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BRAIN Publication-derived

APOE Deficiency Impacts Neural Differentiation and Cholesterol Biosynthesis in Human iPSC-Derived Cerebral Organoids

Source Zhao et al. · Department of Neuroscience, Mayo Clinic, Jacksonville, FL · 10.1101/2022.06.30.498241;

👤 Jing Zhao, Tadafumi C. Ikezu, Wenyan Lu, Jesse R. Macyczko, Yonghe Li, Laura J. Lewis-Tuffin, Yuka A. Martens, Yingxue Ren, Yiyang Zhu, Yan W. Asmann, Nilüfer Ertekin-Taner, Takahisa Kanekiyo, Guojun Bu ⏱ 90 days 📋 6 phases 🧫 Human iPSC

Abstract

This protocol generates three-dimensional cerebral organoids from human iPSCs (parental and APOE-deficient lines) to model neural differentiation and cholesterol biosynthesis dysregulation relevant to Alzheimer's disease. The organoids are characterized by single-cell RNA sequencing, immunostaining, and molecular validation to define cell-type-specific effects of APOE deficiency on neurogenesis, lipid metabolism, and stress pathways.

Cell source
Human iPSC
Application
Disease modeling; Alzheimer's disease pathogenesis

Protocol overview

27 steps across 6 phases

Embryoid Body Formation Day 0–4
  1. 1 Dissociate iPSC colonies into single-cell suspension
  2. 2 Dispense cells into a 96-well plate with ultra-low-attachment surfaces and U-shaped bottom wells.
  3. 3 Add medium supplementation on Day 2
  4. 4 Add medium supplementation on Day 4
Embryoid Body Maturation in Low-Attachment Plates Day 5–9
  1. 1 Transfer embryoid bodies to 48-well low-attachment plates
  2. 2 Culture in Medium B for 3–5 days
Matrigel Embedding and Organoid Initiation Day 10–13
  1. 1 Embed embryoid bodies in Matrigel
  2. 2 Culture embedded organoids in Medium C+D
Orbital Shaker Culture and Neuronal Maturation Day 13–60 (and beyond for ISRIB treatment)
  1. 1 Transfer organoids to orbital shaker culture
  2. 2 Switch to neuronal maturation medium after 4 weeks
  3. 3 Continue culture until Day 90
ISRIB Treatment (Optional, for EIF2 Pathway Inhibition) Day 60–90
  1. 1 Initiate ISRIB treatment at Day 60
  2. 2 Replace medium every 3 days
  3. 3 Harvest organoids at Day 90
Single-Cell Dissociation and RNA-Sequencing Library Preparation Day 90 (harvest day)
  1. 1 Prepare Papain + DNase solution
  2. 2 Transfer organoids to dissociation dish
  3. 3 Mince organoids into small pieces
  4. 4 Enzymatic dissociation in shaking incubator (first incubation)
  5. 5 Mechanical dissociation by pipetting
  6. 6 Enzymatic dissociation in shaking incubator (second incubation)
  7. 7 Complete mechanical dissociation and stop enzymatic reaction
  8. 8 Transfer cell suspension to stop solution
  9. 9 Resuspend cells in PBS-BSA buffer
  10. 10 Assess cell viability
  11. 11 Load cells onto 10x Chromium chip
  12. 12 Construct single-cell libraries
  13. 13 Sequence libraries on Illumina HiSeq 4000

Full SOP

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Attribution

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

This wording is awaiting legal review.

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