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Animal Model |
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From the Istituto di Patologia Sperimentale,*
Ospedale
Oncologico "A Businco," University of Cagliari, Cagliari,
Italy, and The Marion Bessin Liver Research
Center,
Division of Gastroenterology,
Hepatology and Nutrition
and the
Departments of Medicine,§
Cell
Biology,¶
and Pathology,||
Albert
Einstein College of Medicine of 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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Recently, two experimental models of extensive liver repopulation have been described: the urokinase-type plasminogen activator transgenic mouse14-16 and the fumarylacetoacetate hydrolase (Fah)-null mouse,17 the latter serving as a model for hereditary tyrosinemia type I. In the urokinase-type plasminogen activator model, endogenous hepatocytes that have lost expression of the transgene14 and transplanted normal hepatocytes15,16 proliferate extensively because of continuous proteolytic destruction of resident hepatocytes expressing the urokinase-type plasminogen activator transgene.14 Similarly, in Fah-null mice, transplanted wild-type hepatocytes exhibit a growth advantage over enzyme-deficient cells in the recipient liver, because the latter accumulate toxic intermediates in the tyrosine metabolic pathway.17 Both models have convincingly documented that transplanted hepatocytes have enormous proliferative potential.15-17 In the Fah model, most recent studies report that transplanted wild-type hepatocytes can be serially transplanted through six or seven hosts with full repopulation from 10,000 donor cells in each recipient.18 However, because both of these models rely on unique genetic backgrounds, their application as general models for hepatic cell transplantation is rather limited.
We reasoned that if we could develop an alternative method to provide a selective growth advantage to transplanted cells over endogenous hepatocytes, this could represent a generally useful approach to hepatocyte repopulation. The present study describes a new strategy for selective proliferation of transplanted cells by interfering with the proliferative capacity of resident hepatocytes, using the pyrrolizidine alkaloid retrorsine and then transplanting normal cells in conjunction with partial hepatectomy. The validity of this approach was tested by tracking genetically marked transplanted cells in an inbred strain of Fischer (F) 344 rats not expressing a specific marker gene, dipeptidyl peptidase IV (DPPIV), and by monitoring the ability of transplanted hepatocytes to replace hepatocyte mass. The proliferation, expansion, and integration of transplanted cells into the hepatic parenchymal structure was determined by morphological and histochemical analysis, and the biochemical function of transplanted hepatocytes was determined by analysis of liver tissue for expression of hepatocyte-specific proteins, albumin, and glucose-6-phosphatase, as well as glycogen synthesis and storage. Using these methods, we have observed replacement of 40 to 60% of hepatic mass in female rats for up to 1 year. Near-total replacement of hepatocyte mass was observed in male rats (98 to 99%) and persisted for 9 months after transplantation, the duration of experiments conducted.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DPPIV- F344 rats were provided by the Special Animal Core of the Liver Research Center at the Albert Einstein College of Medicine. Donor DPPIV+ F344 rats were purchased from Charles River Laboratories (Wilmington, MA). All animals were maintained on daily cycles of alternating 12-h light/darkness with food and water available ad libitum. Animals were given Purina Rodent Lab Chow diet (No. 5001; Dyets, Inc., Richmond, IN) throughout the experiments. F344 DPPIV- rats weighing 90 to 140 g were given two injections of retrorsine (Sigma Chemical Co., St. Louis, MO), 30 mg/kg each, intraperitoneally, 2 weeks apart. This protocol exerts a strong and persistent inhibition of hepatocyte cell division that lasts for at least several months (E. Laconi and P. Pani, unpublished observation). Four weeks after the last injection, two-thirds partial hepatectomy was performed, and each animal received 2 x 106 freshly isolated hepatocytes via portal vein infusion. All studies were conducted in accordance with U.S. federal guidelines (NIH Publication 86-23, revised 1985), under protocols approved by the Animal Care Use Committee of the Albert Einstein College of Medicine.
