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Hepatology Research 36 (2006) 237–247

Review

Hepatocyte transplantation: State of the art

Flavio Henrique Ferreira Galvao a,∗, Dahir Ramos de Andrade Junior b,Dahir Ramos de Andrade b, Bruno Costa Martins c, Allan Garms Marson c,

Christiano Vinicius Bernard c, Sania Alves dos Santos b, Telesforo Bacchella a,Marcel Cerqueira Cesar Machado a

a Laboratorio de Transplante Experimental da Disciplina de Transplante e Cirurgia do Fıgado (LIM 37),Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, Brazil

b Laboratorio de Bacteriologia e Microbiologia Celular (LIM-54), Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazilc Residente de Cirurgia da Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, Brazil

Received 21 October 2005; received in revised form 29 June 2006; accepted 17 August 2006Available online 2 October 2006

bstract

Advances in biotechnology have allowed hepatocyte transplantation as a relevant proposition to treat liver disease. This procedure mayhange the crescent mortality in liver transplantation waiting lists due global organ shortage. Recent clinical trials have described promisingesults of hepatocyte transplantation for acute, acute-on-chronic and metabolic liver disease. In this report, we discuss progresses regardingepatocyte culture, cryopreservation systems, hepatocyte immortalization, suitable recipient site for hepatocyte engraftment, cell differentiationnd fusion into hepatocytes, current clinical trials, and summarize the bioartificial liver systems. These progressions motivate expectationoncerning hepatocyte transplantation as a consistent therapy for liver disease.

2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Hepatocyte transplantation; Liver transplantation; Xenotransplantation; Cell culture; Liver failure

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2382. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2383. Hepatocyte isolation and culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2384. Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2395. Hepatocyte immortalization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

5.1. Permanent telomeric replication by telomere/telomerase mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2395.2. Gene therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2395.3. Replicative senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2405.4. Reversible immortalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

6. Hematopoietic stem cells and peripheral blood monocytes differentiation and fusion into hepatocytes . . . . . . . . . . . . . . . . . . . . . . . 2407. Suitable recipient site for hepatocyte implantation and engraftment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

7.1. Intraportal and intrasplenic infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2417.2. Ectopic sites for hepatocyte transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

∗ Corresponding author at: Rua Dona Antonia de Queiroz 551, Apt 31, Higienopolis, Sao Paulo 01307-010, SP, Brazil. Tel.: +55 11 98666680/32576478;x: +55 11 30667000.

E-mail address: fgalvao@usp.br (F.H.F. Galvao).

386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.hepres.2006.08.008

10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Liver transplantation is the only therapeutic option for end-tage liver disease. Organ shortage has impaired transplan-ation activity causing crescent waiting list mortality every-here. Recently, hepatocyte transplantation has emerged aspromising and less aggressive method to treat liver disease

1–10].Difficulty to obtain freshly isolated hepatocytes and bar-

iers in cell managing difficult usual application of this pro-edure; however, novel researches describe creative mannershat ascend capable expectations for hepatocyte transplanta-ion practice [1–10].

This review discusses the progresses in hepatocyte trans-lantation like cultured hepatocyte protocols, cryopreserva-ion systems, cell immortalization, cell differentiation andusion into hepatocytes, suitable site for hepatocyte engraft-ent, current clinical trails and summarizes the bioartificial

iver systems.

. Methods

We systematically reviewed seven hundred and fortyeven articles related to the theme throughout Cochraneepato-Biliary Group Controlled Trails Register, Cochraneibrary, MEDLINE and EMBASE. We excluded therticles based on lack of close relation to the subjectnd discussed 89 articles in the manuscript that wasncluded in the reference. We distributed the data fromhese articles as followed: hepatocyte isolation and culture,ryopreservation, immortalization, hematopoietic stemells and peripheral blood monocytes differentiation intoepatocytes, suitable recipient’s place for hepatocytemplantation, existing clinical trails and bioartificial liverystems.

. Hepatocyte isolation and culture

Berry and Friend described hepatocytes isolation in 196911] and their two-step perfusion method for parenchyma dis-

ociation using colagenase was modified elsewhere [12–28].sing regular methods of isolation and culture, viability of

ultured hepatocyte falls rapidly and vanishes after 1 week.he peculiar hepatocyte metabolism requires special sup-

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

lements to prolong its viability and proliferation in culture11–28].

