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Animal Model |
From the Dipartimento di Scienze Mediche e
Biotecnologie,*
Sezione di Patologia Sperimentale,
Università di Cagliari, Cagliari, Italy; the Ospedale Oncologico
"A. Businco",
Cagliari, Italy; and The
Marion Bessin Liver Research Center,
Albert
Einstein College of Medicine, Yeshiva University, Bronx, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The liver has been among the first targets for strategies based on transplantation of isolated cells.5-7 In recent years, this approach has been extended from experimental animals to humans, with the first reported cases of hepatocyte transplantation to treat familial hypercholesterolemia8 and hyperbilirubinemia associated to Crigler-Najjar syndrome.9 The procedure was shown to be technically feasible; however, studies to date have reported only modest reductions in serum cholesterol and bilirubin, respectively. A major obstacle toward achieving better clinical efficacy is the limited extent to which transplanted cells proliferate in the recipient liver.8,9 This comes as no surprise, given the very low level of cell turnover present in this organ under normal conditions.10
In the past few years, significant progress in this field has been made by the development of several experimental models for extensive liver repopulation via transplantation of isolated hepatocytes.11-14 The uPA transgenic mouse model was the first to demonstrate the biological possibility of massive replacement of a diseased liver via exogenously provided normal cells.11 In this system, endogenous hepatocytes expressing the targeted uPA transgene are selectively deleted and replaced by normal cells. A similar principle of selective survival advantage seems to form the basis for the massive proliferation of transplanted cells in the liver of the FAH-null mouse, a model for human hypertyrosinemia type I.12 In both cases, the essential feature of the model resides in the inherent genetic defect of resident cells which causes toxicity and leads to their selective death and replacement.
Using a novel approach, we have recently reported near-total liver
repopulation by transplanted normal hepatocytes in rats treated with
retrorsine (RS).13
In this system, rats with a normal
genetic background are exposed to RS, a naturally occurring
pyrrolizidine alkaloid which causes a long lasting block of hepatocyte
cell cycle.15-17
Two-thirds partial hepatectomy (PH) is
then performed and is followed immediately by transplantation of normal
hepatocytes isolated from a syngeneic donor. Under these conditions,
>95% liver replacement by donor-derived cells is observed within 2 to
4 months after the operation.13,18
A key component in this
model is the persistent block imposed by RS on endogenous hepatocyte
cell division, which allows for the selective expansion of transplanted
cells.13,19
Consistent with this interpretation, PH
was found to be essential to achieve massive liver replacement within a
few months after transplantation. However, it was repeatedly
observed13,20
that a low, but significant extent of
repopulation (up to 
15% within 6 weeks)20
was also
present in animals not receiving PH. Given the potential relevance of
these findings, we decided to conduct a long-term study to follow the
fate of transplanted hepatocytes in rats previously given RS and in the
absence of any exogenously elicited growth stimulus, such as PH. The
dipeptidyl-peptidase type IV-deficient (DPPIV-)
F344 rat model for hepatocyte transplantation was used, as in previous
reports.13,20
Results indicated extensive (70 to 90%)
replacement of the recipient liver by donor-derived cells at 6 to 9
months after transplantation, in both male and female rats. Moreover,
serum analysis, performed up to 1 year after hepatocyte infusion,
revealed normal values for several parameters related to liver function
in the transplanted, repopulated animals.
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Materials and Methods |
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Hepatocyte transplantation was performed with a modification of our
recently published protocol.13
Briefly, F344
DPPIV- rats, both male and female, weighing 80
to 100 g, were given two intraperitoneal injections of RS (Sigma
Chemical Co., St. Louis, MO), 30 mg/kg each, 2 weeks apart. Two
weeks after the last injection of RS, each animal received 2 x
106
freshly isolated hepatocytes via portal vein
infusion. Hepatocytes were isolated from normal young adult
DPPIV+ F344 donor rats according to a standard
two step collagenase perfusion technique.21
The isolated
cell fraction used for transplantation studies was judged to be 95%
hepatocytes by morphological analysis and cell viability was
consistently between 85 to 95%, as determined by trypan blue dye
exclusion. Control groups received either RS alone or no treatment.
