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

Cell Systems, vol. 12, pp. 41–55.e11

Abstract

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.

Authors 14

1. Jeremy J. Velazquez

   University of Pittsburgh · Arizona State University

   Affiliation as printed

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

   Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

   School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

2. Ryan LeGraw

   University of Pittsburgh · Arizona State University

   Affiliation as printed

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

   Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

   School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

3. Farzaneh Moghadam

   University of Pittsburgh · Arizona State University

   Affiliation as printed

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

   Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

   School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

4. Yuqi Tan

   Johns Hopkins University · Johns Hopkins Medicine

   Affiliation as printed

   Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Institute for Cell Engineering Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

   Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

   Institute for Cell Engineering Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

5. Jacquelyn A. Kilbourne

   Arizona State University

   Affiliation as printed

   Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA

6. Joseph C. Maggiore

   University of Pittsburgh

   Affiliation as printed

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

   Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

7. Joshua Hislop

   University of Pittsburgh

   Affiliation as printed

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA

   Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

   Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

8. Silvia Liu

   University of Pittsburgh

   Affiliation as printed

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

   Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

   Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

9. Davy Cats Aachen Department of Medical Statistics and Bioinformatics

   Leiden University Medical Center

   Affiliation as printed

   Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Einthovenweg, 2333 ZC Leiden, the Netherlands

10. Susana M. Chuva de Sousa Lopes Aachen Department of Anatomy and Embryology

    Leiden University Medical Center

    Affiliation as printed

    Department of Anatomy and Embryology, Leiden University Medical Center, Einthovenweg, 2333 ZC Leiden, the Netherlands

11. Christopher L. Plaisier

    Arizona State University

    Affiliation as printed

    School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

12. Patrick Cahan

    Johns Hopkins University · Johns Hopkins Medicine · Arizona State University

    Affiliation as printed

    Institute for Cell Engineering Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA

    Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA

    Institute for Cell Engineering Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

13. Samira Kiani

    University of Pittsburgh · Arizona State University

    Affiliation as printed

    Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

    Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

    Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

    School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA

14. Mo R. Ebrahimkhani corresponding

    Mayo Clinic · University of Pittsburgh · McGowan Institute for Regenerative Medicine · Arizona State University

    Affiliation as printed

    Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA; School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA; Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA; Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA; Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine and Science, Phoenix, AZ 85054, USA; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA. Electronic address: mo.ebr@pitt.edu

    Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA

    Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA

    Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

    Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine and Science, Phoenix, AZ 85054, USA

    McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA

    Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA

    School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85281, USA