|
Edwin C.
Ouyang, Catherine H. Wu, Cherie Walton,
Kittichai Promrat, George Y. Wu Department
of Medicine, Division of Gastroenterology-Hepatology, University of
Connecticut Health Center, Rm. AM-045, 263 Farmington Avenue,
Farmington, CT 06030
Correspondence to: George Y. Wu, M.D., Ph.D., Department of
Medicine, Division of Gastroenterology-Hepatology, University of
Connecticut Health Center, Rm. AM-045, 263 Farmington Avenue,
Farmington, CT 06030
Telephone:
(860) 679-3158, Fax. (860) 679-3159 Email. wu@nso.uchc.edu
Received: 2001-04-11 Accepted: 2001-04-26
Subject
headings:
liver/cytology; immune tolerance; cell transplantation
Ouyang EC, Wu CH, Walton C, Promrat K, Wu GY. Transplantation of
human hepatocytes into tolerized genetically immunocompetent
rats.World J Gastroenterol, 2001;7(3):324-330
Abstract
AIM: To determine whether normal genetically immunocompetent
rodent hosts could be manipulated to accept human hepatocyte
transplants with long term survival without immunosuppression.
METHODS: Tolerance towards human hepatocytes was established
by injection of primary human hepatocytes or Huh7 human hepatoma
cells into the peritoneal cavities of fetal rats. Corresponding
cells were subsequently transplanted into newborn rats via
intrasplenic injection within 24h after birth.
RESULTS: Mixed lymphocyte assays showed that spleen cells
from non-tolerized rats were stimulated to proliferate when exposed
to human hepatocytes, while cells from tolerized rats were not.
Injections made between 15d and 17d of gestation produced optimal
tolerizaton. Transplanted human hepatocytes in rat livers were
visualized by immunohistochemical staining of human albumin. By dot
blotting of genomic DNA in livers of tolerized rats 16 weeks after
hepatocyte transplantation, it was found that approximately 2.5�105
human hepatocytes survived per rat liver. Human albumin mRNA was
detected in rat livers by RT-PCR for 15wk, and human albumin protein
was also detectable in rat serum.
CONCLUSION: Tolerization of an immunocompetent rat can permit
transplantation, and survival of functional human hepatocytes.
INTRODUCTION
Transplantation
of allogeneic hepatocytes into immunocompetent rodents has been
shown to result in rejection of donor cells by host immune system,
with T- cell activation, and a delayed type hypersensitivity
reaction[1-3]. This can been overcome by generalized
immunosuppression or through the use of genetically immunodeficient
animals[4-8]. Another strategy to avoid rejection is by
inducing immunological tolerance specifically to the transplanted
cells. In this regard, it has been demonstrated that the ability of
the immune system to distinguish between endogenous and exogenous
antigens develops shortly before birth. Studies have shown that if
foreign antigens are introduced during this formative period, when
the animals mature, they will be tolerant to those antigens[9],
permitting the survival of allogeneic (cardiac) transplants in rats[10,11].
We hypothesized that, if human liver cell antigens could be
introduced into normal rodents at the appropriate time during fetal
development, those animals could be rendered tolerant and serve as
suitable hosts for human hepatocyte transplants after birth without
either genetic or pharmacological generalized immunosuppression. If
successful, these animals could serve as convenient animal models
for studying mechanisms of human hepatitis viral infections, serve
as models for testing new antiviral agents, and as means for testing
hepatotoxicity of drugs.
MATERIALS AND METHODS
Animals
Pregnant Sprague-Dawley rats, 250g to
300g of body mass
(Charles River Co., Inc., Wilmington, MA) were maintained on 12h light-dark cycles, and fed ad lib with standard rat
chow in the Center for Laboratory Animal Care at the University of
Connecticut Health Center. All animal procedures were approved by
Institutional Animal Care and Use Committee and conformed to USDA
and NIH animal usage guidelines.
Cells
Cryopreserved human primary hepatocytes were obtained from Clonetics
Corp. (Walkersville, MD) and kept in liquid nitrogen until use.
