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BRAIN Publication-derived

Brain Organoids for Infection and Development

Full SOP with Academic or a plan

Source Li et al., 2017 · The Whitehead Institute for Biomedical Research · 10.1016/j.stem.2016.11.017

Authors: Yun Li, Julien Muffat, Attya Omer, Irene Bosch, Madeline A. Lancaster, Mriganka Sur, Lee Gehrke, Juergen A. Knoblich, Rudolf Jaenisch Duration: 84 days 7 phases Cell source: Human ESC (WIBR1, WIBR2, WIBR3 lines)

Abstract

This protocol describes the generation of human cerebral organoids with enhanced expansion and surface folding through PTEN deletion, which activates the PI3K-AKT pathway and increases neural progenitor proliferation. The resulting organoids recapitulate key features of human cortical development and can be used to model cortical malformations such as those induced by Zika virus infection.

Cell source
Human ESC (WIBR1, WIBR2, WIBR3 lines)
Application
Disease modeling; developmental neuroscience

Protocol overview

23 steps across 7 phases

Phase 1 · ESC Culture and PTEN Gene Targeting Days 0-14
  1. a Prepare hESC lines for CRISPR targeting
  2. b Design and validate CRISPR/Cas9 guide RNA for PTEN exon 1
  3. c Electroporate hESCs with CRISPR/Cas9 construct
  4. d Apply puromycin selection
  5. e Pick and expand individual hESC clones
  6. f Verify PTEN protein ablation by immunoblotting
Phase 2 · Cerebral Organoid Culture and EB Formation Days 0-12
  1. a Dissociate and prepare single hESCs
  2. b Form embryoid bodies (EBs) in ultra-low-attachment plates
  3. c Transfer EBs to neural induction medium
  4. d Embed EBs in Matrigel
Phase 3 · Neural Maturation and Organoid Development Days 12-84
  1. a Culture embedded EBs in neural maturation medium (stationary phase)
  2. b Transfer to orbital shaker
  3. c Maintain organoid culture with medium changes
Phase 4 · Optional: Pre-patterning for Dorsal Forebrain Identity Days 0-14
  1. a Apply dorsomorphin treatment during early organoid development
  2. b Validate forebrain commitment
Phase 5 · Optional: AKT Inhibitor Treatment Days 1-42
  1. a Add AKT inhibitors to culture medium
Phase 6 · Optional: Lentivirus Transduction of hESCs Days -7 to 0
  1. a Produce VSVG-coated lentiviruses
  2. b Transduce hESCs with lentiviral particles
  3. c Validate transgene expression
Phase 7 · Optional: Zika Virus Infection Day 19 or Day 30 post-organoid formation
  1. a Prepare and titer Zika virus stock
  2. b Infect organoids at early stage (day 19)
  3. c Infect organoids at later stage (day 30)
  4. d Monitor ZIKV-induced apoptosis and growth defects

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Attribution

This SOP was written by Organthis based on the published method in Li et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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