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LIVER Publication-derived

Single-cell transcriptomics stratifies organoid models of metabolic dysfunction-associated steatotic liver disease

Source Hess et al., 2023 · Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA · 10.15252/embj.2023113898

👤 Anja Hess, Stefan D Gentile, Amel Ben Saad, Raza-Ur Rahman, Tim Habboub, Daniel S Pratt, Alan C Mullen ⏱ 26 days 📋 11 phases 🧫 Human pluripotent stem cells (hPSCs) - H1 (WA01) and patient-derived iPSC4

Abstract

This protocol establishes a systematic approach to evaluate human liver organoid (HLO) models of metabolic dysfunction-associated steatotic liver disease (MASLD) by comparing three injury induction conditions: oleic acid, palmitic acid, and TGF-β1. Using single-cell RNA sequencing analysis of ~100,000 cells, the study characterizes distinct inflammatory and fibrotic responses, finding that palmitic acid and TGF-β1 more robustly model MASH and fibrosis progression, while oleic acid ameliorates fibrotic signatures.

Cell source
Human pluripotent stem cells (hPSCs) - H1 (WA01) and patient-derived iPSC4
Application
Disease modeling - MASLD (metabolic dysfunction-associated steatotic liver disease) with comparative analysis of inflammatory and fibrotic responses

Protocol overview

62 steps across 11 phases

hPSC maintenance and HLO differentiation setup Days 0-20
  1. 1 hPSC culture maintenance
  2. 2 Initiate HLO differentiation
  3. 3 Confirm loss of pluripotency genes by qRT-PCR
  4. 4 Confirm induction of hepatic markers
HLO characterization and histology Days 16-21
  1. 1 Prepare HLOs for histology
  2. 2 Wash and store fixed HLOs
  3. 3 Perform hematoxylin and eosin (H&E) staining
  4. 4 Perform immunohistochemistry (IHC)
Culture condition optimization for collagen induction Days 21-25
  1. 1 Isolate HLOs from Matrigel
  2. 2 Culture isolated HLOs in four mechanical environments
  3. 3 Treat HLOs with TGF-β1 in Matrigel
  4. 4 Measure Matrigel contraction in response to TGF-β1
  5. 5 Select optimal culture condition
Fatty acid and TGF-β1 injury induction Days 21-25
  1. 1 Prepare palmitic acid (PA) stock solution
  2. 2 Prepare oleic acid (OA) stock solution
  3. 3 Prepare control media for fatty acids
  4. 4 Change medium to injury media on day 21
  5. 5 Culture HLOs with injury induction for 4 days
  6. 6 Harvest HLOs on day 25 for downstream analyses
Sirius red collagen staining and quantification Days 25-26
  1. 1 Prepare and fix HLOs for histology
  2. 2 Wash and store fixed HLOs in ethanol
  3. 3 Perform Sirius red staining
  4. 4 Quantify Sirius red staining using custom pipeline
  5. 5 Calculate Sirius red percentage per condition
Quantitative RT-PCR for gene expression analysis Days 25-26 (during injury) or any timepoint
  1. 1 Harvest HLOs for RNA extraction
  2. 2 Centrifuge and collect HLO pellet
  3. 3 Lyse organoids in Trizol
  4. 4 Extract total RNA by phenol-chloroform extraction
  5. 5 Synthesize cDNA
  6. 6 Design and validate qPCR primers
  7. 7 Perform qPCR reactions
  8. 8 Calculate relative gene expression
  9. 9 Analyze target genes for injury response
Triglyceride quantification Days 25-26
  1. 1 Collect treated HLOs
  2. 2 Homogenize HLOs in lysis buffer
  3. 3 Measure triglyceride content
Lipid droplet visualization by BODIPY staining Days 25-26
  1. 1 Collect HLOs for lipid staining
  2. 2 Stain lipid droplets with BODIPY
  3. 3 Image stained HLOs
  4. 4 Quantify lipid content by image analysis
Single-cell RNA sequencing (scRNA-seq) sample preparation Days 25-26 (sample harvest)
  1. 1 Prepare HLOs for dissociation
  2. 2 Dissociate HLOs to single cells
  3. 3 Wash dissociated cells
  4. 4 Count and assess cell viability
  5. 5 Prepare single-cell libraries with 10× Chromium
  6. 6 Sequence single-cell libraries
Single-cell RNA sequencing (scRNA-seq) computational analysis Post-sequencing
  1. 1 Perform initial QC and alignment
  2. 2 Calculate doublet scores and filter
  3. 3 Load and merge scRNA-seq data
  4. 4 Apply cell-level QC filtering
  5. 5 Normalize and scale data
  6. 6 Compute dimensionality reduction (PCA and UMAP)
  7. 7 Perform clustering and cell type annotation
  8. 8 Score cells for inflammatory and fibrotic signatures
  9. 9 Perform cell-type-specific pathway enrichment analysis
  10. 10 Analyze cell-cell interactions using CellPhoneDB
  11. 11 Perform trajectory inference using Palantir
  12. 12 Score MASLD disease severity signatures
Statistical analysis and data reporting Post-analysis
  1. 1 Perform statistical testing for qRT-PCR and functional assays
  2. 2 Perform statistical testing for scRNA-seq comparisons
  3. 3 Prepare figures and supplementary materials
  4. 4 Archive and share raw data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Hess et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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