Figure 5: Distinct lineage-specific differentiation propensities of Wnt^low and Wnt^high hESCs.

(a,b) hESCs maintained with various levels of Wnt pathway activation were subjected to the neural differentiation protocol for 5 days. Neuralectodermal differentiation was detected by immunostaining for Pax6 (a) and Otx2 (b). Scale bar, 200 μm. (c) FACS-sorted populations of >95% pure TCF-GFP^high or TCF-GFP^low were subjected to the neural differentiation protocol for 5 days. Neural differentiation was detected by PAX6 staining, and compared with the number of DAPI-positive nuclei using an automated counting algorithm. Average ±s.d. from five fields of view, representing three independent experiments. (d) Immunofluorescent staining for endodermal marker FoxA2 in hESCs subjected to the neural differentiation protocol for 5 days. Scale bar, 200 μm. (e) Immunofluorescent staining for endodermal marker Gata4 on day 5 of the endodermal differentiation protocol. Scale bar, 200 μm. (f) Schematic description of cardiogenic differentiation protocol. (g) IWP2- and Wnt3a-treated hESCs in standard hESC media were subjected to the cardiogenic differentiation protocol, and shortened versions thereof. The number of spontaneously beating sectors observed on days 15–18 of the full-length protocol (days 12–15 of the shortest protocol) were averaged. Error bars ±s.d., n=4. (h–m) Hematoxylin and eosin staining of tumours arising 8 weeks after injecting hESCs maintained in N2B27 with Wnt3a and KOSR for five passages under the kidney capsule of nude mice. (h) Neural rosette, (i) pigmented epithelium, (j,k) glandular structures, (l) cartilage and (m) bone. Scale bar, 50 μm.