| Endometrium is a dynamic tissue with high regeneration capacity. The human endometrium undergoes cyclical processes of regeneration, differentiation and shedding under the effect of hormone from ovary as part of the menstrual cycle. Endometrial regeneration also follows parturition, extensive curettage or electrosurgical ablation and in postmenopausal women taking estrogen replacement therapy. In non-menstruating species, there are cycles of endometrial growth and apoptosis rather than physical shedding. The concept that endometrial regeneration is mediated by stem cells located in basalis endometrium, rather than the functionalis or myometrium, was postulated many years ago. Further indirect evidence from primate studies and clinical practices also suggest the existence of endometrial stem/progenitor cells. More evidences show up with the researches moving on, and people consider that it is possible that there may be epithelial and stromal stem/progenitor cells and putative endometrial stem/progenitor cells may play a role in the pathophysiology of diseases such as endometriosis, endometrial hyperplasia, endometrial cancer and adenomyosis. Alterations in the number, function, regulation and location of epithelial and/or stromal endometrial stem/progenitor cells may be responsible for any one of these endometrial diseases. Endometrial stem cell research is still in its infancy. In a recent study, side population (SP) cells were detected from human endometrium. But the progress is limited because some functional assay cannot be applicated in human being and some researches are restricted by ethics. The mouse is a well-established animal model for physiological and pathological researches of human being. Investigations about identify the characteristics and the role of putative side population endometrial stem/progenitor cells in mouse endometrium, will lay the groundwork for further studies to characterize properties of stem cell candidates in vivo, the mechanism involved in regulating epithelial and stromal growth, and will give messages to physiological and pathological researches of human being. It will also offer cell materials for new medicine examinations. In this study, we aim to isolate SP cells from the endometrium of adult murine uterus, to characterize their stemness, culture them in vitro, and investigate the role of estrogen on them. Then we can do further researches about the special properties of endometrial stem cells, the role of endometrial stem cells in the regeneration of endometrium, and offer an animal model for the physiological and pathological researches of human being.Methods of the present study were: 1) enzymatic digestion combined with mechanical isolation was used to isolate endometrial epithelial and stromal cells to identify and quantify the live SP cells in murine endometrium with the Hoechst33342-SP method by flowcytometry; 2) to characterize the phenotypes of SP cells, such as CD34, CD45, CD105, SCA-1 and c-Kit, by flowcytometry and to analyze the component of SP cells by immunofluorescence; 3) to evaluate the stemness of SP cells by colony-forming assays and cell cycle analysis; 4) to analyze the expression of BCRP1/ABCG2 associated with the SP phenotype as to locate epithelial and/or stromal SP cells in murine endometrium by immunofluorescence; and, 5) to investigate the role of estrogen on the regeneration and differentiation of SP cells in vitro.Results showed that: 1) These two methods, used to make endometrial single cell suspension, could get enough cells to be stained by Hoechst. The mechanical trituration method was not suitable for SP cell isolation. SP cells could not be detected in the cell suspension made by this method, and there are many cell chips in the suspension. The enzymatic digestion combined with mechanical isolation method, with fewer cell chips, was fit for the stable SP cell isolaton. 2) SP cells could be stably detected in the cells from postpartum murine endometrium, but not from the endometrium of a uterus undergoing a normal estrus cycle. When pure ICR female mice were mated with pure GFP male mice, the SP cells isolated from the postpartum endometrium of them did not express GFP fluorescence. SP cells were from the maternal tissues but not the fetal tissues.3) The percentage of the SP cells varied with the time going. On the first day after parturition, 0.88±0.54% SP cells were detected. The percentage of SP cells went up gradually, peaked at day 17.5(13.95±3.42%), then went down. On the 60th day after parturition, SP cells could not be detected any more.4) SP cells were a heterogeneous population. By the analysis of epithelial and stromal cell surface markers, CK-18 and Vimentin, we found that SP cells were mainly composed of stromal cells (90.82±1.83%), with a small part of epithelial cells (7.83±2.27%) and perivascular cells expressingαSMA.5) SP cells came from the maternal tissues. Phenotype analysis showed that most SP cells were negative for hematopoietic, endothelial, and mesenchymal stem cell markers. The percentage of SP cells positive for CD34 was 11.75±2.39%, for CD45 was 8.95±5.51%, for CD44 was 4.27±1.30%,and for CD105 was 2.64±0.48%. They were a heterogeneous population, mainly of stromal cells, expressing Sca-1 (41.71±15.05%) and c-kit (2.57±1.67%) at various degrees. 