pluripotent plasticity of stem cells and liver repopulation

cell biochemistry and function

Cell Biochem Funct (2009)

Published online in Wiley InterScience

(www.interscience.wiley.com) DOI: 10.1002/cbf.1630

REVIEWARTICLE

Pluripotent plasticity of stem cells and liver repopulation

Luisa Gennero 1,2,3*, Maria Augusta Roos 1, Kirk Sperber 4, Tetyana Denysenko 2, Paola Bernabei 2,Gian Franco Calisti5, Mauro Papotti6, Susanna Cappia6, Roberto Pagni7, Giuseppe Aimo7, Giulio Mengozzi7,Giovanni Cavallo 8, Stefano Reguzzi 1, Gian Piero Pescarmona 2,3 and Antonio Ponzetto 1**

1Department of Internal Medicine, University of Turin, Turin, Italy2CERMS, Center for Experimental Research and Medical Studies, Turin, Italy3Department of Genetics, Biology and Biochemistry, Turin, Italy4Division of Clinical Immunology, Mount Sinai School of Medicine and Immunobiology Center, New York, NY, USA5Department of Aviation Defense, Rome, Italy6Department of Clinical and Biological Sciences, University of Turin and San Luigi Hospital, Orbassano-Torino, Italy7Laboratory of Clinical Chemistry, San Giovanni Battista Hospital, Turin, Italy8Department of Clinical Physiopathology, University of Turin, Turin, Italy

Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin ofhepatic and/or extra-hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self-repair andreplace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about ayear. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, andsinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types ofepithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stemcell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory boweldiseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety ofclinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential toprovide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio-artificial liver-assisteddevices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver. Copyright # 2009 JohnWiley & Sons, Ltd.

key words—stem cells; liver stem cells; liver; liver cells; autologous transplantation

INTRODUCTION

Stem cells have two important characteristics that dis-tinguish them from all the other types of cells. First, they areunspecialized cells and self-replicate for long periods oftime through cell division. Second, under certain physio-logic or experimental conditions, stem cells can be inducedto become cells with specialized functions such as liver cells,

*Correspondence to: Luisa Gennero, Experimental Medicine Researcher,FIRMS, International Foundation 59 for Research of Experimental Medi-cine (University of Turin and San Giovanni Battista "Molinette" Hospital);CeRMS, Centre for Research of Experimental Medicine, Via Santena n. 5,10126, Turin, Italy. Tel: þ39 0116336886. Fax: þ39 011611190.E-mails: [email protected], [email protected]**Correspondence to: Professor Antonio Ponzetto, Department of InternalMedicine, University of Turin, Corso A. M. Dogliotti 14, 10126 - Torino -Italia. Tel: þ39 011 633 6033. Fax: þ39 011 6336250.E-mail: [email protected]

Copyright # 2009 John Wiley & Sons, Ltd.

cardiac myocytes, neurons, and insulin-producing pancrea-tic b-cells. When a stem cell divides, each new cell has thepotential to either remain a stem cell or differentiate towarda more specialized function.1 Stem cells population has anextensive self-maintaining capability. In this context, theadult liver, having the extensive capability of maintainingparenchymal cell number throughout the life span of theorganism, can be considered as a single lineage stem cellsystem in which the hepatocyte itself is the stem cell.2 Since1990, it was proposed that stem cells include the followingthree categories: actual stem cells, potential stem cells, andcommitted stem cells.3 A typical example of committedstem cells are hepatocytes that appear to be normallyquiescent, but can be activated to produce progeny whoseonly differentiation option is hepatocytic cells.2,4 The abilityof the human body to self-repair and replace the cells andtissues of some organs is often evident (as in the case of theskin). It has been estimated that complete renewal of liver

Received 13 July 2009Revised 15 October 2009

Accepted 16 October 2009

l. gennero ET AL.

tissue takes place in about a year.5,6 Replacement of lostliver tissues is accomplished by proliferation of maturehepatocytes, hepatic oval stem cells differentiation, andsinusoidal cells as support.5,6 Hepatic oval stem cells arecapable of evolving, into hepatocytes or bile duct epithelialcells.6 They display a distinct phenotype and have beenshown to be a bipotential progenitor of two types ofepithelial cells found in the liver, hepatocytes and bileductular cells.7–9

Bone marrow (BM) stem cells have recently been shownnot to be a potential source of the hepatic oval cells; ratherthe source of oval cells and small hepatocytes in the injuredliver are endogenous liver progenitors which do not arisethrough trans-differentiation from BM cells.8–10 Both therodent and human embryonic stem (ES) cell, BMhematopoietic stem cell (HSC), mesenchymal stem cell,umbilical cord blood cell, fetal liver progenitor cell, adultliver progenitor cell as well as the mature hepatocyte havebeen reported to be capable of self-renewal, giving rise todaughter hepatocytes both in vivo and in vitro. These cellscan repopulate livers in animal models of liver injury andseemingly improve liver function. However, significantchallenges still exist before these cells can be used inhumans. These include lack of consensus in immunophe-notype of liver progenitor cells, uncertainty of thephysiological role of reported candidate stem/progenitorcell, practicality in obtaining sufficient quantity of cells forclinical use, and concerns over ethics, long-term efficacy,and safety. Current molecular techniques of stem cellidentification are confounded by cell fusion, horizontal genetransfer, incomplete differentiation, and fetal microchimer-ism. Reports of stem cell transplantation and phase 1 trials ofBM transplantation in humans for liver diseases are excitingbut require more robust verification.11

THE BACKGROUND OF STEM CELLS

Basic and clinical research accomplished during the last fewyears on a variety of stem cells has constituted a revolutionin regenerative medicine and cancer therapies by providingthe possibility of generating multiple therapeutically usefulcell types.12

Stem cell research has been developed around the pivotalconcept of the totipotent cell. The fertilized egg is totipotent(Latin etymology, totus¼ all) because it has the potential tocreate all the cells and tissues that built up an embryo. Thefertilized egg divides and differentiates until it produces acomplete mature organism. Adult mammals, includinghumans, consist of more than 200 kinds of cells includingnerve cells (neurons), muscle cells (myocytes), skin(epithelial) cells, blood cells (erythrocytes, monocytes,lymphocytes, platelets, etc.), bone cells (osteocytes), andcartilage cells (chondrocytes). Other cells, which areessential for embryonic development but are not incorpor-ated into the body of the embryo, include the extraembryonic tissues, placenta, and umbilical cord.1

