Publication
Disease circuit verification
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 the I148M mutation (Isoleucine to methionine at position 148, exon 3). (C) PNPLA3 homozygous knock-out with a 6bp deletion and no frameshift mutation.
Key hepatocyte marker analysis
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
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
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
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