Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids

Pluripotent stem cell (PSC)-derived organoids have emerged as novel multicellular models of human tissue development but display immature phenotypes, aberrant tissue fates, and a limited subset of cells. Here, we demonstrate that integrated analysis and engineering of gene regulatory networks (GRNs) in PSC-derived multilineage human liver organoids direct maturation and vascular morphogenesis in vitro. Overexpression of PROX1 and ATF5, combined with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, reprograms tissue GRNs and improves native liver functions, such as FXR signaling, CYP3A4 enzymatic activity, and stromal cell reactivity. The engineered tissues possess superior liver identity when compared with other PSC-derived liver organoids and show the presence of hepatocyte, biliary, endothelial, and stellate-like cell populations in single-cell RNA-seq analysis. Finally, they show hepatic functions when studied in vivo. Collectively, our approach provides an experimental framework to direct organogenesis in vitro by systematically probing molecular pathways and transcriptional networks that promote tissue development. [Display omitted] •Analysis of gene regulatory network identifies maturation transcription factors•Advancement of maturation and vascularity via synthetic gene circuits and CRISPRa•Computational analyses and benchmarking against human organoids and livers•Modeling of hepatic functions in vitro and in vivo Informed by computational analyses, Velazquez et al, exercised overexpression of ATF5, PROX1 transcription factors, and CRISPR-mediated activation of CYP3A4 to advance the maturity and vascularity of human iPSC-derived fetal liver organoids in vitro. The findings highlight the importance of integrative systems and synthetic biology to engineer multicellular systems.