Skip to content
← Back to browse
SKIN Publication-derived

Characterization of the epidermal-dermal junction in 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 📋 12 phases 🧫 Human iPSC (LUMCi045-A1, LUMCi046-A1, WT2)

Abstract

Protocol for generation and comprehensive characterization of hair-bearing skin organoids derived from human induced pluripotent stem cells. The method produces fully stratified interfollicular epidermis with developed hair follicles by day 130, and employs immunofluorescence and transmission electron microscopy to characterize the epidermal-dermal junction (EDJ), including basement membrane composition and hemidesmosome formation, to assess suitability for disease modeling of epidermolysis bullosa.

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

Protocol overview

42 steps across 12 phases

hiPSC Maintenance and Culture Preparation Days -7 to 0
  1. 1 Maintain hiPSC lines in StemFlex medium
Skin Organoid Generation and Initial Development Days 0–30
  1. 1 Initiate skin organogenesis following Lee et al. protocol
  2. 2 Monitor for bipolar organization
Epidermal Stratification and Hair Follicle Development Days 30–130
  1. 1 Monitor epidermal stratification at day 50
  2. 2 Observe hair placode formation
  3. 3 Monitor hair follicle development through day 130
Sample Preparation for Immunofluorescence Analysis Days 55–130
  1. 1 Prepare organoid cryosections
  2. 2 Block sections with BSA
  3. 3 Incubate with primary antibodies
  4. 4 Incubate with secondary antibodies
  5. 5 Stain nuclei with DAPI
Whole-Mount Immunofluorescence Staining Days 55–130
  1. 1 Process organoids for whole-mount staining
Transmission Electron Microscopy (TEM) Sample Preparation Days 55–130
  1. 1 Fix tissue in Karnovsky's fixative
  2. 2 Perform post-fixation with osmium tetroxide
  3. 3 Wash and stain en bloc with Ultrastain 1
  4. 4 Dehydrate samples in ethanol series
  5. 5 Embed in DDSA/NMA/Embed812 mixture
  6. 6 Section and analyze with electron microscope
Keratinocyte Differentiation and Characterization (Parallel Protocol) Days 0–30 (keratinocyte protocol)
  1. 1 Seed hiPSC clumps on Matrigel-coated plates in KSFM with RA and BMP4
  2. 2 Continue culture in KSFM until day 13
  3. 3 Switch to CnT-07 medium at day 13
  4. 4 Sort integrin α6+/β4+ population by FACS
  5. 5 Maintain sorted keratinocytes in CnT-07
Immunofluorescence of Keratinocytes on Coverslips Days 4–5 of keratinocyte culture
  1. 1 Seed hiPSC-derived and primary keratinocytes on coverslips
  2. 2 Fix keratinocytes with 2% paraformaldehyde
  3. 3 Permeabilize with 0.2% TritonX100
  4. 4 Block with PBS 2% BSA
  5. 5 Incubate with primary and secondary antibodies
  6. 6 Visualize actin with phalloidin
  7. 7 Mount coverslips in Mowiol
Confocal Microscopy Imaging and Analysis Days 55–130
  1. 1 Image cryosections and keratinocytes with Zeiss LSM900 Airyscan2
  2. 2 Image day-55 organoid cryosections and whole mounts with Leica TCS SP5
  3. 3 Acquire maximum intensity projections
  4. 4 Quantify extracellular matrix component expression
Hair Follicle Quantification and Morphological Analysis Day 130
  1. 1 Photograph organoids with Leica M420 macroscope
  2. 2 Quantify percentage of organoids with hair follicles
Histological Analysis Day 55–130
  1. 1 Prepare cryosections for H&E staining
  2. 2 Image H&E-stained sections
Quantitative Reverse Transcription PCR (RT-qPCR) Days 55–130
  1. 1 Extract RNA from organoids and human skin
  2. 2 Synthesize complementary DNA (cDNA)
  3. 3 Perform quantitative PCR
  4. 4 Analyze results with CFX Manager software

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

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.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence