| Project 1. Circulating endometriosis-derived mesencymal stem cells propagate the diseaseOBJECTIVE:Endometriosis is the growth of endometrial tissue outside the uterus and thought to reach ectopic locations primarily through retrograde menstruation and peritoneal seeding. Here we aimed to determine if endometrial cells derived from implanted peritoneal lesions were capable of entering the circulation and contributing to the spread of disease.MATERIALS AND METHODS:Experimental endometriosis (EMS) was established by transplanting endometrial tissue from DsRed+mice into the peritoneal cavity of C57BL/6 mice, either with or without ovariectomy (+/-OVX). Using Flow Cytometry Analysis and Cell Sorting (FACS) we identified DsRed+cells in blood obtained from animals with experimental endometriosis but not in controls. Fluorescent immuno-cytochemistry (FICC) and quantitative real-time PCR (qRT-PCR) were used to characterize these cells.RESULTS:Approximately 97% of the sorted circulating donor cells obtained expressed CXCR4 as well as endometrial mesenchymal stem cell (eMSC) markers CD140B & CD146. The sorted cells also expressed mesenchymal stem/stromal cell (MSC) biomarkers CD90, CD 105, CD9, and Oct3/4, but not hematopoeitic stem cell markers CD34 or CD45. The levels of circulating endometriosis derived cells (EDCs) were significantly higher in the EMS+OVX group at days (D) D2 to D8. Serum SDF-1 levels at D7 after surgery in EMS+OVX were significantly higher than EMS without OVX and both were greater than in sham control mice. Western blotting of protein derived from ectopic lesions also showed increased SDF-1. Ectopic and eutopic endometrium from the transplanted mice revealed single cells expressing DsRed, CXCR4 and the eMSCs marker CD140B & CD 146 in new vessels and the peritoneal wall of recipient mice. In human serum SDF-1 levels in endometriosis patients were significantly higher than a control group, suggesting that the SDF-1/CXCR4 axis is operational in women with spontaneous endometriosis as well.CONCLUSIOS:Stem cells rather than differentiated cells from endometriosis enter the circulation. These cells all express stem cell markers and the SDF-1 receptor (CXCR4). We posit that these cells respond to serum SDF-1, recruiting them preferentially to endometriosis, with increased mobilization after acute loss of estrogen. These circulating endometriosis stem cells contribute to both the endometriosis and angiogenesis at the ectopic sites. Further SDF-1 is a promising biomarker that may translate into a non-invasive clinical method to detect active endometriosis in womenProject 2. SDF-1/CXCR4 axis regulates circulating endometriosis derived stem cells and their migration of to distant organs in mice endometriosis modelOBJECTIVE:Endometriosis most commonly found in and around the pelvic area and can also be found in distal organs, but the pathogenisis remains uncertern. This study is to evaluate whether ectopic endometrial cells can migrate to distant organs and the role of the SDF-1/CXCR4(C-X-C motif chemokine 12/C-X-C motif chemokine receptor 4)axis in the development of disease.MATERIALS AND METHODS:Experimental endometriosis (EMS) was established by transplanting uterine horn fragments from donor DsRed mice into the peritoneal cavity of recipient C57BL6 mice. Control mice underwent sham surgeries. First day after inductions, mice were divided into SDF-1 injection groups, AMD3100 injection groups and vehicle injection groups. Flow Cytometry Analysis and Cell Sorting (FACS) were conducted on blood samples from all the animals for DsRed cells. Lung, liver and spleen were harvested four weeks after inductions, followed with FACS analysis and sort for DsRed cells. Paraffin embedded tissue sections were stained by immunofluorescence with anti-DsRed antibody (DsRed Antibody C-20, Santa Cruz Biotechnology, CA). Visualization of the slides was conducted using a confocal microscope (ZEISS LSM 710).Quantitative real-time PCR (qRT-PCR) was used to characterize the sorted DsRed cells in lung, liver and spleen.RESULTS:The levels of circulating DsRed endometriosis derived cells (EDCs) were significantly higher in the SDF-1 injection group (highest) and EMS group (second highest) during the first 17 days after inductions, but no difference were found in AMD3100 injection group comparing with control group. FACS analysis also revealed significant higher DsRed endometriosis derived cellsin lung, liver and spleen in the SDF-1 injection group (highest) and EMS group (second highest), but DsRed cells were slightly increased during D7-D9 after inductions in AMD3100 injection group. Immunofluorescence staining demonstrated the presence of single cells expressing DsRed in all of the recipient mouse lungs, liver and spleen consistent with results with FACS analysis. QRT-PCR showed that the sorted cells from lung, liver and spleen expressed CXCR4, mesenchymal stem cell markers CD90,CD105,OCT3/4, endometrial mesenchymal stem cell (eMSC) markers CD HOB and CD 146, but not the hematopoeitic stem cell markers CD45 or CD34.CONCLUSIOS:Cells from endometriotic lesions are capable of migrating to sites with high SDF-1 expression including distant organs like lung, liver and spleen and related to the circulating endometriosis derived cells. These cells express stem cell markers and CXCR4 which is known as the SDF-1 receptor. Hematologic dissemination and SDF-1/CXCR4 axis might be one of the potential pathogenesis of endometriosis derived cells migration to distant organs, and blocking of the SDF-1/CXCR4 axis reduces migration of endometriosis-derived stem cells to the distant organs. |
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