Transplant Immunology 12(2004) 289–302

0966-3274/04/$ - see front matter� 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.trim.2003.12.014

Emerging insights into liver-directed cell therapy for genetic and acquireddisorders

Sanjeev Gupta*, Mari Inada, Brigid Joseph, Vinay Kumaran, Daniel Benten

Departments of Medicine and Pathology, Marion Bessin Liver Research Center,Cancer Research Center and General Clinical Research Center, Albert Einstein College of Medicine Ullmann Building, Rm 625,

1300 Morris Park Avenue, Bronx, NY 10461, USA

Abstract

Treatment of acute or chronic liver diseases by cell transplantation is an attractive prospect because organ shortages greatlyrestrict liver transplantation. Moreover, a variety of genetic deficiency states affecting extrahepatic organs are amenable to liver-directed cell therapy. While the initial clinical experience with liver cell transplantation has been encouraging, further advancesin several areas are necessary to improve these results. Insights into how engraftment and proliferation of transplanted cells maybe modulated to obtain therapeutically effective masses of transplanted cells will be important in this pursuit. Studies of celltherapy in animal models of specific diseases have provided insights into the development of clinically relevant strategies forvarious disorders. Also, identification of suitable cell types, including stemyprogenitor cells that could be expanded andmanipulated in cell culture conditions, has begun to provide important new information for cell therapy. Similarly, advances incryopreservation of cells and prevention of allograft rejection offer ways to accomplish cell therapy in an effective manner. Takentogether, these advances indicate that liver-directed cell therapy will be well positioned in the near future to play significant rolesin transplantation medicine.� 2004 Elsevier B.V. All rights reserved.

Keywords: Liver; Cell; Transplantation; Gene therapy; Cell therapy

1. Introduction

Acquired diseases of the liver impose a heavy burden,e.g. chronic viral hepatitis afflicts an estimated 350million people worldwide, and acute liver failure isassociated with unacceptably high mortality rates of upto 90%. While orthotopic liver transplantation(OLT)cures chronic liver disease, as well as a variety ofmetabolic and genetic deficiency disorders, the availa-bility of donor livers has limited OLT in the UnitedStates to fewer than 5000 procedures annually. In con-trast, the number of patients waiting for OLT at present,using fairly stringent criteria for acceptance, is several-fold greater. This disparity between organ supply-and-demand obviously requires new strategies to supplementOLT. The concept is not that OLT can be avoided,which is unrealistic, because acute liver failure andmany complications of chronic liver disease can only

*Corresponding author. Tel.:q1-718-430-2098; fax:q1-718-430-8975.

E-mail address: sanjvgupta@pol.net(S. Gupta).

be managed by OLT. However, OLT for treating condi-tions where the liver itself is healthy, as happens inmany genetic conditions, is certainly quite drastic andshould indeed be substituted by less expensive, simplerand potentially ‘reversible’ modalities, such as celltherapy. However, an important question arises imme-diately: If organs are lacking for even OLT, where wouldone find cells for transplantation? The recent arrival ofhuman embryonic stem(hES) cells seems particularlytimely for addressing this question, although extensivework lies ahead to understand how the potential of suchcells could be harnessed for clinical applications. For-tunately, additional sources of liver cells are also beingdeveloped for cell therapy in people and fetal tissue-derived cells can be utilized more readily.

In the aggregate, our own efforts(and those of manyother laboratories) over the previous 20 years have ledto increasing optimism in the potential of liver-directedcell therapy. This review of recent progress in celltherapy will focus on selected aspects, including theregulation of transplanted cell engraftment and prolifer-

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ation, which we believe are critical for clinical applica-tions of cell therapy. Many intertwined areas of biologyare obviously relevant for developing cell therapy,extending from basic sciences to clinical medicine, butit is not possible to adequately cover all aspects oftransplanted cell biology within the given scope of thiswriting. Therefore, we apologize to the reader, as wellas investigators, if our discussion of liver-cell therapy isnot comprehensive and omits the work of specificgroups.

2. Mechanisms concerning engraftment of transplant-ed cells

A variety of locations have been investigated toestablish where transplanted hepatocytes can survive.The earliest such studies were driven by the difficultiesin distinguishing between transplanted and native hepa-tocytes in the liver, which propelled analysis of trans-planted cells in extrahepatic sites, including the spleen,peritoneal cavity, subcutaneous sites, pulmonary capil-laries, etc.w1x. Although transplanted cells survived tovarious degrees in some of these ectopic sites, especiallyin the peritoneal cavity and the spleen, further studiesestablished that transplanted celts survived most effec-tively in the liver itself.

Of course, it is difficult to replicate the complexstructure of the hepatic acinus in ectopic sites. Forexample, hepatocytes are arranged in single-cell thickcords surrounded by a mixture of portal and hepaticarterial blood in adjacent sinusoids. These sinusoids arelined with endothelial cells and a discontinous layer ofbasement membrane encloses the space of Dissebetween hepatocytes and sinusoidal cells, where hepaticstellate cells residew2x. A variety of soluble signals andcytokines emanate from hepatic stellate cells and per-haps from endothelial cells. Moreover, Kupffer cells,which constitute the body’s largest pool of residentmacrophages, contribute to cell–cell signaling, witheffects on virtually all liver cell types, including hepa-tocytes, hepatic stellate cells, and endothelial cellsw3x.In addition, hepatocytes secrete bile into the biliarysystem and there appears to be a complex relationshipbetween hepatocytes and specialized biliary structures,e.g. the canal of Hering, which is thought to producehepatic progenitor cellsw4x. Therefore, each of thesecell types may influence the function of hepatocytes.Certainly, hepatocytes survive in the peritoneal cavitymuch more effectively when non-parenchymal cells areincluded in the transplanted cell mixturew5x, althoughvirtually nothing is known about the specific mecha-nisms that produce such an effect. Nonetheless, it hasbeen evident that the sinusoidal organization of thespleen shares similarities with the liver and it may bethat extracellular matrix components, blood flow pat-terns, and other unknown properties favor survival of

hepatocytes in the spleenw6x. It is also noteworthy thatgene expression is well preserved in hepatocytes in thespleen compared with other extrahepatic sites, such asthe peritoneal cavity and the dorsal fat padw7x.

