Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells

Abstract

Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.

Figure 1

ESC self-renewal requires Wnt signals. (a–f) The 7xTcf–eGFP reporter is active in a subset (arrow) of ESCs cultured for 2 days on MEFs (a,b); Wnt3a protein activates the reporter in all cells (c,d), whereas Fz8CRD extinguishes it (e,f). (a,c,e) Phase-contrast microscopy; (b,d,f) eGFP. (g) The ability of 7xTcf–eGFP cells to form alkaline phosphatase-positive (AP⁺) colonies in the absence of MEFs correlated with the level of eGFP, and was enhanced by the presence of Wnt3a protein (mean ± s.e.m., n = 3). (h) The expansion of R1 ESCs able to form alkaline phosphatase-positive colonies on MEFs was progressively repressed by increasing concentrations of the Wnt antagonist Fz8CRD. This effect was counteracted by simultaneous addition of Wnt3a protein (mean + s.e.m., n = 3). (i) The expansion of R1 ESCs able to establish alkaline phosphatase-positive colonies on MEFs was repressed by IWP2. This repression was relieved by simultaneous addition of Wnt3a protein (240 ng ml⁻¹) (mean+ s.e.m., n = 3). (j–o) Axin2^LacZ ESCs cultured in the absence of MEFs, untreated (j) or treated for 3 days with IWP2 (l,m), 2 μg ml⁻¹ Fz8CRD (n,o) and/or 200 ng ml⁻¹ Wnt3a (k,m,o) and stained with X-gal and Nuclear Red. (p–u) CGR8 ESCs cultured in the absence of MEFs, untreated (p) or treated for three passages with IWP2 (r,s), 2 μg ml⁻¹ Fz8CRD (t,u) and/or 200 ng ml⁻¹ Wnt3a (q,s,u) and stained for alkaline phosphatase. (v) The expansion of CGR8 ESCs able to form alkaline phosphatase-positive colonies in the absence of MEFs was repressed by IWP2 or 500 ng ml⁻¹ Fz8CRD, and promoted by 200 ng ml⁻¹ Wnt3a protein. Scale bars, 100 μm (a–f, j–o), 500 μm (p–u).

Figure 2

Wnt signals are required to inhibit the differentiation of ESCs into EpiSCs. (a) R1 ESCs cultured for 3 days on MEFs in the presence of IWP2 form flattened colonies that reduce alkaline phosphatase (AP) and Nanog expression, and increase Claudin6 and Otx2 expression. (b) On passaging of R1 ESCs in the presence of IWP2, only alkaline phosphatase-low but Oct4-positive colonies expand (mean+ s.e.m., n = 3). (c) Gene expression profiles of R1 ESCs cultured on MEFs in the presence of IWP2, and of EpiSCs, relative to untreated ESCs (mean± s.e.m., n = 3). (d) After two passages in the presence of IWP2 or vehicle on MEFs, the cells were passaged in the presence of 2 μM SB431542, and the number of alkaline phosphatase- and Oct4-positive colonies determined (mean± s.e.m., n = 3). (e,f) Female ESCs were cultured on MEFs for two passages in the presence of vehicle (e) or IWP2 (f) and immunostained for H3K27me3 (red) and Oct4 (green). A yellow focus indicates the presence of an inactive X chromosome. (g) In the presence of IWP2, either no or virtually all cells of a colony showed the inactive X focus. The number of colonies showing no focus (2X) or X inactivation (Xin) was determined over multiple passages. The absolute numbers are plotted within the bars. Scale bars, 50 μm (a,e,f).

Figure 3

Wnt3a protein is sufficient to inhibit the differentiation of ESCs into EpiSCs. (a) FACS plots of EpiSCs and of R1 cells passaged every 3 days in N2B27 supplemented with LIF, bFGF and ActivinA, in the presence of Wnt3a protein (240 ng ml⁻¹) or IWP2 (2 μM) as indicated, and stained with anti-SSEA1-PE (phycoerythrin) and anti-Pecam1-FITC (fluorescein isothiocyanate) antibodies (10,000 cells/plot). (b) Gene expression profiles of EpiSCs and R1 ESCs cultured in N2B27 supplemented with LIF, bFGF, ActivinA and either Wnt3a (LFAW) or IWP2 (LFAI), relative to untreated ESCs (mean± s.e.m., n = 3). (c–e) The Axin2^LacZ/+ reporter indicates Wnt activity in the E3.5 (c) and E4.5 (d) ICM, but is inactive in E5.5 implanted embryos (e). Scale bars, 25 μm.

Figure 4

LIF and Wnt3a are sufficient to support ESC self-renewal. (a,b) Expansion over multiple passages of alkaline phosphatase-positive (AP⁺) R1 ESC colonies in medium containing serum and LIF (a) or in N2B27 containing LIF (b) (mean + s.e.m., n = 3). (c,d) Alkaline phosphatase-stained R1 ESCs cultured on MEFs (c) or maintained for six passages in N2B27 medium supplemented with LIF and Wnt3a (d). (e) Gene expression profile of R1 ESCs following four passages in the indicated conditions. (f) Expansion of alkaline phosphatase-positive R1 ESC colonies in N2B27 medium. Where indicated, Wnt3a was added at 200 ng ml⁻¹. (g) Expansion of alkaline phosphatase-positive R1 ESC colonies in N2B27 medium containing LIF and PD0325901. (h) MEK activity (indicated by the presence of phospho-ERK (p-ERK; extracellular signal-regulated kinase) on western blot) in R1 ESCs cultured in N2B27 with LIF, was repressed by PD0325901 (PD) but not by Wnt3a, or by withdrawal of Wnt3a for 9 h (−9 h). Uncropped images of blots are shown in Supplementary Fig. S4g. Scale bar, 200 μm (c,d).

Figure 5

Wnt3a supports derivation of non-permissive ESCs. (a–c) Alkaline phosphatase, Oct4 and Nanog stainings, respectively, of the newly derived FVB/N ESC line FN3. Scale bar, 200 μm. (d) Chimaera (black–white spotted) obtained from injection of C57Bl/6 blastocysts with passage 7 ESC line FN3 together with FVB mate and pups showing germline transmission (white pups). (e) Expansion of alkaline phosphatase-positive (AP⁺) colonies from newly derived FVB/N ESCs in N2B27 medium with LIF (mean + s.e.m., n = 3). PD, PD0325901. (f) Expansion of alkaline phosphatase-positive colonies from FVB/N ESCs on MEFs in medium containing serum and LIF (mean+ s.e.m., n = 3).