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VASCULAR Publication-derived

Vascular Organoids for Disease Modelling with Gene Editing

Full SOP with Academic or a plan

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

Authors: 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 Duration: 18 days 15 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Ahn et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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