Cell therapy for the diseased liver: from stem cell ... · of the work we do. For many years, liver biologists were limited to in vitro work on liver cells, or in vivo studies using

IntroductionAs practicing biologists, we are aware of the potential applicationsof the work we do. For many years, liver biologists were limitedto in vitro work on liver cells, or in vivo studies using targetedmutations, induced carcinogenesis and liver regeneration. For along time, liver cell transplantation was just a dream. As we willrelate below, it has become an experimental reality in thelaboratory over the past 15 years. However, liver celltransplantation remains distant from the majority of potentialclinical applications. Experimental biology is an area where youcan keep working on the model until you obtain optimal results;this never applies in the clinic. For fundamental liver biologists,cell transplantation using in vivo models provides an optimal testof the differentiation potential and tissue repair activity of specificcell types. For those oriented towards the study of immediateproblems of human health, liver cell transplantation is providingus with a new generation of models that will, for the first time,permit manipulations of hepatotropic pathogens of man,including hepatitis B virus (HBV), hepatitis C virus (HCV) andPlasmodium falciparum. This review will discuss the evolutionand utility of these model systems.

Initial discoveries from liver regeneration modelsTransplanted hepatic cells can replace a diseased liver: theproliferative potential of adult hepatoctyesThe initial observation that opened up the field of livertransplantation was serendipitous. With the goal of establishing anin vivo system to analyze the coagulation systems, Heckel et al.produced transgenic mice expressing uPA (urokinase-typeplasminogen activator) from a chimeric murine uPA gene underthe control of promoter sequences from the albuminenhancer/promoter to restrict expression to the liver (Alb-uPA)(Heckel et al., 1990). The resulting transgenic mice frequently diedfrom neonatal bleeding. Sandgren et al. reported on the propertiesof transgenic lines, characterized by minimal expression of uPAwith normalization of liver function over the first weeks (Sandgrenet al., 1991). They observed that something happened to the liversof transgenic mice, which were white and fatty, whereas normalizedjuvenile animals had white livers with red spots, and older micehad entirely red livers (Fig. 1A).

Sandgren et al. demonstrated that the transgene was under-represented in DNA from red liver nodules (Sandgren et al., 1991).They concluded that transgene expression was toxic to hepatocytes,and deletion of the transgene was followed by clonal expansion ofthe normalized cells. The existence of only a few ‘cured’ cells wassufficient to ensure replacement of the diseased liver, revealing anunexpectedly high proliferative potential of adult liver cells. Theythen demonstrated, using genetically marked lacZ-expressingdonor cells, that hepatic cell transplantation in young Alb-uPAtransgenic mice resulted in replacement of the diseased liver tissue(Fig. 1B) (Rhim et al., 1994). They calculated that donor hepatocytesachieved 12-16 rounds of doubling during liver replacement. Thetransplanted liver cells underwent expansion only in the Alb-uPAtransgenic mice and not in normal livers. It was concluded that a

PERSPECTIVE

Disease Models & Mechanisms 113

Disease Models & Mechanisms 1, 113-130 (2008) doi:10.1242/dmm.000463

Cell therapy for the diseased liver: from stem cellbiology to novel models for hepatotropic humanpathogensNicolas Brezillon1,2,3, Dina Kremsdorf1,2,3 and Mary C. Weiss1,2,3,4,*

1INSERM, U845, Pathogenèse des Hépatites Virales B et Immunothérapie, Paris75015, France2Université Paris-Descartes, Faculté de Médecine René Descartes, Paris 75015,France3Institut Pasteur, Paris 75015, France4CNRS, URA 2578, Département de Biologie du Développement, Paris 75015,France*Author for correspondence (e-mail: [email protected])

It has long been known that hepatocytes possess the potential to replicate through many cell generations becauseregeneration can be achieved in rodents after serial two-thirds hepatectomy. It has taken considerable time andeffort to harness this potential, with liver regeneration models involving hepatocyte transplantation developingover the past 15 years. This review will describe the experiments that have established the models andmethodology for liver repopulation, and the use of cells other than adult hepatocytes in liver repopulation,including hepatic cell lines and hematopoietic, cord blood, hepatic and embryonic stem cells. Emphasis will beplaced on the characteristics of the models and how they can influence the outcome of the experiments. Finally, anaccount of the development of murine models that are competent to accept human hepatocytes is provided. Inthese models, liver deficiencies are induced in immunodeficient mice, where healthy human cells have a selectiveadvantage. These mice with humanized livers provide a powerful new experimental tool for the study of humanhepatotropic pathogens.

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regenerative stimulus was necessary to obtain clonal expansion ofthe transplanted cells. Finally, Rhim et al. introduced the nude geneinto the Alb-uPA trangenics to demonstrate that xenogenichepatocytes from donor rats could reconstitute diseased livers inmice (Rhim et al., 1995).

These studies laid the foundations for liver cell transplantation.They described the application of a genetic-based animal modelfor competitive liver regeneration, in which exogenous cellsintroduced by transplantation, or endogenous hepatocytes lackinga deleterious transgene, have a selective advantage and can replacethe diseased tissue. They defined the window where endogenousregeneration is on-going, and showed that immune deficiency genespermit xenotransplantation, leading Rhim et al. to speculate thatAlb-uPA mice with ‘humanized’ livers, possessing humanhepatocytes, could be produced to provide models for humandisease (Rhim et al., 1995).

A second mouse system for liver cell transplantation:pharmacological attenuation of a lethal gene deficiencyDeficiency of a liver-specific enzyme, fumarylacetoacetatehydrolase (FAH) causes the human disease, hereditary tyrosinaemiatype I. The same deficiency in the mouse is lethal to neonates(Grompe et al., 1993). As the last enzyme in the tyrosinedegradation pathway, FAH deficiency leads to the accumulation oftoxic metabolites in the liver. Blocking the first steps of the pathway

should interfere with generation of the toxic metabolites, and theappropriate inhibitor, 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3cyclohexanedione (NTBC) was tried on a few human patients(Lindstedt et al., 1992). Grompe et al. described that the same drug,initiated in utero and maintained thereafter, permitted survival ofFAH-deficient animals and normalized their liver function (Grompeet al., 1995). Withdrawal of the drug resulted in weight loss anddeath after two months. With the introduction of NTBC treatment,FAH deficiency in mice had been converted from a lethal disorderin neonates to a conditional lethal disorder.

Overturf et al. reported that transplantation of freshly isolatedhepatocytes from wild-type congenic animals (differing frommutants in only one genetic locus) permitted survival of mutantFah animals after drug withdrawal at the time of transplantation.DNA analysis of the liver revealed the presence of a wild-type Fahgene in animals in which NTBC treatment had been discontinued.Again, a selective advantage of transplanted cells was necessary toobtain repopulation (Overturf et al., 1996). As few as 1000transplanted hepatocytes were sufficient to rescue the majority ofanimals

Serial and competitive repopulation by adult hepatocytesOverturf et al. demonstrated that mouse hepatocytes can be usedfor up to six rounds of serial transplantation of FAH-deficient mice,amounting to 69 rounds of cell doubling (Overturf et al., 1997). Toinvestigate whether all hepatocytes from perfused liver possessequal proliferative potential, competitive repopulation experimentswere performed using DNA markers to track cells (Overturf et al.,1999). Centrifugal elutriation was employed to separate the donorhepatocytes into peaks corresponding to large, medium and smallhepatocytes. For competitive repopulation, known numbers ofisolated cell populations were mixed with known numbers ofunfractionated populations. The medium hepatocytes competedbest, whereas small hepatocytes competed least well, the latter beingthe most likely pool for containing potential liver stem cells.Similar experiments revealed that hepatocytes that had alreadyrepopulated a liver failed to demonstrate a selective advantage ordisadvantage. Taken together, these experiments indicate thatmature hepatocytes, and not liver ‘stem cells’, can repopulate theFAH-deficient liver.

A model for visual assessment of liver repopulation: theDPPIV-deficient Fischer ratAs an alternative to using genetic markers that confer liver toxicity,partial hepatectomy (PH) encourages proliferation of transplantedcells. Laconi et al. combined pretreatment with retrorsine, apyrrolizidine alkaloid that causes persistent inhibition of hepatocytedivision, with PH performed simultaneously with celltransplantation (Laconi et al., 1998). In addition, they used syngenicrats of the Fischer 344 line, of which one sub-strain, deficient inenzyme dipeptidyl peptidase IV (DPPIV) activity, serves as arecipient for wild-type donor cells (Gupta et al., 1995). Ahistochemical stain imparts localized red color to DPPIV-positivecells.

Adult hepatocytes were infused into the portal vein of retrorsine-treated hepatectomized recipients. A few days after transplantation,isolated cells or tiny clusters of DPPIV-positive cells were visualized;after a month, clusters were composed of 100 or more cells (Fig.

dmm.biologists.org114

Hepatic stem cells for liver transplantationPERSPECTIVE

Fig. 1. Morphology of Alb-uPA transgenic mouse livers. (A) Grossappearance of Alb-uPA transgenic mouse liver from 5-week-old males. Left,non-transgenic control; center, hemizygous transgenic liver filled withregeneration nodules; right, homozyous transgenic liver, displaying auniformly white color. Figure reprinted with permission from Elsevier(Sandgren et al., 1991). (B) Livers from an experiment using β-gal-positivedonor hepatocytes and Alb-uPA recipients, all stained for β-gal activity. Toprow, left, non-transgenic control; middle lacZ-positive control; right, controlmouse transplanted with lacZ hepatocytes. Bottom, livers from Alb-uPAtransgenic mice transplanted with lacZ hepatocytes. In addition to stainedblue areas, white areas (w) and endogenous red regeneration nodules (r) areobserved in the liver in the middle. Figure reprinted with permission fromAAAS (Rhim et al., 1994).

