Journal of Hepatology
Volume 36, Issue 4 , Pages 552-564, April 2002

Liver stem cells and model systems for liver repopulation

Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

Received 29 November 2001; accepted 1 January 2002.

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1. General concepts of stem and progenitor cells 

Embryonic stem cells originate from the inner cell mass of the mammalian blastocyst and are totipotent [1]. Under certain conditions, these cells can be propagated in culture as stable, undifferentiated pluripotent stem cell lines. During development, embryonic stem cells give rise to somatic stem cells (reviewed in Refs. [2], [3]). Somatic stem cells differentiate further into multipotent tissue stem cells which have been identified and isolated from the neural crest, bone marrow and central nervous system [2], [3], [4], [5], [6].

The general properties of somatic stem cells have been derived primarily from studies of tissues with continuous proliferative activity and high turnover, such as hepatopoietic cells in the bone marrow and epithelial cells of the intestinal mucosa [7], [8]. As listed in Table 1, the properties of stem cells are contrasted with those of progenitor cells and fully differentiated, tissue- or organ-specific somatic cells. Stem cells are generally considered to exhibit the following characteristic properties: (1) self-renewal or maintenance (do not proliferate rapidly and are slowly cycling); (2) multipotency (capable of producing progeny in at least two lineages); (3) long-term tissue repopulation after their transplantation; and (4) serial transplantability.

Table 1. Classical view of somatic cells regarding lineage potential and repopulation capacity
Stemcells
Self-renewing (usually slowly cycling)
Multipotent (yielding progeny in at least two lineages)
Capable of long-term tissue repopulation
Serially transplantable
Progenitorcells
Incapable of self-renewal (usually rapidly dividing)
Multipotent or unipotent (producing differentiated progeny)
Capable of short-term tissue repopulation
Not serially transplantable
Terminallydifferentiatedcells
Fixed tissue-specific phenotype (with ability to alter specific gene expression levels depending on local environmental conditions)
Inability or very limited ability to proliferate
Not serially transplantable

Progenitor cells are the progeny of stem cells. They are comprised of a spectrum of cells in distinct subpopulations, some with multilineage potential similar to somatic stem cells (early progenitors cells or stem/progenitor cells), and others which appear to have progressed further along the differentiation pathway and produce progeny of only a single lineage (late progenitor cells). In contrast to somatic stem cells, progenitor cells are generally rapidly dividing, but they are capable of only short-term tissue repopulation. Progenitor cells are not self-renewing or capable of serial transplantation and they are often referred to as ‘transit cells’ or ‘transit amplifying cells’.

Fully differentiated, organ- or tissue-specific cells in adult mammalian tissues are generally considered to be non-proliferative (or capable of only very limited proliferation). The hepatocyte is a well-known exception, since under normal circumstances hepatocytes can undergo several rounds of division to replace the liver mass after extensive parenchymal loss [9], [10]. However, in recent years, it has been shown that under specialized circumstances, mature hepatocytes can undergo multiple divisions [11], [12], [13]. Furthermore, fully differentiated hepatocytes can even undergo serial transplantation under very selected circumstances [14]. This property was formerly considered a unique characteristic of stem cells, yet it can also be achieved with a fully differentiated tissue-specific cell, the hepatocyte. Nonetheless, we will retain the classical definitions and terminology in the present review, so that we can present results in the liver within the context of the preexisting stem cell literature.

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2. Stem cells during liver embryologic development 

Classical embryological studies have traced the proliferation and differentiation of endodermal stem cells into the hepatocytic and bile ductular cell lineages during normal liver development [15], [16], [17], [18], [19], [20]. This process begins on embryonal day (ED) 8.5 in the mouse with proliferation of undifferentiated endodermal cells of the ventral foregut and their migration into the septum transversum, where they come into contact with mesenchymal cells (see Fig. 1). At this point, they are already specified to enter the liver lineage (determination) and form the hepatic diverticulum [21]. Mouse foregut-derived cells begin to express α-fetoprotein (AFP) at ED9.0 and then albumin at ∼ED 9.5, followed by placental alkaline phosphatase and intermediate filament proteins, cytokeratins (CK)-14, 8 and 18 [19], [20]. Using an in vitro tissue explant co-culture system comprised of cardiac mesenchyme and ventral foregut endodermal cells, Zaret and coworkers have shown that fibroblast growth factors (FGF's), particularly FGFs 1 and 2, can replace cardiac mesenchyme in inducing albumin gene expression [22]. However, other factors or cell–cell interactions provided by cardiac mesenchyme are required for foregut epithelial cells to grow. The morphology of the cells then changes to that of the hepatoblast (an early progenitor or stem/progenitor cell), expressing in addition to AFP and albumin, γ-glutamyl transpeptidase (GGT), α1-antitrypsin (α1-AT), glutathione-S-transferase (GST)-P and fetal isoforms of aldolase, lactic dehydrogenase and pyruvate kinase (M2-PK) [23], [24], [25], [26], [27]. The cells proliferate rapidly between ED12 and ED16 and subsequently diverge along two distinct lineages, the hepatocyte and the cholangiocyte, beginning just prior to ED16 [20], [28], [29]. This time period has been referred to as a differentiation window in hepatic development [29], [30]. In cell culture prior to ED16, fetal liver epithelial cells (early progenitor cells) appear to have the ability to change their phenotypic gene expression pattern from hepatocytic to ductular or the reverse, depending on the environmental (cell culture) conditions [29], [30]. However, after ED16, the cells are committed to progress along one or the other of these lineages and no longer retain their bipotential properties, although they continue to proliferate (i.e. ‘committed’ or late progenitor cells). However, the irreversible nature of these changes no longer holds, as even phenotypically fully mature cells isolated from the adult liver appear to switch their phenotype in culture between hepatocytic and ductal, depending on experimental conditions [31], [32] (and G. Michalopoulos, pers. commun.)

