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BRAIN Publication-derived

Generation of Homogeneous Midbrain Organoids with In Vivo-like Cellular Composition

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

👤 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 ⏱ 20 days 📋 5 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kwak et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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