Extended Data Fig. 7: Generation and analysis of periportal assembloids.
From: Mouse liver assembloids model periportal architecture and biliary fibrosis
a. Representative immunofluorescence images of periportal assembloids generated using AggrewellTM method. Msc (PDGFRα-H2BGFP,green), cholangiocytes (nuclear-tdTom, magenta), nuclei (DAPI, blue) and F-actin (Phalloidin, grey) are shown. n = 3 independent experiments. Scale bar, 50 µm, zoom-in, 10 µm. b-c. Aggregation efficiency of periportal assembloids. b. Aggregation efficiency compared to all structures observed (regardless whether they contained one or more cell types). Pie chart represents the mean of n = 3 biological replicates from 3 independent experiment. Results are presented as % of a specific structure respective to the total number of structures. Periportal assembloid with 3 cell types chol, hep, Msc; HepOrg, hepatocyte organoids only; Chol-Org, cholangiocyte organoid only; Chol-Msc, cholangiocyte-Msc organoid. c. Aggregation efficiency comparing conditions where HepOrg had been pre-conditioned for 48hrs prior to aggregation with the co-culture medium MM (MM) or not (HM-Wnt). Graph represents mean n = 2 biological replicates. d. Two examples of assembloid formation. Still images from time-lapse imaging analysis of periportal assembloids composed of hepatocytes, mesenchyme (nuc-GFP, green) and cholangiocytes (mem-tdTomato, magenta). Nuclei are stained with SPY620 (grey). Scale bar, 100 µm, zoom-in, 20 µm. e. Aggregation mode representing assembly before or after seeding in Matrigel. Graph represents mean ± SEM of n = 3 biological replicates from n = 3 independent experiments. f. Representative confocal images (n = 2) of periportal assembloids stained for cholangiocyte (SOX9) and mesenchymal (vimentin) markers. Nuclei are stained with DAPI (blue). Scale bar, 30 µm. g. Representative confocal images of periportal assembloids stained for portal fibroblast marker Elastin (white) marker, in combination with PDGFRα-H2BGFP endogenous signal (GFP), and with Msc membranes visualised by membrane-tdTom. (n = 7 replicates from n = 3 independent experiments). Scale bar, 50 µm. h. Schematic representation of the experimental set up. Cultures were collected at day 7 after assembly and submitted for scRNAseq analysis. i. UMAP representing three biological replicates, each visualising the proportion of mesenchymal (green), cholangiocyte (magenta) and hepatocyte (blue) cells. j. UMAP combining 3 biological replicates of scRNAseq assembloid datasets. k-m. GSEA against GO_Biological_Process_2023 (Cyan), GO_Cellular_Component_2023 (Black) and GO_Molecular_Function_2023 (Red) databases for each of the cell types in assembloids: mesenchyme (k), cholangiocytes (l) and hepatocytes (m), vs the other 2 cell types. NES, normalized enrichment score.
