Evaluation of the relationship between mesenchymal stem cells and immune system in vitro conditions

Mesenchymal stem cells (MSCs), are a novel therapeutic option as the most common cell source, play an important role in the immunomodulation. In this study, it was aimed to determine the effect of MSCs on cytokines secreted by the immune system cells. Intracellular cytokine levels (Interleukin-4 (IL-4), Interferon-γ (IFN-γ), and Interleukin-17 (IL-17)) detected by flow cytometry before and after co-culture between peripheral blood mononuclear cells (PBMCs) and MCSs. At the same time, supernatant cytokine levels were measured using the ELISA. In our study, MSCs were isolated from cord blood (CB) and Wharton’s Jelly (WJ), and their surface markers (CD44 (100%), CD73 (99.6%), CD90 (100%), CD105 (88%)) shown by flow cytometry method. Both CB-MSCs and WJ-MSCs were used in co-culture MSC/PBMC ratios of 1/5 and 1/10, incubation times of 24 h and 72 h. In the present study, when we compared co-cultures of CB-MSC or WJ-MSC with PBMCs, intracellular levels of cytokines IFN-γ, IL-17 (pro-inflamatory) and IL-4 (anti-inflamatory) were increased, and supernatant levels were decreased significantly (p < 0.05). The level of transforming growth factor beta (TGF-β) (anti-inflamatory) was significantly decreased for both CB-MSC and WJ-MSC in supernatant (p < 0.05). It was investigated pro-inflammatory and anti-inflammatory effects of CB-MSCs and WJ-MSCs on PBMCs with the obtained results. According to the results, MSCs demonstrated different immunologic effects after the incubation time and ratios. For further studies, it should be known between interaction of MSCs and immune system.


Introduction
Stem cells are a group of cells with a very high potential divided into different categories. They are categorized according to their potential characteristics and whether they are embryonic or not. Mesenchymal stem cells, a unique group of stem cells, are multipotent and nonembryonic. One of the sources of mesenchymal stem cells often used in studies is umbilical cord tissue. Although mesenchymal stem cell (MSC) is obtained from different sources such as; bone marrow, adipose, muscle, liver, umbilical cord derived tissues contain abundant MSCs [1,2]. MSCs have plastic adherence and spindle-shape morphology. They also can be distinguished with surface markers such as; CD73 + , CD90 + , CD105 + and the lack of CD14-, CD34-, and CD45- [3,4].
Mesenchymal stem cells bring new perspective to regenerative medicine and immune system. Mesenchymal stem cells have immunomodulatory properties in addition to being self-renewal, differentiation, and obtainable from many tissues [5][6][7][8][9].They secrete specific secretomes composed of various growth factors and cytokines and have immunomodulatory properties, e.g. through direct cellto-cell contact.
Due to their ability to balance the immune response and regulate its profiles, MSCs are preferred in studies to treat various immune-related diseases [10,11]. Umbilical cord blood derived-MSCs (CB-MSC) and Wharton's jelly derived-MSCs (WJ-MSCs) have a more significant cytokine secretion profile and have the potential for immunosuppressive and trophic effects due to the various growth factors and cytokines they produce [12,13].
This study investigated the effect of MSCs on immune system cells. The aim was to determine the intracellular cytokine levels (IL-4, interferon-γ (IFN-γ) and IL-17A) of CD4 + T cells before and after co-culture of PBMC and MSC by flow cytometry. The supernatant cytokine levels of TGF-β, IL-4, IL-17A, IFN-γ were determined by ELISA. It was also intended to compare the results obtained by the two methods. The ultimate goal of the study was to identify the immunomodulatory properties of MSCs, to investigate the effects of MSCs specifically on diseases of the immune system, and to use the results of the study to complete the first step for further in vitro or in vivo studies.

Materials and methods
This study was approved with the permission of the "Ethics Committee of Istanbul Faculty of Medicine" with the date, and no 27/05/2020-85426. Human umbilical cord blood and umbilical cord tissue used in the study were obtained with the formal approval after the informed consent form of the healthy pregnant women with no chronic diseases in the "Department of the Obstetrics of Istanbul Bakırköy Dr. Sadi Konuk Training and Research Hospital of the T.R. Ministry of Health". In the study, MSCs were obtained from Wharton's jelly and cord blood. 10 µg/ml phytohemagglutinin/72 h were used for cytokine stimulation. MSCs and PBMC were co-cultured. After 24 and 72 h, cytokine measurements were performed by flow cytometry and ELISA.

