Present status and perspectives of cell-based therapies for liver diseases

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Journal of Hepatology 45 (2006) 144–159

Review

Present status and perspectives of cell-based therapies for liver diseases

Andreas Nussler1,*, Sarah Konig2, Michael Ott3, Etienne Sokal4, Bruno Christ5,Wolfgang Thasler6, Marc Brulport7, Geredn Gabelein8, Wiebke Schormann7,

Maren Schulze9, Ewa Ellis10, Matthias Kraemer1, Frank Nocken1, Wolfgang Fleig5,Michael Manns3, Steven C. Strom10, Jan G. Hengstler7,*

1Fresenius Biotech Bad Homburg, Division of Cell Therapy, Bad Homburg, Germany2University of Goettingen, Department of General Surgery, Goettingen, Germany

3Medizinische Hochschule Hanover, Department of Gastroenterology, Hanover, Germany4Cliniques Universitaires St. Luc, Department of Pediatric Hepatology and Liver transplantation, Brussels, Belgium

5Martin-Luther-University Halle-Wittenberg, Division of Molecular Hepatology, Halle-Wittenberg, Germany6LMU Munich, Department General Surgery, Munich, Germany

7University of Leipzig, Center for Toxicology, University of Leipzig, Leipzig, Germany8University Medicine Berlin, Charite, Department of General Surgery and Transplantation, Berlin, Germany

9University Hospital Kiel, Division of Transplantation and Biotechnology, Kiel, Germany10University of Pittsburgh, Department of Pathology, Pittsburgh, USA

In recent years the interest in liver cell therapy has been increasing continuously, since the demand for whole liver trans-

plantations in human beings far outweighs the supply. From the clinical point of view, transplantation of hepatocytes or

hepatocyte-like cells may represent an alternative to orthotopic liver transplants in acute liver failure, for the correction

of genetic disorders resulting in metabolically deficient states, and for late stage liver disease such as cirrhosis. Althoughthe concept of cell therapy for various diseases of the liver is widely accepted, the practical approach in humans often

remains difficult. An international expert panel critically discussed the recent published data on clinical and experimental

hepatocyte transplantation and the possible role of stem cells in liver tissue repair. This paper aims to summarise the pres-

ent status of cell based therapies for liver diseases and to identify areas of future preclinical and clinical research.

� 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

1. Introduction

The progress made in the field of liver organ trans-plantation has revolutionized the treatment of a widespectrum of liver diseases. Nevertheless, cell-based ther-apies are emerging as an alternative to whole-organtransplantation. Hepatocyte transplantation has beenused to bridge patients to whole-organ transplantation[7,69], to decrease mortality in acute liver failure[18,58], and for treatment of metabolic liver disease[3,16,19,22,29,30,47,48,66,67,69]. Cell transplantation

0168-8278/$32.00 � 2006 European Association for the Study of the Liver.

doi:10.1016/j.jhep.2006.04.002

* Corresponding authors.E-mail addresses: [email protected]

(A. Nussler), [email protected] (J.G. Hengstler).

Available online 27 April 2006

is less invasive than whole-organ transplantation andcan be performed repeatedly. However, one major lim-itation of cell-based therapies for liver disease is theavailability of human hepatocytes. A wider use of thesetechniques will not be possible until adequate numbersof functional cells for transplantation become morereadily available. There are at least two possible sourcesthat could meet the needs for transplantation, namelystem and precursor cell derived hepatocyte-like cellsor reversibly replicating hepatocyte cell lines [36]. Inrecent years, numerous articles have reported aboutthe generation of liver cells or ‘hepatocyte-like cells’from different types of extrahepatic stem or precursorcells. At a first glance, this appears to provide excitingnew opportunities for cell therapy, as some types ofstem cells proliferate efficiently in vitro and therefore

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A. Nussler et al. / Journal of Hepatology 45 (2006) 144–159 145

may help to generate a larger supply of human hepato-cytes or precursor cells for transplantation. Withoutdoubt, the wide availability of human hepatocyteswould be considered a major breakthrough and mayopen new perspectives for the treatment of liver disease[60,68]. On the other hand, some studies presentingwith far–reaching conclusions with respect to the capac-ity of stem cell therapy have not yet been reproduced ormay have been interpreted in an over-optimistic man-ner. Here, we review the present status of cell therapiesfor liver diseases and discuss the most promising futurestrategies.

2. Which liver diseases are first line candidates for cell

therapy?

Three categories of liver disease can be distinguishedand principally addressed by liver cell therapy: (i) acuteliver failure, (ii) inherited metabolic liver disease and (iii)end-stage liver disease (cirrhosis). The conditions andrequirements of cell therapy differ for each of theseclasses.

2.1. Acute liver failure

Acute liver failure is characterised by rapid deteriora-tion of liver function and a high mortality. Viral hepati-tis, idiosyncratic drug reactions, acetaminophen andmushroom ingestion are common causes of acute liverfailure. Hepatic encephalopathy, brain edema, coagu-lopathy, septicemia and multi-organ failure are criticalkey events [44,61] during the course of the disease. Celltherapy of acute liver failure should provide rapid sup-port for the failing liver by providing metabolism of livertoxins, the secretion of proteins such as clotting factorsor albumin and stabilisation of haemodynamic parame-ters. In several studies, allogeneic primary hepatocytesisolated from cadaver livers were infused into the splenicartery or the portal vein [53,69,7]. Improvements inammonia levels encephalopathy scores, and prothrom-bin time levels were reported. Thus, the first studies per-formed in patients with acute liver failure demonstratedthe feasibility of hepatocyte transplantation and present-ed strong evidence for its therapeutic efficiency.

Due to the large capacity and accessibility of the per-itoneal cavity, intraperitoneal transplantation of hepato-cytes seems to be a promising alternative strategy tobridge life until the spontaneous regeneration of the liveroccurs. In a few patients with acute liver failure andgrade III encephalopathy fetal hepatocytes were injectedinto the peritoneal cavity to bridge patients to organrecovery. Improved survival rates were reported com-pared to historical controls [26]. Since hepatocytes inthe peritoneum survive only short term, alginate-embed-ded or microcarrier-attached hepatocytes seem to offer a

reasonable alternative. For instance, the transplantationof microcarrier-attached hepatocytes into ratssubjected to a 90% near total hepatectomy markedlyimproved long-term survival rates [14]. In contrast, thetransplantation of hepatocytes alone did not prolongsurvival.

Despite the positive results, it should be consideredthat the evaluation of therapies in acute liver failuremay be difficult. This is due to large variations in thecourse of the disease, multiple aetiologies, complex sup-portive treatment and a spontaneous recovery rate ofapproximately 20% by successful hepatic self-regenera-tion. Thus, the inclusion of adequate controls is difficult.In this respect, studies in patients with inherited meta-bolic diseases are easier to interpret.

2.2. Inherited metabolic liver disease

In comparison with acute liver failure, the course ofmetabolic liver disease usually varies less. In addition,objective parameters such as laboratory data (i.e. bileacid, clotting factors, etc.) can be determined tounequivocally assess the efficacy of the treatment. Onthe other hand, the situation is rarely immediately lifethreatening and often acceptable conventional therapiesare available. Therefore, the potential benefit must becarefully weighed against any possible complications,such as immunosuppression, hepatocyte embolisationof the pulmonary vascular system, sepsis or haemody-namic instability.

The results of hepatocyte transplantation for manymetabolic liver diseases have been encouraging (forreview, see: [8]). For instance, therapeutic benefit hasbeen reported in a girl with Crigler–Najjar SyndromeType I, which is a recessively inherited metabolic disor-der characterized by severe unconjugated hyperbilirubi-naemia [19]. Isolated hepatocytes were infused throughthe portal vein and partially corrected plasma bilirubinlevels for more than 11 months [19]. Similarly, a 9-year-old boy received 7.5 · 109 hepatocytes, infused viathe portal vein, which resulted in a decrease in bilirubinlevel from 530 ± 38 lmol/L (mean ± SD) before to359 ± 46 lmol/L [3]. Hughes et al. [30] also report a40% reduction in bilirubin levels in a Crigler–NajjarSyndrome Type I patient following transplantation withhepatocytes. Although these data demonstrate efficacyand safety, a single course of cell application seemsnot sufficient to correct Crigler–Najjar Syndrome TypeI completely.

