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SKIN Publication-derived

Skin Organoids for Disease Modelling

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

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

Phase 1 · hiPSC Maintenance and Culture Preparation Days -7 to 0
  1. a Maintain hiPSC lines in StemFlex medium
Phase 2 · Skin Organoid Generation and Initial Development Days 0-30
  1. a Initiate skin organogenesis following Lee et al. protocol
  2. b Monitor for bipolar organization
Phase 3 · Epidermal Stratification and Hair Follicle Development Days 30-130
  1. a Monitor epidermal stratification at day 50
  2. b Observe hair placode formation
  3. c Monitor hair follicle development through day 130
Phase 4 · Sample Preparation for Immunofluorescence Analysis Days 55-130
  1. a Prepare organoid cryosections
  2. b Block sections with BSA
  3. c Incubate with primary antibodies
  4. d Incubate with secondary antibodies
  5. e Stain nuclei with DAPI
Phase 5 · Whole-Mount Immunofluorescence Staining Days 55-130
  1. a Process organoids for whole-mount staining
Phase 6 · Transmission Electron Microscopy (TEM) Sample Preparation Days 55-130
  1. a Fix tissue in Karnovsky's fixative
  2. b Perform post-fixation with osmium tetroxide
  3. c Wash and stain en bloc with Ultrastain 1
  4. d Dehydrate samples in ethanol series
  5. e Embed in DDSA/NMA/Embed812 mixture
  6. f Section and analyze with electron microscope
Phase 7 · Keratinocyte Differentiation and Characterization (Parallel Protocol) Days 0-30 (keratinocyte protocol)
  1. a Seed hiPSC clumps on Matrigel-coated plates in KSFM with RA and BMP4
  2. b Continue culture in KSFM until day 13
  3. c Switch to CnT-07 medium at day 13
  4. d Sort integrin α6+/β4+ population by FACS
  5. e Maintain sorted keratinocytes in CnT-07
Phase 8 · Immunofluorescence of Keratinocytes on Coverslips Days 4-5 of keratinocyte culture
  1. a Seed hiPSC-derived and primary keratinocytes on coverslips
  2. b Fix keratinocytes with 2% paraformaldehyde
  3. c Permeabilize with 0.2% TritonX100
  4. d Block with PBS 2% BSA
  5. e Incubate with primary and secondary antibodies
  6. f Visualize actin with phalloidin
  7. g Mount coverslips in Mowiol
Phase 9 · Confocal Microscopy Imaging and Analysis Days 55-130
  1. a Image cryosections and keratinocytes with Zeiss LSM900 Airyscan2
  2. b Image day-55 organoid cryosections and whole mounts with Leica TCS SP5
  3. c Acquire maximum intensity projections
  4. d Quantify extracellular matrix component expression
Phase 10 · Hair Follicle Quantification and Morphological Analysis Day 130
  1. a Photograph organoids with Leica M420 macroscope
  2. b Quantify percentage of organoids with hair follicles
Phase 11 · Histological Analysis Day 55-130
  1. a Prepare cryosections for H&E staining
  2. b Image H&E-stained sections
Phase 12 · Quantitative Reverse Transcription PCR (RT-qPCR) Days 55-130
  1. a Extract RNA from organoids and human skin
  2. b Synthesize complementary DNA (cDNA)
  3. c Perform quantitative PCR
  4. d Analyze results with CFX Manager software

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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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