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LIVER Publication-derived

Generation of human iPSC-derived 3D bile duct within liver organoid by incorporating human iPSC-derived blood vessel

Source Carolina et al., 2024 · The Institute of Medical Science, The University of Tokyo · 10.1038/s41467-024-51487-3

👤 Erica Carolina, Yoshiki Kuse, Ayumu Okumura, Kenji Aoshima, Tomomi Tadokoro, Shinya Matsumoto, Eriko Kanai, Takashi Okumura, Toshiharu Kasai, Souichiro Yamabe, Yuji Nishikawa, Kiyoshi Yamaguchi, Yoichi Furukawa, Naoki Tanimizu, Hideki Taniguchi ⏱ 21 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol generates functional intrahepatic bile ducts from human iPSC-derived hepatic endoderm by co-culturing them around an engineered blood vessel composed of iPSC-derived immature smooth muscle cells and endothelial cells. The resulting bile duct-containing liver organoids (BVLO) exhibit bile transport function and can model Alagille syndrome, a congenital biliary disease.

Cell source
Human iPSC
Application
Disease modeling, Developmental study

Protocol overview

42 steps across 9 phases

hiPSC-derived smooth muscle cell (SMC) differentiation 0–11
  1. 1 Maintain hiPSC culture
  2. 2 Day 0: Initiate mesoderm induction
  3. 3 Day 4: Switch to SMC specification factors
  4. 4 Day 6: Harvest immature SMCs (imSMCs)
  5. 5 Day 11: Optional harvest of mature SMCs
hiPSC-derived endothelial cell (EC) differentiation and expansion 0–14
  1. 1 Initiate EC differentiation from hiPSC
  2. 2 Expand ECs in planar culture
hiPSC-derived hepatic endoderm (HE) and organoid generation 0–7
  1. 1 Differentiate hiPSC into hepatic endoderm (HE)
  2. 2 Co-culture HE with mesenchymal cells (MC) and ECs to form liver organoids
  3. 3 Transfer organoid to air-liquid interface (ALI) culture
  4. 4 Maintain organoid in AJI4/VEC-1 medium
hiPSC-derived blood vessel (hiPSC-BV) assembly 1 day before BVLO co-culture
  1. 1 Prepare collagen-1 scaffold tube with SMCs
  2. 2 Solidify SMC-collagen gel
  3. 3 Remove excess water
  4. 4 Attach SMC-vessel to catheters
  5. 5 Seed endothelial cells into lumen
  6. 6 Distribute ECs evenly within lumen
  7. 7 Fill lumen with culture medium
Blood vessel-incorporated liver organoid (BVLO) co-culture setup 1 day after HE organoid ALI transfer
  1. 1 Overlay organoid around hiPSC-BV
  2. 2 Apply extracellular matrix (ECM) coating
  3. 3 Transfer BVLO to air-liquid interface culture
  4. 4 Maintain BVLO in AJI4/VEC-1 medium
BVLO cultivation and functional maturation 1–21
  1. 1 Day 1–7: Early organoid integration
  2. 2 Day 7–14: BD structure formation
  3. 3 Day 14–21: BD maturation and functional development
Functional assay: Rho123 MDR1-dependent transport 21 (or later)
  1. 1 Prepare Rho123 culture medium
  2. 2 Incubate BVLO with Rho123
  3. 3 Wash out excess Rho123
  4. 4 Prepare BVLO for live imaging
  5. 5 Acquire confocal microscopy images
Functional assay: Forskolin-induced swelling 21 (or later)
  1. 1 Prepare BVLO samples
  2. 2 Fix control sample
  3. 3 Treat second sample with forskolin
  4. 4 Acquire macroscopic images
  5. 5 Fix treated sample
  6. 6 Prepare frozen sections for immunofluorescence
  7. 7 Perform immunofluorescence for BD markers
  8. 8 Quantify lumen area
Enzymatic activity assays: GGT and ALP 21 (or later)
  1. 1 Gamma-glutamyltransferase (GGT) activity assay
  2. 2 Alkaline phosphatase (ALP) activity detection via BCIP/NBT staining
  3. 3 Dehydration and mounting
  4. 4 Examine and document staining

Full SOP

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Attribution

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

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