1 Marion Bessin Liver Research Center, 2 Comprehensive Cancer Research Center, 3 General Clinical Research Center, 4 Department of Medicine, and 5 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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The ability to localize transplanted hepatocytes in the liver offers exciting new opportunities. Transplanted hepatocytes enter liver plates, form hybrid plasma membrane structures with adjacent hepatocytes, express liver genes correctly, and survive indefinitely. The transplanted cell mass is regulated, such that cell proliferation is limited in the normal adult liver, whereas the liver is repopulated extensively when proliferation rates in transplanted and host hepatocytes become dissociated or host hepatocytes are ablated selectively. Transplanted hepatocytes are susceptible to hepatitis viruses. These aspects of transplanted hepatocyte biology indicate that liver repopulation systems can help address questions concerning pathophysiological mechanisms.
hepatocyte; transplantation; hepatitis B virus
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INTRODUCTION |
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FOR REPLACEMENT OF an organ with transplanted cells, one envisages the availability of suitable cells to be delivered into specific anatomic regions by appropriate methods and for these cells to have excellent survival and function. Organ repopulation has captured broad attention for its potential in cell and gene therapy. Advances in cell and molecular biology, including stem cell biology, and in transplantation medicine are beginning to move this area toward clinical applications. Use of cells offers highly attractive alternatives for overcoming shortages of transplantable organs. The liver in particular possesses an extensive capacity for regeneration, which implies that repopulation with cells could offer opportunities for treating acute or chronic liver disease. Moreover, the liver is an excellent gene therapy target because many candidate genes are highly expressed in hepatocytes. In the context of liver-directed cell and gene therapy, critical demonstrations have included integration of transplanted cells in the liver parenchyma, and investigators have shown survival and function of transplanted cells throughout the life of animals. Simultaneously, these findings have provided unique models for addressing fundamental questions in a variety of disciplines, which should be of much interest to investigators.
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REPOPULATION OF THE LIVER |
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Although early studies focused on transplantation of hepatocytes in ectopic sites because localization of transplanted cells in the liver required specific markers, survival of transplanted cells in ectopic sites has been generally limited (8). Nonetheless, a variety of animals with metabolic or genetic deficiencies have been used for testing hepatocyte function in ectopic sites, e.g., in the peritoneal cavity or spleen. Despite limitations concerning analysis of transplanted hepatocyte biology in various ectopic sites compared with the liver itself, life-long survival of syngeneic hepatocytes in the spleen of animals was most remarkable. Also, it was established that allogeneic hepatocytes were cleared rapidly after transplantation, providing additional systems for investigating mechanisms in tolerance.
A significant breakthrough occurred when hepatocytes containing unique transgenes, such as hepatitis B virus surface antigen (HBsAg), were utilized (5, 20). Use of these transgenes allowed localization of transplanted cells in the liver of recipients by in situ methods, as well as analysis of transplanted hepatocyte mass by measuring blood levels of HBsAg, and raised the possibility of addressing the issue of liver repopulation systematically. Of course, transplanted cells should benefit when returned to the hepatic microenvironment, which provides correct extracellular matrix components, growth factors, and substrates and allows them to interact with other liver cells.
In early studies, when hepatocytes were injected into the liver of Gunn
rats with jaundice or rats with liver failure, outcomes improved,
although the mechanisms were undefined because the fate of transplanted
cells was not established. Besides HBsAg, a variety of additional
genetic markers have been helpful in localizing transplanted cells in
animals, including human 1-antitrypsin, which can be
simply measured in blood, bacterial
-galactosidase gene, which can
be localized with enzyme histochemistry, and sex chromosomes, which can
be localized with DNA hybridization (18, 20). If transplanted cells are
permanently transduced with transgenes, e.g., by retroviral vectors, it
is again possible to localize these in animals.
The utilization of dipeptidyl-peptidase IV-deficient (DPPIV)
Fischer 344 rats (F344), which arose through spontaneous mutations, has
been very helpful in establishing insights into the biology of
transplanted cells (10, 11, 21). DPPIV is highly expressed in the bile
canalicular domains of hepatocytes. When cells with normal DPPIV
activity are transplanted into DPPIV
F344 rats, it is possible
to localize transplanted cells in any organ of the body with enzyme
histochemistry or immunostaining. On the other hand, it has also been
possible to localize DPPIV
cells in the normal liver through
histochemical techniques, providing yet more cell transplantation
systems (21).
DPPIV F344 rat-based transplantation systems have been highly
effective in addressing mechanisms concerning cell engraftment in the
liver. Integration of hepatocytes in the liver parenchyma with
resumption of normal polarity is certainly most desirable for
physiological regulation of gene expression and cell proliferation. Similarly, reconstitution of plasma membrane structures, such as gap
junctions and bile canaliculi, is required to restore normal responses
in transplanted cells. All of these parameters have been established in
DPPIV
rats following transplantation of normal syngeneic cells
(10, 11, 21).