Frozen cells were thawed, washed with human hepatocyte medium (Clonetics
Corp.) plus 5g�L-1 insulin
and 0.39mg�L-1 dexamethasone, and then spun at 50�g for
10 min at 4℃.
Cell viability was measured by trypan blue exclusion staining
(approximately 65% of the cells were viable, and 90% were
parenchymal hepatocytes). Human hepatoblastoma cell lines Huh7 and
HepG2, human fibroblast IMR-90 and human kidney 293 cells were grown
in Dulbecco Modified Eagle�s medium (DMEM) with 100mL�L-1
fetal bovine serum (FBS) and antibiotics.
Intrafetal intraperitoneal injections of human hepatocytes
At 15d to 17d of
gestation, groups of pregnant rats were anesthetized by
intramuscular injections of ketamine (40mg�kg-1 body
mass) and xylazine (5mg�kg-1 body mass). Laparatomies
were performed under sterile conditions; gravid uteri were exposed,
and transilluminated by a high intensity lamp (Fiber-lite MI-150,
Dolan-Jenner Industries, Lawrence, MA). Human hepatocytes or Huh7
cells, 1�105 cells in 10μL PBS, were injected
through the uterine wall into the peritoneal cavities of rat fetuses
using a sterile 200μL Hamilton syringe with a 28 gauge beveled
point needle (Hamilton Inc., Reno, NV).
Cell transplantation
Within 24h of birth,
newborn rats were placed on ice for 2-5 min. Under sterile
conditions, left paracostal incisions were made, and primary human
hepatocytes or Huh7 cells, 1�1010 cells�L-1
in 200μL PBS were injected over 30 into the spleen by sterile
Hamilton syringe.
Sample collection
Peripheral blood samples were drawn from tail veins, spun, and serum
stored at -20℃
. Liver samples were collected either by
sacrificing animals or by performing partial hepatectomies. Samples
were fresh frozen in liquid nitrogen, and stored at -80℃.
Mixed lymphocyte assays
The tolerance of host animals towards human hepatocytes was
assessed by mixed lymphocyte assays in which the proliferation of
host spleen cells was measured after exposure to exogenous antigens[12].
Briefly, spleens were removed from tolerized or control animals, 1wk
after cell transplantation or for non-transplanted controls
one week after birth, and dispersed into RPMI1640 medium (Gibco-BRL)
with 50mL�L-1 FBS. Stimulator cells (primary human
hepatocytes, Huh7, IMR-90 and 293 cells) were gamma irradiated with
20Gy to inhibit proliferation. Irradiated stimulator cells, 0.5mL of
3�108�L-1, were mixed with 0.5mL of 1�109�L-1
rat spleen cells, pulse-labeled with 37kBq of 3H-thymidine
(2960TBq�mol-1, Amersham Life Science), and then
incubated at 37℃
with 50mL�L-1 CO2
for 72h . After trichloroacetic acid (TCA) precipitation, cells were
harvested onto Whatman glass fiber filter papers (Whatman), washed
successively with phosphate buffered saline (PBS), TCA and ethanol.
Filter papers were counted in a scintillation counter (Tri-CARB
4530, Parkard). Spleen cells from untreated rats as well as
stimulator cells incubated alone served as controls. All experiments
were performed with triplicate animals, and the results expressed as
x�Sx
in units of nBq�cell-1.
Detection of human albumin gene sequences in rat liver
To detect human hepatocytes that survived transplantation in
rat livers, human albumin gene sequences were sought as specific
markers using a 5� primer (5�-CTGGTCTCACCAATCGGG-3�) and a
3� primer (5�-CTGGTCTCACCAATCGGGGG-3�). Genomic DNA extracted
from Huh7 cells served as a positive control. Genomic DNA from
untreated rats, and rats tolerized without transplantation were used
as negative controls.
Quantitation of human albumin DNA in rat liver
To quantify the number of human hepatocytes present in rat livers,
dot blots using probes specific for the human albumin gene were
performed by modifying the method of Kafatos[13] with a 32P-labeled
1750bp Bam HⅠ/
Bst eⅡ
human albumin DNA fragment excised from palb 3, a plasmid containing
the complete human albumin gene[14].
