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Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Therapy Promotes Functional Recovery of Contused Rat Spinal Cord through Enhancement of Endogenous Cell Proliferation and Oligogenesis

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Numerous studies have shown the benefits of mesenchymal stem cells (MSCs) on the repair of spinal cord injury (SCI) model and on behavioral improvement, but the underlying mechanisms remain unclear. In this study, to investigate possible mechanisms by which MSCs contribute to the alleviation of neurologic deficits, we examined the potential effect of human umbilical cord blood-derived MSCs (hUCB-MSCs) on the endogenous cell proliferation and oligogenesis after SCI. SCI was injured by contusion using a weight-drop impactor and hUCB-MSCs were transplanted into the boundary zone of the injured site. Animals received a daily injection of bromodeoxyuridine (BrdU) for 7 days after treatment to identity newly synthesized cells of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells was evident. Behavior analysis revealed that locomotor functions of hUCB-MSCs group were restored significantly and the cavity volume was smaller in the MSCs-transplanted rats compared to the control group. In MSCs-transplanted group, TUNEL-positive cells were decreased and BrdU-positive cells were significantly increased rats compared with control group. In addition, more of BrdU-positive cells expressed neural stem/progenitor cell nestin and oligo-lineage cell such as NG2, CNPase, MBP and glial fibrillary acidic protein typical of astrocytes in the MSC-transplanted rats. Thus, endogenous cell proliferation and oligogenesis contribute to MSC-promoted functional recovery following SCI.
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Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2012, Article ID 362473, 8pages
doi:10.1155/2012/362473
Research Article
Human Umbilical Cord Blood-Derived Mesenchymal
Stem Cell Therapy Promotes Functional Recovery of Contused
Rat Spinal Cord through Enhancement of
Endogenous Cell Proliferation and Oligogenesis
Sang In Park,1, 2 Jung Yeon Lim,2Chang Hyun Jeong,2Seong Muk Kim,2
Jin Ae Jun,2Sin-Soo Jeun,2and Won Il Oh3
1Institute of Catholic Integrative Medicine (ICIM), Incheon St. Mary’s Hospital, The Catholic University of Korea,
Incheon, Republic of Korea
2Department of Neurosurgery, The Catholic University of Korea, Seoul, Republic of Korea
3Medipost Biomedical Research Institute, Medipost Co., Ltd., Seoul, Republic of Korea
Correspondence should be addressed to Sin-Soo Jeun, ssjeun@catholic.ac.kr
Received 23 June 2011; Accepted 29 September 2011
Academic Editor: Ken-ichi Isobe
Copyright © 2012 Sang In Park et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Numerous studies have shown the benefits of mesenchymal stem cells (MSCs) on the repair of spinal cord injury (SCI) model
and on behavioral improvement, but the underlying mechanisms remain unclear. In this study, to investigate possible mechanisms
by which MSCs contribute to the alleviation of neurologic deficits, we examined the potential eect of human umbilical cord
blood-derived MSCs (hUCB-MSCs) on the endogenous cell proliferation and oligogenesis after SCI. SCI was injured by contusion
using a weight-drop impactor and hUCB-MSCs were transplanted into the boundary zone of the injured site. Animals received
a daily injection of bromodeoxyuridine (BrdU) for 7 days after treatment to identity newly synthesized cells of ependymal
and periependymal cells that immunohistochemically resembled stem/progenitor cells was evident. Behavior analysis revealed
that locomotor functions of hUCB-MSCs group were restored significantly and the cavity volume was smaller in the MSCs-
transplanted rats compared to the control group. In MSCs-transplanted group, TUNEL-positive cells were decreased and BrdU-
positive cells were significantly increased rats compared with control group. In addition, more of BrdU-positive cells expressed
neural stem/progenitor cell nestin and oligo-lineage cell such as NG2, CNPase, MBP and glial fibrillary acidic protein typical
of astrocytes in the MSC-transplanted rats. Thus, endogenous cell proliferation and oligogenesis contribute to MSC-promoted
functional recovery following SCI.
1. Introduction
Recovery following spinal cord injury (SCI) is limited
because of axonal damage [1], demyelination, and scar
formation [2]. In addition to the formation of a central
hemorrhagic lesion devoid of normal neurons and glia,
oligodendrocytes and astrocytes in the white matter near the
impact site are reduced by about 50% by 24h after injury [3].
Recently, the use of stem cell for neurodegenerative
disease has been widely investigated as a therapeutic strategy
[46]. Neural stem cells have been used for the treatment
of neurological diseases such as SCI [7]orstroke[8].
