ABSTRACT
Objective: To establish the approach of isolating, culturing and identifing endometrial stem cells (EnSCs) derived from ectopic lesion of endometriosis patient; and preliminarily examine the biological characteristic of ectopic EnSCs, which provide support for further study on the potential role of ectopic EnSCs in the pathogenesis of endometriosis. Methods: The ectopic lesions of endometriosis were harvested from the patients with the informed consent and transferred to lab as soon as possible. The ectopic lesions were minced, digested by collagenase I and seeded into cell culture flasks for conventional culture (n=10). Expression of vimentin in ectopic EnSCs was examined by immunofluorescence (n = 3). Proliferative capacity of ectopic EnSCs was examined by MTT assay (n = 5). Multilineage differentiation potential of ectopic EnSCs was examined by adipogenic and osteogenic differentiation respectively (n = 3). Immunophenotype analysis of ectopic EnSCs was determined by flow cytometry (n = 3). Production of biological factors in ectopic EnSCs derived conditional medium (n = 6) and expression of adhesion molecules on ectopic EnSCs (n = 7) were examined by protein assays. Results: We successfully isolated EnSCs from ectopic lesions of endometriosis patients, and ectopic 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 ectopic EnSCs showed obvious lipid droplets (adipogenic differentiation) and calcium nodules (osteogenic differentiation). The ectopic EnSCs could secrete high concentration of angiogenic factors [vascular endothelial growth factor (VEGF), angiotensin (ANG) and platelet-derived growth factor (PDGF)-AA]and angiogenesis associated inflammation cytokines [interleukin (IL-6), IL-8 and monocyte chemotactic protein l(MCP-l)]. Additionally, adhesion molecules analysis demonstrated the high expression of activated leukocyte adhesion molecule (ACLAM) and intercellular cell adhesion molecule-1 (ICAM-1) on ectopic EnSCs. Conclusion: We successfully establish the procedure of isolating and culturing ectopic EnSCs and demonstrate that ectopic EnSCs is capable of promoting angiogenesis through secreting high concentration of associated biological factors. The above result confirm the existence of EnSCs in ectopic lesions of endometriosis, which not only supports the stem cell based pathogenesis of endometriosis, but also shows the therapeutic potential of taking ectopic EnSCs as promising targets in the treatment of endometriosis.
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AIM:To investigate the myocardial protective effect of endometrial stem cell ( EnSC)-derived cyto-kine cocktail ( EdCC) on myocardial ischemic reperfusion injury and the MEK-ERK signaling pathway.METHODS: A mouse model of myocardial ischemic reperfusion injury was established.Infarct area, cell apoptosis, and expression of cleaved caspase-3 and phosphorylatied ERK1/2 were determined by TTC/Evans blue staining, TUNEL assay and Western blot, respectively.RESULTS:The mesenchymal characteristics were observed in the EnSCs with expressing CD90 and in absence of CD34 and CD45.EdCC contained (6 811 ±312) ng/g epidermal growth factor (EGF) protein.The phospho-rylation of ERK1/2 markedly increased after injection of EdCC, but was abolished by MEK1 inhibitor PD98059 ( 5 mg/kg) .EdCC decreased the infarct area and apoptotic cell number in the border zone and inhibited caspase-3 activation. However, the effects were abolished by MEK1 specific inhibitor PD98059.EGF did not decrease the infarct area, but the EGF receptor antagonist AG-1487 (6 mg/kg) partly abolished the myocardial protective effect of EdCC.CONCLUSION:EdCC protects the myocardium from ischemic reperfusion injury via activating MEK1-ERK signaling pathway, indicating an essential role in the transmission of stem cell therapy from the cell transplantation to cytokine based strategy.
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Objective To investigate the expression of endometrial developing-related genes,the endometrium-like structure and cells during spontaneous differentiation of hESCs. Methods (1) Embryoid bodies were cultured in suspension for 14 days,fixed in 10% neutral-buffered formalin and embedded in paraffin.Estrogen receptor(ER) was detected by immunocytochemical staining.(2) hESCs were cultured in a 35 mm plastic dish and differentiated spontaneously for 10 days.The expression of ER and Vimentin/Keratin was detected by double immunofluorescence histochemistry.(3) hESCs were cultured in a 35 mm plastic dish and differentiated for 5 days.The expressions of endometrial developing-related genes including Wnt4,Wnt7a,Wnt5a,Hoxa10,Hoxa11 and ER were detected by RT-PCR. Results (1) The endometrium-like structures were detected in 14-day-old EBs and some were positive for ER staining.(2) Some spontaneously differentiated cells from hESCs for 10 days were positive for ER together with Vimentin/Keratin.(3)Wnt7a,Wnt4,Hoxa10,Hoxa11 and ER were detected by RT-PCR in hESCs which were differentiated spontaneously for 5 days.Conclusion During the spontaneous differentiation of hESCs,some cells are liable to differentiate into endometrial stem cells and endometrium-like cells.However,further identifications of the whole development process are needed to be done in the future.