Hepatic stem cells in liver regeneration SNORRI S. THORGEIRSSON Laboratory of Experimental Carcinogenesis, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA The key to understanding liver development, hepato- carcinogenesis, and the ability to regenerate lies in es- tablishing the existence of a liver stem cell population (or populations). By now we should be readily con- vinced that hepatocytes themselves have the ability to exit quiescence and replicate. But the fundamental concept that adult mammalian livers contain stem cells that can replace hepatic parenchyma has been the sub- ject of an ongoing controversy, which continues to be fueled by debate over the role of stem cells in liver growth and repair. Early studies of liver cell lineages assumed the role of stem cells as progenitors for both normal and transformed hepatocytes, including hepato- cellular carcinoma. In recent years, significant ad- vances have furthered our understanding of liver stem cell biology: we are beginning to realize the role of growth factors, transcriptional activators, and cell-cell communication in the regulation of stem cell activa- tion. With the development of in vitro assays, in vivo genetic marking studies, and identification and charac- terization of the critical trans-activating factors respon- sible for cell lineage, we are able to address these issues. We now recognize and distinguish the unipo- tential stem cell system of the hepatocyte from the bipotential hepatoblast from the multipotential facul- tative stem cell system or oval cell. Do you believe that the adult liver contains stem cells? After all, we can induce the formation of pancreatic acini and intes- tinal metaplasia in hepatic tissue. Moreover, pancreatic ductules can give rise to cells that exhibit all the phe- notypic traits and biological properties of differentiated hepatocytes. Most liver injuries are not due to simple 70% partial hepatectomies, but rather to toxic injuries from viruses, chemicals, and other agents. It is impera- tive to understand the role of stem cells in order to ul- timately control the ability of the liver to regenerate. I believe that adult liver stem cells exist, and I wager that you will, too, after reading this last review in the series. -Clifford J. Steer, Coordinating Editor ABSTRACT The concept that the liver contains epithelial celia that share some of the major proper- ties of stem cells of the well-characterized, stem cell-fed lineages found in bone marrow, intestinal epitheium, and epidermis is now well supported. Nevertheless, the population dynamics of the major types of liver epithelial cells, hepatocytes, and bile epithelia display a strildng difference from the popu- lation dynamics of the classic stem cell systems. The focus of this review is on recent studies of the activation and expansion of liver stem cells in vivo and the role these cells may play in regeneration of the liver. The requirement for a selective and sus- tained expression of growth factors during the early stages of stem cell activation is highlighted. In addi- tion, results are presented supporting the hypothesis that after loss of liver mass, both the quiescent stem cells as well as the residual differentiated hepatocytes and bile duct epitheial cells are activated to prolif- erate. However, significant contribution of the stem cells to the regeneration process only occurs under circumstances in which the residual differentiated cells are functionally compromised and/or cannot proliferate.-Thorgeirsson, S. S. Hepatic stem cells in liver regeneration. FASEBJ. 10,1249-1256(1996) Key Words: growth factors . liver stem cell biology stem cell activation hepatocytes bile duct epithelial cells . liver paren- chyma oval cells - partial hepatectomy . prolferwion dfferen- tiation THE UVER IS MITOTICALLY a quiescent organ in adult hu- mans and animals. In spite of this slow cellular turnover, the two parenchyinal cell lineages, hepatocytes and bile epithelial cells, have a remarkable capacity to meet re- placement demands of cellular loss. The best example of the capacity of adult hepatocytes and bile epithelial cells to proliferate is seen after partial hepatectomy (PH)2 in rats and mice, in which the compensatory hyperplasia of these cells in the remaining lobes restores the liver mass. Moreover, this process of liver regeneration can be re- 1Addresa correspondence to Dr. Thorgeusson, at:National Cancer Institute, Bldg. 37, Room 3C28, 37 ConventDr.MSC4255, Bethesda, MD 20892-4255 USA. 2Abbreviations: aFGF, acidic fibroblast growth factor, TGFa, trans- forming growthfactor-alpha; HCF, hepatocyte growthfactorSCF, stem cellfactor; AFP, a-fetoprotein; AAF, 2-acetylaminofluorene; EGF, epi- thelial growth factor. 