Liver cell transplantation: The road to clinical application

Liver cell transplantation: The road to clinical application K. J. ALLEN and H. E. SORIANO MELBOURNE, AUSTRALIA, and CHICAGO, ILLINOIS

Abbreviations: HSS = hepatic stimulatory substance; LCT = liver cell transplantation

L

iver cell transplantation is the transfer of a cell suspension into the liver parenchyma via the portal circulation for the correction of acute, chronic, or genetic liver insufficiency. Recent research has demonstrated the ability of donor liver cells to repopulate the diseased liver in animal models of metabolic liver disease1 and fulminant liver failure.2 These experiments have harnessed the proliferative capacity of the transplanted cells so that they selectively outgrow the endogenous liver cells and produce a chimeric liver of donor and host liver cells. More recent advances have shown the ability of non–liver-derived cells such as bone marrow cells to repopulate the diseased liver with functional hepatocytes.3-5 Although the idea of LCT for the treatment of liver disease was first touted in 1977,6,7 when it was noted that liver cells could be isolated and transplanted into animal models to ameliorate liver insufficiency, clinical reality has lagged behind theoretical promise. Why is this so? The reasons are many and varied and will be explored in this review. Most important is the fact that wholeorgan liver transplantation now has an excellent morbidity and mortality profile. The overall liver transplant recipient 1-year survival rate in the United States is now greater than 85%,8 while the 3-year survival rate is higher than 75%. With these figures in mind, clinical trials in LCT have had to compete with whole-organ transplantation, an established therapy.

From the Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne; and the Chicago Children’s Memorial Hospital, Chicago. Submitted for publication April 17, 2001; revision submitted July 10, 2001; accepted August 1, 2001. Reprint requests: K. J. Allen, Murdoch Children’s Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, 3052, Australia. J Lab Clin Med 2001;138:298-312. Copyright © 2001 by Mosby, Inc. 0022-2143/2001 $35.00 + 0 5/1/119148 doi:10.1067/mlc.2001.119148 298

Fulminant liver failure, metabolic liver disease, and chronic liver disease have evolved as potential candidates for LCT trials. In liver failure, cryopreserved liver cells can be used, if a whole organ is not immediately available, as a bridge to recovery or as a bridge to liver transplantation. In liver-based metabolic disease, cell transplantation provides a less surgically invasive approach for patients who may not wish to undertake whole-organ transplantation. In chronic liver disease, auxiliary liver function might be used to ameliorate the symptoms of a patient awaiting a whole organ or in patients ineligible for major abdominal surgery. LCT has both real and theoretic advantages over whole-organ transplantation as discussed below. For these potential advantages to be fulfilled, clinical trials will need to successfully show that LCT is a safe procedure and can be undertaken in patients who are less ill. However, we believe that the single most important factor limiting the widespread clinical application of LCT is that there is currently no clinically applicable method to expand the liver cell population in vitro or to expand the transplanted cell population in vivo by induction of proliferation. This review will summarize the most recent scientific and clinical advances in LCT, explore the factors that are important for optimizing this therapy, and outline why donor liver cell repopulation in the host liver will be an essential criterion before LCT will fulfill its clinical potential. LIVER CELL TRANSPLANTATION WILL HELP EASE THE CRISIS OF ORGAN DONOR SHORTAGE

An important reason for the efficient therapeutic development of LCT is to help ease the worldwide shortage of donor organs for liver transplantation. In the United States, an estimated 1300 patients die each year while waiting for a liver transplant. More than 14,000 patients with liver insufficiency are listed for liver transplant at any one time, and 50% of those patients will wait over 1 year for an appropriate donor organ.8 Coupled to the unmet demand for liver transplants is the predicted increased need for therapy for

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end-stage liver failure in the future. In particular, hepatitis C is expected to massively increase the number of individuals requiring liver transplantation. An estimated 3.9 million people in the United States are chronically infected with hepatitis C, and of these people, 12,000 die each year. One in 4 persons chronically infected with the disease will develop cirrhosis or endstage liver failure.9 Although liver transplantation is an effective form of therapy, it is expensive, requires major surgery, carries a high incidence of surgical and medical complications, and, some argue, “replaces one disease with another,” because patients need to receive lifelong immunosuppression. In contrast, liver cells can be transplanted as a day procedure, after radiologic or surgical placement of a portal catheter. Cell transplantation is less expensive and less invasive. Unlike whole organs, donor liver cells can be cryopreserved and held “on call,” enabling a ready therapy to be administered at the clinical team’s convenience. Transplanted cells can take residence and function normally within a host liver parenchyma to provide corrective metabolic function. Although immunosuppression has been utilized in the early clinical trials of LCT and is needed for allogeneic transplantation in rodents,10,11 there is also potential to minimize the use of immunosuppression or abrogate the host’s immune response to donor liver cells. If liver cells are used to provide transient support while the patient is awaiting recovery, as is the case in acute liver failure, long-term immunosuppression can be avoided. It is important to note that LCT would enable a more efficient use of the scarce donor supply, because one liver could be harvested to provide cells for a number of recipients. Although ethical issues will need to be carefully assessed, there is the potential for living-related liver donation for cell transplantation as currently occurs for liver transplantation in children and more recently adults. Such procedures will continue to expand the organ donor pool and if utilized for cell transplantation would produce higher quality freshly isolated cells. In addition, LCT could take advantage of in vitro expansion of liver cells or use stem cells or bone marrow cells as sources of donor tissue. Exciting new research has shown that bone marrow–derived cells are able to populate the liver and act as stem cells.3,4,12 Such a potential source of cells would be an outstanding improvement on the current shortage of donor livers. CLINICAL STATE OF THE ART OF LIVER CELL TRANSPLANTATION

Until recently, isolation and cryopreservation of human liver cells have been dogged by persistently low quality and viability of cells. It has been difficult to

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argue that the scarce supply of donor livers should be shared with an experimental therapeutic program such as LCT when they can be more reliably used in a whole-organ program. As a result, liver cell programs not only have difficulty obtaining livers for harvesting and cell banking but the livers that do become available for these programs are frequently of marginal quality, because the whole-organ program has usually rejected them. More recent and promising studies have shown the ability to consistently isolate and cryopreserve good quality hepatocytes for the purpose of cell banking.13,14 Cell transplantation groups are also using discarded liver segments that because of vessel damage or recipient size cannot be used for standard liver transplantation. Although in the long term these measures may help to alleviate the donor organ shortage, liver cell transplant programs will have to demonstrate a clear advantage to compete with whole-organ programs as an established therapy for liver insufficiency. LCT for acute liver failure. Current clinical trials of LCT are underway for the treatment of both fulminant liver failure and metabolic liver disease15-19 (see Table I). These initial trials aim to show that cell therapy is safe as well as effective. These two indications for LCT have evolved for separate reasons. In the case of fulminant failure, if a liver for transplantation is not forthcoming, the patient is unlikely to survive without some heroic form of therapy. LCT offers critical life support when there are few therapeutic alternatives. In the case of metabolic liver disease, the desire to avoid the irreversible step of whole-organ transplantation and its associated surgical risks and requirement for lifelong immunosuppression needs to be balanced with the requirement for the therapy to be safe and simple. Therapy for fulminant liver failure is effective if patient survival is significantly improved. Success may therefore occur through three different outcomes: by bridging patients to whole-organ transplant; by bridging them to recovery of liver function of the native liver with concurrent disappearance of the donor liver cells; or by engraftment and long-term function of the liver cell transplant. Several recent clinical trials of LCT for the treatment of acute liver failure have been reported in the literature. The first, from India,22 describes 7 patients who each received a single infusion of fetal hepatocytes (harvested from 26- to 34-week gestational age fetuses) into the peritoneal cavity via a peritoneal dialysis catheter. The overall survival of patients receiving hepatocytes was 43%, as compared with 33% in matched control subjects. All patients with grade III hepatic encephalopathy who received a liver cell transplant survived, whereas matched control subjects who did not receive transplants had a 50% mortality rate. Unfortu-

