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LIVER Publication-derived

En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH

Source Kimura et al., 2022 · Cincinnati Children's Hospital Medical Center · 10.1016/j.cell.2022.09.031

👤 Masaki Kimura, Takuma Iguchi, Kentaro Iwasawa, Andrew Dunn, Wendy L. Thompson, Yosuke Yoneyama, Praneet Chaturvedi, Aaron M. Zorn, Michelle Wintzinger, Mattia Quattrocelli, Miki Watanabe-Chailland, Gaohui Zhu, Masanobu Fujimoto, Meenasri Kumbaji, Asuka Kodaka, Yevgeniy Gindin, Chuhan Chung, Robert P. Myers, G. Mani Subramanian, Vivian Hwa, Takanori Takebe ⏱ 30 days 📋 6 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human liver organoids (HLOs) from iPSCs of multiple donors with varying NAFLD-associated genetic variants, and their pooled phenotyping under insulin-insensitive conditions to identify genetic drivers of steatohepatitis. The system models non-alcoholic steatohepatitis (NASH) pathology and enables en masse screening for genotype-phenotype associations, with specific focus on the GCKR-rs1260326 variant and its metabolic consequences.

Cell source
Human iPSC
Application
Disease modeling (NASH/NAFLD genotype-phenotype association and mechanistic study)

Protocol overview

42 steps across 6 phases

iPSC Maintenance and Foregut Progenitor Generation Day -20 to Day 0
  1. 1 iPSC culture maintenance
  2. 2 Detach iPSCs for foregut differentiation
  3. 3 Foregut progenitor induction — Day 1 medium
  4. 4 Foregut progenitor induction — Day 2 medium
  5. 5 Foregut progenitor induction — Day 3 medium
  6. 6 Foregut progenitor specification — Days 4–6
  7. 7 Harvest foregut progenitor cells for cryopreservation
Pooled Organoid Panel (PoP) Generation Day 0 to Day 18
  1. 1 Thaw and prepare foregut progenitor mixture
  2. 2 Embed pooled FG cells in Matrigel (Days 0–4)
  3. 3 Medium switch to hepatic specification (Days 4–8)
  4. 4 Final maturation in hepatocyte culture medium (Days 8–18)
Induction of Steatohepatitis Phenotype (sHLO) and Genotypic Analysis Day 18 to Day 30
  1. 1 Isolate HLOs from Matrigel and prepare for steatosis induction
  2. 2 Culture HLOs in steatosis-inducing conditions
  3. 3 Harvest organoids for lipid imaging (Day 21)
  4. 4 BODIPY lipid staining for fluorescent quantification
  5. 5 Live imaging of stained organoids
  6. 6 Quantify lipid accumulation and donor identification
  7. 7 SNP genotyping of organoid-derived DNA
Characterization of GCKR Variant Effects and Gene Editing (Isogenic Comparisons) Day 0 to Day 30 (parallel to Phases 2–3, with gene-edited lines)
  1. 1 Select donor iPSC lines for GCKR variant comparison
  2. 2 Generate GCKR[CC>TT] gene-edited iPSC lines
  3. 3 Validate edited clones and expand
  4. 4 Differentiate GCKR[CC], GCKR[CC>TT], and GCKR[TT] lines into HLOs
  5. 5 Measure glucokinase (GCK) activity in HLOs
  6. 6 Induce steatohepatitis in GCKR variant HLOs and quantify lipid accumulation
Pharmacological Studies and Mitochondrial Characterization Day 18 to Day 30 (for sHLO treatments)
  1. 1 Culture HLOs with GCK activity inhibitor (PFK15)
  2. 2 Culture HLOs with GCK-GCKR binding disruptor (AMG3969)
  3. 3 Measure oxygen consumption rate (OCR) in HLOs and sHLOs
  4. 4 Quantify intracellular ROS production
  5. 5 Measure ATP/AMP ratios by NMR spectroscopy
  6. 6 Treat sHLOs with nicotinamide riboside (NR) and nitazoxanide (NTZ)
  7. 7 Assess NAD+/NADH ratio after NR/NTZ treatment
  8. 8 Re-measure OCR, ROS, and inflammatory markers after NR/NTZ treatment
Transcriptomic and Metabolomic Profiling Day 27–30
  1. 1 Prepare organoid samples for RNA-seq
  2. 2 Single-cell RNA-seq library preparation (10X Genomics)
  3. 3 Sequence 10X libraries (Illumina NovaSeq or similar)
  4. 4 Process RNA-seq data (Cell Ranger and alignment)
  5. 5 Perform bulk RNA-seq on GCKR variant HLO lines
  6. 6 Align and quantify bulk RNA-seq reads
  7. 7 Differential expression analysis (edgeR)
  8. 8 Gene Set Enrichment Analysis (GSEA)
  9. 9 Mass spectrometry-based metabolomics
  10. 10 Compare transcriptomic and metabolomic data across GCKR variants

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kimura et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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