Isolation,culture And Cloning Of Human Endometrial Stem/Progenitor Cells In Vitro

Objective: in this study,endometrial stem / progenitor cells were isolated,cultured and cloned from three different tissues of human endometrium,decidua and menstrual blood to observe whether there were differences among the stem cells from three different tissue sources.At the same time,two different methods,plate colony forming experiment and 96 pore plate limited dilution method,were used to culture the monoclonal of the menstrual blood mesenchymal stem cells(MSCs)to observe whether there were differences between the two methods.Method:1.Immunohistochemistry and immunofluorescence were used to identify the location of e MSCs in vivo.2.The endometrial and decidual stem / progenitor cells were isolated and cultured in vitro by collagenase digestion,filtration with different apertures and differential attachment.3.Menstrual blood MSCs were isolated and cultured from menstrual blood by horizontal centrifugation of human peripheral blood lymphocyte isolate.4.The 96 well plates were coated with different concentrations of human fibronectin to determine the effect of human fibronectin on the adhesion and proliferation of menstrual blood MSCs,so as to determine the optimal concentration of the coating.5.Two different methods,plate colony forming experiment and 96 well plate limited dilution method,were used to culture of menstrual blood MSCs.6.Immunocytochemistry and immunofluorescence were used to identify the surface markers of endometrial and decidual stem / progenitor cells and menstrual blood MSCs.7.Adipogenic and osteogenic differentiation of menstrual blood MSCs with different stem cell phenotypes were studied.Result:1.In vivo,the surface markers of endometrial MSCs are mainly expressed in the basal layer,such as CD140? and CD146 antibodies,and CD146 and CD140? are Co-located around the blood vessels in the extracellular matrix,especially around the blood vessels in the basal layer;CD90 is mainly expressed in the functional layer;there is no significant difference in the expression of SUSD2 between the functional layer and the basal layer.2.In vitro,there were differences in cell morphology between endometrial and decidual MSCs.The primary decidual MSCs showed decidualization.After three passages,their morphology was basically the same as endometrial MSCs,and both MSCs could be passed on for more than30 generations and grew for more than 6 months.The clone ability of primary and passage cells of the two MSCs was basically the same.Endometrial and decidual MSCs could be successfully separated from each other The progenitor cells were cultured,but this experiment was not successful.3.MSCs of menstrual blood can be successfully isolated and cultured by using human peripheral blood lymphocyte separation solution,and can also be passed on for more than 30 generations and grow for more than 6 months.The separation process of menstrual blood is simpler than that of endometrium and decidual tissue.It can be seen from the growth curves of endometrial,decidual and menstrual MSCs P3 that there may be stem cells with stronger proliferation ability in menstrual MSCs.4.Through the effect of different concentrations of human fibronectin on the adhesion and proliferation of MSCs in menstrual blood,the optimal concentration of human fibronectin coated on MSCs in menstrual blood was determined to be 10?g / m L.5.Both the plate colony forming experiment and the modified 96 well plate limited dilution method can successfully clone the MSCs of menstrualblood.The modified 96 well plate limited dilution method is simpler and has higher cloning rate.6.CD140?,CD90 and SUSD2 were expressed in all the large and small clones of MSCs,and CD146 was expressed in some of the large clones.The self-renewal and propagation ability of CD146 positive clones were stronger.7.CD146 and CD140? double positive clones in MSCs of menstrual blood have stronger ability of adipogenic and osteogenic differentiation.Conclusion:1.MSCs were successfully isolated and cultured from endometrium,decidua and menstrual blood,and the process of menstrual blood separation was the simplest.2.In the process of monoclone culture of menstrual MSCs,human fibronectin can promote cell adhesion and proliferation,so as to improve the efficiency of monoclone.3.Compared with the plate colony forming experiment,the modified 96 well plate limited dilution method is simpler and more efficient.4.MSCs with CD146 and CD140? double positive menstrual blood have higher ability of self-renewal,proliferation and passage,adipogenic and osteogenic differentiation.