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Table I. Disease entities for which LCT has been utilized Clinical trials

Acute fulminant failure Acetaminophen overdose15 Non-A, non-E hepatitis15,17,18 Unknown etiology22 Dilantin toxicity23 Metabolic liver disease Hypercholesterolemia24 Crigler-Najjar type 126 Ornithine transcarbamylase deficiency28 Progressive familial intrahepatic cholestasis

Chronic liver disease

nately, the fate of the transplanted hepatocytes was not ascertained, as postmortem examinations could not be performed because of social and legal constraints in India at the time of the study. The second report, by Strom et al,23 describes the use of LCT as a bridge to whole-organ transplantation in 5 patients. Patients received a mixture of between 107 and 109 freshly isolated and cryopreserved liver cells via splenic arterial perfusion. All 5 subjects were critically ill with grade IV hepatic encephalopathy and multisystem organ failure. Four patients with illness of equal severity were used as control subjects. All 4 control patients died within 3 days, despite maximal medical therapy. In contrast, the 5 liver cell transplant–treated patients maintained normal cerebral perfusion and cardiac stability, with withdrawal of medical support 2 to 10 days before whole-organ transplantation. Blood ammonia levels decreased significantly, and 3 of the 5 patients successfully bridged to whole-organ transplant were alive and well at 20 months’ follow-up. Several more trials that have been reported15,17,18 have had varying degrees of success after transplantation of fresh and frozen human hepatocytes into the portal vein of patients with liver failure. At least 2 of this series of critically ill patients recovered spontaneously, while other patients demonstrated some improvement in ammonia, prothrombin time, encephalopathy, cerebral perfusion pressure, and cardiovascular stability. Unfortunately, to date there has been no definitive evidence that the demonstrated engraftment of transplanted liver cells in these patients was responsible for the clinical improvements, because patients with acute liver failure can, on occasion, survive without trans-

Animal models

Galactosamine-induced20 Vascular injury21

Hypercholesterolemia25-Watanabe rabbit Crigler-Najjar type 127-Gunn rat Urea cycle defects29-31-Dalmation dog Progressive familial intrahepatic cholestasis32-Mdr2 knockout mouse Wilson disease33-LEC rat Tyrosinemia34-FAH knockout mouse Histidinemia35 Retrorsine-induced injury36,37 Carbon tetrachloride–induced38,39

plantation. There are several possible reasons why these initial transplants have met with only modest success. As in any phase 1 clinical trial, only the sickest patients would be candidates for this new therapy. Initially, the number of liver cells transplanted has been relatively small. In addition, multi-organ failure present in the host could be far too inhospitable to transplanted cells. Less than optimal liver cell quality and viability after cryopreservation in some of the earlier transplants may have also been a factor. Regardless, these clinical trials have been an important contribution to the field because they have demonstrated the safety, function, and engraftment of transplanted liver cells and opened the field to additional clinical applications. Further prospective randomized control studies will be needed to clarify the outcomes of patients who receive liver cell transplants for acute liver failure. LCT for metabolic liver disease. Metabolic liver disease is an inherited inability of the liver to metabolize a specific product (such as tyrosine in tyrosinemia or bilirubin in Crigler-Najjar), which results in damage and tissue pathology that is either hepatic (eg, tyrosinemia) or mostly extrahepatic (eg, Crigler-Najjar). Therefore in each metabolic disease state the environment of the liver may be drastically different, presenting a challenge for investigators to optimize conditions for liver cell engraftment and function. The clinical use of LCT for metabolic liver diseases is currently limited, because the patient’s life is not necessarily foreshortened, and in many metabolic diseases there are acceptable therapeutic alternatives. For example, dietary restrictions and specific metabolic supplements are used in the management of citrullemia,

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tyrosinemia, and glycogen storage diseases affecting the liver, and phototherapy is the mainstay of treatment for Crigler-Najjar. Although many of these diseases have been treated with liver transplants,40,41 the inconvenience of dietary management is not usually regarded as reason enough to undergo major organ transplantation, even if the latter is curative. By contrast, the transplantation of just a few liver cells may provide enough metabolic support for periods of intercurrent illness when even mainstay dietary measures are inadequate to maintain the health of the individual. The earliest report of liver cell–based treatment of a metabolic disease was by Grossman et al.24 They reported transplantation of autologous hepatocytes transfected with a human low-density lipoprotein– expressing recombinant retrovirus in a patient with familial hypercholesterolemia. Hepatocytes were isolated from the patient’s left lateral segment, and the corrective gene was inserted by using retroviral vectors and infused back into the inferior mesenteric vein. This ex vivo gene therapy trial was the first use of LCT in a clinical trial in the United States. It showed that cells could be harvested and safely infused into the recipient’s liver. Although serum cholesterol levels decreased significantly for a prolonged period (18 months), constituting the “first proof of principle” in the field, the trial was abandoned because of the low efficiency of the retroviral transfection of in vitro, low-hepatocyte engraftment in vivo and because of safety concerns over the use of a retroviral vector and potential oncogenicity. The first allogeneic clinical trial of LCT in metabolic diseases was recently described by Fox et al.26 An 11year-old girl with Crigler-Najjar (a defect in bilirubin metabolism that results in a neurologically damaging buildup of bilirubin metabolites) was transplanted with freshly isolated donor liver cells from an unrelated 5year-old boy. Current therapy for patients with CriglerNajjar is ultraviolet phototherapy for 12 to 18 hours each day. Although this form of therapy is generally effective, bilirubin metabolites can become dangerously high during periods of intercurrent illness. In this patient, 7.5 billion liver cells were transplanted via an intraportal catheter placed radiologically. Three perfusions were performed, each consisting of 1 to 1.5 billion cells over 30 minutes, separated by 4 to 6 hours. After cell transplant, the patient’s total serum bilirubin decreased from 26.1 mg/dL to 14 mg/dL, and bilirubin conjugates measured in bile increased from a trace to 33%. Bilirubin uridyl glucuronyl transferase activity (the enzyme deficient in Crigler-Najjar) measured in a liver biopsy sample increased from 0.4% to 5.5% of normal activity. Furthermore, phototherapy treatment could be reduced from 12 hours to 6 hours per day—

