Cell Transplantation, Vol. 20, pp. 1361–1379, 2011
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DOI: http://dx.doi.org/10.3727/096368910X557155
E-ISSN 1555-3892
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Combination of Multifaceted Strategies to Maximize the Therapeutic
Benefits of Neural Stem Cell Transplantation for Spinal Cord Repair
Dong H. Hwang,* Hyuk M. Kim,* Young M. Kang,* In S. Joo,† Chong-Su Cho,‡
Byung-Woo Yoon,§ Seung U. Kim,¶# and Byung G. Kim*†
*Brain Disease Research Center, Institute of Medical Sciences, Ajou University School of Medicine, Suwon, Republic of Korea
†Department of Neurology, Ajou University School of Medicine, Suwon, Republic of Korea
‡Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea
§Departments of Neurology and Neuroscience Research Center, Clinical Research Institute, Seoul National University Hospital,
Seoul, Republic of Korea
¶Department of Neurology, University of British Columbia, Vancouver, BC, Canada
#Medical Research Institute, Chungang University School of Medicine, Seoul, Republic of Korea
Neural stem cells (NSCs) possess therapeutic potentials to reverse complex pathological processes following
spinal cord injury (SCI), but many obstacles remain that could not be fully overcome by NSC transplantation
alone. Combining complementary strategies might be required to advance NSC-based treatments to the
clinical stage. The present study was undertaken to examine whether combination of NSCs, polymer scaffolds, neurotrophin-3 (NT3), and chondroitinase, which cleaves chondroitin sulfate proteoglycans at the
interface between spinal cord and implanted scaffold, could provide additive therapeutic benefits. In a rat
hemisection model, poly(-caprolactone) (PCL) was used as a bridging scaffold and as a vehicle for NSC
delivery. The PCL scaffolds seeded with F3 NSCs or NT3 overexpressing F3 cells (F3.NT3) were implanted
into hemisected cavities. F3.NT3 showed better survival and migration, and more frequently differentiated
into neurons and oligodendrocytes than F3 cells. Animals with PCL scaffold containing F3.NT3 cells showed
the best locomotor recovery, and motor evoked potentials (MEPs) following transcranial magnetic stimulation were recorded only in PCL-F3.NT3 group in contralateral, but not ipsilateral, hindlimbs. Implantation
of PCL scaffold with F3.NT3 cells increased NT3 levels, promoted neuroplasticity, and enhanced remyelination of contralateral white matter. Combining chondroitinase treatment after PCL-F3.NT3 implantation further enhanced cell migration and promoted axonal remodeling, and this was accompanied by augmented
locomotor recovery and restoration of MEPs in ipsilateral hindlimbs. We demonstrate that combining multifaceted strategies can maximize the therapeutic benefits of NSC transplantation for SCI. Our results may
have important clinical implications for the design of future NSC-based strategies.
Key words: Stem cells; Spinal cord injury (SCI); Tissue scaffold (Pclf polymer); Neurotrophin 3;
Motor evoked potential; Chondroitinase ABC
INTRODUCTION
Spinal cord injury (SCI) interrupts neural connections
between the brain and the rest of the body, leading to a
paralysis and loss of sensation below the level of injury.
The pathological consequences of SCI are multitude:
death of local segmental neurons and glial cells, severance of axons in white matter, formation of fluid-filled
cystic cavities, and demyelination of spared white matter
(6,33,52). All these pathological processes, at least to
some degree, contribute together to the devastating func-
tional outcomes after SCI, warranting the development
of a multifaceted combinatorial approach to achieve
clinically relevant functional recovery (19,50).
Preclinical studies of various animal models of SCI
have shown that neural stem or progenitor cells (NSCs)
possess multiple therapeutic potentials that might be exploited to reverse diverse pathological processes. For example, NSCs can modify the injured microenvironment
and provide neuroprotection (27,41). Grafted NSCs can
replace lost neuronal populations at segmental levels
(15,60), and NSCs or lineage restricted glial progenitor
Received March 22, 2010; final acceptance December 5, 2010. Online prepub date: March 7, 2011.
Address correspondence to Dr. Byung G. Kim, M.D., Ph.D., Brain Disease Research Center, Institute of Medical Sciences, and Department of
Neurology, Ajou University School of Medicine, San 5, Woncheon-Dong, Yeongtong-Gu, Suwon 443-721, Republic of Korea. Tel: 82-31-2194495; Fax: 82-31-219-4530; E-mail: [email protected]
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cells can differentiate into myelinating oligodendrocytes
and augment the remyelination of spared tissues (10,15,
30,31,46). Furthermore, NSC transplantation has been
reported to increase axonal regeneration and/or plasticity
(40,44). For these reasons, it is anticipated that the NSC
transplantation will become a viable and effective therapeutic option to improve functional outcome after SCI
(16,38).
However, many considerable obstacles remain to be
overcome before NSC transplantation becomes available
in clinic. The formation of fluid-filled cystic cavities severely impedes axonal growth in lesion areas, and it has
been suggested that transplanted NSCs are not capable
of bridging these cavitary lesions (51,59). Furthermore,
survival and migration of grafted NSCs are often limited
in the injured spinal cord (26,37,48), and NSCs transplanted into injured spinal cord tissue frequently either
remain undifferentiated or differentiate predominantly
into astrocytes, which can contribute to glial scar formation (11,12). In addition, glial scars and inhibitory extracellular matrices such as chondroitin sulfate proteoglycans (CSPGs) could interfere with migration and axonal
growth of grafted NSCs, reducing the possibility of the
proper integration of NSCs with the host neural circuit
(26,32,35).
Therefore, we reasoned that a combination of complementary strategies aimed at removing specific obstacles
would potentiate the efficacy of NSC-based treatments.
Previous studies have shown that NSC transplantation in
combination with a complementary strategy provided
improved therapeutic benefits as compared with NSC
alone. Teng et al. found that implanting polymer scaffold seeded with NSCs led to an improved functional
recovery (57). Brain-derived neurotrophic factor (BDNF)
or neurotrophin-3 (NT3) coadministered with NSCs or
directly transduced to NSCs improved the survival and
migration of grafted NSCs (7,20,29,49,62). Moreover,
combining chondroitinase with NSC grafts to degrade
inhibitory extracellular matrices enhanced NSC migration (26). What remains to be determined is whether a
combination of multifaceted complementary strategies
could provide therapeutic benefits of greater magnitude
and to what extent a multifaceted combinatorial approach could enhance the therapeutic effects of NSC
transplantation.
In an effort to identify an effective NSC-based treatment strategy for SCI, we tested whether a combinatorial
strategy based on the use of polymer scaffold, NSCs, and
NT3 could improve recovery of locomotor function, and
whether the inclusion of a chondroitinase treatment to degrade CSPGs deposited at the interface between the spinal
cord and implanted scaffold could augment the therapeutic benefits conferred by this combinatorial strategy.
HWANG ET AL.