Proliferation of mature hepatocyte in vitro remains a dif-cult task. These cells subsist in differentiation phase G0nd demands strong in vitro stimulation to convert them intophase (early mitotic cell cycle). Newborn donors are pre-

erred because most of their cells convey S phase, archivingetter-cultured subsistence and function [17]. This is whymbilical cords have been considered an important source ofell supply.

Substances that improve cultured hepatocyte viability areenerally divided into mitogenic and co-mitogenic factors12–28]. Co-mitogenics stimulate mitogenic growing fac-ors and decrease the effect of mitosis inhibitor substances,hereas mitogenics agents stimulate DNA synthesis and cellitosis [12–28].Co-mitogenics substances include insulin, glucagon, cor-

ex adrenal hormones, dimethyl sulphoxide, phenobarbital,icotinamide, vasopressin, angiotensin, thyroid and parathy-oid hormones, norepinephrine, calcium, Vitamin D andinc. Hidrocortisone and insulin improve hepatocyte viabil-ty and biliary canaliculus arrangement, regulate functionsuch lipids and carbohydrates metabolism, protein synthesis,reservation of polyclonal morphology, and inhibit fibrob-astic cells contamination [12–14]. Nevertheless, culturedepatocyte supplemented with these substances achievedust 3–10% of gene-specific transcription expression fromhat observed in vivo [15]. Amino acids, nicotinamide, elec-rolytes and phenobarbital promote enzyme stabilization androlong hepatocyte survival. Unfortunately, the best resultssing these agents have not surpassed 12 days of cell sur-ival. On the other hand, dimethyl sulphoxide emerge aspromising agent and its addition during the first day

f hepatocyte culture was able to prolong its viability forver a month [16]. Glucagon, cortex adrenal hormones,asopressin, angiotensin, thyroid and parathyroid hormones,orepinephrine, calcium, Vitamin D and zinc, representsmportant co-mitogenic supplements

Mitogenic substances for cultured hepatocytes compriseepatocyte growth factor (HGF), epidermal growth fac-or (EGF), transforming growth factor alpha (TGF-�), ker-tinocyte growth factor (KGF), vascular endothelial growth

238 F.H.F. Galvao et al. / Hepatology Research 36 (2006) 237–247

8. Clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2428.1. Acute hepatic failure (AHF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2428.2. Chronic hepatic failure (CHF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2438.3. Metabolic disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

9. Bioartificial liver systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

actor (VEGF), human growth factor (GH) and tumor necro-is factor (TNF) alpha [17–24]. Recent appraisals suggest thatGF is the strongest agent for hepatocyte proliferation and

nclose valuable antifibrotic effect [22,23]. The antifibrotic

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F.H.F. Galvao et al. / Hepato

ffect is attributed to two different mechanisms: 1-hepaticollagenase activity augment, which promotes degradation ofhe ECM components; 2-inhibition of procollagens and TGF-1 mRNA level. Procollagens and TGF-�1 up-regulation

s crucial in liver fibrosis [22,23]. A variant of HGF withve amino-acid residues deleted (dHGF) has been shown asn useful tool for in vitro maintenance of hepatocyte func-ions. Chen el al. revealed that hepatocytes cultured withHGF acquired lower cytotoxicity and superior hepatocyte-ifferentiated functions then that cultured with HGF [23].e et al. recently achieved successful proliferation and dif-

erentiation of hepatic stem cells into mature hepatocytes,ombining HGF and EGF [25]. Furthermore, in vivo admin-stration of HGF was able to remodel the hepatic cirrhoticissue [26]. EGF and dHGF combination may be a poten-ial option for cultured hepatocyte improvement and hepaticirrhotic tissue repairs [22–26].

Mitogenic and co-mitogenic combinations is a rationalethod to preserve hepatocyte in culture. For instance, the

ssociation of insulin and EGF has a synergic effect, stim-lating mature hepatocytes to enter phase S within 25–30 h20,21].

Recently Liu et al. [27] described a novel substance called675, found in a Streptomyces sp. SM675 culture medium.his promising substance stimulates the proliferation and

unction of human liver cell 4 by inhibition of apoptosis-athway during serum-deprivation.