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an outcome that would significantly improve this patient’s quality of life. Long-term evidence of liver cell transplant engraftment and function in this patient was demonstrated for more than 18 months. The importance of this study is manifold. It demonstrated a good safety profile clinically, showed unequivocal phenotypic amelioration of a metabolic liver disease, and demonstrated the proof of principle that donor cells have the potential to survive and function long term in patients. One limitation of this study was that the cells engrafted but were not induced to proliferate and repopulate the host liver, and so further liver cells would need to be infused for further clinical gains to be made in this patient. The same group that successfully treated a patient with Crigler-Najjar has also attempted to treat urea cycle defects with LCT with short-lived success.28 A male infant with ornithine transcarbamylase deficiency received a liver cell transplant of 5.3 × 109 hepatocytes via the portal vein through an umbilical vein catheter in five batches over the first 23 days of life. Between days 20 and 31, metabolic stability was achieved with a normal protein intake and removal of phenylbutyrate treatment. Plasma ammonia and glutamate levels returned to normal. At day 31, metabolic medical management was reinstituted after severe hyperammonemia and immunosuppression was withdrawn. The authors retrospectively postulated that the rebound in ammonia levels was a sign of graft rejection, because it was found that tacrolimus blood levels had become subtherapeutic at the time of crisis. They also theorized that steroid boluses should have accompanied each batch infusion of liver cells to help minimize chances of rejection. Safety concerns. Although these initial clinical aims of safety and effective graft function for both fulminant failure and metabolic liver disease are a necessary first step to a more universal clinical application of LCT, the ideal outcome of liver cell therapy is not just the engraftment but the coordinated and orderly expansion of donor cells so that a new liver can be created in the architecture of the old with the smallest number of donor cells. Alternatively, engraftment without proliferation could provide support for a metabolically inadequate liver, but success of this aim may be hampered by the inability to transplant more than a specific volume of cells. Furthermore, each metabolic liver disease is likely to vary with what liver cell mass is required to effect a phenotypic correction. Thus the mass of liver cells transplanted will need to be titrated to an individual’s clinical need. Cell infusion needs to be balanced on one hand by regard for the patient’s safety and on the other hand by the need to transfer the largest volume of cells to pro-

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vide adequate synthetic or metabolic support. Reported complications of LCT in the setting of acute liver failure include transient hemodynamic instability during intraportal hepatocyte infusion, overwhelming sepsis (which may or may not be related to the transplant procedure), and embolization of hepatocytes into the pulmonary circulation without evidence of pulmonary embolism.16 The risk of portal plugging and associated parenchymal ischemia appears to be minimized by limiting the number of cells per infusion to 30 to 100 million cells per kilogram of body weight, with a maximum infusion speed of 4 to 8 mL/kg/h, and by adding heparin to the cell suspension solution. The quality of the liver cell preparation is also likely to affect the success of cell engraftment after transplantation. Activation of inflammatory cells occurs during the processes of cell isolation. Low-speed centrifugation eliminates a significant proportion of nonparenchymal cells in addition to non-viable cells, and Percoll gradients have also been used in animal models to enrich the cell suspension with viable hepatocytes. In addition, many groups infuse cells with heparin (10 U/mL) to prevent coagulation induced by hepatocytes, which presumably may result from the presence of tissue thromboplastin present in the harvested cell suspension.15,26 One further potential complication of LCT is the poor clearance of immunosuppressive agents by the diseased host liver while the new hepatocytes are engrafting and not yet functioning significantly to metabolize these agents. Evaluating success. There are two end-point analyses that are important to define the efficacy of clinical LCT: assessment of donor liver cell chimerism and analysis of disease correction. Clinical studies are obviously hampered by the invasive procedures associated with proving donor liver cell chimerism. Fisher et al42 have reported a practical method of monitoring liver cell engraftment in patients with acute liver failure. They monitored HLA class 1 antigen by serial transjugular liver biopsies in a patient whose antigen was disparate from the donor liver. LCT was used as a “bridge to recovery” of the patient’s native liver, and immunosuppression was safely weaned several months after LCT when the native liver was shown to have regenerated on liver histology. Evaluation of disease correction can be difficult in a patient with acute liver failure because there is usually concurrent multi-organ failure. Plasma ammonia has been consistently reported to be the first marker of clinical improvement after cell transplant.15,16 Because most patients with acute liver failure are aggressively treated with fresh frozen plasma and other products to prevent bleeding, it can be several days before clear evidence of an improvement in prothrombin times can be

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seen, although plasma support can usually be reduced in the initial period after cell infusion. Bilirubin appears to be the last measure of synthetic function to improve. Because the host liver is still in situ, it is of little value to monitor liver function tests to assess cell transplant function. At this point in time there is no valid measure for evaluating liver cell transplant rejection. LIVER CELL TRANSPLANTATION BIOLOGY Early animal models. Seglen43 opened the field of liver

cell manipulation in 1976 by describing methods to isolate rat hepatocytes. Matas et al44 were the first to describe treatment of an animal model of metabolic liver disease by using hepatocyte transplantation. After the portal vein infusion of hepatocytes heterozygous for diphosphate glucuronyltransferase, these authors demonstrated a decrease in plasma bilirubin in Gunn rats lacking the enzyme. Metabolic support to allow for recovery from drug-induced acute liver failure in rats was reported after transplantation of dispersed hepatocytes either intraperitoneally or via the portal vein,7 as well as after allogeneic or xenogenic LCT.45,46 Ectopic placement of cells. The host liver represents the ideal environment for the survival of transplanted hepatocytes in view of the unique hepatic organization, need for biliary secretion, interactions with nonparenchymal liver cells, and access to local and portal hepatic growth factors. However, until the early 1990s, ectopically transplanted hepatocytes were used in animal models because methods for determining the fate of transplanted hepatocytes in the liver were not available. Before the advent of current molecular methods, histologic visualization was the only method that could be relied on to distinguish donor hepatocytes from recipient tissue, and so liver cells were transplanted into the dorsal fat pad,47 peritoneum,48 lung,49 and spleen50,51 to demonstrate the proof of principle that transplanted liver cells did indeed persist after transplantation, maintain morphology, and provide metabolic support for the insufficient liver. Gupta et al52 have provided an excellent review on various extrahepatic approaches that were used for LCT in animal models. During investigation of these approaches in mouse models, it was noted that liver cells infused into the spleen rapidly migrated into the liver, with subsequent normal engraftment and function.53,54 This is the route now generally used for mouse LCTs, because a larger volume of liver cells can be infused, the spleen is easier to access surgically than the portal vein, and the mortality rate is lower. Although most clinical transplant programs infuse cells into the liver via a surgically or radiologically placed portal catheter, research groups are now harnessing the earlier work of ectopic placement of liver cells in the spleen

Study

Rhim et al, Science 19941

Yoshida et al, Hepatol 199633

Allen et al, J Gastroenterol Hepatol 200067

Overturf et al, Am J Pathol 199734

Ilan et al, Transplant 199727

Gunn rat

Disease model

Albumin-urokinase transgenic mouse

LEC rat

Toxic milk mouse

FAH transgenic mouse

Age of recipient

<2 weeks

7 weeks

6-8 weeks

6 weeks

Proliferative stimulus

Toxic transgenic product

2/3 partial hepatectomy and copper overload

Copper overload

Cessation of NTBC–buildup of disease metabolites

Type and number of cells

105 adult hepatocytes hepatocytes

3 × 106 adult newborn hepatocytes

1 × 106 adult hepatocytes

Donor/recipient (% transplanted)

0.25%

0.375%

Donor/recipient (% engrafted)

N/A

Donor/recipient (% repopulated)

