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BRAIN Publication-derived

Generation and Drug Screening of Alzheimer's Disease-Associated Human iPSC-Derived Cerebral Organoids (iCOs)

Source Park et al. · Department of Biochemistry & Biomedical Sciences, Seoul National University College of Medicine · 10.1073/pnas.1314145110

👤 Jong-Chan Park, So-Yeong Jang, Dongjoon Lee, Jeongha Lee, Uiryong Kang, Hongjun Chang, Haeng Jun Kim, Sun-Ho Han, Jinsoo Seo, Murim Choi, Dong Young Lee, Min Soo Byun, Dahyun Yi, Kwang-Hyun Cho, Inhee Mook-Jung ⏱ 65 days 📋 10 phases 🧫 Human iPSC, Patient-Derived iPSC (Alzheimer's Disease)

Abstract

This protocol describes the generation of human iPSC-derived cerebral organoids (iCOs) from patient peripheral blood mononuclear cells, with characterization of normal and Alzheimer's disease-associated pathological features including amyloid-beta and phospho-tau accumulation. The iCOs are used in a high-throughput drug screening platform to identify therapeutic candidates targeting key AD-associated signaling pathways (ApoE ε4 allele and LPL variants).

Cell source
Human iPSC, Patient-Derived iPSC (Alzheimer's Disease)
Application
Disease modeling and drug screening for Alzheimer's disease

Protocol overview

37 steps across 10 phases

iPSC Generation and Characterization Days 0-25
  1. 1 PBMC Isolation and T Cell Activation
  2. 2 Sendai Virus Reprogramming
  3. 3 iPSC Colony Derivation and Passage
  4. 4 iPSC Characterization
Embryoid Body (EB) Formation Days 0-6
  1. 1 Embryoid Body Initiation
  2. 2 Neural Induction with SMAD Inhibitors
Cerebral Organoid Formation and Expansion Days 6-43+
  1. 1 EB Collection and Organoid Seeding
  2. 2 Early Proliferation Phase (Days 6–15)
  3. 3 Maturation Phase with Reduced EGF/bFGF (Days 16–24)
  4. 4 Neurotrophic Factor Phase (Days 25–42)
  5. 5 Maturation Phase without Growth Factors (Days 43+)
Organoid Quality Control and Selection Days 7, 60, and drug screening period
  1. 1 Manual Quality Control (Days 7 and 60)
  2. 2 Automated Quality Control During Drug Screening
Immunohistochemistry and Pathology Characterization Days 60+
  1. 1 Organoid Fixation and Cryopreservation
  2. 2 Cryosectioning and Permeabilization
  3. 3 Blocking and Primary Antibody Staining
  4. 4 Secondary Antibody Staining and Mounting
  5. 5 Confocal Microscopy and Image Acquisition
3D Tissue Clearing (Ethyl-Cinnamate ECi Protocol) Days 60+
  1. 1 Fixation and Blocking/Permeabilization
  2. 2 Primary Antibody Staining
  3. 3 Washing and Secondary Antibody Staining
  4. 4 Post-Staining Fixation and Dehydration
  5. 5 Index Matching and Imaging
Drug Treatment and Screening Days 60–65 (or as specified)
  1. 1 Organoid Preparation for Drug Treatment
  2. 2 Drug Dose Preparation and Application
  3. 3 Drug Response Readout via Cell Viability (MTT Assay)
  4. 4 High-Content Screening with Automated Imaging
Functional Assays: Calcium Oscillation Analysis Days 60–65
  1. 1 Organoid Preparation and Dye Loading
  2. 2 Calcium Flux Detection
Molecular and Genomic Analyses Days 60+
  1. 1 RNA Extraction and RT-qPCR
  2. 2 RNA Sequencing and Library Preparation
  3. 3 RNA-Seq Analysis: Sequencing and Read Mapping
  4. 4 Differential Gene Expression (DEG) Analysis
  5. 5 Pathway Enrichment and Gene Ontology Analysis
Immunocytochemistry for iPSCs Parallel to iPSC characterization (days 15–25)
  1. 1 Slide Preparation and Cell Fixation
  2. 2 Blocking and Primary Antibody Incubation
  3. 3 Secondary Antibody Staining and Mounting

Full SOP

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Attribution

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

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