6) SP cells showed characteristics of stem/progenitor cells. SP cells were found to reside in quiescence. By FASC, it was found that SP cells isolated directly from tissue were largely in G0/G1 (86.34±5.73%), with 13.15±5.44 in G2 and 0.5±0.34% in S. SP cells had significantly higher colony-forming capacity than main population cells when seeded at extremely low density. SP cells showed greater cloning efficiency (CE) than MP cells (0.37±0.06% vs. 0.14±0.05%, p<0.01) when they were seeded at the density of 300 cells/cm2, and Ces were 0.52±0.13% for SP cells and 0.12±0.02% for MP cells seeded at 500 cells/cm2. The differences were significant. SP cells expressed BCRP1/ABCG2 and were diffusely distributed in the stroma compartment of postpartum murine endometrium. Some of them were observed in the vascular endothelium and glands, but none was seen in the luminal surface epithelium. 7) When seeded under the kidney capsule, SP cells could grow and differentiate into tissues like endometrium. They showed the ability of proliferation and differentiation in vivo like stem /progenitor cells. 8) SP cells expressed estrogen receptor and could be regulated by estrogen in vitro. It was found that 75.18±5.47% of SP cells expressed Estrogen Receptorα(ER-α) when freshly isolated. The expression of ER-αin SP cells was significantly higher than MP cells. Real-time and Western Blot analysis showed that the variance of SP cell percentage was the same as it of estrogen level and ER-αexpression in the postpartum uterus tissues. There are a few cells expressing ER-αin the uterus tissue of day 1 and 3 after parturition. They were mianly stromal cells under luminal epithelium. The expression of ER-αincreased from day 7, and they were mainly distributed in the stroma part of the uterus. Some stromal cells, glandular cells and perivascular cells expressed ER-α. 9) Estrogen could promote both regeneration and differentiation of SP cells through the classical pathway. High concentration had significant effect on the regeneration of SP cells, while low concentration similar to the physical condition showed obvious effect on the differentiation of SP cells. Three different concentrations, 10-6,10-7,10-8M, of estrogen could stimulate the proliferation of SP cells. When the inhibitor of ER-α, ICI, was added, the promotion effect could be blocked. The concentration of 10-6M showed significant promotion effect. When the three different concentrations, 10-6,10-7,10-8M,of estrogen were used, the CEs were 0.24±0.07%, 0.19±0.06% and 0.21±0.04%. The large CEs were 0.07±0.05%, 0.04±0.03% and 0.02±0.01%.The CEs went down and the effects could be blocked by adding ICI. But, interestingly, the large CEs grew up when the concentration of estrogen was higher.Conclusion: 1) The present study successfully establishes a method to isolate SP cells from postpartum murine endometrium. The enzymatic digestion combined with mechanical isolation method was used to isolate endometrial epithelial and stromal cells to identify and quantify the live SP cells in murine endometrium with the Hoechst33342-SP method by flowcytometry. It lays ground work for further study of the SP cell characteristics. The SP cells were a heterogeneous population, mainly composed of stroma cells. They also contained a few epithelial cells and perivascular cells. The percentage of SP cells went up then down after parturition. But they could not be detected in normal estrous cycle.2) The biological characteristics and stemness of the SP cells showed that they expressed stem cell markers, had colony forming capacity, were murine endometrial stem/progenitor candidates from maternal uterus tissues. Further researches about their role in the regeneration of endometrium and the involving regulation mechanisms could be undergoing. 3) SP cells could be regulated by estrogen. It shows that sexual hormones could regulate the proliferation and differentiation of endometrial stem/progenitor cells to take part in the recovery of the endometrium.These data suggest that, like other tissues and organs, the murine endometrium also contains SP cells that could be isolated using the Hoechst33342-SP method. They were proved to be a heterogeneous stem/progenitor cell population. SP cells expressed estrogen receptor and could be regulated by estrogen through the classical pathway in the regeneration and differentiation progress. Their specific role in the regeneration of the endometrium warrants further study. Further researches about the function of endometrial stem cells in murine will help to study the role of human endometrial stem/progenitor cells in the mechanisms of physiological and pathological endometrial regeneration. In the future, these study will offer new breakthrough points in the treatment of disease associated with abnormal endometrial regeneration.
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