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SKIN Publication-derived

Development of Physiologically Relevant Skin Organoids from Human Induced Pluripotent Stem Cells

Source Shafiee et al., 2024 · University of Queensland, Herston Biofabrication Institute · 10.1002/smll.202304879

👤 Abbas Shafiee, Jane Sun, Imaan A. Ahmed, Felicia Phua, Gustavo R. Rossi, Cheng-Yu Lin, Fernando Souza-Fonseca-Guimaraes, Ernst J. Wolvetang, Jason Brown, Kiarash Khosrotehrani ⏱ 125 days 📋 8 phases 🧫 Human iPSC (skin fibroblast-derived and placental CD34+-derived)

Abstract

This protocol describes the generation of physiologically relevant skin organoids (SKOs) from human induced pluripotent stem cells (hiPSCs) through optimized embryoid body formation coupled with modulation of TGF-β, FGF, and BMP signaling pathways. The resulting SKOs develop stratified epidermal layers, pigmented hair follicles, sebaceous glands, and eccrine sweat glands, as well as Merkel cells, enabling studies of skin development, disease modeling, and wound healing.

Cell source
Human iPSC (skin fibroblast-derived and placental CD34+-derived)
Application
Disease modeling and regenerative medicine

Protocol overview

36 steps across 8 phases

iPSC Maintenance and Preparation Before differentiation
  1. 1 Coat culture plates with Matrigel
  2. 2 Maintain hiPSCs in culture
  3. 3 Passage confluent hiPSCs
Embryoid Body Formation and Initial Differentiation (Days 0–6) Days 0–6
  1. 1 Prepare hiPSCs for aggregation
  2. 2 Form embryoid bodies in Ultra-Low Attachment plates
  3. 3 Incubate for EB formation
  4. 4 Apply day 0 differentiation medium
  5. 5 Incubate for 3 days
  6. 6 Apply day 3 differentiation medium
  7. 7 Incubate for 3 more days
Extended Culture and Transfer to Larger Plates (Days 6–18) Days 6–18
  1. 1 Add fresh E6 medium on day 6
  2. 2 Perform media changes on days 8 and 10
  3. 3 Transfer organoids to 24-well plates on day 12
  4. 4 Initiate half media changes from day 18
Maturation and Continued Culture (Days 18–120+) Days 18–120+
  1. 1 Continue half media changes three times per week
  2. 2 Increase media volume from day 80 onwards
  3. 3 Continue culture to day 120 or beyond
Organoid Fixation and Tissue Preparation Days 16, 50, 65, 83, 100, 120
  1. 1 Harvest and wash organoids
  2. 2 Fix organoids with paraformaldehyde
  3. 3 Prepare samples for histology
  4. 4 Prepare samples for immunofluorescence (cryosections)
Immunofluorescent Staining and Imaging After fixation and sectioning
  1. 1 Wash cryosections
  2. 2 Block and permeabilize
  3. 3 Apply primary antibodies
  4. 4 Wash after primary antibodies
  5. 5 Apply secondary antibodies
  6. 6 Wash after secondary antibodies
  7. 7 Apply DAPI staining
  8. 8 Final washing and mounting
  9. 9 Image using confocal microscopy
Immunofluorescent Staining of Paraffin Sections After paraffin embedding
  1. 1 Dewax and rehydrate paraffin sections
  2. 2 Perform antigen retrieval
  3. 3 Proceed with immunostaining as described in Phase 6
Hair Follicle and Placode Quantification Days 72, 83, 93, 104, 115, 125
  1. 1 Visualize organoids under light microscope
  2. 2 Capture images and count hair structures
  3. 3 Document results and calculate averages

Full SOP

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Attribution

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

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