Bone morphogenetic protein (BMP) signaling is known to support differentiation of human embryonic stem cells (hESCs) into mesoderm and extraembryonic lineages, whereas other signaling pathways can largely influence this lineage specification. Here, we set out to reinvestigate the influence of ACTIVIN/NODAL and fibroblast growth factor (FGF) pathways on the lineage choices made by hESCs during BMP4-driven differentiation. We show that BMP activation, coupled with inhibition of both ACTIVIN/NODAL and FGF signaling, induces differentiation of hESCs, specifically to βhCG hormone-secreting multinucleated syncytiotrophoblast and does not support induction of embryonic and extraembryonic lineages, extravillous trophoblast, and primitive endoderm. It has been previously reported that FGF2 can switch BMP4-induced hESC differentiation outcome to mesendoderm. Here, we show that FGF inhibition alone, or in combination with either ACTIVIN/NODAL inhibition or BMP activation, supports hESC differentiation to hCG-secreting syncytiotrophoblast. We show that the inhibition of the FGF pathway acts as a key in directing BMP4-mediated hESC differentiation to syncytiotrophoblast.
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References
1.
ThomsonJA, Itskovitz-EldorJ, ShapiroSS, WaknitzMA, SwiergielJJ, MarshallVS, JonesJM. 1998. Embryonic stem cell lines derived from human blastocysts. Science, 282:1145–1147.
AmitM, SharikiC, MarguletsV, Itskovitz-EldorJ. 2004. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod, 70:837–845.
4.
BeattieGM, LopezAD, BucayN, HintonA, FirpoMT, KingCC, HayekA. 2005. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells, 23:489–495.
5.
GreberB, LehrachH, AdjayeJ. 2008. Control of early fate decisions in human ES cells by distinct states of TGFbeta pathway activity. Stem Cells Dev, 17:1065–1077.
6.
JamesD, LevineAJ, BesserD, Hemmati-BrivanlouA. 2005. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development, 132:1273–1282.
7.
VallierL, ReynoldsD, PedersenRA. 2004. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev Biol, 275:403–421.
8.
XuRH, PeckRM, LiDS, FengX, LudwigT, ThomsonJA. 2005. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods, 2:185–190.
9.
TamPP, LoebelDA. 2007. Gene function in mouse embryogenesis: get set for gastrulation. Nat Rev Genet, 8:368–381.
10.
ArnoldSJ, RobertsonEJ. 2009. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat Rev Mol Cell Biol, 10:91–103.
11.
CirunaB, RossantJ. 2001. FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev Cell, 1:37–49.
12.
KunathT, Saba-El-LeilMK, AlmousailleakhM, WrayJ, MelocheS, SmithA. 2007. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development, 134:2895–2902.
13.
ChaiN, PatelY, JacobsonK, McMahonJ, McMahonA, RappoleeDA. 1998. FGF is an essential regulator of the fifth cell division in preimplantation mouse embryos. Dev Biol, 198:105–115.
14.
ConlonFL, LyonsKM, TakaesuN, BarthKS, KispertA, HerrmannB, RobertsonEJ. 1994. A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development, 120:1919–1928.
15.
D'AmourKA, AgulnickAD, EliazerS, KellyOG, KroonE, BaetgeEE. 2005. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol, 23:1534–1541.
16.
ArmanE, Haffner-KrauszR, ChenY, HeathJK, LonaiP. 1998. Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. Proc Natl Acad Sci U S A, 95:5082–5087.
17.
FeldmanB, PoueymirouW, PapaioannouVE, DeChiaraTM, GoldfarbM. 1995. Requirement of FGF-4 for postimplantation mouse development. Science, 267:246–249.
18.
WilderPJ, KellyD, BrigmanK, PetersonCL, NowlingT, GaoQS, McCombRD, CapecchiMR, RizzinoA. 1997. Inactivation of the FGF-4 gene in embryonic stem cells alters the growth and/or the survival of their early differentiated progeny. Dev Biol, 192:614–629.
19.
YamanakaY, LannerF, RossantJ. 2010. FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development, 137:715–724.
20.
YuP, PanG, YuJ, ThomsonJA. 2011. FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation. Cell Stem Cell, 8:326–334.
21.
GreberB, WuG, BernemannC, JooJY, HanDW, KoK, TapiaN, SabourD, SterneckertJ, TesarP, ScholerHR. 2010. Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell, 6:215–226.
22.
SchulzTC, PalmariniGM, NoggleSA, WeilerDA, MitalipovaMM, CondieBG. 2003. Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci, 4:27.
23.