Hepatocyte Isolation
Hepatocytes were isolated from young adult DPPIV+ F344 donor rats according to a standard two-step collagenase perfusion technique.19 The isolated cell fraction used for transplantation studies was judged to be ~95% hepatocytes by morphological analysis. Cell viability was consistently between 85 and 95%, as determined by trypan blue dye exclusion. Rats were divided into four experimental groups: 1) animals receiving cell transplantation but no other treatment; 2) animals receiving retrorsine and cell transplantation but no partial hepatectomy; 3) animals receiving partial hepatectomy and cell transplantation but no retrorsine; and 4) animals receiving retrorsine, partial hepatectomy, and cell transplantation. Controls consisted of untreated rats and rats treated with retrorsine but neither partial hepatectomy nor cell transplantation. Rats from different experimental groups were killed at various time points postsurgery, starting from day 1 and up to 1 year. Livers were examined grossly, and separate samples from each lobe were either frozen or fixed in buffered formalin and processed by histological, histochemical, or immunohistochemical methods.
Detection of Liver-Specific Markers
DPPIV and ATPase histochemical staining was performed according to previously published techniques.20 Quantitation of DPPIV-positive areas in the liver was performed by computer-assisted image analysis. This consisted of projecting images from DPPIV-stained slides onto a magnetic graphics tablet (NewSketch 1212 HR, Genius, KYE System Corp., Langenfeld, Germany) that converted the information into a computer-readable format from which the areas and percentages were determined by a simple computer program. Albumin and cytokeratin-19 were detected by immunohistochemical methods, also as previously reported,20 using secondary antibodies conjugated to peroxidase and peroxidase enzyme activity detected with 3,3'-diaminobenzidine as substrate. Glycogen was detected by the method of Lillie.21
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retrorsine treatment of DPPIV- F344 rats, hepatocyte
isolation and transplantation, and two-thirds partial hepatectomy were
conducted as noted in Materials and Methods. All procedures were well
tolerated, and most animals (>95%) survived all protocols. After the
acute effects of retrorsine subsided (these included dilatation and
congestion of hepatic sinusoids, mild inflammation, and biliary
epithelial cell proliferation), the hepatic parenchyma exhibited mild
to moderate disruption of the parenchymal cord structure, residual
biliary cell hyperplasia, and scattered hepatocyte megalocytosis
(Figure 1A)
. After partial hepatectomy
(with or without cell transplantation), there was a marked increase in
hepatic megalocytes (Figure 1B)
. These cells have large nuclei,
resulting from DNA synthesis in retrorsine-treated hepatocytes after a
liver proliferative stimulus but with an inability of these cells to
undergo mitosis (22-25
and E. Laconi and P. Pani, unpublished
observations). Focal areas of small hepatocytes in clusters were also
observed (Figure 1B)
. There was no evidence of fibrosis (Figure 1, C and D)
, chronic liver disease, or hepatic neoplasms, all of which occur
with higher doses and/or repeated administration of pyrrolizidine
alkaloids.26,27
At the time of cell transplantation, there
was no evidence of retrorsine toxicity in other organs, including lung,
heart, brain, stomach, small intestine, colon, kidney, pancreas,
adrenal gland, testes, and ovaries.
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One day after transplantation of 2 x 106
hepatocytes
into retrorsine and partial hepatectomy-treated female rats,
DPPIV+ cells were difficult to find. They appeared mostly
as single cells in the hepatic sinusoids in or near the portal triads
(Figure 2A)
. Initially, DPPIV
histochemical staining was diffuse over the cell surface and not
confined to the bile canaliculus, probably reflecting the recent
isolation and transplantation of cells. By the 4th day
posttransplantation, groups of 2 to 4 DPPIV+ cells with
typical bile canalicular staining were discernible (Figure 2B)
, and at
2 weeks, DPPIV+ hepatocytes appeared in clusters of 25 to
50 cells in a two-dimensional cross-sectional area (Figure 2C)
.
