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

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 (Asherman's syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding.The human uterus is a formidable organ. From puberty to menopause, it completely sheds off its internal lining every 28 days or so, creating what is in effect a large open wound. Unlike the skin or other parts of the body, however, this tissue can quickly repair itself without scarring. This fascinating process remains poorly understood, partly because human samples and animal models that mimic human menstruation are still lacking. This makes it difficult to grasp how various types of uterine cells get mobilised for healing. To fill this gap, Kirkwood et al. focused on fibroblasts, a heterogenous cell population which helps to support the epithelial cells lining the inside of the uterus. How these cells responded to the advent of menstruation was examined in female mice genetically manipulated to have human-like periods. A method known as single-cell RNAseq was used to track which genes were active in each of these cells before, one day and two days after period onset. This revealed the existence of a subpopulation of cells which only appeared when wound healing was most needed. These ‘repair-specific’ fibroblasts expressed a mixture of genes; those typical of fibroblasts but also some known to be active in the epithelial cells lining the uterus. This suggests that the cells were in the process of changing their identity so they could remake the uterine layer lost during a period. And indeed, labelling these fibroblasts with a fluorescent tag showed that, during healing, they had migrated from within the uterine tissue to become part of its newly restored internal surface. These results represent the first evidence that fibroblasts play a direct role in repairing the uterus during menstruation. From endometriosis to infertility, the lives of millions of people around the world are impacted by disorders which affect the uterine lining. A better understanding of how the uterus can fix itself month after month could help to find new treatments for these conditions. This knowledge could also be useful for to address abnormal wound healing in the skin and other tissues, as this process often involves fibroblasts.