INTRODUCTION — Advances in the understanding of hepatocyte engraftment and the remarkable proliferative potential of hepatocytes have led to the application of liver cell transplantation for the treatment of inherited and acquired human diseases. Extensive pre-clinical animal experiments have shown that hepatocytes transplanted in the liver or at ectopic sites survive, function, and participate in the regenerative process. Because the host liver architecture remains intact following the integration of the engrafted hepatocytes in the liver cords, hepatocyte transplantation is metabolically less stressful than transplantation of the whole organ, and the consequences of graft loss are much less severe.
Hepatocyte transplantation has many potential applications. Therapeutic genes can be transferred into isolated hepatocytes, and the phenotypically modified cells can then be transplanted for ex vivo gene therapy. Such gene transfer could be used to replace a missing gene product, to prevent immune rejection, or to provide the cells with a proliferative advantage. Hepatocyte transplantation does not interfere with subsequent liver transplantation or gene therapy. Although the safety and clinical efficacy of hepatocyte transplantation have been demonstrated, the shortage of good quality donor livers for hepatocyte isolation has limited the widespread clinical application of this method. Clinical studies are beginning to take advantage of some advances made in preclinical animal experiments.
SCOPE OF HEPATOCYTE TRANSPLANTATION — Potential clinical applications of hepatocyte transplantation are listed in (table 1).
Treatment of inherited metabolic diseases — Missing gene products can be substituted by transplanting normal primary hepatocytes from allogeneic donors. This simple approach holds promise for diseases such as Crigler-Najjar syndrome type 1, urea cycle disorders, and coagulopathies, including hemophilias . (See "Crigler-Najjar syndrome" and "Inborn errors of metabolism: Classification", section on 'Urea cycle disorders' and "Genetics of hemophilia A and B".)
Hepatocytes used for gene therapy can be derived from an allogeneic donor (deceased or living donor) or the recipient can serve as the donor, in which case the genetic defect needs to be corrected before transplantation (autologous transplantation). An advantage of autologous transplantation is that it does not require immunosuppression [2-4].
Management of acute liver failure — Liver cell transplantation can assist in the management of patients with fulminant hepatic failure [5-7]. The transplanted hepatocytes can provide temporary liver support in patients awaiting the availability of a donor liver or spontaneous recovery. In some patients, regeneration of endogenous hepatocytes combined with the transplanted hepatocytes could possibly replace the need for transplantation of the whole liver. Hepatocyte transplantation has been shown to improve some biochemical parameters, such as reduction of blood ammonia levels, as well as improvement of encephalopathy, but patient survival beyond a week without liver transplantation has been rare [8-10]. Whether hepatocyte transplantation is superior to the use of artificial liver assist devices for bridging to liver transplantation remains to be determined. (See "Acute liver failure in adults: Management and prognosis", section on 'Artificial hepatic assist devices'.)
Management of chronic liver failure — Hepatocyte transplantation can provide metabolic support and partially restore liver function in patients with chronic liver failure . Transplanted normal hepatocytes have a survival advantage compared with the host cells in some inherited disorders, such as hereditary tyrosinemia type I (fumarylacetoacetate hydrolase deficiency), and could potentially repopulate the liver over time . (See "Disorders of tyrosine metabolism".)
Most patients with chronic liver failure have already established cirrhosis, and the benefit of hepatocyte transplantation in the presence of cirrhosis has not been established by studies performed to date.
SOURCES OF HEPATOCYTES — In animal experiments, primary hepatocytes derived from congenic (varying in only one gene locus) or syngeneic (immunologically compatible) strains can be transplanted without the need for immunosuppression. Such complete tissue matching is not possible in clinical situations. As mentioned above, when hepatocytes are used as vehicles for ex vivo gene therapy, immunosuppression can be circumvented by isolating hepatocytes from resected liver segments and transplanting them back to the donor after genetic manipulations. However, in most cases, the cells are obtained from allogeneic donors and immunosuppression will be required, unless specific tolerance to the alloantigens can be achieved (see below).