80%

Route

Laconi et al, Am J Pathol 199836

De Vree et al, Gastroenterology 200032

Guo et al, JPGN 200037

Mdr2-knockout mouse

Normal mouse

8-12 weeks

6 weeks

6-8 weeks

Ligation of nontransplanted portal vein branches

Retrorsine and partial hepatectomy

Chronic bile salt–induced damage

Retrorsine and CCl4

104-105 adult hepatocytes

2 × 106 adult hepatocytes

2 × 106 adult hepatocytes

2 × 106 adult hepatocytes

2 × 106 adult hepatocytes

2.5%

0.25%

0.25%

0.25%

5%

5%

N/A

0.01%

0.2%

N/A

N/A

0.5%

4%-20%

15%-25%

70%-95%

20%

Females: 40%-60%, males: 99%

21%

20%

Spleen

N/A

Spleen

Spleen

Portal vein

Portal vein

Spleen

Spleen

Time to repopulation

1-2 months

5 months

4 months

2 months

N/A

N/A

4 months

4 months

Comments

High mortality, high variability

50% untreated rats died compared with only 7% treated

Survival of cells up to 1 year after transplant

Repopulation was stronger with bilesalt–supplemented diet but was associated with tumor development

Both retrorsine and CCl4 needed for repopulation

5%

Serial transplantation – 6 times with continuing repopulation

LEC rat, Long Evan Cinnamon (rat model of Wilson disease); FAH transgenic mouse, fumarylacetoacetate hydrolase (mouse model of tyrosinemia); Gunn rat, rat model of Crigler-Najjar; Toxic milk mouse, estimated host hepatocytes. Estimated hepatocytes in adult mouse, 4 × 107; in adult rat, 8 × 108.

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mouse model of Wilson disease; Mdr2-knockout mouse, mouse model of progressive familial intrahepatic cholestasis type 3; Donor/recipient, number of donor liver cells transplanted as a percentage of

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Table II. Successful rodent models of liver repopulation after LCT

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and peritoneum to provide synthetic function for cirrhotic livers where parenchymal disarray and elevated portal pressure complicate hepatic migration of cells.39,55,56 Detection and function of transplanted hepatocytes in

To demonstrate hepatic engraftment of transplanted cells, Vroemen et al57 performed splenectomy after treating hyperbilirubinemia in Gunn rats with cell transplantation into the spleen. Splenectomy reduced but did not abolish the therapeutic effect, indicating that transplanted hepatocytes had migrated from the spleen and were maintaining function in the enzyme-deficient host liver. A major advance in the study of liver cell transplants was the development of transgenically marked hepatocytes to monitor engraftment and repopulation of donor hepatocytes in the host liver.53,54 These models assessed the secretion of transgene products into the peripheral circulation of congenic recipients to determine graft survival and function. Gupta et al53 used HbsAg-secreting transgenic mouse hepatocytes, and Ponder et al54 used α1-antitrypsin-secreting transgenic mouse hepatocytes. These models unequivocally identified donor transplanted hepatocytes in the host liver. Additional methods were also developed by using fluorescent markers that allowed quick and simple detection of non-transgenic transplanted cells.58 Gupta et al59 analyzed the fate of the splenically transplanted hepatocyte with a rat deficient in dipeptidyl peptidase IV activity. They used colocalization studies of adenosine triphosphatase and dipeptidyl peptidase IV to show gap junctions uniting adjacent transplanted and host hepatocytes in liver plates. Visualization of bile canalicular domains in transplanted and host hepatocytes with dipeptidyl peptidase IV and adenosine triphosphatase activities, respectively, also demonstrated hybrid bile canaliculi. These results indicated that transplanted hepatocytes swiftly overcome mechanical barriers in hepatic sinusoids to enter liver plates and join host cells. Such events are not prevented in disease models of acute liver failure.20 In a rat model of D-galactosamine-induced acute liver failure, transplanted hepatocytes were observed in liver plates at 48 hours, although the time required for synthesis of plasma membrane structures and proliferation of transplanted hepatocytes could be delayed in an environment of acute liver necrosis and inflammation. A further study, using a model of toxin-induced liver disease, demonstrated that donor parenchyma in nonsurvivor mouse livers, after LCT, did not have typical lobular organization despite moderate liver repopulation by donor cells.60 These results indicate that the recreation of functional parenchyma by transplanted the liver.

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hepatocytes requires time, during which donor cells proliferate and then establish normal parenchymal architecture. Liver cell engraftment. The initial engraftment of liver cells after transplantation in animal models is low. Under normal circumstances, engraftment is 0.03% to 0.5% of the total recipient liver cells.54 This is surprisingly low considering that in mice, for example, 2 million cells, constituting 3% to 5% of the total recipient liver cells, are usually infused. Therefore approximately 90% of the transplanted cells are not detected in the recipient liver after infusion. Cell damage related to the isolation procedures and mediated by apoptosis might account for part of this cell loss.61 There is also in vitro evidence that intercellular surface molecules exposed on hepatocytes during collagenase perfusion isolation are recognized by autologous granulocytes and monocytes as “non-self,” and this results in cytotoxic lysis of hepatocytes.62 In situations when the goal of cell transplant is not replacement of liver parenchyma but cure of the disease through provision of synthetic function, large infusions of good quality liver cells may be all that is required. A 10-fold increase in engraftment was shown in rats after repeated infusions of liver cells, suggesting that over 5% engraftment, sufficient to correct some metabolic defects, could be achieved after repeated LCT.63 Host liver repopulation by cell transplantation. The ancient Greeks knew something that has taken modern medicine more than 2000 years to fully appreciate and utilize—that the liver has the most extraordinary capacity to proliferate and regenerate. As the legend goes, Zeus punished Prometheus for disobediently giving the gift of fire to mankind. He was bound to a cliff and eternally damned to have his liver pecked out each day by an eagle. Although early 20th-century studies on repeated hepatectomies in dogs, and surgical observations in human subjects, have strongly suggested that the Prometheus legend had a strong biologic basis, the dramatic proliferative potential of normal liver cells was not established until it was demonstrated that near-total repopulation of a mouse liver could occur after the transplantation of as few as 1000 liver cells.1 The key criteria responsible for successful repopulation of host livers with donor hepatocytes appear to be a combination of a strong hepatocyte proliferation signal coupled to a survival growth advantage of the transplanted cells over endogenous hepatocytes. This breakthrough concept was further established during serial transplantation of hepatocytes in a mouse model of tyrosinemia.34 Donor liver cells were able to repopulate a recipient liver and after repopulation, iso-

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lated cells from the recipient liver could subsequently repopulate other livers. This experiment of serial transplantation suggested that there is a regenerative transplantable hepatocyte (akin to a hematopoietic stem cell) because this procedure was repeated through six consecutive transplants. Using serial transplantation, with limiting numbers of repopulating cells, the authors calculated that repopulating liver cells were able to double at least 69 times without loss of function and without becoming malignant in vivo. These data provide evidence that at least a subpopulation of liver-derived cells constitute a stem cell compartment, because most populations of primary cells have an upper limit to the number of cell divisions (the Hayflick limit) they can undergo before senescence occurs. Primary murine cells are limited to 15 to 20 doublings.34 Several other models of proliferative growth advantage have now been developed (see Table II). Induction of a selective growth advantage. Early studies focused on simply inducing a proliferative stimulus to the liver at the time of transplant. These included partial hepatectomy,64,65 hepatocyte growth factor infusion,66 and vascular injury.21 Although these approaches can demonstrate a modest degree of repopulation, the most outstanding results have been obtained when donor cells have a selective growth advantage over and above that of the endogenous hepatocytes. A selective growth advantage of donor cells over host liver cells and successful repopulation can be established by several routes. Table II lists a number of rodent models that have successfully harnessed one of these mechanisms to demonstrate donor liver cell repopulation in a recipient liver. The regenerative ability of the endogenous hepatocytes may be inhibited— either through a genetic or inherited inability to regenerate (as in the tyrosinemic mouse model34 and uPA mouse model1); through a selected genetic inability to respond to extraordinary environmental stimuli (as in the copper-loaded Wilson disease mouse model67); or through a pre-transplant inhibitory stimulus, as in the retrorsine model of cellular inhibition,36,37 which continues to exert an intracellular effect on the endogenous hepatocytes several weeks to months after the noxious stimuli. Transplanted hepatocytes can also be given an extra proliferative boost through donor cell genetic modification to augment cell survival or gene therapy to the endogenous liver to cause selective apoptosis of recipient cells (eg, Fas-induced apoptosis68). The difficulty with many of these induced forms of selective growth advantage is that they are clinically unacceptable. A recent article demonstrated that transplanted hepatocytes can extensively repopulate and function in a heavily irradiated rat liver.69 Because radiotherapy inhibits liver regeneration, the authors