MATERIALS AND METHODS
Establishment of Human NSCs Modified
to Secrete NT3
Telencephalon tissues from gestational week 15 human fetal brains were utilized to generate an immortalized human NSC line (F3) using a retroviral vector encoding v-myc, as described previously (18,34). The
permission to use fetal tissues was granted by the Clinical Research Screening Committee involving Human
Subjects of the University of British Columbia. The
PG13 mouse packaging cell line was transfected with
plasmid pLHCX-NT3 vector containing full-length human NT3 cDNA (ATCC, Manassas, VA) using lipofectamine. Retroviral supernatants obtained from the packaging cells were applied to F3 NSCs. Hygromycinresistant clones were screened and isolated, and one of
the clones (F3.NT3) was expanded and used for the
transplantation.
Pretreatment of PCL Scaffolds and the Cell
Seeding Procedure
A biocompatible poly(-caprolactone) (PCL) scaffold
was used in this study. The scaffold was produced by
reacting PCL diols (MW = 2,000) with acryloyl chloride
and fabricated using a gas-foaming/salt-leaching method
(36). The dimensions of PCL scaffold were determined
to fit hemisected lesion cavities. Before cell seeding, the
PCL scaffolds were sterilized with 70% ethanol, and
then washed five times in distilled water, after which
they were subjected to vacuum desiccation for 10 min
to remove trapped air bubbles. Before seeding, F3 or
F3.NT3 cells were grown attached to culture dishes, dissociated into single cells by a brief trypsin treatment,
and then suspended at a density of 105 cells/µl. A 2.5µl aliquot of medium containing cells was then slowly
injected using a pipette tip into both sides of each scaffold (a total of 5 × 105 cells/scaffold). The PCL scaffolds
seeded with cells were transferred into a 12-well plate
and incubated on shaker at 37°C for at least 3 days before implantation.
Surgical Procedures
All animal procedures were approved by the Animal
Experiment Review Committee at Ajou University
School of Medicine. Adult female Sprague-Dawley rats
(250–300 g) were used. After being anesthetized with
4% chloral hydrate (400 mg/kg), rats were subjected to
dorsal laminectomy at the seventh thoracic vertebral
level (T7–8). After dura was opened, a chunk of right
side spinal cord was excised using iridectomy scissors.
Remaining tissue was removed by vacuum suction until
the vertebral body at the ventral side was visualized.
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NSC-BASED COMBINATORIAL THERAPY FOR SPINAL INJURY
Special care was taken to confirm that the right side
hemicord was entirely removed and that the lesion did
not extend into the left side spinal cord. A PCL scaffold
seeded with human NSCs was implanted into the lesion
site immediately after hemisection. Animals were randomly allocated to four experimental groups: 1) animals
that received hemisection alone [control (CTL) group,
N = 8), 2) animals implanted with PCL scaffold without
cells (PCL group, N = 7), 3) animals implanted with
PCL scaffold seeded with F3 NSCs (PCL-F3 group, N =
9), and 4) animals implanted with PCL scaffold with
F3.NT3 cells (PCL-F3.NT3 group, N = 9). The above
animals were sacrificed at 9 weeks after all behavioral
and electrophysiological tests had been completed. A
different set of animals was sacrificed at 2 weeks postSCI for ELISA and Western blot. All animals received
daily intraperitoneal cyclosporine (Sandimmun; Novartis, Bern, Switzerland) at a dosage of 10 mg/kg, beginning from 1 day before surgery until sacrifice. Prophylactic antibiotics were injected intraperitoneally on the
next day following each surgery. Bladder care was provided until spontaneous voiding resumed.
Infusion of Chondroitinase ABC
Protease-free chondroitinase ABC (C-ase) was purchased from the Seikagaku Corporation (Tokyo, Japan).
One day prior to infusion, an enzyme aliquot (10 U/ml,
0.2 ml) was loaded into the reservoir of the Alzet 2004
osmotic pump (Durect, Cupertino, CA) and incubated
overnight at 37°C. Immediately after implantation of
PCL scaffold seeded with F3.NT3 cells, a PE-10 tubing
(Intramedic, NJ) attached to the osmotic pump was inserted into the subarachnoid space from the intervertebral space one level rostral to the lesion and slowly advanced under the lamina until the tube opening was
located on top of the implantation site. The osmotic minipumps delivered the enzyme solution at a rate of 0.25
µl/h continuously for 28 days. The weight equivalent of
penicillinase (P-ase; Sigma, St. Louis, MO) was delivered as a control injection. Eighteen animals that received PCL implants seeded with F3.NT3 cells were
randomly divided into C-ase (N = 9) and P-ase (N = 9)
groups. All these animals were sacrificed at 9 weeks
post-SCI after behavioral and electrophysiological assessments had been done. A different set of animals was
sacrificed at 2 weeks post-SCI (N = 3 per group), and
the spinal cord sections of these animals were subjected
to immunohistochemistry with CS56 and C4S antibody
to detect intact and degraded CSPGs, respectively.
ELISA and Western Blot Analysis
The levels of NT3 in culture medium were measured
using a human NT3 ELISA kit (R&D Systems, Minne-
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apolis, MN). The detailed procedure was described in an
instruction manual provided by the manufacturer. The
experiment was replicated three times. NT3 levels in the
spinal cord tissue implanted with PCL scaffolds seeded
with F3.NT3 cells were also measured by ELISA. Briefly,
16 animals were sacrificed with an overdose anesthesia
at 2 weeks post-SCI (N = 4 per group). Spinal cord tissue spanning ±5 mm from the epicenter was dissected
and homogenized in lysis buffer (4 µl/mg tissue) containing protease inhibitor cocktail (Sigma). To measure
NT3 levels only in the host spinal cord tissue, PCL scaffolds were manually detached from the spinal cord tissues and not included in the protein extraction. For
Western blot analysis, spinal cord tissue blocks (spanning 2–3 mm) immediately caudal to the tissues used
for ELISA were homogenized in the same lysis buffer.
The blots were probed with growth associated protein
43 (GAP-43; 1:3000; rabbit; Chemicon; Temecula, CA)
and synaptotagmin (1:3000; mouse; Chemicon) antibodies. All blots were also probed with mouse anti-β-actin
to control for differences in loading amount. After incubation with peroxidase-conjugated appropriate secondary antibodies, antigen–antibody reactions were visualized using a chemiluminescence kit.
Assessment of Locomotor Recovery
Locomotor recovery was assessed using the Basso,
Beattie, and Bresnahan (BBB) locomotor rating scale
and the grid walk test. Animals were assigned with new
identification codes after surgery to ensure that behavioral performances were rated in a blind manner. The
BBB locomotor rating scale was used to measure the
quality of hindlimb function during open field locomotion. Because animals were subjected to hemisection injury, right (ipsilateral) and left (contralateral) hindlimbs
were assessed separately. For the grid walk test, animals
were pretrained for 7 days before surgery to walk on
grid runway (30 × 140 cm with 50 × 50-mm holes) for
a water reward. The test was conducted at 4 and 7 weeks
post-SCI, and animals were retrained for 5 days before
testing. On the day of tests, four runs were recorded
using a three-charge-coupled device (CCD) digital video
camera, and later analyzed frame by frame in slow motion. The average number of limb placement errors per
run was determined for each animal.
