Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inherited liver diseases and liver failure. It is a relatively less complicated surgical procedure, and has the advantage that it can be repeated several times if unsuccessful. Another advantage is that hepatocytes can be isolated from partly damaged livers which are not suitable for liver transplantation. Despite these advantages hepatocyte transplantation is less popular. Important issues are poor engraftment of the transplanted cells and the scarcity of donor hepatocytes. Generation of "hepatocyte like cells"/iHeps from embryonic stem cells (ES) and induced pluripotent stem cells (iPSCs) by directed differentiation is an emerging solution to the latter issue. Direct conversation or trans-differentiation of fibroblasts to "hepatocyte like cells" is another way which is, being explored. However this method has several inherent and technical disadvantages compared to the directed differentiation from ES or iPSC. There are several methods claiming to be "highly efficient" for generating "highly functional" "hepatocyte like cells". Currently different groups are working independently and coming up with differentiation protocols and each group claiming an advantage for their protocol. Directed differentiation protocols need to be designed, compared, analyzed and tweaked systematically and logically than empirically. There is a need for a well-coordinated global initiative comparable to the Human Genome Project to achieve this goal in the near future.

There is an urgent need to search for alternatives to whole organ transplantation. Several methods have been proposed. Among these strategies, cell transplantation is currently one of the most promising. To achieve this aim, in addition to highly differentiated adult hepatocytes, the use of stem cells is considered a highly attractive therapeutic method for the treatment of liver disease and for temporary support of hepatic function until a liver becomes available for organ transplantation. This strategy is based on the ability of stem cells to differentiate into different cellular types according to their environment. Therefore, stem cells could be an unlimited source of hepatic cells for transplantation and gene therapy. Bone marrow is considered the most promising source of adult stem cells, partly due to the versatility of the cells obtained in repairing damaged tissues of several lineages. Several different types of stem cells have been described in bone marrow: hematopoietic, mesenchymal, side population and multipotent adult stem cells. Bone marrow cells have been hypothesized as a third recruitment source in liver regeneration in addition to hepatocytes and endogenous liver stem cells. Consequently, attempts have been made to differentiate them into hepatic lineage for their subsequent use in hepatic cell therapy. The present article reviews the progress made in this field or research.

Isolated hepatocyte transplantation has long been recognized as a potential treatment for life-threatening liver disease. The basis for proceeding with clinical trials has been established by the extensive laboratory work in animal models. Human hepatocyte transplantation has been applied in individual cases and very small, uncontrolled series. Data, although sparse, demonstrate the safety and feasibility of this approach and are supportive, if less than conclusive, of effectiveness. The experience of hepatocyte transplantation in the laboratory and clinical arenas is reviewed and discussion will examine what is believed to be the primary cause for the slow growth of this technology in the clinical setting, namely a severe shortage of usable primary human hepatocytes. The potential of isolated hepatocyte transplantation remains largely untapped and awaits alternate sources of cells for transplantation other than those from discarded human cadaveric livers.

Liver transplantation for inborn errors of metabolism has proved effective in some (mostly liver-associated) inborn errors of metabolism. Significant morbidity and mortality rates have been extensively reported due to disease recurrence or to complications of the immunosuppressive regimen. On the basis of clinical trials in animals as well as in humans, the use of isolated hepatocytes offers a unique opportunity for treating inborn errors of metabolism. The state of art of the technique applied to this field is reviewed here and related practical problems are examined. No final conclusions can be drawn, but further insight into the use of alternative sources of cells, including stem/progenitor cells associated with cryobiology and immunological research, will offer new opportunities for cell therapy for inborn errors of metabolism in the near future.

