MSCs preparation and isolation

After filling out the informed consent letter, human umbilical cords (H-UC) were obtained from Imam Reza hospital from full-term pregnancy mothers. H-UC was transferred by transfer media containing 200 ml PBS (Biosera, france), 1% Pen/Strep (Gibco, USA), 1% diluted Betadine, 1% Amphotericin B, and 2% FBS (Biowest, South America) into GMP clean room. After removing blood vessels, the dissection of Wharton´s jelly, rinsed with PBS, and centrifuged at a speed of 1500–2000 RPM for 5 min. Then, the collagenase dispase enzyme (Boehringer Mannheim GmbH, Germany) was applied and placed for 60 min in a 37ºC incubator. Next, H-UC pieces were centrifuged at 1500 − 200 RPM for 5 min; then trypsin was added and left to incubate for 30 min at 37ºC. Finally, PBS was added before centrifugation at 1500–2000 RPM for 5 min. H-UC slices were cultured into α-MEM media with 20% FBS and 1% Pen/Strep in an incubator with 5% CO2 at 37ºC. After a while, the MSCs were budding from Wharton’s jelly and the slice were discarded. After reaching 90% confluency, cells were sub cultured.

Characterization and identification of MSCs

To evaluate the expression of MSCs markers anti-CD45, Anti-CD34, anti-CD90, and anti-CD105 antibodies were applied (BIO-RAD, USA). The Flow Jo software (version 10.5) was used to analyse the data of flow cytometry. To evaluate the capacity of MSCs to transform into different cell types, Oil Red-O and Alizarin Red (BIO-IDEA, Iran) staining techniques were utilized. Briefly, 3× \(\:^\)cells were placed into 6 well plates. To differentiate into adipocyte cell lineage, the adipocyte differential medium was applied into two wells, and one well was used as a control. The media was changed every three days. After 18–21 days Oil Red-O staining was conducted. To differentiate into osteocytes, the same amount of MSCs was cultured in an osteocyte differential medium for 18–21 days. After mineralization, Alizarin Red staining was done. Then, the result was observed with an optical microscope. The MSCs morphology was determined under the light microscope.

MSC-Exo preparation and isolation

For exosome isolation, the third passage of MSCs was maintained in a serum-free medium for 24 h at 37ºC in a 5% CO2 incubator. Then, the supernatant was harvested and centrifuged at 3000 rpm to discard cell debris. The rest of the protocol was performed according to the EXOCIB kit (Cib biotech, Iran) protocol. After extraction, the MSC-Exo concentration was measured by BCA assay based on color intensity (Pars Tous Biotechnology, Iran).

Characterization and identification of MSCs-Exo

To assess the expression of CD9 and CD81, a western blot analysis was conducted. The size range of exosomes was determined using dynamic light scattering (DLS) analysis. The morphology of exosomes was examined through transmission electron microscopy (TEM). For TEM, exosomes were placed on a formvar-coated grid and treated with 2% paraformaldehyde. Subsequently, the exosomes were negatively stained with 2% uranyl acetate. The stained exosomes were then washed in double-distilled water before capturing images.

Animal study

The 45 male C57BL/6 mice, aged 9 weeks, were acquired from Mashhad University Medical Science. For two weeks, all mice were maintained in a controlled environment with unrestricted access to food and water. This study has been reported in line with the ARRIVE guidelines 2.0.

Ethics approval and consent to participate

This study was performed following the Declaration of Helsinki Ethical Principles and approved by the Ethics Committee of the Mashhad University of Medical Sciences, Mashhad, Iran (IR. MUMS. RES.1401.133).

(1)

Title of the approved project: Evaluation of the effectiveness of umbilical cord mesenchymal stem cells along with the synergistic effect of the Exosomes derived from them on the immune response of an IBD mouse model.

(2)

Name of the institutional approval committee: the Ethics Committee of the Mashhad University of Medical Sciences, Mashhad, Iran.

(3)

Approval number: IR. MUMS. RES.1401.133.

(4)

Date of approval: 06/08/2022.

Study design and colitis induction

The 45 male C57BL/6 mice were randomly assigned to nine groups, with each group inclusive of 5 mice. Except for the first group (normal group), in the other 8 groups, colitis was induced by the following method: for inducing acute colitis, mice received 5% DSS (Cayman, Germany) in drinking water within the 7 days and they drank regular water for the remaining 3 days.

