Update on Liver Cell Transplantation

Hepatocyte transplantation aims to correct inborn or acquired liver function defects by supplying metabolically active cells to the diseased liver. The procedure is emerging from experimental to clinical application, and trials are currently under way in a few centers. Experimentally, hepatocytes can be successfully isolated by collagenase digestion of the liver. Cells can be transplanted in the spleen or in the liver via the portal vein or the hepatic artery. In both sites, cells engraft and become metabolically active, can synthesize albumin, or can correct inborn errors of metabolism. They can prolong survival in models of fulminant liver failure. Hepatocytes from donor origin are detected after stem cell transplantation in humans. Fetal hepatocytes may yield higher engraftment and repopulation. Hepatocyte transplantation has so far been shown to bring partial metabolic control in Crigler-Najjar disease, type I glycogenosis, and Refsum disease. At least the technique can be a bridge to safe transplantation for the high-risk patient. Efforts should concentrate on organizing human trials after identifying the best candidate diseases, cell isolation and cryopreservation procedures, and immunosuppression. Hepatocyte transplantation is a promising new approach for the treatment of liver-based inborn errors of metabolism, or secondary liver function impairment, as is seen in fulminant liver failure or end stage liver disease. Orthotopic liver transplantation (OLT) has been considered to be the ultimate validated treatment for restoring single or global metabolic dysfunction and usually is proposed when conservative treatment cannot maintain vital function or sufficient quality of life. Survival rate in children after OLT now exceeds 90% and has been constantly improving during the last 20 years (1). Waiting list mortality caused by long waiting times and organ shortage has prompted the development of living related liver transplantation. Isolated hepatocytes or liver cell transplantation (LCT) is attempted as an alternative to the transplantation of the whole liver. The procedure is less radical, less invasive, potentially less expensive, and fully reversible. It may, to a certain extent, alleviate the problem of organ shortage. LCT was validated in rodents, in whom transplanted syngeneic hepatocytes have been shown to survive indefinitely. LCT allows the correction of various inherited liver enzyme defects and enhances recipient survival in experimentally induced acute liver failure models. After these experimental successes, LCT has generated increasing interest for the treatment of human diseases and recently has been attempted in adults and children with hepatic failure or inborn errors of liver metabolism. The purpose of this review is to compile recent data in the field of LCT and to highlight some aspects of its current limits. PHYSIOLOGY OF HEPATOCYTE TRANSPLANTATION Injection Site Orthotopic injection site Because the liver is supposed to be the optimal site for transplanting isolated hepatic cells, hepatocyte suspensions are infused directly via the portal vein or spleen. The success of such procedures is limited by the number of transplanted hepatocytes that can be infused and by the portal hemodynamics. Intrasplenic transplantation. In rodents, intrasplenic transplantation of liver cells can recompose liver tissue in the spleen that has similar structure and function as the liver, suggesting that the spleen may contain appropriate factors and environment to maintain viability and functionality of transplanted hepatocytes. Such a model of LCT has remained the most suitable and efficient procedure in animals. Hepatocytes transplanted into the spleen reach the liver via the splenic vein and restore missing enzyme activities (2). In Gunn rats, intrasplenic hepatocyte transplantation is effective in reducing plasma bilirubin (3). In anhepatic rats, intrasplenic transplantation of allogeneic hepatocytes prolongs survival, improves blood chemistry, and lowers blood TGF-beta1 levels (4). In Dalmatian dogs, no difference was observed in the engraftment efficiency when using fresh or cryopreserved hepatocytes (5). In pigs, significant complications, high morbidity, and mortality can be observed if the number of isolated hepatocytes transplanted exceeds 2% of the original liver mass (6). In Wistar Furth rats, proliferation of transplanted hepatocytes occurs in the spleen, indicating that this LCT procedure also may be used as a therapeutic application in the treatment of chronic liver disease (7). Intraportal transplantation. In the late 1970s, intraportal hepatocyte transplantation was shown to correct hyperbilirubinemia in glucuronyltransferase-deficient