regenerare 3

SCIENCE FRONTIER

Liver Regeneration and Repair: Hepatocytes,Progenitor Cells, and Stem Cells

Nelson Fausto1

Studies of liver regenerative processes have gainednew prominence, generated from analyses of genet-ically engineered animal models, the transplanta-tion of human livers, and the surging interest in stem cells.These studies gave rise to expectations regarding the prac-tical applications of research on the mechanisms of liverregeneration that were unthinkable just a few years ago.As the eld expanded to a broader audience, this newknowledge also brought confusion and often misinterpre-tations regarding the cellular mechanisms responsible forliver regeneration in different types of hepatic growthprocesses. This article is a brief review of the role ofhepatocytes, oval cells (see box for nomenclature), andbone marrow cells in liver regeneration and repopula-tion. This topic has generated great excitement, a gooddeal of noise, much controversy, and many surprises.The review of the rapidly expanding literature pre-sented here, particularly as it deals with stem cells, wasguided by a few principles: (1) be wary of dogma1 andof overinterpretation of data; (2) a new discoverymay be a true new nding, but it may simply be therepackaging of an old discovery; and (3) an excitingstory is not necessarily a true story.

What is the Source of Hepatocytes in LiverGrowth Processes?

This question has multiple answers, because the sourceof hepatocytes depends on the nature of the growth pro-cess. In every case, it is necessary to ascertain whetherhepatocytes responsible for liver regeneration originatedfrom the replication of existing hepatocytes, were gener-ated by differentiation of oval cells, or were producedfrom bone marrow cells.2 Replication of mature hepato-cytes in liver regeneration has been documented exten-

sively.35 Differentiation of oval cells has also beenestablished as a mechanism that can generate signicantnumbers of hepatocytes (Table 1).611 However, the con-tribution of bone marrow cells to the generation of hepa-tocytes in liver repopulation and regeneration remainsuncertain, both regarding its extent and the mechanismsinvolved.

As a general rule, replication of existing hepatocytes isthe quickest and most efcient way to generate hepato-cytes for liver regeneration and repair. Oval cells usuallyreplicate and differentiate into hepatocytes only when thereplication of mature hepatocytes is delayed or entirelyblocked (Fig. 1). Bone marrow cells can generate hepato-cytes in transplanted livers but so far, the frequency ofhepatocytes produced by this route is very low, and suchcells are not always detectable. Note however, that intransplanted livers, bone marrow cells are an importantsource of nonparenchymal cells such as Kupffer cells andendothelial cells (discussed later).

The Proliferative Capacity of HepatocytesBecause liver regeneration after 70% hepatectomy re-

quires no more than two rounds of hepatocyte replica-tion, it was generally assumed that the proliferativecapacity of mature hepatocytes is very limited. This viewhas now been drastically changed. First, experiments withcultured hepatocytes isolated from transgenic mice thatexpressed liver growth factors demonstrated that long-term hepatocyte replication is compatible with a differen-tiated phenotype.12 More striking were the results ofhepatocyte transplantation experiments in urokinase-plasminogen activator transgenic mice, showing that liverrepopulation by transplanted hepatocytes involved atleast 12 rounds of replication.13 Subsequent serial trans-plantation experiments performed in fumarylacetoacetatehydrolase (FAH)-decient mice (FAH knockout mice)demonstrated that hepatocytes could replicate 70 or moretimes.14 In the serial transplantation experiments, therewas no evidence that the repopulation capacity was de-pendent on stem cells. This conclusion also is supportedby data demonstrating that diploid, tetraploid, and octo-ploid hepatocytes have roughly the same capacity to re-populate damaged livers.15 Thus, although hepatocytesare quiescent in normal livers and replicate in a limited

Abbreviations: FAH, fumarylacetoacetate hydrolase; AFP, -fetoprotein; HCC,hepatocellular carcinoma; HSC, hematopoietic stem cell; MAPC, multipotent adultprogenitor cell.

From the 1Department of Pathology, University of Washington School of Medi-cine, Seattle, WA.

Received December 16, 2003; accepted March 1, 2004Address reprint requests to: Nelson Fausto, M.D., Department of Pathology,

University of Washington School of Medicine, Box 357470, 1959 NE PacicStreet, Seattle, WA 98195-7705. E-mail: [email protected].

