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BRAIN Publication-derived

COVID-19 Brain Models (2021)

Full SOP with Academic or a plan

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

Authors: Cheng Wang, Mingzi Zhang, Gustavo Garcia Jr., E. Tian, Qi Cui, Xianwei Chen, Guihua Sun, Jinhui Wang, Vaithilingaraja Arumugaswami, Yanhong Shi Duration: 144 days 11 phases Cell source: 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

Phase 1 · Generation of Isogenic iPSC Lines Days 0-30
  1. a Obtain and Culture Parental Fibroblasts
  2. b Reprogramming Fibroblasts to iPSCs
  3. c CRISPR/Cas9 Gene Editing for ApoE Conversion
  4. d Verify Isogenic iPSC Lines
Phase 2 · Differentiation of Neural Progenitor Cells (NPCs) Days 0-7
  1. a NPC Induction from hiPSCs
  2. b Continue NPC Induction
  3. c Harvest NPCs
Phase 3 · Differentiation of Neurons from NPCs Days 0-22
  1. a Prepare NGN2/ASCL1 Viral Induction
  2. b Viral Transduction
  3. c Puromycin Selection
  4. d Neural Induction
  5. e Dissociate and Replate Neurons
  6. f Neuronal Maturation
Phase 4 · Differentiation of Astrocytes from NPCs Days 0-42
  1. a Initial Astrocyte Induction
  2. b Dissociate and Replate NPCs
  3. c Continued Induction
  4. d Switch to Growth Factor Medium
  5. e Astrocyte Maturation
  6. f Optional CD49f+ Sorting for Astrocyte Purification
Phase 5 · Brain Organoid Derivation Days 0-60+
  1. a Embryoid Body Formation
  2. b EB Culture and Medium Change
  3. c Switch to Neural Induction Medium
  4. d Embed in Matrigel and Plate
  5. e Neural Rosette Formation
  6. f Transfer to Bioreactor for Large-Scale Culture
  7. g Optional Astrocyte Induction via Matrigel Treatment
Phase 6 · Neuron-Astrocyte Co-cultures Days 0-21
  1. a Prepare Astrocytes
  2. b Seed Neurons onto Astrocytes
  3. c Remove Proliferating Cells
  4. d Co-culture Maturation
Phase 7 · SARS-CoV-2 Infection Day 0 (24-144 hours post-infection)
  1. a Prepare Viral Inoculum
  2. b Mock Infection Control
  3. c Infect Cells with Viral Inoculum
  4. d Replace Inoculum with Fresh Medium
  5. e Incubate Infected Cultures
  6. f Harvest Cells for Analysis
Phase 8 · Remdesivir Treatment Days -1 to 3 (relative to infection)
  1. a Pre-treat Cells with Remdesivir
  2. b Infect with SARS-CoV-2
  3. c Continue Remdesivir Treatment
  4. d Harvest Treated Cells
Phase 9 · Immunostaining and Analysis Variable (post-infection timepoints)
  1. a Fix Cells for Immunostaining
  2. b Prepare Brain Organoid Sections
  3. c Permeabilize and Block
  4. d Primary Antibody Incubation
  5. e Secondary Antibody Incubation
  6. f DAPI Staining and Mounting
  7. g Imaging and Quantification
Phase 10 · RNA Extraction and qRT-PCR Analysis Variable (post-infection timepoints)
  1. a Extract RNA from Cells or Organoids
  2. b Synthesize cDNA
  3. c Perform qRT-PCR
  4. d Data Analysis
Phase 11 · Statistical Analysis Post-data collection
  1. a Organize Quantitative Data
  2. b Assess Data Distribution
  3. c Perform Statistical Testing
  4. d Calculate Error Metrics
  5. e Generate Summary Tables and Figures

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Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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