Repopulation of Athymic Mouse Liver by Cryopreserved Early Human Fetal Hepatoblasts

Abstract

Transplantation of hepatocytes is a promising alternative to liver transplantation for the treatment of severe liver diseases. However, this approach is hampered by the shortage of donor organs and intrinsic limitations of adult hepatocytes. To investigate whether most of the hurdles faced with adult hepatocytes could be surmounted by the use of human fetal hepatoblasts, we have developed a method to isolate, transduce, and cryopreserve hepatoblasts from human livers at an early stage of development (11-13 weeks of gestation). Cells were characterized in vitro for expression of specific markers, and in vivo for their proliferation and differentiation potential after transplantation into athymic mice. Most of the cells (80-90%) harbored a bipotent phenotype, expressing cytokeratins 8/18, albumin, and CK19. They proliferated spontaneously in culture and were efficiently transduced by a beta-galactosidase-expressing retrovirus (90%). After transplantation, cryopreserved cells engrafted into the liver of athymic mice and proliferated, resulting in up to 10% repopulation. Engrafted cells expressed markers of differentiated adult hepatocytes including albumin, alpha1-antitrypsin, cytochrome P450 3A4, and alpha-glutathione-S-transferase. When retrovirally transduced before transplantation they expressed the transgene in vivo. In summary, early human fetal hepatoblasts engraft, proliferate, and mature in athymic mouse liver, without conditioning the donor.

Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development

“…This has been reported by one of the authors and others in this model. 17 One explanation is that the degree of hepatic cell differentiation is partial 2 months after transplantation and that engrafted cells produce less than the expected amount of albumin. The xenogenic environment could also underestimate the capacity of human progenitors to fully differentiate.…”

“…We previously isolated such immature cells during the early developmental stages of human liver and showed that they were able to differentiate and proliferate in rodent livers. 17 Moreover, the proof of principle that progenitors can be used efficiently for cell-based therapy has been clearly established for other organs such as neuronal stem cells in the context of neurodegenerative diseases. 39 In any case, mouse models will not allow by themselves to determine whether differentiated cells will be safe for clinical applications.…”

“…Human hepatoblasts were isolated and cultured as described. 17 Primary human hepatocytes were isolated from normal liver tissue biopsies obtained during resections (partial hepatectomies) for hepatic or metastatic tumors after informed patient consent. The primary hepatocytes were obtained by a twostep collagenase method and cultured as described.…”

“…The primary hepatocytes were obtained by a twostep collagenase method and cultured as described. 17 Differentiation of hESCs into Hepatic Cells. Cells were harvested using 5 mg/mL collagenase IV (Invitrogen) and then plated into plates (Costar; Corning Life Sciences, Acton, MA) precoated either with fetal bovine serum (FBS) or with 15 g/mL of human Fibronectin (Chemicon, Molsheim, France).…”

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

“…These epithelial cells lose their proliferative capacity after several days in classical tissue culture (Mahieu et al, 2004). Thus, generation of liver progenitor cell lines by means of conditional immortalization is of great interest to study the molecular events involved in their proliferation and differentiation in vitro as well as their fate in vivo after transplantation in the liver of recipient mice.…”