Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

ABSTRACT The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study we used a mouse model of endometrial repair and three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the endometrium. Using scRNAseq we identified a novel population of PDGFRb+ mesenchymal stromal cells that developed a unique transcriptomic signature in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they were stromal fibroblasts in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2/CSPG4+). We demonstrated that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to the rapid restoration of an intact luminal epithelium during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Ashermans syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding. Highlights Single cell RNA sequencing identified a population of PDGFRβ+ mesenchymal cells with a unique transcriptomic signature that arises in response to induction of a menses-like wound The repair-specific mesenchymal cells express genes considered specific to both mesenchymal and epithelial lineages indicative of an intermediate phenotype and a mesenchymal to epithelial transition (MET) in silico trajectory analysis revealed that repair-specific cells originate from the fibroblast cell clusters, were distinct from perivascular cells, and had a predicted trans-differentiation trajectory towards definitive epithelial cells in vivo lineage tracing in adult mice provides definitive evidence that PDGFRα+ endometrial fibroblasts, and not NG2+ perivascular cells, undergo MET and can become incorporated into the luminal epithelium of the post repair tissue Competing Interest Statement The authors have declared no competing interest. Footnotes improvements to flow of text and correction of typographical errors