Copyright 2004 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20214

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and regulated manner during liver regeneration after par-tial hepatectomy, these cells have an enormous prolifera-tive potential that can be unleashed under certainconditions. Nevertheless, there is evidence that the repli-cative activity of hepatocytes diminishes in advanced cir-rhosis in humans and in chronic liver injury in mice,reaching a state of replicative senescence, perhaps as aconsequence of telomere shortening.1619

The Origin of Intrahepatic Bile Ducts inLiver Development and of Oval Cells in theAdult Liver

Both hepatocytes and intrahepatic bile ducts originatefrom endodermal-derived hepatoblasts (Fig. 1) that expressalbumin and -fetoprotein (AFP).20,21 At day 14 (mice) or15 (rats) of embryonic development, hepatoblasts locatednear vascular spaces, the site for portal spaces in later devel-opment, express dual markers of the hepatocyte (albuminand AFP) and biliary (cytokeratins 7 and 19) lineages.22

These hepatoblasts give rise to the primitive intrahepatic bileducts, structures that connect parenchymal hepatocytes withthe larger segments of the biliary system. Primitive intrahe-patic bile ducts correspond to the canals of Hering and ter-minal bile ductules of adult livers which may constitute theniche for intrahepatic stem cells.2325 The embryological or-igin of intrahepatic bile ducts explains some important fea-tures of oval cell proliferation in adult livers.

In adult rat liver, cells of the canals of Hering andterminal bile ductules may express AFP.26,27 Oval cellsthought to be generated from them may express AFP and

may contain isozymes of aldolase, pyruvate kinase, andlactic dehydrogenase present in both adult and fetal livercells, and glucose-6-phosphatase, a typical hepatocytemarker.2834 However, the extent to which these markersare expressed in a population of proliferating oval cellsdepends on the agent that elicited oval cell proliferation.

Analysis of marker expression suggests that popula-tions of proliferating oval cells constitute a heterogeneouscell compartment (or oval cell compartment) containingcells that may differ in their differentiation capacity andstage of differentiation. Some of these cells may functionas hepatocyte progenitors, whereas others may be indis-tinguishable from cholangiocytes, cells that do not expressAFP, or hepatocyte markers. Oval cells and cholangio-cytes share epitopes that react with, among others cyto-keratins 7, 8, 18, and 19, the antibodies OV6 (an anti-cytokeratin 19 antibody), OC2 (anti-myeloperoxidase),and some other members of the OC series, glutamyltransferase, and the antigens A6 and G7.29,30,3537

Relationships Between Oval Cells andHematopoietic Stem Cells

In addition to expressing markers of the hepatocyteand bile duct lineage, oval cells express markers of hema-topoietic stem cells. Among these are Thy-1, CD34,CD45, Sca-1, c-Kit, and t-3,38 all of which can also bedetected in fetal livers. Stemlike cells positive for CD34and c-Kit have been isolated from normal and cirrhotichuman livers.39,40 The normal adult murine liver containshematopoietic cells that are phenotypically similar to cellspresent in the bone marrow,41 including cells of the bonemarrow side population.42 The side population cells arehematopoietic stem cells puried on the basis of theirefux capacity on staining with the dye Hoechst 33342.One of the markers for the side population is the adeno-

Table 1. Origin of Hepatocytes in Liver Regeneration andRepair

Growth processes that depend of the replication of differentiated hepatocytesLiver regeneration after partial hepatectomy2

Hepatocyte regeneration after carbon tetrachloride and acetaminophen(centrolobular) injury131

Conditions in which oval cells proliferate and generate hepatocytesExperimentalInjury caused by galactosamine132

Choline-decient diet combined with ethionine or AAF133,134

Partial hepatectomy combined with AAF or Dipin135,136

Carbon tetrachloride combined with AAF137

3,5-dietoxycarbonyl-1-1, 4-dihydrocollidine (DCC)138

Allyl alcohol9

Human diseaseAtypical ductular reactions in advanced stages of cirrhosis of variousetiologiesFatty liver diseaseSmall cell dysplasiasMassive hepatocyte necrosis17,52,68,139

Conditions in which small hepatocyte precursor cells (SHPC) represent a largefraction of the proliferating cells

Injury caused by retrorsine61,62 and galactosamine63

Abbreviation: AAF, N-2-acetylaminouorene.NOTE. Only representative publications are listed.