CarpenterMK, InokumaMS, DenhamJ, MujtabaT, ChiuCP, RaoMS. 2001. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol, 172:383–397.
24.
ReubinoffBE, ItsyksonP, TuretskyT, PeraMF, ReinhartzE, ItzikA, Ben-HurT. 2001. Neural progenitors from human embryonic stem cells. Nat Biotechnol, 19:1134–1140.
GauldenJ, ReiterJF. 2008. Neur-ons and neur-offs: regulators of neural induction in vertebrate embryos and embryonic stem cells. Hum Mol Genet, 17:R60–66.
27.
Munoz-SanjuanI, BrivanlouAH. 2002. Neural induction, the default model and embryonic stem cells. Nat Rev Neurosci, 3:271–280.
28.
SmithJR, VallierL, LupoG, AlexanderM, HarrisWA, PedersenRA. 2008. Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. Dev Biol, 313:107–117.
BoydNL, DharaSK, RekayaR, GodbeyEA, HasneenK, RaoRR, WestFD3rd, GerweBA, SticeSL. 2007. BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies. Exp Biol Med (Maywood), 232:833–843.
31.
ChadwickK, WangL, LiL, MenendezP, MurdochB, RouleauA, BhatiaM. 2003. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood, 102:906–915.
32.
PeraMF, AndradeJ, HoussamiS, ReubinoffB, TrounsonA, StanleyEG, Ward-van OostwaardD, MummeryC. 2004. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J Cell Sci, 117:1269–1280.
33.
ChenG, YeZ, YuX, ZouJ, MaliP, BrodskyRA, ChengL. 2008. Trophoblast differentiation defect in human embryonic stem cells lacking PIG-A and GPI-anchored cell-surface proteins. Cell Stem Cell, 2:345–355.
34.
DasP, EzashiT, SchulzLC, WestfallSD, LivingstonKA, RobertsRM. 2007. Effects of fgf2 and oxygen in the bmp4-driven differentiation of trophoblast from human embryonic stem cells. Stem Cell Res, 1:61–74.
35.
VallierL, TouboulT, ChngZ, BrimpariM, HannanN, MillanE, SmithersLE, TrotterM, Rugg-GunnP, WeberA, PedersenRA. 2009. Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways. PLoS One, 4:e6082.
36.
BernardoAS, FaialT, GardnerL, NiakanKK, OrtmannD, SennerCE, CalleryEM, TrotterMW, HembergerMet al. 2011. BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell, 9:144–155.
37.
YaoS, ChenS, ClarkJ, HaoE, BeattieGM, HayekA, DingS. 2006. Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. Proc Natl Acad Sci U S A, 103:6907–6912.
38.
XuC, InokumaMS, DenhamJ, GoldsK, KunduP, GoldJD, CarpenterMK. 2001. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol, 19:971–974.
39.
WuZ, ZhangW, ChenG, ChengL, LiaoJ, JiaN, GaoY, DaiH, YuanJ, ChengL, XiaoL. 2008. Combinatorial signals of activin/nodal and bone morphogenic protein regulate the early lineage segregation of human embryonic stem cells. J Biol Chem, 283:24991–25002.
40.
RoodeM, BlairK, SnellP, ElderK, MarchantS, SmithA, NicholsJ. 2012. Human hypoblast formation is not dependent on FGF signalling. Dev Biol, 361:358–363.
41.
GreberB, LehrachH, AdjayeJ. 2007. Fibroblast growth factor 2 modulates transforming growth factor beta signaling in mouse embryonic fibroblasts and human ESCs (hESCs) to support hESC self-renewal. Stem Cells, 25:455–464.
TarradeA, Lai KuenR, MalassineA, TricottetV, BlainP, VidaudM, Evain-BrionD. 2001. Characterization of human villous and extravillous trophoblasts isolated from first trimester placenta. Lab Invest, 81:1199–1211.
44.
HandschuhK, GuibourdencheJ, CocquebertM, TsatsarisV, VidaudM, Evain-BrionD, FournierT. 2009. Expression and regulation by PPARgamma of hCG alpha- and beta-subunits: comparison between villous and invasive extravillous trophoblastic cells. Placenta, 30:1016–1022.
45.
LokeYW, KingA, BurrowsT, GardnerL, BowenM, HibyS, HowlettS, HolmesN, JacobsD. 1997. Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody. Tissue Antigens, 50:135–146.
MuhlhauserJ, CrescimannoC, KasperM, ZaccheoD, CastellucciM. 1995. Differentiation of human trophoblast populations involves alterations in cytokeratin patterns. J Histochem Cytochem, 43:579–589.
48.
BabaieY, HerwigR, GreberB, BrinkTC, WruckW, GrothD, LehrachH, BurdonT, AdjayeJ. 2007. Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells, 25:500–510.
49.
HayDC, SutherlandL, ClarkJ, BurdonT. 2004. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells, 22:225–235.
50.
MatinMM, WalshJR, GokhalePJ, DraperJS, BahramiAR, MortonI, MooreHD, AndrewsPW. 2004. Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells. Stem Cells, 22:659–668.
51.
LiuL, RobertsRM. 1996. Silencing of the gene for the beta subunit of human chorionic gonadotropin by the embryonic transcription factor Oct-3/4. J Biol Chem, 271:16683–16689.
52.
MurohashiM, NakamuraT, TanakaS, IchiseT, YoshidaN, YamamotoT, ShibuyaM, SchlessingerJ, GotohN. 2010. An FGF4-FRS2alpha-Cdx2 axis in trophoblast stem cells induces Bmp4 to regulate proper growth of early mouse embryos. Stem Cells, 28:113–121.
53.
TanakaS, KunathT, HadjantonakisAK, NagyA, RossantJ. 1998. Promotion of trophoblast stem cell proliferation by FGF4. Science, 282:2072–2075.
54.
AbellAN, GrangerDA, JohnsonNL, Vincent-JordanN, DibbleCF, JohnsonGL. 2009. Trophoblast stem cell maintenance by fibroblast growth factor 4 requires MEKK4 activation of Jun N-terminal kinase. Mol Cell Biol, 29:2748–2761.
55.
MesianoS. 2001. Roles of estrogen and progesterone in human parturition. Front Horm Res, 27:86–104.
56.
AsanomaK, MatsudaT, KondoH, KatoK, KishinoT, NiikawaN, WakeN, KatoH. 2003. NECC1, a candidate choriocarcinoma suppressor gene that encodes a homeodomain consensus motif. Genomics, 81:15–25.
YamadaK, OgawaH, HondaS, HaradaN, OkazakiT. 1999. A GCM motif protein is involved in placenta-specific expression of human aromatase gene. J Biol Chem, 274:32279–32286.
CrockerIP, ArthurP, HeazellAE, BakerPN. 2007. The mitotic manipulation of cytotrophoblast differentiation in vitro. Placenta, 28:408–411.
61.
IchikawaN, ZhaiYL, ShiozawaT, TokiT, NoguchiH, NikaidoT, FujiiS. 1998. Immunohistochemical analysis of cell cycle regulatory gene products in normal trophoblast and placental site trophoblastic tumor. Int J Gynecol Pathol, 17:235–240.
62.
WakudaK, YoshidaY. 1992. DNA ploidy and proliferative characteristics of human trophoblasts. Acta Obstet Gynecol Scand, 71:12–16.
63.
JuanG, TraganosF, JamesWM, RayJM, RobergeM, SauveDM, AndersonH, DarzynkiewiczZ. 1998. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry, 32:71–77.
64.
UllahZ, KohnMJ, YagiR, VassilevLT, DePamphilisML. 2008. Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity. Genes Dev, 22:3024–3036.
65.
MomandJ, ZambettiGP, OlsonDC, GeorgeD, LevineAJ. 1992. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 69:1237–1245.
66.
RattsVS, TaoXJ, WebsterCB, SwansonPE, SmithSD, BrownbillP, KrajewskiS, ReedJC, TillyJL, NelsonDM. 2000. Expression of BCL-2, BAX and BAK in the trophoblast layer of the term human placenta: a unique model of apoptosis within a syncytium. Placenta, 21:361–366.
MatsuuraK, JigamiT, TaniueK, MorishitaY, AdachiS, SendaT, NonakaA, AburataniH, NakamuraT, AkiyamaT. Identification of a link between Wnt/beta-catenin signalling and the cell fusion pathway. Nat Commun, 2:548.
69.
NaJ, FurueMK, AndrewsPW. 2010. Inhibition of ERK1/2 prevents neural and mesendodermal differentiation and promotes human embryonic stem cell self-renewal. Stem Cell Res, 5:157–169.
70.
ErlebacherA, PriceKA, GlimcherLH. 2004. Maintenance of mouse trophoblast stem cell proliferation by TGF-beta/activin. Dev Biol, 275:158–169.
ChiuSY, AsaiN, CostantiniF, HsuW. 2008. SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages. PLoS Biol, 6:e310.
73.
ChambardJC, LeflochR, PouyssegurJ, LenormandP. 2007. ERK implication in cell cycle regulation. Biochimica et biophysica acta, 1773:1299–1310.
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