Several methods have been developed to demonstratethe biodistribution of cells injected in various vascularspaces, including fluorescent labels and radioisotopiclabels w8–10x. Use of these labels demonstrated thatmost of the hepatocytes injected into the spleen migrateimmediately into liver sinusoids because splenic blooddrains into the portal vein. This process has been widelyexploited to transplant cells easily and safely into theliver of small animals. While hepatocytes migrate outof the spleen mechanically, based on blood flow patterns,whether adhesion molecules or other anchorage mecha-nisms may differentially regulate ‘homing’ of cells needsinvestigation. Such studies will be relevant for under-standing the biology of various candidate cell typessuitable for liver repopulation and should eventuallyprovide insights into the possibility of cotransplantingvarious liver cell types to promote cell–cell interactions.

More recently, use of transgenic reporters or otherendogenous genetic markers, e.g. sex chromosomes, toidentify transplanted cells in the liver provided valuableinformation concerning liver repopulationw11–13x. Useof mutant animals, where the liver undergoes persistentinjury, e.g. due to urokinase-type plasminogen activatorin alb-uPA transgenic mice and accumulation of tyrosineintermediaries in FAH mutant mice lacking fumarylhydoxylase activity, provided unique insights into trans-planted cell proliferationw13,14x. The dipeptidyl pepti-dase 4(Dpp4) deficient Fischer 344 rat has also beenvery helpful in studies of liver repopulationw15–17x.This rat has a natural missense mutation in the codingregion of the Dpp4 gene(also known as CD26), whichinactivates the biological activity of the protein. TheDpp4 gene has been inactivated in the mouse byhomologous recombination and these knockout mice arealso proving useful for addressing the biology of trans-planted cells. Although CD26 is thought to play signif-icant roles in lymphocyte activation, neitherDpp4-deficient rats nor Dpp4 knockout mice exhibit anyphenotypes. The animals exhibit a normal life spanwithout immunodeficiency or tolerance for allograftedcells. As normal cells with Dpp4 activity are readilyidentified in Dpp4-deficienct recipients, these animalshave been used widely for hepatocyte transplantationstudies.

Several key steps have now been established in howtransplanted cells engraft in the liver. Insights into thesemechanisms are important because ways are nowbecoming available to modulate and to enhance cellengraftment by specific perturbations. The first stepconcerns deposition of cells in hepatic sinusoids, whichleads to the entrapment of transplanted hepatocytes inproximal sinusoids due to the differences in the size of

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hepatocyte (20–40 mm) vs. sinusoids (6–9 mm)w17,18x. This entrapment of hepatocytes in sinusoidsprevents further passage of cells into hepatic veins –inferior vena cava – pulmonary capillaries, which avoidspulmonary embolic complications. Quantitative analysisof intrapulmonary shunting of cells established thatintrasplenically transplanted cells remain within the liverin normal animals, although in the presence of portalhypertension and portasystemic shunting, as observed incirrhosis, large fractions of transplanted cells can trans-locate into pulmonary capillaries and produce compli-cations w19x. However, fortunately, hepatocytes arerapidly destroyed in the pulmonary capillaries and pul-monary hemodynamic perturbations are quite evanes-cent, despite the presence of hepatocytes in very largenumbersw20x.

As soon as transplanted cells enter liver sinusoids,portal pressures rise due to increased portal resistancew18–21x. However, portal hypertension is transient andsubsides within 2–3 h following cell transplantation inassociation with hepatic microcirculatory perturbationsinitially followed by the redistribution of blood flowfrom occluded sinusoids through reopening of furthervascular channelsw18x. Also, embolization of cells inhepatic sinusoids leads to the onset of ischemia-reper-fusion in significant portions of the liver, depending onthe number of cells injectedw21x. Circumvention ofthese changes by pharmacological dilatation of liversinusoids decreased the number of transplanted cellsretained within portal vein radicles and pushed morecells into the hepatic sinusoidsw18x. Moreover, entry ofhepatocytes in liver sinusoids promoted cell entry intoliver cords. Among various vasoactive substances testedfor this purpose, nitroglycerine and phentolamine(alpha-adrenergic blocker) were particularly beneficial, withgreater engraftment of transplanted cells. Also, dilatationof sinusoids during cell transplantation decreased micro-circulatory perturbations in the liver.

Activation of Kupffer cells due to hepatic ischemiaand possibly by the presence of non-viable cells consti-tutes another important early event following cell trans-plantation w22x. Kupffer cells release cytokines thatinterfere with cell engraftment in the liver and participatein the clearance of large fractions of transplanted cells.Simultaneously, Kupffer cell activation is associatedwith the recruitment of lymphocytes(both CD4 andCD8-positive) in proximity to the transplanted cells,which is likely to contribute further cytokine signalsthat may interfere with cell engraftmentw23x. This earlyphagocyte–macrophage response leads to the clearanceof 70–80% of the transplanted cells from the liverwithin 24–48 hw24x. On the other hand, impairment ofthe Kupffer cell response by pretreating animals withgadolinium chloride, which interferes with Kupffer cellactivation, leads to significant improvements in trans-planted cell engraftment and ensuing liver repopulation

w22x. These findings offer ways to further manipulatethe engraftment of transplanted cells, including by inter-fering with specific signals emanating from activatedKupffer cells.

The entry of transplanted cells into the liver paren-chyma is another critical step in cell engraftment. Duringthis process, transplanted cells must translocate fromliver sinusoids into the space of Disse, which requirespenetration of the endothelial cell layer, insinuation ofcells between native hepatocytes, reconstitution of plas-ma membrane structures, and finally incorporation inthe liver parenchymaw24x. After arriving in liver sinu-soids, transplanted cells adhere to endothelial cells. Thiscell adherence is facilitated by hepactocyte surface-associated integrins. For instance, when ligands areincorporated, such as fibronectin, that bind hepatocyteintegrins on the one hand, and endothelial cell receptorson the other hand, transplanted cell anchorage isenhanced and cell engraftment in the liver is signifi-cantly improved. Such a manipulation results in accel-erated kinetics of liver repopulation.

