Extended Data Fig. 9: Hepatocytes, cholangiocytes and mesenchyme in assembloids express markers and establish cell-cell interactions similar to the in vivo portal tissue.
From: Human assembloids recapitulate periportal liver tissue in vitro
a, Immunofluorescence staining of day 4 periportal assembloids using the periportal markers SAA1 and SAA2 (yellow) and the epithelial and periportal enriched marker ECAD (white). Hepatocytes with positive (white arrow, region 1) and negative (magenta arrow, region 2) SAA1/SAA2 staining are shown. Left, robust expression of the portal markers SAA1 and SAA2 (top) and ECAD (bottom) in hepatocytes from assembloids. FIRE-LUT illustrates the different intensity of the corresponding markers. Right, co-localization of SAA1 and SAA2 staining with cholangiocytes (Chol-nGFP, green) and portal Mesenchyme (PFs-nRFP, magenta) highlights that the hepatocytes with strongest expression of portal markers SAA1 and SAA2 localize nearby cholangiocytes surrounded by portal fibroblasts (PFs-nRFP, magenta), confirming the periportal-like nature of assembloids. Portal identity is further contextualized by co-localization with ECAD (E-cadherin; epithelial marker) (bottom panels). Representative images from n = 2 samples from n = 1 donor. Scale bars, 50 µm. b, Mass spectrometry analysis was used to detect Norverapamil, the primary metabolite of Verapamil, in assembloids (6-day culture) and differentiated hepatocyte organoids (h-HepOrgs-DM). Unpaired two-tailed Student’s t-test with Welch’s correction, comparing biological replicates (n = 3). Values for h-HepOrgs correspond to the data in Fig. 3 and are shown here for comparison against assembloids. c, Immunofluorescent images showing the similarity between human assembloid (top) and human liver tissue (bottom). In assembloids, mesenchymal cells are labelled in magenta, Cholangiocytes labelled in green, F-actin and KRT19 are stained in green as well, and nuclei with DAPI (blue), n = 3 independent experiments. The assembloid image corresponds to the one presented in Fig. 5f where KRT19 in white is shown. In human liver tissue, staining was done for cholangiocytes (PCK, magenta), cell borders (F-actin, grey) and nuclei (DAPI, cyan), n = 3 independent experiments from n = 2 independent donors. Yellow arrowheads indicate the presence of ductal structures amidst the hepatocytes in both assembloids and tissue. Scale bar, 50 μm. d, Top, images showing magnified areas and maximum intensity projection of the assembloids (from panel c, top) with hepatocytes (DAPIpos, KRT19neg), cholangiocytes (KRT19pos, nuclei white), and the interface between the two cell-types - indicated by the white arrowhead, n = 3 independent experiments. The images are immunofluorescent images visualized in 3D to enable viewing from another angle. Bottom, immunofluorescent images showing magnified areas of human liver tissue (from panel c, bottom) with hepatocytes (PCKneg), cholangiocytes (PCKpos), and the interface between the two cell-types - indicated by the white arrowhead, n = 3 independent experiments from n = 2 biologically independent donors. Scale bar, 50 μm, 25 μm.
