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BRAIN Publication-derived

Reduced Astrocytic Reactivity in Human Midbrain Organoids with PRKN Mutations: Differentiation and Immunofluorescence Analysis

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

👤 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 ⏱ 35 days 📋 6 phases 🧫 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

iPSC Maintenance and Preparation Days -2 to 0
  1. 1 Prepare iPSC cultures
  2. 2 Passage iPSCs with Accutase
Embryoid Body Formation and Ventralization (Days 1–8) Days 1–8
  1. 3 Form embryoid bodies (EBs)
  2. 4 Add small molecule inducers to EB medium
  3. 5 Incubate EBs in formation medium
  4. 6 Harvest and transfer EBs
  5. 7 Replace medium on day 2
  6. 8 Add ascorbic acid on days 3 and 4
  7. 9 Collect neuroepithelial outgrowths on day 8
Expansion of Ventralized Neuroepithelial Stem Cells (Days 8–Maintenance) Days 8 onwards
  1. 10 Maintain neuroepithelial stem cells
  2. 11 Passage cells for organoid seeding
Midbrain Organoid Generation and Maturation (Days 1–35) Days 1–35
  1. 12 Seed organoid precursors
  2. 13 Initiate patterning by withdrawing morphogenic inhibitors (day 7)
  3. 14 Reduce CHIR concentration (day 10)
  4. 15 Switch to neuronal maturation medium (day 9 of differentiation; Day 16 overall)
  5. 16 Culture organoids under static conditions with medium changes
  6. 17 Harvest organoids at day 35
Organoid Preparation and Immunofluorescence Staining Day 35 onwards
  1. 18 Fix organoids in paraformaldehyde
  2. 19 Wash fixed organoids with PBS
  3. 20 Embed organoids in low-melting-point agarose
  4. 21 Section organoids using vibratome
  5. 22 Block sections
  6. 23 Incubate primary antibodies
  7. 24 Wash sections after primary antibody incubation
  8. 25 Incubate secondary antibodies
  9. 26 Wash sections after secondary antibody incubation
  10. 27 Mount sections in mounting medium
  11. 28 Image organoid sections using confocal microscopy
Image Analysis and Quantification After imaging
  1. 29 Analyze immunofluorescent images with automated algorithms
  2. 30 Quantify nuclear density and neuronal markers
  3. 31 Quantify glial markers (GFAP and S100β)
  4. 32 Quantify dopaminergic neuron degeneration via TH fragmentation
  5. 33 Perform quality control filtering on image data
  6. 34 Aggregate and normalize data across organoid sections

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kano et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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