Zinc(II) niflumato complex effects on MMP activity and gene expression in human endometrial cell lines

Endometriosis is a chronic inflammatory disease which increasingly affects young women under 35 years of age and leads to subfertility even infertility. Analysis of the cytotoxic effect of zinc(II) niflumato complex with neocuproine ([Zn(neo)(nif)2] or Zn-Nif) on immortalized human endometriotic cell line (12Z) and on control immortalized human endometrial stromal cell line (hTERT) was performed using xCELLigence technology for approximately 72 h following the treatment with Zn-Nif as well as cell viability Trypan Blue Assay. 12Z cell line proliferated more slowly compared to unaffected cells, whereas hTERT cells did not show similar behavior after treatment. The complex probably reduces the effect of pro-inflammatory pathways due to the effect of NSAID, while presence of zinc might reduce the level of ROS and regulate ER2 levels and MMP activity. The observed effects and high selectivity for rapidly proliferating cells with increased inflammatory activity suggest a good prognosis of successful decrease of endometriosis stage with this complex.

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

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.

Estrogen-decreased hsa_circ_0001649 promotes stromal cell invasion in endometriosis

Endometriosis (EMs) is an estrogen (E2)-dependent inflammatory disorder. Although EMs is considered a benign disease, it presents with malignant characteristics, such as migration and invasion. An increasing number of studies have shown that aberrantly expressed circular RNAs (circRNAs) play an essential role in disease development and progression. However, the mechanisms by which circRNAs exert their pathological effects in EMs remain unclear. Hsa_circ_0001649, a novel cancer-associated circRNA, has been previously reported to be downregulated in several cancer types and related to cell migration and invasion. In the present study, real-time PCR (qRT-PCR) was carried out to measure hsa_circ_0001649 levels in human tissues, human primary endometrial stromal cells (ESCs) and a human endometrial stromal cell line (ThESCs). Matrix metalloproteinase 9 (MMP9) levels in ESCs and ThESCs were assessed by qRT-PCR and Western blotting, and the migration and invasion capacities of ThESCs were evaluated by transwell assay. As a result, hsa_circ_0001649 expression was significantly decreased in ectopic and eutopic endometrial samples compared with that in normal endometrial samples. E2 decreased hsa_circ_0001649 expression but increased MMP9 expression in ESCs and ThESCs. Furthermore, ThESCs were more invasive under E2 stimulation. However, these effects disappeared when ICI or hsa_circ_0001649 transfection was used. Collectively, our findings reveal that decreased hsa_circ_0001649 expression plays a role in E2-increased MMP9 expression through E2 receptors (ERs), which have critical functions in EMs.

Overactivated sonic hedgehog signaling aggravates intrauterine adhesion via inhibiting autophagy in endometrial stromal cells

Autophagy can be dynamically induced in response to stresses and is an essential, ubiquitous intracellular recycling system that impacts the fate of damaged resident cells, thereby influencing wound healing. Endometrial fibrosis is a form of abnormal wound healing that causes intrauterine adhesion (IUA) and infertility. We previously demonstrated that overactivated sonic hedgehog (SHH) signaling exacerbated endometrial fibrosis, but the role of autophagy in this process is still unknown. Here, we report that impaired autophagy participates in SHH pathway-induced endometrial fibrosis. Endometrial stroma-myofibroblast transition accompanied by autophagy dysfunction was present in both endometrial biopsies of IUA patients and Amhr2cre/+R26-SmoM2+/− (AM2) transgenic mouse. Mechanistically, SHH pathway negatively regulated autophagy through pAKT-mTORC1 in a human endometrial stromal cell line (T-HESCs). Furthermore, SHH pathway-mediated fibrosis was partly counteracted by autophagy modulation in both T-HESCs and the murine IUA model. Specifically, the impact of SHH pathway inhibition (GANT61) was reversed by the pharmacological autophagy inhibitor chloroquine (CQ) or RNA interference of autophagy-related gene ATG5 or ATG7. Similar results were obtained from the murine IUA model treated with GANT61 and CQ. Moreover, promoting autophagy with rapamycin reduced fibrosis in the AM2 IUA model to baseline levels. In summary, defective autophagy is involved in SHH pathway-driven endometrial fibrosis, suggesting a potential novel molecular target for IUA treatment.

Endometrial and decidual stromal precursors show a different decidualization capacity

Endometrial stromal cells (EnSCs) and decidual stromal cells (DSCs) originate from fibroblastic precursors located around the vessels of the human nonpregnant endometrium and the pregnant endometrium (decidua), respectively. Under the effect of ovarian or pregnancy hormones, these precursors differentiate (decidualize), changing their morphology and secreting factors that appear to be essential for the normal development of pregnancy. However, the different physiological context – that is, non-pregnancy vs pregnancy – of those precursors (preEnSCs, preDSCs) might affect their phenotype and functions. In the present study, we established preEnSC and preDSC lines and compared the antigen phenotype and responses to decidualization factors in these two types of stromal cell line. Analyses with flow cytometry showed that preEnSCs and preDSCs exhibited a similar antigen phenotype compatible with that of bone marrow mesenchymal stem/stromal cells. The response to decidualization in cultures with progesterone and cAMP was evaluated by analyzing changes in cell morphology by microscopy, prolactin and IL-15 secretion by enzyme immunoassay and the induction of apoptosis by flow cytometry. In all four analyses, preDSCs showed a significantly higher response than preEnSCs. The expression of progesterone receptor (PR), protein kinase A (PKA) and FOXO1 was studied with Western blotting. Both types of cells showed similar levels of PR and PKA, but the increase in PKA RI subunit expression in response to decidualization was again significantly greater in preDSCs. We conclude that preEnSCs and preDSCs are equivalent cells but differ in their ability to decidualize. Functional differences between them probably derive from factors in their different milieus.

Artificial thymic organoids represent a reliable tool to study T-cell differentiation in patients with severe T-cell lymphopenia

The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαβ+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.

Establishment of a FDC-P1 murine cell line with human KIT N822K gene overexpression

The mechanism of resistance of leukemia cells to chemotherapeutic drugs remains poorly understood. New model systems for studying the processes of malignant transformation of hematopoietic cells are needed. Based on cytokine-dependent murine acute myeloid leukemia (AML) FDC-P1 cells, we generated a new cell line with ectopic expression of the KIT gene encoding mutant human receptor tyrosine kinase (N822K). We investigated the role played by overexpression of the mutant KIT in the survival of leukemia cells and their sensitivity to therapeutic drugs. We also generated a co-culture system consisting of FDC-P1 murine leukemia cells and a HS-5 human stromal cell line. Our data can be used for a further comprehensive analysis of the role of KIT N822K mutation in the cellular response to anti-leukemic drugs, growth factors, and cytokines. These data are of interest in the development of new effective therapeutic approaches to the treatment of acute leukemia.