Liver regeneration and repair: Hepatocytes, progenitor cells, and stem cells

Many different terms are used to refer to cells originating in the terminal branches of he bile ductular system and the canals of Hering in rodents and humans, which can function as progenitors for hepatocytes and cholangiocytes, the mature forms of the two hepatic epithelial cell lineages. In rats and mice, in experimental situations in which large amounts of these cells proliferate, the term oval cells is used for single cells or for clusters of cells that form a ductule. Intermediate forms between ductular cells and hepatocytes are often referred to as ductular hepatocytes, and the term neocholangiole has been used to describe the structures that contain these cells. Small hepatocytes that are not completely differentiated, which probably originate from oval cells, often are referred to as small hepatocyte precursor cells. Cell lines obtained from normal rodent liver, which have progenitor cell capabilities, are known as liver epithelial cell lines. Editor's note: The reader is referred to page 1739: “Nomenclature of the finer branches of the biliary tree.” For human liver, the term hepatic progenitor cells frequently is used to refer to cells that are equivalent to oval cells in rodents. The ductular reaction that involves these cells is known as an atypical ductular reaction (to distinguish from typical ductular reactions that do not involve the generation of hepatic progenitor cells). The term ductular hepatocyte also is used commonly to describe intermediate forms, particularly for cells appearing in the repopulation process after massive hepatic necrosis. In rodents and humans, no special name has been given to the cells located in the terminal branches of the ductular system and in the canals of Hering. Because they are likely to be the cells that most commonly give rise to oval cells, they are often referred to as intrahepatic stem cells or ductular stem cells. In this article, the term oval cell is used interchangeably with hepatic progenitor cell. The terms atypical ductular reaction and ductular hepatocytes are used to describe, respectively, ductular reactions involving oval cells and intermediate forms between ductular cells and hepatocytes. FAH, fumarylacetoacetate hydrolase; AFP, α-fetoprotein; HCC, hepatocellular carcinoma; HSC, hematopoietic stem cell; MAPC, multipotent adult progenitor cell. This question has multiple answers, because the source of hepatocytes depends on the nature of the growth process. In every case, it is necessary to ascertain whether hepatocytes responsible for liver regeneration originated from the replication of existing hepatocytes, were generated by differentiation of oval cells, or were produced from bone marrow cells.2 Replication of mature hepatocytes in liver regeneration has been documented extensively.3-5 Differentiation of oval cells has also been established as a mechanism that can generate significant numbers of hepatocytes (Table 1).6-11 However, the contribution of bone marrow cells to the generation of hepatocytes in liver repopulation and regeneration remains uncertain, both regarding its extent and the mechanisms involved. As a general rule, replication of existing hepatocytes is the quickest and most efficient way to generate hepatocytes for liver regeneration and repair. Oval cells usually replicate and differentiate into hepatocytes only when the replication of mature hepatocytes is delayed or entirely blocked (Fig. 1). Bone marrow cells can generate hepatocytes in transplanted livers but so far, the frequency of hepatocytes produced by this route is very low, and such cells are not always detectable. Note however, that in transplanted livers, bone marrow cells are an important source of nonparenchymal cells such as Kupffer cells and endothelial cells (discussed later). Because liver regeneration after 70% hepatectomy requires no more than two rounds of hepatocyte replication, it was generally assumed that the proliferative capacity of mature hepatocytes is very limited. This view has now been drastically changed. First, experiments with cultured hepatocytes isolated from transgenic mice that expressed liver growth factors demonstrated that long-term hepatocyte replication is compatible with a differentiated phenotype.12 More striking were the results of hepatocyte transplantation experiments in urokinase-plasminogen activator transgenic mice, showing that liver repopulation by transplanted hepatocytes involved at least 12 rounds of replication.13 Subsequent serial transplantation experiments performed in fumarylacetoacetate hydrolase (FAH)-deficient mice (FAH knockout mice) demonstrated that hepatocytes could replicate 70 or more times.14 In the serial transplantation experiments, there was no evidence