Distinct gene expression programs can be readily distinguished in ‘committed’ hepatic progenitor cells. In addition to the proteins mentioned above, antigens HBD-1, H.2 and H.4, transferrin receptor, c-CAM and HES6 are expressed in hepatocyte progenitors [20], [26], [27], [30], [33], [34] and CKs-7 and 19, OC-2, OC-3, BDS7, OV-6 and BD-1 are expressed in biliary epithelial cell progenitors [20], [23], [27], [29], [30], [33], [34], [35]. As organogenesis proceeds, the intrahepatic bile ducts are formed in the vicinity of large portal vein branches, beginning on ∼ED17. This is evidenced by formation of ductular structures containing CK-19 positive epithelial cells that have the appearance of ‘strings of pearls’ [36]. The basic lobular structure is then formed, although the hepatic parenchymal plates or cords do not become fully mature until several weeks after birth.

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3. Liver regeneration 

The ability of the liver to regenerate is a property that is unique among solid organs in mammalian species. Following two-thirds partial hepatectomy (PH), there is compensatory growth by the remaining liver, resulting in restoration of the total parenchymal cell number and mass within 1–2 weeks [9], [10]. This process is not really regeneration, because the lost anatomic structures are not replaced, but the remaining tissue expands to its original mass by proliferation of preexisting cells. The final size of the liver appears to be tightly controlled by the size of the animal (there is a fixed ratio of liver to body weight), but the precise mechanism regulating liver mass has not been determined [10].

Radioactive labeling with [3H]thymidine and morphologic studies in the 1960s showed that many hepatocytes are actively engaged in DNA synthesis and mitosis during liver regeneration, and it was estimated that 70–90% of hepatocytes undergo at least one round of cell division following two-thirds PH [37], [38]. Extensive research has also been conducted to elucidate cellular and molecular events occurring during liver regeneration and reveals that this is a highly organized, complex, multistep process involving growth factors and cytokines, gene transcription factors, cell signaling pathways and cell cycle regulating, as well as regulated, genes (for reviews, see Refs. [10], [39]). On the basis of these studies, it has been concluded that the proliferative activity of adult hepatocytes is sufficient to repopulate the liver following PH and that the participation of stem/progenitor cells is not required [40].

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4. Are there stem cells in the adult liver? 

The existence of stem cells in the adult liver, a highly controversial topic [41], was postulated initially more than 40 years ago by Wilson and Leduc [42]. This was based on studies in rodents in which it appeared that cells in the distal cholangioles of the bile ducts were responsible for restoration of liver mass after dietary injury. At that time, it was also established that bile duct epithelial cells and hepatocytes are of common embryologic origin, derived from hepatoblasts emanating from the endoderm of the ventral foregut [15], [16]. Therefore, a potential precursor/product relationship between cells of the distal cholangioles and hepatocytes seemed reasonable. Specific identification of these cells has been problematic, because unique markers for liver stem cells have not yet been identified. However, studies conducted during the last decade clearly establish the existence of cells both within and outside the liver that exhibit properties of hepatic stem cells and can differentiate into mature hepatocytes and/or bile duct epithelial cells after their transplantation and engraftment in the liver.

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5. ‘Oval cells’ as hepatocyte progenitors 

