Skip to content
← Back to browse
BRAIN Publication-derived

Differentiation of Patient-Derived iPSCs to Midbrain Dopaminergic Neurons and Organoids for Electrochemical Cytometry Analysis

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

👤 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 ⏱ 50 days 📋 7 phases 🧫 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

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

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 Zhu et al., 2022. 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