Adipose tissue-derived mesenchymal stem cells ameliorate experimental acute colitis in rats

Complete treatment of ulcerative colitis (UC) is still dicult, while conventional therapies have various adverse effects. Mesenchymal stem cells (MSCs) have anti-inammatory and immunomodulatory properties to be a therapeutic candidate for UC. We evaluated therapeutic potential of adipose tissue-derived mesenchymal stem cells (AdSCs) in treatment of an acute colitis rat model using histological and molecular assessments. Thirty male Sprague Dawley acetic acid-induced (2 mL of 3%) acute colitis rat models were randomly divided into three equal groups of control receiving 0.5 mL/kg of normal saline, sulfasalazine group receiving 500 mg/kg sulfasalazine and AdSCs group transplanted transrectally with 2×10 6 MSCs. They were evaluated histologically and by real time PCR for expression of apoptotic genes until 21 days. MSCs were spindle shape and positive for osteogenic and adipogenic differentiation. They displayed mesenchymal and lacked hematopoietic markers. In control group, severe inammation, edema, ulcer, necrosis and inltration of leukocytes were noticed. In sulfasalazine group, a moderate inammation, edema, ulcer, necrosis and inltration of leukocytes were visible; and in AdSCs group, mild inammation, congestion, and inltration of leukocytes were observed with a mild edema, but necrosis was absent in colonic tissue. A stronger decrease in expression of Bax, together with a higher increase in Bcl-2 was noted in AdSCs group. Based on histological and molecular ndings, AdSCs were effective to ameliorate colitis lesions through their anti-inammatory and anti-apoptotic activities showing that transplantation of AdSCS can be a potentially useful strategy in treatment of colitis. cells sulfasalazine attenuates trinitrobenzene sulfonic acid induced colitis in the S1P pathway.


Introduction
In ammatory bowel diseases (IBDs) are a group of digestive diseases including ulcerative colitis (UC) and Crohn's disease (CD), while environmental, genetics, and immune factors have important roles in occurrence of the disease 1 . In 21st century, IBD was reported a global burden of prevalent diseases, and hundreds of studies were conducted on incidence and prevalence of IBD throughout the world 2 . The prevalence of IBD has more stabilization in Western Europe and North America, but with a rise in Asia and developing countries 3 . Iran as one of the largest Asian countries in the Middle East which is adjacent to Turkey, Afghanistan, Pakistan, Central Asian and Persian Gulf countries, and they are a fertile ground for investigation for IBD 3 .
UC is a chronic and idiopathic in ammation in colon affecting many patients who suffer from a relapsing and remitting course 4 . Both male and female are equally in uenced, specially enrolling 30-40 years old adults 4 . In ammation of the lining of the colon happens in UC leading to several symptoms such as abdominal pain, diarrhea, and rectal bleeding 4 . The acute form of UC is a severe and life-threatening condition that conventional medications include anti-in ammatory agents such as systemic corticosteroids, topical corticosteroids, and 5-aminosalicylic acids (5-ASA), as well as immunomodulators like cyclosporine, azathioprine, 6-mercaptopurine (6-MP), and methotrexate 5 . It has endoscopic and surgical treatments too 6 , but unfortunately; complete treatment of UC is still di cult; because 74% of patients were reported to experience at least one relapse during a 5-year period 4 . Taking conventional medications may lead to the occurrence of various adverse effects that can make nearly one fourth of patients discontinue their treatments 4 . Therefore, the global climbing trend of IBD would need a tandem effort to prevent the disease and to innovate the delivery of healthcare to patients with IBD and to introduce new therapeutic targets such as transplantation of mesenchymal stem cells (MSCs) to achieve ameliorative e cacy without a risk of incontinence 4 .
MSCs are multipotent stem cells that were widely investigated over the past few decades in several diseases 7 . They can be easily isolated and ampli ed from various tissues including bone marrow 8 and adipose tissues 9 . MSCs can regulate immune responses, when exposed to in ammatory environment, thus promoting the repair and regeneration of damaged tissues 10 . They have been successfully used for treatment of UC in human and animals 4 . However, there are still very few studies regarding the e ciency of adipose tissue derived stem cells (AdSCs) as a candidate therapeutic strategy for UC. Therefore, the objective of this study was to determine the healing effect of AdSCs in experimental acute colitis of rat model that were evaluated histologically and quantitatively by real time PCR.

