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SKIN Publication-derived

Coordinated Development of Immune Cell Populations in Vascularized Skin Organoids from Human Induced Pluripotent Stem Cells

Source Mostina et al., 2025 · The University of Queensland, Brisbane, QLD, Australia · 10.1002/adhm.202502108

👤 Mitchell Mostina, Jane Sun, Seen Ling Sim, Imaan A. Ahmed, Fernando Souza-Fonesca-Guimaraes, Ernst J. Wolvetang, Jason Brown, Snehlata Kumari, Kiarash Khosrotehrani, Abbas Shafiee ⏱ 115 days 📋 5 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of vascularized skin organoids (VSKOs) from human induced pluripotent stem cells by coordinating the differentiation of skin and vascular lineages on an air-liquid interface. The resulting VSKOs recapitulate complex vascular structures with endothelial cells, pericytes, smooth muscle cells, and resident immune cell populations including macrophages, Langerhans cells, and neutrophils, providing a physiologically relevant platform for studying skin biology and immune-mediated skin disorders.

Cell source
Human iPSC
Application
Disease modeling, drug screening, regenerative medicine

Protocol overview

20 steps across 5 phases

Generation and Maintenance of Human Induced Pluripotent Stem Cell Lines Prior to differentiation
  1. 1 Prepare hiPSC culture
Skin Organoid (SKO) Differentiation Days 0–115
  1. 1 Generate hiPSC-derived skin organoids
Vascular Organoid (VO) Differentiation Days -2 to 115
  1. 1 Aggregate hiPSCs for VO formation (Day -2)
  2. 2 Initiate mesodermal differentiation (Day 0)
  3. 3 Apply TGFβ inhibition and growth factors (Day 2)
  4. 4 Switch to endothelial cell growth medium (Day 5)
  5. 5 Add Matrigel droplet and transfer to culture plates (Day 7)
  6. 6 Switch to organoid maturation medium (Day 15)
  7. 7 Monitor VO development
Vascularized Skin Organoid (VSKO) Co-Culture on Air-Liquid Interface Days 1–115
  1. 1 Coordinate SKO and VO differentiation start (Day 1 of SKO)
  2. 2 Prepare early VO culture with initial growth factors (Day 10 of SKO)
  3. 3 Assemble VSKO on air-liquid interface (Day 18 of SKO)
  4. 4 Establish control groups (Day 18 of SKO)
  5. 5 Maintain VSKO culture with scheduled medium changes (Days 18–115)
  6. 6 Monitor VSKO development via brightfield microscopy
Organoid Fixation and Characterization Days 42 and 115 of SKO differentiation
  1. 1 Harvest and fix organoid samples for immunofluorescence (Day 115)
  2. 2 Perform cryosection immunofluorescence microscopy
  3. 3 Perform whole-mount immunofluorescence microscopy
  4. 4 Perform hematoxylin and eosin staining
  5. 5 Perform flow cytometric analysis of vascular organoids (Day 42 of VO differentiation)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Mostina et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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