Endoscopic submucosal injection of adipose-derived mesenchymal stem cells ameliorates TNBS-induced colitis in rats and prevents stenosis
Martín Arranz et al. Stem Cell Research & Therapy
Endoscopic submucosal injection of adipose-derived mesenchymal stem cells ameliorates TNBS-induced colitis in rats and prevents stenosis
Eduardo Martín Arranz 1 2
María Dolores Martín Arranz 1 2
Tomás Robredo 2 4
Pablo Mancheño-Corvo 2 3
Ramón Menta 2 3
Francisco Javier Alves 2 6
Jose Manuel Suárez de Parga 1 2
Pedro Mora Sanz 1 2
Olga de la Rosa 2 3
Dirk Büscher 2 5
Eleuterio Lombardo 0 2 3
Fernando de Miguel 0 2 4
0 Equal contributors
1 Gastroenterology Department, La Paz University Hospital , Paseo de la Castellana 261 4th floor, 28046 Madrid , Spain
2 Berlin and IFATS 2013 11th conference in New York , USA
3 Tigenix SAU, Tres Cantos , Madrid , Spain
4 Cell Therapy Laboratory, La Paz Hospital Institute for Health Research , Madrid , Spain
5 Grifols SA, Sant Cugat del Vallés , Barcelona , Spain
6 Pathology Department, La Paz University Hospital , Madrid , Spain
Background: Mesenchymal stem cells have potential applications in inflammatory bowel disease due to their immunomodulatory properties. Our aim was to evaluate the feasibility, safety and efficacy of endoscopic administration of adipose-derived mesenchymal stem cells (ASCs) in a colitis model in rats. Methods: Colitis was induced in rats by rectal trinitrobenzenesulfonic acid (TNBS). After 24 h ASCs (107 cells) or saline vehicle were endoscopically injected into the distal colon. Rats were followed for 11 days. Daily weight, endoscopic score at days 1 and 11, macroscopic appearance at necropsy, colon length and mRNA expression of Foxp3 and IL-10 in mesenteric lymph nodes (MLN) were analyzed. Results: Endoscopic injection was successful in all the animals. No significant adverse events or mortality due to the procedure occurred. Weight evolution was significantly better in the ASC group, recovering initial weight by day 11 (− 0.8% ± 10.1%, mean ± SD), whereas the vehicle group remained in weight loss (− 6.7% ± 9.2%, p = 0.024). The endoscopic score improved in the ASC group by 47.1% ± 5.3% vs. 21.8% ± 6.6% in the vehicle group (p < 0.01). Stenosis was less frequent in the ASC group (4.8% vs. 41.2%, p < 0.01). Colon length significantly recovered in the ASC group versus the vehicle group (222.6 ± 17.3 mm vs. 193.6 ± 17.9 mm, p < 0.001). The endoscopic score significantly correlated with weight change, macroscopic necropsy score and colon length. Foxp3 and IL-10 mRNA levels in MLN recovered with ASC treatment. Conclusions: ASC submucosal endoscopic injection is feasible, safe and ameliorates TNBS-induced colitis in rats, especially stenosis.
Inflammatory bowel disease; Cell therapy; Mesenchymal stem cells; Endoscopic treatment
Inflammatory bowel disease (IBD) includes Crohn’s disease
(CD) and ulcerative colitis (UC); it is characterized by
chronic and relapsing inflammation of the intestinal tract.
