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

Abstract

The objective of this study was to investigate the expression of carbamoylphosphate synthetase I (CPS) and glutamine synthetase (GS) in small hepatocyte colonies and whether the heterogeneous expression of the enzymes could be induced during the maturation of small hepatocytes. Small hepatocytes isolated from an adult rat liver were cultured and proliferated to form colonies. The expression of CPS and GS was examined using immunocytochemistry and immunoblotting. In this culture more than 99% of morphologically hepatic cells were positive for CPS and all small hepatocytes were negative for GS at day 5. CPS-positive cells dramatically decreased with time in culture, whereas GS-positive ones appeared and their number increased in the colonies. Two to 3 weeks after plating, colonies with rising and piled-up cells appeared and the number of such colonies reached about 25% of all colonies at day 30. In most rising and piled-up cells in colonies both proteins were strongly expressed, whereas many small hepatocytes in monolayer colonies did not express either protein. When small hepatocytes in monolayer colonies were 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 and piled-up cells appear to be induced by the extracellular matrix produced by hepatic nonparenchymal cells.

Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells

“…1C). The number of colonies with rising/piled‐up cells reached about 25% of all colonies at day 30 (Mitaka et al, 2001). Other colonies that were not surrounded by NPCs developed faster than the colonies surrounded by them and maintained a monolayer (Fig.…”

Coordinated Movement of Bile Canalicular Networks Reconstructed by Rat Small Hepatocytes

“…35 One-fourth of the SH colonies displayed the piled-up cells that formed BC by day 30. 19 BC networks were detected as translucent belts under phase-contrast microscopy [ Fig. 1(A)].…”

Expression of cytochrome P450 enzymes in hepatic organoid reconstructed by rat small hepatocytes

“…Moreover, the addition of Matrigel, which is derived from Engelbreth‐Holm‐Swarm sarcoma, could induce the alteration of the cell shape, from flat to rising/piling‐up and from small to large. The most important change was the dramatic improvement of hepatic differentiated functions such as serum protein secretions and the expression of amino acid metabolizing and urea cycle enzymes 4,6 . In addition, the changes were correlated to the increased expression of liver‐enriched transcriptional factors (LETF) such as hepatocyte nuclear factor (HNF) 4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, and C/EBPβ 5 …”