Collection of the cord blood samples, mesenchymal stem cell isolation and culture
After cesarean delivery, the blood was transferred into heparinised tubes by an authorized clinician. On average, up to 50-60 cc (7-8 heparinised tubes of 8-cc) blood was taken for each sample from the doctor in charge of the maternity hospital. The samples which were transferred to the cell culture laboratory in the possible shortest time were processed in a sterile cabinet. Cord blood samples were taken in anticoagulants (heparinised) involving approximately10 tubes, and mononuclear cells were isolated using Ficoll paque gradient method within 24 h. The cells were planted on 75 cm 2 flasks, and previously prepared 10% FBS, 1% pen/strep antibiotic DMEM medium was added and was left to expand in the incubator at 37 0 C, 5% CO 2 , and the medium was replenished 1 3 as required. The medium was replenished to remove other mononuclear cells from the cord blood 24 h after planting.

Collection of cord tissue (Wharton's jelly), mesenchymal stem cell isolation and culture
The tissue was taken into a sterile container and MSCs were isolated using explant method within 24 h. Tissue fragments were washed with a mixture of pre-prepared antibiotics (Streptomycin, Penicillin, Amphothericin B) with transfering and shaking procedure into 4 different 50 ml falcons. The last falcon was incubated in a shaker at 37 °C for 1 h. The tissue fragments were placed carefully and in regular intervals in the 6-well with the help of pliers. A round glass lamella was closed over the dried tissue fragments and left to dry. 10% FBS involving 4 ml of DMEM were added, and were left for MSC growth at 37 °C, 5% CO 2 incubator. After MSC proliferation, MSCs were placed into 75 cm 2 flask. The cells were removed by trypsinization and prepared for characterization when they were 80-90% confluent.

Characterization of the mesenchymal stem cell
After trypsinization, MSCs were washed once with PBS (centrifugation at 2000 rpm for 10 min). The Human MSC Analysis Kit was tested in accordance with the (BD-AB_2869404) protocol and analyzed by flow cytometry (Beckman Coulter-Navios).

Isolation and culture of peripheral blood mononuclear cells
Peripheral blood of about 20-30 cc taken from healthy individuals into heparinised tubes was processed in a sterile cabinet. PBMCs were isolated using the Ficoll-paque gradient method.
Before starting the experimental groups, the most appropriate dose and time for IL-4, IFN-γ, and IL-17 cytokine stimulation (STI) of the phytohemagglutinin (PHA) were determined as 10 µg/ml PHA-72 h. Cytokine relase level were measured by ELISA from supernatants and by flow cytometry methods for intracellular cytokine.

The isolated CB-MSC, WJ-MSC and PBMC co-culture
After the characterization, MSCs were expanded until the third passage at 37 °C, 5% CO 2 The PBMCs isolated by Ficoll-paque gradient density method and incubated at 37 °C in a humidified atmosphere with 5% CO 2 in the incubator for 24 h and 72 h at different doses with both CB-MSC and WJ-MSC. The counted cells were adjusted as 1/5 and 1/10 ratio (MSC/PBMC) and co-cultured on 6 well plate culture plates.
Determination of the intracellular cytokine was performed by Flow Cytometry and Human Th1/Th2/Th17 Phenotype Kit (BD-AB_2869360) was used. For cytokine determination of the supernatant, [human IFN-gamma ELISA (Diaclone-950.000.192), the Human TGF-β ELISA (Abbkine-Ket6030]; Human IL-4 ELISA (Diaclone-950.020.192); Human IL-17A ELISA (Diaclone-850.940.192) kits were used. Intracellular cytokine levels were analyzed by flow cytometry as percentaged (%) value. The cytokine levels measured from the supernatant were calculated and evaluated in pg/ml unit by taking absorbance values from the ELISA plaque reader.

Statistical analysis
In descriptive statistics of the data, the mean, standard deviation, median lowest, highest, frequency and ratio values were used. The distribution of the variables was measured by the Kolmogorov-Smirnov test. The Mann-Whitney U test was used for the analysis of quantitative independent data, and the Wilcoxon test was used for the analysis of dependent quantitative data. The Statistical Package for the Social Sciences (SPSS) 28.0 program was used for the analysis.

Characterization of the mesenchymal stem cell
The characterization of the mesenchymal stem cells that were isolated and expanded from cord blood and cord tissue were identified under microscopy for adherent property, and specific surface markers (CD44, CD90, CD105, CD73) were identified by using the flow cytometry analysis (Fig. 1). All experiments were performed in triplicate.

