Detection of viral RNA fragments in human iPSC cardiomyocytes following treatment with extracellular vesicles from SARS-CoV-2 coding sequence overexpressing lung epithelial cells

  1. Youjeong Kwon
  2. Sarath Babu Nukala
  3. Shubhi Srivastava
  4. Hiroe Miyamoto
  5. Nur Izzah Ismail
  6. Jordan Jousma
  7. Jalees Rehman
  8. Sang-Bing Ong
  9. Won Hee Lee
  10. Sang-Ging Ong

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Abstract

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global pandemic. The prevalence/severity of COVID-19 is higher among patients with cardiovascular risk factors. Despite the expression of angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV-2 infection, in cardiomyocytes, there has been no conclusive evidence of direct viral infection although the presence of viral genome within COVID-19 patients’ hearts has been reported. Here, we overexpressed SARS-CoV-2 genes in A549 lung epithelial cells. We then isolated extracellular vesicles (EVs) and detected the presence of viral RNA within these EVs. We observed that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are receptive to these EVs, and viral genes were detectable in the cardiomyocytes. Accordingly, the uptake of viral RNA-harboring EVs led to an upregulation of inflammation-related genes in hiPSC-CMs. Thus, our findings indicate that SARS-CoV-2 RNA containing EVs represents an indirect route of viral RNA entry into cardiomyocytes.

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  1. Version published to 10.1186/s13287-020-02033-7
  2. Version published to 10.1101/2020.05.14.093583v3 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.05.14.093583: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: All procedures conformed to the UIC institutional review board-approved protocol.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies used include CD63 (Thermo Fisher, 10628D), CD81 (Thermo Fisher, 10630D), TSG101 (Thermo Fisher, MA123296), calnexin (Thermo Fisher, MA3027) and albumin (Proteintech, 16475-1-AP).
    CD63
    suggested: (Thermo Fisher Scientific Cat# 10628D, RRID:AB_2532983)
    CD81
    suggested: (Thermo Fisher Scientific Cat# 10630D, RRID:AB_2532984)
    TSG101
    suggested: (Thermo Fisher Scientific Cat# MA1-23296, RRID:AB_2208088)
    calnexin
    suggested: (Thermo Fisher Scientific Cat# MA3-027, RRID:AB_2069043)
    Experimental Models: Cell Lines
    SentencesResources
    Cell Culture: HEK293T cells were purchased from TakaraBio and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Thermo Fisher, 11995-065) supplemented with 10% serum.
    HEK293T
    suggested: None
    A549 cells were purchased from ATCC and cultured in Ham’s F-12K medium (Thermo Fisher, 21127022).
    A549
    suggested: None

    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: We detected the following sentences addressing limitations in the study:
    There are several limitations in our study. Although the precipitation method used in our study for isolating EVs leads to high recovery, it is associated with recovery of non-EV components such as proteins. However, our additional experiments including immuno-magnetic isolation of EVs, RNase/protease treatment and GW4869 treatment support the enrichment of viral genes within EVs. The ExoGlow dye used cannot fully distinguish among binding and internalization of EVs. It should also be noted that the viral genes used in our study are codon-optimized and expressed at a supraphysiological level. Further experiments are needed to validate if EVs are capable of transferring actual viral RNAs or virions at a physiological level, and if the transferred RNAs are biologically active. Overall, our results collectively demonstrated that lung epithelial cells expressing SARS-CoV-2 genes can secrete EVs containing viral RNA fragments that can be detected in cardiomyocytes suggesting an indirect route of viral RNA delivery into cardiac cells via EVs. Transfer of viral RNA via EVs should be considered when studying the widespread multi-organ effects of a SARS-CoV-2 infection that has been reported10, because it indicates that cells which do not express the SARS-CoV-2 receptor ACE2 might still be vulnerable via the uptake of EVs. Further work is needed to clarify whether the entry of SARS-CoV-2 RNA via EVs is sufficient to induce cell injury and inflammation.

    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.

  4. Version published to 10.1101/2020.05.14.093583v2 on bioRxiv
  5. Version published to 10.1101/2020.05.14.093583v1 on bioRxiv