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hypothesized that non-irradiated transplanted hepatocytes would proliferate preferentially in a partially resected and irradiated liver, providing metabolic support. Rats were subjected to hepatic radiotherapy and partial hepatectomy followed by a single intrasplenic hepatocyte transplantation. Survival improved in rats that were transplanted with syngeneic hepatocytes as compared with control subjects that received radiotherapy and partial hepatectomy only. This article demonstrated induction of a selective growth advantage in a clinically applicable method, and the authors postulated that this application may be appropriate for patients after resection of liver cancers. Unfortunately, LCT is unlikely to be valuable for the treatment of patients with cancer in the near future because of the current requirement to immunosuppress patients after cell transplant and because of the potential to activate micro-metastases. Using a rat model of an osteogenic disorder, because of the deficiency of an ascorbic acid biosynthetic enzyme, Nakazawa et al70 assessed the functional ability of HSS to effect hepatocyte proliferation after LCT. The average bromodeoxyuridine labeling index and hepatocyte-occupied ratio in the spleen of HSS-treated rats were significantly higher than those in HSSuntreated rats. All of the rats in the HSS-untreated/LCT groups died by 14 weeks after transplant, whereas survival rates in the HSS-treated/LCT groups were 60% to 80%. Confirming the metabolic effect of the HSStreated transplanted hepatocytes was the finding that the average serum ascorbic acid level of the surviving rats in the HSS-treated/LCT groups was significantly higher than that in the HSS-untreated/LCT groups. Because many of the transplanted hepatocytes appeared to remain in the spleen, HSS may have been providing an intrasplenic stimulus to these cells that they would not otherwise have received. Such proliferative stimuli may one day provide a medical basis for stimulating the proliferative ability of the cells placed in the spleen in the case of therapy for cirrhosis. Regenerative signals for repopulation. The type of regenerative stimulus used is likely to dictate the response of the transplanted hepatocytes. Different proliferative signals are induced by different noxious stimuli, and therefore it is likely that components of a liver cell transplant will respond uniquely to these presumably soluble proliferative signals. After partial hepatectomy, “compensatory hyperplasia” occurs, and normally quiescent hepatocytes exit G0 and undergo one or several rounds of DNA synthesis, enabling the liver to regain its original weight within several weeks. In contrast, hepatotoxins, such as the alkylating agent dipin, inhibit mature hepatocyte cell division and result in activation of the putative stem-cell compartment and oval cell proliferation. Review of liver regeneration is beyond

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the scope of this article and has been well summarized by others.71,72 ANIMAL MODELS OF DISEASE CORRECTION AFTER LCT Models of liver insufficiency: Metabolic or acute.

Numerous studies have reported successful transplantation of hepatocytes with demonstration of function. However, not all have conclusively shown long-term correction of a liver-related metabolic defect. Selden et al35 were the first to show complete correction of a defective biochemical phenotype achieved by hepatocellular transplantation. They transplanted liver cells into the peritoneum of mice lacking histidase as a model for the treatment of the autosomal recessive disorder histidinemia. Recipient mice showed a dramatic decrease, by more than 75%, in urinary histidine levels throughout the 3-month course of the experiment, resulting in levels within the normal range for wild-type mice. Immunohistochemical evidence showed expression of histidase in the ectopic liver tissue in the abdominal wall, pancreas, and peritoneal connective tissue. Moscioni et al73 also reported long-term correction of a liver-related metabolic defect. Nagase analbuminemic rats were immunosuppressed with cyclosporin, and portal vein branches were ligated to induce liver cell regeneration. They demonstrated dramatic elevations in serum albumin to near-normal levels (1.78 ± 0.20 g/dL) and maintenance of these levels until the end of the experiment, more than 3 months after LCT. More recently, a large animal model of urea cycle defects in Dalmatian dogs (hyperuricosuria) was used to assess the effect of multiple, sequential intrasplenic transplants of fresh and cryopreserved hepatocytes. The urinary uric acid excretion decreased an average of 54% after the first hepatocyte transplantation. However, the effect was transient and lasted an average of 22 days (range, 19 to 50 days). Subsequent intrasplenic hepatocyte transplantation with cryopreserved hepatocytes resulted in similar decreases in urinary uric acid excretion, but the result was unsustained. Unfortunately, several reports utilizing this model have been unable to demonstrate a sustained effect of LCT in this dog model, whether or not immunosuppression was used.29-31 The reasons for the lack of success in this larger animal model are unclear, and of course these results have implications for human LCT clinical trials. It is likely that host immune surveillance of transplanted tissue is likely to play a role in rejection of donor liver cells, and it may be that the immunosuppressive regime used in this animal model was not optimal. A more sophisticated animal model of acute liver insufficiency has been experimentally induced by the overexpression of Mad1 using an adenoviral vector.74

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LCT rescued a significant proportion of mice after this acute, noxious stimulus. The findings indicated that Mad1 overexpression perturbed hepatocyte survival and suggested a central role for specific cell cycle regulators in acute liver toxicity. This model will be valuable for further investigation of pathophysiologic mechanisms of acute liver failure and the use of LCT in bridging patients with fulminant failure to whole-organ transplantation. Chronic liver disease. Partial correction of chronic encephalopathy in rodents after intrasplenic hepatocellular transplantation was first demonstrated by Ribeiro et al.2 In an experimental rat model of chronic liver failure induced by end-to-side portacaval shunt, spontaneous activity and nose-poke exploration by rats, 3 months after intrasplenic hepatocellular transplantation, was the same as those of sham-operated rats without induced chronic liver disease. Although increases in plasma ammonia levels after portacaval shunt were not corrected by LCT, amino acid imbalance and bile acid concentration in plasma were partially corrected. One theoretic problem with the use of LCT in chronic liver disease is the lack of endothelial fenestrations in the cirrhotic liver. In spite of the vascular pathology during cirrhosis, Gagandeep et al38 demonstrated that, in a carbon tetrachloride–induced cirrhotic rat model, after splenic infusion, cells were distributed in periportal areas, fibrous septa, and regenerative nodules of the cirrhotic liver. One week after LCT, cells were surprisingly fully integrated in the liver parenchyma and expressed liver-specific genes. In addition, transplanted cells were able to proliferate and survive for more than 1 year in the recipient cirrhotic liver. Interestingly, some studies suggest transplanted hepatocytes might have a protective effect against the development of chronic liver lesions.75 A further study, which also addressed the role of LCT in treating decompensated liver cirrhosis, induced cirrhosis in rats by using phenobarbital and carbon tetrachloride.55 After intrasplenic LCT, body weight and serum albumin levels remained normal, whereas they deteriorated over time in all control animals. Prothrombin time, total bilirubin, serum ammonia, and hepatic encephalopathy score were also significantly improved toward normal in animals receiving intrasplenic hepatocyte transplantation. More importantly, survival was prolonged after a single infusion of hepatocytes, and a second infusion prolonged survival from 15 to 128 days. This study demonstrated that hepatocyte transplantation could improve liver function and prolong the survival of rats with irreversible, decompensated cirrhosis. A fascinating experiment by Rudolph et al76 was described recently when they demonstrated inhibition