All assays were performed in triplicate, and the results were
expressed as mean�SD.
Genomic DNA from known numbers of Huh7 cells was measured in an
identical fashion.
Detection of human albumin mRNA in rat livers
To determine whether transplanted human hepatocytes retained
liver-specific transcription, the presence of human albumin mRNA was
sought by RT-PCR after extraction according to the method of
Chomczynski[15] using primers for human albumin (sense
5�-CCTTGGTGTTGATTGCCTTGCTC--3�, antisense
5�-CATCACATCAACCTCTGGTCTCACC-3�) and rat albumin (sense
5�-CGGTTTAGGGACTT-AGGAGAACAGC-3�, antisense
5�-ATAGTGTCCCA-GAAAGCTGGTAGGG-3�). The expected size of PCR
products for human and rat albumin mRNA were 315bp and
388bp , respectively.
Detection of human albumin in rat liver
Sixteen weeks post-transplantation, groups of rats were sacrificed,
and livers sectioned into 5μm slices
in tissue freezing medium (Triangle biomedical Sciences, Durham,
NC). Immunofluorescence staining was performed using a modification
of the method of Osborn[16] using monoclonal mouse
anti-human albumin antibody (Sigma, St. Louis, MO) and goat
anti-mouse IgG second antibody conjugated with Texas Red (Amersham
Pharmacia Biotech). Immunohistochemical staining for human albumin
was done according to the method of Kieran[17]. Tissue
samples were examined using confocal laser scanning microscopy
(LSM-410, Zeiss, Germany).
Assays for human albumin in rat serum
To measure human albumin in rat serum, Western blotting was
performed in a manner similar to the method of Gershoni[18]using
monoclonal mouse anti-human albumin antibody (Sigma, St. Louis, MO)
and rabbit anti-mouse IgG second antibody conjugated with
horseradish perioxidase (HRP) (Sigma, St. Louis, MO). The signal was
detected by an enhanced chemiluminescence method (ECL kit, Amersham)
and exposed to film.
RESULTS
Mixed lymphocyte assays were used to detect changes in immune
response as a result of intrafetal injections. In these assays,
spleen (responder) cells taken at wk1 after
birth were mixed with irradiated stimulator cells (primary human
hepatocytes, or controls consisting of Huh7 human hepatoblastoma
cells, IMR-90 human fibroblasts, or 293 human kidney cells). Figure
1, lane 1 shows that spleen cells from animals that were not
injected with hepatocytes intrafetally, incubated alone (without any
stimulator cells) had baseline uptake of (1446�111) nBq�cell-1.
Irradiated hepatocytes incubated alone only took up (222�100) nBq�cell-1.
Lane 2, in contrast, the spleen cells from lane 1, from rats with no
intrafetal injection, but subsequently exposed to irradiated human
hepatocytes, lane 3, were stimulated to take up (10842�1585) nBq�cell-1,
a 7.5-fold increase. But, when spleen cells from rats that had
intrafetal injection of primary human hepatocytes, were subsequently
exposed to irradiated primary human hepatocytes, lane 4, they were
not stimulated as uptake was only (1390�139) nBq�cell-1.
To determine whether the lack of stimulatory effect was hepatocyte-specific,
spleen cells from animals injected intrafetally with primary
hepatocytes were exposed to human IMR-90 fibroblasts. In contrast to
hepatocyte stimulator cells, the spleen cells were stimulated by the
fibroblasts, taking up (14373�1473) nBq�cell-1, lane 5,
which was similar to the uptake of cells from rats not intrafetally
injected with hepatocytes, (13956�2085) nBq�cell-1,
lane 7. In another control, uptake by 293 human kidney cells was
also stimulated in spleen cells from rats either intrafetally
injected with hepatocytes, lane 6, or not, lane 8. Irradiated
IMR-90, lane 9, and 293 cells, lane 10, incubated alone had
negligible uptake, indicating that the contribution of these cells
could not account for the observed increases in uptake results found
in lanes 5 and 7.
