Preliminary Exploration On Establishing Porcine Na?ve Embryonic Stem Cells

Na?ve embryonic stem cells(ESCs)are currently regarded as the authentic totipotential stem cells,which have great applications in the fields of cell differentiation,genetic modification and regenerative medicine.However,the na?ve ESCs have not been established successfully in all species but mouse and rat.Pigs are increasingly becoming an important model for studying human diseases and the potential source of organs for xenotransplantation due to the striking similarities to human anatomy,physiology and genetics.Recently,some researches had shown that porcine na?ve-like ESCs had been established by culturing ICM in culture solution including leukemia inhibitory factor(LIF)and 2i(CHIR99021 and kenpaullone),which were transfected with lentiviral vectors bearing the open reading frames of transgenes.But the different blastomeres of ICM were probably exposed in the heterogeneity of different vectors with this method.Other studies had discovered culture system also had important roles of establishing na?ve ESCs as well as the reprogramming factors.Therefore,in this study,firstly,the traditional ESCs culture method was combined with i PSCs technique.The porcine embryonic fibroblasts(PEF)were transfected with lentiviral vector bearing Tet-inducible reprogramming factors followed by somatic nuclear transfer(SCNT)for getting blastocysts.The ICM of blastocysts were isolated and seeded in different mediums with doxycycline hyclate(DOX).The method of establishing porcine na?ve ESCs would be preliminarily investigated.Secondly,the cell lines(PEF-p LIF)of PEF effectively expressing recombinant porcine LIF(p LIF)were established in order to improve the traditional ESCs culture method for establishing stable porcine na?ve ESCs.Objective This study aimed at establishing porcine na?ve ESCs via combination of traditional ESCs culture method and i PSCs technique;The cell lines(PEF-p LIF)of PEF effectively expressing recombinant p LIF also were established in order to improve the traditional ESCs culture method for establishing stable porcine na?ve ESCs.Methods1.Establishing porcine na?ve ESCs via combination of traditional ESCs culture method: validity of Tet O-FUW-OSKM vector and FUW-M2 rt TA vector identified by restricted endonucleases digestion and DOX-induce;The PEF co-transfected with Tet O-FUW-OSKM vector and FUW-M2 rt TA vector were selected by zeocin;blastocysts were obtained by SCNT;Porcine blastocysts were stripped of trophectoderm by immunosurgery to expose ICM;The ICM of blastocysts were isolated and seeded in different mediums with DOX;Passaging and identifying the ESCs.2.Establishing the cell lines of PEF effectively expressing recombinant p LIF(PEF-p LIF): the p LIF genes were obtained from the total RNA of PEF by polymerase chain reaction.The p LIF c DNA fragment was inserted into the eukaryotic expression vector p CAGDNA3 to generate the eukaryotic expression plasmid p CAGDNA3-p LIF.After nuclear transfection of the plasmid p CAGDNA3-p LIF into PEF,the cell lines that effectively express p LIF stably were screened by G418.The expression of the LIF gene and recombinant p LIF was detected by RT-PCR and western blotting.The functions of PEF-p LIF maintaining the characteristics of stem cells were investigated by mouse embryonic stem cells(m ESCs)culture,the alkaline phosphates expression detection and immunocytofluorescent.Results1.Establishing porcine na?ve ESCs via combination of traditional ESCs culture method: tet O-FUW-OSKM vector and FUW-M2 rt TA vector were validated by restricted endonucleases digestion and DOX-induce identifying and could be used in future experiments;Five cell strains of PEF expressing reprogramming factors were selected by zeocin and 343 blastocysts obtained after SCNT;The ICM of blastocysts were isolated and seeded in different mediums with DOX,here are results:1)there were no ESCs obtained in MEF/2i/LIF/ VPA/5-AZA culture system.2)There were 3ESCs clones in MEF/2i/LIF culture system,two of them were compact and dome-shaped colonies and one of them was flattened-shaped colony.None of them was stably passaged.3)Three clones in MEF/FGF/LIF/2i culture system showed the flattened shape.None of them was stably passaged.4)There was one flattened-shaped ESCs passaged 15 in FGF/STO culture system.But it was given up for culture because the low normal diploid karyotype percentage.2.Establishing the cell lines of PEF effectively expressing recombinant p LIF(PEF-p LIF): the results showed that the eukaryotic expression plasmid p CAGDNA3-p LIF was constructed successfully.The cell lines expressing effectively the recombinant p LIF(PEF-p LIF)were successfully established and m ESCs could maintain formal morphology with them as feeder cells.Conclusion1.Porcine na?ve ESCs had not been established successfully via combination of traditional ESCs culture method till now,and it is necessary to improve the culture method for porcine na?ve ESCs.2.As feeder cells,the derived PEF-p LIF cell lines could keep mouse embryonic stem cells at undifferentiated state.The PEF-p LIF cell lines could be possibly helpful for establishing stable porcine na?ve ESCs in the future.