643 Mutations in Cel and Hras1 Are Associated With Obesity-Associated Hepatocellular Carcinoma

Abstract

Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSCs), with their potential capacity to phenotypically model the influence of genetic variation, could be a useful in vitro hepaticmodel to aidmechanistic understanding of drug-induced liver injury in humans. However, the generation of HLCs with a functionally similar phenotype to the gold standard in vitro model of human primary hepatocytes remain elusive. Furthermore there is a significant variation reported for the differentiation propensities of various hiPSC lines towards the hepatic lineage, although the precise reasons are unclear. Recent reports have highlighted the potential importance of the parental cell used for cellular reprogramming, as hiPSCs may retain an epigenetic memory of their original cell type, with a propensity to differentiate back to its lineage of origin. Therefore, we hypothesize that HLCs generated from hepatocyte-derived hiPSCs may exhibit an improved functional phenotype compared to non-hepatocyte-derived hiPSCs. To control for genotypic differences, variations in the reprogramming techniques, and differences in the hepatic differentiation protocols, we derived hiPSC lines from primary hepatocytes and dermal fibroblasts from the same donors using a non-integrative reprogrammingmethod, and generated HLCs using the same differentiation protocol. The hepatocyte-derived hiPSC (H-hiPSC) lines and dermal fibroblast-derived hiPSC (F-hiPSC) lines successfully generated from the same donors were positive for the pluripotency markers OCT4, SOX2, NANOG, TRA-1-60 and SSEA4, and could spontaneously differentiate into cells of all three germ layers. HLCs were differentiated from all the hiPSC lines and were shown to be positive for the endodermal lineage-specific markers of SOX17, HNF4A and ALB. The HLCs were generated using hiPSCs at low passage numbers (<10) to avoid any loss of epigenetic memory which could arise as a result of continued passage in culture. Separate cultures of HLCs generated from the H-hiPSC and F-hiPSC lines were compared against the donors' primary hepatocytes using real-time PCR for expression of liver-related genes, immunoblotting and assays of key hepatic metabolic function. Our resource of having H-hiPSC and F-hiPSC lines to generate HLCs, with the availability of primary hepatocytes from the same donor as the most appropriate comparator, allows for a definitive assessment of the role of epigenetic memory of the cell origin used for cellular reprogramming, in determining the propensity for hepatic differentiation.