Tissue engineering models using polymers scaffolds in theedium is another talented line of research for cell develop-ent and expansion. These extracellular matrices are fun-

amental for long hepatocyte viability in culture, allowingonvenient progression of cellular adhesion, DNA synthesisnd opportune gene expression [24].

The decisive hepatocyte culture medium varies from cen-er to center. Manner of hepatocyte achievement, amount ofttained cells, period of cell culture and the site employed forell engraftment are important concerns for the right choicef hepatocyte media.

. Cryopreservation

Cryopreservation systems permit long hepatocyte storage.his procedure may be useful for hepatocyte transplantationuring urgent liver dysfunction. Current methods of hepato-yte cryopreservation utilize cryoprotector agents and systemor cell freezing, maintenance and defreezing.

Dimethyl sulphoxide in concentration of 14% for ratsnd 10–12% for humans is the most effective cryoprotec-or for cultured hepatocytes [6,29–32]. Glucose added tohe medium improves cellular viability after defreezing [29].sing current cryopreservation technology, Chesne et al. was

ble to expand viability and replication of the hepatocytes upo 32 months [31]. Intracellular frosty is a critical event duringreezing process. It occurs when the cell reaches tempera-ure between −15 and −20 ◦C, and may cause hepatocyte

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esearch 36 (2006) 237–247 239

amage. Slow freezing rate (between 1 and 6 ◦C/min) mayinimize cell damage. When temperature reaches −70 ◦C,

epatocytes may be stored in liquid nitrogen. The thaw pro-ess is less complex and do not require special condition.

. Hepatocyte immortalization

Hepatocytes immortalization technique attempts to cre-te cell clones that replicate indefinitely. The most acceptedethods for hepatocyte immortalization include:

.1. Permanent telomeric replication byelomere/telomerase mechanism

Telomere constitutes the ends of most eukaryotic chro-osomes. It constitutes a reiterated hexanucleotides (5′-TAGGG-3′) responsible for specific mechanisms that con-

rol cell life length and replication. Telore also avoid end-erminal fusions and protects chromosomal tips from degra-ation and gross genetic rearrangements that may cause cellycle disruption and apoptosis [34]. Normal somatic cellsose telomeric DNA at a rate of 50–200 bp per population dou-ling; consequently, cells from older organisms have shorterelomeres than younger organisms. In addition, senescentells have shorter telomeres than stem cells [35].

Telomerase is a reverse transcription ribonucleoproteinesponsible for telomere length conservation. This enzymexpresses high activity in gametes and cells that continu-usly replicate like totipotent stem cells, but is not presentn normal human somatic cells. The telomere/telomerase

echanisms of cell immortalization consist of permanentelomeric replication in chromosome end [35]. Wege et al.escribed an elegant immortalization method using trans-uced fetal human hepatocytes with the catalytic subunitf telomerase reverse transcriptase (hTERT). Telomerase-econstituted immortalized hepatocytes preserved elongatedelomeres without disrupting their differentiation potential36].

.2. Gene therapy

This technique inserts specific genes into hepatocyteenome by means of viral and non-viral vectors. Simian virus0T antigen (SV40T) causes hepatocyte immortalization bynactivation of anti-oncogen p53 and retinoblastoma proteins32]; however, at some stage in 12th passage, this immortal-zed hepatocyte reaches the catastrophic M2 phase (crisis)ausing chromosomal instability and cell death. Co-infectionith telomerase produce hepatocyte clones achieving over a

l. inserted the C/EBPbeta gene into cultured hepatocytes andbserved great hepatocyte regeneration and survival in CCl4-nduced liver injury. These authors affirmed that this proce-ure might have therapeutic function against liver injury [33].

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40 F.H.F. Galvao et al. / Hepat

.3. Replicative senescence

Regular cells comprise a pre-determined and finite numberf divisions called replicative senescence state [32–35]. Theell senescence occurs in phase I of mortality (M1), in whichhe cell remains blocked in G1 phase and cannot reenter intoormal replication cycle.

Retinoblastome protein (pRB) and p53 activity sustain1 phase. Inactivation of these regulators by SV40T antigen

eads the cell to skip M1 checkpoint and achieve M2 or phaseof mortality, restarting cell cycle proliferation. Multipleutations and genomic disarrangement are common during

his period. The clones that are able to survive the crisis (byutations in telomerase activity) frequently acquire cancer-

us alterations [32–36]. However, Wege et al. [36] detectedo oncogenicity and no c-Myc ontogeny up-regulationn human fetal hepatocytes that restored telomerasectivity.

