INTRODUCTION

TOP ABSTRACT INTRODUCTION REPOPULATION OF THE LIVER USES OF SPECIFIC MODELS CONCLUSIONS REFERENCES

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.

	REPOPULATION OF THE LIVER

TOP ABSTRACT INTRODUCTION REPOPULATION OF THE LIVER USES OF SPECIFIC MODELS CONCLUSIONS REFERENCES

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.

	USES OF SPECIFIC MODELS

TOP ABSTRACT INTRODUCTION REPOPULATION OF THE LIVER USES OF SPECIFIC MODELS CONCLUSIONS REFERENCES

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.

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.

View larger version (137K): [in this window] [in a new window]   Fig. 1.   A: transplanted cells in the liver of a dipeptidyl-peptidase IV-deficient (DPPIV) Fischer 344 (F344) rat following histochemical localization of cells. Transplanted cells contain DPPIV in bile canalicular domains (red color, arrows) and are localized in a portal area (p). CV, central vein. Tissue was stained simultaneously for bile canalicular ATPase activity in host hepatocytes (brown color, arrowheads). Development of bile canalicular networks between transplanted and host hepatocytes is one way to demonstrate integration of transplanted cells in the liver parenchyma. B: liver of animal shown in A after 3 cycles of carbon tetrachloride treatment, which causes perivenular hepatic injury while sparing transplanted cells in portal areas. Number of transplanted cells has increased significantly along with a shift in the position of cells toward perivenous areas. Transplanted cells remain joined with host hepatocytes, as shown by staining for DPPIV and ATPase activities in bile canaliculi. The change in the position of hepatocytes has been used effectively for analyzing regulation of gene expression in the liver. C: illustrative example of extensive hepatocyte proliferation in the liver of a DPPIV F344 rat, which was treated before cell transplantation with retrorsine and two-thirds partial hepatectomy. Liver is virtually completely replaced by transplanted hepatocytes, which contain DPPIV in bile canaliculi (red color). D: glucose-6-phosphatase expression in transplanted cells with DPPIV activity (red color, arrow) following dual-enzyme histochemistry. E: glycogen expression in transplanted hepatocytes (arrows) following dual histochemical staining for DPPIV and glycogen.

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).

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.

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.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION REPOPULATION OF THE LIVER USES OF SPECIFIC MODELS CONCLUSIONS REFERENCES

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.