Human hepatocyte transplantation: state of the art

doi: 10.1111/j.1365-2796.2009.02152.x

Human hepatocyte transplantation: state of the art

E. Fitzpatrick1, R. R. Mitry2 & A. Dhawan1,2

From the 1Paediatric Liver Centre; and 2Institute of Liver Studies, King’s College London School of Medicine at King’sCollege Hospital; London, UK

Abstract. Fitzpatrick E, Mitry RR, Dhawan A (Paediatric

Liver Centre; and King’s College London School of

Medicine at King’s College Hospital; London, UK).

Human hepatocyte transplantation: state of the art

(Review). J InternMed 2009; 266: 339–357.

Hepatocyte transplantation is making its transition

from bench to bedside for liver-based metabolic disor-

ders and acute liver failure. Over eighty patients have

now been transplanted world wide and the safety of

the procedure together with medium-term success has

been established. A major limiting factor in the field

is the availability of good quality cells as hepatocytes

are derived from grafts that are deemed unsuitable for

transplantation. Alternative sources of cell, including

stem cells may provide a sustainable equivalent to

primary hepatocytes. There is also a need to develop

techniques that will improve the engraftment, survival

and function of transplanted hepatocytes. Such devel-

opments may allow hepatocyte transplantation to

become an accepted and practical alternative to liver

transplantation in the near future.

Keywords: hepatocyte transplantation, liver disease,

stem cell transplantation.

History

Liver transplantation is the standard of care for end-

stage liver disease and many liver-based metabolic con-

ditions. Techniques involve whole organ replacement,

split or reduced donor liver and auxiliary liver trans-

plantation. Organs are in short supply, however, and

deaths still occur whilst awaiting transplantation. Hepa-

tocyte transplantation is an alternative to orthotopic or

auxiliary liver transplantation in the management

of liver-based metabolic conditions and liver failure

[1–7]. Advantages include allowing the native liver to

remain in place; in the case of acute liver failure, this

allows the possibility of native liver regeneration over

time; in metabolic liver disease, the native liver serves

as a back-up in case of graft failure and remains

available for the future possibility of gene therapy. The

procedure is considerably less invasive than organ

transplantation and cryopreserved cells are available

immediately for treatment of fulminant liver failure.

Hepatocytes are derived from organs that would other-

wise be rejected for transplantation. In general, these

often compromised livers may be steatotic or from non

heart beating donors [8]. Occasionally, a split liver seg-

ment or section IV may be used for isolation of hepato-

cytes [9]. The nature of the livers obtained means that

cell quality is often poor. Innovative methods to

improve cell quality and cryopreservation methods

have been a focus of research. The effective engraft-

ment of hepatocytes into the host liver also remains a

challenge. The optimal immunosuppression regime is

also unknown. Cells are generally lost from after

6–9 months and it is not clear if this is due to rejection,

apoptosis or other causes. The single most important

obstacle to hepatocyte transplantation is the limited

availability of hepatocytes. This has encouraged inves-

tigation into alternative sources of cell including stem

cells, immortalized cells and xenotransplantation each

with its own challenges.

Animal models

In the late 1970s Mito et al. demonstrated the survival

of rat hepatocytes transplanted into spleen [10].

Preclinical studies investigated the technique in animal

ª 2009 Blackwell Publishing Ltd 339

Symposium |

models of inborn errors of metabolism including the

Gunn rat for Crigler-Najjar syndrome type 1 [11],

Spf–ash mice for ornithine transcarbamylase defi-

ciency [12], the long-Evan’s rats for Wilson’s disease

[13], Nagase analbuminaemic rats for hypoalbumina-

emia [14], fumarylacetoacetate hydrolase knock out

mice for tyrosinaemia type 1[15], Mdr2 knock outs for

PFIC [15, 16], dogs with hyperuricosaemia [17], mice

with histidemia [18] and Watanabe rabbits with hyper-

cholesterolaemia [19]. These models demonstrated

medium to long-term improvement in biochemical

abnormalities. The success of breeding immunodefi-

cient mice crossed with knock out mouse models of

various metabolic diseases has allowed further study

of the therapeutic potential of human hepatocyte

transplantation.

In addition, rodent models of hepatocyte transplantation

in acute liver failure induced by d-galactosamine [20],

dimethylnitrosamine [21], 90% hepatectomy [22] or

ischaemic injury [23] demonstrated improved survival.

Many questions remain, however, and effects demon-

strated in these rodent models are not always transfer-

able to human studies. It is clear that larger animal

models are needed to bring innovations closer to the

bedside.

Indications

In all there have been over 80 case reports of clinical

human hepatocyte transplantation from 13 centres.

Metabolic disease

Almost 30 children and adults who received hepato-

cyte transplantation for liver-based metabolic liver

disease are reported in literature, (Table 1) with

demonstrable, although short-term correction of

metabolic deficiency in majority of cases. The aim of

hepatocyte transplantation in metabolic disease is to

replace the missing function without the need to

replace the whole organ. Of course this is dependant

on engraftment. It is not known how many cells are

needed to correct a deficit. For example in Crigler

Najjar syndrome, approximately 12% of liver mass is

needed [24]; however, fewer cells may produce an

effect in other conditions such as Ornithine transcar-

bamylase deficiency (OTC) or Glycogen Storage dis-

ease(GSD) 1a. Limited numbers of hepatocyte may be

infused at a time and achieving effective therapeutic

mass may require repeated infusions.

Grossmann first described gene therapy using autolo-

gous hepatocyte transplantation for familial hypercho-

lesterolemia. Autologous hepatocytes were transduced

with LDL receptor expressing retroviral vector and

transplanted back into patient. Unfortunately, a useful

reduction in LDL levels was not achieved [25]. Autol-

ogous transplantation allows the elimination of the

need for immunosuppression; however, effective

genetic manipulation of diseased autologous cells has

not yet become a therapeutic reality.