Its pathogenesis is not completely understood, but a
dysregulation of the innate and adaptive immune system,
genetic influences and environmental factors are suggested
to intervene in the development of the disease. With
current treatments, there is still a substantial proportion of
patients in whom remission cannot be achieved, leaving
unmet needs in the treatment of IBD and leading to the
emergence of new treatments [
Mesenchymal stem cells (MSCs) are cells that can be
isolated from several adult tissues, including bone
marrow (BM-MSCs) and adipose tissue (ASCs) [
MSCs have been proposed as a potential treatment for
several diseases, including immune-based treatments,
due to their multilineage differentiation capabilities that
could allow MSCs to repair damaged tissues [
their capacity to modulate the function of the majority
of immune cells [
]. This treatment could promote
the regulation of the inflammatory cascade by inducing
tolerogenic phenotypes in antigen-presenting cells (APCs) [
while inhibiting the proliferation of cytotoxic T-cells and
promoting differentiation toward regulatory phenotypes in
lymphocytes (regulatory T cells) and macrophages (M2
]. The immunomodulatory properties of
MSCs rely not only on cell-to-cell interactions but also on
effects mediated by a variety of soluble factors [
MSCs are considered to have low immunogenicity
due to a low expression of major histocompatibility
complex (MHC)-I and an absence of MHC-II and
classic costimulatory molecules, allowing allogeneic use in
the clinical setting or even xenogeneic use for research
Regarding IBD cell therapy, MSCs from different
sources have reported efficacy through various routes of
administration (systemic and local) in animal studies on
experimental colitis [
]. Thus, MSC treatment reduced
the clinical and histopathological severity in
experimental animal models of colitis by downregulating the Th1
and Th17-driven inflammatory response (i.e. reduction
of pro-inflammatory cytokines such as tumor necrosis
factor (TNF), interferon gamma (IFNγ), interleukin (IL)-6,
IL-12 or IL-17), increasing the levels of anti-inflammatory
cytokines such as IL-10, and promoting the generation of
immune cells with immunomodulatory properties such as
FoxP3 regulatory T cells, regulatory B cells, and M2
]. The engraftment of MSCs on the
inflamed gut might play an important role for their
therapeutic effect on IBD models [
13, 14, 19, 20
preactivation of MSCs with inflammatory mediators such
as IFNγ, IL-1β, IL-25 or poly (I:C) increase their
therapeutic capacity in vivo by increasing their
immunomodulatory properties and capacity to home to the site of
]. Noteworthy, efficacy on the
treatment of experimental colitis has been also obtained using
components of MSCs, such as conditioned medium and
extracellular vesicles, showing that MSC secreted factors
can ameliorate colitis [
]. Another aspect of interest
is that exposure of MSCs to drugs commonly used in the
treatment of IBD do not appear to affect MSC function in
]. Furthermore, MSC-based therapies have
been already used in human studies on fistulizing [
and luminal CD [
]. Importantly, Panés et al.,
recently reported for the first time the significant efficacy
of the intralesional treatment with allogeneic ASCs
(Cx601) of complex perianal fistulas in Crohn’s
patients, in a randomized, placebo-controlled phase III
clinical trial .
Although promising, MSC therapy for IBD still raises
some questions, such as the best route of administration
to optimize bioavailability in the damaged areas.
Conflicting results have been reported to date in terms of
intravenous efficacy, and local approaches used in
animal experiments, such as intraperitoneal inoculation or
colon injection through laparotomy, are impractical for
human use [
]. In this study, we propose a new
administration route for IBD cell therapy: injecting cells into
the colon submucosa using endoscopy, allowing direct
cell delivery to the damaged area, while enabling the
evaluation of disease severity and evolution.
The aim of this study was to evaluate the feasibility,
safety and efficacy of submucosal endoscopic injection
of human ASCs in a 2,4,6-trinitrobenzenesulfonic acid
(TNBS)-induced colitis model in rats, and the utility of
endoscopy to evaluate and follow the course and severity
of the disease over time.
All the animal experiments were performed following
approval from the Animal Experimental and Welfare
Ethics Committee of La Paz University Hospital (CEBA
24-2010), and in accordance with the guidelines of the
directive 2010/63/EU from the European Parliament
and of the Council on the protection of animals used
for scientific purposes and the corresponding Spanish
Immunocompetent SD-OFA male rats (Charles River,
Barcelona, Spain), weighing 375–400 g were used, and
were kept in standard stabulation conditions (12 h light/
dark daily cycle) in our facilities with pellet chow and
water ad libitum throughout the experiment.
For ASC isolation, lipoaspirates were obtained from
human adipose tissue after informed consent from
otherwise healthy adult male and female donors undergoing
The ASC isolation was performed as previously
]. Briefly, the lipoaspirates were washed twice
with phosphate-buffered saline (PBS) to remove
contaminating debris and red blood cells and digested at 37 °C
for 30 min with 0.075% collagenase (Type I, Invitrogen,
Carlsbad, CA, USA) in the PBS. The digested sample
was washed with 10% fetal bovine serum (FBS), treated
with 160 mM ammonium chloride to eliminate the
remaining red blood cells, suspended in culture medium
(Dulbecco’s modified Eagle medium), containing 10%
FBS, and filtered. The cells were seeded onto tissue
culture flasks and expanded at 37 °C and 5% carbon
dioxide. The culture medium was changed every 3 to 4
days. The cells were passed to a new culture flask when
the cultures reached of confluence, and were
phenotypically characterized according to their capacity to
differentiate into chondro-, osteo-, and adipogenic lineages
]. In addition, ASCs were verified by flow
cytometry staining for specific surface markers: positive for
HLA-I, CD73, CD90 and CD105; and negative for
HLAII, CD14, CD19, CD34 and CD45. A pool of six various
ASC samples from male and female donors (population
doubling 12–14) were used in the study.
