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LIVER Publication-derived

Generation of liver organoids from human induced pluripotent stem cells as liver fibrosis and steatosis models

Source Tsang et al., 2021 · MOE Key Laboratory for Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong · 10.1101/2021.06.29.450347;

👤 Hoi Ying Tsang, Paulisally Hau Yi Lo, Kenneth Ka Ho Lee ⏱ 21 days 📋 8 phases 🧫 Human iPSC

Abstract

This protocol describes the differentiation of human induced pluripotent stem cells (hiPSC) into hepatoblasts, mesenchymal stem cells, hepatic stellate cells, and endothelial cells, which self-organize into vascularized liver organoids exhibiting hepatic functions and architecture. The organoids can be induced with thioacetamide or free fatty acids to model liver fibrosis and non-alcoholic fatty liver disease (NAFLD), enabling high-throughput drug screening.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

46 steps across 8 phases

hiPSC culture and hepatic cell differentiation Days 1-21
  1. 1 hiPSC culture
  2. 2 Differentiate hiPSCs to hepatoblasts (HBs)
  3. 3 Differentiate hiPSCs to mesenchymal stem cells (MSCs)
  4. 4 Differentiate hiPSCs to hepatic stellate cells (HSCs)
  5. 5 Differentiate hiPSCs to endothelial cells (ECs)
Liver organoid differentiation and formation Days 1-14
  1. 1 Trypsinize and count differentiated cells
  2. 2 Prepare organoid culture medium
  3. 3 Seed cells into ultra-low attachment 96-well plates
  4. 4 Add Y-27632 supplement (days 1-2)
  5. 5 Add FGF2 supplement (days 1-4)
  6. 6 Medium replacement (3 times per week)
  7. 7 Harvest organoids at day 14
Thioacetamide (TAA) treatment for fibrosis induction Days 1-7 (treatment phase)
  1. 1 Prepare TAA solutions
  2. 2 Expose organoids to TAA
  3. 3 Collect media and organoids
  4. 4 Assess cell viability
Free fatty acid (FFA) treatment for steatosis induction Days 1-7 (treatment phase)
  1. 1 Prepare FFA-BSA conjugates
  2. 2 Expose organoids to FFA
  3. 3 Collect media and organoids
  4. 4 Assess cell viability
Liver function assays Day 14 (or post-treatment)
  1. 1 Albumin secretion ELISA
  2. 2 Indocyanine green (ICG) uptake and excretion
Molecular analysis - Real-time quantitative PCR (qPCR) Day 14 (or post-treatment)
  1. 1 RNA extraction
  2. 2 RNA quantification and purity assessment
  3. 3 Reverse transcription
  4. 4 Real-time qPCR
Immunological analysis - Whole-mount immunofluorescent staining Day 14 (or post-treatment)
  1. 1 Wash organoids
  2. 2 Fixation
  3. 3 Storage
  4. 4 Permeabilization
  5. 5 Blocking
  6. 6 Primary antibody incubation
  7. 7 Primary antibody wash
  8. 8 Secondary antibody and DAPI incubation
  9. 9 Secondary antibody wash
  10. 10 Clearing solution treatment
  11. 11 Confocal imaging
Histological analysis Day 14 (or post-treatment)
  1. 1 Fixation
  2. 2 Embedding in agarose-gelatin gel
  3. 3 Secondary fixation
  4. 4 Dehydration
  5. 5 Paraffin embedding
  6. 6 Sectioning
  7. 7 Hematoxylin-Eosin (H&E) staining
  8. 8 Periodic acid-Schiff (PAS) staining
  9. 9 Pico-sirius red (PS Red) staining

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Tsang et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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