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BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids for Drug Screening

Full SOP with Academic or a plan

Source Jarazo et al., 2022 · Developmental and Cellular Biology, Luxembourg Centre for Systems Biomedicine, University of Luxembourg · 10.1002/mds.28810

Authors: Javier Jarazo, Kyriaki Barmpa, Jennifer Modamio, Claudia Saraiva, Sònia Sabaté-Soler, Isabel Rosety, Anne Griesbeck, Florian Skwirblies, Gaia Zaffaroni, Lisa M. Smits, Jihui Su, Jonathan Arias-Fuenzalida, Jonas Walter, Gemma Gomez-Giro, Anna S. Monzel, Xiaobing Qing, Armelle Vitali, Gerald Cruciani, Ibrahim Boussaad, Francesco Brunelli, Christian Jäger, Aleksandar Rakovic, Wen Li, Lin Yuan, Emanuel Berger, Giuseppe Arena, Silvia Bolognin, Ronny Schmidt, Christoph Schröder, Paul M.A. Antony, Christine Klein, Rejko Krüger, Philip Seibler, Jens C. Schwamborn Duration: 30 days 17 phases Cell source: Patient-Derived iPSC (PINK1 mutations)

Abstract

This protocol differentiates patient-derived iPSCs carrying PINK1 mutations into neuroepithelial stem cells (NESCs) and dopaminergic neurons in 2D and 3D organoid formats to model Parkinson's disease phenotypes. Treatment with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) restores impaired dopaminergic differentiation and improves mitophagy capacity, providing a disease model suitable for compound screening and therapeutic evaluation.

Cell source
Patient-Derived iPSC (PINK1 mutations)
Application
Disease modeling and drug screening

Protocol overview

72 steps across 17 phases

Phase 1 · NESC Derivation Days 0-14 (initial setup and expansion)
  1. a Prepare hiPSC culture plates
  2. b Derive neuroepithelial stem cells (NESCs)
Phase 2 · Two-Dimensional Neuronal Differentiation Days 1-21 post-differentiation induction
  1. a Prepare differentiation media 1
  2. b Induce neuronal differentiation with media 1
  3. c Switch to differentiation media 2
  4. d Monitor differentiation efficiency
Phase 3 · Three-Dimensional Midbrain Organoid Differentiation Days 0-30 of organoid culture
  1. a Seed NESCs in ultra-low attachment plates
  2. b Differentiate organoids to day 30
  3. c Sample organoids at key timepoints
Phase 4 · Microfluidic 3D Culture (Optional) Days 0-21 of differentiation
  1. a Seed NESCs in OrganoPlate
  2. b Differentiate in microfluidic environment
Phase 5 · HP-β-CD Treatment Days 1-30 (for organoids) or Days 1-21 (for 2D cultures)
  1. a Prepare HP-β-CD stock solution
  2. b Add HP-β-CD to differentiation media
  3. c Assess HP-β-CD effect on TFEB nuclear translocation
Phase 6 · Gene Correction via CRISPR/Cas9 Variable (typically 2-4 weeks post-transfection)
  1. a Design guide RNAs and donor constructs
  2. b Transfect hiPSCs with CRISPR components
  3. c Select successfully transfected cells by fluorescence
  4. d Remove PSM via transposase excision
  5. e Select and expand gene-corrected clones
Phase 7 · Immunocytochemistry (2D Cultures) Performed at days 7, 14, and 21 of differentiation
  1. a Fixation
  2. b Permeabilization
  3. c Blocking
  4. d Primary antibody incubation
  5. e Secondary antibody incubation
  6. f Mount and image
Phase 8 · Immunohistochemistry (3D Organoids) Performed on organoids harvested at days 10, 20, and 30
  1. a Fixation and processing
  2. b Perform immunofluorescence on organoid sections
  3. c Image organoid sections
Phase 9 · Western Blotting Performed on cells or organoids at specified timepoints (typically day 21 or 30)
  1. a Cell/organoid lysis
  2. b Protein quantification
  3. c Gel resolution
  4. d Protein transfer to membrane
  5. e Membrane blocking
  6. f Primary antibody incubation
  7. g Secondary antibody incubation
  8. h Chemiluminescent detection
Phase 10 · Extracellular Flux Analysis (Seahorse) Performed at specified timepoints during differentiation (typically days 0, 3, 6)
  1. a Prepare Seahorse XF 96-well plate
  2. b Seed cells for Seahorse analysis
  3. c Prepare Seahorse instrument and assay reagents
  4. d Perform baseline and compound-injected measurements
  5. e Data analysis and comparison
Phase 11 · Microelectrode Array (MEA) Measurements Performed on neurons at day 21-30 of differentiation
  1. a Prepare MEA plates with neurons
  2. b Record spontaneous neuronal activity
  3. c Data processing with Axion software
  4. d Statistical comparison
Phase 12 · Rosella Mitophagy/Autophagy Reporter Assay Days 0-14 of differentiation (or basal measurements on hiPSCs)
  1. a Generate or obtain Rosella reporter lines
  2. b Measure basal autophagy in hiPSCs (LC3-Rosella)
  3. c Measure mitophagy during neuronal differentiation (ATP5C1-Rosella)
  4. d Assess effect of rapamycin on autophagy
  5. e Assess effect of chloroquine on mitophagy and dopaminergic differentiation
  6. f Assess effect of HP-β-CD on autophagy/mitophagy
Phase 13 · Immuno-Based Protein-Profiling (Antibody Microarray) Organoids sampled at days 10, 20, and 30
  1. a Harvest and prepare organoid samples
  2. b Quantify protein and prepare lysates
  3. c Perform antibody microarray analysis
  4. d Data analysis and pathway mapping
Phase 14 · MPTP Mouse Model and HP-β-CD Treatment Day 0 to Day 20 (5 days MPTP, 15 days HP-β-CD treatment)
  1. a Generate MPTP-induced Parkinson's disease mice
  2. b Begin HP-β-CD treatment (preventive protocol)
  3. c Monitor behavior and clinical signs
  4. d Harvest brain tissue and assess dopaminergic neuronal loss
  5. e Quantify neuroprotection by HP-β-CD
Phase 15 · Image Acquisition and Analysis Throughout protocol (performed on samples from all phases)
  1. a Automated high-content imaging
  2. b High-resolution imaging for Rosella and subcellular analysis
  3. c Quantitative image analysis using MatLab
  4. d Generate heatmaps and clustering analysis
Phase 16 · RNA Isolation and qPCR Gene Expression Analysis Performed on samples at specified timepoints (typically day 21-30 of differentiation)
  1. a RNA isolation from cells or organoids
  2. b DNase treatment
  3. c Reverse transcription to cDNA
  4. d Quantitative real-time PCR (qRT-PCR)
  5. e Data analysis and fold-change calculation
Phase 17 · Statistical Analysis Final analysis phase (performed after all data collection)
  1. a Perform statistical tests
  2. b Multiple testing correction
  3. c Prepare results tables and figures

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Attribution

This SOP was written by Organthis based on the published method in Jarazo et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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