|
Stephen
M. Riordan1, 2and Roger Williams1
1Institute
of Hepatology, University College London and Universit
y College London Hospitals, London, England
2Department of Gastroenterology, The Prince of Wales
Hospital, Sydney, Australia
Correspondence to: Professor Roger Williams, Institute of
Hepatology, University College London, 69-75 Chenies Mews, London
WC1E, 6HX, England
Telephone:
0044-20-7679-6511，Fax.
0044-20-7380-0405
Email. roger.williams@ucl.ac.uk
Received: 2000-07-17 Accepted: 2000-08-01
Subject
headings: liver failure, acute; liver
diseases; liver transplantation; gene therapy; animals, laboratory;
transfering growth factor beta
Riordan SM, Williams R. Transplantation of primary and reversibly
immortalized human liver cells and other gene therapies in acute
liver failure and decompensated chronic liver disease.World J
Gastroentero,2000;6(5):636-642
Studies performed in experimental small animals with hepatic-based
metabolic dis
orders but no structural liver disease, including Gunn and
analbuminaemic rats a
nd rabbits with inherited low-density lipoprotein receptor
deficiency, have shown that up to 95% of hepatocytes transplanted
into the spleen or liver remain
in these sites, with improvement in metabolic function of recipients
[1-4]. The feasibility of hepatocyte transplantation as a
clinically-relevant therapeutic tool has subsequently been
demonstrated in a small number of patients with disorders such as
Crigler-Najjar syndrome type 1[5], ornithine
transcarbamoylase deficiency[6] and familial
hypercholesterola
emia[7], in whom the delivery of numbers of primary
hepatocytes approximately equivalent to only 5% or less of
the normal liver cell mass led to satisfactory, if incomplete,
correction of the
metabolic defect. Hepatocyte transplantation has several real and
potential adv
antages over conventional orthotopic liver transplantation (OLT). In
addition to
the relative simplicity, less invasive nature and lower associated
cost of the
former intervention, the ability to cryopreserve primary liver cells
without sub
stantial loss of viability or physiological function on subsequent
thawing, prov
ided that they are attached to microcarriers or gel-entrapped as
spheroids
[8-10], offers the potential that, through liver cells stored
in centralise
d banks, it will be possible one day to have them widely available
for clinical
use within a few hours of a decision to institute such therapy.
A
particularly important issue is whether liver cell transplantation
has a role
in the management of patients with severe liver damage resulting in
acute liver
failure (ALF), where only short term support may be required given
the potentia
l for regeneration of the native liver[11], or those with
decompensated
chronic liver disease, in whom on-going support over a longer period
may be ne
cessary. The availability of effective liver support
“on
demand”
would constitute an important advance in the management of such
patients, given the ra
pidity with which irreversible clinical deterioration often occurs
in these settings. In this review we consider clinical experiences
with hepatocyte transp
lantation in each of these liver failure syndromes reported to date,
as well as
discussing issues pertaining to the ideal cell type for clinical
use. The potent
ial applications of adjuvant and other gene therapies, as recently
described exp
erimentally, which may overcome two of the major limitations of
liver cell trans
plantation at present, namely the relative unavailability of primary
human hepat
ocytes and the requirement for pharmacological immunosup-pression
in the post-transplantation period, are also considered.
Liver cell transplantation in experimental animals with ALF
due to chemically-induced hepatic necrosis or surgical models of
hepatic ischaemia or resection has, to date, generally involved the
use of primary hepatocytes. Such treatment has been associated with
improved survival, even when small numbers of cells, in
the order of only 0.5% to 3% of the normal hepatocyte mass, are used[12-1
9]. These small numbers of cells may be sufficient to enhance
hepatic regener
ation, on which recovery ultimately depends, possibly due at least
in part to re
duction in levels of transforming growth factor (TGF)β1,
a potent inhibitor of this process[20]. However, cell
transplantation h
as been performed prior to the induction of ALF in some surgical
models, a scenario clearly incongruous to the clinical setting.
