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BRAIN Publication-derived

Alzheimer's Disease Cerebral Organoids (2020, No. 2)

Full SOP with Academic or a plan

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

Authors: 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 Duration: 28 days 10 phases Cell source: 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

Phase 1 · Generation of iPSCs from human skin fibroblasts Day 0-28
  1. a Fibroblast culture and preparation
  2. b Electroporation of episomal vectors
  3. c Post-transfection plating
  4. d Medium replacement
  5. e iPSC colony isolation and expansion
Phase 2 · Validation of iPSC pluripotency Week 3-6
  1. a Pluripotency marker immunostaining
  2. b Trilineage differentiation assessment
  3. c Karyotype analysis
Phase 3 · Cerebral organoid generation and early differentiation Day 0-12
  1. a iPSC dissociation and seeding
  2. b Embryonic body expansion
  3. c Neural induction
  4. d Matrigel embedding and neural expansion
  5. e Transfer to orbital shaker
Phase 4 · Organoid maturation and neuronal differentiation Week 4-12
  1. a Medium replacement at week 4
  2. b Continued orbital shaker culture
  3. c Organoid monitoring and collection
Phase 5 · Tissue processing and analysis preparation Week 4-12 (at analysis timepoints)
  1. a Organoid harvest and RIPA lysis
  2. b Sequential fractionation
  3. c Protein concentration determination
  4. d Sample storage
Phase 6 · Immunostaining analysis Week 4-12 (at analysis timepoints)
  1. a Organoid fixation
  2. b Cryoprotection and embedding
  3. c Section permeabilization and blocking
  4. d Primary antibody incubation
  5. e Secondary antibody incubation
  6. f Mounting and microscopy
Phase 7 · Western blotting analysis Week 4-12 (at analysis timepoints)
  1. a Sample loading and gel electrophoresis
  2. b Protein transfer
  3. c Membrane blocking
  4. d Primary antibody incubation
  5. e Secondary antibody and detection
  6. f Quantification
Phase 8 · ELISA quantification of proteins Week 4-12 (at analysis timepoints)
  1. a Aβ and phospho-tau measurement
  2. b APP derivative measurement
  3. c ApoE measurement by custom ELISA
  4. d Data normalization
Phase 9 · RNA extraction and RT-qPCR analysis Week 4-12 (at analysis timepoints)
  1. a RNA extraction
  2. b RNA quality assessment
  3. c cDNA synthesis
  4. d Real-time qPCR
  5. e Data analysis
Phase 10 · RNA-sequencing and transcriptomic analysis Week 12
  1. a Sample preparation for RNA-seq
  2. b RNA sequencing
  3. c Read quality control and alignment
  4. d Data normalization
  5. e Differential gene expression analysis
  6. f Pathway analysis
  7. g Weighted Gene Co-expression Network Analysis (WGCNA)

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Attribution

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

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