Fig. 1. Cell lineages in the liver. During embryonic development,hepatoblasts give rise to the two epithelial lineages of the liver, producinghepatocytes and cholangiocytes. Oval cells originate in association withthe intrahepatic biliary system, formed by hepatoblasts located nearportal spaces. In adult livers, hepatocytes and cholangiocytes can rep-licate. Oval cells form a bipotential reserve compartment capable ofgenerating hepatocytes whenever hepatocyte replication is blocked (redlines).

1478 FAUSTO HEPATOLOGY, June 2004

sine triphosphate-binding cassette transporter ABCG2/BCRP1, which also has been detected during hepatic ovalcell proliferation.43 On the basis of the existing data, theexpression of hematopoietic markers in the normal adultliver and after hepatic injury leading to oval cell prolifer-ation may be interpreted in at least two different ways:

A very small number of hematopoietic stem cellspresent in fetal livers may remain in adult livers. Thesecells may be distinct from oval cells, but are induced toproliferate by the same conditions that cause oval cellproliferation. In this case, hematopoietic stem cells wouldbe a component of the oval cell compartment but consti-tute a distinct population, which does not acquire mark-ers of the hepatocyte lineage but shares general stem cellmarkers with oval cells originating from the canals ofHer-ing.

Hematopoietic cells located in the adult liver may bepluripotent stem cells present in the hepatic tissue, func-tioning as the equivalent of embryonic stem cells capableof generating multiple lineages, including hepatocytes. Ifthis view is correct, hematopoietic stem cells located in theliver (perhaps in periductular spaces) would differentiateprogressively,9 rst into oval cells and ultimately intohepatocytes, under stimuli known to cause an oval cellresponse.

The relationships between oval cells and bone marrowcells would be understood more easily if it could be dem-onstrated that oval cells can be derived from hematopoi-

etic cells. This has been shown to occur in someexperimental models,44 but in these experiments, the pro-portion of hepatocytes generated through this route wasvery small, representing approximately 0.15% of hepato-cytes in the liver. More recent data using the FAH modelof liver injury demonstrated that in this model, oval cellsdo not originate from bone marrow precursors but aregenerated intrahepatically.45 Another study, using threedifferent models of rat liver injury, showed that bonemarrow cells were not the source of oval cells that repop-ulated these livers.46

Oval Cell Differentiation in MassiveHepatic Necrosis in Humans

An extensive ductular reaction occurs after massive (orsubmassive) hepatic necrosis in humans.4753 In this typeof injury, ductular proliferation involves mature cholan-giocytes and ductular hepatocytes. The latter, located atthe periphery of portal tracts, proliferate and expresscholangiocyte and hepatocyte markers. Ductular hepato-cytes are considered to be an intermediate form betweenductular cells and hepatocytes, resembling ductal platecells in the developing human liver. Such cells are alsopresent in massive hepatic necrosis in rats.54 It is notknown with certainty whether the generation of hepato-cytes from ductular hepatocytes leads to complete re-population of injured human livers, but at least one well-

Whats in a Name?

Many different terms are used to refer to cells origi-nating in the terminal branches of he bile ductular sys-tem and the canals of Hering in rodents and humans,which can function as progenitors for hepatocytes andcholangiocytes, the mature forms of the two hepatic ep-ithelial cell lineages. In rats and mice, in experimentalsituations in which large amounts of these cells prolifer-ate, the term oval cells is used for single cells or for clus-ters of cells that form a ductule. Intermediate formsbetween ductular cells and hepatocytes are often referredto as ductular hepatocytes, and the term neocholangiole hasbeen used to describe the structures that contain thesecells. Small hepatocytes that are not completely differen-tiated, which probably originate from oval cells, often arereferred to as small hepatocyte precursor cells. Cell linesobtained from normal rodent liver, which have pro-genitor cell capabilities, are known as liver epithelial celllines.

Editors note: The reader is referred to page 1739: Nomenclature ofthe ner branches of the biliary tree.

For human liver, the term hepatic progenitor cellsfrequently is used to refer to cells that are equivalent tooval cells in rodents. The ductular reaction that in-volves these cells is known as an atypical ductular reac-tion (to distinguish from typical ductular reactionsthat do not involve the generation of hepatic progen-itor cells). The term ductular hepatocyte also is usedcommonly to describe intermediate forms, particularlyfor cells appearing in the repopulation process after mas-sive hepatic necrosis. In rodents and humans, no specialname has been given to the cells located in the terminalbranches of the ductular system and in the canals ofHer-ing. Because they are likely to be the cells thatmost com-monly give rise to oval cells, they are often referred to asintrahepatic stem cells or ductular stem cells.

