Establishment and characterization of a new immortalized human adenomyosis epithelial-like cell line, tAEC21

Adenomyosis occurs when endometrial glands and stroma grow within the uterine myometrium. As a clinically significant disease, adenomyosis causes substantial pelvic pain and heavy menstrual bleeding. It remains understudied due to a lack of translational research tools and model systems. This study aimed to develop a telomerase-transformed, epithelial-like cell line derived from the eutopic endometrium of a subject with focal adenomyosis. De-identified endometrial tissue was processed through mechanical and enzymatic digestion. Epithelial and stromal populations were separated by selective adhesion, followed by fluorescence-activated cell sorting using an epithelial cellular adhesion molecule (EpCAM). EpCAM+ cells were effectively immortalized with the human telomerase reverse transcriptase gene. Analyses confirmed the cells were human, free of mycoplasma contamination, and exhibited a unique 16-marker short tandem repeat profile. Cytogenetic analysis of G-banded metaphase spreads revealed polyploidy with multiple chromosomal rearrangements. The cell line, designated tAEC21, expressed epithelial markers cytokeratin-5 and N-cadherin but not the stromal marker CD10. Cells responded strongly to tumor necrosis factor-alpha stimulation by upregulating interleukin-6, C-X-C motif chemokine ligand 8, C-C motif chemokine ligand 2, and mucin 1 gene expression. In a heterotypic, three-dimensional spheroid model, tAEC21 formed a biologically relevant structure by creating an epithelial shell around the stromal cell core. In both 2D monolayer and 3D culture, tAEC21 cells responded to 17β-estradiol (E2) but did not respond to progesterone (P4), consistent with the expression of estrogen (ESR1) and progesterone (PGR) receptors. This new epithelial-like, adenomyosis-derived cell line, tAEC21, will be an impactful, biologically plausible research resource.