Furthermore, since mediato
rs such as interleukin-6 and tumor necrosis factor-αproduced
by activated Kupffer cells in ALF contribute to both progressive
liver damage and the development of multi-organ failure[21-23],
while the liver is also the source of TGF-β1
and
other inhibitors of liver regeneration[24,25], the
relevance of animal
models in which the liver is removed to the clinical situation, in
which it remains in situ, is uncertain.
Nevertheless, several preliminary studies have already been
carried out on the clinical efficacy of hepatocyte transplantation
in adult and paediatric patients with ALF. No randomised, controlled
data is yet available and the impact of such treatment on clinical
course is uncertain. Strom et al[26]reported their
experience at the Medical College of Virginia with transplantatio
n of small numbers (0.1% or less of the normal hepatocyte mass) of
ABO-matched
, freshly isolated or
cryopreserved primary human hepatocytes via injection into the
splenic artery i
n two adults with ALF due to hepatitis B virus infection and
phenytoin hepatotox
icity, respectively. Both patients were in grade Ⅳ
encephalopathy prior to th
e treatment. Immunosuppression was with methylprednisolone and
cyclosporin. Redu
ce
d blood ammonia levels along with improvement in encephalopathy
grade and revers
al of haemodynamic instability were noted. Other evidence of
metabolic function
was unimpressive, with the serum bilirubin level increasing in both
patients and
the prothrombin time rising in one and falling only marginally in
the other. Bo
th patients underwent OLT after three and 10 days, respectively,
with subsequent
full recovery. None of three control patients, for whom either fresh
or frozen
hepatocytes were not available or consent for hepatocyte
transplantation could n
ot be obtained, survived. Of an additional five adult patients with
ALF who unde
rwent hepatocyte transplantation in this way at the same centre,
three (60%) in
grade Ⅲ
to Ⅳ
encephalopathy (due to acetaminophen toxicity: n=1; hepatitis B
virus infection: n=1 and idiopathic: n=1) were successfully
“bridged”
to OLT between one and five days later. Another patient in grade Ⅳ
coma (due to herpes simplex virus infection and/
or sodium valproate hepatotoxicity) died of sepsis-related
cardiovascular instability on day 5 before OLT could be performed,
while the remaining patient with acute hepatitis B virus infection
recovered without OLT but had only low-grade in grade Ⅰ
encephalopathy prior to the procedure[27]. Bilir et al[28]
from the University of Colorado subsequently transplanted by
percutaneous injection larger numbers of cryopreserved primary human
hepatocytes (in the order of 5% of the normal hepatocyte mass) in
five adult patients with ALF and grade Ⅳ
encephalopathy who were not candidates for OLT. Three (60%) patients
survived more than 72 hours, with evidence of improved
encephalopathy score,
serum ammonia levels and prothrombin times. A delay in the order of
24 to 72 hou
rs was apparent between hepatocyte transplantation and the first
biochemical or
clinical sign of improvement, possibly reflecting the time required
for effectiv
e engraftment, as demonstrated in experimental animals[29].
However, no patient survived for more than seven weeks. The same
group reported technical
success in achieving engraftment via the transjugular route in an
additional comatose patient with ALF, although no clinical benefit
could be demonstrated[30].
Transplantation
of comparably small numbers of hepatocytes has been performed in
six children with ALF, aged six months to 15 years, with aetiologies
including
acetaminophen hepatotoxicity (n=1), other drug reactions (n=2),
idiopath
ic (n=2) and sepsis/total parenteral nutrition (n=1)[26,27,31]
. Five (83%) of these children were in grade
Ⅳ
encephalopathy at the time of
treatment. Of these, four (80%) died within seven days, despite
instances of red
uction in serum ammonia levels and requirement for coagulation
factor support, w
hile the other patient recovered without OLT, having received
multiple infusions
of hepatocytes over a three day period. Another child, in whom
hepatocyte trans
plantation was performed when in grade Ⅰ
encephalopathy, underwent OLT two days later.
