Hepatocyte FH

Genetic Validation and Cell Viability

Def-HEP FH cells have been validated and verified for the E101K genetic mutation in the LDLR gene. Typical viabilities of the thawed hepatocytes are >70% upon receipt.

Disease circuit verification

Figure 2. Sequence confirmation of LDLR E101K mutation in Def-HEP FH cells.

Functional Test

The in vivo functional implications of LDL receptor deficiency are conserved in our model, as shown by immunostaining and FACS analysis demonstrating that Def-HEP FH iPSC hepatocytes have an impaired ability to incorporate LDL receptor-specific binding of Dil-LDL is followed by internalization of the bound complex and lysosomal hydrolysis of the ligand. Increase in the fluorescence intensity per cell is hence used as a measure of Dil-LDL uptake and, implicitly, as an indication of LDL-R presence. These results demonstrate that CRISPR generated disease-specific human iPSC can successfully be used to model FH.

Figure 3. These microscopy images demonstrate that uptake of LDL was impaired in the LDLR mutation line in comparison to WT control.

Figure 4. Quantitative LDL receptor assays based on fluorescence Dil-LDL internalization have demonstrated that Def-HEP FH iPSC-derived hepatocytes have a significantly impaired ability to incorporate LDL relative to the isogenic control Def-HEP WT over a range of LDL substrate ranges.