Most investigators use the term pluripotent (derived fromthe Latin plures¼many) to describe stem cells that can give


rise to cells derived from all three embryonic germ layers –mesoderm, endoderm, and ectoderm.13 The germ layers arethe embryonic source of all cells of the human body. All ofthe different types of specialized cells are derived from oneof these germ layers, a property observed in the naturalcourse of embryonic development under certain laboratoryconditions.Some studies have shown the pluripotency of ES cells

after long-term culture. ES cells that have been maintainedfor a long period of time in vitro can behave as pluripotentcells in vivo. They can participate in normal embryogenesisby differentiating into any cell type in the body, and they canalso differentiate into a wide range of cell types in an adultanimal. However, normal mouse ES cells do not generatetrophoblast tissues in vivo.14

Unipotent (Latin word unus¼ one) stem cell, a term thatis usually applied to a cell in adult organisms, means that thecells in question are capable of differentiating along onlyone lineage. Undamaged tissues are typically unipotent andgive rise to one cell type under normal conditions.14

However, if the tissue becomes damaged and thereplacement of multiple cell types is required, pluripotentstem cells may become activated to repair the damage.14,15

The adult stem cell is an undifferentiated (unspecialized)cell that is found in a differentiated (specialized) tissue; itcan self-replicate and become specialized to yield all of thespecialized cell types of the tissue from which it originated.Adult stem cells are capable of self-replication during thelifetime of the organism. Adult stem cells have been found inthe BM, peripheral blood, umbilical cord, liver, skin,gastrointestinal tract, pancreas, cornea and retina of the eye,and the dental pulp of teeth.1,15–18

THE LIVER

The liver is probably the best example for a recycling system.Both parenchymal and non-parenchymal liver cells partici-pate in the clearance activities. Among the non-parenchymalcells Kupffer cells, sinusoidal endothelial cells, and naturalkiller (NK) lymphocytes exert cellular defense functions forthe whole body but also for the liver itself. Furthermore, eachcell type of the liver, including the hepatocytes, possesses itsown defense apparatus.19 The primary structural andfunctional unit of the mature liver is the acinus, that is,divided into three zones: zone 1, the periportal region; zone 2,the midacinar region; and zone 3, the peri-central region.Proliferative potential depends on liver-specific genes and arecorrelated with zonal location.20–22 The liver cells areorganized as cell plates lined on both sides by fenestratedendothelia, lining a network of sinusoids that are contiguouswith the portal and central vasculature. Recent data haveindicated that the Canals of Hering, small ducts locatedaround each of the portal triads, branch into tiny ductules thatextend and splice into the liver plates throughout zone 1forming a pattern similar to that of a bottle brush.23 TheSpace of Disse, separates the endothelia from hepatocytesall along the sinusoid. As a result of this organization,hepatocytes have two basal domains, each of which faces a


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sinusoid, and an apical domain which is defined by the regionof contact between adjacent hepatocytes. The basal domainscontact the blood, and are involved in the absorption andsecretion of plasma components, while the apical domainsform bile canaliculi, specialized in the secretion of bile salts,and are associated through an interconnecting network withbile ducts. Blood flows from the portal venules and hepaticarterioles through the sinusoids to the terminal hepaticvenules and the central vein.20,23 Gradients in the concen-tration of blood components, including oxygen, across theacinus, and following the direction of blood flow from theportal triads to the central vein, are responsible for some ofthis zonation, for example, the reciprocal compartmentationof glycolysis and gluconeogenesis.22 In contrast to hepato-cytes that occupy almost 80% of the total liver volume andperform the majority of numerous liver functions, non-parenchymal liver cells, which contribute only 6.5% to theliver volume, but 40% to the total number of liver cells, arelocalized in the sinusoidal compartment of the tissue. Thewalls of hepatic sinusoid are lined by three different celltypes: sinusoidal endothelial cells, Kupffer cells, and hepaticstellate cells (formerly known as fat-storing cells, Ito cells,lipocytes, perisinusoidal cells, or vitamin A-rich cells).Additionally, intrahepatic lymphocytes (IHL), including pitcells, i.e., liver-specific NK cells, are often present in thesinusoidal lumen.24–26 Although hepatocytes comprisealmost 80% of the liver, there are at least another ten celltypes, many of which provide ‘‘cross-talk’’ and playimportant functional roles in the normal and diseasedliver.25,26 It has been increasingly recognized that both undernormal and pathological conditions, many hepatocytefunctions are regulated by substances released fromneighboring non-parenchymal cells.24,27 Liver sinusoidalendothelial cells constitute the lining or wall of the hepaticsinusoid. They perform important filtration functions dueto the presence of small fenestrations that allow freediffusion of many substances, but not of particles of the sizeof chylomicrons, between the blood and the hepatocytesurface. Sinusoidal endothelial cells show huge endocyticcapability for many ligands including glycoproteins,components of the extracellular matrix (ECM; such ashyaluronate, collagen fragments, fibronectin, or chondroitinsulfate proteoglycan), immune complexes, transferrin, andceruloplasmin. Sinusoidal endothelial cells may function asantigen-presenting cells (APCs) in the context of both MHC-I and MHC-II restriction with the resulting development ofantigen-specific T-cell tolerance. They are also active in thesecretion of cytokines, eicosanoids (i.e., prostanoids andleukotrienes), endothelin-1, nitric oxide, and some ECMcomponents.24,26

Kupffer cells are intrasinusoidal macrophages with apronounced endocytic and phagocytic capability. They arein constant contact with gut-derived particulate materialsand soluble bacterial products so that a sub-threshold levelof their activation in the normal liver may be anticipated.Hepatic macrophages secrete potent mediators of theinflammatory response (reactive oxygen species, eicosa-noids, nitric oxide, carbon monoxide, TNF-a, and other


cytokines), and thus control the early phase of liverinflammation, playing an important part in innate immunedefense. High exposure of Kupffer cells to bacterialproducts, especially endotoxin (lipopolysaccharide, LPS),can lead to the intensive production of inflammatorymediators, and ultimately to liver injury. Besides typicalmacrophage activities, Kupffer cells play an important rolein the clearance of senescent and damaged erythrocytes.Liver macrophages modulate immune responses via antigenpresentation, suppression of T-cell activation by antigen-presenting sinusoidal endothelial cells via paracrine actionsof IL-10, prostanoids, and TNF-a, and participation in thedevelopment of oral tolerance to bacterial super-antigens.Moreover, during liver injury and inflammation, Kupffercells secrete enzymes and cytokines that may damagehepatocytes, and are active in the remodeling of ECM.24,26