In this article, the term oval cell is used interchange-ably with hepatic progenitor cell. The terms atypicalductular reaction and ductular hepatocytes are used todescribe, respectively, ductular reactions involvingoval cells and intermediate forms between ductularcells and hepatocytes.

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documented case has been described in a patient whorecovered from massive liver necrosis. Fujita et al.52 per-formed sequential biopsies on the natural liver of a patientwith massive necrosis after receiving an auxiliary partialorthotopic liver transplant. Complete regeneration of thenatural liver was observed 12 to 14 months after trans-plantation, through a process that involved an initialductular reaction followed by hepatocyte differentiationfrom ductular hepatocytes.

Oval Cells in Liver Tumorigenesis andAdvanced Cirrhosis

As oval cells proliferate in response to treatment withboth carcinogenic and noncarcinogenic agents, the detec-tion of oval cells in a carcinogenic process is not proof ofthe role of oval cells as cancer progenitors. Nevertheless, ithas been demonstrated that oval cells can generate hepa-tocellular carcinoma (HCC), cholangiocarcinoma, andhepatoblastoma in rodents.5557 Although oval cell prolif-eration is fairly common in experimental models of hepa-tocarcinogenesis, in some models, oval cell and ductularproliferation is not apparent (for instance, in carcinogen-esis induced by overexpression of growth factors such astransforming growth factor 58). Indeed, there is no spe-cial reason to support the notion that HCC is generatedexclusively from oval cells.59 Mature hepatocytes also canfunction as tumor precursors, as long as they are in aproliferative state. It is also important to note that ingeneral, oval cells do not seem to produce tumors directly,but do so through the generation of hepatocytes, althoughthese cells merge into hepatocytes of neoplastic nodulesin the liver of rats fed the carcinogen 3-methyl-4-di-methylaminoazobenzene.60 Whether hepatocytes gener-ated from oval cells in adult liver are abnormal, immature,or have a high risk for transformation remains to be es-tablished. In retrorsine-induced hepatocellular injurycombined with partial hepatectomy, the great majority ofproliferating cells are incompletely differentiated hepato-cytes called small hepatocyte precursor cells.61,62 Suchcells, which also have been detected in galactosamine-induced liver injury,63 presumably originate from ovalcells. Their preferential accumulation in some types ofinjury probably reects a variable transit ux betweencellular compartments containing ductular stem cells,oval cells, small hepatocyte precursor cells, and maturehepatocytes.64

Oval cells, commonly referred to as hepatic progenitorcells, have been detected in human livers in small celldysplastic foci, hepatocellular adenomas, chronic viraland alcoholic hepatitis, nonalcoholic fatty liver disease,hemochromatosis, primary biliary cirrhosis, and cirrhosisassociated with primary sclerosing cholangitis, conditions

associated with an increased risk of neoplastic develop-ment.18,6570 Oval cell markers such as AFP and cytoker-atins 7 and 19 are expressed in approximately 50% ofsmall cell dysplastic foci and in HCC, suggesting the pos-sible origin of HCC from cells that express these mark-ers.65 This conclusion is strengthened by the nding thatsuch markers are not detected in foci of large cell dyspla-sia, lesions that are not considered to be tumor precursors.Hepatocyte generation from oval cells occurs at late stagesof cirrhosis, apparently at a time at which hepatocyte rep-lication has diminished.17 However, this process does notlead to extensive parenchymal regeneration and is essen-tially ineffective in restoring the normal parenchyma.Based on the experimental data discussed above, it may besuggested that generation of hepatocytes from oval cells inseverely damaged cirrhotic livers produces hepatocytesthat have a high risk for transformation. Answers to thisquestion may come from studies of populations of cellsisolated from small cell dysplastic foci andHCCs. In bothanimals and humans, chromosomal abnormalities presentin tumors have been found in these foci.71 It would beimportant to know whether the cells that harbor suchchromosomal defects in dysplastic foci express oval cellphenotypes.