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of experimental liver cirrhosis in mice by telomerase gene delivery. Accelerated telomere loss has been proposed to be a factor leading to end-stage organ failure in chronic diseases of high cellular turnover such as liver cirrhosis. To test this hypothesis directly, telomerase-deficient mice were subjected to genetic, surgical, and chemical ablation of the liver. Telomere dysfunction was associated with defects in liver regeneration and accelerated the development of liver cirrhosis in response to chronic liver injury. Adenoviral delivery of telomerase RNA into the livers of telomerase-deficient mice restored telomerase activity and telomere function, alleviated cirrhotic pathology, and improved liver function. This study highlights the fact that development of strategies to enable the use of LCT in patients with cirrhosis will be vital to a more universal application of LCT, because the consequences of cirrhosis comprise the leading clinical indication for whole-organ transplantation. CELL SOURCES FOR TRANSPLANTATION Mixed liver cell population. Most studies to date have used an unfractionated suspension of liver cells.34 It is not yet clear whether a pure fraction of hepatocytes would have the same repopulating ability as a mixed cell population. Percoll has been used to purify hepatocyte suspensions, and repopulation has still been successful.32,77 Because even a single stem cell can potentially repopulate a liver, it is not yet clear whether hepatocyte “purification” may be diluting downward the number of putative stem cells in a liver cell transplant. Fetal hepatocytes. Fetal hepatocytes have several characteristics that make them potentially suitable as donor cells. In contrast to adult hepatocytes, fetal hepatocytes are thought to be highly proliferative, which may facilitate engraftment and expansion of transplanted cell population. Lilja et al78 took fetal rat (20 days of gestation) and adult rat hepatocytes and cultured them in serum-free medium at low densities as well as treating them with a variety of growth factors including hepatocyte growth factor, epidermal growth factor, and transforming growth factor-α. Their results showed that in contrast to adult hepatocytes, fetal hepatocytes have high spontaneous proliferative activity independent of growth factors and are relatively resistant to the inhibitory effect of transforming growth factor-β1. It is interesting in the light of clinical application that it was also found that cyclosporine suppressed proliferation of cultured fetal hepatocytes. Pancreatic cells. Recently, cells with hepatocellular properties have been identified in the adult pancreas. Wang et al79 transplanted suspensions of wild-type mouse pancreatic cells into syngeneic recipients defi-

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cient in fumarylacetoacetate hydrolase and manifesting tyrosinemia. Four of 34 mutant mice were fully rescued by donor-derived cells and had normal liver function. Ten additional mice showed histologic evidence of donor-derived hepatocytes in the liver. Their results demonstrated for the first time that adult mouse pancreas contains hepatocyte progenitor cells capable of significant therapeutic liver reconstitution. Although this might have been predicted—because in embryonic development, the liver and pancreas both originate from the same location in the ventral foregut80—this study serves to highlight that stem cell biology of the liver is still not fully understood and is likely to result in many important clinical applications in the future. Bone marrow–derived cells. Several recent articles have investigated the possibility that pluripotent stem cells reside in the bone marrow and can contribute to liver regeneration after noxious insults to the liver.3-5,72 The first of these studies used rats treated with 2-acetylaminofluorene to block hepatocyte proliferation and then liver injury to induce oval cell proliferation.3 Rats then received cross-sex or cross-strain bone marrow to trace the origin of the repopulating liver cells. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated liver cells were shown to be donorderived, demonstrating that a stem cell associated with the bone marrow has epithelial cell lineage capability. A second study investigated whether such a process occurs in human subjects.4 Archival autopsy and biopsy liver specimens were obtained from 2 female recipients of therapeutic bone marrow transplantations with male donors and from 4 male recipients of orthotopic liver transplantations from female donors. Using immunohistochemical staining to identify hepatocytes and cholangiocytes and fluorescence in situ hybridization for X and Y chromosomes, they demonstrated that between 4% and 43% of hepatocytes and cholangiocytes can be derived from extrahepatic circulating stem cells, probably of bone marrow origin. They further concluded that such “transdifferentiation” can replenish large numbers of hepatic parenchymal cells. The third study also investigated the incidence of bone marrow–derived cells with hepatocyte morphology residing in the liver after sex-mismatch bone marrow transplantation. Although the incidence of bone marrow donor–derived hepatocytes was low (0.5% to 2%), the authors postulated that chronic damage of the livers in the bone-marrow transplant patient could promote colonization and amplification of exogenous hematopoietic stem cells, providing a rationale for experimental models of LCT with bone marrow stem cells.72

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Lagasse et al5 reported the intravenous injection of adult bone marrow cells in the tyrosonemic mouse model and demonstrated rescue of the mouse and restoration of biochemical function of its liver. These studies raise the exciting possibility that bone marrow stem cells, a readily available source of cells, could be clinically utilized for LCT. Immortalized cells. Several groups have investigated the use of conditionally immortalized hepatocytes for LCT because a tightly regulated human hepatocyte cell line that grows economically in culture and exhibits differentiated liver functions would be an attractive alternative to primary human hepatocytes.81-83 These cell lines are based on the use of the SV40 Large T gene that can induce temperature-sensitive immortalization through the production of a protein that is active at 33°C but is conformationally inactivated at 39°C. Thus cell lines maintained in culture at 33°C are immortalized but revert to the non-immortalized state when the SV40 Large T protein is inactivated. However, there is some concern that these conditionally immortalized hepatocytes become de-differentiated with in vitro passage84 and would not therefore be suitable for clinical use because of the risk of tumorigenesis. More recently, conditionally immortalized human hepatocyte cell lines have been constructed with an additional herpes simplex virus–thymidine kinase so that ganciclovir can be used to destroy any SV40Tagexpressing cells should the control of these cells become unregulated.85 Further levels of control have been attained with genes encoding SV40TAg flanked by loxP recombination target sites. These cells can then have the immortalizing gene subsequently excised by Cre/Lox site-specific recombination.86,87 These studies offer hope for the use of a tightly regulated conditionally immortalized human hepatocyte cell line for clinical use, although the concern for genetic drifting in vitro remains. BANKING OF LIVER CELLS

Until recently, methods for cryopreservation of primary human hepatocytes did not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. Adams et al13 described a protocol for efficient cryopreservation of primary human hepatocytes that used University of Wisconsin solution, fetal bovine serum, and dimethyl sulfoxide. They assessed the viability of differentiated primary human hepatocytes 8 months after cryopreservation and found viability to be >90% as measured by Trypan blue dye exclusion. Although they demonstrated that hepatocyte morphology was preserved, as was liver-specific gene expression (α1 antitrypsin) and replication, methods of via-

