3 minute lightning oral presentation (and poster) ESA-SRB 2023 in conjunction with ENSA

Determining the pathogenesis of endometriosis by utilising a single cell atlas of endometrial stem/progenitor cells (#206)

Harriet C Fitzgerald 1 2 , Sally Mortlock 3 , Caitlin Filby 1 2 , Fiona Cousins 1 2 , Brett McKinnon 3 , Elizabeth Marquez-Garcia 1 2 , Grant W Montgomery 3 , Caroline Gargett 1 2
  1. Department of Obstetrics and Gynaecology, Monash University, Clayton, VIC, Australia
  2. The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC, Australia
  3. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia

Adult stem cells present in human endometrium; N-cadherin+ (CDH2) epithelial progenitors and SUSD2+ mesenchymal stem cells (eMSC) are shed during menstruation and refluxed into the peritoneal cavity. SSEA1+ basalis epithelial cells resurface the denuded endometrium to generate the luminal epithelium. Alterations in the gene expression of these cells may enable them to initiate endometriotic lesions. Our aim was to determine the gene expression atlas of single endometrial stem/progenitor cells and identify new markers of endometrial stem cell populations.

Human hysterectomy endometrium (n=5) was digested to single cells for 6-way FACSorting to collect stem/progenitor cell subpopulations and their differentiated progeny using N-Cadherin+/-, SSEA1+/- and SUSD2+/-. For single cell RNA sequencing (scRNA seq), the 6 cell fractions were combined, processed using 10X Genomics, libraries generated, and analysed using Cell Ranger and Seurat pipelines. Immunofluorescence on full thickness endometrium examined the co-localisation of SSEA-1, N-Cadherin and Indian Hedgehog.

scRNA seq identified 16 cell clusters; 10 of epithelial and 6 of mesenchymal origin. CDH2+SOX9+ cells localised to two clusters suggesting a basalis glandular epithelial population, with high expression of IHH. SUSD2 expression was localised to two main clusters, representing MSC with high MYH11 expression, transitioning to more mature stromal fibroblast clusters. Immunofluorescence identified all 4 endometrial stem/progenitor cell subpopulations and IHH+ basalis epithelial cells. Ligand-receptor analysis indicated key interactions between IHH in the basalis progenitor epithelial cells with its co-receptors BOC and CDON in the basalis stromal and smooth muscle cells respectively, reflective of a potential new stem cell niche environment in the human endometrium.

The identification of endometrial stem/progenitor cell subgroups and investigation of their gene expression from patients with endometriosis may assist our understanding of endometriosis pathogenesis for developing new treatments targeting the disease.