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LIVER Publication-derived

Modeling pathogenesis and progression of metabolic dysfunction-associated steatotic liver disease and therapeutic drug screening using hESC-derived mature polarized hepatocyte organoids

Source Qin et al., 2025 · Department of Gastroenterology and Hepatology, Guangzhou Digestive Disease Center, the Second Affiliated Hospital, School of Medicine, South China University of Technology · 10.1186/s13287-025-04865-7

👤 Changlu Qin, Haibin Wu, Peilin Liao, Wenjiao Yan, Xinyi Shi, Jinghe Xie, Shoupei Liu, Sen Chen, Xiangting Cao, Yongjian Zhou, Yuyou Duan ⏱ 15 days 📋 7 phases 🧫 Human ESC (H9 line)

Abstract

This protocol describes the differentiation of human embryonic stem cells (hESC) into mature polarized hepatocyte organoids (P-hep-orgs) and their use as an in vitro model of metabolic dysfunction-associated steatotic liver disease (MASLD). P-hep-orgs are exposed to free fatty acids (FFAs) to recapitulate key pathological features of MASLD progression including lipid accumulation, oxidative stress, apoptosis, and hepatic dysfunction. The model is validated for drug screening applications using known antioxidant and lipid-lowering agents.

Cell source
Human ESC (H9 line)
Application
Disease modeling; Drug screening

Protocol overview

18 steps across 7 phases

hESC Culture and Maintenance Ongoing
  1. 1 hESC Culture Setup
  2. 2 hESC Passaging
Definitive Endoderm (DE) Differentiation Days 0–3
  1. 1 hESC Dissociation and Aggregate Formation
  2. 2 Y-27632 Removal
  3. 3 DE Differentiation Day 0–1
  4. 4 DE Differentiation Day 1–2
  5. 5 DE Differentiation Day 2–3
Hepatoblast (HB) Sphere Formation Days 3–9
  1. 1 HB Sphere Medium Preparation and Culture
Hepatoblast Organoid (HB-org) Expansion Days 9+
  1. 1 HB Sphere Dissociation and HB-org Expansion Medium Preparation
  2. 2 Matrigel Addition and ECM Support
  3. 3 Daily Medium Replacement and Long-term Culture
  4. 4 HB-org Passaging
Polarized Hepatocyte Organoid (P-hep-org) Maturation Days 9–15 (6 days maturation)
  1. 1 Initiation of P-hep-org Maturation
  2. 2 Medium Replacement During Maturation
MASLD Induction with Free Fatty Acids (FFAs) Variable (2–10 days based on experimental endpoint)
  1. 1 FFA Preparation and Application
  2. 2 MASLD Phenotype Development
Drug Treatment and Efficacy Assessment Variable (concurrent with FFA treatment)
  1. 1 Drug Preparation and Cytotoxicity Testing
  2. 2 Co-treatment with FFAs and Drugs

Full SOP

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Attribution

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

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