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

Abstract

SUMMARY 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.

Synthetic living machines: A new window on life

“…For instance, inducible and transient expression of GATA6 transcription factor was used to trigger symmetry breaking, co-development of endoderm and mesoderm germ layers, and formation of human fetal liver bud ( Figure 3 D) ( Guye et al., 2016 ). In a follow-up study, multilineage maturation was also achieved through the synthetic control of GRNs ( Velazquez et al., 2020 ). Recent studies have also used orthogonal differentiation of genetically engineered stem cells to produce multicellular tissues such as vascularized brain organoids ( Skylar-Scott et al, 2020 ).…”

“…To implement this approach in multilineage liver tissue development, Velazquez et al. have recently investigated whether a set of drivers can be identified and engineered to direct fetal liver organoids across developmental trajectory and toward adult human livers ( Figure 5 ) ( Velazquez et al., 2020 ). They used a machine learning-based algorithms ( Cahan et al., 2014 ; Roost et al., 2015 ) to quantitatively compare starting and target GRNs to predict a set of genetic targets ( Figure 5 A).…”

“…Genetic circuits to program cell and tissue fates are predicted, engineered, and delivered to synthetic tissue to program developmental trajectories and cellular interactions. (B and C) (B) Example of genetic circuits enabled directing development of liver organoids from pluripotent stem cells; (C) self-vascularized human liver organoids generated via genetic design and engineering (taken with permission from Velazquez et al., 2020 ). Part of this figure is created using BioRender ( BioRender.com ).…”

Novel synthetic biology approaches for developmental systems

“…In a multicellular context, one of the first examples of organoid protocols improved by genetic engineering showed that complex liver organoids could be generated by overexpression of Gata6 transcription factors in a non-uniform fashion in a population of stem cells (reviewed also in Ebrahimkhani and Ebisuya, 2019 ; Guye et al., 2016 ; Johnson et al., 2017 ). In a follow-up study, informed by computational analyses to identify key factors, genetic manipulation of human induced pluripotent stem cells (hiPSCs) improved the maturity and vascularity of fetal liver organoids in vitro ( Velazquez et al, 2021 ). Interestingly, the overexpression of a factor from the same family, Gata4, has been used in an embryo model system to generate a more faithful multi-lineage egg-cylinder model (see more detail in section “ challenges and opportunities in applying synthetic developmental biology to embryo models ”).…”

Recent Advances and Future Prospects in Immune Checkpoint (ICI)-Based Combination Therapy for Advanced HCC

“…Patient-derived xenografts are missing human immune cells that play key roles in ICI [101]. Recent progress in HCC organoids has advanced our understanding of drug screening on the tumor [102]. A study testing drug screen compounds identified an ERK inhibitor, SCH772984, as a potential therapeutic agent for patients with primary HCC [103].…”