Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids.
Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids.
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DOI：
10.1016/j.cels.2020.11.002
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发表时间：
2021-01-20
期刊：
影响因子：
9.3
通讯作者：
Ebrahimkhani MR
中科院分区：
文献类型：
--
作者：
Velazquez JJ;LeGraw R;Moghadam F;Tan Y;Kilbourne J;Maggiore JC;Hislop J;Liu S;Cats D;Chuva de Sousa Lopes SM;Plaisier C;Cahan P;Kiani S;Ebrahimkhani MR
多能干细胞（PSC）衍生的类器官已成为人类组织发育的新型多细胞模型，但显示出不成熟的表型、异常的组织命运和有限的细胞亚群。在这里，我们证明了PSC衍生的多谱系人类肝脏类器官中基因调控网络（GRNs）的综合分析和工程化在体外直接成熟和血管形态发生。PROX 1和ATF 5的过表达，结合内源性CYP 3A 4的靶向CRISPR转录激活，重新编程组织GRNs并改善天然肝功能，如FXR信号传导，CYP 3A 4酶活性和基质细胞反应性。与其他PSC衍生的肝类器官相比，工程化组织具有上级肝特性，并在单细胞RNA-seq分析中显示存在肝细胞、胆管、内皮和星状细胞群。最后，它们在体内研究时显示肝功能。总的来说，我们的方法提供了一个实验框架，通过系统地探测促进组织发育的分子通路和转录网络来指导体外器官发生。根据计算分析，Velazquez等人进行了ATF 5、PROX 1转录因子的过表达和CRISPR介导的CYP 3A 4激活，以促进体外人iPSC衍生的胎肝类器官的成熟和血管分布。这些发现强调了整合系统和合成生物学对工程多细胞系统的重要性。
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. 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.
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