Long-term culture of postnatal mouse hepatic stem/progenitor cells and their relative developmental hierarchy

Few studies on the long-term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum-free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature alpha-fetoprotein-positive cells along with hepatocytic and cholangiocytic lineage-committed cells. These cells expressed CD49f but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP-binding cassette transporter ABCG2, and sca-1+ cells. As mice aged, the frequency of SP and sca-1+ cells decreased along with the ability of cultured cells to expand. Approximately 20%-40% of the SP cells expressed sca-1, but only a few sca-1+ cells were also SP cells. Analysis of colonies derived from single SP or sca-1+ cells revealed that, although both cells had dual differentiation potential and self-renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca-1+ cells, whereas sca-1+ cells generated only sca-1+ progeny. Thus, SP cells are more characteristic of stem cells than are sca-1+ cells. In regenerating livers, ABCG2+ cells and sca-1+ cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self-renew and differentiate.