Comparison of enzymatic and non-enzymatic isolation methods for menstrual blood mesenchymal stem cells in maintenance of stemness

Endometrium is a thin epithelial layer located on the inside of the uterus and provides the ground for the embryo. Endometrium layer becomes suitable for the embryo every month for pregnancy, sheds with menstruation in cases where there is no pregnancy and prepares the endometrial layer for the pregnancy again. Presence of endometrial mesenchymal stem cells (eMSCs) in the endometrial layer brought about the possibility of stem cells in the menstrual blood (MenSCs). Early studies that identified MenSCs stated that these cells showed mesenchymal stem cell characteristics and hence possessed potential to be used in clinical studies due to their ease in isolation. Although different methods were employed in the isolation of MenSCs in the literature, up to now none of these isolation methods have not been compared in terms of preserving the stem cell properties. In this context, the purpose of this thesis is to compare the three different isolation methods including non-enzymatic, collagenase type 1 and dual digestion method and investigate the potential of MSCs isolated from menstrual blood to maintain their stemness until late passages. No difference in cell morphology was observed when the isolation methods were compared. Similarly, analysis of surface markers by flow cytometry showed that stem cell characteristics were preserved in early and late passage MenSCs and there was no difference between the isolation methods. In terms of colony forming feature for MenSCs, cells isolated with dual digestion method demonstrated an advantage compared to the other protocols at early passage. When cell proliferation rate was analysed using WST-1 assay, it was seen that the best isolation method in early passage cells was the non-enzymatic method. However, the decrease in the proliferation rate recorded for the late passage was evident in all MenSCs regardless of the isolation method they were derived from. As a result, for the first time in the literature, isolation methods of MenSCs were compared in terms of stemness characteristics and our results suggested non-enzymatic approach as the best isolation method.