Fig. 3

Differentiation of TXNIP^−/− and TXNIP^+/+ H1-hESCs into hepatocyte-like cells. A Timeline and schematic representation of multi-stage differentiation protocol for generating TXNIP^−/− and TXNIP^+/+ HLCs from H1-hESCs. B Representative immunofluorescence images showing the expression of stage-specific differentiation markers for definitive endoderm (SOX17), hepatoblast (HNF4A and AFP), and mature hepatocytes (HNF4A and ALB) confirming successful differentiation in TXNIP^−/− and TXNIP^+/+ HLCs. Scale bar, 50 μm. C ELISA quantification showing albumin secretion over time shows reduced secretion TXNIP^−/− HLCs at later stages in the supernatant over HLC differentiation starting at day 13. (n = 4, mean ± SEM, *p  < 0.05, ordinary two-way ANOVA, Tukey’s multiple comparison test). D Cell viability assessment shows no significant differences between TXNIP^−/− and TXNIP^+/+ HLCs. (n = 3, mean ± SEM, unpaired student’s t-test). E Relative gene expression levels of NANOG, HNF4A, and ALB at days 0, 4, 9, 17 and 25 show consistent differentiation dynamics across genotypes. TATA-box-binding protein (TBP) 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 immunoblots and quantifications showing stage-wise expression of TXNIP, ALB, BiP, and lipid metabolism markers (ACC1, FASN, CD36, and PPARγ) in TXNIP^−/− and TXNIP^+/+ H1-hESCs differentiation (n = 3–5, mean ± SEM, *p  < 0.05, **p  < 0.01, ***p  < 0.001, ****p  < 0.0001, ordinary two-way ANOVA, Tukey’s multiple comparison test). Full-length blots are presented in Supplementary Figure S5C