Candidate markers of epithelial stem/progenitor cells in human endometrium and endometrial cancer

The human endometrium is the highly regenerative mucosal lining of the uterus which undergoes cyclical regeneration following menstruation and parturition. Atrophic endometrium also regenerates when post-menopausal women take estrogen therapy. A rare population of endometrial epithelial stem/progenitor cells are likely involved in this remarkable regeneration. However, their precise origin and location are unknown. Endometrial cancer is the most common gynaecological malignancy in women. Genetic alterations involving the stem cell compartment are thought responsible for this cancer. At the beginning of this study, there were no known markers for the prospective isolation of human endometrial epithelial stem/progenitor cells. The overall aim of this thesis therefore, was to identify surface markers to purify endometrial epithelial stem/progenitor cells and endometrial cancer stem cells. It was hypothesised that the epithelium in the basalis of pre-menopausal endometrium and post-menopausal endometrium are similar and contain stem/progenitor cells with common gene expression profiles. To enable the identification of endometrial epithelial stem/progenitor cells, a robust isolation and characterisation techniques using enzyme digestion, differential filtration and flow cytometry sorting was developed. A transcriptional study was then undertaken which identified differential expression of 22 Wnt signalling pathway genes between epithelial cells isolated from pre- and post-menopausal endometrium. One gene, Axin2, a negative regulator of the Wnt pathway was specifically localised to the nucleus of epithelial cells in the basalis of pre-menopausal endometrium and post-menopausal endometrium, possibly to regulate cytoplasmic β-catenin and stabilise Wnt downstream target genes. The surface marker, H2NM selected from the gene list, enriched for a small population of endometrial epithelial cells with stem cell activity. H2NM was localised to epithelial glands deep in the basalis layer adjacent to the myometrium, with a gradient of reducing expression through to the functionalis and luminal epithelium. A common gene pathway was identified in the epithelium of the basalis of pre-menopausal endometrium and in post-menopausal endometrium, confirming the likely basalis location of resident epithelial stem/progenitor cells. This thesis also investigated known cancer stem cell markers (H2M4, H4M9F, H2M9, H4M4) for their potential to isolate cancer stem cells from primary endometrial cancer and endometrial cancer cell lines. The levels of surface marker expression greatly varied between primary endometrial cancer cells and the cell lines. Sorted endometrial cancer cell lines exhibited colony-forming activity and sphere formation more readily compared to primary endometrial cancer cells. However, the markers examined did not enrich for cancer stem cell activity. Stromal feeder layers were necessary to support clonal cultures of sorted primary endometrial cancer cells, however further work is required to identify potential endometrial cancer stem cell markers. Future studies should also examine H2NM for its ability to isolate endometrial cancer stem cell population. This investigation supports the concept that endometrial epithelial stem/progenitor cells exist in pre- and post-menopausal endometrium which can be partially purified using a single marker H2NM. Identification of this marker lays the foundation for further characterisation of the molecular and cellular phenotype of human endometrial epithelial stem/progenitor cells and examines their role in endometrial regeneration and in gynaecological disorders such as endometrial cancer.