Fig. 4. Liver organoids contain the parenchymal cell types of the liver.

A Immunostaining showing the expression of various maturity- and polarity-associated hepatocyte markers in the outer most layer of the organoids (whole mount except for ASGR1/HNF4α which are from 50 μm thick cryosections). Scale bars are 100 μm, and the highlighted area is magnified in Supplementary Fig. 9. B Electron micrograph revealing ultrastructural features associated with hepatocytes, including epithelial cells lining a luminal structure arranged in one layer connected with tight junctions (circles) indicative of polarization. The surface facing the lumen contains numerous microvilli, whereas the abluminal surface facing the extracellular matrix remains smooth and attached to the underlying basal lamina (arrowheads). Scale bar 2 µm. C Immunohistochemical staining showing CK19-positive cells; the arrow denotes cholangiocytes surrounding the lumen. Scale bar is 200 µm. D Immunostaining of an organoid cryosection showing a later developmental cholangiocyte marker (CK7)-positive population of cells separate from the hepatocyte population. These form smaller ring-like structures as well as lining large luminal or cyst-like spaces within the organoids; scale bars are 100 µm. E Paraffin-embedded sections of organoids stained with alcian blue and counterstained with nuclear red. The lumen of this organoid shows cells containing pale blue cytoplasm, indicating the presence of mucopolysaccharides. Scale bar 50 µm. All of the above experiments were performed with the hiPSC line AG27.