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Liver stem cell (LSC) transplantation is a promising alternate approach to liver transplantation for patients with end-stage liver disease. However, the precise origin of LSCs remains unclear. Herein we determine if bone marrow mesenchymal stem cells (BMSCs) isolated from rats in acute hepatic failure (AHF) possess hepatic characteristics and have differentiation potential. BMSCs were isolated from AHF and sham-operated rats, and primary hepatocytes were isolated from normal rats for comparison. The transcriptomic profile of BMSCs and primary hepatoyctes was analyzed using the Affymetrix GeneChip Rat Genome 230 2.0 Array. BMSCs isolated from AHF and normal rats were induced to differentiate into hepatocytes in vitro and the degree of hepatic differentiation was assessed using quantitative real time RT-PCR, immunohistochemistry, and biochemical assays. AHF-derived BMSCs had a significantly different gene expression profile compared to control BMSCs. Thirty-four gene/probe sets were expressed in both AHF-derived BMSCs and primary hepatocytes, but were absent in control-derived BMSCs, including 3 hepatocyte-specific genes. Forty-four genes were up-regulated more than 2-fold in AHF-derived BMSCs compared to controls, including 3 genes involved in hepatocyte metabolism and development. Furthermore, AHF-derived BMSCs expressed more hepatocyte related genes than control BMSCs. Additional experiments to validate the differentiation of AHF-derived BMSCs, compared to control-derived BMSCs, showed that several hepatocyte-specific genes and proteins [such as albumin (ALB) and alpha fetoprotein (AFP)] were expressed earlier, and at higher levels, after 1 week of differentiation. Hepatocyte-specific metabolic functions were also significantly higher in the AHF group compared to control cells. Conclusion: AHF-derived BMSCs had a hepatic transcriptional profile and expressed hepatocyte specific genes early during differentiation, and possessed greater hepatogenic potency in vitro compared to cells isolated from control animals, further confirming their potential as a stem cell-based therapy for end-stage liver disease.

Hepatocyte transplantation and use of bioartificial liver support systems have been suggested as potential therapies for fulminant hepatic failure. Cryopreservation in liquid nitrogen is presently the major method of long-term storage of isolated hepatocytes. However, cryopreservation can result in low cell recovery and reduction in differentiated function. Several possible mechanisms of cell death during cryopreservation have been proposed. The most important mechanisms appear to be oxidative stress and apoptosis. In this study, we isolated fresh rat hepatocytes and cryopreserved them in three media: University of Wisconsin (UW) solution, an antioxidant-containing medium, and medium containing a caspase inhibitor. Viability and function of hepatocytes cryopreserved in these media were examined. Cryopreservation conditions had no effect on hepatocyte viability after thawing. However, after culture we found significant improvements in viability and function in both antioxidant- and caspase inhibitor-treated hepatocytes at 6 and 24 h.

Background. This study analyzes the prognostic significance of serum phosphorus in patients with acute liver failure (ALF). Methods. We performed a retrospective analysis of 112 patients with ALF. Univariate and bivariate analyses based on Kaplan-Meier recovery curves and a multivariate Classification Tree Structure Survival Analysis were performed to identify independent predictors of outcome. The variables analyzed were age, gender, race, ABO blood group, etiology of liver disease, grade of encephalopathy, serum bilirubin, prothrombin time, creatinine, serum phosphorus, phosphorus administered, phosphorus binders, and hemodialysis. Results. The median follow-up time was 5 days, the median age was 28 years, and 62% of the patients were female. The patients' routcomes were as follows: 28% recovered, 52% required orthotopic liver transplantation, and 20% died. White patients showed the best prognosis (58% recovered in the first week), and Hispanics showed the worst prognosis (0.3% recovered at 1 week) (P=0.0001). Encephalopathy and bilirubin were significant predictors of recovery (P

