Opti-HEP Functionality
DefiniGEN’s proprietary differentiation protocols permit the largescale generation of iPSC-derived hepatocytes (Opti-HEP) with fieldleading purity and functionality. Importantly, Opti-HEP successfully recapitulate key aspects of disease pathophysiology across a wide range of conditions that affect different aspects of liver function.
Advantages
- Demonstrate characteristic hepatocyte cobblestone morphology
- Express comparable levels of liver maturity markers to primary human hepatocytes
- Express higher levels of urea cycle markers and secrete higher levels of urea compared to liver carcinoma cell lines
- Demonstrate a functional gluconeogenesis pathway
- Demonstrate comparable levels of CYP450 markers and CYP3A4 activity to primary human hepatocytes
- Demonstrate functional localization and function of ASGR1 for GalNAc-dependent drug deliveries
- Standardized cell product containing iPSC-derived human hepatocytes producing reproducible and biologically relevant data
Morphology
DefiniGEN Opti-HEP demonstrate the characteristic hepatocyte cobblestone morphology.
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Figure 1: Representative cell morphology pictures of induced pluripotent stem cells (iPSCs), hepatocellular carcinoma HepG2 cells, Defi niGEN Opti-HEP, and primary human hepatocytes (PHH). The pictures reveal the characteristic cobblestone morphology of Opti-HEP, and the presence of a uniform monolayer following >3 weeks of iPSC differentiation. Objective: 20x.
Maturity marker analysis
DefiniGEN Opti-HEP express similar levels of liver maturity markers compared to primary human hepatocytes (PHH).
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Figure 2: Representative immunocytochemistry pictures and protein quantification showing expression levels of the hepatocyte maturity markers albumin (red), alpha-1-antitrypsin (A1AT; green), HNF4A (green), and AFP (red) in liver carcinoma HepG2 cells, DefiniGEN Opti-HEP, and primary human hepatocytes (PHH; 3 donors). Cells were counterstained with DAPI, and data are presented as mean±SEM of n=3-4 independent experiments.
Urea cycle marker analysis
DefiniGEN Opti-HEP express higher levels of urea cycle markers and secrete higher levels of urea compared to liver carcinoma cell lines.
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Figure 3: A) Protein expression levels of the urea cycle enzymes OTC, ASS1, ASL, CPS1, and ARG1 in liver carcinoma HepG2 cells and DefiniGEN Opti-HEP. B) Urea secretion in liver carcinoma HepG2 cells and DefiniGEN Opti-HEP. Data are presented as mean±SEM of n=3-4 independent experiments.
Functional gluconeogenesis
DefiniGEN Opti-HEP demonstrate a functional gluconeogenesis pathway and respond to gluconeogenesis inducers.
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Figure 4: A) Simplified schematic of the gluconeogenesis pathway within human liver. B) G6PC mRNA levels in liver carcinoma HepG2 cells, DefiniGEN Opti-HEP, and primary human hepatocytes (PHH). C) G6PC mRNA levels in liver carcinoma HepG2 cells and DefiniGEN Opti-HEP treated with 0.1mM dbcAMP (gluconeogenesis inducer). D) Glucose secretion in dbcAMP-treated liver carcinoma HepG2 cells and DefiniGEN Opti-HEP upon pyruvate challenge. Data are presented as mean±SEM of n=3-4 independent experiments. mRNA expression data were normalized to 18S rRNA.
CYP450 expression and activity
DefiniGEN Opti-HEP demonstrate comparable levels of CYP450 markers and CYP3A4 activity to primary human hepatocytes.
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Figure 5: A) mRNA expression levels of Phase I CYP450 genes in liver carcinoma HepG2 cells, DefiniGEN Opti-HEP, and primary human hepatocytes (PHH). B) Basal CYP3A4 activity in liver carcinoma HepG2 cells, DefiniGEN Opti-HEP, and PHH. mRNA data were normalized to the housekeeping gene 18S rRNA and are presented as mean±SEM of n=3-4 independent experiments. CYP3A4 activity data were normalized 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.
ASGR1 expression and function
DefiniGEN Opti-HEP demonstrate functional membrane localization and activity of the Asialoglycoprotein receptor 1 (ASGR1).
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Figure 6: A) Representative immunocytochemistry pictures showing the localization of ASGR1 in the Opti-HEP membrane. Cells were counterstained with the membrane marker E-cadherin and DAPI. B) The effect of ASGR1 in the transport of GalNAc-siRNA conjugate targeting GAPDH in Opti-HEP using GalNAc-Cy3 staining and qPCR. Data are presented as mean±SEM of n=3-4 independent experiments. mRNA expression data were normalized to 18S rRNA. NTC: non-template control.