Human mesenchymal stem/stromal cells suppress spinal inflammation in mice with contribution of pituitary adenylate cyclase-activating polypeptide (PACAP)
Tsumuraya et al. Journal of Neuroinflammation
Human mesenchymal stem/stromal cells suppress spinal inflammation in mice with contribution of pituitary adenylate cyclase-activating polypeptide (PACAP)
Tomomi Tsumuraya 0
Hirokazu Ohtaki 0
Dandan Song 0
Atsushi Sato 0
Jun Watanabe 0
Tomoya Nakamachi 0
Zhifang Xu 0
Kenji Dohi 0
Seiji Shioda 0
0 Department of Anatomy, Showa University School of Medicine , 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555 , Japan
Background: Adult human mesenchymal stem/stromal cells (hMSCs) from bone marrow have been reported to exhibit beneficial effects on spinal cord injury (SCI). A neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) is known to decrease neuronal cell death and inflammatory response after ischemia, SCI, and other neuronal disorders. Recently, we found that expression of the gene for mouse PACAP (Adcyap1) was greater in animals receiving hMSCs with neural injury such as ischemia. However, the association of PACAP with hMSCs to protect nerve cells against neural injuries is still unclear. Methods: Wild-type and PACAP-gene-deficient (Adcyap1+/) mice were subjected to spinal cord transection, and hMSCs (5 105 cells) were injected into the intervertebral spinal cord on day 1 post-operation (p.o.). Locomotor activity, injury volume, retention of hMSCs, mouse and human cytokine genes (which contribute to macrophage (M) and microglial activation), and Adcyap1 were evaluated. Results: hMSCs injected into wild-type mice improved locomotor activity and injury volume compared with vehicle-treated mice. In contrast, non-viable hMSCs injected into wild-type mice, and viable hMSCs injected into Adcyap1+/ mice, did not. Wild-type mice injected with hMSCs exhibited increased Adcyap1 expression, and observed PACAP immunoreaction in neuron-like cells. Gene expression levels for IL-1, tumor necrosis factor (TNF), interleukin-10 (IL-10), and transforming growth factor (TGF) decreased, while that for interleukin-4 (IL-4) increased, in hMSC-injected wild-type mice. In contrast, IL-1, TGF, and IL-4 gene expression levels were all abolished in hMSC-injected Adcyap1+/ mice on day 7 post-operation. Moreover, the mice-implanted hMSCs increased an alternative activating macrophage/microglial marker, arginase activity. The human gene profile indicated that hMSCs upregulated the gene of IL-4 and growth factors which were reported to enhance Adcyap1 expression. Finally, we demonstrated that hMSCs express human ADCYAP1 and its receptor gene after the inflammation-related interferon- (IFN) in vitro. Conclusions: These results suggest that hMSCs attenuate the deleterious effects of SCI by reducing associated inflammatory responses and enhancing IL-4 production. This effect could be mediated in part by cell-cell cross-talk involving the neuropeptide PACAP.
Spinal cord injury; Human mesenchymal stem/stromal cells (hMSCs); Pituitary adenylate cyclase-activating polypeptide (PACAP); Macrophage; Microglia
Every year, more than 10,000 people in the United States
are victims of spinal cord injury (SCI) caused by traffic,
sports, and other trauma-related accidents. While
medication during the acute injury period involves the
administration of large amounts of steroids and other
anti-inflammatory drugs, the recovery of neurological
function relies on neural plasticity and compensatory
mechanisms specific to each patient. Many of these
patients end up being permanently paralyzed [1,2].
Observations made on animal models suggest that a
potential therapy for disorders of the central nervous
system (CNS) is the administration of adult
mesenchymal stem/stromal cells (MSCs) from bone marrow [3-6].
While human MSCs (hMSCs) initially attracted interest
for their ability to differentiate into multiple cellular
phenotypes in vitro and in vivo, part of the interest in
them is due to their anti-inflammatory and
immunosuppressive properties [4,7-10]. MSCs also cross-talk with
host tissues to enhance protective factors and the
microenvironment, inducing the release of a range of cytokines
and growth factors from those tissues [4,9]. We reported
that hMSCs injected into the mouse hippocampus after
ischemia improved the neurological symptom, and that
hMSCs increased the recipient microglia/macrophage
(M) to alternatively activated M2 type (AAM) ,
which are known to promote repair and regeneration
after injury . We also determined by microarray
analysis the implanted hMSC-suppressed
interferonrelated genes. Recently, we reanalyzed the microarray data
and found that the expression of the gene for mouse
pituitary adenylate cyclase-activating polypeptide (PACAP;
gene: Adcyap1) increased approximately ten fold in
animals receiving hMSCs after ischemia compared with
animals receiving the vehicle after ischemia, or hMSCs
after sham-operation (see Additional file 1: Figure S1).
However, no evidence has thus far been provided to
demonstrate the involvement of neuropeptides in the
neuroprotective properties exerted by hMSCs.
PACAP, which was first isolated from the ovine
hypothalamus, belongs to the secretin/glucagon/vasoactive
intestinal peptide (VIP) superfamily. PACAP exerts
multiple functions through three specific receptors - PACAP
receptor 1 (PAC1R) and two VIP/PACAP receptors [13,14].
Exogenous and endogenous PACAP decreases neuronal
cell death after ischemia, SCI, and other neuronal disorders
[15-20]. PACAP also contributes to suppress inflammatory/
immune responses. Adcyap1-deficient mice exhibited
increased deterioration in an experimental model for
multiple sclerosis . Severe combined immunodeficiency
(SCID)-type immune-deficient mice showed decreased
Adcyap1 expression after facial nerve-crush.
Adcyap1deficient mice also exhibited increased levels of
proinflammatory cytokines such as IL-6, tumor necrosis factor
(TNF), and interferon- (IFN) and decreased levels of
interleukin-4 (IL-4) . Although a synergistic protective
effect in response to co-treatment with hMSCs and PACAP
after SCI has been reported , no evidence has been
shown that hMSCs regulate the expression of PACAP.
We hypothesized here that the anti-inflammatory effect
of hMSCs in response to CNS damage could involve
Adcyap1 regulation. We transplanted hMSCs into the
spinal cord after a SCI in wild-type (WT) and Adcyap1+/
mice and compared the determined neurological
symptoms and mouse Adcyap1 and Adcyap1r1 (PAC1R gene)
expression after SCI. Moreover, the effects of mouse- and
human-specific cytokine gene expression to determine
mechanisms underlying the anti-inflammatory action of
hMSCs were also examined.
Wild-type C57BL/6 mice were purchased from Sankyo
Lab Service Corporation (Tokyo, Japan). Adcyap1+/ mice
on a C57BL/6 background were originally provided by
Dr. Hashimoto of Osaka University . All mice were
housed in the specific pathogen-free animal facility at
Showa University and had free access to food and water.
In all experiments, adult male mice (8 to 12 weeks old,
weighing 17 to 25 g) were used. All experimental
procedures involving animals were approved by the Institutional
Animal Care and Use Committee of Showa University
(#00168 and 01150).