1250 Vol. 10 September 1996 The FASEBJournal THORGEIRSSON peated several times in experimental animals. The in- creased use and success of liver transplantation in clini- cal medicine have shown that these animal models correctly reflect the capacity of the human liver to regen- erate (1). Due to this well-established trait of adult hepa- tocytes and bile epithelial cells to repeatedly regenerate the liver after either surgically or toxically induced loss of the tissue, the existence of hepatic stem cells that may participate in liver regeneration has been somewhat con- troversial. The major part of the hepatic stem cell contro- versy may, however, be due to the failure to recognize that the adult organism contains many kinds of stem cells, and that these may exist at different stages of dif- ferentiation and have very different capacities for gener- ating multilineage progeny. Self-maintenance is a fundamental and common trait of all stem cells. A cell population that has an extensive self-maintaining capacity is perhaps the only definition that applies to all stem cells (2). In this context, the adult liver, having the extensive capacity for maintaining parenchymal cell number throughout the life span of the organism, can be viewed as a single lineage stem cell system in which the hepatocyte is the stem cell. Recent data from hepatic cell transplantation experiments in a transgenic mouse model (3) have demonstrated the tre- mendous growth potential of adult hepatocytes (12-16 doublings per donor cell), further supporting the notion of the liver parenchyma as a single lineage (or unipotential) stem cell system. On the other hand, if the classic defi- nition of stem cells proposed by Potten and Loeffler (4) is used, one would differentiate between actual stem cells, potential stem cells, and committed stem celLs. Actual stem cells are defined as undifferentiated cells capable of 1) proliferation, 2) self-maintenance, 3) production of a large number of differentiated progeny, 4) regeneration of the tissue after injury, and 5) flexibility in the use of these options. Potten and Loeffler (4) consider that po- tential stem cells are latent or quiescent counterparts of the actual stem cells that may be reactivated to become functional stem cells, and that committed stem cells may correspond to the dividing transit cells (e.g., dividing- transit enterocytes), sharing with the terminally differenti- ated cells the ability to execute tissue-specific functions. Applying the definition of stemness used by Potten and Loeffler (4), the hepatocytes appear to be committed stem cells that are normally quiescent, but can be acti- vated to produce progeny whose only differentiation op- tion is hepatocytic. The fact that the early fetal hepatocytes or hepatoblasts are progenitors for both adult hepatocytes and bile epi- thelial cells suggests that the hepatoblasts are at least bipotential precursors (5). The question then arises whether either or both of the cell lineages derived from the hepatoblast retain the bipotential capacity of the precursor cells. There is at present no substantial evi- dence indicating that adult hepatocytes are more than a unipotential committed stem cell system. The possible exception to this generalization is the concept of ductular metaplasia or transformation of hepatocytes into ductules. This topic has been extensively discussed by Desmet and his colleagues (for review, see ref 6). Studies of chronic cholestatic diseases in humans using both enzyme histo- chemistry and cytokeratin immunohistochemistry have provided evidence for gradual transformation of hepato- cytes into bile duct-type cells (7). However, evidence showing that these bile duct-type cells also exhibit func- tional characteristics of normal bile epithelium is still lacking. It is therefore questionable, but still possible, that ductular metaplasia of hepatocytes seen in choles- tatic diseases may reflect a multipotential (or at least bipotential) capacity of the hepatocytes. In contrast to the hepatocyte system, there is strong evidence indicating that the bile epithelium harbors a compartment of cells that can produce progeny (oval cells) capable of differentiating into several lineages in- cluding bile epithelia, hepatocytes, intestinal epithelia, and possibly exocrine pancreas (8-11). Also, as pointed out by Sell (10), there may exist a periductal system of stem cells capable of differentiating into all the hepatic lineages. These ductular/periductal cells are frequently referred to as the hepatic stem cell compartment. There is also strong evidence that oval cells, the early progeny from the hepatic stem cell compartment (8-11), are more primitive and more poorly differentiated than are hepato- cytes or bile epithelial cells, and the multiple differentia- tion options of oval cells are firmly established. Therefore, oval cells meet more of the stem cell criteria of Potten and Loeffler (4) than do hepatocytes: 1) they are poorly differentiated, 2) they demonstrate extensive proliferation, and 3) they have multiple differentiation options, including hepatocytes and bile ductular epi- thelial cells. Whether oval cells possess the capacity to proliferate and differentiate throughout the life span of an animal is still uncertain, as appropriate studies have not been done. Nevertheless, the immediate precursors of oval cells appear to approximate Potten and Loefflers definition of potential stem cell, whose functions of stem- ness are latent but can be reactivated by appropriate con- ditions. This definition of a potential stem cell corresponds to the hypothesis of facultative liver stem cell proposed earlier by Grisham (12), whose activation he predicted would lead to the proliferation of oval cells. In light of these observations, I should like to propose that the liver be viewed as composed of two stem cell systems: the unipotential committed stem cells (hepato- cytes) and the multipotential nonparenchymal epithelial (ductular) systems (Fig. 1). A comprehensive discussion of the founding of hepatocytes and bile duct epithelial cells from hepatoblasts has recently been published (5), and will not be included in this review. HEPATIC STEM CELL COMPARTMENT The existence of cells with stem-like properties that may differentiate into hepatocytes was first postulated nearly HEPATIC STEM CELLS 1251 Figure 1. Schematic diagram showing the development of cell lineages in the liver. 60 years ago by Kinosita (13), and later suggested as a possibility by other investigators (14-16). Wilson and Leduc (16) postulated, based on experiments involving liver regeneration in the mouse after chronic injury, that indifferent cholangiole cells proliferated to form oval cells that might differentiate to form both hepatocytes and biliary epithelial cells. The major support for the existence of hepatic stem cells has, perhaps not surprisingly in light of the earlier work by Wilson and Leduc (16), come from extensive TABLE 1.Markers commonly used to assess differention and totracelineageofliverepithelial cell? Markers Hepatoblasts Oval cells Hepatocytes Bileductcells References CK7 - - - + 17,56,57 CK8 + + + + 17,56 CK18 + + + + 17,56 CK19 -1- + - + 17,56,58 CK14 [+] [+1 - - 17,59,60 1UJ3 + +1- + - 17,56,58 AFP + + - - 17,56,58 GGT + + - + 17,56 OV-6 (+) + . + 61 OV-1 (+) + - 4- 61 BDS7 + 4- - + 58,62 BD1 - - - + 63 BPC5 + - - - 64 HES6 - - + - 58,62 OC.2 4- + - 4 27,65,66 OC.3 + + - 4- 27,65,66 H.1 - - 4 - 27,65,66 H.2 +(Transient) - - - 27,65,66 HBD.1 4- - 4- 4 27,65,66 A6 AFro,,, Crishain +1- and Thorgeirsson (ref 5)Wi + th permission. - + 67,68 1252 Vol. 10 September 1996 The FASEBJournal THORGEIRSSON Activationof Facultative jj StemCells Figure 2. Schematicrepresentation illustrating thehypothesis that oval cells develop from facultative stem cells located in the canal of Hering (biliary ductule). The scheme shows thedevelopmentaloptionsthatoval cellspossess, including differentiation into hepatocytes and biliary epi- thelium,formation ofintestinal epithelium, and pancreatic acinarepithe- hum by differentiation and/or metaplasia, and death. studies in hepatic carcinogenesis (9-11, 17). Several im- portant experimental models, mostly in the rat, have emerged from these studies. The most commonly used models in the rats are produced by 1) treatment with azo- dyes (18), 2) feeding of a choline-deficient diet, with or without supplements of ethionine (19) or 2-acety- laminofluorene (20), 3) treatment with 2-acetylaminofluo- rene and partial hepatectomy (AAF/PH) (21), and 4) treatment with D-galactosamine (22). Central to all these experimen.tal models is the extensive destruction and/or compromised function of hepatocytes, coupled with the apparent inability of the residual hepatocytes to prolifer- ate. A common cellular response in rats subjected to the experimental systems listed above is the proliferation of small periportal cells with scant cytoplasm and ovoid nu- clei, which have been termed oval cells (17, 23, 24). The existence of similar cells has been reported in the human liver (25, 26). These oval cells are thought to represent a progeny of the hepatic stem cell compartment, and at least in some instances to be a precursor for the hepatic tumors (10, 11, 27). The precise anatomical location of the hepatic stem cell compartment in normal liver is still unclear, but present data suggest that the terminal duc- tule cells connecting the canals of Hering with the bile canaliculi and/or a distinct population of periductal cells constitute the hepatic stem cell compartment (vide infra; 9-11, 16, 28, 29). Recent studies in the rat liver have shown that oval cells are capable of differentiating, in ad- dition to bile epithelium, into at least two lineages in vivo, including hepatocytes (24, 30) and intestinal type epithelium (31). Furthermore, isolated oval cells in cul- ture can be induced to differentiate into both hepatocyte- like and biliary type of cells (32-34). Markers commonly used to assess differentiation and to trace lineages of oval cells are listed in Table 1. These data and other results not reviewed here have supported the notion that oval cells have lineage options similar to those displayed by hepatoblasts in early stages of liver development. As such, the oval cells can be regarded as bipotential pre- cursors for the two hepatic parenchymal cell lineages. However, oval cells may, particularly when the hepatic microenvironment is drastically disrupted, exhibit a ca- pacity to differentiate into nonhepatic lineages (Fig. 2) (35). LIVER REGENERATION VIA HEPATIC