Four to six animals were killed at various intervals during the
experiment, as indicated in the Results section. Where indicated, a
single injection of 5'-bromodeoxyuridine (BrdU) (50 mg/kg i.p.; Sigma
Chemical Co.) was given to the animals 2 hours before sacrifice. Liver
samples were fixed in 10% buffered formaldehyde or snap-frozen.
Histochemical determination of DPPIV enzyme activity and quantitation
of DPPIV-positive areas in the liver were performed as
described.13,20
Three random sections were cut from each
liver lobe of each animal and stained for DPPIV enzyme activity.
Sections were then projected into a magnetic graphics tablet (NewSketch
1212 HR; Genius, KEY System Corp., Langenfeld, Germany), and
quantitated with the help of a computer-assisted image analyzer. Liver
DNA content was estimated according to published
techniques,20
whereas serum analysis was performed using
automated Synchron Clinical System CX7 Delta (Beckman Coulter).
Glutathione-S-transferase 7-7 (GST 7hyphen]7) and
BrdU-labeling were detected by immunohistochemical
methods.22
Statistical analysis was performed using the
Student’s t-test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals were treated according to the standard protocol for
hepatocyte transplantation with RS;13
however, a major
modification was introduced, in that no PH was performed at the time of
cell infusion; moreover, in the absence of PH, (which cannot be
performed before 4 weeks after treatment with RS, because it causes
significant animal loss), we decided to perform cell transplantation at
2 weeks after the last dose of RS, reasoning that it was no longer
necessary to maintain a 4-week gap. All groups tolerated well the
experimental procedure. At 2 weeks after cell transplantation, numerous
clusters (30 to 40/cm2) of
DPPIV+ donor hepatocytes were present throughout
the DPPIV- recipient liver; they comprised 5 to
10 cells/cluster/cross section, occupying 3.3 ± 0.9% of the
total liver area (Figure 1
and Figure 2A
). Clusters were composed of
approximately normal-sized hepatocytes, whereas the recipient liver was
extensively megalocytic.13,17
At 1 month,
DPPIV+ clusters had enlarged in size to 10 to 40
cells/cross section and comprised 9.1 ± 1.6% of the total area
(Figure 1
and Figure 2B
). They were mostly round or oval in shape;
however, some appeared elongated along the hepatic plates. No signs of
compression were evident in the surrounding parenchyma. Surrounding
(DPPIV-) parenchymal cells were still
megalocytic; however, collections of small
DPPIV- hepatocytes (20 to 30 cells/focus) were
evident, with a frequency of approximately one in 10 compared to
DPPIV+ clusters. These have been referred to as
"regenerative nodules"13,17,23
and seem to be similar
to those recently described by Gordon and colleagues24
during liver regeneration in rats given RS.
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and Figure 2C
) and was 96.1 ± 5.3% in animals treated
with RS and killed 1 year after cell transplantation (Figure 1
and
Figure 2D
). In the latter group, only residual scattered megalocytes
were visible, along with rare (1.3 ±
0.9/cm2) islands of small
DPPIV- hepatocytes comprising 100 to 300 cells
and representing <3% of the total area. By contrast, no growth of
DPPIV+ hepatocytes was seen at any time point (up
to 1 year after cell transplantation) when cells were transplanted into
normal untreated DPPIV- recipients (data not
presented), in agreement with our previous findings.13
In a parallel study, we investigated whether the liver of female rats
treated with RS would also be replaced by normal transplanted
hepatocytes when no exogenous proliferative stimulus was provided. This
was a relevant issue to address, in light of a reported lower
sensitivity of female versus male animals to the acute
effects of RS.25
DPPIV- female rats
were injected with RS followed by DPPIV+
hepatocyte transplantation, as outlined above, and killed 2 months or 8
months thereafter. Surprisingly, results were very similar to those
observed in male animals, with 33.9 ± 5.2% repopulation by
DPPIV+ hepatocytes at 2 months and 81.4 ±
7.6% at 8 months (Figure 2E)
. Moreover, as revealed by double labeling
for DPPIV enzyme activity and BrdU immunohistochemical staining,
proliferating DPPIV+ hepatocytes were still
detected in the liver of female animals even 8 months after
transplantation (Figure 2F)
. This observation suggests that the process
of liver repopulation was still active for many months after
transplantation.