Hepatic stellate cells are present in the perisinusoidalspace. They are characterized by abundance of intracyto-plasmic fat droplets and the presence of well-branchedcytoplasmic processes, which embrace endothelial cells andprovide focally a double lining for sinusoid. In the normalliver hepatic stellate cells store vitamin A, control turnoverof ECM, and regulate the contractility of sinusoids. Acutedamage to hepatocytes activates transformation of quiescentstellate cells into myofibroblast-like cells that play a key rolein the development of inflammatory fibrotic response.24,26

Pit cells represent a liver-associated population of largegranular lymphocytes, i.e., NK cells, which fulfill functionsin pathogen defense, T-cell recruitment, and modulation ofliver injury.24 They spontaneously kill a variety of tumorcells in an MHC-unrestricted way, and this anti-tumoractivity may be enhanced by the secretion of interferon-g .Besides pit cells, the adult liver contains other sub-populations of lymphocytes such as gd T-cells, and both‘‘conventional’’ and ‘‘unconventional’’ ab T-cells, the lattercontaining liver-specific NK T-cells.24

To the key mediators involved in the intercellularcommunication in the liver belong prostanoids, nitric oxide,endothelin-1, TNF-a, interleukins, and chemokines, manygrowth factors (transforming growth factor-beta (TGF-b),PDGF, IGF-I, HGF), and reactive oxygen species(ROS).24,25

Sinusoidal blood flow is, to a great extent, regulated byhepatic stellate cells that can contract due to the presence ofsmooth muscle a-actin. The main vaso-active substancesthat affect constriction or relaxation of hepatic stellate cellsderive both from distant sources and from neighboringhepatocytes (carbon monoxide, leukotrienes), endothelialcells (endothelin, nitric oxide, prostaglandins), Kupffer cells(prostaglandins, NO), and stellate cells themselves(endothelin, NO). The cellular cross-talk reflected by thefine-tuned modulation of sinusoidal contraction becomesdisturbed under pathological conditions, such as endo-toxemia or liver fibrosis, through the excess synthesisof vasoregulatory compounds and the involvement ofadditional mediators acting in a paracrine way.24,27

Hepatic stellate cells may affect turnover of hepatocytesthrough the synthesis of potent positive as well as negative


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signals such as hepatocyte-growth-factor or TGF-b.Although hepatocytes seem not to produce TGF-b, apleiotropic cytokine synthesized and secreted in the latentform by Kupffer and stellate cells, they may contribute to itsactions in the liver by the intracellular activation of latentTGF-b, and secretion of the biologically active isoform.24

In the liver, prostaglandins synthesized from arachidonicacid mainly in Kupffer cells in response to various inflam-matory stimuli, modulate hepatic glucose metabolism byincreasing glycogenolysis in adjacent hepatocytes.24

The functional heterogeneity of the various liver cell typesprovides new insights into our understanding of thedevelopment, prevention, and treatment of liver disease.27

Overview of liver alterations

The ethiological factors of liver diseases cannotunambiguously be identified from the tissue alterations.28,29

The basic reaction types of the liver alterations can bedivided into well-defined categories: adaptive changes,degeneration and intracellular storage, cell death (necrosis/apoptosis), inflammation, fibrosis, and structural reorgani-zation, as well as regeneration/proliferation (cirrhosis).29

Apoptosis occurs in several liver diseases, with a strikingform appearing to be the Councilman-like body seen inalcoholic but also in viral hepatitis. It may have role in thepathogenesis of cholestasis, biliary atresy, the ‘‘vanishingbile duct syndrome,’’ and alcoholic liver injury.28

The liver cirrhosis is characterized by nodular regener-ation of hepatocytes and diffuse fibrosis. It is caused byparenchymal necrosis followed by nodular proliferation ofthe surviving hepatocytes. The regenerating nodules andaccompanying fibrosis interfere with blood flow through theliver and result in portal hypertension, hepatic insufficiency,jaundice, and ascites.28–30

Primary biliary cirrhosis is an autoimmune liver diseasethat predominantly affects women and is characterized bychronic progressive destruction of small intrahepatic bileducts with portal inflammation and subsequent fibrosis. Theserological hallmark is the presence of antimitochondrialantibodies, which are found in 95% of patients. It hasallowed detailed immunological definition of the antigenicepitopes, the autoantibodies, and the T-cell response.In cirrhosis, CD34-expressing endothelial cells are

observed essentially at the periphery of the nodules, andwithin the septa. It is accepted that liver fibrosis is reversible,whereas cirrhosis is generally irreversible.26,31

The regenerative ability of the liver is excellent. Theprocess is influenced by several growth factors andcytokines. In the case of chronic liver damage, however,the regeneration equilibrium becomes upset and irregular.TGF-b and pro-inflammatory cytokines are importantinducers of fibro-carcinogenesis.32 Studies on liver regen-eration have led to the recognition of hepatic stem cells rolein the healing process.10,29 These cells of ‘‘oval’’morphologic appearance are built up of a primitive structure,making them suitable for this task. Ductular proliferationoriginating from hepatic stem cells can mostly be recognized


in three disease groups: liver regeneration following massiveor sub-massive liver necrosis, ductular proliferation withoutliver insufficiency, and tumorous liver diseases.9,33

Proliferative bile ductular reactions occur in a variety ofliver diseases in humans. It is a matter of debate whethersuch reactions result from progenitor cells proliferation withbiliary and hepatocytic differentiation, or its due to biliarymetaplasia of damaged hepatocytes.24 Proliferating hepaticparenchymal cells with intermediate (hepatocytic/biliary)morphologic features (oval-like cells) and combinedimmunophenotype can be identified in a variety of acuteand chronic liver diseases.24,27,28 The similarity of bileductular reactions among chronic hepatitis, and biliarydiseases suggests that they result from proliferation of oval-like progenitor cells.33