The worldwide shortage of donor organs severely limits the number of donor livers that can be used for hepatocyte isolation. Investigators have attempted to alleviate this shortage by transplanting hepatocytes isolated from one fragment of a donor liver to several recipients . In addition, livers explanted from patients with a monogenic disorder at the time of liver transplantation have been used to isolate hepatocytes for transplantation into a recipient with a different monogenic disease (domino transplantation) . The shortage of transplantable primary human hepatocytes has led to the search for alternative sources of hepatocytes as follows:
●Immortalized hepatocytes: Hepatocytes derived from animal and human livers have been immortalized by transferring the gene for the large T antigen of the simian virus 40 (Tag), permitting expansion in vivo. To prevent persistent expression of T antigen in vivo, which can promote oncogene-mediated transformation, hepatocytes have been conditionally immortalized by transduction with temperature-sensitive mutant T antigen (tsTag) via retroviral vectors. The transduced cells proliferate in vitro at permissive temperatures (33°C), but tsTag is degraded at physiologic temperatures (37 to 39°C), thereby stopping cell growth. In rodent models, such conditionally immortalized hepatocytes have been used to provide metabolic support in acute or chronic liver failure, and for ex vivo gene therapy . Alternatively, the Tag sequence can be flanked with lox sequences, so that it can be excised using cre recombinase . Human hepatocytes, reversibly immortalized in this manner were used successfully to rescue rats with acute liver failure, induced by 90 percent hepatectomy .
●Xenogeneic hepatocytes: Hepatocytes derived from porcine or other animal sources could be potentially used for providing metabolic support during liver failure , but major immunological hurdles remain. Another concern about these approaches is the potential to transmit agents, such as the porcine endogenous retroviruses, which can infect humans.
●Fetal hepatocytes: Human fetal hepatocytes have been transplanted into patients with acute liver failure with marginal benefit. Transplantation of EpCAM+ve liver progenitor cells isolated from human fetal liver by infusion into the hepatic artery of patients with decompensated cirrhosis was reported to improve synthetic and excretory functions of the liver .
●Hepatocytes derived from pluripotent cells: Several laboratories have developed methods to generate hepatocyte-like cells by in vitro differentiation of human embryonic stem cells or induced pluripotent stem cells (iPSC) produced by reprogramming somatic cells, such as fibroblasts, blood cells, and epithelial cells shed in the urine [20-23]. In an alternative approach, skin fibroblasts were directly reprogrammed to hepatocyte-like cells without initial conversion to iPSCs [24,25]. Hepatocyte-like cells derived from human-induced pluripotent stem cells have been transplanted into genetically analbuminemic rats , UGT1A1-deficient jaundiced Gunn rats (model of Crigler-Najjar syndrome type 1) , and mice expressing mutant human alpha-1-antitrypsin . In each case, there was partial repopulation of the liver with the human cells.
●Adult stem/progenitor cells: Cells that can self-replicate, as well as differentiate into multiple cell types, persist in many adult tissues including the bone marrow , fat, and liver. In animal studies, transplanted bone marrow-derived cells have been shown to give rise to hepatocytes, albeit at a frequency too low for them to be a significant source of hepatocytes for transplantation . Mesenchymal stem cells derived from human adipose tissue  and mouse tail fibroblasts  have been differentiated into cells exhibiting some characteristics of hepatocytes. Benefits of using such cells for the treatment of liver-based disorders have yet to be demonstrated. Liver stem cells derived from adult human liver and expanded in culture have been transplanted in a child with ornithine carbamoyltransferase deficiency . The procedure resulted in partial clinical improvement, as well as reduction of blood ammonium and urinary orotic acid levels. However, limited follow-up precluded the evaluation of duration of clinical benefit.
●Human hepatocytes expanded by repopulation of animal liver: Transplanted human hepatocytes proliferate extensively in the livers of immunodeficient fumarylacetoacetate hydrolase knockout mice, replacing the majority of the mouse hepatocytes with human hepatocytes . This source of mature human hepatocytes (expanded in the liver of mice) could potentially be used for transplantation.
SITES OF HEPATOCYTE TRANSPLANTATION — The liver provides the most supportive environment for engraftment and function of transplanted hepatocytes because of the availability of nutrients and growth factors present in the portal circulation, and interaction with other liver cells and the hepatic matrix. However, the optimum route for hepatocyte transplantation also depends upon the specific application.
Hepatocytes infused into the portal vein or injected into the splenic pulp travel to the hepatic sinusoids and integrate into the liver cords [33,34]. The cells obtain polarity and establish appropriate junctions with the neighboring hepatocytes within 24 hours. Studies in rats, mice, and rabbits have shown that syngeneic or congenic hepatocytes survive and function throughout the life of the host. Hepatocytes equivalent to up to one to five percent of the liver mass could be introduced into normal livers through the portal system with only a transient and reversible increase in the portal pressure, although only a fraction of the cells engraft within the next three to four days. By contrast, hepatocyte transplantation in cirrhotic animals results in portal hypertension and an increase in right atrial pressure .
After injection into the splenic pulp, the majority of the hepatocytes pass the liver, but a fraction of the transplanted cells may survive in the spleen and eventually develop liver cord-like structures, replacing up to 40 percent of the splenic mass. Over time, bile canaliculi, sinusoids, and endothelial cells may develop. Thus, in specific circumstances, the spleen may serve as an important site of hepatocyte engraftment.
For inherited metabolic disorders, implantation into the liver via infusion into a tributary of the portal vein or injection into the splenic pulp has been most effective [36-38]. However, in the presence of cirrhosis, the liver may not be the most desirable site for hepatocyte transplantation because of inefficient engraftment and the abnormal microenvironment. Similarly, a liver that is undergoing massive necrosis may not provide a hospitable environment for the transplanted cells. In the presence of portal hypertension, a large proportion of the cells infused into the portal vein may be translocated to the pulmonary vascular bed. In an effort to avoid this complication, hepatocytes have been transplanted into the spleen by injection into the splenic pulp or infusion into the splenic artery . More study is needed to determine if the transplantation in the splenic artery would augment liver function in patients with cirrhosis.
The peritoneal cavity could be an effective site for transplanting hepatocytes that are encapsulated , attached to collagen-coated microcarriers , or encased in Hydrogel-based hollow fibers . Hepatocytes cryopreserved on these carrier beads could represent a readily available source for transplantation into the peritoneal cavity for the treatment of acute liver failure . Alginate-microencapsulated hepatocytes have been used successfully as a bridge to orthotopic liver transplantation or in lieu of liver transplantation in children with acute liver failure . Other sites, such as the renal subcapsular space or subcutaneous tissue, are less efficient.
PRECLINICAL EVALUATION IN EXPERIMENTAL ANIMAL MODELS — The efficacy of hepatocyte transplantation has been evaluated extensively in natural mutant animals.
Disorders due to single gene defects — Hepatocytes have been transplanted into Gunn rats (a model of Crigler-Najjar syndrome type 1), which have life-long unconjugated hyperbilirubinemia due to the lack of hepatic bilirubin-UDP-glucuronosyltransferase activity, by infusion into the portal vein, injection into the splenic pulp, or attachment to microcarriers, followed by injection into the peritoneal cavity. These procedures resulted in the excretion of bilirubin glucuronides in the bile and amelioration of hyperbilirubinemia [44,45]. The metabolic defect was corrected in subsequent studies in which the rats were first treated with liver resection and radiation, thereby promoting the preferential repopulation of the native liver with the transplanted hepatocytes . Hepatic irradiation appears to disrupt hepatic sinusoidal endothelial cells and inhibit the phagocytic function of Kupffer cells, thereby enhancing hepatocyte engraftment . Similarly, serum albumin levels increased after transplantation of normal hepatocytes into Nagase analbuminemic rats . Hepatocyte transplantation reduced plasma cholesterol levels by up to 50 percent in Watanabe heritable hyperlipidemic rabbits that lack LDL receptors (an animal model of familial hypercholesterolemia) . Hepatocyte transplantation also ameliorates the metabolic abnormality in fumarylacetoacetate hydrolase (FAH)-deficient mice (a model of hereditary tyrosinemia 1) , Long-Evans Cinnamon rats (a model of Wilson disease) , and mdr2 (-/-) mice (a model of progressive familial intrahepatic cholestasis) . (See "Disorders of tyrosine metabolism", section on 'Hereditary tyrosinemia type 1' and "Wilson disease: Epidemiology and pathogenesis" and "Inherited disorders associated with conjugated hyperbilirubinemia", section on 'Familial hepatocellular cholestasis'.)
