Establishment of an immortalized stromal cell line derived from human Endometriotic lesion

• Published in: Reproductive biology and endocrinology 2020-11, Vol.18 (1), p.1-119, Article 119
• Main Authors: Huang, Zhi-Xiong, Wu, Rong-Feng, Mao, Xiao-Mei, Huang, Shao-Min, Liu, Tian-Tian, Chen, Qiong-Hua, Chen, Qing-Xi
• Format: Article
• Language: English
• Subjects: Antibodies; Biotechnology; Cholecystokinin; Chromosomes; Colonies; Cytokeratin; Cytokines; Cytology; Development and progression; Endometriosis; Estrogen; Estrogens; Gynecological diseases; Gynecology; IL-1β; Inflammation; Karyotypes; Laboratories; Lipopolysaccharides; Menstruation; Mesenchyme; Morphology; Physiological aspects; Plasmids; Polymerase chain reaction; Progesterone; Progesterone receptors; Proteins; RNA; RNA-directed DNA polymerase; Stromal cells; Telomerase; Telomerase reverse transcriptase; Tumorigenesis; Tumorigenicity
• ISSN: 1477-7827; 1477-7827
• DOI: 10.1186/s12958-020-00669-x

Background Endometriosis is a benign gynecological disease with obviously feature of estrogen-dependence and inflammatory response. The applications of primary endometriotic stromal cells in research of endometriosis are restricted for short life span, dedifferentiation of hormone and cytokine responsiveness. The objective of this study was to establish and characterize immortalized human endometriotic stromal cells (ihESCs). Methods The endometriotic samples were from a patient with ovarian endometriosis and the primary endometriotic stromal cells were isolated from the endometriotic tissues. The primary cells were infected by lentivirus to establish telomerase reverse transcriptase (hTERT)-induced immortalized cells. Quantification of mRNA and proteins was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot. CCK-8 assay and EdU labeling assay were assigned to assess the growth of ihESCs. Karyotype assay was performed to detect the chromosomes of ihESCs. Colony formation assay and nude mouse tumorigenicity assay were used to evaluate colony-formation and tumorigenesis abilities. Results ihESCs continuously overexpressed hTERT via infection of lentivirus and significant extended the life span reaching 31 passages. The morphology, proliferation and karyotype of ihESCs remained unchanged. The expression of epithelial-mesenchymal transition (EMT) markers, estrogen-metabolizing proteins and estrogen/progesterone receptors (ERs and PRs) were unaltered. Furthermore, the treatment of estrogen increased the proliferation and EMT of ihESCs. Lipopolysaccharides (LPS) and IL-1[beta] remarkably induced inflammatory response. The clonogenesis ability of ihESCs was consistent with primary cells, which were much lower than Ishikawa cells. In addition, nude mouse tumorigenicity assay demonstrated that ihESCs were unable to trigger tumor formation. Conclusion This study established and characterized an immortalized endometriotic stromal cell line that exhibited longer life span and kept the cellular morphology and physiological function as the primary cells. The immortalized cells remained normal feedback to estrogen and inflammatory response. Moreover, the immortalized cells were not available with tumorigenic ability. Therefore, ihESCs would be serviceable as in vitro cell tool to investigate the pathogenesis of endometriosis. Keywords: Immortalized, Endometriotic stromal cells, Estrogen, Inflammation, Tumorigenicity