Gene Expression
Nebulas
Gene Expression NebulasSummary: The current COVID-19 pandemic is caused by the novel coronavirus 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 virus 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 often present with respiratory symptoms including cough, dyspnea, and respiratory distress but upwards of 25% of respiratory dysfunction, many COVID-19 patients have digestive system indications, including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with more severe COVID-19 outcomes1. Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of different 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 types of hESC-derived colon cells, but are highly enriched in hPSC-derivedKRT20+ enterocytes. Distinct cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which is further validated in vivo using a humanized mouse model. Finally, we adapted hPSC-derived COs to a high throughput platform to screen 1280 FDA-approved drugs. Mycophenolic acid was found to block the entry of SARS-Cov-2 pseudo-entry virus in COs, and confirmed to block infection of SARS-CoV-2 virus. In summary, 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 a drug suitable for rapid clinical testing that blocks SARS-CoV-2 infection.
Overall Design: scRNA-seq analysis of human pluripotent stem cell-derived colonic organoids (hPSC-COs) infected with SARS-CoV-2 pseudo-entry virus.
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| Growth Protocol: | The colon progenitor organoids were collected from the initial 2D cultures and embedded in a 100% Matrigel dome in a 24-well plate. Differentiation to mature colonic cell types was achieved by culturing these colon progenitor organoids in differentiation medium containing 600 nM LDN193189 (Axon), 3 CHIR99021 and 100 ng/ml hEGF in Advance DMEM F12 medium supplemented with 1X B27 supplement, 1X GlutaMax, 10 mM HEPES and 1X Pen-Strep. The differentiation medium were refreshed every 3 days for at least 40 days to achieve full colonic differentiation. The colon organoids were passaged and expanded every 10 14 days at 1:6 density. To passage the organoids, the Matrigel domes containing the organoids were scrapped off the plate and resuspended in cold splitting media (Advance DMEM F12 medium supplemented with 1X GlutaMax, 10 mM HEPES and 1X Pen-Strep). The organoids were mechanically dislodged from the Matrigel dome and fragmented by pipetting in cold splitting media. The old Matrigel and splitting media were removed after spinning down cells and the organoids were resuspended in 100% Matrigel. 50 Matrigel containing fragmentized colon organoids were plated in one well of a pre-warmed 24-well plate. |
| Treatment Protocol: | To assay pseudotyped virus infection on colon organoids, COs were seeded in 24 well plates. Pseudotyped virus was added at MOI=0.01 plus polybrene at a final concentration of 8 /mL, and the plate centrifuged for 1 hr at 1200g. At 3i, the infection medium was replaced with fresh medium. |
| Extract Protocol: | hPSC-COs were harvested by cell scraper, mixed with 20 l Matrigel (Corning) and transplanted under the kidney capsule of 7-9 weeks old male NSG mice. At two weeks post-transplantation, SARS-Cov-2 pseudo-entry virus was inoculated locally at 1x10e3 FFU. |
| Library Construction Protocol: | Library was constructed according to the user manual of Chromium Single Cell 3Library & Gel Bead Kit v2 |
| Molecule Type: | Poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-Specific: | Specific |
| 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 |
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