Int J Clin Exp Pathol 2013;6(12):2703-2712
www.ijcep.com /ISSN:1936-2625/IJCEP1309076
Original Article
Therapeutic effect of human umbilical cord-derived
mesenchymal stem cells in rat severe acute
pancreatitis
Hong-Bo Meng*, Jian Gong*, Bo Zhou, Jie Hua, Le Yao, Zhen-Shun Song
Department of Hepatobiliary and Pancreatic Surgery, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072, China. *Equal contributors.
Received September 29, 2013; Accepted October 28, 2013; Epub November 15, 2013; Published December 1,
2013
Abstract: Aim: To investigate the therapeutic effect of umbilical cord-derived mesenchymal stem cells (UC-MSCs)
on rat severe acute pancreatitis (SAP). Methods: Rats were randomly divided into three groups (n = 15 per group):
control group, SAP group, and SAP+MSCs group. SAP was established by retrograde pancreatic duct injection of 3%
sodium taurocholate. In SAP+MSCs group, UC-MSCs at 1×107 cells/kg were injected via the tail vein 12 h after SAP.
Rats (n = 5 per group) were sacrificed on days 1, 3 and 5, and the blood and pancreatic tissues were collected. The
levels of serum amylase, lipase, inflammatory cytokines, and anti-inflammatory cytokines were determined. Pathological changes of the pancreas (HE staining) and apoptotic acinar cells (TUNEL staining) were observed under light
microscope. Results: The levels of serum amylase and lipase in SAP group were significantly higher than those in
control group (P<0.05). The pancreas in SAP group showed significantly massive edema, inflammation, hemorrhage
and necrosis when compared with control group. There were numerous TUNEL-positive apoptotic acinar cells after
SAP. However, in SAP+MSCs group, the levels of serum amylase were significantly reduced on days 1, 3, and 5 after
MSC transplantation (P<0.01). The serum lipase level in SAP+MSCs group was significantly lower than that in SAP
group on days 3 and 5 (P<0.01). The edema formation, inflammatory cell infiltration, hemorrhage, and necrosis
were reduced significantly attenuated in SAP+MSCs group as compared to SAP group (P<0.05). MSCs significantly
reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), but increased the levels of anti-inflammatory cytokines (IL-4 and IL-10) in SAP rats. The number of TUNEL-positive acinar cells was significantly reduced on
days 3 and 5 after MSCs transplantation (P<0.01). Conclusion: Transplantation of UC-MSCs significantly inhibits
inflammation and decreases pancreatic injury secondary to SAP.
Keywords: Umbilical cord-derived mesenchymal stem cells, severe acute pancreatitis, pancreatic pathology,
inflammation, apoptosis
Introduction
Acute pancreatitis (AP) is a clinically common
disease seriously affecting human health and
threatening life of patients. About 15-20% of
patients with AP may develop severe acute
pancreatitis (SAP) which is an acute abdominal
disease with rapid progression, multiple complications and high mortality [1-3]. Among SAP
patients with multiple organ dysfunction syndrome (MODS) and infectious pancreatic necrosis, the overall mortality is as high as 47%. With
the improvement of pharmacotherapy and surgical interventions, the overall mortality reduces to about 17%. However, there are no effective strategies for the treatment of SAP
currently. Thus, it is imperative to develop ideal
therapeutic strategy to reduce the complications and mortality of SAP.