The extent of repopulation found in male and female animals in this study was higher than that described in our earlier reports in RS-treated animals not receiving PH.13,26 However, more recently we observed as high as 15 to 20% repopulation in RS-treated female rats at 6 weeks after cell transplantation, in the absence of PH or any other exogenous stimulus.20 Interestingly, both in the latter20 and in the present study, cell transplantation was performed at 2 weeks after the second dose of RS, whereas in the first report,13 this interval was 4 weeks. This suggests that interval between RS treatment and cell transplantation could be a determinant in the extent of repopulation in this model.
Repopulating Hepatocyte Clusters Do Not Express Increased Levels of GST 7-7
Hepatocytes transplanted into the liver of animals treated with RS
undergo rapid proliferation to form discrete proliferative clusters,
which then expand to eventually replace the bulk of the recipient
liver. Given the long-standing concern regarding a possible association
between repeated cell division cycles (clonal expansion) and increased
risk for cell transformation,27
we tested for increased
expression of GST 7-7 in DPPIV+ hepatocyte
clusters at various times during liver repopulation. Increased
expression of GST 7-7 is in fact a common finding in hepatocyte foci
and nodules developing during chemically induced liver carcinogenesis
in rats, and is considered a marker to identify early focal lesions at
risk for neoplastic transformation.28
Serial sections were
used for either histochemical staining of DPPIV enzyme activity or
immunohistochemical detection of GST 7-7. Liver samples obtained at 2
weeks, and 1, 2, 5, and 8 months after cell transplantation were
examined. Representative results are shown in Figure 3, A and B
. No evidence was found for
increased expression of GST 7-7 in DPPIV+
hepatocytes at any time point considered. Positive GST 7-7 staining was
regularly present in bile ducts17
and was found only in
rare scattered hepatocytes of recipient origin. Histological findings
on hematoxylin and eosin-stained liver sections were similar to those
previously described.13
Figure 3, C and D
, reports liver
histology of untreated (Figure 3C)
or treated with RS and transplanted
(Figure 3D)
female rats, killed 9 months after starting the experiment.
The liver architecture was primarily normal, except for some residual
areas of megalocytosis. No proliferation of bile ductular structures
was evident.
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We have previously documented that transplanted hepatocytes
repopulating the liver of RS-treated rats express a fully
differentiated phenotype, according to a series of biochemical
parameters detected by histochemical and immunohistochemical
methods.13
To further extend these observations at the
whole animal level, several serum parameters related both the
hepatocyte function and integrity (including glucose, bilirubin, total
protein, cholinesterase, alanine aminotransferase, alkaline
phosphatase), were monitored in rats presenting extensive liver
repopulation. In addition, animals exposed to RS treatment with no
transplantation were also examined and compared to normal untreated
rats. Figure 4
presents results obtained
in groups of female animals killed 9 months after starting the
experiment. The extent of repopulation in the transplanted group was
81.4 ± 7.6% (data presented above). As reported in Figure 4
, no
significant alterations in any of the considered parameters were
detected in transplanted animals compared to normal reference levels. A
similar pattern of results was also seen in male and female animals
killed at various time points during the process of repopulation, up to
1 year after cell transplantation. Furthermore, exposure to RS alone,
according to protocol developed for hepatocyte
transplantation,13
was also not associated with any
significant alteration in any of the examined parameters, suggesting
that the protocol per se does not compromise liver function.
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Pyrrolizidine alkaloids, including RS, are known for their ability to impose a persistent block on hepatocyte cell division.17,29,30 This effect becomes particularly evident when the liver is challenged with a vigorous regenerative stimulus, such as that elicited by PH.17 However, some reports have also pointed to a relative inability of the liver exposed to these alkaloids to maintain a balanced mass during normal organ turnover.23 In support of these findings, we have recently observed a decrease in liver weight and liver DNA content in animals receiving two doses of RS (30 mg/kg each, as in the present study) and killed 2 weeks after the second injection.17 This finding may become relevant in light of the results presented above (ie, extensive proliferation of transplanted hepatocytes in rats treated with RS, in the absence of exogenous growth stimuli). In fact, it is reasonable to conceive that the presence of a relatively small liver in animals treated with RS may set the stage for the selective proliferation of transplanted cells, which are not blocked by previous exposure to the alkaloid.
Based on the above considerations, we examined whether there was any
alteration in the size of the liver and/or total liver DNA content in
female animals treated with RS and killed several months later. Three
groups of animals were included in this study: group I, untreated
controls; group II, RS-treated only; and group III, RS plus cell
transplantation (details are reported in the Materials and Methods).
