Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering

SUMMARY Pluripotent stem cell (PSC)-derived organoids are emerging as novel human-based microphysiological models but display immature phenotypes with limited subsets of endothelial or stromal cells. Here we demonstrate that in vitro manipulation of gene regulatory networks (GRNs) in PSC-derived liver organoids selected either through computational analysis or targeted tissue design can advance tissue maturation in vitro . Through an unbiased comparison with the genetic signature of mature livers, we identify downregulated GRNs in fetal liver organoids compared to adult livers. We demonstrate that overexpression of PROX1 and ATF5 , together with targeted CRISPR-based transcriptional activation of endogenous CYP3A4 , drives maturation in vitro . Single cell analyses reveal hepatobiliary-, endothelial-, and stellate-like cell populations. The engineered organoids demonstrate enhanced vasculogenesis, capture native liver characteristics (e.g. FXR signaling, CYP3A4 activity), and exhibit therapeutic potential in mice. Collectively, our approach provides a genetically guided framework for engineering developmentally advanced multilineage tissues from hiPSCs. HIGHLIGHTS In vitro tissue maturation via genetically encoded molecular programs Computational analysis to identify maturation transcription factors in liver organoids Promoting vascularization of organoids via genetically encoded molecular programs Single cell analysis of parenchymal and non-parenchymal cells Modeling of native liver functions and in vivo therapeutic potential Graphical Abstract