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BRAIN Publication-derived

Schizophrenia Brain 2D Cell Cultures

Full SOP with Academic or a plan

Source Yoon et al., 2014 · Institute for Cell Engineering, Johns Hopkins University School of Medicine · 10.1016/j.stem.2014.05.003

Authors: Ki-Jun Yoon, Ha Nam Nguyen, Hongjun Song, Guo-li Ming Duration: 22 days 10 phases Cell source: Patient-Derived iPSC (15q11.2 microdeletion schizophrenia risk carriers)

Abstract

This protocol establishes patient-derived iPSC lines from individuals carrying 15q11.2 microdeletion and differentiates them into neural progenitor cells and cortical neural rosettes to model schizophrenia-associated neural stem cell deficits. The model reveals impairments in adherens junctions and apical polarity due to CYFIP1-haploinsufficiency and WAVE complex destabilization, providing mechanistic insights into neuropsychiatric disorder pathogenesis.

Cell source
Patient-Derived iPSC (15q11.2 microdeletion schizophrenia risk carriers)
Application
Disease modeling

Protocol overview

65 steps across 10 phases

Phase 1 · iPSC Generation and Characterization Continuous culture; ~7 days per passage
  1. a Derive iPSCs from skin fibroblasts
  2. b Culture iPSCs on MEF feeders
  3. c Perform DNA FISH to confirm 15q11.2 microdeletion
  4. d Verify pluripotency by immunostaining
  5. e Perform G-banding karyotype analysis
  6. f Perform teratoma formation assay
Phase 2 · Differentiation to Primitive Neural Progenitor Cells (pNPCs) and Characterization 6 days
  1. a Induce neural differentiation from iPSCs
  2. b Dissociate and replat pNPCs
  3. c Verify pNPC identity by immunostaining
  4. d Assess pNPC proliferation
  5. e Measure mRNA levels of 15q11.2 genes
Phase 3 · Neural Rosette Formation and Polarity Assessment 10-17 days (monolayer method); 19-22 days total (embryoid body method)
  1. a Initiate neural rosette formation (monolayer method)
  2. b Initiate neural rosette formation (embryoid body method)
  3. c Identify and quantify neural rosettes
  4. d Immunostain rosettes for apical polarity marker atypical PKCλ
  5. e Immunostain rosettes for adherens junction marker N-cadherin
  6. f Quantify polarity data
Phase 4 · WAVE Complex Analysis and CYFIP1 Complementation 3 days (western blot); 3 days (lentiviral infection)
  1. a Perform co-immunoprecipitation to identify WAVE complex interactions
  2. b Measure CYFIP1 and WAVE2 protein levels by western blot
  3. c Generate CYFIP1-complemented iPSC lines via lentiviral transduction
  4. d Verify WAVE2 rescue in complemented lines
  5. e Reduce CYFIP1 in control cells via shRNA knockdown
Phase 5 · Functional Rescue of Adherens Junction and Polarity Defects 19-22 days (embryoid body rosette formation + immunostaining)
  1. a Validate polarity defect using embryoid body protocol
  2. b Immunostain rosettes from complemented lines for atypical PKCλ
  3. c Analyze additional polarity markers (PAR3, β-catenin)
  4. d Analyze shRNA knockdown phenotype in control cells
  5. e Quantify and statistically analyze polarity rescue data
Phase 6 · In Utero Electroporation and Analyses of Developing Mouse Cortex E13.5 (electroporation) to E16.5 (analysis); 3 days total
  1. a Generate shRNA vectors against mouse Cyfip1
  2. b Prepare pregnant CD1 mice for electroporation
  3. c Inject plasmid DNA and perform in utero electroporation
  4. d Close surgical incision and allow recovery
  5. e Harvest brains at E16.5 (3 days post-electroporation)
  6. f Prepare brain sections for immunohistochemistry
  7. g Immunostain brain sections for CYFIP1, N-cadherin, and Pax6
  8. h Analyze N-cadherin expression at the ventricular surface
  9. i Perform quantitative analysis of electroporated cells
Phase 7 · Analysis of Ectopic RGC Localization and Proliferation E13.5 (electroporation) to E16.5 (EdU pulse); 3 days total
  1. a Quantify distribution of Pax6[+] cells in VZ vs. non-VZ regions
  2. b Assess rescue with shRNA-resistant CYFIP1 cDNA
  3. c Analyze M-phase marker phospho-Histone H3 to assess mitotic localization
  4. d Pulse embryos with EdU to assess RGC proliferation
  5. e Immunostain sections and quantify EdU incorporation
  6. f Analyze cell-cycle exit by EdU/Ki67 co-staining
Phase 8 · Analysis of IPCs and Cortical Neurons from CYFIP1-Deficient RGCs E13.5 (electroporation) to E16.5 (IPC analysis) and P5 (neuron analysis)
  1. a Analyze intermediate progenitor cell (IPC) distribution at E16.5
  2. b Quantify Pax6[+] and Tbr2[+] cell proportions
  3. c Analyze cortical neuron distribution at postnatal day 5 (P5)
  4. d Quantify distribution of CTIP2[+] and Cux1[+] neurons
  5. e Verify normal neuronal subtype specification
Phase 9 · WAVE Complex Signaling Analysis in Developing Mouse Cortex Variable (E14.5 co-IP; E13.5 electroporation to E16.5 analysis)
  1. a Perform co-immunoprecipitation in developing mouse cortex
  2. b Measure WAVE2 protein levels after Cyfip1 knockdown in mouse NPCs
  3. c Verify WAVE2 reduction at the ventricular surface in vivo
  4. d Generate shRNA vectors against mouse Abi1
  5. e Generate shRNA vectors against mouse Arp2/Arp3
  6. f Perform in utero electroporation with Abi1 or Arp2/3 knockdown
  7. g Quantify RGC distribution and adherens junction integrity
Phase 10 · Human Genetic Association and Epistatic Interaction Analyses Variable (postmortem tissue processing and genotyping: weeks to months)
  1. a Prepare postmortem human brain tissue for RNA sequencing
  2. b Prepare mRNA library and perform RNA-sequencing
  3. c Align sequencing reads and quantify gene expression
  4. d Perform cis-eQTL association analysis
  5. e Assess co-expression of WAVE signaling genes
  6. f Genotype DNA samples from postmortem brains
  7. g Analyze schizophrenia case-control cohorts
  8. h Test single SNP associations with schizophrenia
  9. i Perform pairwise SNP-SNP interaction analysis
  10. j Perform meta-analysis of SNP-SNP interaction
  11. k Analyze genotype-dependent epistatic effects

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Attribution

This SOP was written by Organthis based on the published method in Yoon et al., 2014. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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