Acute and chronic liver failure — Transplantation of hepatocytes significantly improved the survival of animals with acute liver failure induced by D-galactosamine or dimethylnitrosamine administration, hepatic ischemia, or 90 percent hepatectomy [40,52], but not in a mouse model of acute toxic liver failure . Hepatocyte transplantation reversed encephalopathy and prevented intracranial hypertension in animals with ischemic liver failure . In rats with hepatic encephalopathy caused by end-to-side portacaval shunt, intrasplenic transplantation of hepatocytes improved the behavior score, partially corrected amino acid imbalances, and protected the rats from hepatic coma induced by an exogenous ammonia load . In similar experiments, transplantation of hepatocytes that had been immortalized by transfection with the simian virus 40 T antigen was able to improve measures of liver failure and prolong survival . Hepatocyte transplantation in rats with chronic decompensated liver cirrhosis stabilized total bilirubin and prothrombin time, improved serum albumin and encephalopathy scores, and prolonged survival .
Massive repopulation of the liver — The effectiveness of hepatocyte transplantation for inherited liver diseases depends on the number of transplanted cells persisting long-term in the liver. The mass of transplanted hepatocytes depends on several factors:
●Efficiency of initial engraftment: After hepatocyte transplantation by infusion into the portal venous system or injection into the splenic pulp, instant blood-mediated inflammatory reaction (IBMIR) activates thrombin, which produces fibrin clots and activates complements, resulting in death of the injected hepatocytes and their clearance by granulocytes, monocytes, Kupffer cells, and natural killer cells . Antithrombin activator (heparin) and thrombin inhibitor (bivalirudin) inhibit IBMIR in vitro, suggesting that effective inhibition of IBMIR should improve initial engraftment. After arriving into the liver sinusoids, the liver sinusoidal endothelial cells (LSEC) pose a barrier to integration of hepatocytes into the liver plates. Preparative X-irradiation of the liver results in transient disruption of the LSEC, thereby enhancing initial engraftment of transplanted hepatocytes in preclinical [47,57] and clinical studies .
●Proliferation of transplanted hepatocytes: Following initial engraftment, proliferation of the transplanted hepatocytes is desirable for generation of a therapeutically effective mass. However, normal physiologic mechanisms tend to keep the total hepatocyte mass constant, so that repopulation of the liver occurs by competitive replacement of the host liver cells by the engrafted hepatocytes. This requires a proliferative advantage of the transplanted cells over host hepatocytes. Such proliferative advantage pre-exists in some inherited liver diseases, such as the FAH-deficient mouse model of hereditary tyrosinemia type 1  and MDR3 gene knockout mouse model of progressive intrahepatic cholestasis type 3 . In these cases, markedly reduced hepatocyte life span provides both space and stimulation for preferential proliferation of transplanted hepatocytes, leading to massive hepatic repopulation. In a transgenic mouse model of human alpha-1 antitrypsin disease (AAT-ZZ), transplanted normal hepatocytes were shown to spontaneously proliferate and replace the host hepatocytes . Similar to the majority of human AAT-ZZ patients, liver injury is minimal in these mice, but the stress of production of the misfolded protein (AAT-Z) is sufficient to provide a competitive proliferative advantage to the engrafted normal hepatocytes. (See "Clinical manifestations, diagnosis, and natural history of alpha-1 antitrypsin deficiency".)