All groups were killed 9 months after the initial treatment. The
results obtained confirmed and extended our previous observations. As
reported in Figure 5A, a
significant
reduction in the relative weight of the liver was seen in the groups
receiving RS (groups II and III) compared to controls (group I), with
no intergroup differences between transplanted and nontransplanted
animals (groups III and II, respectively). A similar pattern of results
was seen when liver DNA content was considered. Both total DNA/liver
(Figure 5B)
and liver DNA/100 g body weight (Figure 5C)
were
significantly reduced (up to 35%) in RS-treated groups compared to
untreated controls. Again, there was no statistically significant
difference in either parameter between groups receiving RS or RS plus
cell transplantation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For transplanted cells to proliferate, one needs to provide the necessary space in the recipient organ.4 Thus, both in the uPA transgenic mouse11 and in the FAH-null mouse,12 the selective proliferation of transplanted normal cells is fostered by a process of chronic toxicity and cell death occurring in the resident hepatocyte population. Similarly, in the RS model of liver repopulation in the rat, as originally described, partial removal of liver tissue in a background of persistent mitoinhibition of resident hepatocytes was found to be essential for the rapid and massive expansion of transplanted cells.13
However, our present data cannot be entirely explained within
this conceptual framework. In fact, no exogenous growth stimulus (eg,
PH) was introduced to drive proliferation of transplanted cells, nor
there was any histological (Figure 3D)
or serological evidence of
chronic toxicity leading to necrosis of endogenous hepatocytes, either
during the process of liver repopulation or in RS-treated animals not
receiving cell transplantation (Figure 4, A through F)
. It is still
possible to consider that a mechanism of compensatory growth may
contribute to the extensive proliferation of transplanted cells in the
absence of exogenous stimuli. In fact, both liver weight and liver DNA
content were reduced compared to appropriate controls (Figure 4)
, in
animals treated with RS and killed as long as 8 months later, in line
with previous observations.20
Such a relative deficit of
liver mass persisting throughout a prolonged period may represent a
sufficient growth compensatory stimulus for transplanted cells to
expand, given the persistent cell cycle block exerted by RS on host
hepatocytes. However, no significant recovery of liver mass and/or
liver DNA content was seen in RS-treated and transplanted animals even
in presence of levels of repopulation as high as 85% of the total
liver. This finding suggests that the selective proliferation of
transplanted normal cells is primarily sustained by additional
mechanisms, different from simple compensatory growth.
It is clear from our data that donor-derived cells are not simply growing side by side with endogenous megalocytes blocked by RS, but they are actually replacing resident parenchymal cells. There is evidence indicating an increased susceptibility to the induction of apoptotic cell death in rat liver exposed to RS.26 Most recently, apoptosis associated with enhanced levels of Bax protein was reported in the liver of rats treated with RS followed by PH.31 The relevance of the latter findings to our present results needs to be carefully evaluated, because in this study liver replacement by transplanted cells occurred in the absence of PH. However, it is possible to hypothesize that a prolonged increase in the rate of apoptosis in RS-exposed endogenous hepatocytes may drive the growth of donor-derived cells; alternately, or in combination, the presence of normal transplanted cells may trigger selective deletion of RS-damaged resident hepatocytes, also possibly through apoptosis.
A second interesting issue raised by the present results pertains to the efficiency of the RS model of hepatocyte transplantation in male versus female rats. In fact, female rats were reported to be relatively resistant to the acute toxicity of RS, with an LD50 almost five times as high as that for male rats of the same age.25 It is therefore surprising to find that, using the same standard dose of RS (30 mg/kg twice) in both male and female recipients, and in the absence of exogenous growth stimuli, female rats also undergo massive liver replacement by transplanted cells, with percentages of repopulation similar to those observed in males. These data are of particular significance and suggest that acute toxicity of RS, or at least part of it, can be avoided in a setting designed for liver repopulation through transplanted hepatocytes. Additional studies are in progress to explore this possibility.
