Microenvironment and its regulation on endometrial mesenchymal stromal/stem-like cells

Abstract

Human endometrium undergoes cyclical shedding and remodeling in response to the steroid hormonal levels. The endometrial mesenchymal stromal/stem-like cells (eMSC) play crucial roles during endometrial regeneration and do not vary in frequency during the menstrual cycle. Stem cells often remain quiescent and undifferentiated when residing within the niche. The perivascular location of eMSC suggests that the endometrial stem cell niche is associated with blood vessels. However, the underlying mechanisms of eMSC responses to signals in the microenvironment are poorly understood. In this study, I hypothesized that endometrial stem cells can respond to specific molecules released by the microenvironment for the long-term maintenance of tissue hemostasis. The first objective was to investigate the molecular mechanism of eMSC responses to myometrial secreted WNT5A. Among different WNT receptors, Frizzled -5 (FZD5) was abundantly expressed in CD140b+CD146+ eMSC when compared to unfractionated stromal cells. Co-expression of FZD5 and co-receptor LRP5 was confirmed in majority of eMSC population. Both gene silencing of FZD5 and blockage of LRP5 reduced the combination of eMSC with WNT5A and further inhibited the stimulatory effect of WNT5A on clonogenicity and phenotypic expression of eMSC. The WNT5A-FZD5-LRP5 complex regulated the self-renewal of eMSC through activating of WNT/β-catenin signaling pathway and a similar phenomenon was observed in gestational and postpartum mouse endometrial tissues. The second objective was to evaluate the interplay between WNT/β-catenin and the Hippo signaling in eMSC by assessing the protein expression of their major components. The results showed that activities of Hippo signaling exhibited an antagonistic effect on WNT signaling in eMSC, and Hippo bioactive compounds such as CIL56 affected the proportion of eMSC, indicating a potential role of Hippo signaling on the cell fate of stem cells. The third objective was to investigate the therapeutic potential of eMSC using the mouse endometrial injury model. Endometrial injury was induced using the electrocoagulation method and the regeneration of injured mouse endometrium with or without intrauterine eMSC transplantation were compared. Evaluation of morphological reconstruction revealed that eMSC transplantation increased the endometrial thickness, stimulated cell proliferation, promoted regeneration of endometrial compartments, relieved endometrial fibrosis and regulated inflammatory reactions in mouse endometrium. Fertility test confirmed the better endometrial receptivity of newly emerged endometrial lining with eMSC transplantation. The fourth objective was to explore the role of interleukin-6 (IL-6) in endometrial stem cell regulation. The level of IL-6 secretion from endometrial stromal cells was high at menstruation and under hypoxic conditions. Functional assays revealed the stimulatory effects of exogenous IL-6 on self-renewal, proliferation and migration of eMSC in vitro, suggesting the involvement of IL-6 in endometrial regeneration. Activation of WNT/β-catenin signaling pathway was observed upon IL-6 treatment, suggesting the interplay of IL-6 and WNT signaling in modulation of eMSC biological activities. The therapeutic potential of IL-6 and WNT5A on endometrial regeneration was also uncovered by using the established mouse endometrial injury model. In summary, niche components secrete regulatory molecules upon physiological signals. The responses of eMSC to these molecules result in the alterations of their properties for maintaining endometrial hemostasis involving intricate signaling networks.