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LIVER Publication-derived

Generation of hiHep Organoids and Modeling Liver Cancer Initiation

Source Sun et al., 2019 · Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences · 10.1038/s41556-019-0359-5

👤 Lulu Sun, Yuqing Wang, Jin Cen, Xiaolong Ma, Lei Cui, Lijian Hui ⏱ 60 days 📋 8 phases 🧫 Human iPSC-derived induced hepatocytes (hiHeps) from fibroblasts

Abstract

This protocol describes the generation of liver organoids from directly reprogrammed human hepatocytes (hiHeps) and their genetic engineering to model human liver cancer initiation, including hepatocellular carcinoma (HCC) and intra-hepatic cholangiocarcinoma (ICC). The organoids display improved hepatic architecture, polarity, and functions, and can be used to study cancer initiation mechanisms and identify preventive therapies.

Cell source
Human iPSC-derived induced hepatocytes (hiHeps) from fibroblasts
Application
Disease modeling; Cancer initiation study

Protocol overview

31 steps across 8 phases

hiHep Cell Culture and Preparation Day 0–14
  1. 1 Prepare hiHep cells from fibroblasts
  2. 2 Culture hiHep cells in hepatocyte-maintaining medium (HMM)
Formation of hiHep Organoids Day 0–5
  1. 1 Seed hiHep cells into ultra-low attachment plates
  2. 2 Allow cells to self-aggregate into spheroids
  3. 3 Change medium daily
  4. 4 Culture organoids for maturation
Oncogenic Transfection (HCC Model) Day 0–60
  1. 1 Clone oncogenes into lentivirus vectors
  2. 2 Produce lentiviral particles
  3. 3 Infect organoids with viral particles at seeding
  4. 4 Perform second viral infection
  5. 5 Monitor c-Myc-related gene expression
Oncogenic Transfection (ICC Model) Day 0–20
  1. 1 Clone ICC-enriched oncogenes into lentivirus vectors
  2. 2 Produce lentiviral particles for ICC oncogenes
  3. 3 Infect organoids with ICC oncogene viruses
  4. 4 Monitor ICC-related gene and protein expression
Functional Characterization of Organoids Day 5–60
  1. 1 Perform albumin secretion assay
  2. 2 Measure glycogen storage
  3. 3 Measure APOA1 secretion
  4. 4 Assess testosterone metabolism (CYP activity)
Orthotopic Transplantation and In Vivo Analysis Day 20–60
  1. 1 Prepare organoids for transplantation
  2. 2 Perform orthotopic transplantation
  3. 3 Monitor tumor formation
  4. 4 Harvest and analyze tumors
Treatment with MAM Inhibitor (c-Myc Model) Day 0–10
  1. 1 Prepare Mdivi-1 stock solution
  2. 2 Treat c-Myc organoids with Mdivi-1
  3. 3 Measure c-Myc-related gene expression
  4. 4 Prepare pre-treated organoids for transplantation (optional)
Treatment with Notch and JAK–STAT Inhibitors (RAS Model) Day 0–10
  1. 1 Prepare inhibitor stock solutions
  2. 2 Treat RAS organoids with individual inhibitors
  3. 3 Treat RAS organoids with combined inhibitors
  4. 4 Measure cell number and ICC-related gene expression

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Sun et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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