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SKIN Publication-derived

An optimized protocol for generating appendage-bearing skin organoids from human-induced pluripotent stem cells

Source Ahmed et al., 2024 · The University of Queensland Frazer Institute, The University of Queensland, Brisbane, Australia · 10.1093/biomethods/bpae019

👤 Imaan Ahmed, Jane Sun, Jason Brown, Kiarash Khosrotehrani, Abbas Shafiee ⏱ 90 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of skin organoids (SKOs) bearing complex appendages (hair follicles, sebaceous glands, sweat glands) from human iPSCs via direct embryoid body formation in E6 differentiation media. The optimized method reduces line-dependent variability by seeding 8000 hiPSCs in E6 media for 24 hours prior to stepwise TGFβ/BMP pathway modulation, producing consistent SKO differentiation across multiple hiPSC sources.

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

Protocol overview

32 steps across 9 phases

hiPSC Maintenance and Preparation Days -∞ to -2
  1. 1 Coat 6-well plate with hESC-Qualified Matrigel
  2. 2 Thaw frozen hiPSCs
  3. 3 Plate thawed hiPSCs onto coated wells
  4. 4 Change media after 24 h and maintain hiPSCs
hiPSC Passaging Every 3–4 days when 70–80% confluent
  1. 5 Assess culture and remove differentiated cells if present
  2. 6 Detach hiPSCs with Accutase
  3. 7 Centrifuge and resuspend cells
  4. 8 Plate hiPSCs at split ratio
  5. 9 Incubate and perform first media change
Day -1: Embryoid Body Formation in E6 Media Day -1 (24 h culture)
  1. 10 Prepare hiPSCs for aggregation
  2. 11 Calculate and prepare cell suspension for seeding
  3. 12 Transfer suspension to multichannel reservoir and seed ultra-low attachment plate
  4. 13 Centrifuge plate and incubate for embryoid body formation
Day 0: Induction of Non-Neural Ectoderm Differentiation Day 0 (3-day incubation)
  1. 14 Check embryoid body formation
  2. 15 Prepare Day 0 differentiation medium
  3. 16 Remove old media and add Day 0 differentiation medium
  4. 17 Incubate for 3 days
Day 3: Induction of Cranial Neural Crest Cell Differentiation Day 3 (3-day incubation)
  1. 18 Prepare Day 3 differentiation medium
  2. 19 Add Day 3 differentiation medium to wells
  3. 20 Incubate for 3 days
Days 6–10: Media Changes and Continued Differentiation Days 6–10
  1. 21 Day 6: Add fresh E6 media
  2. 22 Days 8 and 10: Half media changes
Day 12: Transfer to 24-Well Plates Day 12
  1. 23 Assess and select SKOs for transfer
  2. 24 Prepare organoid maturation medium (OMM) with 1% Matrigel
  3. 25 Prepare pipette tips for organoid transfer
  4. 26 Transfer SKOs to 24-well plates
  5. 27 Incubate transferred SKOs
Day 15: First Half Media Change with OMM + Matrigel Day 15
  1. 28 Prepare OMM with 1% Matrigel
  2. 29 Perform half media change
Day 18 Onwards: Continued Media Changes and Volume Increases Day 18 to Day 90+ (continuous culture)
  1. 30 Perform half media changes with OMM without Matrigel
  2. 31 Increase media volume from Day 80 onwards
  3. 32 Monitor for hair follicle and appendage development

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ahmed 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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