title: Autocrine stimulation of human hepatocytes triggers late DNA synthesis and stabilizes long-term differentiation in vitro.
abstract: Isolated human hepatocytes are of great value in investigating cell transplantation, liver physiology, pathology, and drug metabolism. Though hepatocytes possess a tremendous proliferative capacity in vivo, their ability to grow in culture is severely limited. We postulated that repeated medium change, common to most in vitro systems, may prevent long-term maintenance of hepato-specific functions and growth capacity. To verify our hypotheses we compared the DNA synthesis and differentiation status of isolated human hepatocytes, cultured in medium which was renewed every day or was not changed for 3 weeks ('autocrine' setting). Daily medium change led to rapid hepatocellular de-differentiation without any signs of DNA replication. In contrast, the autocrine setting allowed hepatocytes to become highly differentiated, demonstrated by an elevated ASGPr expression level, and increased albumin and fibrinogen synthesis and release. Cytokeratin 18 filaments were stably expressed, whereas cytokeratin 19 remained undetectable. Hepatocytes growing in an autocrine fashion were activated in the presence of hepatocyte growth factor (HGF), evidenced by c-Met phosphorylation. However, HGF response was not achieved when the culture medium was renewed daily. Furthermore, the autocrine setting evoked a late but strong interleukin 6 release into the culture supernatant, reaching maximum values after a 10-day cultivation period, and intense BrdU incorporation after a further 5-day period. Our data suggest that preservation of the same medium creates environmental conditions which allow hepatocytes to control their differentiation status and DNA synthesis in an autocrine fashion. Further studies are necessary to identify the key mediators involved in autocrine communication and to design the optimal culture configuration for clinical application.
authors.affiliation: Charité Universitätsklinikum, Klinik für Allgemein-, Visceral- und Transplantationschirurgie, Berlin, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Leckel
authors.forename: Christoph
authors.initials: C
authors.lastname: Strey
authors.forename: Wolf O
authors.initials: WO
authors.lastname: Bechstein
authors.forename: Kim A
authors.initials: KA
authors.lastname: Boost
authors.forename: Marcus K H
authors.initials: MK
authors.lastname: Auth
authors.forename: Amal
authors.initials: A
authors.lastname: El Makhfi
authors.forename: Eva
authors.initials: E
authors.lastname: Juengel
authors.forename: Steffen
authors.initials: S
authors.lastname: Wedel
authors.forename: Jon
authors.initials: J
authors.lastname: Jones
authors.forename: Dietger
authors.initials: D
authors.lastname: Jonas
authors.forename: Roman A
authors.initials: RA
authors.lastname: Blaheta
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D019898:Autocrine Communication; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D004261:DNA Replication; D066246:ErbB Receptors / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D007633:Keratins / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism
chemical_list: D000418:Albumins; D037263:Asialoglycoprotein Receptor; D015850:Interleukin-6; D007633:Keratins; D005340:Fibrinogen; D066246:ErbB Receptors; D019859:Proto-Oncogene Proteins c-met