Dorothee M. Runge

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

BACKGROUND/AIMS Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. METHODS DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. RESULTS Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. CONCLUSIONS Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

MATRIX INDUCED RE-DIFFERENTIATION OF CULTURED RAT HEPATOCYTES AND CHANGES OF CCAAT/ENHANCER BINDING PROTEINS

Rat hepatocytes de-differentiate and proliferate when cultured on collagen-coated dishes in a chemically defined Hepatocyte Growth Medium in the presence of hepatocyte growth factor and epidermal growth factor. The addition of biomatrix derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma stops this process and leads to re-differentiation of the cells. We monitored DNA binding activity and protein levels of CCAAT/Enhancer Binding Proteins (C/EBPs) during these events by electrophoretic mobility shift assays and western blot analysis. We used plasma protein gene expression as a marker for the proliferation and differentiation phases. During the initial proliferation phase the DNA binding activity of C/EBPs decreased about 5-10 fold, mainly due to reduction of C/EBP alpha protein to nearly undetectable levels. Addition of EHS-gel prevented the further loss of C/EBP alpha protein and established a new steady state level. Since C/EBP beta proteins were affected to a much lesser extent, the C/EBP alpha:C/EBP beta ratio was greater in the presence of EHS-gel. Transferrin, alpha 1-antitrypsin, and albumin mRNA expression increased substantially. Thus stabilized C/EBP alpha expression, an increased C/EBP alpha:C/EBP beta ratio, and increased expression of liver specific mRNAs all correlated with the transition of proliferative to differentiated cells.

Expanding hepatocytes in vitro before cell transplantation: donor age‐dependent proliferative capacity of cultured human hepatocytes

Background: For hepatocyte transplantation as well as experimental purposes, it would be advantageous to be able to expand human hepatocytes in vitro. However, under serum‐free conditions, even with supplements of HGF (hepatic growth factor) and EGF (epidermal growth factor), proliferation of human hepatocytes is hampered. The aim of this study was to identify differences in the proliferative capacity of cultured primary human hepatocytes related to the age of the liver donors. Methods: Proliferation was determined by BrdU‐uptake, ploidy was measured using propidium iodide staining and flow cytometry, and the expression of cell cycle related proteins was determined by Western blotting. Results: During the initial culture, juvenile hepatocytes proliferated better than adult hepatocytes. The proliferation rate declined to barely detectable levels after 8 days in culture in both juvenile and adult hepatocytes. The higher proliferative capacity of juvenile hepatocytes was associated with a larger fraction of diploid cells and a higher viability. The expression of regulatory cell cycle related proteins was higher in juvenile than in adult hepatocytes. Conclusions: The proliferation of human hepatocytes in vitro is critically related to a large fraction of diploid hepatocytes. The expression of regulatory cell cycle proteins reflects the proliferative capacity of cultured human hepatocytes. Juvenile as compared to adult human hepatocytes may be better suited for expansion in culture and could have a stronger repopulation capacity in vivo.