Extended Data Fig. 4: Spontaneous formation of non-physiological hepatocyte-cholangiocyte structures and identification of assembloid co-culture media.
From: Mouse liver assembloids model periportal architecture and biliary fibrosis
a. Brightfield and immunofluorescence images of chimeric hepatocyte-cholangiocyte (Hep-Chol) organoid, where cholangiocyte organoids (CholOrg) spontaneously emerge in the vicinity of hepatocyte organoids (HepOrg). F-actin (Phall, grey), and nuclei (DAPI, cyan). Note the non-physiological ratio between both cell types (compare to Fig. 1a). n > 3 independent experiments. Scale bars, brightfield −100 µm, IF − 50 µm, zoom-in 10 µm. b. Immunofluorescence (IF) staining for CD13 (BC, green) and SOX9 (cholangiocytes, magenta) in chimeric Hep-Chol organoids. DAPI (nuclei, cyan) and Phalloidin (membrane, grey). Note the lack of connection between the bile canaliculi from hepatocytes and bile duct, despite the proximity of both structures. n > 3 independent experiments. Scale bar, 20 µm, zoom-in, 10 µm. c. Left, schematic of experimental approach (reproduced from ref. 20 (CC BY 4.0)). Right, representative images (n = 3 independent experiments) of seeded hepatocytes after isolation from a tamoxifen-injected Prom1-CreERT2 x R26-LSL-ZsGreen mouse livers after 14 days wash-out period. Note the presence of small ZsGreen-labelled cells (arrowhead, left picture), which then expand into ZsGreen-labelled cholangiocyte organoids (arrowhead, right picture). Scale bars, left 200 µm, right 500 µm. d. Sorting strategy to identify how many cholangiocytes are labelled by ZsGreen in our experiments. Representative plots of n > 2 biological replicates are shown. e. Percentage of recombined cholangiocytes from ‘c’. The % of labelled cells is similar between day 0 and day 7 of culture, suggesting that the CholOrg derived from contaminating cholangiocytes in the hepatocyte isolation prep. Graph represents mean (d0 n = 3, d7 n = 2) of 2 independent experiments. f-j. Co-culture media test to obtain medium that prevented cholangiocyte and mesenchyme overgrowth, while preserving hepatocyte polarity and bile canaliculi structure. f. Representative HepOrg brightfield images at passage 1, cultured in HM-Wnt, and switched to specified media for 7 days. Scale bar, 100 µm, zoom-in, 50 µm. g. HepOrg stained with a live/dead cell dye as detailed in methods; culture at passage 1 switched to specified media for 7 days. Scale bar, 50 µm. h. Ratio of live to dead dye intensity, corresponding to pictures from g. Data is presented as box (the interquartile range, 25th and 75th percentile, with line at median) and whiskers (min, max of the data) plot from HM-Wnt n = 5; DM n = 4; MM n = 5 biological replicates. Paired t test, two-tailed. i. qRT-PCR expression analysis of selected marker genes in HM+Wnt (n = 3), MM (n = 3) and DM (n = 2) media; Graph represents mean, with ± SEM when n = 3. Freshly isolated hepatocytes (n = 2) and CholOrg (n = 1) controls are also included. j. Representative images (n = 2 independent experiments) of cholangiocyte organoids (CholOrg) and portal mesenchymal cells (Msc Pdgfra+Sca1+) monoculture, cultured in MM (top) or HM-Wnt (bottom) and stained with a live/dead fluorescent cell dye. k-l. Total bile acid (k) and albumin (l) production from HepOrg grown in HM-Wnt or MM. Data is presented as mean +/- SD from n = 4 replicates from n = 4 independent experiments. Results are expressed as µM concentration (k) or ng/ml (l) normalised to total cell number per condition; scale bar, 50 µm. m. Number of nuclei per cell in HepOrg grown in specified medium, n = 3.