Promising results have also been obtained in a 47-year-old woman suffering from glycogen storage diseasetype 1a, an inherited disorder of glucose metabolismresulting from mutations in the gene encoding the hepat-ic enzyme glucose-6-phosphatase [48]. 2 · 109 ABO-compatible hepatocytes were infused into the portalvein. Nine months after cell transplantation, her

146 A. Nussler et al. / Journal of Hepatology 45 (2006) 144–159

metabolic situation had clearly improved. Successfulhepatocyte transplantation has also been achieved in a4-year-old girl with infantile Refsum disease, an inbornerror of peroxysome metabolism, leading to increasedlevels of serum bile acids and the formation of abnormalbile acids [66]. A total of 2 · 109 hepatocytes from amale donor were given during eight separate intraportalinfusions. Abnormal bile acid production (for instancepipecholic acid) had decreased by 40% after 18 months.Recently, hepatocyte transplantation has been used suc-cessfully to treat inherited factor VII deficiency [16].Two brothers (aged 3 months and 3 years) received infu-sions of 1.1 and 2.2 · 109 ABO-matched hepatocytesinto the inferior mesenteric vein. Transplantation clearlyimproved the coagulation defect and decreased thenecessity for exogenous factor VII to approximately20% of that prior to cell therapy. As with the other met-abolic liver diseases, hepatocyte transplantation hasbeen shown to provide a partial correction of urea cycledefects. Patients showed clinical improvement, reducedammonia levels and increased production of urea[29,47,67,70]. Some hepatocyte transplantation studieswith inherited metabolic liver disease have been summa-rized in Table 1.

Relatively little is known about long-term engraft-ment of the transplanted hepatocytes. An importantobservation of [16] is an increased requirement forrecombinant factor VII six months after cell therapy,suggesting loss or decreased function of the transplantedhepatocytes. Therefore, it will be important to obtainfurther information whether loss of transplanted alloge-neic hepatocytes is inevitable or a consequence of subop-timal immunosuppression.

With respect to long-term engraftment it will beimportant whether the transplanted hepatocytes willgain a selection advantage over the recipient’s cells.Therefore, it could be reasonable to differentiatebetween two groups of metabolic liver diseases. A typi-cal representative of group 1 is inherited clotting factor

Table 1

Overview over some hepatocyte transplantation studies in patients with inherit

Recipient No. of trhepatocyDisease Sex Age

a1-Antitrypsin deficiency Male 18 wFemale 52 y 2.2 · 106

Crigler–Najjar I Male 10 y 7.5 · 109

Ornithine transcarbamylase deficiency Male 5 y 1 · 109

Ornithine transcarbamylase deficiency Male 10 h? 4.5 · 109

Glycogen storage disease type Ia Female 46 y 2 · 109

Refsum disease Female 4 y 2 · 109

Factor VII deficiency Male 3 m 1.1 · 109

Male 2 y, 11 m 2.2 · 109

a OLT, orthotopic liver transplantation.

deficiencies. Secretion of clotting factors is important forthe organism, but deficiency does not influence survivalof hepatocytes. Therefore, a selection advantage of thetransplanted hepatocytes cannot be expected. The situa-tion is different for group 2 metabolic liver diseases. Atypical example is Wilson’s disease that is caused by adefect in the copper transporting ATPase ATP7B pro-tein. As a consequence of the gene defect copper willaccumulate and lead to deterioration of hepatocytes[25]. In this case transplanted wild-type cells may havea selection advantage over the recipient’s hepatocytes.Therefore, higher numbers of transplanted hepatocytes,better cell engraftment and perhaps repeated transplan-tations may be necessary for the treatment of group 1metabolic liver diseases. However, clinical data withrespect to possible differences in long-term engraftbetween group 1 and 2 are not yet available.

2.3. End-stage liver disease (cirrhosis)

Cell therapy of end-stage liver disease is more prob-lematic. Besides loss of functional hepatocytes abnor-malities of the hepatic architecture contribute to thedecrease in liver function. Intrahepatic portal-to-portalvenous shunts may prevent an efficient exchangebetween hepatocytes and blood plasma. In this situa-tion, the benefit of additionally transplanted hepatocytesinto the liver without restoring the normal liver architec-ture may be questionable. An alternative strategy maybe the transplantation of hepatocytes into other sitese.g. the spleen, peritoneum or omentum to support met-abolic function and regeneration. To evaluate theefficiency of intrasplenic transplantation severalresearchers induced liver cirrhosis in rats using pheno-barbital and carbon tetrachloride followed by directinjection of cells into the splenic pulp [9,35,54]. Only ani-mals with stable liver cirrhosis four weeks after the dis-continuation of carbon tetrachloride were subjected tocell therapy. With different cell types applied, namely

ed metabolic liver disease

ansplantationtes

Route Outcome Ref.

Portal OLTa (day 2) [69]OLTa (day 4)

Portal OLTa (3.5 years) [19]Portal Normal ammonia level within 48 h;

died after 43 days[51]

Portal Normal protein intake possible;OLTa (6 months)

[29]

Portal Improved for 3 years [48]Portal Improved for 1 year [66]Portal Improved and decreased requirement

for recombinant factor VII[16]

Portal


rat or porcine hepatocytes [54], syngeneic rat hepato-cytes [35] or immortalized rat hepatocytes [9] intrasplen-ic cell therapy clearly improved liver function andprolonged survival.

The response to hepatocyte transplantation inhumans with end-stage liver disease, however, has notresulted in the same degree of improvement comparedto experimental animal studies [46,51]. One explanationmay be that the hepatocytes in clinical studies weredelivered into the splenic artery and not into the splenicpulp. This view is supported by Nagata and colleagues,who have shown that the route of hepatocyte deliveryinfluences hepatocyte engraftment and function [55].Another open question remains as to whether thehuman spleen is capable of accommodating a sufficientnumber of functional hepatocytes to compensate forthe cirrhotic liver. From the immunological point ofview the spleen represents the ‘lion’s den’, where trans-planted cells could possibly cause greater immuneresponse than in most other ectopic transplantationsites. Because of its large capacity and accessibility, theperitoneal cavity is an alternative site for the cell therapyof end-stage liver disease. However, since hepatocytesuspensions do not survive for longer periods onlyshort-term effects may be achieved. Application of cellsin patients with decompensated chronic liver disease hasresulted in some improvement of laboratory parameters,but was not able to change the natural cause of thedisease (Ott M., unpublished). Encapsulation of hepato-cytes [4,62] or their attachment to microcarriers may bean alternative method to improve efficacy of intraperito-neal cell transplantation. Implantable hepatocyte-baseddevices may represent another alternative for the treat-ment of end-stage liver disease [10]. However, theseapproaches are conceptual and still far from clinicalapplication.

2.4. Route of administration

In metabolic liver disease and acute liver failure theliver architecture is usually intact. In this case the infu-sion of cells into the portal vein or into the inferior mes-enteric vein is adequate. It is known from animal studiesthat infused hepatocytes disperse with the portal bloodflow and finally translocate to the hepatic sinusoids inthe periportal region of the liver lobules [37]. Single cellssucceed in traversing the endothelial barrier and inte-grate into the parenchyma (Fig. 1). After re-establishingintercellular contacts with neighbouring host cells,transplanted hepatocytes start to proliferate. Donorcells and their descendents form gradually increasingclusters, thus finally repopulating the recipient liver(Fig. 1). While monitoring the portal pressure in a4-year-old girl following portal infusion of hepatocytes,a temporary increase was noted, with a return to pre-in-fusion levels shortly after the infusion [66].

The capacity of the liver to incorporate transplantedhepatocytes can be enhanced for instance by ischaemia.In low-density lipoprotein receptor deficient Watanaberabbits, transient ischaemia reperfusion injury of therecipient liver has been shown to improve the therapeu-tic efficacy of allogeneic intraportal hepatocyte trans-plantation [5]. This technique may offer a perspectiveto improve the engraftment of transplanted hepatocytesin patients with metabolic liver disease.

When the liver architecture is deranged, cell infu-sions may cause prolonged portal hypertension andembolization in the lung [71]. Therefore, ectopic sitesfor hepatocyte engraftment are needed. The best stud-ied ectopic site is the spleen. A study in pigs has shownthat direct intrasplenic injection produced engraftmentthat was far superior to that obtained using splenicartery infusion [55]. Splenic artery infusion causedsplenic necrosis due to vascular occlusion with hepato-cytes. In contrast, injection into the splenic pulp wastolerated much better and was associated with onlyclinically insignificant intraabdominal haemorrhage.Therefore, direct intrasplenic injection may be a feasi-ble strategy to support patients with deranged liverarchitecture. Whether the number of engrafted cellsand their metabolic capacity will be sufficient remainsan open question.

In several animal studies cells were directly injectedinto the liver tissue [74,63,6] to avoid the requirementof homing. Using this technique, the transplanted cellswere observed in the liver tissue, but also in central veinsindicating an increased risk of embolization to the lung.Therefore, direct injection of cells into the liver may beused in basic research but not in the clinical setting.The kidney capsule represents a further potential sitefor ectopic hepatocyte transplantation. Althoughengraftment was demonstrated the renal subcapsularspace will accommodate only relatively small numbersof hepatocytes.

2.5. Which would be the most effective

immunosuppression?

