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BRAIN Publication-derived

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

Source Hernández et al., 2022 · The University of Melbourne · 10.1007/s12015-021-10147-5

👤 Damián Hernández, Louise A. Rooney, Maciej Daniszewski, Lerna Gulluyan, Helena H. Liang, Anthony L. Cook, Alex W. Hewitt, Alice Pébay ⏱ 270 days 📋 8 phases 🧫 Human iPSC (reprogrammed from skin fibroblasts)

Abstract

This protocol describes the generation of human induced pluripotent stem cell (iPSC)-derived cerebral organoids from individuals with different APOE genotypes (APOE-ε3/ε3 and APOE-ε4/ε4) using an unguided differentiation method. The protocol produces 6-month-old cerebral organoids suitable for measuring pathological hallmarks of Alzheimer's disease, including APOE expression, β-amyloid deposits, and Tau phosphorylation levels.

Cell source
Human iPSC (reprogrammed from skin fibroblasts)
Application
Disease modeling

Protocol overview

43 steps across 8 phases

iPSC Generation and Maintenance Days 1–14
  1. 1 Reprogramming of skin fibroblasts to iPSCs
  2. 2 Manual colony dissection and clonal selection
  3. 3 iPSC maintenance in feeder-free, serum-free conditions
Generation of Isogenic Lines via CRISPR/Cas9 Editing Days 15–45
  1. 1 Design of sgRNA and single-strand DNA for homology-directed repair
  2. 2 Preparation of ribonucleoprotein (RNP) complex
  3. 3 Electroporation of iPSCs with RNP complex
  4. 4 FACS sorting of ATTO 550-positive cells
  5. 5 Manual clone picking and genotyping
Characterization of iPSC Lines Days 46–75
  1. 1 Virtual karyotype analysis by copy number variation (CNV) analysis
  2. 2 Immunocytochemistry for pluripotency markers
  3. 3 APOE genotyping by Sanger sequencing
Differentiation to Three Germ Layers Days 76–90
  1. 1 Embryoid body (EB) formation and tri-lineage differentiation
  2. 2 Germ layer immunochemistry assessment
Cerebral Organoid Differentiation (Days 0–180) Days 91–270
  1. 1 iPSC dissociation and seeding for EB formation
  2. 2 EB medium changes (Days 1–5)
  3. 3 Transfer to ultra-low attachment 24-well plates (Day 5)
  4. 4 Matrigel embedding and transfer to expansion conditions (Day 7)
  5. 5 Transition to maturation medium and orbital shaking (Day 10)
  6. 6 Medium changes during maturation phase (Days 10–180)
  7. 7 Organoid harvest at 6 months
Western Blotting for Protein Analysis Days 181–190
  1. 1 Organoid lysis
  2. 2 Protein denaturation
  3. 3 SDS-PAGE separation
  4. 4 Protein transfer to PVDF membrane
  5. 5 Membrane blocking
  6. 6 Primary antibody incubation
  7. 7 Wash in PBS-T
  8. 8 Secondary antibody incubation
  9. 9 Membrane stripping and re-probing
  10. 10 Chemiluminescent detection and scanning
  11. 11 Quantification and normalization
ELISA Analysis of APOE, Aβ, and Tau Days 181–200
  1. 1 Sample preparation: organoid lysis and conditioned media collection
  2. 2 APOE ELISA
  3. 3 Aβ42 ELISA
  4. 4 Aβ40 ELISA
  5. 5 Total Tau ELISA
  6. 6 Phosphorylated Tau ELISA (multiple sites)
  7. 7 Plate reading and data analysis
Statistical Analysis Days 201–210
  1. 1 Data compilation and normalization
  2. 2 Statistical testing: two-group comparisons
  3. 3 Statistical testing: multi-group comparisons
  4. 4 Significance levels and reporting
  5. 5 Data visualization and figure generation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Hernández et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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