During the past decade, whole organ transplantation has become the primary method of treating fulminant hepatic failure and chronic liver failure due to specific genetic, hepatocellular, and anatomic defects of liver function. However, wider application of liver transplantation is restricted by shortage of organ donors, high cost, relatively high morbidity, and need for lifelong immunosuppression. As a result, investigators have attempted to develop alternative methods to treat liver insufficiency. These ranged from use of plasma exchange to utilization of detoxification columns and extracorporeal devices loaded with various liver tissue preparations. Recent advances in hepatocyte isolation and culture techniques, improved understanding of hepatocyte-matrix interactions, availability of new biomaterials, improved hollow-fiber technology, and better understanding of flow and mass transport across semipermeable membranes have resulted in the development of a new generation of liver assist devices. Some of these devices, including the one we developed, are currently being tested in the clinical setting. In this article, past experience with biological liver-assist devices is reviewed, the field's present status is examined, and our own experience with a hepatocyte-based bioartificial liver is summarized. Areas in which further research efforts are particularly needed are briefly characterized.

Despite recent advances in medical therapy, patients with fulminant hepatic failure (FHF) have a mortality rate approaching 90%. Many patients die because of failure to arrest the progression of cerebral edema. Liver transplantation has improved survival to 65% to 75%. However, there is a shortage of donors and approximately one half of the patients with FHF will die while awaiting liver transplantation. There is thus a need to develop an extracorporeal liver assist system to help keep these patients alive and neurologically intact until either an organ becomes available for transplantation or the native liver recovers from injury. Such a system could also be used during the period of functional recovery from massive liver resection or to assist patients with decompensated chronic liver disease. Over the years, various methods utilizing charcoal and resin hemoperfusion, dialysis, plasma exchange, and other methods of blood detoxification have been developed and tested, but none have gained wide acceptance. This was due to: (i) incomplete understanding of the pathophysiology of liver failure; (ii) lack of accurate methods of assessment, quantitation, and stratification of the degree of liver dysfunction; and (iii) inadequate numbers of prospective controlled clinical trials examining the effects of specific therapeutic modalities. Liver support systems utilizing liver tissue preparations were developed in the 1950s, but it was not until recently that advances in hepatocyte isolation and culture, better understanding of hepatocyte-matrix interactions, and improved hollow-fiber technology have resulted in the development of a new generation of liver assist devices. Some of these devices are currently being tested in the clinical setting. In a preliminary clinical study, we have used a porcine hepatocyte-based liver support system to treat patients with acute liver failure as well as patients with acute exacerbation of chronic liver disease. Patients in the first group, who were candidates for transplantation, were successfully bridged to a transplant with excellent survival. No obvious benefit from bioartifical liver treatments was seen in the second group. It is possible that, in this group, patients will have to be treated earlier and for longer periods of time. Prospective controlled trials will be initiated as soon as the current phase I study is concluded to determine the efficacy of this system in both patients populations. (c) 1996 John Wiley & Sons, Inc.

Early studies showed that compensatory liver growth after anterior portal branch ligation (aPBL) may restore normoglycemia in streptozotocin (STZ)-diabetic rats, in which a subtherapeutic islet mass was previously transplanted into the liver. We hypothesized that this effect could be related to islet regeneration at the graft site. This study was designed to characterize the proliferative response of the intraportally transplanted islets, shortly after aPBL. Male Wistar-Furth rats were used as syngeneic islet donors and/or recipients. STZ-diabetic rats were divided in four groups: groups 1 and 2 underwent selective 250-islet transplantation (Tx) into the posterior liver lobes, followed by aPBL 10 days later; rats were killed 24 h (n = 9) and 48 h (n = 10) after aPBL, respectively; groups 3 and 4 underwent selective 250-islet Tx into the posterior liver lobes, followed by sham aPBL 10 days later; rats were killed 24 h (n = 3) and 48 h (n = 3) after aPBL, respectively. Two hours before killing, all animals were injected with 5'-bromo-2'-deoxyuridine (BrdU; 50 mg/kg, i.v.). Liver sections were immunostained for insulin and BrdU, and both hepatocyte and islet cell labeling index (LI) were calculated. Islet cell LI was 2.30 ± 1.18% in group 1, 2.23 ± 1.00% in group 2, 0.43 ± 0.29% in group 3, and 0.39 ± 0.21% in group 4 (group 1 vs. group 3: p < 0.02; group 2 vs. group 4: p < 0.01). Hepatocyte LI was 2.50 ± 2.14% in group 1, 15.0 ± 7.6% in group 2, 0.12 ± 0.04 in group 3, and 0.11 ± 0.03% in group 4, respectively (group 1 vs. group 2: p < 0.02; group 1 vs. group 3: p < 0.001; group 2 vs. group 4: p < 0.001). Our study showed that intraportally transplanted islets undergo a concurrent proliferative response after aPBL, although with a lower extent and a different timing when compared with the liver-cell response.