Hepatocyte cultures have been used extensively for a wide variety of physiological, pharmacological and experimental studies. The warm ischaemic period before isolation is kept to a minimum to achieve a high yield of cells isolated and a good viability for culture. We have recently introduced a new concept of liver resuscitation after warm ischaemia that is based on a 3-h reperfusion period with an improved perfusate and simultaneous dialysis. In this study, we applied the new technique for hepatocyte isolation from livers subjected to 80 min of complete ischaemia at 37 degrees C. Cell yield was improved by a resuscitating perfusion from 58% to 73% and viability from 39% to 76%.

The aim of this experimental study was to modify a rat liver-cell harvesting technique and to evaluate the efficacy of allogeneic liver cell transplantation (Tx) using cyclosporin A immunosuppression in rats with N-dimethylonitrosamine (N-DMNA)-induced acute liver failure (ALF). Twenty male Wistar rats, weighing 190-320 g, were used as donors. Hepatocytes were harvested by the use of a modification of the Seglen portal vein collagenase perfusion technique (type V/1.3 mg/ml), which resulted in the isolation of a mean of 8000 viable clusters of hepatocytes per donor (viability was measured using the trypan blue exclusion test). The male Lewis recipients and controls received 20 mg/kg N-DMNA i.v., and were then divided in three groups. Group 1 (n = 5) received no treatment, group 2 (n = 10) received 5000 clusters of freshly isolated hepatocytes (FIH) in the spleen 24 h after the administration of N-DMNA- and group 3 (n = 10) received 5000 clusters of FIH beneath the renal capsule, 24 h after the administration of N-DMNA. All groups were treated with cyclosporin A 20 mg/kg per day i.p.. SGOT and bilirubin values were measured and all surviving rats were sacrificed on day 14. All rats in group 1 died of histologically confirmed liver necrosis within 72 h. The 14-day survival was 60% in group 2 and 50% in group 3. The post-Tx SGOT values reached their maximum on days 3-4 (group 2, mean 754; group 3, mean 529) and were only slightly elevated on day 14 (group 2 = 75, group 3 = 48). The post-Tx bilirubin values reached their maximum on days 3-5 (group 2 = 1.1, group 3 = 1) but failed to return to normal until day 14. Autopsy and histological examination of the surviving animals showed well-preserved hepatocellular spherical aggregates in the spleen and hepatocellular "cords" in the kidney accompanied by signs of regeneration of the native liver. We concluded that the hepatocyte Tx in a rat experimental allo-Tx model improved the survival rate and the SGOT values in cases of toxic ALF. Survival rates between the two different sites of Tx were similar.

Experimental hepatocyte transplantation (Tx) has been shown to improve the survival rate of acute hepatic failure (AHF) in different models. Histological and biochemical data from some studies suggest more satisfactory function of hepatocytes after combined hepatocyte-islet Tx. The aim of the present study was to compare the survival rate between two different sites of Tx (kidney subcapsular and spleen) of hepatocytes alone or combined with islets of Langerhans in rats with surgically induced AHF (90% hepatectomy) accross a major histocompatibility barrier (WAG to Lewis). Rats were divided into five groups (n = 6 in each group). Group 1 consisted of AHF without treatment, group 2, AHF followed by hepatocyte Tx into the spleen, group 3, AHF followed by hepatocyte Tx subcapsular into the kidney, group 4, AHF followed by combined hepatocyte islet Tx into the spleen, and group 5, AHF followed by combined hepatocyte islet Tx subcapsular into the kidney. The number of hepatocytes was 10(7) and the number of islets was 400. All rats received cyclosporin A (CsA) i.v. (20 mg/kg on days 0-4 and 10 mg/kg on days 5-30). Hepatocytes were harvested using a modification of the portal vein collagenase perfusion (type V 1.3 mg/ml) and islets, with the collagenase digestive technique (type XI 1 mg/ml). All Tx took place 24 h after AHF. All rats in group 1 died within 48 h. In groups 2 and 3, the combined survival rate was 33% 1 month after Tx, while in groups 4 and 5, the combined survival rate was 50% at 1 month. All surviving animals were sacrificed and histological examination showed well-preserved hepatocellular aggregates in the spleen and beneath the renal capsule, as well as islets around the clusters of hepatocytes. SGOT and SGPT values were also measured. We concluded that the combined Tx in a rat experimental allo-Tx model in cases of AHF improves the survival rate in comparison with hepatocyte Tx alone. The survival rate at the two different sites for combined Tx was similar.