In
experimental animal models, successful hepatic engraftment of
transplanted hepatocytes is associated with evidence of transient
micro-circulatory damage to the host liver[32]. This
development of reversible portal hypertensi
on precludes the transplantation of larger quantities of
hepatocytes, at least i
n one session[33]. However, transplantation of a
relatively small number
of cells can lead to substantial replacement of the recipient’s
liver mass in the experimental setting in the presence of various
regenerative stimuli. The latter include the concurrent intravenous
injection of hepatocyte growth factor (HGF)[34],
induction of ischemic atrophy of the contralateral liver lobe[3,4]
and in situations in which the recipient’s own liver cells have a
shortened life-span, as occurs with acute chemical injury[35],
transduction with a recombinant adenovirus vector
expressing a non-secreted urokinase[36] or anti-Fas
antibody-induced
apoptosis[37].
The
growth advantage of transplanted cells over native cells
demonstrated in the
se circumstances may be maximised in the ALF setting by the use of
purified hepa
tic stem cells able to replicate at least 100 times without loss of
function or
malignant transformation, as identified in adult rodent liver[38,39].
Repopulation experiments using purified fractions of total liver
cell suspension
s will be required to identify any such cells in the adult human
liver[40]. Of note, a bone marrow-derived stem cell
capable of repopulating the liver with mature hepatocytes following
hepatic injury has recently been describ
ed in rodents. In the order of 1.0×106
hepatocytes (approximately 0.1% of the total hepatocyte mass)
originated from transplanted bone marrow cells by day 13 after liver
injury[41]. This finding raises the exciting possibility
that bone marrow infusion may hav
e therapeutic potential in liver failure, although, even if
extrapolated from th
e animal to the human situation, the time required for engraftment
and cell diff
erentiation would be problematic in the acute setting.
On the premise that the fetal liver contains epithelial cells
that are in differ
ent stages of lineage progression, some of which may exhibit the
full regenerati
ve potential of stem cells, transplantation of fetal hepatocytes has
been sugges
ted as the way forward, rather than use of adult cells. Clinical
data so far are
limited. Habibullah et al[42] in India
transplanted 6×107
blood group-matched, fetal hepatocytes per kilogram body weight by
intraperiton
eal injection in seven patients with ALF of unspecified aetiology
and grade Ⅲ
to Ⅳ
encephalopathy. Three (43%) transplanted patients survived. Those
who survived each had a prothrombin index of 1.5 or less and were in
grade Ⅲ
or Ⅳ
a encephalopathy at the time of hepatocyte transplantation, while
those who died had a more severe illness marked by prothrombin
indices ranging from 2.6 to 3.0 and grade Ⅳb
encephalopathy at this time. Blood ammonia concentrat
ions fell in 5/6 (83%) transplanted patients in whom serial levels
were obtained. Survival in a control group, selected on the basis of
inability to procure consent for the hepatocyte transplantation
procedure, was 33%. Whether fetus-derived hepatocytes will prove
superior to their adult counterparts in terms of regenerative
capacity and functional characteristics, and whether any such
superiority will translate into increased clinical efficacy in the
ALF situation or with chronic liver damage, remain to be determined.
Irrespective
of the fetal or adult nature of transplanted hepatocytes or even th
e use of bone marrow infusion as a source of stem cells capable of
repopulating
the liver, the potential role in the ALF setting of
co-transplantation of non-
parenchymal cells requires consideration. The co-transplantation of
such cells may be advantageous to the viability and function of
transplanted hepatocy
tes in terms of secretion of extracellular matrix, analogous to the
beneficial effects in these regards documented in ex-vivo
culture systems[43-4
5]. However, the inclu
sion of non-parenchymal cells in suspensions for transplantation
might also have deleterious effects if these cells become activated
to produce cytokines such as TGF-β1
and interleukin-1 which promote apoptosis of hepatocytes and
inhibition of liver regeneration[24,25].
Controlled
studies in which liver cell transplantation in its various forms is
c
ompared to standard intensive care will be required in order to
determine the ef
ficacy or otherwise of this intervention in the ALF setting. These
studies shoul
d be performed, at least in the first instance, in the most severely
affected gr
oups fulfilling criteria for OLT but for whom this is unavailable or
contraindic
ated by co-morbidity. Such studies will need to be conducted on a
multicentre basis and using standardised outcome measures if
sufficient numbers of patients
are to be recruited and meaningful results obtained. If benefit is
demonstrated
in such groups, efficacy should then be addressed in patients with
apparently lesser degrees of liver damage not fulfilling OLT
criteria, as recent analyses showing poor negative predictive values
of current selection criteria for OLT indicate that a substantial
number of such patients nevertheless deteriorate with intensive
medical care alone[46-48].
Clinical experience to date with liver cell transplantation
for decompensated ch
ronic liver disease is similarly limited, uncontrolled and confined
to the use o
f primary adult hepatocytes. The feasibility and apparent safety of
hepatocyte transplantation in chronic liver disease was first
reported by Mito and Kusano in Japan[49], who injected
small numbers of freshly isolated human cells into the spleens of 10
patients with cirrhosis or chronic viral hepatitis at laparotomy and
under the influence of epidermal growth factor
. Viable hepatocytes were detected in the spleen by scintigraphy in
almost all patients up to 11 months later. Four patients with
cirrhosis subsequently underwent hepatocyte transplantation via
injection into the splenic artery at the Medical College of Virginia[27].
One of two patients in grade
Ⅳ
encephalopathy survived until OLT was performed on day 2, while a
third patient in grade Ⅱ-Ⅲ
encephalopathy recovered to be discharged from hospital before
relapsing and dying on day 33. Another patient in whom liver failure
with grade Ⅳ
encephalopathy was precipitated by triseg mentectomy died of
cardiovascular instability within 2 days of the procedure. More rece
ntly, Bilir et al[50]in Colorado treated five
patients with Child’s C cirrhosis who were not candidates
for OLT with transplantation of
1×109
to 1×1010
cryopreserved primary human hepatocytes (in the order
of 5% normal liver cell mass), with viability of 52% to 73% after
thawing, via infusion into the splenic artery. Immunosuppression was
with cyclosporin. Indications for hepatocyte transplantation were
refractory encephalopathy with ascites (n=4) and hepatorenal
syndrome (n=1). Improvements in encephalop
athy grade and ascites and renal function, respe
ctively were noted within days of the procedure. Improvements in
serum albumin l
evels and prothrombin times also occurred, but only after a period
of four to si
x months. Aside from one patient who stopped immunosuppression after
five months
, all patients were alive and well two to 15 months
post-transplantation. There
were no complications related to the procedure. In particular, no
overt evidenc
e of increased portal venous pressure, as reported following
hepatocyte transpla
ntation in experimental animals with cirrhosis[51], was
recorded, altho
ugh it is to be noted that transjugular intrahepatic portosystemic
shunts had been placed prior to transplantation in the majority of
patients. No instances of
pulmonary vascular complications were clinically apparent, despite
concerns of i
ncreased pulmonary sequestration of transplanted cells in the
context of portal
-systemic shunting. Results of a randomised trial of hepatocyte
transplantation
in cirrhosis, which is currently in progress, are awaited.
Even if the efficacy of primary human hepatocyte
transplantation was to become f
irmly established in the ALF and decompensated chronic liver disease
settings, t
he limited availability of these cells is likely to represent a
severe, ongoing
impediment to its widespread clinical application. Primary human
hepatocytes are
obtained from resected surgical specimens, unused segments or
end-lobe wedges
of donor organs for OLT and livers from apparently healthy donors
which are ultimately rejected for OLT on account of overt steatosis,
since these are often also fibrotic. Although approximately 109
hepatocytes are readily obtained from a segment of normal liver, the
yield is substantially reduced when fibrosis is present[52].
The high demand for whole organ OLT and the expertise to transplant
portions of a single organ into more than one recipient mean that
completely normal liver is increasingly in short supply for other
uses. A satisfactory number of viable primary human hepatocytes
cannot at presen
t be obtained from needle biopsy sized fragments of liver.