A related issue concerns the most effective and safest way for delivering cells into the portal vascular bed, which is particularly important in developing clinical applications. It is clear that deposition of cells into hepatic sinusoids by direct injection either into the portal vein or via the spleen, especially in small animals, is most effective (8). Distribution of transplanted cells within the liver lobule appears to be directed by mechanical events (11, 21). After arrival via the portal vein branches, transplanted cells are entrapped in sinusoids that are smaller than cells, leading to their deposition predominantly in periportal areas (or zone 1) of the liver lobule. Although the early events following cell transplantation have not been fully defined, there is evidence for ischemia-reperfusion-type changes in the host liver, since transplanted cells serve as emboli with transient occlusion and rerouting of blood flow in the hepatic microcirculation, as well as release of vascular endothelial growth factor, which is well known to increase endothelial permeability (11).
Interestingly, transplanted cells begin to enter liver plates within 20 h through a process involving disruption of the sinusoidal endothelium (11). Subsequently, transplanted cells integrate with host hepatocytes in the liver plates, although this process requires several days for completion. Dual histochemical studies established that transplanted cells develop functionally intact hybrid plasma membrane structures, such as gap junctions and bile canaliculi (10). When integrated into the liver parenchyma, transplanted cells retain normal metabolic functions, including expression of liver genes, such as glucose-6-phosphatase, albumin, and so forth, and excretion of bile salts, copper, bilirubin, and so forth, into the bile.
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USES OF SPECIFIC MODELS |
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A variety of animal models are currently being utilized for developing
and testing suitable strategies for cell and gene therapy (Table
1). The value of liver
repopulation in these situations should be fairly obvious. Efforts are
underway in several laboratories to define the most useful type of cell
for liver repopulation, i.e., mature hepatocytes, facultative
progenitor cells, and so forth, effective means for gene transfer into
cells, the requisite transplanted cell mass in the liver, the safety of
cell transplantation, and other issues concerning preclinical
development. Transplanted cells can even integrate in the liver of
cirrhotic animals, where the sinusoidal endothelial barrier is greatly
augmented, and could potentially interfere with cell entry into the
liver plates (3). In animals with carbon tetrachloride-induced
cirrhosis of the liver, transplanted cells were found to retain normal
function and to proliferate in the long term. These findings have been especially promising for therapies directed at chronic liver disease, especially when disease progression may be arrested by biliary excretion of toxins, e.g., copper in Wilson's disease, or when disease-resistant cells could escape injury and repopulate the liver,
e.g., chronic viral hepatitis. Although these developments are
exciting, equally enthralling are the prospects offered by cell
transplantation systems in the investigation of areas concerning liver
gene regulation, liver growth control, oncogenesis, and viral
hepatitis, as well as stem cell biology.
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Studies of liver gene regulation.
To analyze site-specific differences in gene expression, specific
genetic sequences can be introduced into hepatocytes that are then
transplantated into the liver and into ectopic sites (13).
Inducible promoter/enhancer sequences can be deployed to further test
gene-regulatory mechanisms. Similarly, position-specific gene
expression can be tested within the liver lobule itself because transplanted hepatocytes integrate in periportal areas. For this purpose, the position of transplanted cells can be shifted from, for
example, periportal to perivenous areas, by specific maneuvers such as
the use of carbon tetrachloride (12) (Fig.
1). In these studies, comparison of gene
expression in transplanted cells established conclusively that
position-specific regulation of gene expression is a function of the
hepatic microenvironment.
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Mechanisms in liver growth control. Transplanted cells exhibit regulated proliferation, without proliferating significantly in the normal liver, which is compatible with the absence of liver regenerative activity in this situation. In contrast, after ablation of host hepatocytes subsequent to liver injury by toxins, such as carbon tetrachloride or D-galactosamine, transplanted hepatocytes proliferate significantly (Fig. 1) (9, 11, 13). Similarly, in animals with progressive depletion of host hepatocytes through genetic mechanisms, transplanted cells can proliferate extensively and can repopulate virtually the entire liver. These principles are amply illustrated by cell transplantation studies in transgenic mice containing the urokinase-type plasminogen activator gene driven by the albumin promoter (alb-uPA) and mice with a mutated FAH gene with tyrosinemia (18, 22). The transgene in alb-uPA mice is lethal to hepatocytes, with progressive and continuous depletion of transgene-expressing cells, although cell clones with transgene deletions eventually appear in surviving animals (22). In contrast, FAH mice exhibit hepatic injury due to abnormal tyrosine metabolism and accumulation of lethal toxins in hepatocytes (18). When normal hepatocytes are transplanted in these animals, there is extensive liver repopulation, such that the entire liver may be replaced by transplanted hepatocytes over a few weeks (18, 22). In other situations, induction of apoptosis in host hepatocytes through selective use of toxins, although sparing transplanted hepatocytes, has been successful in significantly increasing liver repopulation (16). Similarly, in other rat models in which proliferation in host hepatocytes is arrested by retrorsine, a DNA-binding pyrrolizidine alkaloid, and two-thirds partial hepatectomy or arrested by whole liver radiation, which causes DNA injury, and two-thirds partial hepatectomy before cell transplantation, transplanted cells may replace virtually the entire liver (6, 14).
