Identification of Drugs Blocking SARS-CoV-2 Infection using Human Pluripotent Stem Cell-derived Colonic Organoids
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
The current COVID-19 pandemic is caused by SARS-coronavirus 2 (SARS-CoV-2). There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with worse COVID-19 outcomes 1 . Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple hESC-derived colonic cell types, but highly enriched in enterocytes. Multiple cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. We used hPSC-derived COs in a high throughput platform to screen 1280 FDA-approved drugs against viral infection. Mycophenolic acid and quinacrine dihydrochloride were found to block the infection of SARS-CoV-2 pseudo-entry virus in COs both in vitro and in vivo , and confirmed to block infection of SARS-CoV-2 virus. This study established both in vitro and in vivo organoid models to investigate infection of SARS-CoV-2 disease-relevant human colonic cell types and identified drugs that blocks SARS-CoV-2 infection, suitable for rapid clinical testing.
Article activity feed
-
SciScore for 10.1101/2020.05.02.073320: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal work was conducted in agreement with NIH guidelines and approved by the WCM Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable In vivo transplantation and drug evaluation: hPSC-COs were harvested by cell scraper, mixed with 20 μl Matrigel (Corning) and transplanted under the kidney capsule of 7-9 weeks old male NSG mice. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Secondary antibodies included donkey anti-mouse, goat, rabbit or chicken antibodies conjugated … SciScore for 10.1101/2020.05.02.073320: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal work was conducted in agreement with NIH guidelines and approved by the WCM Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable In vivo transplantation and drug evaluation: hPSC-COs were harvested by cell scraper, mixed with 20 μl Matrigel (Corning) and transplanted under the kidney capsule of 7-9 weeks old male NSG mice. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Secondary antibodies included donkey anti-mouse, goat, rabbit or chicken antibodies conjugated with Alexa-Fluor-488, Alexa-Fluor-594 or Alexa-Fluor-647 fluorophores (1:500 goatsuggested: Nonerabbitsuggested: (Alomone Labs Cat# ANR-041-AG, RRID:AB_2876808)chicken antibodies conjugated with Alexa-Fluor-488 , Alexa-Fluor-594 or Alexa-Fluor-647 fluorophores ( 1:500suggested: NonePrimary antibodies were detected using Fluorophore-conjugated secondary goat anti-mouse (IRDye 680RD, 926-68070) and goat anti-rabbit (IRDye 800CW, 926-32211) antibodies. anti-mousesuggested: (LI-COR Biosciences Cat# 926-68070, RRID:AB_10956588)Experimental Models: Cell Lines Sentences Resources HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV2-spike (kindly provided by Dr. Peihui Wang, Shandong University, China) using FuGENE 6 (Promega). HEK293Tsuggested: NoneApproximately 2.5 × 105 Vero E6 cells were pre-treated with the indicated compounds for 1 h prior to infection with SARS-CoV-2 at an MOI of 0.01 in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L-glutamine, 10 mM Vero E6suggested: RRID:CVCL_XD71)After reviewing the clusters, we merged four clusters that were likely from stem cell population into a single cluster (LGR5+ or BMI1+ stem cells) and kept the other four clusters (KRT20+ epithelial cells, MUC2+ goblet cells, EPHB2+ TA cells, and CHGA+ NE cells) for further analysis. TAsuggested: RRID:CVCL_4315)Software and Algorithms Sentences Resources Cell Lines: HEK293T (human [Homo sapiens] fetal kidney) and Vero E6 (African green monkey [Chlorocebus aethiops] kidney) were obtained from ATCC (https://www.atcc.org/). https://www.atcc.org/suggested: (ATCC, RRID:SCR_001672)Images were acquired using an LSM880 Laser Scanning Confocal Microscope (Zeiss) and processed with Zen or Imaris (Bitplane) software. Zensuggested: NoneImarissuggested: (Imaris, RRID:SCR_007370)Antibody-mediated fluorescence was detected on a LI-COR Odyssey CLx imaging system and analyzed using Image Studio software ( Image Studiosuggested: (Image Studio Lite, RRID:SCR_013715)We normalized the gene expression UMI counts for each sample separately using a deconvolution strategy24 implemented by the R scran package (v.1.14.1). scransuggested: (scran, RRID:SCR_016944)The list of dissociation-related genes was originally built on mouse data26, we converted them to human ortholog genes using Ensembl BioMart. Ensemblsuggested: (Ensembl, RRID:SCR_002344)We re-identified marker genes for the merged five clusters and selected the top 10 positive marker genes per cluster for heatmap plot using the DoHeatmap function in the R Seurat package25. Seuratsuggested: (SEURAT, RRID:SCR_007322)Results from OddPub: Thank you for sharing your data.
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.
-