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bility analyses more stringent than Trypan blue dye exclusion were not used. Nevertheless, the effectiveness of University of Wisconsin solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The effects of various organ preservation solutions on hepatocyte membrane potentials, intracellular calcium concentrations, and clinical outcome after liver transplantation have also been shown to be important88 and are likely to be sentinel in the improvement of harvesting methodology for liver cell isolation and cryopreservation. One study investigated the effect of four different preservation solutions, including preservation solutions associated with high (normal saline solution), moderate (Euro-Collins), and low (University of Wisconsin solution) risks of reperfusion injury. They found that hepatocytes undergo prompt and marked depolarization after hepatic resection and that the extent of the depolarization correlated with rat survival after whole-organ transplantation.88 Such membrane depolarization is likely to significantly affect the flux of important ionic compounds and is probably part of the initiating mechanism of liver cell damage after cell isolation. There is evidence that apoptosis is an important mediator of cell death during isolation and banking of primary liver cells.61 Recent studies by our group reveal that apoptosis, which occurs during isolating and banking processes, might be due to the change of intracellular calcium homeostasis and mitochondrial damage of primary mouse hepatocytes, (personal communication, H. Soriano). In addition, several extracellular regulators—such as FasL, steroids, and sphingolipids— as well as dissociation from the extracellular matrix have been considered to play roles in the regulation of apoptosis.89 Research into cell damage could contribute in improving the initial engraftment rate after LCT. On thawing, donor hepatocytes need to retain both their metabolic and their proliferative capacity. Jamal et al14 demonstrated in a mouse model that cryopreserved liver cells still demonstrated clonal replication potentials identical to those of fresh hepatocytes, despite decreased overall hepatocyte viability. Even after storage for 32 months in liquid nitrogen, transplanted mouse hepatocytes, constituting only 0.1% of the total hepatocyte number, could repopulate approximately 32% of the recipient liver parenchyma. These findings were confirmed by Dunn et al,30 who showed no difference between the metabolic effects of cryopreserved hepatocytes and those of fresh hepatocytes after transplantation for the treatment of urea acid cycle defects in Dalmation dogs.

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IMMUNOSUPPRESSION FOR LIVER CELL TRANSPLANTATION

One of the theoretic advantages of LCT over wholeorgan transplantation is the potential to minimize or stop immunosuppression in the host. It is generally recognized that the liver is an organ that occasionally achieves immunotolerance after allogeneic transplantation.90 Whole livers transplanted into major histocompatibility complex–disparate mouse strains can survive indefinitely without immunosuppression,91 so it is somewhat surprising that without immunosuppression, allogeneic hepatocytes are rapidly rejected in a few days. Gupta et al53 reported that hepatocytes from hepatitis B virus transgenic mice survived in syngeneic recipients and that hepatitis B surface antigen was detected in serum for 20 weeks, whereas no antigen was detected in allogeneic recipients within 7 days. Although allogeneic hepatocytes are rapidly rejected after transplantation, recent observations have implicated donor leukocyte microchimerism,92 and in particular, a role for donor-derived dendritic cell progenitors in liver transplant tolerance. It would seem therefore that the privileged role of the liver in transplantation immunology results from the non-hepatocyte components of the liver. Indeed, when whole organs are rejected, the inflammation is usually targeted to the portal tract components of bile duct epithelia and endothelial cells. In allogeneic LCT, the professional antigen-presenting cells of the recipient present alloantigens in the context of self–major histocompatibility complex class II antigen, whereas donor liver cells present mostly class I antigens. Alloreactive T cells recognize these antigens via the T cell receptor. Activation of the T cell receptor in the presence of a co-stimulatory signal from CD28 results in T cell clonal expansion and induction of an effector function, such as the production of lymphokines and the induction of cytotoxic activity. Kawahara et al93 were unable to prolong the survival of allogeneic hepatocytes after transplantation by blocking the CD28 co-stimulatory pathways, implying that hepatocyte rejection occurs through a CD28-independent pathway. Further work by this group has concluded that Fasmediated apoptosis plays a critical role in the CD28independent mechanism of allogeneic hepatocytes.94 Further experiments have investigated the effect of hepatocyte purification and cryopreservation on the ability of mouse liver cells to engraft in allogeneic recipients without immunosuppression.95 Liver cell transplants with crude (unpurified), modified (purified or cryopreserved), or dead (irradiated) hepatocyte preparations labeled with fluorescein dye were assessed

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by using histologic examination. No acute rejection in engrafted purified or cryopreserved hepatocytes was noted up to 21 days after transplant, and they appeared ultrastructurally normal, while the numbers of inoculated crude hepatocytes rapidly declined, with signs of dense infiltration of mononuclear cells in the graft that indicated a destructive response. These results suggest that reduced immunogenicity may be responsible for the longer survival time of purified or cryopreserved hepatocytes with no adverse morphologic effects. Fabrega et al96 reported successful induction of donor-specific tolerance to allogeneic hepatocyte transplantation in rats. Allogeneic donor splenocytes were injected into the thymus of Nagase analbuminemic rats, and rabbit anti-rat anti-lymphocyte serum was injected intraperitoneally 1 week later. Allogeneic hepatocytes were transplanted into the spleen of recipients a week later. Serum albumin levels increased by 0.5 to 1.2 mg/mL and remained stable 6 months later, demonstrating a novel approach to induction of liver cell transplant tolerance. A further novel approach was used by Balladur et al,48 who transplanted hepatocytes into the peritoneum, encapsulated in a semi-permeable membrane to protect them from rejection. Viability and function of encapsulated allogeneic hepatocytes were maintained up to 90 days after transplantation, without immunosuppression. Such studies are useful for development of short-term in vivo liver support, but because these cells are placed ectopically, such an approach is unlikely to be useful for long-term management of liver insufficiency. If the requirement for immunosuppression could be diminished or abolished, LCT would exhibit an outstanding advantage over whole-organ transplant and the indications for LCT would widen to include the treatment of liver cancers (after partial hepatectomy) and cancer patients with liver failure who need liver synthetic support. WHAT WILL BE THE KEYS TO SUCCESS FOR LIVER CELL TRANSPLANTATION?

Providing enough cell support, either through transplantation of larger volumes of cells or by harnessing the proliferative ability of the cell transplant and inducing a selective growth advantage of donor cells over host hepatocytes, will be a major clinical step forward for this therapy. The ability to deliver cells to a cirrhotic liver would broaden the application of the therapy. Overcoming the requirement for immunosuppression would clearly position LCT not just as an alternative to whole-organ transplantation but as a superior therapy. Although the road to clinical success of LCT has been slow and fraught with difficulty, several new stud-

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ies herald a more promising era, especially in addressing the problem of donor cell shortage for cell transplant programs. If repopulation of the diseased host liver by donor liver cells can be clearly demonstrated with attendant improvement in host liver function, then the promise of LCT is likely to be fulfilled in the clinical arena. We are currently poised to exploit the powerful proliferative capacity of liver cells and utilize what the ancient Greeks may well have foretold. Although blood transfusions and bone marrow transplants constitute the first successful clinical applications of cell therapies, over the next decade LCT will be among the first cellular therapies to be derived from solid organs. We thank Dr Thomas Green, Mr Patrick Magoon, Dr Bernard Mirkin, Dr William Schnaper, Dr Peter Whitington, and Ms Adrienne Woodworth for their support and encouragement. REFERENCES

