LIVER Publication-derived

Human liver organoids model progressive inflammatory and fibrotic injury in non-alcoholic fatty liver disease

Source Hess et al., 2022 · Massachusetts General Hospital · 10.1101/2022.07.19.500693;

👤 Anja Hess, Stefan D. Gentile, Amel Ben Saad, Raza-Ur Rahman, Tim Habboub, Alan C. Mullen ⏱ 26 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the generation and treatment of human pluripotent stem cell-derived liver organoids (HLOs) to model progressive liver injury. HLOs are treated with palmitic acid to induce steatohepatitis or with TGF-β1 to induce fibrosis, and analyzed via morphological, histological, and single-cell transcriptomic approaches to characterize disease-specific cellular responses and their progression from inflammatory to fibrotic phenotypes.

Cell source

Human iPSC

Application

Disease modeling (NAFLD/NASH and fibrosis)

Protocol overview

47 steps across 9 phases

hPSC maintenance and HLO differentiation Day 0 to Day 21

1 hPSC culture and maintenance
2 Initiate HLO differentiation
3 Confirm pluripotency gene loss and liver marker induction (Day 16 and Day 21)
4 Assess HLO morphology and histology (Day 21)

HLO isolation and culture system preparation Day 20-21

1 Isolate HLOs from Matrigel
2 Plate isolated HLOs into different 3D culture systems

Liver injury induction with TGF-β1 and fatty acids Day 20/21 to Day 25/26

1 Prepare injury media solutions
2 Change medium to injury media (Day 20-21)
3 Culture HLOs under injury conditions

Morphological and contractility assessment Day 25-26

1 Document morphological changes
2 Perform Matrigel contraction assay

Histological analysis and collagen quantification Day 25-26

1 Fix HLOs for histology
2 Perform H&E staining
3 Perform Sirius red staining for collagen quantification
4 Quantify Sirius red staining using optimized pipeline

Gene expression analysis by qPCR Day 25-26

1 Harvest and lyse HLOs for RNA extraction
2 Extract total RNA via phenol-chloroform extraction
3 Synthesize cDNA
4 Design and validate qPCR primers
5 Perform qPCR reactions
6 Analyze qPCR data

Single-cell RNA sequencing sample preparation Day 21 and Day 25-26

1 Culture HLOs on ultra-low attachment plates for scRNA-seq
2 Dissociate HLOs to single cells
3 Prepare cells for library preparation
4 Prepare 10X Chromium libraries
5 Sequence libraries

Single-cell RNA sequencing data processing and analysis Post-sequencing

1 Convert BCL files and assess quality
2 Align reads and generate count matrices
3 Calculate doublet scores
4 Apply quality control filters
5 Merge replicates and normalize data
6 Assign cell cycle scores
7 Perform dimensionality reduction and clustering
8 Correct batch effects
9 Annotate cell clusters using literature markers
10 Annotate cell clusters using database markers
11 Cross-validate with SingleCellNet classifier
12 Calculate inflammation and fibrosis scores
13 Perform differential gene expression analysis
14 Perform pathway enrichment analysis
15 Infer cell-cell interactions using CellPhoneDB
16 Perform trajectory inference
17 Perform MAGIC imputation and gene trajectory analysis
18 Apply NAFLD fibrosis stage signatures

Statistical analysis Post-data collection

1 Perform two-group statistical comparisons
2 Perform multi-group statistical comparisons
3 Determine significance threshold

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 Hess et al., 2022. 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