Myometrial Cells Stimulate Self-Renewal of Endometrial Mesenchymal Stem-Like Cells Through WNT5A/β-Catenin Signaling

Abstract

Human endometrium undergoes cycles of proliferation and differentiation throughout the reproductive years of women. The endometrial stem/progenitor cells contribute to this regenerative process. They lie in the basalis layer of the endometrium next to the myometrium. We hypothesized that human myometrial cells provide niche signals regulating the activities of endometrial mesenchymal stem-like cells (eMSCs). In vitro coculture of myometrial cells enhanced the colony-forming and selfrenewal ability of eMSCs. The cocultured eMSCs retained their multipotent characteristic and exhibited a greater total cell output when compared with medium alone culture. The expression of active β-catenin in eMSCs increased significantly after coculture with myometrial cells, suggesting activation of WNT/β-catenin signaling. Secretory factors in spent medium from myometrial cell culture produced the same stimulatory effects on eMSCs. The involvement of WNT/β-catenin signaling in self-renewal of eMSCs was confirmed with the use of WNT activator (Wnt3A conditioned medium) and WNT inhibitors (XAV939 and inhibitor of Wnt Production-2 [IWP-2]). The myometrial cells expressed more WNT5A than other WNT ligands. Recombinant WNT5A stimulated whereas anti-WNT5A antibody suppressed the colony formation, self-renewal, and T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcriptional activities of eMSCs. Moreover, eMSCs expressed FZD4 and LRP5. WNT5A is known to activate the canonical WNT signaling in the presence of these receptor components. WNT antagonist, DKK1, binds to LRP5/6. Consistently, DKK1 treatment nullified the stimulatory effect of myometrial cell coculture. In conclusion, our findings show that the myometrial cells are niche components of eMSCs, modulating the self-renewal activity of eMSCs by WNT5A-dependent activation of WNT/β-catenin signaling. STEM CELLS 2019;37:1455-1466 SIGNIFICANCE STATEMENTThis study demonstrated for the first time that myometrial cells are niche cells producing WNT5A, modulating the self-renewal activities of endometrial stem cells through activation of WNT/β-catenin signaling. Bone marrow stem cells transplantation is used to treat women with atrophic endometrium. The findings will facilitate in vitro expansion of eMSCs and provide an alternative cell source for autologous transplantation for treatment of atrophic endometrium. Understanding the niche regulation of endometrial stem cells may lead to the use of niche factors or their associated signaling molecules for treatment of women with atrophic endometrium or Asherman syndrome by stimulating self-regrowth of their endometrium.

Understanding the Regulatory Mechanisms of Endometrial Cells on Activities of Endometrial Mesenchymal Stem-Like Cells During Menstruation

“…Since the stimulatory effect of endometrial niche cells did not require direct contact with eMSCs, we examined two mechanisms of actions of endometrial niches cells, namely via WNT ligands and cytokines or chemokines. We have previously demonstrated the involvement of WNT signalling in endometrial stem cells renewal [9,10]. An elevation of total and active β-catenin in the eMSCs after coculture with menstrual phase endometrial cells con rmed the involvement of the WNT/β-catenin signalling in eMSCs regulation.…”

“…Twenty four hours after transfection, the medium was replaced with OptiMEM. The cells were then assayed using the WNT reporter system as described [9]. The knockdown e ciency was assessed by western blotting (Additional le: Figure S1J).…”

“…In mesenchymal stem cells, activation of the canonical WNT signalling pathway promotes proliferation [7] and controls cell fate [8]. We recently demonstrated that myometrial cells is a niche component of eMSCs, modulating their self-renewal activity by activation of WNT/β-catenin signalling [9]. There is an enhanced expression of active β-catenin in proliferating putative stromal stem/progenitor cells in postpartum mouse endometrium [10].…”

Revealing Cellular Heterogeneity andIn VitroDifferentiation Trajectory of Cultured Human Endometrial Mesenchymal-like Stem Cells Using Single-cell RNA Sequencing

“…For example, the WNT signaling pathway was identified in the undifferentiated/less differentiated clusters. Recently, we demonstrate that WNT5A is a niche factor that promotes self-renewal of eMSCs through FZD4 and canonical WNT-signaling [10]. Here other WNT receptors like FZD3 were identified, highlighting the importance of dissecting the heterogeneity of eMSCs.…”

“…Endometrial aspirates from the menstrual phase of three women and four full-thickness endometrium from the secretory phase of four women were used in this study (Table S1). After enzyme dispersion of the tissues, the CD140b + CD146 + cells were obtained from endometrial stromal cells by serial magnetic microbeading [8,10]. They were subjected to scRNA-seq on a 10X genomics platform.…”