Project Junior Associate Professor Institute of Science Tokyo Fujisawa, Kanagawa, Japan
Abstract: Liver-specific capillary plexus runs between radiating hepatic cords and is comprised of liver sinusoidal endothelial cells (LSECs) which are the most potent cell type in coagulation factor VIII secretion and scavenger system. Recently, several directed differentiation protocols generated LSEC-like cells from pluripotent stem cells. However, the induction of three-dimensional liver-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 iLSEP-derived CD32+ LSEC. Single cell RNA-seq analysis including comparison with human liver and other organoid technologies uncovered in vivo relevant unique molecular profiles of divergent endothelial subsets composed of CD32blowCD31high, LYVE1+F8+CD32bhighCD31lowTHBD-, and LYVE1-THBD+ICAM1+ cells. WNT2 mediated sinusoidal-to-hepatic intercellular crosstalk potentiates hepatocyte differentiation and branched endothelial network formation. Intravital imaging revealed iLSEP developed fully perfused 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 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.
