P-796 Single-cell transcriptomics unveils a role for leptin receptor in human endometrial mesenchymal stromal/stem cells
Authors: Fang Y , Cao D , Chan R W S , Lee C L , Chiu P C N , Ng E H Y , Yeung W S B 
Published: June 2023

Abstract

Study question
What are the characteristics of leptin receptor (LepR) expressing cells within the endometrial mesenchymal stromal/stem cells (eMSCs) population?

Summary answer
LepR+ eMSCs display better stem-cell capacities as well as distinctively quiescent properties compared to eMSCs and LepR- eMSCs.

What is known already
Single-cell RNA sequencing (scRNA-seq) reveals that cultured eMSCs are heterogeneous. LepR is highly expressed in bone marrow MSCs, and LepR+ cells contribute towards the formation of fibroblast colony-forming units (CFU-F). Mouse LepR+ MSCs are quiescent and activate upon injuries to form osteoblasts and adipocytes in the bone marrow. In this study, we hypothesized that LepR+ cells play a critical role in the stem cell population in human endometrium.

Study design, size, duration
We performed scRNA-seq on magnetic bead selected eMSCs (CD140b+CD146+ cells). Functional stem cell assays such as clonogenicity and serial subcloning were performed on the different stromal subsets (eMSC, LepR- eMSC, and LepR+ eMSC) isolated by fluorescence-activated cell sorting (FACS) cultured endometrial stromal cells. The cell cycle analysis was evaluated by multicolor flow cytometry on freshly isolated endometrial stromal cells.

Participants/materials, setting, methods
The endometrial samples were collected from women aged 37–53 years with regular menstrual cycles undergoing hysterectomy. The expression and localization of LepR in human endometrium were determined by flow cytometry and immunohistochemistry, respectively. The in vitro colony-forming and self-renewal abilities were assessed on FACS sorted endometrial stromal populations. The cell cycle status of the stromal subsets was determined by flow cytometry and the quiescent property was determined by qPCR (RB1, RBL2, CDKN1A, CDKN1B, and E2F4).

Main results and the role of chance
Clustering analysis of the scRNA-seq data revealed 5 subpopulations (SP0-4) of cultured eMSCs. A high proportion (&gt;90%) of the cells in SP3 and SP4 were at the G1 phase. The majority of the SP3 cells (92%) were located at the root of the cell trajectory and pseudo time inference analysis.
The expression of LepR was highly specific in SP3 suggesting LepR eMSCs are potentially the origin progenitors.The expression of LepR in endometrial stromal cells was 2.61 ± 1.43%, and in eMSCs was 25.59 ± 19.04%. In vitro experiments demonstrated that LepR+ eMSCs exhibit better stem-cell properties, including clonogenic and self-renewal ability, than its negative counterparts.
QPCR validation of cell-quiescence associated genes revealed higher expression of RB1, RBL2, and CDKN1B in the LepR+ eMSCs than eMSCs and LepR- eMSCs. In addition, multicolor flow analysis on freshly isolated endometrial cells revealed significantly higher proportion of LepR+ eMSCs are in the G0 phase of the cell cycle when compared to eMSCs and LepR- eMSCs. The above results indicate in vivo LepR+ eMSCs are relatively more quiescent.

Limitations, reasons for caution
The mechanism involved in maintaining or activating quiescent LepR+ eMSCs remains unknown. The role of LepR+ is likely to be different in human compared to mouse. We also lack an in vivo model for studying LepR+ eMSCs during endometrial repair.

Wider implications of the findings
Our findings extend the understanding of heterogeneous human endometrial mesenchymal stem cells and unveil a new role for LepR in endometrial regeneration.

Trial registration number
not applicatble