Islet cell transplantation is a well-established clinicaloption in the therapy of diabetes. Transplantation ofislet cells is usually performed transcutaneously intothe intrahepatic portal vein system. Therefore, it repre-sents the circumstances of liver cell therapy at least con-cerning cell administration and may serve as apioneering example for cell-based therapies. Neverthe-less, the clinical outcome of islet cell transplantationwith a basal c-peptide >0.5 ng/ml, insulin independenceof less than 40% after one year (www.med.uni-gies-sen.de/itr) cannot reach the success of whole-organtransplantation of the pancreas with 70% insulin inde-pendence one year after transplantation (www.iptr.umd.edu). However, optimization of immunosuppression

http://www.med.uni-giessen.de/itr

http://www.med.uni-giessen.de/itr

http://www.iptr.umd.edu

http://www.iptr.umd.edu

A B

C D

Fig. 1. Transplantation of adult hepatocytes and liver repopulation. Primary hepatocytes were injected into the portal vein of dipeptidylpeptidase IV

(DPPIV) deficient rats pre-treated with retrorsine and subjected to 30% partial hepatectomy to ensure selective donor growth. Donor cell integration and

repopulation was studied by co-localising transplanted cells (DPPIV-positive = red) with hepatic gap junctions (Connexin-32 = green) to visualize the

intact hepatic architecture, cell nuclei (DAPI = blue). Multilayer immunofluorescence imaging, magnification as indicated). (A) Shortly after

transplantation (4 h), donor cells form microemboli in the distal branches of the portal vein and translocate further to the sinusoids. (B) From day 3

onwards, donor cells are fully integrated and display re-established gap junctions with neighbouring cells. (C) Donor cells and descendents form clusters

and grow out from the periportal areas (21 days). (D) Two months following transplantation, donor cell-derived cell clusters are nearly confluent and

range up to 200 cells in number. The integration process is remarkably homogeneous and the architecture of the parenchyma is perfectly maintained

without any sign of displacement.


substantially improved the clinical outcome of islet celltransplantation [17]. Therefore, it can be expected thatthe immunosuppressive regimen also plays a major rolein cell based therapies for liver diseases. Inhibition ofliver resident natural killer cells by specific or localimmunosuppression could additionally improve engraft-ment and proliferation of transplanted cells [20].

The ultimate goal would be an effective immunosup-pression with as little side effects as possible. The mostcommon immunosuppressive protocols consist of calci-neurin inhibitors (CNI, tacrolimus or cyclosporine) withor without steroids in combination with induction ther-apy such as IL-2 receptor antibodies (e.g., basiliximab)or anti-thymocyte globulins (e.g., ATG Fresenius orThymoglobulin). Depending on the underlying diseaseand existing comorbidities of the patient, immunosup-pressive regimens without steroids or reduced doses ofCNI are favoured. The reduction or even removal ofCNI may be achieved by the addition of other immuno-suppressive drugs such as mycophenolate mofetil(MMF) or sirolimus. Since there are few data aboutimmunosuppression after cell transplantation and no

specific protocol can be recommended for these patientsso far, it is advised to use the standard immunosuppres-sive protocol of each respective centre. However, itshould be taken into account that the immunosuppres-sive requirements after cell transplantation may be lowerthan after solid organ transplantation. Encapsulation ofthe transplanted cells reduces their immunogenicity.This may not only prolong survival of the transplantedcells, but may also allow for less immunosuppression ofthe recipient.

2.6. Number of cells and frequency of administration

The human liver consists of approximately 250 · 109

cells that are organized in about 106 hepatic lobuleseach containing approximately 250,000 liver cells.Hepatocytes constitute approximately 65–70% of thecell population of the liver. Therefore, the total numberof hepatocytes in a human liver is approximately175 · 109. As a rule of thumb transplantation of anumber of hepatocytes corresponding to 1–5% of totalliver mass (1.8–8.8 · 109 hepatocytes) can be expected


to have a positive impact. Although a number as highas 8.8 · 109 hepatocytes is an ambitious goal it seemsdesirable to transplant such high numbers of cells. Inseveral published cases lower numbers of hepatocyteswere transplanted, which may explain lack or marginalclinical benefit. About 5% of liver mass correspondingto approximately 8.8 billion hepatocytes can safely betransplanted in one transplant event, whereby thetransplant may be divided into about 5–6 separateinfusions over a number of hours. When portal pres-sures return to normal, or at least decrease to accept-able levels, it is safe to infuse more cells. It mighteven be useful to transplant 5% of liver mass as severalseparate transplants over a prolonged period of time(several weeks to months) so that ultimately one couldtransplant 15–20% of total liver mass at 4 separatesessions.

2.7. Limited availability of hepatocytes: the major hurdle

of liver cell therapy

Already in 1977 hepatocyte transplantation has beenrecognized as an attractive option for the managementof metabolic liver disease [23]. Groth and colleaguesdemonstrated that intraportal hepatocyte transplanta-tion in glucuronosyltransferase-deficient rats improvedhyperbilirubinaemia. Up to the present, most of thepublished articles have reported a positive impact ofhepatocyte transplantation also in human studies.Despite the positive reports application of hepatocytetransplantation in humans is limited to less than 100cases. The reason for this discrepancy is the success oforthotopic liver transplantation and limited availabilityof human hepatocytes. It is reasonable to use all avail-able donor livers for organ transplantation. Thenumbers and/or quality of hepatocytes isolated fromnon-transplantable livers will not allow a widespreadapplication of hepatocyte transplantation. This situationwill remain unaltered unless an alternative to primaryhepatocytes is available. As soon as an easily availablecell type equivalent to primary hepatocytes will be onhand, treatment of acute liver failure, acquired or mono-genetic metabolic liver diseases and perhaps also of end-stage liver disease will be revolutionized within the nearfuture.

2.8. Are human stem and precursor cells promising

candidates for cell therapy of human liver diseases?

Since 1999, numerous articles have reported the gener-ation of hepatocyte-like cells from different types of extra-hepatic stem or precursor cells (review: [27]). At a firstglance this seems to open exciting new opportunities forcell therapy. However, after a careful evaluation of pub-lished preclinical studies and also considering our ownresults we concluded that preclinical data are not yet suf-

ficient to justify clinical studies. Further informationabout the generated hepatocyte-like cells is needed andso are reproducible results in preclinical animal modelsof human liver disease. This will be illustrated in the nexttwo paragraphs where we will critically discuss recentpublications about differentiation of extrahepatic stemor precursor cells to hepatocyte-like cells, either followingtransplantation into animal livers or in vitro followingcytokine and/or liver extract exposures.

3. Experimental evidence for hepatocyte formation from

extrahepatic stem and precursor cells

3.1. Formation of hepatocyte-like cells from extrahepatic

stem cells in vivo

First publications have shown that haematopoieticstem cells were capable of differentiating into hepato-cytes and cholangiocytes yielding in a high degree ofengraftment within injured rodent livers. Petersen et al.[59] used three approaches to demonstrate that bonemarrow cells contribute to liver cells. First, female ratswere lethally irradiated and transplanted with bone mar-row from a male rat. Engrafted females were treatedwith CCl4 and 2-acetylaminofluorene (2-AAF) to simul-taneously induce hepatotoxicity and block endogenoushepatocyte proliferation. Under these conditions,Y-chromosome-positive hepatocytes were observed inthe female recipients. Second, using the same protocolbone marrow cells from dipeptidylpeptidase IV(DPPIV)+ F-344 male rats were injected into DPPIV�F-344 female rats, resulting in DPPIV expression inbile canalicular sites between hepatocytes of theDPPIV� F-344 recipients. Finally, livers from Lewisrats expressing the major histocompatibility complexclass II L21-6 isozyme were transplanted into Brown–Norway rats that do not express L21-6. After theCCl4/2-AAF protocol the recipients (Lewis rats) showedpositive L21-6 staining in livers. Similar results wereobtained in further studies using also different animalmodels and purified cell types for transplantation (forreview: see [27]). However, marker expression of trans-planted extrahepatic cells in combination with hepato-cellular factors does not automatically imply that thetransplanted cells show transdifferentiation into truehepatocytes, but could more likely be induced by cellfusion. Regardless of the underlying mechanisms, in vivogeneration of hepatocytes from bone marrow has onefundamental requirement: the host bone marrow hasto be reconstituted by transplanted bone marrow inadvance. From a clinical point of view, this requirementmay be considered as an impossible option for manypatients suffering from severe hepatic disease.

The promising results obtained after transplantationof extrahepatic rodent cells into livers of rodents stim-

Table 2

Transplantation of human stem and precursor cells into livers of experimental animals: summary of studies

Human cell type Route and number oftransplanted cells

Liver injury Observations Recipient mice (m),sheep (s), goat (g)

Ref.