Cell Characterization
AdSCs in different passages were adherent to the culture plates and were spindle shape (Fig. 1A, B). In osteogenic differentiation media, the cells illustrated calcium deposits after three weeks that were visualized in red color by Alizarin Red staining (Fig. 1C). In adipogenic differentiation media, the cells were stained by Oil Red-O and revealed intracellular lipid droplets in red color (Fig. 1D). Also, AdSCs displayed positive expression of mesenchymal markers (CD73 and CD90) and lacked expression of hematopoietic markers (CD34 and CD45) (Fig. 1E).

Histological Assessment
In control group 1 with colitis receiving normal saline treatment, in ammation, edema, conjestion, ulcer, necrosis and in ltration of leukocytes were severely visible in the colonic tissue ( Fig. 2A) together with weight loss, hematochezia and bleeding, but without any mortality. In sulfasalazine group receiving sulfasalazine after induction of colitis, a moderate in ammation, edema, ulcer, necrosis and in ltration of leukocytes were con rmed with moderate weight loss, hematochezia and bleeding, but without any mortality ( Fig. 2B and C). In cell transplantation group that AdSCs were injected for treatment of UC, mild in ammation, conjestion, and in ltration of leukocytes were observed with a mild edema, but necrosis, weight loss, hematochezia and bleeding were absent denoting to a prominent healing effect of stem cells in ameliorating UC (Fig. 2D-F). The strongest alterations were visible 3 weeks after cell transplantation.

Quantitative Real Time PCR (qPCR)
The signi cant decrease in expression of Bax pro-apoptotic gene was stronger in cell transplanted group when compared with the control and sulfasalazine-treated group. The highest changes were noticed 3 weeks after cell transplantation. The increase in Bcl-2 anti-apoptotic gene in AdSC transplanted group was signi cantly higher than other groups. The highest changes were noticed 3 weeks after cell transplantation ( Fig. 3, P 0.05).

