Fig. 7.

Inhibition of WNT signaling improves human stem cell to beta-like cell differentiation. a Schematic of hESC differentiation to beta-like cell protocol. b Representative flow cytometry plot of day 13 cultures differentiated with DMSO, 9 µM WIKI4, or 2 µm CHIR at stage 4. Cells were stained with anti-PDX1 and anti-NKX6-1. c Kinetics of NKX6-1⁺/PDX1⁺ pancreatic progenitor development from day 8 to day 13 of differentiation in the presence of DMSO, WIKI4, or CHIR (n = 3 independent experiments). Data are means ± SEM. **** denotes p < 0.0001 by two-way ANOVA with Tukey’s multiple comparison test. d Representative flow cytometry plot of day 23 cultures differentiated at stage 4 with DMSO, WIKI4 or CHIR. Cells were stained with anti-PDX1, anti-NKX6-1 (upper panel) and anti-C-peptide (lower panel). e, f Quantification of NKX6-1⁺/PDX1⁺ (e) and NKX6-1⁺/C-Peptide⁺ (f) percentages at day 23 of differentiation, in cultures treated with DMSO, WIKI4 or CHIR at stage 4. Data are means ± SEM. * denotes p < 0.05, ** denotes p < 0.01 by one-way ANOVA with Tukey’s multiple comparison test. g Down-regulation of mesenchymal Hedgehog (Hh) signaling by Sufu and Spop allows for the establishment of a pancreas-specific mesenchymal niche that properly supports epithelial development (left). Improper activation of Hh signaling due to loss of mesenchymal Sufu and Spop leads to a more gastrointestinal-like niche that secretes gastrointestinal factors such as WNT ligands (right). These WNT ligands can then act on the developing pancreatic epithelium to over-activate WNT signaling and impair beta cell differentiation