Background. We earlier described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobe necrosis. In FHF rats, lack of regeneration of the residual liver was associated with delayed expression of HGF and HGF receptor c-met and elevated blood HGF and TGF-β1 levels. We then found that intrasplenic hepatocyte transplantation prolonged survival in FHF rats and triggered hepatocyte proliferation in the native liver. The latter effect was associated with accelerated expression of HGF and c-met mRNA in the liver and lowering of blood HGF and TGF-β1 levels. In the present study we show that in FHF rats, treatment with a bioartificial liver (BAL) had similar effects. Materials and methods. FHF was induced in inbred Lewis rats and after 4 h, Group 1 rats were subjected to a 4-h whole blood perfusion through the BAL loaded with 3 × 108 microcarrier-attached syngeneic hepatocytes, whereas Group 2 control rats were treated with the BAL containing microcarriers only. Results. Compared to sham-BAL-treated rats, the test rats lived longer (28 ± 5 vs 17 ± 2 h; P = 0.0005), had better coagulation parameters, maintained higher body core temperature, and showed decreased plasma TGF-β1 levels. In addition, their liver remnants were HGF positive and showed increased DNA binding of transcription factors engaged in the modulation of hepatocyte proliferation (e.g., STAT3) and liver-specific gene expression (e.g., HNF1, HNF4, C/EBP). Conclusions. This study demonstrates that hepatocyte-based extracorporeal support not only can provide metabolic support by increasing the available functional liver mass but also is capable of modifying humoral and molecular mechanisms which are responsible for proliferation and organ-specific functions of residual hepatocytes.

During orthotopic liver transplantation (OLT) for fulminant hepatic failure (FHF), some patients develop cerebral injury secondary to intracranial hypertension. We monitored intracranial pressure (ICP) and cerebral perfusion pressure (CPP) before and during OLT in 12 FHF patients undergoing transplantation. All four patients who had normal ICP preoperatively maintained normal ICP/CPP throughout OLT. During OLT, four of the eight patients with pretransplant intracranial hypertension had six episodes of ICP increase. These episodes of intracranial hypertension occurred during failing liver dissection (n=3) and graft reperfusion (n=3). At the end of the anhepatic phase, the ICP was lower than the preoperative ICP in all patients, and was below 15 mmHg in all but one patient. These data suggest that in FHF patients who develop intracranial hypertension before OLT, dissection of the native liver and graft reperfusion are associated with a risk of brain injury resulting from intracranial hypertension and cerebral hypoperfusion.

BACKGROUND There are limited experimental data on the nature of the humoral response elicited in humans against pig antigens. In this study, we have examined the xenoantibody (XAb) response in eight patients with acute liver failure exposed to pig hepatocytes after treatment with the bioartificial liver (BAL). METHODS Patients' plasma samples obtained before and after BAL treatment were tested for IgM and IgG XAbs, IgG subclasses, and XAb cytotoxicity, using enzyme-linked immunosorbent assay and flow-cytometric assays. The characterization of pig aortic endothelial cell (PAEC) surface xenoantigens was analyzed by immunoprecipitation. RESULTS We observed by day 10, a strong anti-pig IgG and IgM XAb response in patients undergoing two or more BAL treatments, with a significant increase in all the IgG subclasses; in contrast, XAb titers did not change if the patients received only one BAL treatment. The majority of the XAbs produced to porcine antigens were primarily specific for the alphaGal epitope. Both IgG and IgM XAbs were cytotoxic to PAECs, and the cytotoxic activity of IgG was associated with high levels of IgG1 and IgG3 subclasses, known to be efficient on complement activation. The characterization of porcine surface antigens demonstrated that IgM human XAbs, before and after BAL exposure, recognized xenoantigens on PAECs with similar molecular weights, suggesting that the same population of XAbs were present in the patients before and after exposure to pig antigens. CONCLUSIONS Repetitive exposure of humans to porcine antigens after BAL treatment, results in a strong IgG and IgM XAb responses that are primarily directed against the alphaGal epitope. These XAbs are cytotoxic to PAECs and the IgG toxicity correlates with high IgG1 and IgG3 levels. Our data also suggest that no new XAb specificity emerges after porcine exposure.

