Multilineage communication regulates human liver bud self-organization from pluripotency II

Conventional 2-D differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. 3-D organoids generate complex organ-like tissues, however it is unclear how heterotypic interactions impact lineage identity. Here we use single-cell RNA-seq to reconstruct hepatocyte-like lineage progression from pluripotency in 2-D culture. We then derive 3-D liver bud (LB) organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during LB development. We find that LB hepatoblasts diverge from the 2-D lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark 3-D LBs against fetal and adult human liver scRNA-seq data, and find a striking correspondence between the 3-D LB and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signaling in LBs, and show that VEGF crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.