On day 0, the rats were weighed and colitis was induced.
On day 1, the animals that presented weight loss
underwent endoscopic evaluation under anesthesia. After
colonoscopy, treatment was subsequently applied according
to the assigned experimental group: ASC group (n = 25),
or vehicle (PBS, n = 21). Two additional groups were used:
one without colitis induction was used as a healthy control
(n = 25); and another with induced colitis but without
endoscopy or treatment, the TNBS group (n = 13), was used
as a safety control for the endoscopy and injection.
All the animals were weighed daily. On day 11, a
second colonoscopy was performed under anesthesia for
the assessment of colonic damage; blood was then
obtained by cardiac puncture, and the animals were
euthanized with saturated potassium chloride through
intracardiac injection. A medial abdominal incision was
then performed for macroscopic evaluation.
On day 0, the animals were weighed and anesthetized
with inhaled isofluorane (5% induction and 2%
maintenance), and feces were removed by gentle manual pressure
of the abdomen. While in a supine position, a flexible
plastic intravenous catheter (BD Insyte™ Autoguard™ 18G,
Becton Dickinson, Madrid, Spain) was inserted 5 cm from
the anal verge, and a single bolus of 0.5 ml of TNBS
(Sigma-Aldrich, Tres Cantos, Spain), 30 mg/ml diluted in
50% ethanol, freshly prepared, was delivered slowly. The
rats were kept in a head-down position for 1 min to
prevent immediate expulsion of TNBS, and were then
returned to their cages where they recovered
consciousness shortly thereafter [
Approximately 24 h after colitis induction, the animals
were weighed and anesthetized with inhaled isofluorane.
Prior to the endoscopy, colon cleansing was performed
with a 20-ml room temperature (RT) saline solution
The endoscopy was performed with a videoendoscope
GIF-XP-160 (Olympus Optical Co Ltd, Tokyo, Japan),
with an outer diameter of 5.9 mm, 180°/90° up/down
bending, 100°/100° right/left bending, 103 cm working
length, 120° view field, 2 mm working channel and a
CV-145 processor (Olympus Optical Co Ltd).
While in the supine position, the endoscope was
inserted into the rectum, advancing until the splenic
flexure (8–10 cm). All the endoscopies were digitally
recorded for posterior analysis by two different observers.
To assess colitis severity, we developed an endoscopic
index, adapted from published animal endoscopic
] and human IBD scales [
degree of inflammation, ulceration, stenosis, thickening,
bleeding and extent of disease were scored individually
and a final score was obtained by adding all the
variables, ranging from 0 to 25 (Table 1).
Endoscopic needles (23G × 5 mm, 160 cm length,
Olympus Optical Co Ltd) were used for submucosal injection.
Following endoscopic evaluation, a needle was passed
through the endoscope’s working channel, then
introduced tangentially into the submucosa using the
dynamic endoscopic submucosal injection method [
injecting 0.2 ml PBS containing ASCs or not (total dose
107 ASCs) in four different spots surrounding the
lesions. This dose and number of injections were chosen
taking into account the maximum concentration in
which ASC viability was guaranteed and covering the
extension of the damaged colon while keeping a low
volume of fluid, and thus avoiding potential mucosal
damage caused by over injection.
The complete procedure, including submucosal
injections, usually took no longer than 10 min.
After euthanasia, the abdominal cavity was exposed
through medial abdominal incision, allowing visualization
of the colon and the presence of adherences. Photographs
were taken for evaluation by two different observers.
Macroscopic damage was assessed by the degree of colon
wall vascularization, wall thickening and presence of
adherences, for a final score ranging from 0 to 9 (Table 2).
The colon was removed to measure its entire length
from the colocecal junction to the anal verge, and the
distal part excised and fixed in buffered formalin.
Mesenteric lymph nodes (MLN) were collected,
snapfrozen in liquid nitrogen and stored at − 80 °C for
Distal colonic tissue was fixed in 10% buffered formalin,
embedded in paraffin, cut into 5-μm sections and
stained with hematoxylin-eosin.
