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 alpha (C/EBPalpha) and hepatocyte nuclear factor-4 (HNF-4), cytokine and mitogen activated factors (nuclear factor kappa B) NFkappaB, 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.
Subject(s)
Liver/cytology , Liver/physiology , Transcription Factors/metabolism , Blood Proteins/genetics , Blood Proteins/metabolism , Cell Culture Techniques/methods , Cell Differentiation , Cells, Cultured , Culture Media, Serum-Free , Gene Expression , Humans , Liver/metabolism , Microscopy, Electron , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time Factors , Transcription Factors/geneticsABSTRACT
Recently, we have developed a culture system in which rat hepatocytes dedifferentiate and proliferate and after the addition of EHS-gel redifferentiate. During both developmental stages HNF-4alpha2 mRNA was more abundant than HNF-4alpha1 mRNA. However, Western blot analysis using COS-7 cell-expressed HNF-4alpha1 and HNF-4alpha2 proteins as standards revealed that (i) HNF-4alpha2 protein was not expressed in dedifferentiated hepatocytes and (ii) either HNF-4alpha2 protein or a highly phosphorylated HNF-4alpha1 protein was the dominating isoform in redifferentiated hepatocytes. The changes in HNF4-isoform expression could not be mimicked by DMSO, suggesting them to be matrix specific. Furthermore, DMSO was less efficient than EHS-gel in reinducing liver-specific gene expression. EHS-gel overlay also led to reduction of ARP-1 DNA binding activity, while overall ARP-1 protein levels did not change. These results suggest that EHS-matrix overlay regulates the expression of different HNF-4alpha isoforms on a posttranscriptional level while ARP-1 DNA binding activity is regulated by posttranslational mechanisms.
