## 3 Results
### Impact of CMSCs on Lymphocytes Proliferation  
The immunomodulatory effect of CMSCs on activated PHA lymphocytes that were fluorescently labelled with CFSE was studied. CMSCs interacted with lymphocytes in co-culture in vitro (see Figure 5a,b). Lymphocytes proliferation assays are based on the ability of lymphocytes to proliferative when activated. CFSE has been widely used for investigating lymphocyte division and mitotic activity [34]. Carboxyfluorescein diacetate succinimidyl ester passively diffuses into cells. Each new generation in a population of proliferating lymphocytes is marked by twofold decrements in cellular fluorescence intensity, which can be detected by flow cytometry. Proliferation of three types of groups of CFSE labeled PHA lymphocytes were compared. Groups of lymphocytes and co-cultured with CMSCs _Poly (I:C)_, lymphocytes with CMSCs without stimulation and lymphocytes alone were compared. The inhibitory effect of CMSCs was tested after 4 days of co-culture with PHA lymphocytes. CMSCs were co-cultured at a ratio (1:4) to activated lymphocytes. The ability to influence proliferation was tested in the PHA lymphocyte group. The impact on proliferation was recorded by measuring the change in CFSE fluorescence intensity (see Figure 6).  
A suppressive effect on lymphocyte proliferation was observed after 4 days of co-culture of the following groups of cells. CMSCs were able inhibit the proliferation of activated lymphocyte independently of _Poly (I:C)_ activation. Statistically significant differences were detected between experimental groups lymphocytes (LY), groups: LY/CMSCs _Poly (I:C)_ and LY/CMSCs against the group of lymphocytes cultured alone. Post hoc analysis confirmed the statistical differences in significance level between the compared group of lymphocytes co-cultured LY/CMSC _Poly (I:C)_ (_p_ = 0.0028) and the lymphocytes LY/CMSC (_p_ = 0.0243). In both groups the percentage difference in proliferation was statistically significant and confirmed by the test (ANOVA, \(p\) = 0.0052) (see Figure 7a). No statistical significance was confirmed between lymphocytes and CMSCs _Poly (I:C)_ and lymphocytes with CMSCs (_p_ = 0.6645). The CMSCs groups and the group lymphocytes alone cultured. The \(\Delta\) proliferating fraction (\(\Delta\)PF) was calculated by subtracting the mean background proliferation from the mean proliferating  
Figure 4: (**a**) Phenotype expression of markers of the phytohemagglutinin (PHA) lymphocyte. Histograms represent mean \(\pm\) S.E of seven independent experiments and (**b**) representative flow cytometry analysis for lymphocytes.  
fraction in response to stimulation: \(\Delta\)PF\({}_{1}\) = [%CFSE Ly CMSCs _Poly (I:C)_] - [%CFSE Ly] and \(\Delta\)PF\({}_{2}\) = [%CFSE Ly(CMSCs)] - [%CFSE Ly]. The stimulation index (SI) was calculated as the percentage of Ly CMSCs _Poly (I:C)_ divided by the percentage Ly or calculated as the percentage of Ly (CMSCs) divided by the percentage Ly. Change in proliferation was marked as significant at both the levels \(\Delta\)PF \(\geq\) 1% and SI \(\geq\) 3.0. Measurements found the mean \(\Delta\)PF1 = 29.61 \(\pm\) 18.93% and the mean \(\Delta\)PF2 = 25.75 \(\pm\) 18.49%. Paired t-test showed that statistically there was no significant difference between \(\Delta\)PF1 and \(\Delta\)PF2 (\(p\) = 0.1380) (see Figure 7b).  
Figure 5: (**a**) CMSCs interact with lymphocytes in co-culture in vitro. CMSCs are adherent spindle shapes and lymphocytes have round shape (20\(\times\) magnification of objective). (**b**) Activated PHA lymphocytes cultured separately (20\(\times\) magnification of objective).  
Figure 6: Flow cytometry analysis of carboxyflurescenin succinidyl ester (CFSE) fluorescence intensity of three groups of co-cultured PHA lymphocytes CFSE and CMSCs. Inhibitory effect of CMSCs after 4 days of co-culture. Representative flow cytometry analysis for lymphocytes.