Mesenchymal stem cells (MSCs) belong to adult
stem cells with self-renewal and multilineage
differentiation potentials. It has been confirmed
that MSCs may exert anti-inflammatory, immunoregulatory and repairing effects in animal
models of lung injury, acute kidney failure, acute
myocardial infarction, Crohn’s disease and diabetes [4-8]. MSCs were first identified in the
bone marrow [9, 10]. However, collection of
bone marrow is invasive and the amount of
MSCs reduces over age, which significantly limits the clinical application of MSCs [11-13]. In
UC-MSCs for treatment of severe acute pancreatitis
Table 1. Criteria for pathological scoring of the pancreas
Pathological
findings
0
Scoring
2
1
Edema
No
Infiltration of inflammatory cells
0-1 white blood 2-10 white blood
cells/HPF
cells/HPF
Bleeding
No
Necrosis
No
11-20 white blood
cells/HPF
4
21-30 white blood
cells/HPF
>30 white blood
cells/HPF
Pancreatic paren- Pancreatic parenchychymal bleeding at mal bleeding at 3-4
1-2 sites
sites
Pancreatic parenchymal bleeding at 5-6
sites
Pancreatic parenchymal bleeding at ≥7
sites
1-4 necrotic cells/
HPF
11-16 necrotic cells/
HPF
>16 necrotic cells/
HPF (fused necrosis)
5-10 necrotic cells/
HPF
2003, the umbilical cord blood was found to be
rich in MSCs which have surface markers and
induced differentiation as in MSCs from the
bone marrow. Thus, umbilical cord blood
derived MSCs have been widely applied in studies [14-16]. In the present study, SAP was
established in rats by retrograde pancreatic
duct injection of sodium taurocholate, which
has similar pathogenesis of human SAP [17,
18]. Then, transplantation of umbilical cord
blood derived MSCs was performed in these
rats aiming to investigate the therapeutic efficacy of umbilical cord blood derived MSCs in
SAP. Our findings may provide experimental evidence for the clinical application of stem cells.
Materials and methods
Materials
The umbilical cord was collected from the
Department of Obstetrics of Shanghai 10th
People’s Hospital (2012-07/2012-11) and
MSCs were separated. DMEM-LG, fetal bovine
serum, penicillin, streptomycin, trypsin (Gibco,
USA), antibodies for flow cytometry (BD
Biosciences, USA), medium for differentiation
induction (Cyagen Biosciences, USA), SPF male
SD rats (180-220 g; 6-8 weeks) (Shanghai
SLAC Animal Co., Ltd), sodium pentobarbital
(Beijing Dingguochangsheng Biotech Co., Ltd),
instruments for establishment of animal model
(Shanghai Yuanxiang Medical Devices Co.,
Ltd.), serum amylase and lipase detection kits
(BioVision, USA), ELISA kit for serum inflammatory cytokines (IL-1β, IL-6, TNF-α) and proinflammatory cytokines (IL-4, IL-10) (R&D, USA)
and TUNEL detection kit (Roche, Swiss) were
used in the present study.
Cell culture
Umbilical cord (1 mm3) was cut into pieces
which were maintained in medium followed by
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3
Diffused expansion Diffused expansion of Diffused expansion of Diffused expansion
of lobular septa
lobular septa
acinar septa
of cell gap
collection of primary MSCs. These MSCs were
maintained in DMEM-LG containing 10% fetal
bovine serum, 100 U/ml penicillin and 100 µg/
ml streptomycin at 37 °C in a humidified environment with 5% CO2 for 3 weeks. When the
cell confluence reached about 80%, cells were
digested with 0.25% trypsin and passaging was
done at a ratio of 1:3. Cells of the third generation were subjected to flow cytometry for identification and used for differentiation induction
and transplantation.
Preparation of rat SAP model and cell transplantation
Before surgery, rats were fasted for 12 h but
given ad libitum access to water. Rats were randomly assigned into 3 groups (n = 15 per group)
and anesthesia was done by intraperitoneal
injection of 3% sodium pentobarbital. Normal
control group: animals were anesthetized for 2
h and laparotomy was not performed. SAP
group: A midline incision was made at the abdomen (2 cm in length) and laparotomy was performed. The duodenum was identified along
the pylorus and then taken out of the abdominal cavity. The pancreas was identified and the
duodenal papilla, common bile duct and pancreatic duct were located. A 9/0 nylon thread
was placed at the shared outlet of common bile
duct and pancreatic duct at the head of pancreas. A small clamp was used to clamp the
common bile duct. Then, a needle was inserted
via the duodenal papilla into the middle common bile duct. Once the head of the needle was
located, ligation was done with the pre-existing
thread. Then, 3% sodium taurocholate (1 ml/
kg) was slowly injected at a speed of 5 min/ml.
