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BRAIN Publication-derived

Modeling Synucleinopathy Using hESC-Derived Cerebral Organoids

Source Kim et al., 2025 · Department of Molecular Physiology, College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University · 10.3390/cells14181436

👤 So Jin Kim, Won Hee Jung, Mu Seog Choe, Ye Seong Jeon, Min Young Lee ⏱ 150 days 📋 9 phases 🧫 Human ESC (hESC SNUhES31 line)

Abstract

This protocol describes the generation of cerebral organoids (COs) from hESCs carrying the SNCA[A53T] mutation to model synucleinopathy. The A53T COs exhibit elevated α-synuclein expression, phosphorylation, Lewy body-like aggregations, and tau pathology, making them suitable for studying synucleinopathy mechanisms and screening aggregation inhibitors.

Cell source
Human ESC (hESC SNUhES31 line)
Application
Disease modeling and drug screening for synucleinopathy

Protocol overview

29 steps across 9 phases

hESC Culture and Maintenance Continuous maintenance (prior to modification)
  1. 1 Feeder-dependent hESC culture
  2. 2 Transition to feeder-free culture
Generation of SNCA[A53T] Mutant hESC Line Variable (typically 2–4 weeks from transfection to colony isolation)
  1. 1 Lentiviral vector construction
  2. 2 Virus production via HEK293T transfection
  3. 3 hESC transduction with lentiviral particles
  4. 4 Single-cell cloning and colony selection
  5. 5 Characterization of A53T hESC line
Cerebral Organoid Generation (Days 0–150) Day 0 to Day 150
  1. 1 Embryoid body (EB) formation (Days 0–6)
  2. 2 Neural induction (Days 6–10)
  3. 3 Matrigel culture phase (Days 10–14)
  4. 4 Long-term spinner flask culture (Days 14–150)
Immunocytochemistry (Characterization of hESCs and early differentiation) As needed for pluripotency assessment
  1. 1 Cell fixation and permeabilization
  2. 2 Primary antibody incubation
  3. 3 Secondary antibody incubation and imaging
Immunohistochemistry (Organoid characterization and pathology assessment) At day 150 (mature organoids)
  1. 1 Organoid cryoprotection and sectioning
  2. 2 Antibody staining (general immunofluorescence)
  3. 3 DAB staining for phospho-α-synuclein
Western Blot Analysis At day 150 (mature organoids)
  1. 1 Protein extraction by sonication
  2. 2 SDS-PAGE and membrane transfer
  3. 3 Membrane blocking and primary antibody incubation
  4. 4 Secondary antibody and detection
Enzyme-Linked ImmunoSorbent Assay (ELISA) At day 150 (mature organoids)
  1. 1 Organoid lysis and protein quantification
  2. 2 ELISA for phospho-α-Syn and Aβ
Bielschowsky's Silver Staining for Neurofibrillary Tangles At day 150 (mature organoids)
  1. 1 Silver nitrate incubation
  2. 2 Ammonium silver incubation
  3. 3 Developer and fixation
  4. 4 Dehydration and mounting
Drug Screening: SynuClean D Treatment Day 70 to Day 150
  1. 1 SynuClean D preparation and treatment
  2. 2 Assessment of drug efficacy

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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