Intracranial hypertension leading to brain stem herniation is a major cause of death in fulminant hepatic failure (FHF). Mannitol, barbiturates, and hyperventilation have been used to treat brain swelling, but most patients are either refractory to medical management or cannot be treated because of concurrent medical problems or side effects. In this study, we examined whether allogeneic hepatocellular transplantation may prevent development of intracranial hypertension in pigs with experimentally induced liver failure. Of the two preparations tested—total hepatectomy (n = 47), and liver devascularization (n = 16)—only pigs with liver ischemia developed brain edema provided, however, that animals were maintained normothermic throughout the postoperative period. This model was then used in transplantation studies, in which six pigs received intrasplenic injection of allogeneic hepatocytes (2.5 × 109 cells/pig) and 3 days later acute liver failure was induced. In both models (anhepatic state, liver devascularization), pigs allowed to become hypothermic had significantly longer survival compared to those maintained normothermic. Normothermic pigs with liver ischemia had, at all time points studied, ICP greater than 20 mmHg. Pigs that received hepatocellular transplants had ICP below 15 mmHg until death; at the same time, cerebral perfusion pressure (CPP) in transplanted pigs was consistently higher than in controls (45 ± 11 mmHg vs. 16 ± 18 mmHg; p < 0.05). Spleens of transplanted pigs contained clusters of viable hepatocytes (hematoxylin-eosin, CAM 5.2). It was concluded that removal of the liver does not result in intracranial hypertension; hypothermia prolongs survival time in both anhepatic pigs and pigs with liver devascularization, and intrasplenic transplantation of allogeneic hepatocytes prevents development of intracranial hypertension in pigs with acute ischemic liver failure.

Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation in experimental animal models with genetic disorders of liver metabolism and liver failure. 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, expansion of transplanted cell population, and gene transfer requiring active DNA synthesis. The present study was undertaken to evaluate the proliferative capacity of fetal and adult rat hepatocytes under standardized culture conditions. Fetal (20 days of gestation) and adult hepatocytes were cultured in serum-free media at low densities and treated with growth factors. Proliferation was assessed by [3H]-thymidine incorporation and cell cycle analysis by flow cytometry. In nonstimulated cells, DNA synthesis at 4 h was about x100 higher and after 10 days in culture x20 higher in fetal compared to adult hepatocytes. When epidermal growth factor (EGF) was added, maximal DNA synthesis in fetal hepatocytes was seen at 48 h, whereas in adult hepatocytes at 72 h. For adult hepatocytes, the average increase compared to untreated cells was x13.8 with EGF, x18.5 with transforming growth factor alpha (TGF-alpha), and x7.6 with hepatocyte growth factor (HGF). For fetal hepatocytes, the increase was twofold with either EGF, TGF-alpha or HGF. EGF-, TGF-alpha- and HGF-dependent DNA synthesis was inhibited by transforming growth factor beta-1 (TGF-beta1) in both fetal and adult hepatocyte cultures; this antiproliferative effect was significantly stronger in adult hepatocyte cultures. With cyclosporine, EGF-, TGF-alpha- and HGF-dependent DNA synthesis in fetal hepatocyte cultures decreased by 36-46%, whereas in adult hepatocytes by 19-27 %. These results show that in contrast to adult hepatocytes, fetal hepatocytes have high spontaneous proliferative activity independently of growth factors and are relatively resistant to the inhibitory effect of TGF-beta1. It was also found that cyclosporine suppresses proliferation of cultured fetal hepatocytes.