Numerous studies have reported that the survival and differ-
entiation of grafted cells into neural cells correlate with
behavior improvement. However, these cells are limited for
clinical application because of insucient cell supply, risk of
immune rejection, and ethical problems. Since mesenchymal
stem cells (MSCs) can be readily isolated and their numbers
increased in vitro and dierentiated into several types of
mature cells including neurons, adipocytes, cartilage, and
skeletal hepatocytes under appropriate conditions [9],anew
therapeutic strategy has been a valuable source for central
nervous stem (CNS) disease [10,11]. Human umbilical
cord blood-derived MSCs (hUCB-MSCs) have therapeutic
2Journal of Biomedicine and Biotechnology
potential and are attractive because these cells are readily
available and are less immunogenic as compared to other
sources of stem cells, such as bone marrow or adipose [12].
An alternative strategy of stem cell therapy is protection
of injured cells and promotion of endogenous cell regen-
eration. Several studies have reported that stem cells might
provide a better environment for damaged tissue and save
remaining neurons by neurotrophic factors or cytokines [13,
14]. However, the specific mechanism of the MSCs for these
assertions remains controversial and ill-explored. Neverthe-
less, MSC treatment of SCI has been reported as a candi-
date that supplies angiogenic, antiapoptotic, and mitogenic
factors as well as migration toward damaged tissue [15].
Recently, MSCs have been used in clinical treatment and were
shown to be eective in the treatment of various pathologies
although evidence for distinct therapeutic mechanism was
lacking [16].
The normal spinal cord contains endogenous neural
progenitor cells (NPC) and oligodendrocyte precursor cells
(OPCs) [17]. Nevertheless, production of new neurons and
oligodendrocytes by endogenous cells into the spinal cord
may be very restricted after injury [18]. Furthermore, cell
transplantation studies have demonstrated that exogenous
stem cells dierentiate only very poorly when grafted into the
spinal cord. Thus, the environment of the spinal cord appears
to be highly restrictive for the dierentiation of OPCs.
If this environmental restriction can be changed by hUCB-
MSC in SCI, OPCs may be able to supply new neurons
and oligodendrocytes. However, it is not known whether
survival and dierentiation generated from endogenous cells
are influenced by transplanted hUCB-MSCs.
In the present study, we show that the transplantation
of hUCB-MSCs confers therapeutic eects in a rat experi-
mental SCI model. We investigated whether transplantation
of hUCB-MSCs improved the functional recovery and im-
proved the proliferation and genesis of resident endogenous
cells within the spinal cord by hUCB-MSCs.
2. Materials and Methods
2.1. Human UCB-Derived MSCs. Human UCBs were ob-
tained from normal full-term pregnant woman. The protocol
for human subjects adhered to the guidelines outlined by the
institutional review IRB board of the Catholic University of
Korea (Seoul, Republic of Korea). hUCB-MSCs were isolated
and expanded using a previously described protocol [12].
2.2. Animal Model. All animal protocols were approved by
the Institutional Animal Care and Use Committee of Catho-
lic University Medical School. Forty-five adult male Sprague-
Dawley rats weighting between 270 and 300 g were employed
in our experiments. Surgical techniques were similar to
those described previously [19]. Briefly, rats were deeply
anesthetized with ketamine-xylazine cocktail (80 mg/kg of
ketamine, 10 mg/kg of xylazine). Under a dissecting micro-
scope, the skin and muscles overlying the thoracic cord were
separated and retracted, the T9 vertebral level was removed
by laminectomy, and the underlying spinal cord segment
was exposed by slitting the dural sheath. The impact rod
of the NYU impactor was centered above T9 and dropped
from a height of 25 mm to induce an incomplete partial SCI.
Following lesion, the dorsal back musculature was sutured
and the skin closed with surgical clips. After surgery, the
animals were kept on a thermostatically regulated heating
pad until completely awake. The urinary bladder of all rats
was emptied manually two times per day until recovery of
urinary function.
2.3. Cell Transplantation. Rats were assigned randomly to
one of the following two major groups: one group of rats
were treated with 5 µL phosphate-buered saline (PBS) as the
control group. The second group of rats received transplant-
ed with hUCB-MSCs (3 ×105cells/5 µL). In experiment 1,
the hUCB-MSCs was designed to test the therapeutic
eectiveness (n=26), and, in experiment 2, these cells were
designed to evaluate the proliferation of endogenous cells
after transplantation (n=12).
Initial locomotor scores were equalized between groups.
The weight-drop injury level was based on our experience
with the model to produce spontaneous recovery at a Basso-
Beatti-Bresnahan (BBB) score of 4. Once the 46 rats were
subjected to contusion SCI, they were divided randomly into
the two groups. Using a 25-gauge syringe (Hamilton, Reno,
NV) fixed in a stoelting stereotaxic frame (Dae Jong) at 7
days after injury, hUCB-MSCs were transplanted into the
spinal cord (0.5 mm from the midline, 1.5 mm down from
the dura, and 5 mm rostral from the contusion epicenter).
Each rat received a 5µL injection in the contusion site over
a 10 min period each time. The cannula of the Hamilton
syringe was left in place after injection for an additional
5 min. All animal received antibiotics (Gentamicin sulfate,
30 mg/kg/day) during the first week after transplantation.