Hepatocyte transplantation is a potential therapeutic modality for overcoming the shortage of liver donors, and the clinical application of allogeneic hepatocyte transplantation has been considered. However, there are two major problems with allogeneic hepatocyte transplantation: protection of transplanted hepatocytes from rejection and stimulation of the rapid proliferation of surviving cells. Without immunosuppression, allogeneic hepatocytes are rapidly rejected within a few days after transplantation, even though it is relatively easy to induce immunotolerance after allogeneic whole liver transplantation. Accordingly, different rejection mechanisms seem to operate after allogeneic hepatocyte transplantation and whole liver transplantation. To overcome the rejection of transplanted hepatocytes, induction of donor-specific unresponsiveness to graft without compromising the host immune system would be ideal. We previously reported that the Fas-Fas ligand system plays a critical role in the CD28-independent pathway of hepatocyte rejection. Therefore, blockade of rejection using CTLA4 immunoglobulin (CTLA4Ig) or anti-CD80/86 monoclonal antibodies and anti-FasL monoclonal antibody may prolong the survival of transplanted allogeneic hepatocytes. Furthermore, administration of hepatocyte growth factor (HGF) can promote the proliferation of allogeneic hepatocytes and this may lead to the development of a functioning liver substitute.

Liver replacement provides an effective method of replacing a failing liver, and corrects the underlying defect in many metabolic conditions. Results of liver transplantation for metabolic diseases have been encouraging, with the exception of hereditary hemochromatosis, in which infectious and for which cardiac complications appear to increase posttransplant mortality. An improved understanding of the underlying genetic and molecular defect will lead to advances in medical therapy and perhaps will decrease the need for liver replacement. The prospects of gene therapy are being pursued for many metabolic disorders, however until this research leads to direct clinical application, liver transplantation remains the only effective option for many patients with metabolic liver disease.

The aims of this study were (1) to assess portal hemodynamics during intraportal hepatocyte transplantation (HTX) in dogs, (2) to evaluate a new method for the detection of transplanted hepatocytes using 5-bromo-2'-deoxyuridine (BrdU) incorporation, and (3) to determine the metabolic effects of HTX on an inborn error of the purine metabolism in dalmatian dogs. HTX was performed by intraportal infusion of freshly isolated allogeneic beagle hepatocytes. Portal flow and pressure were monitored continuously during HTX. For the detection experiments, beagles received hepatocytes that had been exposed to BrdU during regeneration of the donor liver, induced by partial hepatectomy. For metabolic studies, dalmatian dogs were used as recipients. Repetitive HTX was performed. As judged by the portal hemodynamics, the number of hepatocytes that could be infused safely varied from 5 x 1O(8) to 8 x 1O(8) in beagles, to 1 x 10(9) in dalmatians. Transaminase levels showed a 5- to 6-fold increase (P=0.05) after HTX, but normalized within 3 weeks. BrdU-positive cells were identified in the recipient livers 2 weeks after HTX and 5-10% of the total amount of transplanted hepatocytes was retrieved. A significant (P=0.05) decrease in serum uric acid was demonstrated after repeated HTX in dalmatians. In conclusion, (1) intraportal HTX is feasible, but portal hypertension limits the maximum amount of hepatocytes that can be infused in one HTX; (2) BrdU labeling is an attractive method for the detection of transplanted hepatocytes in the recipient liver; and (3) after two consecutive hepatocyte transplantations, a temporary correction of the purine metabolism was accomplished in the dalmatian dog.

