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

Abstract

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-1β 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: Endometriotic stromal cells; Estrogen; Immortalized; Inflammation; Tumorigenicity.

Fig. 1

Establishment of immortalized human endometriotic stomal cells (ihESCs) by lentivirus transfection of hTERT. a The observation of red fluorescent from infected cells to estimate infection efficiency. b The expression of hTERT mRNA in ihESCs of 5, 15 and 25 passages. c hTERT protein level of ihESCs from various passages detected by Western Blot. d The morphology of ihESCs observed by using Optical Microscope (upper) and Scanning Electron Microscope (lower). e The life span of primary cells and ihESCs. Data represent the mean ± SEM. **P < .001, ***P < .0001

Fig. 2

The phenotypes and marker proteins of ihESCs kept unchanged compared with primary stromal cells. a The growth curve of ihESCs and primary stromal cells detected by CCK-8 assays. b The protein expression of epithelial-mesenchymal transition from primary stomal cells and ihESCs. c The level of estrogen-metabolizing proteins and estrogen/progesterone receptors proteins in primary cell and ihESCs. d Identification of epithelial and mesenchymal cells assessed by immunocytochemistry (ICC). e Chromosome karyotype analysis of ihESCs and sorting result (right)

Fig. 3

Normal estrogen response and inflammation are found in ihESCs. a The proliferation of ihESCs with stimulation of estrogen. b EdU-labeled assay employed to monitor cell division of ihESCs treated with estrogen. c Quantified analysis of EdU assays. d The protein expression of EMT markers from ihESCs with treatment of estrogen. The mRNA (e) and protein (f) expression of inflammatory factor in ihESCs with stimulation of LPS. The mRNA (g) and proteins (h) of inflammatory factor from ihESCs treated with IL-1β. Data represent the mean ± SEM. *P < .05, **P < .001, ***P < .0001

Fig. 4

ihESCs are not malignant transformed to trigger tumors. a Colony formation assay used to assess the oncogenic potential of ihESCs. b Nude mouse tumorigenicity assay performed to evaluate tumorigenic capacity of ihESCs