Hepatocyte WT

With the ability to reliably generate functionally mature human hepatocytes from iPSCs, DefiniGEN brings all the advantages of iPSC technology to hepatocyte studies - cell models that possess human-relevant physiology and that are available in virtually unlimited amounts with the same genetic background. 

DefiniGEN's human hepatocytes remain functionally stable over a prolonged period of time in culture, making them ideal for drug discovery, drug metabolism, and toxicology-related studies. They also express key hepatitis markers such as CD81, SR-B1, Claudin-1 and Occludin at similar levels to primary human hepatocytes, making them an effective model for hepatitis lifecycle studies. 

• Highly standardized with > 98% functionally mature human hepatocytes
• Reliable, consistent peformance delivers biologically relevant data
• Verified wild-type donor genetics and karyotype
• Stable function in culture over a +20 day window

Normal physiology includes:

• CYP450 induced activities
• Expression of hepatocyte proteins, including A1AT, ALB, HNF4a
• Secretion of physiologically relevant levels of albumin and urea
• Expression of a range of key hepatitis B & C markers
• Uptake of LDL

Hepatocyte cell morphology

When thawed and plated as a monolayer, Def-HEP cells form hepatocytes with characteristic cobblestone morphology and tight cell junctions

Figure 1. Overview of Def-HEP cell morphology. Def-HEP WT cells exhibit typical hepatocyte cobblestone morphology and bi-nucleation.

Hepatocyte maturation markers

QPCR analysis shows Def-HEP cells show key hepatocyte markers at similar levels to PHH. Functional characteristics including albumin secretion, A1AT production, glycogen storage and LDL uptake are also present.

Figure 2. Functional analysis of Def-HEP WT hepatocytes (A) Albumin secretion, 10x magnification (B) Glycogen storage disease shown by PAS staining (C) LDL cholesterol uptake shown by fluoresceinated LDL incorporation.

Functionality

Figure 3. Gene expression analysis demonstrates that Def-HEP express key hepatocyte markers at similar levels to PHH. AFP levels are extremely low in Def-HEP indicating that the cells have attained a functional mature status.

Extended culture time

Figure 4. Extended functional window of use with DefiniGEN hepatocytes. Cryopreserved Def-HEP cells are thawed, plated, and recovered over 7 days to ensure a functional hepatocyte monolayer forms. Subsequently the cells have a +20 day functional window to enable hepatitis disease modelling, and toxicology studies to be undertaken over a longer timeframe than is possible with PHH.

Multiple Inducible CYP450 Activities

Def-HEP cells display CYP450 induced activity profiles that are similar to PHH (CYP1A2 EROD assay, inducer - omeprazole), (CYP3A4 PGlo assay, inducer - rifampicin).

Figure 5. Multiple CYP activities of cryopreserved Def-HEP hepatocyte cells. The results show Def-HEP cells have comparable CYP activity to PHH and induced activity profiles that are highly similar to PHH (CYP1a2 EROD assay, inducer – omeprazole), (CYP3A4 PGlo assay, inducer – rifampicin).

Hepatitis marker analysis

Figure 6. Gene expression analysis of key Hepatitis markers. The analysis indicates the presence of key hepatitis markers in Def-HEP cells including NTCP, Occludin, SR-B1, CD81 and CLDN7. (HepG2 cells do not express all key hepatitis markers).

Mitochondrial Toxicity

As assessed by dosing Def-HEP WT with valinomycin (0.3-600nM) and papverine (0.03 – 60µM) for 48 hrs both compounds caused significant mitochondrial toxicity with concentration ranges recognized in literature. Cell viability was assessed via the CellTiter 96® Aqueous Non-Radioactive Cell. Both valinomycin and papaverine caused significant mitochondrial toxicity

Figure 7. Typical Def-HEP WT mitochondrial toxicology drug response to valinomycin and papverine.