STEM CELLS The AAF/PH model in the rat will be used to illustrate both the cellular biology and growth factor/receptor sys- tems involved in stem cell-energized liver regeneration. In this experimental system, a rapid and extensive prolif- eration of oval cells takes place after PH, first in the periportal area; later, these cells expand into the liver ac- inus and differentiate into small basophilic hepatocytes (Fig. 3) (24, 36). The powerful activation of the stem cell compartment seen in the AAF/PH model is a conse- quence of a close to complete mitoinhibitory effect of AAF on the adult rat hepatocytes that prevents the regen- eration from the remaining liver tissue (21, 24). LOCALIZATION OF HEPATIC STEM CELLS It has been established that proliferation of desmin-posi- tive Ito cells is closely associated with the early stages of oval cell proliferation in the AAF/PH model (37). Early population of oval cells can be identified by the use of the monoclonal antibody OV-6 (37) and distinguished Figure 3. Time course of the development and expansion of the oval cell population in the AAF/PH model. Oval cells are identified by y-GT staining. The time of partial hepatectomy is day 0 (Od); 6d, 9d, and 13d indicate number of days after the operation. Note the rapid expansion of the oval cell population after 6 days and the formation by 13 daysofthe basophihic focus composed of diphoid hepatocytes that are losing the y-GT staining. ///ThNN HGF aFOF TGFa TGFI1 SCF c-kit Stem Transitional Cells HEPATIC STEM CELLS 1253 - 2 9 II 14y#{149} Differentiated Hepatocytes Figure 4. Schematic diagram showing the temporal relationship between expression of AFP, growth factors, and differentiation stages of the hepatic stem cell progeny during liverregeneration intheAAF/PH model. from proliferating desmin-positive Ito cells (38). Results from a detailed time course study of activation of hepatic stem cells in the AAF/PH model, utilizing a combination of immunohistochemistry with OV-6 and desmin antibod- ies and autoradiography after [3H]thymidine administra- tion shortly after the PH, indicate that the earliest population of proliferating OV-6 positive cells is located in the small bile ductules (38). In addition, these early populations of OV-6-positive cells express albumin and a-fetoprotein (AFP) (36). These data clearly show that the majority of thymidine-labeled, OV-6-positive cells first observed after PH in the AAF/PH model reside in the bile ductules. Moreover, at the time when few of the OV-6-positive cells in the large bile ducts become la- beled with thymidine, the ductular-derived OV-6-positive and thymidine labeled oval cells expressing both albu- min and AFP have already started to infiltrate into the liver acinus (36, 38). It therefore seems likely that the major source of oval cells, at least in the AAF/PH model, is derived from the lining cells of the biliary ductules and that these cells constitute the dormant/facultative hepatic stem cell compartment. GROWTH FACTORS INVOLVED IN HEPATIC STEM CELL ACTIVATION AND EXPANSION During normal hepatic regeneration as well as during re- newal from the stem cell compartment, several growth factors appear to affect the proliferation and differentia- tion of hepatic cells (37, 39, 40). The question therefore arises as to whether the same growth factors known to be involved in normal hepatic regeneration are also involved in regeneration from the stem cell compartment. There are three primary growth factors associated with normal liver regeneration: transforming growth fac- tor-alpha (TGF-a), hepatocyte growth factor (HGF), and acidic fibroblast growth factor (aFGF) (41). Each of these growth factors is also capable of inducing replication of primary hepatocytes in vitro (41). In addition, transform- ing growth factor-beta 1 (TGF-1) is also expressed dur- ing hepatic regeneration, and it has been proposed that TGF-1 may provide at least part of the negative growth signals controlling liver size after the compensatory hy- perplasia that occurs after loss of liver mass (42). The first cells entering DNA synthesis after PH in the AAF/PH model are the OV-6 and desmin-positive bile duetular and Ito cells, respectively, in the periportal area (38). Coincident with the appearance of these cells, an increase in the expression of TGF-a, HGF, and TGF-1 is observed, whereas increased expression of aFGF is first seen 24 h later (29). All the growth factors are then expressed at high levels throughout the period of expan- sion and differentiation of the oval cells and return to lev- els seen in normal liver at the end of the regeneration process. The cellular distribution of the growth factor transcripts differs: TGF-a and aFGF transcripts are found both in Ito cells and oval cells (37, 40), whereas the HGF transcripts are only found in Ito cells (39). The TGF-1 transcripts are located mainly in Ito cells, but the early population of oval cells also contain the TGF-f31 transcripts (43). The cellular distribution of the tran- scripts for all the receptors corresponding to the growth factors has revealed that all are