These findings indicate that a variety of issues concerning cell proliferation mechanisms can be tested in cell transplantation systems. For instance, serial repopulation of the liver in FAH mice established that isolated mouse hepatocytes can replicate extensively (18). Similarly, rat hepatocytes were found to be capable of multiple cell divisions following transplantation into animals pretreated with retrorsine and partial hepatectomy or whole liver radiation and partial hepatectomy, as well as following repeated carbon tetrachloride administration (6, 7, 14). Such systems can be used for further analysis of replicative potential in specific hepatocyte subpopulations or after subjecting cells to cell cycle or other perturbations. In this respect, it is noteworthy that transplanted cells can serve as reporters to determine subtle changes in the liver, such as those occurring in the remnant liver after two-thirds partial hepatectomy, which attenuated the replication capacity of host hepatocytes (24).Development of novel animal models for viral hepatitis, including oncogenesis. Persistent hepatitis B virus (HBV) infection remains a major problem worldwide. Although some progress has been made, effective antiviral therapies are lacking at present. Chimpanzees are susceptible to HBV; however, the availability of these animals for testing is limited. Transgenic mice with HBV replication offer an excellent model for antiviral testing (4) but do not reproduce liver disease, since transgenic animals are tolerant of viral products. Moreover, transgenic mice utilize transgenes to produce HBV pregenomic mRNA and thus the livers cannot be cured of the virus.
During natural infection, covalently closed circular (ccc) molecules of HBV DNA are found in the nucleus of infected hepatocytes. These ccc DNA molecules act as a reservoir for the transcription of HBV mRNAs and for pregenomic mRNA synthesis. The ccc HBV DNA pool is believed to be very long lived in the liver. In contrast, ccc HBV DNA is not produced in transgenic mice and therefore these animals cannot be used to evaluate clearance of these critically important molecules (15). To develop a novel animal model based on extensive liver repopulation, we crossed alb-uPA mice with animals deficient in recombination activation gene-2, which causes immunodeficiency (19). The hypothesis was that these animals would tolerate xenografted woodchuck hepatocytes, which would proliferate extensively in the alb-uPA mouse liver. Such chimeric "woodmice," containing both mouse and woodchuck hepatocytes, would be infected with woodchuck hepatitis virus (WHV), establishing a natural infection characterized by production of CCC WHV DNA in the nucleus of infected cells. The presence of transplanted woodchuck hepatocytes in woodmice was identified by the appearance of a unique species of woodchuck albumin and unique repeat sequences of woodchuck DNA in the liver (see Ref. 19 for full details). Abundant WHV replication intermediates and CCC WHV DNA were present in woodmouse livers with virion particles and viral surface and core proteins in the peripheral blood (19). We found that WHV infection was maintained in woodmice for several months, with modulation of viral replication following treatment with interferon-Mechanisms in stem cell biology. The fate of progenitor liver cells can be best examined in the hepatic microenvironment. Studies have been conducted with cell transplantation to show that progenitor cells isolated from the fetal liver, adult liver, as well as the pancreas can differentiate into mature hepatocytes (1, 2, 25, 26). The recent cloning of primate embryonic stem cells and advances in the understanding of cell aging mechanisms have contributed to the increased interest in stem cell biology. Progenitor liver cells have been isolated from the human liver and mouse liver (23), and the availability of well-defined transplantation systems will help in their characterization, establishment of proliferative potential, development of further xenografted animal models, and other applications.
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CONCLUSIONS |
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Elucidation of mechanisms concerning liver repopulation has begun to open new vistas in basic investigations. Transplanted cells can serve as reporters to address mechanisms concerning the host liver itself. Alternatively, specific properties of a host liver can be utilized to define a variety of mechanisms in transplanted cells. Use of immunodeficient hosts can help produce chimeric animals for establishing specific models of human disease. The combination of these elements can offer highly creative solutions to answer important biological questions.
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FOOTNOTES |
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* Sixth in a series on invited articles on Lessons From Genetically Engineered Animal Models.
Address for reprint requests and other correspondence: S. Gupta, Ullmann 625, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: sanjvgupta{at}pol.net).
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