Fig. 1: HepOrgs retain marker expression and cell polarity of tissue.
From: Mouse liver assembloids model periportal architecture and biliary fibrosis
a, Schematic (left) and representative immunofluorescence image (right; n = 5) of the liver periportal region. CD34 marks portal fibroblasts (PFs), osteopontin (OPN) marks ductal cells, phalloidin marks membranes and DAPI labels nuclei. Chol, cholangiocyte; Hep, hepatocyte; PV, portal vein. Scale bar, 10 µm. b, Representative bright-field images of HepOrgs at passage 1 (P1) cultured under the conditions described in Hu et al.15, Peng et al.16 or in HM-FBS, HM-Wnt or HM-WntS (n = 5 experiments). Scale bars: 500 µm (top row), 10 µm (bottom row). FBS, foetal bovine serum. c, Representative BF images of HepOrgs cultured in HM-Wnt at the indicated time points. Scale bars: 50 µm (day 9), 100 µm (day 49), 200 µm (day 203 and day 355). d, Immunofluorescence staining and 3D reconstruction of bile canaliculi (marked by CD13) in HepOrgs cultured in indicated conditions and mouse tissue. HepOrgs cultured in HM-Wnt have longer bile canaliculi compared with previous studies15,16. Cell borders are indicated by filamentous actin (F-actin) staining with phalloidin (Phall). Top, maximum-intensity projections of confocal images. HepOrgs are outlined with dashed lines. Bottom, bile canaliculi segmentation and 3D reconstruction. Scale bars, 50 µm. e, Number of triple junctions in the largest bile canaliculi network in tissue and organoids cultured in indicated conditions. Dots show the total number of triple junctions per structure and the line represents the mean. Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test. NS, not significant. f, Organoid formation efficiency. Dots represent biologically independent samples (n = 5 biological replicates with at least 2 technical replicates) and the line represents the mean. Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test. g, Immunofluorescence staining of tissue (left) and hepatocyte and cholangiocyte organoids (HepOrgs and CholOrg, respectively) grown in HM-Wnt (right), for the hepatocyte marker HNF4α and cholangiocyte markers KRT19 and OPN (n = 2 experiments). Scale bars: 50 µm (main images), 10 µm (smaller images). Phalloidin marks membranes and DAPI labels nuclei. h, Left, schematic showing hepatocyte polarity. CD13 (apical) and ECAD (basolateral) staining in tissue (middle) and organoids (right) shows similar distributions of markers. Yellow arrowheads indicate binucleated cells. Scale bars: 20 µm (left most images), 10 µm (expanded views). All data were obtained from n = 3–5 independent experiments.