The first clinical trial in human gene therapy began in 1989 with the successful introduction of marker genes into peripheral blood cells as tumor-infiltrating lymphocytes (TIL) in order to investigate the biological behavior of manipulated cells in humans. In further studies, it was possible to ameliorate clinical genetic diseases based on only one single genetic defect such as adenosine deaminase deficiency (ADA) by repeated infusion of manipulated peripheral blood cells. Meanwhile, a multitude of clinical gene transfer studies were initiated. Three main strategies have thus far been applied in human cancer gene therapy: (1) Reinforcement of the body's immune response by gene transfer into immunological cells; (2) reinforcement of the immune response by manipulating tumor cells; and (3) transfer of drug-sensitive genes into tumor cells with subsequent drug treatment. The first clinical trial in gene therapy for gastrointestinal diseases was performed in 1992 with the introduction of the low-density protein receptor gene (LDL) into liver tissue. Human cancer gene therapy of gastrointestinal diseases is still only in the initial phase of research.

The small bowel mesentery provides a unique structure of a large vascularized surface area to support hepatocyte transplantation. Cell-seeded polymeric matrices can be juxtaposed in a relatively atraumatic manner between leaves of mesentery such that adequate exchange of nutrients and diffusion of gases can proceed in the interim while neovascularization occurs. Hepatocytes obtained from (RHA) Wistar rats by collagenase perfusion were seeded onto non-woven filamentous sheets of polyglycolic acid 1 x 3 cm in size and 2 mm thickness to a density of 500,000 cells/cm2. Twenty-six recipient Gunn rats (UDP-glucuronyl transferase deficient) underwent laparotomy. Hepatocyte-ladened polymer sheets were placed between leaves of mesentery. Eight sheets were placed per animal and the leaves were approximated, creating a functional implant 1 x 3 x 2 cm. Biopsies between 5-99 days after implantation revealed neovascularization, moderate inflammatory reaction and the presence of viable hepatocytes in 96% (25/26). Immunoperoxidase studies using anti-albumin antibody substantiated hepatocyte specific function in implants. HPLC profiles of bile from Gunn rats transplanted with hepatocytes from congeneic (RHA) rats demonstrated the presence of bilirubin conjugates. There were no conjugation fractions seen in control gunn rats without hepatocyte transplantation. Although total serum bilirubin did not significantly decrease, conjugated bilirubin was identified in 46% (12/26) animals after transplantation with congeneic hepatocytes. We conclude that the mesentery of the small bowel provides a large vascularized surface for cell transplantation. Large numbers of metabolically active hepatocytes can engraft, vascularize, and show function. The mesentery may be a potential bed for clinical hepatocyte transplantation.

A discussion of the treatment of liver insufficiency with extracorporeal (hybrid) liver assist devices (LADs) should address a definition of the types of liver failure susceptible to being treated by these devices as well as the modalities of in vivo and in vitro testing. Relevant to the first subject is the subject of pathogenesis of hepatic coma, which should be the target for the design of these LADs. Although this modality of therapy is new, it can be predicted that these devices will demand minimal safety conditions, i.e., the seeding with cells that are not tumorigenic or carrying viral particles. Among other topics to be considered in the development of LADs is the proper choice of hollow fiber to be used and the testing on proper animal models of hepatic failure. It is our philosophy that the long-term culture of adult mammalian hepatocytes in hollow fibers is the basis for appropriate designs of this type of temporary liver support.