Comparison of the cytokine changes for WJ-MSC group
For all experiment the third passage of MSCs were used. The comparison of the WJ-MSC included groups and control group showed that while intracellular IL-17, IFN-γ, IL-4 levels increased significantly in the first 24 h, supernatant levels decreased at a ratio of 1/5. The level of supernatant TGF-β was found to have significantly increased in the treatment group (p < 0.05). However, in the first 72 h, only the intracellular IFN-γ level significantly decreased, while the level of all cytokines in the supernatant have decreased (p < 0.05). The comparison of the WJ-MSC included groups and control group showed that the intracellular IL-17, IFN-γ, IL-4 measurements at both 24 h and 72 h were significantly increased in the ratio of 1/10. Supernatant cytokine levels were found to be significantly lower at both 24 h and 72 h (p < 0.05) ( Fig. 2A, C).

Cytokine changes of co-cultures of MSC with STI-PBMC at ratio of 1/5 and 1/10 after 24 and 72 hours
Two different ratios were used as 1/5 and 1/10 in direct MSC/PBMC co-culture and different results were obtained. For 24 h incubation, the comparison of two ratios showed that the level of IFN-γ cytokines, for both intracellular and supernatant, decreased significantly at the ratio of 1/10 compared to the ratio of 1/5. At the end of 72 h, a significant increase was observed in the ratio of 1/10 compared to the ratio of 1/5. The comparison for IL-17 cytokine showed that the cytokine level in both intracellular cytokine and supernatant cytokine increased significantly in 1/10 ratio compared to 1/5 ratio for 24 h, and 72 h incubations. The 72 h comparison for IL-4 cytokine showed that the intracellular cytokine level at the ratio of 1/10 compared to the cell ratio of 1/5, and the cytokine level in the supernatant significantly decreased. The comparison for TGF-β cytokine showed that the ratio of 1/10was significantly decreased at the end of 24 h compared to the ratio of 1/5, and significantly increased at the end of 72 h (Tables 1, 2).

