Predictive and functional hepatocyte-like cells

iPSC-derived human disease models for a wide range of metabolic and liver diseases

 

Human-like, robust and reproducible

An ideal model for the study of liver disease would combine the expansion capacity and phenotypic stability of hepatoma cell lines with the functionality and normal karyotype of primary hepatocytes. Human induced pluripotent stem cell-derived hepatocyte-like cells (HLCs) have the potential to fulfil this niche.

 

DefiniGEN’s proprietary differentiation protocol permits large-scale generation of HLCs with field leading purity and functionality. Importantly, the HLCs successfully recapitulate key aspects of disease pathophysiology across a wide- range of conditions that affect different aspects of liver function.

 

For rare forms of liver disease, identifying patients with a genomic mutation of interest is challenging. CRISPR/ Cas9 can be used with high editing efficiency in iPSCs when compared to primary cell types. In practice, this means specific mutations associated with liver disease can be precisely introduced to iPSCs with relative ease, before expansion and subsequent differentiation to HLCs. This approach allows for the study of rare monogenic diseases and polymorphisms that increase susceptibility to types of liver disease previously inaccessible to researchers.

 

Hepatocyte cobblestone morphology

Figure 1. DefiniGEN hepatocyte-like cells (HLCs) demonstrate the characteristic cobblestone morphology, and the presence of a uniformed monolayer following >3 weeks of iPSC differentiation.

mRNA expression and maturity markers

Figure 2: A) mRNA expression levels of albumin (ALB) and alpha-1-antitrypsin (A1AT) in DefiniGEN mature hepatocyte-like cells (HLCs) and primary human hepatocytes (PHH). B) Representative pictures of mature HLCs revealing protein expression of the hepatocyte maturity markers albumin (green) and alpha-1-antitrypsin (red). Data are presented as mean±SD of n=3 technical replicates. mRNA expression data were normalised to PPIA.

Gluconeogenesis pathway

Figure 3: A) Simplified schematic on the gluconeogenesis/glycogenolysis pathways within human liver. B) Media glucose levels from mature HLCs treated with glucose-free media for 1h and 6h with or without glucagon, suggesting the de novo synthesis of glucose from non-lipid precursors, accompanied by functional glycogenolysis pathway additionally contributing to total glucose levels. Data are presented as mean±SD of n=2 technical replicates.

Urea cycle pathway

Figure 4: A) mRNA expression levels of key genes involved in urea cycle in HLCs cultured in either hypoxic or normoxic conditions. B) protein expression levels of key enzymes involved in urea cycle in iPSC, hypoxia-cultured HLCs, normoxia-cultured HLCs and HepG2 cells. Data are presented as mean±SD of n=2 biological replicates.

Urea cycle pathway secrete urea

Figure 5: A) Media urea levels in normoxia-cultured HLCs and HepG2 cells for 24h and 48h. B) Media urea levels in normoxic-cultured HLCs cultured in the presence of either vehicle, 1mM NH4Cl, or 1mM NH4Cl +1mM Ornithine for 24h, suggestive of functional OTC activity. Data are presented as mean±SEM of n=3 biological replicates.

Frequently asked questions

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Do the hepatocyte-like-cells express ASGR1?

Yes, ASGR1 and ASGR2 has been detected by qPCR analysis and ICC and has been shown to localize on the cell membrane. 

Do they have a functional urea cycle?

Yes this has been characterized by gene expression profiling, Western blot analysis and Ornithine stimulated functional analysis.

Can you see CYP expression activity?

Expression of CYPs is detectable by qPCR analysis, but they are lower than Primary Human Hepatocytes (PHH).

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