The Effect Of TGF-? Pathway On The Pluripotency Of Porcine ESC-like Stem Cells

Embryonic stem cells(ESCs)are a kind of cells derived from early embryos and have the ability of self-renewal and multiple differentiation potential in vitro.They are widely concerned in the field of regenerative medicine because of their ability of infinite proliferation and differentiation into various kinds of tissue cells.In 1981,Evans and Martin et.al.successfully isolated the first ESCs from mouse embryos.A few years later,embryonic stem cells were successfully established in human,monkey and rat.Domestic swine are demonstrated to be very similar to human in anatomic,immunologic and physiological characteristics,the isolation of porcine ESC lines has a great potential and no doubt represent with an excellent model in development of biomedical and regeneration medicine in human.During the past more than 30 years,many embryonic-like stem cells have been established.However,if the ability of germline chimerism is taken as the criterion,the establishment of porcine embryonic stem cells has not been successful until now.PeNK6 is a porcine pluripotent stem cell line that derived from day 5.5 embryos in defined culture system.The characteristics of Pe NK6 are similar to those of human ESCs.The clonal morphology is flatted.It can express core transcription factors and obtain typical tissues of three germ layers no matter in vitro and in vivo.Signaling pathway related with pluripotency had been assessed in this cell line,such as JAK/STAT,FGF/MEK,BMP and PI3 K signaling pathways,but the effects of TGF-? signaling pathway had been not fully evaluated.In view of the important role of TGF-? signaling pathway in maintenance of the unique properties of mouse and human ES cells and the exact roles of TGF-? signaling still are controversial in livestock pluripotent stem cells,so it is necessary to further study the role of TGF-? signaling pathway in pluripotency regulation in Pe NK6.In this study,we demonstrated that TGF-? signaling pathway was activated in Pe NK6 by single cell sequencing and Western Blot.In consideration of the expression patterns of TGF-? related genes,that low activity in the cell mass of early blastocysts and up-regulated gradually in the late blastocysts,we speculate that the inhibition of TGF-? signal in Pe NK6 may have positive effect on its pluripotency.Therefore,in this dissertation,we evaluate the effect of TGF-? signal pathway on Pe NK6 through detecting the changes of the pluripotency index after inhibiting these signaling by adding small molecular inhibitors SB431542 and A83-01 to Pe NK6 culture system(KO).The results showed that after inhibiting TGF-? signal,the core transcription factor Sox2 and the naive pluripotency related factors KLF4,KLF5 and TFCP2L1 in Pe NK6 were significantly upregulated.At the same time,DNMT3 B was decreased significantly,which was defined as primed marker.This indicated that the pluripotency of Pe NK6 was partially changed after inhibiting TGF-? signaling pathway,which showed that the expression patterns of parts pluripotency gene changed from primed state to na?ve-like state.At the same time,the inhibition of TGF-? signal pathway also has an effect on the differentiation ability of Pe NK6.After inhibiting TGF-? signaling pathway,the formation ability of embryoid body(EB)in Pe NK6 cells increased,and the size and the change value of diameter of EBs also increased significantly.There was reduction in the expression level of three germ layers in EBs Pe NK6+SB/+A compared with EBs Pe NK6.These results indicate that the inhibition of TGF-? signal pathway has positive effect on the EB formation.In addition,we established a new cell line(Pe WKSB)from D5.5 IVF embryos in the new developed medium with addition of TGF-? inhibitors.This cell line is similar to Pe NK6+SB/+A cell line in the expression patterns of pluripotent factors,and the ability to form EB was improved.This further proves that the inhibition of TGF-? signaling pathway has positive effects on the pluripotency of porcine embryonic stem cells.In summary,the results of this dissertation show that the TGF-? signaling pathway has an important biological role in porcine pluripotent stem cells,and inhibition of this signaling pathway has positive effects on the pluripotency-related characteristics of porcine pluripotent stem cells.This study provides detailed experimental evidence for the in-depth understanding of the role of TGF-? signaling pathway in the regulation of pluripotency of porcine embryonic stem cells,which has important reference value for the establishment of putative porcine embryonic stem cells with the ability of germline chimerism.