The first study demonstrating the existence of small undifferentiated epithelial cells in the adult liver was reported by Farber [43], who treated rats with different carcinogens, such as ethionine, 2-acetylaminofluorene (2-AAF) and 3-methyl-4-dimethylaminobenzene. In these studies, Farber noted proliferation of epithelial cells in the periportal region with scant basophilic cytoplasm and an oval-shaped, pale blue, homogeneously stained nucleus, which he termed ‘oval cells’. Ultimately, he concluded that ‘oval cells’ are not progenitors of hepatocytes [44]. However, Thorgeirsson and coworkers came to the opposite conclusion; namely, that under certain circumstances ‘oval cells’ differentiate into hepatocytes or neoplastic hepatocytes [45], [46]. Rats were treated with 2-AAF, which causes extensive DNA damage in the liver and were then subjected to two-thirds PH. Under these conditions, there is massive proliferation of ‘oval cells’ in the periportal region (zone 1), associated with uptake of [3H]thymidine. This is followed sequentially by the appearance of nodular masses of [3H]thymidine labeled basophilic hepatocytes in the mid parenchyma (zone 2), which are presumably derived from the ‘oval cells’ [45]. The kinetic pattern of expression of bile ductular (CK-7, CK-19 and OV-6) and hepatocytic (AFP and albumin) markers in [3H]thymidine labeled ‘oval cells’ during the passage of the label into hepatocytes also suggested a precursor/product relationship [45], [46]. The pattern of ‘liver-enriched’ transcription factor expression (HNFs and CEBPs) in the liver during activation of ‘oval cells’ in the 2-AAF/PH model also mirrors the pattern expected during cellular differentiation [47]. Other studies have shown activation of c-kit [48], CD-34 [49], flt 3 receptor [50] and LIF [51] during ‘oval cell’ proliferation in the 2-AAF/PH model. These genes are also hyperexpressed in hematopoietic stem cells or their immediate derivatives. The stem cell-like properties of ‘oval cells’ activated in the 2-AAF/PH model are further supported by suppression of their proliferation in rats with deficient c-kit kinase activity [52].

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6. Other models of ‘oval cell’ activation 

Another rat model showing activation of liver progenitor (‘oval’) cells is d-galactosamine (D-galN)-induced liver injury. A single i.p. dose of D-galN (70–80 mg/100 g body weight) traps uridine nucleotides and UDP-glucose in hepatocytes, which leads to inhibition of RNA and protein synthesis and acute hepatocytic necrosis [53]. During uridine triphosphate deprivation, residual hepatocytes cannot proliferate and survival of the animal depends upon activation of immature hepatic epithelial cells that do not metabolize D-galN and are thus resistant to injury. AFP positive ‘oval cells’ begin to expand in the periportal area within 48 h after D-galN administration, extend into the liver parenchyma, form duct-like structures expressing AFP, albumin, GGT, and glucose-6-phosphatase, and then differentiate into mature hepatocytes [54], [55], [56]. In both the 2-AAF/PH and GalN liver injury models, the key factor in activating ‘oval’ or progenitor cells to proliferate is stimulation of liver regeneration under conditions in which hepatocyte proliferation is blocked.

In another model of liver regeneration with blocked hepatocyte proliferation, produced by treating rats with the DNA alkylating agent, retrorsine, followed by PH (see Section 10), both AFP+ ‘oval cells’ [57], [58] and AFP+ small hepatocyte-like progenitor cells [59] have been observed. Other models of ‘oval cell’ proliferation include allyl alcohol treatment of rats [60], naturally occurring copper toxicosis in the Long–Evans Cinnamon (LEC) rat [61], and Dipin [62], or DDC [63] treatment of mice. Dipin is another DNA damaging agent [62] and DDC causes hepatocyte apoptosis and massive bile ductular cell proliferation with differentiation of some of the bile ductular cells into hepatocytes [63].

Another unique model of gut-derived ‘oval cell’ activation is the pancreatic model originally described by Reddy and coworkers [64], in which rats are fed a copper-deficient diet also containing the copper chelating agent, trien. This treatment causes the pancreatic acini to undergo atrophy and pancreatic duct epithelial cells to proliferate; however, during their proliferation, the pancreatic duct epithelial cells begin to differentiate along the hepatocyte lineage pathway and express liver-enriched transcription factors and many liver-specific genes, including AFP and albumin, as well as others [65], [66]. After returning to a normal diet, activated pancreatic ‘oval cells’ differentiate further into hepatocytes [64], [67].

In both the 2-AAF/PH and D-galN models of liver injury/regeneration, AFP expression is markedly induced during ‘oval cell’ proliferation [46], [54], [55], [56]. AFP was originally discovered in the 1960s by Abelev [68]. This protein is very similar in structure to albumin and is located in tandem with albumin on the same chromosome, chromosome 5 in the mouse [69]. Expression of AFP occurs first in the yolk sac and then in the fetal liver in hepatoblasts, which, as indicated previously, are the precursors of hepatocytes and bile duct epithelial cells. AFP expression decreases as albumin expression increases in late gestation [70], [71], and it is turned off nearly completely after birth, only to reappear during liver carcinogenesis.

In the normal quiescent adult liver, AFP expression is very low and it is induced only marginally during liver regeneration following PH or acute CCl4 administration. In situ molecular hybridization studies performed by Fausto and coworkers and our group revealed a very low number of AFP positive cells in the normal adult liver (1/20,000), predominantly in the periportal region [72], [73], [74], [75]. Most of these cells have the morphologic appearance of ‘oval cells’ rather than hepatocytes, although small numbers of AFP positive hepatocytes are also observed. The AFP positive ‘oval cells’ are located either within the bile ducts or in the hepatic parenchyma immediately adjacent to the ducts [72], [73], [74], [75]. There is also no significant increase in the number of AFP positive ‘oval cells’ following PH or acute CCl4 administration. However, ‘oval cells’, expressing AFP, proliferate massively after D-galN [55], [56], [74], [75], 2-AAF/PH [46], retrorsine/PH [58] or allyl alcohol treatment [60].

