J R Soc Med 2005;98:341-345
doi:10.1258/jrsm.98.8.341
© 2005 Royal Society of Medicine
Hepatocyte transplantation for metabolic liver disease: UK experience
Robin D Hughes PhD
Ragai R Mitry PhD
Anil Dhawan FRCPCH
Institute of Liver Studies, King's College London & King's
College Hospital, London, UK
Correspondence to: Dr Anil Dhawan, Paediatric Liver Centre, King's
College Hospital, Denmark Hill, London SE5 9RS, UK E-mail:
anil.dhawan{at}kcl.ac.uk
 |
INTRODUCTION
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For end-stage liver disease and liver-based metabolic conditions
the
accepted treatment is liver transplantation. With developments
in surgical
techniques and immunosuppressive drug therapy the
survival of patients and
grafts is now good. In the conventional
procedure the patient's whole
liver is replaced with a liver
obtained from a braindead or living donor. When
the donor liver
is too big it can be reduced to a compatible size; and,
recently,
split-liver procedures have been performed whereby the right
lobe is
transplanted into an adult and the smaller left lobe
into a
child,
1 thus
increasing the effective donor pool.
Another important advance in surgical technique is the use of auxiliary
liver transplantation for patients with acute liver failure and certain
liver-based metabolic defects such as Crigler–Najjar syndrome type I,
urea cycle defects, and familial hypercholesterolaemia. In this procedure,
part of the patient's liver, often the left lobe, is replaced with part
of a donor liver. In a patient with acute liver failure this leaves open the
possibility of regeneration of the native liver, in which case
immunosuppression can be stopped and the graft allowed to atrophy; and in a
patient with a metabolic disorder the native liver will be available for
future gene therapy. The results of auxiliary liver transplantation in
man2 have supported
observations in animals that small amounts of liver tissue can provide
sufficient function to correct an underlying metabolic defect. This finding
was a spur to work on hepatocyte transplantation for such disorders. The aim
is to repopulate the liver with donor hepatocytes, injected either directly
into the liver or into the spleen, from which they migrate to the liver.
If the technique proves successful, hepatocyte transplantation offers
several potential advantages. In terms of supply, there is the possibility of
using cells from livers that are unsuitable for conventional transplantation
because of steatosis or trauma. The patient does not have to undergo major
surgery; moreover, in metabolic conditions the native liver provides a safety
net in case of failure. One of the most important advantages is the
availability of the liver as a target organ for gene therapy when this becomes
a clinical reality. Experience of hepatocyte transplantation has been gained
in patients with acute liver
failure3,4
and metabolic liver diseases such as Crigler–Najjar syndrome type
I,5 glycogen storage
disease type 1a,6
and urea cycle
defects.7 The
background to this work has been described
elsewhere.8–11
The current paper discusses the sources of hepatocytes, the isolation process,
preclinical studies and clinical experience in the UK, especially in the
treatment of liver-based inborn errors of metabolism.
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SOURCES OF HEPATOCYTES
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Normal hepatocytes do not divide
in vitro, so that hepatocytes
for
transplantation must be isolated directly from liver tissue.
The main source
of these cells for transplantation in man is
unused donor livers or segments
of livers; from these, hepatocytes
of high viability can be
isolated.
12
An additional possible source is the non-heart-beating donor; livers
removed after the heart has stopped beating and respiration has ceased are
already being assessed for orthotopic
transplantation13
and might allow isolation of hepatocytes. To further increase the tissue pool
our unit has pioneered the isolation of cells from segment IV of the liver
dissected from the right lobe after the split-liver
procedure,14 so
that one liver can be used for three recipients.
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ISOLATION OF HEPATOCYTES
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The well-established protocols for isolation of human
hepatocytes
15,16
employ
collagenase digestion of perfused liver tissue at 37°C.
Once the
liver tissue is digested and cells are released, the
hepatocytes are purified
by centrifugation and assessed for
cell viability and yield. Large numbers of
cells can be obtained.
Hepatocytes must be used as soon as possible for cell
transplantation
(within 24 h of isolation) since their function deteriorates
even
at 4°C. For longer-term storage cryopreservation methods
are
available, but the yield of viable hepatocytes on thawing
tends to be
insufficient. The best results are currently obtained
by cryopreservation in a
mixture of University of Wisconsin
solution and 10% dimethyl sulfoxide by use
of a controlled-rate
cell
freezer.
