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BRAIN Publication-derived

Alzheimer's Disease Brain Models with Myelination

Full SOP with Academic or a plan

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

Authors: Joel W. Blanchard, Leyla Anne Akay, Jose Davila-Velderrain, Djuna von Maydell, Hansruedi Mathys, Li-Huei Tsai, Manolis Kellis Duration: 75 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Blanchard et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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