The SCI mouse model was produced according to our
previous report . Anesthesia was induced in mice by
inhalation of 4.0% sevoflurane and maintained with 3.0%
sevoflurane. Under aseptic conditions, an incision was
made along the midline of skin of the back, and the
muscles, soft tissues, and yellow ligaments overlying the
spinal column between T9 and T10 were removed. The
intervertebral spinal cord between T9 and T10 was then
transected with a thin-bladed razor (FEATHER, Osaka,
Japan). After bleeding had stopped and coagulated blood
was removed, the incision was closed and the animals
were given 1.0-mL lactate Ringers solution (s.c., Otsuka,
Tokyo, Japan) to avoid dehydration. Following recovery,
foods were placed on the cage floor and the intake of
the water bottle was lowered to allow for easy access. All
mice were allowed to recover in a room maintained at
24C 1C during the experimental period. To support
urination, the region of the lower abdomen in all mice
was gently stimulated a few times a day.
Preparation of implanted hMSCs
Frozen vials of hMSCs from bone marrow were obtained
from Dr. Prockop (The Center for the Preparation and
Distribution of Adult Stem Cells (http://medicine.tamhsc.
edu/irm/msc-distribution.html)) under the auspices of a
National Institutes of Health (NIH)/National Center for
Research Resources grant (P40 RR 17 447-06). The
experiments were performed with hMSCs from donor 281L
[11,25]. To expand hMSCs, a frozen vial of 1.0 106
passage 3 cells was thawed and plated at 100 cells/cm2 in
multiple 150-mm plates (Nunclon, Thermo Fisher
Scientific, Rochester, NY) with a 20-mL complete culture
medium (CCM) that consisted of -minimal essential
medium (-MEM; Invitrogen, Grand Island, NY), 20%
heat-inactivated fetal bovine serum (FBS, Hyclone;
Thermo Fisher Scientific), 100 units/mL penicillin, 100
g/mL streptomycin (Invitrogen), and 2 mM L-glutamine
(Invitrogen). The cultures were incubated, and the
medium replaced every 3 days for approximately 8 days
until cells were 70% to 80% confluent. The medium was
then discarded, and the cultures plates were washed with
PBS. Adherent cells were harvested with 0.25% trypsin
and 1 mM EDTA (Invitrogen) for 5 min at 37C and were
resuspended at 5 105 cells in 0.5 L of sterile Hanks
balanced salt solution (HBSS; Invitrogen) for injection.
Unviable hMSCs were prepared by repeated freezing and
thawing (three times) of aliquots of these cells.
PKH26-labeled hMSCs were prepared according to
instructions provided with the PKH26 Red Fluorescent
Cell Linker Kit (Sigma-Aldrich, St Louis, MO). In brief,
harvested hMSCs (approximately 1 107 cells) were
washed with -MEM and centrifuged at 1,500 rpm for 7
min. The cells were then suspended for 3 min at 25C in
1.0 mL of Diluent C with 1.0 mL of a PKH26 solution
diluted 250-fold in Diluent C. Two mL of FBS was
added to the suspension and incubated for 1 min at
room temperature. A further 4.0 mL of CCM was added,
and the suspension was centrifuged at 1,500 rpm for 6
min. After discarding the supernatant, the cells were
washed three times with CCM and resuspended finally
with HBSS at 5 105 cells/mL. In a preliminary study, we
confirmed that the cell suspension showed greater red
fluorescence (Ex 544, Em580-10) than naive hMSCs or
HBSS (Additional file 2: Figure S2). The red fluorescence
of the hMSCs was also confirmed with fluorescence
immunocytochemistry with CD59 (BD Bioscience) or HuNu
(Chemicon) antibodies (Additional file 2: Figure S2).
Injection of hMSCs into spinal cord
The day following surgery to invoke SCI, mice were
reanesthetized by inhalation of 4.0% sevoflurane. The animals
were placed face-down and a 29G-needle (HAMILTON,
Reno, NV) with a 5.0-L glass syringe (HAMILTON) was
inserted directly into the intervertebral spinal cord
between T10 and T11. hMSCs (5 105 cells/L) or HBSS
were infused at a rate of 0.5 L/min with an Ultra Micro
Pump (World Precision Instruments, Sarasota, FL). After
infusion, the needle was left in place for 1 min to enable
the solution to diffuse into the tissue. We have shown
that the fate of hMSCs was not different between
immunocompetent and immunodeficient animals after
ischemia . Therefore, the present study did not use
any immunosuppressant after cell implantation.
Assessment of locomotor function
Motor function after SCI was assessed by using an
openfield behavior test that focused on hindlimb function
according to the Basso Mouse Scale (BMS) . The BMS
consists of an open-field locomotor rating scale, ranging
from 0 (complete paralysis) to 9 (normal mobility). Briefly,
individual mice were placed in the center of the open field
(e.g., 50 50 cm ) with a smooth, non-slip floor and
monitored for 4 min. The hindlimb movements, trunk/
tail stability, and forelimb-hindlimb coordination were
assessed and graded. Mice were tested daily until
postoperative (p.o.) day 7. Mice with peritoneal infection,
hindlimb wounds, and/or tail or foot autophagia were
excluded from the study. Scoring was done by randomly
numbering the mice to ensure that the investigators were
not aware of the treatment groups.
Measurement of injury volume
After anesthesia with sodium pentobarbital (50 mg/kg,
i.p.), the animals were perfused transcardially on p.o.
day 7 with 0.9% saline followed by 4% paraformaldehyde
(PFA) in 50 mM phosphate buffer (pH 7.2) and the spinal
cord removed (T7 to L1 vertebrae). Spinal cords were
then post-fixed with 20% sucrose in 0.1 M phosphate
buffer (pH 7.2) for two nights, and then embedded in
an O.C.T. compound (Sakura Finetech, Tokyo, Japan)
for subsequent preparation of frozen blocks. Five spinal
cord sections (5-m thickness) were obtained from
each mouse: at the midline which included the central
canal nearby to the core-injury site, and bilaterally at
30 m and 60 m lateral to the midline (total five sections
from each mouse). The damaged area can be identified by
glial fibrillary acidic protein (GFAP) immunostaining of
the surrounding area, which is considered to be indicative
of glial scarring . The frozen sections were washed
with phosphate-buffered saline (PBS) and incubated in
0.3% H2O2. The sections were blocked with 2.5% normal
horse serum (NHS) in PBS for 1 h at room temperature.
Subsequently, the sections were incubated overnight
with rabbit anti-GFAP antibody (1:10, DAKO, Glostrup,
Denmark). The sections were washed with PBS and
immersed with goat anti-rabbit IgG (1:200, Santa Cruz,
Santa Cruz, CA) for 2 h. They were then incubated in an
avidin-biotin complex solution (Vector, Burlingame, CA)
followed by diaminobenzidine (DAB; Vector) as a
chromogen. Control staining involved carrying out the same steps
without the incubation with the primary antibody. The
injury area consisting of GFAP-immunopositive cells was
measured by DP2-BSW image analysis software (Olympus,
Tokyo, Japan), and the estimated injury volume was
calculated by integration of the injured areas.