The existence of a liver stem cell population has onlygained credence recently, following the results of animalexperiments. It may contribute to hepatocyte regeneration,or even take over this role if the liver injury is severe andassociated with an impairment of hepatocyte proliferation asin cirrhosis or sub-massive/massive necrosis, due to drugs,toxins, or viruses.33–35

At present it is still unclear whether the hepatocyteformation originated from HSCs is a rarely occurringaccidental transformation, or rather, under appropriatecircumstances, a greatly effective ‘‘third’’ protective system.If the latter is true, this can fundamentally change our viewon the pathomechanism of liver diseases, making possiblenumerous novel therapeutic treatments.33,35,36

HEPATIC STEM CELLS: EXISTENCE AND ORIGIN

Stem cells are not only units of biological organization,responsible for the development and the regeneration oftissue and organ systems, but also are units in evolution bynatural selection. It is accepted that there is stem cellpotential in the liver.34–38

There has been recent progress in the isolation andcharacterization of stem/progenitor cells that may differen-tiate toward the hepatic lineage. This has raised expectationsthat therapy of genetic or acquired liver disease might bepossible by transplanting stem/progenitor cells or their liver-committed progeny. However, it is currently impossible todetermine from the many documented studies which of thestem/progenitor cell populations are the best for therapy of agiven disease.39

The liver, in the healthy adult, such as many otherorgans maintains a perfect cells gain and loss balance. Thereare three levels of cells that can respond to loss ofhepatocytes:

(1) M
ature hepatocytes, which proliferate in normal livertissue renewal less severe liver damage and; they areunipotent, ‘‘committed’’ and respond rapidly to liverinjury.
(2) O
val cells, which are activated to proliferate when theliver damage is extensive and chronic, or when prolifer-ation of hepatocytes is inhibited; they lie within or

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immediately adjacent to the canal of Hering; they areless numerous, bipotent, and respond by longer, but stilllimited proliferation.

(3) E
xogenous liver stem cells, which may derive fromcirculating HSCs or BM stem cells; they respond toalcohol-related injury or hepatic-carcinogenesis; theyare multipotent, rare, but have a very long proliferationpotential. They make a more significant contribution toregeneration, and even completely restore normal func-tion in a murine model of hereditary tyrosinemia.38
The worldwide shortage of donor livers to transplant endstage liver disease patients has prompted the search foralternative cell therapies for intractable liver disease. EScells can be readily differentiated into hepatocytes, and theirtransplantation into animals has improved liver function inthe absence of teratoma formation: their use in bio-artificialliver support is an obvious application. In animal models ofliver disease, adopting strategies to provide a selectiveadvantage for transplanted fetal or adult hepatocytes haveproved highly effective in repopulating recipient livers. Thepoor success of today’s hepatocyte transplants can be attrib-uted to the lack of a clinically applicable procedure to force asimilar repopulation of the human liver. The activation ofbipotential hepatic progenitor cells is clearly vital for sur-vival in many cases of acute liver failure, but surprisinglylittle progress has been made with these cells in terms oftransplantation. Finally, there is the controversial subject ofautologous Bone Marrow Stem Cells (BMSCs), and whilethe contribution of these endogenous cells to liver turnoverseems at best trivial, results from a small number of phase 1studies of transplantation of BM to cirrhotic patients havebeen moderately encouraging.40

Hepatocytes

Hepatocytes as hepatic stem cell. The participation ofresident liver stem cells has never been demonstrated.3,4

Replacement of lost liver tissues is accomplished byproliferation of mature hepatocytes (and supporting sinu-soidal cells).5 In the classic partial hepatectomy (PH)experiments, the loss of two-thirds of the rat liver is replacedwithin 2 weeks by proliferation of hepatocytes.41

Although periportal hepatocytes appear to proliferateearly after PH, all hepatocytes, including those immediatelyadjacent to the central vein, may undergo mitosis andproliferate promptly, continuously replenishing the lostcells.42,43

Overturf et al. performed serial transplantation of alimited number of unfractionated adult parenchymalhepatocytes in fumaryl-aceto-acetate hydrolase (FAH)deficiency mice. The results demonstrate that such cellscan divide at least 69 times without loss of functions.43,44 Soit can be concluded that hepatocytes are highly proliferativeand have growth potential similar to that of HSCs.45

A cell population that has an extensive self-maintainingcapability is the definition of stem cells. In this context, theadult liver, having the extensive capability of maintainingparenchymal cell number throughout the life span of the

right # 2009 John Wiley & Sons, Ltd.

organism, can be considered as a single lineage stem cellsystem in which the hepatocyte is the stem cell.42,43

Since 1990, Potten and Loeffler3 proposed for theintestinal epithelium the concept of stem cells as specialcells that may divide without maturation while normalcells approaching functional competence may mature butdo not divide. On the contrary, transit cells divide andmature, showing intermediate properties between stem cellsand mature functional cells. The need to describe thistransition process and the variable coupling betweenproliferation and maturation, leads to formulate a spiralmodel of cell and tissue organization. It is concluded that thesmall intestinal crypts contain 4–16 actual stem cells insteady-state but up to 30–40 potential stem cells (clonogeniccells) which may take over stem cell properties. This impliesthat transit cells can, under certain circumstances, behavelike actual stem cells while they undergo maturation inspecific conditions. There is also evidence that theproliferation and differentiation/maturation processes aresubject to controls that ultimately lead to a stem cells,potential stem cells, and committed stem cells.3 Sohepatocytes appear to be ‘‘committed stem cells’’ that arenormally quiescent, but can be activated to produce progenywhose only differentiation option is hepatocytic (unipotentconcept).42,43

