Self-Renewal Capability of Hepatocytic Parental Progenitor Cells Derived From Adult Rat Liver Is Maintained Long Term When Cultured on Laminin 111 in Serum-Free Medium

In this study, we investigated how the ability of hepatocytic parental progenitor cells (HPPCs) to self-renew can be maintained and how laminin (LN) isoforms play an important role in their self-renewal and maturation. Hepatocytes isolated from adult rat livers were cultured on hyaluronic acid to form colonies consisting of CD44(+) small hepatocytes, which could be passaged on dishes coated with Matrigel. When second-passage cells were plated on Matrigel, LN111, or LN511, HPPCs appeared on Matrigel and LN111 but not on LN511. We identified two types of cells among the second-passage cells: Small, round cells and large, flat ones were observed on Matrigel, whereas the former and latter ones were specifically attached on LN111 and LN511, respectively. We hypothesized that small and round cells are the origin of HPPC colonies, and the binding to LN111 could be key to maintaining their self-renewal capability. Among the integrins involved in LN binding, integrins alpha 3 and beta 1 were expressed in colonies on LN111 more than in those on LN511, whereas beta 4 was more strongly expressed in colonies on LN511. Integrin alpha 3(high)alpha 6 beta 1(high) cells could form HPPC colonies on LN111 but not on LN511, whereas integrin alpha 6 beta 1(low) cells could not on either LN111 or LN511. In addition, neutralizing anti-integrin beta 1 and anti-LN111 antibodies inhibited the passaged cells' ability to attach and form colonies on LN111 by HPPCs. Matrigel overlay induced second-passage cells growing on LN111 to increase their expression of hepatic functional genes and to form 3-dimensional colonies with bile canalicular networks, whereas such a shift was poorly induced when they were grown onLN511. Conclusion: These results suggest that the self-renewal capability of HPPCs depends on LN111 through integrin beta 1 signaling.