However, many inherited liver disorders are not associated with significantly reduced lifespan of host hepatocytes. Two approaches are being explored to enable hepatic repopulation by the transplanted cells through cell competition: preparative manipulation of the host liver to cause mitotic inhibition of host hepatocytes, or increasing the mitotic capacity of donor hepatocytes.
To reduce the proliferative capacity of host hepatocytes, preparative X-irradiation has been applied to all major lobes of the liver , to a single lobe , or to a targeted volume of the liver by stereotactic body radiation therapy (SBRT). In one study that combined stimulation of hepatocyte mitosis (eg, partial hepatectomy) and preparative hepatic irradiation, the transplanted hepatocytes repopulated the irradiated host liver, thereby reducing serum bilirubin to normal levels in UGT1A1-deficient jaundiced Gunn rats  and normalizing urinary oxalate excretion in a mouse model of primary hyperoxaluria . Normal function and histologic architecture of the liver is retained after repopulation of the liver with the transplanted hepatocytes [46,62,63]. Hepatic irradiation targeting a portion of the liver by a non-invasive technique similar to SBRT has also been studied for the management of inherited liver diseases [57,58,64] (see 'Clinical experience' below). X-irradiation-based preclinical studies of hepatocyte transplantation indicate that extensive liver repopulation requires a combination of hepatic irradiation and a mitotic stimulant, such as hepatocyte growth factor or thyroid hormone receptor-beta agonists .
An alternative approach to enable extensive hepatic repopulation involves ex vivo gene transduction of the donor hepatocytes, which provides the engrafted cells with pharmacologically-regulated proliferative capacity .
●Attrition of transplanted hepatocytes through allograft rejection: Early and delayed allograft rejection is a critical factor that impairs the longevity and function of transplanted hepatocytes. The mechanism of hepatocyte allograft rejection is not fully understood, and methods for detecting organ rejection are not sufficient for monitoring for hepatocyte rejection. However, in one study, the emergence of donor-specific antibodies (DSA) were found to be temporally related to loss of hepatocyte allograft in a patient with phenylketonuria, suggesting that DSA could be an indicator of rejection .
Ex vivo gene therapy — Hepatocytes can be transduced in vitro and then transplanted [66,67]. In one report, primary hepatocytes from LDL-receptor deficient Watanabe heritable hyperlipidemic rabbits were harvested from a resected segment of the liver and transduced with a recombinant retrovirus expressing the LDL receptor gene before transplantation into the donor rabbit . Long-term expression of the transgene was observed, and the serum cholesterol levels were reduced by 20 to 30 percent during several months of observation. A clinical trial of this method in human subjects with familial hypercholesterolemia resulted in a modest reduction of serum LDL cholesterol levels, which was not considered to be therapeutically useful. In another study, hepatocytes isolated from a patient with ornithine transcarbamylase (OTC) deficiency were genetically corrected by CRISPR-mediated gene editing. Repopulation of the liver in immunodeficient FAH-knockout mice (FRGN strain) resulted in normal plasma ammonia levels, enhanced clearance of an ammonia challenge, hepatic OTC enzyme activity and reduced urinary orotic acid excretion. In contrast, FRGN mice undergoing liver repopulation with unedited OTC-deficient patient hepatocytes exhibited metabolic characteristics of OTC deficiency .
Metabolic disorders — As most enzymes are normally present in excess, partial replacement of the mutant hepatocyte mass with wildtype hepatocytes is expected to produce a therapeutically meaningful effect. Based on this concept, patients have undergone hepatocyte transplantation for a variety of liver-based monogenic disorders, including alpha-1 antitrypsin deficiency, Crigler-Najjar syndrome type 1, coagulation factor VII deficiency, glycogen storage disease types 1a and 1b, infantile Refsum's disease, phenylketonuria, progressive familial intrahepatic cholestasis, tyrosinemia type 1, primary hyperoxaluria type 1, and several types of urea cycle disorders (table 2). The results have been variable because of differences in the severity of the underlying diseases, the quality and quantity of transplanted hepatocytes, and immune rejection of the transplants. In most instances, the study design included orthotopic liver transplantation (OLT) or auxilliary liver transplantation after a few months to evaluate the metabolic effect of hepatocyte transplantation. Reports of hepatocyte transplantation for metabolic disorders have included:
●One child with the urea cycle disorder OTC received 10 billion hepatocytes. Although some clinical improvement was seen, she died of pneumonia a short time later.