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3A); and after 6 months, clusters had enlarged and coalesced toconstitute a near total replacement of the liver. Transplantedhepatocytes were morphologically normal and functional, asrevealed by immunohistochemical staining of several hepatocyte-specific enzymes. Significant growth of donor cells was notobserved in rats that did not receive retrorsine or PH, againproviding evidence that a proliferative stimulus is necessary.

Bone marrow- and cord blood-derived stem cells for liverrepopulationMuch excitement was generated by reports that hematopoietic stemcells (HSCs) differentiated into hepatocytes in the liver (Theise etal., 2000). Indeed, reports of bone-marrow-derived cellsparticipating in the generation of tissues of mesodermal (Ferrariet al., 1998), ectodermal (Korbling and Estrov, 2003), andendodermal (Petersen et al., 1999) origin all contributed to hopesfor universal stem cell therapy.

The model can drive the biology: cell fusion mediated rescueof a gene deficiency diseaseThe FAH-deficient mouse was employed to test the ability of stemcells to repopulate the liver (Lagasse et al., 2000). Mutant femaleswere subjected to lethal irradiation, and their bone marrow wasreconstituted with cells from male Rosa26 mice ubiquitouslyexpressing β-galactosidase (β-gal). The NTBC was withdrawn tosee whether circulating donor cells could rescue the diseased liver.After 7 months, surviving animals were sacrificed and their liverswere found to contain nodules of β-gal-expressing cells from donoranimals. Further, when HSCs (c-kithi, Thylo, Lin–, Sca+) cells werepurified using fluorescence-activated cell sorting (FACS) andtransplanted into mice for bone marrow reconstitution, togetherwith on/off NTBC drug cycling to improve survival, theregeneration nodules were again of donor origin. In addition, in allanimals where the hematopoietic engraftment was successful,donor-derived ‘hepatocytes’ were present.

More information about how the differentiation plasticity ofHSCs occurs was offered by a kinetic analysis revealing that thetransition of bone marrow cells to hepatocytes is a slow process(Wang et al., 2002). Under optimum conditions, small clusters ofdonor hepatocytes were present at 7 weeks followingtransplantation, and by 22 weeks the clusters had become confluent.The authors estimated a total of around 170 repopulation nodulesper liver (composed of 5�107 hepatocytes); these numbers did notincrease when purified HSCs were used. In contrast, the numbersof repopulating clusters were several hundred-fold higher aftertransplantation of adult hepatocytes compared with bone marrowtransplants, and nodule expansion was much more rapid with 50%repopulation within one month. Finally, in the absence of total bodyirradiation, which is presumably required for bone marrowreplacement by cells carrying a wild-type Fah allele, no cellreplacement was observed even with NTBC drug cycling.

Wang et al. reported experiments designed to test the hypothesisthat bone-marrow-derived hepatocytes were in fact the productsof cell fusion (Wang et al., 2003b). However, two reportsdemonstrated that, upon cell fusion, the resulting hybrid cells couldperfectly mimic the phenotype of one of the parents (Terada et al.,2002; Ying et al., 2002), as a result of re-programming of the geneexpression pattern. Wang et al. tested the hypothesis that bone-

marrow-derived hepatocytes were the result of cell fusion, usingmice with reconstituted bone marrow and mouse strains withmultiple DNA markers. Analysis of repopulated livers revealedpatterns and relative concentrations of DNA markers that wereconsistent with cell fusion. A direct demonstration was obtainedby cytogenetic analysis (Wang et al., 2003b) (Box 1). In addition,Camargo et al. and Willenbring et al. demonstrated thatmyelomonocytic cells are partners in the cell fusion events in theliver (Camargo et al., 2004; Willenbring et al., 2004) (Fig. 2). Finally,Vassilopoulos et al. employed HSCs expressing green fluorescentprotein (GFP) in similar experiments (Vassilopoulos et al., 2003)(Table 1).

What are the general conclusions to be drawn from this seriesof papers? FAH deficiency is a recessive mutation. Any cellproviding a wild-type Fah allele can theoretically rescue such adeficiency, provided that Fah expression can be activated by re-programming in the hybrid cells. Furthermore, in all of theexperiments cited here (Lagasse et al., 2000; Wang et al., 2002;Vassilopoulos et al., 2003; Wang et al., 2003b; Willenbring et al.,2004; Camargo et al., 2004), Fah mutant mice were initially


Hepatic stem cells for liver transplantation PERSPECTIVE

Box 1. Methods for demonstration of cell fusion-mediatedrescue of Fah-deficient bone marrow chimerasWang et al. used Southern blots to detect genotypic markers from threestrains of mice (Wang et al., 2003b). Mice were subjected to lethal irradiationbefore receiving a bone marrow transplant from donor mice [genotype A,hereafter referred to as (A)]. NTBC was withdrawn in the primary Fah–/–

recipient mice [genotype B, hereafter (B)]. Liver cells from these mice werethen transplanted into Fah–/– recipients to obtain secondary transplants,before tertiary transplantation into another group of Fah–/– recipients(genotype C). In these tertiary recipients, the relative contributions of thedonor (A) and the Fah mutant (B) markers were equivalent and under-represented compared with the level of repopulation. This is because thebone-marrow-derived hepatocytes carry alleles from both donor (genotype A)and recipient (genotype B) genotypes, as a result of cell fusion, which createscells that are twice as large as normal hepatocytes (so that 50% repopulationcalculated by area can be equivalent to 25% repopulation by cell number). Thecontribution of the marker allele C from the tertiary recipient wasapproximately reciprocal to the corrected degree of repopulation by bonemarrow hepatocytes (taking into account that 30% of liver cells are non-hepatocytic). This clarifies that cell fusion events only occurred in the primaryrecipients and were not continuously generated.

A direct demonstration of cell fusion was obtained by cytogenetic analysisof bone marrow transplants from female Fah-positive Rosa26 donor mice intoirradiated male Fah–/– recipients, in which NTBC treatment had beenwithdrawn. Chromosome counts from primary hepatocyte cultures werecompatible with the hypothesis that cell fusion occurs when bone-marrow-derived hepatocytes are formed: significant numbers of hepatocytes contained80 chromosomes including three X and one Y chromosome (80XXXY). Theinterpretation of these experiments is complicated by the fact that a largefraction of hepatocytes in adult rodents are tetraploid to begin with, so cellscontaining either 40 or 80 chromosomes, and two or four sex chromosomes,must be taken into account when calculating the karyotypes that areanticipated when cell fusion is, and is not, involved. Furthermore, metaphaseshave a tendency to break when chromosome spreads are air-dried, so a cellwith 80 or 120 chromosomes may have lost variable numbers of itschromosomes.

Additional experiments, such as immunocytochemistry, were carried out todemonstrate that most of the hepatocytes in primary cultures were Fah-positive and contained a Y chromosome. In a few cases, FAH staining wasfollow by in situ hybridization to show that enzyme activity and Ychromosomes were present in the same cells.

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subjected to lethal irradiation and transplanted with bone marrowcells carrying a wild-type Fah allele; therefore, the animals werealready heavily chimeric before NTBC withdrawal.

Direct transplantation of bone marrow or cells derived frombone marrowStudies by Moran-Jimenez et al. made use of a potentiallyinteresting class of recipient animals. They employed mice with atargeted mutation in the Hfe gene, an established model ofhemochromatosis (Moran-Jimenez et al., 2008), which in humansis a disease involving increased absorption of iron and its depositionin parenchymal organs, leading to fibrinogenesis and in some casesdiabetes. This recessive mutation does not cause lethality. Thetransplanted mice showed a slightly reduced iron concentration inthe liver and a measurable increase in the hepatic Hfe mRNAconcentration. (The mechanism underlying this correction is notclear and could be fusion and selective expansion of hybrid cells,or cell fusion where the cells remain as heterokaryons, ortransdifferentiation.) The use of similar models, where a mutationcompromises liver function without causing death, should permitmeaningful comparisons of the relative selective advantages in theliver of donor cells of different origins.

Table 2 summarizes a number of other papers where either bonemarrow or bone-marrow-derived mesenchymal stem cells (MSCs)

were transplanted into mice or rats to analyze their potential forliver engraftment after induced liver damage. However, it is difficultto compare one experiment with another because: (1) in some cases,the transplanted cells were of human origin; (2) different cellnumbers and different transplantation routes were employed; (3)recipients were irradiated and some were immunodeficient; (4) liverdamage was induced by a number of different means; and (5) someinvestigators checked for cell fusion, whereas others did not. In allcases, successful liver engraftment with donor cells was low. Withthe exception of a study by Oh et al. (Oh et al., 2007), none of theexperiments used any of the robust models for liver repopulationpresented above.

Human umbilical cord blood (UCB) stem cellsRecent studies provide evidence that UCB stem cells not onlyparticipate in the development of hematopoietic progenitors, butcan also differentiate in vitro into adipocytes, osteocytes,chondrocytes, cardiomyocytes, neurons and hepatocytes (van deVen et al., 2007). Thus, there is much excitement about use of theeasy-to-obtain UCB stem cells for the treatment of bothhematopoietic and non-hematopoietic diseases. In addition, manygroups have reported the successful generation of hepatocyte-likecells from UCB using in vitro assays.

Liver regeneration models have been used to test entiremononuclear cell preparations, or cells selected for progenitormarkers, by injecting them into immunocompromised mice (Tables3 and 4). Many papers report that UCB cells can develop in theliver as hepatocyte-like cells. However, even when liver injury isinduced by carbon tetrachloride (CCl4), 2-acetylaminofluorene(AAF) and the Fas ligand, the frequency of human cell engraftmentin the liver is extremely low. Despite the low level of liverrepopulation, two studies do report that human UCB celltransplantation significantly reduces the mortality caused byinduced liver injury (Di Campli et al., 2005; Nonome et al., 2005).

A particularly interesting set of experiments involved intra-fetalinjections of human UCB cells into mice, rats, sheep and goats(Almeida-Porada et al., 2004; Turrini et al., 2005; Zeng et al., 2005;Qian et al., 2006; Zeng et al., 2006; Sun et al., 2007) (Table 4). Cellengraftment into fetal liver would be expected to preclude thenecessity of suppression of the immune response and of induced liverdamage to obtain an environment appropriate for cell proliferation.In these models, high levels of human cells were detected in the liversof animals born from injected fetuses for up to two years after birth(Almeida-Porada et al., 2004; Qian et al., 2006; Zeng et al., 2006).

Whereas the published studies provide data on the ability of UCBstem cells to differentiate as non-hematopoietic cells, and inparticular as liver cells, additional research is required to clarifythe potential usefulness of, and the appropriate moment to use,UCB stem cells for tissue repair and in particular for the restorationof liver function.

Hepatic stem cells from fetal or adult liverIntrinsic, long-term proliferative potential of E14 rat fetalliver epithelial progenitor (FLEP) cellsDabeva et al. used DPPIV-negative rat recipients, subjected toretrorsine treatment and PH or PH alone, to reveal the robustrepopulation potential of fetal liver progenitor cells (Dabeva etal., 2000). Sandhu et al. carried out a long-term quantitative study



Fig. 2. Livers stained for β-gal activity. The Rosa26 liver constitutivelyexpresses β-gal in all cells. Rosa26rFAH shows β-gal-expressing nodules aftertransplantation of Rosa26 hepatocytes into Fah–/– mice. LysM-Cre refers to amouse line in which Cre recombinase, under the control of the LysM (lysozymeM) promoter, is expressed is macrophages. If the same mouse also has aRosa26R gene (R=reporter: the gene contains a stop codon, flanked by loxPsites, upstream of the lacZ gene), when Cre recombinase is expressed inmacrophages, all descendants of that cell will express β-gal. For the twoimages here, this proves that macrophages have fused with hepatocytes tocreate blue nodules. Finally, the last image is of LysM-Cre bone marrowinjected into Fah–/– mice which also possess a Rosa26R sequence. Thepresence of blue nodules in this liver directly demonstrates that a macrophagehas fused with a Rosa26R-containing hepatocyte to generate β-gal-positivehybrid cells. Figure reproduced with permission from the American Society forClinical Investigation (Camargo et al., 2004).

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of the repopulation activity of FLEP cells compared with adulthepatocytes (AH), using rats that were stimulated only by PH(Sandhu et al., 2001). Fig. 3B reveals important differencesbetween FLEP cells and AHs that only became clear after long-term observations. Oertel et al. demonstrated that PH mustprecede, or be simultaneous with, cell infusion for effectivegrowth stimulation of donor cells to occur (Fig. 3C) (Oertel etal., 2006).

It is usually assumed that the environment of growth stimulationtriggered by PH lasts for only about 1 month. Unexpectedly – andin the absence of any obvious selective advantage or ongoing liverdegenerative process – FLEP cells, but not AHs, outstripped theendogenous cells of the liver, gradually replacing many of them overthe long-term. This property reinforces the idea that early fetal livercells are genuine stem cells. These important observations havebeen re-investigated and found to result from cell-cell competition(Oertel et al., 2006).

Oval cells for liver replacementHepatic stem cells of the adult liver have been recognized fordecades and are characterized by their bipotentiality and capacityfor self-renewal (Evarts et al., 1987; Fausto, 2004; Shafritz et al.,2006). In adult rodents, hepatic progenitor cells, so-called ‘ovalcells’, are activated to proliferate when replication of hepatocytesis blocked. A few studies have now demonstrated the capacityof these progenitor cells to repopulate the liver. In competitiverepopulation experiments, Wang et al. showed that freshlyderived oval cells (obtained by treating mice with 3,5-diethylcarbonyl-1,4-dihydrocollidine) are at least as efficient asmature hepatocytes in repopulating FAH-deficient mice (Wanget al., 2003a). Song et al. used GFP-expressing liver progenitorcells from mice that were transduced with human α1-antitrypsin(Song et al., 2004). For transplantation, they treated congenicrecipients with monocrotaline and subjected them to PH.Approximately 40-50% of the regenerated liver was GFP positive.

In addition, 5-10% of the repopulating cells expressed human α1-antitrypsin.

Cell lines for liver regenerationThe establishment of progenitor cell lines derived from embryonicand adult liver, showing bipotential capacity to differentiate intohepatocytes and bile duct cells in vitro as well as in vivo, has beendocumented (Strick-Marchand et al., 2004). The advantages of fetalliver as a source of cells with liver repopulation potential are itscapacity to proliferate without selective pressure and to retainbipotentiality (Sandhu et al., 2001; Strick-Marchand et al., 2004;Suzuki et al., 2002). More recently, Oertel et al. transplantedembryonic (day 14) fetal liver cells that had proliferatedcontinuously for 6 months and found that they differentiated intomature hepatocytes and bile ducts, and replaced 23.5% of the totalliver mass (Oertel et al., 2006). In addition, stem cell linesparticipated in liver regeneration of Alb-uPA/severe combinedimmunodeficiency (SCID) transgenic mice to levels approaching5% of the tissue, and with no indication of cell fusion (Strick-Marchand et al., 2004).

Using culture selection, Herrera et al. isolated and characterizedprogenitor cells from normal adult human liver (Herrera et al.,2006). These pluripotent cells do not express oval cell markers(defined in the rodent) and can undergo mesenchymaldifferentiation in culture. In addition, the cells engrafted the liverin SCID mice with acute liver injury (Herrera et al., 2006).

Hepatic progenitor cell lines have also been isolated from humanfetal livers (Dan et al., 2006). The clonal lines, designated hFLMPC(for human fetal liver multipotent progenitor cells), appear torepresent a mesenchymal-epithelial transition cell, probably derivedfrom mesoendoderm. The cells were shown to engraft the liversof immunodeficient mice with induced liver damage. In these mice,human albumin could be measured in the serum and visualizedon liver sections, indicating hepatocyte functional activity of thehuman cells.



Table 1. Liver rescue in the FAH-deficient mouse following hematopoietic reconstitution with wild-type bone marrow

Cell type

Route of

transplantation

Recipient;

liver damage Comments Reference

Unsorted mouse BM cells; Tg for

lacZ gene

Retro-orbital

(1-10 106 cells)

Fah–/– mice

Serial NTBC stop fromthree weeks after Tx

30-50% lacZ+ cells in the liver 7 months after Tx;

Alb+, DPPIV+ and E-cadherin+; liver injury neededfor expansion of BM-derived hepatocytes;

restoration of liver function

(Lagasse et al., 2000;

Wang et al., 2002)

Sorted HSC from mice:

c-kithighThylowLin–Sca-1+; Tg forlacZ gene

Retro-orbital

(10 to 1 103 cells)

Fah–/– mice

Serial NTBC stop

lacZ+ nodules (50 to 1 105 cells) 6 months after Tx;

HSC did not enhance the degree of liverengraftment; detection of Y chromosome; Alb+,

DPPIV+ and E-cadherin+

(Lagasse et al., 2000;

Wang et al., 2002)

Isolated HSC cells from mice;GFP-transduced cells

Unspecified Fah–/– miceSerial NTBC stop

Regenerative liver with FAH+ nodules; restorationof liver function; cell fusion associated with a

more hepatocyte-like morphology

(Vassilopoulos et al.,2003)

Unsorted mouse cells Retro-orbital

(1 106 cells)

Fah–/– mice

Serial NTBC stop fromthree weeks after Tx

20-30% of transplanted cells in the liver 5 months

after Tx; restoration of liver function; hepatocytesderived from BM arise from cell fusion

(Wang et al., 2003b)

Isolated mouse HSC CD45+/

Sca-1+; unsorted cells fromRag2–/– C–/–mice; unsorted cells

from LysM-Cre mice (see

legend of Fig. 2)

Retro-orbital

(1 106 cells)

Fah–/– or Fah–/–/Rosa26R

miceSerial NTBC stop

The different hematopoietic lineages contribute to

the generation of FAH+ nodules; HSC-derivedhepatocytes are primarily derived from mature

myelomonocytic cells by fusion with host

hepatocytes

(Camargo et al.,

2004)

Tg, transgenic; Tx, transplantation; AFP, alpha-fetoprotein.

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Hepatic stem cells identified by patterns of marker expressionOne approach that has been vigorously pursued to identify andselect progenitor cells from embryonic livers involves the analysisof marker expression in freshly isolated cells, and the determinationof their differentiation potential in culture. The pattern of surfacemarker gene expression can then be used to purify desired cell

populations by FACS or by immunoselection with magnetic beads,resulting in pure populations of stem cells for therapeutic purposesas well as biological studies.

