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BRAIN Publication-derived

Generation of LRRK2-G2019S Midbrain 3D Organoids from hiPSCs for Parkinson's Disease Modeling

Source Kim et al., 2019 · Dongguk University · 10.1016/j.stemcr.2019.01.020

👤 Hongwon Kim, Hyeok Ju Park, Hwan Choi, Yujung Chang, Hanseul Park, Jaein Shin, Junyeop Kim, Christopher J. Lengner, Yong Kyu Lee, Jongpil Kim ⏱ 60 days 📋 7 phases 🧫 Human iPSC (hiPSC)

Abstract

This protocol generates isogenic human iPSC-derived 3D midbrain organoids containing a G2019S mutation in LRRK2 to model sporadic Parkinson's disease. The organoids recapitulate key pathological hallmarks of LRRK2-associated PD including α-synuclein aggregation, dopamine neuron loss, and aberrant clearance. The system enables identification of disease mechanisms and testing of therapeutic approaches in a physiologically relevant 3D environment.

Cell source
Human iPSC (hiPSC)
Application
Disease modeling

Protocol overview

30 steps across 7 phases

hiPSC Culture and LRRK2-G2019S Mutation Introduction Passage 6 through clonal selection (approximately 2-4 weeks)
  1. 1 Prepare Control hiPSCs
  2. 2 Generate hiPSCs via Lentiviral Reprogramming
  3. 3 Introduce LRRK2-G2019S Mutation via CRISPR/Cas9
  4. 4 Characterize Pluripotency of Mutant and Control hiPSCs
Embryoid Body Formation and Initial Neural Induction Day 0-5
  1. 1 Dissociate hiPSCs into Single Cells
  2. 2 Form Embryoid Bodies
  3. 3 Embed EBs in Matrigel and Initiate Neural Induction
  4. 4 Add Midbrain Patterning Factors
  5. 5 Confirm Neuroectodermal Differentiation by qRT-PCR
Midbrain-Specific Differentiation and Dopaminergic Maturation Day 15-45
  1. 1 Add Terminal Differentiation Factors
  2. 2 Monitor Dopaminergic Gene Expression
  3. 3 Assess Maturity at Day 45
  4. 4 Quantify Dopamine Release
Extended Culture to Aged Midbrain Phenotype Day 45-60
  1. 1 Switch to Aged Midbrain Culture Conditions
  2. 2 Confirm Post-Mitotic Dopaminergic Neuron Identity
  3. 3 Assess Neuromelanin Accumulation
  4. 4 Measure DNA Damage as Aging Marker
Pathological Characterization of LRRK2-G2019S Organoids Day 60 (analysis of previously cultured organoids)
  1. 1 Assess Dopaminergic Neuron Vulnerability to MPTP
  2. 2 Analyze α-Synuclein Pathology
  3. 3 Quantify α-Synuclein Aggregation via Thioflavin T Staining
  4. 4 Assess Autophagy and Mitophagy
  5. 5 Perform Western Blot Analysis of Pathological Markers
Therapeutic Testing with LRRK2 Kinase Inhibitor Day 60 (treatment and analysis of previously cultured organoids)
  1. 1 Treat LRRK2-G2019S Organoids with LRRK2 Kinase Inhibitor
  2. 2 Assess Reduction of α-Synuclein Pathology
  3. 3 Measure Restoration of Dopaminergic Gene Expression
  4. 4 Assess Reduction in Dopaminergic Neuron Cell Death
Molecular Mechanism Analysis: TXNIP Knockdown Day 60 (analysis of previously cultured organoids with gene knockdown)
  1. 1 Perform TXNIP Knockdown via shRNA
  2. 2 Analyze Reduction of α-Synuclein Aggregation
  3. 3 Assess Lysosomal Dysfunction Recovery
  4. 4 Quantify α-Synuclein Inclusion Area

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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