Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes

Tanja Deurholt,Ruurdtje Hoekstra,Jurgen Seppen,John N Plevris,Igor M Sauer,Ruth Schwartlander,Aniska A Chhatta,Catherine Payne,Niek P Van Til,Lysbeth Ten Bloemendaal,Robert Afm Chamuleau,Ronald Pj Oude Elferink

doi:10.1186/1472-6750-9-89

Abstract

BackgroundA clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications.ResultsCell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion.ConclusionThis novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications.

Highlights

Introduction of hTERT and Green FluorescentProtein genes The cDNA of the human telomerase reverse transcriptase gene, kindly provided by R.L
Expression of hTERT was detected in some of the clonal derivatives analysed, all human fetal liver cells (HFLCs) and clonal derivatives eventually entered a state of terminal growth arrest (Table 2)
After lentiviral introduction of the hTERT gene, the presence of the hTERT cDNA was confirmed in HFLCs and clonal derivatives by PCR on genomic DNA

Summary

Introduction

Introduction of hTERT and Green FluorescentProtein genes The cDNA of the human telomerase reverse transcriptase (hTERT) gene, kindly provided by R.L. The lentiviral vector contained a cytomegalovirus promoter controlling the expression of a reverse tetracycline (Tet) responsive transcriptional activator and a Tet responsive element controlling the expression of the hTERT gene. In this system hTERT transcription can be increased by adding 1 g/mL doxycyclin to the medium. Several cell lines derived from human liver tumours, such as the hepatoma cell line HepG2 [3], as well as in vitro immortalized cell lines, like the NKNT-3 cell line, have been investigated [4,5] These cell lines proliferate adequately, but the levels of hepatocyte-specific functions (e.g. urea production from ammonia and cytochrome p450 detoxification activity) remain disappointedly low

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Discussion

Conclusion