Figure 3: Serum replacement blocks Wnt-mediated differentiation.

(a) Alkaline phosphatase (AP) staining of cells grown in 'standard (std)', 'N2B27' and 'mTeSR1' media for three passages in the continuous presence of Wnt3a protein or control buffer (vehicle). Scale bar, 2 mm. (b) Quantification of Wnt-mediated changes in self-renewal during passage as clumps. The number of AP+ colonies was determined after each passage. The ratio of cumulative number of AP+ colonies in Wnt3a-treated cultures divided by AP+ colonies in vehicle-treated cultures is shown for each passage in each media. 100% represents an equal number of AP+ colonies in Wnt3a- and vehicle (Veh)-treated cultures. The average ±s.d. of three biological replicates is shown, and is representative of two independent experiments. (c) Quantification of Wnt-mediated changes in total cell number during passage as clumps. The ratio of cumulative cell number in Wnt3a-treated cultures divided vehicle-treated cultures is shown on a log scale. The average ±s.d. of three biological replicates is shown, and is representative of two independent experiments. (d) Phase-contrast and OCT4, Brachyury and GATA4 immunofluorescence images of cells treated with Wnt3a for 7 days in standard hESC media, N2B27 or mTeSR1. Scale bar, 200 μm. (e) Phase-contrast images of hESCs treated for 4 days with Wnt3a in N2B27 media alone, or the same supplemented with 20% PBS or 20% knockout serum replacement (KOSR). Scale bar, 200 μm. (f) AP staining of hESC treated for 3 days with Wnt3a in standard hESC media, or the same media in which the KOSR concentration was reduced to 2%. Scale bar, 200 μm. (g) Phase contrast and GFP fluorescence in live colonies after 5 days of growth on N2B27 in the presence of vehicle, 20% PBS or 20% KOSR. Scale bar, 200 μm. (h,i) Gene expression analysis in cells grown in N2B27 plus the indicated combination of vehicle, Wnt3a and KOSR for four passages. Values were normalized to glyceraldehyde 3-phosphate dehydrogenase, and are expressed relative to vehicle-treated cells at passage 1. The average and range of two biological replicates is shown.