located on oval cells (22, 44, 45). These data suggest that the same primary growth factors involved in liver regeneration from existing differ- entiated parenchyma are also involved in regeneration from the stem cell compartment. Recently, a novel liganci/receptor system, the stem cell factor (SCF)/c-kit system, which may be uniquely in- volved in the earliest stages of hepatic stem cell activa- tion, was discovered (46). In the AAF/PH model, the expression of both SCF and c-kit is seen before the ex- pression of AFP (46), and the levels of both the SCF and the c-kit transcripts decline before those of TGF-a, aFGF, HGF, and TGF-31 (46). It has also been shown that in contrast to TGF-a, HGF, aFGF, and TGF-f31, the SCF/c-kit system is only slightly and transiently activated in regeneration after PH in normal liver (46). The SCF/c- kit signal transduction system is believed to play a funda- mental role in the survival, proliferation, and migration of stem cells in hematopoiesis, melanogenesis, and gameto- genesis (47). It appears that in all cases, SCF and c-kit are involved in the early stages of stem cell activation. In the hemopoietic stem cell system, it has also been dem- onstrated that SCF in combination with selective multipo- tential colony-stimulating factors can influence the relative frequency of progenitor cells committed to vari- ous lineages (48). Whether the SCF/c-kit system in the early hepatic stem cell population interacts with other he- patic growth factors so as to influence the frequency of lineage commitment of progenitor cells is not known at present. However, the hepatic expression pattern and cel- lular location of the SCF/c-kit system indicate that this 1254 Vol. 10 September 1996 The FASEBJournal THORGEIRSSON signal transduction system is required only during the early activation and transitional phase of the oval cell dif- ferentiation. Once the oval cells have differentiated into the small basophilic hepatocytes, the expression of both SCF and c-kit is abolished (49). This concept is sche- matically illustrated in Fig. 4. PARTICIPATION OF LIVER STEM CELLS IN REGENERATION After the hepatocyte population is reduced by, for exam- ple, PH, the residual hepatocytes proliferate promptly, continue to cycle until the deficit is repaired, and con- tinue to function while proliferating. Under these condi- tions, no apparent contribution to the regeneration process is provided by the stem cell compartment. Acti- vation of oval cell proliferation and differentiation by in- jury, which is more severe and/or qualitatively different from the simple loss that triggers only hepatocyte prolif- eration, results in transient reestablishment of a hepato- cytic lineage that has all the characteristics of a potential or facultative stem cell system (5). Cells in the normally quiescent stem cell compartment are activated to produce poorly differentiated oval cell progeny. Oval cells prolif- erate extensively to yield a large population of cells that migrate throughout the parenchyma, some of which differ- entiate as they migrate. Hepatocytic progeny of oval cells merge into the functional compartment of mature hepato- cytes and help restore the parenchyma. Similar to the generation of new hepatocytes after simple loss, the pro- duction of hepatocytes via the stem cell (oval cell) mechanism is also episodic and transient. These two dis- tinct mechanisms of hepatocyte formation are both sub- jected to several points of stringent control (5). Controls are required to regulate the reinitiation of hepatocyte for- mation from the normally quiescent hepatocytes, as well as to regulate the activation of potential stem cells that energizes cell flow through the entire lineage. Although the controls may differ between the two mechanisms of hepatocyte formation, it is probable that both pathways are simultaneously activated after loss of liver mass, in- cluding that after simple PH. One of the earliest phenotypic indications of liver stem cell activation is the expression of AFP (5). A transient expression of AFP is also seen after simple PH and simi- lar to that seen in stem cell activation in the AAF/PH model, the AFP transcripts are located in the bile ductu- les (36, 50). Expression of both SCF and c-kit is also transiently elevated after standard PH (46). However, there is no evidence indicating that the stem cell-derived hepatocytes significantly contribute to regeneration of liver mass after simple PH (5). These observations sug- gest that the activation, and in particular, the expansion of liver stem cells, are stringently controlled during hepa- tocyte-driven liver regeneration. Recent studies of the early activation of oval cell pro- liferation in the AAF/PH model have provided important clues toward defining the mechanism controlling the early expansion of oval cells (51, 52). It has been shown that a low dose of AAF (and its analogs 2-AF and N-OH-2- AAF) elicited, in the absence of PH, a mitogenic re- sponse in ductular and periductular cells within 24 h after administration. The compounds also induced the ex- pression of HNF1, HNF3y, AFP, and