Background. In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative, less immunogenic, and resistant to cryopreservation and ischemic injury. These qualities could enhance FH engraftment, proliferation, and gene transfer requiring active DNA synthesis, Methods. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method, Free and cultured cells were studied using electron microscopy, fluorescence-activated cell sorting, and Northern analysis using alpha-fetoprotein and albumin as markers of hepatocyte lineage, DNA synthetic activity was measured in quiescent and mitogen-stimulated fetal and adult hepatocytes by [H-3]thymidine incorporation, Susceptibility of cultured FH to retrovirally mediated gene transfer was studied using an amphotropic retroviral vector carrying the Escherichia coli lac-Z gene, Nagase analbuminemic rats were used as recipients to study the effects of intraportal FH transplantation, Analysis of serum albumin was carried out by enzyme-linked immunosorbent assay. Results, In fetal liver, 87+/-2%? of the cells showed morphological and molecular features of hepatocytes. DNA synthetic activity in nonstimulated cultured FH was 10 times greater than the maximal hepatocyte growth factor-driven response in adult rat hepatocytes., A total of 5-15% FH stained positive for X-gal; results of transduction in adult hepatocyte cultures were negative. In Nagase analbuminemic rat recipients, FH produced significant amounts of albumin only when a hepatic regenerative stimulus was applied. Immunohistochemistry confirmed presence of albumin-positive hepatocytes, Conclusions, Fetal rat liver from the late gestation period is highly enriched with hepatocyte progenitors, They are highly proliferative and susceptible to retroviral transduction and can engraft and function in the adult rat liver if transplanted under a hepatic regenerative stimulus.

To gain insight into liver regenerationmechanisms in fulminant hepatic failure, we comparedgene expression of hepatocyte growth factor, itsreceptor c-met, c-myc, and albumin in human normal (4cases) and fulminant (14 cases) livers by reversetranscription-polymerase chain reaction. In normallivers, hepatocyte growth factor gene was not expressed,whereas c-met, c-myc and albumin genes were always expressed. In fulminant hepatic failure,hepatocyte growth factor gene was expressed in 1 of 14cases, c-met in none of 14 cases, c-myc in 10 of 14cases, and albumin in 3 of 14 cases. Byimmunofluorescence, c-met protein was revealed in normal but not infulminant hepatic failure liver tissue. Liver tissue isunlikely to account for high hepatocyte growth factorplasma levels typical for fulminant hepatic failure. Lack of its receptor (c-met)expression may explain a poor response of fulminanthepatic failure livers to exogenous hepatocyte growthfactor that normally promotes liver growth andregeneration.

Poor engraftment and consequent loss of β-cell mass could be one of the factors that are responsible for function loss after intraportal islet transplantation (Tx). Streptozotocin-diabetic rats were transplanted with syngeneic islets, which were injected into the portal vein via an indwelling catheter connected to a subcutaneous port. In Group I (n = 6), 1,000 islets were injected in a single dose into the liver. In Group II (n = 6), five doses of 200 islets were repeatedly injected over a period of 14 days, for a total of 1,000 islets. In Group III (n = 4), five decreasing doses of islets were injected over a period of 14 days, for a total of 750 islets. Nonfasting blood glucose (n-FBG) and body weight (b.wt.) were determined twice a week and an intravenous glucose tolerance test (IVGTT) was performed at 30 and 90 days. In Group I, n-FBG decreased in 2 wk from the time of first islet injection, averaging 110 ± 21.9 mg/dL at 1 mo (p < 0.05 vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group II, n-FBG was normalized in 2 wk averaging 90.2 ± 25 mg/dL on day 12 (p = NS vs. normal controls) and 75.8 ± 14.6 mg/dL at 1 month (p = NS vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group III, n-FBG decreased to normal values in 2 wk, averaging 77 ± 15.7 mg/dL at 1 mo (p = NS vs. normal controls), but normoglycemia was maintained for 40 days and then followed by a progressive increase. Only in Group II, KG (percent/min decline in glucose level) was not significantly different from that of normal controls (1.702 ± 0.531 at 1 mo and 1.676 ± 0.891 at 3 mo), while it was significantly lower than normal controls in both Group I and III animals. Body weight increase after Tx correlated with the number of transplanted islets and at 90 days, Group III rats showed less increase than Groups I and II (p < 0.05), while no significant differences in b.wt. were recorded between Group I and II. The findings indicate that intraportal islet Tx, injected repeatedly and in small doses, produced better metabolic effects than injection of the same total number of islets in a single dose. Copyright © 1997 Elsevier Science Inc.

