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BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids (2021, No. 2)

Full SOP with Academic or a plan

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

Authors: 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 Duration: 170 days 11 phases Cell source: 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

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

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Attribution

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

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