The Experimental Study Of Construction And Reparation Function Of Adipose Derived Stem Cells Sheet

Backgroud:Since the birth of the first infant that was conceived with the help of in vitro fertilization and embryo transfer?IVF-ET?in 1978.So far,there are more than six million ART?Assisted Reproductive Technology?infants have been born worldwide.However,ART could not solve all problems that cause infertility,for example,Asherman`s syndorome?AS?is one of the hardest challenges that makes the most experienced clinician frowns.Asherman`s syndorome?AS?is characterized by intrauterine adhesions or fibrosis following damage to the basal layer of endometrium.Intrauterine adhesion and scarring often lead to amenorrhea,infertility,and various complications of pregnancy including placental abruption,preterm premature rupture of membranes,and malpresentation as results of loss of normal endometrium.The prevalence of Asherman`s sndrome is reported as 8%in women undergoing routine infertility evaluation.The mainstream is confined to surgical remodeling of the uterine cavity.Regretfully,The final outcome of surgical therapy depends on the endometrial regeneration capability following restoration of uterine patency,no matter what preventive measures were taken,the pregnancy rate was not significantly improved following surgical restoration of uterine patency.Since the endogenous endometrial stem cells that reside in the endometrial basalis layer presumably serve as a source of endometrial regeneration,Intrauterine adhesions?IUA?involving severe damage to the endometrial basal layer may result in the destruction of endogenous endometrial progenitor stem cells and the loss of endometrial regeneration ability.In the circumstance,ADSCs will serve as potential endometrial stem cells,which may constitute a source of cells for endometrial repair.Therefore,we select human ADSCs to construct ADSCs cell-sheet by a continuous culture method,and further study the biological characteristics and restorative function of human ADSCs cell-sheet,to explore the feasibility of human`s ADSCs cell-sheet in the treatment of endometrial injury.Objective:To construct human adipose-derived stem cells?ADSCs?sheet by isolated and purified ADSCs,And to explore its feasibility for the treatment of IUA.Methods:1.Isolation and characterization of human ADSCs and construction of human ADSCs cell-sheet:Fatty tissue was collected from subcutaneous located in the abdomen of young woman.To get human ADSCs,fatty tissue was digested by collagenase,and then isolated and purified by flow cytometry.The self-renewal ability of the obtained human ADSCs was tested by CCK-8.Addtionally,the multipotent differentiation ability was also identified by adipogenic and osteogenic induction.Construction of human ADSCs cell-sheet:Human ADSCs were incubated and cultured in differentiation inducing culture medium and formed cell-sheet.The morphology and structure of cells sheets were observed by scanning electron microscope?SEM?.2.Preliminary study of restorative function of human ADSCs cell-sheet:The expression of several growth factors and stemness related genes in cell-sheets were detected by qRT-PCR and ELISA.3.In vitro study of the potential of human ADSCs differentiating toward endometrial epithelial cells:Human ADSCs were treated as follows:Group 1:ADSCs were cultured alone as control medium??-MEM,5%FBS?;Group 2:ADSCs were cultured in the differentiation inducing medium(?-MEM,5%FBS,1x10^-7mol/L 17?-estradiol,10ng/ml TGF-?1,10ng/ml EGF,10ng/ml PDGF-BB);Group 3:ADSCs were co-cutured with endometial cells in control medium;Group 4:ADSCs co-cutrured with endometial cells in the differentiation inducing medium.Group 5:ADSCs cell-sheet co-cutrured with endometial cells in the differentiation inducing medium.Immunofluorescence and qRT-PCR was carried out 5 days after culturing cells involed in the 5 groups described above to test the expression of epithelial cells`markers,such as cytokeratin-7?CK7?,cytokeratin-18?CK18?,cytokerati-19?CK19?and epithelial membrance antigen?EMA?.Results:1.Human ADSCs were successfully isolated and purified in vitro.The P0 cells were short spindle-shaped and were primarily adherent after being cultured for 4h and completely adherent after being cultured for 24h.ADSCs grew well after 3 times of passage and the morphology was fibroblast-like.Mesenchymal stem cell markers,such as CD29,CD44,CD90 and CD105 were positively expressed on human ADSCs,while CD45?one of hematopoietic stem cells markers?was negative.The expression condition was consistent with characteristics of ADSCs.After osteogenic and adipogenic induction,the calcified nodule and fat drops could be seen using Alizarin red staining and oil O red staining,respectively,which suggested that the human ADSCs had multipotent differentiation potential.After in vitro culturing of human ADSCs for 10-14 days in differentiation inducing media?-MEM,a white membranous sheet was formed and could be detached by slight mechanical force can could detach it.SEM analysis revealed that closedly adherent human ADSCs grew in dense.2.The expression of growth factors incluing TGF-?1,FGF,HGF and VEGF and the stemness-related genes like NANOG and OCT4 was significantly increased at mRNA level in ADSCs sheets compared with the single layer of the adherent ADSCs.In addition,the expression of TGF-?1,FGF2,HGF and VEGF was significantly higher at protein level in the suspernatant of cell-sheet culture medium.3.In immunofluorescence analysis,showed that undifferentiated ADSCs in group 1and 2 were negative for cytokeratin 18 expression.However,the differentiated ADSCs in group 3,4 and 5 were weakly and strongly positive for cytokeratin 18 expression repetively.The results of qRT-PCR showed that the mRNA expression for CK18 and CK19 was significantly higher in group 2,3,4 and 5 than in the control group,and its expression was upregulated in sequence from group 1 to group 5.The mRNA expression level of CK7 and EMA was significantly upregulated in group 2,3,4 and 5 when compared with group 1.Conclusion:1.We successfully isolated human ADSCs and evently constructed the ADSCs Cell-sheet.The ADSCs cell-sheet manifested significant superiorities in the tissue growth promotion and moltipotent differentiation potential.The results have brought us a very promising method for the treatment of severe endometrial injury.2.Human ADSCs could differentiated toward endometrial epithelial cells in a proper induing condition in vitro.Exogenous and endogenous factors may coordinately create the most appropriate microenvironment for the differentiation of ADSCs toward endometrial epithelial cells.