Mice were grouped as follows (Fig. 1):

1.

The normal group: A group of healthy mice were given access with unrestricted availability of food and water and did not receive any treatment.

2.

The DSS group (colitis group): A group of mice were given access to water that contained 5% DSS for a period of 7 days, after which they were allowed to drink regular water for the remaining 3 days. This group did not receive any other treatment as well.

3.

The Mesalazine group: A group of colitis mice receiving 100 mg/kg Mesalazine (Cayman, Germany) from day 3 to day 10 every day by gavaging.

4.

The Exo group: A group of colitis mice receiving150 µg Exo in 200 µl PBS by intraperitoneal (IP) injection on days 3 and 7.

5.

The Exo + Mesalazine group: A group of colitis mice receiving the same amount of Exo on days 3 and 7, and besides they received 100 mg/kg Mesalasin everyday by gavaging.

6.

The MSCs group: A group of colitis mice receiving 1×\(\:^\) cells in 200 µl PBS intraperitoneally on days 3 and 7.

7.

The MSCs + Mesalazine group: A group of colitis mice receiving the same amount of cells and Mesalazine with previous doses, routes, and on the same days.

8.

The Exo + MSCs group: A group of colitis mice receiving the same amount of cells and Exo on days 3 and 7 intraperitoneally with 6 h interval.

9.

The Exo + MSCs + Mesalazine group: A group of colitis mice receiving the same amount of cells, Exo, and Mesalazine with previous doses, routes, and on the same days.

The alteration in body weight changes, bleeding severity, stool consistency, and rectum prolapse were monitored on a daily basis and they achieved daily disease activity index (DAI, Table 1) using alterations in these clinical signs.

Table 1 Scoring system for disease activity index (DAI)Fig. 1

A schematic illustrating the assessment of the therapeutic effectiveness of different interventions in DSS-induced colitis C57BL/6 mice. During the experiment, the mice were subjected to a 7-day exposure to 5% DSS. On days 3 and 7, different groups of mice were intraperitoneally injected with Exosomes, MSCs and any group that contains these substances. Mesalazine is also gavage from the third to the tenth day in related groups. Abbreviations: DSS; Dextran sulfate sodium, MSZ; Mesalazine

Colon and spleen evaluation using microscopic and macroscopic scoring

On the 10th day, the mice were euthanized by injecting 10 mg/kg xylazine (Merck, Germany) and70 mg/kg Ketamine (Merck, Germany) and cervical dislocation. Colon tissue was harvested from the rectum region to the ileocecal valve. After washing and removing stool with PBS, the colon and the spleen weight and length were measured and the photo was taken. After macroscopic observations, the colon was segmented into small pieces, was immersed in 10% formalin to prepare for hematoxylin and eosin (H&E) staining (kianshimi, Iran) and another part was stored at -70 ºC for cytokine evaluation. Histological scores were given to the intensity of inflammation, mucosal destruction, crypt loss, and pathological changes by a light microscope (Table 2).

Table 2 Scoring system for histological changesFlow cytometry analysis of Th17 and Treg cells

As previously described [21]splenic cells were extracted. For stimulation of releasing intracellular cytokine, the cells were subjected to treatment with PMA/ionomycin (Biolegend, USA). Following a 6-hour incubation in a 5% CO2 and 37 ºC, the cells were stained by anti-CD3, anti-CD4, anti-CD25, anti-FOXP3 (for Tregs), and were stained by anti-CD3, anti-CD4, and anti-IL17A antibodies (for Th17 cells) (Biolegend, USA). Labeled cells were analyzed with a FACSCalibur flow cytometer and FlowJo software.

Measuring anti-inflammatory and inflammatory cytokines level

For measuring IL-17 A and IL-10 levels, the homogenized colon tissue was prepared by a homogenizer device (Tissue lyser LT). The supernatant was collected for evaluating the expression of cytokines level and stored at -20 for ELISA assay (R&D Systems DuoSet® ELISA, china).

Statistical analysis

The data was analyzed using GraphPad Prism version 8. For evaluating group comparison one way -ANOVA and LSD test was performed. The results were reported as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 was considered as significant difference.