Long-term Extensive Expansion of Mouse Hepatic Stem/Progenitor Cells in a Novel Serum-Free Culture System

Abstract

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. 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. 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. 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.