●A newborn OTC-deficient child who received hepatocyte transplantation via the umbilical vein had normalization of plasma ammonia and glutamine levels on a normal diet without phenylbutyrate/phenylacetate therapy. However, following repeated transplantation from multiple donors, the cells appeared to be rejected, possibly because of inadequate immunosuppression .
●Transplantation of 240 million viable cryopreserved hepatocytes per kilogram over 16 weeks in a 14-month-old boy with OTC deficiency resulted in significant decrease in mean blood ammonia levels and increase in blood urea levels. This permitted eventual elective OLT, demonstrating the value of hepatocyte transplantation for bridging to OLT .
●One study reported significant reduction in blood ammonia concentration and improved urea production for up to 11 months in a 2.5-month-old child with carbamoyl phosphate synthetase I deficiency, after transplantation of 1.4 × 109 cryopreserved hepatocytes .
●In a patient with argininosuccinate lyase deficiency, who received multiple hepatocyte transplantation over a five-month period, sustained engraftment was demonstrated for up to 15 months, along with restitution of argininosuccinate metabolism to 3 percent of normal, permitting significant psychomotor catch-up .
●One 18-week-old child received hepatocyte transplantation for alpha-1 antitrypsin deficiency, but the patient had to be subsequently treated with orthotopic liver transplantation as the liver biopsy of the patient at the time of cell transplantation revealed cirrhosis .
●The long-term therapeutic benefit of hepatocyte transplantation was demonstrated unequivocally in a 10-year-old patient with Crigler-Najjar syndrome type 1 who received 7.5 billion hepatocytes via a percutaneous portal vein catheter . Duodenal bile samples collected at various time points up to two years showed the excretion of significant amounts of bilirubin monoglucuronide and diglucuronide, demonstrating function of the engrafted hepatocytes in vivo. Hepatic bilirubin-UDP-glucuronosyltransferase activity was also detected on liver biopsy specimens (5.5 percent of the normal compared with 0.4 percent before treatment). Serum bilirubin declined from a pretreatment average of 27 mg/dL to around 11 to 13 mg/dL, and the phototherapy could be reduced from 12 to 6 hours per day.
Analysis of the world-wide experience in hepatocyte transplantation for inherited metabolic liver diseases (table 2) led to the following conclusions:
●Hepatocyte transplantation exhibits beneficial metabolic effects in the absence of established cirrhosis of the liver.
●Although hepatocyte transplantation improves the metabolic state and the clinical condition in many liver-based inherited metabolic disorders, none of these disorders have been fully cured to date. Also, in the absence of preparative manipulations of the host liver, the engrafted hepatocytes do not tend to survive and function beyond a few months to a few years. Most patients have required subsequent liver transplantation for various reasons.
●Hepatocyte transplantation has been particularly useful as a "bridge" to subsequent liver transplantation in newborns or infants with life-threatening metabolic emergencies. For example, several infants with urea cycle disorders showed significant metabolic improvement, permitting them to grow until liver transplantation could be performed safely .
Two factors have limited the therapeutic benefit of hepatocyte transplantation. First, the concept of inducing proliferation of transplanted hepatocytes by limiting the mitotic capacity of the host liver cells have been in the early stages of application. Second, transplanted allogeneic hepatocytes were found to be more susceptible to immune rejection than are liver allografts . As an example, two patients with urea cycle deficiencies had early hepatocyte graft loss. Follow-up immunologic analysis suggested that additional immunosuppression was needed. Thereafter, a 27-year-old patient with classical phenylketonuria received preparative X-irradiation of the liver and anti-lymphocyte induction before hepatocyte transplantation, followed by frequent post-transplant immune monitoring. Liver biopsies demonstrated multiple small clusters of the transplanted cells, and Ki67+ hepatocytes. In this patient, hepatocyte transplantation resulted in reduction of mean peripheral blood phenylalanine concentration compared with pretransplant levels. It should be noted that although low-dose preparative irradiation was used in this study, the protocol did not include mitotic stimulation, which was found to be needed for maximum repopulation in preclinical studies.
