Skip to content
← Back to browse
BRAIN Publication-derived

Generation and Characterization of iPSC-Derived Cerebral Organoids for Alzheimer's Disease Modeling with APOE Genotype Variants

Source Zhao et al., 2020 · Mayo Clinic, Jacksonville, FL · 10.1038/s41467-020-19264-0

👤 Jing Zhao, Yuan Fu, Yu Yamazaki, Yingxue Ren, Mary D. Davis, Chia-Chen Liu, Wenyan Lu, Xue Wang, Kai Chen, Yesesri Cherukuri, Lin Jia, Yuka A. Martens, Lucy Job, Francis Shue, Thanh Thanh Nguyen, Steven G. Younkin, Neill R. Graff-Radford, Zbigniew K. Wszolek, David A. Brafman, Yan W. Asmann, Nilüfer Ertekin-Taner, Takahisa Kanekiyo, Guojun Bu ⏱ 28 days 📋 10 phases 🧫 Patient-Derived iPSC (Alzheimer's disease, APOE ε3/ε3 or ε4/ε4), Human iPSC (Normal cognition, APOE ε3/ε3 or ε4/ε4)

Abstract

This protocol describes the generation and comprehensive characterization of three-dimensional cerebral organoids derived from human induced pluripotent stem cells (iPSCs) with different APOE genotypes (ε3/ε3 and ε4/ε4) from both Alzheimer's disease patients and cognitively normal individuals. The organoids recapitulate key AD pathologies including increased amyloid-beta and phosphorylated tau accumulation, neuronal apoptosis, synaptic loss, and stress granule formation, particularly in APOE4-carrying AD organoids.

Cell source
Patient-Derived iPSC (Alzheimer's disease, APOE ε3/ε3 or ε4/ε4), Human iPSC (Normal cognition, APOE ε3/ε3 or ε4/ε4)
Application
Disease modeling

Protocol overview

48 steps across 10 phases

Generation of iPSCs from human skin fibroblasts Day 0-28
  1. 1 Fibroblast culture and preparation
  2. 2 Electroporation of episomal vectors
  3. 3 Post-transfection plating
  4. 4 Medium replacement
  5. 5 iPSC colony isolation and expansion
Validation of iPSC pluripotency Week 3-6
  1. 1 Pluripotency marker immunostaining
  2. 2 Trilineage differentiation assessment
  3. 3 Karyotype analysis
Cerebral organoid generation and early differentiation Day 0-12
  1. 1 iPSC dissociation and seeding
  2. 2 Embryonic body expansion
  3. 3 Neural induction
  4. 4 Matrigel embedding and neural expansion
  5. 5 Transfer to orbital shaker
Organoid maturation and neuronal differentiation Week 4-12
  1. 1 Medium replacement at week 4
  2. 2 Continued orbital shaker culture
  3. 3 Organoid monitoring and collection
Tissue processing and analysis preparation Week 4-12 (at analysis timepoints)
  1. 1 Organoid harvest and RIPA lysis
  2. 2 Sequential fractionation
  3. 3 Protein concentration determination
  4. 4 Sample storage
Immunostaining analysis Week 4-12 (at analysis timepoints)
  1. 1 Organoid fixation
  2. 2 Cryoprotection and embedding
  3. 3 Section permeabilization and blocking
  4. 4 Primary antibody incubation
  5. 5 Secondary antibody incubation
  6. 6 Mounting and microscopy
Western blotting analysis Week 4-12 (at analysis timepoints)
  1. 1 Sample loading and gel electrophoresis
  2. 2 Protein transfer
  3. 3 Membrane blocking
  4. 4 Primary antibody incubation
  5. 5 Secondary antibody and detection
  6. 6 Quantification
ELISA quantification of proteins Week 4-12 (at analysis timepoints)
  1. 1 Aβ and phospho-tau measurement
  2. 2 APP derivative measurement
  3. 3 ApoE measurement by custom ELISA
  4. 4 Data normalization
RNA extraction and RT-qPCR analysis Week 4-12 (at analysis timepoints)
  1. 1 RNA extraction
  2. 2 RNA quality assessment
  3. 3 cDNA synthesis
  4. 4 Real-time qPCR
  5. 5 Data analysis
RNA-sequencing and transcriptomic analysis Week 12
  1. 1 Sample preparation for RNA-seq
  2. 2 RNA sequencing
  3. 3 Read quality control and alignment
  4. 4 Data normalization
  5. 5 Differential gene expression analysis
  6. 6 Pathway analysis
  7. 7 Weighted Gene Co-expression Network Analysis (WGCNA)

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Zhao 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.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence