Expression of carbamoylphosphate synthetase I and glutamine synthetase in hepatic organoids reconstructed by rat small hepatocytes and hepatic nonparenchymal cells

The objective of this study was to investigate the expression of carbamoylp hosphate synthetase I (CPS) and glutamine synthetase (GS) in small hepatocy te colonies and whether the heterogeneous expression of the enzymes could b e induced during the maturation of small hepatocytes. Small hepatocytes iso lated from an adult rat liver were cultured and proliferated to form coloni es. The expression of CPS and GS was examined using immunocytochemistry and immunoblotting. In this culture more than 99% of morphologically hepatic c ells were positive for CPS and all small hepatocytes were negative for GS a t day 5. CPS-positive cells dramatically decreased with time in culture, wh ereas GS-positive ones appeared and their number increased in the colonies. Two to 3 weeks after plating, colonies with rising and piled-up cells appe ared and the number of such colonies reached about 25% of all colonies at d ay 30. In most rising and piled-up cells in colonies both proteins were str ongly expressed, whereas many small hepatocytes in monolayer colonies did n ot express either protein. When small hepatocytes in monolayer colonies wer e overlayed with Matrigel, the cells gradually piled up and both CPS and GS proteins were dramatically induced. The expression of CPS and GS in small hepatocytes may interact with the extracellular matrix because the rising a nd piled-up cells appear to be induced by the extracellular matrix produced by hepatic nonparenchymal cells.