Factors Associated With Oval CellProliferation and Oval Cell Phenotypes

In both human and rodent livers, oval cells proliferateand differentiate in close proximity to stellate cells withmyobroblast morphological features.48,72,73 In rats, ovalcells form ductules, which are extensions of the canals ofHering and are surrounded by a continuous basementmembrane. Stellate cells penetrate through this basementmembrane and establish direct contact with oval cells inthe ductules.72 Oval cell proliferation is associated withincreased expression of c-KIT, and also of hepatocytegrowth factor, acidic broblast growth factor, and trans-forming growth factor , which also function as growthfactors for hepatocyte replication.74 In both human androdent liver, expression of transcription factors of the he-patocyte nuclear factor family is detectable shortly afterthe start of oval cell proliferation, indicating an early com-mitment to hepatocyte differentiation.53,75,76 It is puz-zling that growth factors that stimulate oval cellproliferation are similar to those that stimulate hepatocytereplication after partial hepatectomy and that both celltypes require signaling through tumor necrosis factor re-ceptor type I.77,78 Yet, as discussed, oval cells and hepato-cytes rarely proliferate simultaneously; oval cellreplication generally occurs when hepatocyte prolifera-tion is blocked. However, the interferon network isstimulated only in oval cell, but not in hepatocyte, prolif-


eration.79 Studies in progress suggest that interactions be-tween interferon and cytokines such as tumor necrosisfactor may inhibit hepatocyte proliferation while theystimulate oval cell replication (Brooling J et al., unpub-lished manuscript, 2001).

Other interesting features of oval cell phenotypes arethe expression of proteins of the drug resistance gene fam-ilies, of adenosine triphosphate binding cassette trans-porter genes, and of neuroendocrine peptides.8084

Bone Marrow Cells and HepatocyteProduction: Differentiation,Transdifferentiation, and Cell Fusion

The tremendous interest generated by stem cells dur-ing the last few years to a great extent is the result of to twonewly discovered properties of these cells.85 Studies ofhematopoietic stem cells (HSC) and bone marrow mes-enchymal stem cells, revealed that they are capable ofgenerating many different types of tissue cells (a propertyknown as transdifferentiation) and can choose multipledifferentiation pathways (a property called differentiationplasticity). Understanding the mechanisms of transdiffer-entiation is key to the eld of stem cell biology and shouldprovide important clues for the use of stem cells in organrepopulation and regeneration. So far, the most impres-sive demonstrations of hepatocyte generation from bonemarrow cells are the production of hepatocytes in culturesof multipotent adult progenitor cells (MAPCs)86,87 andthe repopulation of livers of FAH knockout mice bytransplanted HSCs.88,89 Yet, neither of these conditionscan be considered as examples of transdifferentiation.

In culture, MAPCs can differentiate into cells of me-sodermal, ectodermal, and endodermal lineages. Injectedinto a blastocyst, a single MAPC contributes to the for-mation of all somatic tissues. Thus, MAPC can be con-sidered as equivalent to embryonic stem cells, which havepersisted in adult tissues.86,87 Human, mouse, and ratMAPCs, grown in matrigel in the presence of hepatocytegrowth factor and broblast growth factor-4, differenti-ated into mature hepatocytes with apparently fully func-tional properties.90 IfMAPCs are indeed adult embryonicstem cells, hepatocyte generation from these cells consti-tutes a process of differentiation of pluripotent, uncom-mitted cells, along a specic differentiation path. This issimilar to the differentiation process of embryonic stemcells during development and is quite different fromtransdifferentiation, which implies a change in differenti-ation commitment of an already committed cell. Theseexciting results await conrmation from other laborato-ries. Much needs to be known about the properties ofMAPCs, and most importantly, whether they can gener-ate hepatocytes in vivo.

The other dramatic demonstration of hepatocyte genera-tion from bonemarrow cells is the extensive repopulation ofdamaged livers of FAH knockout mice transplanted withHSC.88,89 So far, this is the only example in animals or hu-mans of extensive repopulation of damaged livers by cellsderived from bone marrow. In this system, the kinetics ofrepopulation by HSC is slow and inefcient compared withthat obtainedbyhepatocyte transplantation, although signif-icant repopulation eventually occurs. The rst hepatocytesgenerated from HSC appeared at approximately 7 weeksafter transplantation. By contrast, transplanted hepatocytesreconstitutedmore than 50%of the liver in approximately 4weeks, leading the authors to conclude that hepatocyte re-placement by bone marrow cells is a slow and rare event.91

