Comparable to primary human hepatocytes while being more mechanistically relevant than HepG2.
Cytochrome P450 expression and activity
The cytochrome P450 (CYP) enzymes are the major players in drug metabolism, being responsible for the metabolism of >90% of the drugs that are currently used in clinic. CYPs are mainly expressed in the liver and catalyse the Phase I reactions, whereupon drugs can be oxidised, reduced, or hydrolysed, downstream leading to the generation of products with either decreased or increased toxicity. To date, 57 enzymes have been discovered, from which the isoforms belonging to the CYP1, CYP2, and CYP3 families contribute to the metabolism of approx. 80% of clinical drugs.
Primary human hepatocytes are considered the gold standard in vitro model in ADME and toxicology screening, however, they come with limitations, including short-life span, rapid loss of function, and limited supply. Due to these limitations, hepatocellular carcinoma cell lines, including HepG2 and HepaRG, are instead used, however, the malignant origin of these models, in addition to the lack of drug metabolising enzyme expression they demonstrate hinders data interpretation.
DefiniGEN’s improved differentiation iPSC protocols now lead to the generation of Opti-HEP with functional CYP450 enzyme expression and activity. In Figure 1, we demonstrate that Opti-HEP express significantly higher mRNA levels of various CYP enzymes compared to HepG2 carcinoma cells, including CYP3A4, CYP2B6, CYP2C9, CYP2C19, and CYP2A6. Importantly, we show the activity levels of CYP3A4 in DefiniGEN Opti-HEP, revealing comparable functionality to that seen in primary human hepatocytes and significantly higher to that of HepG2 cells.
Figure 1: A) mRNA expression levels of Phase I CYP450 genes in liver carcinoma HepG2 cells and DefiniGEN Opti-HEP. B) Basal CYP3A4 activity in liver carcinoma HepG2 cells, DefiniGEN Opti-HEP, and primary human hepatocytes (PHH). mRNA data were normalised to the housekeeping gene 18SrRNA and are presented as mean±SEM of n=3-4 independent experiments. CYP3A4 activity data were normalised to ATP levels and are presented as mean±SEM of n=3-5 independent experiments. For PHH data, cells from 3 independent donors were used.
Cytochrome P450 induction and inhibition
Induction and inhibition of CYP450 enzymes are central mechanisms in xenobiotic metabolism, resulting in pharmacokinetic drug-drug interactions (DDI). Both mechanisms are of particular clinical importance for therapeutic and toxicological reasons, as inhibition of drug metabolism can lead to undesirable elevations in plasma drug concentrations, whilst CYP induction following prolonged drug treatment has been associated with adverse DDI. Thus, access to in vitro models that can accurately predict both mechanisms is crucial in drug therapy.
Here we show that, unlike HepG2 cells, CYP450 activity can be both induced and inhibited in DefiniGEN Opti-HEP at comparable levels to those seen in primary human hepatocytes following treatment with known inducers and inhibitors. As proof-of-concept, we demonstrate CYP3A4 activity in HepG2, Opti-HEP, and primary human hepatocytes following inhibition with the CYP3A4 inhibitor ketoconazole and induction with increasing concentrations of the CYP3A4 inducer 1a,25-hydroxy-vitamin D3 (Figure 2).
Figure 2: CYP3A4 induction and inhibition in liver carcinoma HepG2 cells, DefiniGEN Opti-HEP and primary human hepatocytes (PHH), following 72h of treatment with vehicle, 1μM ketoconazole (CYP3A4 inhibitor), 10-100nM vitamin D3 (CYP3A4 inducer), or a combination of vitamin D3 and ketoconazole. CYP3A4 activity data were normalised to ATP levels and are presented as mean±SEM fold change of n=3-5 independent experiments. For PHH data, cells from 3 independent donors were used.
Efflux transporter expression and cellular localisation
In addition to the central role of CYP450 enzymes in drug metabolism, inhibition of liver-specific efflux transporters, including the bile acid export pump (BSEP/ABCB11) and the multidrug resistance (MDR) proteins (e.g., ABCG2, ABCC2/MRP2) has emerged as a DILI risk factor. Supporting this, in vitro screening for BSEP inhibition in drug discovery is now recommended by the European Medicines Agency on the Investigation of Drug Interactions (2012). Despite the need of physiological hepatic cell systems for the accurate in vitro screening of efflux transporter inhibition, the predominant in vitro models that are currently used include non-hepatic cell lines (e.g., HEK293) that artificially overexpress a single transporter, only, since transporter expression in hepatocellular carcinoma cells is either absent or lack proper cellular localisation.
Here, we show that DefiniGEN’s improved Opti-HEP express functional levels of the efflux transporters required (e.g., ABCB11, ABCG2, MRP2) for large-scale toxicity screening. Crucially, we demonstrate the cellular localisation of these transporters in the hepatocyte membrane, confirming their functional role in bile acid and drug transport (Figure 3).
Figure 3: A) mRNA expression levels of efflux transporter genes ABCB11, ABCG2, and MRP2 in liver carcinoma HepG2 cells and DefiniGEN Opti-HEP. mRNA data were normalised to the housekeeping gene 18SrRNA and are presented as mean±SEM of n=3-4 independent experiments. B) Protein expression of efflux transporter proteins ABCB11 and MRP2 (green), co-stained with the apical marker ZO1 (red) in sandwich-cultured Opti-HEP by immunocytochemistry (ICC). Nuclei counterstaining with DAPI (blue).