DPPIV+ hepatocyte clusters were much larger at 1 month
posttransplantation (Figure 2D)
, ranging from 100 to several hundred
cells, and at 2 months posttransplantation, DPPIV+ clusters
ranged up to 1000 or more cells, with many clusters becoming confluent
(Figure 2E)
. Using computer-based morphometric analysis in female rats
at 2 weeks postsurgery, we found that about 3 to 5% of hepatocytic
mass expressed DPPIV, increasing to 15 to 25% at 1 month and to 40 to
60% at 2 months. This level of hepatocyte replacement remained
essentially constant for up to 1 year, the duration of experiments
conducted. No significant growth of transplanted hepatocytes was seen
in rats receiving partial hepatectomy but not treated with retrorsine
(data not shown). In retrorsine-treated female rats receiving
transplanted hepatocytes but not undergoing partial hepatectomy, there
was also little proliferation of transplanted cells up to 4 months
posttransplantation (Figure 2F)
.
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. As the number of DPPIV+ cells increased, they
formed clusters that were also subsequently visible in the
mid-parenchyma (Figure 2, C and D)
. Whether this represented further
seeding of the liver by cells migrating from primary clusters in the
periportal regions, an increase in the size of clusters so that they
were now more easily detected in other regions of the lobule that were
initially sparsely populated by transplanted cells, or a combination of
both processes is not clear. The average size of hepatocytes in
DPPIV+ clusters was smaller than hepatocytes in the
surrounding liver. As indicated previously, many of the endogenous
hepatocytes (shown as DPPIV- in Figure 2
) became
megalocytic after partial hepatectomy with large hyperchromatic nuclei
(Figure 2, B and C
, arrows), reflecting the effect of the pyrrolizidine
alkaloid.22-25 Incorporation of Transplanted and Proliferating Hepatocytes into the Hepatic Parenchyma
To determine whether transplanted and newly proliferated
hepatocytes become structurally integrated into the hepatic parenchyma,
dual histochemical analysis was conducted with ATPase and DPPIV. In
normal F344 rats, both stains were positive and superimposed (Figure 3A)
. Under these circumstances, only
DPPIV (rust/orange color) was visualized. In control
DPPIV- F344 rats, ATPase was positive (brown
color), but DPPIV was negative (Figure 3B)
. Two months after
DPPIV+ hepatocyte transplantation into DPPIV-
rats, large clusters of DPPIV+ hepatocytes were noted
(Figure 3C)
. There was no marginal separation between transplanted and
endogenous cells; the borders between transplanted and endogenous
hepatocytes were irregular and transplanted cells appeared to be
expanding into areas of surrounding liver. Transplanted cells were
fully integrated into the hepatic parenchymal plates and formed hybrid
canaliculi with adjacent, DPPIV-/ATPase+
endogenous hepatocytes (Figure 3D
, arrows).
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In male F344 rats treated with the same protocol, the
proliferative response of transplanted hepatocytes was even more
dramatic with respect to both the rate and extent of liver replacement.
One day after transplantation of 2 x 106
hepatocytes,
once again isolated single cells were observed primarily in the
periportal areas (usually not more than one or a few cells in one
portal space per field, using either the x4 or x10 objective; Figure 4A
). Two weeks after cell
transplantation, DPPIV+ hepatocytes were already observed
in clusters of 100 or more cells in cross section (Figure 4B)
,
comparable to levels observed in female rats at 1 month. At 1 month
post-cell transplantation in male rats, DPPIV+ hepatocytes
represented approximately 50% of total hepatocyte mass (Figure 4C)
. By
2 months, transplanted hepatocytes had replaced 90 to 95% of the
parenchymal mass (Figure 4D)
, and at 4 months, hepatocyte replacement
was near total (98%; Figure 4E
). This high level of hepatocyte
repopulation, determined by computer-assisted image analysis to be 99%
at 9 months, persisted for the duration of experiments (Figure 4F)
.
Residual endogenous hepatocytes, usually with very large nuclei and
negative for DPPIV enzyme activity, were observed at the margins of the
lobules and in the area immediately adjacent to some central veins
(Figure 4E
, arrows). In the absence of partial hepatectomy,
proliferation of transplanted hepatocytes in male rats was also quite
limited, but in several animals it reached 3 to 5% within 1 month.