Schmelzer et al. characterized two types of pluripotent hepaticprogenitors, hepatic stem cells and hepatoblasts, which can bedistinguished by differential expression of CK19 and surface markers



Table 2. Experimental transplantation of bone marrow into other liver injury models

Cell type

Route of

transplantation

Recipient;

liver damage* Comments Reference

Isolated CD34+ or CD34+,

CD38–, CD7– hHSC

Tail vein

(2000 or 1 105

cells)

NOD/SCID (+/– 2M-null)

mice

Irradiation; CCl4 after Tx,(+/– HGF injection)

hAlb+ at 5 and 30 days, and only after liver injury;

increase in HGF-treated mice(Wang et al., 2003c)

Isolated CD3+ cells from

actin-Cre GFP mice

Intravenous

(1.5 106 cells)

Cre reporter mice

Irradiation

0.1% of hepatocytes derived from BM; no cell

fusion(Harris et al., 2004)

Isolated HSC from mice

(Fr25lin–); homed HSC(Fr25lin– PKH+)

Tail vein

(1 105 cells)

C57BL/6/NCR mice

Irradiation (+/–); CCl4

after Tx (+/–)

Liver injury induces HSC cell conversion

independently of cell fusion; 7 days after Tx,7.6% of transplanted cells found in the liver of

injured mice (E-cadherin+, Alb+ and malechromosome) and restoration of liver function;

homing increases cell conversion

(Jang et al., 2004)

Isolated MSC Flk1+ from

mice

Tail vein

(1 106 cells)

BALB/c mice

Tx at the time of, orafter, CCl4 treatment

Tx of mouse MSC Flk1+ cells immediately after CCl4

challenge protects liver from injury(Fang et al., 2004)

hMSC, CD34+ and non-

MSC/CD34– cells;

expanded or non-expanded cultures

Intrahepatic

(1 106 cells)

Sprague-Dawley rat

allyl alcohol before and

after Tx; cyclosporin A

hAFP+, hAlb+, hCK18/19+ and asialoglycoprotein

receptor positive in MSC Tx; a maximum of 0.5%

of MSC undergo hepatocyte-like differentiation;no cell fusion

(Sato et al., 2005)

Adherent proliferating

rat MSC

Intravenous

(3 106 cells)

Wistar rat

CCl4 or DMN treatmentbefore transplantation

Reduced mortality rate in both liver injury models (Zhao et al., 2005)


hMSC preconditioned

with EGF and HGF

Intrasplenic

(1 106 cells)

Pfp–/–/Rag2–/– mice

PH and propranolol

hydrochloridetreatment before Tx

HepPar1+, hAlb+, PCK1+ and CX32+; preferential

engraftment in periportal area; no cell fusion(Aurich et al., 2007)


rat MSC

Intravenous

(3 106 cells)

Albino rat

CCl4 treatment beforeTx

MSC Tx have potential therapeutic effect against

fibrotic process

(Abdel Aziz et al.,

2007)

Expansion of rat MSC for

hepatocyte-like

differentiation; GFPtransduction

Intrasplenic

(7 107 cells)

Sprague-Dawley rat

Tx 1 week before 90%

hepatectomy

Concomitant immunostaining of Alb and GFP;

persistence of transplanted cells 100 days after

Tx; prevention of fatal liver failure in 30% ofanimals

(Miyazaki et al., 2007)

Unsorted DPPIV+ rat cells Tail vein

(5 107 cells)

DPPIV– rat

Monocrotaline

treatment; irradiation attime of Tx; AAF and 70%

PH after Tx

20% of DPPIV+ transplanted cells were AFP+;

secondary transplantation of DPPIV+ hepatic

oval cells; no cell fusion

(Oh et al., 2007)

Isolated mononuclearcells of C57BL/6J mice

Tail vein(1 107 cells)

Hfe knockouthemochromatotic mice

Irradiation

11% of transplanted cells in the liver (alsoduodenum); reduction of iron overload

(Moran-Jimenez etal., 2008)

Culture expansion of

human MSC

Tail vein

(1 106 cells)

NOD/SCID mice

Irradiation; single orchronic CCl4 treatment

Chronic injury increased hMSCs transplantation,

but differentiation into hepatocyte-like cells is arare event

(di Bonzo et al., 2008)

Culture expansion of

hMSC or hepatocyte-like differentiated cells

(MDH)

Tail vein or

intrasplenic(1.4–4.2 107

cells/kg body

weight)

NOD/SCID mice

CCl4 treatment beforeTx

MSC and MDH differentiated into hepatocytes and

rescued liver failure; intravenous Tx moreeffective in rescuing liver failure; MSC

transplanted animals more resistant to oxidative

stress; MSC promote proliferation ofendogenous hepatocytes, suggesting possible

paracrine effects

(Kuo et al., 2008)

*For irradiation a sub-lethal dosage was used.

h, human; Tx, transplantation; AFP, -fetoprotein; DMN, dimethylnitrosamine; CK, cytokeratin; EGF, epithelial growth factor; HGF, hepatocyte growth factor; MDH, mesenchymal

differentiated hepatocyte.

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such as epithelial cell adhesion molecule (EpCAM). The hepatic stemcells are located in both fetal and adult liver (Schmelzer et al., 2006;Schmelzer et al., 2007), and are thought to repopulate animal modelsof liver injury, but marker analysis of the engrafted cells is currentlylimited to immunostaining for albumin (Schmelzer et al., 2007).

Simper-Ronan et al. used monoclonal antibodies andmicromagnetic immunobeads to isolate populations ofcholangiocyte-marker-positive fetal rat liver progenitor epithelial

cells (CMP-FLEC) for injection into DPPIV-negative, retrorsine-treated rats subjected to PH. These cells showed excellentrepopulation capacity. Interestingly, if cell populations weredepleted of CMP-FLEC, the repopulation potential was severelydiminished (Simper-Ronan et al., 2006). Finally, studies havedemonstrated that immuno-isolated delta-like-1-positive (Dlk-1+)rat fetal liver cells can account for the repopulation capacity of theFLEP (Oertel et al., 2008).



Table 3. Experimental transplantation of human UCB stem cells into the liver of adult animals

Cell type

Route of

transplantation

Recipient;

liver damage* Comments** Reference

Adherent proliferating cells:

hAlb+, vital staining

Intraliver

(2 105 cells)

SCID mice

With or without PH

7 and 21 days after Tx: hAlb+, hAFP–, hGATA4–,

downregulation of h 2-microglobulin;

frequency: not given

(Beerheide et al., 2002)

Isolated Lin–, CD38–, CD34–,ClqRp and Lin–, CD38–,

CD34+, ClqRp cells

Tail vein(500 to 7 104 cells)

NOD/SCID miceIrradiation

HepPar1+, h c-Met8+ and hAlb+ 10 weeks afterTx; frequency: 0.05-0.1%

(Danet et al., 2002)

Unsorted mononuclear cell

preparation

Tail vein

(5 107 cells)

NOD/SCID mice

Irradiation

HepPar1+ in livers 4, 6 and 16 weeks after Tx,

no evidence for cell fusion; frequency:0.008-0.03%

(Newsome et al., 2003)

Isolated CD34+ or CD34+,

CD38–, CD7– cells

Tail vein

(2000 or 1 105 cells)

NOD/SCID (+/– 2M-null)

mice

Irradiation; CCl4 afterTx (+/– HGF injection)

hAlb+ cells at days 5 and 30, only in injured

liver; more hAlb in HGF-treated mice;

frequency: not given

(Wang et al., 2003c)

Adherent proliferating cells:

hAlb+

Portal vein

(1 107 cells)

SCID mice

AAF injection then PH

hAlb+ and HepPar1+ cells from 4-55 weeks;

frequency: 0.1-1%(Kakinuma et al., 2003)

Isolated CD34+/–, CD38+/–,c-kit+/– cells

Portal vein(1.5 104, 4 106

cells)

NOD/SCID miceRetrorsine; CCl4 after

Tx

Higher expression of hAlb in CD34+

transplanted cells; frequency: 1.2-1.7%; cell

fusion in 77% of cells

(Tanabe et al., 2004)

Isolated CD34+, AC133+, c-kit+

cells

Intraperitoneally

(4 105 cells)

NOD/SCID mice

Allyl alcohol before Tx

0.2% of engrafted cells are AFP+; in

transplanted mice, decrease of mortalityfrom 70 to 20%

(Di Campli et al., 2005)

Adherent proliferating cells Tail vein

(5 105 cells)

NOD/SCID mice

Fas ligand or

irradiation

hAlb+, HepPar1+, hAFP+ cells, positive for

glutamine synthetase and transferrin; in

transplanted mice treated with Fas ligand,decrease of mortality from 70 to 0%

(Nonome et al., 2005)

Unsorted mononuclear cells Tail vein

(1 106 cells)

NOD/SCID mice

Irradiation; CCl4 afterTx

hAlb+, hCK18– cells; no protection from liver

damage(Sharma et al., 2005)

Unsorted mononuclear cells

or isolated CD34+ cells; GFP-

transduced cells

Tail vein

(1-3 105 cells)

NOD/SCID mice

Irradiation; CCl4 before

Tx

Less than of 1% of GFP-positive cells in liver;

cell fusion demonstrated for hepatocyte-like

cells

(Kashofer et al., 2006)

Adherent proliferating cells Intraliver or intrasplenic

(2 105 cells)

NOD/SCID mice,

uPA/Rag2–/– mice

Two types of hAlb+ cells: (1) human nucleus:

no hepatocyte-like morphology; (2) only

mouse nucleus detected: hepatocyte-likemorphology (postulated gene transfer)

(Brulport et al., 2007)

Isolated CD34+ cells Tail vein

(2 104 to 1 107

cells)

NOD/SCID/ C mice

Irradiation

Expression of hAlb; frequency: 3.4% of human

hepatocytes 6 months after Tx; cell fusion

demonstrated.

(Fujino et al., 2007)

Unsorted mononuclear cells Intrasplenic(2 106 cells)

NOD/SCID miceAAF and allyl alcohol

before Tx; AAF for 7

days at day 11 post-Tx

hAlb+; rapid human cell repopulation in week1; no difference in mortality rate (14.7%);

frequency: 0.51% of human hepatocytes 6

months after Tx

(Shyu et al., 2007)

Adherent proliferating cells Intrasplenic

(1 106 cells)

SCID mice

PH before

transplantation

hAlb+ and AFP+ until 6 weeks; Alu+

hybridization and human mitochondria

(Campard et al., 2008)

Adherent proliferating CD34+

cells treated with SCF or

HGF 24 hours before Tx

Tail vein(2 105 cells)

NOD/SCID miceIrradiation

SCF reduced long-term cell engraftment inliver of NOD/SCID mice; frequency: 0.3% of

human cells 56 days after Tx

(Wulf-Goldenberg et al.,2008)

*For irradiation, a sub-lethal dosage was used; **Frequency signifies ‘frequency of transplanted cell in the liver’.

h, human; HGF, hepatocyte growth factor; SCF, stem cell factor; Tx, transplantation; CK, cytokeratin.

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Liver repopulation by the progeny of embryonic stem (ES)cellsThe culture and directed differentiation of both mouse and humanES cells (mES and hES cell lines, respectively) holds great promisefor the future of cell-based therapy for tissue repair. Progress towardthe goal of using such cells in liver repopulation models has beenslow. A promising publication from Heo et al. described theisolation of mES cells with the potential to express GFP undercontrol of the albumin promoter/enhancer. The cells were culturedas embryoid bodies, then re-plated and cultured in medium

containing factors favorable for the emergence and proliferation ofhepatocytes. As an original step, the authors used FACS for GFPexpression in an effort to eliminate undifferentiated ES cells thatcould confer malignant growth. Using a second uPA transgenicmouse line [MUP-uPA, where the liver-specific promoter is froma major urinary protein (Mup) gene], they obtained clusters of GFP-positive hepatocytes that enlarged over 3 months and expressed arange of hepatocyte functions; the transplanted cells proliferatedin harmony with host cells when the liver was injured (Heo et al.,2006). Sharma et al. used ES-HPC (mES-derived hepatic progenitor



Table 4. Experimental transplantation of human UCB stem cells in utero

Cell type

Route of

transplantation

Recipient;

liver damage Comments Reference

Isolated CD34+/–, Lin–,

CD38– cellsIntrafetal (2 104 cells) Sheep hAlb+, human hepatocyte Ag+; 1-2% of human

hepatocytes at 2 months; in one animal, around 17%

of human hepatocytes at 11 months of age

(Almeida-Porada et

al., 2004)

Isolated CD34+ cells Intrafetal (3-5 105 cells)intra-blastocyst (15-20

cells)

CD1 or C57BL/6 mice hAlb+, HepPar1+ and -antitrypsin 1+ 4 weeks afterbirth; HepPar1+ clusters around major blood vessels

(Turrini et al., 2005)

Isolated CD34+, Lin– cells Intrafetal (1 105 cells) Goat hAlb+ and hHNF-3 + )5002 ,.la te gneZ(ANRm

Isolated CD34+, Lin– cellstransduced with GFP

Intrafetal Goat hAlb+, human hepatocyte Ag+ and PCNA+. Around 30%of GFP+ cells in the liver after 3 months or 2 years;

microarrays for human gene expression; no cell

fusion

(Zeng et al., 2006)

Unsorted mononuclear

cells; isolated CD34+,

Lin– cells

Intrafetal

(1 106, 1 104 cells)

Kun Ming Bai mice

CCl4 6 months after

birth

hAlb+, HNF-4+, human hepatocyte Ag+ and hAFP+;

protection from CCl4 damage; most positive clusters

were located in the vicinity of vascular structure

(Qian et al., 2006)

Unsorted mononuclearcells

Intrafetal(5 106 cells)

Rat hAlb+, CK19+ and CK18+ (Sun et al., 2007)

h, human; HNF, hepatocyte nuclear factor; CK, cytokeratin.

Fig. 3. DPPIV-positive E14 embryonic liver cells transplanted into DPPIV mutant rats. (A) Left, one week after transplantation, scattered cells diffuselystained red. Right, one month after transplantation, large clusters of fully differentiated hepatocytes showing bile canalicular staining of DPPIV. Reprinted withpermission from the American Society for Investigative Pathology (Dabeva et al., 2000). (B) Table comparing hepatocyte clusters at different times aftertransplantation of either cells from E14 fetal livers or adult hepatocytes. Reprinted with permission from the American Society for Investigative Pathology(Sandhu et al., 2001). (C) Illustration of the effects of PH timing on the presence of E14 rat liver cells in DPPIV mutant rats. Left to right: PH was performed 1 daybefore (left), at the same time as (middle), or 1 day after (right) cell transplantation (Oertel et al., 2006).

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cells) for engraftment, after a similar FACS sorting operation, intoFah–/–/SCID mice. Although engrafted cells were observedthroughout the liver, immunostaining for the FAH protein wasnegative. The authors concluded that the transplanted ES cells donot acquire a fully mature hepatocyte phenotype (Sharma et al.,2008).

It will take time to identify ES cell lines with sufficient propensityto differentiate into adult hepatocytes and the most favorable mousemodel in which to optimize much-needed experimental tests forrobust liver repopulation by ES cells.

Humanization of rodent models: a novel tool to study humanpathogensThe evaluation of pharmacological compounds and theirmetabolism/detoxification must be carried out using human cellsbecause of the evolutionary diversity of hepatic detoxificationenzymes. Furthermore, the study of human-specific liver pathogenshas been, until recently, severely hampered by the absence ofappropriate cell culture systems and animal models (Box 2). Here,we focus on the development of humanized rodent models to studythe three major hepatic pathogens (Fig. 4) HBV, HCV and P.falciparum, which are responsible for millions of deaths each year(Box 3).

Because of their short gestation period, their small size andtheir low maintenance costs, rodents would certainly be adesirable model for biological studies of human-specificpathogens. At least three interesting models to study HBV andHCV already exist: (1) the immunotolerized rat model; (2) theTrimera mouse model; and (3) the Alb-uPA mouse model. TheAlb-uPA model has also been used to study the hepatic stage ofP. falciparum. Moreover, the Fah–/–/Rag2–/–/Il2–/– model appearsto be efficient for liver humanization and should provide anadditional infection model for different classes of humanpathogens (Table 5; Fig. 5).

Immunotolerization of fetal rats harboring transplantedhuman cells: a potential immunocompetent tool to evaluatevaccinesTaking advantage of the fact that the rat immune system does notdevelop until after 15-17 days of gestation, Ouyang et al.immunotolerized rat embryos to allow the transplantation andmaintenance of a human hepatoma cell line (Huh7) or cryopreservedhuman hepatocytes (Ouyang et al., 2001). After birth, the treatedanimals were infected with either HBV or HCV particles (Wu et al.,2001; Wu et al., 2005). As is depicted in Fig. 5, fetal rats were tolerized,to accept the human cells by an intraperitoneal injection ofcryopreserved human hepatocytes into pregnant females on the 17thday of gestation. Twenty-four hours after birth, the same cells weretransplanted intrasplenically into the rats. After 14 days, the humanhepatocytes represented around 6% of total hepatocytes. When theanimals were inoculated with HCV-infected human serum, in thecase of the Huh7 cell line, nearly 30% of the transplanted humancells (2% of total hepatocytes) were positive for the HCV core proteinand subsequently developed biochemical (elevation of alanineaminotransferase concentration) and histological (presence of fociof mononuclear infiltrates) evidence of hepatitis, consistent with thehuman manifestations of HCV infection. Because of theimmunocompetent background of the animals, this rat model ispromising, especially for vaccine evaluation. However, the overallimpact of the model is limited by the small percentage of infectedhuman cells and because the viremia resulting from infection is weak(Table 5). Moreover, validation of these models, for example byconfirmation of the antiviral effects of therapeutic molecules, remainsnecessary.

Human tissue xenotransplants in preconditioned mice tostudy HBV and HCV infection: the Trimera mouse modelThe so-called ‘Trimera’ model involves the development of achimeric mouse with three different tissue sources (Reisner andDagan, 1998). Immunocompetent mice were pre-conditioned bytotal body irradiation before being reconstituted with SCID mousebone marrow and then transplanted with human peripheral bloodmononuclear cells (this final step is optional and is not used forevaluation of antiviral agents). Finally, these mice receivedtransplants of either HBV- or HCV-infected liver fragments, takendirectly from human patients, or ex vivo HBV- or HCV-infectedliver fragments, into the ear pinna or under the kidney capsule (Fig.5) (Galun et al., 1995; Ilan et al., 1999; Ilan et al., 2002; Eren et al.,2006). The transplants were maintained for several weeks, and HBVDNA and HCV RNA were detected in the serum for up to 1 month(Table 5).