From these various studies, the concept of a facultative or reserve stem cell compartment has been established. These rare cells most probably represent a vestigal population of foregut endodermal cells (or their immediate derivatives) and are activated to proliferate, differentiate into hepatocytes and bile duct cells and restore liver mass only under very specialized circumstances, i.e. when hepatocyte proliferation is markedly impaired. Interestingly, in fish, such as rainbow trout, which have a more primitive liver lobular structure, ‘oval cells’ proliferation occurs as part of the normal liver regeneration process [76].

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7. Relationship of ‘oval cells’ to the canals of Hering 

The canals of Hering were original identified by their namesake in 1866 as luminal channels linking the hepatocyte canalicular system to the biliary tree [77]. Transmission electron microscopy has identified small undifferentiated epithelial cells lining these channels [78], [79]. These cells are often in direct physical continuity with hepatocytes at one membrane boundary and bile duct cells at another boundary and together they form duct-like structures enclosing a lumen (i.e. the canal). Most models of the liver structure depict the canals of Hering as very limited and confined to the portal space. However, recent studies in humans using serial sections and CK-19 as a marker for ductular cells have demonstrated that the canals of Hering are much more elaborate than previously thought and extend far into the hepatic parenchyma [80], [81]. In tissue from a patient with massive hepatic necrosis, these structures contained epithelial cells with dual expression of bile ductular and fetal hepatocytic markers and were thus considered to be facultative hepatic stem cells [80].

Most recent studies in the rat 2-AAF/PH model, using a combination of confocal microscopy and electron microscopy, have demonstrated that proliferating ‘oval cells’ are indeed located in the canals of Hering, which are completely enclosed by a laminin containing basement membrane [82]. The proliferating cells are thought to pass into the parenchyma at discontinuities in the ductal basement membrane which may be a consequence of protease activity of progenitor cells when they come into contact with the basement membrane. After passage through the ductular basement membrane, the ‘oval cells’ continue to proliferate in the parenchyma in conjunction with stellate (Ito) cells and then differentiate into hepatocytes [82] or undergo apoptosis [83]. Therefore, since the very first cell undergoing proliferation and subsequent differentiation into hepatocytes is found initially in the canal of Hering, this structure must contain both stem cells and their initial progeny, the ‘oval cells’. However, other undifferentiated cells have also been found in the terminal bile ductules, which do not appear to give rise to ‘oval cells’ or hepatocytes [84]. A separate population of less well-differentiated progenitor cells has also been identified in the periductular space and these cells proliferate following allyl alcohol-induced liver injury [60], [85]. It has been suggested that these cells comprise a second progenitor or stem cell compartment and that they may originate from hematopoietic stem cells [86] (see Section 3). Another recent study has demonstrated that transplanted fetal liver epithelial cells proliferating in the periportal region of the parenchyma can traverse the limiting plate in the opposite direction, differentiate into bile duct cells and become incorporated into preexisting or new bile ducts [87]. These studies, together with the above [80], [81], [82], suggest that there may be bi-directional flow of liver progenitor cells across the biliary tree at the canals of Hering, and that these structures may have important functions not previously recognized.

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8. Studies with liver-derived ‘oval cells’ lines 

Permanent lines of liver epithelial cells have been established that have characteristics similar to ‘oval cells’ and are thought to be derived from epithelial progenitor cells [88], [89], [90], [91], [92]. The best characterized and most extensively studied of these cell lines is that derived by Grisham and coworkers, WB-F344 [90]. When WB-F344 cells were transduced with a β gal gene and then transplanted into the liver of syngeneic rats, the cells integrated into the hepatic plates acquired the morphologic appearance of mature hepatocytes and expressed liver-specific genes [93], [94], suggesting that WB-F344 cells have retained stem cell-like properties (see Section 12 on plasticity and the liver microenvironment). Other ‘oval cell’ lines have also been established which exhibit the ability to differentiate in culture into hepatocytes or biliary epithelial cells depending on the cell culture conditions, indicating dual lineage capability [95]. Epithelial cells from fetal liver have also been isolated and cultured, and these cells exhibit varying phenotypic properties of fetal hepatoblasts, hepatocytes or bile duct epithelial cells, also depending on the cell culture conditions [26], [30], [96], [97]. An epithelial cell line (HBC-3) has been established from ED9.5 mouse ventral foregut that also exhibits properties of liver stem-like cells [98]. Microarray studies have shown specific changes in gene expression during differentiation of HBC-3 cells in culture, including downregulation of genes in the Wnt/beta-catenin pathway, repression of downstream target genes, and altered expression of many cell cycle regulatory genes [99]. However, as mentioned previously, switching of cells between a hepatocytic or ductular epithelial phenotype has also been observed in cultures derived from mature hepatocytes. Therefore, specific conclusions regarding the lineage or stem cell potential of hepatic cells cannot be based solely on their morphologic appearance and gene expression profile in culture. This requires detailed analysis of cellular behavior after transplantation into the liver (see below).

