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BRAIN Publication-derived

Generation and Characterization of Patient-Derived iPS Cells and Isogenic Lines; Differentiation into Forebrain Neurons; and Electrophysiological and Gene Expression Analysis

Source Wen et al., 2014 · Johns Hopkins University School of Medicine · 10.1038/nature13716

👤 Zhexing Wen, Ha Nam Nguyen, Ziyuan Guo, Matthew A. Lalli, Xinyuan Wang, Yijing Su, Nam-Shik Kim, Ki-Jun Yoon, Jaehoon Shin, Ce Zhang, Georgia Makri, David Nauen, Huimei Yu, Elmer Guzman, Cheng-Hsuan Chiang, Nadine Yoritomo, Kozo Kaibuchi, Jizhong Zou, Kimberly M. Christian, Linzhao Cheng, Christopher A. Ross, Russell L. Margolis, Gong Chen, Kenneth S. Kosik, Hongjun Song, Guo-li Ming ⏱ 42 days 📋 10 phases 🧫 Patient-Derived iPSC (Schizophrenia and Major Depression with DISC1 frameshift mutation)

Abstract

This protocol describes the generation of induced pluripotent stem (iPS) cells from four members of a family carrying a frameshift DISC1 mutation that co-segregates with psychiatric disorders, differentiation into forebrain-specific neurons, and characterization of synaptic deficits via electrophysiology, imaging, and transcriptomics. The protocol reveals that mutant DISC1 causes synaptic vesicle release deficits and widespread transcriptional dysregulation in human forebrain neurons, providing insight into the molecular etiopathology of schizophrenia and related psychiatric disorders.

Cell source
Patient-Derived iPSC (Schizophrenia and Major Depression with DISC1 frameshift mutation)
Application
Disease modeling

Protocol overview

95 steps across 10 phases

iPS Cell Generation and Characterization Days 1–42
  1. 1 Prepare mouse embryonic fibroblasts (MEFs)
  2. 2 Obtain skin biopsy fibroblasts
  3. 3 Transfect fibroblasts with reprogramming vectors
  4. 4 Culture and select iPS cell colonies
  5. 5 Passage iPS cells using collagenase
  6. 6 Perform karyotyping analysis
  7. 7 Perform genotyping of DISC1 locus
  8. 8 Perform bisulphite genomic sequencing
  9. 9 Perform teratoma formation assay
  10. 10 Select iPS cell lines for downstream studies
Generation of Isogenic iPS Cell Lines via TALEN-Mediated Gene Editing Days 1–30
  1. 1 Design and construct TALEN vectors
  2. 2 Construct donor DNA vectors
  3. 3 Prepare iPS cells for electroporation
  4. 4 Electroporate TALEN and donor plasmids
  5. 5 Plate and recover electroporated cells
  6. 6 Select positive clones with hygromycin B
  7. 7 Sub-clone and expand resistant colonies
  8. 8 Remove PGK-hygromycin selection cassette
  9. 9 Verify genetic editing by Sanger sequencing
  10. 10 Validate isogenic iPS cell quality
Differentiation of iPS Cells into Forebrain Neural Progenitor Cells (hNPCs) Days 1–22
  1. 1 Initiate embryoid body (EB) formation
  2. 2 Culture EBs with daily medium changes
  3. 3 Transition to neural induction medium
  4. 4 Form neural tube-like rosettes
  5. 5 Maintain rosette cultures
  6. 6 Mechanically pick and suspend neural progenitor rosettes
  7. 7 Characterize hNPC identity
Differentiation of hNPCs into Forebrain Neurons Days 1–42
  1. 1 Dissociate neural progenitor spheres
  2. 2 Plate dissociated neurons on poly-D-lysine/laminin-coated coverslips
  3. 3 Maintain neuronal cultures
  4. 4 Plate neurons on astrocyte layer for electrophysiology
  5. 5 Characterize neuronal identity and subtype composition
Immunocytochemistry and Synaptic Analysis Days 1–5
  1. 1 Fix cells with paraformaldehyde
  2. 2 Permeabilize and block
  3. 3 Incubate with primary antibodies
  4. 4 Incubate with secondary antibodies
  5. 5 Image with confocal or widefield microscopy
  6. 6 Quantify SV2+ synaptic bouton density
  7. 7 Quantify SYN1/PSD95 pair density
Whole-Cell Patch-Clamp Electrophysiology Days 1–2
  1. 1 Prepare recording chamber and perfusion system
  2. 2 Pull patch pipettes
  3. 3 Fill pipettes with internal solution
  4. 4 Form gigaseal and establish whole-cell configuration
  5. 5 Record spontaneous synaptic currents (SSCs)
  6. 6 Apply pharmacological agents if needed
  7. 7 Analyze SSC events
  8. 8 Perform IV relationship measurements
FM1-43 Vesicle Release Imaging Days 1–2
  1. 1 Load neurons with FM1-43 dye
  2. 2 Wash with low-concentration FM1-43 to remove extracellular dye
  3. 3 Final wash with FMIB
  4. 4 Set up FM1-43 imaging system
  5. 5 Acquire baseline FM1-43 fluorescence
  6. 6 Stimulate vesicle release with depolarization
  7. 7 Analyze FM1-43 imaging data
  8. 8 Quantify vesicle release kinetics
RNA Extraction, qRT-PCR, and RNA-Seq Analysis Days 1–5
  1. 1 Harvest neurons for RNA extraction
  2. 2 Extract total RNA
  3. 3 Synthesize cDNA for qRT-PCR
  4. 4 Perform quantitative RT-PCR (qRT-PCR)
  5. 5 Prepare libraries for RNA-seq
  6. 6 Sequence libraries on Ion Proton Torrent
  7. 7 Map reads to reference genome
  8. 8 Perform quality control on alignments
  9. 9 Count reads covering gene coding regions
  10. 10 Perform differential expression analysis
  11. 11 Perform Gene Ontology (GO) enrichment analysis
  12. 12 Identify differentially expressed disease-associated genes
Biochemical Analyses (Western Blot and Immunoprecipitation) Days 1–3
  1. 1 Prepare DNA constructs for DISC1
  2. 2 Culture HEK293 cells
  3. 3 Transiently transfect HEK293 cells
  4. 4 Harvest transfected cells
  5. 5 Lyse cells in RIPA buffer
  6. 6 Remove insoluble fraction by centrifugation
  7. 7 Quantify protein concentration
  8. 8 Prepare protein samples for SDS-PAGE
  9. 9 Perform SDS-PAGE
  10. 10 Transfer proteins to nitrocellulose membrane
  11. 11 Block non-specific binding
  12. 12 Incubate with primary antibodies
  13. 13 Incubate with secondary antibodies
  14. 14 Detect signal with chemiluminescence
  15. 15 Perform co-immunoprecipitation (co-IP)
  16. 16 Wash immunoprecipitate
  17. 17 Elute and analyze co-IP complexes
  18. 18 Analyze ubiquitination in co-IP experiments
Data Collection, Statistical Analysis, and Quality Control Throughout experiment
  1. 1 Replicate experiments
  2. 2 Perform blinded analysis
  3. 3 Report sample sizes and statistics
  4. 4 Perform ANOVA for multiple group comparisons
  5. 5 Use Kolmogorov-Smirnov test for electrophysiology
  6. 6 Assess data normality and variance homogeneity
  7. 7 Report effect sizes and confidence intervals
  8. 8 Control for multiple testing
  9. 9 Validate findings with independent methods
  10. 10 Generate summary statistics and figures

Full SOP

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Attribution

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

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