2.4. 5-Bromo-2-Deoxyuridine (BrdU) Administration. Spra-
gue-Dawley rats (n=12) were injected with 50 mg/kg BrdU
(Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally each
day for 7 days to label the newly generating cells after trans-
plantation. The examined proliferative response focused on
cell genesis occurring within 7 days after transplantation.
2.5. Behavioral Testing. The motor function restoration after
spinal cord contusion was observed by open-field BBB
locomotor ratio scale [20]. The scale used for measuring
hind-limb function with these procedures ranges from a
score of 0, indicating no spontaneous movement, to a
maximum score of 21, with an increasing score indicating
the use of individual joints, coordinated joint movement,
coordinated limb movement, weight-bearing, and other
functions. Behavioral testing was performed weekly on
each hindlimb from the postoperative day to 7 weeks after
SCI. Spinal cord contusion and cell transplantation were
separately performed in double-blinded experiments by
dierent investigators.
2.6. Tissue of Harvest. To study functional recovery and dif-
ferentiation of transplanted hUCB-MSCs, rats from each
group were sacrificed at 1 and 2 weeks (PBS, n=3; Trans-
plantation, n=3) after transplantation and the others were
Journal of Biomedicine and Biotechnology 3
examined by the BBB locomotor test 6 weeks after transplan-
tation (PBS, n=7; transplantation, n=7). Also, to study
endogenous cell proliferation after transplantation, rats from
each group were sacrificed at 2 h and 1 week after the last
BrdU injection (n=5). All the rats were deeply anesthetized
with a ketamine-xylazine cocktail (80 mg/kg of ketamine,
10 mg/kg of xylazine) and then perfused transcardially with
0.01 M PBS (pH 7.4), followed by 4% paraformaldehyde
(PFA) in 0.01M PBS. The spinal cord was removed from
each rat and postfixed in 4% PFA for 4 hours. Postfixed
tissue was cryoprotected in 0.1 M phosphate buer (pH 7.4)
containing 15% and 30% sucrose solution at 4C. The spinal
cordswereembeddedinOCTcompoundandstoredat
70C. To examine the cavity volume, 14 µm thick serial
transverse sections were prepared from 20 mm long spinal
cord stumps (1 mm each for rostral and caudal to the lesion
epicenter). Also, to compare the coexpression of various cell-
type-specific markers and BrdU+cells, 10 µm thick serial
coronal sections were prepared as described above. Coronal
sections were collected from cell transplantation site to the
injury epicenter sites and mounted on gelatin-coated slides.
2.7. Histology and Immunohistofluorescence. Single and dou-
ble fluorescent staining was used. Single staining was used
to identify newly generated cells after transplantation. For
BrdU immunohistochemistry, the sections were warmed for
20 min and washed with 0.01 M PBS for 10 min. Sections
were incubated in 50% formamide-2X standard saline citrate
at 60C for 2 h, subsequently treated with 2 N HCL at 37C
for 30 min to denature deoxyribonucleic acid, and then
incubated in 0.1 mol/L boric acid at room temperature for
10 min to neutralize residual acid. The sections were incu-
bated with rat anti-BrdU (1 : 100; Abcam, Cambridge, UK)
or mouse anti-BrdU (1 : 100; DakoCytomation, Glostrup,
Denmark). Subsequently, sections were incubated for 1 h at
room temperature with fluorescence-conjugated secondary
antibody or biotinylated antibody; the latter was reacted
with avidin peroxidase for 30 min (ABC-kit; Vectastain
Elite; Vector Laboratories, Burlingame, CA) followed by
detection solution (0.25 mg/mL diaminobenzidine, 0.03%
H2O2, 0.04% NiCl).
To determine the fate of newly generated cells after trans-
plantation, double-fluorescent immunolabeling was per-
formed, combining BrdU labeling with one of cell-specific
phenotypic markers listed below. We used mouse anti-Nestin
(1 : 100; Millipore, Billerica, MA) to identify neural stem
progenitor; mouse anti-NG-2 chondroitin sulfate proteo-
glycan (anti-NG-2; 1 : 100; Millipore) to identify oligoden-
drocyte progenitor; mouse anti-2,3-cyclic nucleotide 3-
phosphodiesterase (anti-CNPase; 1 : 100; Millipore), mouse
antimyelin basic protein (anti-MBP, 1 : 100; Millipore), rab-
bit anti-glial fibrillar y acidic protein (anti-GFAP; 1 : 500;
Millipore) to identify astrocytes. After washing, samples
were incubated in Alexa 488-conjugated goat anti-rat IgG
(1 : 200; Vector Laboratories), Alexa 546-conjugated goat
anti-mouse IgG (1 : 200; Vector Laboratories), or Alexa 546-
conjugated goat anti-rabbit IgG (1 : 200; Vector Laboratories)
for 1 h. Fluorescently stained slides were stored at 20C
and observed using a fluorescence microscope equipped
with a spot digital camera or a model LSM 510 confocal
scanning laser microscope (Zeiss, Jena, Gemany). Apoptosis
was detected by the terminal deoxynucleotidyl-transferase-
mediated d-UTP-biotin nick end (TUNEL) assay using the
in situ cell death detection kit (Roche, Indianapolis, IN)
developed using the Cy2-conjugated streptavidin (Jackson
Laboratories, West Grove, PA). The slides were observed
using the aforementioned confocal scanning laser micro-
scope.
