Modulated differentiation of embryonic stem cells into hepatocyte-like cells by coculture with hepatic stellate cells.

We investigated the effects of coculture with hepatic stellate cells (HSCs) on the differentiation of embryonic stem (ES) cells and embryoid bodies (EBs). Rat HSCs were incubated until becoming semi-confluent and adherent to the dish. Undifferentiated mouse ES cells and 4-day EBs were cultured in gelatin-coated or HSC-feeder dishes, then induced hepatocyte-like cells and the remaining undifferentiated ES cells were examined using immunocytochemical and RT-PCR methods. HSCs promoted the differentiation of EBs into hepatocyte-like cells, whereas they inhibited the differentiation of undifferentiated ES cells. Among EB outgrowths cocultured with HSCs, albumin-immunopositive cells were clearly and abundantly observed, while they were faintly and scarcely seen in EB outgrowths without HSCs. mRNA expressions of the hepatocyte-related markers such as albumin, transthyretin, alpha-1-antitrypsin, tryptophan-2,3-dioxygenase, phosphoenolpyruvate carboxykinase, hepatocyte nuclear factor 4α and cytochrome P4507a1 were clearly detected in EB outgrowths cocultured with HSCs, while they were only weakly detected or undetected in spontaneous EB outgrowths without HSCs. In contrast to the promoted hepatic differentiation of EBs by HSCs, undifferentiated ES cells formed cellular colonies in HSC-feeder dishes that were similar to the colonies of undifferentiated ES cells kept in maintenance medium containing leukemia inhibitory factor. In addition, ES cell colonies were immunopositive for Oct-3/4, markers of an undifferentiated state, and there were few ALB-immunopositive cells in the colonies. Thus, HSCs have contrasting effects on EBs undergoing differentiation and undifferentiated ES cells, i.e., positive and negative modulation, respectively.