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. At 6 months in
male rats transplanted with 100 cells, 2 to 6 clusters of
DPPIV+ hepatocytes were observed in cross sections through
the liver. In these animals, clusters of transplanted cells were of
varying sizes on two-dimensional cross section, with larger clusters
containing hundreds of hepatocytes (Figure 4H)
.
The final appearance of the liver after long-term hepatocyte
replacement at 9 months after hepatocyte transplantation in a male F344
rat compared to a normal rat liver is illustrated in Figure 5
. As shown by hematoxylin and eosin
(H&E) staining at low magnification (Figure 5, A and B)
, the lobular
architecture and overall histological appearance were essentially
normal. At higher magnification (Figure 5, C and D)
, the morphology of
hepatocytes, the hepatic plate structure, and the appearance of
sinusoidal lining cells was normal compared to untreated liver. The
only abnormality observed consistently was slight reduplication of the
bile ducts, which occurred in all animals treated with
retrorsine. As demonstrated by DPPIV histochemical staining before and
after cell transplantation (Figure 5, E
versus F), the
extent of hepatocyte replacement was near total, with only a few
remaining DPPIV- cells in small clusters. Figure 5F
also
shows restoration of a normal liver cord structure by transplanted
hepatocytes.
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To study the ability of transplanted cells to perform unique
hepatocyte biochemical functions, albumin synthesis, glucose
metabolism, and gluconeogenesis were examined. As illustrated by serial
sections in Figure 6, 1
month after cell
transplantation, large clusters of hepatocytes that expressed DPPIV in
a canalicular distribution (Figure 6A)
also demonstrated synthesis of
albumin (Figure 6B)
and storage of glycogen (Figure 6C)
. Similarly,
there was high expression of glucose-6-phosphatase in clusters of
transplanted (DPPIV+) hepatocytes (Figure 6, E and F)
.
Transplanted hepatocytes did not express cytokeratin-19 (Figure 6D)
or
-glutamyl transpeptidase (not shown), indicating that these cells
are in the hepatocytic rather than the bile duct epithelial cell
lineage. Transplanted cells were also negative for 
-fetoprotein
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals treated with this retrorsine/partial hepatectomy protocol have been maintained for up to 2 years without development of adenomas, hepatocellular carcinomas, or other hepatic malignancies. We have observed foci of basophilic hepatocytes that were negative for glutathione-S-transferase P and altered hepatic foci that are glutathione-S-transferase P positive, but the incidence (frequency) and number of these foci were not increased compared to untreated control F344 rats of the same age.
Transplanted cells were observed initially as single cells or doublets, and over a 1- to 2-month period, expanded until approximately 40 to 60% of the hepatocyte mass was replaced in female rats and virtually total replacement (99%) occurred in male rats. The cellular growth pattern appeared clonal, with individual cells forming clusters that increased progressively in size over a 1 to 2-month period. For clusters containing approximately 25 to 50 cells in two-dimensional cross section, this corresponds to 125 to 250 cells in three dimensions, assuming that each cluster represents a sphere and that the cross section is through the maximum diameter of the sphere. If we also assume that each cluster is derived from a single transplanted cell, the total proliferated mass in each cluster represents seven to eight cell divisions. This was observed at approximately 7 to 10 days in male rats and at 2 weeks in female rats. When the cross-sectional cluster size reached 1000 or more cells (1 month in male rats and 2 months in female rats), this represented ~12 to 13 cell divisions. The expanding cells integrated into the parenchymal mass without compressing the surrounding structures. Initially, the proliferating, transplanted hepatocytes were smaller in size than endogenous hepatocytes, but within 3 to 4 days after transplantation, these cells exhibited canalicular expression of DPPIV. Hybrid canaliculi were also observed between transplanted and endogenous hepatocytes within several days, and there was no marginal separation between transplanted and host hepatocytes. After 2 to 4 months, transplanted hepatocytes began to reform normal liver plates with endothelial, Kupffer, and Ito cells in the sinusoids. By 9 months, the liver parenchymal structure was restored to normal, except for modest bile duct reduplication.