Nevertheless, by 22 weeks after transplantation, repopula-tion from transplanted HSC constituted approximately30% of the entire liver. It has now been shown that hepato-cytes generated from transplanted HSC in FAH knockoutmice are the product of cell fusion rather than a result oftransdifferentiation.92,93 The fusion process created tet-raploid hepatocytes, 6X hepatocytes, and aneuploid hepato-cytes. The liver cell that fuses with HSCs has not beenidentied, but the hepatocytes produced by the fusion eventdo not expressHSC genes. Recent data from studies ofmus-cle regeneration suggest thatHSC are not capable of directlygeneratingmyogenic progenitors.94 Instead, after muscle in-jury, circulating inammatory cells such asmacrophages andneutrophils fuse with myotubes. A similar mechanism mayoccur in the liver, that is, fusion may occur between bonemarrow-derived macrophages and hepatic cells, triggeringthe proliferation of the fused liver cell.

It is conceivable that fusion between bone marrow andliver cells occurs in FAH knockouts because of the highproliferative pressure imposed by this system. However, itmay be argued that high levels of hepatocyte productionfrom hematopoietic stem cells can be achieved only in asystem in which cell fusion occurs. In any case, theseresults make it clear that any attempt to use HSC toreconstitute livers needs to demonstrate: (1) the mecha-nism by which hepatocytes are formed and (2) that hepa-tocytes generated from bone marrow cells functionnormally, do not have abnormal genomes, and are notprone to malignancy.95

Injected Bone Marrow Stem Cells HaveMinimal Capacity to Generate Hepatocytesin Normal Livers of Mice and in MostModels of Liver Injury

Krause et al.96 injected single, highly puried bonemarrow cells into irradiated mice and obtained engraft-ment in several organs, including skin, lung, and liver.The livers of ve mice examined did not contain HSC-


derived hepatocytes, and three of these mice also did nothave bone marrow-derived cells in bile ducts. The othertwo mice had 0.4% and 2.2% of bile duct cells of bonemarrow origin 11months after transplantation.Wagers etal.97 used a differentmethod of bonemarrow cell isolationand marked cells with green uorescent protein. Single-labeled HSC cells injected into irradiated mice failed tocontribute to brain, kidney, gut, muscle, and liver, al-though they completely reconstituted the bonemarrow ofthese animals. The experiments of Krause et al. and Wa-gers et al. differ in important technical aspects, includingthe type of cells used, but there seems to be completeagreement regarding the generation of hepatocytes frominjected, puried bone marrow cells: no bone marrow-derived hepatocytes were detected in either of these exper-iments. Theise et al.98 transplanted unfractionated bonemarrow cells or puried CD34lin- cells frommale miceinto irradiated female mice and searched for cells contain-ing the Y chromosome and expressing albumin mRNA inthe liver of the recipient mice 1 week to 8 months aftertransplantation. Cells positive for both the Y chromo-some and albumin mRNA were detected from 2 to 8months after transplantation at frequencies of 0.39% to1.1% of total hepatocytes in the liver. The authors used acorrection factor to compensate for potential error sam-pling in detecting the Y chromosome, which raised thereported frequencies by a factor of 2. In all of these exper-iments, the livers of the mice transplanted with bonemar-row cells were morphologically normal and apparentlyundamaged.

It is of great interest to determine whether more ef-cient production of hepatocytes from injected bone mar-row cells leading to liver repopulation can be achieved ininjured livers. Mallet et al.99 reconstituted the bone mar-row of lethally irradiated mice with bone marrow cellsfrom Bcl-2 transgenic mice100 and asked if bone marrow-derived Bcl-2 expressing hepatocytes would repopulatethe liver after hepatic injury elicited by repeated injectionsof Fas agonist antibody. Even with severe hepatic damage,the proportion of bone marrow-derived Bcl-2-expressinghepatocytes found in hepatic tissue was very small, vary-ing from 0.008% to 0.8% of total hepatocytes, dependingon the extent of damage. Kanazawa and Verma101 studiedthe generation of hepatocytes from bone marrow cells inthree different models of liver injury and concluded thatthere was little or no contribution of bone marrow cells tothe replacement of hepatocytes in these models. Fujii etal.102 transplanted green uorescent protein-positivebone marrow cells into green uorescent protein-negativeirradiated recipient mice and identied green uorescentprotein-positive endothelial cells andKupffer cells, but nohepatocytes. A similar result was obtained by Dahlke et

al.103 using a model of acute liver failure. It can be con-cluded from these experiments that bone marrow cellshave a minimal capacity to generate hepatocytes in nor-mal livers and a very low capacity in injured livers. Theexception is the production of hepatocytes from bonemarrow cells in FAH knockout mice, which, as alreadydiscussed, is a consequence of cell fusion.

