Extended Data Fig. 9: Periportal assembloids recapitulate in vivo tissue architecture and functional connection between BC from hepatocytes and bile duct cells.

a. Schematic representation of the interface between hepatocyte bile canaliculi (BC) and lumen of cholangiocyte bile duct (BD). b. Immunofluorescence staining and image reconstruction of hepatocyte-bile duct interface in assembloid (top panel), and liver tissue (bottom panel). Representative images show how the bile canaliculi (CD13, green) from hepatocytes enters into the lumen of bile duct (nuc-tdTomato, PCK, magenta). Right, 3D reconstruction where hepatocytes (red, yellow, cyan) whose bile canaliculi (green) enter the bile duct lumen (magenta) are visualised. Nuclei are counterstained with DAPI (grey). n = 5 biological experiments. Scale bar, 10 µm. c. Schematic of the imaging set up to visualise integration of the BC-BD connection with portal Msc cells. d. Immunofluorescence image of one assembloid containing three physical connections (BC:BD_1, BC:BD_2, BC:BD_3) between hepatocytes and bile duct. Connections are visualized using zonula occludens (ZO-1, green). Nuclei are counterstained with DAPI (blue). n = 6 independent experiments. Scale bar, 50 µm; zoom-in,10 µm. e. Consecutive sections of assembloids containing hepatocytes, cholangiocytes (nuc-tdTom-Chol, magenta) and portal mesenchyme (PDGFRα-H2BGFP-Msc, yellow). Left-to-right, ascending Z-stacks show 2 physical connections between bile canaliculi (CD13, green) from hepatocytes and a single bile duct lumen. Note that portal mesenchyme cells (yellow) are located in close proximity to cholangiocytes (magenta), recapitulating the architectural arrangement of the in vivo tissue (compare to Fig. 1a). Representative image of an entire assembloid (top), and detail (bottom). Nuclei is stained with DAPI (blue) and F-actin with Phalloidin (Phall, grey). n = 3 biological experiments. Scale bar, 50 µm; zoom-in, 10 µm. f. Representative immunofluorescence image (n > 3) of an assembloid containing hepatocytes, cholangiocytes (mem-tdTomato, magenta), and portal mesenchyme (PDGFRα-H2BGFP, green). Zoom-in, detail of two independent connections (1,2) between bile ducts and bile canaliculi (BC). Right, consecutive Z-stacks of Zoom-in number 1 (left) and Zoom-in number 2 (right) showing the BC entering the BD (Z-slice panels: (1) 0.79 µm, 3.95 µm and 5.53 µm; (2) 26.07 µm, 37.92 µm and 57.67 µm). Nuclei are counterstained with DAPI (blue). Scale bar, 50 µm; zoom-in,10 µm. g. Maximum intensity projection (MIP) of an assembloid with two bile duct structures, one embedded in the assembloid and connecting to the bile canaliculi network (A, yellow arrowhead) and one outside of the assembloid and not connected to the bile canaliculi (B, cyan arrowhead). Bile canaliculi (CD13, green), actin (Phalloidin, grey), nuclei (DAPI, blue) are stained, and Msc and cholangiocytes are visualised using the endogenous fluorescence from PDGFRα-H2BGFP (yellow) and nuc-tdTom (magenta), respectively. n = 3 independent experiments with n = 10 total biological replicates. Scale bar, 50 µm; zoom-in, 10 µm. h. Live imaging analysis of the uptake and flow of bile acid analogue (CLF, Fire LUT) from bile canaliculi into the lumen of bile ducts shows functional bile canaliculi-bile duct connection, n = 3. Scale bar, 50 µm. i. CLF (Fire LUT) uptake is not observed in structures with aberrant architecture where cholangiocytes (magenta, mem-tdTomato, magenta arrowhead) are not embedded in the organoid (white arrowhead), n = 3. Scale bar, 50 µm; zoom-in,20 µm. j-k. Dot plots of scRNA gene expression for bile acid transporters (j) and cholangiocyte apical markers (k) indicate that hepatocytes (j) and cholangiocytes (k) functional marker expression are improved upon assembloid culture.