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 (iLSEP) by dictating developmental pathways. By devising an inverted multilayered air-liquid interface (IMALI) culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organized to generate and sustain hepatocyte-like cells neighbored by divergent endothelial subsets composed of CD32b^lowCD31^high, LYVE1⁺STAB1⁺CD32b^highCD31^lowTHBD^-vWF^-, and LYVE1^-THBD⁺vWF⁺ cells. Wnt2 mediated sinusoidal-to-hepatic intercellular crosstalk potentiates hepatocyte differentiation and branched endothelial network formation. Intravital imaging revealed iLSEP developed fully patent human vessels with functional sinusoid-like features. Organoid-derived hepatocyte- and sinusoid-derived coagulation factors enabled correction of in vitro clotting time with Factor V, VIII, IX, and XI deficient patients' plasma and rescued the severe bleeding phenotype in hemophilia A mice upon transplantation. Advanced organoid vascularization technology allows for interrogating key insights governing organ-specific vessel development, paving the way for coagulation disorder therapeutics.