Study On The Optimization Of Inducing System For Human Induced Pluripotent Stem Cells

Induced pluripotent stem cells （iPSCs） are important discovery in the field of stem cells,which have the characteristics of embryonic stem cells （ESCs）, and can avoid the doublepredicament of ethics and technology in the fields of ESCs research.In this paper, human fetus fibroblast cells （hFFCs） were cultured in vitro, at the sametime, the culture system of hFFCs was optimized. Then, pEYK E4, a recombinant retroviralvector, was identified, and the transfection conditions of pEYK·E4for packaging high titerretrovirus was optimized. In addition, the optimum condition of using mouse embryonicfibroblasts （MEFs） to produce feeder layer was investigated. Lastly, recombinant retroviruswas used to infect hFFCs, the changes in morphology of hFFCs were observed. In this paper,the inducing system for producing human induced pluripotent stem cells （hiPSCs） wascomprehensively optimized. The detailed contents and the results are described as follows:Using methods of single cell culture and tissue culture, hFFCs were cultured in vitro.Some factors （including medium, the concentration of fetal bovine serum （FBS）, basicfibroblast growth factor （bFGF）） in the culture system of hFFCs were optimized. The resultsshowed that hFFCs derived from tissue culture exhibited better morphology and had lessdead cells after which were passaged, and the optimum culture system for hFFCs was highglucose DMEM supplemented with8%（v/v） FBS and10ng/mL bFGF. In this optimumculture system, hFFCs exhibited good morphology, had strong proliferative ability and shortpopulation doubling time （about19.3h）, and thawed hFFCs also exhibited good morphology,and suitable to be used as donor cells for producing hiPSCs.By the methods of restriction analysis, PCR and sequencing, pEYK E4, a recombinantretroviral vector, was identified. The results showed that the size of pEYK E4, which wasbased on retroviral vector pEYK3.1and contained the cDNA sequence of four transcriptionfactors of c-myc, Klf4, Sox2and Oct4, was8228bp. Then, by the methods of liposometransfection and calcium phosphate transfection, pEYK·E4was transfected into293T cells,retrovirus were packaged and its titer was measured. Three influencing factors （the seedingdensity of293T cells, the amount of plasmid DNA, the concentration of CO₂in cultureenvironment） in the method of calcium phosphate transfection were optimized. The optimumcondition of calcium phosphate transfection was described as following: the seeding densityof293T cells was5×10⁵/mL, the concentration of CO₂in culture environment was3%andthe amount of plasmid DNA was30μg. Under this optimum transfection condition, the retrovirus titer could reach1.9×10⁴CFU/mL. On the other hand, the retrovirus titer by themethods of liposome transfection could reach4×10⁴CFU/mL.Using methods of single cell culture and tissue culture, MEFs were cultured in vitro.MEFs were treated with mitomycin C, feeder layer cells were produced, and the optimumconcentration and treatment time of mitomycin C were investigated. The results showed thatthe optimum concentration of mitomycin C was10μg/mL, and the optimum treatment timeof mitomycin C was2hours.After infected by recombinant retrovirus twice, hFFCs were inoculated on the feederlayer and cultured in the environment of iPSCs. Then, the changes in hFFCs’ morphologywere observed. Compared with the control group, the morphology of hFFCs infected byretrovirus became from typical long fusiform into roundness, semiellipse, trilateral orirregular morphology. This result indicated that the objective genes that were introduced intohFFCs by recombinant retroviral vector could initiate the changes of hFFCs’ morphology.