Ornithine transcarbamylase deficiency

Figure 1. mRNA expression levels of key genes involved in urea cycle in iPSC, low oxygen-cultured hepatocyte-like cells (HLCs), high oxygen-cultured HLCs, and HepG2 cells. mRNA data were normalised to 18SrRNA and are presented as mean±SEM of n=3 biological replicates. *p<0.05, **p<0.01, ***p<0.001 

Figure 2. Protein expression levels of key enzymes involved in urea cycle in iPSC, low oxygen-cultured HLCs, high oxygen-cultured HLCs, and HepG2 cells. Data were normalised to beta actin and are presented as mean±SEM of n=3 biological replicates. *p<0.05, **p<0.01, ***p<0.001 

DefiniGEN iPSC-derived wild-type hepatocyte-like cells secrete urea in a time-dependent manner

Figure 3. Media urea levels in high oxygen-cultured HLCs and HepG2 cells for 24h and 48h. Data were normalised to total protein levels and are presented as mean±SEM fold change of n=3 biological replicates. **p<0.01

Figure 4. Media urea levels in high oxygen-cultured HLCs cultured in the presence of either vehicle, 1mM NH4Cl, or 1mM NH4Cl +1mM Ornithine for 24h, suggestive of functional OTC activity. Data were normalised to total cell number and are presented as mean±SEM fold change of n=3 biological replicates.  **p<0.01

DefiniGEN CRISPR-derived OTC deficiency (OTCD) HLCs carry the D175V mutation

Figure 5. Sanger sequencing showing healthy wild-type (top sequence) as well as mutated iPSCs (bottom sequence) carrying the D175V mutation (GAT>GTT) on the OTC gene. The codon change is highlighted with yellow.

Figure  6. A) Representative images demonstrating the characteristic hepatocyte cobblestone morphology in wild-type (HEP-003) and OTCD (HEP-003-OTC-a) hepatocyte-like cells (HLCs). B) mRNA expression levels of the hepatocyte maturity markers albumin (ALB) and alpha-
1-antitrypsin (A1AT) in wild-type HLCs (HEP-003), OTCD HLCs (HEP-003-OTC-a), and primary human hepatocytes (PHH). mRNA data were normalised to PPIA and are presented as mean±SEM of n=3 independent experiments. Objective: 10x.

DefiniGEN CRISPR-derived OTCD HLCs demonstrate decreased OTC protein expression and urea secretion compared to 
wild-type HLCs

Figure 7. Protein expression levels of OTC in wild-type HLCs (HEP-003) and CRISPR-derived OTCD HLCs (HEP-003-OTC-a). Data were normalised to beta actin and are presented as mean±SEM of n=3 biological replicates.

DefiniGEN CRISPR-derived OTCD HLCs demonstrate decreased OTC protein expression and urea secretion compared to wild-type HLCs

Figure 8. Media urea levels in wild-type HLCs (HEP-003) and CRISPR-derived OTCD HLCs (HEP-003-OTC-a). Data were normalised to total cell number and are presented as mean±SEM fold change of n=2 biological replicates. 

Conclusion

This case study demonstrates, for the first time, a fully functional urea cycle pathway in wild-type iPSC-derived  hepatocyte-like cells (HLCs) and reveals the superiority of DefiniGEN iPSC-derived HLCs compared to liver carcinoma HepG2 cells. Supporting these findings, we demonstrate functional OTC activity, with a >30% increase in urea secretion when cells are stimulated with 1 mM NH4Cl and 1 mM ornithine (OTC substrate). Informed by these data, and by applying CRISPR gene editing on DefiniGEN wild-type iPSCs, we have successfully generated an OTC deficiency (OTCD) iPSC line carrying the pathogenic, missense mutation D175V. This CRISPR-engineering OTCD iPSC line can differentiate equally well towards HLCs, without any compromise in the expression of hepatocyte maturity markers (ALB, A1AT). Crucially, and upon differentiation, the OTCD HLCs reveal decreased protein expression of OTC and decreased urea formation compared to their isogenic controls, demonstrating their ability to serve as a platform for primary screening activities.