To
determine whether transformed human hepatocytes could be also used
to induce immunological tolerance, Huh7, and HepG2 human
hepatoblastoma cell lines, were compared to primary human
hepatocytes in terms of induction of tolerance. Figure 2 shows that
spleen cells from rats not injected intrafetally with hepatocytes,
and subsequently exposed to primary hepatocytes, lane 1; Huh7 cells,
lane 2; or HepG2 cells, lane 3 all had uptake ratios significantly
and substantially greater than cells from rats intrafetally injected
and subsequently exposed to the corresponding cells, lanes 4, 5 and
6, respectively.
Figure 1(PDF)
Mixed lymphocyte assays. Rat spleen (responder) cells, from 3
rats per group, were incubated either alone, or with (stimulator)
irradiated primary human hepatocytes, IMR-90 human fibroblasts, or
293 human kidney cells in the presence of 3H-thymidine.
The incorporation of radioactivity was used as a measure of
proliferation of rat spleen cells induced by exposure to foreign
cells. When performed, rats were intrafetally injected with primary
human hepatocytes on d16 of
gestation. Mixed lymphocyte assays were performed at wk1 after birth. Spleen cells from rats neither injected
intrafetally with hepatocytes, nor transplanted, lane 1; irradiated
primary human hepatocytes incubated alone, lane 2; spleen cells from
rats neither intrafetally injected nor transplanted, but which were
incubated with irradiated hepatocytes, lane 3; spleen cells from
rats intrafetally injected and transplanted and subsequently exposed
to irradiated hepatocytes, lane 4; responder spleen cells from
intrafetally injected and transplanted, exposed to irradiated IMR-90
fibroblasts, lane 5, and 293 kidney cells, lane 6; spleen cells from
animals neither intrafetally injected nor transplanted, but exposed
to irradiated IMR-90 cells, lane 7, or 293 cells, lane 8; irradiated
IMR-90 and 293 cells incubated alone, lanes 9 and 10, respectively.
Results were expressed as means�S.E. *indicates statistical
significance, P<0.05.
Figure 2(PDF)
Mixed lymphocyte assays for measuring tolerance induced by different
types of human hepatocytes. Rats were intrafetally tolerized with
either primary human hepatocytes (PH), or Huh7 cell, or HepG2 cells.
All assays were performed at wk1 after birth and show radioactive incorporation by spleen cells
from rats that were injected intrafetally with only saline, and
subsequently incubated with primary hepatocytes, hepatoblastoma cell
lines Huh7, or HepG2, lanes 1, 2 and 3, respectively. Radioactive
uptake of spleen cells from rats intrafetally injected with primary
hepatocytes, Huh7 or HepG2 cells and incubated with their
corresponding irradiated cells is shown in lanes 4, 5, and 6,
respectively. The number of rats in each group is indicated on the
top of each column. Results are expressed as percentage of controls
(spleen cells from untreated rats incubated alone) as mean�SD.
Duncan�s test was used to analyze the significance between
different treatment groups. *indicates significant differences
between groups 1 and 4, between 2 and 5; and 3 and 6, P
<0.05.
Because
of the difficulty in obtaining primary human hepatocytes,
immunohistochemical methods for detection of transplanted cells were
established using Huh7 hepatoblastoma cells. To visualize these
cells in livers of rats that were previously tolerized intrafetally
and transplanted with Huh7 cells, staining with monoclonal mouse
antibody against human albumin was performed. Figure 3 shows that
staining was detectable in rat livers on day 1 after transplantation
mostly as single cells, but with occasional pairs, fairly evenly
distributed throughout the parenchyma, panel A and with high
magnification, panel B. Seven days after birth, clusters of 2 and 3
cells each were visible, and single cells less common, panel E and
with higher magnification, panel F. No human albumin was detected in
rats that were tolerized with Huh7, but were not transplanted, panel
C and at higher magnification, panel D, confirming that the antibody
was specific for human albumin and lacked cross reactivity with
endogenous rat albumin.