The preferred therapy for acute and chronic liver insufficiency and severe heritable disorders of liver metabolism is whole-organ transplantation. However, due to the shortage of organ donors and high cost, alternative therapeutic approaches have been proposed, including transplantation of normal allogeneic hepatocytes. Recently, it has been reported that many hepatocytes transplanted into the spleen migrated to the liver. We therefore carried out a series of large-animal experiments to reexamine the intrasplenic route and to develop a method for large-scale hepatocellular transplantation in pigs.Allogeneic porcine hepatocytes were transplanted using the following routes: (1) retrograde injection of cells via the splenic vein, (2) intraarterial injection of cells, (3) direct intrasplenic injection of cells after laparotomy, (4) percutaneous intrasplenic injection of cells under laparoscopic control, (5) laparoscopic intrasplenic injection of cells. The number of cells injected varied from 2 x 10(9) to 10 x 10(9) cells.Of all the methods tested, only direct intrasplenic injection of 2 bln of cells was found to be compatible with survival. However, even with this "small" number of cells (2% original liver mass), there was a significant risk of spleen infarction, perisplenic adhesion formation, and portal vein thrombosis. The laparoscopic approach was found to be reliable, simple, and safe.Even though the spleen is considered by many authors the optimal site for hepatocellular transplantation, transplantation of cells in a number needed to support the failing liver may be associated with significant complications, morbidity, and mortality.

Numerous studies have reported successful allotransplantation of hepatocytes. However, none have shown long-term correction of a liver-related metabolic defect. In this study, we used a method of regional hepatocyte transplantation and subsequent induction of transplanted cell proliferation by regeneration response in the transplant-bearing liver lobes. New Zealand White rabbits were used as cell donors and Watanabe heritable hyperlipidemic (WHHL) rabbits were used as cell recipients (2 x 10(8) cells/rabbit). All recipient rabbits were maintained on daily cyclosporine. Two weeks after baseline serum cholesterol determination, group I WHHL rabbits (n = 7) received an infusion of cells into the right lateral liver lobe, and a loose ligature was placed around the portal venous branch supplying the anterior lobe. After 1 week, to allow engraftment, the portal venous branch was ligated, which resulted in the atrophy of the affected liver parenchyma and induction of hyperplasia in the transplant-bearing liver tissue. Group II rabbits (n = 6) were transplanted with New Zealand White hepatocytes without portal branch ligation (PBL) and group III rabbits (n = 4) were subjected to sham transplantation (saline) and PBL. The experimental period extended to 150 days after transplantation. All WHHL rabbits transplanted with normal hepatocytes showed reduction in serum cholesterol and low-density lipoprotein (LDL) levels. Group I (PBL-stimulated) recipients demonstrated a more pronounced and sustained effect than group II animals (P < 0.05). Group III controls showed only a slight, typical for aging decrease in serum cholesterol. Group I recipient livers perfused with LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) showed much higher numbers of DiI-LDL-positive hepatocytes than those of group II recipients. In conclusion, a liver regeneration stimulus enhanced the population of transplanted hepatocytes and their functional effect in a large animal model of inborn error of liver metabolism.

