Rachel Wah Shan Chan

Background The human endometrium is a regenerative tissue relying on stem/progenitor cells. Endometrial mesenchymal stem cells (eMSCs) are typically enriched using perivascular markers like CD140b and CD146. However, the identity of more primitive and quiescent eMSC subpopulations remains unclear. Methods We performed single-cell RNA sequencing (scRNA-seq) on cultured CD140b⁺CD146⁺ eMSCs and integrated this with published scRNA-seq data of primary human endometrial cells. We identified a LEPR⁺ subpopulation and analyzed its characteristics through in vitro assays, flow cytometry, immunostaining, and bioinformatic tools including cell-cell interaction analysis and pseudotime trajectory inference. Results A LEPR⁺ eMSC subpopulation was found to reside at the root of the differentiation trajectory and showed high expression of Notch receptors. These cells exhibited quiescent features, resided predominantly in the G0 phase, and demonstrated superior clonogenic and self-renewal capacity compared to LEPR⁻ eMSCs and bulk eMSCs. Notch signaling, particularly via JAG1 and DLL1, was implicated in maintaining the LEPR⁺ phenotype and quiescence. Conclusions LEPR⁺ eMSCs represent a primitive, quiescent subset of human endometrial stem cells. Notch signaling maintains their stemness and quiescence, suggesting therapeutic relevance for endometrial regeneration.

The extracellular matrix (ECM) constantly remodels to tailor a temporal spatial specific environment for the residing cells to respond to physiological or pathological stimuli. Endometrial mesenchymal stem cells (eMSC) are excellent therapeutic candidates for treating endometrial problems. In‐depth investigation of the native niche to understand the regulatory mechanisms of the stem cells will enable greater translational potentials in regenerating the thin or damaged endometrium. To understand the ECM niche of eMSC, endometrial ECM from full thickness human endometrial tissues at different menstrual phases are preserved by tissue decellularization and then transformed into hydrogel material (EndoGel). EndoGel exhibits excellent compatibility with eMSC by enhancing the expansion of eMSC in vitro and facilitating the therapeutic regenerative effect in vivo evidenced by the improved fertility outcome. Comparative study of the proliferative to secretory phase EndoGel reveals unique matrisome at specific phase of the human menstrual cycle. The post‐regenerated endometrium shows distinct transcriptomic profile when transplanted with different menstrual phase EndoGel, suggesting the regulatory effect of the tissue matrix is menstrual phase specific. This is the first study comparing the endometrial matrix from specific human menstrual cycle and exploring its therapeutic potentials as a supportive biomaterial for eMSC to enhance endometrial regeneration.

In the human endometrium, studies show the importance of extracellular vesicles in mediating various physiological as well as pathological processes. We have demonstrated that the myometrial cells are candidate niche cells of the endometrial mesenchymal stem/stromal cells (eMSC) modulating their biological function. The Notch signaling pathway regulates the endometrial stem cell functions. Although classical Notch signaling relies on direct cell contract for actions, this pathway can also be activated at a distance by Notch ligands containing extracellular vesicles (EV). We hypothesized that certain Notch ligand(s) are packaged into the myometrial EV to mediate stem cell functions. Endometrial samples were obtained from women undergoing total abdominal hysterectomy. Endometrial MSC (CD140b+CD146+ cells) were cocultured with myometrial EV and the percentage of eMSC was analysed by flow cytometry. Blockage of the secretion of EV was performed by transfection of RAB27 A siRNA. Western blot analysis and gene silencing approach were used to validate the role of Notch signaling in eMSC. The therapeutic features of transplanted eMSC/myometrial EV was determined using a mouse injured endometrium model. EV released from myometrial cells could be internalized by eMSC, leading to a significant stimulatory effect on the self-renewal and clonogenic activity of eMSC. Pharmacological inhibition of Notch signaling with DAPT or silencing of NOTCH 1 nullified the stimulatory effects. Myometrial EV contains a high amount of the Notch ligand – JAG1, thus inducing a strong Notch activity in eMSC. When JAG1 was silenced in the myometrial EV, the self-renewal and clonogenic activity was reduced. Combined transplantation of eMSC with myometrial EV improves the therapeutic effect of eMSC in endometrial regeneration in vivo. The observed therapeutic feature was potentially achieved by elevating the cell proliferation and suppressing apoptosis in the injured mouse endometrium. This study identifies a novel EV mediated communication axis between the myometrial cells and the eMSC, providing new insights into endometrial regeneration. The findings highlight the potential of eMSC and myometrial EV as a therapeutic strategy for women with intrauterine adhesions and other endometrial disorders.

