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VASCULAR Publication-derived

Blood Vessel Organoids Generated by Base Editing and Harboring Notch3 SNV for CADASIL Disease Modeling

Source Ahn et al., 2024 · Korea Research Institute of Bioscience and Biotechnology (KRIBB) · 10.1007/s12035-024-04141-4

👤 Yujin Ahn, Ju-Hyun An, Hae-Jun Yang, Wi-Jae Lee, Sang-Hee Lee, Young-Ho Park, Jong-Hee Lee, Hong J. Lee, Seung Hwan Lee, Sun-Uk Kim ⏱ 18 days 📋 15 phases 🧫 Patient-Derived iPSC (CADASIL)

Abstract

This protocol describes the generation of human blood vessel organoids (hBVOs) from hiPSCs carrying NOTCH3 mutations (R153C or R182C) via CRISPR/Cas9 base editing. The resulting CADASIL-model hBVOs exhibit pathologic features including reduced vessel diameter, mural cell degeneration, increased Notch3ECD accumulation, elevated apoptosis, and cytoskeletal alterations, recapitulating CADASIL pathogenesis in vitro.

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

Protocol overview

83 steps across 15 phases

Design and Construction of Base-Editing Vector Preparatory
  1. 1 Design sgRNA candidates
  2. 2 Ligate sgRNAs to backbone plasmid
  3. 3 Construct base-editing vector
Base-Editor Efficiency Testing and Notch3 Mutation Generation Days 1-7 (approximate)
  1. 1 Culture and prepare HEK293 cells for transfection
  2. 2 Transfect HEK293 cells with base-editor plasmids
  3. 3 Isolate GFP-positive cells and assess editing efficiency
  4. 4 Transfect wild-type hiPSCs with optimized base-editor plasmids
  5. 5 Identify and expand Notch3 mutant hiPSC clones
Pluripotency and Genomic Stability Characterization of Notch3 Mutant hiPSCs Days 1-14
  1. 1 Assess pluripotency markers by flow cytometry
  2. 2 Confirm pluripotency by immunostaining
  3. 3 Perform karyotype analysis
  4. 4 Perform off-target analysis
Maintenance and Expansion of Feeder-Independent hiPSCs Ongoing (every 4-5 days)
  1. 1 Coat culture plates with Matrigel
  2. 2 Culture hiPSCs on Matrigel-coated plates
  3. 3 Passage hiPSCs every 4-5 days
Generation of Spin Embryoid Bodies Day 0
  1. 1 Prepare hiPSCs for embryoid body formation
  2. 2 Centrifuge and resuspend cells
  3. 3 Count live cells
  4. 4 Plate hiPSCs for spin embryoid body formation
  5. 5 Incubate spin embryoid bodies overnight
Differentiation of Human Blood Vessel Organoids (Days 0-15) Days 1-15
  1. 1 Initiate mesoderm induction (Days 0-2)
  2. 2 Vascular specification (Days 3-4)
  3. 3 Embed organoids in extracellular matrix (Day 5)
  4. 4 Vascular sprouting and network formation (Days 7-10)
  5. 5 Isolate and maintain individual blood vessel organoids (Day 10)
Whole-Mount Immunofluorescence Staining of Blood Vessel Organoids Post-differentiation (any timepoint)
  1. 1 Extract organoids from gel and rinse
  2. 2 Remove non-specific background (gel recovery)
  3. 3 Fix organoids
  4. 4 Clear organoids
  5. 5 Permeabilization
  6. 6 Rehydration
  7. 7 Antibody penetration and blocking
  8. 8 Primary antibody incubation
  9. 9 Primary antibody wash
  10. 10 Secondary antibody and DAPI incubation
  11. 11 Secondary antibody wash
  12. 12 Dehydration and final clearing
  13. 13 Confocal imaging
Flow Cytometric Analysis of hBVOs Post-differentiation (typically day 15-18)
  1. 1 Dissociate organoids into single cells
  2. 2 Wash and prepare cells
  3. 3 Surface antibody staining
  4. 4 Intracellular staining (if applicable)
  5. 5 Flow cytometry acquisition
  6. 6 Data analysis
Apoptosis Assessment by TUNEL Assay Post-differentiation (day 15-18)
  1. 1 Prepare organoids for TUNEL staining
  2. 2 Perform TUNEL staining
  3. 3 Co-stain with endothelial and mural cell markers
  4. 4 Confocal imaging and analysis
Cytoskeleton Analysis by Fluorescence Phalloidin Staining Post-differentiation (day 15-18)
  1. 1 Prepare organoids for F-actin imaging
  2. 2 Stain F-actin with fluorescent phalloidin
  3. 3 Wash and prepare for imaging
  4. 4 Confocal imaging and analysis
Transmission Electron Microscopy (TEM) to Assess GOM Deposition Post-differentiation (day 15-18)
  1. 1 Fix organoids for TEM
  2. 2 Post-fixation in osmium tetroxide
  3. 3 Dehydration in graded alcohols
  4. 4 Embedding in resin
  5. 5 Generate semi-thin sections
  6. 6 Cut ultrathin sections
  7. 7 Stain ultrathin sections
  8. 8 Transmission electron microscopy imaging
Mural Cell Differentiation from hiPSCs (2D Culture) Days 1-18
  1. 1 Prepare Matrigel-coated plates
  2. 2 Plate hiPSCs for mural differentiation
  3. 3 Day 1 medium change
  4. 4 Day 2–6 mesodermal induction
  5. 5 Day 6–18 mural cell specification
  6. 6 Assess mural cell differentiation
Propidium Iodide (PI) Staining for Cell Cycle Analysis Post-differentiation (day 18 or later)
  1. 1 Harvest and fix mural cells
  2. 2 Wash fixed cells
  3. 3 Stain with PI and RNase
  4. 4 Flow cytometry analysis
  5. 5 Statistical analysis
Inhibitor Treatment Study (Days 15-18 of hBVO Differentiation) Days 15-18
  1. 1 Prepare organoids and inhibitor solutions
  2. 2 Treat Notch3 mutant hBVOs with inhibitors
  3. 3 Continue treatment until day 18
  4. 4 Assess vascular morphology and cell interactions
  5. 5 Quantify restoration of cell interactions
Quantitative Real-Time Reverse-Transcription PCR (qRT-PCR) Post-differentiation (any timepoint)
  1. 1 Sample collection and RNA extraction
  2. 2 Quantify RNA and assess quality
  3. 3 Reverse transcription
  4. 4 Design or verify qRT-PCR primers
  5. 5 Set up qRT-PCR reactions
  6. 6 Run qRT-PCR and acquire Ct values
  7. 7 Data analysis: relative quantification

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ahn et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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