Background: The human endometrium undergoes cyclical shedding and regeneration during menstruation likely regulated by clonogenic, self-renewing epithelial stem/progenitor cells residing within the deep basalis. These N-Cadherin+ (CDH2) and SSEA-1+ epithelial progenitors, also present in menstrual fluid, are likely involved in initiating endometriosis and adenomyosis. Our aim is to investigate the role of human endometrial stem/progenitor cells in these gynaecological conditions.
Methods: Full thickness hysterectomy endometrial tissue from women with (n=5) and without endometriosis and adenomyosis (n=5) was digested to single cells, FACS sorted into three epithelial progenitor subpopulations (EpCAM+N-Cadherin+SSEA-1-, EpCAM+N-Cadherin+SSEA-1+, EpCAM+N-Cadherin-SSEA-1+), and a mature epithelial population (EpCAM+N-Cadherin-SSEA-1-). Sorted populations were seeded into Matrigel (5000 cells/dome) and cultured as 3D organoids in standard WNT-promoting medium. Growth and morphology were monitored over 3 passages, each 21 days, using live-imaging, immunofluorescence and proliferation assays. N-Cadherin and SSEA-1 were localised in full thickness endometrial tissue containing both basalis and functionalis regions from control, endometriosis and adenomyosis patients using immunofluorescence.
Results: Each human endometrial epithelial organoid subpopulation demonstrated differences in organoid formation and growth. The deepest basalis population (EpCAM+N-Cadherin+SSEA-1-) showed no organoid development within the initial 21-days suggesting a quiescent state consistent with their stem/progenitor cell identity, but following passaging, rapidly developed many large organoids. In comparison, functionalis EpCAM+N-Cadherin-SSEA-1- and EpCAM+N-Cadherin-SSEA-1+ populations proliferated and developed small organoids within the first 21-days of culture. The epithelial cells also differentiated from their original sorted identity during in vitro culture; some EpCAM+N-Cadherin+SSEA-1- cells differentiated into EpCAM+N-Cadherin-SSEA-1+ cells and some EpCAM+N-Cadherin-SSEA-1+ cells de-differentiated into EpCAM+N-Cadherin+SSEA-1- cells at differing rates between adenomyosis and endometriosis.
Conclusion: Changes in phenotype (differentiation state) between the different epithelial progenitor populations demonstrates the cellular hierarchy within endometrial epithelium. N-cadherin and SSEA-1 localisation varied across different pathologies and menstrual cycle phases indicating possible involvement of epithelial progenitors in endometriosis and adenomyosis initiation and pathophysiology.