after SCI are
not yet well understood [46] so care needs to be taken in the
evaluation of cytokines secreted by MSC. For an overall
positive effect on recovery of function, neurotrophic factors
and cytokines secreted by MSC need to be carefully
balanced. Therefore, it seems logical that differences in the
secretion patterns of MSC from different donors would result
in different outcomes.
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
The promising results of previous studies [9,23,60] led to
the suggestion that MSC are an ideal candidate for cell
therapy in SCI. Indeed, MSC can be easily obtained from
bone marrow and both expanded and stored in buniversal
donorQ cell banks, or used for autologous transplantation.
The latter possibility is exciting, as immune suppression and
prevention of the host immune response would not be
necessary. So far, transplantation studies have been done
with MSC derived from a single donor (human or rodent),
and the possibility of variation affecting axonal regeneration
and efficacy of the cell transplant was not addressed. Our
results suggest that such variations among donors exist,
implying that not every patient’s MSC may be beneficial for
autologous treatment of SCI. Similarly, MSC stored in cell
banks will most likely not have identical properties.
A multitude of different factors could potentially
influence variations among donors. Among these are age,
gender, health status, and genetic background of donors. It is
known that variations exist in the growth kinetics and the
potential for osteogenic differentiation among different
human donors and even within the same donor [44,50].
Similar variations have been shown for MSC derived from
different strains of inbred mice [42,43]. These differences
may be derived from variations in the composition of bone
marrow aspirates at the time of harvest. Previous studies
have also shown that yields from different bone marrow
aspirates differ significantly in volume and cell composition
[33,50]. However, no correlation could be made between
age or gender and the above described differences. In
addition, it has also been shown that proliferative activity
and cell yield from human bone marrow varies with the
circadian rhythm [52]. Other factors could also play a role,
for instance, nutrition or stress level. As these factors are
hard to control, it is essential to find specific parameters that
allow quick and reliable selection of MSC with therapeutic
potential.
We focused our study on variations originating in
samples derived from different donors; however, it is
possible that these and additional variations are due to
differences in harvesting and culturing the cells. MSC
consist of a heterogeneous population of cells, and culture
without selection for well-defined cell types may reinforce
already existing variations reflecting the properties of MSC
derived from the bone marrow of different donors.
Experiments using selected subpopulations of MSC with
different phenotypes will need to be conducted to determine
the effects of specific parameters on recovery of function.
This should lead to standardized protocols for the collection
and processing of MSC that should then be strictly
followed and accompanied by quality control assays as
indeed required by FDA for clinical application. Additionally, use of optimized culturing protocols may result in
better defined cell population and reduced variability.
Future studies in our lab are aimed at defining such a
protocol and characterizing MSC with proven efficacy
using various parameters.
83
In summary, MSC for autologous or allogeneic transplantation will have to be tested for specific parameters to
ensure their therapeutic potential; however, as of now, we do
not know which factor(s) or factor combination(s) is/are
essential. It is likely that more than one factor is involved in
promoting recovery; however, it may be more complicated—factor combinations will have to be investigated and
within these combinations, effects may depend on high or
low expression of certain factors in relationship to others. It
will be challenging to find the right parameters to determine
therapeutic success, but having MSC with carefully defined
secretion profiles, possibly genetically engineered to augment certain therapeutic effects, may bring us closer to a
functional cell therapy for SCI.
Acknowledgments
Human marrow stromal cells and ELISA data for this
study were provided by Neuronyx Inc., Malvern, PA. We
thank Drs. Gene Kopen and Joseph Wagner for helpful
suggestions and critical review of the manuscript, and Dr.
Scott Stackhouse for help with the statistical analysis. We
thank Dr. Masata Shibata, Maryla Obrocka, Lee Silver,
Maureen Tumolo, and Guillermo Samper for excellent
technical support. The hybridoma used to produce the
neurofilament antibody was developed by Dr. John Wood
and obtained from the Developmental Studies Hybridoma
Bank maintained by the University of Iowa, Department of
Biological Sciences, Iowa City, IA 52242.
References
[1] Y. Akiyama, C. Radtke, J.D. Kocsis, Remyelination of the rat
spinal cord by transplantation of identified bone marrow stromal
cells, J. Neurosci. 22 (2002) 6623 – 6630.
[2] A.T. Askari, S. Unzek, Z.B. Popovic, C.K. Goldman, F. Forudi, M.
