Trophoblast stem cells (TSCs), the precursors of placental cells, are effective for studying placental formation in vitro. Using a dual inhibition (2i) medium and mixed L-Wnt3a/mouse embryonic fibroblast feeder cells, we previously established the bovine trophoblast cell line BTS-1. In this study, we used bovine fetal fibroblasts and added Wnt3a to the 2i medium to establish another bovine TSC line (BTSW). BTSW cells expressed pluripotency markers, including NANOG, SOX2, OCT4, TRA-1-60, TRA-1-81, SSEA4, CDH1, and KRT18, and TSC markers CDX2, TEAD4, and ESRRB. Methylation sequencing of the promoter regions of NANOG, OCT4, and CDX2 revealed no significant differences between BTS-1 and BTSW cells. Removal of Wnt3a from the culture medium resulted in downregulation (p < 0.05) of NANOG, OCT4, CDX2, and TSC marker genes, and upregulation of TSC differentiation markers, including MASH2, GCM1, and PAG. Western blotting indicated activation of the WNT-YAP/TAZ signaling pathway in BTS-1 and BTSW cells, consequently activating TEAD4 transcription. However, this pathway was not activated in BCFF cells, an established bovine embryonic stem-like cell line that expresses OCT4, SOX2, and NANOG, but not CDX2. Thus, Wnt3a may play a critical role in bovine TSC maintenance by activating and regulating CDX2 expression through the WNT-YAP/TAZ signaling pathway.
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References
1.
AngersS., and MoonR.T. (2009). Proximal events in Wnt signal transduction. Nat Rev Mol Cell Biol, 10, 468.
2.
AntonR., KestlerH.A., and KühlM. (2007). β-Catenin signaling contributes to stemness and regulates early differentiation in murine embryonic stem cells. FEBS Lett, 581, 5247–5254.
3.
BaoS., TangW.W., WuB., KimS., LiJ., LiL., et al. (2018). Derivation of hypermethylated pluripotent embryonic stem cells with high potency. Cell Res, 28, 22–34.
4.
BogliottiY.S., WuJ., VilarinoM., OkamuraD., SotoD.A., ZhongC., et al. (2018). Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. Proc Natl Acad Sci U S A, 115, 2090–2095.
5.
DavidsonK.C., AdamsA.M., GoodsonJ.M., McDonaldC.E., PotterJ.C., BerndtJ.D., et al. (2012). Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc Natl Acad Sci, 109, 4485–4490.
6.
DegrelleS.A., CampionE., CabauC., PiumiF., ReinaudP., RichardC., et al. (2005). Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts. Dev Biol, 288, 448–460.
7.
DenicolA.C., DobbsK.B., McLeanK.M., CarambulaS.F., LoureiroB., and HansenP.J. (2013). Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage. Sci Rep 3,1266.
8.
DuelloT.M., ByattJ.C., and BremelR.D. (1986). Immunohistochemical localization of placental lactogen in binucleate cells of bovine placentomes. Endocrinology 119,1351.
9.
ElizaM.Z., KamilaH., MaciejO., PiotrP., and AnnaP. (2015). WNT/β-catenin signaling affects cell lineage and pluripotency-specific gene expression in bovine blastocysts: prospects for bovine embryonic stem cell derivation. Stem Cells Dev, 24, 2437–2454.
10.
ErlebacherA., PriceK.A., and GlimcherL.H. (2004). Maintenance of mouse trophoblast stem cell proliferation by TGF-β/activin. Dev Biol, 275, 158–169.
11.
FléchonJ.E., LaurieS., and NotarianniE. (1995). Isolation and characterization of a feeder-dependent, porcine trophectoderm cell line obtained from a 9-day blastocyst. Placenta, 16, 643–658.
12.
Gardiner-GardenM., and FrommerM. (1987). CpG islands in vertebrate genomes. J Mol Biol, 196, 261–282.
13.
GodfreyK.M. (2002). The role of the placenta in fetal programming-a review. Placenta 23,S20.
14.
GreensteinJ.S., MurrayR.W., and FoleyR.C. (1958). Observations on the morphogenesis and histochemistry of the bovine preattachment placenta between 16 and 33 days of gestation. Anat Rec 132,321.
