Long-term extensive expansion of mouse hepatic stem/progenitor cells in a novel serum-free culture system |
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Authors: | Tsuchiya Atsunori Heike Toshio Fujino Hisanori Shiota Mitsutaka Umeda Katsutsugu Yoshimoto Momoko Matsuda Yasunobu Ichida Takafumi Aoyagi Yutaka Nakahata Tatsutoshi |
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Institution: | Department of Pediatrics, Graduate School of Medicine, Kyoto University, Japan. |
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Abstract: | BACKGROUND & AIMS: The liver has high regenerative potential. We attempted to establish a novel culture system for extensive expansion of fetal mouse hepatic stem/progenitor cells and to characterize cultured cells. METHODS: Hepatic spheroids collected from 6-day floating cultures were cultured on collagen-coated dishes in serum-free conditions in medium containing growth factors. Cultured cells were mainly characterized by immunocytochemistry and flow cytometry or transplanted into adult mice. RESULTS: Approximately 400 expanding hepatic spheroids were generated from every 1 x 10(6) fetal liver cells. Subsequently, highly replicative colonies were subcultured with maintaining colony formation on collagen-coated dishes. These colonies consisted of small immature alpha-fetoprotein-positive cells and hepatocytic and cholangiocytic lineage-committed cells. The immature alpha-fetoprotein-positive cells could be expanded in a reproducible manner at least 5 x 10(5)-fold (which involved at least 30 passages over >6 months) without losing differentiation potential. Flow cytometric analysis showed that all cultured cells expressed CD49f, but not CD34, Thy-1, c-kit, or CD45. Nearly 15% of the cells expressed Sca-1, and approximately 5%-20% of the cells were side population cells. Both sorted side population cells and Sca-1-positive cells (especially side population cells) produced a large number of alpha-fetoprotein-positive cells and lineage-committed cells. Expanded cells had bidirectional differentiation potential and improved serum albumin levels in mice with severe liver damage. CONCLUSIONS: Long-term extensive expansion of transplantable hepatic stem/progenitor cells was reproducibly achieved in a novel serum-free culture system. Moreover, this culture system yielded side population and Sca-1-positive cell populations that included hepatic stem/progenitor cells with differentiation and proliferation properties. |
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Keywords: | AFP α-fetoprotein ALB albumin bFGF basic fibroblast growth factor BGP biliary glycoprotein CK cytokeratin CPSI carbamoyl phosphate synthetase I EGF epidermal growth factor FACS fluorescence-activated cell sorter FCS fetal calf serum FITC fluorescein isothiocyanate β-gal β-galactosidase HGF hepatocyte growth factor KSR knockout serum replacement MP main population OSM Oncostatin M PCR polymerase chain reaction SCC small cell cluster SP side population TAT tyrosine amino transferase TO tryptophan-2 3-dioxygenase |
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