Focal bleeding and diffuse edema were
observed in the pancreas. The needle was withdrawn, and the thread and the clamp were
removed. When there was no active bleeding in
Int J Clin Exp Pathol 2013;6(12):2703-2712
UC-MSCs for treatment of severe acute pancreatitis
Figure 1. Identification of umbilical cord derived MSCs. A: Morphology of MSCs (×40); B: Proliferation of MSCs; C: Surface markers of MSCs; D: Osteogenesis (×100),
adipogenesis (×200) and chondrogenesis (×40) of MSCs.
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Int J Clin Exp Pathol 2013;6(12):2703-2712
UC-MSCs for treatment of severe acute pancreatitis
Figure 2. Pancreatic pathological findings in different groups (HE, ×200). A-C: Pancreatic pathology on days 1, 3 and
5 in SAP group; D-F: Pancreatic pathology on days 1, 3 and 5 in SAP+MSCs group; G-I: Pancreatic pathology on days
1, 3 and 5 in control group.
the pancreas and duodenum, the viscera were
placed back into the abdominal cavity followed
by wound closure. In SAP+MSC group, MSCs
(1×107/ml) were injected via the tail vein at
1×107/kg at 12 h after surgery.
Morphological examination of the pancreas
At 1, 3 and 5 days after surgery, rats (n = 5 per
group) were anesthetized, and 5 ml of blood
was collected from the heart and anti-coagulated. The pancreas was harvested, fixed in 4%
paraformaldehyde over night and embedded in
paraffin. The paraffin-embedded tissues were
cut into sections (4 µm) followed by HE staining
and observation under a light microscope. Ten
fields were randomly selected, and pathological scoring was done according to the pathological criteria for pancreatitis (Table 1).
collected. Then, 5 µl of serum was added into
96-well plates and distilled water was added
with the final volume of 50 µl in each well.
Subsequently, detection buffer (50 µl) and substrate solution (50 µl) were independently
added to each well, and the absorbance (OD)
was measured at 405 nm immediately (T0).
Then, OD was also measured at different time
points (T1). The serum amylase and lipase levels were determined according to the standard
curves. Serum amylase/lipase level = B/
[V×(T1-T0)] mU/ml, where B is the nitrophenol
concentration (nmol) in the standard curve
when the OD405 was measured at T1; V is the
volume of samples (5 µl); T1-T0 refers to the time
from immediately after addition of substrate to
a specific time point.
Detection of serum amylase and lipase
Detection of serum pro-inflammatory cytokines
and anti-inflammatory cytokines
The anti-coagulated blood was centrifuged at
250 g for 25 min at 4 °C, and the serum was
The ELISA kits (R&D, USA) were used to detect
the content of IL-1β, IL-6, IL-4, IL-10 and TNF-α
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Int J Clin Exp Pathol 2013;6(12):2703-2712
UC-MSCs for treatment of severe acute pancreatitis
Figure 3. Scoring of pancreatic pathology: A: Score of pancreatic edema; B: Score of inflammatory cell infiltration; C:
Score of pancreatic bleeding; D: Score of pancreatic necrosis; aP<0.05; bP<0.01.
Detection of acinar cell apoptosis
Figure 4. Serum levels of amylase and lipase in different groups. A: Serum level of amylase; B: Serum
level of lipase, aP<0.05; bP<0.01.
in the serum according to manufacturer’s
instruction.
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The paraffin embedded sections were deparaffinized and dehydrated, and then treated with
protease K at 20 µg/ml for 20 min to increase
the tissue permeability. After washing in PBS
twice, 100 μL of balance buffer was added to
each section followed by incubated at room
temperature for 5-10 min. Then, the biotin conjugated nucleoside mixture buffer (100 µl) and
Terminal Deoxynucleotidy1 Transferase (100
µl) were added to each section followed by incubation at 37 °C for 60 min. The sections were
subsequently immersed in SSC solution for 15
min to stop reaction. After washing in PBS
thrice, sections were washed in 0.3% hydrogen
peroxide solution for 5 min to inhibit the endogenous catalase. Following washing in PBS
thrice, 100 µl of horseradish peroxidase
solution was added, followed incubation for 30
min. After washing in PBS thrice, visualization
was done with DAB followed by mounting.
