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Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering

Jeremy J. Velazquez, Ryan LeGraw, Farzaneh Moghadam, Yuqi Tan, Jacquelyn Kilbourne, Joshua Hislop, S Liu, Davy Cats, Susana M. Chuva de Sousa Lopes, Christopher Plaisier, Patrick Cahan, Samira Kiani, Mo R. Ebrahimkhani
doi: https://doi.org/10.1101/2020.05.10.087445
Jeremy J. Velazquez
1Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
3School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA
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Ryan LeGraw
1Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
3School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA
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Farzaneh Moghadam
1Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
3School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA
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Yuqi Tan
4Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA
5Institute for Cell Engineering Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Jacquelyn Kilbourne
6Biodesign Institute, Arizona State University, Tempe, AZ, USA
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Joshua Hislop
1Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
7Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA
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S Liu
1Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
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Davy Cats
8Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands
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Susana M. Chuva de Sousa Lopes
9Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
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Christopher Plaisier
3School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA
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Patrick Cahan
5Institute for Cell Engineering Johns Hopkins University School of Medicine, Baltimore, MD, USA
6Biodesign Institute, Arizona State University, Tempe, AZ, USA
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Samira Kiani
1Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
3School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA
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Mo R. Ebrahimkhani
1Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
3School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, USA
6Biodesign Institute, Arizona State University, Tempe, AZ, USA
7Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA
10Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine and Science, Phoenix, AZ, USA
11McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
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  • For correspondence: mo.ebr@pitt.edu
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SUMMARY

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.

HIGHLIGHTS

  • In vitro tissue maturation via genetically encoded molecular programs

  • Computational analysis to identify maturation transcription factors in liver organoids

  • Promoting vascularization of organoids via genetically encoded molecular programs

  • Single cell analysis of parenchymal and non-parenchymal cells

  • Modeling of native liver functions and in vivo therapeutic potential

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Competing Interest Statement

M.R.E, S.K., P.C., J.J.V., and R.L. have submitted a patent (WO2019237124) for the work included in this publication.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering
Jeremy J. Velazquez, Ryan LeGraw, Farzaneh Moghadam, Yuqi Tan, Jacquelyn Kilbourne, Joshua Hislop, S Liu, Davy Cats, Susana M. Chuva de Sousa Lopes, Christopher Plaisier, Patrick Cahan, Samira Kiani, Mo R. Ebrahimkhani
bioRxiv 2020.05.10.087445; doi: https://doi.org/10.1101/2020.05.10.087445
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Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering
Jeremy J. Velazquez, Ryan LeGraw, Farzaneh Moghadam, Yuqi Tan, Jacquelyn Kilbourne, Joshua Hislop, S Liu, Davy Cats, Susana M. Chuva de Sousa Lopes, Christopher Plaisier, Patrick Cahan, Samira Kiani, Mo R. Ebrahimkhani
bioRxiv 2020.05.10.087445; doi: https://doi.org/10.1101/2020.05.10.087445

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