Figure 5.
WNT signaling inhibits binucleation of human hepatocytes in an E2F7/8-dependent manner. (A) Percentage of binucleated cells in Hep-Org 1 line with GFP-NLS and stained with CellMask Orange in control medium and after 3 days of CHIR99021 removal (N = 3 experiments, 200–300 cells analyzed per experiment, *P < 0.05, Student’s t test, two-tailed). (B) Representative brightfield images of Hep-Org 1 in control medium and after 3 days of CHIR99021 removal. Scale bars represent 50 µm. (C) Base-editing strategy for introduction of a premature stop codon in E2F7 and E2F8 open reading frames with Sanger sequencing chromatographs of wildtype and mutant alleles, confirming homozygous base changes (highlighted in red) in E2F7Q206X and E2F8Q462X Hep-Org 1 lines. Protospacer adjacent motif (PAM) sequences are highlighted in blue. (D) Representative brightfield images of wildtype, E2F7Q206X, and E2F8Q462X Hep-Org 1 lines in control medium and after 3 days of CHIR99021 removal. Scale bars represent 50 µm. (E) Relative expression of E2F7 or E2F8 in wildtype and mutant lines as measured by RT-qPCR in control medium and after 3 days of CHIR99021 removal (N = 3 experiments, ns = not significant, *P < 0.05, **P < 0.01, Student’s t test, two-tailed). Error bars represent standard deviation. (F) Percentage of binucleated cells in wildtype and mutant lines expressing GFP-NLS and stained with CellMask Orange in control medium and after 3 days of CHIR99021 removal. Each dot represents the average percentage of binucleated cells per experiment (N = 3 experiments, 200–300 cells analyzed per experiment, ns = not significant, *P < 0.05, **P < 0.01, Student’s t test, two-tailed). (G) Representative images of Hep-Org 1 line expressing GFP-NLS (green) and E-cadherin-tdTomato (magenta) grown in control medium and after 3 days of CHIR99021 removal. Scale bars represent 50 µm. (H) Representative images of Hep-Org 1 line expressing E-cadherin-tdTomato (magenta) and stained with DRAQ5 (gray) to visualize DNA, after growth for 7 days in either control medium or differentiation medium (DM). Scale bars represent 50 µm. (I) Percentage of binucleated cells per organoid in Hep-Org 1 line expressing E-cadherin-tdTomato and stained with DRAQ5 after growth for 7 days in either control medium (n = 84 organoids) or differentiation medium (DM, n = 83 organoids). Each dot represents the percentage of binucleated cells per organoid, and the black line represents the median (N = 2 experiments, 73–93 organoids analyzed per experiment, ns = not significant, Mann–Whitney test). (J) Percentage of binucleated cells per organoid in Hep-Org 1 line expressing GFP-NLS/E-cadherin-tdTomato grown for 1 day in control medium (n = 21 organoids) or in medium supplemented with insulin (n = 37 organoids). The black line represents the median. See material and methods section for more information on media composition (N = 2 experiments, ns = not significant, Mann–Whitney test). (K) Percentage of binucleated cells per organoid in Hep-Org 1 line expressing GFP-NLS/E-cadherin-tdTomato grown for 7 days in control medium (n = 15 organoids) or in medium without insulin (n = 19 organoids). The black line represents the median. See Materials and methods section for more information on media composition (N = 2 experiments, ns = not significant, Mann–Whitney test).

WNT signaling inhibits binucleation of human hepatocytes in an E2F7/8-dependent manner. (A) Percentage of binucleated cells in Hep-Org 1 line with GFP-NLS and stained with CellMask Orange in control medium and after 3 days of CHIR99021 removal (N = 3 experiments, 200–300 cells analyzed per experiment, *P < 0.05, Student’s t test, two-tailed). (B) Representative brightfield images of Hep-Org 1 in control medium and after 3 days of CHIR99021 removal. Scale bars represent 50 µm. (C) Base-editing strategy for introduction of a premature stop codon in E2F7 and E2F8 open reading frames with Sanger sequencing chromatographs of wildtype and mutant alleles, confirming homozygous base changes (highlighted in red) in E2F7Q206X and E2F8Q462X Hep-Org 1 lines. Protospacer adjacent motif (PAM) sequences are highlighted in blue. (D) Representative brightfield images of wildtype, E2F7Q206X, and E2F8Q462X Hep-Org 1 lines in control medium and after 3 days of CHIR99021 removal. Scale bars represent 50 µm. (E) Relative expression of E2F7 or E2F8 in wildtype and mutant lines as measured by RT-qPCR in control medium and after 3 days of CHIR99021 removal (N = 3 experiments, ns = not significant, *P < 0.05, **P < 0.01, Student’s t test, two-tailed). Error bars represent standard deviation. (F) Percentage of binucleated cells in wildtype and mutant lines expressing GFP-NLS and stained with CellMask Orange in control medium and after 3 days of CHIR99021 removal. Each dot represents the average percentage of binucleated cells per experiment (N = 3 experiments, 200–300 cells analyzed per experiment, ns = not significant, *P < 0.05, **P < 0.01, Student’s t test, two-tailed). (G) Representative images of Hep-Org 1 line expressing GFP-NLS (green) and E-cadherin-tdTomato (magenta) grown in control medium and after 3 days of CHIR99021 removal. Scale bars represent 50 µm. (H) Representative images of Hep-Org 1 line expressing E-cadherin-tdTomato (magenta) and stained with DRAQ5 (gray) to visualize DNA, after growth for 7 days in either control medium or differentiation medium (DM). Scale bars represent 50 µm. (I) Percentage of binucleated cells per organoid in Hep-Org 1 line expressing E-cadherin-tdTomato and stained with DRAQ5 after growth for 7 days in either control medium (n = 84 organoids) or differentiation medium (DM, n = 83 organoids). Each dot represents the percentage of binucleated cells per organoid, and the black line represents the median (N = 2 experiments, 73–93 organoids analyzed per experiment, ns = not significant, Mann–Whitney test). (J) Percentage of binucleated cells per organoid in Hep-Org 1 line expressing GFP-NLS/E-cadherin-tdTomato grown for 1 day in control medium (n = 21 organoids) or in medium supplemented with insulin (n = 37 organoids). The black line represents the median. See material and methods section for more information on media composition (N = 2 experiments, ns = not significant, Mann–Whitney test). (K) Percentage of binucleated cells per organoid in Hep-Org 1 line expressing GFP-NLS/E-cadherin-tdTomato grown for 7 days in control medium (n = 15 organoids) or in medium without insulin (n = 19 organoids). The black line represents the median. See Materials and methods section for more information on media composition (N = 2 experiments, ns = not significant, Mann–Whitney test).

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