The Role of Notch Signaling in Endometrial Mesenchymal Stromal/Stem-like Cells Maintenance

Abstract Background: Human endometrium undergoes cycles of regeneration in reproductive women and endometrial mesenchymal stromal/stem like cells (eMSC) contribute to this process. Notch signaling is essential for the homeostasis of somatic stem cells, however its role in eMSC remains largely unknown.Methods: RT-qPCR was used to compare the activity of Notch signaling between endometrial fibroblast (eSF) and eMSC. Flow cytometry, immunofluorescence, western blotting, in vitro clonogenicity and proliferation assays were used to assess the properties of eMSC after Notch signaling activation or inhibition. The crosstalk between Notch and Wnt signaling in eMSC was determined by western blotting, in situ proximity ligation assay and luciferase assay. Using a label-retaining cell method, hematoxylin-eosin staining, immunohistochemistry, immunofluorescence and western blotting were applied to characterize the features of label-retaining stromal cells (LRSC) in mouse menstrual-like model. Intrauterine injection of Notch inhibitor was performed at the time of breakdown to explore the potential role Notch signaling in endometrial regeneration in vivo.Results: The gain and loss function showed that activation of Notch signaling promoted eMSC maintenance, while inhibition displayed opposite effect. Activation of Notch pathway better maintained eMSC in a quiescent state. However, these quiescent eMSC can re-enter into the cell cycle depending on Notch and Wnt activity in the microenvironment, suggesting a crosstalk between two signaling pathways. In a mouse menstrual-like model, Notch signaling was highly involved in the dynamic endometrial remodeling event. Suppression of Notch signaling significantly reduced the proliferation of Notch1+ label-retaining stromal cells and consequently delayed the endometrial repair.Conclusions: These results demonstrate the importance of Notch signaling in regulating the endometrial stem/progenitor cells in vitro and in vivo. Understanding the precise mechanism of endometrial stem/progenitor cells provides a promising therapeutic approach for women with atrophic endometrium or Asherman syndrome.