15.
Grigor'evaE.V., ShevchenkoA.I., MazurokN.A., ElisaphenkoE.A., ZhelezovaA.I., ShilovA.G., et al. (2009). FGF4 independent derivation of trophoblast stem cells from the common vole. PLoS One 4,e7161.
16.
HaoJ., LiT.G., QiX., ZhaoD.F., and ZhaoG.Q. (2006). WNT/beta-catenin pathway up-regulates Stat3 and converges on LIF to prevent differentiation of mouse embryonic stem cells. Dev Biol, 290, 81–91.
17.
HashizumeK., ShimadaA., NakanoH., and TakahashiT. (2006). Bovine trophoblast cell culture systems: a technique to culture bovine trophoblast cells without feeder cells. Methods Mol Med, 121, 179–188.
18.
HayashiK., BurghardtR., BazerF., and SpencerT. (2007). WNTs in the ovine uterus: potential regulation of periimplantation ovine conceptus development. Endocrinology, 148, 3496–3506.
19.
HeS., PantD., SchiffmacherA., MeeceA., and CarolL.K.P.D. (2008). Lymphoid enhancer factor 1-mediated Wnt signaling promotes the initiation of trophoblast lineage differentiation in mouse embryonic stem cells. Stem Cells, 26, 842–849.
20.
HimenoE., TanakaS., and KunathT. (2008). Isolation and manipulation of mouse trophoblast stem cells. Curr Protoc Stem Cell Biol Chapter 1, Unit 1E. 4.
21.
HuangX., HanX., UyunbiligB., ZhangM., DuoS., ZuoY., et al. (2014). Establishment of bovine trophoblast stem-like cells from in vitro-produced blastocyst-stage embryos using two inhibitors. Stem Cells Dev, 23, 1501–1514.
22.
KaH., JaegerL.A., JohnsonG.A., SpencerT.E., and BazerF.W. (2001). Keratinocyte growth factor is up-regulated by estrogen in the porcine uterine endometrium and functions in trophectoderm cell proliferation and differentiation 1. Endocrinology, 142, 2303–2310.
23.
KazuyoshiH., KoichiU., PatelO.V., KeiichiroK., KeiI., OsamuY., et al. (2007). Gene expression and maintenance of pregnancy in bovine: roles of trophoblastic binucleate cell-specific molecules. Reprod Fertil Dev, 19, 79–90.
24.
KrivegaM., EssahibW., and Van de VeldeH. (2015). WNT3 and membrane-associated β-catenin regulate trophectoderm lineage differentiation in human blastocysts. Mol Hum Reprod, 21, 711–722.
25.
La BonnardiereC., FlechonJ.E., BattegayS., FlechonB., DegrouardJ., and LefevreF. (2002). Polarized porcine trophoblastic cell lines spontaneously secrete interferon-gamma. Placenta, 23, 716–726.
26.
LarsenF., GundersenG., LopezR., and PrydzH. (1992). CpG islands as gene markers in the human genome. Genomics, 13, 1095–1107.
27.
LiP., TongC., Mehrian-ShaiR., JiaL., WuN., YanY., et al. (2008). Germline competent embryonic stem cells derived from rat blastocysts. Cell, 135, 1299–1310.
28.
LuW., TuZ., WangS., LuJ., WangQ., WangW., et al. (2013). Spatiotemporal expression of Wnt signaling pathway components during bovine placental development. Theriogenology 80,893.
29.
MeekS., WeiJ., SutherlandL., NilgesB., BuehrM., TomlinsonS.R., et al. (2013). Tuning of β-catenin activity is required to stabilize self-renewal of rat embryonic stem cells. Stem Cells, 31, 2104–2115.
30.
MiyabayashiT., TeoJ.L., YamamotoM., McmillanM., NguyenC., and KahnM. (2007). Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc Natl Acad Sci U S A, 104, 5668–5673.
31.
MiyazakiH., ImaiM., HirayamaT., SaburiS., TanakaM., MaruyamaM., et al. (2002). Establishment of feeder-independent cloned caprine trophoblast cell line which expresses placental lactogen and interferon Tau. Placenta, 23, 613–630.