Gunn rats (8,9). In dogs, portal hypertension represents a limiting factor for the number of hepatocytes that can be used for a single transplantation. Thus, repetitive transplantations are proposed to correct metabolic alterations (10), and this can be achieved via an indwelling catheter. Nagase analbuminemic rats subjected to repeated cell infusions acquire high plasma albumin levels and normal liver histologic findings. Repeated infusions will allow successful engraftment of a large number of isolated hepatocytes (11). After LCT in the low density lipoprotein (LDL)-receptor deficient Watanabe rabbit, a dose-dependent reduction of serum LDL is observed that lasts for 2 to 3 weeks (12). Recently, Muraca et al. (13) have explored the effects of intraportal hepatocyte transplantation on portal, pulmonary, and systemic hemodynamics in pigs and concluded that as much as 2.4% of the total hepatocyte mass can be infused in pig model but with a risk of significant thrombotic complications. Transplantation via hepatic artery. Direct hepatocyte transfer via the hepatic artery in the cirrhotic rat model improves liver function, increases the degradation of liver collagen, and reverses liver cirrhosis (14). In Long-Evans Cinnamon rats, an animal model of Wilson disease, LCT by the arterial route leads to a reduction in hepatic copper deposits (15). The phenotype of donor hepatocytes depends on the surrounding environment, and transplanted hepatocytes originally expressing high levels of glutathione S-transferase placental form (GST-P) and low levels of cytochrome P-450, turn GST-P negative within 5 to 10 days, and express cytochrome P-450 at a level comparable to that in surrounding host hepatocytes after 2 days (16). Ectopic injection sites Intraperitoneal transplantation The intraperitoneal route exposes hepatocytes to cell loss before they reach the host liver. Techniques aiming to protect hepatocytes against the host immune response include encapsulation or microencapsulation of cells within synthetic semipermeable membranes (17–19). Encapsulated hepatocyte spheroids showed better metabolic activity after intraperitoneal transplantation than did isolated hepatocytes and improved survival rates in 90% of hepatectomized rats (20). However, the survival of human encapsulated hepatocytes remains limited after their intraperitoneal transplantation in rats (21). Hepatocyte Suspension Liver cells are obtained from livers using the technique of two-step collagenase perfusion (22), modified by Seglen (23). Cells can be infused to animals or patients immediately after isolation, being kept in a cold solution (solution from the University of Wisconsin). At this stage LCT is not a sufficiently validated technique to use a conventional donor from the current organ allocation systems. Thus, in humans, the availability of freshly isolated cells does not match with simultaneous recipient presentation, especially in the case of fulminant liver failure. For these reasons, the challenge is to maintain isolated hepatocytes viable and functional until a recipient candidate presents and vice versa. Primary culture of hepatocytes Hepatocytes isolated from liver can be maintained in culture ex vivo for several days and have the ability to organize as pseudo liver tissue (24). Animal hepatocyte culture systems also have been shown to supply biologically active substances crucial for the regeneration or the repair of the damaged liver. In addition, significant progress in cell culture technology allows the assembly of these cells as extracorporeal bioartificial liver support systems, to be used as temporary support devices for patients with acute liver failure. Primary cultures of human hepatocytes transplanted in mice after 48 hours culture in vitro did not alter the isolated cell functionality or the expression of liver specific proteins (25). Immortalized hepatocytes Xenogenic primary cells, human hepatoma cells, immortalized hepatocytes, and stem cells have been used in isolated cell transplantation. However, xenogenic hepatocytes may transmit infectious pathogens and produce immunologic and physiologic incompatibilities between animals and humans. Human hepatocyte cell lines may become widely available and can be obtained using currently available cell cultures and gene transfer technology (26). A clonal hepatocyte cell line that could be grown economically in vitro and that exhibits a differentiated, nontransformed phenotype after transplantation would be an attractive solution, provided that such cell lines divide indefinitely while maintaining characteristics of differentiated hepatocytes. Different oncogenic, viral, or chemical agents can induce cell immortalization. Hepatocyte immortalization using the