that the repopulation capacity was dependent on stem cells. This conclusion also is supported by data demonstrating that diploid, tetraploid, and octoploid hepatocytes have roughly the same capacity to repopulate damaged livers.15 Thus, although hepatocytes are quiescent in normal livers and replicate in a limited and regulated manner during liver regeneration after partial hepatectomy, these cells have an enormous proliferative potential that can be unleashed under certain conditions. Nevertheless, there is evidence that the replicative activity of hepatocytes diminishes in advanced cirrhosis in humans and in chronic liver injury in mice, reaching a state of “replicative senescence,” perhaps as a consequence of telomere shortening.16-19 Both hepatocytes and intrahepatic bile ducts originate from endodermal-derived hepatoblasts (Fig. 1) that express albumin and α-fetoprotein (AFP).20, 21 At day 14 (mice) or 15 (rats) of embryonic development, hepatoblasts located near vascular spaces, the site for portal spaces in later development, express dual markers of the hepatocyte (albumin and AFP) and biliary (cytokeratins 7 and 19) lineages.22 These hepatoblasts give rise to the primitive intrahepatic bile ducts, structures that connect parenchymal hepatocytes with the larger segments of the biliary system. Primitive intrahepatic bile ducts correspond to the canals of Hering and terminal bile ductules of adult livers which may constitute the niche for intrahepatic stem cells.23-25 The embryological origin of intrahepatic bile ducts explains some important features of oval cell proliferation in adult livers. Cell lineages in the liver. During embryonic development, hepatoblasts give rise to the two epithelial lineages of the liver, producing hepatocytes and cholangiocytes. Oval cells originate in association with the intrahepatic biliary system, formed by hepatoblasts located near portal spaces. In adult livers, hepatocytes and cholangiocytes can replicate. Oval cells form a bipotential reserve compartment capable of generating hepatocytes whenever hepatocyte replication is blocked (red lines). In adult rat liver, cells of the canals of Hering and terminal bile ductules may express AFP.26, 27 Oval cells thought to be generated from them may express AFP and may contain isozymes of aldolase, pyruvate kinase, and lactic dehydrogenase present in both adult and fetal liver cells, and glucose-6-phosphatase, a typical hepatocyte marker.28-34 However, the extent to which these markers are expressed in a population of proliferating oval cells depends on the agent that elicited oval cell proliferation. Analysis of marker expression suggests that populations of proliferating oval cells constitute a heterogeneous cell compartment (or oval cell compartment) containing cells that may differ in their differentiation capacity and stage of differentiation. Some of these cells may function as hepatocyte progenitors, whereas others may be indistinguishable from cholangiocytes, cells that do not express AFP, or hepatocyte markers. Oval cells and cholangiocytes share epitopes that react with, among others cytokeratins 7, 8, 18, and 19, the antibodies OV6 (an anti-cytokeratin 19 antibody), OC2 (anti-myeloperoxidase), and some other members of the OC series, γ glutamyl transferase, and the antigens A6 and G7.29, 30, 35-37 A very small number of hematopoietic stem cells present in fetal livers may remain in adult livers. These cells may be distinct from oval cells, but are induced to proliferate by the same conditions that cause oval cell proliferation. In this case, hematopoietic stem cells would be a component of the oval cell compartment but constitute a distinct population, which does not acquire markers of the hepatocyte lineage but shares general stem cell markers with oval cells originating from the canals of Hering. Hematopoietic cells located in the adult liver may be pluripotent stem cells present in the hepatic tissue, functioning as the equivalent of embryonic stem cells capable of generating multiple lineages, including hepatocytes. If this view is correct, hematopoietic stem cells located in the liver (perhaps in periductular spaces) would differentiate progressively,9 first into oval cells and ultimately into hepatocytes, under stimuli known to cause an oval cell response. The relationships between oval cells and bone marrow cells would be understood more easily if it could be demonstrated that oval cells can be derived from hematopoietic cells. This has been shown to occur in some experimental models,44 but in these experiments, the proportion of hepatocytes generated through this route was very small, representing approximately 0.15% of hepatocytes in the liver. More recent data using the FAH model of liver injury demonstrated that in this model, oval cells do