Fig. 4: The tunable differentiation system provides both transcriptional and morphological insights into modeling human pancreas diseases caused by PDX1 and RFX6 mutations.

Representative flow plots (a) and quantification of Stage 1 cells examined for FOXA2 and SOX17 (b), Stage 3-4 cells examined for PDX1 and NKX6.1 (c, d) from HUES8 wildtype (WT), PDX1 KO and RFX6 KO hESC-derived cultures. Both bulk and budding differentiation pipelines were applied. n = 4 independent differentiations, ns not significant, **p < 0.01, one-way ANOVA with Dunnett test for multiple comparisons to WT controls. e Representative images of typical Stage 4-7 clusters stained for pancreatic progenitor markers (PDX1, NKX6.1), endocrine marker (CHGA), and islet cell types (INS, GCG, SST, PPY) Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. f Quantification of CHGA+ cells in Stage 5 clusters. The percentage of CHGA+ cells was normalized to the total number of PDX1+ cells. n = 4 independent differentiations, ns not significant, **p < 0.01, one-way ANOVA with Dunnett test for multiple comparisons to WT controls. g Summary of the presence of islet cell types in WT, PDX1 KO, and RFX6 KO hESC-derived Stage 7 clusters. h Schematic summary of our stem cell modeling with WT, PDX1 KO, and RFX6 KO hESC lines. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Bulk and budding differentiation were induced by 100 ng/mL GDF8 plus 3 μM and 1.5 μM CHIR99021 for Stage 1 Day 1, respectively, using the indicated hESC lines. Source data are provided as a Source Data file.