| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. We found methods by which SHs could differentiate into mature hepatocytes(MHs) and form three-dimensional structures. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled-up. Although this morphological change occurs when hepatic nonparenchymal cells such as stellate cells invade under SH colonies and produce an extracellular matrix, an overlay of Matrigel^<TM> on SHs can induce similar phenomena. The differentiated SHs express hepatocyte nuclear factor (HNF) 4, HNF6, and CCAAT/enhancer binding protein α, which are expressed in MHs. In addition, not only albumin and transferrin but also carbamoylphosphate synthetase (CPS) I and glutamine synthetase, which are key enzymes of urea synthesis and ammonia metabolism, are also expressed. Furthermore, isozymes of cytochrome P450 can be induced by specific agents. When SH colonies were cultured in a collagen sponge, SHs differentiated into MHs and bile ducts and capillaries were also formed within the scaffold. Now, we are earring out the experiments that the in vitro reconstructed hepatic tissues should be transplant ed to the livers of nude mice or syngeneic rats.
SH colonies could be cryopreserved for more than 6 months and the maximum period of the storage was about 90 weeks. About 60 % of SH colonies survive and attached on the dishes. The surviving SHs could proliferate and the average area of SH colonies was about 7.5 times larger at day 15 than at day 1. An amount of albumin secretion into culture medium was about 5 times larger at day 15 than at day 1. In addition, the cells produced other serum proteins such as transferrin and fibrinogen, and expressed CPS I and tryptophan 2,3-dioxygenase. The cryopreserved SHs could differentiate into MHs by an overlay of Matrigel.^<TM>
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