Abstract
BACKGROUND & AIMS: Intercellular orchestration across hepatic and immune lineages governs critical liver development and disease processes. Existing in vitro human liver models limit immune lineage outputs partly due to the lack of an endogenous niche for blood co-development. By modeling a developmental niche, we developed human fetal liver-like organoids (FLOs) harboring a multipotent hematopoietic system to model and study the complex multilineage interaction in liver development and injury. METHODS: We generated FLOs from human pluripotent stem cells to study cross-lineage self-organization by co-developing a hemogenic mesoderm and hepatic endoderm. Hematopoietic progenitor cell and epithelial lineage potential and dynamics were assessed through single-cell and bulk transcriptomics, immunophenotyping, and functional assays. RESULTS: FLOs established a bona fide niche that supports the simultaneous emergence of hepatobiliary, endothelial, and mesenchymal lineages, along with multipotent hematopoietic progenitor cells exhibiting predominant myeloid lineage commitment while retaining the potential for fetal B and T cell differentiation. Within the FLO microenvironment, hepatic and hematopoietic lineages continued to mature in the absence of extrinsic differentiation factors. Enriching hematopoietic derivatives with a small molecule-cytokine cocktail generated divergent immune cell populations, including granulocytes and polarized macrophages equipped with immunoreactive and lineage-specific function. Finally, FLOs mechanistically revealed an IL-8-mediated neutrophil-driven injury response upon steatotic-lipotoxic injury, highlighting key liver-intrinsic immune mechanisms. CONCLUSIONS: FLOs provide a physiologically relevant model for multi-lineage hemato-hepatogenesis and innate immunity in the liver. By recapitulating critical fetal liver tissue functions, FLOs offer a translational platform for studying human fetal liver development, immune-mediated hepatic injury, and regenerative therapies. IMPACT AND IMPLICATIONS: This study establishes a human pluripotent stem cell-derived fetal liver-like organoid (FLO) system that reconstitutes hepato-hematopoietic co-development and innate immune function. FLOs offer a human platform to dissect cross-lineage signaling during liver development and immune-mediated injury. Clinically and translationally, FLOs enable mechanistic modeling of pediatric liver diseases and regenerative interventions, offering a tractable in vitro system for preclinical screening and precision-medicine approaches.