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BRAIN Publication-derived

Zika Virus Infection of Human Cerebral Organoids and Analysis of TLR3-Mediated Neural Progenitor Depletion

Source Dang et al., 2016 · University of California San Diego School of Medicine · 10.1016/j.stem.2016.04.014

👤 Jason Dang, Shashi Kant Tiwari, Gianluigi Lichinchi, Yue Qin, Veena S. Patil, Alexey M. Eroshkin, Tariq M. Rana ⏱ 15 days 📋 5 phases 🧫 Human ESC (H9)

Abstract

This protocol describes the generation of human embryonic stem cell-derived cerebral organoids and their infection with Zika virus to model microcephaly-associated neural progenitor depletion. Organoids recapitulate early trimester fetal brain development and show regionalization of forebrain, midbrain, hippocampus and retinal tissues. ZIKV infection causes organoid growth attenuation and neural progenitor cell depletion through activation of the innate immune receptor TLR3.

Cell source
Human ESC (H9)
Application
Disease modeling

Protocol overview

25 steps across 5 phases

hESC Culture and Embryoid Body Formation Day 0-2
  1. 1 Prepare hESC for differentiation
  2. 2 Collect and dissociate hESC colonies
  3. 3 Form embryoid bodies by hanging drop method
  4. 4 Culture embryoid bodies in hanging drops
  5. 5 Transfer embryoid bodies to petri dishes
Neural Induction and Organoid Formation Day 2-15
  1. 1 Initiate neural induction
  2. 2 Embed organoids in Matrigel
  3. 3 Transfer organoids to bioreactors
  4. 4 Maintain long-term organoid culture
ZIKV Expansion and Organoid Infection Variable
  1. 1 Prepare ZIKV inoculum
  2. 2 Collect viral supernatant
  3. 3 Quantify viral titer
  4. 4 Infect organoids with ZIKV
  5. 5 Monitor organoid growth and viral kinetics
TLR3 Pathway Modulation and Analysis Variable
  1. 1 Treat neurospheres with poly(I:C)
  2. 2 Treat organoids with TLR3 competitive inhibitor
  3. 3 Co-treat organoids with ZIKV and TLR3 inhibitor
  4. 4 Measure organoid size changes
  5. 5 Assess immunostaining for ZIKV and neural markers
Gene Expression and Pathway Analysis Variable
  1. 1 Extract RNA from organoids and neurospheres
  2. 2 Quantify viral RNA by one-step RT-qPCR
  3. 3 Analyze TLR3 and target gene expression by RT-qPCR
  4. 4 Perform RNA-seq transcriptome analysis
  5. 5 Conduct gene ontology and pathway analysis
  6. 6 Correlate organoid transcriptome with fetal brain development

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Dang et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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