Epidermal growth factor- and hepatocyte growth factor-receptor activity inserum-free cultures of human hepatocytes

Background/Aims: Serum-free primary cultures of hepatocytes are a useful to ol to study factors triggering hepatocyte proliferation and regeneration. W e have developed a chemically defined serum-free system that allows human h epatocyte proliferation in the presence of epidermal growth factor and hepa tocyte growth factor. Methods: DNA synthesis and accumulation were determined by [H-3]thymidine i ncorporation and fluorometry, respectively. Western blot analyses and co-im munoprecipitations were used to investigate the association of proteins inv olved in epidermal growth factor and hepatocyte growth factor activation an d signaling: epidermal growth factor receptor, hepatocyte growth factor rec eptor (MET), urokinase-type plasminogen activator and its receptor, and a m ember of the signal transducer and activator of transcription family, STAT- 3. Results: Primary human hepatocytes proliferated under serum-free conditi ons in a chemically defined medium for up to 12 days. Epidermal growth fact or-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activa tor receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to var ying 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 facto r and epidermal growth factor can stimulate growth and differentiation by i nteracting with their receptors and initiating downstream signaling. This i nvolves complex formation of the receptors with other plasma membrane compo nents for MET (urokinase-type plasminogen activator in context of its recep tor) and activation of STAT-3 for both receptors.