Single-cell transcriptomics reveals notch regulation in quiescent LEPR⁺ endometrial mesenchymal stem cells

Abstract

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.