Skip to content
← Back to browse
LIVER Publication-derived

Human ESC-derived expandable hepatic organoids (hEHOs) for hepatic differentiation and alcoholic liver disease modeling

Source Wang et al., 2019 · Beijing Institute of Health Service and Transfusion Medicine · 10.1038/s41422-019-0242-8

👤 Shuyong Wang, Xuan Wang, Zuolong Tan, Yuxin Su, Juan Liu, Mingyang Chang, Fang Yan, Jie Chen, Tao Chen, Chuanjiang Li, Jie Hu, Yunfang Wang ⏱ 36 days 📋 7 phases 🧫 Human ESC

Abstract

This protocol describes the generation of human embryonic stem cell-derived expandable hepatic organoids (hEHOs) using chemically defined, serum-free media. The hEHOs maintain bipotential hepatic stem/progenitor cell phenotypes, expand for over 20 passages, and can differentiate into functional hepatocytes or cholangiocytes. When co-cultured with human fetal liver mesenchymal cells (hFLMCs), the resulting hFLMC/hEHO model system recapitulates alcoholic liver disease-associated pathophysiology including oxidative stress, steatosis, inflammation, and fibrosis.

Cell source
Human ESC
Application
Disease modeling and therapeutic liver repopulation

Protocol overview

30 steps across 7 phases

Definitive Endoderm (DE) Formation 0–3 days
  1. 1 Maintain H9 human ESCs in feeder-free conditions
  2. 2 Induce definitive endoderm differentiation—Day 1
  3. 3 Continue DE induction—Days 2–3
Hepatic Specification (HS) Lineage Formation 3–8 days
  1. 1 Initiate hepatic specification with BMP4 and FGF2
  2. 2 Dissociate HS cells into single cells
3D Hepatic Organoid (hEHO) Formation and Expansion 8–20+ days (20 passages)
  1. 1 Embed HS cells in Matrigel droplets
  2. 2 Add organoid formation medium (NBNiGNaEWRAF)
  3. 3 Monitor organoid formation
  4. 4 Passage hEHOs every 7–9 days
  5. 5 Cryopreserve hEHOs
In Vitro Differentiation of hEHOs into Mature Hepatocytes (hEHO-Heps) 20–36 days (relative to start of organoid formation)
  1. 1 Pretreat hEHOs with growth factors
  2. 2 Dissociate pretreated hEHOs and form 3D aggregates
  3. 3 Culture aggregates in hepatocyte maturation medium—Phase 1 (3 days)
  4. 4 Culture aggregates in hepatocyte maturation medium—Phase 2 (8 days)
In Vitro Differentiation of hEHOs into Mature Cholangiocytes (hEHO-Clcs) 10–24 days (from start of organoid formation)
  1. 1 Dissociate hEHOs into single cells
  2. 2 Resuspend cells in type I collagen and prepare culture wells
  3. 3 Culture in cholangiocyte differentiation medium
Co-culture of hEHOs with Human Fetal Liver Mesenchymal Cells (hFLMCs) for ALD Modeling Days 0–14 (organoid formation and maturation)
  1. 1 Isolate and prepare hFLMCs
  2. 2 Coat wells with sterile agarose
  3. 3 Dissociate hEHOs and hFLMCs; resuspend at 2:1 ratio
  4. 4 Seed cells in agarose-coated wells and culture—Phase 1 (7 days)
  5. 5 Switch to maturation medium—Phase 2 (7 days)
  6. 6 Treat hFLMC/hEHO organoids with ethanol (EtOH) for ALD modeling
  7. 7 Assess ALD-related pathophysiology at day 7 of EtOH treatment
In Vivo Transplantation of hEHOs into FRG Mice Variable (3 months post-transplantation for analysis)
  1. 1 Prepare FRG mice and NTBC treatment
  2. 2 Dissociate hEHOs and prepare cell suspension
  3. 3 Transplant hEHOs into liver parenchyma
  4. 4 Initiate NTBC withdrawal schedule
  5. 5 Monitor survival, weight, and serum markers
  6. 6 Harvest liver samples at 3 months post-transplantation

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 Wang et al., 2019. 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