Reprogramming Porcine Somatic Cells Into Induced Pluripotent Stem Cells With Defined Transcription Factors

Several porcine pluripotent stem cells have been established not long before, however,these cells were various each other and did not meet all the criterion of pluripotent stem cells.Meanwhile, the molecular mechanism for the reprogramming of porcine somatic cells is stillunclear. Besides, the research of porcine embryonic stem cells is not improved, and becauseof no appropriated culture system, authentic porcine ES cell still has not been established. Inthis study, porcine fibroblasts were reprogrammed into pluripotency through ectopicexpression of mouse Oct4/Sox2/Klf4/c-Myc. We detected the expression behaviors ofimportant genes during the process of porcine fibroblasts reprogramming, and revealed thekinetics mechanism of porcine fibroblasts reprogramming. Meanwhile, ROCK specificinhibitor Y-27632was used for porcine iPS cells to improve the cryopreserved survival andpassage effect of porcine iPS cells. The results showed below:1. Generation of porcine induced pluripotent stem cellsPorcine embryonic fibroblasts and porcine ear skin fibroblasts were isolated, and wereinfected with retrovirus containing mouse Oct4/Sox2/Klf4/c-Myc. Then the infected cellswere cultured in specific culture medium for reprogramming, and generated porcine iPS cells.Finally, these porcine iPS cells were detailed analyzed for characterization of pluripotent stemcells.The results revealed that these porcine iPS cell held normal karyotype, and representeddemethylation in the promoter of OCT4. The quantitive RT-PCR, immunofluorescence andmicroarray analysis suggested porcine iPS greatly expressed pluripotent genes and stem cellspecific surface antigens, and their transcription profiles were successfully reversed andrepresented very different with porcine fibroblasts. Finally, the pluripotency examination ofporcine iPS cells revealed these cells held multi-lineages differentiation potential and couldincorporate in early porcine embryos.All above results suggested that porcine fibroblasts werereprogrammed into pluripotency, and these cells represented typical pluripotent stem cellfeatures and were able to self-renew in vitro and held multi-lineages differentiation potential. 2. Kinetics analysis of porcine fibroblasts reprogramming towards pluripotency by definedfactorsThe reprogramming porcine fibroblasts from day4to day10were collected and used forexamination of the expression of OCT4, NANOG and TERT though Realtime PCR, and thesepluripotency genes gradually increased in7days after infection. The maternal imprintedgenes GTL2greatly decreased after exogenous genes introduction, yet paternal imprintedgenes DLK1and DIO3did not change notably. Meanwhile, apoptosis related gene BAXincreased during the reprogramming process. AP staining and immunofluorescence analysisrevealed that AP activity and SSEA4were re-activated about6days after infection, andgradually enhanced along with the reprogramming process. The expression of exogenous GFPsuggested that transgenes represented increased in3days, and slightly decreased in10daysafter infection and finally silenced in some full reprogrammed porcine iPS cells. Besides, thepluripotency genes did not increased in three factors Oct4, Sox2and Klf4triggeredreprogramming, but DLK1, GTL2showed greatly decreased in this process. The expressionlevel of apoptosis related genes was lower than the expression of them in reprogrammingporcine cells induced by OSKM four factors. The above results revealed the kineticsmechanism of porcine fibroblasts reprogramming into pluripotency, and suggested thetranscription behaviors of important genes during the process of reprogramming and c-Mycplays an important role in this process.3. ROCK inhibitor Y-27632improves the cryopreserved survival and passage effect ofporcine iPS cellROCK specific inhibitor Y-27632was used for porcine induced pluripotent stem cells,and it was found that Y-27632was able to enhance cryopreserved survival and colonyformation of porcine iPS cells, and did not affect cells' pluripotency. Besides, Y-27632couldinhibit the apoptosis of porcine iPS cells when cells were in stress, and improve porcine iPScells to incorporate into porcine early embryos. So Y-27632used for porcine iPS cells as asupplymentary small molecules can improve the cryopreserved survival and coloniesformation, and reduce apoptosis.