Other conditions such as OTC deficiency are candi-

dates for hepatocyte transplantation. The neonatal

form of OTC deficiency can lead to death or severe

neurological consequences of hyperammonemia.

Experience in patients has shown successful use of

hepatocyte transplantation as a bridge to liver trans-

plantation in a number of children [6, 9, 26, 27].

Crigler-Najjar disease, a rare autosomal defect of the

UGT1A1 gene complex leads to severe unconjugated

hyperbilirubinaemia secondary to impaired glucuroni-

dation of bilirubin. Unconjugated bilirubin can cross

the blood brain barrier and cause severe neurological

damage and death. The mainstay of management is

with daily phototherapy though the inconvenience and

risk of hyperbilirubinaemia during intercurrent illness

means a permanent replacement of function with liver

transplantation is appealing. Hepatocyte transplanta-

tion has been successfully used as an alternative to

whole organ transplantation with a transient decrease

in bilirubin for up to 11 months [4, 28–33].

Hepatocyte transplantation for Glycogen storage dis-

ease 1a and 1b in an adult and a child respectively

resulted in an improvement in glucose control in both

cases [5, 34]. The peroxisomal disorder, Refsum dis-

ease was treated with hepatocyte transplantation in

4 year old girl with good biochemical and neurologi-

cal outcome [35].

E. Fitzpatrick et al. | Symposium: Human hepatocyte transplantation

340 ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 339–357

Table 1 Human hepatocyte transplantation for metabolic liver disease

Disease Age Reference Cells Outcome

Urea cycle

OTC 5 yr Strom et al.[26] 1 · 109 fresh, intra-portal Died day 43, decreased ammonia initially

OTC Day2 Mitry et al.[9] 1.9 · 109 fresh ⁄ frozen Decreased ammonia; Auxiliary

transplant at 6 months

OTC Day1 Horlsen et al.[6] 4 · 109 fresh 11 days ammonia controlled; OLT at 6 months

OTC 14 m Stephenne et al.[27] 2.4 · 109 frozen Decreased ammonia; OLT at 6 months

ASL 3 yr Stephenne et al.[36] 3.4 · 109 Decreased ammonia, FISH for donor cells;

OLT at 18 months

OTC 10 wk Meyburg et al.[37] Some stabilization

OTC 3 y Meyburg et al.[37] 3 · 109 Some stabilization

Citrullinemia 2 y Fisher and Strom[38] Decreased ammonia from 2 weeks to 6 months

Crigler-Najjar I 10 y Fox et al.[4] 6 · 109 fresh 60% decrease in bilirubin up to 11 months

8 y Darwish et al.[28] 7.5 · 109 fresh ⁄ frozen 40% decrease bilirubin up to 6 months;

OLT at 20 months

9 y Ambrosino et al.[39] 7.5 · 109 fresh 50% decrease bilirubin weeks; OLT

18 m Dhawan et al.[30] 4.3 · 109 50% decrease in bilirubin to 7 months;

OLT at 8 months

3 y Dhawan et al.[30] 2.1 · 109 30% decrease in bilirubin; OLT at 18 months

8 y K. Allen (unpub) 1.5 · 109 30% decrease in bilirubin 5 months;

lost to follow up

2 y Khan et al.[31] 15 million HFH EpCAM+ 50% decrease in bilirubin at 2 months

1 y Lysy et al.[32] 1.5 · 109 50% decrease in bilirubin at 5 months

GSD1a 47 y Muraca et al.[5] 2 · 109 fresh Better fasting time, decrease in triglyceride

up to 18 months

GSD1b 18 y Lee et al.[34] 6 · 109 fresh ⁄ frozen Improvement in glucose control, normal enzyme

levels on biopsy

Refsum 4 y Sokal et al.[35] 2.1 · 109 fresh ⁄ frozen Partial clearance abnormal bile acids, development

improved to 18 m

Factor VII def. 3 mo Dhawan et al.[7, 30] 1.1 · 109 frozen Requirement for recombinant factor VII 20% of

pre transplant level; OLT (venous access) at 7 and

8 months; 6 months function declined

3 y 2.2 · 109 fresh ⁄ frozen

3 mo A. Dhawan (unpub) 3 · 109 fresh ⁄ frozen OLT

PFIC1 16 mo Dhawan et al. [30] 2.3 · 108 fresh No benefit; OLT 5 and 14 months later

3 yr 3.7 · 108 fresh

Hyper-

Cholesterolemia

Grossman et al.[25] 1.1 · 109 fresh 20% decrease in cholesterol, LDL, ApoB to 28 month

1.3 · 109 fresh No effect

1 · 109 fresh 6% decrease in cholesterol, LDL, ApoB to 19 months

3.2 · 109 fresh Minor effect

1.5 · 109 fresh 20% decrease in cholesterol up to 7 months

OLT: orthotopic liver transplant, F ⁄ up: follow up, Tx: transplant, HFH: human foetal hepatocytes, EpCAM: Epithelial Cell Adhesion Molecule,ApoB: apolipoprotein B, GSD: glycogen strorage disease, OTC: ornithne transcarbamylase deficiency ASL: arginine succinyl lyase deficiency,PFIC: progressive familial intrahepatic cholestasis, FISH: fluorescent in situ hybridization, LDL: low density lipoprotein


ª 2009 Blackwell Publishing Ltd Journal of Internal Medicine 266; 339–357 341

Factor VII deficiency is a rare autosomal recessive

disorder, three boys from the same kindred underwent

hepatocyte transplantation resulting in a decrease in

requirement for recombinant factor VII at 6 months to

20% of pretransplant levels. However, as the boys

were still dependent on recombinant factor and central

line thrombus and sepsis were considerable issues, all

three have since had OLT [30].

Progressive familial intrahepatic cholestastis was dem-

onstrated to respond to hepatocyte transplantation in a

mouse model though there was no benefit found in

two children [30]. The lack of benefit was thought to

be due to the fact that as fibrosis was already estab-

lished in these children, the disrupted liver architec-

ture did not allow effective engraftment.

