| Co-Investigator(Kenkyū-buntansha) |
KOJIMA Takashi Sapporo Medical University, School of medicine, Assistant Professor, 附属がん研究所, 教授 (30260764)
MITAKA Toshihiro Sapporo Medical University, Cancer Research Institute, Assistant Professor, 附属がん研究所, 助教授 (50231618)
HIRATA Koichi Sapporo Medical University, School of medicine, Professor, 医学部, 教授 (50136959)
TAKEDA Hiroshi Sapporo Medical University, Cancer Research Institute, Instructor, 附属がん研究所, 助手 (00333310)
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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 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 mature hepatocytes. 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 drugs. On the other hand, 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. Albumin production increased with time in culture. In addition, the cells produced other serum proteins such as transferrin and fibrinogen, and expressed CPS I and tryptophan 2,3-dioxygenase.
Human hepatocytes were isolated and we found the proliferation of SHs in the culture. In addition, when the cells were cultured in a collagen sponge, the proliferation of biliary epithelium was observed and mature hepatocytes were maintained with three-dimensional structures for a long time.
We found that interstitial cells derived from rat bone marrow could support the maintenance of differentiated functions of primary hepatocytes.
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