Adherently proliferatingcells from cord blood

Injection of 2 · 105 cellsdirectly into the livertissue

None Expression of human albumin 7and 21 days after transplantation(IHC, RT-PCR), but noexpression of a-fetoprotein andGATA4 (RT-PCR)

m [6]

Lin�CD38�CD34�ClqRp+

and Lin�CD38�D34+

ClqRp+ cells isolated from

cord blood and bonemarrow

Tail vein injectionof 500 to 7 · 104 cells

3.75 Gy Expression of HepPar1 antigenand human c-met 8–10 weeksafter transplantation (IHC),human albumin (RT-PCR).Mouse liver suspensions showeda rare population of humanHLA- ABC+ and CD45� cells(flow cytometry)

m [13]

Unsorted mononuclear cellpreparations from humancord blood

Tail vein infusion of50 · 106 cells

2.5 Gy Expression of the HepPar1antigen (IHC) in livers 4, 6 and16 weeks after transplantationinto SCID/NOD mice. Noevidence for cell fusion (FISH)

m [56]

CD34+ or CD34+, CD38�,CD7� cells isolated byfrom cord blood

Tail vein injection of 2000CD34+ or 1 · 105 CD34+,CD38�, CD7� cells

3 Gy CCl4 Expression of human albumin(WB, RT-PCR, IHC) 5 and 30days after liver injury. PositiveRT-PCR for CK19. Negativeresults without CCl4 inducedliver injury

m [76]

CD34+ cells isolated fromcord blood and fromperipheral blood

Tail vein injection of2 · 105 cells

3.75 Gy CCl4 Human albumin positive cellspreferentially around bile ducts(IHC, RT-PCR, WB).Neutralization of the SDF-1receptor CXCR4 abolishedhoming of human stem cells tothe mouse liver, whereas localinjection of SDF-1 into themouse liver increased homing

m [39]

Adherently proliferating cellsfrom cord blood

Injection of 10 · 106 cellsinto the portal vein

2-AAF andone.thirdhepatectomy

Expression of human albumin(RT-PCR, IHC) and HepPar1antigen’’ (IHC) in liver, humanalbumin detection in serum (WB)and detection of human Xchromosome centromers (FISH)

m [33]

CD34+ or CD45+ cells fromcord blood


5-Fluorouraciland anti-mousec-kit

Expression of human albumin(RT-PCR), HepPar1 antigen(IHC) and human centromers(FISH)

m [31]

Nestin-positive islet-derivedadherently proliferatingprecursor cells

Injection of 0.15, 1.5,and 7.5 · 105 directlyinto the liver tissue

None Expression of human albuminbut not of mouse albumin inindividual cells (IHC, RT-PCR)3 and 12 weeks aftertransplantation. Negative resultsafter injection of 0.15 · 105 cells,but positive for 1.5 and 7.5 · 105

injected cells

m [74]

CD34+ cells isolated fromcord blood

3–5 · 105 for intra-fetaland 15–20 cells for intra-blastocyst injection

None Expression of human albumin,HepPar1 antigen, and humana1-antitrypsin (IHC, RT-PCR)1 and 4 weeks after birth

m [72]


Table 2 (continued)

Human cell type Route and number oftransplanted cells

Liver injury Observations Recipient mice (m),sheep (s), goat (g)

Ref.

CD34+Lin�CD38� cellsfrom human bonemarrow, cord blood andmobilized peripheral blood

Intra-fetal injection of2 · 104 cells

None Expression of human albumin(IHC, ELISA), ‘‘humanhepatocyte antigen’’ (IHC) anddetection of Alu-sequences (FISH)

s [1]


Intra-fetal injectionof 1500 cells in sheep

None Expression of human albuminand ‘‘human hepatocyte specificantigen’’ in livers of sheep(IHC, WB)

s [38]

Human blood monocytederived cells

Injection of 7.5 · 105

cells directly into theliver tissue

None Expression of human albuminbut not of mouse albumin inindividual cells (IHC) 3 weeksafter transplantation

m [63]


Tail vain injection of5 · 104 cells

Fas-ligand 1.5Gy

Expression of human albuminand HepPar1(IHC) as well asalbumin, a-fetoprotein,glutamine synthetase andtransferrin (RT-PCR)

m [57]

CD34+lin� cells derivedfrom cord blood

Intra-fetal injection None Expression of human-specificserum albumin and hHNF-3bmRNA

g [78]

Unsorted mononuclearcell preparations fromhuman cord blood


2.5 Gy Expression of human albumin,but no expression of humanCK18 (IHC, RT-PCR) 4 weeksafter induction of liver injury and8 weeks after cell transplantation

m [64]


ulated a relatively large number of independent groupsto study the fate of different types of human stem andprecursor cells in livers of experimental animals (Table2). Without doubt differentiation of human stem cellsto genuine hepatocytes or even to liver tissue wouldbe an enormous progress with high clinical relevance.In all experiments published so far a similar strategywas used. Extrahepatic stem cells were introduced intothe liver of experimental animals by different routes(Table 2). After periods usually ranging between 3weeks and 6 months expression of human hepatocytemarkers were analysed by immunohistochemistry,RT-PCR or in situ hybridization. Although differenthuman cell types, routes of injection and recipients(mice, sheep, goat) have been tested, similar resultswere obtained. In 14 of the 15 published studies(Table 2) expression of human albumin was observedin the recipients. In one of the studies [56] listed inTable 2, human albumin was not analysed, but humanspecific antigen (HepPar1) was detected. In most ofthese studies the positive immunostaining data havebeen confirmed by RT-PCR analysis (Table 2). Twostudies included analysis of cell fusion and did notfind evidence for this mechanism [56,38]. Other param-eters relevant for hepatocytes, including activities ofdrug metabolizing enzymes, clotting factors andcomplement, were not yet tested in these experiments(Table 2).

Without doubt, the observation of human albumin-or HepPar1-positive cells in livers of animals aftertransplantation of human stem cells is intriguing. Never-theless, it is still questionable if these cells representgenuine human hepatocytes that could take over allhepatocellular functions. Although the data obtainedby several groups are comparable (Table 2) the interpre-tation and discussion remain controversial. Manyauthors tend to give an optimistic view. For instance[38] transplanted adherently proliferating cells isolatedfrom human cord blood into livers of fetal sheep. Theauthors observed expression of albumin and humanhepatocyte-specific antigen after transplantation andconcluded that the human cord blood cells differentiatedto human parenchymal hepatic cells. Newsome demon-strated expression of the HepPar1 human hepatocyte-specific antigen, and concluded that cells from humancord blood ‘become mature hepatocytes’ in livers ofSCID/NOD mice [56]. Ishikawa detected human albu-min and the HepPar1 antigen in livers of immunodefi-cient mice, postulating that the engrafted cells fromhuman cord blood ‘functioned as hepatocytes’ [31].Considering these interpretations one might ask whythese cells are not yet used in the clinical setting.

However, some challenges may have been underesti-mated. In addition there is reason to suspect publicationbias. At least in recent years a simple interpretation ofan albumin- and/or HepPar1-positive stem cell derived


cell type as ‘‘hepatocyte’’ could be published easier thana ‘‘problematic’’ study about intermediate cell typesexpressing some but not all hepatocellular markers. Itshould also be considered that bone marrow-derivedcells as a source for hepatocyte regeneration have alsobeen critically discussed. Cantz et al. [11] investigatedthe contribution of intrasplenic bone marrow trans-plants or in vivo mobilized haematopoietic stem cellsto the formation of hepatocytes in normal and injuredliver. Direct intrasplenic injections of bone marrowmononuclear cells, Scal+/lin� haematopoietic stem cellsand highly purified ‘‘side population’’ hematopoieticstem cells derived from enhanced green fluorescent pro-tein (EGFP)-transgenic mice were performed in normalC57Bl/6 mice and in C57Bl/6 mice following two-thirdshepatectomy. The results demonstrate that there is littleor no contribution of bone marrow-derived cells to theregeneration of normal and injured liver in the animalmodels used [11]. Since a realistic assessment is crucialfor further progress in this field we will focus on possiblelimitations and problems in the next paragraphs. Itshould be considered that albumin expression has beenobserved in most published studies. However, expres-sion of albumin was not automatically associated withexpression of further hepatocyte markers. For instanceGATA4, a-fetoprotein and CYP3A4 [6] as well as cyto-keratin 18 and DPPIV [64] were negative despite posi-

Fig. 2. Morphology of extrahepatic stem cells after transplantation into liver

transplantation of purified human Lin�CD38�CD34�or+C1qRp+ cells isolated

recovered 8–10 weeks posttransplant and immunostained for human albumi

mononuclear cell preparations of human cord blood and immunostaining for He

from human umbilical cord blood and immunostaining for human albumin. Ma

isolated from human cord blood. Immunostaining for human albumin. Magn

proliferating cells isolated from human cord blood. Immunostaining for HepPar

islet-derived adherently proliferating cells. Immunostaining for human albumin.

positive islet-derived adherently proliferating cells. Immunostaining for human

et al. [63]: transplantation of primary human hepatocytes (left side) and human

for albumin. Bar: 50 lm. (I) Sharma et al. [64]: transplantation of unsorted hu

tive staining for human albumin. Thus, mixed orchimeric cell types may occur after stem or precursor celltransplantation. Their impact on liver physiologyremains unexplored.

3.1.1. Single cells but no tissue formationTypically single cells or small clusters of hepatocyte-

like cells were observed after transplantation of thehuman stem and precursor cells into mouse livers(Fig. 2). However, no human liver tissue formationcould be observed in the mouse livers (all mouse studiesin Table 2). In contrast ‘more than 20% albumin-pro-ducing human parenchymal hepatic cells’ have beenreported after transplantation of adherently proliferat-ing cord blood cells into fetal sheep [38]. This is surpris-ing and requires independent confirmation beforefurther conclusions can be drawn. Thus, formation ofhuman liver tissue after transplantation of human stemcells into animal models has not yet been demonstrated(see Fig. 3).