We have previously documented that normal hepatocytes
transplanted into RS-treated liver express a differentiated phenotype
during the process of liver repopulation and perform normal biochemical
functions.13,18
Results presented in this report confirm
and extend the above conclusion; in fact, both RS-treated and
RS-treated and repopulated livers tested normal according to several
serum parameters related to hepatocyte function and integrity (Figure 3)
. It is noteworthy that RS treatment per se, when given according to
the present protocol for hepatocyte transplantation, does not seem to
cause significant impairment of liver function several months after
exposure, despite the persistence of extensive areas of megalocytosis.
It is still debatable whether repeated cell proliferation per se
represents a risk factor for the emergence of the neoplastic
phenotype.27,32
Under the experimental conditions
described in the present studies, in which transplanted cells undergo a
process of clonal expansion involving many cell divisions, no evidence
for increased focal expression of GST 7-7 was found at any time point
during the repopulation process (Figure 3, A and B)
. To the extent that
this enzyme is a reliable marker of neoplastic precursor
lesions,26
this result indicates that liver repopulation
with transplanted normal hepatocytes in this model is not associated
with the emergence of phenotypically altered cells on the pathway
toward neoplasia. However, additional studies with longer periods of
observation are warranted to settle this issue.
In conclusion, the present results provide important insights toward developing strategies for effective liver repopulation by transplanted hepatocytes with reduced toxicity for the host and potential clinical applicability. Extensive liver replacement could in fact be demonstrated in RS-treated animals in the absence any of exogenous growth stimuli, such as PH, thereby avoiding any additional treatment. Furthermore, acute toxicity of RS, including hepatocyte necrosis, seems unnecessary to set the stage for liver repopulation to occur in this model. A better understanding of the relevant mechanisms which are driving the growth of transplanted normal hepatocytes in RS-treated rat liver will help in designing better approaches toward the applicability of hepatocyte transplantation as an effective therapeutic strategy.1-4
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Acknowledgements |
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Footnotes |
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Supported in part by Telethon, Italy (to P. P.), the Roche Organ Transplantation Research Foundation (to E. L.), and by grants RO1 DK17609 and P30 DK41296 from the National Cancer Institute, Bethesda, MD (to D. A. S.).
Accepted for publication October 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein mRNA expression in the retror-sine model for hepatocyte transplantation. Cancer Res 1998, 58:5825-5834This article has been cited by other articles:
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  F. Marongiu, S. Doratiotto, S. Montisci, P. Pani, and E. Laconi Liver Repopulation and Carcinogenesis: Two Sides of the Same Coin? Am. J. Pathol., April 1, 2008; 172(4): 857 - 864. [Abstract] [Full Text] [PDF]
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 F. C. Popp, P. Slowik, E. Eggenhofer, P. Renner, S. A. Lang, O. Stoeltzing, E. K. Geissler, P. Piso, H. J. Schlitt, and M. H. Dahlke No Contribution of Multipotent Mesenchymal Stromal Cells to Liver Regeneration in a Rat Model of Prolonged Hepatic Injury Stem Cells, March 1, 2007; 25(3): 639 - 645. [Abstract] [Full Text] [PDF]
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M. H. Walkup and D. A. Gerber Hepatic Stem Cells: In Search of Stem Cells, August 1, 2006; 24(8): 1833 - 1840. [Abstract] [Full Text] [PDF]
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 S. Eventov-Friedman, H. Katchman, E. Shezen, A. Aronovich, D. Tchorsh, B. Dekel, E. Freud, and Y. Reisner Embryonic pig liver, pancreas, and lung as a source for transplantation: Optimal organogenesis without teratoma depends on distinct time windows PNAS, February 22, 2005; 102(8): 2928 - 2933. [Abstract] [Full Text] [PDF]
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A. R. Migliaccio, R. Quarto, and W. Piacibello Cell Therapy: Filling the Gap Between Basic Science and Clinical Trials October 15-17, 2001, Rome, Italy Stem Cells, May 1, 2003; 21(3): 348 - 356. [Abstract] [Full Text] [PDF]
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S. Laconi, P. Pani, S. Pillai, D. Pasciu, D. S. R. Sarma, and E. Laconi A growth-constrained environment drives tumor progression invivo PNAS, June 20, 2001; (2001) 131210498. [Abstract] [Full Text] [PDF]
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S. Laconi, P. Pani, S. Pillai, D. Pasciu, D. S. R. Sarma, and E. Laconi A growth-constrained environment drives tumor progression invivo PNAS, July 3, 2001; 98(14): 7806 - 7811. [Abstract] [Full Text] [PDF]
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