Electrophysiological Assessment
The integrity of the motor pathway through the spinal
cord was assessed by the transcranial magnetic motor
evoked potentials (39). After all the behavioral tests had
been completed (at 8 weeks post-SCI), transcranial magnetic stimulation (Magstim model 2002, Magstim, Wales,
UK) was delivered around the vertex of animals anes-
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thetized with chloral hydrate (400 mg/kg). A customgenerated double coil (diameter 25 mm) was used to
elicit focal stimulation. Recording needle electrodes
were inserted into the gastrocnemius muscle. Stimulation was started from subthreshold intensities and the
stimulus intensity was progressively increased until
maximal evoked potentials were obtained. Final stimulation intensities ranged from 80 to 100 A/µs. Motor
evoked potentials (MEPs) were recorded at both contralateral and ipsilateral gastrocnemius muscles. MEP amplitude in each animal was determined by averaging the
results of 7–10 trials.
Tissue Processing and Immunohistochemistry
For histological analysis, animals were anesthetized
with an overdose of chloral hydrate and perfused with
heparinized saline (0.9%), followed by 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Spinal
cords were then dissected, postfixed in 4% paraformaldehyde at room temperature for 2 h, and then cryoprotected in a graded series of sucrose solutions. Longitudinal or transverse 20-µm sections were cut using a cryostat
(Leica) in a 1:10 series and thaw-mounted onto Super
Frost plus slides. For immunohistochemistry, spinal cord
sections were incubated overnight at 4°C with antihuman mitochondria (1:300; mouse and rabbit; Chemicon), anti-APC-CC1 (adenomatous polyposis coli; 1:
200; mouse; Calbiochem, La Jolla, CA), anti-GFAP
(glial fibrillary acidic protein; 1:500; rabbit; Chemicon),
anti-MAP2 (microtubule associated protein; 1:200; rabbit; Chemicon), anti-neurofilament-M (150 kDa) (1:200;
rabbit; Chemicon), anti-CS56 (chondroitin sulfate; 1:
200; mouse; Sigma), anti-C4S (degraded CSPGs; 1:200;
mouse; Chemicon), and rabbit 5-HT antibodies (5hydroxytryptamine; 1:5000; Immunostar, Hudson, WI).
Spinal cord sections were then washed and incubated
with fluorophore-tagged appropriate secondary antibodies. For fluorescence staining, cover slips were mounted
onto glass slides using Gelvatol and examined under an
Olympus confocal laser scanning microscope.
Quantitative Measurements of Cell Numbers
and Axonal Growth
Human mitochondria-positive cells in the entire longitudinal spinal cord sections were counted using unbiased stereology. Cell counting was performed on an
Olympus BX51 Microscope that was coupled with the
Stereo Investigator 8 software (MBF Bioscience, Williston, VT). Human mitochondria-positive cells within optical dissectors randomly placed in regions of interest
were counted using stereological criteria. Stereological
estimation was performed systematically using the formula embedded in the software. To determine the percentage of grafted human NSCs that were colocalized
HWANG ET AL.
with differentiation markers GFAP, APC-CC1, and
MAP2, two longitudinal sections showing the highest
graft survival were chosen for each animal. The numbers
of human NSCs colocalized with these markers were
counted in the entire sections, and these were then divided by the total numbers of human mitochondria-positive cells. Averages of these two sections were taken
to represent final percentage values. To determine the
maximal migration distance of grafted human NSCs, a
montage image of the entire longitudinal spinal cord
section showing the greatest migration distance was created using Photoshop software. Distances between the
scaffold/spinal cord interfaces and the cells that migrated most were measured in each montage image and
averaged for each group.
To measure the extent of neurofilament (NF)-positive
axon growth within PCL scaffolds, two consecutive longitudinal spinal cord sections that contain the largest lesion areas were chosen. Three regions of interest (ROIs)
measuring 1422 × 1249 µm were placed inside PCL
scaffolds along the rostral, caudal, and medial borders
in each section. NF-positive axons within these ROIs
were manually traced at 200× magnifications using the
Metamorph software 7.01 (Universal Imaging Corporation, Downingtown, PA). Numbers and lengths of axons
in three ROIs were calculated using Metamorph software and averaged for the two sections in each animal.
To quantify 5-HT axons in the caudal spinal cord, three
transverse sections spaced 200 µm apart from each other
were chosen from spinal cord blocks caudal to lesion
sites. Left and right ventral horn regions in each section
were imaged at 200×. ROIs were then drawn using a
freehand selection tool to cover the entire ventral horn.
The numbers of pixels occupied by the serotonergic (5HT) fibers at the ventral horns were quantified. Serotonergic fiber densities of right side were normalized versus
those of left side and percentage serotonergic innervations of lesioned versus nonlesioned sides were calculated and averaged for the three sections per animal.
Extent of Myelination in White Matter
Tissue sections were immersed for 8 min in a staining
solution consisting of 240 ml of 0.2% eriochrome cyanine RC (Sigma) and 10 ml of 10% FeCl3ⴢ6H2O (Sigma)
in 3% HCl. Sections were then washed with running tap
water followed by differentiation in 1% aqueous
NH4OH. In each animal, the eriochrome-stained spinal
cord section containing the largest lesion area was chosen, and the two more sections 200 µm dorsal and ventral each to the chosen section were included in the analysis (three consecutive sections per animal). Stained
sections were viewed on an optical microscope (Olympus BX51) and images were captured using a CCD camera (JVC digital camera KY-F1030). A single ROI
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NSC-BASED COMBINATORIAL THERAPY FOR SPINAL INJURY
(120 × 90 µm) per section was chosen in contralateral
white matter. The areas of myelin staining above a predetermined threshold were quantified using Image J
software (National Institutes Health). Percentages of
ROIs stained by myelin were averaged for three sections
per animal.
Statistical Methods
Statistical analysis was performed using SPSS version 12.0 (Chicago, IL) or GraphPad Prism software
version 4.0 (San Diego, CA). The unpaired Student’s ttest or one-way ANOVA followed by Tukey’s post hoc
test was used to compare group means at single time
points. Repeated measures two-way ANOVA with Tukey’s post hoc test was used to compare mean values at
multiple time points (BBB scores) or different locations
from the epicenter (cell numbers). Numerical values are
presented as mean ± SEM.
RESULTS
Implantation of PCL Scaffolds Seeded With Human
NSCs Into Hemisected Spinal Cord
A total of 5 × 105 parental human NSCs (F3) or NT3
overexpressing human NSCs (F3.NT3) were seeded into
PCL scaffolds and cultured for at least 3 days before
implantation. Scanning EM showed that seeded NSCs
were attached to the inner walls of the PCL scaffold
and packed into its micropores (Fig. 1A). To determine
whether seeded F3.NT3 cells survive and function properly inside PCL scaffolds, levels of NT3 in culture me-
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dia were measured. As expected, freely grown F3.NT3
(dissociate culture) secreted almost 10-fold more NT3
into culture media than parental F3 cells. Although
NT3 levels were reduced by almost a half, F3.NT3 cells
seeded into PCL scaffolds still secreted substantial
amounts of NT3 (Fig. 1B).