17 The
frozen hepatocytes can then be stored at
-140°C until required. Cells from
the hepatocyte bank can
be thawed for immediate use.
An aseptic environment is required to prepare cells on a large scale in
conditions of 'good manufacturing practice'. Air entering the
laboratory passes through HEPA filters to remove any particles and an
air-handling unit maintains a temperature-controlled environment. A gradient
of air pressures provides the highest pressure in the aseptic room, where the
tissue processing is performed. Standard operating procedures are followed for
all aspects of work in the cell isolation unit. A comprehensive quality
control system, which monitors all aspects of laboratory performance, is in
operation.
All donated organs/tissues are screened for viral infection, including
hepatitis and HIV, as with whole organ transplantation. The hepatocytes are
released for transplantation only if the viability exceeds 60% and there is no
microbial contamination (Figure
1).
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PRECLINICAL STUDIES
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Work in animals has explored hepatocyte transplantation to different
sites
including liver, spleen, pancreas, peritoneal cavity,
and renal subcapsule,
and in models of acute, chronic and metabolic
liver disorders. (This paper
concentrates on metabolic liver
disease, but the results in acute liver
injury, induced either
surgically
18 or by
toxins,
19 are
encouraging.) For human metabolic disorders
there are several animal
models—e.g. Gunn rats for Crigler–Najjar
disease,
20 spf-ash
mice for congenital ornithine transcarbamylase
deficiency,
21
Long–Evans
cinnamon rats for Wilson's
disease,
22 Nagase
analbuminaemic
rats for
hypoalbuminaemia,
23
fumarylacetoacetate hydrolase —/—
knockout mice for hereditary
tyrosinaemia type
I,
24 Mdr2
—/—
knockout mice lacking biliary phospholipid excretion for
progressive
familial intrahepatic
cholestasis,
25 dogs
with
hyperuricosuria,
26
mice
with
histidinaemia,
27
Watanabe rabbits with
hypercholesterolaemia.
28
In
all these, hepatocyte transplantation has yielded long-term
improvement of
the biochemical abnormalities.
|
CLINICAL STUDIES
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Clinically, hepatocyte transplantation was first tried in patients
with
liver failure, who have a high mortality and in whom liver
transplantation is
the only proven treatment. Experience in
30 patients with liver failure, from
six centres in the USA,
was reviewed by Strom
et al. in
1999.
29
Hepatocytes, either
freshly isolated or thawed after cryopreservation, were
infused
into the splenic artery or portal vein of patients. Reductions
in
ammonia and bilirubin were recorded, with improvements in
hepatic
encephalopathy. The maximum amount of cells infused
was 5% of normal liver
mass; it is questionable whether this
is sufficient to replace the lost
function in acute liver failure.
In inherited metabolic liver diseases, where the aim is to replace a single
deficient enzyme, fewer cells may be needed. One of the key early reports was
from Fox and colleagues in
1998,5 who reported
the case of a 10-year-old girl with Crigler–Najjar syndrome type I in
whom liver expression of bilirubin UDP-glucuronosyltransferase activity
continued for up to nine months after hepatocyte transplantation. The overall
experience of hepatocyte transplantation in liver-based metabolic liver
disorders, mainly in children and including that at King's College
Hospital, London, is shown in Table
1.
Clinical experience in the UK
Only one centre in the UK, at King's College Hospital, London, is
currently performing hepatocyte transplantation. Over the 4 years since the
start of the project, 90 livers or liver segments have been processed for
preparation of hepatocytes including, from conventional donors, 15 whole
livers, 35 right lobes, 16 left lobes or left lateral segments and 6 liver
segment IVs or caudate lobes. Mean viability of the hepatocytes was 65% (range
13–98%). In addition, isolations were made from 18 livers or segments
obtained from non-heart-beating donors, perfused by the methods used for
conventional donor livers; these gave a lower mean cell viability (50%, range
1–81%), but higher viability has lately been obtained by reducing both
cold and warm ischaemia times before processing.
