Construction And The Massive Self-induced Hepatic Differentiate Evaluation Of Human-derived Tet Regulated Stably Expressing Cell Lines

Part 1:Construction of Tet-on regulated stably expressing HGF/FGF4 human MSCsAIM:Construct Tet-on regulated stably expressing HGF/FGF4 cell lines from human MSCs through lentiviral transfection.METHODS:HGF and FGF4 genes were synthesized and then spliced into lentiviral vectors plenti6.3/TO-HGFand plenti6.3/TO-FGF4.Firstly,we transfected Lenti3.3/TR into UE7T-13 cells to construct UE7T-13-TR cell line possessing Tet-on gene swift.And then use plenti6.3/TO-HGFand plenti6.3/TO-FGF4 transfecting UE7T-13-TR cells respectively to construct UE7T-13-TR-HGF cell line that can be regulated by Tet stably expressing HGF and UE7T-13-TR-FGF4 regulated stably expressing FGF4.Check the expression of target genes through QPCR,Western Blot and Elisa in the level of gene,protein and secretion respectively.RESULTS:We successfully constructed UE7T-13-TR-HGF and UE7T-13-TR-FGF4 cell lines.Through QPCR we verified that HGF gene expressed in UE7T-13-TR-HGF with Tet was 78 folds than without Tet,and FGF4 was more than 20 thousands.Western Blot and Elisa test confirmed these results and verified that HGF and FGF4 can be synthesized in protein level and secreted outside cell membrane.CONCLUSION:We successfully constructed UE7T-13-TR-HGF and UE7T-13-TR-FGF4 cell lines through lentiviral transfection,laid the foundation for further study.Part 2:Self-induced hepatic differentiate evaluation of human-derived Tet regulated stably expressing HGF/FGF4 cell linesAIM:Self-induced hepatic differentiate evaluation of cell lines constructed in part 1.METHODS:Firstly,we verificated cell surface markers of UE7T-13-TR-HGF and UE7T-13-TR-FGF4 cells.And then we evaluated the multiplication capacity of these cells.Hepatic differentiation test were divided into 4 groups(Group A:UE7T-13-TR-HGF/UE7T-13-TR-FGF4 cells were added with extrinsic induced factors of 35 ng/ml HGFand 35 ng/ml FGF-4;Group B:UE7T-13-TR-HGF cells added with 1?g/ml Tet;Group C:UE7T-13-TR-FGF4 added with 1?g/ml Tet;Group D:co-culture of UE7T-13-TR-HGFcells and UE7T-13-TR-HGF cells added with 1?g/ml Tet(group D were divided into 3 subgroups:2:1;1:1;1:2,according to different proportions)?RESULTS:About 90 percent cells were CD90+CD44+CD45-,indicated that UE7T-13-TR-HGF/UE7T-13-TR-FGF4 cells retain MSCs surface markers.MTT test proved that UE7T-13-TR-HGF/UE7T-13-TR-FGF4 cells still possess strong proliferative capacity,but would be inhibited by Tet.Hepatic differentiation test confirmed that UE7T-13-TR-HGF/UE7T-13-TR-FGF4 cells retain hepatic differentiate ability,and can be induced by high concentration of HGF and FGF4 secreted by themselves.Group D with 1:2 had the best self-induced hepatic differentiate results.CONCLUSION:The cells constructed in part 1 can be self-induced into hepatocyte-like cells in the presence of Tet,and the co-culture of UE7T-13-TR-HGF and UE7T-13-TR-FGF4 cells with 1:2 was the best collocation,could be expected to applied into liver tissue engineering and regenerative medicine in the furture.Part 3:Massive self-induced hepatic differentiate evaluation of human-derived Tet regulated stably expressing HGF/FGF4 human stem cell linesAIM:Massive self-induced hepatic differentiate evaluation of human-derived Tet regulated stably expressing HGF/FGF4 human stem cell lines constructed in the part 1.METHODS:UE7T-13-TR-HGF and UE7T-13-TR-FGF4 cells were rapidly proliferated by microcarrier vehicle Cytodex3 in a short period of time,observation of cells growth and count.Using the method of large-scale proliferation in the first step and add with Tet to be induced into hepatocyte-like cells in the second step to get large-scale hepatocyte-like cells.Electron microscopic observation of cell morphology change was applied.Immunofluorescence test and functional identification of differentiated cells were used.RESULTS:Cell density reached its peak on the 7th day of culture which was 1.76±0.12×109/L.cell morphology change was observed on the 8th day after Tet was added,and differentiated cells were identified as hepatocyte-like cells through Immunofluorescence test and functional identification.CONCLUSION:Cultured with microcarrier vehicle Cytodex3,a sufficient amout of human stem cells were obtained and successfully differentiated into functional hepatocyte-like cells,could be expected to applied into liver tissue engineering and regenerative medicine in the furture.Thus,we established an effective progam of getting Large-scale functional hepatocyte-like cells from human stem cells.