Simian virus SV40 DNA does not alter the ability of these cells to express a number of acute-phase genes and to respond to external stimuli (27–31) while drug-metabolizing enzyme activities also are maintained (32). Immortalized cells promote in co-culture survival of both fresh fetal human hepatocytes and adult mouse hepatocytes (33). In rats, intrasplenic transplantation of immortalized hepatocytes protect against CCl4-induced acute liver injury (34). The unlimited growth of hepatocyte cell lines in vivo raises the possibility of a subsequent risk of malignant tumor growth in transplant recipients. To develop methods to prevent this undesirable possibility, primary Lewis rat hepatocytes were conditionally immortalized with a thermolabile mutant SV40 large T antigen. Such cells proliferate in culture at 33°C and stop growing at 37°C to 39°C thus preventing excessive proliferation after transplantation. Intrasplenic transplantation of these hepatocytes in portacaval-shunted rats protects recipients from hyperammonemia-induced hepatic encephalopathy (35). The same strategy has been shown to improve survival in rats subjected to 90% hepatectomy (36). Transfected cells expressing herpes simplex virus thymidine kinase sequences can subsequently be inhibited by ganciclovir (37). Immortalized primary hepatocytes of Gunn rats, transduced with a recombinant retrovirus expressing human bilirubin-UDP-glucuronosyl-transferase, were injected into the spleens of syngeneic Gunn rats and led to decreasing serum bilirubin levels as long as 4 months after cell transfer (38). These studies and others concerning liver expressed proteins (39) confirm the possible use of immortalized cell lines for transplantation, at least in animals (40). Human clonal immortalized cell lines obtained from human fetal hepatocytes transplanted in rats subjected to a 90% hepatectomy protect animals from hyperammonemia and the associated hepatic encephalopathy (41). Other immortalized human cell lines have been shown to provide metabolic support during acute liver failure in rats (41). Cryopreserved hepatocytes Cryopreservation of freshly isolated hepatocytes is required for the long-term storage of liver cells. The use of cryopreserved hepatocytes for transplantation was first documented in rat model in the early 1980s (42). Only small numbers of cryopreserved hepatic cells are detected in the host liver after transplantation (43). Good quality cryopreserved hepatocytes are crucially dependent on the components of the medium in which they are frozen, including the dimethylsulfoxide (DMSO) concentration needed to avoid osmotic shock and prevent formation of ice crystals within cells. Studies suggest that freshly isolated and cryopreserved transplanted hepatocytes have similar behaviors after their intrasplenic transplantation (44) and that long-term cryopreservation has no effect on cell growth and the hepatic-differentiated activity of small hepatocytes (45). Long-term preservation does not alter the ability of the cryopreserved hepatic cells to repopulate the damaged liver (5,46,47). Moreover, in a recent study, no major differences were observed in many hepatic-specific parameters when hepatocytes were immediately cryopreserved or after short-term culture (48). However, other studies have failed to demonstrate the efficacy of cryopreserved hepatocytes to engraft and to produce albumin (49). Survival of transplanted cryopreserved hepatocytes is inversely correlated with immunogenicity, as demonstrated in mice (50). Apoptosis may contribute to cell death in cryopreserved hepatocytes (51), an effect that seems dependent on caspase-3 protease activation (52,53). Genetically engineered hepatocytes Isolated hepatocyte transduction is an alternative approach to in vivo gene transfer. This strategy aims to increase transgene expression per hepatocyte to efficiently repopulate host liver. Hepatocytes were first transduced using retroviral vectors. In dogs, transduced hepatocytes reach the liver and survive in vivo (54). Transplanted hepatocytes can be subject to apoptosis induced by host cytotoxic T lymphocytes, which limit the transplantation efficiency. For this reason, hepatocytes were retrovirally transduced to express Bcl-2, an antiapoptotic gene. The transduced hepatic cells are resistant to Fas-induced apoptosis, suggesting a plausible of cell and gene in the liver hepatocytes also have been used for transplantation in mice and showed a comparable of of the liver as that seen when using hepatocytes the use of the gene was into rat hepatocytes, and the cytotoxic effect was both in vivo and in This in an increase in hepatocyte survival via an of caspase-3 activation The low efficiency of gene transfer to indefinitely a