2.8. Cell Counts. The counting of BrdU+cells was done by
previously described [21]. BrdU+cells were counted within
a reticule of a specified area (0.0682 mm2) positioned in
the ependymal and parenchymal region (dorsal (above the
corticospinal tract), lateral, and ventromedial region of the
residual white matter) in sections. White matter regions were
counted in six randomly chosen sections per 1 mm2length of
spinal cord, and the numbers were averaged.
2.9. Measurement of the Cavity Volume. For measurement of
the cavity volume, rats at 6 weeks after transplantation were
used. The transverse sections were stained with hematoxylin-
eosin (HE). The area of the cavity in the damaged spinal
cord was measured in images of the sections using ImageJ
version 1.38 image analyzer software (National Institutes of
Health, Bethesda, MD) on consecutive sections at an interval
of 70 µm. The volume of the cavity was then calculated by
multiplying the average area by the depth of the spinal cord.
2.10. Statistical Analysis. The BBB score and cell counts
were subjected to the paired t-test or one-way ANOVA for
transplantation and PBS-treated groups of rats. Data are
presented as mean ±SE. Value of P<0.05 was considered
statistically significant.
3. Results
3.1. Behavioral Assessment and Measurement of the Cavity
Volume. We assessed the recovery of hindlimb function with
the BBB locomotor scale from 1 day to 6 weeks after SCI. In
the case of SCI rats, BBB scores were low (<9). The motor
function scores of MSCs-injected rats (11.07 ±0.3) were
significantly higher than the PBS-injected rats (9.25 ±0.3)
at 7 weeks after SCI. The behavioral data from the BBB
locomotor scores demonstrated that MSCs-treated rats were
dramatically improved in neurological function (P<0.005,
Figure 1). In addition, the spinal cords of MSCs-injected rats
had cavities much smaller than those of the PBS-injected rats.
The cavity volume of MSCs-treated rat was 0.82 ±0.14 mm3
on average, whereas the PBS-treated rats showed a volume
of 2.12 ±0.28 mm3. These results for cavity volume were
significantly dierent between the MSCs-treated and PBS-
treated rats. Thus, MSC transplantation led to a significant
improvement of behavior as well as reduction of cavity
volume after SCI.
3.2. Proliferation of Endogenous Generated Cells. MSCs pro-
moted the functional recovery and reduced the cavity volume
following transplantation in SCI (Figure 1). Since an eect
4Journal of Biomedicine and Biotechnology
0
1
2
3
4
5
6
7
8
9
10
11
12
13
BBB score
Days after trauma
hUCB-MSCsPBS
0
0.5
1
1.5
2
2.5
3
Cavity volume (mm3)
1 7 14 21 28 35 42 49 56
Transplantation
PBS
hUCB-MSCs
(a) (b)
(c)
(d)
hUCB-MSCs
PBS
Figure 1: BBB scores of rats with SCI before and after hUCB-MSCs transplantation at 7 days after SCI. (a) hUCB-MSC transplantation group
displayed significantly improved scores compared with control at 6 weeks after transplantation. (b) Cavity volume between the hUCB-MSC
and control groups at 6 weeks after transplantation. The values of the cavity volume of the hUCB-MSC group were lower than those of the
control group. (c) and (d) HE-stained sections of transplantation group and control group, P<0.05.
Transplantation
(a)
(b)
(c) (d)
Ependymal region Parenchymal region
PBS hUCB-MSCs
500
400
300
200
100
0
50
100
150
200
250
PBS hUCB-MSCs
Number of BrdU +cells/0.0682 mm2
Number of BrdU +cells/0.0682 mm2
Figure 2: Quantitative analysis of BrdU-labeled cells in the ependymal and parenchymal regions. (a) Result of immunohistochemistry using
anti-BrdU antibody. (b) Enlargement of the boxed region in (a), showing BrdU-labeled cells in the parenchymal region. (c) Average number
of BrdU-labeled cells per white matter area from all five white matter areas in the parenchymal region. (d) Average number of BrdU-labeled
cells per ependymal region in grey matter. At 14 days after transplantation, proliferation of endogenous cells was significantly increased from
injury site to cell transplantation site in hUCB-MSCs-transplanted group compared with control group, P<0.05.
of hUCB-MSCs was evident, we investigated whether newly
generated cells were enhanced by the transplanted cells [22].