Discussion
Mesenchymal stem cell is a valuable cell group that is promising in the field of cellular therapy due to their selfrenewal, immunomodulatory properties, and multi-potential differentiation capacities. MSCs therapy offers a promising treatment option for autoimmune diseases, sepsis, and in transplant surgery. Although the in situ characteristics and biological functions of MSCs have not yet been elucidated, the in vitro cells were shown to have the potential to differentiate into specific cell lineages. Furthermore, the studies have shown that MSCs have a different capacity to effect on immune system and can modulate immune system interactions and suppress some inflammatory diseases. The researches have shown the therapeutic effect of MSCs on acute graft versus host disease (aGvHD) after stem cell transplantation [29].
In many studies, the researchers have been focused on the mechanism as secretion of cytokine and chemokine to detect anti-inflammatory and immunomodulatory effects of MSCs [30,31]. Cytokines act in an interrelated immune cascade with IFN-γ playing a central role. Blocking IFN-γ with MSCs is the safest and most effective method of treatment of Th1-mediated autoimmune diseases, although blocking other cytokines in the cascade may lead to some therapeutic effect as well. Most of the autoimmune diseases belong to Th1 and combined Th1/Th2 types, while most of the allergic diseases belong to Th2 variety. The response of MSC to proand anti-inflammatory cytokines such as IFN-γ, IL-1, TNF, or IL-4 has been shown to be due to its mediators it secretes (TGF, IDO, PGE2) and with the conversion of CD4 + T helper cells into the Treg cell phenotype [32]. T cells are key components, which are responsible for adaptive immune system and provides protection against malignancies, infections, autoimmune diseases. Many studies have been shown the effect of MSCs on T cells through inhibition [33]. In the current study, we aimed to investigate the effect of CB-MSC and WJ-MSC on PBMCs. Therefore, we determined the intracellular cytokine levels before and after co-culture of MSCs and PBMCs by flow cytometry and ELISA.
Luz-Crawford et al. have reported that MSCs suppressed the secretion of IL-17, IFN-γ however induced the IL-10 production of the T cells by antagonizing the differentiation of Th17, and Th1 cells and thus induced the Treg formation [34]. Yang et al. have demonstrated that IFN-γ was significantly suppressed in the comparison of WJ-MSCs administered PBMCs and PBMCs stimulated with PHA without WJ-MSC in the examination of immune system stimulants detected in cells and supernatants, which were collected after co-culture of PBMCs with WJ-MSC [26]. Riazifar et al. have found that in the presence of MSC-derived exosomes, the levels of several pro-inflammatory Th1 and Th17 cytokines including notably IL-6 (Th17 cytokine), IL-12p70 (Th1), IL-17AF (Th17), and IL-22 (Th17) were significantly reduced in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model [35]. In our study, the IFN-γ level in the supernatant after co-culture was found to have decreased significantly compared with the STI-PBMCs that were not performed WJ-MSC at the end of the hour 24, and hour 72. For the determination of the intracellular cytokine, PBMCs were obtained from culture plates and studied, and for the supernatant, the culture fluid containing MSC and PBMC after co-culture was collected and studied. We estimate that MSCs inhibited the cytokine release out of the cell from PBMCs as the reason for the difference in the amount of intracellular cytokine and supernatant cytokine amount released out of the cell. The MSCs isolated from Wharton's Jelly inhibited the intracellular release of the pro-inflammatory cytokine IFN-γ at the end of hour 72 in the 1/5 co-culture of WJ-MSC/PBMC. When comparing intracellular and supernatant IFN-g from CB-MSC, intracellular IFN-g levels increased significantly while supernatant IFN-g levels decreased compared to controls for both 24 h and 72 h incubation. The addition of CB-MSC on PBMC inhibited the IFN-γ cytokine values only in the supernatant.
Th17 cell subsets in the immune response express the chemokine receptor CCR6 and the transcription factor RARassociated receptor T (RORyT) as a distinguishing factor of Th17-specific T cells, in addition to expressing the cytokines IL-17A, IL-17F and IL-22. Th17 cells, which are involved in the pathogenesis of various inflammatory and autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus, Type 1 diabetes, and rheumatoid arthritis, play a role in insufficient or deficient T cell immune regulation. Although the mechanisms by which MSCs modulate the response of the Th17 cells subgroups, some studies have been reported to date but that is not enough to explain all mechanisms [36][37][38][39]. Mesenchymal stem cells can directly or indirectly inhibit Th17 differentiation and functions [40,41]. MSCs can inhibit Th17 differentiation by increasing the expression of CCL2, PD-1, IL-10, or SOCS3. Some studies show that MSCs may also promote Th17 proliferation [42][43][44]. Guo et al. have shown that MSCs isolated from bone marrow promote IL-17 expression and Th17 cell differentiation [45]. In our study, we also observed that WJ-MSC added at different rates on stimulated PBMC significantly increased the intracellular expression of IL-17 after co-culture at different times. As the incubation times of PBMC and MSC co-culture increased, the intracellular cytokine expression decreased. However, the cord-derived MSC showed the opposite effect and the intracellular level of IL-17 cytokines increased higher by 1/5 ratio of the cell. The examination of the IL-17 cytokine levels in the supernatant of MSC and PBMC co-culture showed significant inhibition in both WJ, and CB. Compared with the conducted studies, the level of IL-17 supernatant cytokines may be inhibited by MSC. On the other hand, further studies are needed for the intracellular cytokine level. It is suggested that where MSCs were isolated from, and the difference in their ratio to T cells may change their effect on cytokine levels.
