Fig. 5
Differentiation of TXNIP−/− and TXNIP+/+ H1-hESCs into SC-islets and their interferon response. A Schematic representation of the multi-stage differentiation protocol for generating TXNIP−/− and TXNIP+/+ SC-islets from H1-hESCs. Cells are differentiated as a 2D planar culture until stage 4 (pancreatic progenitors) and are then differentiated as aggregates in static microwells (3D). B Representative immunofluorescence images showing the expression of stage-specific differentiation markers for definitive endoderm (SOX17), pancreatic progenitors (PDX1/NKX6.1), and SC-islets (INS/GCG/SST), confirming successful stage-specific differentiation. Scale bar, 50 μm. C Quantification of the percentage of SC-islet marker-positive cell populations of insulin (INS +), glucagon (GCG +), somatostatin (SST +), and polyhormonal (PH) show comparable endocrine lineage specification in TXNIP−/− and TXNIP+/+ SC-islets (n = 6, mean ± SEM, unpaired student’s t-test). D Cell viability assessment at stage 4 pancreatic progenitor cells and stage 7 SC-islets shows no significant differences between TXNIP−/− and TXNIP+/+ (n = 6, mean ± SEM, unpaired student’s t-test). E Relative gene expression levels of NGN3 and INS at stages S4, S5, S6, and S7 show consistent differentiation dynamics across genotypes. Cyclophilin was used as the endogenous housekeeping gene for ΔCt calculation, and ΔΔCt values were normalized to undifferentiated TXNIP+/+ H1-hESCs to determine relative gene expression (mean ± SEM, unpaired student’s t-test). F Representative western blot and quantification showing stage-wise TXNIP expression over β-like cell differentiation. (n = 3, mean ± SEM, ****p < 0.0001, ordinary two-way ANOVA, Tukey’s multiple comparison test). Full-length blots are presented in Supplementary Figure S7A. G Representative western blot showing STAT1 signalling response in TXNIP−/− and TXNIP+/+ SC-islets following pulse and chase treatment with IFNα and IFNγ (NP = no pulse; n = 2). Full-length blots are presented in Supplementary Figure S7B