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9. Cell transplantation models for liver repopulation 

Initial studies demonstrated that after transplantation to the spleen, hepatocytes migrate to the liver and become functionally incorporated into the parenchymal plates [100], [101], [102]. Transplanted hepatocytes were localized primarily in the periportal regions and it was quite surprising that these rather large cells (25–30 μm diameter) could actually cross the liver sinusoids. However, transplanted hepatocytes do not repopulate the normal liver significantly after two-thirds PH [103]. This is not surprising because the number of endogenous hepatocytes exceeds the number of transplanted cells by two to three orders of magnitude and if both populations respond similarly to a liver proliferative stimulus, then a proportionate expansion of both populations will not lead to significant repopulation by transplanted hepatocytes.

In the early to mid 1990s, some very unexpected findings were observed in two genetically modified mouse model systems which have revolutionized our thinking regarding the proliferative capacity of differentiated somatic cells. Sangren et al. [11] developed a transgenic mouse model to study protease function by inserting the urokinase plasminogen activator gene (uPA) into the liver under control of the albumin promoter to direct its expression exclusively in the liver. In alb-uPA transgenic mice, most of the uPA was secreted into the blood stream, where it was being studied as a thrombolytic agent. However, some of the protease remained in the liver and caused severe inflammation. The liver underwent continuous injury leading to extensive necrosis, and most animals died. However, some animals survived and in these animals large clusters of normal liver cells were observed, sometimes replacing almost the entire liver [11]. The normal cells had deleted the uPA transgene and then proliferated extensively in response to continuous liver injury. Isolated, single cell suspensions of genetically marked wt hepatocytes were then transplanted into uPA transgenic mice and these cells also repopulated the liver [12] and it was calculated that during liver repopulation, each transplanted cell underwent ∼12–14 divisions. This result dispelled the widely held concept that mature differentiated hepatocytes had only limited proliferative capacity.

A second model for liver repopulation, the fumaryl acetoacetate hydrolase (FAH) null mouse, was subsequently developed by Grompe and coworkers [13] to study the human disorder, hereditary tyrosinemia type 1 (HT1). In this model, tyrosine metabolism is blocked at the last step in tyrosine catabolism, conversion of fumarylacetoacetate to fumatate, acetoacetate and succinate. This leads to accumulation of the upstream metabolic intermediates that are toxic and cause continuous liver injury, chronic liver disease, cirrhosis and hepatocellular carcinoma [104]. Similar to findings in uPA transgenic mice, humans with HT1 occasionally show clusters of normal hepatocytes in an otherwise extensively diseased liver, again suggesting spontaneous genetic reversion with selective proliferation/survival of hepatocytes exhibiting a wt phenotype [105]. As observed with uPA transgenic mice, transplantation of wt hepatocytes into FAH null mice leads to extensive liver repopulation and restoration of normal liver architecture and function [13]. In the FAH null model, as few as 10,000 wt hepatocytes can be serially transplanted through seven generations of mice with total liver repopulation in each mouse [14]. If one assumes that all hepatocytes have equal proliferative capacity in these serial transplantation studies, it was concluded that hepatocytes have the ability to undergo a minimum of 77 cell divisions, equivalent to the most robust of stem cells. The innate regenerative power of fully mature parenchymal cells in the liver is, therefore, essentially infinite. The results of studies in uPA transgenic and FAH null mouse model systems have completely revolutionized our thinking concerning what is a stem cell and what might be possible in terms of organ repopulation by transplanted cells. Together with studies in bone marrow, brain, skeletal muscle and heart, this research has spawned an entirely new field of tissue engineering.

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10. Retrorsine/PH model for liver repopulation 

In both of the above models, a unique combination of experimental conditions in the host environment permits repopulation of the liver by transplanted hepatocytes: (1) the liver is under a constant state of massive injury/regeneration; and (2) the transplanted cells have an enormous selective advantage for survival compared to host hepatocytes. However, both uPA transgenic mice and FAH null mice are perinatal lethal and comparable conditions will be found only rarely in humans. An alternative strategy to achieve selective repopulation by transplanted cells would be to disrupt the proliferative capacity of host hepatocytes, and then transplant cells. Laconi et al. developed such a strategy by treating rats with retrorsine, a pyrrolizidine alkaloid that is taken up by the liver and metabolized in hepatocytes to a biologically active form, which alkylates cellular DNA and prevents hepatocytes from proliferating by disrupting their progression through the cell cycle [57]. Treatment with retrorsine creates a long-lasting mito-inhibitory environment in the liver, so that when retrorsine-treated rats are subjected to PH, endogenous hepatocytes are unable to proliferate but transplanted cells are unaffected.