Mesenchymal stem cells support the maturation, differentiation of Th2 cells and secretion of IL-4 from Th2 cells through the expression of IDO. Gieseke et al. have indicated that MSCs might directly suppress the T cell proliferation [46,47]. MSCs have a pro-inflammatory enhancing effect and an anti-inflammatory suppressive effect on the immune system. Aggarwal et al. have found in their study evaluating the supernatant values after co-culture of the MSCs isolated from the bone marrow with the T cells isolated from the BMC by 1/10, that the IL-4 level significantly increased [48]. In our study, the intracellular IL-4 ratio significantly increased in each group in the groups where CB-MSC was used. The level of IL-4 in the supernatant was significantly suppressed in the groups where both sources of MSCs were used.
T regulatory (Treg) cells are essential for the immune homeostasis by preventing autoimmunity. MSCs facilitate the formation of Tregs in vitro and in vivo. TGF-β1neutralization studies have shown that the generation of Tregs is TGF-β1-mediated and that MSCs constitutively secrete TGF-β1 [46,49,50]. TGF-β inhibits the expression of IL-2, MHC-II (major histocompatibility complex II) and T cells. Both Th1 differentiation and Th2 differentiation can be suppressed by TGF-β. In addition, the immunosuppressive effects of bone marrow-derived MSCs stimulated by TGF-β, IFN-γ and TNF-α were shown to have been eliminated by inhibiting the expression of iNOS and IDO [51,52]. Yang et al. have shown that WJ-MSCs and PHA-PBMC stimulated by co-culture supernatant detected in collected PGE2, TGF-ß1 and IL-10 Treg immune system on examining stimuli, WJ-MSCs with PBMC with WJ-MSCs without PBMC stimulated with PHA when compared to PGE2, TGF-ß1 and IL-10 have shown that greatly increases the number of Treg cells. Along with this finding, they emphasized that WJ-MSCs have an immunosuppressive effect by increasing the production of suppressive cytokines [26]. Wang et al. have found that the Th1 and Th17 cells were suppressed, and the count of the Th2 and Treg cells increased after 5-day co-culture of 1/5 ratio of stimulated WJ-MSC/stimulated PBMC [53]. In our study, the TGF-β cytokine level was determined by ELISA from the supernatant after co-culture in all experimental groups. The level of TGF-β1 cytokines in the supernatant showed an important and significant increase after 24-h co-culture of WJ-MSC/PBMC by 1/5 ratio. The study of Wang et al. supports our study and the level of TGF-β cytokines secreted from Treg cells increased significantly by 1/5 ratio [53]. We suggest that the reason for obtaining different results than other studies could be due to the use of stimulated MSCs, and co-cultures times were longer, and were suppressed in our other groups.
Deuse et al. have emphasized that the use of higher ratio of MSC inhibits T cell proliferation, and the use of MSC in smaller ratio contributes positively to T cell proliferation. They suggested that the ratios of PBMC and MSC in co-culture may have different immunomodulation effects by affecting T cell proliferation, and that cell-cell interaction is not essential in their study [37]. In our study, we used at 1/5 and 1/10 ratios in direct MSC/PBMC co-culture, and different results were obtained at the ratios. Our study supports the results of Krampera et al. They emphasized that a higher amount of MSC has a greater ability to suppress T cell [54]. The comparison of these two different ratios showed that the level of IL-17 cytokine was significantly increased both in intracellular cytokine and supernatant cytokine at the end of 24 h and 72 h at 1/10 cell ratio compared to the levels at 1/5 cell ratio. The levels of IL-4 intracellular cytokine and TGF-β cytokine in the supernatant significantly increased at 72 h 1/10 ratio compared with the levels at 1/5 ratio. We suggest that administration of a higher amount of MSC on PBMC increases cytokine production and secretion by increasing T cell proliferation. In our study, while the count of MSC remained constant, the PBMC count was proportionally increased. In co-cultures where MSC was relatively higher, it stimulated T cell proliferation, which led to higher cytokine expression and secretion.
The MSCs derived from adipose tissue and bone marrow are generally used in the studies of mesenchymal stem cells on the immune system. In this study, the use of Wharton's Jelly and cord blood as a source of MSC and the investigation of its effect on the immune system provided a different perspective to the literature. The effect of different sources of MSC on the immune system in the study was evaluated in different combinations in the form of using 2 different cell ratios of 1/5 and 1/10, and two different incubation periods of 24 h and 72 h.
Despite all this promising research, the presence of ambiguous results in clinical studies with MSC, caused by the variability of MSC populations on the immune system hinders the progress of the studies. Therefore, we believe that examining both intracellular and supernatant levels of pro-and anti-inflammatory cytokines in our study brings a new perspective to this question. There is no available previous study on how MSC affects the level of intracellular cytokines in the cells of the immune system; the study is original in this respect. However, our study has some limitations. We only explored the levels of cytokines intracellular and in supernatant to determined the immunomodulatory function of CB-and WJ-MSCs but did not address the relation between all mechanism for immunoregulation. Furthermore, the results of this study can provide a novel theoretical basis for the understanding of the relationship between immune system and MSC and provide a new strategy improve the therapeutic efficacy of MSCs in inflammatory diseases.
The interaction of MSC and immune system cells initiates a complex process. In order to elucidate this complex process, the correct and appropriate selection of the source of MSC can be possible by specifically identifying the subgroups of cells belonging to the immune system, as well as in in vitro and in vivo studies. MSCs are very heterogeneous and can change significantly with pro-inflammatory or antiinflammatory stimuli. Therefore, it is difficult to determine how MSC variability influences their induced immunomodulatory effects. It is possible that viable MSCs provoke more complex immunomodulatory mechanisms because of their intact secretome.
Conclusion, the immunomodulatory function of WJ-MSCs could be more benefit for potential clinical immunotherapy than CB-MSCs. The immunomodulatory effects of WJ-MSC in different autoimmune diseases need further research. The hallmark of this study is the identification of putative mechanisms by which different MSCs cause immunemodulation.