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LIVER Publication-derived

Hepatoblastoma Liver Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Yang et al., 2022 · State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai · 10.1007/s13238-021-00893-0

Authors: Li Yang, Jin Chen, Jianqing Liang, Yufeng Zhang, Qingzhe Wang, Xiaojun Ren, Jinsong Wei, Qianchun Gong, Jiting Zhang, Ning Jiang, Xinhua Lin, Jin Li, Bing Zhao Duration: 120 days 9 phases Cell source: Human fetal hepatoblasts (8-12 weeks post-coitum)

Abstract

This protocol establishes human hepatoblastoma organoids by lentiviral transfection of YAP1(5SA) into fetal liver organoids derived from 8-12 weeks post-coitum human liver tissue. The YAP1-activated organoids develop malignant features including hepatoblastoma signature gene expression and, when transplanted into NSG mice, generate xenograft tumors with spontaneous lung metastasis, recapitulating clinical hepatoblastoma progression.

Cell source
Human fetal hepatoblasts (8-12 weeks post-coitum)
Application
Disease modeling; hepatoblastoma oncogenesis and tumorigenesis

Protocol overview

27 steps across 9 phases

Phase 1 · Fetal Liver Organoid Culture Initiation Day 0-7
  1. a Isolation and culture of human fetal liver cell clusters
  2. b Maintain organoid growth using initial growth-factor cocktail
Phase 2 · Organoid Expansion with Small-Molecule Cocktail Day 7-21
  1. a Replace growth factors with small-molecule cocktail at day 7
  2. b Culture organoids and verify hepatoblast marker expression
Phase 3 · Lentiviral Transfection and Transformation Day 21-60
  1. a Prepare lentiviral vectors carrying GFP (control), β-catenin(Δex3), or YAP1(5SA)
  2. b Transfect fetal liver organoids with lentiviral constructs
  3. c Assess organoid transformation phenotype at day 60
Phase 4 · Molecular Characterization of YAP1-Activated Organoids Day 42-60
  1. a Perform RNA extraction and transcriptome analysis
  2. b Verify hepatoblastoma signature gene expression via qRT-PCR
  3. c Perform Gene Set Enrichment Analysis (GSEA)
Phase 5 · In Vivo Xenograft Transplantation Day 60–post-transplant
  1. a Prepare YAP1-activated organoids for orthotopic transplantation
  2. b Perform orthotopic liver capsule transplantation into NSG mice
  3. c Monitor tumor growth and metastasis
  4. d Verify tumor and metastasis identity via immunofluorescence
Phase 6 · Wnt-β-Catenin Signaling Modulation Studies Day 21-28
  1. a Treat organoids with IWP-2 Wnt inhibitor
  2. b Assess growth and gene expression after IWP-2 treatment
Phase 7 · Metabolomic Analysis and Methyltransferase Studies Day 60-120
  1. a Perform LC-MS metabolomic analysis
  2. b Treat organoids with pan-methyltransferase inhibitor ADOX
  3. c Screen histone methyltransferase inhibitors
  4. d Validate G9a (EHMT2) expression in YAP1-activated organoids
  5. e Determine G9a catalytic requirement via wild-type vs. enzyme-dead mutant
Phase 8 · EHMT2 Genetic Manipulation and Rescue Studies Day 21-60
  1. a Generate EHMT2 knockdown organoids
  2. b Assess downstream gene expression after EHMT2 knockdown
  3. c Perform EHMT2 rescue by shRNA-resistant overexpression
Phase 9 · Therapeutic Intervention Studies Day 40-60 (ex vivo); post-transplant (in vivo)
  1. a Treat YAP1-activated HB organoids with ADOX
  2. b Treat xenograft tumors with G9a inhibitor BIX 01294 in vivo
  3. c Assess tumor response to BIX 01294 treatment

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Attribution

This SOP was written by Organthis based on the published method in Yang et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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