Right-sided spinal cord hemisection was performed
at the T7 level and PCL scaffolds with or without seeded
human NSCs were implanted immediately into hemisected cavities to bridge lesion sites. At 9 weeks post-SCI,
PCL scaffolds were found to adhere well to residual spinal cord tissues and to form a rostrocaudal bridge across
lesion cavities (Fig. 2A, B). Surviving human NSCs
were identified by their immunoreactivities against human-specific mitochondrial antigen. Both F3 and F3.NT3
cells migrated into residual spinal cord tissues across
scaffold–spinal cord interfaces (Fig. 2C–F). Numbers of
human NSCs were comparable for F3 and F3.NT3 cells
within scaffolds, but F3.NT3 cell numbers in residual
spinal cord tissues were markedly greater than those of
F3 cells. Stereological counting of grafted cells revealed
that F3.NT3 cells were more widely distributed in a rostrocaudal direction, and the differences between the two
cells were maintained at up to ±6 mm from the epicenter
(Fig. 2G). The total number of human NSCs in the entire
rostrocaudal extent was also significantly higher for
F3.NT3 than F3 cells (29,256 ± 9,291 and 14,507 ±
3,696, respectively; p < 0.001), indicating that the overall
survival was better in F3.NT3 cells. We also found that
the mean migration distance was significantly greater in
animals with PCL-F3.NT3 grafts (p < 0.01) (Fig. 2H).
Figure 1. Seeding human neural stem cells (NSCs) into a poly(-caprolactone) (PCL) scaffold.
(A) Scanning electron microscope image of a PCL scaffold seeded with human NSCs overexpressing NT3 (F3.NT3). NSCs were found attached to the walls of the PCL scaffold and packed into
its micropores. (B) Comparison of NT3 levels in culture media between NSCs in dissociated
culture and seeded in PCL scaffolds. The diagrams below the abscissa illustrate different culture
conditions. Although NT3 levels in culture media were lower than those from freely grown F3.NT3
cells, F3.NT3 cells seeded in PCL scaffolds produced significant amounts of NT3 in media as
determined by ELISA. Gray bar: F3, black bar: F3.NT3. ***p < 0.001 by Student’s t-test.
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HWANG ET AL.
Figure 2. Survival and migration of human neural stem cells (NSCs) grafted inside PCL scaffolds. (A, B) Representative longitudinal spinal cord sections from PCL-F3 (A) and PCL-F3.NT3 groups (B) at 9 weeks post-spinal cord injury (SCI). Spinal cord
sections were stained with glial fibrillary acidic protein (GFAP; green) and human-specific mitochondria antibody (red) to detect
grafted NSCs of human origin. PCL scaffolds are nicely adhered to residual spinal cord tissue and formed a rostrocaudal bridge
across the lesion cavities. Rostral direction is leftwards. Dotted lines demarcate scaffold borders. Scale bar: 250 µm. (C–F) Higher
magnification images of boxed regions in the longitudinal section of PCL-F3 (C, D) or PCL-F3.NT3 (E, F) groups. The number
of F3.NT3 cells that migrated into the spinal cord was markedly higher than that of F3 cells. DAPI-stained nuclei are shown in
blue. Scale bar: 50 µm. (G) Stereological counting of grafted cells. *p < 0.05, **p < 0.01, and ***p < 0.001 by repeated measures
two-way ANOVA followed by Tukey’s post hoc analysis. Error bars represent mean ± SEM. (H) Quantification of maximal migration distance. Each triangle or circle represents maximal migration distance in each animal, and each line represents group mean
value. **p < 0.01 by Student’s t-test. N = 9 per group.
F3 or F3.NT3 cells that migrated into remaining spinal cord tissues appeared to acquire mature neural cell
phenotypes (Fig. 3). F3.NT3 cells observed in remaining
spinal cord tissue were frequently colocalized with neuronal marker MAP-2 (Fig. 3D, G). On the other hand,
only a small proportion of F3 cells differentiated into
MAP-2-positive neurons (Fig. 3A, G). F3.NT3 cells also
more frequently differentiated into oligodendrocytes
than F3 cells (Fig. 3B, E, G). Approximately 10% of F3
and F3.NT3 cells that migrated into remaining spinal
cord were found to be positive for GFAP, and there was
no difference between the two cells (Fig. 3C, F, G).
However, in contrast to F3 and F3.NT3 cells observed
in remaining spinal cord tissues, human NSCs that remained within the scaffolds showed no evidence of differentiation into mature neural cells.
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NSC-BASED COMBINATORIAL THERAPY FOR SPINAL INJURY
Assessment of Functional Recovery
BBB locomotor and grid walk tests were performed
to assess locomotor recovery. All groups showed similar
BBB scores immediately after injury (Fig. 4A, B). Locomotor deficits rapidly recovered during the first two
weeks in all animals. Animals in PCL-F3.NT3 group
exhibited clearly better locomotor recovery than those
in the other groups at 3 weeks and continued to show
better improvement until 7 weeks post-SCI. Locomotor
scores also improved in PCL-F3 group (mean BBB
score at 7 weeks post-SCI, 12.7 ± 1.7 and 13.9 ± 1.8 for
ipsi- and contralateral, respectively), but the mean BBB
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score at this time point was significantly less than that in
PCL-F3.NT3 group (15.1 ± 1.5 for ipsilateral and 16.6 ±
1.3 for contralateral). At 7 weeks post-SCI, animals in
PCL-F3.NT3 group fully regained coordinated plantar
stepping with improved ankle positioning. Implantation
of PCL scaffold alone provided slight functional benefits
compared to the injury alone (CTL) group. Repeated
measures two-way ANOVA revealed a highly significant treatment effect over time in both hindlimbs (p <
0.001 for both hindlimbs). Tukey’s post hoc analysis
showed significant differences between PCL-F3.NT3
and the other three groups (Fig. 4A, B). The difference
between PCL-F3 and CTL groups was also statistically
Figure 3. Phenotypic differentiation of NSCs after implantation into hemisected spinal cord.
Grafted NSCs were detected by immunoreactivity against human mitochondrial antigen (red) at 9
weeks post-SCI. Arrows indicate the colocalizations of cell type-specific markers (A–F). Merged
cells are shown in yellow. Compared to F3 cells (A, D), F3.NT3 cells were frequently colocalized
with neuronal (microtubule-associated protein 2; MAP-2) and oligodendrocytic (CC1) markers (B,
E). (C, F) Small percentage of F3 or F3.NT3 cells differentiated into GFAP-positive astrocytes.
(G) Quantification of percent NSCs that differentiated into neurons (MAP-2), oligodendrocytes
(CC1), or astrocytes (GFAP). Scale bar: 25 µm. **p < 0.01 and ***p < 0.001 by Student’s t-test.
N = 9 animals for each group. Error bars represent mean ± SEM.
