Biological Characteristics Of Endometrial Stem Cells Isolated From Ectopic Lesions Of Endometriosis

BackgroundThe pelvic pain,infertility and higher postoperative recurrence rate caused by endometriosis(EMs)seriously affect the quality of life of patients,but the detailed mechanism is still unknown.Also,our preliminary research findings showed that there were significant differences on the function and gene expression between the eutopic EnSCs isolated from control group and the ectopic EnSCs isolated from EMs patient.Based on the previous results,we suppose that the dysfunction of eutopic EnSCs plays a critical role on the onset of EMs,which directly causes the aberration of endometrium.Subsequently,retrograde menstruation delivers abnormal endometrium to the location outside the uterus where it implants and grows,and forms ectopic lesions finally.This study intends to detect the biological characteristics and functional differences between eutopic and ectopic EnSCs by routine biological methods.It not only improves the "stem cell theory" in the mechanism of EMs,but also provides a theoretical basis for the development of potential EMs diagnostic and therapeutic targets.Objective1.To establish methods for isolation,culture and identification of eutopic and ectopic EnSCs.2.To determine the biological activity and functional differences of EnSCs in the EMs patients and the control group.Methods1.The ectopic lesions of endometriosis and the eutopic endometrium of control group were harvested from the patients with the informed consent.The ectopic lesions and the eutopic endometrium were minced,digested by collagenase I and seeded into cell culture flasks for conventional culture.2.Proliferative capacity of eutopic and ectopic EnSCs were examined by MTT assay;multilineage differentiation potential of eutopic and ectopic EnSCs were examined by adipogenic and osteogenic differentiation respectively;immunophenotype analysis of eutopic and ectopic EnSCs were determined by flow cytometry.3.Functional differences of EnSC isolated from control group and EMs patient,such as migration,adhesion,and promotion of angiogenesis,were tested by conventional methods.4.Production of biological factors in the eutopic and ectopic EnSCs derived conditional medium and expression of adhesion molecules on the eutopic and ectopic EnSCs were examined by protein assays.5.RNA-sequence was performed to screen differentially expressed genes between the eutopic and ectopic EnSCs.Results1.EnSCs were successfully isolated from ectopic lesions of endometriosis patients and eutopic endometrium of people without endometriosis.EnSCs were positive for vimentin and typical markers of mesenchymal stem cell(CD29,CD73,CD90 and CD105),and negative for the markers of hematopoietic stem cell(CD34 and CD45).The induced EnSCs showed obvious lipid droplets(adipogenic differentiation)and calcium nodules(osteogenic differentiation).2.The ectopic EnSCs had stronger ability of proliferation,migration,adhesion and angiogenesis than the eutopic EnSCs.3.The ectopic EnSCs could secrete higher concentration of angiogenic factors(VEGF,ANG and PDGF-AA)and angiogenesis associated inflammation cytokines(IL-6、IL-8 and MCP-1)than the eutopic EnSCs.Additionally,adhesion molecules analysis demonstrated ALCAM and ICAM-1 in the ectopic EnSCs were more highly expressed than in the eutopic EnSCs.4.There were 523 different genes which expressed in the eutopic and ectopic EnSCs by high-throughput sequencing analysis,and the number of up-regulated genes were 244 while the down-regulated genes were 279.Conclusion1.EnSCs were successfully isolated from control group and EMs patients,and their biological activity detection found that they meet the criteria for mesenchymal stem cell identification.2.There were significant differences in the biological characteristics and functions of the eutopic EnSCs isolated from control group and the ectopic EnSCs isolated from EMs patient.