Fig. 4: Cystic spheroid formation from hiHepPC-derived cholangiocytes.

a Representative images of 3D cultures of HUVECs and hiHepPCs at day 7 and passage (P) 5 and those of adult human liver-derived cholangiocytes at P3 after initiation of 3D culture with Matrigel. Scale bars, 100 µm. b Co-immunofluorescence staining of E-CAD with EZRIN or CFTR, of CK19 with SOX9 or HNF1B, and of EpCAM with α-TUBULIN and immunofluorescence staining of ZO-1 with phalloidin staining of F-actin were conducted for hiHepPC spheroids. DNA was stained with DAPI. Scale bars, 50 µm. c Representative ultrastructural images of hiHepPC spheroids (n = 10 spheroids). Black arrowheads indicate intracellular tight junctional complexes. Scale bars, 10 µm (left panel) and 500 nm (right panel). d PCA was performed using CEL-seq2 data for HUVECs, hiHepPC monolayer cultures, hiHepPC spheroids, and human fetal cholangiocyte-derived spheroids. e GSEA of CEL-seq2 data for hiHepPC spheroids and HUVECs and of those for hiHepPC spheroids and hiHepPC monolayer cultures were performed using the set of top 100 genes specifically upregulated in human fetal cholangiocyte-derived spheroids. f Representative bright-field and fluorescence images showing uptake of rhodamine 123 (green) into the luminal space of hiHepPC spheroids and inhibition of rhodamine 123 transport after treating the spheroids with verapamil before the addition of rhodamine 123. Scale bars, 50 µm.