3.1.2. No functional improvement reportedMany animal models of liver disease are available and

seem to be adequate for preclinical studies (Table 3).Usually clear criteria are available to evaluate thesuccess of therapy, such as liver copper content for theATP7B-deficient mouse [21] or clotting factor VIII in

s of immunodeficient mice from published studies. (A) Danet et al. [13]:

from human umbilical cord blood. Livers from transplanted mice were

n. Bar: 10 lm. (B) Newsome et al. [56]: transplantation of unsorted

pPar1. Bar: 20 lm. (C) Wang et al. [76]: transplantation of CD34+ cells

gnification: 100·. (D) Kollet et al. [39]: transplantation of CD34+ cells

ification: 100·. (E) Kakinuma et al. [33]: transplantation of adherently

1. Bar: 10 lm. (F) Von Mach et al. [74]: transplantation of nestin-positive

Magnification: 400·. (G) Von Mach et al. [74]: transplantation of nestin-

(red) and mouse (green) albumin. Magnification: 630-fold. (H) Ruhnke

blood monocyte derived hepatocyte-like cells (right side). Immunostaining

man cord blood cells. Immunostaining for albumin. Magnification: 40·.

Fig. 3. Heterogeneity of human albumin positive cell types. Adherently

proliferating cord blood cells derived from a single colony were directly

injected into the left liver lobe of SCID/NOD mice followed by

immunohistochemistry visualizing human albumin 3 weeks after trans-

plantation. (A) Human albumin positive cells with hepatocyte-like

morphology. Magnification: 200·. (B) Human albumin positive cells that

do not show a hepatocyte like morphology, but rather resemble monocytes

Magnification: 400·. (Hengstler and Brulport, unpublished data).


a factor VIII-deficient mouse [42]. Several of thesemouse models have been evaluated by transplantationof primary hepatocytes or precursors. For instanceintrasplenic transplantation of embryonic hepatocytesisolated from 14-day fetal mouse livers into a mousemodel of Wilson’s disease reduced toxic copper accumu-lation [65]. However, to our knowledge an improvementof liver function in these mouse models (Table 3) by celltherapy with human extrahepatic stem cells has not yetbeen reported.

3.1.3. The wrong strategy?

Many scientists transplanted human stem cells intolivers of immunodeficient mice to study the differentia-tion capacity. An advantage of this strategy is the gen-uine liver microenvironment which is difficult toimitate in vitro. Many human growth and differentia-tion factors are known to be efficient also in humanhepatocytes (and vice versa). Therefore, it is notimplausible to assume that the mouse liver microenvi-ronment may promote differentiation also of humanstem cells to hepatocytes. However, when negativeresults or, for instance, mixed cell types are observedit is not clear, whether this is due to limitations ofthe specific stem cell type used or to interspeciesincompatibilities.

To avoid this dilemma an alternative strategy mightbe applied. When possible, identical types of stem orprecursor cells should be isolated from human andmouse tissue and should be tested in the same mousemodel. The worst case scenario is that both, mouseand human cell types, fail to improve liver function. Inthis case clearly the limited differentiation capacity ofthe tested stem cell type is responsible for the negativeresult. However, if the mouse cells improve liver func-tion, whereas the respective human cells remain nega-tive, further, more adequate models must be appliedfor the human cells. To our knowledge this strategy(comparing human and allogeneic stem cells in the sameanimal model) has not yet been performed.

On the other hand, studies are available whererodent stem cells have been transplanted into rodents.A well-known example has been published by Janget al. [79], who used a relatively complex method ofisolating haematopoietic stem cells including threesteps: (i) isolation of a small-sized cell population frommale C57Bl6 mice by counter-flow elutriation of bonemarrow cells; (ii) depletion of lineage-positive cells;and (iii) labelling of the resulting cell fraction withthe red fluorescence dye PKH26 and isolation afterinjection into lethally irradiated female C57Bl6 mice.These cells were intravenously injected into CCl4 pre-treated mice (100,000 cells per animal). The authorsreport that liver function was restored already 2–7 daysafter transplantation. For instance, mean fibrinogenplasma concentrations were reported to be 129 mg/dlin mice two days after treatment with CCl4 comparedto 252 mg/dl in mice two days after treatment withCCl4 plus stem cells. Negative control cells have notbeen transplanted in this study, which would be impor-tant to prove an advantage of the rigorously purifiedcell fraction. In addition studies in wild-type rodent liv-ers may be difficult, due to the extremely fast spontane-ous regeneration. Thus, the data of Jang et al. [79] arepromising, but confirmation by independent groupsand evaluation in further animal models (for instance,those suggested in Table 3) are needed.

Table 3

Animal models of inherited liver disease adequate for evaluating functionality of transplanted stem and precursor cells

Name/disease Defective gene Ref.

Toxic milk mouse, Wilson disease ATP7B [25]Gunn rat, Crigler–Najjar type I UDP-glucuronosyltransferase 1A1 (UGT1A1) [50]Nagase analbuminemic rats Mutation of the 5 0 splice site of the intron HI leads to skipping

of the albumin exon H[52,28]

Watanabe hyperlipidemic rabbit, familial hypercholesterolemia Deficiency of the low density lipoprotein receptor gene(in frame deletion in the ligand binding domain)

[12,43]

mdr2-knockout mouse, familial intrahepatic cholestasis (PFIC)type 3

mdr2-knockout [15]

FAH mouse, hereditary tyrosinaemia type I Fumarylacetoacetate hydrolase (FAH) deficient [75,73]Spf(ash) mouse, ornithine transcarbamylase (OTC) deficiency Inactivation of the spf(ash) gene (causing OTC deficiency) [34]FVII-deficient mice Replacement of exons 2–8 of the FVII gene with a neomycin

phosphotransferase (neo) gene[49]

FVIII-deficient mice, haemophilia A FVIII knockout mice [40]


We also noticed that human stem cells have not yetbeen tested in livers of pigs, which seems to be mandato-ry. The rodent liver is capable of extremely rapid regen-eration, which is much more efficient compared to pigand human. Therefore, the pig liver may represent amore realistic model with respect to human liver regen-eration. In addition size and anatomical structure of piglivers more closely resemble the human situation thanrodent livers. This will also be important for evaluationof cell application techniques and adverse effects.

3.1.4. Which stem and precursor cell types are most

promising?

Based on the available data it is difficult to com-pare the different human cell types with respect oftheir capacity to differentiate to hepatocytes in vivo.Human cell types studied so far are (references inTable 2): (i) adherently proliferating cells from humancord blood. These cells are isolated from the mononu-clear cell fraction by adhesion to culture dishes andrepeated passaging in culture after trypsinization toremove monocyte contaminations. These cells have afibroblast-like morphology and proliferate for at leastten passages with 1:5 splitting. (ii) The mononuclearcell fraction from cord blood, either as a crude frac-tion or after FACS sorting for markers, such asCD34. These cells differ from the adherently prolifer-ating cord blood cells because they do not adhere tocell culture vessels and (usually) do not proliferatein vitro. (iii) Haematopoietic cells from bone marrow,isolated by FACS sorting for well-established markers[32]. As well, these cells do not adhere to culture dish-es and cannot be multiplied in vitro by proliferation.(iv) Nestin-positive pancreatic islet cells. These cellsare isolated after outgrowth from pancreatic isletsin vitro and selected for high nestin expression. Thesecells have a fibroblast-like morphology and proliferatewell in vitro. (v) Hepatocyte-like cells isolated fromperipheral blood monocytes by a two-step dedifferenti-ation/differentiation in vitro protocol. After differenti-

ation the cells stop proliferating and adopt ahepatocyte-like morphology. (vi) Amniotic epithelialcells that develop from the epiblast by 8 days after fer-tilization and have the capacity to differentiate tohepatocyte-like cells [45]. Of course many more prom-ising extrahepatic stem and precursor cell types areavailable. To our knowledge these stem and precursorcells have not yet been tested for differentiation intohepatocytes in vivo. As mentioned above it is difficultto compare the capacity of different human cell types.Most scientists have concentrated on a specific celltype. Comparative in vivo studies of several humanstem or precursor cell types to evaluate hepatocellulardifferentiation capacity under standardized conditionsare not available. However, concerning the publishedstudies in which human cells have been transplantedinto mice, the results appear remarkably similar forthe different transplanted cell types. Almost all authorsobserved albumin and/or HepPar1-positive single cellsor small cell clusters, but no tissue formation (Table2). However, as discussed above it is not yet clear,whether this reflects the true differentiation capacityof the cells or a limitation of the mouse model.

With respect to the implementation of cell therapy,there are clear differences between the above-mentionedcell types. An advantage of the adherently proliferatingcells is the availability of large amounts of cells. Howev-er, especially for the proliferating cell types it is crucialto exclude malignant transformation after transplanta-tion. An advantage of the monocyte derived hepato-cyte-like cells [63] is the opportunity to generate thesecells from the recipient’s own blood, thus avoidingimmune suppressive medication.

