Characterization Of HESC-derived And Adult Mesenchymal Stem Cells After Lentivirus Transduction Of GFP Gene

Objective:1.PLVX-IRES-Puro-GFP lentivirus was used to transfer human embryonic stem cell derived mesenchymal stem cells,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells.The transduction efficiency of lentivirus to three kinds of human mesenchymal stem cells was analyzed by flow cytometry.2.Mesenchymal stem cells derived from human embryonic stem cells successfully labeled with green fluorescent protein gene,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells were subcultured.the effects of subculture on the morphology and fluorescence expression of green fluorescent protein gene positive cells were analyzed.3.The proliferation,differentiation and cell phenotype of human embryonic stem cell-derived mesenchymal stem cells,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells before and after fluorescent protein labeling were measured.The effects of lentivirus-mediated green fluorescent protein transduction on the proliferation,differentiation and phenotype of human mesenchymal stem cells were analyzed,which laid a foundation for in vivo tracing of human mesenchymal stem cells.Methods:1.pLVX-IRES-Puro-GFP lentivirus with the same amount was used to transfer human embryonic stem cell derived mesenchymal stem cells,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells.The lentivirus transduction efficiency of three kinds of human mesenchymal stem cells from different sources was detected by flow cytometry.2.The mesenchymal stem cells derived from human embryonic stem cells labeled with green fluorescent protein gene,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells were cultured and passaged,and the changes of cell morphology and the expression of green fluorescent protein gene were observed.The proportion of green fluorescent protein positive cells after 10 generations of cell proliferation was detected by flow cytometry.3.The proliferation of cells before and after green fluorescent protein labeling was measured by Cell Counting Kit-8.Adipogenic,osteogenic and chondrogenic differentiation was induced by directed differentiation medium.The differentiated cells were stained with ORO,Alizarin Red and Alcian Blue Stain Kit,pH1.0 staining solution.and the expression of cell surface antigens CD73,CD90,CD105,CD34,CD11 b,CD19,CD45 and HLA-DR were determined by flow cytometry.Results:1.There were significant differences in the efficiency of pLVX-IRES-Puro-GFP lentivirus transducing mesenchymal stem cells derived from human embryonic stem cells,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells.The results showed that the lentivirus transduction efficiency of mesenchymal stem cells derived from human embryonic stem cells was the highest,followed by human umbilical cord mesenchymal stem cells,and human adipose mesenchymal stem cells had the lowest transduction efficiency.2.Mesenchymal stem cells derived from human embryonic stem cells labeled with green fluorescent protein gene,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells were cultured and propagated for 10 generations.Almost all cells have green fluorescence under inverted fluorescence microscope.Under natural light,it can be seen that the cells adhere to the wall into a fusiform,the cytoplasm is rich,and the cells grow in a whirlpool as a whole,all of which maintain a normal and good cell morphology,and there is no significant difference compared with the same generation of untransduced stem cells.3.The proliferation curves of mesenchymal stem cells derived from human embryonic stem cells labeled with green fluorescent protein,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells were similar to those of unlabeled cells,but the OD values measured at 450 nm wavelength had no significant difference.Human embryonic stem cell derived mesenchymal stem cells,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells can still differentiate into adipocytes,osteoblasts and chondrocytes after labeling with green fluorescent protein gene.Flow cytometry showed that the positive rates of surface antigens CD73,CD90 and CD105 were all more than 95%,and CD34,CD11b,CD19,CD45 and HLA-DR were almost not expressed(<0.2%).The results were consistent with the results of surface antigen expression of untransduced stem cells of the same generation.Conclusion:1.There were significant differences in the efficiency of pLVX-IRES-Puro-GFP lentivirus transducing mesenchymal stem cells derived from human embryonic stem cells,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells with the same MOI value(P< 0.01).The analysis may be related to the different sources of mesenchymal stem cells or cell proliferation.2.Mesenchymal stem cells derived from human embryonic stem cells labeled with green fluorescent protein gene,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells were cultured and propagated for 10 generations,and the expression of green fluorescent protein did not attenuate.it is suggested that green fluorescent protein gene can be expressed stably in cells for a long time and is suitable for long-term labeling of stem cells.3.Lentivirus GFP gene transduction has no adverse effects on the growth,proliferation,differentiation and cell phenotype of human embryonic stem cell-derived mesenchymal stem cells,human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells,indicating that lentivirus-mediated green fluorescent protein transduction is non-toxic and can be stably expressed in cells,so it is an ideal method for stem cell labeling.