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BRAIN Publication-derived

Neuroinvasion of SARS-CoV-2 in Human Brain Organoids and Mouse Models

Source Song et al., 2021 · Yale School of Medicine · 10.1084/jem.20202135

👤 Eric Song, Ce Zhang, Benjamin Israelow, Alice Lu-Culligan, Akiko Iwasaki ⏱ 68 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol establishes methods to investigate SARS-CoV-2 neuroinvasion using three complementary approaches: human brain organoids derived from induced pluripotent stem cells, transgenic mice expressing human ACE2, and postmortem analysis of COVID-19 patient brain tissue. The organoid model enables single-cell transcriptional profiling to characterize viral tropism, cellular metabolic changes, and neuronal death without robust interferon responses.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

71 steps across 12 phases

iPSC Culture and Neural Differentiation Days 1-12
  1. 1 Culture human iPSC lines
  2. 2 Differentiate iPSCs to neural progenitor cells
  3. 3 Characterize NPCs by immunostaining
Cerebral Organoid Generation Days 13-63
  1. 1 Prepare embryoid bodies
  2. 2 Generate cerebral organoids
  3. 3 Confirm dorsal cortical identity
SARS-CoV-2 Virus Production Days 1-14
  1. 1 Generate P1 viral stock
  2. 2 Generate working viral stock
  3. 3 Concentrate virus by PEG precipitation
  4. 4 Harvest and resuspend precipitated virus
  5. 5 Determine viral titer by plaque assay
SARS-CoV-2 Infection of Brain Organoids Days 64-67
  1. 1 Prepare organoids for infection
  2. 2 Infect organoids with SARS-CoV-2
  3. 3 Collect organoids at multiple time points
ACE2 Blocking and Neutralization Studies Days 64-67
  1. 1 Prepare organoids for ACE2 blocking
  2. 2 Infect antibody-pretreated organoids
  3. 3 Prepare cerebrospinal fluid (CSF) from COVID-19 patient
  4. 4 Perform ELISA for anti-SARS-CoV-2 antibodies
  5. 5 Perform neutralization assay
Immunostaining and Microscopy Analysis Days 64-68
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Prepare cryostat sections
  3. 3 Block and permeabilize sections
  4. 4 Perform primary antibody staining
  5. 5 Perform TUNEL staining
  6. 6 Apply secondary antibodies and mount
  7. 7 Acquire confocal images
Single-Cell RNA Sequencing Days 64-68
  1. 1 Prepare single-cell suspension
  2. 2 Load cells onto 10x Chromium
  3. 3 Prepare single-cell RNA-seq libraries
  4. 4 Sequence libraries on NovaSeq
  5. 5 Demultiplex and align reads
  6. 6 Generate count matrices and integrate samples
  7. 7 Perform dimensionality reduction and clustering
  8. 8 Annotate cell types
  9. 9 Identify infected cells
  10. 10 Perform differential expression analysis
Electron Microscopy Days 64-68
  1. 1 Fix organoid tissue
  2. 2 Dehydrate tissue
  3. 3 Embed and section tissue
  4. 4 Image with transmission electron microscopy
In Vivo Infection - Mouse Models Days 1-7
  1. 1 Prepare transgenic mice
  2. 2 Perform intranasal SARS-CoV-2 infection
  3. 3 Collect brain tissue at defined time points
  4. 4 Quantify viral RNA by RT-qPCR
  5. 5 Determine viral titer by plaque assay
Whole-Brain Imaging and Analysis Days 7-10
  1. 1 Prepare infected brains for iDISCO+
  2. 2 Perform dichloromethane and peroxide bleaching
  3. 3 Rehydrate and permeabilize brains
  4. 4 Block and immunostain with primary antibodies
  5. 5 Apply secondary antibodies
  6. 6 Clear brains and prepare for imaging
  7. 7 Perform light sheet microscopy imaging
  8. 8 Analyze viral distribution with ClearMap
AAV-Mediated hACE2 Expression and Tissue-Specific Infection Days 1-10
  1. 1 Prepare AAV-hACE2 virus
  2. 2 Perform intratracheal AAV injection
  3. 3 Perform intracisternal magna AAV injection
  4. 4 Administer post-operative care
  5. 5 Allow AAV expression time
  6. 6 Perform intranasal SARS-CoV-2 infection of lung-hACE2 mice
  7. 7 Perform intraventricular SARS-CoV-2 infection of brain-hACE2 mice
  8. 8 Monitor weight and survival
Human Postmortem Brain Analysis Variable
  1. 1 Obtain human autopsy tissues
  2. 2 Prepare FFPE sections
  3. 3 Perform antigen retrieval
  4. 4 Stain with H&E
  5. 5 Block and permeabilize tissue sections
  6. 6 Perform primary antibody staining
  7. 7 Apply secondary antibodies
  8. 8 Quench lipofuscin autofluorescence
  9. 9 Mount and image sections
  10. 10 Perform immunohistochemistry for semiquantitative analysis

Full SOP

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Attribution

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

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