Biological Characteristics Of Different Tissue-specific Amniotic Fluid-derived Stem Cells And Their Treatment In Septic Mouse Model

Background:Pluripotent progenitor cells called mesenchymal stromal / stem cells(MSCs)can be isolated from a variety of human tissues and organs,and their research has developed rapidly in the past decade,providing a continuous driving force for the field of cell therapy and regenerative medicine.The most widely studied sources of MSCs include bone marrow,fat,umbilical cord and placenta.According to the standard definition of MSC by the International Association of Cell Therapy(ISCT),cells are required to adhere to plastic plates and express corresponding differentiated cluster(CD)markers(such as CD73,CD90 and CD105 markers),and can be adipogenic,chondrogenic and osteogenic differentiation in vitro.The cell therapeutic potential of MSCs is mainly reflected in its differentiation potential and immunomodulatory ability,especially the immunomodulatory ability makes it show a good effect in different types of inflammatory diseases in many systems of the whole body.Cells that are highly consistent with the biological characteristics of MSCs can also be isolated from amniotic fluid,which are usually defined as amniotic fluid mesenchymal stem cells(AFMSCs)or amniotic fluid-derived stem cells(AFSCs).It is generally believed that AFSCs is between embryonic stem cell(ESCs)and MSCs,and has more good biological characteristics than traditional MSCs,such as faster proliferation,wider differentiation potential and stronger immunomodulatory ability compared with bone marrow and adipose MSCs.However,AFSCs has significant heterogeneity,and many reports have shown that AFSCs is unstable in many aspects,such as cell morphology,molecular phenotype and differentiation ability.The difference of biological characteristics caused by cell heterogeneity will further expand this difference in the process of cell proliferation in vitro,and then affect the stability of clinical cell therapy.The heterogeneity of AFSCs is mainly due to the existence of different tissue and organ sources.Some studies have pointed out that specific markers of bone marrow,skin,kidney,liver,lung,heart and other tissues and organs can be detected in amniotic fluid cells.Up to now,although different tissue-specific AFSCs,have been isolated from international studies,there is no in-depth detection of their biological characteristics and evaluation of clinical therapeutic efficacy.For this reason,we intend to isolate different tissue-specific AFSCs,to evaluate their biological characteristics and therapeutic efficacy from multiple angles,so as to provide feasible ideas for solving the heterogeneity of AFSCs and provide a more comprehensive and in-depth theoretical basis for the clinical application of AFSCs.PartⅠ:Isolation of stem cells from amniotic fluid by different culture methods and analysis of their biological characteristics Objective:Establish appropriate primary and subculture programs of AFSCs,evaluate their biological characteristics,and compare them with umbilical cord mesenchymal stem cells,so as to lay an experimental foundation for follow-up research.Method:1.Establish three culture models,commercial medium for primary and subculture(AFC),traditional mesenchymal stem cells for primary and subculture(MSC),commercial medium for primary culture and traditional mesenchymal stem cell subculture(AFC-MSC);2.The culture efficiency of different culture methods was evaluated by colony formation test,CCK-8 test and population doubling time test;3.Detection of CD markers on cell surface by flow cytometry;4.Osteogenic,adipogenic and chondrogenic differentiation of cells obtained from different culture methods,tissue specific staining and expression of differentiation marker genes were used to evaluate the effect of differentiation;5.The cells were co-cultured with activated peripheral blood mononuclear cells((PBMCs)).The inhibition ability of cells to PBMCs was evaluated by quantitative analysis of CFSE proliferation,expression of proliferation marker protein and expression of pro-inflammatory factors.Result:1.AFSCs cells can be obtained by all three culture methods,but the efficiency of AFC culture is higher,while that of MSC is cheaper,and the efficiency and cost of AFC-MSC culture are between the two;2.There were differences in the expression of CD105 in AFSCs obtained from different culture methods,but it did not affect the main biological characteristics such as cell morphology,expression of pluripotent genes,differentiation