title: Elevated expression of hormone-regulated rat hepatocyte functions in a new serum-free hepatocyte-stromal cell coculture model.
abstract: The specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal (stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the 2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto stromal cells precultured for 4-14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells. Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could be observed in cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30% and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for the investigation of stroma-derived differentiation factors.
authors.affiliation: Department of Biochemistry and Molecular Cell Biology, University of Göttingen, Medical School, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Ries
authors.forename: P
authors.initials: P
authors.lastname: Krause
authors.forename: M
authors.initials: M
authors.lastname: Solsbacher
authors.forename: P
authors.initials: P
authors.lastname: Schwartz
authors.forename: K
authors.initials: K
authors.lastname: Unthan-Fechner
authors.forename: B
authors.initials: B
authors.lastname: Christ
authors.forename: P M
authors.initials: PM
authors.lastname: Markus
authors.forename: I
authors.initials: I
authors.lastname: Probst
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002469:Cell Separation; D002470:Cell Survival; D018920:Coculture Techniques / Q000379:methods; D016895:Culture Media, Serum-Free; D003907:Dexamethasone / Q000378:metabolism* / Q000494:pharmacology; D005934:Glucagon / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000166:cytology*; D008297:Male; D008954:Models, Biological; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger; D051381:Rats; D017208:Rats, Wistar; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology
chemical_list: D016895:Culture Media, Serum-Free; D007328:Insulin; D012333:RNA, Messenger; D003907:Dexamethasone; D005934:Glucagon; D019764:Phosphoenolpyruvate Carboxykinase (ATP)