The regulatory role of Notch signaling in the maintenance of endometrial mesenchymal stromal/stem-like cells (EMSC)

Abstract

In reproductive women, endometrium undergoes cycles of regeneration and endometrial mesenchymal stromal/stem like cells (eMSC) are responsible for this process. A fundamental mechanism of stem cell regulation is signals from their immediate microenvironment or niche. There are very limited studies investigating the role of niche signals in regulating the properties of endometrial stem/progenitor cells. A variety of intracellular signaling pathways such as Notch, BMP, Wnt, JAK/Stat have been implicated to play a role in stem cell regulation. In this project, I hypothesized that Notch signaling is a key regulator in endometrial stem/progenitor cells.

In the first Chapter, I investigated the role of Notch signaling in the maintenance of eMSC activities. The gain and loss function of Notch signaling showed that activation of Notch signaling promoted phenotypic expression of eMSC, while inhibition displayed the opposite effect. The stimulatory effect of Notch signal by JAG1 was reduced after gene silencing of Notch1 in eMSC. Activation of Notch pathway better maintained eMSC in a quiescent state, which can be reversed by WNT/β-catenin pathway. Using a mouse model of stimulated menses, I further demonstrated the action of Notch signaling in vivo during dynamic endometrial remodeling events. DAPT – a Notch inhibitor significantly reduced the proliferation activity of Notch1+ label retaining stromal cells (LRSC) and consequently delayed endometrial repair process.

In the second Chapter, I demonstrated for the first time that extracellular vesicles (EVs) participated in the intercellular communications between eMSC and their niche cells. Myometrial derived EVs significantly enhanced the self-renewal and clonogenicity of eMSC while stromal derived EVs showed little effect. Further investigation demonstrated that the stimulatory effect of myometrial EVs on eMSC maintenance was mediated by the Notch ligand JAG1 packaged into these EVs. Pharmacological inhibition of Notch signaling or suppression of JAG1 in EVs reversed their positive regulating effect on eMSC activities. Moreover, transplantation of eMSC with myometrial derived EVs promoted endometrial regeneration in a mouse intrauterine injury model.

In the third chapter, I explored whether eMSC resided in a hypoxic niche and investigated the role of hypoxia in stem cell maintenance. In a mouse model of simulated menses, the relationship between LRSC and endometrial hypoxia was evaluated. My findings demonstrated that LRSC localized in a hypoxic microenvironment during endometrial breakdown and early repair. Immunofluorescent staining further revealed that these hypoxic-located LRSC underwent proliferation and co-expressed with Notch1. In vitro studies illustrated that hypoxia significantly facilitated the self-renewal capacity, clonogenicity and proliferation of eMSC by activating Notch signaling. More importantly, HIF-1α played an essential role in the hypoxia-mediated maintenance of eMSC through the activation of Notch signaling.

Overall, findings from this thesis demonstrate the importance of Notch signaling in the maintenance of endometrial stem/progenitor cells in vitro and in vivo. The stemness of eMSC are likely to be mediated by myometrial derived EVs carrying JAG1 through the activation of Notch signaling. During menstruation, the endometrial stem/progenitor cells are exposed to a hypoxic niche. This hypoxic condition can regulate the self-renewal activity of eMSC through the HIF-1α/Notch signaling pathway.