Discussion
People who suffer from IBD are prone to increased risks of developing colorectal cancer, which has a global incidence rate and mortality rate 4 . During the last decade, conventional treatments for IBD were found to provide scarce desired ndings and often with severe complications. Biological agents including transplantation of stem cells have been introduced as a treatment of choice in IBD with a great deal of success in both clinical and experimental models 4 . The ability of MSCs to actively proliferate, undergo plastic differentiation, exhibit low immunogenicity, trigger strong immune regulation, and express abundant trophic factors has con rmed their success in immune interventions and therapies and in regenerative medicine of IBD 4 .
In our study, the transplanted AdSCs were shown to have mesenchymal properties similar to previous reported ndings 9,10 , that were plastic adherent, spindle shape, positive for osteogenic and adipogenic induction, and also positive for expression of CD73 and CD90 as mesenchymal markers and negative for expression CD34 and CD45 as hematopoietic markers. AdSCs can be easily isolated and expanded while maintaining their biological features 9 4,20 , rat in 13 investigations 4,21 , Guinea pig in one study 4,22 and dog in one study 23 . In our research, rat model has similarly been used for induction of colitis and AdSCs as a therapeutic approach with successful results of healing in colonic lesions. Among animal models, different methods were introduced for induction of UC with subsequent transplantation of MSCs in treatment of disease including acetic acid in one study 4,24 , dextran sulfate sodium (DSS) in 7 investigations 4,25 , and trinitrobenzene sulfonic acid (TNBS) in four assays 4,26 . Identically, we utilized acetic acid in our study to induce UC in the rat model resulting in colonic lesions of severe in ammation, edema, ulcer, necrosis and in ltration of leukocytes.
Among MSCs transplanted in animal models for treatment of UC, BMSCs in 24 studies [4,25] In previous reports, the origin and type (autologous or allogeneic) of MSCs, method of administration (dosage, route, schedule, timing of infusions, the intervals between injections, pretreatment with chemokines or cytokines, etc.), animal modeling of colitis were illustrated to affect the treatment outcome 29,37 . The mode of storage can also be an in uencing factor, while the storage in various laboratories regarding the cold chain, lyophilization, and transportation can be different. In our study, 2×10 6 autologous AdSCs were transplanted transrectally that could ameliorate colonic lesions, even there were some limitations in our study which are worthy of consideration including the assessment methods that were histological and molecular, the follow-up time that was for 21 days and the sample size that was 10 animals in each group.
In conclusion, based on histological and molecular ndings, AdSCs were demonstrated to be effective in amelioration of colitis lesions through their anti-in ammatory and anti-apoptotic activities revealing that transplantation of AdSCS can be a potentially useful strategy in treatment of colitis. in 0.5 mL normal saline was administered transrectally after inducing acute colitis. In cell treatment group, 2×10 6 of rat AdSCs of passage 4 th in 0.5 mL volume were transplanted transrectally. Acute colitis was induced via enema using a needle inserted up to 8 cm into the colonic tissue and by injection of 2 mL of 3% acetic acid solution, while rats were kept in a vertical position (head-down) to avoid any leakage of acetic acid as described before 11 . To con rm induction of colitis, rats were checked after 24 hours to suffer from diarrhea or rectal bleeding. Prior to induction of colitis, rats were deprived from food intake for 24 h, but had free access to water.
All treatment interventions were started 24 hours after induction of colitis. During interventions, rats were anaesthetized by intra-peritoneal injection of 3 mg/kg xylazine (2% Rompun, Bayer Co., Germany) and 30 mg/kg ketamine (Imalgène 1000, Merial, Germany). They were kept in separate cages under an ambient temperature of 21±2°C and a 65-70% relative humidity in a good ventilated room. All groups were followed for 21 days. On 7 th , 14 th and 21 st days following treatment measures, the animals were sacri ced, and tissue samples were provided from the distal 10 cm portion of the colon for histological and molecular assessments. The animal studies were undertaken based on NIH Guidelines for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Institutional Animal Care and Use Committee of our University (1398.056). All methods conducted in this study regarding the animals were in accordance with the ethical standards of Institutional Animal Care and Use Committee of Shiraz University of Medical Sciences.

Isolation of AdSCs
Adipose tissues around the testes of rats were used for isolation of AdSCs. The isolated tissue was placed on ice under sterile condition and transferred to stem cell laboratory of Stem Cell Technology Research Center of Shiraz University of Medical Sciences, Shiraz, Iran. Brie y, adipose tissue was minced into tiny segments and was treated with 0.2% collagenase type II (Gibco, U.S.A.) for 40 min at 37°C, while shaken. The lysed tissue was centrifuged at 1500 rpm for 5 min. The precipitate was suspended in 2 mL of Dulbecco's Modi ed Eagles Medium (DMEM; Gibco, USA) in culture asks containing 3 mL DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin and 1% streptomycin (Sigma, USA) and 2 mM L-glutamine (Invitrogen, Netherlands) and were put in an incubator containing 5% CO 2 at 37°C and saturated humidity. After 3 days, the media was refreshed to reach 85% con uence. The cells were later sub-cultured until passage 4 th using 0.25% trypsin (Gibco, USA), while they were later inactivated by adding equal volume of DMEM.
The isolated AdSCs in passage 4 th were later cryopreserved to be used for future cell transplantation purposes using 2×10 6 viable cells/mL in 50% DMEM media, 40% FBS, and 10% dimethyl sulfoxide (DMSO; MP Bio) in sterile labeled cryovials and kept in a nitrogen tank. To prepare for cell transplantation, they were taken out from nitrogen tank and transferred into a 37°C water bath for thawing. Centrifugation was carried out for 5 min at 1500 rpm and the cell pellet was re-suspended in DMEM and placed in a CO 2 incubator at 37°C and saturated humidity until use.

Characterization of AdSCs by Morphology
AdSCs were assessed to be morphologically spindle shape.

