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LIVER Publication-derived

Ubiquitinated hepatitis D antigen-induced CD8+ T-cell responses inhibit HDV replication in HDV-infected liver organoids

Source Shen et al., 2025 · Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine · 10.1016/j.antiviral.2025.106266

👤 Leer Shen, Luying Tian, Qingxin Guo, Rongli He, Yi Zhang, Siyuan Ma, Weiwei Hu, Jie Chen, Soon Seng Ng, Jinmei Chen, Xiaohua Chen ⏱ 28 days 📋 13 phases 🧫 Human iPSC

Abstract

This protocol establishes HBV/HDV superinfected liver organoids derived from human iPSCs differentiated into hepatocyte-like cells and seeded on inverted colloidal crystal scaffolds. The protocol demonstrates that ubiquitinated small hepatitis D antigen (Ub-S-HDAg) transduced into dendritic cells promotes CD8+ T cell activation via the JAK/STAT pathway, leading to significant reduction in HDV viral load and providing a platform for immunotherapy screening.

Cell source
Human iPSC
Application
Disease modeling; Antiviral immunotherapy development

Protocol overview

60 steps across 13 phases

iPSC Culture and Hepatocyte Differentiation Days 0-21
  1. 1 iPSC Culture on Laminin-coated Plates
  2. 2 Initiate Endoderm Formation
  3. 3 Promote Hepatoblast Formation
  4. 4 Differentiate into Hepatocyte-like Cells
Inverted Colloidal Crystal Scaffold Preparation and Organoid Culture Days 15-28
  1. 1 Construct Inverted Colloidal Crystal Scaffold
  2. 2 Remove Polystyrene Microspheres
  3. 3 Coat Scaffold with Collagen I
  4. 4 Seed Hepatocyte-like Cells onto Scaffold
HBV and HDV Production and Infection Days 0-21
  1. 1 Produce HBV in HepAD38 Cells
  2. 2 Produce HDV Particles
  3. 3 Purify and Concentrate HBV/HDV Particles
  4. 4 Infect Liver Organoids with HBV
  5. 5 Superinfect HBV-infected Organoids with HDV
Dendritic Cell and T Cell Isolation and Culture Days 0-14
  1. 1 Differentiate PBMCs into Dendritic Cells
  2. 2 Isolate and Culture T Cells
Lentiviral Vector Construction and DC Transduction Days 0-10
  1. 1 Synthesize and Clone Ub-S-HDAg Sequence
  2. 2 Produce Lentiviral Particles
  3. 3 Transduce DCs with Lentiviral Vector
  4. 4 Culture Transduced DCs
  5. 5 Establish Experimental Groups
DC and T Cell Co-culture Days 5-8
  1. 1 Co-culture DCs and T Cells
  2. 2 Harvest CD8+ T Cells
Flow Cytometry Analysis of DC Maturation Days 3-5
  1. 1 Prepare Cells for Flow Cytometry
  2. 2 Stain with DC Maturation Markers
  3. 3 Assess Cell Viability
  4. 4 Analyze via Flow Cytometry
Cytokine Measurement and Immune Function Assays Days 5-8
  1. 1 Culture CD8+ T Cells Post-isolation
  2. 2 Collect Supernatants
  3. 3 Measure Cytokine Levels by ELISA
  4. 4 Perform IFN-γ ELISPOT Assay
Gene Expression Analysis and JAK/STAT Pathway Evaluation Days 5-10
  1. 1 Extract Total RNA
  2. 2 Assess RNA Integrity
  3. 3 Synthesize cDNA
  4. 4 Perform qRT-PCR for JAK/STAT Genes
  5. 5 Prepare Libraries for Whole Transcriptome Sequencing
  6. 6 Perform Whole Transcriptome Sequencing and Analysis
Cell Proliferation and Cytotoxicity Evaluation Days 5-8
  1. 1 Assess Cell Proliferation via CCK-8 Assay
  2. 2 Measure Absorbance at 450 nm
  3. 3 Prepare Effector and Target Cells for Cytotoxicity Assay
  4. 4 Measure Lactate Dehydrogenase Activity
Western Blot Analysis of JAK/STAT Pathway Components Days 5-8
  1. 1 Extract Total Protein
  2. 2 Clarify Protein Extract
  3. 3 Determine Protein Concentration
  4. 4 Prepare Protein Samples
  5. 5 Run SDS-PAGE Gel Electrophoresis
  6. 6 Transfer Proteins to Membrane
  7. 7 Probe with Primary Antibodies
  8. 8 Incubate with Secondary Antibodies
  9. 9 Visualize and Analyze Blots
HDV Infection and Antiviral Treatment Assessment Days 0-21
  1. 1 Establish Five Experimental Treatment Groups
  2. 2 Perform Interventions on HDV-infected Organoids
  3. 3 Collect Culture Supernatants
  4. 4 Extract Viral Nucleic Acids
  5. 5 Quantify Viral Load via qRT-PCR
Immunofluorescence and Immunohistochemistry Analysis Throughout organoid culture
  1. 1 Prepare Samples for Immunofluorescence
  2. 2 Incubate with Primary Antibodies
  3. 3 Incubate with Secondary Antibodies
  4. 4 Mount Coverslips
  5. 5 Image and Analyze Samples
  6. 6 Perform Immunohistochemical Staining

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Shen et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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