Acute and chronic liver failure

In acute liver failure, hepatocyte transplantation may

act as a bridge to recovery and regeneration of the

injured native liver or alternatively to orthotopic liver

transplantation once an organ becomes available

(Table 2). The procedure may also be used in

patients who are not candidates for organ transplan-

tation. A major advantage of hepatocyte transplanta-

tion is the immediate availability of cryopreserved

cells. Sufficient cell mass (approximately 10–15% of

liver cell mass) is needed to provide enough function

to sustain metabolic function [24]. The mass of cells,

which can be transplanted into the liver, is limited

by the effect on portal hypertension. Other options

include intrasplenic or intraperitoneal transplantation,

which allow a larger volume of cells. The spleen

has been used successfully in animal [40, 41] and

human transplantation [2]; however, in view of the

number of immunologically active cells located in

the spleen, rejection or destruction of the nonnative

cells needs consideration.

Hepatocyte transplantation in patients with ALF has

resulted in a reduction in ammonia and bilirubin with

improvements in hepatic encephalopathy and cardio-

vascular instability [2, 38, 42]. In the absence of any

randomized controlled trials, it is difficult to comment

on the true efficacy of the intervention.

In chronic liver disease, or acute on chronic dysfunc-

tion, the aim of hepatocyte transplantation is both to

replace function and to allow the cells engraft and

repopulate the liver (Table 3). As the liver architecture

is disrupted in chronically damaged liver, there are

difficulties with engraftment. This indication for hepa-

tocyte transplantation has been the least successful to

date, though there was some improvement in a patient

with acute on chronic alpha 1 anti-trypsin deficient

liver disease where it served as a bridge to OLT [26].

Again there was some improvement in a patient with

hepatitis C; however, this patient did not survive to

organ transplant [26].

Procedure

Isolation

Hepatocytes are isolated from livers, which are

donated but unsuitable for organ transplantation. In

particular, these are the steatotic livers, those from

nonheart beating donors (DCD or donation after car-

diac death) and those with long cold ischemic times

or otherwise ‘marginal’ organs. In general, this means

that these livers of poor quality, but can, however,

still yield reasonable numbers of good quality cells.

In addition, segment IV that may be isolated and not

used in split liver procedures can be a valuable source

of cell for hepatocyte transplantation [9].

In order to isolate hepatocytes, a collagenase perfu-

sion technique [51] with modifications [52] is used

(Fig. 1). The tissue is weighed and cannulated with

1–4 cannulae. Perfusion is undertaken using flow

rates of 20–80 mL min)1 and all buffers (Cambrex,

Berkshire, UK) are maintained at 37 �C. The first buf-

fer consists of Hank’s Balanced Salt Solution contain-

ing 0.5 mmol L)1 of the Ca2+chelator ethylene

glycol-bis (2-aminoethylether)-tetraacetic acid and

5 mmol L)1 4-(2 hydroxyethyl)-1-piperazineethane-

sulphonic acid; the second buffer consists of Hank’s

Balanced Salt Solution only and then Minimum

Essential Medium with Eagle’s Balanced Salt Solution

(EMEM) containing collagenase (SERVA, Heidelberg,

Germany). Hepatocytes are purified by washing three

times with ice-cold EMEM containing 2% human



Table 2 Human hepatocyte transplantation for acute liver failure

Indication Ref Patients Cells Outcome

Drug Strom et al.[3] 27 y dilantin 2.8 · 107 OLT d10

Strom et al. [42] 26 y acetaminophen OLT d2

Strom et al.[42] 13 y dilantin Died d4

Strom et al.[42] 43 y halothane Died d35

Soriano et al.[43] 15 y paracetamol Died d2

Soriano et al.[43] 12 y isoniazid Died d4

Bilir et al [2] 32 y polysubstance 1.2 · 109 IS Died d14

Bilir et al.[2] 35 y polysubstance 1 · 1010 IS Died d20

Bilir et al.[2] 55 y chloroform ⁄C2H5 3.9 · 1010 IS Died 6 hrs

Soriano et al.[43] 10 y phenytoin 2.8 · 109 Died d7

Fisher and Strom[38] 14 y tegretol 2.5 · 109 D1 OLT

Fisher and Strom[38] 21 y polysubstance 1.8 · 109 IS Died d1

Fisher and Strom[38] 35 y polysubstance 5.4 · 109 IP Died d18

Fisher and Strom[38] 35 y polysubstance 3.7 · 109 Full recovery no OLT

Fisher and Strom[38] 51 y polysubstance 3.9 · 109 Died d3

Viral Strom et al.[3] 28 y acute HBV 1.9 · 107 OLT d3

Fisher et al.[44] 37 y acute HBV 8.8 · 108 Recovery no OLT

Strom et al.[42] 37 y HSV ⁄ valproate Died d5

Strom et al.[42] 43 y acute HBV OLT d1

Fisher and Strom[38] 54 y HBV lymphoma Died d7

Fisher and Strom[38] 4 y viral 1.7 · 109 ICH died d2

Bilir et al.[2] 29 y HSVII 1 · 1010 Died 18 hr

Bilir et al.[2] 65 y HBV 3 · 1010 Died d52

Habibullah et al.[45] 40 y HBV Died d13

Meyburg et al.[37] 3 weeks congenital HSV 6 · 107 ⁄ kg IP Died

Miscellaneous Strom et al.[42] 23 y idiopathic OLT d5

Strom et al.[42] 69 y post trisegmentec. 1.7 · 108 Died d2

Strom et al.[42] 5 month old idiopathic Died d2

Soriano et al.[43] 3 y old idiopathic Full recovery; No OLT

Soriano et al.[43] 5 y old idiopathic 2 · 109 OLT d4

Sterling et al.[46] 3 months old idiopathic 1.8 · 108 OLT d1

Fisher et al.[38] 48 y Reyes 7.5 · 108 Died d1

Schneider et al.[47] 64 y mushroom 4.9 · 109 Recovered; No OLT

Habibullah et al.[45] 8 yr 6 · 107 ⁄ kg IP Recovered

Habibullah et al.[45] 32 y 6 · 107 ⁄ kg IP Died 30 hr



Habibullah et al.[45] 20 y 6 · 107 ⁄ kg IP Recovered

Habibullah et al.[45] 24 y 6 · 107 ⁄ kg IP Recovered

OLT: orthotopic liver transplant, C2H5: alcohol, IP: intraperitoneal, IS: intrasplenic, HBV: hepatitis B virus, HSVII: herpes simplex II, triseg-mentect.: trisegmentectomy.



serum albumin (centrifugation at 50 g for 5 min at

4 �C). The cell number and viability are determined

using a haemocytometer and trypan blue exclusion

technique. On average 4–5 · 106 hepatocytes per

gram of liver tissue are isolated.

