The Nucleocapsid Protein of SARS-CoV-2 Abolished Pluripotency in Human Induced Pluripotent Stem Cells

  1. Zebin Lin
  2. Zhiming Wu
  3. Jinlian Mai
  4. Lishi Zhou
  5. Yu Qian
  6. Tian Cai
  7. Zhenhua Chen
  8. Ping Wang
  9. Bin Lin

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Abstract

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging across the world, leading to a global mortality rate of 3.4% (estimated by World Health Organization in March 2020). As a potential vaccine and therapeutic target, the nucleocapsid protein of SARS-CoV-2 (nCoVN) functions in packaging the viral genome and viral self-assembly. To investigate the biological effects of nCoVN to human stem cells, genetically engineered human induced pluripotent stem cells (iPSC) expressing nCoVN (iPSC-nCoVN) were generated by lentiviral expression systems, in which the expression of nCoVN could be induced by the doxycycline. The proliferation rate of iPSC-nCoVN was decreased. Unexpectedly, the morphology of iPSC started to change after nCoVN expression for 7 days. The pluripotency marker TRA-1-81 were not detectable in iPSC-nCoVN after a four-day induction. Meanwhile, iPSC-nCoVN lost the ability for differentiation into cardiomyocytes with a routine differentiation protocol. The RNA-seq data of iPSC-nCoVN (induction for 30 days) and immunofluorescence assays illustrated that iPSC-nCoVN were turning to fibroblast-like cells. Our data suggested that nCoVN disrupted the pluripotent properties of iPSC and turned them into other types of cells, which provided a new insight to the pathogenic mechanism of SARS-CoV-2.

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    SciScore for 10.1101/2020.03.26.010694: (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.

    Table 2: Resources

    Antibodies
    SentencesResources
    These primary antibodies were [target, dilution, species, company, product number]: Troponin T Cardiac Isoform, 1:100, mouse, Thermo Fisher, MA5-12960; alpha-smooth muscle actin, 1:100, mouse, Bioss, bsm-33187M; S100A4, 1:100, rabbit, Bioss, bs-3759R; SSEA4, 1:250, mouse, Invitrogen, 14-8843-80;
    MA5-12960; alpha-smooth muscle actin
    suggested: None
    S100A4
    suggested: (LSBio (LifeSpan Cat# LS-C10045-250, RRID:AB_800396)
    SSEA4
    suggested: (Thermo Fisher Scientific Cat# 14-8843-80, RRID:AB_657847)
    Cells were washed three times with PBS containing 0.1% Triton X-100, then incubated with the Alexa Fluor 488 goat anti-mouse or Alexa Fluor 555 goat anti-rabbit IgG secondary antibodies at 37°C for 1 hour.
    anti-mouse
    suggested: None
    anti-rabbit IgG
    suggested: None
    Software and Algorithms
    SentencesResources
    The data were analyzed and plotted using GraphPad Prism 6. 2.5 Immunofluorescence Staining: Cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes and washed three times with 1× PBS.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Images were obtained by using the DMi6000 B inverted microscope (Leica) or the FV1000 confocal laser scanning microscope (Olympus), then were analyzed by using ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    We employed Kallisto (v0.46.0) (Bray et al., 2016) to determine the read count for each transcript and quantified transcript abundance as transcripts per kilobase per million reads mapped (TPM), using gene annotation in the GENCODE database (v32, GRCh38) (Frankish et al., 2019).
    Kallisto
    suggested: (kallisto, RRID:SCR_016582)
    GENCODE
    suggested: (GENCODE, RRID:SCR_014966)
    DESeq2 (v1.26.0) (Love et al., 2014) was used to identify differentially expressed genes (DEGs) (false discovery rate (FDR) <0.05 and abs(log2FoldChange) >3).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    The pathway analysis was performed by ToppGene Suite (Chen et al., 2009).
    ToppGene
    suggested: ( ToppGene Suite , RRID:SCR_005726)

    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: 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

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  2. Version published to 10.1101/2020.03.26.010694v3 on bioRxiv
  3. Version published to 10.1101/2020.03.26.010694v2 on bioRxiv
  4. Version published to 10.1101/2020.03.26.010694v1 on bioRxiv