Fig. 1. Characterization of β-catenin deficient murine embryonic stem cells. (A) RT-PCR analysis indicates that β-catenin −/− cells do not express β-catenin. (B) Wildtype E14.1 and β-catenin −/− ES cells grow on feeder layer cells (FL). (C) Wildtype ES cells as well as β-catenin −/− cells show alkaline phosphatase staining. (D) RT-PCR analysis to monitor components of the canonical Wnt-pathway as well as expression of stemness marker genes in wildtype and β-catenin knock-out ES cells. HPRT was used as loading control for RT-PCR analyses. RT-PCR panels were assembled from different experiments but from identical experiments for individual genes. (E) Intracellular localization of Oct-3/4 and β-catenin in wildtype E14.1 ES cells as visualized through immunfluorescence (IF). DAPI counterstaining (DAPI) and an overlay are shown as well. (F) Immunfluorescence of Oct-3/4 and β-catenin in co-cultures of wildtype E14.1 and β-catenin −/− ES cells.

Fig. 2. Activation of the Wnt/β-catenin pathway in murine ES cell. (A) Murine ES cells were transfected with TOPFlash reporter gene and treated with Wnt3a, Wnt5a or LiCl at indicated concentrations for 24 h. At least three independent measurements per time point were performed. (B) Untreated E14.1 ES cells show only low TOPFlash activity if at all. n = 18. Data were normalized to FOPFlash reporter. Oct-3/4, nanog and Wnt11 can reduce LiCl induces TOPFlash activity in ES cells (n = 13) or stabilized β-catenin induced TOPFlash activity in HEK293 cells (n = 6–9). Data were normalized to TOPFlash values in untreated cells. (C) siRNA against Wnt11 reduces endogenous Wnt11 RNA levels. RT-PCR panel was assembled from the same experiment. In transfection experiments Wnt11 siRNA increases TCF/β-catenin mediated TOPFlash activity (n = 8). siRNA against Wnt5a was not effective. HPRT was used as internal control for RT-PCR experiments. siRNA against Wnt11 did not change FOPFlash activity as control (n = 5). Data were normalized to TOPFlash or FOPFlash values, respectively, in control siRNA cells. Error bars indicate S.E. of the mean.

Fig. 3. Activation of the Wnt/β-catenin pathway by LiCl in differentiating embryoid bodies prolongs expression of stemness genes and delays the expression of differentiation marker genes. The expression of marker genes in differentiating embryoid bodies was analyzed by RT-PCR at days 2, 4, 6, 8 and 10 of differentiation and compared between untreated (left) and LiCl treated embryoid bodies (right). RT-PCR panel was assembled from different experiments but from identical experiments for individual genes.

Fig. 4. Activation of the Wnt/β-catenin pathway delays differentiation in embryoid bodies. Brachyury-GFP (Bry-GFP) knock-in ES cells were used to monitor differentiation. (A) LiCl treatment from days 0 to 4 of differentiation reduces the expression GFP in day 5 embryoid bodies. (B) Quantification of GFP-expressing Bry-GFP ES cells by FACS at day 2 and 5 of differentiation in untreated and LiCl treated (days 0–4) ES cells. (C) Kinetics of GFP expression in Bry-GFP cells is altered by different timeframes of LiCl incubation. (D) Quantitative analysis of GFP expression in Bry-GFP cells using days of maximal GFP expression (upper panel, n = 14) and the location in time of the center of gravity (CoG) of the area under the curve (lower panel, n = 13). Statistical evaluation was done with a two-sided paired exact Wilcoxon rank sum test. CGR-8-MHC-GFP cells were used to monitor formation of terminally differentiated cardiomyocytes as indicated by a contractile phenotype (n = 4) (E) or cardiac MHC driven GFP expression (F) after treatment with LiCl from days 0 to 4 in comparison to untreated controls. In F a representative view is given. Error bars indicate S.E. of the mean.

Fig. 5. (A) Treatment of Bry-GFP knock in ES cells with Wnt3a (100 ng/ml) from days 0 to 4 or days 2 to 4 reduces formation of a contractile phenotype at day 10, whereas treatment from days 0 to 2 enhanced cardiomyocyte formation. Data of nine representative experiments as well as the average are shown. (B) Treatment of Bry-GFP knock in murine ES cells with Wnt3a (100 ng/ml) slightly increases number of Bry-positive cells at day 4 (n = 16) at all tested conditions. (C) This effect is more clearly seen when selecting differentiation experiments in which the number of Bry-positive cells at day 4 was below 50% (n = 9, low velocity). (D) This effect could not be seen in experiments with a high number of GFP-positive cells at day 4 (n = 7, high velocity). (E) Treatment with a higher concentration of Wnt3a (400 ng/ml) between day 0 and 2 results in a modest decrease of Bry-positive cells at day 4 (n = 9). Error bars indicate S.E. of the mean.