Retrorsine/PH treatment was then combined with a cell transplantation model originally developed by Hixson, Faris and coworkers [106] in which dipeptidylpeptidase IV (DPPIV)+ wild type hepatocytes are transplanted into the liver of a DPPIV− mutant rat (see Fig. 2). The fate and phenotype of the transplanted cells is then determined by enzyme histochemistry. Transplanted hepatocytes become fully integrated into the hepatic plates without disturbing the surrounding parenchyma and form hybrid bile canaliculi with endogenous hepatocytes. In this model, in collaboration with Laconi, we showed near total (up to 99%) repopulation of the retrorsine-treated liver with adult hepatocytes [57]. The liver mass returned to near normal size and the transplanted cells were fully active biochemically and physiologically. The latter was demonstrated by correcting serum albumin levels in the genetically deficient Nagase analbuminemic rat, using the retrorsine/PH model [107].

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  • Fig. 2. 

    The Fischer (F) 344 rat model of liver repopulation by transplanted cells using dipeptidylpeptidase IV (DPPIV) as a detection marker. Isolated cells or cell lines from wild type F344 rats are transplanted to the liver of DPPIV− mutant F344 rats via injection into the splenic pulp or infusion into the portal vein. After engraftment and proliferation, transplanted cells can be detected and their phenotype (hepatocytic or bile duct epithelial) and amount of repopulation determined by enzyme histochemistry for DPPIV. An example of histochemical detection of DPPIV+ hepatocytes and bile duct structures in the liver 1 year after transplantation of fetal liver epithelial cells from a wt F344 rat is shown in the inset.

Irradiation has also been used as another method to block proliferation of endogenous hepatocytes and allow transplanted wt hepatocytes to fully repopulate the liver [108]. Apoptosis of DNA damaged endogenous hepatocytes probably plays a role in long-term liver repopulation in both the retrorsine and irradiation models [108], [109], [110], [111]. In this regard, injection of Fas antibody (Jo2) into mice has also been used to induce apoptosis in the host liver and to allow proliferation of transplanted hepatocytes transduced with an adenovirus containing the anti-apoptotic gene, bcl2 [112]. Using repeated antibody injection over a 10-week period, transplanted hepatocytes transduced in vitro with a retrovirus containing a bcl2 gene can repopulate up to 85% of the host liver [113]. Bcl-xL transgenic hepatocytes can also repopulate up to 10% of the normal mouse liver, using anti-Fas antibody selection [114]. Other models in which hepatocyte transplantation has been used successfully are the LEC rat, an animal model for Wilson's disease [115], [116], and the mdr-2 null mouse, a model for progressive familial intrahepatic cholestasis [117]. In the latter case, feeding the mice a 0.03% cholate supplemented diet was used to augment liver repopulation [117].

Rogler and colleagues have developed an additional model for liver repopulation in which recipient mice can accept xenographs. This required cross-breeding of uPA transgenic mice (with constant liver injury) to Rag(−/−) mice, which are T and B cell-deficient and are immunotolerant to transplanted cells, even across species barriers. In uPA(+/+)/Rag2(−/−) mice, transplanted woodchuck hepatocytes can repopulate the host liver [118]; human hepatocytes can also proliferate and partially repopulate the liver of uPA(+/+)/Rag2(−/−) mice [119]. The NOD/scid mouse, which is even more immunotolerant than the Rag2(−/−) null mouse, has also been cross bred to the uPA(+/+) transgenic mouse, and this model also accepts human liver cell xenographs [120]. Human hepatocyte-transplanted uPA(+/+)/Rag2(−/−) mice have been successfully infected with human hepatitis B virus (HBV) [119], and human hepatocyte transplanted uPA(+/+)/NOD/scid mice have been infected with hepatitis C virus (HCV) [120]. This provides a unique opportunity to study antiviral therapies for HBV and HCV infection in animal systems, which cannot normally be infected with these viruses.

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11. Transplantation of ‘oval cells’, cell lines and fetal liver epithelial (stem/progenitor) cells 

With the development of an effective hepatic cell transplantation and detection system, it was hoped that liver repopulation could be achieved by transplanting hepatic progenitor (‘oval’) cells or cell lines. Since these cells or cell lines would be expected to have a higher proliferative rate or capacity than mature hepatocytes and should have the ability to differentiate into both hepatocytes and bile duct cells, it was hoped that effective liver repopulation and generation of complete new liver lobules could be achieved in the absence of harsh liver treatments or strong selection conditions, as needed for adult hepatocytes. Initial studies were conducted with ‘oval cells’ obtained from D-galN-treated rat liver and Cu2+-deficient rat pancreas, and showed differentiation of ‘oval cells’ into hepatocytes and incorporation of transplanted cells into the hepatic cords [121]. However, there was only very limited proliferation of these cells, even after two-thirds PH. With the WB-344 liver epithelial (progenitor) cell line, there was also differentiation of transplanted cells into hepatocytes, but proliferation was again quite limited [93], [94], [122]. In contrast, a small percentage of FAH null mice transplanted with primary cells isolated from the normal pancreas differentiated into hepatocytes and extensively repopulated the liver [123]. This suggested that strong selection is still necessary for liver repopulation by hepatic progenitor cells or cell lines. However, different results were obtained with preparations of rat fetal liver epithelial cells.

