Gene Expression
Nebulas
Gene Expression NebulasSummary: Recent clinical data has suggestedsed a bi-directional relationship between Coronavirus disease 19 (COVID-19) and diabetes. Here, we showdemonstrateed the detection of SARS-CoV-2 in pancreatic endocrine cells in autopsy samples derived fromof COVID-19 patients. Single cell RNA-seq and immunostaining confirmed that multiple types of pancreatic islet cells can be infected byare susceptible to SARS-CoV-2, eliciting a cellular stress response and the induction of chemokines. SARS-CoV-2 infection causes the increase of chemokine response, cell stress, and interferon signal. Upon SARS-CoV-2 infection, beta cells show a the decreased expression of insulin and the increased expression of alpha and acinar cell markers, including glucagon and , and acinar cell markers, including PRSS1/trypsin1, respectfully, suggesting which suggests that infected beta cells undergocellular dedifferentiation. This was furtherCorroboration of these findings could be further validated using theinex vivo using single cell sequencing of pancreatic tissue from autopsy of COVID-19 patients autopsies. Trajectory analysis identifiedindicated that the EIF2 pathway that changess along withmediates beta cell dedifferentiation. Furthermore, a, and a high content screen identified trans-integrated stress response inhibitor (trans-ISRIB) that as decreasinges poly-hormonal cells. Finally, trans-ISRIB treatmentwhich rescueds beta cell dedifferentiation upon SARS-CoV-2 exposure. Together, it, suggestings that SARS-CoV-2 infection causes EIF2 pathway-mediated beta cell dedifferentiation. Altogether, tThis study provides a potential mechanism of new onset diabetes in upon the development of COVID-19.
Overall Design: Human islets were infected with SARS-CoV-2 and analyzed using scRNA-seq
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| Growth Protocol: | The pancreatic organs were obtained from the local organ procurement organization under the United Network for Organ Sharing (UNOS). The islets were isolated in the Human Islet Core at University of Pennsylvania following the guidelines of Clinical Islet Transplantation consortium protocol (Ricordi et al., 2016). Briefly, the pancreas was digested following intraductal injection of Collagenase & Neutral Protease in Hanks’ balanced salt solution. Liberated islets were then purified on continuous density gradients (Cellgro/Mediatech) using the COBE 2991 centrifuge and cultured in CIT culture media and kept in a humidified 5% CO2 incubator. |
| Treatment Protocol: | Approximately 1 × 10^6 untreated, control or integrated stress response inhibitor treated (ISRIB) treated, and Sal003 treated primary human pancreatic islet cells were infected with passage-3 SARS-CoV-2 at an MOI of 1 for 24 h in CIT culture media. After 24 hours, infected and mock infected pancreatic islets were dissociated into a single cell suspension using Accutase cell detachment solution (Innovative Cell Technologies) and incubation at 37C for ~20 minutes followed by gentle pipetting to break apart groups of cells. |
| Extract Protocol: | Target cell inputs of 10,000 cells for each condition were then loaded into a Chromium Controller using Chromium Next GEM (Gel Bead-In Emulsion) single Cell 5’ Library & Gel Bead Kit v1.1 (10x Genomics) according to manufacturer’s instructions. After generation of GEMs, cDNA synthesis and subsequent library preparation of all samples was completed using the Chromium Single Cell 5’ Library Kit v1.1 (10x Genomics) according to manufacturer’s instructions. |
| Library Construction Protocol: | The 10X Libraries (Single Cell 5’ R2-only library) were sequenced on the Illumina NovaSeq6000 sequencer with pair-end reads (28 bp for read 1 and 91 bp for read 2). |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | PAIRED |
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| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-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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