Human Alu (hAlu) real-time PCR assays
Immediately following, and then at 7 and 14 days after
injection of hMSCs, mice were anesthetized with sodium
pentobarbital (50 mg/kg, i.p.) and the spinal cord was
dissected. The tissue was snap frozen in liquid nitrogen
and stored at 80C until use. Genomic DNA was extracted
(n = 4; DNeasy, Qiagen, Valencia, CA) and total DNA was
assayed by UV absorbance. Real-time PCR was performed
with 100 ng of target DNA, hAlu-specific primers, and a
fluorescent probe (Model 7700; Applied Biosystems, Foster
City, CA). The primers were as follows: Alu forward,
5-CAT GGT GAA ACC CCG TCT CTA-3; Alu reverse,
5-GCC TCA GCC TCC CGA GTA G-3; Probe,
5-FAMATT AGC CGG GCG TGG TGG CG-TAMRA-3.
Standard curves were prepared by adding 1 102 to 1 106
hMSCs to samples of spinal cord from uninjured mice.
Spinal cords (T7 to L1 segment) from immediately after the
injection of hMSCs and on p.o. day 7 were obtained and
frozen sections prepared as described above. Microscope
slides containing attached hMSCs in culture were washed
three times with HBSS, fixed with 4% PFA for 15 min, and
immersed in PBS containing 0.1% Tween 20 (PBST).
Tissue sections or microscope slides were washed
several times with PBST and incubated in 2.5% NHS/PBST
for 1 h. Subsequently, the sections were incubated
overnight with primary antibodies. The sections were then
rinsed with PBST and immersed with appropriate
fluorescently labeled secondary antibodies for 2 h. Control
staining involved carrying out the same procedures but
without the incubation with primary antibodies. The
primary antibodies were used as follows: rabbit
anti-2microglobulin (B2M, 1:1000; LifeSpan Biosciences, Seattle,
WA), rabbit anti-PACAP (1:1000; Peninsula Laboratories,
San Carlos, CA), and rabbit anti-type1 PACAP receptor
(PAC1R, 1:400). The rabbit anti-PAC1R antibody was
raised by using the N-terminal residue as an antigen
[15,27]. The secondary antibody used was goat anti-rabbit
Alexa 488 (1:400; Invitrogen). Some sections were stained
with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI,
1:10,000; Roche, Manheim, Germany) to identify cell nuclei.
Fluorescence was detected using an Axio Imager optical
sectioning microscope with ApoTome (Carl Zeiss, Inc.;
Isolation of RNA
Immediately after injection of hMSCs, or on p.o. days 3
or 7, mice were anesthetized with sodium pentobarbital
(50 mg/kg, i.p.) and the spinal cord was dissected (T7 to
L1 vertebrae). The excised tissue was snap frozen in
liquid nitrogen and stored at 80C until use. The total
RNA was isolated from the cultured hMSCs or the spinal
cord samples using the TRIZOL Reagent (Invitrogen,
Carlsbad, CA) according to the manufacturers
instructions. In brief, cultured hMSCs (1 106 cells) or spinal
cord tissue samples (40 mg) in 1.0 mL of TRIZOL Reagent
were homogenized using a Dounce tissue grinder
(WHEATON, Millville, NJ). Added to the homogenized samples
was 0.2 mL of chloroform per 1 mL of TRIZOL Reagent.
Following centrifugation, the aqueous phase (containing
RNA) was separated from the mixture. Added to this
aqueous phase was 0.5 mL of isopropyl alcohol per 1 mL of
TRIZOL Reagent. After centrifugation, RNA precipitate
was formed on the bottom of sample tube. The RNA
precipitate was washed with 75% ethanol and dried
completely at room temperature. The RNA was dissolved in
RNase-free water. The purity and concentration of
extracted RNA were determined spectrophotometrically
(NanoDrop, Wilmington, DE). Extracted RNA was stored
at 80C until use.
cDNA was then synthesized with a TaKaRa PrimeScript
RT reagent Kit (TaKaRa BIO Inc., Shiga, Japan), using
3 g of the total RNA. The synthesized cDNA was
made up to a volume of 30 L with sterile distilled water.
Real-time PCR was performed as previously reported
by our group , with minor modifications. Reverse
transcription PCR (RT-PCR) for tumor necrosis factor
-induced protein 6 (TSG-6) was performed on a
Taqman system , while for the others, SYBR Green was
used. All human- or mouse-specific primers and probes
were designed as described in Table 1.
Assay for arginase activity
Arginase is a marker for AAM, and its activity was
measured according to our previous report . Briefly,
spinal cord sections containing the T5 and L1 vertebrae
from p.o. day 7 animals were removed. The tissues were
homogenized with a lysis buffer (10 mM Tris-HCl (pH
7.4), 0.15 M NaCl and 1% Triton X-100, 1 mM ethylene
glycol tetraacetic acid (EGTA), 50 mM NaF, 2 mM
sodium orthovanadate, 10 mM sodium pyruvate, and
protease inhibitor cocktail (Sigma-Aldrich)) and
centrifuged at 800 g for 10 min at 4C, and the supernatant
was collected. Protein concentration in the samples was
determined using the BCA protein assay kit (Thermo
The homogenate was mixed with an equal volume of
pre-warmed 50 mM Tris-HCl, pH 7.5 containing 10 mM
MnCl2 and incubated for 15 min at 55C for activation.
The mixture was then incubated in 0.25 M L-arginine for
Table 1 Primers and probe to use for real-time PCR
Official symbol Species Forward (5 to 3)
Adcyap1 mouse AACCCGCTGCAAGACTTCTATGAC
GTGAGGTAAGCAAGCTCCAACAGAC CTCGATCTGATTGCTGGGTGAA Cyber green (TaKaRa) AAGCAGGGTCTGGCAAATACAAGC
ATCCATCCAGCAGCACAGACATGA Taqman (Japan Bio Service) probe:FAM-TTTGAAGGCGGCCATCTCGCAACTT-TAMRA
BDNF brain-derived neurotrophic factor, IL interleikin, NGF nerve growth factor, NT-3 neurotrophin 3, PACAP pituitary adenylate cyclase-activating polypeptide,
PAC1R pituitary adenylate cyclase-activating polypeptide specific receptor, RPLP1 large ribosomal protein P1, TGF transforming growth factor , TNF tumor
necrosis factor , TSG-6 tumor necrosis factor-induced protein 6.
60 min at 37C to hydrolyze urea from L-arginine, and the
reactions were stopped by adding Stop solution (H2SO4/
H3PO4/H2O, 1:3:7). A 1% (final concentration) solution
1phenyl-1,2-propanedione-2-oxime (ISPF, Wako, Tokyo,
Japan) in ethanol was then added to the solution, which
was heated at 100C for 45 min. The reaction between
urea and ISPF produced a pink color, and absorption
was measured at 540 nm. Data are presented as specific
activity (nmol/min/mg of protein).
Stimulation of hMSCs with IFN
hMSCs were plated at 2 105 cells/well in 6-well plates.
The next day, the cells were washed twice with PBS ()
and incubated in an experimental medium (-MEM
supplemented with 1% FBS, 100 units/mL penicillin,
100 g/mL streptomycin, and 2 mM L-glutamine). Then
the cells (n = 3 plates for each phenotype) were exposed to
a recombinant mouse IFN (10 ng/mL, PeproTech, Rocky
Hill, NJ) or vehicle. Forty-eight hours later, the cells were
collected and stored at 30C until analysis.