The methods for hepatocyte transplantation used at King's have been
modified from those reported by Strom et
al.29 in the
USA. All the cell infusions were with ABO compatible hepatocytes. Up to 100
million cells per kg body weight are infused into the liver via the portal
vein, with monitoring of portal pressure. Repeated infusions are performed
until the donor hepatocyte cell mass is about 10% of recipient liver mass. At
present there is no way to determine the proportion of administered cells that
survive and engraft. The immunosuppression regimen used is similar to that
given to whole-organ transplantation recipients, currently based on tacrolimus
and prednisolone. Probably this needs to be modified for cell transplantation;
also there are no laboratory tests to monitor cell allograft rejection.
Once the cell isolation methods had been established and validated, the
first patient was treated. In September 2002 a boy with an antenatal diagnosis
of severe ornithine transcarbamylase deficiency had hepatocytes infused via an
umbilical vein catheter (the umbilical vein extends into the left portal
vein). After transplantation of 1.9 billion hepatocytes he improved in terms
of blood ammonia (maintained at low levels to prevent neurotoxicity) and urea
(increased) while he was on normal protein
intake.14 Longterm
uncertainties about the efficacy of hepatocyte transplantation prompted
auxiliary left lobe orthotopic liver transplantation at seven months of age.
The patient was well at 2
years of age with normal neurodevelopment
and growth.
The next patients were two brothers with severe inherited coagulation
factor VII deficiency—the first to have hepatocyte transplantation in
such a condition. 1.1 and 2.2 billion hepatocytes were infused through a
Hickman line. The coagulation defect improved, such that the requirement for
exogenous factor VII (rFVIIa) became less than 20% of that before cell
transplantation.33
Importantly, one of the patients received exclusively cryopreserved
hepatocytes—so it seems that function related to clotting factors, at
least, is maintained after cryopreservation. Six months after transplantation,
both patients were needing higher rFVIIa doses, suggesting gradual loss of
transplanted hepatocyte function. These two brothers later had successful
orthotopic liver transplantation.
Two of the other children treated in 2003 had progressive familial
intrahepatic cholestasis (PFIC2), a genetic
disease34 in which
the liver lacks the bile salt export pump. As a result of this defect, bile
flow is severely impaired and cirrhosis develops rapidly. These children with
PFIC2 each received 300 million fresh hepatocytes through a portal vein
catheter, the rationale being that the injected hepatocytes would have a
selective advantage over the native hepatocytes to repopulate the recipient
liver (as shown in the mouse model of progressive familial intrahepatic
cholestasis,25
where up to 70% of host hepatocytes were replaced with donor cells after nine
months). However, no benefit was seen and both patients had whole-liver
transplants five and fourteen months later. A possible explanation is that
fibrosis in the hepatic sinusoids impaired engraftment, in which case earlier
treatment, before the onset of fibrosis, might improve engraftment.
The most recently completed treatment was in a child with
Crigler–Najjar syndrome type I who received a total of 4.3 billion fresh
and cryopreserved hepatocytes with nine infusions over two weeks and a further
infusion three months later. There was an encouraging reduction in serum
bilirubin, still present nine months later. Overall 6 patients have been
treated by hepatocyte transplantation without serious complications. The unit
plans to extend the technique to children with acute liver failure.
|
THE FUTURE
|
|---|
Although considerable progress has been made in hepatocyte transplantation,
the
promise from animal experiments has not yet been fully borne
out. There
are several areas for improvement and development.
In terms of the limited supply of organs for isolation of hepatocytes, one
potential source is livers rejected for clinical transplantation because of
steatosis; we need better ways to isolate and purify hepatocytes from fatty
livers so that their viability is good enough for transplantation. We also
need to improve methods of hepatocyte storage, both for longer periods in the
cold so that they can be used fresh after a few days and also cryopreservation
for the longer term. Some progress has been made in our laboratories in
preventing loss of hepatocyte function after cryopreservation by means of
cryo/cytoprotectant
agents.35 It is
clear, also, that many injected cells do not engraft into the recipient liver
and are either cleared by the reticuloendothelial system or lose viability
during this early phase. The outcome of hepatocyte transplantation would
benefit from methods to enhance engraftment and subsequent repopulation of the
liver, although the options for this in man will be limited.
Clearly, one way to overcome the difficulties with hepatocyte supply and
engraftment would be to use stem cells or stem-cell-derived
hepatocytes.36
Possible sources are fetal liver, cord blood, embryos and bone marrow. Liver
stem cell biology is under scrutiny worldwide but there are many hurdles to be
jumped before clinical application. In theory, stem cells could be made to
differentiate into all types of liver cell, could be frozen and thawed without
harm and would be less immunogenic than donor cells (or non-immunogenic if
autologous). As another approach, hepatocytes could be genetically manipulated
in vitro to upregulate gene expression to enhance enzymatic activity
and function—e.g. ornithine transcarbamylase, bilirubin
glucuronosyltransferase—or render them immunotolerant. Methods to
transfect hepatocytes are available and those employing non-viral vectors are
of particular interest.
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Acknowledgments
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We thank the Children's Liver Disease Foundation and King's
College
Hospital Charitable Trust for financial support. This work would
not
have been possible without the contributions of Mr Nigel
Heaton and Mr Mohamed
Rela, transplant surgeons, Dr John Korani,
consultant radiologist, and the
other members of the liver transplant
surgical team and staff at the
Paediatric Liver Centre.
|
REFERENCES
|
|---|
- Rela M, Vougas V, Muiesan P, et al. Split liver
transplantation: King's College Hospital experience. Ann
Surg 1998;227:282
-8[CrossRef][Medline]
- Pereira SP, McCarthy M, Ellis AJ, et al. Auxiliary partial
orthotopic liver transplantation for acute liver failure. J
Hepatol 1997;26:1010
-17[CrossRef][Medline]
- Strom SC, Fisher RA, Thompson MT, et al. Hepatocyte
transplantation as a bridge to orthotopic liver transplantation in terminal
liver failure. Transplantation1997; 63:559
-69[Medline]
- Bilir BM, Guinette D, Karrer F, et al. Hepatocyte
transplantation in acute liver failure. Liver
Transplant 2000;6:32
-40[Medline]
- Fox IJ, Chowdhury JR, Kaufman SS, et al. Treatment of the
Crigler–Najjar syndrome type I with hepatocyte transplantation.
N Engl J Med1998; 338:1422
-6[Free Full Text]
- Muraca M, Gerunda G, Neri D, et al. Hepatocyte
transplantation as a treatment for glycogen storage disease type 1a.
Lancet2002; 359:317
-18[CrossRef][Medline]
- Horslen SP, McCowan TC, Goertzen TC, et al. Isolated
hepatocyte transplantation in an infant with a severe urea cycle disorder.
Pediatrics2003; 111:1262
-7[Abstract/Free Full Text]
- Horslen SP, Fox IJ. Hepatocyte transplantation.
Transplantation2004; 77:1481
-6[CrossRef][Medline]
- Fox IJ, Roy-Chowdhury J. Hepatocyte transplantation. J
Hepatol 2004;40:878
-86[CrossRef][Medline]
- Fox IJ, Chowdhury JR. Hepatocyte transplantation. Am J
Transplant2004; 4(suppl 6):7
-13
- Najimi M, Sokal E. Update on liver cell transplantation.
J Pediatr Gastroenterol Nutr2004; 39:311
-19[CrossRef][Medline]
- Mitry RR, Hughes RD, Aw MM, et al. Human hepatocyte
isolation and relationship of cell viability to early graft function.
Cell Transplant2003; 12:69
-74[Medline]
- Muiesan P. Can controlled non-heart-beating donors provide a
solution to the organ shortage? Transplantation2003; 75:1627
-8[CrossRef][Medline]
- Mitry RR, Dhawan A, Hughes RD, et al. One liver, three
recipients—segment IV from split liver procedures as a source of
hepatocytes for cell transplantation. Transplantation2004; 77:1614
-16[CrossRef][Medline]
- Strom SC, Dorko K, Thompson MT, Pisarov LA, Nussler AK. Large scale
isolation and culture of human hepatocytes. In: Franco D, Boudjema K, Varet B,
eds. Îlots de Langerhans et hépatocytes.
Paris: Editions INSERM, 1998:195
- Mitry RR, Hughes RD, Dhawan A. Progress in human hepatocytes:
isolation, culture and cryopreservation. Semin Cell Dev
Biol 2002;13:463
-7[CrossRef][Medline]
- Diener B, Utesch D, Beer N, Durk H, Oesch F. A method for the
cryopreservation of liver parenchymal cells for studies of xenobiotics.
