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BRAIN Publication-derived

Schizophrenia Brain Organoids with Gene Editing

Full SOP with Academic or a plan

Source Qu et al., 2023 · National University of Singapore, Yong Loo Lin School of Medicine · 10.1016/j.isci.2023.108497

Authors: Yinghua Qu, Jonathan Jun-Yong Lim, Omer An, Henry Yang, Yi-Chin Toh, John Jia En Chua Duration: 120 days 11 phases Cell source: Human ESC, Human iPSC

Abstract

This protocol describes the generation and characterization of FEZ1-knockout human cerebral organoids (hCOs) to model disrupted cortical development and neurodevelopmental disorders. FEZ1 deletion causes abnormal expansion of HOPX− outer radial glial cells, altered actin dynamics, ectopic neuroprogenitor migration, and cortical lamination defects, providing a model for understanding FEZ1-associated neurodevelopmental pathologies including schizophrenia and Jacobsen syndrome.

Cell source
Human ESC, Human iPSC
Application
Disease modeling

Protocol overview

82 steps across 11 phases

Phase 1 · FEZ1-Knockout hPSC Generation via CRISPR-Cas9 D0-D21
  1. a Design sgRNA and construct lentiviral vector
  2. b Produce lentivirus
  3. c Infect hPSCs with lentivirus
  4. d Select transduced cells with puromycin
  5. e Generate single-cell clones
  6. f Pick and expand individual clones
  7. g Genomic sequencing of FEZ1-KO clones
  8. h Karyotype analysis
Phase 2 · 2D Neural Epithelium (hNE) Induction D0-D6
  1. a Prepare hPSCs for differentiation
  2. b Prepare N2B27 differentiation medium
  3. c Culture hPSCs in N2B27 with daily medium changes
  4. d Harvest hNE cells
Phase 3 · Human Cerebral Organoid (hCO) Generation D0-D120
  1. a Generate embryoid bodies (EBs)
  2. b Culture EBs (D0-D5)
  3. c Transfer to induction medium (D5-D7)
  4. d Embed in Matrigel and initiate expansion (D7)
  5. e Replace with maturation medium (D10)
  6. f Maintain hCOs with medium changes (D10-D120)
  7. g Harvest hCOs at designated timepoints
Phase 4 · Immunofluorescence Staining and Imaging D10-D60
  1. a Fix hCOs
  2. b Wash and permeabilize hCOs
  3. c Block hCOs
  4. d Incubate with primary antibodies
  5. e Wash hCOs
  6. f Incubate with secondary antibodies and DAPI
  7. g Final wash and clearing
  8. h Mount and image on confocal microscope
Phase 5 · RNA Extraction and qPCR Analysis D5-D60
  1. a Harvest hCOs or hNE cells
  2. b Extract total RNA
  3. c Synthesize cDNA
  4. d Design and validate qPCR primers
  5. e Perform real-time qPCR
  6. f Analyze qPCR data
Phase 6 · Bulk RNA-Sequencing (RNA-seq) and Analysis D10
  1. a Harvest D10 hCOs
  2. b Extract high-quality RNA
  3. c Prepare RNA-seq libraries
  4. d Perform single-end or paired-end sequencing
  5. e Align reads and quantify gene expression
  6. f Perform differential expression analysis
  7. g Perform Gene Ontology and pathway enrichment analysis
Phase 7 · Single-Cell RNA Sequencing (scRNA-seq) and Analysis D28
  1. a Dissociate D28 hCOs into single cells
  2. b Prepare scRNA-seq libraries
  3. c Perform single-cell sequencing
  4. d Align reads and quantify gene expression
  5. e Normalize and batch correction
  6. f Dimensionality reduction and clustering
  7. g Identify cell clusters and major populations
  8. h Compare cell proportion changes between WT and FEZ1-KO
  9. i Perform differential gene expression between HOPX+ and HOPX− oRG
  10. j Perform trajectory inference analysis
Phase 8 · Actin Dynamics Analysis (G-actin/F-actin Assay) D28
  1. a Dissociate D28 hCOs
  2. b Plate cells on coverslips
  3. c Fix cells
  4. d Permeabilize and stain for G- and F-actin
  5. e Co-stain with PAX6 marker
  6. f Image and quantify G- to F-actin ratio
  7. g Perform statistical analysis
Phase 9 · Cell Migration Analysis (Time-Lapse Imaging) D10
  1. a Prepare D10 hCOs for viral infection
  2. b Mount hCOs for live imaging
  3. c Perform time-lapse imaging
  4. d Reconstruct cell tracks
  5. e Quantify migration parameters
  6. f Perform statistical analysis
Phase 10 · EdU Pulse-Labeling for Cell Proliferation and Migration Analysis D23-D28
  1. a Pulse-label hCOs with EdU
  2. b Remove EdU and continue culture
  3. c Fix hCOs at D28
  4. d Perform Click-iT EdU detection
  5. e Co-stain with PAX6 and TUJ1
  6. f Define VZ-like and SVZ-like regions
  7. g Quantify EdU+ postmitotic cells in VZ-like vs SVZ-like
  8. h Perform statistical analysis
Phase 11 · Protein Analysis via Immunoblotting D0-D120
  1. a Harvest cells or organoids
  2. b Prepare protein lysates
  3. c Quantify protein concentration
  4. d Prepare samples for SDS-PAGE
  5. e Perform SDS-PAGE electrophoresis
  6. f Transfer proteins to membrane
  7. g Block and incubate with primary antibody
  8. h Wash and incubate with secondary antibody
  9. i Detect bands using chemiluminescence
  10. j Quantify band intensity
  11. k Perform statistical analysis

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Attribution

This SOP was written by Organthis based on the published method in Qu et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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