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BRAIN Publication-derived

Autism Spectrum Disorder Cortical Models

Full SOP with Academic or a plan

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

Authors: Mariani J, Coppola G, Kim SH, Rao A, Szuber SE, Huttner A, Bhatt R, Chen HH, Nairn AC, Bhatt RS, Vaccarino FM Duration: 42 days 10 phases Cell source: 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

Phase 1 · iPSC Preparation and Embryoid Body Formation Day 0-2
  1. a Pre-treatment of iPSC Colonies
  2. b Dissociation to Single Cells
  3. c Embryoid Body Formation on AggreWell Plates
  4. d EB Cultivation in Growth-Promoting Medium (Days 0-2)
Phase 2 · Neural Rosette Formation and Selection Day 3-6
  1. a Transfer EBs to Matrigel-Coated Dishes
  2. b Supplement with Growth Factors (Day 4)
  3. c Manual Dissection of Neural Rosettes
  4. d Transfer Rosettes to Suspension Culture
Phase 3 · Expansion and Organoid Maturation Day 7-11
  1. a Suspension Culture for 5 Days (Day 7-11)
  2. b Transfer to Ultra Low-Attachment 96-Well Plates
  3. c Initiate Terminal Differentiation (Day 11, Terminal Differentiation Day 0)
Phase 4 · Organoid Maturation and Characterization Day 12-42
  1. a Medium Changes During Terminal Differentiation
  2. b Characterization at Terminal Differentiation Day 11 (TD11)
  3. c Characterization at Terminal Differentiation Day 31 (TD31)
Phase 5 · RNA Isolation and Transcriptome Analysis Parallel to Phases 1-4
  1. a Organoid Pooling for RNA Extraction
  2. b RNA Extraction
  3. c RNA-seq Library Preparation
  4. d Paired-End RNA Sequencing
  5. e RNA-seq Data Processing and Differential Expression Analysis
Phase 6 · qPCR Validation and Gene Network Analysis Parallel to Phase 5
  1. a cDNA Synthesis
  2. b Quantitative Real-Time PCR
  3. c Weighted Gene Co-Expression Network Analysis (WGCNA)
  4. d Functional Annotation of Gene Modules
Phase 7 · Immunostaining and Cellular Quantification Parallel to Phases 1-4
  1. a Organoid Fixation and Cryopreservation
  2. b Embedding and Cryosectioning
  3. c Immunostaining: Blocking and Primary Antibody Incubation
  4. d Immunostaining: Washing and Secondary Antibody Incubation
  5. e Special Preparation: BrdU Staining
  6. f Image Acquisition
  7. g Stereological Quantification
  8. h Quantification of Neurite Density and Synaptic Puncta
Phase 8 · Electrophysiology TD11–TD31
  1. a Preparation of Neurons for Patch-Clamp Recording
  2. b Patch-Clamp Recording Setup
  3. c Measurement of Voltage-Activated Currents
  4. d Action Potential Recording
  5. e Recording of Spontaneous Synaptic Currents
  6. f Analysis of Steady-State Inactivation
  7. g Data Analysis
Phase 9 · Functional Validation: FOXG1 RNA Interference Variable (integrated with Phases 1-4)
  1. a Lentiviral Vector Selection and Production
  2. b Preparation of iPSCs for Lentiviral Transduction
  3. c Lentiviral Transduction
  4. d Selection of Transduced iPSCs
  5. e Verification of FOXG1 Knockdown
  6. f Immunostaining Validation of FOXG1 Knockdown
  7. g Analysis of GABAergic Markers After FOXG1 Knockdown
  8. h Analysis of Proliferation After FOXG1 Knockdown
Phase 10 · Phenotype Correlation Analysis After Phase 5 (RNA-seq and Analysis)
  1. a Clinical Phenotype Data Collection
  2. b Correlation of Gene Expression with Clinical Phenotype
  3. c FOXG1 Expression Correlation with Clinical Phenotype
  4. d Interpretation and Hypothesis Generation

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Attribution

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

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