albumin in the ductular cells, similar to the observations shown earlier in the complete AAF/PH model (53). In contrast, initia- tion of bile duct proliferation by ligation of the common bile duct had no effects on the expression of these genes in ductal cells. In addition to eliciting a mitogenic re- sponse, administration of AAF also induced apoptosis of cells in the portal areas, a process that contributed to the overall retention of liver morphology. As no significant increase in the cellularity of the portal areas was ob- served at this time, it is probable that an equilibrium be- tween mitosis and apoptosis exists to maintain a constant number of ductal cells. This observation is in striking contrast to the condition seen in the complete AAF/PH Figure 5. Schematic depiction of liver regeneration illustrating the contribution of hepatic stem cells. Induction of reparative renewal of the epithelial cell populations in the liver after both simple PH and certain types of more severe liver injury involves activation of both the normally quiescent hepatocytes and the potential (facultative) liver stem cells. As discussed inthe text, the essential requirement for the sustained activa- tion of the stem cells and their progeny required for generating the differentiated cell lineages needed for liver regeneration is a prolonged expression of a set of growth factors, including those known to be involved in liver regeneration after simple partial hepatectomy. HEPATIC STEM CELLS 1255 model, in which the oval cells continuously proliferate and expand into the liver acinus. Furthermore, admini- stration of AAF alone results in only a modest and tran- sient increase in expression of hepatotrophic growth factors, again in contrast to the intense and sustained ex- pression of growth factors seen in the AAF/PH model (vide supra). The importance of some of the growth factors that are highly expressed during the activation and expansion of oval cells in the AAF/PH model is beginning to emerge. It has now been demonstrated that infusion of epidermal growth factor (EGF) and HGF, either alone or in combi- nation, can partially substitute for the PH in the AAF/PH model (52). The results from this study demonstrate that although both EGF and HGF increase the number of pro- liferating cells after AAF administration, they preferen- tially act on different cell populations. Although administration of AAF alone or in combination with infu- sion of HGF resulted in proliferation of almost equal numbers of ductal and Ito cells, infusion of EGF and/or combination of EGF and HGF resulted in 75-80% of the proliferating cells having a ductal phenotype. Also, infu- sion of EGF and HGF resulted in a decreased number of cells undergoing apoptosis in response to AAF. These re- sults have important implications for our understanding of both hepatic stem cell activation and expansion as well as for the mechanism of liver regeneration. Based on the data discussed above, the following model of liver regen- eration can now be proposed (Fig. 5). Induction of re- parative renewal of the epithelial cell populations in the liver after simple PH and certain types of more severe liver injury involves activation of both the normally qui- escent hepatocytes and the potential (facultative) liver stem cells. In a healthy liver, the reparative renewal of the hepatocyte and biliary epithelial cell populations is accomplished in most instances by proliferation of resid- ual differentiated cells of each types, resulting in only a transient activation of the stem cells. However, under conditions in which the hepatocytes are unable to re- spond to the regenerative stimuli and/or are functionally compromised, a sustained activation of the stem cells and their progeny ensues, generating the differentiated cell lineages needed for the liver regeneration. An essential requirement for the stem cell-driven liver regeneration is a sustained expression of a set of growth factors, includ- ing those known to be involved in liver regeneration after simple PH. Several implications emerge from this integrated model of liver regeneration. Perhaps the most important predic- tion arising from the model suggests that in chronic liver diseases such as viral hepatitis, a condition in which the hepatocytes are compromised and a continuous regen- eration exists, the stem cells would be activated and might contribute to the regenerative process. The obser- vations that oval-type cells occur in livers harboring hepatitis B virus-associated liver tumors (26) and that these cells can be infected by the hepatitis B virus at early stage of differentiationhave significantimplications for human hepatocarcinogenesis (54, 55). Also, the possi- bility of isolating liver stem cells from which hepatic lineages can be generated in vitro holds promise for fu- ture approaches to gene therapy of liver-related diseases. REFERENCES 1. Van Thiel, D. H.,Gavaler, J.S.,Kam, L, Francavilla, A.. Polimeno, L, Schade, P.R., Smith, J., Diven, W., Penkmt, R. J., and Staral, T. E. 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