The Role Of Wnt3a In The Establishment And Maintenance Of Bovine Trophoblast Stem Cells

As a temporary organ,the placenta is responsible for the supply of nutrients during embryonic development after implantation.The malfunction of placental will seriously affect embryonic development and cause pregnancy failure.Trophoblast stem cells(TSCs),which are precursor cells of the placental trophoblast,have the ability to differentiate into all placental trophoblast cells in vitro.Therefore,trophoblast stem cells are a powerful cellular model for studying trophectoderm(TE)differentiation,trophoblast(TR)lineage establishment and placental formation.And the establishment of the trophoblast stem cells with stable characteristics and strong self-renewal ability in vitro is essential for the further understanding of the placenta differentiation.The 2i culture system contains two inhibitor molecules,PD0325901 and CHIR99021,the former is the MEK/ERK signaling pathway inhibitor and the latter is the GSK-3? inhibitor.This system was originally reported as the culture system of mouse and rat ESCs in vitro.We successfully established a bovine TSC cell line(BTS-1)by using this system in combination with L cells that continuously secrete Wnt3 a protein and MEF as a mixed feeder layer.To explore the role of Wnt3 a and its corresponding WNT signaling pathway in the establishment of bovine TSCs in vitro,we conducted the following studies: 1.The establishment of bovine trophoblast stem cell and embryonic stem-like cell lines by different culture systems.We developed 11 culture systems composed by combination of three kinds of culture media(2i,CHI,KOSR),four kinds of additives(mLIF,bFGF,forskolin,Wnt3a),two feeder cells(L-Wnt3a/MEF mixed feeder cells,BFF)and Matrigel,and then attempted to establish bovine trophoblast stem cells and embryonic stem cell(ESC)-like cells from bovine in vitro fertilization blastocysts.The results showed that mouse ESC-like cells could not be efficiently obtained in all 2i culture system,but could be found in two systems such as the KOSR-BFF culture system supplemented with human recombinant bFGF and the CHI-Matrigel culture system supplemented with bFGF and forskolin.Both cell lines could maintain the morphology of mouse ESC-like cells during in vitro passaging,but the cell proliferation rate in KOSR-BFF culture system gradually slowed down.In contrast,the cells in the CHI(bFGF+forskolin)-Matrigel culture system were efficiently passaged and could be subcultured to the 20 th generation while preserving the mouse ESC-like morphology and the cell line was named BCFF.Also,two bovine TSC-like cell lines were obtained in the 2i-BFF system supplemented with human recombinant Wnt3 a.One cell line already has been passaged for more than 120 generations in vitro with stable characteristics,and the colony edge was not easy to differentiate.We named it as BTSW.2.The identification of characteristics of the cell line(BTSW)in 2i+Wnt3a-BFF culture system.The expression levels of pluripotency genes and TSC marker genes in BTSW were comprehensively identified through alkaline phosphatase staining,immunofluorescence staining and reverse transcription PCR method.The results showed that BTSW cells not only expressed a variety of pluripotency markers(OCT4,SOX2,NANOG,SSEA4,TRA-1-60,TRA-1-81,etc.),but also expressed a series of mouse TSC markers(CDX2,TEAD4,ELF5,ETS2,SMARCA4,KRT18,etc.).Moreover,BTSW has a higher expression level of pluripotency genes(OCT4,NANOG)and TSC marker gene CDX2 than those in bovine TSC cell line(BTS-1)obtained under 2i-L/M system.The methylation analysis of gene promoter regions in CDX2,OCT4,NANOG,and SOX2 showed that BTSW has lower methylation level than BTS-1.In vitro differentiation assays confirmed that BTSW can efficiently differentiate into placental binuclear trophoblast cells with secretory function in vitro.Therefore,the results indicate that BTSW is a true bovine TSC cell line.3.The study on the mechanism of Wnt3 a in bovine TSC.The changes of morphology,pluripotent genes,TSC marker genes,and differentiation marker genes were detected in BTSW cells in a culture system with or without Wnt3 a.After removal of Wnt3 a in the culture system,BTSW cells gradually differentiated along with significantly decreased of pluripotent(OCT4,NANOG)gene expression.The expression levels of TSC marker genes(CDX2,EOMES,ESRRB,GATA3,ETS2,ELF5,SMARCA4,KRT18)also decreased whereas differentiation marker genes(MASH2,PAG,GCM1,and PL-I)were highly expressed.The major members of the WNT-YAP /TAZ signaling pathway were tested by western blot.The results showed that WNT-YAP /TAZ pathway was active in three bovine TSC lines.The bovine TSCs(BTS-1,BTSA)with continuously secreted Wnt3 a in 2i-L/M culture system were in more active states than bovine TSCs(BTSW)which were added a fixed amount of Wnt3 a in 2i-BFF culture system.However,this pathway was inactivated in bovine embryonic stem cell line(BCFF,the culture system did not contain Wnt3a).The detection of ?-catenin protein showed that BTSW contained the least amount in all three bovine TSC cells,while the content in bovine embryonic stem cell lines was significantly lower than that in BTSW.The differences in contents of signaling pathway members might be caused by the different concentrations of Wnt3 a protein in the culture system.Then after the removal of Wnt3 a in the culture system,most members of this pathway(WNT-YAP/TAZ pathway)were down-regulated.In conclusion,Wnt3 a effectively activates WNT-YAP /TAZ signaling pathway in bovine trophoblast stem cells and sustains its expression of TEAD4 and CDX2.The study indicates that Wnt3 a can efficiently promote the establishment and culture of bovine trophoblast stem cells in vitro,and maintain characteristics of trophoblast stem cells utilizing both WNT-YAP/TAZ and WNT/?-Catenin signaling pathways.These findings provide new ideas for understanding the mechanism of ruminant trophoblast stem cell formation.