Cells are used as soon as possible for transplantation

since function deteriorates over hours. Cryopreserva-

tion techniques have been developed (see below) and

cells may be stored at )140 �C and can be thawed for

immediate use. This ready availability of cryopreserved

cells is of particular importance for the treatment of

acute liver failure.

Good manufacturing practice (GMP) processing

In order to minimize risk to the patient and to comply

with human tissue authority (HTA) directives, an

aseptic environment is needed in which to prepare

cells on a large scale. Good manufacturing practice

conditions are adhered to and standard operating

practices are in place for all steps of the procedure.

Table 3 Human hepatocyte transplantation for acute on chronic liver disease

Indication Reference Patient Cells Outcome

Alpha 1 anti-trypsin deficiency Strom et al.[26] 52 y 2.2 · 107 IS OLT d2

Strom et al.[42] 18 weeks OLT d4

Viral Strom et al. [26] 40 y Hepatitis C 7.5 · 106 IC bleed died

Miscellaneous Strom et al. [26] 6 months NEC ⁄ sepsis 5.2 · 107 IC bleed died

Mito et al.[48] 10 patients: autotransplant Age 46 – 78 y From 2 · 107 – 2 · 108 Detected at 6 mo

Soriano et al.[43] 3 weeks;cryptogenic 7 · 108 OLT d7

Soriano et al.[43] 3 months idiopathic 5 · 108 OLT 6 weeks

Baccarani et al. [49] 56 yr graft failure 2 · 109 frozen Died d2

Khan et al.[50] 1 yr old biliary atresia hepatic artery Decreased bilirubin

Alcohol Strom et al.[42] 62 y Died d33

Strom et al.[42] 46 y Died d50

Bilir et al.[2] Alive

Bilir et al.[2] Alive

Bilir[2] Alive

OLT: orthotopic liver transplant, IS: intrasplenic, HBV: hepatitis B virus, HSII: herpes simplex II, IC bleed: intracranial haemorrhage, NEC:necrotizing enterocolitis.

Fig. 1 Isolation of hepatocytesfor transplantation.



A HEPA filter removes particles from the air and an

air-handling unit maintains a temperature controlled

environment within the HTA approved cell isolation

unit. A gradient of air pressures provides the highest

pressure in the aseptic room where the tissue

processing is performed. Quality control issues

include strict environmental microbiological monitor-

ing. All donated organs are screened for viral

infection HIV ⁄Hepatitis B and C. For clinical

transplantation, hepatocytes should have a viability

>60% and an absence of microbiological contamina-

tion [53].

In order to improve the quality of cells obtained,

N-acetyl cysteine may be used as perfusion agent

prior to collagenase digestion in order to improve the

quality of yield [54]. Protocols for transplantation are

based on those from Strom et al. [42]. Transplanted

hepatocytes are ABO compatible. Up to 100 million

cells per kg body weight or 2.4 · 106 per gram native

liver may be transplanted via portal vein as a single

procedure. As the average adult liver contains

2.8 · 1011 hepatocytes, no more than 1% of liver

mass can be transplanted at any one infusion [55].

Repeated infusions can be undertaken at intervals

monitoring portal pressure until the transplanted hepa-

tocyte mass reaches 10% of recipient mass. Unfortu-

nately, at present, there is no effective method of

determining the proportion of administered cells that

survive and engraft.

Embryonic stem cells Haematopoietic stem cells

Liver-specific progenitor cellsProgenitor/oval cellsLiver derived MSC

Foetal progenitor cells

Mesenchymal stem cells

Potential sources ofcell for hepatocyte

transplantation

- Unlimitedexpansion/availability

- Incomplete function- Potential fortumorigenisis

- Less immunogenic

- Organ specific/committedprogentiors- Proliferativepotential

- Tissue difficult toattain

- Incomplete function

- Availability,expandable- Less immunogenic

- Possibility forautotransplantation

-Antiinflammatory

- Fibrogenic potential- Incomplete differentiation

- Uncertainty aboutwhich cell to target –stem cell or hepatoblast

Pros Cons- Availability of cells - Potential for fusion

and nucleardisruption- Tumorigeneisis

- Functiondemonstrated inanimal models

Pros Cons

- Availability of cells - Potential for fusionand nucleardisruption- Tumorigeneisis

- Functiondemonstrated inanimal models

Pros Cons

Pros Cons

Pros Cons

Fig. 2 Potential alternative sources of hepatocytes for transplantation.



Transplantation techniques

There are different approaches to transplantation of

hepatocytes and a number of different sites have been

studied. Probably, the most widely accepted in clinical

practice is intraportal delivery of hepatocytes. The

cells move across the sinusoids and ideally engraft in

the liver. Pulmonary infiltrates have been reported in

some cases suggesting that some cells may also lodge

in pulmonary capillary bed [2]. The number of

hepatocytes that may be transplanted into the portal

vein at a time is also limited by portal pressure thus

intrasplenic transplantation has been suggested as an

alternative. The spleen has good capacity as a site of

transplantation and may be particularly useful in

chronic liver disease where the deranged liver archi-

tecture prevents hepatocyte engraftment. The exposure

of hepatocytes to the immunological environment of

the spleen is a disadvantage. Injection into the splenic

artery has also been associated with splenic necrosis

[56].