Stimulation of M-differentiated U937 with LPS and RNA
Human monocyte-like cell line U937 was obtained from
the RIKEN Cell Bank (Tsukuba, Japan). For routine
maintenance, cells were cultured in RPMI 1640 medium
(Invitrogen) supplemented with 10% FBS and 1.0%
penicillin/streptomycin in 5% CO2 at 37C in a humidified
chamber. Cell concentrations were maintained between
2 105 and 2 106 cells/mL. For differentiation into
M, 5 105 cells were placed into the wells of a 12-well
plate and treated with 20 nM phorbol myristate acetate
(PMA, Sigma-Aldrich) for 24 h to induce the M-like
adherent phenotype. Subsequently, the medium was
replaced with fresh RPMI 1640 medium and cells
cultured for a further 48 h. PMA-induced U937 cells were
stimulated with 0.1 g/mL of lipopolysaccharide (LPS,
Sigma-Aldrich) for 24 hours as a positive control.
RT-PCR for human PACAP and PAC1R
RT-PCR was performed as reported previously .
Briefly, the total RNA was extracted from cell pellets
(hMSCs, hMSCs stimulated with IFN, M-like
differentiated U937, M-like differentiated U937 stimulated
with LPS) by TRIZOL reagent (Invitrogen) according to the
manufacturers instructions. The purity and concentration
of extracted RNA were determined spectrophotometrically
(NanoDrop, Wilmington, DE). The cDNA was then
synthesized with an AffinifyScript QPCR cDNA Synthesis Kit
(Stratagene, Agilent Technologies, La Jolla, CA), using 1 g
of the total RNA. The synthesized cDNA was made up to a
volume of 50 L with sterile water supplied with the kit.
The reaction mixture contained 0.6 L of the first-strand
cDNA, 7 pmols of each primer set and 6.0 L of the
Emerald Amp PCR Master Mix (2X premix) (TaKaRa)
in a total volume of 12 L. The primers were as follows:
hPACAP forward, 5-GAAACAAATGGCTGTCAAGA
AA-3; hPACAP reverse, 5- TCTGTGCATTCTCTA
GTGCTTTG-3; hPACAPR1forward, 5-GTTACTTCG
CTGTGGACTTCAA-3; hPACAPR1 reverse, 5-GGA
CCAGTACCAAAACAAGGAG-3; hGAPDH forward,
Thermal-cycling parameters were set as follows: 97C
for 5 min for an initial denaturation, then a cycling
regime of 40 cycles at 95C for 45 s, 60C for 45 s, and
72C for 1 min. At the end of the final cycle, an
additional extension step was carried out for 10 min at
72C. Three microliters of each reaction mixture were
loaded for 1.6% agarose gel electrophoresis and bands
were visualized with ethidium bromide.
Each mouse was assigned a random number, and all data
were collected and analyzed without investigator
knowledge of group identities. Data are expressed as mean
SEM for in vivo experiments and as mean SD for
in vitro experiments. Statistical comparisons were made
by Students t-test for two groups and by one-way
ANOVA following non-parametric multiple comparison
as indicated in each figure legend. A value of P < 0.05
was considered to indicate statistical significance.
Injection of hMSCs improved locomotor activity and
suppressed SCI-related damage
We first determined locomotor activity and lesion size
after injecting hMSCs into the spinal cord. This
intervention induced severe locomotor deficits (BMS of
around 1.5 points; see the Methods section)  within
a few hours. On p.o. day 1, hMSCs (5 105 cells/0.5 L)
were injected into the intervertebral spinal cord one
vertebra rostral to the injury site in WT or Adcyap1+/
mice. Another set of mice were injected with repeat
freeze-thawed unviable hMSCs. WT mice implanted
with viable hMSCs (hMSC/WT mice) exhibited
significantly improved BMS on p.o. days 3 and 7 compared with
vehicle-treated WT one (HBSS/WT mice). However, WT
mice implanted with unviable hMSCs (unviable hMSC/
WT mice) and Adcyap1+/ mice implanted with viable
cells (hMSC/Adcyap1+/ mice) had significantly lower
BMS scores than the hMSC/WT mice (Figure 1A).
We next compared lesion areas on p.o. day 7. hMSC/
WT mice exhibited significantly reduced lesion area
compared with HBSS/WT mice, whereas unviable hMSC/WT
Fate of hMSCs in the spinal cord
We next observed the fate of hMSCs with human hAlu
quantitative real-time PCR and estimated number of
hMSCs after implantation. Within 10 min of injection of
hMSCs, 326,058 62,153 hMSCs cells (>65%) were
counted from the injection site, 424 173 cells were
measured from the rostral site, and 12,322 9,126 cells
from the caudal site (Figure 2A). The number of hMSCs
decreased rapidly to 3,879 2,704 and 250 169 (1/100
and 1/10,000 immediately after implantation) on p.o. day
7 and 14, respectively. The estimate numbers of hMSCs
in the spinal cord from the non-injured WT and injured
Adcyap1+/ mice on p.o. day 7 after hMSCs treatment
were not different from that in the injured WT mice
Migration of hMSCs toward the injection site was
confirmed by PKH26-fluorescence labeling. On the day of cell
injection (p.o. day 1), red fluorescence was clustered
around the injective site and merged with human B2M
immunoreactivity, but was not observed at the injury site.
The fluorescence migrated toward the injury site and
merged with the B2M immunoreactivity in both of the
injection and peri-injury sites by p.o. day 7 (Figure 2C, D).
Adcyap1 and Adcyap1r1 expression were induced by hMSCs
We reported previously that Adcyap1+/ mice increased
lesion size to compare with the WT mice after brain
ischemia  and contusion-induced SCI .
Therefore, failure of hMSCs to induce tissue protection in
Adcyap1+/ mice could have been due to the deletion of
Adcyap1. For this reason, we next examined mouse
Adcyap1 and Adcyap1r1 induction after the injection of
hMSCs (Figure 3A, B). In HBSS/WT, Adcyap1 expression
in the spinal cord was temporarily increased on p.o. day 1
and then decreased. Adcyap1 expression in hMSC/WT
mice was significantly greater than that in HBSS/WT and
hMSC/Adcyap1+/ mice. Adcyap1r1 expression levels in
the spinal cord did not differ among any of the
experimental groups. PACAP and PAC1R immunoreactivities
were mainly observed in neuron-like cells (Figure 3C).
Alteration of mouse cytokine profiles in spinal cords
injected with hMSCs
Several factors have been reported concerning hMSCs
capacity to decrease inflammation via M; hMSCs
increase AAM via the induction of IL-4 , they
suppress classical activating M (CAM) which are induced
by IFN [25,29], and they increase deactivating M
(DAM) which are induced by interleukin-10 (IL-10) .
