The Key Technology Study Of Reprogramming Human Cells Into Induced Pluripotent Stem Cells In Vitro

Objective Lentivirus vector which includes the four genes as Oct4、Sox2、c-Myc、Klf4infect hPFF to investigate the optimum condition of target gene over-expressionlentivirus particle infecting hPFF which were reprogrammed into induced pluripotentstem cell in vitro. To establish the process of culturing primary human umbilical veinendothelial cells (HUVE) line and the HUVE were infected by Lentivirus vector ofmicroRNA-302-367. Lentivirus vector which includes the genes asmicroRNA-302-367infect HUVE to investigate the optimum condition of target geneover-expression lentivirus particle infecting hPFF which were reprogrammed intoinduced pluripotent stem cell in vitro.Methods First: The hPFFs were reprogrammed into iPSCs by the best virusMultiplicity of infection (MOI) in vitro. The iPSCs were identified by cellmorphology, alkaline phosphatase staining and teratoma tumor formation experiment.Second: Using0.1%collagenase I digestion human umbilical vein endothelial toseparate and culture HUVE, Observing cells morphology under the invertedmicroscope, measuring cells viability by trypan blue assay indentify cells byimmunocy to chemical staining of Strept a ctividin-biotin complex (SABC). TheOptional planting density of cell was determined according to the cellular growthcurve detected by MTS colorimetric assay. Third: Using lentivirus particle expressingGFP infect HUVE observe fluorescence efficiency under the inverted microscope toget the best infecting condition and multiplicity of infection (MOI); Forth: Randomlydividing HUVE in good condition into three groups: control group, lentivirus particleexpressing GFP group, target gene infected group infect HUVE. The second day,giving HUVE condition of stem cells culture, observing the morphological changesand analyze three group cells to get the best laboratory condition.Results First: The hPFFs were infected with lentivirus under the best MOI40. After7 days, cells pseudopod contraction and shape from long fusiform to round. After25days, the cell clones were selected; alkaline phosphatase staining was positive; andthe teratoma tumor formation test confirmed iPSC have inward differentiationpotential to the three germ layers. Second:0.1%collagenase I digestion culturetechnique can found stable HUVE lines in vitro. After3days, cells overspread thebottom,0.25%tryptic finished digestion and passage. The indentified result was rightby immunocytochemical staining of Strept actividin-biotin complex. Third: fishingout the best infecting multiplicity of infection was10in our laboratory condition;microRNA-302-367overexpression lentiviral particles can effectively infect HUVEby PCR tecnology.Conclusion This study successfully reprogrammed the hPFF into iPSC andestablished HUVE lines in vitro. The experiments show that lentiviral particles cansuccessfully infect hPFF and HUVE.