These sections were observed done under light
microscope. In detection of acinar cell
apoptosis, 5 fields were randomly selected
Int J Clin Exp Pathol 2013;6(12):2703-2712
UC-MSCs for treatment of severe acute pancreatitis
Figure 5. Serum levels of proinflammatory cytokines and antiinflammatory cytokines: MSCs
transplantation reduced serum
levels of pro-inflamamtory cytokines (TNF-α, IL-1β, IL-6) and
increased serum levels of anti-inflamamtory cytokines (IL-4, IL-10)
a
P<0.05; bP<0.01.
from each section at a high magnification
(×400), and the apoptotic cells were counted.
Statistical analysis
Statistical analysis was done with SPSS version
14.0. Data were expressed as mean ± standard deviation (SD). Comparisons were done
with one way analysis of variance and a value of
P<0.05 was considered statistically significant.
Results
Culture and identification of MSCs
MSCs were long spindle-shaped (Figure 1A)
and had potent proliferation activity. Flow
cytometry showed umbilical cord derived MSCs
had no expressions of CD45, CD34, CD11b,
CD19 and HLA-DR (0.61%) but high expressions of CD44 (99.99%), CD73 (99.98%), CD90
(99.99%) and CD105 (99.97%) (Figure 1C).
Experiment of induction differentiation showed
the MSCs had the capabilities of osteogenesis,
adipogenesis, chondrogenesis (Figure 1D).
These findings suggest these cells are MSCs.
Pathological examination of pancreas after
MSC transplantation
At different time points, there were no edema,
infiltration of inflammatory cells, bleeding and
necrosis in the pancreatic lobules in control
group. In SAP group, the pancreatic edema was
observed immediately after surgery. One day
after surgery, evident expansion of alveolar
septum, parenchymal bleeding and infiltration
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of inflammatory cells were noted. Three days
after surgery, the pancreatic parenchymal
necrosis (focal patchy necrosis) deteriorated.
Five days after surgery, the necrotic area
merged, and tubular complexes were observed.
In SAP+MSCs group, pancreatic edema, bleeding, necrosis and infiltration were also noted,
but these pathological changes were milder
than in SAP group. Moreover, the pathological
changes in SAP+MSCs group improved over
time, and a small amount of fibrous tissues
were observed (Figure 2). On the basis of pancreatic parenchymal necrosis, pathological
scoring was performed (Figure 3). Results
showed the pathological scores of pancreatic
edema, pancreatic parenchymal bleeding and
necrosis and infiltration of inflammatory cells in
SAP group were markedly higher than those in
control group (P<0.01). In SAP+MSCs group,
the pancreatic scores were significantly
reduced when compared with SAP group
(scores of edema and bleeding on days 1, 3
and 5: P<0.01; scores of inflammatory cell infiltration and acinar cell necrosis on day 1:
P<0.05; scores of inflammatory cell infiltration
and acinar cell necrosis on days 3 and 5:
P<0.01).
Serum amylase and lipase levels after MSCs
transplantation
On day 1, the serum amylase level increased
markedly (2100±245 mU/ml), and further elevated on days 3 and 5 (2380±194 mU/ml and
2920±231.5 mU/ml) when compared with control group (P<0.01). This, together with pathological findings, suggests the SAP animal model
Int J Clin Exp Pathol 2013;6(12):2703-2712
UC-MSCs for treatment of severe acute pancreatitis
Figure 6. Apoptosis of pancreatic acinar cells (TUNEL, ×400). A-C: Apoptosis of pancreatic acinar cells on days 1, 3
and 5 in SAP group; D-F: Apoptosis of pancreatic acinar cells on days 1, 3 and 5 in SAP+MSCs group; G-I: Apoptosis
of pancreatic acinar cells on days 1, 3 and 5 in control group.
