Skip to content
← Back to browse
LIVER Publication-derived

Modeling drug-induced liver injury and screening for anti-hepatofibrotic compounds using human PSC-derived organoids

Source Wu et al., 2023 · Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences · 10.1186/s13619-022-00148-1

👤 Xiaoshan Wu, Dacheng Jiang, Yi Yang, Shuang Li, Qiurong Ding ⏱ 25 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human liver organoids (HLOs) from human induced pluripotent stem cells (iPSCs) and their application in modeling drug-induced liver injury (DILI) and screening for anti-hepatofibrotic compounds. HLOs contain multiple cell types including hepatocytes, stellate cells, and Kupffer cells, enabling the recapitulation of steatosis, fibrosis, and immune responses induced by drugs such as acetaminophen, fialuridine, methotrexate, and TAK-875. A high-content imaging system enables high-throughput screening of candidate anti-fibrotic compounds, with SD208 and Imatinib identified as effective suppressors of fibrogenesis.

Cell source
Human iPSC
Application
Drug-induced liver injury modeling and anti-fibrotic drug screening

Protocol overview

45 steps across 10 phases

Definitive Endoderm (DE) Differentiation Day 0-6
  1. 1 Seed hiPSCs on Geltrex-coated plates
  2. 2 Day 1 medium change with Activin A and BMP4
  3. 3 Day 2 medium change with Activin A and 0.2% FBS
  4. 4 Day 3 medium change with Activin A and 2% FBS
  5. 5 Days 4-6 culture in Advanced DMEM/F12 with growth factors
HLO Generation - Standard Protocol (Days 6-20) Day 6-20
  1. 1 Dissociate foregut cells and embed in Matrigel
  2. 2 Culture in RA medium for 4 days
  3. 3 Switch to liver maturation medium (Days 10-20)
HLO Generation - 5F Expansion Protocol (Days 6-25) Day 6-25
  1. 1 Dissociate foregut cells with accutase and embed in 5F medium
  2. 2 Maintain in 5F medium for 4 days
  3. 3 Switch to RA medium for 4 days
  4. 4 Harvest and re-embed organoids
  5. 5 Culture in liver maturation medium for 10 days
Drug Treatment - APAP-induced toxicity 7 days of treatment
  1. 1 Prepare APAP treatment medium
  2. 2 Treat HLOs with APAP for 7 days
  3. 3 Harvest organoids for downstream analysis
Drug Treatment - FIAU-induced steatosis and mitochondrial dysfunction 10 days of daily treatment
  1. 1 Prepare FIAU treatment medium
  2. 2 Treat HLOs with FIAU for 10 days (daily dosing)
  3. 3 Harvest and stain for lipid accumulation and mitochondrial potential
Drug Treatment - Methotrexate-induced steatosis and fibrosis 7 days of treatment
  1. 1 Prepare MTX treatment medium
  2. 2 Treat HLOs with MTX for 7 days
  3. 3 Harvest for lipid, collagen, and gene expression analysis
Drug Treatment - TAK-875-induced oxidative stress and immune response 7 days of treatment
  1. 1 Prepare TAK-875 treatment medium
  2. 2 Treat HLOs with TAK-875 for 7 days
  3. 3 Harvest and analyze ROS, cytokine release, and albumin
Fibrosis Induction - TGFβ or LPS treatment 3 days of treatment
  1. 1 Prepare fibrogenic stimulus medium
  2. 2 Treat HLOs with fibrogenic stimulus for 3 days
  3. 3 Harvest for Collagen I staining and gene expression analysis
Anti-fibrotic compound screening and validation 3-9 days (depending on fibrosis model)
  1. 1 Prepare HLOs with fibrogenic stimulus and test compounds
  2. 2 Culture for 3 days (TGFβ model) or 9 days (MTX or LPS models)
  3. 3 Stain for Collagen I and acquire high-content images
  4. 4 Extract features and perform tSNE analysis
  5. 5 Validate lead compounds with gene expression analysis
  6. 6 Confirm anti-fibrotic effects with Collagen I staining
Functional characterization assays End of differentiation or post-treatment
  1. 1 Albumin secretion by ELISA
  2. 2 ATP content measurement
  3. 3 Reactive Oxygen Species (ROS) staining with CellROX
  4. 4 Mitochondrial membrane potential staining with TMRM
  5. 5 Lipid accumulation staining with Bodipy
  6. 6 Immunofluorescence staining for cell-type markers
  7. 7 CDCFDA uptake and release assay for bile canaliculi function
  8. 8 LDL uptake assay
  9. 9 Glycogen accumulation assessment by PAS staining
  10. 10 Gene expression analysis by RT-qPCR
  11. 11 Cytokine release measurement (MCP-1, IL-6) by ELISA

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 Wu et al., 2023. 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