Generation of Hepatocytes by Bone MarrowCells and Cell Chimerism in Recipients ofLiver and Bone Marrow Transplants

Theise et al.104 investigated, in patients receiving bonemarrow or liver transplants, whether hepatocytes could begenerated from the bone marrow cells of the transplantrecipient. The frequency of hepatocytes that were consid-ered to be bone marrow derived varied from 1% to 3.6%in ve patients and was 8% in one patient. The authorsmultiplied these values by a factor of approximately 5 tocorrect for sampling errors in the detection of the Y chro-mosome and reported that hepatocyte engraftmentranged from 4% to 43%. Alison et al.105 also examinedthe livers of patients who received bone marrow or livertransplants from sex-mismatched donors. The frequencyof bone marrow-derived hepatocytes in the liver of therecipients was estimated to be 0.5% to 2%. Korbling etal.106 reported that the frequency of hepatocytes gener-ated from the bone marrow of recipients of sex-mis-matched liver or bonemarrow transplants varied from 4%to 7% and was unrelated to liver injury and to the timeafter transplantation. Two other studies of liver transplantpatients did not detect bone marrow-derived hepatocytesor found only occasional cells.107,108 Several studies haveaddressed the question of cellular chimerism in liver trans-plants. Hove et al.109 examined the livers of 16 trans-planted patients to identify cells originating from therecipient and reported chimerism of endothelial cells in14 patients, bile duct epithelial cell chimerism in ve pa-tients, and hepatocyte chimerism in one patient. Ng etal.110 recently reported that the vast majority of recipient-derived cells present in transplanted livers were macro-phages or Kupffer cells and that only 1.6% of the totalrecipient cells detected in these livers were hepatocytes(this corresponded to 0.62% of the total number of hepa-tocytes in the liver). Finally, Kleeberger et al.111 detected91% chimerism frequency in liver transplant recipients,and the presence of hepatocyte chimerism in two of ninepatients in samples obtained 4 weeks after transplantationand in ve of nine patients at 12 months or longer (aquantitative analysis of the percentage of recipient hepa-tocytes in the chimeric livers was not reported).

How can these results be interpreted? First, the lack ofconsistency of the results may be a consequence of the use


of different techniques to identify recipient-derived hepa-tocytes in transplanted livers. For instance, factors oftenare used to correct for the inability to examine the com-plete surface of hepatocyte nuclei to detect the Y chromo-some. This type of correction, which involves themultiplication of observed values by factors that vary fromless than 2 to more than 5 can introduce signicant errorsand uncertainties in the reported data. Another difcultyis that large numbers ofmesenchymal cells in transplantedlivers originate from the recipients bone marrow. Al-though various markers can be used to identify hepato-cytes, the precision of the methods used for thisidentication is variable and not always optimal. Super-imposition of images without confocal microscopy maylead to errors caused by the detection of reaction productsor in situ hybridization signals in endothelial or Kupffercells that are in close apposition to a hepatocyte. Theuncertainties about the techniques used in some of theseexperiments have been discussed.112,113 Until more den-itive data are obtained, it can be concluded that the gen-eration of bone marrow-derived hepatocytes occurs in thelivers of some but not all transplant patients, that it is ahighly inefcient process, and that, because of its very lowfrequency, its physiological relevance remains unproven.

