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BRAIN Publication-derived

Cerebral Organoids Reveal Early Cortical Maldevelopment in Schizophrenia: Differentiation and Analysis Protocol

Source Stachowiak et al. · <UNKNOWN>

👤 E.K. Stachowiak, C.A. Benson, S.T. Narla, A. Dimitri, L. Bayona Chuye, S. Dhiman, K. Harikrishnan, S. Elahi, D. Freedman, K.J. Brennand, P. Sarder, M.K. Stachowiak ⏱ 20 days 📋 7 phases 🧫 Human ESC (H9, HUES8) and Patient-Derived iPSC (Control and Schizophrenia)

Abstract

This protocol describes the generation of cerebral organoids from human embryonic stem cells (H9, HUES8) and induced pluripotent stem cells to model early cortical development and investigate maldevelopment associated with schizophrenia. The protocol includes neural induction, organoid formation, immunocytochemical analysis, and computational morphological assessment of proliferation and neuronal organization.

Cell source
Human ESC (H9, HUES8) and Patient-Derived iPSC (Control and Schizophrenia)
Application
Disease modeling

Protocol overview

48 steps across 7 phases

hESC Maintenance and Neural Induction to NPCs Day 0 to Day 7 (EB formation and SMAD inhibition)
  1. 1 Seed hESCs on Matrigel
  2. 2 Prepare Embryoid Bodies (EBs)
  3. 3 EB Formation and SMAD Inhibition
  4. 4 Plate EBs on Polyornithine/Laminin-Coated Plates
  5. 5 Rosette Selection
  6. 6 Culture NPCs in NPC Medium
hESC Cerebral Organoid Generation Day 0 to Day 20+ (EB formation through organoid maturation)
  1. 1 Collagenase Treatment and EB Formation
  2. 2 Feed EBs with SMAD Inhibitors
  3. 3 Transfer EBs to 24-Well Low-Attachment Plates
  4. 4 Neuroepithelial Tissue Formation
  5. 5 Transfer Neuroepithelia to Matrigel Droplets
  6. 6 Stationary Culture (No Vitamin A)
  7. 7 Transfer to Orbital Shaker and Continued Culture
  8. 8 Harvest and Processing
  9. 9 Cryosectioning
iPSC Cerebral Organoid Generation Day 0 to Day 20+ (culture to organoid maturation)
  1. 1 Maintain iPSCs and HUES8 on MEF Feeder Plates
  2. 2 EB Formation (First 4–5 Days)
  3. 3 SMAD Inhibition (4–5 Days)
  4. 4 Neural Induction (4 Days)
  5. 5 Matrigel Droplet Culture (4 Days Stationary)
  6. 6 Orbital Shaker Culture with Vitamin A
  7. 7 Harvest and Cryopreservation
  8. 8 Cryosectioning
Immunocytochemical Staining and Microscopy Day 0 to Day 2 (staining and imaging)
  1. 1 Rehydration and Permeabilization
  2. 2 Blocking
  3. 3 Primary Antibody Incubation
  4. 4 Secondary Antibody Incubation
  5. 5 Double Immunostaining (if required)
  6. 6 Quality Control and Verification
  7. 7 Fluorescence Microscopy
NPC Differentiation and Neuronal Commitment (for transfection and RNAseq) Day 0 to Day 2 (NPC to neuronal committed cell differentiation)
  1. 1 Culture hESC H9-Derived NPCs
  2. 2 Initiate Neuronal Differentiation
  3. 3 Harvest Neuronal Committed Cells (NCCs)
Transfection of FGFR1 Constructs and RNAseq Day 0 to Day 3 (transfection through harvest for RNAseq)
  1. 1 Prepare FGFR1 Constructs
  2. 2 Transfect hESC-Derived NPCs
  3. 3 Post-Transfection Incubation (Non-Differentiated NPCs)
  4. 4 Post-Transfection with Neuronal Differentiation (NCCs)
  5. 5 Pool Transfected Cells for RNAseq
  6. 6 RNA Extraction
  7. 7 RNA Library Preparation
  8. 8 Sequencing on Illumina Hi-seq 2500
  9. 9 cDNA Synthesis for Independent mRNA Assays
  10. 10 RNAseq Data Analysis
Quantitative Image Analysis and Fluorescence Intensity Measurement Variable (performed post-immunostaining)
  1. 1 Reelin Concentration Measurement
  2. 2 Pan-Neu Neuronal Marker Measurement
  3. 3 Fluorescence Intensity Measurement (FIM)
  4. 4 BrdU, FGFR1, and TBR1 Nuclear Counting
  5. 5 Multi-Sample Analysis

Full SOP

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Attribution

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

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