Human endometrium is highly regenerative, undergoing 400 cycles of growth, differentiation and shedding during a woman’s reproductive life. Following menses, ~1 cm tissue grows from the remaining basalis to generate a new functionalis into which an embryo implants. Mice do not menstruate, however the endometrium grows and regresses each estrus cycle. We hypothesised that endometrial epithelial progenitor cells and mesenchymal stem/stromal cells (MSC) located in the basalis mediates the remarkable, cyclical regenerative capacity of glands and vascularised stroma, respectively.
We discovered rare colony-forming epithelial and stromal cells in human endometrium and demonstrated their ability for self-renewal, proliferation and differentiation, key stem/progenitor cell properties. Large epithelial clones differentiated into gland-like structures, and stromal clones into smooth muscle, adipocyte, osteoblast and chondrocyte lineages, indicating their MSC phenotype. Label-retaining cells which drive estrogen-stimulated endometrial regeneration were identified as stem/progenitor cells in mice.
Specific surface markers for human endometrial MSC (eMSC) were identified by combining several bone marrow MSC candidates, which showed CD146 and PDGFR-β co-expression enriched for clonogenic eMSC, revealing their perivascular/pericyte location in both the basalis and functionalis. Screening with perivascular antibodies identified SUSD2 as a single eMSC marker. Our unbiased gene microarray approach, comparing highly purified epithelial cells from pre- and post-menopausal basalis-like endometrium, identified CDH2 (N-Cadherin) as a surface marker that enriched for clonogenic, self-renewing epithelial cells that differentiated into extensive glandular structures in organoid cultures. N-cadherin was localised in the bases of horizontal interconnecting glands in the basalis, adjacent to the myometrium.
These fundamental studies allowed us to examine the role of endometrial stem/progenitor cells in the pathogenesis of gynecological disorders associated with abnormal endometrial proliferation; endometriosis, adenomyosis and endometrial cancer. Conversely, defective function or lack of endometrial stem/progenitor cells may result in thin unresponsive endometrium unable to implant embryos, or Asherman’s syndrome where scar tissue destroys epithelial progenitor niches.
With our markers, we demonstrated that SUSD2+ eMSC and N-cadherin+ epithelial progenitors were shed into menstrual fluid of women with and without endometriosis and that both were retrogradely shed into the peritoneal cavity during menstruation, predominantly in women with endometriosis. Here, they could initiate endometriotic lesions. We identified epithelial progenitors in single cell RNAseq studies and our current work is detecting novel molecules regulating their niches.
We are translating our eMSC research to develop an autologous eMSC-based therapy for treating and preventing Pelvic Organ Prolapse (POP). We developed a culture protocol that maintains eMSC function using a TGFβR inhibitor and characterised their molecular properties. With CSIRO, we developed new non-degradable vaginal meshes with mechanical properties matching human vagina. Our eMSC/mesh constructs promoted angiogenesis, modulated inflammatory responses to mesh and improved outcomes in rat and mouse models. We developed a sheep model of POP and trans-vaginally implanted autologous ovine eMSC/mesh demonstrating eMSC retention. New directions are the rational design of degradable 3D printed meshes for use with bioprinted eMSC. In summary, our discovery of human endometrial stem/progenitor cells has underpinned our ability to impact gynaecological disease through increased understanding of endometriosis causation and developing an eMSC-based therapy for POP.