We then assayed expression levels of mouse
inflammatory cytokines in the spinal cord. The mouse interleukin-1
Figure 1 Injection of viable hMSCs suppresses SCI symptoms in wild-type (WT) mice. (A) Motor function determined by BMS improved
significantly after injection of viable hMSCs (filled circles, n = 67) compared with that of HBSS (open circles with solid line, n = 47) in WT mice. This
improvement was diminished when unviable hMSCs were injected into WT mice (open circles with dashed line, n = 18) or when viable hMSCs
were injected into Adcyap1+/ mice (filled triangles, n = 17). Data are expressed as mean SEM. **P < 0.01 (Tukey post hoc t-test). (B) Typical images of
lesion site after SCI. Injection of viable hMSCs (right upper) into WT mice decreased the lesion area compared with that in HBSS-treated control (left
upper). Unviable hMSCs injected into WT mice (left bottom) and viable hMSCs injected into Adcyap1+/ mice (right bottom) did not reduce the lesion
size. R: rostral, C: caudal, D: dorsal, V: ventral. Scale bar is 500 m. (C) Quantification of GFAP-unstained injury volume at 7 days. Data are expressed as
mean SEM. *P < 0.05, **P < 0.01 (Tukey post hoc t-test). BMS Basso Mouse Scale, HBSS Hanks balanced salt solution, MSC mesenchymal stem/stromal
cell, n.s. no significant, PA+/ Adcyap1 (PACAP gene) heterozygous mice, SCI spinal cord injury, wt wild-type.
Figure 2 Distribution of implanted hMSCs in the spinal cord and distribution of PKH26-labeled hMSCs after hMSC injection, with higher
magnification images. Distribution of implanted hMSCs in the spinal cord (A, B). (A) Real-time PCR assays for hAlu after injection of hMSCs (5 105
cells) into spinal cord (n = 4). Data are expressed as mean SEM. **P < 0.001 (Dunnet post hoc t-test vs injection site). (B) Temporal profile of hMSC
survival after injection in animals with or without SCI. The survival of hMSCs in wild-type (WT) mice with SCI decreased drastically with time. Data are
expressed as mean SEM. **P < 0.01 (Dunnet post hoc t-test). (C) Distribution of PKH26-labeled hMSCs (red) after hMSC injection. Within 10 min of
injection (day 1), the cells were observed as a cluster in the injection site (injection). At 7 days after SCI, the cells are seen to have migrated along the
spinal cord toward the injury site (injury) and detected the PKH26 signals in peri-injury site (arrowhead). Blue: DAPI counterstaining. R: rostral, C: caudal,
D: dorsal, V: ventral. (D) Higher magnification images of (C) with human B2M staining (green) shows co-labeling with PKH26-stained cells. One day
after SCI and immediately after hMSC injection, no PKH26 signals can be observed in the peri-injury site. However, PKH26-red signals merged with
B2M-positive reactions (green) can be observed on the peri-injury site on post-operative day 7. B2M 2-microglobulin, DAPI 4,6-Diamidine-2-phenylindole
dihydrochloride, hMSC human MSC, n.s. no significant, PA+/ Adcyap1 (PACAP gene) heterozygous mice, SCI spinal cord injury, wt wild-type.
Figure 3 Implanted hMSCs induced increased mouse Adcyap1 expression after SCI. Mouse Adcyap1 (A) and Adcyap1r1 (B) expression levels
in spinal cord after injury and in the presence and absence of injected hMSCs. (A) Although Adcyap1 expression in HBSS-treated animals decreases
after SCI, in hMSC-treated animals it increases significantly. Gene expression levels are similar between HBSS-treated WT mice and hMSC-treated
Adcyap1+/ mice at 7 days. (B) Adcyap1r1 expression is similar for all experimental groups. Rplp1 is the large ribosomal protein P1 as a house keeping
gene. Data are expressed as mean SEM. **P < 0.01 (Tukey post hoc test). (C) Multiple-immunostaining of PACAP or PAC1R (green) and nuclei (blue;
DAPI). DAPI 4,6-Diamidine-2-phenylindole dihydrochloride, HBSS Hanks balanced salt solution, MSC mesenchymal stem/stromal cell, PA+/ Adcyap1
(PACAP gene) heterozygous mice, PACAP pituitary adenylate cyclase-activating polypeptide, PAC1R pituitary adenylate cyclase-activating polypeptide
specific receptor, wt wild-type.
(IL-1) gene (Il1b) and TNF gene (Tnf ) in HBSS/WT
mice showed greater expression after SCI. hMSC/WT mice
suppressed significantly the expression levels. However, in
hMSCs/Adcyap1+/ mice, the level of Il1b expressed was
similar to that in the HBSS/WT. (Figure 4A, B). Expression
levels for the IL-10 (Il10) and TGF1 (Tgfb1) genes
increased in the HBSS/WT mice after SCI and decreased
significantly in the hMSC/WT mice. hMSC/Adcyap1+/
mice exhibited a similar level of expression of Tgfb1 as that
seen in HBSS/WT, while Il10 expression was not modified
(Figure 4C, D). Low expression levels of the IL-4 gene (Il4)
were seen in HBSS/WT on p.o. day 7; in contrast,
drastically increased levels were seen in the hMSC/WT mice,
while no expression was seen in the hMSC/Adcyap1+/
mice (Figure 4E). We then assayed for arginase activity, an
AAM marker, in WT mice with or without injected hMSCs
and demonstrated that the activity was significantly
increased in the hMSC/WT mice compared with the HBSS/
WT mice (Figure 4F).
Genetic profile of hMSCs in the spinal cord
PACAP has been reported to induce the gene expression
of some growth factors and Th2-type cytokines. We next
determined selected human gene expression levels
according to previous studies [22,31,32].
Gene expression levels of growth factors such as the
nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF), and neurotrophin 3 (NTF3) were
temporarily but significantly increased in the hMSC/WT
mice compared with cultured hMSCs on p.o. days 3 or 7
(Figure 5A, B, C). The expression of genes for Th2-type
cytokines such as IL4, IL10, and TGFB1 was also
significantly increased on p.o. day 7 (Figure 5E, F, G).
Moreover, we assayed TSG-6 (TNFAIP6) as well. TSG-6 from
hMSCs has been reported to decrease inflammatory
responses in peritoneal and traumatic brain injury [29,33]
and was found here to be significantly increased in the
spinal cord of hMSC/WT on p.o. day 7 compared to that
in HBSS/WT (Figure 5D).
Figure 4 Implantation of hMSCs reduced mouse pro-inflammatory gene profiles and favored the M2-type alternative activating
microglial/M (AAM) environment. Mouse cytokine gene expressions were determined in spinal cords after injury. hMSC-treated wild-type
(wild) mice (black) manifested suppressed levels of mouse proinflammatory cytokines gene expression such as IL-1 (Il1b; (A)) and TNF (Tnf; (B))
compared with HBSS-treated mice (white). The cell treatment also decreased IL-10 (Il10; (C)) and TGF (Tgfb1; (D)) levels. However, hMSC-treated
wild-type mice exhibited increased levels of gene expression for anti-inflammatory cytokine such as IL-4 (Il4; (E)). For Il1b, Tnf, Tgfb1, and Il4, these
were diminished in hMSC-treated Adcyap1+/ (PA+/) mice (gray) 7 days after SCI. Cont means intact spinal cord in wild-type mice. Data show
mean SE (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 (Tukey post hoc test). Injection of hMSCs significantly increased arginase activity 7 days after
SCI (F). Data show mean SE (n = 7). *P < 0.05 (Students t-test). HBSS Hanks balanced salt solution, MSC mesenchymal stem/stromal cell, n.s. no
significant, PA+/ Adcyap1 (PACAP gene) heterozygous mice.
In the hMSCs/Adcyap1+/ mice, while gene expressions
for growth factors and IL10 tended to decrease in
Adcyap1+/ mice, no significant differences were found to
compare with the WT mice (Figure 5A, B, C, F). TNFAIP6
was not altered in the Adcyap1+/ mice (Figure 5D);
however, the IL4 and TGFB1 expressions were significantly
down-regulated (Figure 5E, G).
The possibility exists that PACAP acts directly on
hMSCs, most likely via PAC1R. Although we initially
assayed the ADCYAP1 and ADCYAP1R1 gene expressions
in the spinal cord of hMSC/WT, these were not detected
(data not shown). Expression of these genes was then
examined in vitro on hMSC cultures where we added
10 ng/mL IFN (or vehicle alone for control) to mimic
inflammatory conditions (Figure 5H). IFN-treated hMSCs
exhibited increased ADCYAP1 and ADCYAP1R1 at 24 h,
whereas the vehicle alone did not express the genes.
We demonstrated here that when hMSCs were injected
on p.o. day 1 into the spinal cord of WT mice, subsequent
improvements were seen in various parameters which
suggested to reduce SCI, but inviable hMSCs did not exert
these effects. We observed also that these findings could
not be reproduced in Adcyap1+/ mice. Because the
retention of hMSCs was no different between the hMSC/WT
and hMSC/Adcyap1+/ mice, we considered that the
beneficial effect of hMSCs was due to cross-talk between
the hMSCs and recipient tissues involving the action of
PACAP. PACAP is a well-documented neuropeptide that
suppresses cell death in ischemia, SCI, and other CNS
disorders [15-17,19-21]. We previously demonstrated the
exacerbation of cell death in Adcyap1+/ mice to compare
with a WT mouse after ischemia and SCI [15,19]. In the
present study, we however could not see significant
difference in the neural damage in the HBSS-injected animals
both for the WT and Adcyap1+/ mice. Therefore, it was
considered that it may be only competing between an
increase of the cell death in Adcyap1+/ and suppression
of the cell death by hMSCs. Then, we examined the
expression of recipient mouse Adcyap1 and Adcyap1r1 in
the spinal cord after implanted hMSCs and demonstrated
clearly that hMSC transplantation exhibited an increase of
mouse Adcyap1. These findings strongly suggest that
hMSCs may contribute to neuroprotection with PACAP
To determine how hMSCs induced Adcyap1
expression, we studied recipient mouse and donor hMSC gene
expressions. So far, it is reported that PACAP has been
induced by cyclic AMP, amyloid -protein, CREB,
progesterone, growth factors such as BDNF and NGF, and
PACAP itself in vitro and in vivo experiments [31,32,34].
The expression of Adcyap1 might be also influenced by
immune/inflammatory stimuli, in particular IL-4-related
Figure 5 Human gene profiles after injection of hMSCs into injured spinal cord. Real-time RT-PCR assays with human-specific primers of naive
hMSCs and injured spinal cord after injection of hMSCs into wild-type (wild) and Adcyap1+/ (PA+/) mice. Human BDNF (BDNF; (B)) and NT3 (NTF3;
(C)) increased 3 days after SCI. Human NGF (NGF; (A)), TSG-6 (TNFAIP6; (D)), IL-4 (IL4; (E)), IL-10 (IL10; (F)), and TGF (TGFB1; (G)) increased 7 days after
SCI. Injection of hMSCs into Adcyap1+/ mice with SCI resulted in a decrease of human IL4 and TGFB1 expression. Naive means aliquot of cultured
hMSCs before transplantation. Data are expressed as mean SE (n = 8). *P < 0.05, **P < 0.01 (Tukey post hoc test). Human ADCYAP1 and ADCYAP1R1
expression in hMSCs stimulated with IFN (in vitro) (H). hMSCs exposed to IFN for 48 h showed increased ADCYAP1 and ADCYAP1R1 expression.
M-like differentiated U937 was used as a positive control for the gene expression. BDNF brain-derived neurotrophic factor, B2M 2-microglobulin,
NGF nerve growth factor, n.s. no significant, PA+/ Adcyap1 (PACAP gene) heterozygous mice.
stimuli, given the decreased expression seen in SCID
mice after nerve injury . In the present study, we
observed that the hMSCs after transplantation increased
growth factors such as NGF, BDNF, and NTF3. These
have been suggested to increase Adcyap1 expression
[31,32,34] and are released from hMSCs after implantation
. hMSCs increased an expression of anti-inflammatory
cytokines such as IL4 and IL10, and TGFB1 as well. Indeed,
mouse Il4 and an AAM marker, arginase activity, were
greater in the hMSC/WT of the spinal cord. It also suggests
that microenvironment PACAP expressions are produced
in the spinal cord after hMSCs implantation.
We next investigated how hMSCs suppressed SCI and
how PACAP associated the effect. Several studies including
ours suggested that hMSCs modulated the inflammatory
response in recipient tissues, in particular that of
microglia/M activation. Microglia/M show different types of
activation - CAM, AAM, and DAM - depending on the
cytokine stimuli involved [12,24]. After hMSCs
implantation, a mouse proinflammatory cytokine gene such as Il1b
and Tnf is significantly suppressed, suggesting that hMSCs
modulated CAM in the spinal cord. We have reported that
a hMSC-mixed culture with mouse microglial cells under
IFN stimuli decreased the level of nitric oxide in a
hMSCnumber-dependent fashion . We reported also that
hMSCs increased the expression of TNFAIP6 in the
present study and the traumatic brain injury model .
TNFAIP6 (also known as TSG-6) is a candidate factor to
be involved in hMSCs anti-inflammation. TNFAIP6
increased from hMSCs after the implantation in the cardiac
infarction, global ischemia, and peritoneal inflammation
[11,29,36] and suppressed TNF [29,36,37]. Conversely,
hMSC implantation increased both human and mouse
IL4 gene expression and arginase activity in the recipient
tissue, suggesting that hMSCs increased AAM [12,38,39]
which consisted with global ischemia . It has been
reported that hMSCs increased CAM mediated by IL-10
and transforming growth factor (TGF)  in a
hippocampal organotypic culture  and sepsis mouse .
However, our observation showed a decrease in the
Il10 and Tgfb1 expression, probably due to a decrease of
proinflammatory cytokine. Like these, we suggest that
hMSCs decreased CAM and inflammation and induced
a resolution by AAM. Our results interestingly suggested
that hMSCs modulated mouse cytokine profile at least
via two different pathways: PACAP-dependent and
PACAP-independent pathways. Il1b, Il4, and Tgfb1, and
part of Tnf, were abolished in Adcyap1+/ mice,
suggesting PACAP worked as a mediator between recipient
tissue and donor hMSCs. On the other hand, Il10 and
most of Tnf were independent with endogenous PACAP.
We reported previously that Il4 and AAM decreased in
Il1a- and Il1b-deficient mice after SCI although the
injury area was suppressed in the deficient mice. Like
these, the cytokines form a complicated network during
the disease .
