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. 2011 Mar 4;8(3):326-34.
doi: 10.1016/j.stem.2011.01.001.

FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation

Pengzhi Yu  1 , Guangjin Pan, Junying Yu, James A Thomson
Affiliations

FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation

Pengzhi Yu et al. Cell Stem Cell. .

Abstract

Here, we show that as human embryonic stem cells (ESCs) exit the pluripotent state, NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ESCs into extraembryonic lineages. Here, we find that FGF2, acting through the MEK-ERK pathway, switches BMP4-induced human ESC differentiation outcome to mesendoderm, characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation, that forced NANOG expression results in FGF-independent BMP4 induction of mesendoderm, and that knockdown of NANOG greatly reduces T induction. Together, our results demonstrate that FGF2 signaling switches the outcome of BMP4-induced differentiation of human ESCs by maintaining NANOG levels through the MEK-ERK pathway.

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Figures

Figure 1
Figure 1. FGF2 signaling switches the outcome of BMP4 induced differentiation of human ES cells
(A) Microarray data showing expression levels of selected lineage marker genes from H1 cells treated with different concentrations of BMP4 for 5 days, w/ or w/o FGF2. Fold change values, shown in log2 scale, were normalized against data from H1 ES cells grown in mTeSR medium. (B) Expression levels of selected genes during a 5 ng/mL BMP4 time course, detected by quantitative RT-PCR. Error bars represent standard deviations from 3 replicates. mTeSR cultured H1 ES cell control data (red) was set as 1 for each gene except T, whose expression level was normalized against data from 36h +FGF2+BMP4 treated cells. (C) FGF receptor inhibitor PD173074 inhibited T induction at the 36 h time point shown by quantitative RT-PCR analysis. Average expression level from +FGF2+BMP4 group was set as 1 for each gene. (D) T protein detected by immunostaining. Top row: DAPI nuclei staining; bottom row: FITC channel showing T staining; Scale bar: 0.1mm. (E) Flow cytometry data revealed a uniform shift from −FGF2+BMP4 population (shaded grey) to +FGF2+BMP4 population (solid black) for T staining.
Figure 2
Figure 2. MEK-ERK pathway is required for mesendoderm induction
(A) Quantitative RT-PCR analysis confirmed transgene expression in DUSP6 and DUSP10 overexpressing cells. Messenger RNA levels were normalized to that from the non-transfected human ES cells. Error bars represent standard deviations from 3 replicates. (B) DUSP6 overexpression reduced ERK phosphorylation compared to non-transfected H1 ES cells, detected by western blot. (C and D) DUSP6 overexpression suppressed the induction of T upon +FGF2+BMP4 treatment, confirmed by both quantitative RT-PCR (C), and flow cytometry (D). mRNA levels were normalized to that of the non-treated control cells in (C). (E and F) Flow cytometry and immunostaining for T induction with inhibitors. FGF receptor inhibitor PD173074 and MEK inhibitor U0126 blocked the induction of T upon +FGF2+BMP4 treatment, while other inhibitors: SB203580 against p38; SP600125 against JNK; and LY294002 against PI3K, failed to block T induction. Scale bar: 0.1mm, in (F). (G) Only constitutively activated MEK1 mutant (MAP2K1-CC) upregulated T expression in the −FGF2+BMP4 conditon compared to wild type MEK1 (MAP2K1-W1) control and EGFP control. A single plasimid with doxycycline inducible elements was used to express transgenes in H9 cells. mRNA levels were normalized against that from EGFP control in -DOX condition.
Figure 3
Figure 3. NANOG substitutes for the requirement of FGF2 in switching the outcome of BMP4 induced differentiation
(A) NANOG constitutively expressed cells induced T expression after 36h of −FGF2+BMP4 treatment, detected by quantitative RT-PCR. Error bars represent standard deviations from 3 replicates. mRNA levels of T and NANOG were normalized against +FGF2+BMP4 treated H1 cells and mTeSR cultured H1 cells respectively. (B) Most NANOG overexpressed cells were T positive detected by flow cytometry at 48h of BMP4 treatment in both +FGF2 and −FGF2 groups. Non-treated H1 ES cells served as negative control (shaded grey). The non-transduced ES cell control panel is the same as in Figure 2D, since the data were from the same set of expriment with the same control samples. (C) NANOG overexpressing cells had an extended T expression time window at day 5 of differentiation. NANOG overexpressing cells cultured in mTeSR were included as negative control (shaded grey). (D) NANOG knockdown by siRNA reduced T expression level at 36h from +FGF2+BMP4 treated cells. siRNAs were introduced at the same time when cells were treated with BMP4. 0.1 μM PD173074 was used to block endogenous FGF signaling in the −FGF2+BMP4 condition in all sub panels in this figure.
Figure 4
Figure 4. Simplified model for the mechanism of the FGF2 switch on BMP4 induced differentiation outcomes
Arrow represented activation or induction, dashed arrow represented indirect activation with multiple steps involved, hammer-ended line represented inhibition, and arrow with question mark represented induction with mechanism undiscovered. Red labeled inhibitors on the MEK-ERK pathway that blocked T induction.
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References

    1. Arkell RS, Dickinson RJ, Squires M, Hayat S, Keyse SM, Cook SJ. DUSP6/MKP-3 inactivates ERK1/2 but fails to bind and inactivate ERK5. Cell Signal. 2008;20:836–843. - PubMed
    1. Armstrong L, Hughes O, Yung S, Hyslop L, Stewart R, Wappler I, Peters H, Walter T, Stojkovic P, Evans J, et al. The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis. Hum Mol Genet. 2006;15:1894–1913. - PubMed
    1. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005;122:947–956. - PMC - PubMed
    1. Brons IG, Smithers LE, Trotter MW, Rugg-Gunn P, Sun B, Chuva de Sousa Lopes SM, Howlett SK, Clarkson A, Ahrlund-Richter L, Pedersen RA, et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature. 2007;448:191–195. - PubMed
    1. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 2003;113:643–655. - PubMed

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