In the development of liver, the early fetal hepatocytes orhepatoblasts are progenitors for both adult hepatocytes andbile epithelial cells, which suggests that hepatoblasts are atleast bipotential precursors.45 The question then ariseswhether either or both of the cell lineages derived from thehepatoblast retain the ‘‘bipotential capability’’ of theprecursor cells. There is, at present, no substantial evidenceindicating that adult hepatocytes are more than a uni-potential committed stem cell system, while adequate datahave been collected that show the existence of ‘‘oval cells’’in adult liver. Oval cells have lineage options similar to thosedisplayed by hepatoblasts in early stages of liver develop-ment.46 Those oval cells can be regarded as bipotentialprecursors ‘‘for the two hepatic parenchymal cell lineages.’’They are able to differentiate into mature hepatocytes orcholangiocytes in response to various types of stress orinjury.52 Although a few cases of allogenic transplantationhave resulted in long-term engraftment and function, only apartial and transient correction of the disease was achieved.This may partly result from a lack of proliferation oftransplanted cells.47

LIVER OVAL CELLS

Hepatic oval stem cells are bipotent stem cells

Morphologically, oval cells are small in size (approximately10mm), with a large nucleus-to-cytoplasm ratio, with anoval-shaped nucleus (hence their name).38 They are thedescendants of the stem cells and are found in the portal andperiportal regions in experimental animals within days fromliver injury. These cells proliferate to form narrow ductules,which may stain positively for biliary cytokeratins CK 19,


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and radiate out into the damaged parenchyma.34 Probably,these cells, deriving from the BM or (from) the cellpopulation associated with the BM, can be activated inhepatic stem cell. The morphological characteristic of thehepatic stem cells is similar to the oval cells and itsphenotypic profile is c-kit/CD45/TRE19 and OV-6positive.38,46,48 In Libbrecht et al.’s experiments,49,50 thesmall OV-6-positive oval cells are analogous to those seen inrat models and may represent human liver progenitor cellsthat can differentiate into OV-6-positive ductal cells orlobular hepatocytes. The most commonly recognized tissuereaction in support of oval cells in human is the appearanceof ‘‘ductular reactions.’’50 Ductular reaction is the prolif-erative response to many types of liver injury in humans,characterized by an increase of bile duct-like structures. Theanatomic location of oval cell has also been a subject ofcontroversy. Results from a detailed time course study ofactivation of hepatic stem cells indicated that the earliestpopulation of proliferating OV-6 positive cells is located inthe small bile ductules. In addition, these early population ofOV-6 positive cells express albumin and a-fetoprotein.Therefore, it seems likely that the major source of oval cellsis derived from the lining cells of the biliary ductules andthat these cells constitute the dormant/facultative hepaticstem cell compartment.50

Theise et al.’s23 experimental work suggests that ovalcells lie within or immediately adjacent to the canal ofHering, which is the anatomic juncture of the hepatocytecanalicular system, and the terminal branches of the biliarytree. Oval cells express similar markers to hepatocytes orbile duct cells. High levels of certain mRNAs like a-fetoprotein and stem cell factor can also be expressed byoval cells. OV-6 identifies a cytokeratin of molecular weight56 000, with epitopes shared on cytokeratins 14 and 19. It ispresent in rat liver on bile ducts, oval cells, and nodularhepatocytes as well as transitional hepatocytes.51 In humanliver, OV-6 identifies cells in the ductal plate, oval cells andbile ducts and ductules in fetal tissue, and oval cells found infocal nodular hyperplasia.49,50

Full length a-fetoprotein has been shown to be expressedin oval cells and small basophilic hepatocytes during theearly stages of carcinogenesis.52,53 Hence, AFP expressioncan be used as an indicator for an early hepatic lineage, andhas also served as an important marker for the activation ofthe hepatic stem cell compartment. AFP is stronglyexpressed in many patients with hepatocellular carcinoma,and high levels of a-fetoprotein expression have beenreported as an independent prognostic factor. Poor prognosisassociated with high a-fetoprotein is due to high cellproliferation, high angiogenesis, and low apoptosis.54

C-kit ligand (SCF/c-kit system, CD117), encodes a trans-membrane tyrosine kinase protein and belongs to the sub-family of platelet derived growth factor. Studies have shownthat both SCF and c-kit are expressed in the bile duct cellsand the expression of both genes is increased in oval cells inthe 2-acetylaminofluorene and PH model.38,46,55 Gp130-mediated IL-6 signaling may play a role in oval cellproliferation in vivo. Levels of IL-6 are elevated in livers of


mice treated with a choline-deficient ethionine-supple-mented (CDE) diet that induces oval cells, and there is areduction of oval cells in IL-6 knock-out mice. The CDE dietrecapitulates characteristics of chronic liver injury inhumans.56

Hepatic oval cells display a distinct phenotype andhave been shown to be a bipotential progenitor of twotypes of epithelial cells found in the liver, hepatocytes,and bile ductular cells.37,45 In fact, in vitro and in vivoanimal studies now suggest that oval cells are bipotentstem cells and, indeed, can differentiate into bile ductularcells or hepatocytes to allow repopulation of the injuredliver.34,37,57

Oval cells are activated to proliferate after hepatocyte lossin the mature liver. This phenomenum happens when theliver damage is extensive and chronic, or if proliferationof hepatocytes is inhibited, such as by viral infection orchemicals. Then their progeny extended across the liverlobule and differentiated into either hepatocytes or bile ductcells, rebuilt the liver.38,57

Moreover, extensive studies in rodent models of hepatic-carcinogenesis and other non-carcinogenic injury modelssuggest that oval cells may represent a facultative hepaticprogenitor/stem cell compartment. These cells not only canbe activated to proliferate but also differentiate themselvesinto mature hepatocytes and biliary epithelial cells undercertain conditions.55,58

As the oval cells differentiate into hepatocytes they mayshow positive staining for pyruvate kinase isoenzyme L-PK,albumin, and a-fetoprotein.38,45 Other investigators suggestthat they are the progeny of a hepatic stem cell, also referredto as adult liver stem cells.45,59 The mechanisms by whichthese cells are activated to proliferate and differentiateduring liver regeneration is important for the development ofnew therapies to treat liver disease.59

Hepatic oval cell activation, proliferation, and differen-tiation has been observed under certain physiologicalconditions, mainly when the proliferation of existinghepatocytes has been inhibited as a consequence of a severehepatic injury.60 In addition to the effects seen in theliver, oval cells or oval like-cells are implicated in thearchitecture of the regenerated pancreas after injury61,62 andthey may be involved in the regeneration of other organsas well.60

