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

Gene Expression Nebulas

A data portal of transcriptome profiles across multiple species

PRJNA715767: hPSC-derived cells to model Macrophage-mediated inflammation in COVID19 Hearts 

Source: NCBI / GSE169241
Submission Date: Mar 19 2021
Release Date:
Update Date: Apr 16 2021

Summary: hPSC-derived cells to model Macrophage-mediated inflammation in COVID19 Hearts 

Overall Design: We systematically compared autopsy samples from non-COVID-19 donors and COVID-19 patients using RNA-seq and immunohistochemistry. We observed strikingly increased expression levels of CCL2 as well as macrophage infiltration in heart tissues of COVID-19 patients. We generated an immuno-cardiac co-culture platform containing human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) and macrophages. We found that macrophages induce increased reactive oxygen species (ROS) and apoptosis in CMs by secreting IL-6 and TNF-α after SARS-CoV-2 exposure. Using this immuno-cardiac co-culture platform, we performed a high content screen and identified ranolazine and tofacitinib as compounds that protect CMs from macrophage-induced cardiotoxicity. We established an immuno-host co-culture system to study macrophage-induced host cell damage following SARS-CoV-2 infection, and identified FDA-approved drug candidates that alleviate the macrophage-mediated hyper-inflammation and cellular injury.

GEN Datasets:
GEND000502
Strategy:
Species:
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Protocol
Growth Protocol: For monolayer-based differentiation of cardiomyocytes (CMs), hPSCs were cultured between P20-P40 and seeded at a density of 3x105 cells/well of a 6-well plate and grown for 48 hr 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 µM 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 µM 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; Macrophages were derived from hPSCs by adapting a previously reported protocol28. hPSCs were cultured between P20-P40. First, H9 or H1 cells were lifted with ReLeSR (STEMCELL Technologies) as small clusters onto Matrigel-coated 6-well plates at low density. On day 0, IF9S medium was supplemented with 50 ng/ml BMP-4, 15 ng/ml Activin A and 1.5 µm CHIR99021. On day 2, medium was refreshed with IF9S medium with 50 ng/ml VEGF, 50 ng/ml bFGF, 50 ng/ml SCF (R&D Systems) and 10 µm SB431542 (Cayman Chemical). On day 5 and da y7, medium was changed to IF9S with 50 ng/ml IL-6 (R&D Systems), 12 ng/ml IL-3 (R&D Systems), 50 ng/ml VEGF, 50 ng/ml bFGF, 50 ng/ml SCF and 50 ng/ml TPO (R&D Systems). On day 9, cells were dissociated with TrypLE (Life Technologies) and re-suspended in IF9S medium with 50 ng/ml IL-6, 12 ng/ml IL-3 and 80 ng/ml M-CSF (R&D Systems). On day 13 and day 15, medium was changed and supplemented with 50 ng/ml IL-6, 12 ng/ml IL-3 and 80 ng/ml M-CSF. All differentiation steps were cultured at 37°C, 5% CO2.
Treatment Protocol: To analyze the gene expression of CMs co-cultured with H9-derived macrophages using RNA-seq, we used trans-well plates. CMs were seeded on the lower plate of 24 well plates and macrophages were seeded onto the upper chamber. After 24 hr, drug treatments were done to CMs and macrophages in the upper chamber were infected with SARS-CoV-2 (MOI=0.1). The trans-well chamber will allow cytokines to go through but not macrophages. 24 hpi, CMs were collected to extract total RNA. For analyzing macrophages infected with virus, macrophages were seeded onto 24 well plates and infected with SARS-CoV-2 (MOI=0.1). 24 hpi, macrophages were collected for total RNA.
Extract Protocol: Total RNA was extracted in TRIzol (Invitrogen) and DNase I treated using Directzol RNA Miniprep kit (Zymo Research) according to the manufacturer’s instructions.
Library Construction Protocol: RNAseq libraries of polyadenylated RNA were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) or TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions.
Sequencing
Molecule Type: polyA(+) RNA
Library Source:
Library Layout: PAIRED; SINGLE
Library Strand: Forward
Platform: ILLUMINA
Instrument Model: Illumina NovaSeq 6000
Strand-Specific: Specific
Samples
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Analysis:
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
Longitudinal single-cell epitope and RNA-sequencing reveals the immunological impact of type 1 interferon autoantibodies in critical COVID-19.
bioRxiv : the preprint server for biology . 2021-03-10 [PMID: 33758859]
An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts.
Circulation research . 2021-04-15 [PMID: 33853355]