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BRAIN Publication-derived

Idiopathic Parkinson's Disease Midbrain Organoids

Full SOP with Academic or a plan

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

Authors: Paula Chlebanowska, Anna Tejchman, Maciej Sułkowski, Klaudia Skrzypek, Marcin Majka Duration: 60 days 7 phases Cell source: 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

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

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Attribution

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

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