Hepatic Progenitors of the Liver and Extra-Hepatic Tissues

The liver has a tremendous capacity to regenerate at all developmental stages (for reviews, see (1-3)). Liver cell mass can be restored even after repeated partial hepatectomies as well as after toxic injury. The contribution of stem cells to these processes is still under debate. Adult liver cells have been shown to regenerate liver tissue repeatedly when transplanted serially (4). However, hepatocytes cannot be considered stem cells because they are unipotent (for a glossary of terms, see Table 1). This chapter describes various liver progenitors that have been found by different researchers in humans and other mammalian species. Intra and extra-hepatic progenitors are discussed that can give rise to liver lineages. Intra-hepatic progenitors of non-hepatic lineages, such as endothelial or hematopoietic restricted progenitors, are not discussed. Although the focus of this chapter is on progenitors that have been characterized in normal, non-pathological conditions of the liver, oval cells will be described briefly.

Table 2. Progenitors with hepatic potential isolated from human livers.Details are given in the respective sections.

Human liver multipotent progenitors
Dan et al. isolated liver stem cells co-expressing endodermal and mesenchymal phenotypes from human fetal liver by culture selection on feeder cells (19).These cells could differentiate not only into hepatocytes and bile duct cells, but also into fat, bone, cartilage, and endothelial cells.Because of their multilineage differentiation potential, these cells were termed human fetal liver multipotent progenitor cells (hFLMPC).The in vivo percentage of this progenitor was not given, as these cells were isolated by culture selection.Cell surface and intracellular markers included: CD34, CD90, CD117, CD326 (also called epithelial cell adhesion molecule (EpCAM)), c-met, SSEA4, CK18, CK19, CD44h, and vimentin.Cells were negative for albumin, CD133, CD45, and AFP.They could be cultured monoclonal and longterm for up to 100 population doublings.Cells had population doubling times of 46h.Early and late passages demonstrated identical morphology, differentiation potential, and telomere length.Cultured cells formed typical clusters with cells having a high nuclear to cytoplasm ratio.The morphology of these clusters resembled hepatic stem cells colonies described by Schmelzer et al. (12).When transplanted into immunotolerant Rag2 -/ --/ -mice (using a modified retrorsine/carbon tetra-chloride model), human-specific albumin in mouse serum and human-specific albumin in sections of the liver could be detected.Liver sections of transplanted mice demonstrated clusters of human hepatocytes.A repopulation of 0.8-1.7% was estimated.The multipotential differentiation potential and resemblance to hepatic stem cell colonies suggests that hFLMPC represent mesendodermal precursors of hepatic stem cells.
Herrera et al. isolated a similar population from human adult livers (22) using culture selection.These cells also expressed hepatic and mesenchymal markers.Cell surface and intracellular markers included albumin, AFP, CD29, CD73, CD44, CD90, vimentin and nestin; however, there was a negative expression of CD34, CD45, CD117, CD133, and CK19, and a weakly positive expression of CK8 and CK18.The cells were different from those described by Dan et al., as albumin and AFP expression could be observed and hematopoietic markers CD34 and CD117 were absent.In vitro, progenitors differentiated not only into hepatocytes, but also into osteogenic, endothelial, and islet-like, insulin-producing structures.Adipogenic differentiation could not be induced.As these cells were cultureselected, percentages of their in vivo occurrence were not established.Cells in vitro demonstrated exponential growth rates.When transplanted, human cells could be localized in vivo within the liver parenchyma of severe-combined immunodeficient (SCID) mice treated with N-acetyl-p-aminophen.
Mesenchymal progenitors isolated from adult human livers were investigated for their potential to differentiate into hepatocytes (20,21).Mesenchymal-like cells were obtained by selective culture (not sorting) of total liver cells.FACS analyses of cultured cells revealed a phenotype similar to mesenchymal stem cells with positive expression for CD90, CD73, CD29, CD44, CD13, and HLA-class I, but negative expression for CD105, CD133, CD117, CD45, CD34, and HLA-DR; cells were weakly positive for CD49e and CD49b, and only a minor fraction expressed CD49f.When cells were intrasplenically transplanted into uPA + / + -SCID mice, human albumin and AFP positive cells could be observed and human albumin secretion was detected.When transplanted into SCID mice with and without 70% hepatectomy, human albumin gene expression could be measured in mice livers that had undergone hepatectomy, and human albumin positive cells could be detected in mouse liver sections in both models.Potential fusion events were not analyzed.When cells were induced to hepatic lineages in vitro (21), hepatic functions were increased compared to noninduced controls, but lower than those of freshly isolated adult liver cells.