1 and was 3 folds higher than that in control
group since day 3 (day 3: 4010±357.7 mU/ml;
day 5: 4560±441 mU/ml; P<0.01). One day
after transplantation, the serum lipase level
remained unchanged (P>0.05), but reduced
markedly since day 3 (day 3: 1880±204 mU/
ml; day 5: 2160±387.8 mU/ml) when compared
with SAP group (P<0.01; Figure 4B).
Serum levels of inflammatory cytokines after
MSC transplantation
Figure 7. Counting of apoptotic pancreatic acinar
cells, bP<0.01.
was stable and reliable. After MSC transplantation, the serum amylase level reduced to
1080±172 mU/ml, 1020±283.5 mU/ml and
1180±278.6 mU/ml on days 1, 3 and 5, respectively, which were significantly lower than those
in SAP group (P<0.01; Figure 4A). In SAP group,
the serum lipase level increased slightly on day
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When compared with SAP group, the serum
TNF-α content reduced in SAP+MSCs group,
and significant difference was noted since day
3 day 3: P<0.05; day 5: P<0.01). In SAP+MSCs
group, the serum IL-1β and IL-6 contents were
markedly lower than those in SAP group on day
1 (P<0.05), and they further reduced over time
(day 5: P<0.01). In addition, MSC transplantation significantly increased the serum IL-4 and
IL-10 contents which were 0.0812±0.0153 ng/
Int J Clin Exp Pathol 2013;6(12):2703-2712
UC-MSCs for treatment of severe acute pancreatitis
ml and 0.306±0.052 ng/ml on day 3 and 5,
respectively and significantly higher than those
in SAP group (P<0.01) (Figure 5).
Apoptosis of pancreatic acinar cells after
MSCs transplantation
TUNEL staining showed there was almost no
apoptotic cells in control group (Figure 6G-I). In
SAP group, massive apoptotic cells were
observed in the pancreas, and the number of
apoptotic cells increased over time (Figure
6A-C). After MSCs transplantation, the number
of apoptotic acinar cells reduced since day 3,
and these apoptotic cells were scattered
(Figure 6D-F). In SAP+MSCs group, the number
of apoptotic cells was markedly lower than that
in SAP group on days 3 and 5 (P<0.01, Figure
7).
Discussion
AP is one of common acute abdominal diseases and characterized the self-digestion, edema,
bleeding and necrosis (features of inflammation) due to the activation of trypsin in the pancreas. The incidence of AP increases with the
elevation of living standard [19]. Less than 20%
of patients with AP may develop SAP which has
a lot of complications and high mortality. Thus,
SAP has been a dangerous and refractory acute
abdominal disease [1]. Currently, SAP is managed with traditional supportive therapy or surgical interventions, and no effective strategy
has been developed for the treatment of SAP to
date. Cell therapy with MSCs has been found to
promote the repairing of acute and chronic injury, regulate immune function and attenuate
inflammatory response [8]. It has been confirmed that MSCs can improve the inflammatory response in the liver, kidney and lung, and
have favorable promise in clinical application in
myocardial infarction, Crohn’s disease and
organ transplantation. Studies have shown that
multiple inflammatory cytokines are involved in
the pathogenesis of SAP including pro-inflammatory cytokines (IL-1, IL-6, TNF-α) and antiinflammatory cytokines (IL-4, IL-10) [20, 21].
Thus, MSCs have the potential for the treatment of pancreatitis due to the anti-inflammatory effect of MSCs. In addition, MSCs can be
induced to differentiate into different cell types
including insulin secreting cells and endothelial
cells [22, 23]. Thus, it is feasible to promote
pancreatic regeneration and repairing of microvascular endothelium of the pancreas with
MSCs.
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Retrograde cholangiopancreatic injection of
sodium taurocholate was employed to induce
acute obstructive pancreatitis which has similarity in the pathogenesis of clinical pancreatitis. This animal model is easy to establish and
feasible. The pathological examination and
pancreatic enzyme detection showed this animal model was stable. The main pathological
findings were pancreatic edema, infiltration of
massive inflammatory cells, pancreatic parenchymal bleeding and acinar cell necrosis. The
umbilical cord derived MSCs were injected into
SAP rats via the tail vein, and results showed
the serum levels of amylase and lipase reduced,
pancreatic pathology and acinar cell apoptosis
improved, serum levels of pro-inflammatory
cytokines (TNF-α, IL-1β, IL-6) reduced, and
those of anti-inflammatory cytokines increased
(IL-4, IL-10). These findings suggest that MSCs
have favorable therapeutic effects on SAP.
