Generation of human bronchial organoids for SARS-CoV-2 research

  1. Tatsuya Suzuki
  2. Yumi Itoh
  3. Yusuke Sakai
  4. Akatsuki Saito
  5. Daisuke Okuzaki
  6. Daisuke Motooka
  7. Shohei Minami
  8. Takeshi Kobayashi
  9. Takuya Yamamoto
  10. Toru Okamoto
  11. Kazuo Takayama

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Abstract

Coronavirus disease 2019 (COVID-19) is a disease that causes fatal disorders including severe pneumonia. To develop a therapeutic drug for COVID-19, a model that can reproduce the viral life cycle and evaluate the drug efficacy of anti-viral drugs is essential. In this study, we established a method to generate human bronchial organoids (hBO) from commercially available cryopreserved human bronchial epithelial cells and examined whether they could be used as a model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research. Our hBO contain basal, club, ciliated, and goblet cells. Angiotensin-converting enzyme 2 (ACE2), which is a receptor for SARS-CoV-2, and transmembrane serine proteinase 2 (TMPRSS2), which is an essential serine protease for priming spike (S) protein of SARS-CoV-2, were highly expressed. After SARS-CoV-2 infection, not only the intracellular viral genome, but also progeny virus, cytotoxicity, pyknotic cells, and moderate increases of the type I interferon signal could be observed. Treatment with camostat, an inhibitor of TMPRSS2, reduced the viral copy number to 2% of the control group. Furthermore, the gene expression profile in SARS-CoV-2-infected hBO was obtained by performing RNA-seq analysis. In conclusion, we succeeded in generating hBO that can be used for SARS-CoV-2 research and COVID-19 drug discovery.

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  1. ScreenIT

    SciScore for 10.1101/2020.05.25.115600: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    A549 culture: A549 cells were cultured with Ham’s F12 medium (Thermo Fisher Scientific) containing 10% FBS, 1×GlutaMAX (Thermo Fisher Scientific), and penicillin-streptomycin.
    A549
    suggested: None
    The virus was plaque-purified and propagated in Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    SARS-CoV-2 virus plaque assays: VeroE6/TMPRSS2 cells (JCRB1819, JCRB Cell Bank) 17 were seeded on 12 well plates (1.7×105 cells/well) and incubated for 24 hr.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    The trimmed reads were mapped to the human reference genome sequences (hg19) using HISAT2 ver 2.1.0.
    HISAT2
    suggested: (HISAT2, RRID:SCR_015530)
    The raw counts were calculated using featureCounts ver 2.0.0 and used for heatmap visualization with integrated differential expression and pathway analysis (iDEP, (http://ge-lab.org/idep/)) 18.
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    Access to raw data concerning this study was submitted under Gene Expression Omnibus (GEO) accession number GSE150819.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.05.25.115600 on bioRxiv