Gene Expression
Nebulas
Gene Expression NebulasSummary: Heart injury has been reported in up to 20% of COVID-19 patients, yet the cause of myocardial histopathology is unknown. We examined heart tissue from autopsies of healthy and COVID-19 patients using RNA-seq and identified a strikingly increased CCL2 expression and macrophage population in COVID-19 heart sample. Transwell assays using hESC-derived cardiomyocytes (CMs) and macrophages revealed that SARS-CoV-2 infected CMs secrete CCL2, which recruits macrophages, and this was validated using adult human CMs. Macrophages recruited by CCL2 from infected CMs secrete IL-6 and TNF-δΌͺ, which causes increased ROS and cell apoptosis of CMs. Finally, a high content chemical screen using FDA-approved drugs identified ranolozine and tofacitinib, which rescue SARS-CoV-2 infected CMs from macrophages-induced cardiotoxicity.
Overall Design: scRNA-seq of three conditions: hESC-derived cardiomyocytes+SARS-CoV-2 Pseudo-entry virus; hESC-derived cardiomyocytes+macrophages+SARS-CoV-2 Pseudo-entry virus
| Strategy: |
|
| Species: |
|
| Healthy Condition: |
|
| Cell Type: |
|
| Growth Protocol: | For monolayer-based cardiomyocyte (CMs) differentiation, hESCs were passaged at a density of 3x105cells/well of 6-well plate and grown for 48 hours to 90% confluence in a humidified incubator with 5% CO2 at 37 On day 0, the medium was replaced with RPMI 1640 supplemented with B27 without insulin and 6 CHIR99021. On day 1, the medium was changed to RPMI 1640 supplemented with B27 without insulin for 48 hr. Day 3, medium was refreshed to RPMI 1640 supplemented with B27 without insulin and 2 C59 for another 48 hr. On day 5, the medium was changed back to RPMI-B27 without insulin for 48 hr, and then switched to RPMI 1640 plus normal B27 until day 12 with a medium change every 2 days. On day 12, the medium was transiently changed to RPMI 1640 without D-glucose containing ascorbic acid, human albumin and DL-Lactate for two days to allow metabolic purification of CMs. From that day on, fresh RPMI 1640-B27 was changed every two days. On day 21, cells were dissociated with Accutase at 37followed by resuspending with fresh RPMI 1640-B27 plus Y-27632 and reseeding into 96-well plates. After 24 hr, then switched to RMPI 1640-B27 without Y-27632 for following virus confection tests. |
| Treatment Protocol: | hPSC-derived cardiomyocytes were dissociated with Accutase at 37 followed by resuspending with fresh RPMI 1640-B27 plus Y-27632 and reseeding into 96-well plates. After 24 hr, the medium was switched to RMPI 1640-B27 without Y-27632. After 24h recovery, macrophages were added into hPSC-derived cardiomyocytes and co-cultured for another 24 hr before following analysis. Adult cardiomyocytes were also seeded into plates for 48-96 hr and co-cultured with macrophages for 24 hr before following analysis. |
| Extract Protocol: | null |
| Library Construction Protocol: | Library was constructed according to the user manual of Chromium Single Cell 3 Library & Gel Bead Kit v2 |
| Molecule Type: | Poly(A)+ RNA |
| Library Source: | |
| 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 |
|---|