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Establishment of an Immortalized Endometriotic Stromal Cell Line from Human Ovarian Endometrioma.

ABSTRACT: Endometrial-like stromal cells, one of the main components of endometriotic lesions, are an important in vitro model for studying cellular and molecular mechanisms associated with lesion development in endometriosis. However, the short life span of primary endometriotic stromal cells (Ec-ESCs) limits their use. Human telomerase reverse transcriptase (hTERT) plasmids can be used to develop immortalized cell lines. Here we aimed to establish an endometriotic stromal cell line by hTERT immortalization. Primary Ec-ESCs were obtained from a human ovarian endometriotic cyst. The purity was assessed by morphology and the expression of vimentin, cytokeratin, and human interferon-inducible transmembrane protein 1 (hIFITM1). Cells were infected with hTERT lentiviral vector and selected with hygromycin. hTERT mRNA levels were confirmed by RT-qPCR. Immortalized Ec-ESCs (iEc-ESCs) were characterized by examining the expression of morphological markers and key genes of interest, TP53, estrogen receptor β (ERβ), progesterone receptor (PR), and steroidogenic factor-1 (SF-1). Karyotyping and in vitro decidualization studies were also performed. Ec-ESCs were positive for vimentin and hIFITM1 and negative for cytokeratin, indicating that they were representative of Ec-ESC. The fibroblast-like morphology, expression of TP53, ERβ, PR, and SF-1 did not change before and after hTERT immortalization. iEc-ESCs showed an impaired decidualization response like primary Ec-ESCs when compared to normal eutopic stromal cells. Karyotyping showed that 15/19 cells had normal female karyotype, while 4/19 cells had partial trisomy 11q. Collectively, we successfully established and characterized an immortalized endometriotic stromal cell line. It is potentially useful as an in vitro experimental model to investigate endometriosis biology.

SUBMITTER: Song Y

PROVIDER: S-EPMC7529860 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC) decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. METHODS: Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. RESULTS: St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82). Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. CONCLUSION: St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural progesterone. St-T1b cells therefore serve as a useful model for primary ESC.

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Establishment of an Immortalized Endometriotic Stromal Cell Line from Human Ovarian Endometrioma.

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