Fig. 10: High activation of the canonical Wnt pathway in hESC-derived organoids causes suppression of ChPe markers.

a A schematic presentation of the protocol for the treatment of organoids with the canonical Wnt agonist CHIR. b, c The ChP marker TTR is present in the periphery of a day 25 organoid exposed to 3 μM, but not when exposed to 12 μM CHIR, from day 18. c N = 4 organoids (3 μM CHIR) and 5 organoids (12 μM CHIR treated); were used for immunohistochemistry analysis. d High magnification images (boxed region shown in b) of serial sections of a day 40 organoid exposed to 3 μM reveal the presence of TTR, AQP1, and OTX2 in the periphery. This labeling is reduced or undetectable upon exposure to higher concentrations of CHIR (6/9/12 μM). d N = 14 organoids (3 μM CHIR), 9 organoids (6 μM CHIR), 5 organoids (9 μM CHIR treated), and 14 organoids (12 μM CHIR treated) from 2 independent experiments were used for analysis (each organoid was considered as an independent biological replicate). Additional examples are provided in Supplementary Fig. S10. e q-PCR analysis shows that the expression of the ChP markers TTR, AQP1, and OTX2 is upregulated in organoids at day 30 organoids exposed to 3 μM CHIR from day 18. This increased expression is lost upon treatment with 12 μM CHIR for the same period. (f) LEF1 expression displays a stepwise increase upon 3 μM and 12 μM CHIR treatment, consistent with increased activation of the canonical Wnt pathway. Bar graphs (e, f) represents mean ± SEM. Group means were compared using one-way ANOVA followed by post hoc Tukey’s multiple comparison test, p < 0.0001(e, f), *p value<0.05, **p value<0.01, ***p value<0.001; (e, f) N = 8 biologically independent replicates examined over 3 independent experiments. (g) Heatmap and hierarchical clustering comparing normalized RNA-seq reads for 3 μM and 12 μM CHIR-treated organoids. Several ChP-enriched genes are downregulated with increased activation (12 μM CHIR) of canonical Wnt signaling (N = 3 biologically independent replicates). Scale bars: 100 μm (all panels in c and d). Source data are provided as a Source Data file.