The intraperitoneal route has been used to accommo-

date sufficient cell mass to sustain function in acute

liver failure [45]. The attack of the host immune sys-

tem and lack of anchorage are the major challenges

encountered with this mode of delivery. Encapsulation

in a bio-inert material such as alginate may protect

the cells from the host immune response and provide

a scaffold to sustain survival and function [57].

The renal capsule, which has been used as a site of

transplantation in animal studies, can allow relatively

easy access for biopsy to monitor survival of trans-

planted cells [58]. The small space available may not

be sufficient for transplantation of a meaningful cell

mass in humans however.

Our experience has been with intraportal transplanta-

tion. In neonates, umbilical vein cannulation appears

to be an ideal route of entry into portal vein. The

position of the catheter is confirmed with contrast

prior to the transfusion. If the ductus venosus is still

patent, there is a risk of pulmonary embolization of

hepatocytes, but selective right portal vein infusion is

possible under fluoroscopy. In older children, surgical

or radiological line placement is necessary with radio-

logical catheter guidance into portal vein. An alterna-

tive approach is the middle colic vein via the inferior

mesenteric vein [37]. Portal venous pressure is mea-

sured during the infusion.

Complications include transient portal hypertension

(lasts 2–3 hours post procedure), sepsis, haemody-

namic instability during the infusion, sepsis and

embolization to pulmonary capillary bed. This last

complication may present with hypoxemia and chest

X-ray infiltrates [3–5] and post mortem examination

of hepatocyte recipients has shown hepatocytes in the

alveoli [59]. The risk of portal plugging and ischemia

is minimized by limiting the number of cells per infu-

sion to 30–100 million cells per kg weight with an

infusion speed of 4–8 mL)1kg)1h)1 and with the

addition of heparin to infusion [4].

Cell engraftment

The fate of the hepatocytes following transplantation

is dependent on their interaction with the local micro-

environment; the extracellular matrix, soluble media-

tors such as cytokines and growth factors and the

immunological response of the host [60]. Depending

on the indication for the transplant, either immediate

short-term function or longer term engraftment and

repopulation may be required. The quality of cells

may affect success of engraftment after transplanta-

tion.

The hepatocyte is roughly 20–40 lm in diameter; it

becomes entrapped in sinusoids which have 6–9 lmfenestrations. This causes portal hypertension and

transient ischaemia-reperfusion injury. This effect on

the microvasculature stimulates Kuppfer cells and

TNF alpha release thus inducing vascular permeability.

Hepatocytes stick to the activated endothelium and

translocate through the sinusoidal fenestrations where

they integrate into the liver parenchyma forming gap

junctions and bile canaliculi between transplanted and

host hepatocytes [61]. Hepatic remodelling occurs in

rodents in 3 to 7 days where the transplanted cells

become histologically indistinguishable from host cells

and transplantation of 3–5% host liver mass results in



0.5% engraftment of the host liver [60]. A survival

advantage [62] or regenerative stimulus is required for

any significant repopulation to occur [63].

Cell-to-cell interactions between transplanted hepato-

cytes, Kuppfer cells and stellate cells modulate hepato-

cyte engraftment [64, 65]. The extracellular matrix

also has an important role, experimental infusion of a

fibronectin polymer increases hepatocyte engraftment in

a rodent model [66].

Various experimental techniques have been described

to improve engraftment of transplanted hepatocytes.

Animal experiments often use partial hepatectomy as

a regenerative stimulus together with retrorsine to

block regeneration of native hepatocytes. Though this

is not clinically applicable, temporary embolization of

the portal vein can generate ischaemia reperfusion

injury and simulate such a regenerative stimulus [67].

Macaca monkeys underwent partial reversible emboli-

zation of the portal vein and subsequent autologous

hepatocyte transplantation resulting in replacement of

10% of liver mass [68].

Liver irradiation (25Gy) has been investigated in a rat

model and the arrest of native hepatocyte proliferation

induced by irradiation may promote preferential pro-

liferation of transplanted hepatocytes following partial

hepatectomy [69].

Administration of growth factors such as TGF alpha

can also accelerate hepatocyte repopulation after

hepatocyte transplantation. This is evident from rodent

experiments where hepatocytes are harvested from

transgenic mice which over-express TGF alpha [70].

The cells from the transgenic animals have more

proliferative activity and are more resistant to Fas

mediated apoptosis once transplanted.

Tracking engraftment

At present, tracking cell engraftment in a clinical

context is problematic. Animal experiments make use

of cell labelling using green fluorescent protein (GFP)

transfection and Hoechst dye. Cell expansion may be

assessed using BrdU which is a synthetic nucleoside

that is incorporated into the daughter cells of proliferat-

ing cells. Dyes may leak and dilute further with cell

division however. Local scavengers such as Kuppfer

cells may take up particles leading to false positive

results. Cell function may also be affected by labelling.

In vivo tracking is more difficult. Obviously markers of

successful engraftment include evidence of disease

correction. The degree of liver chimerism has been

assessed in a patient by serial transjugular biopsy by

monitoring a HLA class 1 antigen in a patient with

mismatched HLA hepatocyte transplantation [44].

New techniques using iron oxide particle labelling

with in vivo MRI tracking are under development

[71]. Again there are issues with the potential toxicity

of the marker and specificity to the transplanted he-

patocytes rather than scavenging cells.

Immunosuppression

Originally hepatocytes were thought to be less immu-

nogenic than whole organ transplants and it was

speculated that immunosuppression may be unneces-

sary [60], although this idea was quickly abandoned

[72]. Even with immunosuppression, there are still

problems with long-term survival of hepatocytes. It is

yet unclear whether the mechanism of cell loss is

secondary to nonspecific mechanisms such as apopto-

sis from lack of engraftment or specific cell-mediated

or humoral rejection. Current immunosuppressive

protocols have been informed by experience in both

islet transplantation and liver transplantation. Initially

intravenous methylprednisolone with cyclosporine or

tacrolimus were favoured [30]. Now an IL2 receptor

monoclonal antibody (daclizumab) with low dose

tacrolimus and sirolimus is increasing in popularity.

