Previously, we reported a system to enrich mouse fetal hepatic progenitor cells (HPCs) by forming cell aggregates. In this study, we sorted two cell populations, CD49f ؉ Thy1 ؊ CD45 ؊ cells (CD49f-postive cells) and CD49f ؎ Thy1 ؉ CD45 ؊ cells (Thy1-positive cells), from the cell aggregates using a flow cytometer. CD49f-positive cells stained positive for endodermal specific markers such as ␣-fetoprotein (AFP), albumin (ALB), and cytokeratin 19 (CK19), and are thus thought to be HPCs. However, Thy1-positive cells were a morphologically heterogeneous population; reverse-transcription polymerase chain reaction (RT-PCR) and immunocytochemical analyses revealed the expression of mesenchymal cell markers such as ␣-smooth muscle actin, desmin, and vimentin, but not of AFP, ALB, or CK19. Therefore, Thy1-positive cells were thought to be of a mesenchymal lineage. When these two cell populations were co-cultured, the CD49f-positive colonies matured morphologically and stored a significant amount of glycogen. Furthermore, real-time RT-PCR demonstrated an increased expression of tyrosine amino transferase and tryptophan oxygenase mRNA, and transmission electron microscopy confirmed that co-cultured cells produced mature hepatocytes. However, when CD49f-positive cells were cultured alone or when the two populations were cultured separately, the CD49f-positive cells did not mature. These results indicate that CD49f-positive cells are primitive hepatic endodermal cells with the capacity to differentiate into hepatocytes, and that Thy1-positive cells promote the maturation of CD49f-positive cells by direct cell-to-cell contact. In conclusion, we were able to isolate CD49f-positive primitive hepatic endodermal cells and Thy1-positive mesenchymal cells and to demonstrate the requirement of cell-to-cell contact between these cell types for the maturation of the hepatic precursors. (HEPATOLOGY 2004;39:1362-1370.) B ecause of a shortage of donors for liver transplantation, cell transplantation has been explored as a useful bridge or alternative therapy. Hepatocyte transplantation can improve liver function sufficiently to extend the waiting time for liver transplantation. [1][2][3][4] However, using this as an effective clinical therapy requires the development of a cell source other than donated organs. Therefore, research is currently being conducted on hepatic stem and progenitor cells. In general, progenitor cells are highly expandable in vitro, easily cryopreservable, and quite resistant to hypoxic conditions. 5 Hepatic progenitor cells (HPCs) mature rapidly into adult hepatocytes in quiescent liver 6 and have far greater regenerative capacity than do adult hepatocytes in retrorsine-treated liver. 7 However, very little functional analysis of transplanted HPCs has been performed, and it is currently unknown whether transplanted HPCs can improve liver dysfunction. To transplant fully functional cells, it may be necessary for immature cells to be matured in vitro before transplantation. Therefore, the development of an in vitro maturation system is important.
Many studies of the maturation of primitive hepatic endodermal cells in vitro have demonstrated the requirement for maturation not only of soluble factors, such as