therapeutic gene in a large number of hepatocytes is a major limiting for modified hepatocyte transplantation. The long-term of in vivo is limiting Liver stem cells development in stem cell has prompted in hepatic stem cells for transplantation for several The use of hepatic stem cells in liver transplantation of their use would the for immunosuppression. Moreover, cells that can be from the blood have been proposed to be a plausible candidate for some genes needed to liver failure or disease of their ability to the liver is considered to be an of hepatic cells, suggesting its in normal cell liver Such which have been documented both in and have new therapeutic some have liver after transplantation others have that of stem cells into hepatocytes is in vivo the of of cells to the Recently, et al. have using a model of type that transplanted hepatocytes from from cell between donor and host cells and not by of stem cells. Such data were by et al. in the same liver disease recent data to the use of stem cells in transplantation, especially if many concerning the of hepatic stem cells have to be before the therapeutic of stem cells. Fetal hepatic cells Fetal liver cells have a high are less are resistant to injury during cryopreservation and resistant to These could their their proliferation increases gene transfer efficiency. These cells can be isolated with a collagenase to et al. In fetal hepatocytes have a high activity with adult hepatocytes In Gunn rats, transplanted fetal hepatocytes serum bilirubin levels for to weeks after transplantation transplanted fetal hepatocytes maintain their metabolic activity for after transplantation In Nagase analbuminemic rats, fetal hepatocytes used for intraportal or intrasplenic transplantation increase albumin levels In the 90% hepatectomized rats, human fetal hepatocytes transplanted improve survival to weeks after transplantation, gene expression that liver cells an adult phenotype Fetal hepatocytes are and have an active DNA a crucial required for gene transfer and for a high of and expression in some liver Fetal can as efficient for ex vivo gene have been used to fetal human hepatocytes activity in human fetal hepatocytes has led to immortalization of the cells with no in their of transplantation efficiency was first in models in which animals were subjected to partial or total A animal model is the 90% hepatectomy Liver diseases were also by using such as or a review and in have prompted studies on small or animal models but no large animal or models are the number of animal models and of hepatocytes, are far from a in humans, especially in long-term The most experimental models that have been used for LCT are in Animal models of metabolic liver diseases used for isolated hepatocyte OF LCT LCT has recently from an animal experimental procedure to its first clinical with the of patients with fulminant liver end stage or inborn errors of liver metabolism. The first in have demonstrated at least that the procedure is and The procedure a but is by far less than After the is LCT can be in a is by and which is by to the the of a into the portal This can be or by For single procedures, the can be but if repeated infusions are considered for the treatment of inborn errors of liver of an is have used the a A directly into a of the Such can be in for several weeks or to infusions when cells become In the intraportal can be directly into the portal by of the the and as long as the remains in the portal are to portal can be provided that the of the is in a large in a small portal may not be new the of the is if by into the and their the portal vein and the liver, using the of Human Hepatocytes LCT currently is not a validated clinical treatment procedure and can not with the procedure of OLT as far as organ is of supply remains a major problem in LCT and with and organ allocation systems is The liver in which the whole organ is to allow its transplantation in a a large of as many as cells, in The technique was widely used at the of the liver transplant to provide a solution to the problem of organ for small children However, this technique is now widely by the liver transplantation to an adult recipient with the of the liver and to a with the no large for hepatocyte However, may to provide the usually during the Such a may provide as many as hepatocytes, sufficient for cell transplant into a or for cell in a 10 can be for the specific purpose of hepatocyte Other of hepatocytes include damaged livers for whole organ for OLT of the quality of the donor may be for cell isolation, but this may not be the same quality should for LCT donor and recipients as for The viability of cells isolated from livers for OLT cells with with a of with a of when cells were isolated