Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium

  1. Fadi Jacob
  2. Sarshan R. Pather
  3. Wei-Kai Huang
  4. Feng Zhang
  5. Samuel Zheng Hao Wong
  6. Haowen Zhou
  7. Beatrice Cubitt
  8. Wenqiang Fan
  9. Catherine Z. Chen
  10. Miao Xu
  11. Manisha Pradhan
  12. Daniel Y. Zhang
  13. Wei Zheng
  14. Anne G. Bang
  15. Hongjun Song
  16. Juan Carlos de la Torre
  17. Guo-li Ming

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Abstract

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  1. Version published to 10.1016/j.stem.2020.09.016
  2. ScreenIT

    SciScore for 10.1101/2020.07.28.225151: (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 AuthenticationContamination: All hiPSC lines were confirmed free of Mycoplasma, Acholeplasma, and Spiroplasma with a detection limit of 10 CFU/ml by targeted PCR (Biological Industries).

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 virus was expanded and titered using Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Brain organoid sections were imaged as z stacks using a Zeiss LSM 810 confocal microscope or a Zeiss LSM 710 confocal microscope (Zeiss) using a 10X, 20X, 40X, or 63X objective with Zen 2 software (Zeiss).
    Zen
    suggested: None
    Images were analyzed using either Imaris 7.6 or ImageJ software.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Images were cropped and edited using Adobe Photoshop (Adobe) and Adobe Illustrator (Adobe)
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)
    Bioinformatics Analyses: Choroid plexus organoid raw sequencing data were demultiplexed with bcl2fastq2 v2.17.1.14 (Illumina) with adapter trimming using Trimmomatic v0.32 software (Bolger et al., 2014).
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Alignment was performed using STAR v2.5.2a (Dobin et al., 2013).
    STAR
    suggested: (STAR, RRID:SCR_015899)
    A combined reference genome consisting of GENCODE human genome release 28 (GRCh38.p12) and Ensembl SARS-CoV-2 Wuhan-Hu-1 isolate genome (genome assembly: ASM985889v3, GCA_009858895.3; sequence: MN908947.3) was used for alignment.
    GENCODE
    suggested: (GENCODE, RRID:SCR_014966)
    Ensembl
    suggested: (Ensembl, RRID:SCR_002344)
    Transcript lengths were retrieved from GTF annotation files using GenomicFeatures v1.36.4 (Lawrence et al., 2013) and raw counts were converted to units of transcripts per million (TPM) using the calculateTPM function in scater v1.12.2 (McCarthy et al., 2017).
    GenomicFeatures
    suggested: (GenomicFeatures, RRID:SCR_016960)
    Differential gene expression analysis for choroid plexus organoids between 72 hpi vehicle, 24 hpi SARS-CoV-2 infection, and 72 hpi SARS-CoV-2 infection was performed using DESeq2 v1.24.0 (Love et al., 2014).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Volcano plots for 24 hpi SARS-CoV-2 and 72 hpi SARS-CoV-2 were plotted using EnhancedVolcano v1.7.8.
    EnhancedVolcano
    suggested: (EnhancedVolcano, RRID:SCR_018931)
    Shared and unique signatures for upregulated and downregulated genes between 24 hpi SARS-CoV-2 and 72 hpi SARS-CoV-2 were visualized using VennDiagram v1.6.20 (Chen and Boutros, 2011).
    VennDiagram
    suggested: (VennDiagram, RRID:SCR_002414)
    Upregulated and downregulated gene lists for each timepoint were used for gene ontology (GO) (Ashburner et al., 2000; The Gene Ontology, 2019) enrichment analysis using PANTHER v15.0 (Mi et al., 2019).
    PANTHER
    suggested: (PANTHER, RRID:SCR_004869)

    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:
    Limitations of in vitro Neural Cultures to Model Infection: Our study demonstrates clear susceptibility of hiPSC-derived neurons, astrocytes, and choroid plexus cells, as well as primary astrocytes and choroid plexus epithelial cells, to SARS-CoV-2 infection in vitro, however, a thorough analysis of brain samples from patients with COVID-19 is necessary to confirm neural susceptibility in vivo. Our methodology has a number of constrains that limit result interpretation. First, since brain organoids more closely resemble the developing fetal brain than the mature adult brain, there may exist important differences in SARS-CoV-2 susceptibility between immature and mature cells. There has been recent evidence of vertical transmission of SARS-CoV-2 from mother to fetus (Dong et al., 2020; Vivanti et al., 2020; Zeng et al., 2020), but impact on the fetal brain remains unclear. Brain organoids may serve as a useful model system to explore the potential effects of fetal SARS-CoV-2 exposure on brain development. Second, direct exposure of brain organoid cultures to SARS-CoV-2 in the culture medium may not accurately recapitulate the physiological amount and duration of viral exposure in humans. Brain organoid models also lack an intact blood-brain-barrier or blood-CSF-barrier which may modulate SARS-CoV-2 access to the brain. Third, our brain organoids lack additional cell types, such as oligodendrocytes, stromal cells, immune cells, and endothelial cells, which may also modulate susc...

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 32, 27, 36 and 30. 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.

  3. ScreenIT

    SciScore for 10.1101/2020.07.28.225151: (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

    Antibodies
    SentencesResources
    Monolayer hiPSC-derived cortical neurons, microglia, and astrocytes were exposed to either SARS-CoV-2 virus isolate or vehicle control for 12 hours and analyzed at 24, 48, and 120 hours post-infection (hpi) for immunolabelling using convalescent serum from a patient with COVID-19 or SARS-CoV-2 nucleoprotein antibodies (Figures S1A-C).
    SARS-CoV-2 nucleoprotein
    suggested: (Sino Biological Cat# 40143-R019, AB_2827973)
    Experimental Models: Cell Lines
    SentencesResources
    Because SARS-CoV-2 can affect multiple organs, the currently widely used cellular model systems, such as Vero E6 cells or human cancer cell lines, do not adequately recapitulate the cellular diversity and breadth of the disease.
    Vero E6
    suggested: CVCL_XD71
    Software and Algorithms
    SentencesResources
    Our finding that dysregulated gene expression varies widely among hepatocyte, intestinal, and choroid plexus organoids infected with SARS-CoV-2 suggests unique responses in different cell types and highlights the need for diverse cellular model systems when studying the disease.
    SARS-CoV-2
    suggested: (Active Motif Cat# 91345, AB_2847847)
    Neuron to neuron propagation has been described for other coronaviruses (Dube et al., 2018; Netland et al., 2008), but it was not obvious in our study.
    Neuron
    suggested: (NEURON, SCR_005393)

    Data from additional tools added to each annotation on a weekly basis.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.07.28.225151 on bioRxiv