Characterization by Osteogenic Induction
Approximately 5×10 4 AdSCs were transferred into 6-well plates, while the media was refreshed with osteogenic medium at 90% con uence containing the culture media supplemented with 15% FBS, 100 nM dexamethasone (Sigma-Aldrich, USA), 50 µM ascorbic acid (Merck, Germany), and 10 mM glycerol 3phosohate (Merck, Germany) for 21 days. The media change was done every 3 days and after 21 days, 10% formalin was added for 20 min to x the cells. After 3 washes with deionized water, the differentiation was assessed by alizarin red staining (Sigma-Aldrich, USA) bound to calcium mineralized deposits and revealed a red color.

Characterization by Adipogenic Induction
Around 5×10 4 AdSCs were seeded in 6-well plates, while media change happened with adipogenic medium at 90% con uence using culture media supplemented with 15% FBS, 100 nM dexamethasone, 100 µM ascorbic acid, and 200 µM indomethacin (Sigma-Aldrich, USA) for 21 days. After 3 weeks, 10% formalin was added for 20 min to x the cells and after 3 washes with deionized water, fresh 0.5% Oil Red-O dye (Sigma-Aldrich, USA) dissolved in 2-propanol solution (Merck, Germany) for 2 h was added for staining of differentiated cells. Oil red O staining reveals red color droplets when adipogenic induction is positive.

Characterization of AdSCs by Flow Cytometry
CD73 and CD90 (Dako, Denmark) were used to con rm the positive expression of mesenchymal surface markers and CD34 and CD45 (Dako, Denmark) to verify the absence of hematopoietic surface markers.

Histological Evaluation
The removed colonic tissue provided from animals was transferred into 10% buffered formaldehyde for 72 h. Then, dehydration was done by cold ethanol, and clearing was undertaken using cold xylene. The tissue samples were later embedded in para n at 53°C and a 5-µm thickness tissue section was prepared serially, dried at 37°C for an hour and stained by hematoxylin and eosin (H&E). All slides were visualized under a light microscope and photography was undertaken.

RNA Extraction of AdSCs
RNA extraction of AdSCs was done using an RNA extraction kit (Cinna Gen Inc., Tehran, Iran). The RNA was assessed at optical density ratio of A260/A280 and A260/A230 using a Nanodrop spectrophotometer (Nanodrop; Thermo Fisher Scienti c,Waltham, USA). cDNA was prepared using 1000 ng total RNA applying the Revert Aid rst strand cDNA synthesis kit (Thermo Fisher Scienti c, Waltham, USA). Collection of the samples for analyzing the gene expression was based on methods described before 12 .

Quantitative Real Time Polymerase Chain Reaction (qPCR)
The targeted apoptotic genes were Bax and Bcl-2 and B 2 m was considered as an endogenous control gene. The sequences of these genes were determined by NCBI database and primer sets were designed by primer3 software (Table 1). Real time PCR was carried out using SYBR Green I as reporter dye and Step One Real-Time PCR reactions (Applied Biosystems, Waltham, USA). In each reaction, 200 nM of each primer was added to target the speci c sequence. The PCR condition was set at 94°C for 10 min followed by 40 cycles at 94°C for 15 s, at 60°C for 60 s, and melting curve analysis ramping from 65 to 95°C. The ampli cation signals of different samples were normalized to B 2 m cycle threshold (Ct), and then the 2-DDCt method was applied to compare mRNA levels of various groups, which represented a fold-change in data analysis 12 . Table 1 The Bax, Bcl-2 and B2m gene sequences designed by primer3 software  A. In control group receiving just normal saline for treatment, severe in ammation, edema, ulcer, necrosis and in ltration of leukocytes were noticed in the colonic tissue (H&E, 40X). B and C. In sulfasalazinetreated group, a moderate in ammation, edema, ulcer, necrosis and in ltration of leukocytes were noted in the colonic injured tissue (H&E, 40X). D-E. In AdSCs transplantation group, mild in ammation, congestion, and in ltration of leukocytes were observed with a mild edema, but necrosis was absent denoting to the prominent healing effect of stem cells in ameliorating UC. Figure 3