Skip to content
← Back to browse
BRAIN Publication-derived

FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron

Source Mariani et al. · Yale University School of Medicine, New Haven, CT, USA · 10.1016/j.cell.2015.06.034

👤 Mariani J, Coppola G, Kim SH, Rao A, Szuber SE, Huttner A, Bhatt R, Chen HH, Nairn AC, Bhatt RS, Vaccarino FM ⏱ 42 days 📋 10 phases 🧫 Patient-Derived iPSC (Autism Spectrum Disorder)

Abstract

Mariani et al. generated telencephalic organoids from iPSCs derived from patients with idiopathic Autism Spectrum Disorder, revealing FOXG1 overexpression and a shift in excitatory/inhibitory neuron balance favoring GABAergic neuron overproduction. This Cell 2015 study established a paradigm for using patient-derived organoids to uncover mechanisms underlying neurodevelopmental disorders.

Cell source
Patient-Derived iPSC (Autism Spectrum Disorder)
Application
Autism Spectrum Disorder Modeling

Protocol overview

50 steps across 10 phases

iPSC Preparation and Embryoid Body Formation Day 0–2
  1. 1 Pre-treatment of iPSC Colonies
  2. 2 Dissociation to Single Cells
  3. 3 Embryoid Body Formation on AggreWell Plates
  4. 4 EB Cultivation in Growth-Promoting Medium (Days 0–2)
Neural Rosette Formation and Selection Day 3–6
  1. 5 Transfer EBs to Matrigel-Coated Dishes
  2. 6 Supplement with Growth Factors (Day 4)
  3. 7 Manual Dissection of Neural Rosettes
  4. 8 Transfer Rosettes to Suspension Culture
Expansion and Organoid Maturation Day 7–11
  1. 9 Suspension Culture for 5 Days (Day 7–11)
  2. 10 Transfer to Ultra Low-Attachment 96-Well Plates
  3. 11 Initiate Terminal Differentiation (Day 11, Terminal Differentiation Day 0)
Organoid Maturation and Characterization Day 12–42
  1. 12 Medium Changes During Terminal Differentiation
  2. 13 Characterization at Terminal Differentiation Day 11 (TD11)
  3. 14 Characterization at Terminal Differentiation Day 31 (TD31)
RNA Isolation and Transcriptome Analysis Parallel to Phases 1–4
  1. 15 Organoid Pooling for RNA Extraction
  2. 16 RNA Extraction
  3. 17 RNA-seq Library Preparation
  4. 18 Paired-End RNA Sequencing
  5. 19 RNA-seq Data Processing and Differential Expression Analysis
qPCR Validation and Gene Network Analysis Parallel to Phase 5
  1. 20 cDNA Synthesis
  2. 21 Quantitative Real-Time PCR
  3. 22 Weighted Gene Co-Expression Network Analysis (WGCNA)
  4. 23 Functional Annotation of Gene Modules
Immunostaining and Cellular Quantification Parallel to Phases 1–4
  1. 24 Organoid Fixation and Cryopreservation
  2. 25 Embedding and Cryosectioning
  3. 26 Immunostaining: Blocking and Primary Antibody Incubation
  4. 27 Immunostaining: Washing and Secondary Antibody Incubation
  5. 28 Special Preparation: BrdU Staining
  6. 29 Image Acquisition
  7. 30 Stereological Quantification
  8. 31 Quantification of Neurite Density and Synaptic Puncta
Electrophysiology TD11–TD31
  1. 32 Preparation of Neurons for Patch-Clamp Recording
  2. 33 Patch-Clamp Recording Setup
  3. 34 Measurement of Voltage-Activated Currents
  4. 35 Action Potential Recording
  5. 36 Recording of Spontaneous Synaptic Currents
  6. 37 Analysis of Steady-State Inactivation
  7. 38 Data Analysis
Functional Validation: FOXG1 RNA Interference Variable (integrated with Phases 1–4)
  1. 39 Lentiviral Vector Selection and Production
  2. 40 Preparation of iPSCs for Lentiviral Transduction
  3. 41 Lentiviral Transduction
  4. 42 Selection of Transduced iPSCs
  5. 43 Verification of FOXG1 Knockdown
  6. 44 Immunostaining Validation of FOXG1 Knockdown
  7. 45 Analysis of GABAergic Markers After FOXG1 Knockdown
  8. 46 Analysis of Proliferation After FOXG1 Knockdown
Phenotype Correlation Analysis After Phase 5 (RNA-seq and Analysis)
  1. 47 Clinical Phenotype Data Collection
  2. 48 Correlation of Gene Expression with Clinical Phenotype
  3. 49 FOXG1 Expression Correlation with Clinical Phenotype
  4. 50 Interpretation and Hypothesis Generation

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

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

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence