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BRAIN Publication-derived

Modeling a Genetic Risk for Schizophrenia in iPSCs and Mice Reveals Neural Stem Cell Deficits Associated with Adherens Junctions and Polarity

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

👤 Ki-Jun Yoon, Ha Nam Nguyen, Hongjun Song, Guo-li Ming ⏱ 22 days 📋 10 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Yoon et al., 2014. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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