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HWANG ET AL.
Figure 4. Behavioral assessment of locomotor recovery. (A, B) Recovery of Basso, Beattie, and Bresnahan (BBB) locomotor scores
over the 7-week period after injury for ipsilateral (right) (A) and contralateral (left) (B) hindlimbs. Animals in PCL-F3.NT3 group
showed best recovery. *p < 0.05, **p < 0.01, and ***p < 0.001 by repeated measures two-way ANOVA followed by Tukey’s post
hoc analysis. (C, D) Grid walk. Average numbers of ipsilateral (C) and contralateral (D) hindlimbs placement errors on grid per
run were counted at 4 and 7 weeks post-SCI. Animals in PCL-F3.NT3 group showed highest accuracy on grid walk. *p < 0.05,
**p < 0.01, and ***p < 0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. N = 8, 7, 9, and 9 for control (CTL),
PCL, PCL-F3, and PCL-F3.NT3 groups, respectively, for all graphs. Error bars represent mean ± SEM.
significant. In grid walk, animals in CTL group made
significantly more frequent errors in both hindlimbs than
those in the other groups (Fig. 4C, D). Implantation of
PCL scaffold alone markedly reduced the number of errors (p < 0.001 compared to CTL). These errors were
further reduced in PCL-F3 group. Animals in PCLF3.NT3 groups showed the highest accuracy in hindlimb placement. One-way ANOVA followed by Tukey’s post hoc analysis revealed a statistically significant difference between PCL-F3.NT3 and PCL-F3
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groups. Taken together, these results suggest that PCLF3.NT3 implantation provided the greatest behavioral
benefits.
We further examined whether the improvements in
hindlimb locomotor function were accompanied by electrophysiological restoration of motor evoked potentials
(MEPs). In sham-operated animals, transcranial magnetic stimulation of the vertex readily elicited MEP responses at both hindlimbs of mean amplitudes 7.2 ± 1.6
and 7.3 ± 1.2 mV (ipsilateral and contralateral, respec-
1369
tively) (Fig. 5A, B). MEP responses were almost absent
in CTL group in both hindlimbs. None of PCL, PCLF3, and PCL-F3.NT3 groups showed ipsilateral MEP response with the amplitude of more than 1 mV (Fig. 5A,
C). In the contralateral side, none of PCL alone group
and only two of PCL-F3 group (22%, 2/9) exhibited appreciable MEP response (>1 mV). In PCL-F3.NT3
group, 67% (6/9) animals showed MEP responses with
the amplitude of more than 1 mV (Fig. 5B). Mean amplitude of PCL-F3.NT3 groups in the contralateral side
Figure 5. Recovery of motor evoked potentials in response to transcranial magnetic stimulation.
Motor evoked potentials (MEPs) were recorded at 8 weeks post-SCI in both ipsilateral and contralateral gastrocnemius muscles in response to transcranial magnetic stimulation over the vertex at
intensities of 80–100 A/µs. (A, B) Representative MEP recordings of ipsilateral (A) and contralateral (B) hindlimbs. The MEP responses disappeared in both hindlimbs after injury. Significant
MEP recovery was observed only in PCL-F3.NT3 group in the contralateral side. (C, D) Quantitation graphs of MEP amplitudes in ipsilateral (C) and contralateral (D) hindlimbs. *p < 0.001 by
one-way ANOVA followed by Tukey’s post hoc analysis. N = 4, 8, 7, 9, and 9 for sham, CTL,
PCL, PCL-F3, and PCL-F3.NT3 groups, respectively. Error bars represent mean ± SEM.
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1370
was 2.6 ± 1.7 mV and significantly higher than those of
the other groups (p < 0.001) (Fig. 5D).
Mechanism of Functional Recovery Mediated
by PCL-F3.NT3 Implantation
First, we determined whether implantation of PCL
scaffolds with F3.NT3 increased the concentration of
NT3 in host spinal cord tissue at 2 weeks after injury
(Fig. 6A). ELISA showed that levels of NT3 in the spinal cord of PCL-F3.NT3 group were significantly higher
than in the other three groups (p < 0.05 vs. PCL-F3, and
p < 0.01 vs. PCL and CTL groups). We also examined
whether PCL-F3.NT3 implantation promoted neuroplasticity in the spinal cord caudal to lesions where the spinal locomotor center is located at the 2 weeks after injury when rapid behavioral improvement was observed.
GAP-43 and synaptotagmin were chosen as a marker for
axonal plasticity and synaptogenesis, respectively. Western blot showed that PCL-F3.NT3 implantation markedly increased the expression of GAP-43 and synaptotagmin at 2 weeks post-SCI (Fig. 6B). In fact, GAP-43
levels in PCL-F3.NT3 group were almost fourfold
higher and synaptotagmin levels were threefold higher
than in CTL group (Fig. 6C, D). GAP-43 expression was
also significantly increased in PCL-F3 group compared
to CTL and PCL groups (Fig. 6B, D), but levels of
GAP-43 expression in PCL-F3.NT3 group was significantly higher than in PCL-F3 group (p < 0.01). Consistent with the Western blot data, GAP-43 immunoreactivity in neuropil was found to increase in the caudal gray
matter (lumbar level), especially in the intermediate regions and ventral horns, of animals in PCL-F3.NT3
group (Fig. 6G, H). In contrast, only subtle immunoreactivity was observed in CTL group (Fig. 6E, F).
Electrophysiological assessment showed that electrical transmission through contralateral (left) spinal cord
HWANG ET AL.
was also compromised after right side spinal cord hemisection (Fig. 5). This was consistent with the finding of
a recent study in which a decline in electrical transmission through contralateral uninjured fibers was suggested to be due to chronic progressive demyelination
(1). Because NT3 has been known as a strong signal
to promote myelination (43,53), we reasoned that the
recovery of contralateral MEP responses in PCL-F3.
NT3 group was due, at least in part, to improved myelination in contralateral white matter. Eriochrome cyanine
(EC) staining of myelin in contralateral white matter of
CTL group showed a coarse arrangement of myelinating
fibers with frequent unstained foci interspersed with myelin fragments (Fig. 6I), and the staining pattern was
similar with PCL and PCL-F3 groups (Fig. 6J, K). In
contrast, myelinating fibers were more compactly arranged in PCL-F3.NT3 group without apparent demyelinating foci (Fig. 6L). Quantification revealed significant increases of EC-stained areas in PCL-F3.NT3 group
compared to the other groups (p < 0.001) (Fig. 6M).