Kiedrowski, A. Rovner, S.G. Ellis, J.D. Thomas, P.E. DiCorleto, E.J.
Topol, M.S. Penn, Effect of stromal-cell-derived factor 1 on stem-cell
homing and tissue regeneration in ischaemic cardiomyopathy, Lancet
362 (2003) 697 – 703.
[3] D.M. Basso, M.S. Beattie, J.C. Bresnahan, A sensitive and reliable
locomotor rating scale for open field testing in rats, J. Neurotrauma 12
(1995) 1 – 21.
[4] M.S. Beattie, J.C. Bresnahan, J. Komon, C.A. Tovar, M. Van Meter,
D.K. Anderson, A.I. Faden, C.Y. Hsu, L.J. Noble, S. Salzman, W.
Young, Endogenous repair after spinal cord contusion injuries in the
rat, Exp. Neurol. 148 (1997) 453 – 463.
[5] L.I. Benowitz, P.J. Apostolides, N. Perrone-Bizzozero, S.P.
Finklestein, H. Zwiers, Anatomical distribution of the growthassociated protein GAP-43/B-50 in the adult rat brain, J. Neurosci.
8 (1988) 339 – 352.
[6] X. Chen, Y. Li, L. Wang, M. Katakowski, L. Zhang, J. Chen, Y. Xu,
S.C. Gautam, M. Chopp, Ischemic rat brain extracts induce human
marrow stromal cell growth factor production, Neuropathology 22
(2002) 275 – 279.
[7] J. Chen, Y. Li, M. Katakowski, X. Chen, L. Wang, D. Lu, M. Lu, S.C.
Gautam, M. Chopp, Intravenous bone marrow stromal cell therapy
reduces apoptosis and promotes endogenous cell proliferation after
stroke in female rat, J. Neurosci. Res. 73 (2003) 778 – 786.
84
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
[8] M. Chopp, Y. Li, Treatment of neural injury with marrow stromal
cells, Lancet Neurol. 1 (2002) 92 – 100.
[9] M. Chopp, X.H. Zhang, Y. Li, L. Wang, J. Chen, D. Lu, M. Lu, M.
Rosenblum, Spinal cord injury in rat: treatment with bone marrow
stromal cell transplantation, NeuroReport 11 (2000) 3001 – 3005.
[10] M.D. Christensen, A.W. Everhart, J.T. Pickelman, C.E. Hulsebosch,
Mechanical and thermal allodynia in chronic central pain following
spinal cord injury, Pain 68 (1996) 97 – 107.
[11] W. Deng, M. Obrocka, I. Fischer, D.J. Prockop, In vitro differentiation
of human marrow stromal cells into early progenitors of neural cells
by conditions that increase intracellular cyclic AMP, Biochem.
Biophys. Res. Commun. 282 (2001) 148 – 152.
[12] E. Eliav, U. Herzberg, M.A. Ruda, G.J. Bennett, Neuropathic pain
from an experimental neuritis of the rat sciatic nerve, Pain 83 (1999)
169 – 182.
[13] F. Facchiano, E. Fernandez, S. Mancarella, G. Maira, M. Miscusi, D.
D’Arcangelo, G. Cimino-Reale, M.L. Falchetti, M.C. Capogrossi, R.
Pallini, Promotion of regeneration of corticospinal tract axons in rats
with recombinant vascular endothelial growth factor alone and
combined with adenovirus coding for this factor, J. Neurosurg. 97
(2002) 161 – 168.
[14] I. Fischer, Y. Liu, Gene therapy strategies, in: N. Ingoglia, M. Murray
(Eds.), Axonal Regeneration in the Central Nervous System, Marcel
and Dekker, New York, 2001, pp. 563 – 601.
[15] R. Grill, K. Murai, A. Blesch, F.H. Gage, M.H. Tuszynski, Cellular
delivery of neurotrophin-3 promotes corticospinal axonal growth and
partial functional recovery after spinal cord injury, J. Neurosci. 17
(1997) 5560 – 5572.
[16] B.C. Hains, K.M. Chastain, A.W. Everhart, D.J. McAdoo, C.E.
Hulsebosch, Transplants of adrenal medullary chromaffin cells reduce
forelimb and hindlimb allodynia in a rodent model of chronic central
pain after spinal cord hemisection injury, Exp. Neurol. 164 (2000)
426 – 437.
[17] B.C. Hains, K.M. Johnson, D.J. McAdoo, M.J. Eaton, C.E.
Hulsebosch, Engraftment of serotonergic precursors enhances
locomotor function and attenuates chronic central pain behavior
following spinal hemisection injury in the rat, Exp. Neurol. 171
(2001) 361 – 378.
[18] B.C. Hains, A.W. Everhart, S.D. Fullwood, C.E. Hulsebosch, Changes
in serotonin, serotonin transporter expression and serotonin denervation supersensitivity: involvement in chronic central pain after spinal
hemisection in the rat, Exp. Neurol. 175 (2002) 347 – 362.
[19] S.S. Han, Y. Liu, C. Tyler-Polsz, M.S. Rao, I. Fischer, Transplantation
of glial-restricted precursor cells into the adult spinal cord: survival,
glial-specific differentiation, and preferential migration in white
matter, Glia 45 (2004) 1 – 16.
[20] K. Hargreaves, R. Dubner, F. Brown, C. Flores, J. Joris, A new and
sensitive method for measuring thermal nociception in cutaneous
hyperalgesia, Pain 32 (1988) 77 – 88.
[21] E.L. Herzog, L. Chai, D.S. Krause, Plasticity of marrow-derived stem
cells, Blood 102 (2003) 3483 – 3493.
[22] B.T. Himes, M.E. Goldberger, A. Tessler, Grafts of fetal central
nervous system tissue rescue axotomized Clarke’s nucleus neurons in
adult and neonatal operates, J. Comp. Neurol. 339 (1994) 117 – 131.
[23] C.P. Hofstetter, E.J. Schwarz, D. Hess, J. Widenfalk, A. El Manira,
D.J. Prockop, L. Olson, Marrow stromal cells form guiding strands in
the injured spinal cord and promote recovery, Proc. Natl. Acad. Sci.
U. S. A. 99 (2002) 2199 – 2204.
[24] K. Ishii, M. Toda, Y. Nakai, H. Asou, M. Watanabe, M. Nakamura, Y.
Yato, Y. Fujimura, Y. Kawakami, Y. Toyama, K. Uyemura, Increase of
oligodendrocyte progenitor cells after spinal cord injury, J. Neurosci.
Res. 65 (2001) 500 – 507.
[25] K. Jin, D.A. Greenberg, Tales of transdifferentiation, Exp. Neurol. 183
(2003) 255 – 257.
[26] W.M. Jurney, G. Gallo, P.C. Letourneau, S.C. McLoon, Rac1mediated endocytosis during ephrin-A2- and semaphorin 3A-induced
growth cone collapse, J. Neurosci. 22 (2002) 6019 – 6028.
[27] D. Kim, T. Schallert, Y. Liu, T. Browarak, N. Nayeri, A. Tessler, I.
Fischer, M. Murray, Transplantation of genetically modified fibroblasts expressing BDNF in adult rats with a subtotal hemisection
improves specific motor and sensory functions, Neurorehabilitation
Neural Repair 15 (2001) 141 – 150.
[28] B.J. Kim, J.H. Seo, J.K. Bubien, Y.S. Oh, Differentiation of adult bone
marrow stem cells into neuroprogenitor cells in vitro, NeuroReport 13
(2002) 1185 – 1188.
[29] T. Kinnaird, E. Stabile, M.S. Burnett, C.W. Lee, S. Barr, S. Fuchs,
S.E. Epstein, Marrow-derived stromal cells express genes encoding a
broad spectrum of arteriogenic cytokines and promote in vitro and in
vivo arteriogenesis through paracrine mechanisms, Circ. Res. (2004)
678 – 685.
[30] G.C. Kopen, D.J. Prockop, D.G. Phinney, Marrow stromal cells
migrate throughout forebrain and cerebellum, and they differentiate
into astrocytes after injection into neonatal mouse brains, Proc. Natl.
Acad. Sci. U. S. A. 96 (1999) 10711 – 10716.
[31] M.A. LaBarge, H.M. Blau, Biological progression from adult bone
marrow to mononucleate muscle stem cell to multinucleate muscle
fiber in response to injury, Cell 111 (2002) 589 – 601.