potential and immunosuppressive energy;3.Compared with UCMSCs,AFSCs has higher osteogenic and chondrogenic differentiation ability and stronger immunomodulatory ability.Conclusion:The difference of AFSCs,CD105 expression which can be effectively obtained by the three culture methods does not affect other major biological characteristics,and has stronger differentiation potential and immunosuppressive ability than UCMSCs.Considering the time and capital cost,it is more appropriate to choose the commercial medium for primary culture and traditional mesenchymal stem cell subculture.Part Ⅱ: In the second part,different tissue-specific amniotic fluid-derived stem cells were isolated and their biological characteristics were analyzed.Objective:AFSCs,with different tissue specificity was isolated and its biological characteristics were analyzed from many angles.Methods:1.The clones formed by single cells were isolated by mechanical cloning and subcultured;2.The tissue marker genes of the isolated clones were detected,and different tissue specific AFSCs were identified;3.Cell morphology was observed under microscope,proliferation ability was analyzed by population doubling time,β-galactosidase staining and cell cycle regulation marker genes were used to analyze the degree of senescence;4.The expression of embryonic stem cell factor was analyzed qualitatively by immunofluorescence,and the expression of cellular antigenic determinant and pluripotent factor was quantitatively analyzed by flow cytometry;5.Osteogenic,adipogenic and chondrogenic differentiation was carried out,and the degree of differentiation was evaluated by qualitative and quantitative analysis of differentiation specific staining and detection of differentiation marker gene expression;6.Scratch test,Transwell test and marker genes to detect the ability of migration and chemotaxis to evaluate the ability of cell migration;7.The cells were co-cultured with activated PBMCs,and the inhibition ability of CFSE cells to PBMCs was evaluated by cell proliferation test,the expression of proliferation marker protein and the expression of pro-inflammatory factor gene.Results:1.AFSCs cells in amniotic fluid from 18 to 22 weeks old can express lung and kidney tissue specific markers.By isolating and identifying single clones,kidney specific AFSCs(AFSCs-K),lung specific AFSCs(AFSCs-L)and unknown tissue specific AFSCs(AFSCs-X)can be isolated.2.There were differences in biological characteristics of different tissue-specific AFSCs.Compared with the other two groups,AFSCs-K had faster proliferation rate,higher expression level of CD90,CD105 and CD117,better mesoderm triplet differentiation ability and stronger immunosuppressive ability.3.AFSCs did not express OCT4,SOX2 and NANOG.AFSCs-K and AFSCs-X were strongly positive for SSEA4,and weakly positive for TRA-1-81.AFSCs-L weakly positive expression SSEA4,negative expression of TRA-1-81.4.AFSCs-K has stronger horizontal and vertical migration ability and expresses more migration marker genes,but AFSCs-L expresses more chemokine receptors.Conclusion:Different tissue-specific cells can be isolated from AFSCs,but the biological characteristics are different.Generally speaking,the property of AFSCs-X is better.The consistency of cells separated by tissue specificity is higher,and the isolation of different tissue specific AFSCs is a feasible way to solve the problem of cell heterogeneity.Part Ⅲ: Therapeutic efficacy of different tissue-specific amniotic fluid-derived stem cells on septic mice.Objective:To evaluate the therapeutic efficacy of different tissue-specific AFSCs in septic mouse model.Methods::1.Establishment of sepsis mouse model by cecal puncture.2.Carry on the survival analysis to the mouse,construct the survival cancellation.3.Bacterial culture of blood and peritoneal fluid to evaluate the antibacterial efficacy of AFSCs.The expression of pro-inflammatory factors in blood was analyzed by 4.Elisa,and the anti-inflammatory efficacy of AFSCs was analyzed.Results:1.Different tissue-specific AFSCs can significantly improve the survival rate of septic mice.2.Different tissue-specific AFSCs can help the body to remove bacteria,but the effect of AFSCs-X is more significant.3.Compared with AFSCs-L,AFSCs-K and AFSCs-X,the expression of pro-inflammatory cytokines was more inhibited,but there was no significant difference between the two groups.Conclusions:on the whole,AFSCs can improve the survival rate of sepsis and has the effect of antibacterial and anti-inflammation,among which the effect of AFSCs-X is more obvious.