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BRAIN Publication-derived

Differentiation of Patient-Derived iPSCs to GABAergic Interneurons and Ventral Forebrain Organoids for Major Depressive Disorder with Suicide Behavior (sMDD) Disease Modeling

Source Lu et al., 2023 · Institute for Stem Cell and Neural Regeneration, State Key Laboratory of Reproductive Medicine, School of Pharmacy, Nanjing Medical University · 10.15252/emmm.202216364

👤 Kaiqin Lu, Yuan Hong, Mengdan Tao, Luping Shen, Zhilong Zheng, Kaiheng Fang, Fang Yuan, Min Xu, Chun Wang, Dongya Zhu, Xing Guo, Yan Liu ⏱ 70 days 📋 14 phases 🧫 Patient-Derived iPSC (Major Depressive Disorder with Suicide Behavior)

Abstract

This protocol generates GABAergic interneurons (GINs) and ventral forebrain organoids from iPSCs derived from patients with major depressive disorder and suicide behavior (sMDD). The differentiated sMDD GINs display increased neurite complexity, heightened neural firing, and weakened calcium signaling compared to controls. Transcriptomic analysis reveals decreased expression of the 5-HT2C receptor, which can be reversed using FDA-approved small molecule agonists or genetic approaches, providing a human cellular model for studying sMDD pathogenesis and therapeutic targets.

Cell source
Patient-Derived iPSC (Major Depressive Disorder with Suicide Behavior)
Application
Disease modeling

Protocol overview

105 steps across 14 phases

iPSC Generation from Patient Peripheral Blood Day 0 to Day 12
  1. 1 PBMC Isolation
  2. 2 Sendai Virus Reprogramming
  3. 3 Transfer to MEF Feeder Cells
  4. 4 Switch to hiPSC Medium
  5. 5 Monitor Colony Formation
  6. 6 Transition to Feeder-Free Culture
iPSC Maintenance and Passage Day 12 onwards
  1. 1 Maintain iPSC Cultures
  2. 2 Passage iPSCs
  3. 3 Change Medium After Passage
Neural Differentiation - Embryoid Body Formation and Neural Induction Day 0 to Day 16
  1. 1 Dissociate iPSCs into Embryoid Bodies
  2. 2 Suspend EBs in Neural Induction Medium
  3. 3 Attach EBs to Culture Plates
  4. 4 Replace with Pure NIM and Monitor Rosette Formation
  5. 5 Isolate Rosette-Containing Colonies
GABAergic Interneuron Specification and Differentiation Day 10 to Day 35
  1. 1 Apply Sonic Hedgehog Agonist (SAG)
  2. 2 Maintain Neurosphere Culture
  3. 3 Continue NIM Culture Post-SAG
  4. 4 Dissociate Neurospheres to Single Cells
  5. 5 Plate Single Cells for Maturation
  6. 6 Culture GINs to Day 35
Ventral Forebrain Organoid Generation Day 10 to Day 30
  1. 1 Maintain Organoid in Suspension with SAG
  2. 2 Continue Culture Post-SAG
  3. 3 Use Organoids at Day 30
Characterization - Immunocytochemistry Day 35 onwards
  1. 1 Fix Cells in PFA
  2. 2 Wash with PBS
  3. 3 Permeabilization
  4. 4 Blocking
  5. 5 Primary Antibody Incubation
  6. 6 Wash After Primary
  7. 7 Secondary Antibody Incubation
  8. 8 Nuclear Staining
  9. 9 Mount and Image
Electrophysiology - Whole-Cell Patch-Clamp Recording Day 45–70
  1. 1 Prepare Recording Setup
  2. 2 Prepare Bath Solution
  3. 3 Prepare Internal Recording Solution
  4. 4 Pull Recording Pipettes
  5. 5 Select and Prepare GINs
  6. 6 Establish Whole-Cell Configuration
  7. 7 Record Sodium and Potassium Currents
  8. 8 Record Action Potentials
  9. 9 Data Acquisition and Storage
  10. 10 Complete Recording and Withdraw Pipette
Calcium Imaging Analysis Day 40–65
  1. 1 Plate GINs or Organoids on Imaging Dishes
  2. 2 Load Fluo-4 AM Calcium Indicator
  3. 3 Wash Cells
  4. 4 Prepare Live-Cell Imaging Solution
  5. 5 Acquire Baseline Images
  6. 6 Stimulate with High-KCl Solution
  7. 7 Analyze Calcium Dynamics
  8. 8 Quantify Results
Drug Treatment and Pharmacological Rescue Day 35–45
  1. 1 Prepare Drug Solutions
  2. 2 Treat GINs with Drugs
  3. 3 Perform Calcium Imaging on Drug-Treated Cells
  4. 4 Analyze Calcium Response to Drug Treatment
  5. 5 Extended Treatment for Electrophysiology
Viral Infection and Genetic Rescue Day 30–37
  1. 1 Prepare Viral Stocks
  2. 2 Set Up Injection Apparatus
  3. 3 Transfer Organoids to Injection Dish
  4. 4 Prepare Virus for Injection
  5. 5 Inject Virus into Organoids
  6. 6 Incubate Post-Injection
  7. 7 Transfer to Culture Flasks
  8. 8 Dissociate Infected Organoids
  9. 9 Perform Assays on Infected Neurons
RNA Sequencing and Transcriptomic Analysis Day 35–45
  1. 1 Harvest Cells for scRNA-seq
  2. 2 Remove Cell Debris
  3. 3 Prepare Single Cells for 10X Genomics
  4. 4 Perform Droplet-Based scRNA-seq
  5. 5 Perform Reverse Transcription
  6. 6 Prepare cDNA Libraries
  7. 7 Perform Library Sequencing
  8. 8 Process Raw Sequencing Data
  9. 9 Perform Quality Control Filtering
  10. 10 Normalize and Correct Batch Effects
  11. 11 Perform Dimensionality Reduction and Clustering
  12. 12 Annotate Cell Types
  13. 13 Perform Differential Expression Analysis
  14. 14 Perform Gene Ontology and Pathway Enrichment
  15. 15 Identify Candidate Drug Targets
Bulk RNA Sequencing and Validation Day 35
  1. 1 Harvest Cells for Bulk RNA-seq
  2. 2 Extract Total RNA
  3. 3 Enrich mRNA
  4. 4 Construct RNA-seq Libraries
  5. 5 Align Sequencing Reads
  6. 6 Perform Differential Expression Analysis
  7. 7 Perform Pathway Analysis
  8. 8 Validate HTR2C Expression
Quantitative PCR Validation Day 35–45
  1. 1 Harvest Cells and Extract RNA
  2. 2 Synthesize cDNA
  3. 3 Design qPCR Primers
  4. 4 Perform qPCR
  5. 5 Perform Thermocycling
  6. 6 Analyze qPCR Results
Western Blotting for Protein Validation Day 35–45
  1. 1 Harvest Cells and Prepare Lysates
  2. 2 Quantify Protein
  3. 3 Prepare Loading Buffer and Denature
  4. 4 Perform SDS-PAGE
  5. 5 Transfer to Membrane
  6. 6 Block Non-Specific Binding
  7. 7 Incubate with Primary Antibody
  8. 8 Wash Membranes
  9. 9 Incubate with Secondary Antibody
  10. 10 Final Wash
  11. 11 Detect Protein Bands with ECL
  12. 12 Quantify Band Intensity

Full SOP

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Attribution

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

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