Proliferative bile ductular reactions occur in a variety ofliver diseases in humans. It is a matter of debate whethersuch reactions result from progenitor cell proliferation withbiliary and hepatocytic differentiation, versus biliarymetaplasia of damaged hepatocytes. Proliferating hepaticparenchymal cells with intermediate (hepatocytic/biliary)morphologic features and combined immunophenotype canbe identified in a variety of acute and chronic liver diseases.The similarity of bile ductular reactions among chronichepatitis, alcoholic, and biliary diseases suggests thatthey result from proliferation of oval-like progenitorcells.46,48,63

Oval cell isolation and culture techniques, together withstem cell transplantation strategies, may in the future


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provide novel treatments for individuals with inherited andacquired hepatic disorders or pancreatic diseases.57,61,62

In the past, there was also evidence that BM stem cellscontributed to liver regeneration.37 Recently, BM stem cellshave been shown not to be a potential source of the hepaticoval cells that sources of oval cells and small hepatocytes inthe injured liver are endogenous liver progenitors and thatthey do not arise through trans-differentiation from BMcells.8

Numerous in vivo and in vitro studies have documented acentral role of oval cells in liver biology and carcinogen-esis.45,46,64–66 In a number of morphological studies, thepresence of cells with a distinctive ‘‘small’’ or oval cell-likeappearance have been reported in diseased human livertissue.46,48,63 This includes severe hepatic necrosis, alco-holic cirrhosis, focal nodular hyperplasia, hepatoblastoma,and biliary diseases such as primary biliary cirrhosis orbiliary atresy.46,48

The possible involvement of hepatic stem cells in thedevelopment of dysplastic nodules, hepatocellular carci-noma, and cholangiocarcinoma has been suggested butremains highly controversial.37,64,65

However, other than understanding a potential originof these cells and some of the markers that characterizethem, it still remains unclear the way these cells migrate(‘‘home’’) into the damaged areas and how they begintheir differentiation into mature and functioning hepaticcells.37

OXIDATIVE STRESS AND OVAL CELL

In animals, the combination of oxidative liver damage andinhibited hepatocyte proliferation increases the numbers ofhepatic progenitors (oval cells). Different murine models offatty liver disease (FLD) and patients with non-alcoholicfatty liver disease (NAFLD) or alcoholic liver disease showthat oval cells increase in fatty livers.28,65–67

To varying degrees, all mouse models exhibit excessivehepatic mitochondrial production of H2O2, a known inducerof cell-cycle inhibitors. In mice with the greatest H2O2

production, mature hepatocyte proliferation is inhibitedmost, and the greatest number of oval cells accumulates.These cells differentiate into intermediate hepatocyte-likecells after a regenerative challenge.24,25,28,65

Animal data indicate that oval cells are activated whenoxidative stress inhibits the regenerative capability of moremature hepatocytes.66,68 Oxidative stress is thought to play amajor role in the pathogenesis of both alcoholic andNAFLD.69,70 The replicative activity of mature hepatocytesis also known to be inhibited in patients with alcoholichepatitis,71 rodents with alcohol-induced fatty livers72 and insome animal models of NAFLD.73,74 Although thecombination of oxidative liver damage and inhibitedhepatocyte proliferation provides a strong stimulus for ovalcell accumulation in the liver, whether or not this cellpopulation is expanded in FLD has never been studied.28


During viral-and steatosis-induced liver disease the contri-bution of BM cells to hepatocytes, either via oval cells or byindependent mechanisms, is minimal and the majority ofoval cells responding to this injury are sourced from theliver.74 Hepatic oval cells are also increased significantly inpatients with NAFLD and alcoholic liver disease.28,65 Inhumans, fibrosis stage and oval cell numbers, as well as thenumber of intermediate hepatocyte-like cells, are stronglycorrelated.9,10,26,27,35

However, cirrhosis is not required for oval cellaccumulation in either species. Rather, as in mice,progenitor cell activation in human FLDs is associatedwith inhibited replication of mature hepatocytes.37

The activation of progenitor cells during FLD mayincrease the risk for hepatocellular cancer, similar to the oneobserved in the Solt–Farber model of hepatic-carcinogenesisin rats.35–38,42,60

In rodent models for hepatocarcinogenesis, small ovalcells that express both hepatocyte and cholangiocytemarkers accumulate in the liver before cancerous nodulesdevelop.70,72,73,75 Similar oval cell(s) accumulation has alsobeen described in human liver, close to hepatitis B-associated hepatocellular carcinoma,74 in hepatoblastoma,76

in regenerating liver,77 and in cholestatic liver diseases.78

The putative progenitor cells accumulation in the livers ofpatients with chronic hepatitis C appears to be anacknowledged risk factor for hepatocellular carcinoma.79,80

A direct correlation between the oval cell response and thedegree of hepatic inflammation suggests that inflammatorycytokines and the resultant oxidant stress play a role in theactivation of human progenitor cells during chronic viralhepatitis.9,10,35–37

Although the expression of ATP-binding cassette proteinsMRP-1, MRP-3, and MDR-1 is increased in the progenitorcell compartment in various human liver diseases, thesetransporters are known to be cytoprotective.81 MRP-1 helpscells to secrete GSSG or the GSH conjugates of 4-hydroxynononeal, products of oxidative stress reactions.82

Therefore, the phenotype of oval cells may convey asurvival advantage that permits them to accumulate inconditions that severely damage more mature hepato-cytes.9,10,35–37 This helps to explain the positive relationshipbetween oval cells and accumulation or reductions in maturehepatocyte mass through stem hepatocyte-like cells way. Infact oxidant stress reduces the viability and expansion ofmature hepatocytes, while sparing less mature hepaticprogenitors.35,36

Yang et al.’s experimental work suggests that senescentmature hepatocytes exhibited hepatic accumulation ofliver progenitor oval cells and oval cell numbers increasedwith the demand for hepatocyte replacement. Therefore,although hepatic oxidant production and damage aregenerally increased in fatty livers, expansion of hepaticprogenitor cell populations helps to compensate forthe increased turnover of damaged mature hepatocytes.Indeed, the induction of mechanisms to replace damagedhepatocytes is important for limiting the progression ofFLD.83


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A similar paradigm has been implicated during neoplastictransformation.64 Thus, it is tempting to speculate thathepatocellular carcinoma develops because chronic oxi-dative stress exerts a selection pressure that favors theoutgrowth of progenitor cell clones that are most resistant tooxidant damage.36,37 Further studies are needed to evaluatethis possibility.