In this report the history and clinical results of heterotopic liver transplantation (HLT) are reviewed and some special aspects of current research on HLT are high-lighted. The first laboratory experiments on liver transplantation were performed with auxiliary heterotopic grafts. The initial clinical results of HLT, however, were disappointing and orthotopic liver transplantation (OLT) evolved to be the procedure of choice. Of all the patients who received a heterotopic graft before 1980, only two survived. Since 1980, 50 HLTs are known to have been performed on 48 patients. Results of HLTs after 1986 are clearly better than earlier ones, and survival rates come within the range of those reported for OLT. Intraoperative fibrinolysis is found in the anhepatic phase of OLT, something which is absent in HLT. Tissue-type plasminogen activator (t-PA) is said to be responsible for this phenomenon, as well as for the postreperfusion hyperfibrinolysis. Parallel to the hemostatic changes, the intraoperative hemodynamic stability may be impaired by deleterious substances that arise during liver transplantation. Furthermore, the interaction between the two livers, the effect of HLT on portal pressure and hypersplenism, and the possible role of HLT in inborn errors of hepatic metabolism are described. Special attention is given to the treatment of acute hepatic failure. OLT, in an early phase of the disease, negates the possibility of spontaneous recovery, while delay of the decision to transplant may lead to further deterioration of the patient's clinical condition. As the procedure of HLT is reversible, the decision to transplant can be made more quickly. The clinical experience with HLT for acute liver failure is reported in detail.

The minimal hepatic mass necessary to reverse the metabolic defect of unconjugated hyperbilirubinemia in the rat model of Crigler-Najjar type I deficiency was determined using heterotopic (auxiliary) partial liver transplantation (HLT) and orthotopic liver transplantation (OLT). In HLT, the donor graft consisted of the right upper and/or right lower hepatic lobe(s) depending on the final mass of liver tissue desired for transplantation. The mass of the donor graft ranged from 12% to 23% of the whole organ (n = 12). The serum unconjugated bilirubin levels decreased quickly after HLT from a preoperative value of 8.98 +/- 0.34 mg/dL to 0.63 +/- 0.11 mg/dL in 24 hours, which was similar to OLT in which the levels decreased from a preoperative value of 8.20 +/- 0.44 mg/dL to 0.24 +/- 0.07 mg/dL in 24 hours. Conjugated bilirubin was excreted from the graft liver shortly after OLT and also from both the host and graft livers after HLT. This study demonstrates that using as little as 12% of the whole liver mass in HLT reduces serum bilirubin significantly in 24 hours in a fashion similar to whole-organ OLT. The clinical application of alternative therapies to whole-organ OLT such as HLT or hepatocyte transplantation may provide sufficient replacement therapy in metabolic disease.

One approach to gene therapy for hepatic diseases is to remove hepatocytes from an affected individual, genetically alter them in vitro, and reimplant them into a receptive locus. Although returning hepatocytes to the liver itself would be advantageous, the feasibility of this approach has never been evaluated due to the inability to distinguish donor from host hepatocytes. To unambiguously identify transplanted hepatocytes after transplantation, and to better quantitate their number and degree of liver function, two transgenic mouse lines were generated in a C57BL/6 background. The first expresses the Escherichia coli beta-galactosidase gene from the relatively liver-specific human alpha 1-antitrypsin (hAAT) promoter and allows transgenic hepatocytes to be readily identified after 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining; the second produces the hAAT protein under control of the same promoter, which enables hepatocyte survival and maintenance of liver function to be quantitated by measuring the serum levels of hAAT. Hepatocytes isolated from transgenic donors were transplanted into nontransgenic C57BL/6 recipients by intrasplenic injection. Surprisingly, a large fraction of these cells were identified within the liver parenchyma but not the spleen at 2 months after transplantation. The high levels of serum hAAT detected in transplant recipients were stable for greater than 6 months, suggesting that established cells will survive indefinitely. These results have important implications for liver organogenesis and hepatic gene therapy.

A method for large-scale production of hepatocytes on microcarriers have been developed for the purpose of bioartificial liver support system. Hepatocytes obtained by collagenase treatment of rat liver were efficiently attached and spread on a microcarrier surface in the presence of O2-saturated perfluorodecalin. In order to compare the metabolic activities of hepatocytes under long-term cultivation on microcarriers with those of cells under conventional monolayer culture, some liver-specific functions were investigated. Microcarrier-attached hepatocytes cultured in the absence of serum for 8 days synthesized and secreted albumin and fibronectin. Moreover, hepatocytes on microcarriers retained the ability to conjugate bilirubin for 4-5 days. With respect to these specific metabolic properties, microcarrier-attached hepatocytes were comparable to those from routine dish culture. These results suggest that this method developed for large-scale production of hepatocytes on microcarriers will allow one to obtain metabolically active cells suitable for extracorporeal liver support systems.