.4. Reversible immortalization

Since immortalized hepatocytes may become more sen-itive to oncogenes [32–37], researchers have aimed expandepatocyte clones up to a normal limit, avoiding bizarre hep-tic development and neoplasic transformation. Fox et al. [38]ontrolled the cell growth by using a termolabil mutant ofV40T antigen. Permissive temperatures surround the max-

mum expansion of this antigen, which is rapidly inactivatedhen the temperature rises to physiological levels. Kobayashi

t al. [39] used a Cre-Lox system to remove the immortal-zing antigen. SV40T antigen marked with the Lox site ofecognition immortalized the cells. After cell immortality,he antigen was completely removed by transitory transin-ection using a vector with Cre recombinase expression, thats capable of recognize and remove the Lox site. This methodas able to prolong survival of rats with acute hepatic failure

nd may have clinical importance [39].

. Hematopoietic stem cells and peripheral bloodonocytes differentiation and fusion into hepatocytes

Murase et al. [40] and Ye et al. [41] observed close rela-ionship between bone marrow and liver cell components.n lethally irradiated rats, they revealed that singeneic liverransplantation was capable to reconstitute recipient’s bone

arrow and prolong animal survival. This situation was notresent when heart or kidney was transplanted into lethallyrradiated rats. They suggest that the passenger leucocytesrom transplanted liver contain sufficient amount of stemoval) cells to reconstitute recipient’s bone marrow. On thether hand, Petersen et al. transplanted syngeneic bone mar-

ow into rats with severe hepatic injury and observed hepato-ytes, biliary and oval cells of donor origins in the receptors’iver. Furthermore, the animals submitted to bone marrowransplantation obtained a life expectancy improvement [42].

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esearch 36 (2006) 237–247

hese experiments suggested that bone marrow cells haveherapeutic potential in chronic and acute hepatic failure.

Indeed, under certain circumstances, stem cells frommbilical cord, bone marrow and even peripheral bloodonocytes can differentiate not only into hepatocytes, but

lso into cholangiocytes and epithelial cell [3,4,8]. Theseources of hepatocytes are an appealing proposal for the lackf donors and may amplify the therapeutic applicability ofhis procedure.

Kakinuma et al. [43] showed that human umbilical cordlood is a relevant in vivo and in vitro source of trans-lantable hepatic progenitor cells and, after 21 days in aovel primary culture system, 50% of these cells coex-ressed hepatocyte lineage markers and release albumin.hese observations have been confirmed by other correlated

nvestigations [44–48]. In addition, human umbilical cordlood transplantated into severe liver-injured immunodefi-ient mice engrafted as functional hepatocytes in the livernd were able to released human albumin into recipient sera43].

Recently Ruhnke et al. [8] evaluated the differentia-ion potential of human peripheral blood monocytes intoepatocyte-like cells. They treated peripheral monocytesith macrophage colony-stimulating factor and interleukin 3

or 6 days, followed by incubation with hepatocyte-specificifferentiation media. These programmable cells retainedonocytic characteristics (CD14 and transcription factorU expression), but were capable of differentiating intoeohepatocytes that resemble primary human hepatocytesorphology, markers expression, and specific hepatocyte

unctions. Transplantation of this neohepatocytes into severeombined immunodeficiency disease/nonobese diabetic miceells engraft into recipient’s liver and enclose similar mor-hology and albumin expression of primary human hepa-ocytes transplanted under identical conditions. The abilityo reprogram, expands, and differentiate peripheral blood

onocytes in large quantities opens a real possibility forlinical application of the programmable neohepatocytes forepatic repair and regeneration.

Porcine hepatocyte is also a promising source, since itould be readily available and was able to rescue rats present-ng hepatic failure; nevertheless, further investigation usingorcine hepatocytes into primate should be performed formprovement of this model, before clinical application [28].

