Serum-Free, Long-Term Cultures of Human Hepatocytes: Maintenance of Cell Morphology, Transcription Factors, and Liver-Specific Functions

doi:10.1006/bbrc.2000.2215

Biochemical and Biophysical Research Communications

Volume 269, Issue 1, 5 March 2000, Pages 46-53

https://doi.org/10.1006/bbrc.2000.2215 Get rights and content

Abstract

Since human hepatocytes are available only in limited number, the development of a serum-free culture system for long-term cultivation of differentiated and functional hepatocytes is of great importance. Here we describe the culture of human hepatocytes in a chemically defined serum-free medium for up to 5 weeks. Cell morphology was assayed by light and electron microscopy and revealed a well-preserved cellular morphology. Marker proteins for epithelial and bile duct cells, cytokeratin (CK) 18 and 19, and liver-specific proteins, like phosphoenolpyruvate carboxykinase-2 (PCK2) and serum proteins, were expressed. Liver-enriched transcription factors CCAAT/enhancer binding protein α (C/EBPα) and hepatocyte nuclear factor-4 (HNF-4), cytokine and mitogen activated factors (nuclear factor kappa B) NFκB, and activator protein-1 (AP-1) were maintained and active for several weeks in our cultures. In summary, our serum-free culture system allows the culture of differentiated human hepatocytes for several weeks. It may serve as a model system for metabolic, pharmacologic–toxicologic studies, and studies on human pathogens under defined chemical conditions.

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The discovery of the wide plasticity of most cell types means that it is now possible to produce virtually any cell type in vitro. This concept, developed because of the possibility of reprogramming somatic cells toward induced pluripotent stem cells, provides the opportunity to produce specialized cells that harbor multiple phenotypical traits, thus integrating genetic interindividual variability. The field of hepatology has exploited this concept, and hepatocyte-like cells can now be differentiated from induced pluripotent stem cells. This review discusses the choice of somatic cells to be reprogrammed by emergent new and nonintegrative strategies, as well as the application of differentiated human induced pluripotent stem cells in hepatology, including liver development, disease modeling, host-pathogen interactions, and drug metabolism and toxicity. The actual consensus is that hepatocyte-like cells generated in vitro present an immature phenotype. Currently, developed strategies used to resolve this problem, such as overexpression of transcription factors, mimicking liver neonatal and postnatal modifications, and re-creating the three-dimensional hepatocyte environment in vitro and in vivo, are also discussed.
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Moreover, the cultured hepatocytes from early isolation experiments dedifferentiated quickly in culture, within a few hours losing hallmark features of in vivo liver function, such as albumin secretion and biotransformation activity. As dedifferentiation occurs, the cuboidal networks of cells often flatten and lose expression of specialized structures such as bile canaliculi, as well as distinct cell–cell contacts [55,56]. Another limiting factor of the technique is the conservation and storage of isolated cells.
There are several issues to be considered to reduce the risk of rejection and minimize side effects associated with liver cell transplantation in chronic liver diseases. The source and the condition of stem cell proliferation and differentiation ex vivo and the transplantation protocols are important safety considerations for cell based therapy. The biochemical and molecular markers are important tools for safety evaluation of different processes of cell expansion and transplantation. Studies show that hepatocytes differentiated from adult and embryonic stem cells exhibit biochemical and metabolic properties resembling mature hepatocytes. Therefore these assays can help to assess the biological and metabolic performance of hepatocytes and progenitor stem cells. The assays also help in testing the contribution of transplanted hepatocytes in improving the repair and function of damaged liver in the recipient.
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The aim of this study was to determine suitable culture conditions for maintaining the activity of cytochrome p450 (CYP) 3A4 and drug transporters in primary cultured human hepatocytes. Human hepatocytes were isolated using the two-step collagenase perfusion technique and were cultured with four different media, serum-free William’s E medium (serum-free WEM), WEM containing fetal calf serum (FCS-WEM), WEM with human serum (HS-WEM), and Lanford’s medium. The albumin levels were maintained for 7 days in hepatocytes. Although CYP3A4 mRNA levels gradually decreased from 3 days, CYP3A4 and hepatocyte nuclear factor-4α alpha protein levels and activities were maintained for 7 days in hepatocytes cultured with serum-free WEM and Lanford’s but not in those with FCS-WEM and HS-WEM. Furthermore, CYP3A4 protein levels were significantly increased by the addition of rifampicin and dexamethasone to the culture media, indicating that the induction potential was maintained. The protein levels of P-glycoprotein, multi-drug-resistance-2, and breast cancer-resistance protein were maintained for 7 days in all media. Serum-free WEM and Lanford’s also maintained protein levels of CYP2C19, CYP2D6, and organic anion transporter polypeptide in the hepatocytes. Serum-free WEM and Lanford’s may be appropriate culture media for maintaining CYP3A4 and drug transporter protein levels in primary cultured hepatocytes.
Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes
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Briefly, RT-PCR analysis was performed on freshly isolated hepatocytes, and hepatocytes cultured for 72 hours, the time frame for our experiments. Albumin (ALB), transferrin (TFN), hepatocyte nuclear factor 4 (HNF4), and connexin 26 (CNX26) were used as accepted markers of differentiated hepatocytes (Block et al., 1996; Gearhart and Bouchard, 2010; Piechocki et al., 1999; Runge et al., 2000). Additionally, we examined connexin 43 (CNX43), which is expressed in dedifferentiated hepatocytes, Kupffer cells, stellate cells, and liver sinusoidal endothelial cells.
Chronic HBV infections cause hepatocellular carcinoma (HCC). Activities of the HBV HBx protein regulate HBV replication and may contribute to the development of HCC. We previously reported that HBx causes primary rat hepatocytes to exit G0 but stall in G1 phase of the cell cycle; entry into G1 stimulated HBV replication. We now report that the activity of the mitochondria permeability transition pore is required for HBx regulation of cell cycle proteins and HBV replication in primary rat hepatocytes, that progression from G0 to G1 stimulates HBV polymerase activity, and that HBV replication is facilitated by the HBx-induced G1 arrest. HBx stimulation of HBV replication was linked to elevation of the R2 subunit of ribonucleotide reductase. Our studies suggest that HBx uses mitochondrial-dependent calcium signaling to cause hepatocytes to exit G0 but stall in G1 and that this HBx activity alters the cellular environment and stimulates HBV replication.
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¹: To whom correspondence should be addressed at Department of Pathology, University of Pittsburgh, South 410 BST, Pittsburgh, PA 15261. Fax: 412-648-9846. E-mail: [email protected].

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Regular ArticleSerum-Free, Long-Term Cultures of Human Hepatocytes: Maintenance of Cell Morphology, Transcription Factors, and Liver-Specific Functions

Abstract

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Hepatol.

Chem. Biol. Interact.

Chem. Biol. Interact.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Hepatology

J. Biol. Chem.

Arch. Biochem. Biophys.

Cell

Biochem. Biophys. Res. Commun.

Exp. Cell Res.

J. Lipid Res.

J. Biol. Chem.

Drug. Metab. Dispos.

Brit. J. Nutr.

Biochem. J.

Regular Article
Serum-Free, Long-Term Cultures of Human Hepatocytes: Maintenance of Cell Morphology, Transcription Factors, and Liver-Specific Functions