Box 2. Limitations of available models for studies of hepaticpathogensThe discovery of prophylactic and curative treatments for HBV, HCV and P.falciparum has been hampered by the lack of cell culture systems and smallanimal models. In vitro studies have shown that primary human hepatocytes(PHH) are susceptible to infection by HBV (Gripon et al., 1988), HCV (Fournier etal., 1998) and by sporozoites (the hepatic form of P. falciparum) (Mazier et al.,1985). However, these systems are limited because PHH rapidly revert toundifferentiated hepatocytes (the loss of differentiation leads to resistance toinfection) and there are difficulties in obtaining fresh cells. HBV and HCV celllines can be used in stable (Sells et al., 1987; Wakita et al., 2005) or transienttransfection strategies, resulting in production and secretion of infectiousvirions, but cannot be used to study the entry steps of these viruses. In 2002,the human HepaRG cell line was described (Gripon et al., 2002) as a tool tostudy the entry steps of HBV infection. In vivo, the chimpanzee constitutes thebest non-human primate for use in HBV, HCV and P. falciparum studies (Dandriet al., 2005b; Kremsdorf and Brezillon, 2007; Moreno et al., 2007). Except forthis higher primate, the only non-human primate that is permissive for HCVinfection is the marmoset (Kremsdorf and Brezillon, 2007), whereas the rhesusmonkeys, cynomoglus monkeys, gibbons and orangutans are susceptible toHBV infection. In addition, Tupaia, a member of the tree shrew genus, can beinfected with both HBV and HCV. However, these models are hampered bymultiple drawbacks, including: (1) the inability to produce numerous progenyin a short time because of long gestation periods, (2) exorbitant housing andbreeding costs, and (3) scarcity of the species threatened by extinction. HBV-and HCV-related viruses, such as GBV-B (GB virus B, a hepatotrophic virus ofthe Flaviviridae family) have been used to infect tamarins and marmosets tobetter characterize HCV replication and to test HCV antiviral drugs (Bright etal., 2004; Nam et al., 2004; Rijnbrand et al., 2005). For HBV, WM-HBV (woollymonkey HBV), WHBV (woodchuck HBV), GSHV (ground squirrel hepatitis virus),ASHV (artic squirrel hepatitis virus), DHBV (duck HBV) and HHBV (heron HBV)have all been used to improve understanding of the life cycle of theHepadnaviridae family. Finally, transgenic mice have enabled numerousdiscoveries to be made in the field of HBV and HCV virology. However, theserodents cannot be used to study the entry step of HBV or HCV and are notsuitable for evaluating the efficacy or toxicity of treatments.

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This model can produce monoclonal antibodies (Ilan et al., 2002)and was validated as a tool for testing antiviral components. ForHBV therapy, it has been used to study specific T-cell responses,vaccination strategies (Bocher et al., 2000; Bocher et al., 2001) andthe therapeutic effect of monoclonal antibodies directed againstHBV epitopes (Eren et al., 2000; Galun et al., 2002). In addition,concerning HCV, an inhibitor of the internal ribosomal entry siteand an anti-HCV monoclonal antibody were demonstrated to actas HCV inhibitors (Ilan et al., 2002) using this model. More recently,two monoclonal antibodies directed against HCV envelope proteinE2 were produced and characterized in this model. Following invitro validation of the ability of these antibodies toimmunoprecipitate HCV particles, an inhibitory effect on HCVinfection was confirmed (Eren et al., 2006). Indeed, in the HCV-Trimera model, both monoclonal E2 antibodies were shown toinhibit ex vivo HCV infection of human liver fragments and bothwere effective for treating HCV-infected Trimera mice (Eren et al.,2006).

In conclusion, although the Trimera mouse model appears tobe well-suited to produce antibodies, as well as to evaluate theinhibitory capacity of drugs, this approach does involve the use

of heterotopic and xenogenic grafts, so the physiological relevanceof observations need to be validated with other models. Moreover,this kind of transplantation does not allow the durable persistenceof the human tissue, and gives rise to only low-level transientviremia.

Humanized FAH-deficient mice: a tool under developmentAs detailed above, the lethal condition of FAH deficiency can bealleviated by administering NTBC to the mice. Withdrawal of thedrug after transplantation of FAH-expressing hepatocytes leads toefficient liver repopulation and counters the effects of FAHdeficiency in endogenous cells. Until recently, the transplantationof human hepatocytes into this mouse was not successful:Fah–/–/nude, Fah–/–/non-obese diabetic (NOD)/SCID orFah–/–/Rag1–/– mice did not permit persistence and repopulationby human hepatocytes (Azuma et al., 2007) (Box 4). Although nudemice lack T cells and Rag1–/– mice lack both B and T cells, bothstrains do still have some competent immune cells, leaving thepotential for immune rejection of transplanted hepatocytes.Although Azuma et al. specified that human cell grafts do survivein the Fah–/–/NOD/SCID model, the efficiency is not great enoughfor the animals to tolerate withdrawal of NTBC treatment (Azumaet al., 2007).

Recently two different groups have used a similar strategy tocreate a novel mouse model to study human liver celltransplantation. They backcrossed Rag2–/–/Il2–/– mice with Fah–/–

mice to generate a new mouse model that could successfullyreceive transplants and be repopulated by human hepatocytes,presumably because the mice lacked not only B and T cells, butalso natural killer (NK) cells (Azuma et al., 2007; Bissig et al.,2007). From these studies, differences have emerged in themethods used to obtain satisfactory repopulation. One groupspecified the necessity of treating the mice, by pre-injection, withan adenovirus encoding the uPA protein (Azuma et al., 2007) toallow engraftment of human cells, and found that additionaltreatment to control innate immunity was unnecessary. Anothergroup did not use uPA treatment, but found that concomitanttreatment of mice with the protease inhibitor nafamostat mesilate(to prevent human complement activation) and liposomemolecules encapsulating a Kupffer cell toxin (clodronate) allowed



2001 2006

HBV infection of uPA/Rag2-/-

humanized mouse (Dandri et al.)

HCV infection of uPA/

SCID/Bg humanized mouse

(Mercer et al.)

Evidence of the liver

stage of Plasmodium falciparum in uPA/SCID

humanized mice

(Morosan et al.)

HBV infection in the

Trimera mouse model

(Ilan et al.)

1999 2000

HBV and HDV infection of

primary human hepatocytes

transplanted under the kidney

capsule of NOD/SCID mice

(Ohashi et al.)

HBV stably transfected in primary

human and immortalized (T

SV40) hepatocytes were

transplanted into Rag2-/- mice

(Brown et al.)

HBV infection of

immunocompetent rats (Wu et al.)

HCV infection of

immunocompetent

rats (Wu et al.)

2005

HCV infection

in the Trimera

mouse model

(Ilan et al.)

2002

Fig. 4. Steps in the creation of humanized rodent models for infection by liver pathogens. The arrow represents a timeline. Each box represents anindependent initial study describing the infection of a humanized model by a hepatotropic pathogen. Boxes connected to the timeline through the same lineindicate that the studies were published in the same year.

Box 3. Epidemiology of three major hepatic pathogensHepatitis B virus: around two billion people are infected worldwide, 350million of whom are chronically infected (95% of infected adults resolve theinfection whereas 95% of vertically-infected children became chronicallyinfected), resulting in 0.5-1.2 million deaths/year due to chronic hepatitis,cirrhosis and hepatocellular carcinoma (HCC) (Lavanchy, 2004). HBVtransmission results from exposure to infectious blood or body fluids – verticaltransmission from chronically infected mother to child occurs in 20% of cases.An effective and safe prophylactic vaccine does exist.Hepatitis C virus: around 130 million people are chronically infected. 60-85%of infected patients become chronically infected, and HCV accounts for anestimated 27% of cirrhosis and 25% of HCC worldwide (Alter, 2007).Transmission results from blood to blood contact. Vertical transmission andtransmission during the birth process can occur rarely (in 6% of births) fromwomen with viremia at the time of delivery. No vaccine is available.Malaria (Plasmodium falciparum): around 500 million people worldwide areinfected with malaria resulting in one million deaths/year from direct orindirect causes (Greenwood et al., 2008). The female Anopheles mosquito isthe vector for human malaria. No vaccine is available.

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for transplanted cell survival (Bissig et al., 2007). In both cases,the Fah–/–/Rag2–/–/Il2–/– mouse model was successfullytransplanted and repopulated, with up to 90% of the mouse liverpopulated by human hepatocytes by 3 months post-transplantation. However, assuming that highly repopulatedanimals should possess at least 1 mg/ml of human albumin intheir serum, only 16% of transplanted animals met this criterion,and this level of success was only found in the Azuma et al. studyusing uPA administration prior to transplantation. Interestingly,immunochemistry and histological staining provided evidencethat human hepatocytes were interspersed among mousehepatocytes and did not form individualized clones (Bissig et al.,2007). Moreover, highly humanized mice permitted long-termexpansion and maintenance of human cells, and could be usedto perform serial transplantation of human hepatocytes from onehumanized mouse to a second generation, without requiring anew batch of human cells. Finally, the humanized livers of themice expressed a broad range of human markers, includingdetoxification enzymes (Azuma et al., 2007), and thus should beuseful for pharmacological studies.

The Alb-uPA immunodeficient mouse model: an efficient toolto study hepatotropic pathogensBased on their proven utility as hosts for liver repopulation, Alb-uPA transgenic mice were backcrossed onto an immunodeficientbackground [SCID, Rag2–/– or Rag2–/–/Pfp–/– (perforin 1 geneknockout), see Box 4] to obtain a mouse model that tolerated thexenotransplantation of hepatocytes from humans, woodchucks andTupaia belangeri (a tree shrew) (Dandri et al., 2001; Dandri et al.,2005a; Meuleman et al., 2005; Petersen et al., 1998; Rhim et al.,1994; Tateno et al., 2004). Because of the observed transgenedeletion in mice heterozygous for the Alb-uPA transgene, optimumliver repopulation requires intrasplenic transplantation of highquality hepatocytes into immunodeficient 1–4-week-old mice thatare homozygous for the Alb-uPA transgene (Mercer et al., 2001)(Table 5; Fig. 5). In these conditions, human hepatocytes engraftedand repopulated the mouse parenchyma. The resulting chimericliver showed satisfactory hepatic architecture and intermingling ofthe mouse and human subcellular structures, indicating aphysiological integration of transplanted cells (Meuleman et al.,2005; Tateno et al., 2004).

