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BRAIN Publication-derived

ApoE-Isoform-Dependent SARS-CoV-2 Neurotropism and Cellular Response

Source Wang et al., 2021 · Beckman Research Institute of City of Hope · 10.1016/j.stem.2020.12.018

👤 Cheng Wang, Mingzi Zhang, Gustavo Garcia Jr., E. Tian, Qi Cui, Xianwei Chen, Guihua Sun, Jinhui Wang, Vaithilingaraja Arumugaswami, Yanhong Shi ⏱ 144 days 📋 11 phases 🧫 Human iPSC

Abstract

This protocol uses hiPSC-derived neurons, astrocytes, and brain organoids to model SARS-CoV-2 neurotropism and characterize ApoE isoform-dependent differences in viral infection and cellular response. The protocol generates isogenic ApoE3/3 and ApoE4/4 neural cell types to investigate susceptibility to viral infection and demonstrates that remdesivir can inhibit SARS-CoV-2 infection in brain cells.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

56 steps across 11 phases

Generation of Isogenic iPSC Lines Days 0-30
  1. 1 Obtain and Culture Parental Fibroblasts
  2. 2 Reprogramming Fibroblasts to iPSCs
  3. 3 CRISPR/Cas9 Gene Editing for ApoE Conversion
  4. 4 Verify Isogenic iPSC Lines
Differentiation of Neural Progenitor Cells (NPCs) Days 0-7
  1. 1 NPC Induction from hiPSCs
  2. 2 Continue NPC Induction
  3. 3 Harvest NPCs
Differentiation of Neurons from NPCs Days 0-22
  1. 1 Prepare NGN2/ASCL1 Viral Induction
  2. 2 Viral Transduction
  3. 3 Puromycin Selection
  4. 4 Neural Induction
  5. 5 Dissociate and Replate Neurons
  6. 6 Neuronal Maturation
Differentiation of Astrocytes from NPCs Days 0-42
  1. 1 Initial Astrocyte Induction
  2. 2 Dissociate and Replate NPCs
  3. 3 Continued Induction
  4. 4 Switch to Growth Factor Medium
  5. 5 Astrocyte Maturation
  6. 6 Optional CD49f+ Sorting for Astrocyte Purification
Brain Organoid Derivation Days 0-60+
  1. 1 Embryoid Body Formation
  2. 2 EB Culture and Medium Change
  3. 3 Switch to Neural Induction Medium
  4. 4 Embed in Matrigel and Plate
  5. 5 Neural Rosette Formation
  6. 6 Transfer to Bioreactor for Large-Scale Culture
  7. 7 Optional Astrocyte Induction via Matrigel Treatment
Neuron-Astrocyte Co-cultures Days 0-21
  1. 1 Prepare Astrocytes
  2. 2 Seed Neurons onto Astrocytes
  3. 3 Remove Proliferating Cells
  4. 4 Co-culture Maturation
SARS-CoV-2 Infection Day 0 (24-144 hours post-infection)
  1. 1 Prepare Viral Inoculum
  2. 2 Mock Infection Control
  3. 3 Infect Cells with Viral Inoculum
  4. 4 Replace Inoculum with Fresh Medium
  5. 5 Incubate Infected Cultures
  6. 6 Harvest Cells for Analysis
Remdesivir Treatment Days -1 to 3 (relative to infection)
  1. 1 Pre-treat Cells with Remdesivir
  2. 2 Infect with SARS-CoV-2
  3. 3 Continue Remdesivir Treatment
  4. 4 Harvest Treated Cells
Immunostaining and Analysis Variable (post-infection timepoints)
  1. 1 Fix Cells for Immunostaining
  2. 2 Prepare Brain Organoid Sections
  3. 3 Permeabilize and Block
  4. 4 Primary Antibody Incubation
  5. 5 Secondary Antibody Incubation
  6. 6 DAPI Staining and Mounting
  7. 7 Imaging and Quantification
RNA Extraction and qRT-PCR Analysis Variable (post-infection timepoints)
  1. 1 Extract RNA from Cells or Organoids
  2. 2 Synthesize cDNA
  3. 3 Perform qRT-PCR
  4. 4 Data Analysis
Statistical Analysis Post-data collection
  1. 1 Organize Quantitative Data
  2. 2 Assess Data Distribution
  3. 3 Perform Statistical Testing
  4. 4 Calculate Error Metrics
  5. 5 Generate Summary Tables and Figures

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wang et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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