[32] S. Lacroix, L. Chang, S. Rose-John, M.H. Tuszynski, Delivery of
hyper-interleukin-6 to the injured spinal cord increases neutrophil and
macrophage infiltration and inhibits axonal growth, J. Comp. Neurol.
454 (2002) 213 – 228.
[33] H.M. Lazarus, S.E. Haynesworth, S.L. Gerson, N.S. Rosenthal, A.I.
Caplan, Ex vivo expansion and subsequent infusion of human bone
marrow-derived stromal progenitor cells (mesenchymal progenitor
cells): implications for therapeutic use, Bone Marrow Transplant. 16
(1995) 557 – 564.
[34] Y. Liu, M.S. Rao, Transdifferentiation—fact or artifact, J. Cell.
Biochem. 88 (2003) 29 – 40.
[35] Y. Liu, D. Kim, B.T. Himes, S.Y. Chow, T. Schallert, M. Murray, A.
Tessler, I. Fischer, Transplants of fibroblasts genetically modified to
express BDNF promote regeneration of adult rat rubrospinal axons
and recovery of forelimb function, J. Neurosci. 19 (1999) 4370 – 4387.
[36] Y. Liu, B.T. Himes, M. Murray, A. Tessler, I. Fischer, Grafts of
BDNF-producing fibroblasts rescue axotomized rubrospinal neurons
and prevent their atrophy, Exp. Neurol. 178 (2002) 150 – 164.
[37] Y. Liu, Y. Wu, J.C. Lee, H. Xue, L.H. Pevny, Z. Kaprielian, M.S. Rao,
Oligodendrocyte and astrocyte development in rodents: an in situ and
immunohistological analysis during embryonic development, Glia 40
(2002) 25 – 43.
[38] S. Lou, P. Gu, F. Chen, C. He, M. Wang, C. Lu, The effect of bone
marrow stromal cells on neuronal differentiation of mesencephalic
neural stem cells in Sprague–Dawley rats, Brain Res. 968 (2003)
114 – 121.
[39] I. Mocchetti, J.R. Wrathall, Neurotrophic factors in central nervous
system trauma, J. Neurotrauma 12 (1995) 853 – 870.
[40] M. Ohta, Y. Suzuki, T. Noda, Y. Ejiri, M. Dezawa, K. Kataoka, H.
Chou, N. Ishikawa, N. Matsumoto, Y. Iwashita, E. Mizuta, S. Kuno,
C. Ide, Bone marrow stromal cells infused into the cerebrospinal fluid
promote functional recovery of the injured rat spinal cord with
reduced cavity formation, Exp. Neurol. 187 (2004) 266 – 278.
[41] D. Orlic, Adult bone marrow stem cells regenerate myocardium in
ischemic heart disease, Ann. N. Y. Acad. Sci. 996 (2003) 152 – 157.
[42] A. Peister, J.A. Mellad, B.L. Larson, B.M. Hall, L.F. Gibson, D.J.
Prockop, Adult stem cells from bone marrow (MSCs) isolated from
different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential, Blood 103 (2004) 1662 – 1668.
[43] D.G. Phinney, G. Kopen, R.L. Isaacson, D.J. Prockop, Plastic
adherent stromal cells from the bone marrow of commonly used
strains of inbred mice: variations in yield, growth, and differentiation,
J. Cell. Biochem. 72 (1999) 570 – 585.
[44] D.G. Phinney, G. Kopen, W. Righter, S. Webster, N. Tremain, D.J.
Prockop, Donor variation in the growth properties and osteogenic
potential of human marrow stromal cells, J. Cell. Biochem. 75 (1999)
424 – 436.
B. Neuhuber et al. / Brain Research 1035 (2005) 73–85
[45] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas,
J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak,
Multilineage potential of adult human mesenchymal stem cells,
Science 284 (1999) 143 – 147.
[46] P.G. Popovich, T.B. Jones, Manipulating neuroinflammatory reactions
in the injured spinal cord: back to basics, Trends Pharmacol. Sci. 24
(2003) 13 – 17.
[47] J. Sanchez-Ramos, S. Song, F. Cardozo-Pelaez, C. Hazzi, T. Stedeford,
A. Willing, T.B. Freeman, S. Saporta, W. Janssen, N. Patel, D.R.
Cooper, P.R. Sanberg, Adult bone marrow stromal cells differentiate
into neural cells in vitro, Exp. Neurol. 164 (2000) 247 – 256.
