NAFLD TM6SF2 disease modelled hepatocytes

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. There are a number of genetic factors which contribute to the occurrence of NAFLD, one of these is the transmembrane protein 6 superfamily member 2 (TM6SF2), in which loss of function has a significant associated risk for NAFLD. This validated disease model is one of a number of NAFLD genetic variants produced by DefiniGEN to support research and drug development in this important area.

• Disease circuit verified: Def-HEP NAFLD TM6SF2
• Display multiple key hepatocyte markers A1AT, ALB, HNF4a
• Phenotypic analysis verified fatty acid accumulation
• Genetic manipulation does not negatively affect differentiation capacity into hepatocytes

Disease circuit verification

Figure 1. Sanger sequencing of the edited TM6SF2 gene using CRISPR/Cas9 in the reference hiPSC line. (A) TM6SF2 gene in the wild-type line. (B) TM6S2 homozygous knock-in clone with E167K mutation (Glutamic acid to Lysine at position 167, exon 6).

Hepatocyte marker analysis

Figure 2. Functional analysis of Def-HEP TM6SF2 cells. QPCR analysis indicates that the TM6SF2 CRISPR-modified cells can be successfully differentiated into hepatocyte cells that express key hepatocyte markers at similar levels to primary human hepatocytes (PHH).

NAFLD marker analysis

Figure 3. Functional analysis of Def-HEP TM6SF2 cells. QPCR analysis indicates that the TM6SF2 CRISPR-modified hepatocytes express few key NAFLD markers at higher levels to Def-HEP Wild type cells.

Hepatocyte cell morphology

Figure 4. Def-HEP NAFLD TM6SF2 cells exhibit typical hepatocyte cobblestone morphology.

Fatty acid accumulation

Figure 5. Fatty acid accumulation in Def-HEP TM6SF2 cells. Both Oil Red O and BODIPY staining show a higher accumulation of lipids in Def-HEP TM6SF2 E167K when cultured in low-lipid media.