The endothelial cell layer must be breached duringthe entry of transplanted cells into the space of Disse,which could well be aided by Kupffer cell activationand cytokine release, as well as release of vascularendothelial growth factor(VEGF) from transplanted andnative hepatocytesw24x. Studies showed that VEGF isreleased several hours after cell transplantation but priorto the entry of transplanted cells into the liver parenchy-ma, which requires 16–20 h after cell transplantation.If penetration of the endothelial cell barrier is animportant mechanism, prior disruption of hepatic endo-thelial cells should facilitate cell engraftment. Thispossibility has been examined in detailed studies, wheredisruption of hepatic endothelial cells with cyclophos-phamide significantly accelerated the entry of transplant-ed cells in the liver parenchyma and enhanced liverrepopulationw25x. Additional studies with monocrota-line, which injures endothelial cells in the liver andother vascular beds, also demonstrated several-foldgreater cell engraftment in recipients pretreated with thechemical.

Finally, the recipient liver parenchyma and transplant-ed cells must undergo remodeling to reconstitute cellpolarity. This process appears to be accompanied bydisruption of gap junctions in native hepatocytes shortlyafter cell transplantation due to ischemic eventsw21x.Although disruption of gap junctions should help sepa-rate native hepatocytes and facilitate insinuation oftransplanted cells in liver cords, the significance of thisprocess in cell engraftment is unclear. For instance,transplanted cells integrate without any difficulty despitethe abrogation of ischemic injury and decrease in gapjunction disruption following nitroglycerine-induced pre-vention of hepatic ischemia.

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Table 1Specific mechanisms regulating transplanted cell engraftment in liver

Regulator of cell Intervention Effect on cell engraftmentengraftment

Vasomotor tone of Nitroglycerine or Sinusoidal dilatation, less microcirculatoryhepatic sinusoidal bed1 phentolamine impairment, more transplanted cells in

sinusoids, greater engraftmentKupffer cells2 Gadolinium chloride Kupffer cell impairment, greater transplanted

cell engraftmentEndothelial cells3 Cyclophosphamide or Endothelial cell disruption, superior

monocrotaline transplanted cell engraftmentTransplanted cell Ligands binding Transplanted cell anchorage to endothelium;

adhesion to endothelial hepatocytes and activation of focal adhesion complexes,cells4 endothelial cells greater transplanted cell engraftment

(Slehria S., et al. Hepatic sinusoidal vasodilators improve transplanted cell engraftment and ameliorate microcirculatory perturbations in the1

liver. Hepatology 2002; 35: 1320–1328).(Joseph B, Malhi H, Bhargava KK, Palestro CJ, McCuskey RS, Gupta S. Kupffer cells participate in early clearance of syngeneic hepatocytes2

transplanted in the rat liver. Gastroenterology 2002; 123: 1677–1685).(Malhi H., et al. Cyclophosphamide disrupts hepatic sinusoidal endothelium and improves transplanted cell engraftment in rat liver. Hepatology3

2002; 36: 112–121).(Kumaran V, Joseph B, Benten D, Gupta S. Hepatic integrin-dependent extracellular matrix(ECM) component interactions regulate hepatocyte4

engraftment in the liver. Hepatology 2003; 38: 216A).

Reconstitution of the plasma membrane structures,including gap junctions and bile canaliculi, requires3–7 days for completionw15,24x. During this period,the bile canalicular network is fully restored and trans-planted cells become indistinguishable from adjacentnative hepatocytes. Reconstitution of bile canaliculi isanother critical event because transplanted cells cannotfunction physiologically without an excretory apparatusand correction of diseases characterized by abnormalexcretion of toxins requires the functional integrity ofthe biliary apparatus. Hepatic stellate cells may partici-pate in this process because evidence has recently beenobtained for their perturbation 1–3 days after celltransplantation. As remodeling of the liver plate structureis likely to be associated with matrix type metallopro-teinase-dependent processes, as well as de novo synthe-sis of extracellular matrix components, it stands toreason that hepatic stellate cells will play roles inhepatocellular engraftment. The potential interactionsbetween Kupffer cells, specific cytokine signals andother perturbing events capable of regulating hepaticstellate cell activation are being studied at present.

It should be important to note that transplanted hepa-tocytes complete the above process of engraftment inthe liver despite significant hepatic injury. In animalswith acute liver failure induced by hepatotoxins, trans-planted cells have been demonstrated to survive, engraftand proliferatew26,27x. However, transplanted cell pro-liferation is dissociated from hepatic DNA synthesisobserved in the native liver because of the 3–7 dayperiod required for completion of cell engraftment inthe liver w26x. Also, it is worth noting that transplantedcells can engraft in the liver with chronic disease orcirrhosis and extensive fibrosisw28x. These findings are

interesting because cirrhosis is associated with ‘capillar-ization’ of the sinusoidal endothelium, implying depo-sition of collagenous matrix components, which suggeststhat transplanted cells may possess protease activitycapable of dissolving extracellular matrix componentsunder these circumstances, although further study isrequired of such mechanisms.

Therefore, taken together, these manipulations havebegun to provide multiple ways to decrease the initiallosses of transplanted cells and improve cell engraftment(Table 1). As the efficiency of cell engraftment isreflected in the rate by which the liver can be repopu-lated, further exploration of these mechanisms shouldbe appropriate for clinical applications.