32.
MyersD.A., and ReimersT.J. (1988). Purification and endocrine evaluation of bovine binucleate trophoblastic cells. J Tissue Culture Methods, 11, 83–88.
33.
NishiokaN., InoueK., AdachiK., KiyonariH., OtaM., RalstonA., et al. (2009). The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev Cell 16,398.
34.
NishiokaN., YamamotoS., KiyonariH., SatoH., SawadaA., OtaM., et al. (2008). Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos. Mech Dev, 125, 270–283.
OzawaM., SakataniM., YaoJ., ShankerS., YuF., YamashitaR., et al. (2012). Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst. BMC Dev Biol 12,33.
37.
ParkH.W., KimY.C., YuB., MoroishiT., MoJ.S., PlouffeS.W., et al. (2015). Alternative Wnt signaling activates YAP/TAZ. Cell, 162, 780–794.
38.
PlassC., and SolowayP.D. (2002). DNA methylation, imprinting and cancer. Eur J Hum Genet 10,6.
39.
RalstonA., CoxB.J., NishiokaN., SasakiH., CheaE., Rugg-GunnP., et al. (2010). Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2. Development 137,395.
40.
RamsoondarJ., ChristophersonR.J., GuilbertL.J., and WegmannT.G. (1993). A porcine trophoblast cell line that secretes growth factors which stimulate porcine macrophages. Biol Reprod, 49, 681–694.
SimmonsD., and CrossJ. (2005). Determinants of trophoblast lineage and cell subtype specification in the mouse placenta. Dev Biol, 284, 12–24.
43.
SondereggerS., PollheimerJ., and KnoflerM. (2010). Wnt signalling in implantation, decidualisation and placental differentiation—review. Placenta, 31, 839–847.
44.
SpencerT., and BazerF. (2004). Uterine and placental factors regulating conceptus growth in domestic animals. J Anim Sci, 82, E4–E13.
45.
TalbotN.C., CapernaT.J., EdwardsJ.L., GarrettW., WellsK.D., and EalyA.D. (2000). Bovine blastocyst-derived trophectoderm and endoderm cell cultures: interferon tau and transferrin expression as respective in vitro markers. Biol Reprod, 62, 235–247.
46.
TanakaS., KunathT., HadjantonakisA.-K., NagyA., and RossantJ. (1998). Promotion of trophoblast stem cell proliferation by FGF4. Science, 282, 2072–2075.
47.
TanakaS., OdaM., ToyoshimaY., WakayamaT., TanakaM., YoshidaN., et al. (2001). Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer1. Biol Reprod, 65, 1813–1821.
48.
ten BergeD., KurekD., BlauwkampT., KooleW., MaasA., ErogluE., et al. (2011). Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells. Nat Cell Biol, 13, 1070–1075.
49.
ThompsonJ., McNaughtonC., GasparriniB., McGowanL., and TervitH. (2000). Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro. J Reprod Fertil, 118, 47–55.
50.
VandevoortC.A., ThirkillT.L., and DouglasG.C. (2007). Blastocyst-derived trophoblast stem cells from the rhesus monkey. Stem Cells Dev, 16, 779–788.
51.
WimsattW.A. (1951). Observations on the morphogenesis, cytochemistry, and significance of the binocleate giant cells of the placenta of ruminants. Am J Anat, 89, 233–281.
52.
XuZ., RobitailleA.M., BerndtJ.D., DavidsonK.C., FischerK.A., MathieuJ., et al. (2016). Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells. Proc Natl Acad Sci U S A, 113, E6382–E6390.
53.
YangJ., RyanD.J., WangW., TsangJC.-H., LanG., MasakiH., et al. (2017a). Establishment of mouse expanded potential stem cells. Nature, 550, 393–397.
54.
YangY., LiuB., XuJ., WangJ., WuJ., ShiC., et al. (2017b). Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell, 169, 243–257 e225.
55.
ZhuD., GongX., MiaoL., FangJ., and ZhangJ. (2017). Efficient induction of syncytiotrophoblast layer II cells from trophoblast stem cells by canonical Wnt signaling activation. Stem Cell Rep, 9, 2034–2049.
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