THE ROLE OF HEPATOCYTES AND OVAL CELLSIN LIVER REGENERATION AND GROWTHFACTORS

Liver regeneration after PH is a very complex and well-orchestrated phenomenon. It is carried out by theparticipation of all mature liver cell types.84 Normallyquiescent hepatocytes undergo one or two rounds ofreplication to restore the liver mass by a process ofcompensatory hyperplasia.85 In situations when hepatocytesor biliary cells are blocked from regeneration, these celltypes can function as facultative stem cells for each other.Liver manages to restore any lost mass and adjust its size tothat of the organism, while at the same time providing fullsupport for body homeostasis during the entire regenerativeprocess.84 A large number of genes are involved in liverregeneration, but the essential circuitry required for theprocess may be categorized into three networks: cytokine,growth factor, and metabolic. There is much redundancywithin each network, and intricate interactions exist betweenthem. Thus, loss of function from a single gene rarely leadsto complete blockage of liver regeneration. The innateimmune system plays an important role in the initiation ofliver regeneration after PH, and new cytokines and receptorsthat participate in initiation mechanisms have beenidentified. Hepatocytes primed by these agents readilyrespond to growth factors and enter the cell cycle.Presumably, the increased metabolic demands placed onhepatocytes of the regenerating liver are linked to themachinery needed for hepatocyte replication, and mayfunction as a sensor that calibrates the regenerative responseaccording to body demands. In contrast to the regenerativeprocess after PH, which is driven by the replication ofexisting hepatocytes, liver repopulation after acute liverfailure depends on the differentiation of progenitor cells.Such cells are also present in chronic liver diseases, but theircontribution to the production of hepatocytes in thoseconditions is unknown.85

In addition to hepatocytes and non-parenchymal cells, theliver contains intrahepatic ‘‘stem’’ cells which can generatea transit compartment of precursors named oval cells.64–66

Local hepatic microenvironment may participate in theoval cell-mediated liver regeneration through the cell–celland cell–matrix interactions. In the liver, normally quiescentdifferentiated cells replicate rapidly after tissue resection,while oval cells proliferate and generate lineage only insituations in which hepatocyte proliferation is blocked ordelayed.57,64,86,87


Many studies have now revealed the importance of factorssuch as fibroblast growth factors (FGFs) and bonemorphogenic protein-4 (BMP-4) in the liver specifica-tion.88,89 After such commitment, the resident cells of theprimitive liver bud undergo balanced events includingproliferation, apoptosis, and differentiation to eventuallyconstitute a functioning organ.90

Several signaling pathways such as the Wnt/b-cateninpathway, jagged/notch pathway, and sonic hedgehog path-way have been shown to play a role in maintenance andexpansion of stem cells elsewhere. They are involved in thestringent regulation of the self-renewal and/or differen-tiation of adult stem cells.89,91 Of particular importance isthe Wnt/b-catenin pathway because of its involvement inliver growth, regeneration, and cancer as well as its role inepithelial–mesenchymal transitions.92–95 High levels of b-catenin protein expression is present in liver at embryonicday 10 (E10) through E14 of liver development.96 Duringearly liver development, the role of b-catenin/Wnt-3A hasbeen clarified in cell proliferation, apoptosis, and differen-tiation using embryonic liver culture model.96,97 In addition,up to 40% of hepatocellular carcinomas are clonal,potentially arising from stem cells and increased activationof multiple pathways including IL-6/STAT3, WNT, CDK4,and hedgehog, as well as loss of response to the TGF-bsignaling pathway.98 Pharmacological activation of thecanonical Wnt/b-catenin signaling induced proliferation ofcultured hepatic stem/progenitor cell lines. Increase of b-catenin protein was observed in oval cell compartments.87

Embryonic liver cultures have also been used forexploring the effect of exogenous growth factors and toinvestigate function of novel genes by in vitro knockoutstudies.99–101 Normally E10 embryonic liver in mouse ispredominantly composed of c-kit-positive cells (90%) thatdrops to 25% at E13.5 to E14 indicating their differen-tiation.90

FGFs are known to induce the expression of liver-specificgenes in the adjacent foregut endoderm, thus initiating liverbud formation through the selective proliferation ofhepatoblasts. FGF10 has been implicated in the proliferationor differentiation of various stem/progenitor cell popu-lations. Mesenchymally expressed FGF10 regulates differ-entiation of the foregut epithelial cells toward hepatic orpancreatic cell lineages in zebrafish, suggesting a significantrole for FGF10 in the differentiation of liver precursor cells.FGFs bind in a promiscuous manner to seven differenttyrosine kinase FGF receptors (FGFRs) encoded by fourgenes to activate the RAS-RAF-mitogen activated proteinkinase (MAPK) pathway. Similar pleiotropic phenotypes ofnull mutants for either FGF10 or the FGFR2-IIIb isoform(FGFR2b) indicate that this ligand and receptor are eachother’s main – although not exclusive – binding partners.Interestingly, FGFR2B may be crucial to postnatal liverregeneration, because adult mice expressing a soluble,dominant-negative form of FGFR2B display decreased cellproliferation after PH.102

Sekhon et al.’s90 experimental work suggests that theeffect of FGF1 and FGF4 were overall similar on the