This protocol is derived from islet transplantation

and is used in part to spare the diabetogenic high dose

steroids [73].

Monitoring rejection is a challenge. In the case of ini-

tially demonstrable disease correction, loss of function

may indicate rejection. Markers such as granzyme

B and perforin have been postulated as effective

markers of rejection but have yet to be widely

accepted [74].



There are few studies of hepatocyte immunology in

the context of hepatocyte transplantation. Allen et al.examined graft loss following hepatocyte transplanta-

tion in a patient with Crigler-Najjar type 1 [75]. They

demonstrated a gradual reduction of graft function

due to cell loss. The transplanted cells demonstrated

good viability and plating efficiency initially, suggest-

ing that anoikis was unlikely to be the main mecha-

nism. They found no evidence for humoral rejection

and concluded that cell mediated rejection was the

most likely cause of graft loss based on the patient’s

CD8 + T-cell alloreactivity directed against a donor

HLA antigen.

Differences in the immunological response to hepato-

cytes and to whole organs may include the absence of

co-transplanted immunological cells (APCs which are

important in priming immune response and inducing

T-regulatory cells involved in tolerance induction) and

alterations in surface markers of hepatocytes during

isolation [76]. At present, this is speculative.

Wesolowska et al. used a model of sublethal whole

body irradiation and reconstitution with syngenic BM

cells with elimination of NK cells using anti-

asialoGM1 antiserum to study the response to trans-

planted hepatocytes [77]. This work concluded that the

innate immune response has an important role in the

rejection of transplanted hepatocytes. Inhibition of

liver-resident NK cells by specific or local immunosup-

pression could improve engraftment and proliferation.

Novel techniques have been used to avoid need for

immunosuppression. Encapsulation of cells into algi-

nate beads should protect the cell from the host

immune response [78]. Co-transplantation with immu-

nomodulatory cells may also prove beneficial [79].

This technique is suitable for acute liver failure rather

than metabolic disease as long term engraftment and

function of the encapsulated cells is not expected.

Cryopreservation and cell banking

The importance of immediate availability of cells

is especially relevant to treatment of acute liver

failure. Both animal models and clinical case reports

have demonstrated the feasibility and success of

transplanting previously cryopreserved hepatocytes.

This is a distinct advantage of hepatocyte over liver

transplantation.

Following isolation, hepatocytes are kept at 4 �C and

are cryopreserved as soon as possible. The cryopreser-

vation protocol optimized in our unit is based on that of

Diener et al. [80] with some modifications in a

controlled-rate freezer (Kry 10, Series III; Planer PLC,

Middlesex UK). Cryopreservation medium consists of

University of Wisconsin solution to which dimethyl-

sulfoxide is added to give a final concentration of 10%

(v ⁄v). Glucose is added to an overall concentration of

10%. Cells are frozen at high density (1.0–1.5 · 107

cells mL)1) to allow large numbers to be thawed

quickly. After cryopreservation, the hepatocytes are

immediately transferred to the vapour phase of liquid

nitrogen for storage until thawing. Preincubation of

hepatocytes with cytoprotectants such as glucose,

fructose and alpha lipoic acid prior to cryopreservation

can improve viability and function on thawing [81, 82],

as can cryopreservation as a three-dimensional structure

in an alginate gel capsule [83].

Thawing is rapid at 37 �C with slow dilution of the

cytoprotectant at 4 �C. Hepatocytes are then washed

well. The process of isolation and cryopreservation is

known to disrupt many cellular functions and the aim

of pretreatment of cells, optimum freezing solutions

and thawing protocols is to minimize this damage.

Postthaw hepatocyte function is generally poorer than

fresh with a decrease in viability of 20–30% [84].

Cells demonstrate a low attachment efficiency follow-

ing cryopreservation which may be of importance in

engraftment. Loss of cellular adhesion molecules may

be implicated in impaired hepatocyte attachment [85].

Source of hepatocytes

The supply of good quality livers for hepatocyte

isolation is a major issue. There is a pressing need for

an easily available cell equivalent to primary hepato-

cytes. Stem or progenitor cells have many advantages

as an alternative source of cell for hepatocyte

transplantation (Fig. 2). They are readily available and



may be effectively expanded in vitro or in vivo.Autologous cells (from bone marrow for example)

may be used, eliminating the need for immunosup-

pression [86]. It has also been suggested that stem

cells may be more resistant to cryopreservation [87]

and are less immunogenic [88, 89].

Stem cells are cells which self renew and produce

daughter cells that differentiate along different lin-

eages whereas progenitor cells are cells capable of

self renewal, which may be induced to differentiate

into a specific mature cell type under certain condi-

tions. The major criteria for stem cells also include

the ability to repopulate an organ in vivo. The terms

are often used interchangeably. Stem ⁄progenitor pop-

ulations under investigation for cellular therapy of

liver disease include ‘oval cells’ in adult liver [90],

foetal hepatocytes [91], embryonic stem cells [92],

bone marrow derived haematopoietic (HSC) and

mesenchymal stem cells (MSC) [93, 94], and also

MSC from peripheral blood [95], umbilical cord [96]

and adipose tissue [97].

