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BRAIN Publication-derived

Schizophrenia and Neurodevelopmental Disorder Cerebral Organoids

Full SOP with Academic or a plan

Source Johnstone et al., 2019 · Centre for Clinical Brain Sciences, University of Edinburgh · 10.1038/s41380-018-0292-1

Authors: Mandy Johnstone, Navneet A. Vasistha, Miruna C. Barbu, Owen Dando, Karen Burr, Edward Christopher, Sophie Glen, Christelle Robert, Rana Fetit, Kenneth G. Macleod, Matthew R. Livesey, David St. Clair, Douglas H. R. Blackwood, Kirsty Millar, Neil O. Carragher, Giles E. Hardingham, David J. A. Wyllie, Eve C. Johnstone, Heather C. Whalley, Andrew M. McIntosh, Stephen M. Lawrie, Siddharthan Chandran 8 phases Cell source: Patient-Derived iPSC (chromosome 16p13.11 microduplication)

Abstract

This protocol generates cerebral organoids and neural precursor cells (NPCs) from patient-derived and control induced pluripotent stem cells (iPSCs) carrying chromosome 16p13.11 microduplication to model neurodevelopmental deficits and test interventions targeting the NFκB p65 signaling pathway. The differentiation produces 3D cortical structures mimicking human brain development with reduced proliferation and altered radial glial cell division orientation in patient cells that can be rescued by pharmacological or genetic NFκB p65 activation.

Cell source
Patient-Derived iPSC (chromosome 16p13.11 microduplication)
Application
Disease modeling

Protocol overview

43 steps across 8 phases

Phase 1 · iPSC generation and validation Variable
  1. a iPSC generation from skin fibroblasts using episomal methods
  2. b iPSC quality control and karyotyping
  3. c Three germ layer differentiation validation
Phase 2 · Neural precursor cell (NPC) derivation and characterization 7-14 days
  1. a iPSC neuralisation to generate anterior neural precursor cells
  2. b NPC monolayer culture with rosette patterning
  3. c NPC characterization by immunocytochemistry
Phase 3 · NPC proliferation assays 7-10 days pre-assay plus assay day
  1. a Plate NPCs in default media (mitogen removal)
  2. b EdU labeling of proliferating cells
  3. c EdU chase and fixation
  4. d Cell permeabilization
  5. e EdU detection using Click-IT kit
  6. f Immunolabeling for NPC markers
  7. g Mounting and imaging
Phase 4 · Cerebral organoid generation and culture 30+ days
  1. a iPSC aggregate formation (Phase I)
  2. b Embryoid body formation and rosette patterning (Phase EB1)
  3. c EB maturation and ventricle-like cavity formation (Phase EB2)
  4. d Extended organoid culture (Phase EB3 onwards)
  5. e Organoid harvesting and processing
Phase 5 · Molecular analyses: RNA-seq and RPPA Variable based on processing
  1. a RNA isolation from NPCs
  2. b RNA-sequencing library preparation
  3. c Reverse-phase protein array (RPPA) sample preparation
  4. d RPPA antibody probing
  5. e RPPA data analysis and visualization
Phase 6 · Pharmacological NFκB p65 activation and rescue experiments 24-48 hours
  1. a Preparation of NFκB p65 activator compounds
  2. b Treatment of case and control NPCs with compounds
  3. c Assessment of NFκB p65 nuclear translocation by immunofluorescence
  4. d Validation of NFκB p65 activation by immunoblotting
  5. e Proliferation rescue assay
  6. f Statistical analysis of rescue experiments
Phase 7 · Genetic rescue: NFκB p65 lentivirus expression 48-72 hours post-infection
  1. a Lentivirus production
  2. b Lentiviral transduction of NPCs
  3. c Media replacement and recovery
  4. d Verification of NFκB p65 overexpression
  5. e Proliferation rescue assay post-lentiviral infection
  6. f Statistical validation of genetic rescue
Phase 8 · Transfection with NDE1 construct 48-72 hours
  1. a NDE1 construct generation
  2. b NPC preparation for transfection
  3. c Electroporation of NPCs
  4. d Post-transfection recovery
  5. e Confirmation of NDE1 overexpression by immunofluorescence
  6. f Quantification of NDE1 overexpression by immunoblotting
  7. g Proliferation assay in NDE1-overexpressing cells
  8. h Statistical analysis of NDE1 effects

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Attribution

This SOP was written by Organthis based on the published method in Johnstone et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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