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BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids (2020)

Full SOP with Academic or a plan

Source Kwak et al., 2020 · Konkuk University, Department of Stem Cell Biology, School of Medicine, Seoul, Republic of Korea · 10.1002/stem.3163

Authors: Tae Hwan Kwak, Ji Hyun Kang, Sai Hali, Jonghun Kim, Kee-Pyo Kim, Chanhyeok Park, Ju-Hyun Lee, Ha Kyun Ryu, Ji Eun Na, Junghyun Jo, Hyunsoo Shawn Je, Huck-Hui Ng, Jeongwoo Kwon, Nam-Hyung Kim, Kwon Ho Hong, Woong Sun, Chi Hye Chung, Im Joo Rhyu, Dong Wook Han Duration: 20 days 5 phases Cell source: Human ESC

Abstract

This protocol describes the robust generation of homogeneous midbrain-like organoids (DAC3.0 MOs) from human embryonic stem cells using optimized dual SMAD inhibition (dorsomorphin + A83-01) combined with WNT gradient manipulation (3 μM CHIR99021). The resulting organoids display structural and functional maturity with in vivo-like cellular composition including dopaminergic neurons, multiple neuronal subtypes, and functional glial cells (astrocytes and oligodendrocytes), enabling MPTP-based Parkinson's disease modeling.

Cell source
Human ESC
Application
Disease modeling (Parkinson's disease)

Protocol overview

17 steps across 5 phases

Phase 1 · Embryoid Body Formation and Mesencephalon Specification Days 1-7
  1. a Plate hESCs as single cells
  2. b Add Brain Organoid Generation Medium (BGM) at 24 hours
  3. c Treat with dual SMAD inhibitors and CHIR99021 for mesencephalon specification
  4. d Replace medium every other day
  5. e Harvest embryoid bodies at day 7
Phase 2 · Mesencephalic Floor Plate Induction and Ventralization Days 8-12
  1. a Treat with FGF8 and SAG for ventralization
  2. b Continue ventralization treatment for 5 days
Phase 3 · Basal-Apical Lamination and Maturation Days 12-onwards (up to 20 weeks)
  1. a Embed organoids in growth factor-reduced Matrigel droplets
  2. b Transfer to 6-cm petri dishes with appropriate maturation medium
  3. c Culture on orbital shaker with maturation medium
  4. d Monitor organoid growth and maturity markers
Phase 4 · WNT Gradient Optimization (Alternative to Standard CHIR99021 Approach) Days 1-12 (concurrent with Phase 1-2)
  1. a Establish in vitro WNT gradient using CHIR99021 and IWP2
  2. b Screen CHIR99021 concentrations from 0 to 3 μM
Phase 5 · Functional Validation and Parkinson's Disease Modeling (MPTP Treatment) Weeks 8-onwards
  1. a Prepare DAC3.0 MOs at 8 weeks maturation (8WM)
  2. b Treat organoids with MPTP at four concentrations
  3. c Assess MPTP-induced cell death after 48 hours
  4. d Quantify dose-dependent cell death response

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Attribution

This SOP was written by Organthis based on the published method in Kwak et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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