Early experimental evidence for progenitor cells was

demonstrated by Petersen et al. [94], since that time

there have been numerous studies showing expression

of liver specific genes by differentiated stem cells in

vitro and in vivo. Proof of true functionality of these

cells has been less frequent. The major question

remains: what characterizes a hepatocyte. Clearly this

is not merely the ability of a cell to express albumin

but the ability to provide a variety of other hepatocyte

functions in order to be suitable for clinical use. Cyto-

chrome P450 enzyme expression, glycogen storage,

the ability to synthesis urea and conjugate bilirubin

are amongst the functions required. Most in vivo

experimental data comes from work with either syn-

genic or immunosuppressed rodents. Interpreting these

results in the context of interspecies differences is dif-

ficult. In addition, xenotransplantation of human cells

into rodents is likely not reflective of clinical poten-

tial. It is not clear as yet whether transdifferentiation

or fusion is responsible for change in phenotype of

the cells. For example, with severe selection pressure

such as in the FAH ) ⁄ ) mouse (model for tyrosina-

emia type 1), successful repopulation occurs with

human HSC mainly because of HSC fusion with host

hepatocytes [98]. The tumorigenic potential of modifi-

cations to the genome need to be considered. Stem

cells for cellular therapy of liver disease have also

been the subject of a number of excellent reviews

[86, 99, 100].

Embryonic stem cells

Human embryonic stem cells (hESC), isolated in

1998 [101] have generated great interest and contro-

versy over the last decade. Despite their undoubted

therapeutic promise, their effective clinical application

has remained elusive to date.

Armed with knowledge of liver embryology, a physi-

ological approach can be taken to the differentiation

of hESC cells. The induction of hepatic lineage mark-

ers has been demonstrated using factors involved in

early hepatic specification and maturation. These

include Activin A, FGF, BMP, HGF, Oncostatin M

and dexamethasone [92].

Mouse ESC which have been differentiated to hepato-

cyte like cells were transplanted into models of liver

injury demonstrating evidence of improved survival

without malignancy [102, 103]. Human ESC lines

have been effectively differentiated to hepatocyte line-

age with evidence of repopulation of immunosup-

pressed mouse model of liver injury [104, 105].

However, undifferentiated human ESC transplanted

into immunodeficient mice have also resulted in

teratoma formation [102, 106]. This issue with safety

is a major obstacle to clinical translation.

Human embryonic stem cells are thought to be less

susceptible to immune rejection than more mature

cells because of their low immunostimulatory poten-

tial [107], although the true capacity of hESC to

evade the immune response remains unknown.

Hepatoblasts and foetal liver progenitor cells

The foetal liver is a source of more committed pro-

genitor cells in the form of hepatoblasts. These cells

retain their proliferative capacity but are ‘directed’



specifically towards development of hepatocytes and

cholangiocytes. There has been success in using

progenitor cells from foetal liver in animal models

[87] and clinical case reports in acute liver failure

[45] and Crigler-Najjar syndrome [31].

Previous studies have distinguished between the hepa-

tic stem cell, which is present at constant level

throughout life, and the hepatic progenitor cell (or

hepatoblast), which is abundant in foetal liver at

certain stages of development [108].

Results from animal experiments show successful

repopulation of immunodeficient mouse and rat liver

with rodent foetal hepatocytes following host liver

injury [109, 110]. Human foetal liver cells have also

been transplanted into hepatectomized, immunodefi-

cient mice. These cells proliferated spontaneously in

culture, had a bipotent phenotype for hepatocytes

and cholangiocytes and resulted in up to 10%

repopulation in vivo. The engrafted cells expressed

human albumin, had the ability to store glycogen

and inducible cytochrome P450 activity [87, 111,

112]. Experiments using human foetal cells suggest

that it is possible to establish long-term cultures of

cells [112, 113]. Progenitor cells which could

clonally expand insufficient number for clinical use

and differentiate in vivo are an ideal source for

hepatocyte transplantation [108, 111, 114]. Again

there is some concern regarding the ability of

progenitor cells to differentiate into functionally

mature hepatocytes [115]. The most appropriate

microenvironmental conditions for this have not yet

been fully defined. An ‘adult’ microenvironment

may be conducive to differentiation. An increase in

liver-specific function was found in foetal rat liver

cells which were co-cultured with adult rat

hepatocytes [116]. Foetal liver cells are thought to

be relatively hypoimmunogenic, expressing MHC I

poorly and no MHC II [88]. This could potentially

be useful in transplantation.

Problems with this source include difficulty accessing

tissue with uncertainty around the most appropriate

gestational age to target. There are also some ethical

concerns surrounding the harvesting of foetal tissue.

By contrast, ‘adult’ stem cells combine the ability to

differentiate into liver cells with proliferative capacity.

Use of this source is less ethically complex and closer

to the clinic than embryonic or foetal liver stem cells.

Endogenous liver stem cells

It is well known that the liver has the ability to regen-

erate by division of mature hepatocytes. This is dem-

onstrated to good effect following partial hepatectomy

[117]. Ordinarily hepatocytes turn over once or twice

a year [100]. When stimulated by the release of vari-

ous cytokines, growth factors and other mitogens full

regeneration of the liver can occur following 2–3

cycles of mature hepatocytes [118]. A second com-

partment of facultative stem cells become active in

the presence of long standing injury or impaired

proliferation of mature hepatocytes. This occurs when

replicative senescence is reached. These cells are

known as oval cells in the rodent and are located in

the intrahepatic biliary tree within the canals of

Herring [90]. They have been identified in the human

liver and give rise to bipotent transient amplifying

cells, which have the potential to become both

cholangiocytes and hepatocytes. Mesenchymal like

cells have also been isolated from human livers,

which demonstrate the ability to differentiate into

hepatocyte like cells in vitro and in vivo [119, 120].

Bone marrow stem cells; HSC and MSC

Interest in bone marrow stem cells as a potential

source of hepatocyte arises from early work by Peter-

sen [94]. Following bone marrow transplantation in

irradiated mice, donor cells were shown to engraft in

recipient’s liver and differentiate into hepatocyte like

cells. These experiments challenged the assumption

that hepatocytes may only be derived from endoder-

mal sources. Theise et al. described the presence of

male hepatocytes in the livers of two women who

were recipients of bone marrow transplant from male

donors [121].

