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BRAIN Publication-derived

Zika Virus Depletion of Neural Progenitors in Human Cerebral Organoids through TLR3 Activation

Source Dang et al., 2016 · University of California San Diego, Department of Pediatrics · 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 ⏱ 30 days 📋 7 phases 🧫 Human ESC (H9 hESC, WA09)

Abstract

This protocol describes the generation of human embryonic stem cell-derived cerebral organoids to model early fetal brain development and investigate Zika virus (ZIKV) infection mechanisms. The protocol demonstrates ZIKV infection of neural progenitor cells via TLR3-mediated innate immune activation, leading to dysregulation of neurogenesis and increased apoptosis, recapitulating microcephaly-like phenotypes.

Cell source
Human ESC (H9 hESC, WA09)
Application
Disease modeling - Zika virus-induced microcephaly

Protocol overview

20 steps across 7 phases

hESC Preparation and Embryoid Body Formation Day 0 to Day 2
  1. 1 hESC Culture and Passaging
  2. 2 Hanging Drop Embryoid Body Formation
  3. 3 Transfer Embryoid Bodies to Culture Dishes
Neural Induction and Organoid Formation Day 2 to Day 15
  1. 4 Transfer to Neural Induction Media
  2. 5 Matrigel Embedding and Differentiation Media Transfer
  3. 6 Transfer to Bioreactors and Long-Term Culture
ZIKV Expansion and Viral Inoculation Day -3 to Day 0 (before infection experiment)
  1. 7 ZIKV Stock Expansion in Vero Cells
  2. 8 Viral Titer Quantification
  3. 9 Organoid Infection with ZIKV
Post-Infection Monitoring and Analysis Day 0 to Day 5 (post-infection)
  1. 10 Organoid Size Tracking
  2. 11 Viral Kinetics Assessment by RT-qPCR
  3. 12 Immunohistochemistry for ZIKV and Neural Markers
TLR3 Activation and Inhibition Studies Concurrent with Phase 4, Day 0 to Day 5
  1. 13 TLR3 Agonist Treatment with poly(I:C)
  2. 14 TLR3 Competitive Inhibitor Treatment
  3. 15 Size Quantification of Neurospheres and Organoids
Gene Expression Analysis Throughout infection (Day 0 to Day 5)
  1. 16 Total RNA Extraction
  2. 17 RT-qPCR Analysis of TLR3 and Target Genes
  3. 18 RNA-Seq Transcriptome Analysis
  4. 19 Pathway and Gene Ontology Analysis
Functional Validation - Calcium Imaging Day 20–30 of organoid culture (optional, for functional characterization)
  1. 20 Calcium Dye Imaging of Neural Activity

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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