Extended Data Fig. 1: Intracellular redox management enables CPS1+ hepatocyte specification in HLOs.
From: Multi-zonal liver organoids from human pluripotent stem cells
a) H&E histology, GLS2 IHC, and GS IHC images of liver sections are shown in panels from ODS od/od (GULO mutant) rat treated with 0.2% Ascorbic acid (AsA), ODS od/od rat treated without AsA. Scale bars indicate 100 µm. 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. Data points are shown for 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. 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 D20 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) RT-qPCR of genes for Z1-HLOs. (mean ± SD and n = 9 independent experiments). 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 and primary liver. Scale bar indicates 200 µm. (n = 3 independent experiments). In c-e, g data are represented as boxplots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper and lower whisker extends from the hinge to the largest and smallest value respectively no further than 1.5 × IQR from the hinge (where IQR is the inter-quartile range). c-e, f, g, one-way ANOVA with multiple comparisons and Tukey’s correction.