To detect ASCs by immunohistochemistry, tissue
sections were dewaxed and rehydrated, and then
microwaved for 20 min in 0.01 M trisodium citrate buffer pH
6.0. After cooling to RT, endogenous peroxidase was
blocked by incubating the tissues for 10 min in 1% H2O2
in methanol. After rinsing with TBST buffer, the samples
were blocked for 1 h at RT with 5% normal goat serum
and 1% bovine serum albumin in TBST, then incubated
overnight at 4 °C with anti-human mitochondria
antibody (113-1, Abcam, Cambridge, UK), 1:500 in blocking
solution. Detection was performed with goat anti-mouse
biotinylated IgG (Life Technologies, Carlsbad, CA, USA)
1:250 in blocking solution for 1 h at RT, followed by the
ABC kit (HRP-based, Vector Laboratories, Burlingame,
CA, USA), for 1 h at RT, and DAB as the chromogen.
Sections were counterstained with hematoxylin, and
mounted with DPX.
RNA isolation, reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR
Total RNA was isolated from frozen MLN with TRIzol
reagent (Ambion, Life Technologies), according to the
manufacturer’s recommendations. The average yield was
1.91 ± 0.06 μg RNA per mg MLN. Complementary
DNA synthesis was performed with MultiScribe Reverse
Transcriptase and oligo (dT)16 (Applied Biosystems,
Foster City, CA, USA), according to the manufacturer’s
recommendations. Standard PCR was performed with 1u
DNA polymerase (Biotools, Madrid, Spain) and 0.5 μM
each oligonucleotide for 35 cycles of 30 s at 95 °C, 30 s
at 60 °C and 30 s at 72 °C. The sequences of the
oligonucleotides were as follows: Foxp3, forward, 5’-CAG CTG
CCT ACA GTG CCC CTA G-3′, reverse, 5’-CGT TTG
CCA GCA GTG GGT AG-3′; IL-10, forward, 5′-GGA
TCC AAC GCA GCC TTG CAG AAA C-3′, reverse,
5’-ACG CGT ATT TTT CAT TTT GAG TGT CAC
GTA GGC -3′; ß-Actin, forward, 5′-AGA GGG AAA
TCG TGC GTG-3′, reverse, 5’-CTG GGT ACA TGG
TGG TGC-3′. The PCR products were analyzed on 1.
6% agarose gels in 0.5 × TBE buffer (Tris-Borate-EDTA)
containing 1 × REALSafe (Durviz, Valencia, Spain) and
with a digital photodocumentation system (Alliance 2.7,
UVitec, Cambridge, UK).
For the quantitative PCR, we used a CFX96 Touch
system (Bio-Rad, Hercules, CA, USA) and Quantimix
Easy kit (Biotools, Madrid, Spain) containing 0.3 μM
each oligonucleotide and 0.5 × SYBR Green (Life
Technologies), for 40 cycles of 20 s at 95 °C, 20 s at 60 °C, 30
s at 72 °C and 2 s at 80 °C (when fluorescence was
acquired). The oligonucleotide sequences were as follows:
Foxp3, forward, 5′-TGG CAG GGA AGG AGT GTC
AG-3′, reverse, 5’-GGC TGA CTT CCA AGT CTC
GT3′; IL-10, forward, 5’-GGC TCA GCA CTG CTA TGT
TGC C-3′, reverse, 5’-AGC ATG TGG GTC TGG CTG
ACT G-3′; GAPDH, forward, 5′- CGT GGA GTC TAC
TGG TGT CTT CAC C-3′, reverse, 5′- GAT GGC
ATG GAC TGT GGT CAT GAG C-3′. We used the
standard 2-ΔΔCt method to quantitate expression levels.
The statistical analyses were performed with SPSS 20.0
(SPSS Inc., Chicago, IL, USA) and Prism 5.01 (GraphPad
Software Inc., San Diego, CA, USA). The statistical level
of significance was p < 0.05, corrected for multiple
comparisons where appropriate.
The quantitative data are expressed as mean ± SD.
The differences between continuous and qualitative
variables were calculated with non-parametric tests: the
Kruskal-Wallis or the Mann-Whitney U test. The
Wilcoxon signed-rank test was used for paired analysis.
For the frequency analysis between qualitative
variables we used the chi-squared test or Fisher’s exact test,
when necessary (if n < 20 or any value in the expected
value table was < 5).
Correlations were analyzed by Pearson’s correlation
coefficient. Slopes of linear regression were compared
with the beta coefficient of the simple linear regression
and its standard error.
