The Study Of Isolation, Culture And Biological Characteristics Of Endometrial Stem Cells From Human Decidual Tissue

Research found that endometrial stem cells exist in human endometrial function layer and the base layer. There is a significant correlation between endometrial stem cells with endometrial repair, endometriosis, endometrial polyp, and so on. Endometrial stem cell also has been widely used in endometrial reconstruction, pelvic organ prolapse, bladder tissue repair tissue repair, and all that. At present, endometrial stem cells mainly obtained from ectopic endometrium and normal endometrium, however, we found that is difficult to access endometrial stem cells from the patients whose endometrium was severe injury by curettage, endometrial inflammation. Study suggested that endometrial damage (such as severe intrauterine adhesions) is mainly caused by Endometrial stem cell deficiency or functional defect. So we try to extract endometrium stem cells from the decidua and storge as we can. This way has the advantages of convenient operation and without ethical restrictions, which can provide reliable basis for the diseases. The study was separation, purification, amplification, culturing endometrial stem cell from deciduas, and then identify endometrial stem cells. And observe endometrial stem cell multidirection differentiation properties in vitro. Provide a model cells for endometrial injury, pelvic organ prolapse, vaginal reconstruction, endometriosis, endometrial polyps, endometrial cancer and other diseases related to uterus endometrial stem cell.Objiective1. To investigate isolation, culture and identification of Human Endometrial Stem Cells in decidua.2. To investigate the biological characteristics of the endometrial stem cells in decidual, and observe the differentiation potential of endometrial stem cell to osteoblast and smooth muscle cells in vitro.Methods1. Human endometrial stem cells were digested and isolated from decidua using pancreatin and collagenase II, and cultured with DMEM/F12 in very low density in 10 cm dish, and then specific clones were isolated, Morphology and clone of the Human Endometrial Stem Cell was observed under phase-contrast microscope.2. The growth curve and doubling time was analysed by CCK-8 assay.3. Cell cycle percentage and surface antigen (CD29、CD31、CD34、CD44、CD45、 CD73、CD90、CD105、CD146)was detected by flow cytometry.4. The endometrial stem cell was iduced by osteogenic induction and smooth muscle induction liquid respectively, to study its potential to osteoblast and smooth muscle cells in vitro.5. Alkaline phosphatase staining and alizarin red staining were used to identify osteoblast induced from endometrial stem cell and western blot and immunofluorescence were used identify smooth muscle cells induced from endometrial stem cell.Results1. Endometrial Stem Cells were spindle-like shapes, fibroblast like cells, which had different thicknesses long protuberances, arranged tightly in a vortex-like appearance when grew up to 80-90%. The clone formation rate of the Endometrial Stem Cells was 3.91% to 9.14%.2. The growth curve was "S" shaped with 48 to 60 hour to logarithmic phase and the ninth days to plateau and the culture doubling time was 37-52h.3. Flow cytometry analysis showed that Human Endometrial Stem Cells were positive for CD29(99.13%), CD44(97.39%), CD73(99.68%), CD90(96.76%), CD105(89.64%) and CD146(77.96%), but they were negative for CD31(1.30 %), CD34(0.68%), CD45(1.26%).4. Cell cycle analysis showed that 61.79% of the Human Endometrial Stem Cells was in the G0/G1 phase,22.37% of the cells was in S+G2/M phase.5. The osteogenic induction medium and smooth muscle induction medium can induce endometrial stem cells to differentiate into osteoblasts and smooth muscle cells.ConclusionHuman Endometrial Stem Cells were successfully isolated from monoclonal endometrial cells by tissue digestion method, and osteogenic induction medium...