It is not known whether hepatocyte like cells arise from

bone marrow cells through true trans-differentiation,

fusion or horizontal gene transfer. This is still a



matter of debate [122]. The presence of selective

pressure in the liver through injury or deficiency may

lead to cell fusion and disruption of the nucleus of

the host cell with potentially tumorigenic conse-

quences.

It is not clear whether unsorted bone marrow [123],

HSC or MSC [124, 125] is the optimal source for

cellular therapy. HSC are readily identifiable by the

markers CD34 and CD31 and may be isolated form

bone marrow, umbilical cord blood and occasionally

from peripheral blood. Lagasse et al. were success-

ful in correcting the defect in FAH ) ⁄ ) mice (model

for tyrosinaemia type 1) by transplantation using

mouse bone marrow haematopoietic cells [93]. Wang

et al. showed that in presence of liver injury, trans-

planted human HSC had the capacity to produce

albumin synthesizing cells in the mouse liver detect-

able in mouse serum with evidence of cell fusion

[98]. By contrast, MSC have not been associated

with fusion. These cells may be derived from bone

marrow [126], umbilical cord blood and tissue [125,

127–130], placental tissue [131] and adipose tissue

[132] and combine their multipotential with anti-

inflammatory and immunosuppressive properties

[133–135]. They are identified by certain cell sur-

face markers and have the ability to grow exten-

sively on plastic tissue culture dishes in the

laboratory in addition to the ability to differentiate

into bone, cartilage and fatty tissue [136]. MSC

have been shown to differentiate into neurons, car-

diomyocytes and hepatocytes [126, 129, 137, 138].

Thus, MSC are potential hepatocyte progenitor cells

that are readily available and can be expanded in

experimental conditions whilst retaining the potential

to differentiate into liver cells.

Morphological, immunophenotypical and functional

differentiation of MSC along a hepatocyte lineage has

been demonstrated in vitro [139–141], although ques-

tions about the mechanism and efficacy of the process

remain. In vivo work has described the expression

of human liver-specific proteins when unmodified

MSC from bone marrow were transplanted into

preimmune foetal sheep [140]. Unconditioned MSC

from cord blood have also been transplanted into

mice and foetal sheep giving rise to up to 20%

repopulation with demonstrable hepatocyte function

[96, 142].

There has been particular interest in the capacity of

MSC to avoid rejection. Indeed MSC have been

shown to exert immunosuppressive effects whilst

escaping recognition by recipient lymphocytes [143].

The mechanism by which they exert this immunomod-

ulatory effect is not yet known but may be secondary

to soluble factors, which are secreted by the cell [89,

144, 145]. Also of interest is the effect of MSC in

acute liver injury, their anti-inflammatory effects pos-

sibly mediating liver recovery or stimulating liver

regeneration [141, 146]. Although engraftment was

not seen, this was not withstanding a significant sur-

vival and regeneration benefit for the treated group.

Experiments have also shown that culturing MSC in

proximity to rat-liver derived hepatocytes improves

hepatocyte function, probably because of soluble

factors produced by MSC [147].

The clinical application of bone marrow stem cells in

liver disease has been recently reviewed [148]. To

date, 11 trials have been reported. Some of these

involve the use of granulocyte-colony stimulating

factor (G-CSF) to mobilize bone marrow stem cells

and induce faster hepatocyte regeneration without

harvesting cells, demonstrating improvement in

encephalopathy and Child-Pugh score [149]. Infusion

of autologous BMC in 27 patients in four trials

demonstrated a variable improvement in albumin and

coagulopathy [150–153]. Gordon et al. demonstrated

that mobilization and reinfusion of BMC produced a

reduction in bilirubin ⁄ improvement in albumin [154,

155].

Despite some initial success in the therapy of liver

disease with bone marrow stem cells, the mechanism

of effect remains to be elucidated. Safety concerns

still arise and in particular concerns about the profibr-

ogenic potential of MSC which may worsen liver dis-

ease [156]. Improved understanding of the influence

of these stem cells on the injured liver is required

before the appropriate randomized trials can be

designed.



Immortalization

In order to establish a sustainable source of cells for

transplantation, immortalization of hepatocytes has

been described. Clonal cell lines of human and rat

hepatocytes have been transduced with retroviral vector

expressing the immortalizing simian virus 40 large

T antigen gene [157]. Despite control mechanisms

for switching immortalization on and off, concerns

regarding safety remain.

Conclusion

In all, the last decade has seen considerable clinical

success in the field of hepatocyte transplantation. In

particular, short-term efficacy and safety have been

demonstrated for both liver-based metabolic disease

and acute liver failure. Techniques for isolation,

cryopreservation and transplantation of hepatocytes

under GMP conditions have been well established

but barriers remain to the establishment of hepato-

cyte transplantation as a main stream therapeutic

modality. The availability of good quality cells is a

major limiting factor. The successful engraftment of

cells and effective repopulation of the host liver

remain elusive and optimal immunosuppression is

unknown at present.

New developments within the field include the devel-

opment of new cell sources. Stem cells, in particular

‘adult-derived’ progenitors are an exciting prospect

for the future of hepatocyte transplantation and may

prove a sustainable alternative source of cell. Issues

regarding tumorigenicity remain a concern. Enriching

the quality of current cell sources is also an area of

investigation. Use of N-acetyl cysteine in hepatocyte

isolation and improved cryopreservation techniques

have resulted in better cell viability. Transient liver

injury using partial portal embolization and irradiation

may be useful in enhancing cell engraftment. Encap-

sulation in a bioinert material such as alginate may

allow evasion of the immune response. Transplanted

cells may also be tracked using newly developed

imaging techniques including MRI. The future of

hepatocyte transplantation relies on further progress

with these techniques and ideas.

Conflict of interest statement

No conflict of interest was declared.

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Correspondence: Anil Dhawan, King’s College London School

of Medicine – Paediatric Liver Centre, King’s College Hospital,

Denmark Hill, London SE5 9PJ, UK.

(fax: 0044 203 2994228; e-mail: [email protected]).



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Human hepatocyte transplantation: state of the art

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