3. Regulation of transplanted cell proliferation andliver repopulation

The ability of transplanted hepatocytes to engraft inthe liver has provided reporter systems to address phys-iological mechanisms concerning regulation of geneexpression and fate of liver cells. These studies estab-lished that transplanted hepatocytes do not proliferate inthe normal liver throughout the adult life, although somecell proliferation is evident in the liver of very youngrats w29x. Also, in aging F344 rats, transplanted cellsbegin to proliferate but this may reflect an unexplainedtendency of this animal strain to exhibit hepatocellularproliferation and adenoma formation during old age.The absence of transplanted cell proliferation in thenormal liver is in agreement with the replicative quies-cence of normal hepatocytes and implies that from atherapeutic standpoint the magnitude of transplanted cellmass achieved with a single session of cell transplanta-

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tion (0.5–2% of the normal rodent liver) will beinadequate. While repeated cell transplantation is safeand can increase the transplanted cell mass progressively,such that after 3 sessions of cell transplantation, 5–7%of the liver mass can be replaced in rodents, this toomay be insufficient for cell therapy in most disordersw30x. Therefore, additional manipulations to increase thetransplanted cell mass have been investigated.

Studies of selective hepatic ablation with chemicaltoxins that spared transplanted hepatocytes, such ascarbon tetrachloride to induce perivenous liver injury,generated the idea that transplanted cell could proliferatein the liver w27x. The use of alb-uPA transgenic mice asrecipients provided very effective evidence for thecapacity of transplanted cells to proliferate extensivelyand repopulate large areas of the liverw14x. As indicatedabove, this was related to the onset of extensive liverinjury following hepatotoxicity from the uPA transgene.Similarly, transplantation of hepatocytes into FAHmutant mice was shown to be associated with extensiveliver repopulation, again because transplanted cells werespared from injury affecting native cellsw13x. The FAHmouse has been highly effective for a variety of furtherstudies to demonstrate mechanisms concerning the rep-lication potential of primary hepatocytes, oval cellsisolated from the pancreas or liver, as well as plasticityof extrahepatic stem cells, e.g. hematopoietic stem cells(see below). Since cell cycle check point controls canbe circumvented by genetic deletion of suppressor genes,it is possible to determine whether increased cell cyclingwill provide ways to repopulate the liver faster. Instudies using p27 null mouse hepatocytes, it was dem-onstrated that these cells proliferated more rapidly com-pared with normal hepatocytes in FAH micew31x. Ofcourse, cell cycle manipulations are not without the riskof additional oncogenic perturbations, which requirescareful consideration.

The concept of selective injury in native hepatocytesto induce transplanted cell proliferation was furtherstrengthened in studies using the murine Fas agonist,J0-2 antibody, to induce hepatic apoptosis, whereastransplanted cells were made resistant to apoptosis byexpressing a human Bcl-2 transgenew32x. These studiesshowed that the liver could be repopulated in a gradedfashion, in agreement with the magnitude of apoptosisin the native liver. In another approach, retrorsine, aDNA-binding alkaloid, was used to inhibit proliferationand survival in native hepatocytesw33x. Retrorsineinduces polyploidy in the liver, which is further accel-erated by two-thirds partial hepatectomy. Studies inanimals subjected to partial hepatectomy, which inducescompensatory hepatic growth, showed that transplantedhepatocytes proliferated only when cells were transplant-ed subsequent to partial hepatectomy. Interestingly, par-tial hepatectomy was found to impair the survival andproliferation capacity of hepatocytes in the remnant liver

w34x, which offers further explanations of its synergisticactivity with retrorsine and related perturbations.

Transplantation of hepatocytes in rats pretreated withretrorsine and partial hepatectomy led to near-total liverreplacement with transplanted cellsw33x. This systemhas subsequently been modified in a variety of ways,including substitution of partial hepatectomy by repeatedadministration of the thyroid hormone tri-iodothyronine(T3), which is a regulator of hepatic polyploidy, as wellas of carbon tetrachloridew35,36x. Recently, monocro-taline, which is another pyrrolizidine alkaloid, was foundto have additional effects on hepatocytes, similar toretrorsine, and produced extensive liver repopulation incombination with both carbon tetrachloride and partialhepatectomy(Fig. 1). In Dpp4-deficient rats pretreatedwith monocrotaline, cell transplantation resulted in supe-rior cell engraftment due to monocrotaline-inducedendothelial disruption. Moreover, monocrotaline per-turbed native hepatocytes, as elucidated by additionalinjury with carbon tetrachloride, which led to extensivereciprocal proliferation of transplanted cells, since thesecells were unaffected by toxic injuries. In contrast,control animals that did not receive monocrotaline pre-treatment showed slight proliferation, as expected withlimited liver regeneration induced by CC14 alone.Monocrotaline appears to be more effective than retror-sine in mice compared with rats. Unfortunately, retror-sine, monocrotaline or other such toxins are unsuitablefor clinical use in people.

However, the principle of genotoxicity in native hepa-tocytes to perturb their survival has been adopted fordeveloping additional strategies that should be moreapplicable for clinical use. In initial studies, radiationwas found to induce extensive liver injury when animalswere subjected to partial hepatectomyw37x. Examinationof whether hepatocyte transplantation could help amelio-rate this radiation-induced liver disease established thattransplanted cells proliferated extensively in animalstreated with liver radiation and partial hepatectomyw37x.The combination of radiation and T3 has also beeneffective, as would have been predicted by studies usingretrorsine and partial hepatectomy, and produced signif-icant liver repopulation. Again, the combination ofradiation and partial hepatectomy is not amenable toclinical applications. Therefore, in further studies, partialhepatectomy was substituted with ischemia-reperfusionon the basis of findings indicating that partial hepatec-tomy produces extensive oxidative DNA damage, simi-lar to radiationw38x. Hepatic ischemia-reperfusion is amost potent source of oxidative stress and has been usedin clinical medicine to treat liver cancer. The combina-tion of ischemia-reperfusion and radiation was highlyeffective in promoting transplanted cell proliferation andthe rat liver was repopulated virtually completely overa period of 3 months. Among important aspects of thisliver repopulation were that native hepatocytes was lost

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Table 2Induction of liver repopulation in animals with iatrogenic mechanisms

