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

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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.


  • 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

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  1. Evaluation Summary:

    This is an important and delicately designed study that uses integrated tools to reveal underlying mechanisms of repair of the endometrium at menstruation. It combines single cell sequencing analysis and lineage tracing technologies to strongly prove that repair-specific cells originate from the fibroblast cell clusters and PDGFRα+ endometrial fibroblasts undergo MET and can become incorporated into the luminal epithelium of the post repair tissue.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Using a mouse model of menstruation the authors have investigated the contribution of stromal mesenchyme cell populations to the restotation of the luminal epithelium. This work has been performed by combining the strengths of trajectory analysis in single cell RNAseq data with lineage tracking of cells using reporter constructs. This approach is an excellent example of integrating bioinformatic analysis with in vivo modelling to achieve a synergy between the two different types of data. The findings are clear and well presented with careful consideration of confounding issues. The understanding developed of the restoration of the luminal epithelium using this model system helps to define the mechanisms involved in the rapid nature of this event. This understanding is of obvious relevance to a number of related human pathologies. As yet the comparison between the mouse model data and human systems is preliminary.

  3. Reviewer #2 (Public Review):

    In the manuscript, Kirkwood et al prove that an MET program of fibroblast is responsible for repair of the endometrium at menstruation. The strongest point is that this study combines single cell RNA sequencing and lineage tracing technology to provide strong support to their hypothesis, which will undoutedly help us understand how endometrium repair, not only at menstruation, but also when it is damaged during pathogenic conditions. It may also contribute to the development of novel therapeutic strategies for patients with endometrium-related diseases.