Skip to content
← Back to browse
SKIN Publication-derived

EV-A71 Infection of Human iPSC-Derived Skin Organoids and Antiviral Drug Screening

Source Li et al., 2025 · Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College · 10.1038/s41467-025-57610-2

👤 Jun Li, Jie Ma, Ruiyuan Cao, Qiyu Zhang, Mansheng Li, Wenwen Wang, Yujie Wang, Wei Li, Yunping Zhu, Ling Leng ⏱ 143 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human induced pluripotent stem cell-derived skin organoids (hiPSC-SOs) and their infection with enterovirus 71 (EV-A71) to model hand, foot, and mouth disease. The organoids contain multiple cell types including epidermal cells, hair follicles, fibroblasts, and nerve cells, enabling investigation of viral tropism and pathogenesis for identification of antiviral drug candidates.

Cell source
Human iPSC
Application
Disease modeling and drug screening

Protocol overview

115 steps across 12 phases

hiPSC Culture and Skin Organoid Generation Days 0-143
  1. 1 Thaw and Culture hiPSCs
  2. 2 Form hiPSC Aggregates
  3. 3 Initiate Differentiation (Days 0-3)
  4. 4 Secondary Differentiation (Days 3-6)
  5. 5 Nutrition Support (Days 6-12)
  6. 6 Transfer to Organoid Maturation Medium (Day 12)
  7. 7 Medium Replenishment (Days 15-18)
  8. 8 Epidermal Enhancement (Day 75)
  9. 9 Mature Organoid Harvest
EV-A71 Virus Preparation and Cell Culture Prior to infection experiments
  1. 1 Culture RD and Vero Cell Lines
  2. 2 Propagate EV-A71 Virus
  3. 3 Clarify Viral Culture Medium
  4. 4 Purify Virus by Ultracentrifugation
  5. 5 Resuspend Purified Virus
  6. 6 Determine Viral Titer
EV-A71 Infection of hiPSC-SOs Days 0-8 post-infection
  1. 1 Prepare Organoids for Infection
  2. 2 Infect Organoids with EV-A71 (Standard Infection)
  3. 3 Replace Medium Post-Infection
  4. 4 Maintain Infected Organoids
  5. 5 Collect Samples at Timepoints
Drug Treatment with NSC167409 During EV-A71 infection (0-72 hpi)
  1. 1 Prepare NSC167409 Stock Solution
  2. 2 Treat Cells with NSC167409 (Immediate Treatment)
  3. 3 Treat hiPSC-SOs with NSC167409 (Delayed Treatment)
  4. 4 Assess Antiviral Efficacy
In Vivo Xenograft Infection and Drug Evaluation Days 1-30 (including pre-transplant recovery period)
  1. 1 Prepare Immunocompromised Mice
  2. 2 Implant Skin Organoids
  3. 3 Post-Operative Care and Recovery
  4. 4 Wait for Organoid Integration
  5. 5 Divide Mice into Treatment Groups
  6. 6 Administer Prophylactic Drug Treatment
  7. 7 Wait Post-Drug Administration
  8. 8 Inoculate Organoids with EV-A71
  9. 9 Monitor Viral Infection and Drug Efficacy
Immunofluorescence and Histological Analysis Following sample collection
  1. 1 Fix Tissue Samples
  2. 2 Dehydrate Samples
  3. 3 Clear with Xylene
  4. 4 Embed in Paraffin
  5. 5 Section Paraffin Blocks
  6. 6 Deparaffinize and Rehydrate Sections
  7. 7 Perform Antigen Retrieval
  8. 8 Block Non-Specific Binding
  9. 9 Apply Primary Antibodies
  10. 10 Wash Sections
  11. 11 Apply Secondary Antibodies
  12. 12 Final Wash and DAPI Staining
  13. 13 Mount Slides
  14. 14 Image Sections
  15. 15 Analyze Images
Proteomics Sample Preparation Following organoid collection
  1. 1 Harvest Organoid Tissue
  2. 2 Prepare Urea Lysis Buffer
  3. 3 Lyse Organoid Tissue
  4. 4 Homogenize Tissue
  5. 5 Clarify Lysate
  6. 6 Reduce Proteins
  7. 7 Alkylate Cysteines
  8. 8 Digest with Lys-C
  9. 9 Digest with Trypsin
  10. 10 Desalt Peptides
  11. 11 Dry Peptide Samples
  12. 12 Prepare Pooled Library Fractions
  13. 13 Collect and Merge Fractions
  14. 14 Dry Pooled Fractions
  15. 15 Reconstitute Peptides for Mass Spectrometry
Mass Spectrometry Analysis Following peptide preparation
  1. 1 Set Up UHPLC-MS System
  2. 2 Acquire Data-Dependent Acquisition (DDA)
  3. 3 Acquire Data-Independent Acquisition (DIA)
  4. 4 Apply Fragmentation Parameters
  5. 5 Monitor and Verify Acquisition
Proteomic Data Processing and Analysis Following mass spectrometry runs
  1. 1 Process DDA Data
  2. 2 Generate DIA Library
  3. 3 Merge Libraries
  4. 4 Process DIA Data
  5. 5 Apply Modifications
  6. 6 Configure Quantification Parameters
  7. 7 Enable Interference Correction
  8. 8 Set False Discovery Rate
  9. 9 Extract Protein Intensities
  10. 10 Statistical Analysis of Differentially Expressed Proteins
  11. 11 Analyze Temporal Expression Patterns
  12. 12 Perform Functional Enrichment Analysis
  13. 13 Generate Data Visualizations
Single-Cell RNA Sequencing (scRNA-seq) and Analysis Following organoid collection
  1. 1 Prepare Single-Cell Suspension
  2. 2 Dissociate Organoids
  3. 3 Neutralize Enzyme and Filter
  4. 4 Count and Assess Viability
  5. 5 Prepare Cell Suspension for Library Preparation
  6. 6 Generate scRNA-seq Libraries
  7. 7 Sequence Libraries
  8. 8 Process Raw Sequencing Data
  9. 9 Quality Control Filtering
  10. 10 Normalize and Select Variable Features
  11. 11 Scale and Remove Batch Effects
  12. 12 Identify Cell Clusters
  13. 13 Identify Differentially Expressed Genes
  14. 14 Perform Functional Enrichment Analysis
  15. 15 Generate Visualizations
HMGB1 Knockdown Experiments During EV-A71 infection (0-48 hpi)
  1. 1 Prepare siRNA Transfection Mixture
  2. 2 Transfect RD Cells
  3. 3 Allow Transfection Recovery
  4. 4 Infect with EV-A71
  5. 5 Harvest Samples for Analysis
  6. 6 Prepare Cell Lysates
  7. 7 Clarify Lysates
  8. 8 Quantify Protein Concentration
Western Blot Analysis Following cell lysis
  1. 1 Prepare Samples for SDS-PAGE
  2. 2 Prepare and Load SDS-PAGE Gel
  3. 3 Run Electrophoresis
  4. 4 Transfer Proteins to Membrane
  5. 5 Block Non-Specific Binding
  6. 6 Incubate with Primary Antibodies
  7. 7 Wash Membrane
  8. 8 Incubate with Secondary Antibody
  9. 9 Final Wash
  10. 10 Detect Protein Bands
  11. 11 Quantify Band Intensities

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 Li et al., 2025. 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