When compared with SAP group, MSCs transplantation significantly reduced serum levels of
amylase and lipase (amylase: 48% on day 1;
55% on day 3; 60% on day 5; lipase: 20% on
day 1, 50% on day 3, 53% on day 5), suggesting
that MSCs transplantation may protect the
structural integrity of acinar cells. MSCs transplantation facilitates the recovery of pancreatic
pathology which was characterized by improved
pancreatic edema, reduction of inflammatory
cells and attenuation of pancreatic parenchymal bleeding and necrosis. However, the specific mechanisms are still poorly understood.
Previous studies have shown that bone marrow
derived MSCs can migrate into the injured pancreas and differentiate into pancreatic cells to
repair the injured pancreas. In the SAP, more
MSCs migrate into the pancreas. This suggests
that the homing ability of MSCs is related to the
severity of injury [24]. However, in recent years,
MSCs are found to secret some cytokines (IL10, TGF-β, IL-1Reg and HGF) in a paracrine
dependent manner to exert the anti-inflammatory effect [25]. More recent studies reveal that
the TNF-α and IL-1 may activate MSCs which
then secret anti-inflammatory protein TSG-6,
which is found to be involved in the anti-inflammatory effect of MSCs [26-28]. Our results
showed MSCs transplantation reduced the
serum levels of pro-inflammatory cytokines and
increased those of anti-inflammatory cytokines, which was consistent with the antiinflammatory effect of TSG-6. Thus, the therapeutic effect of MSCs transplantation might be
associated with the TSG-6 secreted by MSCs.
Int J Clin Exp Pathol 2013;6(12):2703-2712
UC-MSCs for treatment of severe acute pancreatitis
Pancreatic acinar cell apoptosis play an important role in the pathogenesis of SAP. The initiation of pancreatic acinar cell apoptosis is
regarded as a protective response to inflammation related stimulation in cells [29]. The apoptosis and necrosis of pancreatic acinar cells
have reciprocal transformation. Thus, to reduce
the apoptotic acinar cells may also attenuate
the necrosis of acinar cells to a certain extent
[30]. In the present study, a lot of apoptotic acinar cells were observed in the pancreas of SAP
rats, and the number of apoptotic acinar cells
increased over time. However, in SAP+MSCs
group, the apoptotic acinar cells reduced significantly when compared with SAP. This suggests that MSCs transplantation attenuate the
pancreatitis and inhibit the apoptotic initiation,
which prevent the further attack on acinar cells
and improve the acinar cell necrosis.
In the present study, umbilical cord derived
MSCs are transplanted into rats with SAP.
Results demonstrate that MSCs transplantation may protect the structural integration of
acinar cells, improve the pancreatic pathology,
regulate inflammatory response and attenuate
acinar cell apoptosis. The specific mechanism
underlying the therapeutic effect of MSCs
transplantation may be attributed to the homing and transdifferentiation of MSCs or the
paracrine effect of MSCs. However, more studies are required to investigate the exact mechanism underlying the therapeutic efficacy of
stem cells, which may provide evidence for a
novel strategy for the treatment of SAP.
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Acknowledgements
The study was supported by National Natural
Science Foundation of China (No. 81170436);
and Foundation of Shanghai Municipal Health
Bureau (No. 2010015). We thank Fei Yin and
Qianglin Duan for their kind help.
Disclosure of conflict of interest
[11]
[12]
None.
Address correspondence to: Dr. Zhen-Shun Song or
Dr. Hong-Bo Meng, Department of Hepatobiliary and
Pancreatic Surgery, Shanghai Tenth People’s
Hospital, Shanghai 200072, China. Tel: +862166301051; E-mail: [email protected]
(Zhen-Shun Song); [email protected] (HongBo Meng)
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