We hypothesized first that PACAP acts downstream of
hMSCs and that it does not influence the human gene
profile. However, our results indicated that the
endogenous mouse PACAP might modulate the hMSCs function
because hMSC/Adcyap1+/ mice influenced human gene
expression. For example, IL4 and TGFB1 were influenced
by PACAP, whereas TNFAIP6 and IL10 were not the same
as mice gene profiles. These indicated that recipient tissue
communicated between hMSCs and PACAP or a factor
mediated by PACAP. To the present time, no studies have
reported that hMSCs express PAC1R. We firstly examined
human ADCYAP1 or ADCYAP1R1 in the implanted
spinal cord. However, we failed to detect the gene
expression. Then, we examined the in vitro study and observed
slight increases of them after IFN stimulation in vitro.
This result suggests that hMSCs could express PAC1R in
response to inflammatory conditions, thus enabling hMSCs
to communicate with recipient tissues via autocrine and
paracrine processes partially mediated by PAC1R. The
contribution of human ADCYAP1 in vivo is still unclear
because we could not detect human ADCYAP1 in the
spinal cord. Using RNA interference or other techniques,
we need to clarify how much human PACAP contributed
to the communication. This synergistic cross-talk may
enhance anti-inflammatory processes and give rise to an
AAM environment. Further studies are needed to clarify
the central player(s) in this communication and the
complicated cytokine network.
A summarized putative schematic diagram of how hMSCs
suppressed the effects of SCI is given in Figure 6. (a)
Injected hMSCs migrate toward the peri-injury site. (b)
There, the hMSCs might be activated or stimulated by
inflammatory and/or injury stress-related factors including
INF. (c) Stimulated hMSCs produce and release human
anti-inflammatory factors such as TSG-6, IL-10, TGF,
and growth factors. (d) Simultaneously, hMSCs and
recipient tissues might co-produce IL-4 and induce an
AAM environment. (e) Together with reinforcing the
anti-inflammatory response, the environment and growth
Figure 6 Schematic illustration of the putative neuroprotective
mechanism underlying the effect of cross-talk between hMSCs and
PACAP in animals subjected to SCI (see the Discussions section)
AAM alternatively activated (activating) macrophage, GFs growth factors,
hMSCs human MSCs, IFN interferron-, IL interleikin, MG/M microglia
and/or macrophage, PACAP pituitary adenylate cyclase-activating
polypeptide, PAC1R pituitary adenylate cyclase-activating polypeptide
specific receptor, TGF transforming growth factor , TNF tumor necrosis
factor , TSG-6 tumor necrosis factor -induced protein 6.
factors from hMSCs induce the production of recipient
PACAP, which may then feed back to PAC1R on hMSCs
and could further enhance the hMSCs rescue signals in a
positive manner. (f ) Finally, by cross-talk processes, these
signals both in the donor hMSCs and in the recipient
induce neural rescue and repair at the injury site.
Additional file 1: Figure S1. Expression of mouse Adcyap1 in
hippocampus after transient global ischemia. C57/BL6 mice were
subjected to 15 min of common carotid artery occlusion and were
injected with hMSCs (1 105 cells) or vehicle (HBSS) into each dentate
gyrus the next day. One day after cell implantation, the hippocampi were
extracted and analyzed by a mouse microarray system. The same
procedures were performed in sham-operated animals not subjected to
ischemia. DNA microarray data were reanalyzed for our previous work.
Additional file 2: Figure S2. Validation of PKH-labeled hMSCs.
PKH26labeled hMSCs were characterized prior to injection. (A) PKH-labeled hMSCs
were stained with either anti-CD59 (Beckman) or anti-Human Nuclei
(Chemicon) antibodies to validate that the labeled cells were hMSCs.
The blue color represents DAPI staining of nuclei. (B) The fluorescence
intensity (Ex 544, Em580) of cells was also measured. While non-labeled
control cells (cont) did not show any change in signal intensity, labeled
cells clearly showed an increased fluorescence signal.
AAM: Alternatively activated (activating) macrophage; BDNF: Brain-derived
neurotrophic factor; BMS: Basso Mouse Scale; CAM: Classical activating
macrophage; CCM: Complete culture medium; CNS: Central nervous system;
DAM: Deactivating macrophage; DAPI: 4,6-Diamidine-2-phenylindole
dihydrochloride; GFAP: Glial fibrillary acidic protein; HBSS: Hanks balanced salt
solution; hMSCs: Human mesenchymal stem/stromal cells; IFN: Interferon-;
IL-10: Interleukin-10; IL-1: Interleukin-1; IL-4: Interleukin-4; M: Macrophage;
NGF: Nerve growth factor; NTF3: Neurotrophin 3; PAC1R: Pituitary adenylate
cyclase-activating polypeptide specific receptor; PACAP: Pituitary adenylate
cyclase-activating polypeptide; PMA: Phorbol myristate acetate; SCI: Spinal
cord injury; TGF: Transforming growth factor ; TNF: Tumor necrosis factor ;
TSG-6: Tumor necrosis factor-induced protein 6; VIP: Vasoactive intestinal
peptide; WT: Wild-type.
TT, HO, TA, and SS conceived and designed the experiments. TT, HO, DS,
AS, TN, ZX, and KD performed the experiments. TT, HO, DS, JW, ZX, and KD
analyzed and interpreted the data. HO, AS, YH, and SS supported finance.
HH provided study materials. TT, HO, DS, and SS wrote the paper. All authors
have read and approved the final version of the manuscript.
1. Nesathurai S. Steroids and spinal cord injury: revisiting the NASCIS 2 and NASCIS 3 trials . J Trauma . 1998 ; 45 : 1088 - 93 .
2. Rolls A , Shechter R , Schwartz M. The bright side of the glial scar in CNS repair . Nat Rev Neurosci . 2009 ; 10 : 235 - 41 .
3. Owen M , Friedenstein AJ . Stromal stem cells: marrow-derived osteogenic precursors . Ciba Found Symp . 1988 ; 136 : 42 - 60 .
4. Prockop DJ . Stemness does not explain the repair of many tissues by mesenchymal stem/multipotent stromal cells (MSCs) . Clin Pharmacol Ther . 2007 ; 82 : 241 - 3 .
5. Wright KT , El Masri W , Osman A , Chowdhury J , Johnson WE . Concise review: bone marrow for the treatment of spinal cord injury: mechanisms and clinical applications . Stem Cells . 2011 ; 29 : 169 - 78 .
6. Uccelli A , Milanese M , Principato MC , Morando S , Bonifacino T , Vergani L , et al. Intravenous mesenchymal stem cells improve survival and motor function in experimental amyotrophic lateral sclerosis . Mol Med . 2012 ; 18 : 794 - 804 .
7. Parr AM , Tator CH , Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury . Bone Marrow Transplant . 2007 ; 40 : 609 - 19 .
8. Shi Y , Su J , Roberts AI , Shou P , Rabson AB , Ren G . How mesenchymal stem cells interact with tissue immune responses . Trends Immunol . 2012 ; 33 : 136 - 43 .
9. Uccelli A , Moretta L , Pistoia V. Mesenchymal stem cells in health and disease . Nat Rev Immunol . 2008 ; 8 : 726 - 36 .
10. Nmeth K , Leelahavanichkul A , Yuen PS , Mayer B , Parmelee A , Doi K , et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)- dependent reprogramming of host macrophages to increase their interleukin-10 production . Nat Med . 2009 ; 15 : 42 - 9 .
