Hepatocyte (NAFLD) Liver Disease Model - Definigen

NAFLD disease modelled hepatocytes

DefiniGEN disease modelled NAFLD hepatocytes represent an optimized model for drug discovery applications and a principal tool for elucidating the underlying mechanisms of the disease.
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Product Overview

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of fat within the liver that can lead to inflammation, fibrosis, and hepatocellular carcinoma. The most common disease implicated genetic variant is I148M in the gene coding for Patatin-like phospholipase domain-containing protein 3 (PNPLA3). DefiniGEN disease modelled NAFLD hepatocytes represent an optimized model for drug discovery applications and a principal tool for elucidating the underlying mechanisms of the disease.

Benefits
  • Disease circuit verified three product variants available: Def-HEP PNPLA3-I148M, Def-HEP PNPLA3 KO1, Def-HEP PNPLA3 KO2 plus WT isogenic control
  • Display multiple key hepatocyte markers A1AT, ALB, HNF4a, and TTR
  • Phenotypic analysis verified fatty acid accumulation
  • Genetic manipulation does not negatively affect differentiation capacity into hepatocytes
  • Mutations in PNPLA3 have been demonstrated to interfere with lipid metabolism and induce a steatotic-like phenotype
Technical Data
Disease circuit verification
NAFLD Sequencing
Figure 1. Sanger sequencing of the edited PNPLA3 gene using CRISPR/Cas9 in the reference hiPSC line. (A) PNPLA3 gene in the wild-type line. (B) PNPLA3 homozygous knock-in clone with I148M mutation (Isoleucine to methionine at position 148, exon3). (C) PNPLA3 homozygous knock-out with a 6bp deletion and no frameshift mutation.
Key hepatocyte marker analysis

Key hepatocyte marker analysis DefiniGEN NAFLD disease modelled hepatocytes
Figure 2. Functional analysis of Def-HEP PNPLA3 cells. QPCR analysis indicates that the PNPLA3 CRISPR-modified cells can be successfully differentiated into hepatocyte cells that express key hepatocyte markers at similar levels to primary human hepatocytes (PHH).
Fatty acid accumulation
Fatty acid accumulation in DefiniGEN NAFLD cells
Figure 3. Fatty acid accumulation in Def-HEP PNPLA3 cells. BODIPY staining shows a higher accumulation of lipids in Def-HEP I148M when cultured in low-lipid media. When cells are treated with 0.25mM of Oleic acid for 7 days all cell variants demonstrate an increase in lipid accumulation. A moderate increase in lipid accumulation can be observed with treatment of 0.25mM of Palmitic acid for 7 days.

Principal component analysis

PCA Analysis DefiniGEN NAFLD disease modelled hepatocytes
Figure 4. Principal component analysis of lipid metabolism-related genes. PCA analysis of Def-HEP cells using a subset of 129 lipid metabolism associated genes demonstrates that the wild-type control and CRISPR modified PNPLA3 disease model hepatocyte cells cluster differently independently of fatty acid
Gene Ontology
Gene Ontology DefiniGEN NAFLD disease modelled hepatocytes
Figure 5. Gene ontology of differentially expressed genes in PNPLA3 I148M relative to Def-HEP WT. Gene ontology analysis was performed using large-scale genome and gene function analysis using the PANTHER classification system. Pathways related to lipid, cholesterol and triglyceride metabolism are observed to be affected.
Heatmap
Heatmap DefiniGEN NAFLD disease modelled hepatocytes
Figure 6. Heatmap analysis showing the differential expression of relevant lipid metabolism and accumulation genes. PNPLA3 I148M variant shows a significantly altered gene expression pattern when compared to Def-HEP WT.