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CARDIAC Publication-derived

SARS-CoV-2 Cardiac Tropism and Injury in iPSC-CM

Source Marchiano et al. · Department of Pathology, University of Washington, Seattle, USA · 10.1016/j.stemcr.2021.02.005

👤 Marchiano S, Hsiang TY, Khanna A, Bhatt S, Higashi T, Whitmore LS, Bargehr J, Davaapil H, Chang J, Smith E, Ong LP, Colzani M, Murry CE ⏱ 82 days 📋 8 phases 🧫 iPSC-Derived (Human iPSCs)

Abstract

Marchiano et al. demonstrated that SARS-CoV-2 directly infects iPSC-derived cardiomyocytes via ACE2, causing sarcomeric disruption, beating cessation, and innate immune activation. The model confirmed direct cardiac tropism independent of immune infiltration and validated remdesivir as a cardioprotective antiviral agent.

Cell source
iPSC-Derived (Human iPSCs)
Application
COVID-19 Cardiac Research

Protocol overview

44 steps across 8 phases

iPSC Preparation and Plating Day 0
  1. 1 Dispase Treatment and Colony Dissociation
  2. 2 Generate Smaller Colony Clumps
  3. 3 Plate on Matrigel-Coated Wells
Definitive Endoderm (DE) and Anterior Foregut Endoderm (AFE) Differentiation Days 1–12
  1. 4 Initiate DE Differentiation
  2. 5 Specify to Anterior Foregut Endoderm (AFE)
  3. 6 Characterize AFE by Immunostaining and qRT-PCR
Lung Organoid (LORG) Development Days 12–60
  1. 7 Differentiate AFE to Lung Progenitor Cells
  2. 8 Generate 3D Lung Organoids (LORGs)
  3. 9 Confirm LORG Morphology by H&E Staining
  4. 10 Characterize Cell Type Composition by Immunostaining
  5. 11 Confirm Gene Expression by qRT-PCR
  6. 12 Confirm Protein Expression by Western Blot
Cerebral Organoid (CORG) and Neural Cell Differentiation Days 1–80
  1. 13 Generate Cerebral Organoids (CORGs)
  2. 14 Characterize CORG Cell Type Composition
  3. 15 Differentiate iPSCs into Neural Progenitor Cells (NPCs)
  4. 16 Differentiate NPCs into Neurons
  5. 17 Differentiate iPSCs into Astrocytes
  6. 18 Quantify ACE2 and TMPRSS2 Expression in Neural Cells
SARS-CoV-2 Pseudovirus Infection Studies Days 60–65 (LORGs); Day 80+ (CORGs/Neural Cells)
  1. 19 Prepare SARS-CoV-2 Pseudovirus
  2. 20 Infect LORGs with SARS-CoV-2 Pseudovirus (GFP)
  3. 21 Visualize GFP Signal in LORGs
  4. 22 Confirm Viral Co-localization with Receptors
  5. 23 Quantify Pseudovirus Infection by Luciferase Activity
  6. 24 Infect CORGs, NPCs, and Neurons with GFP Pseudovirus
  7. 25 Assess Cell-Type-Specific GFP Infection
  8. 26 Compare Pseudovirus Infection Efficiency: LORGs vs. CORGs
SARS-CoV-2 Virus Infection and Drug Inhibition Studies Days 62–66 (LORGs for drug testing); Day 80+ (CORGs/Neural Cells for infection)
  1. 27 Treat LORGs with Viral Entry Inhibitors
  2. 28 Infect Inhibitor-Treated LORGs with SARS-CoV-2 Pseudovirus
  3. 29 Quantify Inhibition of Viral Infection
  4. 30 Test Inhibitors in Lung Epithelial Cell Line (Calu-3)
  5. 31 Assess Nafamostat in Patient-Derived SARS-CoV-2 Isolate
Live SARS-CoV-2 USA-WA1/2020 Infection and Host Response Analysis Days 62–65 (LORGs); Day 80–82 (NPCs/Neurons/Astrocytes)
  1. 32 Prepare Patient-Derived SARS-CoV-2 USA-WA1/2020 Inoculum
  2. 33 Infect Day 60 LORGs with USA-WA1/2020 Virus
  3. 34 Quantify Viral RNA in LORG Supernatant
  4. 35 Quantify Viral RNA in LORG Cellular Fraction
  5. 36 Analyze Immune Response Gene Expression in Infected LORGs
  6. 37 Analyze Inflammasome Pathway Gene Expression in Infected LORGs
  7. 38 Infect NPCs, Neurons, and Astrocytes with USA-WA1/2020
  8. 39 Quantify Viral RNA in Neural Cell Supernatant and Cellular Fractions
  9. 40 Analyze Innate Immunity Gene Expression in Infected Neurons
  10. 41 Analyze Complement System Gene Expression in Infected Neurons
  11. 42 Analyze Neuronal Dysfunction and Apoptosis Gene Expression
Optional: Large-Scale Organoid Culture in Bioreactors Days 80+
  1. 43 Transfer Organoids to Bioreactor System
  2. 44 Maintain Bioreactor Culture

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Marchiano et al.. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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