Fibroblast growth factors promote hepatocyte stem cell renewal in embryonic liver cultures

Abstract

Fibroblast growth factors (FGFs) have been shown to play a definite role in hepatic induction. Stage and tissue specific expression of FGF 1,4 and 8 are necessary for initiation of hepatic bud from the foregut endoderm FGF is also routinely used to maintain and propagate embryonic stem ceils in culture. The aim of this study was to investigate the effect of FGF 1, 4 and 8 on liver development utilizing an in vitro embryonic liver culture system and recotnbinant FGF proteins. Livers were harvested from day 10 embryos (El0) and cultured as an organ in DMEM supplemented with 10% FCS (control) with or without additional FGF 1 or FGF 4 or FGF 8. After 72 hours, the cultured livers were fixed and processed and 4/zm sections were utilized for histologic and immunohistochemical evaluation. Proliferation (PCNA and Ki-67) and apoptosis (TUNEL) assay was also performed. The major difference in the histology of the FGF 1/4/8 treated cultures was in the arrangement of cells in sheet like architecture only as compared to the controls that displayed cells in both ductular and sheet like arrangements. There was no significant change in number of apoptotic cells in presence of FGF 1/4 but there was a decrease in number of apoptotic nuclei in FGF8 treated cultures. Almost all cells in all FGF cultures exhibited PCNA positivity and a sigmficant number of cells were Ki-67 (S-phase) positive. There was a striking difference in the number of c-kn positive stem cells in the presence of any of the three FGFs used as compared to the controls. These stem cells were also positive for a-fetoprotein and albumin and very few were positive for CK-19. There was an overall decrease in number of CK-19 positive cells in presence of the FGFs. To conclude, FGF 1, 4 and 8 are excellent growth factors to maintain and propagate liver stem cells in development. These factors support prohferation and affect apoptosis and thus promote liver stem cell renewal. Also, lineage analysis shows a preponderance of unipotential stem cells (more hepatocytic than biliary) in the FGF treated cultures that suggest existence of selective hepatocyte stem ceils, h appears that FGFs promotes self-renewal of such a unipotential stem cell population in the liver.