The Relationship Between Porcine Embryonic Stem Cells And Pluripotency-related Signaling Pathways

Pigs are not only livestock animals,but also have high similarities with humans in organ size and morphology,physiology,metabolism and immunology.So the establishment of pig embryonic stem cells is a great significance for agricultural production and biological research.However,people attempt to establish a porcine embryonic stem cell line(p ESC)in the past ten years after the successful establishment of mouse and human embryonic stem cells,the true porcine embryonic stem cell line has not yet been obtained.One of the main problems is that the most suitable vitro culture environment for p ESC has not been determined.And pluripotency and self-renewal of embryonic stem cells depends mainly on the regulation of pluripotent signaling pathways.It is known that mouse embryonic stem cells(na?ve state)are mainly dependent on the LIF/STAT3 signaling pathway,and human embryonic stem cells rely on FGF and TGF?/Activin/Nodal signaling pathways.Rats and other rodents can establish the na?ve embryonic stem cell line by inhibiting the FGF signaling pathway and GSK3?-activated WNT signaling pathway,and can weaken the dependence on the LIF signaling pathway.Therefore,these pluripotent signaling pathways are an important research basis in the process of establishing embryonic stem cells.In this paper,the existing pig embryonic stem cells in the laboratory were used as research objects,and the expressions of the above-mentioned several signal pathway related factors were compared and analyzed with reference to the sequencing data of early blastocysts.After conclusion,To verify whether the pluripotency was improved,the signals in the porcine embryonic stem cell line were inhibited or activated,respectively.Therefore,we studied the above signaling pathways LIF/STAT3,FGF,TGF?/Activin/Nodal and WNT signaling pathways,and finally obtained the following results.(1)RNA-Seq data show that STAT3 is specifically expressed in the ICM of early blastocysts At the same time,the result shows that p STAT3 is also expressed.However,in the study of LIF/STAT3 in cells,it was found that STAT3 could not be activated by LIF and IL-6 and pluripotency was maintained without the addition of LIF.And Only KO-like pig embryonic stem cell lines can be obtained by the two small factors during establishing cell line.This also indicates that the porcine embryonic stem cells obtained under the current system do not depend on the JAK/STAT3 signaling pathway,and it is difficult to reprogram the cells to the na?ve state by simply using JAK/STAT3 activators.(2)Sequencing data show that the expression pattern of FGF signaling pathway in porcine embryonic stem cells is similar to ICM.So We conclude that activation of the FGF signaling pathway is a key factor in maintaining pluripotency.The results confirmed that the addition of b FGF was indispensable in the process of establishing pig embryonic stem cells.However,the continued addition of FGF causes embryonic stem cells to have the molecular state of the post-implantation embryo.(3)RNA-Seq data show that Porcine embryonic stem cells express highly TGF-?/Activin/Nodal signaling pathway-related factors relative to early blastocysts in the sequencing data.This indicates that inhibition of this signaling pathway during the establishment of porcine embryonic stem cells may be an important factor for obtaining na?ve embryonic stem cells.Indeed,Inhibition of TGF-?/Activin/Nodal signaling pathway improves pluripotent status of porcine embryonic stem cells.(4)From the sequencing results,we can find that the WNT signaling pathway target gene TCF3 is specifically expressed relative to porcine embryonic stem cells.So we conclude that activation of the WNT signaling pathway may be a key factor in maintaining pluripotency.in porcine embryonic stem cells.Cytoplasmic retention of ?-catenin does not translocate nuclear to the activation of WNT/TCF in p ESCs.Instead,it enhances E?cadherin expression on the cell membrane,thereby enhancing biological activity.We also further understood the relationship between these pluripotency-related signaling pathways and porcine embryonic stem cells,although we could not get the pig embryonic stem cell line with complete na?ve state through changing any of signaling pathway.At the same time,the signal pathway that maintains the pluripotency should be a network,and the roles of these signal pathways in stem cells are also intertwined.In our study,we can find that the addition of b FGF during the establishment of p ESC can promote the formation of primary clones.But it will promote the generation of primed embryonic stem cells.Using SB431542,an inhibitor of the TGF-?/Activin/Nodal signaling pathway,could inhibit the characteristics of primed embryonic stem cells.However,the obtained cell lines showed no significant improvement in cell growth ability.Research on the WNT signal path just compensates for this Missing.Therefore,our research laid the foundation for obtaining the culture conditions of pig naive embryonic stem cells and provided a new idea for obtaining the na?ve cell line.