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BRAIN Publication-derived

Young-Onset Parkinson's Disease Midbrain Organoids with Single-Cell Profiling

Full SOP with Academic or a plan

Source Zhu et al., 2022 · School of Pharmacy, Nanjing Medical University, China · 10.1039/d2sc00809b

Authors: 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 Duration: 50 days 7 phases Cell source: Patient-Derived iPSC (Young-onset Parkinson's disease, PINK1 mutation)

Abstract

This protocol describes the differentiation of human iPSCs derived from a YOPD patient into midbrain dopaminergic neurons and 3D midbrain organoids for single-cell electrochemical analysis of vesicular dopamine storage. The method enables detection of catecholamine content at the single-vesicle level using nano-tip microelectrode-based intracellular vesicle impact electrochemical cytometry (IVIEC), allowing assessment of disease pathology and drug efficacy in a human disease model.

Cell source
Patient-Derived iPSC (Young-onset Parkinson's disease, PINK1 mutation)
Application
Disease modeling and drug screening for Parkinson's disease

Protocol overview

33 steps across 7 phases

Phase 1 · iPSC Maintenance and Neural Induction (2D Culture) Day 0-24
  1. a Culture iPSC lines (control H9 and PD52) in pluripotency state
  2. b Initiate neural differentiation protocol
  3. c Assess neural progenitor markers at day 18
  4. d Assess midbrain specification markers at day 24
  5. e Assess dopaminergic neuron marker at day 45
Phase 2 · 3D Midbrain Organoid Differentiation Day 0-45
  1. a Initiate organoid protocol from established 2D differentiation method
  2. b Characterize nascent neural organoids at day 24
  3. c Assess functional maturity via whole-cell patch-clamp electrophysiology at day 45
  4. d Verify dopaminergic neuron maturity and pathology at day 45
  5. e Assess apoptosis and neuronal morphology at day 45
Phase 3 · Nano-Tip Microelectrode Fabrication for IVIEC Prior to experiments
  1. a Fabricate conical nano-tip electrodes
  2. b Validate electrode performance prior to cell experiments
Phase 4 · Single-Cell Electrochemical Cytometry of Vesicular Dopamine Content (2D Neurons) Day 42-50 post-differentiation
  1. a Prepare neurons for IVIEC at 6-8 weeks post-differentiation
  2. b Mount neurons on nano-tip microelectrode for IVIEC measurement
  3. c Record amperometric traces of vesicle release events
  4. d Quantify vesicular catecholamine content using Faraday's Law
  5. e Compare vesicular content between control and disease neurons
Phase 5 · Single-Cell Electrochemical Cytometry of Vesicular Dopamine in Organoids (Untreated) Day 42-50 post-differentiation
  1. a Prepare midbrain organoids for IVIEC at day 45
  2. b Mount individual neurons from organoids onto nano-tip electrode
  3. c Record amperometric traces from organoid-derived DA neurons
  4. d Quantify dopamine molecules per vesicle in organoid neurons
  5. e Compare dopamine content between control and PD organoids
Phase 6 · Drug Treatment: Amantadine (AMA) Rescue Experiment Day 44-45 post-differentiation
  1. a Prepare organoid cultures for AMA treatment
  2. b Incubate organoids with AMA for 12 hours
  3. c Prepare AMA-treated organoids for IVIEC
  4. d Record vesicle content from AMA-treated neurons via IVIEC
  5. e Quantify dopamine rescue by AMA in PD organoids
Phase 7 · Alpha-Synuclein Assessment and PMA Treatment Day 44-45 post-differentiation
  1. a Assess α-synuclein expression in untreated neurons and organoids
  2. b Prepare organoid cultures for PMA treatment
  3. c Incubate organoids with PMA for 12 hours
  4. d Assess α-synuclein downregulation following PMA treatment
  5. e Record vesicle content from PMA-treated organoid neurons
  6. f Quantify dopamine rescue by PMA in PD organoids

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Attribution

This SOP was written by Organthis based on the published method in Zhu et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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