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LIVER Publication-derived

Vascularized and Functional Human Liver from an iPSC-Derived Organ Bud Transplant

Source Takebe et al., 2013 · Yokohama City University · 10.1038/nature12271

👤 Takanori Takebe, Keisuke Sekine, Masahiro Enomura, Hiroyuki Koike, Masaki Kimura, Takunori Ogaeri, Ran-Ran Zhang, Yasuharu Ueno, Yun-Wen Zheng, Naoto Koike, Shinsuke Aoyama, Yasuhisa Adachi, Hideki Taniguchi ⏱ 60 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of vascularized and functional human liver tissue from human induced pluripotent stem cells (iPSCs) through in vitro liver-bud formation and subsequent transplantation into immunodeficient mice. The iPSC-derived liver buds recapitulate organogenetic interactions between hepatic endoderm, endothelial, and mesenchymal cells, resulting in three-dimensional tissue that matures upon vascularization to exhibit adult liver-like structure and function including albumin production, drug metabolism, and rescue of drug-induced liver failure.

Cell source
Human iPSC
Application
Disease modeling; Regenerative medicine; Drug metabolism evaluation

Protocol overview

50 steps across 12 phases

Endodermal Differentiation of Human iPSCs Days 0–5 to 6
  1. 1 Maintain undifferentiated human iPSCs
  2. 2 Seed iPSCs on Matrigel for endodermal differentiation
  3. 3 Add human activin A for endoderm induction
Hepatic Specification and iPSC-Derived Hepatic Endoderm Generation Days 5–6 to 8–10
  1. 1 Treat differentiated endodermal cells for hepatic specification
  2. 2 Culture for hepatic specification
  3. 3 Verify hepatic marker expression
In Vitro Liver-Bud Formation by Co-Culture Days 0–6 of co-culture (approximately 4–6 days)
  1. 1 Prepare cell suspension for liver-bud formation
  2. 2 Supplement cell suspension with growth factors and hormones
  3. 3 Prepare pre-solidified Matrigel substrate
  4. 4 Plate cell suspension on Matrigel
  5. 5 Monitor self-organization into 3D liver buds
  6. 6 Collect matured liver buds
In Vivo Transplantation of Liver Buds Day of transplantation (after 4–6 day culture period)
  1. 1 Prepare recipient mice
  2. 2 Transplant liver buds into cranial window (primary site) or mesentery (secondary site)
  3. 3 Monitor engraftment and vascularization in vivo
Intravital Imaging and Vascular Characterization Days 1–60 post-transplantation (variable; representative checkpoints)
  1. 1 Prepare fluorescent dextran for vessel visualization
  2. 2 Inject fluorescent tracers via tail vein
  3. 3 Perform confocal microscopy and image acquisition
  4. 4 Analyze vascular parameters using image-processing software
Gene Expression and Microarray Analysis Day 4–6 of in vitro culture and day 60 post-transplantation
  1. 1 Harvest cells and extract total RNA
  2. 2 Perform quantitative PCR (qPCR) for hepatic marker genes
  3. 3 Perform microarray hybridization
  4. 4 Scan microarray and extract intensity data
  5. 5 Analyze and compare expression profiles
Flow Cytometric Characterization Day 4–6 of in vitro culture and day 60 post-transplantation
  1. 1 Prepare single-cell suspension from liver buds or transplants
  2. 2 Stain cells with primary antibodies
  3. 3 Wash and acquire flow cytometry data
  4. 4 Analyze flow cytometry data
Immunohistochemistry and Tissue Characterization Days 60 post-transplantation and in vitro day 4–6
  1. 1 Tissue harvest and fixation
  2. 2 Whole-mount immunostaining (for thick sections/cranial implants)
  3. 3 Paraffin embedding and sectioning (for standard histology)
  4. 4 Antigen retrieval and immunostaining (paraffin sections)
  5. 5 Image acquisition and analysis
ELISA-Based Protein Production Analysis Days 10–45 post-transplantation and as specified
  1. 1 Collect blood samples from transplant-bearing mice
  2. 2 Prepare serum
  3. 3 Measure human albumin (ALB) by ELISA
  4. 4 Measure human alpha-1-antitrypsin (AAT) by ELISA
  5. 5 Analyze ELISA results and compare with controls
Drug Metabolism Studies Day 60 post-transplantation (or as specified for drug challenge)
  1. 1 Administer test drug (ketoprofen or debrisoquine)
  2. 2 Collect urine and serum samples
  3. 3 Analyze metabolite formation by LC-MS or HPLC
  4. 4 Compare human-specific metabolite formation in iPSC-LB transplants vs. controls
Metabolomic Profiling Day 60 post-transplantation
  1. 1 Harvest transplant tissue and prepare for metabolomic analysis
  2. 2 Extract metabolites
  3. 3 Perform CE-TOFMS analysis
  4. 4 Compare metabolic profiles and identify liver-specific metabolites
Liver Failure Rescue Experiment (Mesenteric Transplantation) Days 0–30 post-transplantation; drug administration at days 7 and 10
  1. 1 Prepare TK-NOG mice and perform mesenteric transplantation
  2. 2 Administer ganciclovir to induce selective hepatic injury
  3. 3 Monitor survival and collect clinical samples
  4. 4 Generate Kaplan-Meier survival curves and statistical analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Takebe et al., 2013. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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