Immortalization of a primate bipotent epithelial liver stem cell

Abstract

Liver regeneration after partial hepatectomy results primarily from the simple division of mature hepatocytes. However, during embryonic and fetal development or in circumstances under which postnatal hepatocytes are injured, organ regeneration is believed to occur from a compartment of epithelial liver stem or progenitor cells with biliary and hepatocytic bipotentiality. The ability to identify, isolate, and transplant epithelial liver stem cells from fetal liver would greatly facilitate the treatment of hepatic diseases currently requiring orthotopic liver transplantation. Here we report the identification and immortalization by retrovirus-mediated transfer of the simian virus 40 large T antigen gene of primate fetal epithelial liver cells with a dual hepatocytic biliary phenotype. These cells grow indefinitely in vitro and express the liver epithelial cell markers cytokeratins 8͞18, the hepatocyte-specific markers albumin and ␣-fetoprotein, and the biliary-specific markers cytokeratins 7 and 19. Bipotentiality of gene expression was confirmed by clonal analysis initiated from single cells. Endogenous telomerase also is expressed constitutively. After orthotopic transplantation via the portal vein, Ϸ50% of the injected cells integrated into the liver parenchyma of athymic mice without tumorigenicity. Three weeks after transplantation, cells having seeded in the liver parenchyma expressed both albumin and ␣-fetoprotein but had lost expression of cytokeratin 19. These results provide strong evidence for the existence of a bipotent epithelial liver stem cell in nonhuman primates. This unlimited source of donor cells also should enable the establishment of a model of allogenic liver cell transplantation in a large animal closely related to humans and shed light on important questions related to liver organogenesis and differentiation.

Long-term controlled immortalization of a primate hepatic progenitor cell line after Simian virus 40 T-Antigen gene transfer

“…Previously, we documented that the retrovirus-mediated expression of T-Ag in a model of non-human primate foetal hepatic stem cells led to the isolation of one clone with an unlimited proliferation, after a 7-month period of replicative senescence (Allain et al, 2002). This clone (IPFLS) arose at a frequency of 5 Â 10 À7 , consistent with published results (Zhu et al, 1999) for human cells.…”

“…IPFLS cells had bypassed the M1 senescence checkpoint (Allain et al, 2002) and we show that their telomerase was upregulated to a level exceeding that in a normal counterpart and this level remained stable over 90 passages. These results suggest that IPFLS cells had also bypassed the M2 crisis.…”

“…Previously, we showed that primary hepatic foetal cells display telomerase activity immediately after thawing, and that this activity drops after 2 days of culture (Allain et al, 2002). …”

“…Cells were blocked with 1% gelatin and incubated for 1 h with goat polyclonal anti-p53 antibody (N-19; 1 : 200) For immunohistochemistry, liver samples were snap-frozen. Simian cells were detected as described previously using monoclonal anti-human proteins (Allain et al, 2002). Briefly, several random sections were cut from each liver lobe of each animal and stained for albumin and cytokeratin 19 (CK19).…”

“…During early passages, up to population doubling (PD) 20, the cells were bipotent and expressed markers of hepatocytes and biliary cells. After transplantation into nude mice, T-Ag-expressing cells were not tumorigenic, integrated the hepatic parenchyma and expressed hepatocyte functions (Allain et al, 2002). The goal of this work was to develop this model system of hepatic progenitor cell line to study further the role of T-Ag and the genetic events underlying long-term immortalization in these cells.…”