Laser
scanning confocal microscopy was used to visualize transplanted
hepatocytes by staining with monoclonal antibody against human
albumin. Figure 4 shows that in tolerized rats transplanted with
human hepatocytes, human albumin was detected at week 16
post-transplantation, panel B. In tolerized rats without
transplantation, no staining was detected, panel C. Rat liver
sections stained with secondary antibody alone, panel D demonstrated
that the staining was not an artifact due to non-specific
interaction with second antibody. As expected samples stained with
monoclonal goat anti-rat albumin antibody, panel A, resulted in
positive signals for rat albumin in virtually every parenchymal cell
in the liver.
Figure 3 Immunohistochemistry for detecting human albumin in
rat livers. Antibody against human albumin was visualized using a
DAB method as described in Materials and Methods. Fifteen SD fetal
rats were tolerized with Huh7 cells. Ten newborn rats were
subsequently transplanted with Huh7 cells on d1 after
birth, and the rest were not transplanted. Panel
A, a representative
rat that was tolerized and transplanted with Huh7 cells, sacrificed
on d1 post-transplantation,
magnification �125; panel
B, same as panel A,
�250; panel
C, a representative
rat that was tolerized with Huh7 cells, but without transplantation,
�125; panel
D, same as panel C,
�250; panel
E, a representative
rat that was tolerized and transplanted with Huh7 cells, sacrificed
on d7 after birth, �125;
panel
F, same as panel B,
�250.
Figure 4 Confocal immunofluorescence microscopy for detection
of human albumin in rat livers at wk16 post-transplantation. Panel A,
a representative rat intrafetally injected with primary hepatocytes
and subsequently transplanted with those same cells, stained with
monoclonal goat anti-rat albumin. Panel
B, a section from
the same sample stained with monoclonal mouse anti-human albumin
antibody. Panel
C, a representative
rat intrafetally injected with primary human hepatocytes, but not
transplanted with hepatocytes, stained with anti-human albumin
antibody. Panel
D, the same sample
as in Panel A stained with only second antibody. Magnification, �250.
To
estimate of the number of human hepatocytes present in rat liver
transplanted with human hepatocytes, human albumin DNA sequences
were detected by amplification of rat liver genomic DNA by PCR.
Figure 5, lanes 3-5 show human albumin DNA extracted from 104,
103 and 102 Huh 7 cells, respectively. PCR
produced expected 307bp products
with decreasing intensities of signals. DNA from a rat intrafetally
injected with primary human hepatocytes and transplanted with those
cells, lane 6 produced a band at the expected position. In contrast,
a littermate intrafetally injected with human hepatocytes, but
without hepatocyte transplantation, lane 7, produced no detectable
human albumin DNA signal. Bands at the bottom of the gels are due to
excess primers. Neonatal rats sustained intrasplenic transplantation
of human hepatocytes well, with a mortality rate of about 5%.
Figure 5 Detection
of human albumin DNA in rat liver genomic DNA 16wk post-transplantation. From livers of animals treated as
described in Figure 4, DNA was extracted, and assayed for the
presence of human albumin sequences by PCR as described in Materials
and Methods. Lane 1, molecular markers; lane 2, liver from untreated
rats; lanes 3 to 5, 104, 103 and 102
Huh7 cells, respectively; lane 6, a representative rat intrafetally
injected with primary human hepatocytes, and transplanted with those
cells; lane 7, a rat intrafetally injected with primary hepatocytes,
but not transplanted. The expected position of the amplified human
albumin sequence is indicated by the arrow corresponding to 307bp based on the DNA molecular markers in lane 1.
To
quantitate the amount of human albumin gene present, dot blotting
for human albumin DNA was performed. In Figure 6, the upper row of
panel A, shows that liver samples from a representative rat
intrafetally injected and transplanted with primary human
hepatocytes had positive signals for human albumin DNA at 6wk and
16wk post-transplantation, with little obvious change in signal
between the time points. In contrast, a littermate tolerized with
human hepatocytes, but not transplanted, lower row, had no signals
at the same time points indicating that the human albumin signal
detected in the upper panel were not due to residual DNA from the
tolerization procedure or cross reactivity with rat sequences. In
panel B, the plasmid palb3, was loaded in serial dilutions as
standards for human albumin DNA. Based on the amount of human
albumin DNA in 10 (g rat liver DNA, the number of surviving human
hepatocytes was calculated to be 2.5�105 cells per whole
adult liver 16wk post-transplantation.
