Skip to content
Back to browse
BRAIN Publication-derived

Microcephaly Cerebral Organoids for Disease Modelling

Full SOP with Academic or a plan

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

Authors: Jason Dang, Shashi Kant Tiwari, Gianluigi Lichinchi, Yue Qin, Veena S. Patil, Alexey M. Eroshkin, Tariq M. Rana Duration: 15 days 5 phases Cell source: 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

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

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Dang et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence