Normal Hepatocytes Correct Serum Bilirubin After

Repopulation of Gunn Rat Liver Subjected to

Irradiation/Partial Resection
Chandan Guha,1,2 Bhupesh Parashar,1 Niloy J. Deb,1 Madhur Garg,1 Giridhar R. Gorla,1 Anupam Singh,2
Namita Roy-Chowdhury,2,3,4,5 Bhadrasain Vikram,1 and Jayanta Roy-Chowdhury2,3,4,5
The treatment of inherited metabolic liver diseases by hepatocyte transplantation (HT) would be
greatly facilitated if the transplanted normal hepatocytes could be induced to proliferate prefer-
entially over the host liver cells. We hypothesized that preparative hepatic irradiation (HIR)
should inhibit host hepatocyte proliferation in response to partial hepatectomy (PH). Normal
nonirradiated hepatocytes transplanted in this setting should have a selective growth advantage
over the host liver cells and should progressively repopulate the liver. To test this hypothesis, we
transplanted 5 million hepatocytes from normal Wistar-Roman High Avoidance (RHA) rats into
the livers of congeneic bilirubin-uridine 5ⴕ-diphosphoglucuronate glucuronosyltransferase
(UGT1A1)-deficient jaundiced Gunn rats by intrasplenic injection after one of the following
treatments: (1) 68% PH, (2) HIR (50 Gy), or (3) HIR ⴙ PH. In rats receiving either PH or HIR
alone before HT, serum bilirubin concentrations declined by 25% to 30% in 28 weeks. In
contrast, serum bilirubin levels were normalized completely in rats receiving HIR ⴙ PH before
HT. Massive repopulation of the Gunn rat liver by the UGT1A1-positive Wistar-RHA hepato-
cytes was shown by UGT1A1 enzyme assay, immunoblot analysis, and immunohistochemical
staining of the recipient liver. High-performance liquid chromatography analysis of the bile collected
from Gunn rats 5 months after PH, HIR, and HT showed normalization of the pigment profile, with
bilirubin diglucuronide and monoglucuronide as the predominant pigments. In conclusion, a pre-
parative regimen of HIR ⴙ PH results in massive repopulation of the liver with functionally normal
transplanted hepatocytes, resulting in complete correction of a metabolic deficiency. Noninvasive
strategies to replace PH for providing proliferative stimuli to the transplanted cells should make this
regimen valuable in augmenting the effects of HT for the treatment of liver diseases. (HEPATOLOGY 2002;
36:354-362.)

H
epatocyte transplantation (HT) is currently be-
ing evaluated as a treatment strategy for patients
with acute and chronic liver failure and to re-
place metabolic liver functions in inherited liver diseases.1
Abbreviations: HT, hepatocyte transplantation; PH, partial hepatectomy; HIR,
hepatic irradiation; UGT1A1, bilirubin-uridine 5⬘-diphosphoglucuronate glucu-
ronosyltransferase; RHA, Roman High Avoidance; BrdU, 5-bromo-2⬘-deoxyuri-
dine.
From the Departments of 1Radiation Oncology, 3Medicine, 4Pathology, and
5Molecular Genetics and 2The Marion Bessin Liver Research Center, Albert Ein-
stein College of Medicine, Bronx, NY.
Received February 17, 2002; accepted May 6, 2002.
Supported in part by National Institutes of Health grants RO1-DK 46057 (to
J.R.-C.) and RO1-DK 39137 (to N.R.-C.), Liver Research Core Center grant
DK-P30-41296 (Director, David A. Shafritz), and American Cancer Society grant
RPG-00-066-01-CCE (to C.G.).
Presented at the 1999 annual meeting of the American Association for the Study
of Liver Diseases in Dallas, TX.
Address reprint requests to: Chandan Guha, M.D., Ph.D., Department of Ra-
diation Oncology, Montefiore Medical Center, 111 East 210 St., Bronx, NY
10467. E-mail: cguha@montefiore.org; fax: 718-231-5064.
Copyright © 2002 by the American Association for the Study of Liver Diseases.
0270-9139/02/3602-0012$35.00/0
doi:10.1053/jhep.2002.34516
354
HT has been used in the treatment of inherited metabolic
diseases, such as Crigler-Najjar syndrome type I,2 and for
hepatocyte-based ex vivo gene therapy in experimental
animals3-5 as well as in patients with low-density lipopro-
tein receptor deficiency.6 However, the clinical applica-
tion of HT is limited by the availability of human
hepatocytes and the number of liver cells that can be
transplanted safely at one time. An important consider-
ation is whether a sufficient number of hepatocytes can be
engrafted to achieve the desired metabolic correction
without causing portal hypertension or other adverse ef-
fects. Therefore, a method to induce preferential prolifer-
ation of a relatively small number of engrafted
hepatocytes in vivo could markedly enhance the applica-
bility of HT.
We hypothesized that preparative irradiation of the
liver along with a strong mitotic stimulus provided by a
maneuver such as partial hepatectomy (PH) should dam-
age the host hepatocyte DNA, causing cell cycle arrest.
Subsequently transplanted normal, nonirradiated hepato-
cytes should proliferate preferentially in response to the
HEPATOLOGY, Vol. 36, No. 2, 2002
mitotic stimulus provided by PH, thereby progressively
repopulating the liver. Preparative irradiation is routinely
used for bone marrow transplantation,7 but this approach
has not been evaluated for facilitation of HT. Addition-
ally, hepatic irradiation (HIR) is associated with poten-
tially lethal radiation-induced liver disease.8 Previously,
we have shown that transplantation of syngeneic, nonir-
radiated hepatocytes in F344 rats that had undergone
PH ⫹ HIR resulted in protection of the liver from radi-
ation injury.9 These findings have encouraged us to test
the feasibility of this method in correcting an inherited
metabolic abnormality of the liver. Here we report the
effect of preparative HIR, PH, and HT in jaundiced
Gunn rats, an animal model of Crigler-Najjar syndrome
type I, which is caused by inherited bilirubin-uridine 5⬘-
diphosphoglucuronate glucuronosyltransferase (UGT1A1)
deficiency. The lack of bilirubin glucuronidation due to
UGT1A1 deficiency results in inefficient biliary excretion
of bilirubin, accumulation of unconjugated bilirubin in
plasma, and consequent bilirubin encephalopathy. The
purpose of this study was to determine whether the repop-
ulating engrafted hepatocytes retain normal detoxifying
activity and are functionally connected to the biliary sys-
tem. We also determined the necessary HIR dose for re-
population of rat liver by the engrafted cells and tested the
ability of allogeneic hepatocytes to repopulate the livers of
cyclosporine-treated rats.
Materials and Methods
Animals. Inbred Gunn rats and normal Wistar-Ro-
man High Avoidance (RHA) rats (congeneic to the Gunn
rats) were obtained from our colony at the Albert Einstein
College of Medicine, where they were bred and main-
tained on standard laboratory chow and kept on 12-hour
light/dark cycles. All animal protocols were approved by
the institutional Animal Care and Use Committee.
Hepatocyte Isolation and Perfusion. Hepatocytes
were isolated from male Wistar-RHA rats by in situ col-
lagenase perfusion by a modification of the procedure of
Berry and Friend.10,11 Hepatocytes with greater than 90%
viability, as determined by trypan blue dye exclusion,
were used for transplantation.
Partial Hepatectomy. Anesthesia was induced by in-
traperitoneal injection (1 mL/kg) of a mixture of ket-
amine (100 mg/mL) and xylazine (20 mg/mL) in a ratio
of 4:3 (by volume). After aseptic preparation, 68% PH
was performed through a midline incision between 8 AM
and 12 PM.12
Hepatic Irradiation. Immediately after PH, the ani-
mals were placed in a supine position on a polystyrene
(Aquaplast) platform. A jig with a 5 ⫻ 7– cm irradiation
GUHA ET AL. 355
port was aligned to this platform. Two 3 ⫻ 4 – cm lead
shields, each 2 mm thick, were wedged under the liver to
protect the stomach and intestines, taking care not to
compress the hepatic and aortic vessels. A Philips 320
MGC orthovoltage unit (Phillips Electronic Instruments,
Alpharetta, GA) was used (320 kVP, 10 mA, 0.5 mm
copper filtration, 35 cm SSD; dose rate, 3.2 Gy/min). The
dose delivered to the liver was verified by thermolumines-
cent dosimetry using a liver phantom within the jig. The
abdomen was then closed in 2 layers.
Hepatocyte Transplantation. HT was performed as
previously described.9 Briefly, 4 days after PH or HIR ⫹
PH, rats were anesthetized by ether inhalation and the
lower pole of the spleen was exposed by a left flank inci-
sion. Hepatocytes, freshly isolated by collagenase perfu-
sion of the liver, of congeneic Wistar-RHA rats were
suspended in 0.5 mL RPMI 1640 culture medium
(GIBCO-BRL, Grand Island, NY) and injected into the
splenic pulp.13 Homeostasis was achieved by ligation of
the splenic tip using a 2-0 silk suture. In initial pilot
experiments, we determined the optimal number of hepa-
tocytes for intrasplenic transplantation. Rats transplanted
with up to 5 million hepatocytes survived without obvi-
ous ill effect, but transplantation of 10 million hepato-
cytes resulted in death in most of the animals within one
week of transplantation. Liver sections of these animals
showed occlusion of the portal vein by aggregates of trans-
planted cells. Therefore, in subsequent experiments, we
transplanted 5 million viable hepatocytes. The time of
HT was determined by another pilot study in which DNA
synthesis in response to PH was measured by 5-bromo-
2⬘-deoxyuridine (BrdU) uptake experiments. In rats sub-
jected to PH without irradiation, the DNA synthesis
peaked in 24 to 36 hours, whereas this peak was delayed,
blunted, and prolonged over several days in rats that re-
ceived PH ⫹ HIR. Based on this information, we trans-
planted the hepatocytes 4 days after PH ⫹ HIR.
Experimental groups include rats receiving HT alone
(n ⫽ 3) as well as HT after one of the following regimens:
HIR alone (n ⫽ 4), PH alone (n ⫽ 4), and PH ⫹ HIR
(n ⫽ 9). For allogeneic HT, 5 million freshly isolated
F344 hepatocytes were injected intrasplenically into
Gunn rats in the following groups: PH alone (n ⫽ 2),
Allo-HT alone (n ⫽ 4), and PH ⫹ HIR (n ⫽ 3). Follow-
ing HT, the animals received daily intraperitoneal injec-
tions of cyclosporine (10 mg/kg intraperitoneally) to
suppress immune rejection of allogeneic donor cells.
Serum Bilirubin Concentration. Approximately 0.2
to 0.4 mL of blood was collected through the tail vein at
various time points, and serum bilirubin levels were de-
termined using an automated clinical microchemistry sys-
tem (Bayer Chem One; Bayer Corp., Tarrytown, NY).
356 GUHA ET AL.
Analysis of Pigments Excreted in Bile. At the com-
pletion of the serial blood collection (14-28 weeks after
HT), the bile ducts of the Gunn rats were cannulated with
polyethylene cannulae (PE10) under ether anesthesia.
The rats were placed in restraining cages. After the rats
regained consciousness and their body temperature re-
turned to normal, bile was collected on ice in 0.1-mL
aliquots in a darkened room. Bile samples were analyzed
by reverse-phase high-performance liquid chromatogra-
phy using a ␮-Bondapak C-18 column (Millipore-Wa-
ters, Milford, MA) as described.14 Peaks were detected by
absorbance at 436 nm, identified by retention time using
authentic pigment standards, and quantified by electronic
integration of the peak areas.
Histomorphologic Analysis. After bile collection for
bile pigment analysis, the liver was embedded in tissue-
freezing medium (TBS; Triangle Biomedical Sciences,
Durham, NC), frozen in liquid nitrogen, and stored at
⫺70°C until use or was fixed in formalin for paraffin
embedding and hematoxylin-eosin staining.
Incorporation of BrdU. Two rats in each group were
killed 24 and 48 hours after treatment with PH or PH ⫹
HIR, respectively. Two hours before the animals were
killed, they were injected intraperitoneally with BrdU
(100 mg/kg body weight) for determination of DNA syn-
thesis. Incorporation of BrdU was determined by im-
munohistochemical staining of sections of paraffin-em-
bedded liver tissues using a BrdU staining kit (Oncogene,
Boston, MA).
Immunotransblot Analysis for UGT1A1 Content of
the Liver. Liver tissue was minced and homogenized in a
glass-Teflon motorized homogenizer. Twenty percent
homogenates were produced using a buffer containing
0.25 mol/L sucrose and 10 mmol/L Tris-HCl, pH 7.4, as
well as protease inhibitors (1 mmol/L phenylmethylsulfo-
nyl fluoride, 3 ␮g/mL leupeptin, and 1 ␮L/mL aproti-
nin). Protein concentrations in homogenates were
determined using the Bio-Rad protein assay (Bio-Rad
Laboratories, Richmond, CA). For immunoblot analysis,
proteins (100 ␮g/lane) were resolved by sodium dodecyl
sulfate/7.5% polyacrylamide gel electrophoresis and elec-
troblotted to polyvinylidene difluoride membranes (Mil-
lipore Corp., Bedford, MA). The membranes were
probed with a monospecific rabbit antiserum raised
against a 31-mer polypeptide corresponding to the
unique amino-terminal domain of rat UGT1A1. The
membranes were then incubated with horseradish peroxi-
dase– conjugated goat anti-rabbit immunoglobulin G
(Sigma Chemical Co., St. Louis, MO) followed by devel-
opment with enhanced chemiluminescence (ECL Plus;
Amersham, Arlington Heights, IL).
HEPATOLOGY, August 2002
UGT1A1 Activity. Rat UGT1A1 enzyme activity was
assayed with bilirubin as the aglycone substrate as de-
scribed previously.15
Immunohistochemical Analysis of Hepatic Repopu-
lation by Transplanted Wild-type Hepatocytes. At the
termination of the experiments, fragments of the liver
were fixed in 40% formaldehyde and sections of the par-
affin-embedded tissues were stained using the rabbit poly-
clonal anti-rat UGT1A1 antibody. The antibody binding
was visualized using an ABC staining kit (Santa Cruz
Biotechnology, Santa Cruz, CA).
Statistical Analysis. Data are expressed as mean ⫾
SD. To assess the amelioration of hyperbilirubinemia in
Gunn rats by various preparative regimens, Student’s t
test was performed between pretreatment and posttreat-
ment serum bilirubin concentrations for each treatment
group and also between the posttreatment serum biliru-
bin levels of various treatment regimens, expressed as per-
cent of pretreatment serum bilirubin levels. A P value of
less than .05 was considered statistically significant.
Results
HIR Delayed and Blunted DNA Synthesis in the
Host Liver After PH. DNA synthesis was determined by
immunohistochemical staining of BrdU incorporated in
the replicating DNA of regenerating hepatocytes after PH
with or without HIR. In the nonirradiated animals, there
was an increase in BrdU-positive cells in livers 24 (Fig.
1A) and 48 hours after PH. In contrast, PH in animals
receiving HIR resulted in only a modest incorporation of
BrdU in hepatocytes in the first 24 hours (Fig. 1B), indi-
cating that hepatocytes in the irradiated liver do not ex-
hibit this normal DNA synthesis peak in response to PH.
Instead, this peak is delayed, blunted, and prolonged over
several days (not shown in Fig. 1).
Restoration of the Liver Mass. Because the BrdU
incorporation study showed a delay in the onset of DNA
synthesis, we determined the extent of eventual restora-
tion of liver mass by determining the liver weights at the
end of the study. The mean liver weight ⫾ SD in rats
subjected to HIR ⫹ PH ⫹ HT was 36.4 ⫾ 3.6 g/kg body
weight, which was not significantly different from rats
that received PH ⫹ HT (39.8 ⫾ 5.0 g/kg body weight)
(P ⬎ .2).
Immunoblot Analysis of Rat UGT1A1 Protein. As
expected, livers from nontransplanted Gunn rats (Fig. 2,
lane 1) lacked the 52-kd immunoreactive UGT1A1 band
that is seen in normal Wistar-RHA rats (lane 6). Trans-
planted Gunn rats treated with HIR alone (lane 2) or PH
alone (lanes 3-4) showed a faint UGT1A1 band that was
derived from the small number of transplanted hepato-
HEPATOLOGY, Vol. 36, No. 2, 2002
Fig. 1. BrdU staining of rat livers. BrdU was injected at various time points after (A) PH or (B) PH ⫹ HIR, and the rats were killed 2 hours later.
The field shown is representative of 10 sections of the liver from experiments in 3 rats 24 hours after PH.
cytes that remain engrafted in the host liver in these ani-
mals. Among the various experimental groups, Gunn rats
transplanted with congeneic normal hepatocytes after
preparative HIR ⫹ PH showed a strong immunoreactive
UGT1A1 band 5 months after HT (lane 5).
Immunohistochemical Staining. Liver biopsies were
performed as terminal experiments. Five months after
HT, immunohistochemical staining showed clusters of
UGT1A1-staining cells. Based on examination of 50
fields at ⫻200 magnification from different liver lobes,
60% to 80% repopulation of the host liver by the en-
grafted hepatocytes was seen in rats receiving preparative
Fig. 2. Immunoblot analysis of UGT1A1 in liver extracts of Gunn rats.
The liver was homogenized and the proteins were resolved by sodium
dodecyl sulfate/7.5% polyacrylamide gel electrophoresis, and immuno-
blot was performed as described in Materials and Methods. Lane 1,
control Gunn rat (no HT); lane 2, HIR ⫹ HT; lanes 3-4, PH ⫹ HT; lane
5, PH ⫹ HIR ⫹ HT; lane 6, Wistar-RHA.
GUHA ET AL. 357
HIR ⫹ PH. A representative field is shown in Fig. 3.
Consistent with our previous finding in dipeptidyl pepti-
dase IV–negative recipient F344 rats, the cluster of hepa-
tocytes derived from the transplanted cells was scattered
throughout the liver acinus rather than limited to the
periportal region.
UGT Activity Toward Bilirubin. To determine the
extent of repopulation of the Gunn rat livers by the func-
tional donor hepatocytes, UGT1A1 activity was deter-
mined in vitro in the liver homogenates with bilirubin as
the acceptor substrate. Homogenates of normal Wistar rat
livers showed UGT1A1 activity of 2.62 ⫾ 0.15 ␮mol/g
liver/h. The activity was undetectable in untreated Gunn
rats. In Gunn rats that were transplanted with normal
hepatocytes after preparative HIR ⫹ PH, the mean
UGT1A1 activity was 1.87 ⫾ 0.32 ␮mol/g liver/h
(71.4% ⫾ 11.9% of the levels found in the normal
Wistar-RHA rats). UGT1A1 activity in the transplant-
recipient Gunn rats that had been pretreated with HIR or
PH only was 0.11 ⫾ 0.05 and 0.10 ⫾ 0.02 ␮mol/g
liver/h, respectively.
Excretion of Bilirubin Glucuronides in Bile. To
determine the bilirubin glucuronidating activity of the
repopulating normal donor hepatocytes in vivo, we ana-
lyzed the pigments excreted in bile. High-performance
liquid chromatography of bile from normal Wistar-RHA
rats showed the characteristic bilirubin monoglucuronide
and diglucuronide peaks and only minor amounts of un-
conjugated bilirubin (Fig. 4A). The monoglucuronide
and diglucuronide peaks were absent in the bile of the
untreated Gunn rats, and a prominent unconjugated bil-
irubin peak was seen (Fig. 4B). In contrast, bile collected
from Gunn rats 5 months after HIR ⫹ PH ⫹ HT showed
358 GUHA ET AL. HEPATOLOGY, August 2002

Fig. 3. Repopulation by transplanted UGT1A1-positive hepatocytes. Immunohistochemical staining was performed on paraffin sections using a
polyclonal UGT1A1-specific anti-peptide antibody. (A) Normal Wistar-RHA rat, (B) control nontransplanted Gunn rat, (C) rat treated with PH ⫹ HIR ⫹
HT (5 months after HT).

complete normalization of the pigment profile (Fig. 4C).
The results indicated a major degree of repopulation of
the recipient Gunn rat livers by the functional engrafted
UGT1A1-proficient donor hepatocytes. The concentra-
tion of bilirubin conjugates in bile in Gunn rats treated
with HIR ⫹ PH ⫹ HT was 7.39 ⫾ 2.2 mg/dL, which was
not significantly different from that in the bile of RHA-
Wistar rats (7.50 ⫾ 2.5 mg/dL). This was significantly
higher than that of Gunn rats receiving HT after PH
(0.51 ⫾ 0.34 mg/dL) or HIR (0.33 ⫾ 0.24 mg/dL) alone
(P ⫽ .006).
Serum Bilirubin Levels. To determine whether re-
population of the Gunn rat livers by the engrafted nonir-
radiated cells could correct the bilirubin glucuronidation
defect in Gunn rats, serum bilirubin levels were deter-
mined serially after transplantation of 5 ⫻ 106 congeneic

Fig. 4. Excretion of bilirubin glu-

curonides in bile 20 weeks after HT.
Bile was collected through a bile
duct cannula as a terminal experi-
ment. Bile pigments were analyzed
by high-performance liquid chroma-
tography as described in Materials
and Methods. The data are from
single animals representative of the
various groups. (A) Wistar-RHA rat,
(B) Gunn rat, (C) HIR ⫹ PH ⫹
HT–treated Gunn rat, (D) PH-treated
Gun rat. UCB, unconjugated biliru-
bin; BMG, bilirubin monoglucu-
ronide; BDG, bilirubin diglucuronide.
HEPATOLOGY, Vol. 36, No. 2, 2002
Fig. 5. (A) Dose response of preparative liver irradiation. Partially
hepatectomized Gunn rats received liver irradiation at 15, 30, or 50 Gy.
The bars show mean serum bilirubin concentrations ⫾ SD expressed as
percent of pretreatment values 5 weeks after HT. (B) Serum bilirubin
levels in Gunn rats receiving PH ⫹ HT (n ⫽ 4, 䊐), HIR ⫹ HT (n ⫽ 4,
●), or HIR ⫹ PH ⫹ HT (n ⫽ 9, ‚).
normal hepatocytes. Initially, the dose response of prepar-
ative HIR was determined by transplanting the hepato-
cytes in partially hepatectomized Gunn rats that received
HIR at 15 to 50 Gy. As shown in Fig. 5A, serum bilirubin
levels were reduced to 64.18% ⫾ 3.92% (P ⬍ .004),
52.61% ⫾ 14% (P ⬍ .004), and 29.97% ⫾ 13.96% (P ⬍
.001) of the pretreatment bilirubin concentrations at 5
weeks after 15, 30, and 50 Gy of preparative HIR, respec-
tively. The reduction in serum bilirubin levels was signif-
icantly greater after 50 Gy HIR compared with 15 Gy
(P ⬍ .003) and 30 Gy (P ⬍ .04) HIR doses.
Because 50-Gy HIR resulted in the highest reduction
of serum bilirubin levels, experiments to determine the
time course of change of plasma bilirubin concentrations
were performed at this dose of HIR. The effect of HT on
serum bilirubin concentrations in various treatment
groups is shown in Fig. 5B. Twenty-eight weeks after HT,
the serum bilirubin concentrations decreased to 78.96%
⫾ 9.43% of the pretreatment levels in the PH ⫹ HT
GUHA ET AL. 359
group (P ⬎ .05), 71.05% ⫾ 3.72% in the HIR ⫹ HT
group (P ⬍ .02), and 5.38% ⫾ 1.73% in the PH ⫹
HIR ⫹ HT group (P ⬍ .0002). The trend in reduction of
serum bilirubin levels in the PH ⫹ HT, HIR ⫹ HT, and
HT alone groups probably resulted from the engraftment
of hepatocytes without significant amplification. In con-
trast, the greatest reduction and, in fact, complete nor-
malization of serum bilirubin levels occurred in rats that
received preparative HIR and PH before HT. In this
group (n ⫽ 9), serum bilirubin concentrations decreased
from initial levels of 10.4 ⫾ 2.3 mg/dL to completely
normal levels (0.64 ⫾ 0.16 mg/dL) by 12 weeks. Serum
bilirubin levels remained normal (0.56 ⫾ 0.18 mg/dL) in
5 rats from this group that were followed up for up to 7
months.
Histomorphology of the Liver. Five and 7 months
after transplantation, hematoxylin-eosin–stained liver
sections from the transplant-recipient Gunn rats that re-
ceived HT after PH ⫹ HIR showed normal hepatic ar-
chitecture (Fig. 6). The morphology of the hepatocytes
and bile duct epithelial cells was normal. This is consistent
without our previous studies in F344 rats, which showed
that HT prevented both short-term and long-term radia-
tion-induced liver damage. Compared with animals that
received PH ⫹ HT (3.87 ⫾ 0.25 mg/dL), the serum
albumin levels in animals receiving PH ⫹ HIR ⫹ HT
(3.54 ⫾ 0.44 mg/dL) were unchanged. Serum ␣-fetopro-
tein level was undetectable at the completion of the ex-
periments. The normalization of serum bilirubin levels,
maintenance of normal serum albumin levels despite ex-
tensive replacement of the host hepatocytes by the trans-
planted cells, and maintenance of the body weight and
general health of the transplant-recipient rats indicate that
the repopulating transplanted hepatocytes maintained
their normal physiologic function in the irradiated host
liver. The spleen, kidney, and lung of these rats were also
normal histologically and did not show any engrafted
hepatocytes (data not shown).
Effect of Transplantation of Allogeneic Hepato-
cytes. We also examined whether allogeneic HT could
reduce serum bilirubin levels following treatment with
PH ⫹ HIR in Gunn rats treated with cyclosporin A to
prevent allograft rejection. As seen in Fig. 7, there was
significant reduction in serum bilirubin levels by 4 weeks
in rats that received PH ⫹ HIR (2.9 ⫾ 0.13 mg/dL)
compared with rats that received PH alone (4.6 ⫾ 0.4
mg/dL) following allogeneic HT (P ⬍ .003). This indi-
cated that allogeneic hepatocytes could be used to repop-
ulate the liver if immunosuppressive therapy is used and
that administration of cyclosporine did not prevent the
proliferation of the engrafted hepatocytes.
360 GUHA ET AL.
Fig. 6. Hematoxylin-eosin staining of liver biopsy specimens from Gunn rats 5 months after PH ⫹ HIR ⫹ HT. (Original magnification: A, ⫻100;
B, ⫻200.)
Discussion
The purpose of this study was to determine whether
preparative HIR could be used to correct a metabolic
disorder by preferential repopulation of the liver by trans-
planted normal hepatocytes. This is the first report of a
preparative HIR protocol that permits engraftment and
massive proliferation of functional donor hepatocytes, re-
sulting in complete, long-term correction of an inherited
liver disease. Using Gunn recipient rats, a rodent model of
Crigler-Najjar syndrome type I, we show that the trans-
planted hepatocytes not only engraft and proliferate in the
Fig. 7. Serum bilirubin levels after allogeneic HT in Gunn rats receiving
no preparative treatment (n ⫽ 3, ●), PH (‚), or HIR ⫹ PH (Œ). Each
data point is the mean ⫾ SD.
HEPATOLOGY, August 2002
irradiated host liver but also maintain their normal phys-
iologic function on a long-term basis. Importantly, con-
sistent with our previous results, HT prevented the
delayed radiation-induced liver injury, manifested by bile
duct proliferation, fibrosis, and deterioration of liver
function.9
Another new finding of this report was the determina-
tion of the relationship between HIR dose and the extent
of functional hepatic repopulation by the engrafted cells.
This determination is critical for potential translation of
the present results to clinical application. Although the
50-Gy dose of HIR permits an extensive repopulation,
this dose would cause severe liver damage in rodents if, for
any reason, the transplanted hepatocytes failed to engraft.
This level of irradiation would also result in radiation-
induced liver injury in humans if the transplanted hepa-
tocytes failed to proliferate. Therefore, it was important to
determine an HIR dose that would permit amelioration
of hyperbilirubinemia but would not be toxic to rodents
or humans in case the hepatocyte engraftment failed. We
show that a preparative irradiation dose of 15 and 30 Gy
resulted in reduction in serum bilirubin levels by 30% and
50%, respectively, in 5 weeks. This allows us to design
effective preparative HIR regimens in which the irradia-
tion dose would be kept below the threshold for the in-
duction of radiation-induced hepatopathy. Preferential
proliferation of transplanted hepatocytes over the host
liver cells requires that hepatocytes in the recipient liver
HEPATOLOGY, Vol. 36, No. 2, 2002
have a shortened life span or fail to respond to a prolifer-
ative stimulus. Using this principle, other investigators
have shown hepatic repopulation by engrafted hepato-
cytes in transgenic or gene-disrupted animals in which the
host hepatocytes die spontaneously or fail to proliferate.
In other cases, hepatotoxic agents have been used to pre-
vent the regeneration of host hepatocytes.
Most methods described heretofore for hepatic re-
population have used animal models in which the host
hepatocytes undergo spontaneous cell death. The prolif-
erative potential of transplanted mature hepatocytes has
been amply shown in recipient urokinase-type plasmino-
gen activator transgenic mice,16 fumarylacetoacetate hy-
drolase– deficient tyrosinemic mice,5 and mdr2 knockout
mice.17 In each of these situations, hepatocytes of the
recipient liver undergo necrosis or apoptosis at a high rate,
providing proliferative stimuli to transplanted normal
hepatocytes, which progressively replace the host hepato-
cytes. Similar repopulation of the host liver by trans-
planted hepatocytes occurs in Long Evans Cinnamon rats
with hepatocellular copper overload (a model of human
Wilson’s disease).18 Obviously, spontaneous hepatic re-
population by transplanted hepatocytes, as previously de-
scribed, occurs in only specific circumstances. However,
principles learned from these studies are being used in
designing strategies for more general application. Hepa-
tocytes from Bcl-2 transgenic donor mice or hepatocytes
transduced with a retrovirus expressing bcl-2 were trans-
planted into the livers of normal mice. Administration of
an agonistic anti-Fas antibody was used to induce apopto-
sis of host hepatocytes, while the bcl-2– expressing trans-
planted cells were protected.19,20 This method requires
long-term expression of an oncogene, bcl-2, in the trans-
planted cells, which may be associated with significant
risk. In other studies, lasiocarpine, a pyrrolizidine alka-
loid, was used to inhibit host liver regeneration and PH
was used to stimulate the proliferation of the transplanted
hepatocytes.21 This regimen was used to correct albumin
deficiency in genetically analbuminic rats.22 However, la-
siocarpine is carcinogenic and undesirable for human ap-
plication. Because of the inherent limitations of existing
methods of hepatic repopulation, we have explored pre-
parative irradiation of the liver as a method for inhibiting
host liver proliferation.
Preparative HIR, which was used in this study, is not
associated with increased incidence of cancer. There was
no histologic evidence of hepatocarcinogenesis with un-
detectable serum ␣-fetoprotein levels in these rats. In a
clinical setting, the liver could be subjected to conformal
irradiation without significant damage of other organs.
Studies are underway to replace PH as a proliferative stim-
ulus by noninvasive vascular or pharmacologic manipula-
GUHA ET AL. 361
tions of the liver. The absence of any histologic
abnormality of the liver after long-term follow-up of the
experimental group suggests that, in a modified form,
preparative HIR could be potentially useful in assisting
repopulation of the human liver with transplanted hepa-
tocytes for the treatment of inherited liver diseases.
To examine the mechanisms of hepatocyte repopula-
tion, we have determined the effect of irradiation on hep-
atocellular proliferation following PH. The proliferative
effect of PH was shown by incorporation of BrdU by
hepatocytes. The enhancement of BrdU incorporation
after PH was not seen in rats that were subjected to
HIR ⫹ PH 24 and 48 hours after the procedure. How-
ever, despite the delay in DNA synthesis, the liver mass
was eventually restored, as shown by similar liver weights
in the PH ⫹ HT and HIR ⫹ PH ⫹ HT groups at the end
of the study. The post-regeneration liver weights in these
groups were similar to those found in rats subjected to PH
alone in a separate study. The initial delay in the prolifer-
ative response of host hepatocytes to PH may have
provided a growth advantage to the nonirradiated trans-
planted hepatocytes. Therefore, this regimen fulfills the 2
requirements of massive repopulation of the liver: sup-
pression of the proliferative response of the recipient
hepatocytes by HIR and a strong mitotic stimulus to the
transplanted nonirradiated hepatocytes by PH.
The hepatic repopulation was progressive over 12
weeks. After this, serum bilirubin levels remained normal
throughout the 7-month duration of the study, indicating
that the correction of the metabolic defect is likely to be
lifelong. The near-complete disappearance of the uncon-
jugated bilirubin peak in the high-performance liquid
chromatography analysis of bile pigments of the trans-
plant-recipient Gunn rats was consistent with the massive
repopulation of the liver by the normal hepatocytes, as
shown by immunohistochemical staining.
In a patient with Crigler-Najjar syndrome type I who
was transplanted with allogeneic hepatocytes representing
approximately 5% of the liver mass, a 50% reduction was
observed, with some clinical improvement and reduction
of the required duration of daily phototherapy.2 Our re-
sults show that HIR at a dose as low as 15 Gy was suffi-
cient to achieve significant reduction of serum bilirubin
levels 5 weeks after HT in the partially hepatectomized
Gunn rats. Therefore, in humans, a lower radiation dose
to the whole liver or a precisely targeted and controlled
ablation of host hepatocytes in a specific liver lobe by
conformal irradiation may be sufficient to achieve liver
repopulation. In future clinical applications, the source of
hepatocytes is likely to be allogeneic donors. Therefore,
we also determined whether liver repopulation would be
possible with allogeneic hepatocytes in recipients immu-
362 GUHA ET AL.
nosuppressed with cyclosporine. The results showed that
Gunn rat livers could be repopulated with allogeneic rat
hepatocytes using preparative HIR ⫹ PH, and the re-
population was not inhibited by cyclosporine therapy.
Acknowledgment: The authors thank Dr. David
Neufeld of the Molecular Genetics and Cell Culture core
of the Marion Bessin Liver Research Center for isolation
of primary hepatocytes.
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