Extended Data Fig. 2: The starting number of initiating cells dictates HepOrg morphology.
From: Mouse liver assembloids model periportal architecture and biliary fibrosis
a. Maximum intensity projection of images from live imaging of membrane-tdTomato HepOrg seeded as >2 hepatocyte cell clusters (top) or as single cells (bottom). Seeding density, 200 cells/μl from n = 2 independent experiments. Scale bar, 50 µm. b-d. DETECT calculations on HepOrg morphological variation integrating all time points (d0-d13). (b) t-SNE plot visualizes the DETECT results. Dot, individual organoid coloured according to shape type. (c) Heatmap of the DETECT metric distance for the DETECT results of each HepOrg pair integrating all time points (d0-d13). (d) K-means clustering applied to DETECT calculations following PCA reduction to two principal components. Dot, individual organoid; crosses, centroid of the respective clusters; colour, cluster assignment for the organoid. e-f. Analysis of the HepOrg shape according to the number of cells originating the structure. e, Schematic illustration. f, Bar graph shows the shape-type according to the initial number of cells in the structure. Results are presented as percentage from a total of n = 30 structures per experiment and expressed as mean ± SEM from n = 2 independent experiments. g. qRT-PCR expression analysis of the indicated markers in HepOrg (passage 2) hand-picked according to their ball-shape or bubbly/grape-shape morphology. Graph represents the mean ± SEM from n = 5 independent experiments for most genes, aside from Cyp2e1, tested n = 1 for ball-shape and n = 2 for bubbly-shape. Each dot is a biological replicate. The differences between ball-shape and bubbly-shape are not significant (Mann-Whitney test). Chol, cholangiocyte; Hep, hepatocyte; BAtrans, bile canaliculi transport. h. Viability assay (CellTiter-Glo) performed on ball-shape and bubbly/grape-shape HepOrg. Graph represents mean ± SEM of n = 3 biological replicates from 3 independent experiments, with dot colour denoting each independent experiment; Mann-Whitney test, two-tailed. i-j. Albumin (i) and Cytochrome activity (j) measurements of ball-shape versus grape-like/bubbly-shape HepOrg show non-significant but marked reduction in functionality of ball-HepOrg. Graph represents mean ± SEM of n = 5 biological replicates from 3 independent experiments; Mann-Whitney test, two-tailed. k. Brightfield and immunofluorescence images of bubbly/grape-like-shape (top) and ball-shape (bottom) HepOrg, stained for apical polarity marker CD13 (green) and apoptosis marker (cleaved caspase 3, grey; also shown in Fire LUT for easy visualization). n = 3 independent experiments. Scale bar, 50 µm. l. Still images of live cell imaging analysis of bile acid analogue uptake (CLF, green in the middle panel, fire LUT in most right panel) in membrane-tdTomato HepOrg (mTom, magenta) with bubbly/grape-shape (left) or ball-shape (right). SiR-actin (grey) labels cell borders. Note that bubbly/grape-like organoids uptake and release CLF into their bile canaliculi while ball-shape organoids accumulate it in hepatocytes. n = 2 independent experiments. Left, schematic of experimental set up. Scale bar, 20 µm.
