Localization of Liv2 as an Immature Hepatocyte Marker in EB Outgrowth

Abstract

The objective of this study was to establish Liv2, a surface marker of mouse immature hepatocytes (hepatoblasts), as a selection tool for embryonic stem (ES) cell–derived immature hepatocytes by acquiring basic data on Liv2 in normal mouse embryos and by confirming Liv2 expression in mouse ES-derived cells. The estimated molecular weight of Liv2 was 40–45 kDa, and immunoreactivity was definitively detected in the cell membrane of fetal hepatocytes on embryonic day (E) 9.5, declined gradually until E12.5, and subsequently became undetectable. Liv2 was localized on and close to the cell membrane. Embryoid bodies (EB) were formed from mouse ES cells whose undifferentiated state was confirmed with immunostaining of Nanog by the hanging drop method. A few Liv2-positive cells occurred as a cluster in EB outgrowth on day 7, but only some of these were albumin (ALB)-positive on day 13. These cells had the same pattern of immunoreactivity, i.e., localization on the cell membrane, as immature hepatocytes in the developing liver, although there were other types of cells with a different pattern of immunoreactivity that were seen only as a granular pattern in the cytoplasm and without ALB or the neuronal marker nestin. These results suggest that Liv2 may be useful as a surface marker for immature hepatocytes derived from ES cells. This application would allow for the sole selection of immature hepatocytes and provide a useful tool for regenerative medicine.

Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture

“…In addition to these four markers, we examined the expression pattern of several other highly relevant hepatic genes during the culture period to better characterize the transformation process. We looked at the expression of mature hepatocyte markers HepPar1 and HNF‐4α, immature hepatocyte marker Liv2,24 biliary ductal/oval cell marker CK19, and liver progenitor/embryonic liver marker Sall425 in a time‐dependent manner. IF staining and quantitative analysis of the images revealed a pattern (Supporting Fig.…”

Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

“…One of the major concerns about using EBs for differentiation, however, is the heterogenous population of cells that is generated and the presence of remaining undifferentiated cells [ 28 ]. In order to avoid teratoma formation in vivo , we sorted the hepatocyte precursors by using Liv2 as a fetal liver cell surface marker which can be used to isolate immature hepatocytes [ 17 , 29 ]. The advantage of using immature compared to fully mature hepatocytes for in vivo colonisation experiments is that the former have a higher proliferative capacity with respect to the latter [ 30 ].…”

Liver Development, Regeneration, and Carcinogenesis

“…The phenotype of foetal human liver progenitor cells remains controversial. A range of cell markers based on rodent studies, such as Liv2 [ 39 , 40 ], E-cadherin [ 41 ], and delta like kinase-1 (Dlk-1) [ 42 ], have only been characterised in human livers by immunodetection methods in vitro [ 43 , 44 ]. To date, the only convincing evidence to show that liver progenitors can be isolated from human foetal livers comes from immunoselection for epithelial cell adhesion molecule (EpCAM)-positive cells [ 45 ].…”