Hepatic stem cells in the human liver
Hepatic stem cells can be isolated from fetal, neonatal, pediatric, and adult human livers with identical characteristics (12,18) (24).Stem cells could be selected by MACS sorting as well as under selective culture conditions, which included serum-free medium and culture on plastic.Under these culture conditions, hepatic stem cell colonies formed.These colonies (Figure 1) exhibit a typical epithelial morphology of densely packed, small cells with high nucleus-to-cytoplasm ratio.Stem cell colonies are positive for CD326 (Figure 2), CD44h, CD56, and weakly express albumin, but are negative for AFP.
Cells were capable of self-renewal, as shown by clonogenic expansion for more than 150 population doublings.0.5 -2.5% of all liver cells from all ages were positive for CD326 expression.Hepatic stem cells have a small diameter of about 9 μm.In vivo, they are located in the ductal plates in fetal and neonatal livers and in the Canals of Hering in pediatric and adult livers.The Canal of Hering has been previously described as the reservoir of stem cells in postnatal livers (25,26).Carpentier et al. recently studied lineage tracing by using a Cre recombinase Sox9 mouse model and confirmed that ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells (27).

Hepatoblasts in human liver
Hepatoblasts are the main parenchymal cell type of the fetal liver and are defined by their expression of AFP.AFP positive cells are rare in normal adult livers, except in livers with severe injury or disease (30-32) (for review, see (33)).Hepatoblasts can give rise to hepatocytes and cholangiocytes, and are therefore also named bipotential progenitors (15).AFP-negative hepatic stem cells are the precursors to hepatoblasts that can mature into AFPpositive hepatoblasts (12).Human fetal hepatoblasts could be cultured long-term and clonally, and contributed to liver parenchyma when transplanted into SCID mice (23).Hepatoblasts express biliary and hepatocyte markers such as CK19, CK14, gamma glutamyl transpeptidase, glucose-6-phosphatase, glycogen, albumin, AFP, E-cadherin (34), -1 microglobulin, HepPar1, glutamate dehydrogenase, and dipeptidyl peptidase IV (15,18).

49
Human hepatoblasts do not express the mesenchymal or hematopoietic markers CD90, vimentin, and CD34 (34).In mice, hepatoblasts express the surface marker Dlk-1 (35)(36)(37), which was subsequently demonstrated to be expressed by human fetal hepatoblasts as well (34).Mouse fetal liver cells sorted for Dlk-1 can be cultured long-term; transplantation of Dlk-1 positive cells into the spleen gives rise to hepatocytes in the liver.Several signaling pathways and transcription factors contribute towards differentiation into either cell type.In mice, Notch signaling controls differentiation towards biliary epithelium by upregulation of HNF1 but downregulation of HNF1 , HNF4, and C/EBP (38), and, in turn, suppression of C/EBP expression in periportal hepatoblasts is suggested to induce biliary epithelial differentiation by increasing HNF6 and HNF1 expression (17).

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)(42)(43)(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)(47)(48)(49)(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 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)(36)(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.

Oval cells
Oval cells were first described in rodents, emerging when the liver is exposed to certain toxins (for review, see ( 63)) (64).Termed "oval cells" because of their oval shaped nucleus, these small cells have a diameter of less than 10 μm.They are located near the portal triads and expand in the livers of animals exposed to oncogenic insults.The term "oval cells" frequently refers to liver stem cells or progenitors.However, oval cells can be distinguished from normal hepatic progenitors phenotypically and in their growth regulatory requirements (65).Several protocols have been shown to lead to the emergence of oval cells: administration of 2-acetylamino fluorine or dipin in combination with partial hepatectomy; administration of carbon tetrachloride, 3-methyl-diaminobenzidine, galactosamine, furane, or 3,5-diethoxycarbonyl-1,4-dihydrocollidine; etluonine addition to a choline-deficient diet; or transgenic albumin-urokinase-type plasminogen-activator mice.

Hepatic progenitors found in various mammalian species
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 were used to establish colonies of pluripotent progenitors (76,77).

