Telocytes Enhanced In Vitro Decidualization And Mesenchymal-epithelial Transition In Endometrial Stromal Cells Via Wnt/β-catenin Signaling

objective:Decidualization of endometrial stromal cells(ESCs)is the key to endometrial receptive preparation and successful embryo implantation.It is regulated by the estrogen and progesterone during the menstrual cycle,and this process is accompanied by interstitial to epithelial Conversion.Telocytes(TCs)are widely present in various tissues and organs of the human body.Previous studies have shown that telocytes(TCs)also exist in the female reproductive system,and their cell membranes are rich in estrogen and progesterone receptors.The mechanism of maturation and differentiation of endometrial stromal cells is unclear.This study further explored the regulatory effects of TCs-derived WNTs on decidualization and mesenchymal epithelial transformation(MET)of ESCs.The aim is to further understand the role played by TCs in reproduction.Thus provided a promising way of cell therapy for certain gynecologic conditions and related reproductive problems associated with decidualization insufficiency.Methods:Sexually mature 8-week-old SPF-grade BALB/c female mice at 4 days post coitum(d p.c.)of were used to extract primary uterine tertiary cells and endometrial stromal cells.1.Select 8-week-old male and female mice of sexual maturity to close the cage at 2:1 the day before yesterday at 8 o’clock in the evening.A vaginal plug was observed as the first day of pregnancy at 8 o’clock in the morning.2.The mice were sacrificed by intraperitoneal injection of excessive chloral hydrate.The uterine tissue was removed from the sterile ultra-clean bench,and the uterine horns on both sides were cut longitudinally.The method was digested with collagenase and the method of differential adherence to extract and purify the endometrial matrix.Cells and uterine special cells.3.Identification of endometrial stromal cells by immunofluorescence method,and ESC-based immunofluorescence identification,determination of the purity of uterine endometrial stromal cells,cytokeratin(Cytokeratin)staining was negative,and vimentin staining(Vimentin)Is positive.Three different areas were randomly selected under the microscope and the ratio of positive cells to total cells was counted.4.Double immunofluorescence method was used to identify uterine special collateral cells,vimentin staining,CD34 double positive.The typical structure of TCs was observed with an inverted microscope.5.Differential expression of WNTs ligands in mouse uterine ESC and TC by QPCR Array6.TCs primary cells were cultured for 2 days and then subcultured for 24 hours.The serum-free basal medium was added,and the supernatant was collected after 48 hours of culture,which is the conditioned medium(TCM)of telocyte cells,7.TCM cultured ESC was used as the experimental group,E2+P4+cAMP in vitro decidualization medium was used as the positive control group,and blank medium was used as the negative control group to establish the experimental condition model.Relevant indicators of endometrial cell decidualization and mesenchymal epithelial transformation were measured by WB and RT-PCR techniques at 3 and 5 days,respectively,and the corresponding changes in cell morphology were observed.8.Use shRNA to silence the CTNNB1 gene of endometrial stromal cells(ie,express[3-catenin protein).TCM cultured ESCs that silenced sh-catenin and scramble were used as the experimental group,and TCM cultured ESCs were used as the control group.The cells were harvested for 3 days and the relevant indicators of endometrial cell decidualization and mesenchymal epithelial transformation were measured by WB and RT-PCR techniques,and the corresponding changes in cell morphology were observed.Results:1.ESCs showed a vimentin-positive/Cytokeratin-negative immunophenotype under a fluorescence microscope.The purity of endometrial stromal cells was determined by immunofluorescence.Three different regions were randomly selected under a microscope to calculate the ratio of positive cells to total cells.The results showed that the purity of endometrial stromal cells was(96.2±2.5)%.2.Microscopic uterine TCs of mice have a typical structure:small cell bodies with thick and thin cytoplasmic protrusions(Tps).Vimentin and CD34 were positive under fluorescence microscope.3.QPCR array to detect the relative expression of Wnt ligands and pathway-related genes in TCs and ESCs with a total of 85 mRN A expression profiles(TCs/ESC).Compared with ESCs,TCs contain a relatively high abundance of a series of Wnts ligand and pathway gene expressions.,Including high level:FZD4,WIF1,SFRP4,SFRP1,FZD8,CCND1,WNT4,WNT2,TCF711,LRP5,SFRP2,NFATC1,DKK1,CTNNB1,CTNNBIP1,AES,RUVBL1,FZD1,KREMEN1,CCND2,DVL2,FOS11,FRXB,FZD9;low level:WNT9A,WNT16,TCF7,WNT5A,FZD6,AXIN2,LEF1,MYC,NLK,WISP1,FOXN1,SOX174.Decidualization-related proteins(cyclin-D3 and desmin),MET-related epithelial cell markers(E-cadherin),and β-catenin-related proteins(β-catenin,foxo1)in ESC in the experimental group showed an increasing trend.MET-related stromal cell markers(N-cadherin)showed a downward trend.Observe the cell morphology under the microscope:In the blank control,ESCs showed a typical stromal cell shape:the shape of a slender spindle without obvious intercellular connections.In the TCM treatment group,ESC gradually transformed into an epithelial cell phenotype,manifested as multinucleated circular cells,with secretion,translucency,and abundant cytoplasm.In the positive decidualization control group of E2+P4+cAMP in vitro,ESCs were closely linked to each other,and changed from the spindle shape of typical stromal cells to the typical shape of round epithelial cells.5.Infected cells were divided into two groups by flow cytometry;β-catenin protein in ESC knockdown system was measured by WB and RT-PCR.The results showed that β-catenin protein was reduced,which verified that the shRNA system was successfully constructed.(P<0.05)6.Knocking down β-catenin will cause morphological changes of ESC,which are round or oval,and the decidual cells in the negative control have epithelial characteristics.At the same time,downregulation of β-catenin by shRNA1 and shRNA2 in ESCs leads to a decrease in decidualization markers(cyclin-D3,Desmin),epithelial cell markers(E-cadherin),and downstream migrating protein(foxo1),and stromal cells.Increase in marker(N-cadherin),(P<0.05)Conclusions:This study provides the first evidence that TCs can enhance decidualization and MET of ESCs through the WNT/β-catenin signaling pathway.It therefore provides a promising cell therapy for certain gynecological diseases and reproductive problems associated with decidual insufficiency.