It has been suggested that oligogenesis [23]byendogenous
OPCs and survival of these cells can contribute to self-repair
after myelin loss [24]. With the thought that these processes
might be stimulated recovery to CNS injury, an experiment
was done to investigate the proliferation endogenous gen-
erated cells by daily injection of BrdU during the 7 days
after transplantation. BrdU-positive cells were counted in the
ependymal and parenchymal regions (Figures 2(a) and 2(b))
as previously described [23,25]. Proliferation of the newly
generated cells increased greatly in hUCB-MSCs-treated rats
Journal of Biomedicine and Biotechnology 5
Nestin+/ BrdU+cells/0.0682 mm2
GFAP+/ BrdU+cells/0.0682 mm 2
Ependymal region Parenchymal region
BrdU/NG2
PBS hUCB-MSCs PBS hUCB-MSCs
(a) (b)
(c) (d)
(e) (f)
(g) (h)
(i) (j) (k)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
12
(week)
PBS
hUCB-MSCs
PBS
hUCB-MSCs
12
(week)
0
5
10
15
20
25
30
35
40
45
50
12
(week)
PBS
hUCB-MSCs
0
5
10
15
20
25
30
BrdU/Nestin BrdU/Nestin
BrdU/GFAPBrdU/GFAP BrdU/NG2
BrdU/NG2BrdU/NG2
1 week
2 week
1 week
1 week
NG2+/ BrdU +cells/0.0682 mm2
Figure 3: Endogenous neurogenesis induced by transplantation. Endogenous stem cells were assessed quantitatively by double staining of
BrdU with nestin, GFAP, and NG2 at 1 and 2 weeks after transplantation in both the ependymal and parenchymal regions. (a)–(d) At 1
and 2 weeks following transplantation, BrdU/nestin-labeled cells as well as BrdU/GFAP-labeled astrocytes were present in ependyma. (e)
and (h) BrdU-labeled NG2 cells were coexpressed at 1 and 2 weeks in the parenchyma. (i) and (j) The numbers of BrdU-labeled ependyma
coexpressing GFAP/nestin were quantified at 1 and 2 weeks after transplantation. (k) The numbers of BrdU-labeled parenchyma coexpressing
NG2 were quantified at 1 and 2 weeks after transplantation. P<0.05, scale bars =10 µmin(a)(d);20µmin(e)(h).
as compared with PBS-treated rats (Figure 2(c)). This data
demonstrated that hUCB-MSCs could enhance proliferation
of endogenous cells within the spinal cord.
3.3. Characterization of Endogenous Stem Cells. Functional
recovery in response to therapeutic grafting of stem cells
after SCI is related to the dierentiation of grafted cells
into glial cells, including astrocytes or oligodendrocytes [24].
Appropriately, an experiment was done to examine if the
transplantation of MSCs could enhance the dierentiation
of endogenous OPCs into astrocytes or oligodendrocytes
by performing immunostaining for BrdU and several phe-
notype markers including dierentiating oligodencrocyte
markers NG2, CNPase, the mature oligodendrocyte marker
6Journal of Biomedicine and Biotechnology
BrdU/CNP
BrdU/MBP
BrdU/GFAP BrdU/GFAP
(a) (b)
(c) (d)
(e) (f)
(g)
PBS
hUCB-MSCs
Parenchymal region
30
25
20
15
10
5
CNPase/BrdU MBP/BrdU GFAP/BrdU
PBS hUCB-MSCs
BrdU/CNP
BrdU/MBP
Number of BrdU+cells/0.0682 mm2
Figure 4: Quantitative analysis of endogenous oligogenesis by hUCB-MSCs. At 2 weeks after cell transplantation, BrdU and cell-specific
markers were observed up to the edge of the SCI region. (a) and (b) BrdU/CNPase-labeled cells were present. (c) and (d) BrdU/MBP-labeled
cells. (e) and (f) BrdU/GFAP-labeled cells. (g) Quantity of BrdU/CNPase, MBP, and GFAP-labeled cells. P<0.05. Scale bars: 10 µm.
MBP, GFAP typical of astrocytes, and the neural stem cell
marker nestin. Cells in the ependymal and parenchymal
region were counted in sections from the injury epicenter
to cell transplantation site. One and 2 weeks after cell
transplantation, the numbers of BrdU positive cells were sig-
nificantly increased compared with the PBS group (Figure 3).
In the ependymal region, BrdU-labeled nestin and GFAP
cells were increased compared with the PBS group at 1
and 2 weeks (Figure 3). The numbers of BrdU-labeled NG2
positive cells were also significantly increased compared with
the PBS group in the parenchymal region (Figure 3). Also,
BrdU-labeled cells displaying strong immunoreactivities for
CNPase, MBP, or GFAP in the cell transplantation group
were evident. But these immunoreactivities were weak for
those rats treated with PBS (Figure 4). These data sug-
gest that hUCB-MSCs are an influential microenvironment
within the spinal cord.
