TY - JOUR T1 - Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering JF - bioRxiv DO - 10.1101/2020.05.10.087445 SP - 2020.05.10.087445 AU - Jeremy J. Velazquez AU - Ryan LeGraw AU - Farzaneh Moghadam AU - Yuqi Tan AU - Jacquelyn Kilbourne AU - Joshua Hislop AU - S Liu AU - Davy Cats AU - Susana M. Chuva de Sousa Lopes AU - Christopher Plaisier AU - Patrick Cahan AU - Samira Kiani AU - Mo R. Ebrahimkhani Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/05/10/2020.05.10.087445.abstract N2 - 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.HIGHLIGHTSIn vitro tissue maturation via genetically encoded molecular programsComputational analysis to identify maturation transcription factors in liver organoidsPromoting vascularization of organoids via genetically encoded molecular programsSingle cell analysis of parenchymal and non-parenchymal cellsModeling of native liver functions and in vivo therapeutic potentialCompeting Interest StatementM.R.E, S.K., P.C., J.J.V., and R.L. have submitted a patent (WO2019237124) for the work included in this publication. ER -