Degradation of CSPGs Enhanced Migration
of Transplanted Cells and Axonal Remodeling
It has been shown that CSPGs deposition around the
injury sites inhibits axonal regeneration and/or cellular
migration (17,26,45). Therefore, we asked if the combined use of CSPG degrading enzyme could provide additional therapeutic benefits in animals with implantation of PCL scaffold with F3.NT3 cells. Chondroitinase
ABC (C-ase) or a control penicillinase (P-ase) was infused using osmotic minipumps in animals with PCLF3.NT3 implantation. C-ase infusion almost completely
abolished CS56 staining (intact CSPGs), which was
densely observed around PCL scaffolds in P-ase control
animals (Fig. 7). C-ase treatment also revealed immunoreactivity against C4S (degraded CSPGs), indicating that
FACING PAGE
Figure 6. Mechanism of functional recovery mediated by implantation of PCL scaffold with F3.NT3 NSCs. (A) ELISA of NT3
levels in host spinal cord tissue at 2 weeks post-SCI. The levels of NT3 in the spinal cord of PCL-F3.NT3 group were significantly
higher than those in the other groups. N = 4 per group. (B–D) Western blot analysis of caudal spinal cord tissue at 2 weeks postSCI. (B) Representative blots of growth-associated protein-43 (GAP-43) and synaptotagmin. PCL-F3.NT3 implantation markedly
increased the expression of GAP-43 and synaptotagmin. (C, D) Quantification of GAP-43 (C) and synaptotagmin (D) expressions.
GAP-43 levels in PCL-F3.NT3 group were almost fourfold higher and synaptotagmin levels were threefold higher than in CTL
group (D). *p < 0.05, **p < 0.01, and ***p < 0.001 by one-way ANOVA followed by Tukey’s post hoc analysis. N = 4 per group.
(E–H) Representative images of GAP-43 immunofluorescent staining in the transverse lumbar sections (lesioned side). GAP-43
immunoreactivity (green) was regionally increased in PCL-F3.NT3 group (G). Apparent GAP-43 immunoreactivity was observed
only in dorsal horn region in CTL group (E), and there was very subtle neuropilic immunoreactivity in the ventral motor region
(F). In contrast, GAP-43-positive axon terminals were readily observed in the ventral motor region of PCL-F3.NT3 group (H). The
boxed regions in (E) and (G) are magnified in (F) and (H), respectively. DAPI-stained nuclei are shown in blue. Scale bars: 500
µm (E, G), 50 µm (F, H). (I–L) Representative eriochrome cyanine (EC)-stained longitudinal spinal cord sections from CTL (I),
PCL (J), PCL-F3 (K), and PCL-F3.NT3 (L) groups. The images were taken at the white matter contralateral to spinal lesions. Scale
bars: 100 µm. (M) Quantification of percent EC-stained areas in the contralateral white matter. *p < 0.05 and ***p < 0.001 by oneway ANOVA followed by Tukey’s post hoc analysis. N = 8, 7, 9, and 9 for CTL, PCL, PCL-F3, and PCL-F3.NT3 groups,
respectively. All error bars represent mean ± SEM.
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1371
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1372
HWANG ET AL.
Figure 7. Chondroitinase ABC abolished chondroitin sulfate proteoglycans (CSPGs) at the interface between PCL scaffolds and
residual spinal cord tissue. Animals with PCL-F3.NT3 implants were treated with penicillinase (P-ase, A–D) or chondroitinase
ABC (C-ase, E–H), respectively. The effectiveness of C-ase to degrade CSPGs was examined using antibodies against intact (CS56,
green) and digested (C4S, red) chondroitin sulfate. C-ase infusion almost completely abolished CS56 staining, which was densely
observed around the PCL scaffold in P-ase control. Scale bar: 250 µm (A, E), 50 µm (B–D, F–H).
C-ase successfully degraded CSPGs at the interface between PCL scaffolds and spinal cord tissue. Degradation
of CSPGs promoted the migration of F3.NT3 cells into
residual spinal cord tissue (Fig. 8A, B). In P-ase group,
F3.NT3 cells migrated across the interface between scaffolds and spinal cord, but a large proportion of migrating
cells did not cross the GFAP-positive glial scars or remained adjacent to glial scar fronts (Fig. 8C, D). Combination of C-ase increased the extent of F3.NT3 cells that
crossed glial scar fronts, especially in the caudal direction (Fig. 8E, F). The total number of cells was not significantly different between C-ase and P-ase groups
(34,228 ± 17,706 and 25,695 ± 4,226, respectively; p =
0.18). However, numbers of F3.NT3 cells that migrated
into rostrocaudal spinal cord were significantly higher at
several sites in C-ase group (Fig. 8G), and the longest
migration distance from the implant was also significantly greater in C-ase group (p < 0.01). The extent of
differentiation of F3.NT3 cells into mature neural cell
phenotypes was not different between P-ase and C-ase
groups (data not shown).
The extent of axonal growth into PCL scaffolds (Fig.
9A–D) was also evaluated. In P-ase group, only a small
number of short neurofilament-positive axons were observed inside PCL scaffolds, and these were was mostly
limited to the very vicinity of the interface (Fig. 9A, B).
In contrast, the number of axons observed inside PCL
scaffolds was markedly higher in C-ase group (Fig. 9C,
D, I), and the lengths of ingrowing axons were also
greater in C-ase group (Fig. 9J). We also compared the
extent of lumbar ventral horn innervation by serotonergic fibers (Fig. 9E–H), which are thought to play an
important role in locomotor recovery after SCI (8). Although serotonergic (5-HT) axon areas in the contralateral ventral horn tended to be higher in C-ase group, the
difference was not statistically significant (6.1 ± 4.9%
and 9.6 ± 6.0%. in P-ase and C-ase groups, respectively;
p = 0.19). In P-ase group, mean serotonergic axon density in the ipsilateral ventral horn was only 32.8 ± 8.8%
of contralateral side (Fig. 9K). C-ase treatment substantially increased the extent of serotonergic innervation in
the ipsilateral side, reaching 56.2 ± 11.5% of the contralateral side (Fig. 9H, K).
Degradation of CSPGs Augmented the Functional
Recovery Mediated by PCL-F3.NT3 Implantation
BBB locomotor test was performed to examine
whether C-ase treatment further improved the quality of
hindlimb locomotion in animals implanted with PCLF3.NT3. Animals treated with C-ase (N = 9) showed behavioral recovery superior to P-ase group (N = 9) in
both hindlimbs (Fig. 10A, B). Repeated measures twoway ANOVA revealed a significant treatment effect
over time (p < 0.05 for both hindlimbs). Locomotor performance was also evaluated at 4 and 7 weeks post-SCI
using the grid walk test. C-ase treatment significantly
improved the accuracy of hindlimb placement compared
to P-ase treatment in both hindlimbs (Fig. 10C, D).
MEP responses to transcranial magnetic stimulation
were measured to compare electrophysiological recovery (Fig. 10E, F). C-ase treatment in animals implanted
with PCL-F3.NT3 recovered recognizable MEP responses
in ipsilateral hindlimbs, where no significant MEP recovery was observed by PCL-F3.NT3 implantation (Fig.
