Skip to content
← Back to browse
LIVER Publication-derived

iPSC-Induced Multilineage Liver, Small Intestinal, and Brain Organoid Infection Models for Hepatitis E Virus Propagation and Antiviral Drug Evaluation

Source Liu et al., 2025 · State Key Laboratory of Drug Regulatory Science, National Institutes for Food and Drug Control, Beijing, China · 10.1136/gutjnl-2025-336105

👤 Fan Liu, Tianxu Liu, Xi Wu, Zihao Yu, Jincheng Tong, Lili Wang, Chenyan Zhao, Jianhui Nie, Youchun Wang, Kai Wang, Lin Bai, Pengyan Xia, Guochao Wei, Zi-Zheng Zheng, Di Wu, Weijin Huang, Lin Wang ⏱ 60 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol establishes iPSC-derived multilineage organoids (liver, intestinal, and brain) that support the complete life cycle of wild-type hepatitis E virus (HEV) genotypes 1, 3a, and 4. The models recapitulate HEV-induced pathophysiology including hepatocyte injury, intestinal barrier dysfunction, and neuronal damage, providing platforms for HEV virology research and antiviral therapeutic evaluation.

Cell source
Human iPSC
Application
Disease modeling; Antiviral drug screening; Viral tropism and pathogenesis research

Protocol overview

38 steps across 10 phases

iPSC Maintenance and Culture Preparation Days -7 to 0
  1. 1 iPSC Culture Setup
Hepatic Organoid (hLO) Differentiation Days 0-25
  1. 1 Endoderm-Mesoderm Co-induction
  2. 2 Posterior Foregut Specification (Days 4-8)
  3. 3 Spheroid Formation (Day 8)
  4. 4 Progenitor Patterning (Days 8-14)
  5. 5 Hepatic Maturation (Days 14-25)
Intestinal Organoid (hIO) Differentiation Days 0-35
  1. 1 Endoderm-Mesoderm Co-induction and Mid/Hindgut Specification
  2. 2 Spheroid Formation and Intestinal Patterning (Days 8-14)
  3. 3 Matrigel Embedding and Maturation (Days 11-35)
  4. 4 Polarity Reversal (Days 28-35)
Brain Organoid (hBO) Differentiation Days 0-60
  1. 1 Embryoid Body Formation (Days 0-2)
  2. 2 Neural Induction (Days 2-10)
  3. 3 Neural Expansion (Days 10-22)
  4. 4 Neuronal Maturation (Days 22-60)
Virus Extraction and Purification from Faecal Samples Days before infection (preparation)
  1. 1 Initial Sample Preparation and Clarification
  2. 2 Initial Centrifugation Steps
  3. 3 Ultracentrifugation
  4. 4 Iodixanol Density Gradient Centrifugation
  5. 5 Viral Load Quantification
Organoid Infection with HEV Infection day (Day 0 of infection protocol)
  1. 1 Organoid Recovery from Matrigel
  2. 2 Viral Inoculation
  3. 3 Post-Infection Washing and Medium Exchange
  4. 4 Secondary Inoculation (Reinfection Validation)
Antiviral Drug Treatment Days 1-6 post-infection (concurrent with infection phase)
  1. 1 Ribavirin Treatment
  2. 2 NITD008 Treatment (Alternative/Comparative Antiviral)
  3. 3 Viral Load Monitoring During Treatment
Cross-Organ Transmission (Liver-Intestinal System) Days 6-14 post-infection
  1. 1 Gut-to-Liver Transmission (hIO Supernatant to hLO)
  2. 2 Liver-to-Gut Transmission (hLO Supernatant to hIO)
  3. 3 Sequential Passage in Cross-Organ System
Molecular and Cellular Characterization Days 3-6 post-infection (tissue collection for analysis)
  1. 1 Sample Collection for RNA Analysis
  2. 2 Quantitative RT-PCR for Viral and Host Gene Expression
  3. 3 Immunofluorescence Staining for Viral and Cellular Markers
  4. 4 Secondary Antibody Incubation and Imaging
  5. 5 Quantitative Image Analysis
Functional Assays for Hepatocyte and Organoid Function Days 3-6 post-infection (concurrent with characterization phase)
  1. 1 Preparation of Culture Supernatants for ELISA
  2. 2 ELISA Measurements (Albumin, Factor IX, IL-6, ALT, AST)
  3. 3 Cell Viability Assessment (Antiviral Toxicity Testing)
  4. 4 CellTiter-Glo Luminescence Assay

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 Liu 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