Transplantation of hematopoietic stem cells (HSC) from adult sheep into fetal lambs results in hematopoietic chimerism and graft-versus-host disease (GVHD). To evaluate the role of T cells in HSC engraftment and GVHD we depleted adult marrow HSC of T cells and observed the incidence of chimerism and GVHD in the fetal recipients. Using a naturally occurring polymorphism of the beta-globin locus to detect engraftment, bone marrow obtained from homozygous type A hemoglobin adult sheep were transplanted (2 x 10(9) cells/kg fetal weight) into 90 days' gestation fetal lambs homozygous for type B hemoglobin. Donor HSC were T-cell depleted by treatment with antisera (raised in rabbits against sheep thymocytes) in the presence of complement. T-cell depletion resulted in significant decrease in hematopoietic colony formation by donor HSC in vitro (305 +/- 49 v 134 +/- 21 colonies/10(5) cells) that normalized by the addition of autologous T cells (433 +/- 32 colonies/10(5) cells). Marrow depleted of T cells exhibited reduced engraftment in the recipient fetuses. When T cells were added back to donor HSC (depleted of T cells) at near-normal concentrations, engraftment improved but the lambs also developed GVHD. The addition of T cells to donor HSC (depleted of T cells) at concentrations below that present in unprocessed bone marrow resulted in significant engraftment but not GVHD. T cells play an important role in both the engraftment of adult HSC in fetal recipients and the development of GVHD in chimeric newborns. The elimination of T cells prevents GVHD but markedly reduces engraftment.(ABSTRACT TRUNCATED AT 250 WORDS)

Pediatric liver transplantation is successful but donor scarcity is a major limitation. We are studying hepatocyte transplantation as an alternative to provide functional hepatic replacement. This report details the study of rat liver perfusion for optimal harvest of hepatocytes and cell implantation. We performed 128 rat liver perfusions using a technique modified from the two-step enzymatic perfusion described by Seglen. We examined variations in the perfusion, rate, time, antegrade versus retrograde, pulsatile versus continuous flow, temperature, collagenase type, and variables of buffer composition. We have found optimal cell yield and viability under the following conditions: in situ perfusion, continuous flow at 25 cc/min, retrograde perfusion via the inferior vena cava, water bath temperature 38 degrees C, Boerhinger-Mannheim collagenase using a nonoxygenated HEPES based perfusion buffer, pH 7.4, for the initial perfusion and the same buffer with 4.8 mmol/L CaCl2 for the collagenase perfusion. These conditions consistently generate cell harvests of 500 to 700 x 10(5) cells/g of liver tissue with cell viability between 85% and 95%.

Isolated rat hepatocytes were transplanted into the splenic parenchyma of syngeneic animals. The effects on the degree of colonization by the transplanted cells of three forms of experimental liver injury in recipient animals were studied. Significant colonization was observed in animals with carbon tetrachloride (CCl4)-induced injury combined with portacaval shunt (PCS) and in animals with common bile duct (CBD) ligation but not in control animals or in animals with CCl4-induced injury alone. Transplanted cells in the CCl4/PCS group resembled normal hepatocytes. In contrast, in the CBD group, the intrasplenic hepatocytes exhibited a pattern of 'ductular metaplasia' similar to that observed in the obstructed liver of the recipients. Transplanted syngeneic hepatocytes can thus proliferate in the spleen in response to liver injury in the recipient. The morphological appearances of the transplanted cells can be modified depending on the nature of the liver injury.