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SKIN Publication-derived

Skin Organoids for Disease and Development

Full SOP with Academic or a plan

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

Authors: 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 Duration: 130 days 9 phases Cell source: 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

Phase 1 · hiPSC Maintenance and Expansion Ongoing, as needed
  1. a Culture hiPSCs in StemFlex medium
  2. b Passage cells using Gentle Cell Dissociation Reagent
Phase 2 · Skin Organoid Generation (Days 0-30) Days 0-30
  1. a Initiate organoid differentiation
  2. b Monitor organoid bipolar organization
Phase 3 · Organoid Maturation and Hair Follicle Development (Days 30-130) Days 30-130
  1. a Continue organoid culture
  2. b Monitor epidermal stratification at day 55
  3. c Observe hair placode formation around day 60
  4. d Complete hair follicle development by day 130
Phase 4 · Immunofluorescence (IF) Characterization of Organoid Structure Day 130 (or as indicated)
  1. a Prepare organoid cryosections
  2. b Fix and permeabilize cryosections
  3. c Block nonspecific binding
  4. d Incubate with primary antibodies
  5. e Wash and apply secondary antibodies
  6. f Optional: Stain filamentous actin and nuclei
  7. g Mount sections for confocal microscopy
  8. h Image sections using confocal microscopy
Phase 5 · Immunofluorescence of Cultured Keratinocytes Day 4 of keratinocyte culture
  1. a Culture hiPSC-derived and primary keratinocytes on glass coverslips
  2. b Fix keratinocytes
  3. c Permeabilize keratinocytes
  4. d Block nonspecific binding
  5. e Incubate with primary antibodies
  6. f Wash and apply secondary antibodies and phalloidin
  7. g Stain nuclei and mount coverslips
  8. h Image keratinocytes
Phase 6 · Whole-Mount Immunofluorescence Day 130
  1. a Prepare organoid whole mounts
  2. b Stain whole-mount organoids with integrin antibodies
  3. c Apply secondary antibodies to whole mounts
  4. d Image whole-mount organoids
Phase 7 · Transmission Electron Microscopy (TEM) Day 130
  1. a Primary fixation in Karnovsky's fixative
  2. b Post-fixation with osmium tetroxide
  3. c Wash tissue samples
  4. d En bloc staining with Ultrastain 1
  5. e Ethanol dehydration series
  6. f Resin embedding
  7. g Ultramicrotomy and section collection
  8. h Electron microscopy imaging
Phase 8 · Histological Analysis (H&E Staining) Day 55, Day 130
  1. a Prepare cryosections from organoids
  2. b Perform hematoxylin and eosin (H&E) staining
  3. c Image H&E-stained sections
Phase 9 · Organoid Quantification and Analysis Day 130
  1. a Quantify hair follicle development
  2. b Quantify ECM protein expression (mean intensity analysis)
  3. c Quantify collagen VII expression in cultured keratinocytes
  4. d Statistical analysis

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Attribution

This SOP was written by Organthis based on the published method in Ramovs et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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