It was subsequently shown that this humanized mouse modelcould not only be used to recapitulate human infection, but alsoused to study various aspects of the viral life cycle of HBV andHCV. It is known that various genotypes of the HBV induce liverdisease of differing severity (Schaefer, 2005). In order to improvethe definition of virological differences among HBV genotypes,Sugiyama and colleagues used Alb-uPA/SCID mice as a tool toevaluate HBV DNA levels produced by different genotypes ofvirus, and succeeded in confirming their earlier in vitro resultsshowing higher replication of the C versus Ae genotype (Sugiyamaet al., 2006). A second group took advantage of immunesuppression in the Alb-uPA/SCID mouse model to demonstratethat liver disease induced by HBV can be, at least in part, directlymediated by the virus, and is not solely the result of immunesystem activation (Meuleman et al., 2006). It should be noted thata drastic cytopathic effect was observed in this study since they



Box 4. Genetic mutations conferring an immunotolerantbackgroundSCID mutation: this spontaneous mutation in the prkdc locus leads torecombination defects, resulting in a lack of B and T cells (Bosma et al., 1983).Beige mutation: spontaneous mutation leading to an impairment of NK-cellfunction and defective cytotoxic-T cells and macrophages.Rag2 knockout: targeted mutation disrupting the recombinase 2 gene (Rag2)and leading to a lack of mature T and B cells (Shinkai et al., 1992).pfp knockout: targeted mutation disrupting the perforin 1 gene (Prf1,previously known as pore-forming protein) and leading to severe depletion ofNK cells (Walsh et al., 1994).Il2 (or γC) knockout: targeting mutation disrupting the Il2 receptor gammachain gene, the knockout creates a lack of functional receptors for manycytokines (IL-2, IL-4, IL-7, IL-9 and IL-15) leading to impaired lymphocytedevelopment and a lack of NK cells (DiSanto et al., 1995).

Table 5. Characteristics of HBV and HCV infection in three rodent models

Rodent model Humanization Virus Viremia

Duration of viral

infection Comments Publications

HBV Positive (non-

quantitative PCR)At least 60 days (Wu et al., 2003; Wu et

al., 2001)Rat Immunotolerization

and transplantation

of a humanhepatoma cell line

HCV 1-2 104 copies/ml Minimum 4 months

with peak viremiaat 3 months

Low viremia;

transplantation of cells

from a hepatoma line;immunocompetent rat

(Wu et al., 2005)

HBV Up to 3 105

copies/ml

Around 1 month

with peak viremia

at 18 days

(Ilan et al., 1999)Trimera mice Xenograft of human

liver tissue

HCV Around 7 104

copies/mlAround 1 month

with peak viremia

at 18 days

Low viremia;

difficult to access human

liver tissues;survival of xenografted

tissue only in anheterotopic place;

immunosuppressed mice

(Ilan et al., 2002; Eren etal., 2006)

HBV Up to 2 1010

copies/mlAt least 15 weeks (Meuleman et al., 2005)Alb-uPA mice

combined witheither: SCID,

SCID/Bg, Rag2–/–,or Rag2–/–/pfp–/–

trait

Human hepatocyte

transplantation

HCV 1 104 to 8 107

copies/ml

Up to 9 months with

maximum viremiafrom 1 month

High viremia;

difficult to access humanliver tissues;

real engraftment and

proliferation of human cellsin mouse liver;

immunosuppressed mice

(Mercer et al., 2001;

Meuleman et al., 2005;Kneteman et al., 2006)

Bg, beige mutation.

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used genotype E, which is a highly pathogenic strain of HBV,isolated from a patient with fulminant hepatitis. A third teamdemonstrated the ‘infectability’ of Alb-uPA/Rag2–/–/Pfp–/– mice,after either repopulation by Tupaia hepatocytes and infection withwoolly monkey HBV, or repopulation by human hepatocytes andinfection with human HBV. In both systems, the authors showedthat treatment of repopulated mice with acylated HBV preS-derived lipopeptides prevented infection. From these results, theyestimated that less than 100 μg/day of lipopeptides, administratedintracutanously, should be enough to treat patients (Petersen etal., 2008). This alternative approach could benefit patientsundergoing liver transplantation to prevent vertical transmissionas well as re-infection. It might also be effective in a post-exposureprophylactic strategy.

Studies in HCV-infected humanized mice demonstrated theantiviral activity of two compounds that had already been shownto be effective during clinical trials: interferon α2b (IFNα2b) and

an anti-protease agent (BILN-2061) that significantly reducedHCV viremia. The antiviral effect depended on the viral genotype,but was independent of the source of human hepatocytes(Kneteman et al., 2006). Human hepatocytes, in the context ofthe Alb-uPA/SCID liver, maintain their ability to expressnumerous enzymes implicated in metabolic and detoxification(cytochrome p450 family) pathways (Katoh et al., 2007). Underthese conditions the Alb-uPA model is well suited to evaluate boththe antiviral potential of drugs and the potential hepato-toxicityof compounds. Recently, an independent group confirmed theantiviral properties of BILN-2061, but noted cardiotoxic sideeffects (Vanwolleghem et al., 2007).

As with HBV, it is clear that the immune response to viralinfection plays a major role in the outcome of liver disease duringHCV infection. To study the involvement of the innate immunesystem against viral infection, Walters et al. used theimmunotolerant Alb-uPA/SCID mouse model to analyze



Fig. 5. Steps in the creation of four humanized rodent models. (A) The rat model: fetal rats are immunotolerized by in utero injection of human hepatocytes,new born rats receive human hepatocyte transplants and young chimeric rats are inoculated with HBV- or HCV-positive human serum. (B) The Trimera model:mice are irradiated and their bone marrow is reconstituted with immunodeficient cells, then they receive transplants with either HCV-infected or ex vivo-infectedhuman liver fragments. (C) The FAH model: mice may or may not be injected with an adenoviral vector encoding the uPA protein. They all receive humanhepatocyte transplants into the spleen and NTBC is withdrawn. (D) The Alb-uPA model: human hepatocytes are isolated, homozygous mice are thentransplanted with human hepatocytes into the spleen and infected with HBV- or HCV-positive human serum.

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transcriptome profiles of HCV-infected versus non-infected mice(Walters et al., 2006). HCV infection in the Alb-uPA/SCID mousemodel activates the transcription of IFN-stimulated genes thatare implicated in establishing the innate immune response, andthus active in the inhibition of HCV replication. As previouslyshown in HCV-infected patients and HCV transgenic mice,Walters et al. confirmed the relationship between severe HCVinfection and perturbation of lipid metabolism in the liver of Alb-uPA/SCID mice. These observations indicate that the innateimmune response plays a fundamental role in the pathogenesisof HBV and HCV infection, rather than liver disease beingmediated exclusively by an HCV-specific adaptive immuneresponse. Since we know that vertical transmission of viralinfection occurs more frequently with HBV than HCV (Box 3),Alb-uPA/SCID mice should provide an adequate model toevaluate the role of the innate immune response against this modeof infection by both hepatitis viruses.

Infection by the P. falciparum parasite is restricted to humansand closely related species. Numerous studies have used modelsin which humanized mice carry human erythrocytes (for a review,see Moreno et al., 2007). Recently, sporozoites, which are thehepatic form of the parasite [for a review of the P. falciparum lifecycle see Greenwood et al. (Greenwood et al., 2008)], were usedto infect chimeric livers of humanized Alb-uPA/SCID mice. Thereduction of the innate immune response by anti-macrophage andanti-NK cell treatments both enhanced the humanization of Alb-uPA/SCID mouse livers and allowed the infection of humanhepatocytes by sporozoites, while promoting the maturation ofthe pathogen (Morosan et al., 2006). This new model should

permit the evaluation of drugs specifically directed against thehepatic stage of the infection. Moreover, this model provides astarting point to create a future humanized model allowing studyof the entire parasite cycle.

Overview of the information on liver replacement and rodentmodels for humanized liversRequirement for on-going liver damage to stimulate cellproliferationA common feature of all of these models (Table 6) is the failure oftransplanted cells to proliferate in the absence of a proliferationstimulus. The necessary stimulus is most easily achieved by PHbut is short-lived (2-4 weeks). In addition, the liver resection mustoccur simultaneously with, or precede, cell transplantation (Oertelet al., 2006). Alternatively, in mouse models created by FAHdeficiency or in Alb-uPA transgenic mice, liver damage iscontinuous and does not require experimental intervention. Theduration of liver damage in Alb-uPA animals is a function of themouse genotype. Most investigators have worked with animals thatare hemizygous for the transgene; in this case, rare hepatocytesdelete the transgene and the resulting corrected cells are selectedfor in the diseased liver, repopulating the liver within 12 weeks andresulting in an environment of competitive regeneration. Correctedcells are more rare in animals that are homozygous for the transgeneand essentially do not compete with transplanted cells. This effecthas become important for successfully obtaining humanized livers.For homozygotes, the absence of transplanted liver cells is usuallylethal and depends on the rare occurrence of transgenedeletion/silencing (Table 6).