Manipulation Mechanism Efficacy

Retrorsine alone1 Genotoxic liver injury Moderate liver repopulation; slow onsetRetrorsineqpartial Genotoxic liver injury, Extensive liver repopulation; relatively

hepatectomy, T3 or CC142–4 oxidative stress rapid onset(better in rats than mice)Fas ligandyBcl-2 Apoptosis in native Moderate to high liver repopulation;

expression5 hepatocytes relatively rapid onsetRadiationqpartial Genotoxic liver injury, Extensive liver repopulation; slow onset

hepatectomy or ischemia- oxidative stressreperfusion6,7

MonocrotalineqCC148 Genotoxic liver injury, Extensive liver repopulation; relativelyoxidative stress rapid onset

(Laconi S, Pillai S, Porcu PP, Shafritz DA, Pani P, Laconi E. Massive liver replacement by transplanted hepatocytes in the absence of1

exogenous growth stimuli in rats treated with retrorsine. Am J Pathol 2001; 158: 771–777).(Laconi E, et al. Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am J Pathol2

1998; 153: 319–329).(Oren R, et al. Role of thyroid hormone in stimulating liver repopulation in the rat by transplanted hepatocytes. Hepatology 1999; 30: 903–3

913).(Guo D, Fu T, Nelson JA, Superina RA, Soriano HE. Liver repopulation after cell transplantation in mice treated with retrorsine and carbon4

tetrachloride. Transplantation 2002; 73: 1818–1824).(Mignon A, et al. Selective repopulation of normal mouse liver by FasyCD95-resistant hepatocytes. Nat Med 1998; 10: 1185–1188).5

(Guha C, et al. Amelioration of radiation-induced liver damage in partially hepatectomized rats by hepatocyte transplantation. Cancer Res6

1999; 59: 5871–5874).(Malhi H, Gorla GR, Irani AN, Annamaneni P, Gupta S. Cell transplantation after oxidative hepatic preconditioning with radiation and7

ischemia-reperfasion leads to extensive liver repopulation. Proc Natl Acad Sci USA 2002; 99: 13114–13119).(Joseph B, Kumaran V, Bhargava K, Palestro C, Gupta S. Monocrotaline perturbs hepatic endothelial cells and hepatocytes but not Kupffer8

cells to promote transplanted cell engraftment and proliferation. Hepatology 2003; 38: 270A).

gradually, which decreases the possibility of liver failure,and that soluble signals were likely involved in directinghepatic injury following ischemia-reperfusion, but thisneeds further study.

Examination of the proliferation potential of hepato-cytes has also utilized animals subjected to other pertur-bations, including hepatic injury withD-galactosamine,Mad1 transcription factor, thymidine kinase based prod-rug activation, etc.w26,39,40x. Efforts to induce prolif-eration in transplanted cells with the administration ofhepatocyte growth factor were unsuccessfulw27x. There-fore, while more study is needed for understanding howthe liver can be repopulated with transplanted hepato-cytes, recent insights into specific mechanisms in thisprocess are quite exciting(Table 2). Use of radiationand ischemia-reperfusion to induce hepatic damage priorto cell transplantation is clinically applicable and willhopefully benefit cell therapy applications in people.

4. Liver-directed cell therapy for specific disorders

Numerous animal models are now available for stud-ying the requirements and efficacy of cell therapy inspecific disorders. Monogenetic disorders, such as thosethat may cause metabolic abnormalities or protein defi-ciencies are excellent candidates for cell therapy, becausethe efficiency of interventions can be analyzed byspecific assays. Similarly, cell therapy is appropriate foracute or chronic liver failure, especially for the formerif survival could be prolonged to bridge toward OLT

(Table 3). Of course, under the best circumstances, OLTmight be avoided altogether if transplanted cell helpedthe liver to regenerate after failing acutely. However,patients with chronic liver failure could potentially betided over from disabling complications, such as hepaticencephalopathy or coagulopathy, if a supplemental massof transplanted liver cells could be created. However, todemonstrate whether cell therapy will be effective inacute or chronic liver failure, careful analysis of out-comes is necessary in controlled studies, which presentscomplex logistical issues.

Many types of animal models have been used todemonstrate whether cell therapy could be effective foracute liver failurew41x. Although early studies showedthat cell transplantation could improve mortality, mor-tality was also altered following injection of conditionedmedium from cultured cells, fragmented cells, and evenxenogeneic cells that were promptly rejectedw42x, cast-ing doubt on the mechanisms of improved survival.More recently, studies in two different genetic modelsof acute liver failure in mice established that animalswith liver failure can be rescued by cell transplantationw39,40x. In one of these models using HSV-TK inducedganciclovir hepatotoxicity, transplanted cells were shownto repopulate the liver variablyw40x. In the other model,where expression of the Mad transcription factor in theliver caused hepatotoxicity, transplanted cells proliferat-ed and associations were demonstrated betweenimproved outcomes and proliferation of transplanted

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Table 3Disorders considered amenable to liver-directed cell therapy

Liver damaged No liver injury

● Fulminant liver failure ● Congenital hyperbilirubinemia,● Chronic hepatitis, cirrhosis e.g. Crigler–Najjar syndrome● Alpha-1 antitrypsin deficiency ● Familial hypercholesterolemia● Wilson’s disease ● Apolipoprotein E deficiency● Tyrosinemia, type 1 ● Hyperammonemia syndromes● Erythropoietic protoporphyria ● Defects of carbohydrate● Lipidoses, e.g. Gaucher’s disease, metabolism

Niemann-Pick disease ● Coagulation defects, e.g. FactorVIII or IX deficiency

● Refsum’s disease● Hereditary angioedema● Oxalosis

cells w39x. Also, cell transplantation has been shown toprevent the onset of intracranial hypertension in pigswith acute liver failurew43x. These studies are promising,although the early clinical experience in a handful ofpatients with acute liver failure treated with intraperito-neal, splenic arterial or intraportal injection of cells issomewhat difficult to interpret in terms of the efficacyof cell therapy, although some patients survived longenough to undergo OLT, and in others, improvements inhepatic encephalopathy and cerebral perfusion pressurewere ascribed to cell transplantationw44–46x.

