Self-organization of sinusoidal vessels in pluripotent stem cell-derived human liver bud organoids

The induction of tissue-specific vessels in in vitro living tissue systems remains challenging. Here, we directly differentiated human pluripotent stem cells into CD32b⁺ putative liver sinusoidal progenitors by dictating developmental pathways. By devising an inverted multilayered air-liquid interface culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organize to generate and sustain hepatocyte-like cells neighboured by divergent endothelial subsets composed of CD32b^lowCD31^high, LYVE1⁺STAB1⁺CD32b^highCD31^lowTHBD^-vWF^- and LYVE1^-THBD⁺vWF⁺ cells. WNT2 mediates sinusoidal-to-hepatic intercellular crosstalk potentiating hepatocyte differentiation and branched endothelial network formation. Intravital imaging reveals the iPS-cell-derived putative liver sinusoidal endothelial progenitor develops fully perfused human vessels with functional sinusoid-like features. Organoid-derived hepatocyte- and sinusoid-derived coagulation factors enable correction of in vitro clotting time with Factor V-, VIII-, IX- and XI-deficient plasma, and rescues the severe bleeding phenotype in haemophilia A mice on transplantation. Advanced organoid vascularization technology allows for interrogating key insights governing organ-specific vessel development, paving the way for coagulation disorder therapeutics.