Skip to content
← Back to browse
LIVER Publication-derived

Hepatobiliary organoids differentiated from hiPSCs relieve cholestasis-induced liver fibrosis in nonhuman primates

Source Li et al., 2024 · School of Life Science, Beijing University of Chinese Medicine · 10.7150/ijbs.90441

👤 Hongmei Li, Jingyi Li, Ting Wang, Ke Sun, Guangrui Huang, Yulin Cao, Fenfang Wu, Anlong Xu ⏱ 56 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol establishes the differentiation of human induced pluripotent stem cells (hiPSCs) into functional hepatobiliary organoids (HBOs) over approximately 35 days in vitro, followed by transplantation into a cynomolgus monkey model of cholestasis-induced liver fibrosis via subcapsular and submesenteric routes. The study evaluates the safety and efficacy of HBO transplantation in alleviating liver fibrosis through antiapoptotic, antifibrotic, and CTSV-mediated mechanisms.

Cell source
Human iPSC
Application
Disease modeling - cholestasis-induced liver fibrosis

Protocol overview

30 steps across 10 phases

hiPSC Culture and Preparation Days 1-5 pre-differentiation
  1. 1 Plate hiPSCs on Matrigel-coated wells
  2. 2 Verify pluripotency markers
Hepatobiliary Organoid Differentiation - Endoderm Induction Days 1-4
  1. 3 Dissociate hiPSCs and passage onto fresh Matrigel plates
  2. 4 Initiate differentiation with activin A and BMP4
  3. 5 Add mTeSR supplement and continue culture
  4. 6 Transition to endoderm specification
Hepatobiliary Organoid Differentiation - Hepatic Progenitor Formation Days 5-16
  1. 7 Induce liver bud formation with BMP2 and FGF4
  2. 8 Culture with HGF and KGF for hepatic progenitor maturation
Hepatobiliary Organoid Differentiation - Hepatobiliary Maturation Days 17-36
  1. 9 Hepatocyte and cholangiocyte differentiation with oncostatin M and cholesterol MIX
  2. 10 Mature HBO culture with ascorbic acid and transferrin supplement
  3. 11 Select Day 35 HBOs for transplantation
Nonhuman Primate Bile Duct Ligation and HBO Transplantation - Preparation Day -5 (pre-transplant)
  1. 12 Initiate immunosuppressive therapy
Bile Duct Ligation Surgery and HBO Transplantation - Day 0 Day 0 (surgery day)
  1. 13 Anesthetize monkey and prepare surgical field
  2. 14 Collect pre-surgery liver biopsy and verify normal liver
  3. 15 Dissect hepatic vasculature and ligate common bile duct
HBO Transplantation - Liver Subcapsular Route Day 0 (within 30 minutes post-BDL)
  1. 16 Prepare Day 35 HBOs for subcapsular transplantation
  2. 17 Inject HBOs under liver capsule at 5 sites
  3. 18 Close liver incision and abdominal wall
HBO Transplantation - Submesenteric Route (Alternative to Subcapsular) Day 0 (within 30 minutes post-BDL, alternative route)
  1. 19 Prepare HBOs for submesenteric transplantation
  2. 20 Identify colon mesentery and select 6 transplant sites
  3. 21 Graft HBOs into mesenteric sites and suture
  4. 22 Close abdominal wall
Post-Transplantation Monitoring and Sample Collection Days 1-56 (8 weeks post-transplantation)
  1. 23 Collect baseline blood sample (Pre-Tx)
  2. 24 Weekly blood sampling and biochemical analysis (Weeks 1-8)
  3. 25 Abdominal ultrasound imaging
  4. 26 CT scan imaging at baseline, 4 weeks, and 8 weeks
Terminal Procedures and Sample Collection Day 56 (8 weeks post-transplantation)
  1. 27 Euthanasia and gross organ examination
  2. 28 Quantify liver and spleen volumes
  3. 29 Tissue sampling for histopathology
  4. 30 Collect final blood sample for coagulation and safety parameters

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 Li et al., 2024. 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