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observed tissue histology. In the presence of FGF8, no ductformation was observed, the cells arranged in sheets onlyand the apoptosis data reveals only marginal effect of FGF1and FGF4, in contrast FGF8 significantly promoted cellsurvival in the embryonic liver culture. This observationsuggests that the decrease in the ductal structures (in thepresence of FGF1/4) or their complete absence (FGF8) wasnot because of any increase in cell death of the ductal cellsbut might be directly augmenting the sheet-like architec-ture.90 Cell proliferation of resident cells increasedsignificantly in the presence of any of the tested FGFsindicating an overall morphogenetic effect on the cells inembryonic liver cultures. The balance between these twocellular events of apoptosis and proliferation is crucial forphysiological growth in multiple organs and tissuesincluding the hepatic growth during early liver develop-ment.101 Moreover aberrations in their balance result inpathologies ranging from hyperplasia to neoplasia.103 Datasuggest that FGF signaling is one such pathway that isdictating a balance of these events as they affect apoptosis(especially FGF8) as well as promote resident cellproliferation inside the in vitro organ cultures, suggestingan overall hepatotrophic effect.90 FGF8 treatment induced amore dramatic liver phenotype: 80–90% of the entire organculture was constituted by c-kit, a-fetoprotein, and albumin-positive hepatic progenitors. However, these cells lackedCK-19 positivity precluding them from being true bipoten-tial progenitors. Also, failure of formation of any ductalstructures is also supporting this observation. This suggeststhat FGF8 does induce progenitor enrichment, but inaddition, also induces a one step-differentiation of theseprogenitors toward hepatocytic lineage Thus FGF8 enrichesthe culture for unipotential hepatocytic progenitors bypromoting cell proliferation and survival.90 Their exper-imental work also suggests that FGF8 induce thesignificantly higher b-catenin staining and its nuclearlocalization. Also, a substantially higher number of residentcells demonstrates membranous localization of b-cateninthat might be indicative of a more differentiated cell type,reflecting a contributory mechanism for the one-stephepatocytic differentiation of the bipotential hepaticprogenitors. These observations pertaining to the effect ofFGF8 on the enrichment and differentiation are crucialbecause the molecular mechanisms that dictate bifurcationof the differentiation of the bipotential hepatic precursors areprimarily obscure.61

FGFs on ex vivo liver development can be applied tohepatic stem cell biology and tissue culture applications.Hepatic tissue engineering for cell therapy or bioreactorapplications is being investigated in liver failure for short-term or long-term relief because of an obvious paucity ofdonor organs for orthotopic liver transplantation. Howeverthis area faces several challenges including the absence of asuitable cell source.104–106 Use of a hepatic progenitor cellpopulation is an attractive alternative because these cellspossess not only a higher plasticity and survival capabilitybut also a renewal ability. The positive impact of FGFs onhepatic progenitor pool expansion might find application in


cell therapies, hepatic tissue engineering, and artificial liverdevices.90,107

POTENTIAL APPLICATIONS OF STEM CELLSIN HEPATOLOGY

It has been hypothesized that liver stem cells may be usefulfor treating liver chronic diseases and other inflammatorydiseases that results in healing of the organ damage. Ingastroenterology and hepatology, the first attempts totranslate stem cell basic research into novel therapeuticstrategies have been made for the treatment of severaldisorders, such as inflammatory bowel diseases, diabetesmellitus, celiachy, and acute or chronic hepatopaties.108

Most liver diseases lead to hepatic dysfunction with organfailure. Liver transplantation is the best curative therapy, butit has some limitations such as donor shortage, possibility ofrejection, and maintenance of immunosuppressant. Newtherapies have been actively searched for over severaldecades, primarily in the form of artificial liver supportdevices and hepatocyte transplantation, but both of thesemodalities remain experimental. Stem cells have recentlyshown promise in cell therapy because they have thecapacity for self-renewal and multi-lineage differentiation,and are applicable to human diseases. Very recent reports ofunexpected plasticity in adult BM have raised hopes of stemcell therapy offering exciting therapeutic possibilities forpatients with chronic liver disease.109

The promise of liver stem cells lie in their potential toprovide a continual and readily available source of liver cellsthat can be used for gene therapy, cellular transplant, bio-artificial liver-assisted devices, drug toxicology testing, anduse as an in vitro model to understand the developmentalbiology of the liver.5,6 Reproducible stem cell programming,either embryonic or adult, will open a variety of clinicalapplication strategies for the treatment of tissue injuries,degenerated organs, and body components, respectively, inthe future. The use of stem cells in humans, presentbiological advantages and disadvantages related to differenttypes of stem cells and ethical aspects connected to the ESuse.28 Successful application of stem cell-associatedtherapies in man will be closely related with patient risktransplantation profiles, stringent indications, long-termpatient outcomes, and will be determined by cost-benefitefficacy.28,29

IN THE FUTURE

In the future, pluripotent plasticity of stem cells will open avariety of clinical application strategies for the treatment oftissue injuries, degenerated organs, and body components.Clinical application of hepatocyte transplantation is limitedby good quality donor livers for the isolation of cells,independently from their mesenchymal or hematopoieticorigin. When the microenvironment is balanced, differenttypes of stem cells have all a role in liver regeneration duringand after several liver diseases.110,111 When the micro-environment is not balanced, different types of stem cells


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have all a role in liver fibrous reactive repair.112 It has beenestimated that complete renewal of liver tissue takes place inabout a year.111 The ability of the human body to self-repairand replace the liver cells and liver tissue is often evident,independently from the origin of hepatic and/or extra-hepatic stem cells during reactive liver repopulation.111

Recent publications thus focus on stem cells as suitablesources for hepatocytes and liver repopulation strategies thatcould reduce the number of transplanted cells.110

The awareness of the complexities and heterogeneity ofthe liver will add to a greater understanding of liver functionand disease processes that lead to toxicity, cancer, and otherdiseases. The study of liver stem cells and hepatic oval stemcells has demonstrated that the in vivo differentiation andproliferation of mature hepatocytes with sinusoidal cellssupport is also possible in vitro.16,42,46,48 This new frontier ofliver research can represent a neo-adjuvant method to liversurvival in attended of the transplant or special therapy.5,6

ACKNOWLEDGEMENTS

This research is supported by Grants from ‘‘Istituto Super-iore di Sanita’’, ‘‘Ministero dell’Educazione e dellaRicerca’’; ‘‘Regione Piemonte’’; ‘‘CRT Foundation’’, ViaXX Settembre, 10100 Turin, Italy; ‘‘FIRMS, FondazioneInternazionale per la Ricerca di Medicina Sperimentale orInternational Foundation for Research of ExperimentalMedicine (University of Turin and San Giovanni Battista‘‘Molinette’’ Hospital) and ‘‘CeRMS Centre for Research ofExperimental Medicine or Centro per la Ricerca di MedicinaSperimentale), Via Santena n. 5, 10126, Turin, Italy’’.

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pluripotent plasticity of stem cells and liver repopulation

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