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NSC-BASED COMBINATORIAL THERAPY FOR SPINAL INJURY
5A, C). Only 11% (1/9) of animals in P-ase group exhibited ipsilateral MEP response with the amplitude of
more than 1 mV, whereas this percentage was elevated
in C-ase group (67%, 6/9). The mean amplitude was
also significantly different in C-ase group (1.4 ± 0.7 mV
in C-ase and 0.6 ± 0.5 mV in P-ase group, p < 0.05)
(Fig. 10E). However, MEP amplitude was not significantly changed in contralateral hindlimbs (Fig. 10F),
where implantation of PCL scaffolds with F3.NT3 already significantly recovered MEP responses (Fig. 5B,
D). Collectively, these data indicated that degradation of
1373
CSPGs augmented the behavioral and electrophysiological recovery mediated by PCL-F3.NT3 implantation.
DISCUSSION
The present study demonstrated that the implantation
of polymer scaffold seeded with NT3 overexpressing
NSCs provided marked behavioral and electrophysiological recovery in a rat hemisection SCI model. Degradation of CSPGs deposited at the interface between
scaffolds and spinal cord tissue further enhanced the
therapeutic benefits of PCL-F3.NT3 implantation. The
Figure 8. Chondroitinase ABC treatment promoted migration of F3.NT3 NSCs. (A, B) Representative longitudinal spinal cord
sections of penicillinase (P-ase, A) and chondroitinase ABC (C-ase, B) treated animals with PCL-F3.NT3 implants at 9 weeks postSCI. Spinal cord sections were stained with GFAP (green) and human-specific mitochondria antibody (red) to detect grafted NSCs
of human origin. F3.NT3 cells in C-ase group showed more frequent migration in a rostrocaudal direction. Rostral direction is
leftwards. Dotted lines demarcate scaffold borders. Scale bar: 250 µm. (C–F) Higher magnification images of boxed regions in the
longitudinal section of P-ase (C, D) or C-ase (E, F) groups. Note that F3.NT3 cells more readily cross glial scar fronts. DAPIstained nuclei are shown in blue. Scale bar: 50 µm. (G) Stereological counting of grafted cells. *p < 0.05 and **p < 0.01 by
repeated measures two-way ANOVA followed by Tukey’s post hoc analysis. Error bars represent mean ± SEM. (H) Quantification
of maximal migration distance. Each triangle or circle represents maximal migration distance in each animal, and each line represents group mean value. **p < 0.01 by Student’s t-test. N = 9 per group.
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1374
HWANG ET AL.
Figure 9. Chondroitinase ABC treatment in combination with PCL-F3.NT3 implantation promoted axonal remodeling. (A–D)
Longitudinal spinal cord sections of animals with PCL-F3.NT3 implants treated with penicillinase (P-ase) (A, B) or chondroitinase
ABC (C-ase) (C, D). (B) and (D) are magnified images of the boxed regions in (A) and (C), respectively. Neurofilament (NF)positive axons are shown in green. NF axons were found to grow into PCL scaffolds (arrows) in C-ase treated group. PCL scaffolds
were often visualized by background staining (arrowheads, red) with anti-human mitochondria antibody. Dotted lines indicate
scaffold borders. (E–H) Serotonin (5-HT) axons were visualized by immunofluorescence staining in transverse sections of the
caudal spinal cord of animal treated with P-ase (E, F) or C-ase (G, H). 5-HT axon innervation in the ventral gray matter in the
lesioned side (right) (F, H) was markedly reduced compared to that in the contralateral side (left) (E, G). In C-ase group, ipsilateral
5-HT axonal densities were higher than those in P-ase group (H). (I–K) Quantitation graphs of the numbers (I) and lengths (J) of
NF axons growing into PCL, and the percent ipsilateral 5-HT axon density compared to contralateral side (K). ***p < 0.001 by
Student’s t-test. Error bars represent mean ± SEM. Scale bars: 100 µm.
full combination enabled the injured rats to attain wellcoordinated plantar stepping accompanied by improved
ankle positioning and toe clearance (mean BBB score,
16.0 ± 1.5 and 17.4 ± 1.3 for ipsi- and contralateral
hindlimbs, respectively) and reduced paw placement errors on grid to a minimum (3.5 ± 1.0 and 2.1 ± 1.0 per
run for ipsi- and contralateral hindlimbs, respectively).
Furthermore, animals with the full complement of combinatorial strategies regained bilateral MEP responses to
transcranial magnetic stimulation. Therefore, our study
highlights the additive benefits conferred by combining
the following complementary strategies: 1) transplantation of NSCs with polymer scaffold, 2) incorporation of
neurotrophic factor gene, and 3) modulation of inhibitory extracellular matrix. The combination of all these
strategies together was feasible and led to greatest functional recovery, providing an applicable and promising
framework for future NSC-based transplantation therapies targeting spinal cord repair.
The implantation of artificial scaffolds has been employed to bridge lesion cavities and to create a favorable
tissue environment for axonal growth (9,21). Various
scaffolding materials including biodegradable polymers
have been tested in animal models of SCI (24,47,54). In
the present study, we found that the polymer scaffold
itself could provide some therapeutic benefits in terms
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NSC-BASED COMBINATORIAL THERAPY FOR SPINAL INJURY
of behavioral recovery. Animals with PCL scaffold
alone exhibited significantly higher precision in hindlimb placement during the grid walk than those with injury alone. A previous study also reported a significant
functional improvement by implantation of polymer
scaffold (57). Implantation of polymer scaffolds may
provide mechanical support and prevent instability-related
functional impairments. Furthermore, implanted poly-
1375
mer scaffolds may deter the infiltration of inflammatory
cells and fibroblasts into the lesion cavities with suppression of glial scar formation (57). In addition to their
own positive contribution, bioengineered polymer scaffolds may find their versatile utility in integrating combinatorial strategies, especially cell-based interventions
(47,61). As also shown in the present study, polymer
scaffolds have proved to be compatible with a variety of
Figure 10. Functional recovery following chondroitinase ABC treatment in combination with PCL-F3.NT3 implantation. (A, B)
Recovery of BBB locomotor scores over the 7-week period after injury for ipsilateral (right) (A) and contralateral (left) (B)
hindlimbs. Chondroitinase (C-ase, N = 9) treatment significantly improved recovery of BBB scores over time in animals with PCLF3.NT3 implantation compared with penicillinase (P-ase, N = 9) group. *p < 0.05 by repeated measures two-way ANOVA. (C, D)
Grid walk. Average numbers of hindlimb placement errors per run were counted at 4 and 7 weeks (4W and 7W) post-SCI for
ipsilateral (C) and contralateral (D) hindlimbs. C-ase treatment significantly decreased the number of errors in both hindlimbs. *p
< 0.05 and **p < 0.01 by Student’s t-test. N = 9 per group. (E, F) Comparison of the mean amplitudes of motor evoked potentials
(MEPs) recorded in ipsilateral (E) and contralateral (F) gastrocnemius muscles at 8 weeks post-SCI. C-ase treatment significantly
improved the mean amplitude of MEPs in ipsilateral hindlimbs. *p < 0.05 by Student’s t-test. N = 9 per group for all graphs. Error
bars represent mean ± SEM.
