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Hepatocyte NAFLD

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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.

Disease circuit verification

A)                                                              B)                                                              C)

Sanger sequencing of CRISPR-modified, iPSC-derived hepatocytes containing the I148M mutation in the PNPLA3 gene

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

CRISPR-modified NAFLD hepatocytes exhibit comparable levels of ALB, A1AT and HNF4a gene expression when compared with wild-type primary human 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

BODIPY staining of fatty acid accumulation in wild-type, PNPLA3 knockout and PNPLA3 I148M hepatocytes

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

Principal component analysis of lipid metabolism-related genes

 

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 treatment.

Gene ontology

Gene ontology of differentially expressed genes in PNPLA3 I148M relative to Def-HEP WT

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 analysis showing the differential expression of relevant lipid metabolism and accumulation genes

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.

 

A subset of potential causal gene variants have been determined which are currently a significant focus on NAFLD studies

 

Generation of custom NAFLD disease modelled hepatocytes

 

NAFLD gene variants

PNPLA3 rs738409 impaired hepatocellular triglycerides hydrolysis and increased lipogenesis associated to the 148 M allele

GCKR rs1260326 increased glycolysis favours an increase in triglyceride levels

TM6SF2 rs58542926 impaired mobilization of neutral lipids for very low-density lipoprotein (VLDL) assembly and secretion by the liver in E167K carriers

MBOAT7 rs641738 variant causing decreased MBOAT7 expression, predisposes to NAFLD/NASH by affecting the acyl remodelling of phosphatidylinositol in the liver

 

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