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BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids (2021)

Full SOP with Academic or a plan

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

Authors: 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 Duration: 90 days 4 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Jo et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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