The ratio of human to rat hepatocytes that were present, 16 weeks
post-transplantation, was calculated to be approximately 1 human
cell per 6�103 rat hepatocytes.
Figure 6
Quantitation of human albumin DNA in rat livers by dot blotting.
Panel A, upper row, shows DNA extracted from liver samples from a
intrafetally injected, and transplanted rat at wk6 and 16
post-transplantation, respectively. Lower row shows results from a
rat tolerized but without transplantation at the same time points.
All dots were hybridized with a 32P-labeled probe for
human albumin DNA. Panel B shows a plasmid, palb-3, containing the
complete human albumin gene, applied in decreasing amounts, 100 pg,
10 pg and 1 pg as standards.
To
assess albumin gene transcription in rat livers, RT-PCR of albumin
mRNA was performed. In Figure 7, panel A, lane 3 shows that human
albumin mRNA extracted from Huh7 cells was detected by RT-PCR by the
presence of a product with the expected size of 315bp . As expected,
the same sample failed to generate a signal when rat albumin primers
were used indicating that the human primers were specific for the
detection human albumin mRNA, lane 7. In tolerized rats 16wk after
human hepatocyte transplantation, human albumin mRNA was also
detected as a 315bp band, lane 4. However, no human albumin mRNA was detected in a
littermate intrafetally injected, but without transplantation, lane
5, or from a non-fetally injected, and non-transplanted rat, lane 2.
However, a band corresponding to the rat albumin amplification
product of 388bp was
demonstrated in a representative rat neither intrafetally injected,
nor transplanted, lane 6; a rat intrafetally injected, and
transplanted, and sampled 16 weeks after transplantation, lane 8;
and a rat intrafetally injected, but without transplantation, lane
9. As standards, RNA extracts from Huh7 cells were amplified with
primers for human albumin, and as expected produced decreasing
signals of 315 bp products, lanes 10-12.
The
transcriptional activity of transplanted human hepatocytes as a
function of time was measured over the entire experimental period of
16wk . Figure 7, panel B, lane 3 shows that human albumin mRNA
extracted from Huh7 cells in culture and detected by RT-PCR produced
a product with the expected size of 315bp . Human albumin mRNA
levels from rats were not obviously different at wk2 , 6, and 16
after transplantation, lanes 4, 5 and 6, respectively, suggesting
that, within the limits of the assay, the function of transplanted
human hepatocytes, at least with regard to albumin production,
remained unchanged for at least 16 weeks.
Figure
8 shows Western blots of serum from a representative rat tolerized
and transplanted with primary human hepatocytes. A band with
migration corresponding to 56 ku, the expected size of human serum
albumin, as shown by standard human albumin in lane 1 was found in
serum from a rat intrafetally injected and transplanted with primary
human hepatocytes 1wk post-transplantation,
lane 3, and remained detectable at 2 and 3wk post-transplantation, lanes 4 and 5, respectively. There was
no cross reactivity of antibody with rat liver not transplanted with
human cells as shown in lane 2. These data support the conclusion
that human hepatocytes can be transplanted, and survive in the
livers of intrafetally tolerized rats, and remain sufficiently
active to secrete detectable amounts of human serum albumin into the
circulation.