We hypothesized that repopulation might be more effective with bipotential epithelial progenitor cells isolated from fetal liver prior to commitment, i.e. before ED16 compared to adult hepatocytes. Since fetal liver epithelial cells are rapidly proliferating between ED12 and ED16, we reasoned further that these cells might exhibit other stem cell-like properties and effectively repopulate the normal liver after their transplantation. We, therefore, isolated cells from ED12–ED14 fetal rat liver and studied their expression of hepatocytic markers, AFP and albumin, and the biliary epithelial cell marker, CK-19 [124].

We determined that there are at least three distinct subpopulations of epithelial progenitors within the fetal liver at ED12–ED14, potentially bipotent cells simultaneously expressing AFP, albumin, and CK-19 and unipotent cells expressing either albumin/AFP (hepatocyte-specific) or CK-19 (biliary epithelial cell-specific) [124]. After their transplantation, ED14 fetal liver epithelial cells proliferated in the host liver and new hepatic cords and bile ducts were formed. With ED12–ED14 fetal liver epithelial cells, we observed up to nine to ten cell divisions in very large clusters of transplanted cells at 6 months after transplantation. Using double label immunohistochemistry/in situ hybridization, transplanted cells were shown to be still proliferating 6 months after transplantation, and we could achieve up to 10% liver repopulation [87]. Under the same conditions, mature hepatocytes showed proliferative activity for only 2–4 weeks following their transplantation, and there was no significant liver repopulation. Most of the liver repopulation with fetal liver epithelial cells occurred between 2 and 6 months following transplantation, long after the stimulus for cell proliferation (two-thirds PH) had abated. Both new hepatic plates and bile ducts were formed by transplanted cells and the majority of the proliferating cell clusters contained both hepatocytes and bile ducts, suggesting bipotency of cells producing these clusters. Thus, fetal liver epithelial cells transplanted into the normal adult rat liver exhibited three major properties of stem cells; namely, bipotency, proliferation for an extended period and long-term tissue repopulation.

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12. Plasticity and the role of the liver microenvironment 

As evident from recent studies in brain, bone marrow, liver and pancreas, somatic cells exhibit considerable flexibility in their gene expression properties and cells derived from one organ can differentiate into cells of another organ after their engraftment into the latter site (see Fig. 3). This property is referred to as plasticity. In the liver, when fetal liver stem/progenitor cells engraft in the portal space, they differentiate into bile duct cells and when they engraft into the parenchyma, they differentiate into hepatocytes [124]. Another example of strong phenotype control exerted by the liver microenvironment is seen with chemically transformed WB-344 cells. When these cells are transplanted into the subcutaneous tissue, they are highly malignant. However, when they are transplanted into the liver of an adult rat, they stop proliferating and differentiate into mature hepatocytes [93], [94]. Furthermore, when WB-344 cells are transplanted into the liver of aged rats, they remain transformed [125]. Perhaps the most spectacular observation regarding plasticity and control exerted by the tissue microenvironment is the recent finding that WB-344 cells can differentiate into other tissue-specific phenotypes when transplanted into other organs, including myocytes when transplanted into the heart [126], hematopoietic cell lineages in the bone marrow and glandular epithelial cells in the prostate [127]. Thus, the tissue microenvironment ‘instructs’ WB-344 cells to acquire a specific differentiation state [127].

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  • Fig. 3. 

    Examples of plasticity of somatic stem cells or stem/progenitor cells isolated from specific organs as determined by their ability to differentiate into different tissue-specific phenotypes when transplanted into different organs.

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13. Hematopoietic stem cells and liver repopulation 

Until recently, it was thought that stem cells in adult organs (somatic or tissue-determined stem cells) were restricted to a single embryonic germ layer, but recent studies with hematopoietic and brain stem cells indicate differentiation across germ cell layers [128], [129], [130], [131], [132], [133], [134], [135], [136], [137]. As part of these studies, Petersen et al. [132] and Theise et al. [133] transplanted either crude bone marrow or purified hematopoietic stem cells into lethally irradiated rats and mice, respectively, and searched for donor-derived cells in the liver. In both instances, donor cells identified as wt DPPIV+ cells in DPPIV− F344 rats or Y chromosome positive male cells in female rats or mice differentiated into hepatocytes. Liver repopulation in these systems was estimated to be ∼0.1% by Petersen and 1–2% by Theise. Lagasse et al. [134] conducted similar studies with purified hematopoietic stem cells in FAH null mice. Repopulation was much higher in this case (up to 30% at 6 months), because selection for transplanted cells could be performed by first maintaining mice on 2(2-nitro-4 trifluoromethylbenzoyl)-1,3 cyclohexane dione (NTBC), which blocks accumulation of toxic tyrosine catabolites in the liver and permitted hematopoietic cells to seed both the bone marrow and liver, and then cycling the animals off and on NTBC twice to produce massive liver injury, so that transplanted cells that had seeded the liver would be stimulated to proliferate and differentiate into hepatocytes.

