Background: There is an urgent need to develop assisted reproduction technologies (ARTs) to combat the devastating rate of extinction facing Australian marsupials. A core requirement to develop ARTs is a deep understanding of the endometrium and its secretions. This is of heightened significance in marsupials as a significant proportion of gestation occurs unattached to the endometrium. However, there are limited tools to examine endometrial secretions in vitro, with no endometrial cell lines commercially available. We therefore aimed to develop a cell line to address this need, utilising conserved markers of endometrial progenitor cells.
Methods: SSEA1 was profiled in the fat-tailed dunnart endometrium using RT-qPCR (FUT4, n=3), immunohistochemistry (n=10), and flow cytometry (n=10). Isolated SSEA1+ cells were assessed for clonogenicity using a colony formation assay (n=3) and immortalised using SV40T antigen technology. A clonal cell line was derived, treated with gestational hormones (estradiol, progesterone, cAMP), and known hormone-responsive transcripts assessed by RT-qPCR (n = 3).
Results: FUT4 transcript was detected in the dunnart uterus, and SSEA1 was localised to the endometrial epithelium. At estrus, SSEA1+ cells comprised ~7% of endometrial cells, with a clonogenicity of 0.06%, 0.06%, and 0.15% for bulk, negative, and SSEA1+ populations respectively (P = 0.38).
The clonal line maintained epithelial morphology, and expressed appropriate markers including KRT8, ESR1 and PGR. Hormonal response was confirmed by the modulation of PGR (P=0.0003), IHH (P=0.028), and PTGS2 (P=0.016) by gestational hormones. Ongoing RNA-sequencing analyses will define marsupial-specific features of SSEA1+ cells and their response to hormones.
Conclusion: We have generated an immortal SSEA1+ endometrial epithelial cell line enabling the culture and functional interrogation of this population. This tool creates new opportunities to study tissue remodelling, develop marsupial in vitro endometrial models and characterise uterine secretions underpinning histotrophic marsupial reproduction, thereby accelerating ART development for the conservation of Australian marsupial biodiversity.