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SKIN Publication-derived

Epidermolysis Bullosa Skin Organoids

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 12 phases Cell source: Human iPSC (LUMCi045-A1, LUMCi046-A1, WT2)

Abstract

This protocol describes the generation of hair-bearing skin organoids from two independent human iPSC lines, followed by comprehensive characterization of the epidermal-dermal junction (EDJ) using immunofluorescence and transmission electron microscopy. The organoids develop stratified epidermis with hair follicles and sebaceous glands, though they exhibit markedly reduced type VII collagen expression compared to mature human skin, limiting their applicability for modeling type VII collagen-dependent forms of epidermolysis bullosa.

Cell source
Human iPSC (LUMCi045-A1, LUMCi046-A1, WT2)
Application
Disease modeling (Epidermolysis bullosa), Developmental study, Tissue characterization

Protocol overview

60 steps across 12 phases

Phase 1 · hiPSC Maintenance and Preparation Day -7 to Day 0
  1. a Culture hiPSC lines on vitronectin-coated plates
  2. b Passage hiPSC cells
Phase 2 · Skin Organoid Initiation and Early Development Day 0 to Day 30
  1. a Initiate directed differentiation toward skin organoids
  2. b Monitor bipolar organoid formation
Phase 3 · Epidermal Stratification and Hair Follicle Development Day 50 to Day 130
  1. a Assess epidermal stratification at day 55
  2. b Observe hair placode formation around day 60
  3. c Allow hair follicle maturation to day 130
  4. d Quantify hair follicle development at day 130
Phase 4 · Organoid Preparation for Microscopy and Analysis Day 130
  1. a Embed organoids in sucrose-gelatin solution for cryosectioning
  2. b Prepare 10 μm cryosections
Phase 5 · Immunofluorescence Microscopy Day 130 (Tissue Preparation and Staining)
  1. a Fix cryosections in acetone
  2. b Block sections with bovine serum albumin
  3. c Incubate with primary antibodies
  4. d Wash sections in PBS
  5. e Incubate with secondary antibodies
  6. f Perform Nile Red staining for lipid visualization (optional)
  7. g Stain nuclei with DAPI
  8. h Visualize filamentous actin (optional)
  9. i Mount sections for microscopy
  10. j Image sections using confocal microscopy
  11. k Process images using Fiji/ImageJ
Phase 6 · Transmission Electron Microscopy (TEM) Preparation Day 130
  1. a Fix tissue samples in Karnovsky's fixative
  2. b Post-fix with osmium tetroxide
  3. c Wash tissue samples
  4. d Perform en bloc staining with Ultrastain 1
  5. e Dehydrate tissue through ethanol series
  6. f Embed tissue in DDSA/NMA/Embed812 mixture
  7. g Polymerize resin blocks
Phase 7 · Transmission Electron Microscopy (TEM) Imaging Day 130 (After Resin Polymerization)
  1. a Section polymerized blocks
  2. b Mount sections on TEM grids
  3. c Image sections with transmission electron microscope
Phase 8 · Preparation and Analysis of hiPSC-Derived Keratinocytes Day -2 to Day 4
  1. a Differentiate hiPSCs to keratinocytes
  2. b Seed keratinocytes on coverslips
  3. c Culture keratinocytes for 4 days
  4. d Fix keratinocytes with paraformaldehyde
  5. e Permeabilize cells
  6. f Block with bovine serum albumin
  7. g Incubate with primary antibodies
  8. h Wash cells and incubate with secondary antibodies
  9. i Stain actin filaments with phalloidin
  10. j Mount coverslips for microscopy
  11. k Image keratinocytes using confocal microscopy
  12. l Quantify type VII collagen expression using image analysis
Phase 9 · Primary Human Keratinocyte Isolation and Culture Day -5 to Day 4 (Tissue Harvest to Analysis)
  1. a Obtain breast tissue explants
  2. b Isolate primary keratinocytes from tissue explants
  3. c Culture primary keratinocytes
  4. d Seed primary keratinocytes on coverslips
  5. e Perform immunofluorescence analysis (See Phase 8, Steps 4-12)
Phase 10 · Histological Analysis Day 130
  1. a Prepare cryosections for histology
  2. b Stain sections with hematoxylin and eosin (H&E)
  3. c Image H&E-stained sections
Phase 11 · Whole-Mount Immunostaining of Organoids Day 130
  1. a Prepare organoids for whole-mount staining
  2. b Perform whole-mount immunofluorescence staining
  3. c Image whole-mount organoids with confocal microscopy
  4. d Process and present confocal images
Phase 12 · Statistical Analysis and Data Interpretation After all experimental phases
  1. a Compile experimental data
  2. b Perform two-sample comparisons
  3. c Perform multi-group comparisons using one-way ANOVA
  4. d Set significance threshold
  5. e Prepare data presentation and figures

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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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