Background: The identification of endometrial stem/progenitor cells in a high turnover rate tissue suggests that a well-orchestrated underlying network controls the behaviour of these stem cells. The thickness of the endometrium can grow from 0.5-1 mm to 5-7 mm within a week indicating the need of stem cells for self-renewal and differentiation during this period. The cyclical regeneration of the endometrium suggests specific signals can activate the stem cells during or shortly after menstruation.
Methods: Endometrial mesenchymal stem-like cells (eMSCs) were cocultured with endometrial epithelial or stromal cells from different phases of the menstrual cycle; the clonogenicity and the phenotypic expression of eMSC markers (CD140b and CD146) were assessed. The functional role of WNT/β-catenin signalling on eMSC was determined by western blot analysis, immunofluorescent staining, flow cytometry, quantitative real-time PCR and small interfering RNA. The cytokine levels in the conditioned medium of epithelial or stromal cells cocultured with eMSCs were evaluated by enzyme-linked immunosorbent assays.
Results: Coculture of endometrial cells (epithelial or stromal) from the menstrual phase enhanced the clonogenicity and self-renewal activities of eMSCs. Such phenomenon was not observed in niche cells from the proliferative phase. Coculture with endometrial cells from the menstrual phase confirmed an increase in expression of active β-catenin in the eMSCs. Treatment with IWP-2, a WNT inhibitor, suppressed the observed effects. Anti-R-spondin-1 antibody reduced the stimulatory action of endometrial niche cells on WNT/β-catenin activation in the T cell factor/lymphoid enhancer-binding factor luciferase reporter assay. Moreover, the mRNA level and protein immunoreactivities of leucine-rich repeat-containing G-protein coupled receptor 5 were higher in eMSCs than unfractionated stromal cells. Conditioned media of endometrial niche cells cocultured with eMSCs contained increased levels of C-X-C motif ligand 1 (CXCL1), CXCL5 and interleukin 6. Treatment with these cytokines increased the clonogenic activity and phenotypic expression of eMSCs.
Conclusions: Our findings indicate a role of WNT/β-catenin signalling in regulating activities of endometrial stem/progenitor cells during menstruation. Certain cytokines at menstruation can stimulate the proliferation and self-renewal activities of eMSCs. Understanding the mechanism in the regulation of eMSCs may contribute to treatments of endometrial proliferative disorders such as Asherman's syndrome.
Keywords: Cytokines; Endometrium; LGR5; Menstruation; RSPO1; Stem cells; WNT signalling.