not originate from bone marrow precursors but are generated intrahepatically.45 Another study, using three different models of rat liver injury, showed that bone marrow cells were not the source of oval cells that repopulated these livers.46 An extensive ductular reaction occurs after massive (or submassive) hepatic necrosis in humans.47-53 In this type of injury, ductular proliferation involves mature cholangiocytes and ductular hepatocytes. The latter, located at the periphery of portal tracts, proliferate and express cholangiocyte and hepatocyte markers. Ductular hepatocytes are considered to be an intermediate form between ductular cells and hepatocytes, resembling ductal plate cells in the developing human liver. Such cells are also present in massive hepatic necrosis in rats.54 It is not known with certainty whether the generation of hepatocytes from ductular hepatocytes leads to complete repopulation of injured human livers, but at least one well-documented case has been described in a patient who recovered from massive liver necrosis. Fujita et al.52 performed sequential biopsies on the natural liver of a patient with massive necrosis after receiving an auxiliary partial orthotopic liver transplant. Complete regeneration of the natural liver was observed 12 to 14 months after transplantation, through a process that involved an initial ductular reaction followed by hepatocyte differentiation from ductular hepatocytes. As oval cells proliferate in response to treatment with both carcinogenic and noncarcinogenic agents, the detection of oval cells in a carcinogenic process is not proof of the role of oval cells as cancer progenitors. Nevertheless, it has been demonstrated that oval cells can generate hepatocellular carcinoma (HCC), cholangiocarcinoma, and hepatoblastoma in rodents.55-57 Although oval cell proliferation is fairly common in experimental models of hepatocarcinogenesis, in some models, oval cell and ductular proliferation is not apparent (for instance, in carcinogenesis induced by overexpression of growth factors such as transforming growth factor α58). Indeed, there is no special reason to support the notion that HCC is generated exclusively from oval cells.59 Mature hepatocytes also can function as tumor precursors, as long as they are in a proliferative state. It is also important to note that in general, oval cells do not seem to produce tumors directly, but do so through the generation of hepatocytes, although these cells “merge” into hepatocytes of neoplastic nodules in the liver of rats fed the carcinogen 3′-methyl-4-dimethylaminoazobenzene.60 Whether hepatocytes generated from oval cells in adult liver are abnormal, immature, or have a high risk for transformation remains to be established. In retrorsine-induced hepatocellular injury combined with partial hepatectomy, the great majority of proliferating cells are incompletely differentiated hepatocytes called small hepatocyte precursor cells.61, 62 Such cells, which also have been detected in galactosamine-induced liver injury,63 presumably originate from oval cells. Their preferential accumulation in some types of injury probably reflects a variable transit flux between cellular compartments containing ductular stem cells, oval cells, small hepatocyte precursor cells, and mature hepatocytes.64 Oval cells, commonly referred to as hepatic progenitor cells, have been detected in human livers in small cell dysplastic foci, hepatocellular adenomas, chronic viral and alcoholic hepatitis, nonalcoholic fatty liver disease, hemochromatosis, primary biliary cirrhosis, and cirrhosis associated with primary sclerosing cholangitis, conditions associated with an increased risk of neoplastic development.18, 65-70 Oval cell markers such as AFP and cytokeratins 7 and 19 are expressed in approximately 50% of small cell dysplastic foci and in HCC, suggesting the possible origin of HCC from cells that express these markers.65 This conclusion is strengthened by the finding that such markers are not detected in foci of large cell dysplasia, lesions that are not considered to be tumor precursors. Hepatocyte generation from oval cells occurs at of cirrhosis, at a at which hepatocyte replication has However, this process does not to extensive parenchymal regeneration and is in the normal on the experimental data it may be that generation of hepatocytes from oval cells in damaged livers hepatocytes that have a high risk for to this question may from of populations of cells isolated from small cell dysplastic foci and In both and humans, present in tumors have been in these It would be important to whether the cells that such in dysplastic foci express oval cell In both human and rodent livers, oval cells proliferate and differentiate in to cells with In oval cells form which are of the canals of