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1376
HWANG ET AL.
cell types and successfully delivered cells of therapeutic
neurons receive supraspinal inputs). This suggests that
interests to the lesioned spinal cord (23,47,54,56). The
neuroplasticity was enhanced in a regionally specific
PCL scaffold in our study appeared to function like a
manner and that synaptic connections were actively rereservoir supplying migratory NSCs to the host spinal
modeled only in motor pathway, but not in behaviorally
cord. Polymer scaffolds are primarily expected to funcirrelevant regions. It is also possible that F3.NT3tion as a bridge filling tissue defects (9). Although the
derived neurons migrating to host spinal cord contribPCL scaffolds physically filled the lesion cavity by prouted to the observed locomotor recovery by participating
viding structural support, they were not able to guide
in the formation of new intraspinal relay circuits (2).
axonal growth without the addition of C-ase infusion. It
NT3 can directly enhance oligodendrocyte precursor
has been suggested that glial scars forming on- and offproliferation and survival (3,4) and also promotes myramps of polymer scaffolds can impede their bridging
elination by endogenous oligodendrocytes (43). In this
function by preventing axonal growth in and out of polyregard, it was intriguing to find that MEP response was
mer scaffolds (21).
not elicited in the contralateral hindlimbs after hemisecParental NSCs (F3 cells) grafted with PCL scaffolds
tion injury. A recent study demonstrated that electrical
survived the transplantation and migrated to host spinal
transmission through contralateral spared white matter
cord. Although implantation of PCL scaffold seeded
was impaired, and this was ascribed to a decrease in
with F3 cells appeared to improve accuracy of hindlimb
the number of myelinated axons (1). Our data strongly
placement compared to PCL alone group, behavioral resuggested that the absence of MEP response in concovery assessed by BBB score was not significantly diftralateral hindlimb (Fig. 5B, D) was due to the demyeferent between PCL-alone and PCL-F3 groups. The inlination of contralateral white matter and that MEP recorporation of NT3 gene into F3 cells substantially
covery could be explained by greater myelination in
improved cell survival and migration. F3.NT3 cells were
PCL-F3.NT3 group.
more numerous than F3 cells along the rostrocaudal exAlthough PCL-F3.NT3 implantation resulted in subtent from the implantation site. Furthermore, the maxistantial recovery of locomotor function, we hypothemal distance of F3.NT3 migration was also markedly
sized that CSPGs surrounding PCL scaffolds would still
greater than that of F3 cells. A previous study reported
limit the extent of functional benefits. The present study
that NT3 treatment, by acting through its cognate recepshowed that degradation of CSPGs by intrathecal infutor TrkC, enhanced the survival and migration of transsion of C-ase enhanced migration of F3.NT3 cells to the
planted NSCs in the rat spinal cord (13). In addition,
spinal cord and increased the extent of axonal growth
we found that F3.NT3 cells differentiated into mature
into PCL scaffolds. This enhancement in migration inneurons and oligodendrocytes more frequently than F3
duced by C-ase could increase the number of F3.NT3cells. Therefore, the incorporation of NT3 gene could
derived neurons or oligodendrocytes located in host
positively regulate the intrinsic properties of grafted
spinal cord, allowing more chances for intraspinal relay
NSCs such as survival, migration, and differentiation in
circuit formation or replacing lost oligodendrocytes. Bean autocrine or paracrine fashion. As a result, a much
cause surrounding CSPGs inhibit axonal growth into
larger number of new neurons and oligodendrocytes descaffolds, PCL scaffolds would not function adequately
rived from NSCs were supplied to the host spinal cord
as an actual bridge without C-ase (21). Therefore, degraby PCL-F3.NT3 implantation.
dation of CSPGs may be essentially required for the
The animals implanted with PCL-F3.NT3 exhibited
proper bridging functions either by polymer scaffold or
better locomotor recovery than those in the other three
other biological grafts (14,32). The increases in ipsilatstudy groups. We speculate that several mechanisms
eral 5-HT innervation may explain our observation that
may be responsible for PCL-F3.NT3-mediated funcMEP responses were significantly recovered by C-ase
tional recovery. Increased NT3 concentration in host
in ipsilateral hindlimbs (Fig. 10E), where PCL-F3.NT3
spinal cord tissue (Fig. 6A) could promote neuroplasticimplantation failed to restore recognizable MEP reity in the caudal spinal cord, which is directly responsisponses (Fig. 5A, C). Combination of C-ase treatment
ble for hindlimb locomotion. It has been previously
could promote the establishment of new axonal connecdemonstrated that NT3 enhances the sprouting of cortitions to the ipsilateral lumbar motor neurons by supportcospinal axons and that NT3 signaling plays a critical
ing axon growth directly through implanted PCL scafrole in synaptogenesis (25,42,55). The increases in the
folds. Alternatively, C-ase treatment might promote the
expression of GAP-43 and synaptotagmin indicated that
sprouting of contralateral spared axons to the ipsilateral
PCL-F3.NT3 implantation markedly promoted neulumbar regions (5,8). At any rate, these findings indiroplasticity in the caudal spinal cord. Intriguingly, the
cated that combination of C-ase treatment compleincrease in GAP-43 immunoreactivity was found in the
mented the PCL-F3.NT3 implantation by strengthening
intermediate regions (where corticospinal fibers termiipsilateral supraspinal connections to caudal ventral
nate) and ventral motor regions (where ventral motor
horn.
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NSC-BASED COMBINATORIAL THERAPY FOR SPINAL INJURY
One caveat in this study is that we use the hemisection SCI model, which has less clinical relevance compared to contusion or compression injury. We chose this
model because it creates a defined cavity into which the
solid PCL scaffold can be implanted without additional
surgical damages. Because contusion or compression injury is accompanied by progressive cavitation that is
characteristic of human SCI (58), these models would be
more suited for a preclinical testing of a new therapeutic
strategy. However, irregular and unpredictable shape
and dimensions of lesion cavities would make it almost
impossible to implant the solid PCL scaffold. Injectable
hydrogels that can cast into irregular cavities would
allow us to test the current combinatorial strategy in a
more clinically relevant model (22,28). The timing of
the combinatorial treatment also needs to be addressed.
Because our acute treatment paradigm cannot be instituted for human patients, it would be desirable to test
the treatment efficacy in a chronic injury setting. In this
regard, a recent study that demonstrated the therapeutic
effects of the combinatorial strategy involving hydrogel
scaffold and mesenchymal stem cells appears to be
highly encouraging (23).
Summarizing, this study demonstrated that combining multifaceted strategies could maximize the therapeutic benefits of NSC transplantation to repair the injured
spinal cord. We believe that our results have important
clinical implications regarding the future design of NSCbased therapeutic strategies. Developing an appropriate
combinatorial strategy would accelerate the clinical
translation of NSC-based transplantation approaches for
human victims suffering from traumatic SCI.
ACKNOWLEDGMENTS: This study was supported by Korea
Research Foundation Grant (KRF-2006-E00439, B.G.K),
Brain Science Fundamental Technology Development Project
(2009-0081466, B.G.K), Ajou University School of Medicine
(2007–2008, B.K.G.), and 21st Century Frontier Research
Fund (SC-3111, B.W.Y). The authors declare no conflicts of
interest.
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15.
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