Survival curves were made using the Kaplan-Meier
method and the log-rank test.
Twenty-four hours after a TNBS enema, the rats
suffered significant weight loss (− 3.9% ± 2.4% vs. 1.0% ± 1.
4% in the control group, p < 0.001); diarrhea and other
signs of discomfort were also evident in the colitic
animals. The endoscopic score increased significantly in
these animals compared with the control group (16.1 ±
4.9 vs. 1.3 ± 1.9, p < 0.001). The selection of rats to
either treatment group, PBS or ASC, was not biased, given
neither weight loss (− 3.6% ± 2.8% vs. -3.0% ± 1.9%,
respectively, p = 0.21) nor endoscopic score (17.6 ± 4.3 vs.
15.0 ± 5.0, respectively, p = 0.08) was statistically
Feasibility and safety
The endoscopic injection was successful in all the rats,
with formation of a visible submucosal bleb (Fig. 1a-c).
Neither significant adverse events nor mortality due to
the procedure occurred.
We assessed the correct location of the cell injection
using immunohistochemistry with anti-human
mitochondria antibody, which allows detection of human
ASCs. In a healthy animal in which ASCs were injected
following the same method of the experiment, a dense
accumulation of positive cells was observed in the colon
submucosa 24 h after the injection, with no signs of
immune reaction (Fig. 1d).
In the colitic rats, we also detected ASCs in the colon
submucosa 24 h after cell injection, with intense
inflammatory infiltrate and edema (Fig. 1e). At the end of the
experiment, however, we could not detect ASCs by
immunohistochemistry within the submucosa, even in the
animals that showed signs of improvement: the extensive
areas that were seen in the submucosa of these animals
showed cells with a fibroblastic phenotype, less
inflammatory infiltrate and almost absent edema (Fig. 1f ).
There was no difference in overall survival rate with
either treatment, (85.7% PBS vs. 83.3% ASCs vs. 84.6%
TNBS, p is not significant). As expected, the healthy
control group suffered no deaths (data not shown).
All the control rats gained weight steadily throughout
the course of the experiment, reaching a significant
increase of 9.9% ± 3.4% (p < 0.001) compared with
their initial weight (Fig. 2a, b). Regarding the colitic
rats, the TNBS group suffered rapid weight loss,
reaching − 11.9% ± 2.1% at day 3; they then began a slight
recovery, with mean weight loss at the end of the
experiment of − 8.7% ± 8.2% (p = 0.021) (Fig. 2a). The
PBS-treated rats also exhibited a similar pattern, with
significant weight loss that reached a maximum on the
fifth day after induction with the hapten (− 11.3% ± 4.2%).
After this day, the rats gained weight slightly, and, on
average, did not reach initial values (− 6.7% ± 9.2% on day 11,
p = 0.024) (Fig. 2a).
The ASC-treated rats also suffered an initial weight
loss that reached its peak on day 3 (− 8.4% ± 4.2%), after
which, unlike the other colitic groups, weight recovery
was evident, reaching, on average, initial values by the
end of the experiment (− 0.8% ± 10.1% on day 11, p = 0.
741) (Fig. 2a). In fact, the ASC-treated animals gained
weight with a slope comparable to the one observed in
the control group (0.93 ± 0.08 vs. 0.89 ± 0.04, respectively,
p = 0.601), whereas the slopes for the TNBS and the
PBStreated groups were significantly flatter (0.52 ± 0.09, p = 0.
001, and 0.41 ± 0.09, p < 0.001, respectively) (Fig. 2a).
Weight recovery was statistically significant in the
ASCs group over the PBS group on day 5 (− 6.5% ± 5.
86% vs. −11.32% ± 4.18%, respectively, p = 0.009), day 9
(− 3.7% ± 9.1% vs. −8.5% ± 6.8%, respectively, p = 0.034),
day 10 (− 1.9% ± 9.4% vs. −8.4% ± 8.6%, respectively,
p = 0.013) and day 11 (−0.8% ± 10.1% vs. −6.7% ± 9.2%,
respectively, p = 0.037).
Individually, some animals from every colitic group
recovered initial weight by the end of the experiment, but
the frequency of weight recovery was higher in the
ASCtreated animals compared with the TNBS and PBS
groups: 60.0% (12/20), 27.3% (3/11) and 38.9% (7/18),
respectively (p is not significant) (Fig. 2b).