Hering and are by a cells through this and with oval cells in the Oval cell proliferation is associated with increased expression of and also of hepatocyte growth growth and transforming growth factor which also function as growth factors for hepatocyte In both human and rodent liver, expression of factors of the hepatocyte factor is after the of oval cell an to hepatocyte It is that growth factors that oval cell proliferation are to that hepatocyte replication after partial hepatectomy and that both cell types through tumor necrosis factor type as oval cells and hepatocytes proliferate oval cell replication generally occurs when hepatocyte proliferation is However, the γ is only in oval but not in in that between γ and such as tumor necrosis factor may hepatocyte proliferation they oval cell replication et features of oval cell are the expression of of the of and of The generated by stem cells during the to a great extent is the of to two of these of hematopoietic stem cells and bone marrow stem cells, that they are capable of generating different types of cells known as and can multiple differentiation called differentiation the mechanisms of is to the of stem cell and important for the of stem cells in repopulation and far, the most of hepatocyte generation from bone marrow cells are the of hepatocytes in of multipotent adult progenitor cells and the repopulation of livers of FAH knockout mice by transplanted of these conditions can be considered as of In can differentiate into cells of and lineages. into a a single to the of Thus, can be considered as equivalent to embryonic stem cells, which have in adult and rat in in the of hepatocyte growth factor and growth differentiated into mature hepatocytes with If are adult embryonic stem cells, hepatocyte generation from these cells a process of differentiation of cells, a differentiation This is to the differentiation process of embryonic stem cells during and is different from which a in differentiation of an cell. These results from other to be known the of and most whether they can generate hepatocytes in The other of hepatocyte generation from bone marrow cells is the extensive repopulation of damaged livers of FAH knockout mice transplanted with far, this is the only in or humans of extensive repopulation of damaged livers by cells derived from bone In this system, the of repopulation by is and with that obtained by hepatocyte transplantation, although significant repopulation The first hepatocytes generated from at approximately 7 after transplanted hepatocytes more than 50% of the liver in approximately the to that by bone marrow cells is a and Nevertheless, by after transplantation, repopulation from transplanted approximately of the liver. It has now been shown that hepatocytes generated from transplanted in FAH knockout mice are the of cell than a of The process hepatocytes, hepatocytes, and hepatocytes. The liver cell that with has not been but the hepatocytes produced by the do not express data from of regeneration that are not capable of generating after injury, cells such as and with A mechanism may occur in the liver, that may occur between bone and hepatic cells, the proliferation of the liver cell. It is that between bone marrow and liver cells occurs in FAH because of the high proliferative by this system. However, it may be that high of hepatocyte from hematopoietic stem cells can be only in a system in which cell In case, these results it that to to livers to the mechanism by which hepatocytes are formed and that hepatocytes generated from bone marrow cells function do not have and are not to et bone marrow cells into mice and obtained in including and liver. 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The used a factor to for potential in the which the by a factor of In of these experiments, the livers of the mice transplanted with bone marrow cells were normal and It is of great to whether more efficient of hepatocytes from bone marrow cells to liver repopulation can be in injured livers. et the bone marrow of mice with bone marrow cells from transgenic and if bone hepatocytes would repopulate the liver after hepatic injury elicited by of with hepatic the proportion of bone hepatocytes in hepatic was very small, from to of hepatocytes, on the extent of and the generation of hepatocytes from bone marrow cells in three different models of liver injury and that there was or no contribution of bone marrow cells to the of hepatocytes in these et transplanted bone marrow cells into mice and endothelial cells and Kupffer cells, but no hepatocytes. 