The ex-vivo
expansion of hepatocytes recovered from surgical specimens repr
esents a potential means of overcoming the problem of limited
hepatocyte availab
ility for transplantation. A number of proliferating human cell
lines which have become immortalised by virtue of cultural
conditions, without the use of oncogenes o
r carcinogens, have now been maintained in continuous culture for up
to several
years, although considerable limitations in their spectra of
metabolic activity
may inhibit their clinical utility were they to be used in the liver
failure set
ting[53-56]. An alternative approach to achieving
population expansion
of hepatocytes ex-vivo, without necessarily compromising
their differentiat
ed functional capability and the prospect of obtaining meaningful
clinical support following their subsequent transplantation, is to
transfect the cells with a replication deficient retrovirus carrying
a temp erature-sensitive variant of the simian virus 40 large tumor
(SV40 large-T) antigen gene. This gene binds to the cell cycle
control protein, p53, and produces cell lines which
proliferate at 33℃
but cease proliferating and develop enhanced differentiated
function, including upregulated synthesis of α-1-antitrypsin
and inducible activity of some cytochrome P450 isoforms, at 39℃[57,58]
.
Hepatocytes from
Gunn rats immortalised by transfection with the SV40 large-T antigen
have been used successfully for ex-vivo gene therapy, in
which the
cells were also transduced with the gene for bilirubin uridine
diphosphoglucur
onate (UDP)-glucoronosyltransferase, expanded in vitro by
culturing at 33℃
and finally transplanted into syngeneic animals. Long-term reduction
of serum bilirubin in association with the appearance of bilirubi
n glucuronides ensued[59]. In addition, transplantation
of SV40 large T-antigen-immortalised hepatocytes in port
o-caval shunted rats and those with surgically-induced ALF has been
shown to improve resultant hepatic encephalopathy and survival,
respectively[60,61]. Several non-clonal human hepatocyte
cell lines successfully transfected with an amphotropic mouse
retrovirus containing this gene have remained stable in long-term
continuous culture[58]. Nonetheless, the potential
clinica
l applicability of such cell lines has been questioned following
reports in experimental animals that hepatocellular carcinoma may
develop in hepatocytes expressing the SV40 large-T antigen[62].
Kobayashi et
al[63] have recently investigated the feasibility of
o
vercoming the otherwise unacceptable risk of tumorigenicity of SV40
large-T-im
mortalised cel
ls, using a technique of reversible immortalisation in which cells
are initially
transduced to proliferate in vitro prior to the excision of
the T-antigen-encoding DNA sequence before transplantation.
Specifically, a highly different
iated cell line, NKNT-3, was generated by retroviral transfer to
primary adult
human hepatocytes of the SV40 large-T gene flanked by LoxP
recombination target
s, with concurrent expression of a fusion protein conferring
sensitivity to ganc
yclovir. NKNT-3 cells became immortalised without an apparent growth
crisis and
double in numbers every 48 hours. The immortalising and gancyclovir
sensitivity
-conferring genes could subsequently be completely excised in
vitro by tran
sient transduction with a replication deficient recombinant
adenovirus expressin
g the Cre recombinase, which resulted in Cre/Lox site-specific
recombination.
After removal of the SV40 large-T gene in this way, NKNT-3 cells
stopped proliferating and appeared more differentiated, with nucleus
to cytoplasm ratios
and cytogranules resembling those of normal primary hepatocytes.
While mRNA’s for bilirubin-UDP-glucur-onosyltransferase and
glutamine synt
hetase were each detectable in NKNT-3 cells before reversal of
immortalisation, levels increased
significantly after excision of the SV40 large-T gene by
recombination, with mRNA’s for albumin and coagulation factor X
becoming detectable only in this latter circumstance.