Recently, Cowan et al. [49] investigated the use of embry-nic stem cells as an alternative to oocytes for reprogram-ing human somatic nuclei. They used human embryonic

tem cells fused with human fibroblasts, resulting in hybridells that retain a steady tetraploid DNA content and acquireorphology, growth rate, and antigen expression pattern

f human embryonic stem cells. Furthermore, analysis ofenome-wide transcriptional activity, gene activation, allele-

pecific gene expression, and DNA methylation demon-trated that the somatic genome was reprogrammed to anmbryonic state. Thus, according to these authors’, humanmbryonic stem cells can reprogram the transcriptional state

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f somatic nuclei and provide a system for investigating thenderlying fusion mechanisms. This new and revolutionaryechnology may solve the dilemma of stem cells shortage forepatocyte transplantation.

Controversy has recently arisen as to whether hematopoi-tic stem cells (HSCs) contribute to the hepatocyte lineagen liver injury via transdifferentiation alone or by adoptinghe phenotype of hepatocytes after spontaneous cell fusion50]. In support of transdifferentiation, some groups haveemonstrated that HSCs can differentiate into hepatocytes51,52] without any evidence of cell fusion. In the transd-fferentiation, donor HSCs undergo genetic reprogramming,witch lineage, and generate hepatocytes upon exposure tohe hepatic environment. The frequency of this occurrenceepends on diverse factors, including the type and extent ofiver injury. In the cell fusion, donor HSCs fuse with matureepatocytes and eventually redifferentiate into terminally dif-erentiated hepatocytes. It has been reported that bone mar-ow cells are capable to fuse with hepatocytes [53–55], butot with skeletal muscle, gut, kidney, or lung tissues [55].n the two-step process, donor HSCs engrafts and differenti-te into hepatocytes, which then undergo fusion with matureative hepatocytes. If transdifferentiation is truly responsi-le, then there could be of wide-ranging utility for acquirediver diseases, whereas if cell fusion is responsible, thenhis could be exploited to deliver corrective genes for hep-tic metabolic disorders, as long as genetic stability in theeprogrammed cells could be assured. Additionally, if HSCsre capable of engrafting directly as hepatocytes, then directntraorgan injection may facilitate better response and obviatehe need for irradiation to deplete the recipient’s bone mar-ow. Alternatively, if marrow engraftment is a prerequisite,arrow preparation and manipulation with hepatic microen-

ironment may be necessary to attract HSCs [56]. There is novidence that all marrow-derived hepatocytes were formed byusion. Thus, given that hepatocytes themselves are known touse in pathological conditions, it is important to recognizehat although fusion may occur, it does not explain most newepatocytes obtained [56].

. Suitable recipient site for hepatocyte implantationnd engraftment

Evidently, the optimal site for hepatocytes engraftmenthould be the liver; however, cirrhoses process providesad environment to engraftment and researches have beeneeking alternative (ectopic) sites of implantation. Sites ofmplantation include hepatic, intraportal, peritoneal cavity,ubcutaneous fat tissue, renal capsule and spleen.

.1. Intraportal and intrasplenic infusion

The most used and accepted sites for hepatocyte trans-lantation are the intraportal and intrasplenic via. The liverrovides the optimal conditions for transplanted hepatocyte

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esearch 36 (2006) 237–247 241

ifferentiation, proliferation, engraftment and survival. Thisnvironment supports natural interaction of transplanted hep-tocyte with mesenchymal, immunocompetent other hepaticell lineages, extracellular hepatic matrix and specific hepato-yte growth factors provided by the portal flow. Hepatocytesransplantation into the liver is usually performed by intra-ortal infusion. An alternative method performs hepatocytesnfusion directly into the hepatic parenchyma. The incon-enience of this technique is the possibility of pulmonarymboli due an unadvised injection of the cells into liverenous system [58].

Intraportal infusion technique initiates transplanted hepa-ocytes deposition in the sinusoids [57–59]. Although mostells are depurated in the sinusoids, some of them migratehroughout sinusoid endothelium permeability to the Dissepace. Few days after cell transplantation, reconstitution ofiliary canaliculus between native and transplanted hepato-ytes has been observed.

