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. 2023 Sep 1;55(9):2005–2024. doi: 10.1038/s12276-023-01074-1

Fig. 5. Liver organoids are de novo vascularized.

Fig. 5

A Max projection and cross section of whole mount immunostained organoids showing overlapping populations expressing one or both of the endothelial markers CD54 and CD31. Cross-section reveals stronger CD54 expression toward the outer surface contrasted with stronger CD31 expression toward the center of the organoid. Additionally, visible are multiple conjoined luminal spaces bounded by the positive cells. Scale bar, 100 μm; the highlighted area is magnified in Supplementary Fig. 9. B Immunofluorescence of CD31-expressing endothelial cells (green) under high magnification. Plane image and 3D volume rendering are shown. Hoechst 33342 (blue) dye was used to counterstain nuclei. Right panels show 3D volume rendering and a plane image of the selected region. Insert, visualization of vessel cross-section. C Immunostaining of a 50 μm cryosection showing adjacent and overlapping expression of CD34 and CD31 in a small structure indicative of neo-vascularization. Scale bar 50 μm, the highlighted area is magnified in Supplementary Fig. 9. D Whole mount staining of LYVE1, a sinusoidal endothelial marker, showing positive cells demarking a diversity of lumen shapes and sizes. Scale bar, 100 μm. E Immunostaining of two 50 μm thick cryosections showing co-localization of liver endothelial-associated FVIII and endothelial cell marker CD31 and luminal spaces in the organoids. Scale bar, 100 μm; the highlighted area is magnified in Supplementary Fig. 9. F Immunofluorescence analysis of AcLDL-488 and FSA-FITC binding and uptake in CD54+ endothelial cells within the organoids. Scale bar 10 μm, highlighted area is magnified in Supplementary Fig. 9. All of the above experiments were performed with the hiPSC line AG27 on organoids from Day 20 to Day 27.