11. Ohtaki H , Ylostalo JH , Foraker JE , Robinson AP , Reger RL , Shioda S , et al. Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inflammatory/immune responses . Proc Natl Acad Sci U S A . 2008 ; 105 : 14638 - 43 .
12. Gordon S. Alternative activation of macrophages . Nat Rev Immunol . 2003 ; 3 : 23 - 35 .
13. Arimura A , Shioda S. Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors: neuroendocrine and endocrine interaction . Front Neuroendocrinol . 1995 ; 16 : 53 - 88 .
14. Vaudry D , Gonzalez BJ , Basille M , Yon L , Fournier A , Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions . Pharmacol Rev . 2000 ; 52 : 269 - 324 .
15. Ohtaki H , Nakamachi T , Dohi K , Aizawa Y , Takaki A , Hodoyama K , et al. Pituitary adenylate cyclase-activating polypeptide (PACAP) decreases ischemic neuronal cell death in association with IL-6 . Proc Natl Acad Sci U S A . 2006 ; 103 : 7488 - 93 .
16. Ohtaki H , Nakamachi T , Dohi K , Shioda S. Role of PACAP in ischemic neural death . J Mol Neurosci . 2008 ; 36 : 16 - 25 .
17. Fang KM , Chen JK , Hung SC , Chen MC , Wu YT , Wu TJ , et al. Effects of combinatorial treatment with pituitary adenylate cyclase activating peptide and human mesenchymal stem cells on spinal cord tissue repair . PLoS One . 2010 ; 5 : e15299 .
18. Reglodi D , Kiss P , Lubics A , Tamas A. Review on the protective effects of PACAP in models of neurodegenerative diseases in vitro and in vivo. Curr Pharm Des . 2011 ; 17 : 962 - 72 .
19. Tsuchikawa D , Nakamachi T , Tsuchida M , Wada Y , Hori M , Farkas J , Yoshikawa A , Kagami N , Imai N , Shintani N , et al. Neuroprotective effect of endogenous PACAP on spinal cord injury . J Mol Neurosci . 2012 ; in press.
20. Tsuchida M , Nakamachi T , Sugiyama K , Tsuchikawa D , Watanabe J , Hori M , et al. PACAP Stimulates Functional Recovery after Spinal Cord Injury through Axonal Regeneration . J Mol Neurosci . 2014 ; 54 : 380 - 7 .
21. Tan YV , Abad C , Lopez R , Dong H , Liu S , Lee A , et al. Pituitary adenylyl cyclase-activating polypeptide is an intrinsic regulator of Treg abundance and protects against experimental autoimmune encephalomyelitis . Proc Natl Acad Sci U S A . 2009 ; 106 : 2012 - 7 .
22. Armstrong BD , Abad C , Chhith S , Cheung-Lau G , Hajji OE , Nobuta H , et al. Impaired nerve regeneration and enhanced neuroinflammatory response in mice lacking pituitary adenylyl cyclase activating peptide . Neuroscience . 2008 ; 151 : 63 - 73 .
23. Hashimoto H , Shintani N , Tanaka K , Mori W , Hirose M , Matsuda T , et al. Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) . Proc Natl Acad Sci U S A . 2001 ; 98 : 13355 - 60 .
24. Sato A , Ohtaki H , Tsumuraya T , Song D , Ohara K , Asano M , et al. Interleukin-1 participates in the classical and alternative activation of microglia/macrophages after spinal cord injury . J Neuroinflammation . 2012 ; 9 : 65 .
25. Song D , Ohtaki H , Tsumuraya T , Miyamoto K , Shibato J , Rakwal R , et al. The anti-inflammatory property of human bone marrow-derived mesenchymal stem/stromal cells is preserved in late-passage cultures . J Neuroimmunol . 2013 ; 263 : 55 - 63 .
26. Basso DM , Fisher LC , Anderson AJ , Jakeman LB , McTigue DM , Popovich PG . Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains . J Neurotrauma . 2006 ; 23 : 635 - 59 .
27. Suzuki R , Arata S , Nakajo S , Ikenaka K , Kikuyama S , Shioda S. Expression of the receptor for pituitary adenylate cyclase-activating polypeptide (PAC1-R) in reactive astrocytes . Brain Res Mol Brain Res . 2003 ; 115 : 10 - 20 .
28. Nakamachi T , Tsuchida M , Kagami N , Yofu S , Wada Y , Hori M , et al. IL-6 and PACAP receptor expression and localization after global brain ischemia in mice . J Mol Neurosci . 2012 ; 48 : 518 - 25 .
29. Choi H , Lee RH , Bazhanov N , Oh JY , Prockop DJ . Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-B signaling in resident macrophages . Blood . 2011 ; 118 : 330 - 8 .
30. Hori M , Nakamachi T , Rakwal R , Shibato J , Nakamura K , Wada Y , et al. Unraveling the ischemic brain transcriptome in a permanent middle cerebral artery occlusion mouse model by DNA microarray analysis . Dis Model Mech . 2012 ; 5 : 270 - 83 .
31. Hashimoto H , Hagihara N , Koga K , Yamamoto K , Shintani N , Tomimoto S , et al. Synergistic induction of pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression by nerve growth factor and PACAP in PC12 cells . J Neurochem . 2000 ; 74 : 501 - 7 .
32. Glorioso C , Sabatini M , Unger T , Hashimoto T , Monteggia LM , Lewis DA , et al. Specificity and timing of neocortical transcriptome changes in response to BDNF gene ablation during embryogenesis or adulthood . Mol Psychiatry . 2006 ; 11 : 633 - 48 .
33. Watanabe J , Shetty AK , Hattiangady B , Kim DK , Foraker JE , Nishida H , et al. Administration of TSG-6 improves memory after traumatic brain injury in mice . Neurobiol Dis . 2013 ; 59 : 86 - 99 .
34. Ha CM , Kang JH , Choi EJ , Kim MS , Park JW , Kim Y , et al. Progesterone increases mRNA levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and type I PACAP receptor (PAC(1)) in the rat hypothalamus . Brain Res Mol Brain Res . 2000 ; 78 : 59 - 68 .
35. Munoz JR , Stoutenger BR , Robinson AP , Spees JL , Prockop DJ . Human stem/ progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice . Proc Natl Acad Sci U S A . 2005 ; 102 : 18171 - 6 .
36. Lee RH , Pulin AA , Seo MJ , Kota DJ , Ylostalo J , Larson BL , et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell . 2009 ; 5 : 54 - 63 .
37. Prockop DJ , Oh JY . Mesenchymal stem/stromal cells (MSCs): role as guardians of inflammation . Mol Ther . 2012 ; 20 : 14 - 20 .
38. Martinez FO , Helming L , Gordon S. Alternative activation of macrophages: an immunologic functional perspective . Annu Rev Immunol . 2009 ; 27 : 451 - 83 .
39. Gordon S , Martinez FO. Alternative activation of macrophages: mechanism and functions . Immunity . 2010 ; 32 : 593 - 604 .
40. Foraker JE , Oh JY , Ylostalo JH , Lee RH , Watanabe J , Prockop DJ . Cross-talk between human mesenchymal stem/progenitor cells (MSCs) and rat hippocampal slices in LPS-stimulated cocultures: the MSCs are activated to secrete prostaglandin E2 . J Neurochem . 2011 ; 119 : 1052 - 63 .