PerspectivesDuring the last few years, work with stem cells became

one of the most exciting areas of biomedical research,generating much enthusiasm from scientists, clinicians,and the general public. Having as an endpoint the re-population and regeneration of tissues and organs, thepromise of this research is far reaching. After the initialphase of excitement and the publication of ndings thatoften deed long-accepted knowledge, it is now time toassess the progress made, to point out pitfalls and misin-terpretations, and, most importantly, to project a realisticlook into the future. It is a sobering thought that deni-tions for stem cells are still being debated.114 Does theexpression Seeing is not being apply to stem cells?115 Isa stem cell an entity or a function,116 a contextual cate-gory,1 the rst, dened, component of a hierarchical sys-tem, or a functionally plastic entity (the chiaroscuro stemcell117)? Can this entity be isolated and studied in culturein a biologically meaningful way, or are we dealing withHeisenbergs uncertainty?1 Carried to their most extremeimplications, and crudely interpreted, some of these ideasmay lead us to an anything goes intellectual environ-ment, in which all results even the most discordant, areaccepted uncritically. Great progress in stem cell researchhas beenmade andwill continue to bemade by the carefulscrutiny of experimental data, by examining the reliabilityand reproducibility of methods to identify stem cells, and

by addressing the issue of the biological relevance of thendings. Progress in the eld is inextricably linked tothe understanding of mechanisms of cell differentia-tion, proliferation, and transdifferentiation and knowl-edge about the interactions between cells andextracellular matrix components in normal and injuredtissues. Future work may show that umbilical cordcells, embryonic stem cells, or fetal liver cells are bettersources of hepatocyte precursors for hepatic repopula-tion than bone marrow cells.118130

The emphasis on bone marrow stem cells often ob-scures the fact that the remarkable regenerative capacity ofthe liver primarily is the result of to the replication ofmature hepatocytes. When hepatocyte replication is slowor inhibited, intrahepatic stem cells give rise to oval cells,which replicate and differentiate into hepatocytes (Fig. 1).The proliferative capacity of normally quiescent, highlydifferentiated hepatocytes is unique among differentiatedcells inmammalian tissues. As paradoxical as it may soundto stem cell biologists, the hepatocyte is the most highlyefcient stem cell for the liver.

Where Do We Go From Here?A rst observation is that the identication of bone

marrowderived hepatocytes needs to be carried out ac-cording to rigorous criteria. Methodological accuracy andreproducibility are critical factors that determine the reli-ability of the data reported. From the review of the liter-ature presented here, I conclude that the generation ofhepatocytes from bone marrow cells is a very rare event inliver transplantation and repopulation after injury andthat such hepatocytes are produced by cell fusion ratherthan by a transdifferentiation mechanism (Fig. 2). Thispremise does not exclude a potential functional role forbone marrow-derived cells in hepatic homeostasis and, infact, suggests experimental approaches to study this issue.

Fig. 2. Possible mechanisms for the generation of hepatocytes frombone marrow cells. Of the mechanisms shown in the gure, only cellfusion (#4) has been shown to occur in vivo. Generation of hepatocytesfrom pluripotent bone marrow stem cells (#2) can occur in culture.90(Diagram redrawn from Wagers AJ, Weissman IL.140)


Does cell fusion generate functionally intact hepato-cytes that, despite their abnormal ploidies, may not carrya high risk for transformation? Can cell fusion techniquesbe applied to liver repopulation in a clinical setting? Maythis approach be more successful for liver repopulationthan hepatocyte, oval cell, or hepatic embryonic stem celltransplantation?

The role of the bone marrow in generating non-parenchymal cells in liver regeneration and repopulationseems to be much more signicant than the generation ofhepatocytes. Would blockage of the migration of HSC orbone marrow-derived leucocytes into the liver interferewith liver regeneration and repopulation? Is the hepaticseeding of bone marrow-derived endothelial and Kupffercells essential for normal liver homeostasis? Do these cells(or perhaps even the small number of bone-marrow-de-rived hepatocytes) produce special cytokines and growthfactors that are required for hepatocyte replication?

The derivation of oval cells (albeit at very low levels)from the bone marrow has been reported, but so far notconrmed in other laboratories. This very importantquestion must be settled denitively. If oval cells are notgenerated by bonemarrow cells, could they (or cells in thecanals of Hering) fuse with bone marrow cells to generatehepatocytes?

Do MAPCs differentiate in vivo and generate lin-eages that populate the liver in adult humans or animals?Can cultures ofMAPCs be used to produce large amountsof human hepatocytes suitable for transplantation?

Great progress has been achieved in the puricationof stem cells from embryonic liver and umbilical cordblood. Would transplantation of these types of cells intoinjured livers reconstruct both hepatocytes and bile ducts?Are these cells more efcient than fetal or adult hepato-cytes for the repair of liver injury?

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