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32. De Vree JML, Ottenhoff R, Bosma PJ, Smith AJ, Aten J, Oude Elferink PJ. Correction of liver disease by hepatocyte transplantation in a mouse model of progressive familial intrahepatic cholestasis. Gastroenterology 2000;119:1720-30. 33. Yoshida Y, Tokusashi Y, Lee GH, Ogawa K. Intrahepatic transplantation of normal hepatocytes prevents Wilson’s disease in Long-Evans cinnamon rats. Gastroenterology 1996; 111:1654-60. 34. Overturf K, al-Dhalimy M, Ou CN, Finegold M, Grompe M. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 1997;151: 1273-80. 35. Selden C, Calnan D, Morgan N, Wilcox H, Carr E, Hodgson HJ. Histidinemia in mice: a metabolic defect treated using a novel approach to hepatocellular transplantation. Hepatology 1995;21:1405-12. 36. Laconi E, Oren R, Mukhopadhyay DK, Hurston E, Laconi S, Pani P, et al. Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am J Pathol 1998;153:319-29. 37. Guo D, Fu T, Crawford SE, Superina R, Soriano HE. Enhanced liver cell engraftment in a non-surgical, non-transgenic murine model. J Pediatr Gastroenterol Nutr 2000;31: S282. 38. Gagandeep S, Rajvanshi P, Sokhi RP, Slehria S, Palestro CJ, Bhargava KK, et al. Transplanted hepatocytes engraft, survive, and proliferate in the liver of rats with carbon tetrachloride-induced cirrhosis. J Pathol 2000;191:78-85. 39. Jiang B, Sawa M, Yamamoto T, Kasai S. Enhancement of proliferation of intrasplenically transplanted hepatocytes in cirrhotic rats by hepatic stimulatory substance. Transplantation 1997;63:131-5. 40. Jan D, Laurent J, Lacaille F, Jouvet P, Poggi F, Rabier D, et al. Liver transplantation in children with inherited metabolic disorders. Transplant Proc 199527:1706-7. 41. Burdelski M, Rodeck B, Latta A, Latta K, Brodehl J, Ringe B, et al. Treatment of inherited metabolic disorders by liver transplantation. J Inherit Metab Dis 1991;14:604-18. 42. Fisher RA, Bu D, Thompson M, Tisnado J, Prasad U, Sterling R, et al. Defining hepatocellular chimerism in a liver failure patient bridged with hepatocyte infusion. Transplantation 2000;69:303-7. 43. Seglen PO. Preparation of isolated rat hepatocytes. Methods Cell Biol 1976;13:30-8. 44. Matas AJ, Sutherland DER, Steffes MW, Mauer SM, Lowe A, Simmons RL, et al. Hepatocellular transplantation for metabolic deficiencies: decrease of plasma bilirubin in Gunn rats. Science 1976;192:892-4. 45. Makowka L, Rotstein LE, Falk RE, Falk JA, Langer B, Nossal NA, et al. Reversal of toxic and anoxic induced hepatic failure by syngeneic, allogeneic, and xenogeneic hepatocyte transplantation. Surgery 1980;88:244-53. 46. Makowka L, Rotstein LE, Falk RE, Falk JA, Nossal NA, Langer B, et al. Allogeneic and xenogeneic hepatocyte transplantation in experimental hepatic failure. Transplantation 1980;30:429-35. 47. Gupta S, Vemuru RP, Lee CD, Yerneni PR, Aragona E, Burk RD. Hepatocytes exhibit superior transgene expression after transplantation into liver and spleen compared with peritoneal cavity or dorsal fat pad: implications for hepatic gene therapy. Human Gene Therapy 1994;5:959-67. 48. Balladur P, Crema E, Honiger J, Calmus Y, Baudrimont M, Delelo R, et al. Transplantation of allogeneic hepatocytes without immunosuppression: long-term survival. Surgery 1995;117:189-94.

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49. Sandbichler P, Erhart R, Herbst P, Vogel W, Herold M, Dietze D, et al. Hepatocellular transplantation into the lung in chronic liver failure following bile duct obstruction in the rat. Cell Transplant 1994;3:409-12. 50. Ebata H, Onodera K, Sawa M, Mito M. A study of liver regeneration using fetal rat liver tissue transplanted into the spleen. Jpn J Surg 1988;18:540-7. 51. Mito M, Ebata H, Kusano M, Onishi T, Hiratsuka M, Saito T. Studies on ectopic liver utilizing hepatocyte transplantation into the rat spleen. Transplantation Proc 1979;11:585-91. 52. Gupta S, Rajvanshi P, Bhargava KK, Kerr A. Hepatocyte transplantation: progress toward liver repopulation. Prog Liver Dis 1996;14:199-222. 53. Gupta S, Chowdhury NR, Jagtiani R, Gustin K, Aragona E, Shafritz DA, et al. A novel system for transplantation of isolated hepatocytes utilizing HBsAg-producing transgenic donor cells. Transplantation 1990;50:472-5. 54. Ponder KP, Gupta S, Leland F, Darlington G, Finegold M, DeMayo J, et al. Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation. Proc Natl Acad Sci U S A 1991;88:1217-21. 55. Kobayashi N, Ito M, Nakamura J, Cai J, Gao C, Hammel JM, et al. Hepatocyte transplantation in rats with decompensated cirrhosis. Hepatology 2000;31:851-7. 56. Kobayashi N, Ito M, Nakamura J, Cai J, Hammel JM, Fox IJ. Hepatocyte transplantation improves liver function and prolongs survival in rats with decompensated liver cirrhosis. Transplant Proc 1999;31:428-9. 57. Vroemen JP, Buurman WA, Heirwegh KP, van der Linden CJ, Kootstra G. Hepatocyte transplantation for enzyme deficiency disease in congenic rats. Transplantation 1986;42: 130-5. 58. Soriano HE, Lewis D, Legner M, Brandt M, Baley P, Darlington G, et al. Use of DiI marked hepatocytes to demonstrate orthotopic intraheptic engraftment following hepatocellular transplantation. Transplantation 1992;54:717-23. 59. Gupta S, Rajvanshi P, Lee CD. Integration of transplanted hepatocytes into host liver plates demonstrated with dipeptidyl peptidase IV-deficient rats. Proc Natl Acad Sci U S A 1995;92:5860-4. 60. Braun KM, Degen JL, Sandgren EP. Hepatocyte transplantation in a model of toxin-induced liver disease: variable therapeutic effect during replacement of damaged parenchyma by donor cells. Nat Med 2000;6:320-6. 61. Fu T, Guo D, O’Gorman M, Crawford S, Soriano HE. Glucose prevents apoptosis of cryopreserved primary mouse hepatocytes. Cell Transplantation 2001;10:59-66. 62. Olszewski WL, Hiwot H, Interewicz B, Rudowska A, Szyper E, Mecner B. Hepatocyte transplantation: in vitro cytotoxic reaction of autologous granulocytes and mononuclears to isolated hepatocytes. Ann Transplant 1999;4:11-6. 63. Rajvanshi P, Kerr A, Bhargava KK, Burk RD, Gupta S. Efficacy and safety of repeated hepatocyte transplantation for significant liver repopulation in rodents. Gastroenterology 1996;111:1092-102. 64. Gupta S, Johnstone R, Darby H, Selden C, Price Y, Hodgson HJ. Transplanted isolated hepatocytes: effect of partial hepatectomy on proliferation of long-term syngeneic implants in rat spleen. Pathology 1987;19:28-30. 65. Jirtle RL, Michalopoulos G. Effects of partial hepatectomy on transplanted hepatocytes. Cancer Res 1982;42:3000-4. 66. Kato K, Onodera K, Sawa M, Imai M, Kawahara T, Kasai S, et al. Effect of hepatocyte growth factor on the proliferation of intrasplenically transplanted hepatocytes in rats. Biochem Biophys Res Commun 1996;222:101-6.

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