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LIVER Publication-derived

Fatty Liver Disease Organoids (2022)

Full SOP with Academic or a plan

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

Authors: Anja Hess, Stefan D. Gentile, Amel Ben Saad, Raza-Ur Rahman, Tim Habboub, Alan C. Mullen Duration: 26 days 9 phases Cell source: 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

Phase 1 · hPSC maintenance and HLO differentiation Day 0 to Day 21
  1. a hPSC culture and maintenance
  2. b Initiate HLO differentiation
  3. c Confirm pluripotency gene loss and liver marker induction (Day 16 and Day 21)
  4. d Assess HLO morphology and histology (Day 21)
Phase 2 · HLO isolation and culture system preparation Day 20-21
  1. a Isolate HLOs from Matrigel
  2. b Plate isolated HLOs into different 3D culture systems
Phase 3 · Liver injury induction with TGF-β1 and fatty acids Day 20/21 to Day 25/26
  1. a Prepare injury media solutions
  2. b Change medium to injury media (Day 20-21)
  3. c Culture HLOs under injury conditions
Phase 4 · Morphological and contractility assessment Day 25-26
  1. a Document morphological changes
  2. b Perform Matrigel contraction assay
Phase 5 · Histological analysis and collagen quantification Day 25-26
  1. a Fix HLOs for histology
  2. b Perform H&E staining
  3. c Perform Sirius red staining for collagen quantification
  4. d Quantify Sirius red staining using optimized pipeline
Phase 6 · Gene expression analysis by qPCR Day 25-26
  1. a Harvest and lyse HLOs for RNA extraction
  2. b Extract total RNA via phenol-chloroform extraction
  3. c Synthesize cDNA
  4. d Design and validate qPCR primers
  5. e Perform qPCR reactions
  6. f Analyze qPCR data
Phase 7 · Single-cell RNA sequencing sample preparation Day 21 and Day 25-26
  1. a Culture HLOs on ultra-low attachment plates for scRNA-seq
  2. b Dissociate HLOs to single cells
  3. c Prepare cells for library preparation
  4. d Prepare 10X Chromium libraries
  5. e Sequence libraries
Phase 8 · Single-cell RNA sequencing data processing and analysis Post-sequencing
  1. a Convert BCL files and assess quality
  2. b Align reads and generate count matrices
  3. c Calculate doublet scores
  4. d Apply quality control filters
  5. e Merge replicates and normalize data
  6. f Assign cell cycle scores
  7. g Perform dimensionality reduction and clustering
  8. h Correct batch effects
  9. i Annotate cell clusters using literature markers
  10. j Annotate cell clusters using database markers
  11. k Cross-validate with SingleCellNet classifier
  12. l Calculate inflammation and fibrosis scores
  13. m Perform differential gene expression analysis
  14. n Perform pathway enrichment analysis
  15. o Infer cell-cell interactions using CellPhoneDB
  16. p Perform trajectory inference
  17. q Perform MAGIC imputation and gene trajectory analysis
  18. r Apply NAFLD fibrosis stage signatures
Phase 9 · Statistical analysis Post-data collection
  1. a Perform two-group statistical comparisons
  2. b Perform multi-group statistical comparisons
  3. c Determine significance threshold

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Attribution

This SOP was written by Organthis based on the published method in Hess et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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