Patient-derived eutopic and ectopic endometrial stromal cells: characterization and development of immortalized lines

Ovarian endometriosis is an estrogen-dependent inflammatory disorder in which endometrial stromal cells are key cellular contributors to hormone-immune crosstalk and lesion persistence. Here, we isolated paired eutopic (NESC) and ectopic (EESC) endometrial stromal cells from patients with ovarian endometriosis, compared their proliferation, migration/invasion and decidual responsiveness, and profiled their transcriptomes by RNA sequencing. EESCs displayed enhanced proliferative and migratory/invasive capacity and an attenuated decidual response. RNA-seq revealed an inflammatory transcriptional program with enrichment of cytokine-cytokine receptor interaction and MAPK-related pathways and increased expression of chemokines and pro-inflammatory cytokines. Steroid receptor analyses showed reduced ERα and progesterone receptor expression with relative ERβ predominance, consistent with progesterone-resistance-like features. We then generated SV40 large T antigen-immortalized NESC and EESC lines. These lines showed stable growth and retained stromal identity and several disease-relevant phenotypic features, while also acquiring immortalization-associated transcriptomic remodeling involving cell-cycle, DNA-replication and proliferation-related programs. These paired primary and immortalized stromal cell models provide a practical platform to investigate endocrine-immune mechanisms in endometriosis and to facilitate preclinical screening of therapies targeting inflammatory and steroid signaling.