|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1999 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1998 : ¥1,900,000 (Direct Cost : ¥1,900,000)
We isolated a kind of hepatic progenitor cells, small hepatocytes, from an adult rat liver. A small hepatocyte can clonally proliferate for more than 3 months when the cells are cultured in DMEM supplemented with 10% FBS, 10mM nicotinamide, 1mM ascorbic acid 2-phosphate, 10 ng/ml epidermal growth factor, 10ィイD1-7ィエD1 M dexamenthasone, 0.5 μg/ml insulin, and antibiotics. The cell divides to form a colony and the number of the cells reaches to more than 100 cells within 20 days. Some colonies consist of more than 1000 cells in the culture. Small hepatocytes immunocytochemically show positivity for albumin, transferrin, cytokeratin 8 and 18. In contrast, α-fetoprotein, cytokeratin 14, OC2, and glutathione S-transferase P, which are markers of immature cells, are rarely observed in the early culture period. When small hepatocytes are cultured with hepatic nonparenchymal cells (NPCs) including liver epithelial cells Kupffer cells sinusoidal endothelial cells and stellate cells, piled up cells often appear in the small hepatocyte colonies two to three weeks after plating. In those colonies the invasion of NPCs under the colonies and deposits of extracellular matrix such as type I collagen, type IV collagen, and laminin are observed. The piled up cells show the alteration of cell shape from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin connexin 32 &26, glutamine synthetase, and carbamoylphosphate synthetase are induced. In addition, overlapping NPCs on the piled up cells induced the formation of duct- or cyst-like structures consisting of mature hepatocytes. Thus, small hepatocytes may be "committed progenitor cells" that can further differentiate into mature hepatocytes.