The efficacy of
transplantation of NKNT-3 cells, before or after recombination,
was then assessed in a rodent model of ALF in which 5×107
cells, equivalent to approximately 5% of the total number of
hepatocytes in an adult rat, were delivered by intrasplenic
injection one day prior to 90% hepatectomy under immun
osuppression with tacrolimus[63]. Transplanted animals
showed substanti
al improvements in total bilirubin, prothrombin time and blood
ammonia levels and survival compared to controls, with a trend
towards a particular survival advantage in those animals which
received the reverted NKNT-3 cells rather than immortalised
counterparts. Taken together, these findings demonstrate the
feasibility of controlling the ex-vivo expansion of primary
human hepatocyt
es by transfection with the SV40 large-T antigen gene and
Cre/Lox-based reve
rsible immortalisation, with the removal of the oncogene prior to
transplantatio
n promoting a level of differentiated function adequate to sustain
short-term s
urvival in an experimental ALF setting. Future studies should assess
the in vi
vo efficacy of reverted NKNT-3 cells when attached to
microcarriers or gel-entrapped as hepatocyte spheroids,
modifications which would render them amenable to cryopreservation
and storage in cell banks for clinical use as required.
Adjuvant genetic
manipulation of transplanted human liver cells may also be used
to overcome the current necessity for pharmacological immunosup-pression
to pr
event allograft rejection[64]. This is an important issue
since, althoug
h reduction or withdrawal of pharmacological immunosuppression in
the post-OLT
setting has been associated with lower incidences of metabolic,
infective and ne
oplastic complications, side effects once developed such as chronic
nephrotoxici
ty and hypertension may be irreversible[65-69]. One
approach to prevent
ing rejection of transplanted cells without the need for
immunosuppressive drugs
relates to the use of cells previously transfected with the
adenoviral E3 gene,
which encodes several proteins that are known to inhibit host T cell
reactivity
[70,71]. Preliminary data in rodents suggests that these
proteins have t
he capacity to protect transplanted hepatocytes from rejection. In
particular, Brown-Norway hepatocytes expressing E3 proteins
persisted for up to 6 weeks following transplantation into
allogeneic Gunn rats, as reflected by reduced serum bilirubin
levels, while no reduction in this parameter, indicative of rapi
d allograft rejection, occurred in control Gunn rats receiving a
comparable numb
er of untreated Brown-Norway cells[72]. It remains to be
determined wh
ether strategies designed to induce tolerance that are currently
under investiga
tion or consideration in the post-OLT situation, such as the
adjunctive infusion of donor-derived bone marrow or soluble class Ⅰ
antigens, the use of anti-CD4, anti-CD25 and anti-CD54 monoclonal
antibodies, blockade of the CD28-B7 T cell co-stimulatory pathway
with CTLA41g,pre-treatment of the recipient with IL-10 or even the
withholding of all immunosuppression for the first 24 to 48 hours[73-80],
are of relevance
when hepatocytes only, rather than with additional non-parenchymal
cells and donor-derived leukocytes (as with OLT), are transplanted.
Another potential clinical application of gene therapy in the
liver failure sett
ing does not involve the transplantation of liver or other cells,
but rather the
repeated in vivo transfection of skeletal muscle with the
gene for human HG
F. Ueki et al[81] recently reported that such an
approach in a roden
t model of cirrhosis, induced by repeated administration of
dimethylnitrosamine,
led to significantly increased plasma levels of human as well as
endogenous rat HGF and increased tyrosine phosphorylation of the HGF
receptor. Inhibition of fibrogenesis and hepatocellular apoptosis
were noted, possibly consequent to reduction in levels of TGF-β1.
Gene transfer into the rats’ skeletal muscle was achieved using
weekly injections of liposomes contain
ing the haemagglutinating virus of Japan and human HGF cDNA inserted
into the EcoRI and NotI sites of the pUC-SRa expression vector.
Survival of the animals
was significantly improved compared to that in phosphate buffered
saline-treate
d controls, with the cirrhotic lesion completely r
eversed in all transfected animals within 50 days of the toxic
insult. Such HGF
gene therapy would seem less likely to have substantial impact in
ALF, since pla
sma HGF levels are already substantially elevated in this
circumstance[82,
83]. The prospect of maximising liver regeneration via gene
therapy in this
latter situation will likely depend, in the first instance, on a
more comprehensive understanding of both the expression of cell
surface receptors for stimulatory and inhibitory growth factors and
the integrity of down-stream
effector and adaptor mechanisms, allowing targeted over-expression
or inhibitio
n, respectively, of key elements in these signalling pathways.
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