Animals with established liver disease offer otherways to demonstrate whether cell therapy would bebeneficial. Two types of animal models have beenutilized for this purpose. Induction of cirrhosis withrepeated administration of hepatotoxins or of hepaticencephalopathy with portacaval shunts constitute onetype of animal models. Animals with genetically inducedliver disease, e.g. LEC rats with Wilson’s disease, whereextensive liver disease occurs due to copper toxicosis,mdr2 knockout mice, where deficiency of biliary phos-pholipid transport produces biliary disease, or FAHmutant mice with tyrosinemia, represent another type ofsuitable animals.

In rats with end-to-side portacaval shunt, intrasplenictransplantation of hepatocytes improved hepatic enceph-alopathyw47x. Similarly, hepatic encephalopathy in por-tacaval-shunted rats burdened with additional loads ofammonia improved following hepatocyte transplantationw48x. Therefore, provision of supplemental hepatocytemasses is thought to improve metabolic complicationsof chronic liver disease. However, in animals withcirrhosis induced by repeated carbon tetrachlorideadministrationw28x, cell transplantation in the liver didnot show any differences in mortality over a 12-monthperiod. On the other hand, intrasplenic cell transplanta-tion in near-terminally ill cirrhotic rats improved livertests, coagulopathy and survivalw49x. The most effectivedemonstrations of disease correction were in LEC rats,mdr2 knockout mice, as well as FAH mutant mice,

where transplanted cells can engraft and proliferate dueto progressive liver disease in recipientsw13,50,51x.Under these circumstances, even established diseasewith extensive liver fibrosis and architectural disorgani-zation was found to revert completely with return tonormal liver histology following removal of copper bytransplanted cells in LEC ratsw50x. Similar findingshave been observed in mdr2 knockout mice and FAHmutant mice, where liver histology become normalfollowing extensive liver repopulation with healthy cellsw13,51x. Although several patients with chronic liverdisease have been transplanted with hepatocytes in Japanand the United States, it is difficult to determine whethercell transplantation improved outcomes, partly becauselimited numbers of patients were treated in uncontrolledstudies, although some patients eventually underwentOLT w52x.

Treatment of metabolic diseases with cell therapy hasbeen investigated in laboratory animals. Gunn rats withhyperbilirubinemia, similar to Crigler–Najjar syndrometype-1, and Nagase analbuminemic rats, have beenshown to be highly amenable to liver cell therapyw53,54x. Similarly, the Watanabe heritable hyperlipidem-ic rabbit, which models familial hypercholesterolemia,has been useful to demonstrate how genetically modifiedautologous cells, allografted cells, or xenogeneic(por-cine) cells can ameliorate this disorderw55–57x. Hepa-tocyte transplantation has been effective in correctinghypercholesterolemia in a mouse model of apolipopro-tein E deficiencyw58x.

In other circumstances, correction of the bleedingabnormality in hemophilia A, which is due to geneticdeficiency of factor VIII, represents an attractive targetfor liver cell therapy. The liver plays a major role infactor VIII synthesis as shown by cure of hemophilia Afollowing OLT w59x. However, although factor VIII isexpressed abundantly in hepatocytes and endothelialcells in the liver, spleen or elsewhere in the body, it hasbeen unclear as to which of these cell types can actuallycorrect this disorder. Recently, we found that transplan-

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tation of hepatocytes was ineffective, whereas endothe-lial cell transplantation cured the bleeding diathesis infactor VIII gene knockout mice with severe hemophiliaA.

Early studies in patients have begun to reproduce theexperience in several of these animal models, includingin patients with familial hypercholesterolemia, ornithinetranscarbamylase(OTC) deficiency, a-1-antitrypsindeficiency, Crigler–Najjar syndrome type-1, glycogenstorage disease, and Refsum’s diseasew60–63x. How-ever, this initial experience also indicates the importanceof adequate liver repopulation if significant therapeuticbenefits or cures are to be achieved and it is here thatintegration of insights obtained in experimental studiescited above with clinical efforts will likely be mostfruitful.

5. Stemyprogenitor cells and liver repopulation

The area of stem cells has recently undergone aparadigm shift following the demonstration of stem cellplasticity, as well as the recognition of cellular mecha-nisms, such as the telomere hypothesis, which regulateterminal replicative senescence and immortalizationw64x. While certain organs, including the liver, havelong been known to exhibit considerable regenerativepotential, it has only been relatively recently that wehave begun to understand the potential of specific stemyprogenitor cell types in this process. Nonetheless, thepossibility of deriving mature liver cells from hES cells,fetal stemyprogenitor cells, and extrahepatic organs,especially hematopoietic stem cells derived from thebone marrow, peripheral blood, or umbilical cord blood,has elicited considerable interest. The acid test of allthese cell populations will of course concern the dem-onstration of their engraftment and function in animalmodels compared with mature hepatocytes. Nonetheless,early studies have begun to demonstrate some potentialof ES cells in generating hepatocyte-like cells both invitro and in vivow65,66x. Embryoid bodies derived fromthese ES cells expressed albumin anda-fetoprotein andshowed urea synthesis. Also, transplantation of ES cell-derived hepatocytes into mice pretreated with carbontetrachloride showed cell engraftment in the liverw67x.

The intriguing proliferation capacity exhibited byhepatocytes, as shown by Grompe and colleagues, whereserial transplantation of normal adult liver cells in theFAH mouse, never exhausted the replication potentialof transplanted cells despite)80 divisions per cell,indicates that hepatocytes behaved like stem cellsw68x.These important experiments simultaneously establishedthat the biological behavior of hepatocytes was markedlydifferent in intact animals compared with in cell culture.For instance, primary adult hepatocytes are generallyunable to divide or proliferate in cell culture conditions.

