Fig. 3: In vitro and in vivo functionality characterization of BVLO.

A 3D imaging demonstrates BVLO tubular structure. Scale bar: 100 μm. B TEM image of BVLO at varying magnifications. Scale bar: 50 μm, 10 μm, and 1 μm. Experiments were repeated independently two times with similar results. C Immunostaining of MDR1. Scale bar: 50 mm. Experiments were repeated independently two times with similar results. D Rho123 transport in hiPSC-BD without and with hiPSC-BV and the Verapamil. Scale bars: 50 μm. Experiments were repeated independently two times with similar results. E Immunostaining after Rho123 incubation showing luminal accumulation of Rho123 in the absence of verapamil. Scale bar: 50 μm. Experiments were repeated independently three times with similar results. F Immunostaining of CFTR. Scale bar: 100 μm. Experiments were repeated independently three times with similar results. G Immunofluorescence pre- and post-forskolin treatment. Scale bar: 50 μm. H Quantification of lumen area pre- and post-forskolin (Two-tailed Mann-Whitney test, Statistical significance ****P-value < 0.0001). Lumen area for BVLO with Forskolin (n = 30) and without (n = 26). Error bars represent SEM. I GGT activity assay of BVLO (Two-tailed Mann-Whitney test, Statistical significance **P-value of 0.0093). GGT activity of LO (n = 7) and BVLO (n = 8) was determined. Error bars represented SEM. J Alkaline phosphatase activity of hiPSC-BD after BCIP/NBT staining. Scale bar: 50 μm. Experiments were repeated independently more than three times with similar results, K Schematic overview of liver transplantation procedure. L Histological analysis of liver specimen rom NOG mice post-transplantation, H&E staining. Scale bar: 100 µm. Experiments were repeated independently more than three times with similar results. M Immunofluorescence analysis of LO, or BVLO post-transplantation. Cholangiocyte (CK19, SOX9, CK7, Ac. Tub, hOPN), epithelial junction and polarity markers (βCAT, RDX), human cell marker (Ku80) and a hepatocyte marker (HNF4A). Scale bar: 50 μm. Experiments were repeated independently more than three times with similar results. N Schematic illustration of carbon ink injection process from EHBD post-transplantation. O Bright field and fluorescence of carbon ink injection showing hCK7⁺ human and mOPN⁺ mouse BDs in the boundary transplanted area and the recipient area. Scale bar: 100 μm. Experiments were repeated independently more than three times with similar results.