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BRAIN Publication-derived

Use of 3D Organoids as a Model to Study Idiopathic Form of Parkinson's Disease

Source Chlebanowska et al., 2020 · Jagiellonian University Medical College, Faculty of Medicine, Institute of Pediatrics, Department of Transplantation, Kraków, Poland · 10.3390/ijms21030694

👤 Paula Chlebanowska, Anna Tejchman, Maciej Sułkowski, Klaudia Skrzypek, Marcin Majka ⏱ 60 days 📋 7 phases 🧫 Patient-Derived iPSC (Idiopathic Parkinson's Disease), Human iPSC

Abstract

This protocol describes the generation and characterization of midbrain organoids from patient-derived iPSCs to model idiopathic Parkinson's disease. iPSCs were generated from peripheral blood mononuclear cells of healthy volunteers and PD patients via Sendai viral vector reprogramming, then differentiated into three-dimensional midbrain organoids displaying dopaminergic neuron networks. The model identified dysregulation of key neuronal markers (TH, PTX3, LMX1A, FOXA2) associated with disease pathology.

Cell source
Patient-Derived iPSC (Idiopathic Parkinson's Disease), Human iPSC
Application
Disease modeling

Protocol overview

34 steps across 7 phases

iPSC Generation and Culture Preparation Days -7 to 0
  1. 1 Prepare Mouse Embryonic Fibroblasts (MEFs) Feeder Layer
  2. 2 Isolate PBMCs from Blood
  3. 3 Culture PBMCs in Expansion Medium
Sendai Viral Vector Reprogramming (Days 0–30) Days 0 to 30
  1. 1 Transduce PBMCs with Sendai Viral Vector
  2. 2 Wash Out Viral Vectors (Day 1)
  3. 3 Plate Transduced Cells on iMEF Feeder Layer (Day 2)
  4. 4 Medium Transition Phase (Days 5–9)
  5. 5 iPSC Colony Formation and Picking (Days 21–28)
iPSC Characterization and Pluripotency Validation (Days 30–60) Days 30 to 60 (concurrent with passages 1–10)
  1. 1 Culture and Expand iPSC Clones
  2. 2 Alkaline Phosphatase Staining
  3. 3 RT-PCR for Endogenous Pluripotency Markers
  4. 4 Flow Cytometry for Surface Markers
  5. 5 Teratoma Formation Assay in Immunodeficient Mice
  6. 6 Select Representative Clone for Organoid Differentiation
Embryoid Body Formation (Days 0–4) Days 0 to 4
  1. 1 Prepare iPSCs for Embryoid Body Formation
  2. 2 Dissociate iPSC Colonies
  3. 3 Form Embryoid Bodies (EBs)
  4. 4 Generate Large Batch of Embryoid Bodies
Midbrain Organoid Formation and Differentiation (Days 4–49) Days 4 to 49
  1. 1 Neural Induction Phase (Days 4–7)
  2. 2 Midbrain Patterning Phase (Days 7–10)
  3. 3 Matrigel Embedding and Tissue Growth Induction (Days 10–11)
  4. 4 Transition to Non-Adherent Culture (Day 11)
  5. 5 Final Organoid Maturation and Dopaminergic Differentiation (Days 11–49)
  6. 6 Organoid Sampling for Analysis
Gene Expression Analysis by RT-qPCR (Days 0, 4, 17, 27, 39, 49) Concurrent with organoid culture (collected on days 0, 4, 17, 27, 39, 49)
  1. 1 RNA Extraction
  2. 2 Reverse Transcription
  3. 3 Quantitative Real-Time PCR Setup
  4. 4 Data Analysis and Normalization
Immunofluorescence Staining and Protein Analysis (Day 39) Day 39 post-organoid initiation
  1. 1 Organoid Fixation and Embedding
  2. 2 Antigen Retrieval and Blocking
  3. 3 Primary Antibody Incubation
  4. 4 Secondary Antibody and Hoechst Staining
  5. 5 Mount and Image Organoid Sections
  6. 6 Image Analysis and Colocalization Assessment

Full SOP

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Attribution

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

This wording is awaiting legal review.

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