Cryobiology1993; 30:116
-27[CrossRef][Medline]
- Demetriou AA, Reisner A, Sanchez J, Levenson SM, Moscioni AD,
Chowdhury JR. Transplantation of microcarrier-attached hepatocytes into 90%
partially hepatectomized rats. Hepatology1988; 8:1006
-9[Medline]
- Sutherland DE, Numata M, Matas AJ, Simmons RL, Najarian JS.
Hepatocellular transplantation in acute liver failure.
Surgery1977; 82:124
-32[Medline]
- Matas AJ, Sutherland DE, Steffes MW, et al. Hepatocellular
transplantation for metabolic deficiencies: decrease of plasma bilirubin in
Gunn rats. Science1976; 192:892
-4[Abstract/Free Full Text]
- Michel JL, Rabier D, Rambaud C, et al. Intrasplenic
transplantation of hepatocytes in spf-ash mice with congenital ornithine
transcarbamylase deficiency. Chirurgie1993
–94;119:666
-71[Medline]
- Yoshida Y, Tokusashi Y, Lee GH, Ogawa K. Intrahepatic
transplantation of normal hepatocytes prevents Wilson's disease in
Long–Evans cinnamon rats. Gastroenterology1996; 111:1654
-60[CrossRef][Medline]
- Rozga J, Holzman M, Moscioni AD, Fujioka H, Morsiani E, Demetriou
AA. Repeated intraportal hepatocyte transplantation in analbuminemic rats.
Cell Transplant1995; 4:237
-43[CrossRef][Medline]
- Overturf K, Al-Dhalimy M, Ou CN, Finegold M, Grompe M. Serial
transplantation reveals the stem-cell-like regenerative potential of adult
mouse hepatocytes. Am J Pathol1997; 151:1273
-80[Abstract]
- De Vree JM, Ottenhoff R, Bosma PJ, Smith AJ, Aten J, Oude Elferink
RP. Correction of liver disease by hepatocyte transplantation in a mouse model
of progressive familial intrahepatic cholestasis.
Gastroenterology2000; 119:1720
-30[CrossRef][Medline]
- Kocken JM, Borel Rinkes IH, Bijma AM, et al. Correction of
an inborn error of metabolism by intraportal hepatocyte transplantation in a
dog model. Transplantation1996; 62:358
-64[CrossRef][Medline]
- Selden C, Calnan D, Morgan N, Wilcox H, Carr E, Hodgson HJ.
Histidinemia in mice: a metabolic defect treated using a novel approach to
hepatocellular transplantation. Hepatology1995; 21:1405
-12[CrossRef][Medline]
- Wiederkehr JC, Kondos GT, Pollak R. Hepatoctye transplantation for
the low-density lipoprotein receptor-deficient state. A study in the Watanabe
rabbit. Transplantation1990; 50:466
-71[CrossRef][Medline]
- Strom SC, Chowdhury JR, Fox IJ. Hepatocyte transplantation for the
treatment of human disease. Semin Liver Dis1999; 19:39
-48[Medline]
- Grossman M, Rader DJ, Muller DW, et al. A pilot study of
ex vivo gene therapy for homozygous familial hypercholesterolaemia.
Nat Med1995; 1:1148
-54[CrossRef][Medline]
- Strom SC, Fisher RA, Rubinstein WS, et al. Transplantation
of human hepatocytes. Transplant Proc1997; 29:2103
-6[CrossRef][Medline]
- Sokal EM, Smets F, Bourgois A, et al. Hepatocyte
transplantation in a 4-year-old girl with peroxisomal biogenesis disease:
technique, safety, and metabolic follow-up.
Transplantation2003; 76:735
-8[CrossRef][Medline]
- Dhawan A, Mitry RR, Hughes RD, et al. Hepatocyte
transplantation for inherited factor VII deficiency.
Transplantation2004; 78:1812
-14[CrossRef][Medline]
- Thompson R, Strautnieks S. BSEP: function and role in progressive
familial intrahepatic cholestasis. Semin Liver Dis2001; 21:545
-50[CrossRef][Medline]
- Terry C, Dhawan A, Mitry RM, Lehec SC, Hughes RD. Preincubation of
rat and human hepatocytes with cytoprotectants prior to cryopreservation can
improve viability and function on thawing. Liver
Transplant (in press)
- Fausto N. Liver regeneration and repair: hepatocytes, progenitor
cells, and stem cells. Hepatology2004; 39:1477
-87[CrossRef][Medline]
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INTRODUCTION
SOURCES OF HEPATOCYTES
