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LIVER Publication-derived

A human multi-lineage hepatic organoid model for liver fibrosis

Source Guan et al., 2021 · Stanford University, Department of Anesthesiology, Pain and Perioperative Medicine · 10.1038/s41467-021-26410-9

👤 Yuan Guan, Annika Enejder, Meiyue Wang, Zhuoqing Fang, Lu Cui, Shih-Yu Chen, Jingxiao Wang, Yalun Tan, Manhong Wu, Xinyu Chen, Patrik K. Johansson, Issra Osman, Koshi Kunimoto, Pierre Russo, Sarah C. Heilshorn, Gary Peltz ⏱ 90 days 📋 9 phases 🧫 Human iPSC (Patient-Derived iPSC and Isogenic Controls)

Abstract

This protocol generates human multi-lineage hepatic organoids from iPSCs via sequential growth factor stimulation, enabling the study of ARPKD-induced liver fibrosis. ARPKD organoids engineered with the PKHD1 T36M mutation develop key features of congenital hepatic fibrosis—including abnormal bile ducts and extensive collagen deposition—within 21 days, providing a human-based in vitro system to model and test anti-fibrotic therapies.

Cell source
Human iPSC (Patient-Derived iPSC and Isogenic Controls)
Application
Disease modeling - Congenital hepatic fibrosis and liver fibrosis mechanisms

Protocol overview

53 steps across 9 phases

iPSC generation and CRISPR-mediated genome editing Days 0-2 (editing); culture-dependent
  1. 1 iPSC line preparation
  2. 2 Design guide RNA sequences for CRISPR targeting
  3. 3 Construct homology-directed repair (HDR) targeting vectors
  4. 4 Introduce PKHD1 T36M mutation via piggyBac-CRISPR system
  5. 5 Verify homozygous PKHD1 T36M mutations
Hepatic organoid differentiation from iPSCs Days 0-21
  1. 1 Initial iPSC expansion
  2. 2 Differentiate iPSCs to hepatoblasts
  3. 3 Culture hepatoblasts to form hepatic organoids
  4. 4 Harvest hepatic organoids at day 21
Histological and immunohistochemical characterization Days 21-28
  1. 1 Prepare organoid tissue sections
  2. 2 Perform H&E staining
  3. 3 Perform Trichrome staining
  4. 4 Perform immunofluorescence staining for cell markers
  5. 5 Quantify collagen and bile duct regions
Single-cell RNA sequencing and transcriptomic analysis Days 21-60 (sample preparation to data analysis)
  1. 1 Dissociate hepatic organoids into single cells
  2. 2 Prepare single-cell suspension for 10× Chromium capture
  3. 3 Capture single cells using 10× Chromium system
  4. 4 Perform library preparation and sequencing
  5. 5 Demultiplex samples by donor using demuxlet
  6. 6 Pre-process scRNA-Seq data in Seurat
  7. 7 Perform clustering analysis
  8. 8 Identify differentially expressed genes and biomarkers
  9. 9 Perform pathway enrichment analysis
Second harmonic generation (SHG) and CARS microscopy for collagen quantification Days 21-28
  1. 1 Prepare organoids for SHG/CARS imaging
  2. 2 Perform SHG/CARS microscopy imaging
  3. 3 Image processing and collagen fiber quantification
  4. 4 Quantify collagen fiber diameter distribution
  5. 5 Statistical analysis of collagen measurements
Primary cilium imaging and cilia abundance quantification Days 21-28
  1. 1 Clone ARL13B-GFP fusion protein construct
  2. 2 Produce lentiviral vectors expressing ARL13B-GFP
  3. 3 Transduce hepatic organoids with ARL13B-GFP lentivirus
  4. 4 Stain for endogenous ciliary markers
  5. 5 Image primary cilia using fluorescence microscopy
  6. 6 Quantify primary cilium abundance and length
Mass cytometry (CyTOF) analysis of cell populations Days 21-28
  1. 1 Prepare single-cell suspensions for CyTOF
  2. 2 Fix and stain cells with metal-conjugated antibodies
  3. 3 Stain DNA and prepare for CyTOF measurement
  4. 4 Perform CyTOF measurement and data processing
  5. 5 Generate t-SNE maps and identify cell populations
PDGFRB tyrosine kinase inhibitor treatment and anti-fibrotic efficacy testing Days 12-22 (treatment protocol)
  1. 1 Generate single organoid cultures in microplates
  2. 2 Add PDGFRB and control inhibitors on day 12
  3. 3 Maintain organoid cultures with drug treatment
  4. 4 Harvest organoids and isolate for collagen quantification
  5. 5 Perform whole-mount organoid immunostaining
  6. 6 Quantify fibrosis score from immunostained organoids
  7. 7 Measure collagen 1A mRNA by RT-PCR
  8. 8 Quantify collagen content by hydroxyproline LC-MS
  9. 9 Assess organoid viability after inhibitor treatment
Gene set enrichment analysis (GSEA) and transcriptome correlation Days 60-90 (data analysis only)
  1. 1 Construct myofibroblast-specific gene expression signature
  2. 2 Obtain publicly available liver gene expression datasets
  3. 3 Perform Gene Set Enrichment Analysis (GSEA)
  4. 4 Compare myofibroblast signatures between ARPKD and fibrotic human livers
  5. 5 Generate correlation plots and statistical summary

Full SOP

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Attribution

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

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