Spleen is an important site for hepatocyte transplanta-ion due its analogous liver extracellular matrix architecturend portal vascularization supply. This technique can be per-ormed by direct intraparenchymal injection or infusion intoplenic artery. In both models, a relevant number of hepato-ytes migrate to the liver through portal flow. Transplantedepatocytes that stay in the spleen migrate to red pulp andnitiate the process of splenic hepatization [57]. Our researchroup has experienced hepatocyte transplantation in hepatec-omized rats throughout cell injection into the inferior pole ofhe spleen (Medical Investigation Laboratories—LIM 54 andIM 37). We observed that the occlusion of the blood flow

n the splenic artery and vein during the hepatocyte injec-ion avoid immediate course of cells to the liver during cellnfusion. We also observed that cell injection directly intopleen is technically easer then infusion into splenic arteryunpublished data).

Hepatocytes transplanted into rat spleen achieve citoplas-atic irregularities and mitochondrial edema in the first

–3 weeks, but normal hepatocellular structure tends to beestored into cellular reorganization near the normal hep-tic architecture and function [8,57,60]. After 21 months ofntrasplenic hepatocytes infusion, the graft tends to be orga-ized into recognizable hepatic plates, pursuing glandularosettes, acini, space of Disse, sinusoidal structure and biliaryanaliculus [8,57,60,61]. Continuous proliferation of hepa-ocytes can substitute over 40% of the spleen cells in few

onths [60].Several studies demonstrated metabolism of glycogen,

lbumin, glucose-6-phosphatase and urea cycle enzymes inhe transplanted hepatocytes [36,60,62,63]. Metabolic stud-es demonstrated the cytochrome p-450 activity [36,62],lbumin secretion [63], bilirubin glucorinization [63], andightening of organic acids such as 99mTc-HIDA are pre-

erved in this cells [64]. Vroemen et al. demonstrated inyperbilirubinemc Gunn rat improvement of bilirubin UDP-lucuronyl transferase after intrasplenic hepatocyte trans-lantation [65,66].

242 F.H.F. Galvao et al. / Hepatology R

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The restricted amount of hepatocyte that can be trans-lanted without complication is the main limitation of intra-ortal or intrasplenic site. It has been shown that only 2–5%f the total amount of donor hepatocyte infused will engraft inhe host [6]. Malhi et al. showed that disruption of the hepaticinusoidal endothelium using cyclophosphamide was asso-iated with accelerated entry and integration of transplantedells into the liver parenchyma [67].

The main complication of both intraportal and intrasplenicnfusion is thrombosis [6,57,60]. Thrombosis of portal veinrovokes hepatic failure, severe portal hypertension and hem-rrhage, and pulmonary emboli due cell migration.

.2. Ectopic sites for hepatocyte transplantation

Ectopic sites for hepatocyte transplantation are placesther than liver and spleen. Usually it is performed in dor-al fat, peritoneal cavity, renal capsule, lungs and pancreasTable 1). The advantage of these sites include low compli-ations rate and allowance of large amount of donor cells.urthermore, this approach simplifies the possibility of dis-

inction between donor hepatocytes and host cells, facilitatinghe recognition of the graft. Nevertheless, transplanted hep-tocytes into these ectopic sites reach considerable lowerurvival (2–3 months) compared to intraportal or intrasplenicnfusion [57]. Moreover, lack of hepatotrofic factors and otherubstances provided by portal flow results in poor neovascu-arization and proliferation of ectopicly transplanted hepa-ocytes [6,57]. Hepatocyte transplantation in ectopic sites isresented in Table 1.

Advances in hepatocyte biology has detected that extracel-ular matrix components (ECM) are important to improve thedequate engraftment of the donor cells [57,68,69]. There-ore, various methods have appeared to provide ECM com-onents. Hepatocytes attached to devices (microcarriers, syn-hetic fibers, biocompatible materials or bioabsorbable arti-cial polymers) as matrices, may survive for a longer period68–73]. Ajioka et al. [74] transferred the endothelial growthactor gene to the cultured hepatocytes and transplanted themo the peritoneal cavity. These authors observed genesis ofarge hepatocyte colonies and remarkable vascular net sur-ounding the graft [74].

. Clinical trials

Clinical hepatocyte transplantation has been indicated inhree circumstances: acute hepatic failure, chronic hepaticailure and metabolic disorders.