Background: At menstruation, the functional layer of the human endometrium sheds off due to the trigger of the release of inflammatory factors, including interleukin 6 (IL-6), as a result of a sharp decline in progesterone levels, leading to tissue breakdown and bleeding. The endometrial mesenchymal stem-like cells (CD140b⁺CD146⁺ eMSC) located in the basalis are responsible for the cyclical regeneration of the endometrium after menstruation. Endometrial cells from the menstruation phase have been proven to secrete a higher amount of IL-6 and further enhance the self-renewal and clonogenic activity of eMSC. However, the IL-6-responsive mechanism remains unknown. Thus, we hypothesized that IL-6 secreted from niche cells during menstruation regulates the proliferation and self-renewal of eMSC through the WNT/β-catenin signaling pathway.Methods: In this study, the content of IL-6 across the menstrual phases was first evaluated. Coexpression of stem cell markers (CD140b and CD146) with interleukin 6 receptor (IL-6R) was confirmed by immunofluorescent staining. In vitro functional assays were conducted to investigate the effect of IL-6 on the cell activities of eMSC, and the therapeutic role of these IL-6- and WNT5A-pretreated eMSC on the repair of injured endometrium was observed using an established mouse model.Results: The endometrial cells secrete a high amount of IL-6 under hypoxic conditions, which mimic the physiological microenvironment in the menstruation phase. Also, the expression of IL-6 receptors was confirmed in our eMSC, indicating their capacity to respond to IL-6 in the microenvironment. Exogenous IL-6 can significantly enhance the self-renewal, proliferation, and migrating capacity of eMSC. Activation of the WNT/β-catenin signaling pathway was observed upon IL-6 treatment, while suppression of the WNT/β-catenin signaling impaired the stimulatory role of IL-6 on eMSC activities. IL-6- and WNT5A-pretreated eMSC showed better performance during the regeneration of the injured mouse endometrium.Conclusion: We demonstrate that the high level of IL-6 produced by endometrial cells at menstruation can induce the stem cells in the human endometrium to proliferate and migrate through the activation of the WNT/β-catenin pathway. Treatment of eMSC with IL-6 and WNT5A might enhance their therapeutic potential in the regeneration of injured endometrium.

Background: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. Methods: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. Results: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. Conclusions: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

Human endometrium undergoes cycles of regeneration in women of reproductive age. The endometrial mesenchymal stromal/stem cells (eMSC) contribute to this process. Notch signaling is essential for homeostasis of somatic stem cells. However, its role in eMSC remains unclear. We show with gain- and loss-of-function experiments that activation of Notch signaling promotes eMSC maintenance, while inhibition induces opposite effect. The activation of Notch signaling better maintains eMSC in a quiescent state. However, these quiescent eMSC can re-enter the cell cycle depending on the Notch and Wnt activities in the microenvironment, suggesting a crosstalk between the two signaling pathways. We further show that the Notch signaling is involved in endometrial remodeling event in a mouse menstrual-like model. Suppression of Notch signaling reduces the proliferation of Notch1⁺ label-retaining stromal cells and delays endometrial repair. Our data demonstrate the importance of Notch signaling in regulating the endometrial stem/progenitor cells in vitro and in vivo.

Human endometrium is a unique adult tissue that undergoes cyclical shedding, repair, and regeneration during a woman’s reproductive life. Over the past 2 decades, tremendous progress has been made towards the identification and characterization of endometrial stromal stem/progenitor cells . The substantial regeneration of vascularized stroma in the endometrium during the proliferative stages of each menstrual cycle is likely to be mediated by endometrial mesenchymal stromal/stem cells. This review focuses on the perivascular niche for CD140b⁺CD146⁺ pericytes and SUSD2⁺ perivascular cells. The identity, characteristics, and underlying mechanisms of uterine regeneration are also discussed.

Human endometrium is an incredibly dynamic tissue undergoing cyclic regeneration and shedding during a woman’s reproductive life. Endometrial mesenchymal stromal/stem-like cells (eMSC) contribute to this process. A hypoxic niche with low oxygen levels has been reported in multiple somatic stem cell types. However, the knowledge of hypoxia on eMSC remains limited. In mice, stromal stem/progenitor cells can be identified by the label-retaining technique. We examined the relationship between the label-retaining stromal cells (LRSC) and hypoxia during tissue breakdown in a mouse model of simulated menses. Our results demonstrated that LRSC resided in a hypoxic microenvironment during endometrial breakdown and early repair. Immunofluorescence staining revealed that the hypoxic-located LRSC underwent proliferation and was highly colocalized with Notch1. In vitro studies illustrated that hypoxia activated Notch signaling in eMSC, leading to enhanced self-renewal, clonogenicity and proliferation of cells. More importantly, HIF-1α played an essential role in the hypoxia-mediated maintenance of eMSC through the activation of Notch signaling. In conclusion, our findings show that some endometrial stem/progenitor cells reside in a hypoxic niche during menstruation, and hypoxia can regulate the self-renewal activity of eMSC via Notch signaling.