Liver failure — While the effect of hepatocyte transplantation can be readily evaluated in the treatment of inherited metabolic disorders, the greatest need for hepatocyte transplantation is for acute, acute-on-chronic and chronic liver failure. In an early study, hepatocytes were injected into the spleen of several patients with chronic hepatitis and cirrhosis . Epidermal growth factor was injected into the splenic artery to stimulate hepatocyte proliferation. The transplanted hepatocytes were detected in 88 percent of patients 1 to 11 months after transplantation, although the metabolic benefit was not clear.
Another group injected 5 billion pooled human fetal (24- to 34-week gestation) hepatocytes into the peritoneal cavity of patients with fulminant hepatic failure (FHF) of less than two weeks in duration and grade III to IV encephalopathy . A greater proportion of patients who received cell transplantation survived compared with those who did not (48 versus 33 percent), but the difference was not statistically significant because of the small number of patients. Best results were obtained when the transplantation was performed during earlier stages of liver failure. In the survivors, plasma ammonia and serum bilirubin levels decreased in 48 hours after hepatocyte transplantation.
In the United States, hepatocyte transplantation has been performed in an effort to "bridge" patients awaiting OLT for FHF or chronic liver failure with acute decompensation (table 3 and table 4). Following transplantation by infusion into the splenic artery, 66 percent of patients with FHF survived long enough to receive OLT . In the patients with chronic liver failure with acute decompensation, only one of four survived long enough to receive OLT. In the transplant recipients, there was improvement in plasma ammonia levels, grade of encephalopathy, cerebral perfusion pressure, and prothrombin time, but the differences from the values observed in controls were not statistically significant. As most survivors had received OLT, it is difficult to be sure whether hepatocyte transplantation provided a significant clinical benefit. However, in one 37-year-old woman with hepatitis B virus-induced liver failure, there was sufficient recovery after hepatocyte transplantation to permit discharge from the hospital without OLT.
In another study, human hepatocytes encapsulated in alginate microbeads were injected into the peritoneal cavity of eight children with acute liver failure who were awaiting a suitable allogeneic donor liver for orthotopic liver transplantations  (table 4). Four of the eight children recovered without liver transplantation, while three children were successfully bridged to liver transplantation, and one child died. The microbeads retrieved during liver transplantation were free of host cell adherence and contained functional hepatocytes.
Ex vivo gene therapy — Ex vivo gene therapy has been performed in patients with familial hypercholesterolemia (LDL receptor deficiency). Hepatocytes were isolated by collagenase perfusion of resected liver segments from patients. The cells were transduced with LDL receptor gene in culture using recombinant Moloney murine leukemia retrovirus (MoMLV) vectors, and were transplanted back into the donors. Although persistence of a small number of transgene expression was demonstrated , the serum cholesterol reduction was modest and probably not therapeutically beneficial . In this study, the low efficiency of transduction of quiescent hepatocytes limited the therapeutic effect. In addition, no host preparation or post-transplant mitotic stimulation was used.
BARRIERS AND ADDITIONAL RESEARCH — As discussed above, one major barrier to extensive clinical application of hepatocyte transplantation is the shortage of donor organs for hepatocyte isolation. Hepatocytes are usually obtained from donor organs that have been rejected for transplantation because of extensive steatosis. Cells obtained from these livers are of variable viability and their survival after cryopreservation is unpredictable. The best quality hepatocytes are obtained from segmental transplants or reduced liver grafts, which have the shortest duration of cold-ischemia.
