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BRAIN Publication-derived

HSV-1 Infection of hiPSC-Derived Neural Stem Cells and Cerebral Organoids for Neurodevelopmental Disorder Modeling

Source Qiao et al., 2020 · Department of Biomedical Engineering, Wuhan University School of Basic Medical Sciences · 10.1371/journal.ppat.1008899

👤 Haowen Qiao, Moujian Guo, Jia Shang, Wen Zhao, Zhenyan Wang, Nian Liu, Bin Li, Ying Zhou, Ying Wu, Pu Chen ⏱ 72 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of hiPSC-derived monolayer neurons, 3D neuroepithelial buds, and cerebral organoids, followed by HSV-1 infection to model neurodevelopmental disorder-associated neuropathological changes. The infected models recapitulate impaired neurogenesis, dysregulated neural differentiation, abnormal microglial activation, and dysregulated brain regionalization.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

57 steps across 9 phases

Generation of Monolayer Neuronal Differentiation Models Day 1–21
  1. 1 Culture hiPSCs in pluripotent state
  2. 2 Initiate neural induction
  3. 3 Harvest and expand neural stem cells
  4. 4 Seed NSCs for neuronal differentiation
  5. 5 Culture NSCs in SFM medium
  6. 6 Switch to neural differentiation medium
  7. 7 Change differentiation medium regularly
  8. 8 Add dibutyryl cAMP supplement
Generation of Human Cerebral Organoids Day 0–45+
  1. 1 Dissociate hiPSCs to single cells
  2. 2 Form embryoid bodies
  3. 3 Transfer EBs to ultra-low-attachment plates
  4. 4 Feed EBs every other day
  5. 5 Transfer EBs to low adhesion 24-well plates
  6. 6 Embed EBs in Matrigel
  7. 7 Culture embedded EBs in neural expansion medium
  8. 8 Transfer to orbital shaker
Viral Propagation and Titer Determination Variable
  1. 1 Propagate HSV-1 strain F in Vero cells
  2. 2 Harvest and freeze-thaw virus stocks
  3. 3 Filter virus stocks
  4. 4 Store virus stocks at -80°C
  5. 5 Perform plaque assay for titer determination
  6. 6 Incubate plates for plaque development
  7. 7 Fix and stain plaques
  8. 8 Calculate viral titer
Infection of Monolayer Neuronal Models Day 7–21 (post-differentiation)
  1. 1 Prepare cells for infection
  2. 2 Infect cells with HSV-1
  3. 3 Remove inoculum
  4. 4 Wash cells with preheated medium
  5. 5 Culture in fresh medium
Infection of 3D Brain Organoid Models Day 15–45
  1. 1 Prepare organoids for infection
  2. 2 Infect organoids with HSV-1
  3. 3 Maintain infected organoids in culture
Immunofluorescence Analysis Post-infection timepoints
  1. 1 Fix cells with paraformaldehyde
  2. 2 Block with serum
  3. 3 Permeabilize and block simultaneously
  4. 4 Incubate with primary antibodies
  5. 5 Incubate with secondary antibodies
  6. 6 Counterstain with DAPI
  7. 7 Image samples
  8. 8 Quantify fluorescence intensity
Real-Time PCR Analysis Post-infection timepoints
  1. 1 Extract total mRNA
  2. 2 Synthesize cDNA
  3. 3 Set up qRT-PCR reaction
  4. 4 Run qRT-PCR cycling program
  5. 5 Normalize and analyze expression
Flow Cytometry Apoptosis Analysis 24 hours post-infection
  1. 1 Harvest cells
  2. 2 Stain with Annexin V and PI
  3. 3 Analyze by flow cytometry
  4. 4 Calculate apoptotic percentage
RNA Sequencing and Pathway Analysis 72 hours post-infection
  1. 1 Infect NSCs with HSV-1
  2. 2 Remove inoculum
  3. 3 Culture in fresh medium
  4. 4 Harvest cells for RNA extraction
  5. 5 Extract RNA
  6. 6 Perform RNA sequencing
  7. 7 Perform GO and KEGG pathway analysis
  8. 8 Map genes to network databases

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Qiao et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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