3.1.5. What are the requirements for clinicians to use

hepatocyte-like cells in patients?

Several requirements have to be considered to be metin order to use hepatocyte-like cells in patients. Cellsmust be produced following GMP criteria in order toensure safety (transmission of infections such as hepati-


tis), stability and continuous quality of the graft.Engraftment, survival and functionality of the cells arecertainly important factors, since they predict the fre-quency of recurrent treatments and thereby directlyinfluence therapy success and costs. Cryopreservationof the cells is mandatory in order to ensure permanentavailability in regard to the distribution within the clin-ical centres. Comparable to solid organ transplantationprocedures, the transplanted cells should carry the sameABO antigens as the recipient. Therefore, cells of thevarious blood groups should be kept on stock.

4. Cell fusion and ‘‘fusogenic cell therapy’’

The mechanism underlying the conversion of stemcells has been heavily debated. It is generally acceptedthat some stem cell types can fuse with the recipient’scells [2], thus leading to cytoplasmic mixing and repro-gramming of cell fate. Alternatively, it seems that stemcells can be instructed by factors of the host’s micro-environment to adopt a hepatocyte fate. To differenti-ate between cell fusion and transdifferentiation is offundamental importance and has several practicalimplications. For instance, fusogenic cells may be usedas vehicles that might deliver their own wild-typegenes to the deficient genome of the recipient’shepatocytes.

To our knowledge the efficiency of ‘‘fusogenic celltherapy’’ so far has only been demonstrated in thefumarylacetoacetate hydrolase-deficient mouse model(FAH�/�): an animal model of fatal tyrosinaemiaType 1. FAH�/� mice suffer from severe liver damageas a consequence of accumulation of the hepatotoxicmetabolites, fumarylacetoacetate and its precursormaleylacetoacetate. Due to the deterioration of hepato-cytes, FAH-deficient mice cannot survive unless they aretreated with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione (NTBC), which prevents productionof the toxic metabolites. Due to permanent deteriorationof hepatocytes the FAH�/� mice represent an animalmodel with an extremely high selection pressure forwild-type (i.e., FAH+/� or FAH+/+) hepatocytes. Atleast three articles (from two independent groups) haveconvincingly demonstrated that in the FAH�/� mousemodel the transplanted stem cells fuse with the host’shepatocytes, thus leading to liver regeneration[75,73,77]. Wang et al. [75] transplanted bone marrowcells from female FAH wild-type LacZ transgenic miceinto male FAH�/� recipients. Cytogenetic analysis dem-onstrated 80 XXXY karyotypes, indicating cell fusionbetween two diploid cells. Similarly, 120 XXXXYYkaryotypes demonstrated cell fusion events between atetraploid recipient’s hepatocyte to a diploid donor bonemarrow cell. Vassilopoulos et al. [73] analysed genomicDNA of FAH-expressing liver nodules after transplan-

tation of FAH+/+ bone marrow cells. Interestingly, thenodules contained more mutant (host) than wild-type(donor) FAH alleles. If donor bone marrow hadtransdifferentiated into hepatocytes the FAH-expressingliver nodules should have contained mostly donorDNA. Willenbring et al. [77] concentrated on the celltype responsible for therapeutic cell fusion. They dem-onstrated that differentiated macrophages (obtainedfrom the mononuclear fraction of OSA26+/� [R26R],FAH+/+-mice) could act as fusion partners forFAH�/� hepatocytes. In contrast, hepatocyte–hepato-cyte fusion did not occur or was extremely rare, whichhas been demonstrated by transplantation of FAHwild-type hepatocytes into FAH�/� mice.

Therefore, the experiments with FAH�/� mice clearlyprovide a proof of principle that a monogenetic liver dis-ease can be cured by ‘‘fusogenic cell therapy’’. Onemight argue that the extreme selective pressure inFAH�/� livers may create a situation that facilitates cellfusion. Usually, such an extreme selection pressure can-not be found in human inherited metabolic liver diseas-es. Therefore, efficiency of gene transfer by cell fusionmay be insufficient for a significant improvement of clin-ical outcome in human metabolic liver disease. At pres-ent, cell fusion remains a rare event not considered asthe principal mechanism of liver repopulation. Never-theless, future developments might increase the ‘‘fuso-genic potential’’ of cells for ‘‘fusogenic therapy’’. Goodcandidates for fusogenic cell therapy are inherited meta-bolic liver diseases that are caused by defects in a singleor a limited number of genes, which have been discussedabove (Table 2). However, it should be considered thatfusogenic therapy could have serious consequences. Cellfusion leads to aneuploidy and eventually to chromo-some instability and loss of chromosomes. Therefore,serious consequences including neoplasia must be care-fully excluded before fusogenic therapy can be appliedin patients.

5. Technical approaches related to human cell

transplantation

In order to track the fate of human stem cells follow-ing transplantation, the use of suitable animal models isnecessary. However, the analysis of human stem cellsafter their transplantation into the livers of laboratoryanimals is technically demanding. The experimentaltransplantation of hepatic cells aiming to repopulatethe host liver has two fundamental requirements: First-ly, donor cells need to be identified within the recipienttissue and secondly, selective proliferation stimulus isnecessary, so that transplanted cells proliferate in prefer-ence to host cells.

Several techniques are available to identify trans-planted cells, such as fluorescent dyes, nanoparticles,


and genetic markers (Fig. 4A). After identification of thetransplanted cells in the recipient tissue by these mark-ers, their human origin should be confirmed, forinstance through in situ hybridization with alu- andmouse major satellite probes that allow differentiationbetween mouse and human nucleic acids (Fig. 4B–D).A next milestone is the demonstration that previouslysilent hepatocyte markers become expressed in the trans-planted cells (Fig. 4E). For this purpose combination ofin situ hybridization using alu-probes with immuno-staining for human hepatocyte markers may identifycells of human origin that express, for instance, albumin(Fig. 4F). Relying on the species specificity of an anti-body alone may be problematic, since it is difficult toestablish conditions that guarantee a 100% species spec-

A B

C

E F

Fig. 4. Milestones in defining hepatocyte-like cells: (A) Marking of cells befo

marked by red fluorescent nanoparticles. (B–D) Identification of cells of human o

tissue after in situ hybridization with mouse major satellite probes (visualized by

alu-probes (visualized by green fluorescence). (D) Visualization of human hep

combined in situ hybridization with alu- and mouse major satellite probes [80]

albumin, demonstrating that the transplanted cell expresses human but not mo

expressing cell. Human albumin is immunohistochemically detected (visualiz

hybridization with alu-probes (visualized by green fluorescence). Blue fluoresce

ificity. After identification of cells expressing humanhepatocyte markers it is recommended to confirmexpression by an independent technique, for instanceby RT-PCR with species specific primers. If confirma-tion is positive an important milestone has beenachieved. Some examples of cells that fulfil these criteriaat least partially have been shown in Fig. 2.

To allow preferential proliferation of donor cells, theregeneration capacity of the recipient liver can beimpaired. In most animal models, chemotoxins or carcin-ogens targeting the liver (e.g. retrorsine, 2-acetylamino-fluorene, CCl4) are widely used for this purpose [41].However, owing to the severe systemic side effects, theseagents are not suitable for human application. Therefore,less harmful stimuli as an alternative to prime the host liv-

D

re transplantation. In this example human fetal hepatocytes have been

rigin in mouse livers by in situ hybridization. (B) SCID/NOD mouse liver

pink fluorescence). (C) Human liver tissue after in situ hybridization with

atocytes after transplantation into the liver of a SCID/NOD mouse by

. (E) Double immunohistochemistry for human (red) and mouse (green)

use albumin. (F) Confirmation of the human origin of a human albumin

ed by red fluorescence), whereas human DNA is identified by in situ

nce: nuclear staining with DAPI. Magnification: A,E,F: 630·; D: 200·.


er have to be studied. It has been recently shown that liverirradiation may be a promising alternative as a potentialpreparative regimen for hepatocyte transplantation [24].

A question of relevance is, whether these cells can beregarded as genuine, functional hepatocytes. Somerecent studies suggested that expression of albuminand an epithelial morphology alone do not guaranteeexpression of all functions that make up a hepatocyte[64,6]; review: [27]. For this purpose functional studiesin animal models of liver disease (Table 3) are needed.An important further criterion is whether the hepato-cyte-like cells of human origin can form liver tissue whengiven a selection advantage over the recipient’s hepato-cytes. Finally, hepatocyte-like cells of human origincould be isolated after collagenase digestion to be ableto characterize enzyme activities and further hepatocel-lular functions to cultured primary hepatocytes, asrecently recommended [27]. In conclusion, many scien-tists have demonstrated hepatocyte-like cells after trans-plantation of human stem and precursor cells into liversof experimental animals. However, as a worst case sce-nario, it cannot yet be excluded that these cells are inter-mediate cell types expressing only a small number ofhepatocyte markers. In terms of clinical applicationfunctional analysis in animal models of human liver dis-ease is imperative.

