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SKIN Publication-derived

Generation and Characterization of Hair-Bearing hiPSC-Derived Skin Organoids

Source Ramovs et al., 2022 · Leiden University Medical Center, Department of Anatomy and Embryology · 10.1016/j.stemcr.2022.04.008

👤 Veronika Ramovs, Hans Janssen, Ignacia Fuentes, Amandine Pitaval, Walid Rachidi, Susana M. Chuva de Sousa Lopes, Christian Freund, Xavier Gidrol, Christine L. Mummery, Karine Raymond ⏱ 130 days 📋 9 phases 🧫 Human iPSC (hiPSC lines LUMCi045-A1, LUMCi046-A1, WT2)

Abstract

This protocol generates hair-bearing skin organoids from human induced pluripotent stem cells that develop a fully stratified interfollicular epidermis with hair follicles and sebaceous glands. The organoids are characterized for epidermal-dermal junction (EDJ) formation using immunofluorescence and transmission electron microscopy, revealing type I hemidesmosomes and integrin-mediated adhesion complexes, though with reduced collagen VII expression compared to adult skin.

Cell source
Human iPSC (hiPSC lines LUMCi045-A1, LUMCi046-A1, WT2)
Application
Disease modeling (Epidermolysis Bullosa), developmental study of epidermal-dermal junction

Protocol overview

43 steps across 9 phases

hiPSC Maintenance and Expansion Ongoing, as needed
  1. 1 Culture hiPSCs in StemFlex medium
  2. 2 Passage cells using Gentle Cell Dissociation Reagent
Skin Organoid Generation (Days 0–30) Days 0–30
  1. 1 Initiate organoid differentiation
  2. 2 Monitor organoid bipolar organization
Organoid Maturation and Hair Follicle Development (Days 30–130) Days 30–130
  1. 1 Continue organoid culture
  2. 2 Monitor epidermal stratification at day 55
  3. 3 Observe hair placode formation around day 60
  4. 4 Complete hair follicle development by day 130
Immunofluorescence (IF) Characterization of Organoid Structure Day 130 (or as indicated)
  1. 1 Prepare organoid cryosections
  2. 2 Fix and permeabilize cryosections
  3. 3 Block nonspecific binding
  4. 4 Incubate with primary antibodies
  5. 5 Wash and apply secondary antibodies
  6. 6 Optional: Stain filamentous actin and nuclei
  7. 7 Mount sections for confocal microscopy
  8. 8 Image sections using confocal microscopy
Immunofluorescence of Cultured Keratinocytes Day 4 of keratinocyte culture
  1. 1 Culture hiPSC-derived and primary keratinocytes on glass coverslips
  2. 2 Fix keratinocytes
  3. 3 Permeabilize keratinocytes
  4. 4 Block nonspecific binding
  5. 5 Incubate with primary antibodies
  6. 6 Wash and apply secondary antibodies and phalloidin
  7. 7 Stain nuclei and mount coverslips
  8. 8 Image keratinocytes
Whole-Mount Immunofluorescence Day 130
  1. 1 Prepare organoid whole mounts
  2. 2 Stain whole-mount organoids with integrin antibodies
  3. 3 Apply secondary antibodies to whole mounts
  4. 4 Image whole-mount organoids
Transmission Electron Microscopy (TEM) Day 130
  1. 1 Primary fixation in Karnovsky's fixative
  2. 2 Post-fixation with osmium tetroxide
  3. 3 Wash tissue samples
  4. 4 En bloc staining with Ultrastain 1
  5. 5 Ethanol dehydration series
  6. 6 Resin embedding
  7. 7 Ultramicrotomy and section collection
  8. 8 Electron microscopy imaging
Histological Analysis (H&E Staining) Day 55, Day 130
  1. 1 Prepare cryosections from organoids
  2. 2 Perform hematoxylin and eosin (H&E) staining
  3. 3 Image H&E-stained sections
Organoid Quantification and Analysis Day 130
  1. 1 Quantify hair follicle development
  2. 2 Quantify ECM protein expression (mean intensity analysis)
  3. 3 Quantify collagen VII expression in cultured keratinocytes
  4. 4 Statistical analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ramovs et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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