Molecular and functional characterization of leptin receptor-positive mesenchymal stem cells in human endometrium

Abstract

The human endometrium is a highly dynamic tissue with periodic regeneration, a process primarily facilitated by endometrial stem cells. In contemporary research, single-cell RNA sequencing (scRNA-seq) not only highlights cell heterogeneity but also unveils novel subsets within a population of cells. Leptin receptor (LEPR) has been well-studied in bone marrow MSCs. In this project, I hypothesized that LEPR positive subset is a key component within endometrial stem cells. The first aim of this study was to employ scRNA-seq to characterize heterogeneity within cultured and primary endometrial mesenchymal stem cells (eMSCs), identifying distinct subpopulations. Notably, 5 and 6 distinct clusters were identified in cultured and primary eMSCs, respectively. In cultured eMSCs, a primitive subpopulation denoted as SP3, exhibited an exclusive LEPR expression. In addition, this LEPR-enriched subpopulation also manifested its presence within primary eMSCs. Furthermore, LEPR subpopulation were found to be the multifaceted in processes involved in endometrial development and immune regulation. CellPhoneDB analysis revealed that the LEPR+ eMSCs were the most communicative subpopulation compared to the LEPR- eMSCs and non-eMSCs. The second objective was to confirm the protein expression of LEPR in stromal cells and evaluate their stem-cell abilities. These cells displayed enhanced colony formation, self-renewal, and adipogenic differentiation compared to LEPR- eMSCs and general eMSCs. Flow cytometry and qPCR supported the quiescent characteristic of freshly isolated LEPR+ eMSCs. The third objective further examine in-vitro methods to maintain the quiescent state of LEPR+ eMSCs. Two distinct quiescence models were explored: one induced by serum starvation and the other by activating NOTCH signaling. LEPR+ eMSCs subjected to this approach displaying lower levels of apoptosis compared to those in the serum starvation-induced model. To reveal the underlying mechanisms of quiescence, NOTCH-related molecules within LEPR+ eMSCs were studied. The analysis unveiled strikingly elevated expression levels of HES1, NOTCH1, and NOTCH4 within this subset, findings that were subsequently corroborated through independent in-vitro experiments. The fourth objective was to investigate the association between LEPR+ eMSCs and endometriosis. Given this context, a higher proportion of LEPR+ eMSCs emerged within ectopic endometrial tissue compared to their eutopic counterparts in patients with endometriosis. Additionally, NOTCH signaling was discovered through in-depth analysis of gene signatures associated with LEPR+ eMSCs in different patient groups. Major biological processes such as cell cycle regulation, angiogenesis, cell adhesion, and immune response are closely related to LEPR+ eMSCs. In summary, this thesis demonstrated the significance of the LEPR-positive subset within endometrial mesenchymal stem cells. The distinctiveness of this subset is evident in both cultured and primary eMSCs, where it remains quiescent in normal endometrium yet emerges as a highly communicative cell population. In-vitro experiments demonstrate its superior stem cell abilities, and in the context of endometriosis, its proportion increases, accompanied by diverse biological processes. These findings emphasize the crucial role of LEPR+ eMSCs in endometrial biology.