Figure 7
Detection of human albumin mRNA in rat livers by RT-PCR. Panel A,
RT-PCR of RNA extracts from liver samples collected at wk16
post-transplantation. Lane 1, 1 kb plus molecular markers; lanes 2
and 6, non-intrafetally injected, non-transplanted rats; lanes 3 and
7, Huh7 cells as positive control; lanes 4 and 8, rats intrafetally
injected, and transplanted with primary human hepatocytes; lanes 5
and 9, rats intrafetally injected, but not transplanted with human
hepatocytes. For lanes 2 to 5, samples were amplified with primers
for human albumin DNA, and for lanes 6 to 9, samples were amplified
with primers for rat albumin DNA. In lanes 10 to 12, DNA from 104,
103 and 102 cultured Huh7 cells were amplified
with primers for human albumin. The expected positions of human and
rat albumin mRNA products at 315bp and
388bp , respectively, are indicated by arrows. Panel B, Time course
of human albumin mRNA expression in the rat livers. Lane 1, 1kb plus
molecular markers; lane 2, samples from a non-tolerized and
non-transplanted rat; lane 3, Huh 7 cells as a positive control;
lanes 4 to 6, samples from a representative rat tolerized and
transplanted with primary human hepatocytes collected at wk2 , 6 and
16 post-transplantation, respectively.
Figure 8
Detection of human albumin protein in rat serum by Western blots.
Western blots of serum samples from a representative rat
intrafetally injected and transplanted with primary human
hepatocytes at post-transplantation wk1 , lane 3; wk2 , lane 4; and
wk3 , lane 5. Lane 1: standard human albumin and lane 2: standard
rat albumin.
DISCUSSION
The developing immune system in the fetus allows a unique
opportunity for the induction of tolerance to specific cells without
generalized suppression of the immune system. In utero
injection of donor cells directly into the peritoneal cavities of
fetuses during gestation has been shown previously to result in a
donor-specific tolerance[10,11,19]. With regard to the
timing of cell injections for induction of tolerance, previous
studies in normal mice demonstrated that in utero injections
of splenocytes on days 14 to 16[20] were satisfactory for
induction of donor-specific tolerance and prolongation of the
survival of allogeneic skin grafts. In our experiments, intrafetal
injections performed on any of three days, from day 15 to 17 of
gestation, resulted in equal effectiveness in establishing tolerance
to human hepatocytes.
Livers
of tolerized rats transplanted with human hepatocytes was found to
contain the human albumin gene up to 16 weeks post-transplantation,
the duration of the experiment, Figures 5 and 6. The transplanted
cells were functional as indicated by the presence of human albumin
mRNA, Figure 7, and human albumin gene product, Figures 4 and 8. The
presence of antibodies to human albumin was not detected (data not
shown).
Chimeric
liver models have been described previously, but in
immunocompromised hosts. For example, normal adult woodchuck
hepatocytes were transplanted into uPA/RAG2 knock out mice via
intrasplenic injection. These cells eventually reconstituted 90% of
those mouse livers[6]. Similarly, immortalized primary
human hepatocytes have been transplanted into RAG-2 knock out mice[7].
Recently, primary human hepatocytes were transplanted under the
kidney capsule of SCID/NOD mice[8]. The results
demonstrate that when transplanted into immunocompromised hosts,
human hepatocytes can not only survive, but also maintain
differentiated cellular function in foreign hosts and even in
unnatural locations. Our current data further indicate that general
immune deficiency or suppression is not required, and intrafetal
tolerization is sufficient to prevent rejection. Furthermore, the
technique could be used to generate tolerance to other cell types,
that might result in development of other useful animal models.
The
mechanism of tolerance induction via intrauterine exposure to
foreign antigen has been ascribed to T- cell clonal
deletion[20]. Recently, Knolle and colleagues[21,22]
described another method of inducing tolerance in the liver to
foreign antigens: direct injection of foreign antigens into portal
circulation resulted in the presentation of foreign antigens by
liver endothelial cells to CD8+ T- cells. Such a model
for tolerance has not been tested specifically for induction of
tolerance to xenografted human hepatocyte transplantation.
ACKNOWLEDGMENTS This work was supported in part by grants
from the NIDDK: DK-42182 (GYW), Connecticut Innovations, Inc. (CHW),
and the Herman Lopata Chair in Hepatitis Research (GYW). The authors
would like to thank Dr. Thiruchandurai V. Rajan for kind
co-operation in animal surgery, Ms. Nancy Ryan for assistance in
preparing tissue sections, and Dr. Ravi Kondapalli for technical
assistance.
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