Extra-hepatic sources of potential liver progenitors
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 here; further literature can be found in reviews (78)(79)(80)(81)(82).
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 recipients; up to 2.2% (bone marrow) or about 0.7% (CD34 + lin -) Y-chromosome positive cells 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-chromosome.Lagasse et al. (87) intravenously injected adult wild type bone marrow cells in FAH -/ -mice, an animal model of tyrosinemia type I.The mice were rescued and biochemical functions were regained.Only purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration from total bone marrow cells.However, subsequently published studies revealed that the majority of those liver cells, which were assumed to be donor derived differentiated bone marrow cells, are instead rather the product of donor cells fusing with host liver cells (88,89).Other studies demonstrated bone marrow cells contributed nothing or very little to liver lineages in vivo (90)(91)(92).Jang et al. (93) and Harris et al. (94) could show, however, that a minor percentage (up to 0.1%) of bone marrow cells can contribute to liver cells in vivo without fusion.Most evidence to date indicates that only a minority of the observed trans-differentiation events is actually due to differentiation of bone marrow cells into liver lineages and the majority of observed trans-differentiated cells are indeed fusion events.
Similar to the findings of the above described in vivo studies, in vitro studies of the hepatic differentiation potential of hematopoietic stem cells produced contradicting findings (95)(96)(97)(98)(99).
Overall, results from in vitro studies suggest that bone marrow hematopoietic stem cells can differentiate only barely, if at all, into hepatic lineages.
Lee et al. (119) transplanted green fluorescent protein mouse gallbladder epithelial cells into non-fluorescent SCID mice that had undergone retrorsine treatment and either partial hepatectomy before transplantation or carbon tetrachloride treatment following transplantation.Within one to four months after transplantation, green fluorescent protein positive cells could be detected within the recipient mice.These cells expressed mostly biliary markers, but cells positive for hepatic markers could be detected as well.
Zhao et al. isolated hematopoietic stem cells from peripheral blood (120) and demonstrated their in vitro multilineage differentiation potential; treatment of cultures with HGF induced cells to acquire a round or oval-like flattened morphology.Most of the cells were positive for intracellular albumin and AFP expression; some cells demonstrated CK7 expression.Sun et al. (121) showed that human umbilical blood cells integrated into livers of rat chimeras, and these cells were positive for human hematopoietic, biliary, and hepatic proteins.Crema et al. isolated CD133 + cord blood cells (122).Transplantation into liver-damaged SCID mice resulted in clusters of human-derived cells expressing human leucocyte antigen-class I, HepPar1, and OV6 antigens.Within these clusters, human albumin, AFP, and CK19 could be detected.Human umbilical blood cells demonstrated in vitro hepatocyte-like differentiation and expression of hepatic proteins when transplanted in rodents with induced liver damage (107,123,124).
Conclusively, it appears that extra-hepatic progenitors integrate into the liver only to a very minor percentage and only when severe liver damage is induced.The majority of these events appear to be due to fusion and not differentiation.The observed improvements of liver functions by mesenchymal cells could be attributed to their secretion of growth factors and cytokines and immunosuppressive properties (111,(125)(126)(127).

Conclusion
Although there is still some debate about the detailed characteristics that identify hepatic progenitors, much progress has been achieved during recent years in defining, isolating, characterizing, and transplanting various types of progenitors.This is especially the case for hepatic progenitors isolated from human livers.Hepatic progenitors represent a population with potential advantages over total liver cell suspensions or hepatocytes for cell transplantation in patients (29,128), for review see (129).Because of their high proliferation and differentiation potential a major advantage for transplantation of stem cells over total liver cell suspensions would be the requirement for less cell numbers to inject, which would decrease the risks associated with transplanting high cell numbers.In addition, because of their proliferation and differentiation potential, progenitors could be used in applications such as extracorporeal liver support systems (130,131), and may be used as an alternative cell source in pharmacological screening models.Cultures of progenitors also provide an easy in vitro tool to study principles of developmental biology.
Furuyama et al.  (28)  demonstrated that adult intestinal cells, hepatocytes and pancreatic acinar cells are physiologically supplied from Sox9-expressing progenitors using Cre-based lineage tracing in mice.In CCl 4 mediated liver injury, Sox9-positive progenitors contributed to liver regeneration.Hepatic stem cells have been shown to differentiate into biliary and hepatocytic lineages in vivo and in vitro(12).Freshly isolated cells or stem cells expanded in culture developed into mature liver tissue expressing human-specific proteins when transplanted into NOD/SCID mice, and lost their expression of stem cell marker CD326, CD133, and CK19.Whether those cells also possess multilineage differentiation potential beyond endodermal fates, i.e. mesodermal or ectodermal, has not yet been investigated.Khan et al. transplanted human fetal liver derived CD326 + sorted progenitors into patients with liver fibrosis(29).Patients demonstrated improvements in clinical and biochemical parameters and a decrease in mean MELD (model for end-stage liver disease) score at sixmonth follow-up.

Fig. 1 .
Fig. 1.Human hepatic stem cell colony in culture, established from fetal liver cell suspensions as described in (12); phase contrast microscopy (A), and fluorescence microscopy (B) of proliferating cells with positive nuclei for incorporated thymidine analog bromodeoxyuridine (B-I) and corresponding total nuclei stained with 4',6-diamidino-2phenylindole (B-II).
, as described by Schmelzer et al.