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BRAIN Publication-derived

Parkinson's Disease Midbrain Assembloids

Full SOP with Academic or a plan

Source Kim et al., 2025 · Dongguk University · 10.1002/advs.202412548

Authors: Hongwon Kim, Soi Kang, Byounggook Cho, Saemin An, Yunkyung Kim, Jongpil Kim Duration: 20 days 5 phases Cell source: Human fibroblasts; iPSC-derived astrocytes

Abstract

A protocol for generating 3D induced dopaminergic (iDA) neuron organoids directly from human fibroblasts via lentiviral transduction with key transcription factors (ASCL1, PITX3, NURR1, LMX1A), bypassing the pluripotent state to preserve age-related epigenetic signatures. Extension to 3D iDA-astrocyte assembloids enables modeling of complex neuron-glia interactions in Parkinson's disease, including α-synuclein pathology and neuroprotective or neurotoxic astrocyte effects.

Cell source
Human fibroblasts; iPSC-derived astrocytes
Application
Disease modeling - Parkinson's disease pathogenesis and drug screening

Protocol overview

17 steps across 5 phases

Phase 1 · Lentiviral Transduction and Aggregate Formation Day 0-7
  1. a Prepare human fibroblasts
  2. b Infect fibroblasts with lentivirus
  3. c Embed infected fibroblasts in Matrigel
  4. d Culture organoid aggregates
  5. e Transition to neural differentiation medium
Phase 2 · Dopaminergic Maturation and Synuclein Induction Day 7-20
  1. a Culture organoids on orbital shaker
  2. b Induce mutant α-synuclein (optional for PD modeling)
  3. c Maintain culture and change medium
Phase 3 · Generation of 3D iDA-Astrocyte Assembloids (Optional) Day −14 to Day 20
  1. a Differentiate iPSCs to induced astrocytes (iAs)
  2. b Prepare iDA neurons and astrocytes for co-embedding
  3. c Culture assembloids in N3 medium (5 days)
  4. d Transition assembloids to neural differentiation medium and apply orbital shaking
Phase 4 · Optional: Advanced PD Modeling with Mutant Astrocytes Day −7 to Day 20
  1. a Transduce astrocytes with A53T α-synuclein lentivirus
  2. b Co-embed control iDA neurons with A53T mutant astrocytes
  3. c Culture and induce synuclein expression
Phase 5 · Neurotoxicity Assessment (Optional: MPTP Treatment) Day 0-20 (parallel to differentiation)
  1. a Prepare MPTP or DMSO control
  2. b Treat organoids or assembloids with MPTP

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Attribution

This SOP was written by Organthis based on the published method in Kim et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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