Elevated expression of hormone-regulated rat hepatocyte functions in a newserum-free hepatocyte-stromal cell coculture model

The specific performance of the adult hepatic parenchymal cell is maintaine d and controlled by factors deriving from the stromal bed; the chemical nat ure of these factors is unknown. This study aimed to develop a serum-free h ierarchical hepatocyte-nonparenchymal (stromal) cell coculture system. Hepa tic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal growth factor, basic fibroblast growth factor, and hepatocy te-conditioned medium; cell type composition changed during the 2-wk cultur e period. During the first wk, the culture consisted of proliferating sinus oidal endothelial cells with well-preserved sieve plates, proliferating hep atic stellate cells, and partially activated Kupffer cells. The number of e ndothelial cells declined thereafter; stellate cells and Kupffer cells beca me 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 t op of the hepatocytes; Kupffer cell extensions established multiple contact s to hepatocytes and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte di fferentiation became evident after 48 h; a strong improvement of cell respo nsiveness toward hormones could he observed in cocultured hepatocytes. Hier archial hepatocyte coculture enhanced the glucagon-dependent increases in p hosphoenolpyruvate carboxykinase activity and messenger ribonucleic acid (m RNA) content three- and twofold, respectively; glucagon-activated urea prod uction was elevated twofold. Coculturing also stimulated glycogen depositio n; basal synthesis was increased by 30% and the responsiveness toward insul in and glucose was elevated by 100 and 55%, respectively. The insulin-depen dent rise in the glucokinase mRNA content was increased twofold in cocultur ed hepatocytes. It can be concluded that long-term signals from stromal cel ls maintain hepatocyte differentiation. This coculture model should, theref ore, provide the technical basis for the investigation of stroma-derived di fferentiation factors.