.1. Acute hepatic failure (AHF)

In 1994, Habibullah et al. [7] published their hepatocyteransplantation experience in seven AHF patients presentingepatic encephalopathy grade III and IV, with less than 2eeks of evolution. They used human fetal hepatocytes

F.H.F. Galvao et al. / Hepatology R

Tabl

e2

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esearch 36 (2006) 237–247 243

njected in a single intraperitoneal dose of 6 × 107 cells/kgf body weight by a dialysis catheter. All patients achievedomplete recovery of hepatic encephalopathy, while inontrol groups the survival rate was 50% in patients withncephalopathy grade III and 33% in encephalopathy gradeV.

Bilir et al. [2] performed hepatocyte transplantation inve patients with acute hepatic failure with contraindication

o liver transplantation. In this study, 0.1 × 1010 to 4.0 × 1010

epatocytes were injected into the spleen and two of theecipients got an additional intraportal infusion. Theybserved remission of encephalopathy from grades IV to IIr 0 and survival ranging between 14 and 52 days in threef the five patients. Furthermore, these patients achievedmprovement in protrombin time, ammonia serum level anderebral edema. Two early deaths due hypoxemia occurredithin 18 h after the transplantation in the patients that got

dditional hepatocyte infusion via portal. The necropsybserved hepatocytes embolus in the lungs of both patients.

The Medical College of Virginia [75] has the larger expe-ience in hepatocyte transplantation for AHF. Nine patientsere treated by this procedure and the results are summa-

ized in Tables 2 and 3. Seven out of nine patients wererought up successfully into liver transplantation or achievedegeneration of native liver. One patient died 5 days afterransplantation due sepsis by herpes virus. Another patientied of cardiopulmonary complications 2 days after trans-lantation. The seven remaining patients presented full recov-ry from their neurological and clinical status. One patientchieved ‘spontaneous’ recovery after hepatocyte transplan-ation, being dismissed from the waiting list for liver trans-lantation.

Recently two cases of hepatocyte transplantation wereeported. Khan et al. [76] described a case of a 26-year-ld patient diagnosed as acute fatty liver of pregnancynd encephalopathy grade IV. The patient achieved 3 × 108

uman fetal hepatocytes and the level of consciousnessmproved in 24 h. The patient recovered completely withindays. Baccarani et al. [77] described hepatocyte transplan-

ation in a liver transplant recipient with acute graft dysfunc-ion, not eligible for retransplantation. The patient underwentntraportal infusion of two billion viable cryopreserved ABOdentical human allogenic hepatocytes. Portal vein thrombo-is was identified 8 h after hepatocyte infusion leading theatient to death.

.2. Chronic hepatic failure (CHF)

In 1992, Mito et al. [9] performed the first clinical trialf hepatocyte transplantation. This trial included 10 patientsith CHF child A–C. Hepatocytes were obtained from liver

esection of the left lobe segment. The cells were infusedirectly in the splenic artery by an open-air surgical proce-ure. There were clinical improvement in only two patientsnd the authors did not credit it to the hepatocyte transplant.

244 F.H.F. Galvao et al. / Hepatology Research 36 (2006) 237–247

Table 3Hepatocyte transplantation for chronic hepatic failure at University of Virginia and University Hospital of Cincinnati

Patient Sex/age Co-morbities Improvement after hepatocyte transplantation Evolution

1 Fem/52 years Terminal hepatic failure ↓NH3 hemodynamic stability ↓ISP OLT 2nd day FR2 Fem/40 years Hepatitis C ↓NH3 hemodynamic stability Death in the 4th day by

complications due ISP3 Masc/6.5 months Colestase hepatic fibrosis enterocolitis ↓NH3 Intraskull hemorrhage4 Masc Mesenteric thrombosis ↓NH3 hemodynamic stability extubation Hospital discharge death after

33 days by self-suspension of

N ll recov

phtoopaT

8

mdeemw

i[eh

oct[wuahd3btp

9

b

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ote. OLT: orthotopic liver transplantation; ISP: intraskull pressure; FR: fu

The Medical College of Virginia and University Hos-ital of Cincinnati described four patients with CHF whoad received hepatocyte transplantation [75]. In this study,he cells were obtained by non-cirrhotic livers donors, cry-preserved for a variable period and infused intraportalr intrasplenic site by percutaneous arterial catheters. Allatients achieved coma, hemodynamically unstable, anuric,nd under breathing support. The results are summarized inable 3.

