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LIVER Publication-derived

Modeling Hepatoblastoma Development with Human Fetal Liver Organoids Reveals YAP1 Activation is Sufficient for Tumorigenesis

Source Yang et al. · Obstetrics and Gynecology Hospital of Fudan University

👤 Yang et al. ⏱ 21 days 📋 14 phases 🧫 Human Fetal Liver Hepatoblasts

Abstract

This protocol describes the isolation and culture of human fetal liver organoids from elective abortion tissues, followed by lentiviral-mediated manipulation to model hepatoblastoma development. YAP1 activation in organoids recapitulates hallmarks of hepatoblastoma and enables in vivo tumorigenesis studies in xenograft models.

Cell source
Human Fetal Liver Hepatoblasts
Application
Disease modeling (Hepatoblastoma)

Protocol overview

63 steps across 14 phases

Tissue Collection and Primary Hepatoblast Isolation Day 0
  1. 1 Tissue Collection and Cold Storage
  2. 2 Tissue Mincing
  3. 3 Tissue Washing
  4. 4 Enzymatic Digestion
  5. 5 Digestion Arrest and Cell Recovery
  6. 6 Cell Filtration
  7. 7 Cell Pelleting and Washing (First Round)
Human Fetal Liver Organoids Culture - Growth Factor Phase Day 0–7
  1. 1 Final Washing and Pellet Resuspension
  2. 2 Organoid Formation with Growth Factor Medium
  3. 3 GF-Medium Change (Days 1–7)
Human Fetal Liver Organoids Culture - Chemical-Defined Phase Day 7 onwards
  1. 1 Medium Transition to Chemical-Defined Medium
  2. 2 5C-Medium Refreshment (Day 7 onwards)
  3. 3 Organoid Cryopreservation (Optional)
Lentiviral Vector Construction, Production, and Organoid Infection Day 7
  1. 1 Lentiviral Vector Plasmid Construction
  2. 2 HEK293T Cell Transfection for Lentivirus Production
  3. 3 Viral Supernatant Harvest
  4. 4 Viral Concentration
  5. 5 Lentivirus Infection of Organoids
Immunofluorescence and Histological Analysis Variable (post-culture)
  1. 1 Organoid Fixation and Agarose Embedding
  2. 2 Paraffin Embedding
  3. 3 Tissue Sectioning
  4. 4 Section Hydration and Antigen Retrieval
  5. 5 Blocking and Primary Antibody Staining
  6. 6 Secondary Antibody Staining and Nuclear Counterstaining
  7. 7 Imaging
Immunoblotting Analysis Variable (post-culture)
  1. 1 Sample Lysis and Protein Extraction
  2. 2 Protein Quantification
  3. 3 SDS-PAGE and Protein Transfer
  4. 4 Membrane Blocking
  5. 5 Primary Antibody Incubation
  6. 6 Membrane Washing and Secondary Antibody Incubation
  7. 7 Protein Detection and Imaging
Functional Analysis of Fetal Liver Organoids Variable (post-culture)
  1. 1 LDL Uptake Assay
  2. 2 Bile Canaliculi Function Assay
Mouse Xenograft Studies Variable (post-organoid culture)
  1. 1 Organoid Preparation for Transplantation
  2. 2 Orthotopic Liver Transplantation
RNA Isolation and Quantitative Reverse Transcription PCR (qRT-PCR) Variable (post-culture)
  1. 1 Total RNA Extraction
  2. 2 Reverse Transcription
  3. 3 qRT-PCR Amplification
High-Throughput Screening Day 7–12 (or Day 14–21)
  1. 1 Organoid Seeding in 96-Well Microplate
  2. 2 Chemical Compound Treatment
  3. 3 Nuclear Staining and Image Acquisition
  4. 4 Image Analysis
mRNA Sequencing and Bioinformatic Analysis Variable (post-culture)
  1. 1 Total RNA Extraction and Library Preparation
  2. 2 Sequencing
  3. 3 Read Alignment and Transcript Quantification
  4. 4 Differential Expression Analysis
  5. 5 Heatmap Visualization and Clustering
Organoid Hydrophilic Metabolite Extraction and Targeted Metabolomic Analysis Variable (post-culture)
  1. 1 Organoid Homogenization
  2. 2 Solvent Extraction (First Round)
  3. 3 First Centrifugation and Supernatant Collection
  4. 4 Solvent Extraction (Second Round)
  5. 5 Supernatant Pooling
  6. 6 Sample Aliquoting and Drying
  7. 7 Sample Storage
  8. 8 Sample Resuspension and Injection
  9. 9 Hydrophilic Interaction Chromatography (HILIC) Separation
  10. 10 Mass Spectrometry Data Acquisition
Gene Set Enrichment Analysis (GSEA) Variable (post-sequencing)
  1. 1 Data Preparation for GSEA
  2. 2 GSEA Analysis with Custom Gene Sets
  3. 3 GSEA Result Interpretation
Statistical Analysis and Data Presentation Variable (final analysis)
  1. 1 Statistical Testing
  2. 2 Data Presentation

Full SOP

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Attribution

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

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