3.4. Apoptotic Phenomena of Endogenous Cells. To investigate
whether transplantation of MSCs have a protected injured
spinal cord cells from apoptosis, a TUNEL assay was per-
formed on sections obtained from the injury site on 2 weeks
after transplantation. Numerous TUNEL-positive (green)
cells were observed at the injury site in PBS-treated rats.
The number of TUNEL positive cells was significantly lower
in MSC-treated rats than in PBS-treated rats (Figure 5(b)).
Taken together, these results indicate that hUCB-MSCs
not only promote oligogenesis in the spinal cord but also
have a neuroprotective eect relative with cavity volume
(Figure 1(c)).
4. Discussion
In this study, hUCB-MSCs that were transplanted after SCI
survived in and around the injured site and were able to
ameliorate some of the behavior eects of SCI, as measured
by spontaneous limb movement in an open-field test, hind
limb extension, and toe spread. In addition, the cavities
of MSC-treated rats were much smaller than PBS-injected
rats. Cavity formation is a characteristic of progressive tissue
necrosis, which follows the initial primary cell destruction in
SCI. Therefore, reduction of the cavity volume means that
transplanted MSCs after SCI have a neuroprotective eect.
The presently indicated therapeutic eect of hUCB-MSCs in
SCIagreeswithpreviousdata[26], but the exact mechanisms
to improve the functional deficits remain to be elucidated.
A prior study showed that transplanted cells ameliorated
the functional recovery through the integration into spinal
cord tissue and establishment of some connections within
the injured area of the spinal cord [27]. However, the
transplantation of hUCB-MSCs could not solely account
for functional recovery after SCI. Other possibility may
be various beneficial actions of endogenous neurogenesis
or oligogenesis within the adult spinal cord which is
largely mediated via trophic influences. Previous studies
have indicated that MSCs could produce trophic factors,
cytokines, and other neuroprotective factors in stroke or
traumatic brain injury [28,29]. These factors and cytokines
can then promote the regrowth of interrupted nerve fiber
tract. BMS cells secrete more than 20 cytokines in vitro,
and hUCB-MSCs can secrete a number of cytokines and
Journal of Biomedicine and Biotechnology 7
0
100
200
300
400
500
PBS hUCB-MSCs
TUNEL positive cell
PBS hUCB-MSCs
TUNEL
TUNEL
DAPIDAPI
(a) (b)
(c) (d) (e)
Figure 5: Protection of apoptosis by hUCB-MSCs as revealed by TUNEL assay in the injury site at 2 weeks after transplantation. (a)–(d)
TUNEL staining (green) and staining with 4,6-diamidino-2-phenylindole (blue) indicate undergoing apoptotic cell death. (e) Quantity of
TUNEL positive cells. The number of TUNEL positive cells was significantly reduced in cell transplantation group than in control group.
P<0.05, scale bars denote: 10 µm.
chemokines [30]. Therefore, these factors and some of the
other cytokines secreted by hUCB-MSCs may function as
survival and dierentiation factors for neural progenitor cells
and then play an important role in the proliferation and
dierentiation of neural tissue and in the increase of central
nerve system plasticity [31,32].
To understand whether the transplanted hUCB-MSCs
are capable of restoring the production of endogenous cells,
we studied the mechanisms that contributed to functional
recovery by determining the endogenous cell proliferation
and dierentiation into glial cells following transplantation.
Compared to the control group, transplanted cells increased
endogenous cell division within the SCI area and a subpop-
ulation of newly dividing cells. Also, in the received, the
transplanted cells, immature and mature oligodendrocytes,
and astrocytes were stimulated. These observations support
the possibility that factors produced by hUCB-MSCs activate
nearby oligogenesis, and that activation of the astrocytes
increases in oligogenesis, since astrocytes are located in close
proximity to neural stem cells and express several factors that
independently increase oligogenesis. In addition, some of
transplanted cells were BrdU-positive cell. It has been shown
that transplanted cells might proliferate in the spinal cord.
But, these cells are not dierentiated neural lineage markers.
In agreement with the present findings, a previous study
reported not only extensive oligogenesis of newly born cells
after SCI but also that MSCs promote oligogenesis in neural
stem cells in vitro [24,33].
Presently, the majority of hUCB-MSCs progressed to
apoptotic cell death. However, MSC-treated rats displayed
markedly reduced apoptotic cell death in the injured site.
These results suggest that functional recovery might result in
endogenous oligogenesis and neuroprotection stimulated by
trophic factors secreted into transplanted cells.
The collective results support the view that hUCB-MSCs
transplantation is beneficial in SCI by virtue of their growth
factor secretion and ability to provide physical support to
growing axons. Further studies are needed to confirm that
the benefit obtained from hUCB-MSCs persists at later time
points and/or to improve the ecacy of the transplanted
hUCB-MCSs. Also, the mechanisms underlying functional
recovery after transplantation of hUCB-MSCs remain to be
further investigated.
