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BRAIN Publication-derived

Midbrain Dopaminergic Organoids from Human Pluripotent Stem Cells

Source Jo et al. · Sloan Kettering Institute / Weill Cornell Medicine, New York, USA · 10.1016/j.stem.2016.07.005

👤 Jo J, Xiao Y, Sun AX, Cukuroglu E, Tran HD, Göke J, Tan ZY, Saw TY, Tan CP, Lokber H, Lee Y, Kim D, Ko HS, Kim SO, Park JH, Cho NJ, Hyde TM, Bhatt JE, Bhatt AH, Bhatt RM, Bhatt KJ, Bhatt NP, Bhatt SM, Bhatt RS, Ng HH ⏱ 60 days 📋 4 phases 🧫 Human iPSC / Human ESC

Abstract

Jo et al. generated midbrain-specific organoids containing functional dopaminergic neurons that produce neuromelanin-like granules and release dopamine, closely mimicking the substantia nigra. This Cell Stem Cell 2016 protocol provides a critical platform for Parkinson disease modeling and dopaminergic drug screening.

Cell source
Human iPSC / Human ESC
Application
Parkinson Disease Modeling

Protocol overview

16 steps across 4 phases

Embryoid Body Formation and Neural Induction Day 0–4
  1. 1 Dissociate hPSCs to single cells and form uniform embryoid bodies
  2. 2 Apply dual-SMAD inhibition and Wnt activation for neuroectodermal induction
  3. 3 Add midbrain patterning factors
Matrigel Embedding and Organoid Growth Day 7–8
  1. 1 Embed neuroectodermal spheroids in Matrigel
  2. 2 Culture in tissue growth induction medium with midbrain patterning factors
  3. 3 Transfer organoids to ultra-low-attachment 6-well plates
Long-term Organoid Maturation and Differentiation Day 8–60+
  1. 1 Culture organoids in final differentiation medium on orbital shaker
  2. 2 Perform regular medium changes
  3. 3 Monitor organoid growth and morphology
  4. 4 Optional: Accelerate neuromelanin production with L-DOPA or dopamine treatment
Characterization and Functional Analysis Day 35–60+
  1. 1 Prepare organoid cryosections for immunohistochemistry
  2. 2 Perform immunohistochemistry with midbrain and DA markers
  3. 3 Detect neuromelanin using Fontana-Masson staining
  4. 4 Perform flow cytometry to quantify dopaminergic neuronal populations
  5. 5 Record electrophysiology from hMLO neurons in situ
  6. 6 Measure dopamine content by HPLC

Full SOP

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Attribution

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

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