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BRAIN Publication-derived

Generation and Characterization of Cerebral Cortex Organoids from 22q11.2 Deletion Syndrome Patient iPSCs

Source Rao et al., 2025 · Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University · 10.1038/s41467-025-62187-x

👤 Sneha B. Rao, Zhixiong Sun, Francesco Brundu, Yannan Chen, Yan Sun, Huixiang Zhu, Robert J. Shprintzen, Raju Tomer, Raul Rabadan, Kam W. Leong, Sander Markx, Steven A. Kushner, Bin Xu, Joseph A. Gogos ⏱ 150 days 📋 10 phases 🧫 Patient-Derived iPSC (22q11.2 deletion syndrome with schizophrenia)

Abstract

This protocol generates dorsal forebrain cerebral organoids from patient-derived iPSCs carrying the canonical 3-Mb 22q11.2 deletion and schizophrenia diagnosis. The organoids recapitulate abnormal neurodevelopmental trajectories including delayed cortical neuron maturation, increased neural progenitor proliferation, and reduced mature neuron proportions, serving as a model to study disease mechanisms in early human brain development.

Cell source
Patient-Derived iPSC (22q11.2 deletion syndrome with schizophrenia)
Application
Disease modeling and neurodevelopmental study of 22q11.2 deletion syndrome-associated schizophrenia

Protocol overview

51 steps across 10 phases

iPSC Line Preparation and Characterization Pre-experiment
  1. 1 iPSC generation via reprogramming
  2. 2 Confirm iPSC stemness markers
  3. 3 Validate genomic stability and genotype
Dorsal Forebrain Organoid Generation DIV 0-40
  1. 1 Initiate forebrain patterning
  2. 2 Transition to maintenance media (DIV 40)
Early Organoid Development and Characterization DIV 40-70
  1. 1 Maintain organoids in N2/FBS media
  2. 2 Confirm forebrain identity via immunostaining (DIV70)
  3. 3 Identify cell type composition markers
  4. 4 Measure organoid growth rate (DIV14-DIV140)
Mid-Stage Organoid Maturation and Electrophysiological Assessment DIV 70-150
  1. 1 Continue organoid culture with enriched media (DIV 80+)
  2. 2 Assess neuronal activity via Ca²⁺ imaging (DIV 259)
  3. 3 Quantify Ca²⁺ peak amplitude
Single-Cell RNA Sequencing Sample Preparation DIV 70 and DIV 150
  1. 1 Prepare single-cell suspensions
  2. 2 Perform viability assessment and cell counting
  3. 3 Load cells onto 10x Chromium platform
  4. 4 Perform library preparation and sequencing
Quality Control and Cell Type Annotation Post-sequencing
  1. 1 Aggregate and normalize raw sequencing data
  2. 2 Perform quality control filtering
  3. 3 Normalize and integrate data
  4. 4 Perform clustering and UMAP visualization
  5. 5 Annotate cell types using SingleR
  6. 6 Verify 22q11.2 locus gene expression
Neural Progenitor Cell Proliferation Analysis DIV 20-70
  1. 1 Immunostaining for Ki67 and SOX2 (DIV70 organoids)
  2. 2 Secondary antibody incubation
  3. 3 Image acquisition and analysis
  4. 4 Quantify and compare NPC populations
  5. 5 PIP-FUCCI cell cycle phase analysis (DIV20 NPCs)
  6. 6 Quantify cell cycle phase distribution
  7. 7 FACS-based neurogenesis assay (DIV20-30)
  8. 8 FACS analysis and quantification
Neuronal Morphology Analysis DIV 55-70
  1. 1 Transduction with AAV-hSYN-eGFP (DIV 55-57)
  2. 2 Organoid immobilization (DIV 67)
  3. 3 Fixation and immunostaining (DIV 70)
  4. 4 Whole-mount imaging and 3D neurite tracing
  5. 5 Quantify morphological parameters
  6. 6 Verify neuronal cell identity
Bulk RNA-seq of Sorted Cortical Neurons DIV 50-70
  1. 1 AAV-hSYN-GFP transduction (DIV 50-55)
  2. 2 Organoid dissociation and FACS sorting
  3. 3 RNA extraction and quality control
  4. 4 Library preparation and sequencing
  5. 5 Read alignment and quantification
  6. 6 Quality control and PCA analysis
  7. 7 Gene ontology enrichment analysis
  8. 8 Synaptic gene enrichment analysis
microRNA Sequencing and Analysis DIV 70
  1. 1 miRNA-enriched RNA extraction
  2. 2 RNA quality assessment
  3. 3 Small RNA library preparation
  4. 4 Sequencing
  5. 5 Differential miRNA expression analysis
  6. 6 miRNA target enrichment analysis
  7. 7 Assess overlap between miRNA targets and dysregulated genes

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Rao et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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