Identification of Candidate COVID-19 Therapeutics using hPSC-derived Lung Organoids
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Abstract
The SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre- or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDA-approved drugs that might be repurposed and should be considered for COVID-19 clinical trials.
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SciScore for 10.1101/2020.05.05.079095: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Primary 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)anti-rabbitsuggested: (LI-COR Biosciences Cat# 926-32211, RRID:AB_621843)Experimental Models: Cell Lines Sentences Resources The hESC line RUES2 was cultured on irradiated mouse embryonic fibroblasts (Global Stem, cat. … SciScore for 10.1101/2020.05.05.079095: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Primary 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)anti-rabbitsuggested: (LI-COR Biosciences Cat# 926-32211, RRID:AB_621843)Experimental Models: Cell Lines Sentences Resources The hESC line RUES2 was cultured on irradiated mouse embryonic fibroblasts (Global Stem, cat. no. GSC-6001G) at a density of 20,000-25,000 cells/cm2 in a medium of DMEM/F12, 20% knockout serum replacement (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma Aldrich) and 20 ng/ml bFGF (R&D Systems), and medium was changed daily. hESC cultures were maintained in an undifferentiated state at 37 °C in a 5% CO2/air environment until stem cells reached about 90% confluence. hESC differentiation into endoderm was performed in serum-free differentiation (SFD) medium of DMEM/F12 (3:1) (Life Technologies) supplemented with N2 (Life Technologies), B27, 50 μg/ml ascorbic acid, 2 mM Glutamax, 0.4 μM monothioglycerol, 0.05% BSA at 37 °C in a 5% CO2/5% O2/95% N2 environment. RUES2suggested: RRID:CVCL_B810)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/). HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Approximately 2.5 × 105 Vero E6 cells were pre-treated with DMSO, 10 μM imatinib, 3 μM MPA or 4.5 μM QNHC 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)Experimental Models: Organisms/Strains Sentences Resources Xenograft formation: 1 million hESC-derived cells at lung progenitor stage (at day 25) were subcutaneously injected into 6-8 weeks old NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJ (NSG) mice (Jackson Laboratory, Bar Harbor, Maine) NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJsuggested: NoneSoftware 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)Antibody-mediated fluorescence was detected on a LI-COR Odyssey CLx imaging system and analyzed using Image Studio software (LI-COR). qRT-PCR: Total RNA samples were prepared from cells/organoids using TRIzol and Direct-zol RNA Miniprep Plus kit (Zymo Research) according to the manufacturer’s instructions. Image Studiosuggested: (Image Studio Lite, RRID:SCR_013715)We normalized the gene expression UMI counts using a deconvolution strategy 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 data 29; we converted them to human ortholog genes using Ensembl BioMart. Ensemblsuggested: (Ensembl, RRID:SCR_002344)We scaled the normalized counts and performed PCA on the highly variable genes using the ScaleData and RunPCA functions in the R Seurat package 28. Seuratsuggested: (SEURAT, RRID:SCR_007322)The rest plots were generated using the R ggplot2 package. ggplot2suggested: (ggplot2, RRID:SCR_014601)The resulting single end reads were checked for quality (FastQC v0.11.5) and processed using the processed using the nf-core RNA-seq (v.1.4.2) workflow. FastQCsuggested: (FastQC, RRID:SCR_014583)Samples had adapters trimmed using Trim Galore (v0.6.4) then ribosomal reads removed using SortMeRNA (v.2.1)before being aligned to human reference genome (GRCh38) using STAR aligner30 (v.2.6.1). Trim Galoresuggested: (Trim Galore, RRID:SCR_011847)SortMeRNAsuggested: (SortMeRNA, RRID:SCR_014402)STARsuggested: (STAR, RRID:SCR_015899)Raw gene counts were quantified using Subread (v.1.6.4) featureCounts. featureCountssuggested: (featureCounts, RRID:SCR_012919)Principal component analysis was performed using Log2 CPM values and gene set analysis was run with WebGestalt 32. WebGestaltsuggested: NoneHeatmaps and bar plots were generated using Graphpad Prism software, version 7.0d. Graphpad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)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.
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