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BRAIN Publication-derived

Parkinson's Disease Brain Organoids

Full SOP with Academic or a plan

Source Ha et al., 2020 · Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea · 10.3389/fcell.2020.594090

Authors: Jeongmin Ha, Ji Su Kang, Minhyung Lee, Areum Baek, Seongjun Kim, Sun-Ku Chung, Mi-Ok Lee, Janghwan Kim Duration: 30 days 8 phases Cell source: Human iPSC; Patient-Derived iPSC (Parkinson's disease, LRRK2 G2019S)

Abstract

This protocol establishes simplified brain organoids (simBOs) generated from expandable hPSC-derived primitive neural stem cells (pNSCs) in approximately 2 weeks. The simBOs are structurally simpler but more homogeneous than conventional brain organoids and can be rapidly specified into midbrain-like organoids for Parkinson's disease modeling and drug screening.

Cell source
Human iPSC; Patient-Derived iPSC (Parkinson's disease, LRRK2 G2019S)
Application
Disease modeling and drug screening

Protocol overview

40 steps across 8 phases

Phase 1 · hiPSC Maintenance and Differentiation into Primitive Neural Stem Cells (pNSCs) Day 0-7 (initial induction)
  1. a Maintain hiPSCs in TeSR-E8 medium
  2. b Seed hiPSCs on Geltrex-coated dishes for neural induction
  3. c Switch to Neural Induction Medium-I (NIM-I) for 2 days
  4. d Switch to Neural Induction Medium-II (NIM-II) for 5 days
  5. e Dissociate cells and transfer to Geltrex-coated dishes with Neural Stem Cell Maintenance Medium
Phase 2 · Spontaneous Differentiation of pNSCs in 2D Culture (Control/Reference) Day 0-30 (optional, for comparison)
  1. a Dissociate pNSCs into single cells
  2. b Plate cells on Geltrex-coated 2D culture plates
  3. c Switch to differentiation medium and feed every other day
Phase 3 · Generation of Simplified Brain Organoids (simBOs) Day 0-20 (organoid differentiation)
  1. a Seed pNSCs on ultra-low attachment 96-well plates for self-organization
  2. b Culture for 4 days to allow sphere formation
  3. c Transfer 96 spheres to bioreactor with differentiation medium
  4. d Culture in bioreactor with medium changes every 3 days
  5. e Harvest organoids at day 10-20
Phase 4 · LRRK2 Inhibitor Treatment (PD Modeling) Day 10-13 (3-day treatment window)
  1. a Apply PFE-360, an LRRK2 kinase inhibitor, to midbrain-like simBOs.
  2. b Maintain PFE-360 treatment for 3 days
  3. c Harvest organoids for analysis
Phase 5 · Immunohistochemistry / Immunocytochemistry Variable (performed on harvested organoids or 2D cultures)
  1. a Fix organoids with 4% paraformaldehyde overnight
  2. b Cryoprotect organoids with sucrose gradient
  3. c Embed in OCT compound and freeze
  4. d Cryosection organoids and 2D cultures
  5. e Block and permeabilize samples
  6. f Incubate with primary antibodies
  7. g Wash with dilute BSA/DPBS and incubate with secondary antibodies
  8. h Acquire fluorescent images
Phase 6 · Quantitative Real-Time PCR (qRT-PCR) Variable (performed on harvested organoids)
  1. a Lyse organoids and extract total RNA
  2. b Synthesize cDNA from total RNA
  3. c Perform quantitative real-time PCR
  4. d Analyze Ct values and normalize expression data
Phase 7 · Western Blotting Variable (performed on harvested organoids)
  1. a Prepare whole-cell protein extracts
  2. b Quantify protein concentration
  3. c Separate proteins on MP TGX Precast Gels
  4. d Transfer proteins to PVDF membrane
  5. e Block membrane and incubate with primary antibodies
  6. f Wash and incubate with HRP-conjugated secondary antibodies
  7. g Develop blot and acquire band images
  8. h Quantify band intensity
Phase 8 · Dopamine Secretion Assay Day 13 post-differentiation (24-hour culture medium collection)
  1. a Culture midbrain-like simBOs to day 13
  2. b Collect 24-hour culture medium
  3. c Perform enzyme-linked immunosorbent assay (ELISA) for dopamine
  4. d Quantify dopamine concentration

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Attribution

This SOP was written by Organthis based on the published method in Ha et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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