[48] M. Schwartz, I. Cohen, O. Lazarov-Spiegler, G. Moalem, E. Yoles,
The remedy may lie in ourselves: prospects for immune cell therapy in
central nervous system protection and repair, J. Mol. Med. 77 (1999)
713 – 717.
[49] E.D. Schwartz, J.S. Shumsky, S. Wehrli, A. Tessler, M. Murray, D.B.
Hackney, Ex vivo MR determined apparent diffusion coefficients
correlate with motor recovery mediated by intraspinal transplants of
fibroblasts genetically modified to express BDNF, Exp. Neurol. 182
(2003) 49 – 63.
[50] I. Sekiya, B.L. Larson, J.R. Smith, R. Pochampally, J.G. Cui, D.J.
Prockop, Expansion of human adult stem cells from bone marrow
stroma: conditions that maximize the yields of early progenitors and
evaluate their quality, Stem Cells 20 (2002) 530 – 541.
[51] J.S. Shumsky, C.A. Tobias, M. Tumolo, W.D. Long, S.F. Giszter, M.
Murray, Delayed transplantation of fibroblasts genetically modified to
secrete BDNF and NT-3 into a spinal cord injury site is associated
with limited recovery of function, Exp. Neurol. 184 (2003) 114 – 130.
[52] R. Smaaland, R.B. Sothern, O.D. Laerum, J.F. Abrahamsen, Rhythms
in human bone marrow and blood cells, Chronobiol. Int. 19 (2002)
101 – 127.
[53] S. Song, J. Sanchez-Ramos, Brain as the sea of marrow, Exp. Neurol.
184 (2003) 54 – 60.
85
[54] S. Song, S. Kamath, D. Mosquera, T. Zigova, P. Sanberg, D.L. Vesely,
J. Sanchez-Ramos, Expression of brain natriuretic peptide by human
bone marrow stromal cells, Exp. Neurol. 185 (2004) 191 – 197.
[55] A. Tessler, Neurotrophic effects on dorsal root regeneration into the
spinal cord, Prog. Brain Res. 143 (2004) 147 – 154.
[56] C.A. Tobias, J.S. Shumsky, M. Shibata, M.H. Tuszynski, I. Fischer, A.
Tessler, M. Murray, Delayed grafting of BDNF and NT-3 producing
fibroblasts into the injured spinal cord stimulates sprouting, partially
rescues axotomized red nucleus neurons from loss and atrophy, and
provides limited regeneration, Exp. Neurol. 184 (2003) 97 – 113.
[57] A.N. Tullio, P.C. Bridgman, N.J. Tresser, C.C. Chan, M.A. Conti, R.S.
Adelstein, Y. Hara, Structural abnormalities develop in the brain after
ablation of the gene encoding nonmuscle myosin II-B heavy chain, J.
Comp. Neurol. 433 (2001) 62 – 74.
[58] L. Wang, Y. Li, J. Chen, S.C. Gautam, Z. Zhang, M. Lu, M. Chopp,
Ischemic cerebral tissue and MCP-1 enhance rat bone marrow
stromal cell migration in interface culture, Exp. Hematol. 30 (2002)
831 – 836.
[59] J. Widenfalk, A. Lipson, M. Jubran, C. Hofstetter, T. Ebendal, Y. Cao,
L. Olson, Vascular endothelial growth factor improves functional
outcome and decreases secondary degeneration in experimental spinal
cord contusion injury, Neuroscience 120 (2003) 951 – 960.
[60] S. Wu, Y. Suzuki, Y. Ejiri, T. Noda, H. Bai, M. Kitada, K. Kataoka, M.
Ohta, H. Chou, C. Ide, Bone marrow stromal cells enhance
differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord, J. Neurosci. Res. 72 (2003) 343 – 351.
[61] R.P. Yezierski, S. Liu, G.L. Ruenes, K.J. Kajander, K.L. Brewer,
Excitotoxic spinal cord injury: behavioral and morphological characteristics of a central pain model, Pain 75 (1998) 141 – 155.
[62] C. Zhong, Z. Qin, C.J. Zhong, Y. Wang, X.Y. Shen, Neuroprotective
effects of bone marrow stromal cells on rat organotypic hippocampal
slice culture model of cerebral ischemia, Neurosci. Lett. 342 (2003)
93 – 96.