Poster Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Immortalised endometrial stromal cells as a potential tool for endometriosis studies (#462)

Eliza M Colgrave 1 , Premila Paiva 1 , Jenny N Fung 2 , Leonie Cann 1 , Jane E Girling 1 , Grant W Montgomery 2 , Peter AW Rogers 1 , Sarah J Holdsworth-Carson 1
  1. Department of Obstetrics and Gynaecology, RWH, The University of Melbourne, Melbourne, Victoria, Australia
  2. The Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

INTRODUCTION: Endometriosis is a common gynaecological disorder that causes pelvic pain and subfertility, which occurs due to a combination of environmental and genetic factors. The limited number and size of endometrial biopsies hinders research into genes associated with increased endometriosis risk identified by genome-wide association studies (GWAS). We aimed to create several human immortalised endometrial stromal cell lines (ESCs), of known genotypes, as tools to investigate endometriosis-associated genes.

METHODS: Endometrial samples were collected from women undergoing laparoscopy. Primary ESCs from patients with genotypes of interest were isolated following standard protocols. ESCs (passage-1) were immortalised using a human telomerase reverse transcriptase (hTERT) lentiviral vector (pLenti-G111-CMV-GFP-2A-Puro). Cell purity was determined in puromycin-selected hTERT ESCs (n=14), a commercial ESC line (tHESC) (n=1) and early passage primary ESCs (n=6) by immunocytochemistry (ICC). Oestrogen (ERα/β) and progesterone (PRA/B) receptor levels were compared using Western Blots and RT-PCR before and after in vitro treatment with steroid hormones E2 and MPA, as well as cAMP.

RESULTS: ICC confirmed our primary ESCs, hTERT ESCs and tHESCs were vimentin-positive and cytokeratin-negative. hTERT ESCs displayed elevated hTERT mRNA expression, while matched primary cells or cells transfected with control vector have undetectable hTERT.  ERα, ERβ and PR mRNA and protein were detected in primary ESCs, hTERT ESCs and tHESCs; however, both hTERT ESCs and the tHESC line have reduced steroid receptor expression relative to primary cells.  mRNA expression levels of PRL and IGFBP1 increase in decidualised primary ESCs and tHESCs but, unlike primary cells, tHESCs do not undergo decidualisation-associated morphological changes.

PLANNED FUTURE STUDIES: We will utilise our multiple hTERT-immortalised ESC lines for mechanistic studies of genes linked with increased endometriosis risk.  Using lines homozygous for each of the GWAS-identified “risk” alleles will allow us to investigate how variations in individual genes contribute to the aetiology of endometriosis.