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BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids with Astrocytes

Full SOP with Academic or a plan

Source Kano et al., 2020 · Juntendo University Graduate School of Medicine · 10.1038/s41531-020-00137-8

Authors: Masayoshi Kano, Masashi Takanashi, Genko Oyama, Asako Yoritaka, Taku Hatano, Kahori Shiba-Fukushima, Makiko Nagai, Kazutoshi Nishiyama, Kazuko Hasegawa, Tsuyoshi Inoshita, Kei-ichi Ishikawa, Wado Akamatsu, Yuzuru Imai, Silvia Bolognin, Jens Christian Schwamborn, Nobutaka Hattori Duration: 35 days 6 phases Cell source: Patient-Derived iPSC (Parkinson's disease - PRKN mutations)

Abstract

This protocol differentiates patient-derived induced pluripotent stem cells (iPSCs) from individuals with PRKN mutations into midbrain organoids (MOs) over 35 days of culture. The resulting organoids recapitulate reduced glial fibrillary acidic protein (GFAP)-positive astrocytes and increased tyrosine hydroxylase (TH)-positive neuron fragmentation compared to age- and sex-matched healthy controls, enabling in vitro modeling of PRKN-associated Parkinson's disease pathology and astrocytic dysfunction.

Cell source
Patient-Derived iPSC (Parkinson's disease - PRKN mutations)
Application
Disease modeling

Protocol overview

34 steps across 6 phases

Phase 1 · iPSC Maintenance and Preparation Days -2 to 0
  1. a Prepare iPSC cultures
  2. b Passage iPSCs with Accutase
Phase 2 · Embryoid Body Formation and Ventralization (Days 1-8) Days 1-8
  1. a Form embryoid bodies (EBs)
  2. b Add small molecule inducers to EB medium
  3. c Incubate EBs in formation medium
  4. d Harvest and transfer EBs
  5. e Replace medium on day 2
  6. f Add ascorbic acid on days 3 and 4
  7. g Collect neuroepithelial outgrowths on day 8
Phase 3 · Expansion of Ventralized Neuroepithelial Stem Cells (Days 8–Maintenance) Days 8 onwards
  1. a Maintain neuroepithelial stem cells
  2. b Passage cells for organoid seeding
Phase 4 · Midbrain Organoid Generation and Maturation (Days 1-35) Days 1-35
  1. a Seed organoid precursors
  2. b Initiate patterning by withdrawing morphogenic inhibitors (day 7)
  3. c Reduce CHIR concentration (day 10)
  4. d Switch to neuronal maturation medium (day 9 of differentiation; Day 16 overall)
  5. e Culture organoids under static conditions with medium changes
  6. f Harvest organoids at day 35
Phase 5 · Organoid Preparation and Immunofluorescence Staining Day 35 onwards
  1. a Fix organoids in paraformaldehyde
  2. b Wash fixed organoids with PBS
  3. c Embed organoids in low-melting-point agarose
  4. d Section organoids using vibratome
  5. e Block sections
  6. f Incubate primary antibodies
  7. g Wash sections after primary antibody incubation
  8. h Incubate secondary antibodies
  9. i Wash sections after secondary antibody incubation
  10. j Mount sections in mounting medium
  11. k Image organoid sections using confocal microscopy
Phase 6 · Image Analysis and Quantification After imaging
  1. a Analyze immunofluorescent images with automated algorithms
  2. b Quantify nuclear density and neuronal markers
  3. c Quantify glial markers (GFAP and S100β)
  4. d Quantify dopaminergic neuron degeneration via TH fragmentation
  5. e Perform quality control filtering on image data
  6. f Aggregate and normalize data across organoid sections

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Attribution

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

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