Hepatic cell lines are widely used by pharmaceutical companies and have a key role in drug research and development. However, their relevance as hepatic models is mostly dependent on the banking of the initial cells which is also the first source for distribution of cells with highly stable properties.
Our aim was to set up a new strategy based on reprogramming towards stem cells of the parental HepaRG cell line, allowing establishment of a new indefinite source for differentiated HepaRG cells. We then used the “stem cell like” “Hepa-SC” to reconvert them into hepatic and other differentiation lineages.
As the first step, stem-like Hepa-SC cells were obtained by submitting parental progenitors to mechanical forces induced by shape constraint which favoured occurrence of stemness properties, including self-renewal, plasticity and pluripotentiality. These cells were subsequently stabilized by epigenetic factors able to modulate the methylation/acetylation status, and they were expanded to produce the master and working banks.
In a second step, we successfully demonstrated that Hepa-SC cells from the banks were able to lose their stem-like status and retrieve the hepatic lineage. This strategy has led us to obtain new cell lines called “Hepa-RP” which rapidly recover the unique characteristics of the parental cells, including a bipotent progenitor stage corresponding to a proliferative stage, and a differentiation stage leading to the organization of mature hepatocyte colonies with a polarized accumulation of F-actin at the biliary poles. Further analyses have revealed that Hepa-RP cells: i)- grow faster than HepaRG cells; ii)- express high levels of drug metabolism enzymes such as CYP3A4, as well as the glycolytic enzyme, aldolase B, and hepatic lipid metabolism enzymes such as APOA1, APOB, and APOC; iii)- exhibit functional transporter expression e.g. the canalicular MRP2 s efflux of the fluorescent MRP2-substrate, CDFA. Finally, incubation with specific substrates of CYP2B6, CYP3A4 and CYP1A2 has confirmed that the levels of these enzymes are the same in both Hepa-RP and HepaRG cells. By contrast, as expected, an analysis of Hepa-SC cells showed that these stem-like cells do not express any liver markers but expressed stem markers including OCT3/4 and Sox17.
In conclusion, we succeeded in producing stem-like cells, Hepa-SC, which constitutes an indefinite source for establishing new Hepa-RP cell lines that preserve all main hepatic characteristics of the parental HepaRG cell line. This technology ensures the sustainability unlimited supply of this robust and valuable cell line for use in ADME and toxicology assays.
