Skip to content
← Back to browse
BRAIN Publication-derived

Generation of Lewy Body-like Inclusions in Human Midbrain Organoids with GBA1 and α-Synuclein Perturbations

Source Jo et al., 2021 · Genome Institute of Singapore; National Neuroscience Institute; Duke-NUS Medical School · 10.1002/ana.26166

👤 Junghyun Jo, Lin Yang, Hoang-Dai Tran, Weonjin Yu, Alfred Xuyang Sun, Ya Yin Chang, Byung Chul Jung, Seung-Jae Lee, Tzuen Yih Saw, Bin Xiao, Audrey Tze Ting Khoo, Lai-Ping Yaw, Jessica Jiaxin Xie, Hidayat Lokman, Wei-Yi Ong, Grace Gui Yin Lim, Kah-Leong Lim, Eng-King Tan, Huck-Hui Ng, Hyunsoo Shawn Je ⏱ 90 days 📋 4 phases 🧫 Human ESC; Patient-Derived iPSC (Parkinson disease)

Abstract

This protocol generates human midbrain-like organoids (hMLOs) from pluripotent stem cells carrying glucocerebrosidase (GBA1) and α-synuclein (SNCA) perturbations to model Parkinson disease pathology. The resulting organoids recapitulate α-synuclein aggregation, Lewy body-like inclusion formation, and selective dopaminergic neurodegeneration, providing a 3D system for studying PD mechanisms and screening therapeutics.

Cell source
Human ESC; Patient-Derived iPSC (Parkinson disease)
Application
Disease modeling

Protocol overview

27 steps across 4 phases

hPSC Culture and Cell Line Generation Day 0 (preparation)
  1. 1 Maintain hESC and iPSC cultures
  2. 2 Generate GBA1[−/−] isogenic lines using CRISPR/Cas9
  3. 3 Generate doxycycline-inducible SNCA overexpression lines
Generation of hMLOs Day 0–7
  1. 1 Dissociate hPSCs and initiate neural induction
  2. 2 Switch to floorplate induction medium
  3. 3 Embed organoids in Matrigel
  4. 4 Culture on orbital shakers
Characterization and Analysis (Days 30–90) Day 30–90
  1. 1 Harvest hMLOs for enzyme activity assay
  2. 2 Measure lysosomal enzyme activity
  3. 3 Perform lipidomics analysis
  4. 4 Analyze lipids by LC–MS
  5. 5 Fix and prepare hMLO sections for immunofluorescence
  6. 6 Immunofluorescence staining
  7. 7 Immunohistochemistry on paraffin sections
  8. 8 Transmission electron microscopy (TEM) preparation
  9. 9 TEM sectioning and imaging
  10. 10 Intact cell cross-linking assay
  11. 11 Thioflavin S staining
  12. 12 Flow cytometry analysis
  13. 13 Cell viability assay
  14. 14 Protein misfolding cyclic amplification (PMCA) assay
  15. 15 Proteinase K digestion of α-syn samples
  16. 16 Seeded polymerization and thioflavin T binding assay
  17. 17 TEM imaging of α-syn fibrils
  18. 18 RNA sequencing and analysis
Optional: Pharmacological GCase Inhibition Day 30–90 (chronic treatment)
  1. 1 Treat hMLOs with conduritol-b-epoxide (CBE)
  2. 2 Assess LBLI formation in CBE-treated 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 Jo et al., 2021. 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