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BRAIN Publication-derived

A logical network-based drug-screening platform for Alzheimer's disease using human iPSC-derived cerebral organoids (iCOs)

Source Park et al., 2021 · Seoul National University, Department of Biochemistry and Biomedical Sciences, College of Medicine · 10.1038/s41467-020-20440-5

👤 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 ⏱ 150 days 📋 6 phases 🧫 Human iPSC, Patient-Derived iPSC (Sporadic Alzheimer's disease), CRISPR-Cas9-edited ApoE isogenic lines

Abstract

This protocol describes the generation of human iPSC-derived cerebral organoids (iCOs) from sporadic Alzheimer's disease (sAD) patients and healthy controls, including CRISPR-Cas9-edited ApoE4 isogenic lines. The organoids recapitulate AD pathological features (amyloid-beta deposition, phosphorylated tau, neuronal loss) and are used as a platform for high-content screening of FDA-approved drug candidates identified through mathematical network modeling.

Cell source
Human iPSC, Patient-Derived iPSC (Sporadic Alzheimer's disease), CRISPR-Cas9-edited ApoE isogenic lines
Application
Drug screening and disease modeling for Alzheimer's disease

Protocol overview

35 steps across 6 phases

iPSC generation and characterization Days 1–30
  1. 1 Recruitment and brain amyloid imaging of participants
  2. 2 Generate induced pluripotent stem cells (iPSCs) from patient-derived cells
  3. 3 Characterize iPSCs for pluripotency markers
  4. 4 Generate CRISPR-Cas9 ApoE isogenic iPSC lines
Cerebral organoid (iCO) generation Days 1–60
  1. 1 Generate embryoid bodies (EBs)
  2. 2 Differentiate EBs into cerebral organoids (iCOs)
  3. 3 Quality control of iCOs on Day 60
  4. 4 Characterize iCO pathological features on Day 60
Secreted protein quantification and RNA analysis Days 60–75
  1. 1 Collect conditioned media from iCOs
  2. 2 Quantify secreted AD hallmark proteins by ELISA
  3. 3 Perform alternative quantification by xMAP technology (optional)
  4. 4 Extract RNA and perform RNA sequencing
  5. 5 Perform differential gene expression (DEG) and Gene Ontology (GO) analysis
  6. 6 Validate transcriptomic data against public databases
Tissue clearing and HCS imaging Days 75–90
  1. 1 Prepare iCOs for tissue clearing
  2. 2 Perform ethyl cinnamate (ECi) 3D tissue clearing
  3. 3 Perform 3D confocal HCS imaging
  4. 4 Analyze HCS images for Aβ and p-tau quantification
  5. 5 Validate pathology localization by confocal microscopy
Mathematical network modeling and drug target identification Days 90–120 (computational)
  1. 1 Construct molecular regulatory network model for AD
  2. 2 Establish Boolean logical rules for network nodes
  3. 3 Validate network model with oxidative stress simulations
  4. 4 Validate network against experimental pathway data
  5. 5 Perform in silico single-node perturbation analysis
  6. 6 Perform in silico double-node (combination) perturbation analysis
  7. 7 Map perturbation targets to FDA-approved drugs
  8. 8 Filter drug candidates by pharmacological properties
Drug validation and HCS screening Days 120–150
  1. 1 Prepare high-quality iCOs for drug screening
  2. 2 Prepare drug stock solutions and working concentrations
  3. 3 Treat organoids with candidate drugs
  4. 4 Assess neuronal cell viability by MTT assay
  5. 5 Perform tissue clearing and HCS imaging of drug-treated organoids
  6. 6 Quantify drug efficacy on Aβ and p-tau reduction
  7. 7 Validate efficacy across organoid genotypes
  8. 8 Summarize drug-screening results

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Park et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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