5. Conclusion
We have shown that stem cell therapy of hUCB-MSCs
may provide more of functional recovery in spinal cord
injury such as reduction of cavity volume, increasing of cell
proliferation and endogenous oligogenesis, and decreasing of
apoptosis. Therefore, the author suggests that promotion of
oligogenesis by hUCB-MSCs may provide a scientific basis
for the potential use of these cells as a therapeutic tool for the
treatment of other disease.
Conflict of Interests
The authors declare that there is no conflict of interests.
Acknowledgments
This study was supported by the Basic Science Research
program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology (2010–0022845), Republic of Korea. This study
was supported by a grant of the Korea Healthcare technology
R&D Project, Ministry of Health, Welfare& Family Aairs,
Republic of Korea (A092258).
8Journal of Biomedicine and Biotechnology
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... Mesenchymal stem cells (MSCs) are relatively a better option since they are easy to isolate, multiply in vitro, and differentiate into several types of mature cells that include neurons, adipocytes, cartilage, and skeletal hepatocytes under appropriate conditions. 3 This therapeutic strategy has been a valuable therapy source for central nervous stem (CNS) diseases and injuries, and has proven to make a significant impact on the recovery process. Human umbilical cord blood-derived MSCs (hUCB-MSCs) have significant therapeutic potential and are preferred because of their availability and poor immunogenicity compared to other sources of stem cells, such as bone marrow or adipose. ...
... Human umbilical cord blood-derived MSCs (hUCB-MSCs) have significant therapeutic potential and are preferred because of their availability and poor immunogenicity compared to other sources of stem cells, such as bone marrow or adipose. 3 Another strategy of stem cell therapy is the protection of injured cells and the promotion of endogenous cell regeneration. Stem cells provide a better environment for damaged tissue and protect the remaining neurons by neurotrophic factors or cytokines. ...
... In this study, hUCB-MSCs that were transplanted after SCI, survived in and around the injured site. 3 Also, the cavities of the MSC -treated rats were much smaller than those of the PBS-injected rats. Transplanted MSCs after SCI demonstrated a neuroprotective effect by reduction of the cavity volume. ...
... BM-MSCs group: Rats received one single injection I.V. into the tail vein with (1 × 10 7 ) cells in 0.2 mL labeled with PKH26 fluorescent linker dye in phosphate buffer saline (PBS) and after 12 weeks the rats were sacrificed. 16 CP + BM-MSCs group: Following the dosage of Larginine hydrochloride, rats were treated with one injection as in the BM-MSCs group. At the end of the experimental period of 12 weeks, rats were anaesthetized by I.P. injection of urethane (1.2 g/kg b.w), 15 then sacrificed by cervical decapitation. ...
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Background and objective: Living organisms respond to physical, chemical, and biological threats with a potent inflammatory response which alters organ cell signaling and leads to dysfunction. We evaluated the therapeutic effect of bone marrow-based mesenchymal stromal cell (BM-MSC) transplanted in rats to preserve tissue integrity and to restore homeostasis and function in the pancreatitis experimental pattern. Methods: This study involved 40 adult male Wister rats. Repeated L-arginine injections caused chronic pancreatitis (CP), leading to the development of pancreatic damage and shifting the intracellular signaling pathways. Rats were then infused with BM-MSC labeled with PKH26 fluorescent linker dye for 12 weeks. Results: Cell-surface indicators of BM-MSCs such as CD 90 and CD29 were expressed with the lack of CD34 expression. BM-MSC treatment considerably improved the alterations induced in a series of inflammatory markers, including IL-18, TNF-α, CRP, PGE2, and MCP-1. Furthermore, improvement was found in digestive enzymes and lipid profile with amelioration in myeloperoxidase activity. BM-MSC treatment also regulated the (TGF-/p-38MPAK/SMAD2/3) signaling factors that enhances repair of damaged pancreatic tissue, confirmed by reversed alteration of histopathological examination. Conclusion: our results further bring to light the promise of cell transplant therapy for chronic pancreatitis.
... There is a specific interest in them as a suitable source of stem cells because they are easily available from a patient's bone marrow [6]. It is believed that MSCs could restore the damage to the spinal cord by reducing the damage size or replacing the destroyed cells [7][8][9]. Also, some researchers have proposed that stem cells can provide growth factors to increase the survival of the residual host cells following an injury [10]. Also, researchers have reported that transplantation of MSCs may have a therapeutic role because of their neural differentiation-inducing effects or by their direct shift to neuronal cells [11,12]. ...
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Spinal cord injury (SCI) is a complex neuropathological condition that represents a major challenge for clinicians and scientists due to patient's functional dysfunction and paralysis. Several treatments have been proposed including biological factors, drugs and cells administered in various ways. Stem cells arise as good candidates to treat SCI since they are known to secrete neurotrophic factors, improving neuroregeneration, but also due to their role in modulating the inflammatory process, favoring a pro-regenerative status. There are several types of cells that have been tested to treat SCI in experimental and clinical studies, but we still face many unanswered questions; one of them is the type of cells that can offer the best benefits and, also the ideal dose and administration routes. This review aimed to summarize recent research on cell treatment, focusing on current delivery strategies for SCI therapy and their effects in tissue repair and regeneration.