In human studies by Theise et al. [135] and Alison et al. [136], the presence of Y chromosome positive hepatocytes has been found in the liver of female recipients of male bone marrow cells and in male recipients of orthotopic liver transplants from female donors. The presence of donor-derived bile duct cells has also been reported [135], [136] and in one of the human studies, estimates of liver repopulation by hematopoietic cells were as high as 35–40% [135]. In most recent studies in mice [137], donor-derived epithelial cells have been identified in multiple organs, including the lung, gastrointestinal tract, liver (bile ducts), and hair follicles, after transplantation of a single hematopoietic stem cell into sublethally irradiated animals. The implications of these findings are enormous, although it is unclear at present whether bone marrow transplantation will become a practical method for liver repopulation in most clinical settings.

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14. Enriched populations of liver stem/progenitor cells 

The first studies reporting enrichment of fetal liver stem/progenitor cells were those of Reid and coworkers [97] using panning and fluorescence activated cell sorting methods with monoclonal antibodies reacting with cell surface proteins expressed on oval cells and bile duct epithelial cells. However, even today, specific selection markers for fetal liver stem/progenitor cells have not been identified. Fetal liver cells have been sorted by Suzuki et al. [138] for those that are positive for expression of α6 (CD49f) integrin and β1 (CD29) integrin, which are known to be present on hepatocytic cells but not on hematopoietic cells, and negative for CD45 and Ter119, which are specific markers for hematopoietic cells. Interestingly, cells showing the greatest enrichment for liver progenitor cells in a colony forming assay were negative for c-kit [138], which is a positive sorting marker for hematopoietic stem cells and is also detected in liver ‘oval cells’ [48]. In contrast, c-kit (or CD34) positive selection has been used by Strain and coworkers [139] to enrich for human liver stem-like progenitor cells that can differentiate in culture into biliary epithelial cells.

Kubota and Reid [140] have used entirely different genes (MHC class 1, OX18 and ICAM-1) to select for ED13 rat fetal liver cells that in culture express phenotypic markers for hepatocytes (AFP and albumin) and bile duct epithelial cells (CK-19). Most recently, Demetriou and coworkers [141] have isolated beta-2 microglobulin (β2m)−/Thy-1+ cells from the bone marrow that express liver-specific genes, and after intraportal infusion, these cells differentiate into mature hepatocytes. Interestingly, in several of these studies [138], [139], [140], [141], cells with similar marker gene expression patterns were identified or isolated from the normal adult liver, albeit at reduced numbers, suggesting that tissue-specified stem/progenitor cells may be present in the mature organ. Transplantation of these cells will hopefully identify those that are true stem cells with the greatest potential for liver repopulation.

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15. Summary 

During the past decade, it has become clear that the mammalian liver contains stem/progenitor cells. These cells are derived embryologically from the foregut endoderm (see schematic diagram, Fig. 4) and seed the bone marrow, giving rise to hematopoietic stem cells. They also generate undifferentiated epithelial cells which are maintained in the canals of Hering, situated at the terminal webs of the bile ductules. Both hematopoietic stem cells and canals of Hering cells can give rise to hepatic ‘oval cells’, which can differentiate into hepatocytes and bile duct epithelial cells and are thus considered stem/progenitor cells. A role for stem/progenitor cells in maintaining the liver parenchyma during normal liver cell turnover or in restoring liver mass during liver regeneration has not been established. However, these cells can be induced to proliferate under circumstances in which the proliferative capacity of mature hepatocytes is blocked. Both hepatocytes and stem/progenitor cells can repopulate the liver under highly selective conditions of severe continuous liver injury or disruption of endogenous hepatocytes proliferation, but only stem/progenitor cells can do this in a normal liver environment. Recently, it has been discovered that hematopoietic stem cells can migrate to the liver and differentiate into hepatocytes in both rodent models and in humans; however, a role for these cells in liver regeneration or repopulation remains to be established. All of these findings provide hope that under selected circumstances cell transplantation will become a therapeutic reality in the treatment of inherited or acquired chronic liver diseases.

  • View full-size image.
  • Fig. 4. 

    Current status of our understanding of the origin of different endodermal-derived cell populations in the liver and their role in liver development and maintenance of liver mass.

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Acknowledgements 

The authors would like to thank Anna Caponigro and Emily Bobe for secretarial assistance in preparing this manuscript and our many students and postdoctoral fellows for their contributions to studies from our laboratory cited in this review.

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PII: S0168-8278(02)00013-2

doi:10.1016/S0168-8278(02)00013-2

Journal of Hepatology
Volume 36, Issue 4 , Pages 552-564, April 2002

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