Fig 1: Intracellular redox management enables CPS1+ hepatocyte specification in HLOs.a) H&E histology, GLS2 IHC, and GS IHC images of liver sections are shown in panels (ODS od/od (GULO mutant) rat treated with 0.2% Ascorbic acid (AsA), ODS od/od rat treated without AsA. Scale bars indicate 100µm. The graph shows the GLS2 or GS positive area ratio versus the hematoxylin positive area in ODS od/od (GULO mutant) rat treated with 0.2% Ascorbic acid (AsA) and ODS od/od rat treated without AsA (n=2; Mean +-SD, Mann-Whitney U test, p = 4.507e-08) Data points are shown GLS2 area at portal vein (37 portal vein sections of + AsA rats, 27 portal vein sections of - AsA rats) and GS area in 8 images of +/− AsA rat for analysis with Fiji.b) Schematic for development of Z1-HLOs and doxycycline induction to induce CPS1+ hepatocyte specification (left). Brightfield and fluorescence images of mCherry expression in ascorbate depleted Dox (100 ng/ml) treated Z1-HLOs compared to HLOs with ascorbic acid depletion at day 20 and control HLOs (right).c) ELISA for mGULO protein concentration in Dox treated Z1-HLOs compared to control HLOs. (n = 9 independent experiments)d) Cellular Antioxidant concentration in Dox treated Z1-HLOs compared to control HLOs. (n = 9 independent experiments)e) ROS levels in Dox treated and extracellular ascorbate induced Z1-HLOs compared to control HLOs. (n = 9 independent experiments)f) Heatmap of Zone 1 genes from RNAseq dataset for Dox treated Z1-HLOs compared to control.g) Albumin ELISA for Z1-HLOs treated with Dox compared to control HLOs and PHH normalized by cell viability. (n = 9 independent experiments)h) Immunofluorescence images of Dox treated Z1-HLOs for CPS1, ACSS2 and CDH1 compared to control HLOs. Scale bar indicates 200 µm.