Gradient-Based Regulation of Activin A Directs Multilineage Liver Organoid Development from Human Pluripotent Stem Cells

Morphogen gradients critically regulate the coordinated emergence of endodermal and mesodermal lineages during liver bud formation, yet current human hepatic organoid models inadequately replicate these spatial cues. Here we demonstrate that precise modulation of Activin A concentrations within a dynamic 3D vertical-wheel bioreactor system enables the co-emergence and spatial organization of hepatic epithelium and septum transversum mesenchyme-like populations from human pluripotent stem cells. High Activin levels induce uniform definitive endoderm and enhanced hepatic maturation, whereas low levels promote epithelial tube–like structures encased by mesenchymal cells, recapitulating early liver diverticulum architecture. This gradient-based approach autonomously generates parenchymal and non-parenchymal hepatic lineages, providing a scalable platform to model human liver organogenesis and engineer ventral foregut–derived tissues.