References

[1] Almeida-Porada G, Porada CD, Chamberlain J, Torabi A,Zanjani ED. Formation of human hepatocytes by human hema-topoietic stem cells in sheep. Blood 2004;104:2582–2590.

[2] Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, LeeHO, Pfeffer K, et al. Fusion of bone-marrow-derived cells withPurkinje neurons, cardiomyocytes and hepatocytes. Nature2003;425:968–973.

[3] Ambrosino G, Varotto S, Strom SC, Guariso G, Franchin E,Miotto D, et al. Isolated hepatocyte transplantation for Crigler–Najjar syndrome type 1. Cell Transplant 2005;14:151–157.

[4] Aoki T, Jin Z, Nishino N, Kato H, Shimizu Y, Niiya T, et al.Intrasplenic transplantation of encapsulated hepatocytes decreas-es mortality and improves liver functions in fulminant hepaticfailure from 90% partial hepatectomy in rats. Transplantation2005;79:783–790.

[5] Attaran M, Schneider A, Grote C, Zwiens C, Flemming P, GratzKF, et al. Regional and transient ischemia/reperfusion injury inthe liver improves therapeutic efficacy of allogeneic intraportalhepatocyte transplantation in low-density lipoprotein receptordeficient Watanabe rabbits. J Hepatol 2004;41:837–844.

[6] Beerheide W, von Mach MA, Ringel M, Fleckenstein C,Schumann S, Renzing N, et al. Downregulation of beta2-micro-globulin in human cord blood somatic stem cells after transplan-tation into livers of SCID-mice: an escape mechanism of stemcells?. Biochem Biophys Res Commun 2002;294:1052–1063.

[7] Bilir BM, Guinette D, Karrer F, Kumpe DA, Krysl J, Stephens J,et al. Hepatocyte transplantation in acute liver failure. LiverTransplant 2000;6:32–40.

[8] Burlina AB. Hepatocyte transplantation for inborn errors ofmetabolism. J Inherit Metab Dis 2004;27:373–383.

[9] Cai J, Ito M, Nagata H, Westerman KA, LaFleur D, ChowdhuryJR, et al. Treatment of liver failure in rats with end-stage cirrhosis

by transplantation of immortalized hepatocytes. Hepatology2002;36:386–394.

[10] Chan C, Berthiaume F, Nath BD, Tilles AW, Toner M, YarmushML. Hepatic tissue engineering for adjunct and temporary liversupport: critical technologies. Liver Transplant2004;10:1331–1342.

[11] Cantz T, Sharma AD, Jochheim-Richter A, Arseniev L, Klein C,Manns MP, et al. Reevaluation of bone marrow-derived cells as asource for hepatocyte regeneration. Cell Transplant2004;13:659–666.

[12] Chowdhury JR, Grossman M, Gupta S, Chowdhury NR, BakerJr JR, Wilson JM. Long-term improvement of hypercholesterol-emia after ex vivo gene therapy in LDLR-deficient rabbits. Science1991;254:1802–1805.

[13] Danet GH, Luongo JL, Butler G, Lu MM, Tenner AJ, SimonMC, et al. C1qRp defines a new human stem cell population withhematopoietic and hepatic potential. Proc Natl Acad Sci USA2002;99:10441–10445.

[14] Demetriou AA, Reisner A, Sanchez J, Levenson SM, MoscioniAD, Chowdhury JR. Transplantation of microcarrier-attachedhepatocytes into 90% partially hepatectomized rats. Hepatology1988;8:1006–1009.

[15] De Vree JM, Ottenhoff R, Bosma PJ, Smith AJ, Aten J, OudeElferink RP. Correction of liver disease by hepatocyte transplan-tation in a mouse model of progressive familial intrahepaticcholestasis. Gastroenterology 2000;119:1720–1730.

[16] Dhawan A, Mitry RR, Hughes RD, Lehec S, Terry C, Bansal S,et al. Hepatocyte transplantation for inherited factor VII defi-ciency. Transplantation 2004;78:1812–1814.

[17] Ryan Edmond A, Lakey Jonathan RT, Rajotte Ray V, KorbuttGregory S, Kin2 Tatsuya, Imes Sharleen, et al. Clinical outcomesand insulin secretion after islet transplantation with the Edmon-ton protocol. Diabetes 2001;50:710–719.

[18] Fisher RA, Bu D, Thompson M, Tisnado J, Prasad U, Sterling R,et al. Defining hepatocellular chimerism in a liver failure patientbridged with hepatocyte infusion. Transplantation2000;69:303–307.

[19] Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, ChowdhuryNR, Warkentin PI, et al. Treatment of the Crigler–Najjarsyndrome type I with hepatocyte transplantation. N Engl J Med1998;338:1422–1426.

[20] Francavilla A, Starzl TE, Barone M, Zeng QH, Porter KA, ZeeviA, et al. Studies on mechanisms of augmentation of liverregeneration by cyclosporine and FK 506. Hepatology1991;14:140–143.

[21] Fuentealba IC, Aburto EM. Animal models of copper-associatedliver disease. Comp Hepatol 2003;2:5.

[22] Goss JA, Stribling R, Martin P. Adult liver transplantation formetabolic liver disease. Clin Liver Dis 1998;2:187–210.

[23] Groth CG, Arborgh B, Bjorken C, Sundberg B, Lundgren G.Correction of hyperbilirubinemia in the glucuronyltransferase-deficient rat by intraportal hepatocyte transplantation. TransplantProc 1977;9:313–316.

[24] Guha C, Parashar B, Deb NJ, et al. Liver irradiation: a potentialpreparative regimen for hepatocyte transplantation. Int J RadiatOncol Biol Phys 2001;49:451–457.

[25] Harada M, Kawaguchi T, Kumemura H, Terada K, Ninomiya H,Taniguchi E, et al. The Wilson disease protein ATP7B resides inthe late endosomes with Rab7 and the Niemann-Pick C1 protein.Am J Pathol 2005;166:499–510.

[26] Habibullah CM, Syed IH, Qamar A, Taher-Uz Z. Human fetalhepatocyte transplantation in patients with fulminant hepaticfailure. Transplantation 1994;58:951–952.

[27] Hengstler JG, Brulport M, Schormann W, Bauer A, Hermes M,Nussler AK, et al. Generation of human hepatocytes by stem celltechnology: definition of the hepatocyte. Expert Opin Drug MetabToxicol 2005;1:61–74.


[28] Hitomi Y, Sugiyama K, Esumi H. Suppression of the 50 splice sitemutation in the Nagase analbuminemic rat with mutatedU1snRNA. Biochem Biophys Res Commun 1998;251:11–16.

[29] Horslen SP, McCowan TC, Goertzen TC, Warkentin PI, Cai HB,Strom SC, et al. Isolated hepatocyte transplantation in aninfant with a severe urea cycle disorder. Pediatrics2003;111:1262–1267.

[30] Hughes RD, Mitry RR, Dhawan A. Hepatocyte transplantationfor metabolic liver disease: UK experience. J R Soc Med2005;98:341–345.

[31] Ishikawa F, Drake CJ, Yang S, Fleming P, Minamiguchi H,Visconti RP, et al. Transplanted human cord blood cells give riseto hepatocytes in engrafted mice. Ann NY Acad Sci2003;996:174–185.

[32] Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD,Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cellsderived from adult marrow. Nature 2002;418:41–49.

[33] Kakinuma S, Tanaka Y, Chinzei R, Watanabe M, Shimizu-Saito K,Hara Y, et al. Human umbilical cord blood as a source oftransplantablehepatic progenitor cells. Stem Cells 2003;21:217–227.

[34] Kiwaki K, Kanegae Y, Saito I, Komaki S, Nakamura K,Miyazaki JI, et al. Correction of ornithine transcarbamylasedeficiency in adult spf(ash) mice and in OTC-deficient humanhepatocytes with recombinant adenoviruses bearing the CAGpromoter. Hum Gene Ther 1996;7:821–830.

[35] Kobayashi N, Ito M, Nakamura J, Cai J, Gao C, Hammel JM,et al. Hepatocyte transplantation in rats with decompensatedcirrhosis. Hepatology 2000;31:851–857.

[36] Kobayashi N, Westerman KA, Tanaka N, Fox IJ, Leboulch P. Areversibly immortalized human hepatocyte cell line as a sourceof hepatocyte-based biological support. Addict Biol2001;6:293–300.

[37] Koenig S, Stoesser C, Krause P, Becker H, Markus PM. Liverrepopulation after hepatocellular transplantation: integration andinteraction of transplanted hepatocytes in the host. Cell Trans-plant 2005;14:31–40.

[38] Kogler G, Sensken S, Airey JA, Trapp T, Muschen M, FeldhahnN, et al. A new human somatic stem cell from placental cordblood with intrinsic pluripotent differentiation potential. J ExpMed 2004;200:123–135.

[39] Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, DabevaMD, et al. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J ClinInvest 2003;112:160–169.