Table 6. Characteristics of the models for liver regeneration/replacement

Model and its nature

Proliferation induction

(window for use)

Selective advantage

for exogenous

hepatocytes/cells Advantages/caveats Reference

Alb-uPA tg mice: intrinsic

hepatocyte toxicity from Tgproduct

Tg hemizygous (2-6 weeks);

Tg homozygous:(permanent until

lethality)

Strong transitory;

strong permanent

Competition with Tg-deleted

endogenous hepatocytes;

reproductive problems forhomozygotes

(Rhim et al., 1994)

Fah –/– mice: conditionally lethal

(NTBC blocks toxicity)Permanent Strong permanent Recessive mutation: selects for

products of cell fusion if wild-

type Fah allele in transplantedcells

(Overturf et al., 1996)

DPPIV-deficient rats and mice

(to receive DPPIV+ donor cells)Must be provided None Powerful positive staining

highlights even rare cells(Gupta et al., 1995)

enoN)xT erofeb( htnom 1≤HP Can reveal long-term intrinsicselective cellular advantage

(Laconi et al., 1998)

PH + retrosine or AAF ≤1 month + persistent

inhibition of hepatocyte

proliferation

Strong permanent Host liver histology abnormal (Laconi et al., 1998)

PH + CCl4 or allyl alcohol ≤1 month + hepatocytetoxicity

Strong Hepatocyte toxicity level can becontrolled by dose; repeated

treatments possible

Recipient mouse sensitive to Jo2

MAB, donor resistant toapoptosis (Bcl2

overexpressed)

Within 24 hours of MAB

administrationStrong Variability of antibody activity (Mignon et al., 1998)

Intrafetal injection Intrinsic in fetus None Absence of immune response (Almeida-Porada et al.,2004)

Tg, transgene or transgenic; Tx, transplantation; MAB, monoclonal antibody.

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One exception to the requirement for liver damage to stimulatedonor hepatocyte proliferation has been observed with bonemarrow to hepatocyte transition in FAH-deficient animals.However, the recipients’ hematopoietic system needed to berescued after total body irradiation, and it was the progeny of thesetransferred cells that then conferred a wild-type Fah allele by cellfusion.

Stem cells versus adult hepatocytes or embryonic liver cellsfor liver repopulationAt first, it might seem that stem cells would be more appropriateto regenerate a liver than a distinct population of liver tissue cells.However, the pioneering work of Sandgren et al. and Rhim et al.demonstrated that some cells of the liver, presumably hepatocytes,possessed sufficient proliferative capacity to replace the liver tissue(Sandgren et al., 1991; Rhim et al., 1994). Judging from the numbersof regeneration nodules, it was calculated that 12-16 cell generationswere required for replacement. This number of doubling eventswas significantly enhanced in serial repopulation experiments byOverturf et al., who estimated that a minimum of 69 rounds ofhepatoctye doubling were required to repopulate a single liverthrough 6 serial transfers (Overturf et al., 1997). Thus, it is not anintrinsic limitation of replication capacity that limits the usefulnessof hepatocytes for liver regeneration, but rather their restrictedavailability, especially with human hepatocytes.

The surprising proliferative potential of rat E14 (FLEP)embryonic liver cells was brought to light by the work of Sandhuet al. (Sandhu et al., 2001). Using just PH as a proliferation stimulus,they compared the growth of E14 and adult liver cells. Thedifferences were most apparent several months after hepatectomy,when the proliferative stimulus was no longer operative – adulthepatocytes had ceased growing, whereas embryonic cellscontinued to expand throughout the 6-month experiment,replacing healthy endogenous host hepatocytes. This intrinsicdifference in growth potential between transplanted adult and fetal

liver cells is of great interest, and the conservation of thisphenomenon in human cells should be explored.

Liver-derived stem cell linesStem cell lines are beginning to be used with success for livertransplant experiments and it remains to be seen whether they willprove to be as robust as freshly isolated liver cells for long-termrepopulation. For the preparation of humanized liver models, itwould be a major advantage to be able to use cells from a tissueculture flask rather than await availability of fresh humanhepatocytes. The emergence of malignant cells in stem cell lines isa disconcerting possibility but should not preclude their use forthe development of experimental animals.

Differentiation plasticity and transdifferentiation of stem cellsThere has been enormous enthusiasm about the possibility of usingstem cells, particularly from humans, to promote tissue repair. Thedemand for donor liver greatly exceeds the supply. Alternatively,stem cells could be obtained from the afflicted individualsthemselves, or from an immunotyped stem cell bank. The lessonobtained with the FAH model and liver replacement by HSCs hasput us in a stronger position to devise therapeutic strategies in arational manner. In particular, future experiments must be designedwith appropriate genetic markers to address the question:transdifferentiation or cell fusion?

Although direct transplantation of bone marrow stem cells, cordblood cells and derivatives of ES cells, induced to undergo liverdifferentiation, is currently under investigation, it is too early tojudge the effectiveness and future potential of these approaches. Itwill be particularly important to attempt such experiments usingthe best available models.

Advantages and drawbacks of humanized rodent modelsThe recent development of small animal models for experimentalHBV, HCV or Plasmodium infection has opened new avenues for



1993 1990 2000

Identification of the Fah

mutation as a cause of liver

failure (Grompe et al.)

Creation of the uPA

transgenic mouse

(Heckel et al.)

1991

Identification of the

uPA transgenic mice as

a model for liver

regeneration (Sandgren

et al.)

1997 1999 1994 1995

Repopulation of uPA

liver by syngenic

healthy hepatocytes

(Rhim et al.)

Pharmacological correction of

the lethal hepatic dysfunction

in the Fah-/- model (Grompe et

al.)

2001 2007

Repopulation of

Fah-/-/Rag2-/-/Il2-/-

by human

hepatocytes (Bissig

et al.)

Use of uPA adenovirus to

enhance repopulation of

Fah-/-/Rag2-/-/Il2-/- by human

hepatocytes (Azuma et al.)

Serial transplantation of syngenic

mouse Fah+/+ hepatocytes into the

Fah-/- model: stem-cell-like

regenerative potential of adult

hepatocytes (Overturf et al.)

Mature hepatocytes are

responsible for repopulation

of the Fah-/- mouse liver

(Overturf et al.)

1998

Anti-Fas resistant

hepatocyte can repopulate

mouse liver treated by Fas

ligand (Mignon et al.)

Repopulation of uPA/nude liver by xenogenic (rat) hepatocytes (Rhim et al.)

1996

Fah-/- mice as a model

for retroviral therapy of

the liver (Overturf et al.)

Use of the DPPIV marker and the

retrorsine pretreatment conjugated

with PHx as a model for liver

regeneration in rat (Laconi et al.)

Fetal liver progenitor cell

(FLEP) repopulate the liver of

the DPPIV-/- rat pretreated

with retrorsine and submitted

to PHx (Dabeva et al.)

FLEP can repopulate the rat liver

which was submitted to PHx but

without pretreatment by retrorsine

(Sandhu et al.)

Fig. 6. Steps in the creation of rodent models for liver cell transplantation. The arrow represents a timeline. Each box represents an independent initial studydescribing a rodent model to study liver cell transplantation. Boxes connected to the timeline through the same line indicate that the studies were published inthe same year.

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the evaluation of novel therapeutic and/or prophylactic compoundsagainst these pathogens. Indeed, the rodent models are trulypromising and each one has its own advantages and drawbacks.All are relatively complicated to use, but rodent models presentthe unquestionable advantage of being cheaper, and easier tomaintain and breed than primates. The rat model may be the mostaccessible, notably because of their immunocompetency and thelarge number of reproducibly infected animals that could beobtained following transplantation of human cells from a cell line.The three mouse models described above are more physiologicallyrelevant in that they are based on transplantation of freshly isolatedhuman tissue or primary hepatocytes. The Alb-uPA and FAHmodels are certainly the most closely related, physiologically, tohumans. Even if these models are developed in differentimmunotolerant settings, the humanized liver may contain up to90% of human hepatocytes compared with just 6% of hepatomacells in the rat (Table 5).

Until recently, the FAH model did not tolerate the transplantationof human cells. Backcrosses of the Fah–/– mouse onto theRag2–/–/Il2–/– immunotolerant background resolved this problem(Fig. 6) and led to the emergence of a novel humanized mousemodel for the study of pharmacological compounds. Severalproperties favor this new model over the Alb-uPA mouse. First,the transgenic Alb-uPA model can lose the transgene through DNArecombination, whereas in the Fah–/– model, exon 5 of the Fah geneis deleted, so the knockout mouse is not subject to spontaneousreversion. The Fah–/– mice can receive transplants at any time,whereas Alb-uPA mice must receive transplants during a narrowtime window (between 4-30 days after birth). Another disadvantageof the Alb-uPA model is its poor breeding efficacy, although thiscan be overcome by conducting syngeneic wild-type hepatocytetransplants, thus correcting the hepatic defect of Alb-uPA mice(Brezillon et al., 2008).

In the FAH model, NTBC treatment is simple (it is added todrinking water), but experience is required for dealing withtreatment cycles to ensure optimum repopulation; and to date,only a few laboratories have used this model. Above all, no HBV,HCV or P. falciparum infecitons has been reported in theFah–/–/Rag2–/–/Il2–/– model. However, in the near future, wepredict that this model should provide an additional tool fortherapeutic studies. To date, HBV and HCV viremias clearly lastlonger and achieve higher levels in Alb-uPA/SCID mice than inother models (Table 5). Moreover, the tractability of this modelfor studying the hepatic stages of P. falciparum is clear. Atpresent, all of these observations lead us to conclude that the Alb-uPA transgene, combined with the appropriate immunodeficiencygenes, appears to constitute the most relevant humanized modelfor providing a bridge between in vitro research and clinical trials.ACKNOWLEDGEMENTSThe authors acknowledge numerous fruitful discussions on liver repopulation withcolleagues, in particular Hélène Gilgenkrantz, Jacques-Emmanuel Guidotti, SerbanMorosan and Hélène Strick-Marchand.

COMPETING INTERESTSThe authors declare no competing financial interests.

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Cell therapy for the diseased liver: from stem cell ... · of the work we do. For many years, liver biologists were limited to in vitro work on liver cells, or in vivo studies using