**4. Murine and rat hepatic progenitors in the fetal and adult liver**

Various surface markers have been applied to identify hepatic stem or progenitor populations in rodents.

Germain *et al*. described the bipotential capacity of fetal rat liver cells to differentiate into hepatic and biliary cells *in vitro* (39), as did Kubota *et al*. using clonal cultures (40). Small hepatocytes were detected in non-parenchymal fractions of adult rat liver cells (41-44). These small hepatocytes produced colonies that expressed hepatic and biliary markers. A similar type of colony could be obtained when adult liver cell clusters were placed into culture (45). Suzuki *et al*. sorted progenitor populations from fetal mice and rat livers with a phenotype of c-met+, CD49f+, CD117- , CD45-, and TER119- (46-50). Sorted cells developed into albumin and glycogen positive cells when transplanted into retrorsine-treated adult rats that had undergone two-third partial hepatectomy. Cells negative for c-met or positive for CD45 could not repopulate recipient livers. These progenitors could be also cultured clonally. Feng *et al*. (51) demonstrated that these cells could also undergo pancreatic differentiation in culture as well as *in vivo* when transplanted into alloxan-induced diabetic

Hepatic Progenitors of the Liver and Extra-Hepatic Tissues 51

Few data have been published on hepatic progenitors from species other than human or rodent. In general, pigs are used as an animal model closely resembling human physiology and metabolic functions. This makes the pig model more favorable than the rodent model. However, this model is scarcely used due to obvious constraints in keeping animals. Kano *et al*. (72, 73) investigated hepatic progenitors isolated by culture selection from nonparenchymal liver cell suspensions of six-seven months old pigs. Cell clusters in culture were positive for the hepatic markers AFP, albumin, transferrin, CK18, CK7, and c-met, but did they not express biliary markers such as gamma-glutamyltransferase, CK19, and CK14, although they were positive for oval cell marker OV6. Duct-like structures emerged from clusters expressing biliary epithelial markers. Clonal cell growth could be established (74). Comparable cells could be obtained (75) by isolating small liver cells from pigs that had undergone partial hepatectomy. In addition to the hepatic markers albumin and AFP, these cells also expressed biliary marker CK19 and were positive for OV6. In culture, cells were positive for stem-cell factor, CD117, CD90, AFP, CK19, and OV6. Fetal porcine liver cells

Several extra-hepatic sources have been described to harbor progenitors able to differentiate into hepatic lineages *in vitro* and *in vivo*. It is widely debated whether cells of extra-hepatic origin are able to differentiate into hepatic cell types or if they fuse with the recipient's liver cells when transplanted. Tissue sources include bone marrow, adipose tissue, umbilical cord, and peripheral blood. Hepatic differentiation potentials of embryonic stem cells (ESC), placenta derived stem cells, or induced pluripotent stem cells (iPS cells) are not discussed

Bone marrow cells or bone marrow derived hematopoietic stem cells have been suggested to be able to trans-differentiate into hepatic lineages. Petersen *et al*. performed initial experiments with cross-strain and cross-sex bone marrow and liver transplantations in rats (83). When male bone marrow was transplanted into female recipients and liver damage was induced, Y-chromosome positive cells could be detected in the female livers. Also, when male dipeptidyl peptidase (DPPIV) positive bone marrow was transplanted into female DPPIV negative recipients and liver damage was induced, DPPIV positive cells could be detected in the female livers. A further approach included transplantations of major histocompatibility complex class II L21-6 isozyme negative whole livers into positive enzyme expressing rats; after induction of liver damage, positive enzyme expressing cells could be detected. Alison *et al*. (84) investigated human female livers from patients who had received male bone marrow transplants. Y-chromosome positive cells that co-expressed CK8 were detected in the female livers. About 0.5 – 2% of all livers cells were Y-chromosome positive. Theise *et al*. described further *in vivo* experiments on the possible contribution of bone marrow cells towards hepatic lineages in mice (85) and humans (86). Whole bone marrow cells or CD34+lin- sorted cells from male mice were transplanted into female

could be detected within the female livers. In human patients who had undergone cross-sex bone marrow transplantation, Y-chromosome positive cells could be observed in female livers. 4 – 43% of cholangiocytes and 4 – 38% of hepatocytes were positive for Y-

) Y-chromosome positive cells

**6. Hepatic progenitors found in various mammalian species** 

were used to establish colonies of pluripotent progenitors (76, 77).

**7. Extra-hepatic sources of potential liver progenitors** 

here; further literature can be found in reviews (78-82).

recipients; up to 2.2% (bone marrow) or about 0.7% (CD34+lin-

mice. Similarly, Nierhoff *et al*. (35) demonstrated that fetal mouse liver epithelial cells positive for AFP or E-cadherin did not express hematopoietic stem cell markers CD34, CD117, Ter119, or CD45, but were positive for progenitor markers Sca-1 and Pancytokeratin. Both E-cadherin positive sorted as well as unsorted fetal liver cell fractions from wild type mice gave rise to liver parenchyma when transplanted into retrorsine treated DPPIV-/- mice.

As described for human hepatoblasts above, mouse fetal liver hepatoblasts have been shown to express the surface marker Dlk-1 (35-37). Dlk-1 positive sorted mouse fetal liver cells can be cultured long-term and, when transplanted into the spleen, give rise to hepatocytes in the liver. Dabeva *et al*. (52) described the re-population potential of wild type fetal rat liver cells when transplanted into DPPIV-/- rat models. These models included knockouts that had undergone two-third partial hepatectomy and were either treated with retrorsine or not. In rats treated with retrorsine, which blocked proliferation of endogenous hepatocytes, mainly bipotential, transplanted progenitors were observed expressing AFP, albumin, and CK19. In non-treated rats, transplanted cells expressed mainly either hepatocytic or biliary markers.

The positive expression of aldehyde dehydrogenase (ALDH) has been used as a feature to select progenitors from adult mouse liver (53). ALDH+ cells were shown to have stem cell characteristics and to express markers of human hepatic stem cells such as CD326, CK19, CD133, and Sox9.

Various hepatic progenitor cell lines have been developed from normal, genetically modified, or toxin treated rodents (54-62). Several of these lines were described as bipotential *in vitro* or when transplanted *in vivo*.