.3. Metabolic disorders

Hepatocyte transplantation can also be employed to treatetabolic disorders such ornithine transcarbamoylase (OTC)

eficiency, �1-antitrypsin deficiency, glycogen storage dis-ase type 1a, Crigler–Najar Syndrome type I, among oth-rs [1,78–81]. Hepatocyte transplantation may correct theetabolic defect and theoretically treat the metabolic diseaseithout liver transplantation.For OTC deficiency treatment, a report described repeated

ntraportal hepatocyte infusions during a period of 30 days1]. However, the responses were insufficient to treat the dis-ase, probably due the insufficient amount of genetic healthyepatocytes [1].

The best results of hepatocyte transplantation werebserved in the Crigler–Najar Syndrome. This disorder isharacterized by enzymatic deficiency in bilirubin conjuga-ion and excretion, leading to its accumulation in the organism81]. Current treatment is based in phototherapy sessionsithout satisfactory results and most patients must definitelyndergo OLT [82]. Fox et al. [80] infused via portal veinmount of hepatocytes equivalent to 5% of the normal hostepatocyte mass, into a patient with type 1 Crigler–Najar syn-rome. Bilirubin levels stabilized at 10.6–14.0 mg/dl by day5. Eleven months after hepatocyte transplantation, the biliru-in level remained at 14.0 mg/dl. The authors conclude thathe procedure did not lead to the disease’s cure, but improvedatient’s conditions in the waiting list for OLT.

. Bioartificial liver systems

The bioartificial is not the aim of this report and wille summarized. Briefly, bioartificial liver is an extra corpo-

1

m

dialysis

ery.

eal system that provides a hepatic support to the AHF orHF patient. The essence of this system is the perfusion ofatient’s blood into a bioreactor, which contains hepatocytesxed in a collagen matrix. This system clears the plasmarom toxic substances. Bioartificial liver systems differ inepatocytes source component and system design [83–89].ince the shortage of primary culture of human hepatocytes,orcine hepatocytes and human lineages of hepatoblastomaave been used in some system, which leads to immunolog-cal, infectuous and neoplastic concerns.

Artificial and bioartificial support systems may provide abridge” for patients with severe liver disease to recover orndergo to transplantation. Kjaergard et al. [87] in a system-tic review evaluate the effect of artificial and bioartificialupport systems for acute and acute-on-chronic liver fail-re. This review concludes that artificial support systemseduce mortality in acute-on-chronic liver failure comparedith standard medical therapy; nevertheless, these systemsid not appear to affect mortality in acute liver failure.

For the other hand, Demetriou et al. [88] describedhe safety and efficacy of an extracorporeal porcineepatocyte-based bioartificial liver in 85 patients with ful-inant/subfulminant hepatic failure and primary nonfunction

ollowing liver transplantation and compared with 86 patientsn the same conditions, but the bioartificial support (control).here was no difference in terms of overall survival between

he two groups; however, the subgroup of patient with ful-inant/subfulminant hepatic failure submitted to the bioar-

ificial support archived higher survival rate then patients onontrol group receiving standard supportive therapy.

Recently, the molecular absorbent recycling systems orxtracorporeal albumin dialysis technique appears to be aromising topic of this subject; nevertheless, data testing itsse in acute liver failure is still scant and difficult to assess.owever, in acute on chronic liver failure the extracorpo-

eal albumin dialysis technique has been shown to improveurvival compared to a similar randomized control groupeceiving standard supportive therapy [89].

0. Conclusion

Orthotopic liver transplantation is the contemporary treat-ent for end-stage liver disease. Advances in biotechnol-

logy R

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F.H.F. Galvao et al. / Hepato

gy have allowed hepatocyte transplantation as a promis-ng and less aggressive proposition to treat liver disease.ultured hepatocytes derived from multiple cell resource

adult, fetal, embryonic stem cells, bone marrow, engineeredmmortalized cells clones, tissue engineering devices, non-uman donors) is talented solution for the shortage organs.t is noticeable that existing clinical trials are insufficient toalidate hepatocyte transplantation as the standard manage-ent for serious liver dysfunction; however, the use of this

pproach holds theoretical merit and the remarkable progres-ion in cell therapy apparatus will undoubtedly inspire furtherrials of this modern therapy.

cknowledgement

Medical student Christine Akie Tatai for manuscript assis-ance.

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[

[

[

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[

[

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