Limited data suggest that repeated cell transplantation from multiple donors may increase the risk of allograft rejection. Improving methods of cryopreservation of human hepatocytes may permit repeated transplantation of hepatocytes obtained from a single donor. Human hepatocytes transplanted in the liver of immunodeficient fumaryl acetoacetate hydrolase-(FAH) deficient mice  or Rag-1 knockout rats  have been shown to massively repopulate the liver . Human hepatocytes isolated from these livers express hepatocyte-specific genes and ameliorate hyperbilirubinemia after transplantation into the liver of Gunn rats (model of Crigler-Najjar syndrome type 1). With further development, human hepatocytes expanded in animal livers could be a potential source of hepatocyte-based therapies.
The extent and durability of the therapeutic benefit of hepatocyte transplantation for monogenic liver diseases depends on the extent of repopulation of the liver by the transplanted hepatocytes. In some disorders, such as the classic form of alpha1 antitrypsin (AAT) deficiency, accumulation of the mutant AAT-Z protein in hepatocytes causes a stress in the host liver that promotes spontaneous repopulation by transplanted normal hepatocytes via cell competition . However, in a majority of liver-based inherited metabolic disorders, the longevity and regenerative capacity of host hepatocytes remains normal. In these cases, an alternative solution could be to prepare the host liver to promote repopulation by a relatively small number of transplanted hepatocytes. Preclinical studies have demonstrated that preparative irradiation of the host liver [7,62,78] combined with post-transplantation treatment with mitotic stimulants result in sufficient liver repopulation to cure most monogenic liver diseases. However, this combination has not been tested in clinical trials.
Alternative sources of hepatocytes are also being explored (see 'Sources of hepatocytes' above). A noninvasive method to measure the mass of engrafted cells could greatly facilitate the follow-up and optimization of hepatocyte transplantation methods. Magnetic resonance imaging methods are being developed for three-dimensional quantification of transplanted hepatocytes using genetic marking with creatine kinase, which is not normally expressed in hepatocytes .
Hepatocyte transplantation would be much more useful and attractive if the need for immunosuppression could be circumvented. For this purpose, the immune response of the host to specific alloantigens could be abrogated by interference with costimulation between antigen-presenting cells and cytotoxic lymphocytes, at the time of hepatocyte transplantation, by the administration of CTLA4-Ig alone, or in combination with an anti-CD40 antibody. CTLA4-Ig could also be expressed in vivo by gene transfer for this purpose [80,81]. As an alternative, immunomodulatory genes could be transduced into the donor hepatocytes before transplantation . Some studies in rodents showed that ex vivo transduction with some viral genes can protect transplanted liver cells from allograft rejection . In addition, when allogeneic hepatocytes are used, developing methods for detecting early allograft rejection is important, so that measures to prevent graft loss could be initiated.
●Hepatocyte transplantation has many potential clinical applications including treatment of inherited metabolic diseases and management of acute, acute-on-chronic, and chronic liver failure. With the available methodology, the primary application of hepatocyte transplantation has been as a "bridge" to orthotopic liver transplantation. (See 'Scope of hepatocyte transplantation' above.)
●When hepatocytes are used as vehicles for ex vivo gene therapy, immunosuppression can be circumvented by isolating hepatocytes from resected liver segments and transplanting them back to the donor after genetic manipulations. However, in most cases, the cells are obtained from allogeneic donors and immunosuppression will usually be required. (See 'Sources of hepatocytes' above.)
●The liver provides the most supportive environment for engraftment and function of transplanted hepatocytes because of the availability of nutrients and growth factors present in the portal circulation, as well as interaction with other liver cells and the hepatic matrix. Hepatocytes infused into the portal vein or injected into the splenic pulp travel to the hepatic sinusoids and integrate into the liver cords. (See 'Sites of hepatocyte transplantation' above.)
●Shortage of donor organs for hepatocyte isolation is a major barrier to extensive clinical application of hepatocyte transplantation. Developing methods for host conditioning to promote liver repopulation and reduce the risk of allograft rejection would make hepatocyte transplantation more useful and attractive. (See 'Barriers and additional research' above.)
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