In retrorsine-treated rats, transplanted wild-type hepatocytes appear to undergo selective proliferation compared to endogenous host hepatocytes, which are mitoinhibited by the pyrrolizidine alkaloid. This proliferation is markedly enhanced by partial hepatectomy. This is in contrast to either the urokinase-type plasminogen activator transgenic or Fah-null mouse, in which cellular proliferation in the liver is a continuous process resulting from the toxic effects of genetic manipulation. In these models, both endogenous and transplanted hepatocytes can proliferate, but endogenous hepatocytes are removed by toxic injury, allowing the transplanted cells to become dominant. Although the specific mechanisms of hepatocyte replacement are different, it is interesting that in all three models, hepatocyte replacement is completed in approximately 2 months. How this process is controlled and the role of specific growth factors, hormones, and cytokines are interesting areas for future investigation.
Dilution experiments showed substantial liver repopulation after transplanting 104 cells and the presence of multiple clusters of transplanted hepatocyte in animals receiving as few as 100 cells. These findings are consistent with previous reports indicating that transplanted hepatocytes exhibit a high proliferative capacity under a variety of conditions in which there is a need to replace toxically injured hepatocytes. Overturf et al17 recently showed in their Fah-null mouse model that both wild-type transplanted hepatocytes and mutant hepatocytes, genetically corrected in vivo by repeated intravenous injection of a retrovirus vector containing a normal Fah cDNA, were able to repopulate the liver of Fah-null mice and restore normal tyrosine metabolism. Thus, under appropriate circumstances, either cell transplantation or gene therapy approaches can be used to correct specific genetic deficiencies.
In our protocol, transplanted hepatocytes appeared morphologically, biochemically, and anatomically normal and, after two-thirds partial hepatectomy, expanded to restore hepatic mass. Functional activity of transplanted cells and their progeny was demonstrated by histochemical and immunohistochemical detection of hepatocyte-specific markers at levels comparable to those found in normal hepatocytes, including albumin, glucose-6-phosphatase, glycogen, and DPPIV. These results concerning differentiated hepatocyte function of transplanted cells are consistent with the findings of Overturf et al, in which glutamine synthetase activity was observed in transplanted hepatocytes in the central vein region of hepatic lobules reformed after wild-type hepatocytes were transplanted into Fah-null mice.18
To our knowledge, this study represents the first example of extensive liver repopulation and function in the rat. In principle, the same strategy can be applied to virtually any mammalian species. Furthermore, this model is easily reproduced and offers a suitable system to investigate many aspects of basic liver biology, such as analysis of the lineage and proliferative potential of putative hepatocyte progenitor cells and cell lines. It can also be used to study the role of specific growth and differentiation factors in liver repopulation and to develop new animal models of liver disease using genetically engineered or modified cells. With appropriate modifications, the approach presented in this study could also bear relevance to the management of both acquired and genetically inherited human liver disease. One of the limitations in current studies of ex vivo gene therapy is the small number of transduced cells that ultimately reside in the host liver (estimates range from 0.1 to 1.0% of liver cell mass).36 By coupling ex vivo gene therapy with the current cell transplantation strategy, it should be possible to eliminate this major technical barrier.
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Acknowledgements |
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Footnotes |
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Supported in part by research grants RO1 DK17609 and DK50636 and Digestive Diseases Core Center Grant P30 DK41296, all from the National Institute of Diabetes and Digestive and Kidney Diseases (to DAS), a research grant from Consiglio Nazionale delle Ricerche-Applicazioni Cliniche della Ricerca Oncologica, Rome, Italy (to PP), and the Gail I. Zuckerman Foundation for Research in Chronic Liver Diseases of Children. E.L. is the recipient of a research fellowship from the Italian Foundation for Cancer Research.
Accepted for publication April 16, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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