Gene Expression Nebulas
A data portal of transcriptomic profiles analyzed by a unified pipeline across multiple species

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A data portal of transcriptome profiles across multiple species
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PRJNA643694

PRJNA643694: SARS-CoV-2 infection and replication in human fetal and pediatric gastric organoids

Source: NCBI / GSE153684
Submission Date: Jul 02 2020
Release Date:
Update Date: Dec 02 2021

Summary: Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global public health emergency. COVID-19 typically manifests as a respiratory illness but an increasing number of clinical reports describe gastrointestinal (GI) symptoms. This is particularly true in children in whom GI symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. By contrast, fetuses seem to be rarely affected by COVID-19, although the virus has been detected in placentas of affected women. These observations raise the question of whether the virus can infect and replicate within the stomach once ingested. Moreover, it is not yet clear whether active replication of SARS-CoV-2 is possible in the stomach of children or in fetuses at different developmental stages. Here we show the novel derivation of fetal gastric organoids from 8-21 post-conception week (PCW) fetuses, and from pediatric biopsies, to be used as an in vitro model for SARS-CoV-2 gastric infection. Gastric organoids recapitulate human stomach with linear increase of gastric mucin 5AC along developmental stages, and expression of gastric markers pepsinogen, somatostatin, gastrin and chromogranin A. In order to investigate SARS-CoV-2 infection with minimal perturbation and under steady-state conditions, we induced a reversed polarity in the gastric organoids (RP-GOs) in suspension. In this condition of exposed apical polarity, the virus can easily access viral receptor angiotensin-converting enzyme 2 (ACE2). The pediatric RP-GOs are fully susceptible to infection with SARS-CoV-2, where viral nucleoprotein is expressed in cells undergoing programmed cell death, while the efficiency of infection is significantly lower in fetal organoids. The RP-GOs derived from pediatric patients show sustained robust viral replication of SARS-CoV-2, compared with organoids derived from fetal stomachs. Transcriptomic analysis shows a moderate innate antiviral response and the lack of differentially expressed genes belonging to the interferon family. Collectively, we established the first expandable human gastric organoid culture across fetal developmental stages, and we support the hypothesis that fetal tissue seems to be less susceptible to SARS-CoV-2 infection, especially in early stages of development. However, the virus can efficiently infect gastric epithelium in pediatric patients, suggesting that the stomach might have an active role in fecal-oral transmission of SARS-CoV-2.

Overall Design: Transcriptomic analysis of organoids derived from fetal and pediatric gastric samples infected by SARS-CoV-2 virus at MOI 1, and paired negative controls.

GEN Datasets:
GEND000487
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Protocol
Growth Protocol: Fully grown gastric organoids at day 7 after single cell disaggregation were removed from surrounding extracellular matrix using a modified published protocol. Matrigel was dissolved with 60 min treatment of the droplets with Cell Recovery Solution (Corning) at 4掳C. Organoids were retrieved from the plates using 1% BSA-coated cut-end tips and transferred to 1% BSA-coated 15 mL tubes. Cells were extensively washed with ice-cold PBS and centrifuged at 200 g for 5 min at 4掳C. Supernatant was discarded, the pellet was resuspended in complete medium and transferred to non-tissue culture treated low-adhesive multiwell plates (pre-coated in 1% BSA). Organoids were cultured in suspension for 3 days to allow reversion of polarity, before use in infection experiments.
Treatment Protocol: Reversed-polarity organoids were infected at a MOI of 1 by incubation with 250l of an expansion medium viral suspension for 2 hours. After infection, organoids were washed twice in DMEM to remove unbound virus. RP-GOs were dispersed in a 400 expansion medium at 37 C with 5% CO2. For all organoid cultures 50l of supernatant were harvested at 0, 24, 48, and 72 hours post infection. An equal volume of expansion medium replaced the sampled supernatant at each collection time. An extra sample at 96 hours post infection was collected for the RP-GOs. Samples were stored at -80C before titration through the FFA.
Extract Protocol: Pelletted organoids were extracted in RLT buffer
Library Construction Protocol: -
Sequencing
Molecule Type: rRNA- RNA
Library Source:
Library Layout: PAIRED
Library Strand: -
Platform: ILLUMINA
Instrument Model: Illumina NovaSeq 6000
Strand-Specific: Unspecific
Samples
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Data Resource GEN Sample ID GEN Dataset ID Project ID BioProject ID Sample ID Sample Name BioSample ID Sample Accession Experiment Accession Release Date Submission Date Update Date Species Race Ethnicity Age Age Unit Gender Source Name Tissue Cell Type Cell Subtype Cell Line Disease Disease State Development Stage Mutation Phenotype Case Detail Control Detail Growth Protocol Treatment Protocol Extract Protocol Library Construction Protocol Molecule Type Library Layout Strand-Specific Library Strand Spike-In Strategy Platform Instrument Model Cell Number Reads Number Gbases AvgSpotLen1 AvgSpotLen2 Uniq Mapping Rate Multiple Mapping Rate Coverage Rate
Publications
SARS-CoV-2 infection and replication in human gastric organoids.
Nature communications . 2021-11-16 [PMID: 34785679]