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BRAIN Publication-derived

Generation of Midbrain Dopaminergic Neurons and Organoids from Young-Onset Parkinson's Disease Patient-Derived iPSCs with Single Cell Electrochemical Analysis

Source Zhu et al., 2022 · Nanjing Medical University

👤 Wanying Zhu, Mengdan Tao, Yuan Hong, Shanshan Wu, Chu Chu, Zhilong Zheng, Xiao Han, Qian Zhu, Min Xu, Andrew G Ewing, Xing Guo, Yan Liu ⏱ 56 days 📋 13 phases 🧫 Patient-Derived iPSC (Young-Onset Parkinson's Disease - PINK1 mutation)

Abstract

This protocol describes the generation of midbrain dopaminergic neurons and 3D organoids from patient-derived iPSCs carrying a PINK1 gene mutation associated with young-onset Parkinson's disease. Dopaminergic progenitors are differentiated over approximately 5 weeks, yielding both 2D neurons on coverslips and 3D organoids that exhibit disease-relevant phenotypes including altered vesicular function and α-synuclein accumulation, which are analyzed using intracellular vesicle impact electrochemistry and whole-cell patch-clamp electrophysiology.

Cell source
Patient-Derived iPSC (Young-Onset Parkinson's Disease - PINK1 mutation)
Application
Disease modeling and single cell electrochemical analysis

Protocol overview

79 steps across 13 phases

iPSC Line Generation and Maintenance Days -∞ to Day 0 (ongoing)
  1. 1 Isolate peripheral blood mononuclear cells (PBMCs)
  2. 2 Transfect PBMCs with Sendai virus
  3. 3 Allow reprogramming to proceed
  4. 4 Clone selection and amplification
  5. 5 Establish feeder-free iPSC culture
  6. 6 Passage iPSCs regularly
Midbrain-Like Differentiation - Neural Induction and Patterning Days 1-8
  1. 1 Initiate neural induction
  2. 2 Maintain neural induction phase
Midbrain Specification - Floating Cluster Expansion Days 9-12
  1. 1 Dissociate and expand epithelial colonies
  2. 2 Maintain floating cluster culture
Dopaminergic Progenitor Specification Days 13-19
  1. 1 Switch to FGF8b-based medium
Late Dopaminergic Progenitor Maturation Days 20-34
  1. 1 Maintain FGF8b and SHH signaling
Final Differentiation to Dopaminergic Neurons and Organoid Maturation Days 35-45+
  1. 1 Dissociate neurospheres for 2D plating
  2. 2 Culture 2D dopaminergic neurons
  3. 3 Maintain 3D midbrain organoid culture
  4. 4 Culture organoids until analysis
Fabrication of Nano-Tip Microelectrodes As needed
  1. 1 Prepare glass capillary and carbon fiber assembly
  2. 2 Pull glass capillary into separate electrodes
  3. 3 Cut exposed carbon fiber
  4. 4 Flame etch the carbon fiber
  5. 5 Inspect electrode tip under microscope
  6. 6 Seal electrode with epoxy
  7. 7 Obtain scanning electron microscope images
Single Cell Electrochemical Cytometry (IVIEC) Days 45-47
  1. 1 Set up electrochemical recording apparatus
  2. 2 Prepare recording solution
  3. 3 Configure patch-clamp amplifier
  4. 4 Digitize and record signals
  5. 5 Position electrode at cell membrane
  6. 6 Penetrate into cell body
  7. 7 Conduct independent replicate experiments
Data Analysis for Single Cell Electrochemistry After data acquisition
  1. 1 Filter and process amperometric data
  2. 2 Detect peak events
  3. 3 Extract peak parameters
  4. 4 Quantify vesicle content using Faraday's equation
  5. 5 Pool and normalize data
  6. 6 Perform statistical analysis
Whole-Cell Patch-Clamp Electrophysiology Days 42-56 (6-8 weeks)
  1. 1 Prepare patch-clamp setup
  2. 2 Record action potentials
  3. 3 Record voltage-dependent sodium currents
  4. 4 Record voltage-dependent potassium currents
  5. 5 Prepare patch pipettes
  6. 6 Record voltage and current signals
  7. 7 Digitize and store data
  8. 8 Analyze patch-clamp data
Immunofluorescent Staining - 2D Dopaminergic Neurons Days 35-47
  1. 1 Fix 2D midbrain neurons
  2. 2 Permeabilize and block cells
  3. 3 Incubate with primary antibodies
  4. 4 Incubate with secondary antibodies
  5. 5 Counterstain nuclei
  6. 6 Image stained coverslips
  7. 7 Analyze images
Immunofluorescent Staining - 3D Midbrain Organoids Days 24-45
  1. 1 Fix 3D organoids
  2. 2 Wash organoids
  3. 3 Cryoprotect organoids with 20% sucrose
  4. 4 Cryoprotect organoids with 30% sucrose
  5. 5 Embed organoids in OCT
  6. 6 Cut cryosections
  7. 7 Rehydrate cryosections
  8. 8 Permeabilize and block sections
  9. 9 Incubate with primary antibodies
  10. 10 Incubate with secondary antibodies
  11. 11 Counterstain nuclei
  12. 12 Mount cryosections
  13. 13 Image stained organoid sections
  14. 14 Analyze organoid section images
  15. 15 Pool and compare data
Western Blotting Days 35-45
  1. 1 Lyse neurons or organoids
  2. 2 Sonicate samples
  3. 3 Centrifuge lysates
  4. 4 Measure total protein concentration
  5. 5 Prepare samples for SDS-PAGE
  6. 6 Perform SDS-PAGE
  7. 7 Transfer proteins to membrane
  8. 8 Block membrane
  9. 9 Incubate with primary antibodies
  10. 10 Incubate with secondary antibodies
  11. 11 Visualize protein bands
  12. 12 Analyze band intensities
  13. 13 Pool results from multiple differentiations

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Zhu et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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