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BRAIN Publication-derived

Reconstruction of the Human Blood-Brain Barrier in vitro (iBBB) and Investigation of APOE4-Mediated Cerebral Amyloid Angiopathy

Source Blanchard et al., 2020 · Picower Institute for Learning and Memory, Massachusetts Institute of Technology · 10.1038/s41591-020-0886-4

👤 Joel W. Blanchard, Michael Bula, Jose Davila-Velderrain, Leyla Anne Akay, Lena Zhu, Alexander Frank, Matheus B. Victor, Julia Maeve Bonner, Hansruedi Mathys, Yuan-Ta Lin, Tak Ko, David A. Bennett, Hugh P. Cam, Manolis Kellis, Li-Huei Tsai ⏱ 30 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of a three-dimensional in vitro human blood-brain barrier model (iBBB) from iPSC-derived brain endothelial cells, pericytes, and astrocytes. The iBBB recapitulates key anatomical and physiological properties of the in vivo BBB including tight junctions, efflux transporter polarization, and selective permeability. The model reveals that APOE4 dysregulation in pericytes, via calcineurin/NFAT-signaling, drives increased amyloid-beta accumulation mimicking cerebral amyloid angiopathy pathology.

Cell source
Human iPSC
Application
Disease modeling; Mechanism of action studies for APOE4 in cerebral amyloid angiopathy

Protocol overview

50 steps across 12 phases

iPSC Maintenance and Culture Preparation Ongoing before differentiation
  1. 1 Maintain iPSC and hESC lines
  2. 2 Verify iPSC genotype and quality
Brain Endothelial Cell (BEC) Differentiation Days 1–14
  1. 1 Dissociate iPSCs to single cells
  2. 2 Change medium (Days 2–3)
  3. 3 Apply DeSR2 medium (Days 4–8)
  4. 4 Switch to endothelial specification medium (Days 9–11)
  5. 5 Passage and expand BECs (Day 12)
Mural Cell (Pericyte-like Cell) Differentiation Days 1–7
  1. 1 Dissociate and seed iPSCs for mural differentiation
  2. 2 Apply BMP4 and CHIR99021 (Day 1)
  3. 3 Apply PDGF-BB and Activin A (Days 4–5)
  4. 4 Maintain in N2B27 medium until co-culture
Astrocyte Differentiation Days 1–28
  1. 1 Differentiate neural progenitor cells (NPCs)
  2. 2 Induce astrocyte differentiation
  3. 3 Confirm astrocyte identity
Three-Dimensional iBBB Assembly and Culture Days 1–30
  1. 1 Prepare cell suspension for 3D encapsulation
  2. 2 Seed Matrigel into culture dishes
  3. 3 Culture in complete astrocyte medium with VEGFA (Days 1–14)
  4. 4 Remove VEGFA and culture in astrocyte medium only (Days 15–30)
  5. 5 Fix and stain for imaging (Day 30)
Trans-well Permeability Assays (TEER and Molecular Permeability) Days 1–7
  1. 1 Seed BECs on transwell membrane
  2. 2 Seed pericytes and astrocytes (Day 1, 24 hours post-BEC seeding)
  3. 3 Measure TEER values (Days 2–7)
  4. 4 Perform molecular permeability assay
  5. 5 Collect media and quantify permeability
Efflux Transporter Polarization Assays Days 6–7
  1. 1 Prepare transwell cultures and inhibitors
  2. 2 Measure apical-to-basolateral and basolateral-to-apical transport
  3. 3 Quantify and compare transport rates
Amyloid-Beta Accumulation Assays Days 1–30 (synchronized with iBBB maturation)
  1. 1 Prepare amyloid-beta sources
  2. 2 Expose mature iBBBs to amyloid (Day 30 iBBB)
  3. 3 Fix and immunostain for amyloid
  4. 4 Quantify amyloid accumulation by image analysis
  5. 5 Measure soluble amyloid in media (Optional)
RNA Extraction and qRT-PCR Analysis Days 1–30 (synchronized with experimental endpoints)
  1. 1 Harvest cells and extract total RNA
  2. 2 Reverse transcription (RT)
  3. 3 Quantify gene expression by qRT-PCR
  4. 4 Validate critical genes by RNA-sequencing (optional)
Calcineurin Inhibitor Treatment and APOE Modulation Days 1–28 (iBBB pre-treatment phase)
  1. 1 Prepare calcineurin inhibitor stock solutions
  2. 2 Treat iBBBs or iMCs with calcineurin inhibitors (Days 1–14)
  3. 3 Measure APOE expression by qRT-PCR
  4. 4 Measure APOE protein by Western blot and ELISA
  5. 5 Assess amyloid accumulation post-treatment
APOE Immunodepletion Experiments Days 1–4 (conditioned media preparation)
  1. 1 Prepare conditioned media from iMCs
  2. 2 Immunodeplete APOE from conditioned media
  3. 3 Verify APOE immunodepletion by ELISA or Western blot
  4. 4 Expose iBBBs to immunodepleted media and quantify amyloid
In Vivo Studies (Murine Models) 6–9 weeks (age of mice at treatment initiation)
  1. 1 Prepare cyclosporine A for intraperitoneal injection
  2. 2 Treat APOE4KI x 5XFAD mice with CsA
  3. 3 Perfuse brains and collect tissue
  4. 4 Slice and stain brain tissue
  5. 5 Acquire and analyze images

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Blanchard et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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