After colitis induction, endoscopic signs of damage were
evident, with disappearance of vascular pattern, edema
and ulceration (Fig. 3a, b), which improved over time, in
particular in the ASC group (Fig. 3d).
We evaluated the endoscopic mucosal damage following
the score described in Table 1, with higher scores for more
There is no validated score for endoscopic evaluation
of experimental colitis severity. The most commonly
used variable for follow-up is daily weight; therefore, we
first analyzed the overall correlation between our
colonoscopy score and weight change, finding a strong
correlation at both time points evaluated: at day 1, the
Pearson coefficient was r = − 0.75 (p < 0.001) and at the
end of the experiment at day 11 r = −0.78 (p < 0.001)
(Additional file 1: Figure S1).
At day 11, the final endoscopic score was 9.1 ± 5.6 in
the ASC group compared with 13.3 ± 6.2 in the PBS
group (p = 0.052) (Fig. 3e). As expected in an acute
colitis model, both groups improved; however, this
improvement was significantly greater in the ASC group
compared with the PBS group (−6.6 ± 2.9 vs. −3.5 ± 5
points, respectively, p = 0.011). This represents a mean
improvement of 47.1% ± 5.3% in the ASCs compared
with 21.8% ± 6.6% in the PBS group (p = 0.005).
ASC treatment dramatically decreased the
development of stenosis (defined as the presence of a narrowing
of the lumen that hinders or prevents passage of the
endoscope) when evaluated by endoscopy on day 11
(Fig. 4a, b). While in the PBS group, 7 of 17 evaluable
rats (41.2%) developed stenosis, whereas only 1 of 21
rats (4.8%) did so in the ASC group (p = 0.001) (Fig. 4c).
Macroscopic evaluation of the abdominal cavity was
performed after euthanasia at day 11. Vascular pattern
distortion, colon wall thickening and fat tissue or visceral
adhesions to the colon were assessed (Fig. 5a-d). Both
the TNBS and PBS groups showed a significantly higher
macroscopic damage score compared with the control
group (6.5 ± 2.5, p < 0.001; 6.1 ± 2.7, p < 0.01; 2.2 ± 1.0,
respectively). This total score and all the evaluated
subitems were lower in the ASC group than in the PBS and
TNBS groups, although there was no statistical
significance among them. Nevertheless, the macroscopic
damage score in the ASC group (4.7 ± 2.5) decreased to
values not statistically different from the control group
The macroscopic damage score correlated significantly
with weight change on the day of euthanasia, with a
Pearson correlation r = 0.78 (p < 0.001), and with the
endoscopic index on the same day (Pearson correlation
r = 0.80, p < 0.001) (Additional file 2: Figure S2).
Colitis is known to induce a marked shortening of the
colon. In the TNBS group and the PBS group, colon
length was shorter than in the control group (204.5 ±
24.6 mm, p < 0.001; 193.6 ± 17.9 mm, p < 0.001, vs.
237.2 ± 16.3 mm, respectively). This shortening was
significantly improved in the ASC group, with a mean
length of 222.6 ± 17.3 mm, showing no difference from
the control group (p = 0.2) and statistically longer than
the PBS group (p < 0.001) (Fig. 5f ). Colon length also
correlated significantly with the endoscopic score at the end
of the experiment, with a Pearson correlation r = − 0.33,
p = 0.041 (Additional file 3: Figure S3). Distal colonic
samples stained with hematoxylin-eosin were evaluated
for ulceration and histologic recovery (Fig. 6).
Immunomodulatory effects mediated by ASCs
The possible immunomodulatory effect of the local
injection of ASCs into the colon submucosa was evaluated
at day 11 in mesenteric lymph nodes by measuring the
mRNA expression of Foxp3, a transcription factor
expressed by regulatory T lymphocytes, and of IL-10, an
anti-inflammatory cytokine produced by various cell
As shown in Fig. 7a, Foxp3 expression decreased
dramatically in the untreated colitic rats, being three times
smaller in the TNBS group than in the control group (0.
33 ± 0.1 vs. 1.05 ± 0.3, respectively, p = 0.014). The ASC
group showed a recovered expression (1.27 ± 0.5 times),
which was not statistically different from the control
group (p = 0.46) (Fig. 7a).
Regarding IL-10 mRNA, the expression of IL-10 was
reduced in the TNBS colitic group (0.56 ± 0.2 times)
compared with the healthy control group (1.07 ± 0.4, p = 0.05),
whereas in the ASC-treated animals, IL-10 expression was
similar to the control group (1.05 ± 0.1, p = 0.71) (Fig. 7b).