from For from did not between cell viability and early function donor liver transplantation is an alternative of for OLT but cannot be considered for LCT as long as the procedure has not been For this and other reasons, of active in LCT is to and the of Fetal hepatocytes an alternative of cells for However, are obtained from and may prevent the use of this for therapeutic stem cells from blood or are a of hepatocytes that may the of hepatocyte for After blood stem cells or transplantation, as many as of hepatocytes are of donor and these cells may for as long as after transplantation Human blood cells transplanted into mice also have been shown to into hepatocytes with the of human DNA and albumin and are active in tissue from adult or stem cells, and therapeutic and this increases the that new of hepatocytes will be than in are active in the in to the and liver transplant LCT for Liver hepatic failure has a with a currently from the on the transplant waiting list but the waiting can last several LCT in patients with aims to bring temporary functional the to for a suitable organ for OLT and to of the hepatocytes or from the transplanted hepatocytes. In a from fetal hepatocytes were infused in was an in survival in transplanted patients as recipients with encephalopathy with in the control of fetal hepatocyte transplantation was observed in patients with liver failure and encephalopathy In other studies using the splenic artery route of transplantation has been a survival of patients to the of cannot be that LCT improved survival, these patients needed was seen in several including blood and blood suggesting that the procedure been and may have the bridge to transplantation by the in a better metabolic of a liver cells also has been by after LCT in and LCT may have the of bridge to in this In patients with to transplantation with of LCT for LCT has also been attempted in patients with end stage with of encephalopathy and in of patients LCT may prolong survival until OLT in these LCT remains in this is that LCT a in of However, in diseases with a of the cells, such as Wilson disease or is not that LCT may cirrhosis to to the in animal LCT for of Liver animal models have shown the of LCT to repopulate the liver and restore the in metabolic This is best achieved in models with cell impairment, for the infused liver cells Animal In humans, inborn errors of liver may to cirrhosis In these OLT is the best Other diseases may the quality of liver disease. For this is the case with Crigler-Najjar disease, type I storage disease, and other liver-based metabolic diseases that that is of these metabolic patients are in such that major is may to or metabolic In for patients may have to chronic failure with a In these LCT may the to sufficient to alleviate the disease if to the to OLT have shown a and significant metabolic after LCT in humans. et al. a case of intraportal LCT in a with Crigler-Najjar The hepatocytes and bilirubin levels to by between and to be et al. also demonstrated the of bilirubin in while the activity of liver from to of normal on the after In such disease, the may be less and infusions may increase the of the first The procedure was considered than LCT was attempted in a with The demonstrated metabolic activity with a reduction of and However, the of days and of the transplanted cells et al. showed metabolic control in a with after LCT via the Muraca et al. LCT in a with type I storage disease. 2 cells, to of hepatocyte after which significant in to while improved has liver cell transplantation in The first was in a with Refsum disease, a The 2 cells, of After observed of serum total also to also significant reduction of and The with and was maintained for months after LCT Moreover, the of donor hepatocyte DNA in a recipient liver was demonstrated by of the A from the Crigler-Najjar and a total of cells, a in bilirubin from to The effect for and the was for OLT with The and considered with of hyperammonemia and Because no living donor was the was for organ transplantation and infusions of cells, of while the to with a of encephalopathy and blood clinical and improved a with significant and in other patients have the have not been In have clinical for LCT as treatment inborn errors of and Liver cell transplantation is a promising technique and is its clinical application support of liver function is and short-term can be achieved for The challenge is to long-term efficacy and demonstrate engraftment and of the recipient liver. animal studies have been are many that to be including increasing engraftment and in vitro tissue and alternative of cells such as stem cells, fetal hepatocytes, and allocation should to and liver in to increase the number of patients potentially from this