Robust three-dimensional expansion of human adult alveolar stem cells and SARS-CoV-2 infection

  1. Jeonghwan Youk
  2. Taewoo Kim
  3. Kelly V. Evans
  4. Young-Il Jeong
  5. Yongsuk Hur
  6. Seon Pyo Hong
  7. Je Hyoung Kim
  8. Kijong Yi
  9. Su Yeon Kim
  10. Kwon Joong Na
  11. Thomas Bleazard
  12. Ho Min Kim
  13. Natasha Ivory
  14. Krishnaa T. Mahbubani
  15. Kourosh Saeb-Parsy
  16. Young Tae Kim
  17. Gou Young Koh
  18. Byeong-Sun Choi
  19. Young Seok Ju
  20. Joo-Hyeon Lee

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Abstract

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which is the cause of a present global pandemic, infects human lung alveolar cells (hACs). Characterising the pathogenesis is crucial for developing vaccines and therapeutics. However, the lack of models mirroring the cellular physiology and pathology of hACs limits the study. Here, we develop a feeder-free, long-term three-dimensional (3D) culture technique for human alveolar type 2 (hAT2) cells, and investigate infection response to SARS-CoV-2. By imaging-based analysis and single-cell transcriptome profiling, we reveal rapid viral replication and the increased expression of interferon-associated genes and pro-inflammatory genes in infected hAT2 cells, indicating robust endogenous innate immune response. Further tracing of viral mutations acquired during transmission identifies full infection of individual cells effectively from a single viral entry. Our study provides deep insights into the pathogenesis of SARS-CoV-2, and the application of long-term 3D hAT2 cultures as models for respiratory diseases.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.10.194498: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Appropriate Human Tissue Act (HTA) guidance was followed; For organoids used for viral infection and following analysis, human lung tissue was obtained from patients undergoing lobectomy surgery at Seoul National University Hospital (SNUH) with written informed consent from approval of the ethical committee (approval no. C-1809-137-975).
    IRB: Appropriate Human Tissue Act (HTA) guidance was followed; For organoids used for viral infection and following analysis, human lung tissue was obtained from patients undergoing lobectomy surgery at Seoul National University Hospital (SNUH) with written informed consent from approval of the ethical committee (approval no. C-1809-137-975).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were prepared for flow cytometry with primary antibodies CD31-APC (Biolegend, 303116), CD45-APC (Biolegend, 368512), EpCAM-FITC (Biolegend, 324204) and HTII-280-IgM (Terrace Biotech, TB-27AHT2-280) at 1:40 per 4 million cells for 30 min on ice.
    CD45-APC
    suggested: None
    EpCAM-FITC
    suggested: None
    Following permeabilisation, organoid sections were blocked for 1 hr in 5% normal donkey serum in PBS at RT, and incubated with primary antibody mixes overnight at 4 °C at the following dilutions; rabbit pro-SFTPC (1:500, Millipore, Ab3786), mouse anti-HTII-280 (1:500
    pro-SFTPC
    suggested: None
    anti-HTII-280
    suggested: None
    Antibodies were E-cadherin (R&D, AF748, 1:300), NP (Sino, 40143-MM05, 1:200)
    E-cadherin
    suggested: (R and D Systems Cat# AF748, RRID:AB_355568)
    AF748
    suggested: (R and D Systems Cat# AF748, RRID:AB_355568)
    Experimental Models: Cell Lines
    SentencesResources
    HTII-280− cells were cultured as with HTII-280+, although with a few minor differences.
    HTII-280−
    suggested: None
    After infection, remove infection media and wash the Vero cells with PBS two times, mixed agar and Modified Eagle’s Medium (Thermofisher) were poured on each well.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    Cell sorting was performed on an Aria III fusion (BD Biosciences) using a 100 μM nozzle, and data were analysed with FlowJo software (Tree Star, Inc.).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Single-cell RNA sequencing and data processing: Fastq file was aligned and each UMI count was calculated using Cell Ranger software provided by the manufacturer (10X Genomics).
    Cell Ranger
    suggested: (Cell Ranger , RRID:SCR_017344)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 32 and 26. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2020.07.10.194498 on bioRxiv