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. 2015 Dec 8;6:8989. doi: 10.1038/ncomms9989

Figure 4. Active Wnt signalling is required for expression of stemness factors in the organoids and supports their growth.

Figure 4

(a) Formation efficiency of the organoids determined by quantification in four different media with three independent donors (basic (B), FGF10 (F), noggin (N), EGF (E), RSPO1 (R)). Addition of EGF to the medium almost doubles sphere formation. Addition of RSPO1 strongly increases the size of the organoids, with a much higher number growing to above 300 μm diameter (dark grey fractions). Data are normalized to control conditions (B) ± cumulative s.d. of big and small organoids in duplicate wells. (b) During long-term expansion only organoids growing in complete medium maintain stable growth as seen in phase contrast images at 3 months in culture. (c) Active Wnt signalling is detected in the cells of the growing organoids by the TCF4-GFP reporter (green), which was introduced by lentiviral transduction. All cells containing reporter are labelled red (mCherry). Images are representative stills from a live-cell imaging experiment (Supplementary Movie 3). (d) qPCR analysis shows that addition of RSPO1 and Wnt3A to the medium for 7 days leads to a strong increase in the expression of hTERT and the proliferation marker Ki67 relative to GAPDH (ΔΔCt)2. Increased expression of the Wnt targets AXIN2 and cMYC confirms pathway activation. Data are representative of two independent donor samples, and are represented as mean±s.d. of technical replicates. (e) Addition of 25% Wnt3A conditioned medium to 25% RSPO1 conditioned medium increases expression of Olfactomedin4 and Notch1, compared with control organoids grown in RSPO1 conditioned medium alone, as shown using qPCR. Data are representative of two independent patient samples and represented as mean±s.d. of technical replicates. (f) Phase contrast images of organoids 7 days post seeding in media supplemented with different Wnt pathway ligands. Pictures are representative of three independent patient samples. Notably, addition of Wnt3A on its own failed to improve sphere formation and growth, while RSPO1 greatly increased both number and size of spheres.