Another population of liver cells, designated hepatic‘oval cells’, which originally referred to poorly differ-entiated cholangiolar cells arising after carcinogenicinduction, but also arise during chemical or viral hepaticinjury, has shown progenitor cell propertiesw4x. Ovalcells exhibit a variety of hepatocytic and biliary markers,including albumin,a-fetoprotein, glycogen, glucose-6-phosphatase, and hybrid isoenzymes. Oval cells isolatedfrom the normal rat liver or from the liver of LEC ratswith copper toxicosis can generate mature hepatocytesw69–71x. Similarly, oval cell-like ductular cells isolatedfrom the rat or mouse pancreas have been shown togenerate hepatocytesw72,73x. Oval cells are amenableto significant proliferation in culture conditions and non-oncogenic cell lines with oval cell properties have alsobeen generated, e.g. FNRL cells and WB344 cellsisolated from the F344 rat liverw70,71x. These cell lineshave shown capacity to differentiate along the hepato-cyte lineage. The ability to propagate such cells is ofinterest for cell therapy. In this context, recent studiesof liver-derived oval cells in FAH mice provided impor-tant evidence for the capacity of oval cells to differen-tiate into mature hepatocytes, to repopulate the mouseliver, and to correct tyrosinemia in the micew74x.Isolation of similar oval cells from the human livercould well be significant for clinical applications byproviding an additional source of cells, e.g. from liverexplants.

Much interest remains in transplanting cells isolatedfrom the fetal liver. The fetal liver contains largenumbers of hepatoblasts compared with the adult liver,which has only rare cells witha-fetoprotein expressionor other markers associated with progenitor cellsw75,76x.Studies of the rat liver showed another significantproperty of fetal liver cells, where cells are predomi-nantly diploid w77x. In contrast, hepatocytes in neonatalanimals begin to accumulate non-diploid cells and theliver of young adult rats contains greater fractions ofpolyploid cells instead of diploid cells. Investigatorshave isolated fetal cells from the rodent liver anddemonstrated that such cells can complete differentiationprograms along the hepatocytic and biliary lineages,much more commonly along the former lineage, includ-ing after transplantation in Dpp4-deficient ratsw75,78x.Interestingly, fetal hepatocytes exhibit greater capacityto proliferate in the intact rat liver compared with adulthepatocytes, which is again in agreement with theprogenitor cell type behavior of these cellsw78x.

Clinical use of fetal human hepatocytes should beappropriate, if these cells exhibit similar progenitorproperties. Since the fetal human liver arises at 4 weeksof gestation and matures rapidly with bile formation by12 weeks, it is possible to isolate cells that are alreadydifferentiated along the hepatic lineages. This offerscells that could be used immediately, compared with,say, ES cells, where a variety of limitations, including

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Fig. 1. Regulation of transplanted cell engraftment and proliferation by the endothelial cell and hepatic injury mechanisms. Shown are studies ofcell transplantation in Dpp4-deficient rats following monocrotaline-inducted perturbations. Transplanted syngeneic F344 rat hepatocytes wereidentified by Dpp4 histochemistry(red color product). The number of transplanted cells was much less in control animals(a, arrows) comparedwith monocrotaline-treated rats(b) after 7 days, as a result of endothelial disruption in the latter. Panel(c) shows the effect of monocrotaline ontransplanted cell proliferation following additional liver injury with carbon tetrachloride, which produced significant liver repopulation, whereastransplanted cells proliferated only minimally in control animals treated with carbon tetrachloride alone. Orig. mag., a and b,=200; c,=40.Hematoxylin counterstain.

Fig. 2. Isolation of progenitor cells from the fetal human liver. Cells shown are from a 22-week-old fetal liver. Immediately after isolation, cellsexpressed albumin(a), with some cells expressing it more(arrow) than others, as well as cytokeratin(CK)-19, which is normally expressed inmature bile duct cells(b). The cells attached to tissue culture plastic and proliferated readily, as shown by a confluent cell culture after 10 days,with characteristic epithelial cell morphology(c). The inset in(c) shows a higher magnification view. Albumin and CK-19 expression wasanalyzed by immunostaining using the peroxidase substrate diaminobenzidine for color development. Orig. mag.=200.

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the current requirement of murine feeder cells for expan-sion, potential for teratogenicity, and unknown signalsrequired for inducing differentiation along the hepato-cyte lineage, need to be resolved. Of course, use of fetallivers for isolating cells does not interfere with the organsupply needed for OLT. Finally, fetal tissues and cellshave been transplanted into patients, including withacute liver failurew45x, over the past many years, duringwhich many ethical issues concerning the use of fetaltissues have been addressed.

Analysis of fetal human liver cells has providedvaluable information. A remarkable finding has beenthat fetal cells can proliferate extensively in culture withgene expression profiling consistent with the presenceof multiple oval cell markers, includinga-fetoprotein,gamma glutamyl transpeptidase, plasminogen activatorinhibitor type-1, and various cytokeratinsw76x. Thesecells exhibit extensive replication capacity, while main-taining differentiation potential. Excellent cultures ofepithelial cells can be established from the fetal humanliver (Fig. 2). The cells show characteristic hepatocyticmorphology with rounded nuclei, prominent nucleoliand abundant cytoplasm containing liver proteins, e.g.albumin. In some cells, biliary markers, e.g. CK-19, canbe demonstrated. Indeed, these features of hepatocyticand biliary cell markers in fetal human liver cells areconsistent with cellular bipotentialityw76x Furthermore,such cells can express multilineage markers, includinghematopoietic markers in a small cell subset, which isagain in agreement with the presence of stemyprogenitorcell properties in isolated cells. Another significantfinding has been that fetal human liver cells can becryopreserved with excellent recoveries. Also, whenfetal human liver cells were transplanted into immuno-deficient mice, cells engrafted in the liver parenchymaor the peritoneal cavity and produced mature hepatocy-tes, which is desirable for clinical applications.

