Establishment of a human neonatal hepatocyte cell line

Abstract

Hepatocytes are routinely used to generate and identify drug metabolites and hepatic toxicity. Primary cultures of human hepatocytes are the model cell of choice for most of these pharmacological and toxicological studies. However, major problems are encountered with primary liver cell cultures: the dwindling availability of viable livers, hepatocytes having a limited life span, the loss of liver-specific functions in culture, and the donor to donor variability. These limitations have created a significant need for an in vitro hepatocyte system, which has both the potential for use in toxicological and pharmaceutical studies as well as clinical applications. Ectopic expression of human telomerase reverse transcriptase (hTERT) is one of the major strategies used to develop immortalized cells. Immortalization of primary cells using hTERT allows retention of the original cellular characteristics and functions and avoids some of the genetic and phenotypic instabilities associated with using known oncogenes. In the present study, we developed a cell line from human neonatal hepatocytes by transduction with a recombinant retrovirus expressing the hTERT gene. Induction of stable expression of hTERT in the neonatal cells led to immortalization of these cells. The cell line was cultured continuously for more than 25 passages, equivalent to >25 population doublings, whereas the parental cells senesced within five passages. Analysis of telomerase activity as measured by telomeric repeat amplification protocol assay indicated elevated levels of telomerase activity in immortalized cells compared to the parental cells. These immortalized human hepatocytes cells maintained a normal diploid karyotype as well as the gene expression profile similar to that of human normal neonatal hepatocytes. The data suggest that these immortalized cells preserved some of the biological characteristics of hepatic progenitor cells and might be useful as an in vitro model for pharmacological and toxicity studies.

Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability

“…In contrast to the diffuse cytoplasmic distribution of wild type (WT)-LHBS, preS2-LHBS was usually detected at the cell margin of GGHs in the liver (Figure 1A ). Similarly, WT-LHBS and preS2-LHBS displayed diffuse and marginal localization, respectively, in the hepatic progenitor line NeHepLxHT [ 21 ] (Figure 1B ). As preS2-LHBS is an ER-resident protein, we asked whether the cytoplasmic distribution of bulk ER is affected in these cells.…”

Higher-Order Chromatin Regulation and Differential Gene Expression in the Human Tumor Necrosis Factor/Lymphotoxin Locus in Hepatocellular Carcinoma Cells

“…1C and D). We used standard cell lines: Hep3B, which originates from human HCC, and NeHepLxHT, which is a telomerase-immortalized human neonatal hepatocyte line (51). Both CTCF and RAD21 bound to the TC sites but not to the NC site.…”

Sgo1 is a potential therapeutic target for hepatocellular carcinoma

“…It is plausible that cells with extra chromosomes may need more Sgo1 to preserve sister chromatid cohesion. Over 90% of RPE-1 and NeHepLxHT contain a modal chromosome number of 46 [ 30 , 31 ]. In contrast, HeLa cells contain one extra version of most chromosomes, with up to five copies of some, and an average chromosome number of 82 [ 32 ].…”