In the present study, we hypothesized that local
administration by endoscopy of ASCs in the submucosa of
colitic rats could be an optimal route of administration to
increase the bioavailability of ASCs in the damaged areas
of the colon and reduce the severity of the disease.
Therefore, we evaluated the feasibility, safety and
efficacy of endoscopic ASC administration in an
experimental colitis model, as well as the utility of endoscopy to
follow the course of the disease in rats.
Our results support endoscopy as a useful
administration route for ASC treatment for colitis and its utility for
assessing treatment efficacy and severity of the disease
in an animal model. We also demonstrate that
submucosal injection of human ASCs ameliorates the course of
TNBS colitis in immunocompetent rats.
IBD pathogenesis is complex and involves multiple
mechanisms. One of the most significant is the
disbalance between pathogen recognition and tolerance
against commensals. In this setting, MSC treatment could
help restore this balance due to its diverse
immunomodulatory properties, promoting both differentiation of
lymphocytes and mononuclear cells toward tolerant phenotypes
and suppression of activated lymphocytes [
studies have tried stem cell therapy in colitic animals with
both BM-MSCs and ASCs [
13–15, 19, 36, 50–54
Although promising, MSC therapy has yet to address
several questions, such as optimal doses and
administration routes, selection of patients and elucidation of
mechanisms of action, making animal models essential.
Regarding administration route, it has been
demonstrated that ASCs have homing capabilities toward
damaged areas [
]. While convenient, intravenous use
remains controversial due to concerns about lung
entrapment that could lead to a reduced number of
available cells and therefore reduced efficacy and potential
adverse events [
19, 52, 54, 56
]. Delivering the cells
directly into the damaged area would overcome this issue,
and intraperitoneal use has been used in animals, but
this approach is impractical in humans. Injecting cells
through endoscopy could offer advantages in experimental
studies and translational potential for future human use.
The TNBS chemical-induced model of colitis is
inexpensive, reproducible, easy to handle and widely used
40, 41, 57
]. Although human IBD is better represented
by knock-out mouse models, we chose the TNBS model
because it leads to distal colitis and can be used in rat
strains that can grow enough to make endoscopy a
feasible tool for evaluation and treatment. Therefore, even if
the use of a chemical model is a limitation of our study,
it is useful for the evaluation of the endoscopic
administration route and that efficacy data obtained with this
model is meaningful.
Our data demonstrate that endoscopy in trained hands
is a simple and very effective tool for follow-up and
evaluation of colitic rats, with an excellent safety profile,
given we did not observe any case of perforation or
significant bleeding due to the injection, and no deaths
were attributed to the procedure. Moreover, there were
no significant differences in the evolution of the rats
with colitis without endoscopy (TNBS group) and the
group with endoscopy and PBS injection. This safety
profile is one of the main advantages of the technique,
allowing in vivo repeated assessment of the severity and
evolution of the disease, and also reducing the number
of animals needed.
While it has been used for carcinogenesis [
and colitis [
19, 42, 44, 64
] evaluation, these studies are
heterogeneous in aims, endoscopes and scoring systems.
Although lacking a formal validation, the endoscopic
score developed for this study showed a significant
correlation with body weight changes, macroscopic damage
and colon length, which are commonly used variables in
experimental colitis. Further studies will determine
which is the best score for colitis evaluation in murine
ASC administration demonstrated improvement in
weight recovery, in colon length and in the endoscopic
damage score. In addition, the macroscopic damage
observed after euthanasia was numerically smaller in the
treated group, although it did not reach statistical
significance. These are among the main variables commonly
used in colitic animals, thus supporting the ameliorating
effect of the cells, and confirming the results of previous
13–15, 19, 36
]. Although these studies differ in
protocol, severity of the colitis, animal strains, cell type,
dose and administration routes, all suggest a beneficial
effect of ASC treatment in murine chemical-induced
One of the main results of our study was the decrease
in stenosis development; even though the experiment
was not designed for this purpose, the difference
between the ASC and the vehicle group is remarkable.
Whereas in the PBS group stenosis developed in 41% of
the animals, in the ASC group this occurred in 4.8%.
This is an observation that, in our opinion, deserves
further studies to elucidate whether ASCs are able to
prevent stenosis development in other models and if they
can also help to reverse established stenosis.
A limitation of our study is the lack of histological
scoring; in our hands, microscopic changes in
inflammation and healing were inconsistent.