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Spinal cord injury (SCI) is a debilitating condition which often leads to a severe disability and ultimately impact patient's physical, psychological, and social well-being. The management of acute SCI has evolved over the couple of decades due to improved understanding of injury mechanisms and increasing knowledge of disease. Currently, the early management of acute SCI patient includes pharmacological agents, surgical intervention and newly experimental neuroprotective strategies. However, many controversial areas are still surrounding in the current treatment strategies for acute SCI, including the optimal timing of surgical intervention, early versus delayed decompression outcome benefits, the use of methylprednisolone. Due to the lack of consensus, the optimal standard of care has been varied across treatment centres. The authors have shed a light on the current updates on early treatment approaches and neuroprotective strategies in the initial management of acute SCI in order to protect the early neurologic injury and reduce the future disability.
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DML THESIS Thesis (Ph.D.)-Georgetown University, Dept. of Neuroscience, 2004 Bibliography : leaves 122-129.
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A two-stage procedure to enrich hemopoietic progenitor cells from human umbilical cord blood is described. Mononuclear cells isolated from 60% Percoll gradients were first treated with a panel of monoclonal antibodies and complement to remove lymphoid and myeloid cells and granulocyte-macrophage colony-forming cells (CFU-GM) and their immature progeny. Cells stained with monoclonal antibody RFB-1 were separated on the fluorescence-activated cell sorter and cultured in the mixed-colony assay. Progenitor cells were isolated in a high RFB-1 antigen density cell fraction containing greater than 95% undifferentiated blast cells and 32% +/- 12% colony-forming cells. Pluripotential progenitor cells (CFU mix) were enriched 229-fold and accounted for 3.2% +/- 1.2% of the isolated cells. Erythroid progenitor cells (BFU-E) were enriched 204-fold and accounted for 20.8% +/- 8.4% of the isolated cells. The procedure recovered 114% +/- 32% of CFU mix but the number of CFU mix in unseparated cord blood appears to be underestimated due to the presence of granulocytic cells identified by monoclonal antibody CMRF-7 (CD15) that decrease the cloning efficiency of CFU mix. Removal of CD15+ cells yielded a four- to fivefold improvement in the plating efficiency of CFU mix. Mixed-colony formation was completed in 9-11 days in cultures inoculated with enriched progenitor cell fractions, compared with 14-16 days in cultures of unseparated cord blood. The enriched progenitor cells could be useful for studying the regulation of hemopoiesis by recombinant growth factors.
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Injury reproducibility is an important characteristic of experimental models of spinal cord injuries (SCI) because it limits the variability in locomotor and anatomical outcome measures. Recently, a more sensitive locomotor rating scale, the Basso, Beattie, and Bresnahan scale (BBB), was developed but had not been tested on rats with severe SCI complete transection. Rats had a 10-g rod dropped from heights of 6.25, 12.5, 25, and 50 mm onto the exposed cord at Tl 0 using the NYU device. A subset of rats with 25 and 50 mm SCI had subsequent spinal cord transection (SCI + TX) and were compared to rats with transection only (TX) in order to ascertain the dependence of recovery on descending systems. After 7-9 weeks of locomotor testing, the percentage of white matter measured from myelin-stained cross sections through the lesion center was significantly different between all the groups with the exception of 12.5 vs 25 mm and 25 vs 50 mm groups. Locomotor recovery was greatest for the 6.25-mm group and least for the 50-mm group and was correlated positively to the amount of tissue sparing at the lesion center (p < 0.0001). BBB scale sensitivity was sufficient to discriminate significant locomotor differences between the most severe SCI (50 mm) and complete TX (p < 0.01). Transection following SCI resulted in a drop in locomotor scores and rats were unable to step or support weight with their hindlimbs (p < 0.01), suggesting that locomotor recovery depends on spared descending systems. The SCI + TX group had a significantly greater frequency of HL movements during open field testing than the TX group (p < 0.005). There was also a trend for the SCI + TX group to have higher locomotor scores than the TX group (p > 0.05). Thus, spared descending systems appear to modify segmental systems which produce greater behavioral improvements than isolated cord systems.
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In the vertebrate central nervous system, multipotential cells have been identified in vitro and in vivo. Defined mitogens cause the proliferation of multipotential cells in vitro, the magnitude of which is sufficient to account for the number of cells in the brain. Factors that control the differentiation of fetal stem cells to neurons and glia have been defined in vitro, and multipotential cells with similar signaling logic can be cultured from the adult central nervous system. Transplanting cells to new sites emphasizes that neuroepithelial cells have the potential to integrate into many brain regions. These results focus attention on how information in external stimuli is translated into the number and types of differentiated cells in the brain. The development of therapies for the reconstruction of the diseased or injured brain will be guided by our understanding of the origin and stability of cell type in the central nervous system.