Determination of the role of antioxidant molecules in differentiation processes of wharton's jelly mesenchymal stem cells into endothelial cells

Introduction and aim: The success of stem cell therapy in regenerative medicine is related to the ability of stem cells to survive and to restore damaged tissues in a stressful environment. When differentiating stem cells into functionally mature adult cells, different metabolic processes are involved in the cell. It is suggested that one of the most important factors triggering the process of possible metabolic change in the process of stem cell differentiation is the oxidative stress to which the cells are exposed. However, oxidative metabolism changes during the differentiation of stem cells to endothelial cells have not been clearly defined. In our study, it was aimed to determine the reactive oxygen species (ROS) levels and antioxidative enzyme activity changes in the differentiation process of multipotent mesenchymal stem cells (MSCs) to endothelial cells. Also to determine the effects of the applications that may be effective on oxidative stress on cell viability and differentiation process of WJ-MSCs into endothelial cells were aimed. Methods: MSCs were obtained from Wharton's jelly of the umbilical cord by enzymatic and explant method and their proliferation capacity was compared. The gene and protein expression levels of CD44, CD90, CD105 surface antigens were determined by quantitative PCR, immunohistochemistry and flow cytometry methods. 2 mM N-acetyl cysteine (NAC), 300 uM ascorbic acid (AAP) and 40 nM sodium selenite (NaSe) were applied to investigate the effect of antioxidant molecules on the differentiation of MSCs to endothelial cells. In addition, 200 μM H2O2 was applied to determine the oxidative stress that is thought to affect the differentiation process. Fluorometric measurement was performed by using carboxymethyl dichlorofolorosein diacetate (CM-H2DCFDA) probe to determine ROT level against oxidative stress in WJ-MSCs. MDA analysis was also performed by HPLC method to determine cellular oxidative stress damage. The change in catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities in the differentiation process were evaluated by using colorimetric enzyme activity measurement kits. Antioxidant molecules and changes in gene expression of CAT, SOD and GPx enzymes against H2O2 were analyzed by quantitative PCR. Results: In the experiments conducted under WJ-MSC and entothelial cell characterization, the number of advanced passages (> 4-5) was found to lead to a decrease in MSC characterization. It was observed that the treatment with NAC(2mM) increased the cell viability in WJ-MSCs (p <0.05). In endothelial cells, NAC and ascorbic acid application increased cell viability by ~ 40% and ~ 20% respectively, whereas Na-selenite application did not have a positive effect. It was determined that total ROT and MDA levels in endothelial cells increased by ~ 7 and ~ 2-fold, respectively, according to WJ-MSCs. In the endothelial differentiation process, it was shown that catalase and glutathione peroxidase enzyme activities increased significantly (p <0.05). It was shown that three-hour treatment with H202 (300 uM) decreased 60% of stem cell viability compared to control group , significantly decreased antioxidant enzyme levels ,induced oxidative stress in WJ-MSC. NAC application protected WJ-MSCs against to H202 toxicity, but in the endothelial differentiated cells, oxidative stress level was not to be fully suppressed. Conclusion: As a result, according to our literature reviews, the effects of NaSe, AAP and NAC on WJ-MSCs' self-renewal and endothelial differentiation process were shown for the first time.The isolation protocol of WJ-MSCs was optimized in our laboratory conditions and the MSC characters of the cells were shown at the level of protein and gene expression.The endothelial differentiation protocol was made available in our laboratory conditions and metabolic changes due to oxidative stress were determined in this process. The data and protocols obtained by our study are thought to be the basic step for the projects that can be planned later in the scope of regenerative medicine applications. Keywords: Wharton's Gel, Mesenchymal Stem Cell, Endothelial Differentiation, Oxidative Stress