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LIVER Publication-derived

Modeling metabolic-associated steatohepatitis with human pluripotent stem cell-derived liver organoids

Source Wu et al., 2024 · Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences · 10.1097/hc9.0000000000000585

👤 Xiaoshan Wu, Dacheng Jiang, Yuchen Wang, Xin Li, Chenyu Liu, Yanhao Chen, Wei Sun, Ruikun He, Yi Yang, Xiaosong Gu, Chunping Jiang, Qiurong Ding ⏱ 27 days 📋 9 phases 🧫 Human iPSC

Abstract

A protocol for generating human liver organoids (HLOs) from induced pluripotent stem cells and inducing metabolic-associated steatohepatitis (MASH) features through oleic acid and TGFβ treatment. The M-HLO model demonstrates lipid accumulation, oxidative stress, mitochondrial dysfunction, inflammation, and fibrosis, enabling MASH pathogenesis study and drug screening.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

36 steps across 9 phases

iPSC culture and foregut differentiation Day 1-6
  1. 1 iPSC culture initialization
  2. 2 Day 1 – Definitive endoderm induction
  3. 3 Day 2 – Activin A treatment with reduced serum
  4. 4 Day 3 – Increased serum concentration
  5. 5 Days 4–6 – Foregut cell differentiation
Organoid formation and specification Day 7-14
  1. 1 Day 7 – Foregut dissociation and Matrigel embedding
  2. 2 Days 7–10 – 5F medium culture
  3. 3 Days 10–14 – Retinoic acid-driven maturation
Hepatocyte maturation Day 15-20
  1. 1 Day 15 – Harvest and transfer to hepatocyte maturation medium
  2. 2 Days 16–20 – Complete maturation with medium changes
MASH induction Day 20-27
  1. 1 Day 20 – Plate HLOs for MASH induction
  2. 2 Days 20–27 – OA+TGFβ treatment (4 days OA, then 3 days OA+TGFβ)
  3. 3 Vehicle control treatment
  4. 4 Optional: Herbal extract treatment
Functional and molecular characterization Day 27 onwards (post-harvest)
  1. 1 BODIPY staining for lipid droplet visualization
  2. 2 TMRM staining for mitochondrial membrane potential assessment
  3. 3 CellROX staining for oxidative stress evaluation
  4. 4 TUNEL assay for apoptosis detection
  5. 5 Collagen I immunofluorescence staining for fibrogenesis assessment
RNA extraction and expression analysis Post-harvest
  1. 1 Total RNA isolation
  2. 2 Reverse transcription
  3. 3 Quantitative real-time PCR (RT-qPCR)
ELISA and secretion analysis Post-harvest
  1. 1 Collect culture supernatant
  2. 2 Human albumin ELISA
  3. 3 Human MCP-1 (CCL2) ELISA
  4. 4 Human IL-6 ELISA
RNA-sequencing analysis Post-harvest
  1. 1 RNA extraction for bulk RNA-seq
  2. 2 Library preparation and sequencing
  3. 3 KEGG pathway enrichment analysis
Single-cell RNA sequencing analysis Post-harvest
  1. 1 Single cell dissociation and preparation
  2. 2 scRNA-seq library preparation
  3. 3 Data processing and quality control
  4. 4 Cell-type annotation and clustering
  5. 5 Differential expression analysis
  6. 6 Intercellular communication analysis
  7. 7 Pseudotime trajectory analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wu et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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