Elevated expression of hormone-regulated rat hepatocyte functions in a new serum-free hepatocyte-stromal cell coculture model |
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Authors: | Kati Ries Petra Krause Meike Solsbacher Peter Schwartz Kirsten Unthan-Fechner Bruno Christ Peter M Markus Irmelin Probst |
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Institution: | 1. Department of Biochemistry and Molecular Cell Biology, University of G?ttingen, Medical School, Humboldtallee 23, D-37073, G?ttingen, Germany 2. Department of Surgery, University of G?ttingen, Medical School, Humboldtallee 23, D-37073, G?ttingen, Germany 3. Department of Anatomy, University of G?ttingen, Medical School, Humboldtallee 23, D-37073, Germany
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Abstract: | Summary 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. |
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Keywords: | rat hepatocytes liver strom cells nonparenchymal cells coculture differentiation fenestration |
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