Human hepatocytes undergo canonical, endomitosis, and polyploid M phases in Hep-Orgs. (A) Representative images of Chol-Org and Hep-Org lines expressing GFP-NLS (green) and stained with CellMask Orange (magenta) to mark nuclei and membranes, respectively. Images show one plane in the center of each organoid. Examples of binucleated cells are indicated in the white squares. (B) Percentage of binucleated cells in Chol-Org and Hep-Org lines. Columns depict mean percentages (N = 3 experiments, 200–300 cells analyzed per experiment). (C) Percentage of binucleated cells per organoid plotted against organoid size (N = 3 experiments, 20–30 organoids analyzed per Hep-Org line). Each dot represents one organoid, with the mean depicted in column (ns = not significant, Student’s t test, two-tailed). (D) Stills from live imaging of GFP-NLS/E-cadherin-tdTomato Hep-Org 1 line showing canonical (top) and endomitosis (bottom) M phase. Time is relative to NEB in h:min. White asterisks mark daughter cells. (E) Percentage of types of M phases observed during live-imaging experiments of GFP-NLS/E-cad-tdTomato Hep-Org 1 line and Tubulin-mNeon Hep-Org 2 line (N = 5 experiments, >175 events analyzed per line). Error bars represent standard deviation. (F) Stills from live imaging of GFP-NLS/E-cadherin-tdTomato Hep-Org 1 line showing polyploid M phase. Time is relative to NEB in h:min. White asterisks mark daughter cells. (G) Percentage of polyploid M phases segregating their DNA content to two, three, or four daughter nuclei (N = 5 experiments, >7 events per line). Scale bars in A, D, and F represent 50 µm.