In further studies of fetal human liver cells, it becameapparent that despite their capacity to proliferate overseveral monthsw76x, cells eventually began to showdecreasing replication, although terminal replicativesenescence was not observed, as assessed by senescence-associated beta galactosidase activity or p21 expression.However, the telomere length was found to shorten overtime in cultured cells. When the telomerase reversetranscriptase was introduced into fetal human liver cells,telomere length did not shorten and cells became‘immortal’ with extensive replication()300 populationdoublings compared with 30–50 population doublingsin non-transduced cells) w79x. These immortalized fetalhuman liver cells continued to express similar geneexpression profiles that were observed in parental cells,including liver genes, such as albumin and cytochromeP450s, and synthesized urea in cell culture. Moreover,cells engrafted in the liver and peritoneal cavity ofinununodeficient mice and differentiated into mature

hepatocytes. In additional studies, immortalized fetalliver cells were found capable of expressing insulinfollowing the introduction of Pdx-1 gene, which directspancreatic genesis, beta cell differentiation and insulinexpressionw80x. Under these conditions, cells began tosense glucose and released insulin appropriately. Fur-thermore, transplantation of Pdx-1 expressing immortal-ized cells corrected diabetes mellitus in streptozotocin-treated irnmunodeficient mice. These findings were inagreement with previous demonstrations of the potentialof Pdx-1-induced insulin expression in progenitor livercells and suggest that liver stemyprogenitor cells mayhave additional applications above and beyond thetreatment of liver disease.

Whether hematopoietic stem cells could be used fortherapeutic purposes has been of interest. Extramedul-lary hematopoiesis is a feature of the fetal liver andunder certain circumstances of the adult liver as well.Moreover, progenitor cells isolated from the livershowed presence of selected hematopoietic markers,which led to the initial demonstration in Dpp4-deficientrats of the capacity of bone marrow cells to generatehepatocytesw81x. Subsequently, these observations wereverified in the mouse, as well as humansw82–84x.Studies of hematopoeitic stem cell transplantation in theFAH mouse provided compelling findingsw84x. Trans-plantation of specific subsets of bone marrow cellscharacterized by c-kit and sca-1 antigen expression, lowexpression of Thy-1 antigen, and negative lineage mark-ers, produced extensive liver repopulation with maturehepatocytes and correction of tyrosinemia in the recipi-ents. However, the number of bone marrow-derivedhepatocyte foci was very small, although expansion ofthese foci was eventually therapeutic. However, in thenormal mouse liver and the liver of animals subjectedto a variety of perturbations, including other types ofacute or chronic injuries has not been as successful. Insome studies, only a handful of hepatocytes are thoughtto have originated from transplanted hematopoietic cellsw85x. Far more often, stromal cells or endothelial cellsin the liver were of hematopoietic origin. Also, studiesof bone marrow derived cells by two independentlaboratories using the FAH mouse have shown that thesecells actually produce hepatocytes by fusing with nativecells w86x. This process appears to be associated withpolyploidy and even aneuploidy, which raises concernsof oncogenic transformation, although serial transplan-tation of fused cells was not shown to have beenassociated with overt tumorigenesis. In studies of humancells transplanted into the mouse of gender mismatchedhuman tissue transplantation, bone marrow cells havebeen shown to produce hepatocytes without cell fusionw87x. Whether the liver of FAH mice offers greaterfusogenic conditions for bone marrow derived cells isunclear and studies in additional animal species will

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probably be necessary to better understand the potentialof stem cell plasticity.

6. Further considerations

In people, cells have typically been transplantedimmediately after isolation because it has been generallydifficult to recover human hepatocytes after cryopreser-vation. However, cell therapy will be greatly facilitatedby the development of cryopreservation techniques forlong-term storage of hepatocytes. One difficulty inoptimizing cryopreservation protocols for human hepa-tocytes concerns the quality of discarded donor livers,which is variable because such organs may be subopti-mal (e.g. fatty livers) to begin with or may deteriorateduring storage. While rodent, porcine, and even primatehepatocytes withstand cryopreservation, an importantdistinction between these cells and human liver cellsconcerns the ability to commence liver digestion in situin animals, whereas donor human livers must undergocold ischemia during organ preservation. For short-termstorage, modified University of Wisconsin solution hasbeen reported to maintain hepatocytes at 48C for a fewdays w88x. For longer-term preservation, storage belowy100 8C, such as in liquid nitrogen vapor, with incor-poration of cryoprotectants, including dimethylsulfoxide,serum or plasma, and proprietary mixtures containingnutrients, sources of energy, antioxidants, etc. have beeneffective to various degrees. A specific advantage of theability to cryopreserve hepatocytes is that cells can betested beforehand for their in vitro properties, as well ascapacity for engraftment in animals, before use inpeople.

Induction of tolerance toward allogeneic, and perhapsxenogeneic, hepatocytes offers another relevant chal-lenge. Although primary hepatocytes are thought toexpress class I and II histocompatibility antigens atrelatively low levels, this degree of antigen expressionis sufficient for allograft rejection with first order kinet-ics. Similarly, transplantation of xenogeneic liver cells,e.g. hamster hepatocytes into immunocompetent rats,leads to transplanted cell clearance over minutes tohours, as would be expected. In contrast, use of calci-neurin inhibitors has been effective in prolonging sur-vival of allografted cells. Similarly, thymic implantationof cells has been effective for inducing tolerance towardtransplanted hepatocytes in the absence of third-partygraft tolerance, as demonstrated for other organs as wellw89x. Nonetheless, the host immune response to alloge-neic hepatocytes appears to be different from thatelicited by solid organ allograftsw90,91x. For instance,allogeneic hepatocyte transplantation has been associat-ed with the activation of CD4q, as well as CD8q Tcell responses, implying the potential for additionalimmunosuppressive strategies. In particular, costimula-tory blockade seems to be helpful in prolonging allo-

geneic hepatocyte graft survival and offers furtheropportunities for achieving tolerance for cell therapyw92x. However, recent studies have shown that trans-plantation of porcine hepatocytes in terminally ill ratswith liver cirrhosis was not associated with the clearanceof transplanted cellsw49x. This raises issues concerningthe possibility of cell-specific displays of histocompati-bility antigens playing roles, e.g. it is known that stemyprogenitor liver cells have very weak expression of theseantigensw93x or of undefined recipient-specific mecha-nisms that promote xeno- or allograft tolerance. Furtherinsights into these areas will obviously be of majorsignificance in advancing cell therapy for people.

Acknowledgments

Supported in part by NIH grants R01 DK46952, P30DK41296, and M01 RR12248

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