Iwasawa, K.; Koike, H.; Reza, H. A.; Milton, Y.; Kishimoto, K.; Thorner, K.; Granitto, M.; Saiki, N.; Santangelo, C.; Glaser, K.; Kimura, M.; Bondoc, A.; Lim, H.-W.; Morimoto, M.; Iwafuchi, M.; Wells, J. M.; Zorn, A. M.; Takebe, T.

The embryonic development of the liver is initiated by the emergence of hepatoblasts, originating from the ventral foregut endoderm adjacent to the heart. Here, we identify and characterize a previously unrecognized population of early hepatoblasts at the ventroposterior part of the emerging liver bud, traced from Cdx2-positive endoderm progenitors, which we term primitive hepatoblasts. Mouse and human single-cell transcriptomics reveals the expression of both canonical hepatoblast markers TBX3, FGB, and KRT8/18 and primitive-specific mesenchymal markers ID3, VIM, and GATA4. Lineage tracing revealed the notable contribution up to 12.6% of LIV2+ hepatoblasts at E11.5 but diminishes in late fetal and postnatal development. Epigenetic and functional perturbation studies further uncover that primitive hepatoblast emergence is primed by WNT-suppression on RA-permissive CDX2+FOXA2+ progenitors. Furthermore, human pluripotent stem cell-derived primitive hepatoblast-like cells secrete pleiotrophin and midkine to amplify hepatoblast populations and develop epithelial-mesenchymal hybrid tissues in vivo. Our results provide a new framework for understanding lineage heterogeneity during early hepatogenesis and offer revised insights into strategies to model normal and abnormal liver development.