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SKIN Publication-derived

Establishment of Human Pluripotent Stem Cell-Derived Skin Organoids for SARS-CoV-2 Infection Modeling

Source Ma et al., 2022 · Peking Union Medical College Hospital, Chinese Academy of Medical Sciences · 10.1002/advs.202104192

👤 Jie Ma, Jia liu, Dunqin Gao, Xiao Li, Qiyu Zhang, Luye Lv, Yujie Wang, Jun Li, Yunping Zhu, Zhihong Wu, Hengrui Hu, Yufeng Li, Longda Ma, Qian Liu, Zhihong Hu, Shuyang Zhang, Yiwu Zhou, Manli Wang, Ling Leng ⏱ 144 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of hiPSC-derived 3D skin organoids containing epidermis, dermis, hair follicles, and integrated nervous system. The mature organoids (day 140) are subsequently exposed to SARS-CoV-2 to model pathological mechanisms of COVID-19 skin infection, including hair follicle damage and neuronal infection.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

50 steps across 9 phases

iPSC Preparation and Initial Aggregation Day 0-2
  1. 1 Culture hiPSCs on Matrigel-coated plates
  2. 2 Seed iPSCs into U-bottom low-attachment 96-well plates
  3. 3 First medium change at 24 hours
  4. 4 Continue culture for second 24 hours
Differentiation and Ectoderm Induction (Day 2-12) Day 2-12
  1. 5 Transfer aggregates to differentiation medium
  2. 6 Induce cranial neural crest formation
  3. 7 Medium replacement at day 6
  4. 8 Transfer to organoid maturation medium at day 12
Organoid Maturation and Hair Follicle Formation (Day 12-140) Day 12-140
  1. 9 First medium change at day 15
  2. 10 Routine medium changes from day 15 onward
  3. 11 Add TGFBI on day 75
  4. 12 Continue culture to day 140 for full maturation
SARS-CoV-2 Infection of Mature Organoids Day 140-144
  1. 13 Transfer mature organoids to infection plates
  2. 14 Expose organoids to SARS-CoV-2
  3. 15 Medium change at 48 hours post-infection
  4. 16 Fixation of infected organoids at 96 hours
Sample Preparation for Proteomics Analysis Post-fixation
  1. 17 Tissue lysis in urea buffer
  2. 18 Mechanical disruption by vibration
  3. 19 Clarify lysate by centrifugation
  4. 20 Protein reduction
  5. 21 Protein alkylation
  6. 22 Enzymatic protein digestion with Lys C
  7. 23 Enzymatic protein digestion with Trypsin
  8. 24 Peptide desalting via C18 cartridges
  9. 25 Vacuum drying of peptide samples
Peptide Pre-Fractionation by High-pH HPLC Post-sample preparation
  1. 26 Dissolve and load peptides on C18 column
  2. 27 Peptide elution with linear gradient
  3. 28 Consolidate fractions for mass spectrometry
Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Post-fractionation
  1. 29 Re-dissolve peptides in mobile phase A
  2. 30 Load peptides onto C18 nano-capillary analytical column
  3. 31 Separate peptides by LC gradient (90 min for proteome profiling)
  4. 32 Data-dependent acquisition (DDA) for fractionated pool samples
  5. 33 Data-independent acquisition (DIA) for skin tissue samples
  6. 34 Label-free MS analysis for organoid samples (alternative protocol)
Immunofluorescence and Pathological Analysis Post-fixation
  1. 35 Tissue processing and sectioning
  2. 36 H&E staining
  3. 37 Deparaffinization and antigen retrieval for immunohistochemistry
  4. 38 Blocking and primary antibody incubation for immunohistochemistry
  5. 39 Secondary antibody staining for immunohistochemistry
  6. 40 Immunofluorescence fixation and permeabilization
  7. 41 Blocking and primary antibody incubation for immunofluorescence
  8. 42 Secondary antibody incubation and DAPI counterstaining
  9. 43 Slide mounting and image acquisition
Transmission Electron Microscopy Post-tissue collection
  1. 44 Primary fixation with glutaraldehyde
  2. 45 Post-fixation with osmium tetroxide
  3. 46 Dehydration and infiltration
  4. 47 Resin polymerization
  5. 48 Ultramicrotomy and section preparation
  6. 49 Staining ultrathin sections
  7. 50 Transmission electron microscopy examination

Full SOP

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Attribution

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

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