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BRAIN Publication-derived

FEZ1 Deletion in Human Cerebral Organoids: Protocol for Disease Modeling of Neurodevelopmental Disorders

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

👤 Yinghua Qu, Jonathan Jun-Yong Lim, Omer An, Henry Yang, Yi-Chin Toh, John Jia En Chua ⏱ 120 days 📋 11 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Qu et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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