Variable susceptibility of intestinal organoid–derived monolayers to SARS-CoV-2 infection
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Abstract
Gastrointestinal effects associated with Coronavirus Disease 2019 (COVID-19) are highly variable for reasons that are not understood. In this study, we used intestinal organoid–derived cultures differentiated from primary human specimens as a model to examine interindividual variability. Infection of intestinal organoids derived from different donors with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid–derived monolayers. Susceptibility to infection correlated with angiotensin I converting enzyme 2 ( ACE2 ) expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue, indicating that this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike (S) protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.
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Leandra Santos Baptista, Fabiana Avila Carneiro
Review 1: "Intestinal organoids expose heterogeneity in SARS-CoV-2 susceptibility"
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SciScore for 10.1101/2021.07.16.452680: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Human intestinal tissue specimen collection: Patients with and without IBD were recruited at outpatient colonoscopy performed for colon cancer screening, surveillance, or IBD activity assessment at NYU Langone Health’s Ambulatory Care Center, New York, under an NYU Grossman School of Medicine Institutional Review Board–approved study (Mucosal Immune Profiling in Patients with Inflammatory Bowel Disease; S12-01137). Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Sections were blocked in 5% normal donkey serum (Sigma) in TBS-T at room … SciScore for 10.1101/2021.07.16.452680: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Human intestinal tissue specimen collection: Patients with and without IBD were recruited at outpatient colonoscopy performed for colon cancer screening, surveillance, or IBD activity assessment at NYU Langone Health’s Ambulatory Care Center, New York, under an NYU Grossman School of Medicine Institutional Review Board–approved study (Mucosal Immune Profiling in Patients with Inflammatory Bowel Disease; S12-01137). Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Sections were blocked in 5% normal donkey serum (Sigma) in TBS-T at room temperature (RT) for 1 hr, followed by incubation with rabbit anti-ACE2 antibody (1:100, Abcam, ab15348) in the blocking solution at 4°C overnight. anti-ACE2suggested: (Abcam Cat# ab15348, RRID:AB_301861)The permeabilized organoids were washed 3 times with PBS and incubated with mouse anti-SARS-CoV-2 N antibody (1:1,000, ProSci, 10-605) and rabbit anti-ACE2 antibody (1:500, Abcam, ab15348) diluted in PBS containing 3% BSA overnight at 4°C. anti-SARS-CoV-2 Nsuggested: NoneThe following antibodies were used for immunoblotting studies: mouse anti-β-actin (1:5,000, Sigma, AC-15), rabbit anti-ACE2 (1:1,000, Abcam, ab15348), mouse anti-TMPRSS2 (1:1,000, Santa Cruz, sc-515727). anti-β-actinsuggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)anti-TMPRSS2suggested: (Santa Cruz Biotechnology Cat# sc-515727, RRID:AB_2892118)Secondary antibodies (mouse anti-rabbit and goat-anti mouse, 211-032-171 and 115-035-174, respectively) were purchased from JacksonImmunoResearch RNA deep sequencing and analysis: Organoid monolayers were cultured in differentiation media for 7 days and then were infected with SARS-CoV-2 at MOI of 4 for 24 hrs or 72 hrs before RNA extraction with 2-3 technical duplicates per line. anti-rabbitsuggested: (Jackson ImmunoResearch Labs Cat# 211-032-171, RRID:AB_2339149)mouse, 211-032-171suggested: (Jackson ImmunoResearch Labs Cat# 211-032-171, RRID:AB_2339149)Experimental Models: Cell Lines Sentences Resources A working stock of SARS-CoV-2-mNG was generated by infecting a 90-95% confluent monolayer of Vero E6 cells (ATCC CRL-1586) for 48 hrs at 37°C. Vero E6suggested: NoneRecombinant DNA Sentences Resources Amplicons were cloned into pCR™2.1-TOPO® (Invitrogen). pCR™2.1-TOPO®suggested: NoneSoftware and Algorithms Sentences Resources The crypts were embedded in 30 μl of Matrigel and cultured with Human IntestiCult™ Organoid Growth Medium (OGM) (Basal Medium and Organoid Supplement, Stemcell Technologies) supplemented with 100 IU Penicillin and 100 μg/ml Streptomycin (Corning) and 125 μg/ml Gentamicin (ThermoFisher), herein referred to as expansion medium. ThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)For differentiation, the human organoids were cultured with 1:1 mix of Human IntestiCult™ OGM Basal Medium and DMEM/F-12 (ThermoFisher) in the presence of 100 IU Penicillin and 100 μg/ml Streptomycin, 125 μg/ml Gentamicin and 2 mM L-Glutamine (Corning), herein referred to as differentiation medium. Human IntestiCult™suggested: NoneImages were acquired using an EVOS FL Auto Cell Imaging System (ThermoFisher) and then processed and quantified using ImageJ. ImageJsuggested: (ImageJ, RRID:SCR_003070)Upstream regulators analysis was performed by uploading the differentially expressed genes to Ingenuity Pathway Analysis software (Qiagen). Ingenuity Pathway Analysissuggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)Statistical differences were determined as described in figure legend using either R or GraphPad Prism 9 software (La Jolla, CA, USA). 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.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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