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The is the of hepatocytes from bone marrow cells in FAH knockout mice, as is a consequence of cell et in receiving bone marrow or liver whether hepatocytes could be generated from the bone marrow cells of the The frequency of hepatocytes that were considered to be bone marrow derived from to in and was in one The these by a factor of approximately to for in the detection of the and that hepatocyte from to et also the livers of who bone marrow or liver from The frequency of bone hepatocytes in the liver of the was to be to et that the frequency of hepatocytes generated from the bone marrow of of liver or bone marrow from to and was to liver injury and to the after other of liver not bone hepatocytes or only have the question of cellular in liver et the livers of transplanted to cells originating from the and of endothelial cells in 14 bile epithelial cell in and hepatocyte in one et that the majority of cells present in transplanted livers were or Kupffer cells and that only of the cells detected in these livers were hepatocytes to of the number of hepatocytes in the et detected frequency in liver and the of hepatocyte in two of in obtained after transplantation and in of at 12 months or of the of hepatocytes in the livers was not can these results be First, the of of the results may be a consequence of the of different to hepatocytes in transplanted livers. For instance, factors often are used to for the to the complete of hepatocyte to the This type of which involves the of observed by factors that from than to more than can significant and in the Another is that large numbers of cells in transplanted livers originate from the bone Although markers can be used to hepatocytes, the of the used for this is variable and not always of may to by the detection of reaction or in in endothelial or Kupffer cells that are in to a The the used in some of these experiments have been more data are it can be that the generation of bone hepatocytes occurs in the livers of some but not that it is a and because of its very its remains During the with stem cells one of the most of generating from and the general as an the repopulation and regeneration of and the of this is the initial of and the of that often it is now to the to and most to a into the It is a thought that for stem cells are the expression is not to stem a stem cell or a a the component of a system, or a stem this be isolated and in in a or are with to their most and some of these may to an in which results the most are in stem cell has been and to be by the of experimental by the and of to stem cells, and by the of the of the in the is to the of mechanisms of cell and and the between cells and in normal and injured may that cells, embryonic stem cells, or fetal liver cells are of hepatocyte precursors for hepatic repopulation than bone marrow The on bone marrow stem cells often the that the capacity of the liver is the of to the replication of mature hepatocytes. hepatocyte replication is or intrahepatic stem cells give rise to oval cells, which replicate and differentiate into hepatocytes (Fig. 1). The proliferative capacity of differentiated hepatocytes is among differentiated cells in As as it may to stem cell the hepatocyte is the most efficient for the liver. A first is that the of bone hepatocytes to be to and are factors that the of the data the of the that the generation of hepatocytes from bone marrow cells is a very in liver transplantation and repopulation after injury and that such hepatocytes are produced by cell than by a mechanism (Fig. This does not a potential role for bone cells in hepatic in suggests experimental to this mechanisms for the generation of hepatocytes from bone marrow cells. the mechanisms shown in the only cell has been shown to occur in of hepatocytes from pluripotent bone marrow stem cells can occur in from cell generate hepatocytes their may not a high risk for cell be to liver repopulation in a this be more for liver repopulation than oval or hepatic embryonic stem cell The role of the bone marrow in generating nonparenchymal cells in liver regeneration and repopulation to be more significant than the generation of hepatocytes. of the of or bone into the liver with liver regeneration and the hepatic of bone endothelial and Kupffer cells for normal liver these cells (or perhaps the small number of produce special and growth factors that are for hepatocyte The of oval cells at very from the bone marrow has been but so not in other This very important question be If oval cells are not generated by bone marrow cells, could they (or cells in the canals of with bone marrow cells to generate differentiate in and generate lineages that the liver in adult humans or of be used to produce large amounts of human hepatocytes for has been in the of stem cells from embryonic liver and transplantation of these types of cells into injured livers both hepatocytes and bile these cells more efficient than fetal or adult hepatocytes for the of liver