Extended Data Fig. 2: Low dose bilirubin promotes GLUL+ hepatocyte specification in HLOs.
From: Multi-zonal liver organoids from human pluripotent stem cells
a) Schematic for low dose bilirubin treatment and Z3-HLO development to induce GLUL+ expression. b) Cell viability assay with different concentration of bilirubin to titrate dose for maximal viability. (n = 9 independent experiments). c) Brightfield image of Z3-HLOs treated with low dose bilirubin (1 mg/L) compared to control, and luminal outline using ImageJ, arrows indicate luminal projections that are similar to bile canaliculi found in human liver. Scale bar indicates 200 µm. d) Comparison of size and circularity of lumen of the control and 1 mg/L bilirubin treated Z3-HLOs. (Data is mean ± SD, n = 9 independent experiments). e) Heatmap of Zone 3 genes from RNAseq dataset for bilirubin treated Z3-HLOs compared to control. f) RT-qPCR of genes for Z3-HLOs. (mean ± SD and n = 9 independent experiments). g) CYP3A4 activity assay in response to Rifampicin in control, Z1-, Z3-HLOs, and PHH (left). CYP1A2 activity assay in response to Omeprazole in control, Z1-, Z3-HLOs, and PHH (right). (n = 9 independent experiments). h) Immunofluorescence images of Z3-HLOs for GLUL, NR3C1 and CDH1 compared to control HLOs and primary liver. Scale bar indicates 200 µm. (n = 3 independent experiments). i) Heatmap of Z1- and Z3- HLOs depicting expression of zonal genes and lack of consensus expression of markers such as ARG1 and AKR1C1. j) Caspase 3 activity assay in Z3-HLOs (left) and cell viability assay in Z3-HLOs compared to Z1-HLOs after treatment with Zone 1 toxin (right). (n = 9 independent experiments). k) Caspase 3 activity assay in Z3-HLOs (left) and cell viability assay in Z3-HLOs compared to Z1-HLOs after treatment with Zone 3 toxin (right). (n = 9 independent experiments). In b, g, j, k 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). b,f, g,j,k one-way ANOVA with multiple comparisons and Tukey’s correction. d, Kruskal–Wallis test (left) and unpaired two-tailed Student’s t-test (right).
