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BRAIN Publication-derived

Midbrain Organoids with SNCA Gene Triplication Model - Synucleinopathy

Source Mohamed et al., 2021 · Montreal Neurological Institute-Hospital, McGill University · 10.1093/braincomms/fcab223

👤 Mohamed, N-V, Sirois, J, Ramamurthy, J, Mathur, M, Lépine, P, Deneault, E, Maussion, G, Nicouleau, M, Chen, CX-Q, Abdian, N, Soubannier, V, Cai, E, Nami, H, Thomas, RA, Wen, D, Tabatabaei, M, Beitel, LK, Singh Dolt, K, Karamchandani, J, Stratton, JA, Kunath, T, Fon, EA, Durcan, TM ⏱ 170 days 📋 11 phases 🧫 Patient-Derived iPSC (SNCA triplication), CRISPR-corrected isogenic control iPSC

Abstract

This protocol generates three-dimensional human midbrain organoids (hMOs) from induced pluripotent stem cells (iPSCs) derived from patients carrying a triplication of the SNCA gene, an early-onset Parkinson's disease model. The hMOs recapitulate key pathological features of synucleinopathies, including age-dependent alpha-synuclein aggregation, phosphorylation, and selective loss of dopaminergic neurons, providing a physiologically relevant in vitro system for studying Parkinson's disease mechanisms.

Cell source
Patient-Derived iPSC (SNCA triplication), CRISPR-corrected isogenic control iPSC
Application
Disease modeling - Parkinson's disease and synucleinopathy pathology

Protocol overview

70 steps across 11 phases

iPSC Culture and Quality Control Prior to differentiation
  1. 1 iPSC Maintenance and Passaging
  2. 2 Pluripotency Marker Staining
  3. 3 Karyotype Analysis
  4. 4 Genomic Stability Assessment
  5. 5 SNCA Copy Number Determination by Digital Droplet PCR
  6. 6 Sanger Sequencing of SNCA Regions
Embryoid Body Formation and Early Patterning Days 0–4
  1. 1 Embryoid Body Induction
  2. 2 Centrifuge for Aggregation
  3. 3 First Medium Change
  4. 4 Midbrain Patterning Medium Application
Matrigel Embedding and Tissue Induction Days 7–8
  1. 1 Matrigel Preparation
  2. 2 EB Embedding and Incubation
Organoid Expansion and Long-term Culture Days 9–end point (up to 170+ days)
  1. 1 Transfer to Orbital Shaker Culture
  2. 2 Medium Replacement
  3. 3 Monthly Mycoplasma Testing
  4. 4 Organoid Sampling at Key Timepoints
Characterization – RNA and Protein Analysis At defined timepoints (30, 50, 100, 170 days)
  1. 1 RNA Isolation
  2. 2 cDNA Synthesis
  3. 3 Quantitative Real-Time PCR (qPCR)
  4. 4 hMO Cell Lysis for Immunoblotting
  5. 5 Protein Quantification
  6. 6 SDS-PAGE and Western Blotting
  7. 7 Membrane Fixation for Enhanced α-Synuclein Detection
  8. 8 Membrane Blocking
  9. 9 Primary Antibody Incubation
  10. 10 Membrane Washing and Secondary Antibody Incubation
  11. 11 Chemiluminescent Detection
Immunofluorescence and Pathology Staining At defined timepoints (50, 100, 165–170 days)
  1. 1 hMO Fixation and Cryosectioning
  2. 2 Cryosection Rehydration and Blocking
  3. 3 Primary Antibody Incubation
  4. 4 Primary Antibody Washing
  5. 5 Secondary Antibody Incubation
  6. 6 Secondary Antibody Washing and Hoechst Staining
  7. 7 Mounting and Confocal Imaging
  8. 8 Immunofluorescence Quantification
Synuclein Proximity Ligation Assay (Syn-PLA) At defined timepoints (50, 100, 165–170 days)
  1. 1 Organoid Fixation and Paraffin Embedding
  2. 2 Paraffin Section Dewax and Rehydration
  3. 3 Antigen Retrieval
  4. 4 Blocking and Primary Antibody Incubation
  5. 5 Primary Antibody Washing and Secondary Antibody Application
  6. 6 PLA Probe Ligation
  7. 7 PLA Amplification and Detection
  8. 8 Confocal Microscopy and Quantification
Neuromelanin Staining (Fontana Masson) At defined timepoints (35–45 days, optionally longer)
  1. 1 Dopamine Treatment (Optional)
  2. 2 Organoid Fixation and Paraffin Processing
  3. 3 Fontana Masson Staining
  4. 4 Whole-Organoid Imaging
  5. 5 High-Magnification Imaging
  6. 6 Colorimetric Quantification
Flow Cytometry Analysis At defined timepoints (100, 170 days)
  1. 1 hMO Dissociation to Single-Cell Suspension
  2. 2 Cell Filtering and Centrifugation
  3. 3 Live/Dead Viability Staining
  4. 4 Washing and Fc Receptor Blocking
  5. 5 Extracellular Antigen Staining
  6. 6 Intracellular Fixation and Permeabilization
  7. 7 Intracellular Antibody Staining
  8. 8 Final Washing and FACS Buffer Resuspension
  9. 9 Flow Cytometry Setup and Compensation
  10. 10 Flow Cytometry Data Acquisition
Single-Cell RNA Sequencing At defined timepoints (165–170 days)
  1. 1 Sample Preparation and Pooling
  2. 2 10X Chromium Library Preparation
  3. 3 Illumina Sequencing
  4. 4 Data Processing with Seurat
  5. 5 Variable Gene Selection and Dimensionality Reduction
  6. 6 Cluster Annotation
  7. 7 Gene Expression and Data Visualization
Statistical Analysis Throughout, post-data collection
  1. 1 Descriptive Statistics and Normality Testing
  2. 2 Two-Group Comparisons
  3. 3 Multi-Group Comparisons
  4. 4 P-Value Interpretation and Reporting

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Mohamed et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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