The Induction Of Human Induced Pluripotent Stem Cells And Optimization Of Inducing System

iPS cells, that is induced pluripotent stem cells, can be derived from terminallydifferentiated cells when they are driven reversly back to embryonic stem cell-likecells by introducing some genes(Oct4, Sox2, Klf4, c-Myc). iPS cells have the samecharacteristecs as embryonic stem cells (ESCs), and can differentiate into multipletypes of cells.In this study, virus packaging of lentiviral vectors (FU-tet-o-hSox2,FU-tet-o-hOct4, FU-tet-o-hc-Myc, FU-tet-o-hKLF4, FUdeltaGW-rtTA) and retroviralvector, pEYK E4, have been performed by transfection of calcium phosphate andQuickShuttle transfection reagent. Then, viral titer was measured, at the same time, itstransfection condition was optimized. In addition, human fetus fibroblast cells (hFFCs)were established by primary culture and subculture in vitro, and the methods ofprimary culture were compared. Mouse embryonal fibroblasts (MEFs) were alsoestablished by primary culture and subculture in vitro, and were treated withmitomycin C to prepare the feeder layer. hFFCs were infected by virus, and themorphologic change of hFFCs was observed. Then they were inoculated on feederlayer, and the medium was replaced every day until clones appeared. In this paper, theinducing system for producing human induced pluripotent stem cells was optimized ina certain way, and a plan with simple operation, simple instruments and cost cheapwas provided. The detailed studies and the results are described as follows:Virus packaging of lentiviral vectors (FU-tet-o-hSox2, FU-tet-o-hOct4,FU-tet-o-hc-Myc, FU-tet-o-hKLF4, FUdeltaGW-rtTA) and retroviral vector,pEYK E4, have been performed by transfection of calcium phosphate andQuickShuttle transfection reagent. Then, viral titer was measured. Three influencingfactors (different packaging systems, different transfection time, presence of gelatinpackage) in the method of calcium phosphate transfection and QuickShuttle reagenttransfection were optimized. The optimum condition of calcium phosphatetransfection and QuickShuttle reagent transfection was described as following: pLP1, pLP2and pLP/VSVG3plasmid packaging system was used, and293T cells wereinoculated on petri dish coated with gelatin, then transfection of72h can obtain idealresults. The virus titer of calcium phosphate transfection is600TU/mL, the virus titerof QuickShuttle reagent transfection is7130000TU/mL.Using methods of single cell culture and tissue culture, hFFCs and MEFs werecultured in vitro. The results show that, primary cells derived from tissue cultureexhibited better morphology and had less suspension of dead cells after which werepassaged. MEFs were treated with mitomycin C to prepare the feeder layer. hFFCswere infected by virus, and the morphologic change of hFFCs was observed.Compared with the control group, the morphology of hFFCs infected by viruschanged gradually. Some cells exhibited conical, branches, irregular triangle shape.After6day of infection, hFFCs were inoculated on feeder layer, the stem cell mediumwas replaced every day, at the same time Vc (vitamin C) and VPA (valproic acid)were added, and the morphologic change of cells were observed. When cultured at thefifth day, the cell membrane of some cells became relatively thicker, moreover, cell’sthree-dimensional space overall stereo sense became more obvious. Continued todevelop to11day, ESCs sample clone will appear.