BRAIN Publication-derived

Protection of ZIKV infection-induced neuropathy by abrogation of acute antiviral response in human neural progenitors

Source Liu et al., 2019 · Brain and Spinal Cord Innovative Research Center, Tongji Hospital, Tongji University School of Medicine, Shanghai · 10.1038/s41418-019-0324-7

👤 Ling Liu, Zhenyu Chen, Xin Zhang, Shun Li, Yi Hui, Hexi Feng, Yanhua Du, Guohua Jin, Xiaohui Zhou, Xiaoqing Zhang ⏱ 45 days 📋 12 phases 🧫 Human ESC (H9)

Abstract

This protocol generates forebrain dorsal (FD), forebrain ventral (FV), and hindbrain & spinal cord (H&S) neural progenitor cell (NPC) organoids from human embryonic stem cells (hESCs) for studying Zika virus (ZIKV) infection-induced microcephaly. The protocol establishes regional brain organoids, infects them with ZIKV strain SZ01, and assesses viral pathogenesis through analysis of cell growth arrest, apoptosis, and IFN-independent antiviral gene activation.

Cell source

Human ESC (H9)

Application

Disease modeling

Protocol overview

43 steps across 12 phases

hESC maintenance and preparation Passages 25-45 (ongoing)

1 Culture human ESCs on MEF feeders

Generation of p65 or IRF3 knockout hESC lines (optional for KO studies) Days 0-17 post-electroporation

1 Pre-electroporation treatment with Rho kinase inhibitor
2 Prepare single-cell suspension
3 Electroporate with CRISPR/Cas9 components
4 Puromycin selection
5 Clone selection and genotyping

Neural differentiation to regional NPCs Days 0-23

1 EB formation
2 Neural induction
3 Plating on laminin-coated surface
4 Regional patterning (FD identity - no morphogen)
5 Regional patterning (FV identity - Shh treatment)
6 Regional patterning (H&S identity - RA treatment)
7 Organoid maturation

ZIKV infection of regional organoids Days 17-23

1 Virus preparation and titration
2 Infection of organoids
3 Mock infection control

Analysis of viral infection and organoid growth Days 17-23 post-infection

1 Light microscopy imaging
2 Immunostaining for viral envelope protein
3 BrdU incorporation assay
4 Immunostaining for proliferation markers (Ki67, pH3)

Assessment of apoptosis and cell death Days 17-23 post-infection

1 Annexin V-FITC/PI flow cytometry
2 Immunostaining for cleaved caspase-3

Quantitative RT-PCR analysis of viral RNA and gene expression Days 17-23 post-infection

1 RNA extraction
2 cDNA synthesis
3 Quantitative RT-PCR

Western blot analysis Days 17-23 post-infection

1 Protein extraction
2 SDS-PAGE and transfer
3 Detection and quantification

IFNβ treatment studies Days 17-23

1 IFNβ or IFNγ treatment
2 Assess growth and ISG expression

Analysis of p65 and IRF3 knockout organoids Days 17-23 post-infection

1 Differentiate p65 or IRF3 KO hESCs to regional organoids
2 ZIKV infection of KO and WT organoids
3 Assess ISG expression in KO organoids
4 Measure viral load in KO organoids
5 Assess protection from growth arrest in KO organoids
6 Assess apoptosis in KO organoids

TLR3 pathway investigation Optional; days 17-23 post-infection

1 Assess basal TLR3 expression
2 TLR3 agonist treatment
3 Assess ZIKV pathology with TLR3 inhibition

Transcriptome profiling (RNA-seq) Days 17-23 post-infection; analysis later

1 Sample collection and RNA extraction
2 RNA-seq library preparation
3 Sequencing
4 Bioinformatics analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Liu et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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