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BRAIN Publication-derived

APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes

Source Blanchard et al., 2022 · Picower Institute for Learning and Memory, Massachusetts Institute of Technology · 10.1038/s41586-022-05439-w

👤 Joel W. Blanchard, Leyla Anne Akay, Jose Davila-Velderrain, Djuna von Maydell, Hansruedi Mathys, Li-Huei Tsai, Manolis Kellis ⏱ 75 days 📋 5 phases 🧫 Human iPSC (isogenic APOE3/3 and APOE4/4), Patient-Derived iPSC (APOE4/4 AD), Human post-mortem prefrontal cortex

Abstract

This protocol describes the generation of isogenic iPSC-derived oligodendroglia from APOE3/3 and APOE4/4 backgrounds, along with associated characterization methods including lipidomic profiling, immunostaining, and functional myelination assays. The protocol establishes a cellular model for investigating APOE4-mediated cholesterol dysregulation and impaired myelination in oligodendrocytes, with applications for understanding Alzheimer's disease mechanisms and testing therapeutic interventions.

Cell source
Human iPSC (isogenic APOE3/3 and APOE4/4), Patient-Derived iPSC (APOE4/4 AD), Human post-mortem prefrontal cortex
Application
Disease modeling of APOE4-associated Alzheimer's disease pathogenesis and oligodendrocyte dysfunction

Protocol overview

26 steps across 5 phases

iPSC Differentiation into Oligodendroglia 0-75+
  1. 1 Prepare iPSCs for differentiation
  2. 2 Initiate oligodendrocyte differentiation (Day 0)
  3. 3 Add SAG and continue morphogen treatment (Day 8)
  4. 4 Promote sphere aggregation (Day 12)
  5. 5 Plate spheres for cell migration (Day 30)
  6. 6 Select for NG2+ OPC population (Day 75)
  7. 7 Promote oligodendrocyte maturation
Cholesterol Biosynthesis and Transport Assays Day 75+
  1. 1 Drug treatment of oligodendroglia
  2. 2 Bodipy-cholesterol uptake assay
  3. 3 Filipin staining for total cholesterol localization
  4. 4 Lipidomic profiling of oligodendroglia
In Vitro Myelination Assay 0-42
  1. 1 Generate iNeurons from iPSCs
  2. 2 Encapsulate oligodendroglia-iNeuron co-cultures in 3D matrix
  3. 3 Allow myelination formation (Weeks 2–6)
  4. 4 Assess myelination via immunostaining
  5. 5 Optional: Transmission electron microscopy (TEM) for ultrastructure
Therapeutic Intervention: Cyclodextrin Treatment 0-42 (in vitro) or 0-56 (in vivo)
  1. 1 In vitro cyclodextrin treatment of co-cultures
  2. 2 Assess cholesterol levels after treatment
  3. 3 Assess myelination after cyclodextrin treatment
  4. 4 In vivo cyclodextrin administration to mice
  5. 5 Assess myelination in treated mice
Behavioral Assessment of Learning and Memory Post-treatment (cyclodextrin or control)
  1. 1 Novel object recognition task—Habituation
  2. 2 Novel object recognition task—Sample phase (Day 1)
  3. 3 Novel object recognition task—Test phase (4 hours later)
  4. 4 Puzzle Box task—Habituation (Day 0)
  5. 5 Puzzle Box task—Test phase (Days 1–4)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Blanchard et al., 2022. 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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