Characterization of endometrial mesenchymal-like stem cells activity in different endometrial layers and during the menstrual cycle

Abstract

The human endometrium is a highly dynamic tissue with the ability to cyclically regenerate during the reproductive life. Endometrial mesenchymal-like stem cells (eMSCs) have shown to functionally contribute to endometrial regeneration. However, the properties of eMSCs in different layers of the endometrium (functionalis vs. basalis) and during the menstrual cycle have not been characterized. In this study, we evaluated the percentage and properties of the eMSCs from different layers of the endometrium and in different menstrual phases. Full thickness endometrial tissues were obtained from women undergoing hysterectomy. To isolate eMSCs in different endometrial compartments, functionalis was separated from basalis by gentle scraping on the superficial layer of the tissue. To assess eMSCs across the menstrual cycle, samples were collected from menstruation, proliferative and secretory stage. Single cell suspension was obtained by enzymatically digestion and red blood cells and leukocytes were removed by ficoll-paque and CD45 dynabeads, respectively. Using flow cytometry, the proportion of eMSCs from freshly isolated endometrial cells was identified by co-expression of CD140b and CD146 cells. EMSCs isolated by sequential beading with magnetic beads coated with anti-CD140b and anti-CD146 antibodies were used for functional assays. Clonogenic assay was performed by seeding eMSCs at low density and large colony-forming units were harvested after 15 days in culture for serial sub-cloning and total cell output assays. Assessment of eMSCs revealed that the basalis (1.08±0.52%) contained significantly more eMSCs than the functionalis (0.18±0.09%, n=4, P11, n=5) produced larger cumulative population than the secretory phase (1.10±0.61 x 109, n=4; p In conclusion, more eMSCs reside in the basalis but their stem cell properties are similar throughout the endometrial layers. The percentage and clonogenic ability of eMSCs were constant in the menstrual cycle. However, eMSCs in the menstruating phase exhibited the greatest self-renewal ability and yielded a higher output of cells than those from other phases of the cycle. Our finding suggests that eMSCs become activated in menstruation phase for cyclical regeneration of the endometrium.