[40] Kumaran V, Benten D, Follenzi A, Joseph B, Sarkar R, Gupta S.Transplantation of endothelial cells corrects the phenotype inhemophilia A mice. J Thromb Haemost 2005;3:2022–2031.

[41] Laconi E, Laconi S. Principles of hepatocyte repopulation. SeminCell Dev Biol 2002;13:433–438.

[42] Landskroner KA, Olson NC, Jesmok GJ. Thromboelastog-raphy measurements of whole blood from factor VIII-deficient mice supplemented with rFVIII. Haemophilia2005;11:346–352.

[43] Li J, Fang B, Eisensmith RC, Li XH, Nasonkin I, Lin-Lee YC,et al. In vivo gene therapy for hyperlipidemia: phenotypiccorrection in Watanabe rabbits by hepatic delivery of the rabbitLDL receptor gene. J Clin Invest 1995;95:768–773.

[44] Meier M, Woywodt A, Hoeper MM, Schneider A, Manns MP,Strassburg CP. Acute liver failure: a message found under theskin. Postgrad Med J 2005;81:269–270.

[45] Miki T, Lehmann T, Cai H, Stolz DB, Strom SC. Stem cellcharacteristics of amniotic epithelial cells. Stem Cells 2005 [Epubahead of print].

[46] Mito M, Kusano M, Kawaura Y. Hepatocyte transplantation inman. Transplant Proc 1992;24:3052–3053.

[47] Mitry RR, Dhawan A, Hughes RD, Bansal S, Lehec S, Terry C,et al. One liver, three recipients: segment IV from split-liver

procedures as a source of hepatocytes for cell transplantation.Transplantation 2004;77:1614–1616.

[48] Muraca M, Gerunda G, Neri D, Vilei MT, Granato A, FeltraccoP, et al. Hepatocyte transplantation as a treatment for glycogenstorage disease type 1a. Lancet 2002;359:317–318.

[49] Rosen ED, Chan JC, Idusogie E, Clotman F, Vlasuk G, Luther T,et al. Mice lacking factor VII develop normally but suffer fatalperinatal bleeding. Nature 1997;390:290–294.

[50] Seppen J, van der Rijt R, Looije N, van Til NP, Lamers WH,Oude Elferink RP. Long-term correction of bilirubin UDPglucu-ronyltransferase deficiency in rats by in utero lentiviral genetransfer. Mol Ther 2003;8:593–599.

[51] Strom SC, Chowdhury JR, Fox IJ. Hepatocyte transplantationfor the treatment of human disease. Semin Liver Dis1999;19:39–48.

[52] Moscioni AD, Rozga J, Chen S, Naim A, Scott HS, DemetriouAA. Long-term correction of albumin levels in the Nagaseanalbuminemic rat: repopulation of the liver by transplantednormal hepatocytes under a regeneration response. Cell Trans-plant 1996;5:499–503.

[53] Mito M, Kusano M. Hepatocyte transplantation in man. CellTransplant 1993;2:65–74.

[54] Nagata H, Ito M, Cai J, Edge AS, Platt JL, Fox IJ. Treatment ofcirrhosis and liver failure in rats by hepatocyte xenotransplanta-tion. Gastroenterology 2003;124:422–431.

[55] Nagata H, Ito M, Shirota C, Edge A, McCowan TC, Fox IJ.Route of hepatocyte delivery affects hepatocyte engraftment in thespleen. Transplantation 2003;76:732–734.

[56] Newsome PN, Johannessen I, Boyle S, Dalakas E, McAulay KA,Samuel K, et al. Human cord blood-derived cells can differentiateinto hepatocytes in the mouse liver with no evidence of cellularfusion. Gastroenterology 2003;124:1891–1900.

[57] Nonome K, Li XK, Takahara T, Kitazawa Y, Funeshima N, YataY, et al. Human umbilical cord blood-derived cells differentiateinto hepatocyte-like cells in the Fas-mediated liver injury model.Am J Physiol Gastrointest Liver Physiol 2005.

[58] Ott M, Schmidt HH, Cichon G, Manns MP. Emerging therapiesin hepatology: liver-directed gene transfer and hepatocyte trans-plantation. Cells Tissues Organs 2000;167:81–87.

[59] Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK,Murase N, et al. Bone marrow as a potential source of hepaticoval cells. Science 1999;284:1168–1170.

[60] Petersen J, Ott M, von Weizsacker F. Current status of cell-basedtherapies in liver diseases. Z Gastroenterol 2001;39:975–980.

[61] Jalan Rajiv. Acute liver failure: current management and futureprospects. J Hepatol 2005;42 (Suppl. 1):S115–S123, [Epub 2004Dec 15].

[62] Ringel M, von Mach MA, Santos R, Feilen PJ, Brulport M,Hermes M, et al. Hepatocytes cultured in alginate microspheres:an optimized technique to study enzyme induction. Toxicology2005;206:153–167.

[63] Ruhnke M, Ungefroren H, Nussler A, Martin F, Brulport M,Schormann W, Hengstler JG, et al. Reprogramming of humanperipheral blood monocytes into functional hepatocyte andpancreatic islet-like cells. Gastroenterology 2005;128:1774–1786.

[64] Sharma AD, Cantz T, Richter R, Eckert K, Henschler R, WilkensL, et al. Human cord blood stem cells generate human cytoker-atin 18-negative hepatocyte-like cells in injured mouse liver. Am JPathol 2005;167:555–564.

[65] Shi Z, Liang XL, Lu BX, Pan SY, Chen X, Tang QQ, et al.Diminution of toxic copper accumulation in toxic milk micemodeling Wilson disease by embryonic hepatocyte intrasplenictransplantation. World J Gastroenterol 2005;11:3691–3695.

[66] Sokal EM, Smets F, Bourgois A, Van Maldergem L, Buts JP,Reding R, et al. Hepatocyte transplantation in a 4-year-old girlwith peroxisomal biogenesis disease: technique, safety, andmetabolic follow-up. Transplantation 2003;76:735–738.


[67] Stephenne X, Najimi M, Smets F, Reding R, de Ville de Goyet J,Sokal EM. Cryopreserved liver cell transplantation controlsornithine transcarbamylase deficient patient while awaiting livertransplantation. Am J Transplant 2005;5:2058–2061.

[68] Strom S, Fisher R. Hepatocyte transplantation: new possibilitiesfor therapy. Gastroenterology 2003;124:568–571.

[69] Strom SC, Fisher RA, Thompson MT, Sanyal AJ, Cole PE, HamJM, et al. Hepatocyte transplantation as a bridge to orthotopicliver transplantation in terminal liver failure. Transplantation1997;63:559–569.

[70] Strom SC, Fisher RA, Rubinstein WS, Barranger JA, Towbin RB,Charron M, et al. Transplantation of human hepatocytes.Transplant Proc 1997;29:2103–2106.

[71] Strom SC, Chowdhury JR, Fox IJ. Hepatocyte transplantationfor the treatment of human disease. Semin Liver Dis1999;19:39–48.

[72] Turrini P, Monego G, Gonzalez J, Cicuzza S, Bonanno G, ZelanoG, et al. Human hepatocytes in mice receiving pre-immuneinjection with human cord blood cells. Biochem Biophys ResCommun 2005;326:66–73.

[73] Vassilopoulos G, Wang PR, Russell DW. Transplantedbone marrow regenerates liver by cell fusion. Nature2003;422:901–904.

[74] von Mach MA, Hengstler JG, Brulport M, Eberhardt M,Schormann W, Hermes M, et al. In vitro cultured islet-derived

progenitor cells of human origin express human albumin in severecombined immunodeficiency mouse liver in vivo. Stem Cells2004;22:1134–1141.

[75] Wang X, Montini E, Al-Dhalimy M, Lagasse E, Finegold M,Grompe M. Kinetics of liver repopulation after bone marrowtransplantation. Am J Pathol 2002;161:565a–574a.

[76] Wang X, Ge S, McNamara G, Hao QL, Crooks GM, Nolta JA.Albumin-expressing hepatocyte-like cells develop in the livers ofimmune-deficient mice that received transplants of highly purifiedhuman hematopoietic stem cells. Blood 2003;101:4201–4208[Epub 2003 Jan 30].

[77] Willenbring H, Bailey AS, Foster M, Akkari Y, Dorrell C, OlsonS, et al. Myelomonocytic cells are sufficient for therapeutic cellfusion in liver. Nat Med 2004;10:744–748.

[78] Zeng F, Chen M, Katsumata M, Huang W, Gong Z, Hu W, et al.Identification and characterization of engrafted human cells inhuman/goat xenogeneic transplantation chimerism. DNA CellBiol 2005;24:403–409.

[79] Jang YY, Collector MI, Baylin SB, Diehl AM, Shatkis SJ.Hematopoietic stem cells convert into liver cells within dayswithout fusion. Nat Cell Biol 2004;6:532–539.

[80] Brustle O, Spiro AC, Karram K, Choudhary K, Okabe S, McKayRD. In vitro-generated neural precursors participate in mamma-lian brain development. Proc Natl Acad Sci USA1997;94:14809–14814.

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Present status and perspectives of cell-based therapies for liver diseases

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