Human embryonic stem cell-derived cardiomyocytes express SARS-CoV-2 host entry proteins: screen to identify inhibitors of infection

  1. Thomas L. Williams
  2. Maria T. Colzani
  3. Robyn G.C. Macrae
  4. Emma L. Robinson
  5. Stuart Bloor
  6. Edward J. D. Greenwood
  7. Jun Ru Zhan
  8. Gregory Strachan
  9. Rhoda E. Kuc
  10. Duuamene Nyimanu
  11. Janet J. Maguire
  12. Paul J. Lehner
  13. Sanjay Sinha
  14. Anthony P. Davenport

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Abstract

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2021.01.22.427737: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Slide Scanner Axio Scan.Z1 (Zeiss) Methods: Surgical samples of human tissue were obtained with informed consent from Royal Papworth Hospital Research Tissue Bank and ethical approval (05/Q104/142).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The primary antibody used was sheep anti-SARS-CoV-2 nucleocapsid antibody (DA114, MRC-PPU), which was visualised with AF488 conjugated donkey anti-sheep antibody (Jackson ImmunoResearch #713-545-147) Pseudotyped virus production: Pseudotyped virus infection of hESC-derived cardiomyocytes: For infection of hESC-derived cardiomyocytes, cells in CellCarrier-96 Ultra Plates (PerkinElmer) were incubated for 4 h with pseudotyped viral stock at the desired MOI in media.
    anti-SARS-CoV-2 nucleocapsid antibody (DA114, MRC-PPU)
    suggested: None
    In drug screens, cells were pre-treated for 1 h before infection with either camostat, benztropine, or E64d at a final concentration of 30 μM in media; DX600 at a final concentration of 10 μM in media; ACE2 antibody (AF933; R&D Systems) at 20 μg/mL; a mix of camostat + E64d at a final concentration of 30 μM each; or DMSO at 0.6% (equivalent of the highest concentration included in drug dilutions).
    ACE2
    suggested: (GenWay Biotech Inc. Cat# 18-661-15169-0.1 mg, RRID:AB_514759)
    For viral infection experiments, excitation/emission laser and filter sets for two fluorescent channels were used: 405/435-480 nm (blue) for the Hoechst 33342 nuclear stain, and 488/500-550 nm (green) for GFP or the donkey anti-sheep antibody conjugated to Alexa Fluor 488 (Jackson ImmunoResearch #713-545-147).
    GFP
    suggested: (Jackson ImmunoResearch Labs Cat# 713-545-147, RRID:AB_2340745)
    anti-sheep
    suggested: (Jackson ImmunoResearch Labs Cat# 713-545-147, RRID:AB_2340745)
    Sections were then incubated overnight at 4°C with either: primary goat polyclonal antibody to Human ACE-2 (AF933; R&D; 1:100); primary rabbit monoclonal antibody to TMPRSS2 (ab92323; Abcam; 1:500); primary rabbit monoclonal antibody to B0AT1 (ab180516; Abcam; 1:300); primary rabbit monoclonal antibody to cathepsin B (ab125067; Abcam; 1:100); primary rabbit polyclonal antibody to cathepsin L (ab203028; Abcam; 1:100); or primary rabbit polyclonal antibody to furin (ab3467; Abcam; 1:500), all prepared in PBS with 1% donkey sera, 0.1% Tween-20, and 3.3 mg/mL bovine serum albumin.
    TMPRSS2
    suggested: (Abcam Cat# ab92323, RRID:AB_10585592)
    After 24 h, sections were washed 3x with PBS with 0.1% Tween-20 before incubation with the secondary polyclonal Donkey Anti-Goat IgG H&L antibody conjugated to Alexa Fluor 555 (ab150130; Abcam; 1:200) or Donkey Anti-Rabbit IgG H&L antibody conjugated to Alexa Fluor 555 (ab150066; Abcam; 1:200) prepared at 0.01 mg/mL in PBS with 1% donkey sera, 0.1% Tween-20, and 3.3 mg/mL bovine serum albumin, for 1 h at room temperature.
    Anti-Rabbit IgG
    suggested: (Biorbyt Cat# orb14385, RRID:AB_10735740)
    The second (yellow channel) used an LED-Module 567 nm light source set at 80% intensity and 30 ms exposure time at a depth of focus of 1.88 μm to illuminate the Donkey Anti-Goat IgG antibody or Donkey Anti-Rabbit IgG H&L antibody, both conjugated to Alexa Fluor 555 (max excitation and emission of 555 and 580 respectively for both).
    Anti-Goat IgG
    suggested: None
    The third (far red channel) used an LED-Module 630 nm light source set at 50% intensity and 20 ms exposure time at a depth of focus of 2.09 μm to illuminate the cardiac troponin-T antibody conjugated to APC (max excitation and emission of 650 and 660 respectively).
    APC
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    In total, the stock used was passaged three times in VeroE6 cells, once in Caco2 cells, and once in Calu3 cells.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Caco2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    The titre of the virus stock was determined by TCID50 (Median Tissue Culture Infectious Dose) assay on Huh7 cells transduced with an ACE2 expression vector.
    Huh7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Software and Algorithms
    SentencesResources
    Analysis was performed using FlowJo software (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Acquired images were saved and visualised using ZEN software (Zeiss).
    ZEN
    suggested: None
    Graphical presentation and statistical analyses were performed using GraphPad Prism version 6.07 for Windows (GraphPad Software, La Jolla, California, USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    RNA concentration was determined using a NanoDrop 1000 (ThermoFisher), and RNA samples subsequently stored at −70°C prior to RNA sequencing library preparation.
    ThermoFisher
    suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
    Data Analysis: Processing and analysis of next generation sequencing data: All next generation sequencing data were aligned using HiSAT2 (http://daehwankimlab.github.io/hisat2/) to the Homo sapiens reference genome GRCh38/hg38.
    HiSAT2
    suggested: (HISAT2, RRID:SCR_015530)
    Reads were trimmed prior to alignment using Trim Galore, using Phred quality score for base calling cut-off of 20, corresponding to a maximum error of 1 in 100 bases and with a maximum trimming error rate of 0.1 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)27.
    Trim Galore
    suggested: (Trim Galore, RRID:SCR_011847)
    Phred
    suggested: (Phred, RRID:SCR_001017)
    Trimmed and aligned sequence files were imported as BAM files into SeqMonk (v1.42.0) for visualization and analysis (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/).
    SeqMonk
    suggested: (SeqMonk, RRID:SCR_001913)

    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 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 10.1101/2021.01.22.427737v1 on bioRxiv