We were able to detect the ASCs in colon submucosa
using anti-human mitochondrial antibodies 24 h after
injection, proving the correct location of the cells after
the endoscopic administration. In agreement with
previous studies reporting a short persistence of the cells in
vivo, ASCs were not detected after 11 days. The fate of the
ASCs in vivo remains largely unknown to date [
In order to determine whether the endoscopic
administration of ASCs resulted in immunomodulatory effects
in the colitic rats, we determined the levels of the
antiinflammatory cytokine IL-10 and the transcription factor
Foxp3, characteristic of regulatory T cells. Our results
show that IL-10 and Foxp3 levels in MLN of
ASCtreated rats were elevated, supporting the notion that
ASCs administered endoscopically have an
immunomodulatory mechanism of action, similar to what has
been previously described for MSCs using other routes
of administration [
]. Nevertheless, we did not
investigate in detail the mechanisms underlying the
efficacy of ASCs and, therefore, further studies are
needed to better understand the mechanism of action
of ASCs administered through the endoscopic route of
We cannot completely rule out a potential
immunogenicity using xenogeneic cells; however, it appears that
human ASCs are sufficiently well tolerated in this
proofof-concept model. This immunoprivileged status is
probably due to a lack of MHC molecules, as has been shown
in other recent studies [
If our results are confirmed there would be
translational potential, in humans, endoscopic injection could
be a simple, well-tolerated route of delivering cells directly
into the damaged area, through a technique routinely used
in patients with IBD for both diagnostic and therapeutic
], including pharmacological local injection
]. Cell-based therapies are currently
being tested in different phases in humans, reporting efficacy
of intravenous infusion with various doses and regimens
of BM-MSCs, both autologous and allogeneic [
Other studies have focused on local treatment for
fistulizing disease, showing improved healing with
] or ASCs [
29–31, 35, 70
In conclusion, our study provides evidence that
endoscopy is a safe and reliable method to administer cell
therapy into the colon and to follow up colitis murine
models. ASC treatment ameliorates the course of TNBS
colitis and prevents stenosis development.
Additional file 1: Figure S1. Endoscopic score correlation with weight
change at day 1 (A) and 11 (B). (PPTX 2289 kb)
Additional file 2: Figure S2. The macroscopic damage score correlates
with weight change (A) and with endoscopic score (B). (PPTX 2254 kb)
Additional file 3: Figure S3. The endoscopic score correlation with the
colon length 2. (PPTX 37 kb)
APCs: Antigen-presenting cells; ASCs: Adipose-derived mesenchymal stem
cells; BM-MSCs: Bone marrow mesenchymal stem cells; CD: Crohn’s disease;
FBS: Fetal bovine serum; IBD: Inflammatory bowel disease; IL: Interleukin;
INF: Interferon; MHC: Major histocompatibility complex; MLN: Mesenteric
lymph nodes; MSCs: Mesenchymal stem cells; PBS: Phosphate-buffered saline;
RT: Room temperature; TBST: Tris-buffered saline with Tween 20;
TNBS: Trinitrobenzenesulfonic acid; UC: Ulcerative colitis
The authors would like to thank Jesus Diez for his statistical support, Dr.
Carlota Largo Aramburu for her veterinary support and the Hospital
Universitario La Paz Inflamatory Bowel Disease Unit and Cell Therapy
Laborathory members for their help and support for the study.
This work was partially supported by a grant of the Instituto de Salud Carlos
III to FdM (PI10/0317).
Availability of data and materials
All data generated or analyzed during this study are included in this
published article (and its supplementary information file).
EM, MDM, FdM and EL designed the study, analyzed and interpreted the
data. EM wrote the draft, MDM, FdM, OR, DB, FdM and EL reviewed the draft
and made important contributions to the final manuscript: All authors
collected data and reviewed contributed and approved the final version of
Ethics approval and consent to participate
All the animal experiments were performed following approval from the
Animal Experimental and Welfare Ethics Committee of La Paz University
Hospital (CEBA 24-2010), and in accordance with the guidelines of the directive
2010/63/EU from the European Parliament and of the Council on the protection
of animals used for scientific purposes and the corresponding Spanish
Consent for publication
PM, RM, OR and EL are full-time employees of Tigenix SAU; EM received
consultancy fees from Cellerix SAU (now Tigenix). DB is a full-time employee
of Grifols. All the other authors have no conflicts of interest to disclose.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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