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BRAIN Publication-derived

Schizophrenia endothelial cells exhibit higher permeability and altered angiogenesis patterns in patient-derived organoids

Source Stankovic et al., 2024 · Weill Cornell Medicine, Cornell University · 10.1038/s41398-024-02740-2

👤 Isidora Stankovic, Michael Notaras, Paul Wolujewicz, Tyler Lu, Raphael Lis, M. Elizabeth Ross, Dilek Colak ⏱ 35 days 📋 6 phases 🧫 Patient-Derived iPSC (Schizophrenia)

Abstract

This protocol describes the generation of 3D cerebral organoids from patient-derived and healthy control iPSCs to model endothelial cell dysfunction in schizophrenia. The protocol combines a morphogen-free organoid culture system with single-cell RNA sequencing, immunohistochemistry, and functional permeability assays to identify altered endothelial cell signatures and blood-brain barrier dysfunction in schizophrenia brain tissue.

Cell source
Patient-Derived iPSC (Schizophrenia)
Application
Disease modeling

Protocol overview

38 steps across 6 phases

iPSC Maintenance and Preparation Prior to organoid generation
  1. 1 Obtain and characterize iPSC lines
  2. 2 Culture iPSCs on Vitronectin-coated plates
Three-Dimensional Cerebral Organoid Generation Days 0-35
  1. 1 Generate embryoid bodies (EB stage)
  2. 2 Neural fate specification
  3. 3 Embed organoids in Matrigel droplets
  4. 4 Culture organoids in terminal organoid media without agitation
  5. 5 Continue culture with agitation
  6. 6 Apply quality control criteria at multiple stages
Single-Cell RNA Sequencing Day 30 DIV
  1. 1 Dissociate organoids to single-cell suspension
  2. 2 Load cells into 10x Chromium microfluidic devices
  3. 3 Prepare cDNA libraries
  4. 4 Perform next-generation sequencing
  5. 5 Align reads and perform quality control
  6. 6 Normalize and integrate data
  7. 7 Perform clustering and cell type annotation
Immunohistochemistry and Microscopy Analysis Day 30 DIV
  1. 1 Prepare organoids for cryosectioning
  2. 2 Serially cryosection organoids
  3. 3 Perform antigen retrieval
  4. 4 Incubate primary antibodies
  5. 5 Incubate secondary antibodies
  6. 6 Acquire laser-scanning confocal microscopy images
  7. 7 Quantify endothelial cells and vessel-like structures
Differentiation of iPSCs to Brain Microvascular Endothelial Cells (iBMECs) Days 0-26
  1. 1 Expand iPSCs to confluence
  2. 2 Reduce ROCK inhibitor concentration
  3. 3 Switch to unconditioned medium
  4. 4 Transduce cells with lentiviral vectors
  5. 5 Dissociate and plate cells on coated surfaces
  6. 6 Expand iBMECs to confluency
  7. 7 FACS isolation of pure endothelial populations
  8. 8 Expand sorted iBMECs
FITC-Dextran Paracellular Permeability Assay Day 26 of iBMEC differentiation
  1. 1 Prepare Transwell plates with endothelial cells
  2. 2 Culture cells to confluency
  3. 3 Add anti-VE-cadherin antibody
  4. 4 Add FITC-labeled dextran
  5. 5 Incubate FITC-dextran
  6. 6 Collect media from lower compartment
  7. 7 Measure fluorescence intensity
  8. 8 Calculate FITC-dextran passage

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Stankovic et al., 2024. 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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