Anatomical separation of pancreatic islets in some teleost fish makes them a useful source of pancreatic endocrine tissue. Islets were harvested from tropical Tilapia fish (Oreochromis nilotica) and cultured for 24 hr at 37°C. Eight athymic nude mice were rendered diabetic by streptozotocin (STZ) and transplanted under the kidney capsule with fish islets. After transplantation (Tx), nonfasting blood glucose (n-FBG), which was in all recipients >450 mg/dl, decreased to

Despite recent advances in medical supportive therapy, patients with severe fulminant hepatic failure (FHF) have mortality rate approaching 90%. Investigators have attempted to improve survival by using various extracorporeal liver support systems loaded with sorbents and liver tissue preparations. None of them succeeded in gaining clinical acceptance and orthotopic liver transplantation (OLT) remains a primary therapeutic option for patients with FHF. In this study, authors discuss the systems which utilize isolated hepatocytes. Most of these devices were tested in vitro and in animals with chemically and surgically induced liver failure. In some studies, signficant levels of detoxification and liver functions were achieved. The authors describe their own hepatocyte-based artificial liver (BAL). It is based on plasma perfusion through a hollow-fiber module seeded with matrix-anchored porcine hepatocytes. The BAL was used 14 times to treat 9 patients with acute liver failure. On 10 occasions, a charcoal column was included in the plasma circuit. Each treatment lasted 7 +/- 1 h. All procedures were tolerated well and 8 patients (including 6 patients with FHF) underwent OLT. Five patients with increased intracranial pressure (ICP) and evidence of decerebration had normalization of ICP and enjoyed full neurologic recovery after OLT. Laboratory data showed evidence for bilirubin conjugation, decrease in blood ammonia, maintenance of low lactic acid levels, and increase in the ration between the branched chain and aromatic amino acids. No allergic reactions to xenogeneic hepatocytes were observed. The authors conclude that BAL treatment with porcine hepatocytes appears to be safe and can help maintain patients alive and neurologically intact until a liver becomes available for transplantation. (c) 1994 John Wiley & Sons, Inc.

The purpose of this study was to develop a method of transduction of normal skin fibroblasts with a retroviral vector expressing the human factor IX (hF-IX) gene and transplantation of the transduced cells into rats. A retroviral vector containing the hF-IX gene, as well as a selectable marker (hygromycin B resistance; HB), was used to transduce cultured normal skin (rat and human) fibroblasts. Transduced fibroblasts were selected with HB; resistant clones were propagated and assessed for expression of active hF-IX in vitro. Transduced cells secreted significant amounts of hF-IX in vitro (327.9 +/- 14.2 ng/10(6) cells) which showed measurable clotting activity, indicating terminal processing of the gene-transcribed hF-IX product by the transduced fibroblasts. Integration of the proviral gene into rat and human fibroblast genomic DNA was confirmed by polymerase chain reaction. Transduced rat fibroblasts were attached to collagen-coated microcarriers and transplanted intraperitoneally into syngeneic recipients. Transplanted microcarrier-attached cells formed aggregates in the peritoneal cavity and exhibited positive immunohistochemical staining for hF-IX up to 8 weeks following transplantation, the time of termination of the experiment. These experimental results may have significant implications in developing strategies for future clinical therapy for hemophilia B.

Although significant progress has been achieved in isolated hepatocyte transplantation, the optimal site of cell implantation has not yet been determined. We have developed a novel experimental method of intraportal hepatocyte transplantation that allows easy assessment of the morphology and function of transplanted hepatocytes. Donor hepatocytes were harvested from Sprague-Dawley rats by in situ EDTA/collagenase perfusion. Fifteen recipient Nagase analbuminemic rats (NAR) underwent cannulation of the gastroduodenal vein under ether anesthesia. Either the posterior or anterior liver lobes were selectively infused with cells by occluding the portal venous supply of the nontransplanted liver lobes. Normal donor hepatocytes (2 x 10(7)) suspended in normal saline were infused over 1 min (4 ml). Recipients were treated with cyclosporine for the duration of the experiment. Plasma albumin levels were determined by ELISA, before and at various intervals after transplantation. In NAR rats transplanted with normal hepatocytes, there was a significant (P < 0.003) and sustained (12 weeks) increase in plasma albumin levels. Control NAR rats transplanted with NAR hepatocytes (n = 8) showed no significant changes in plasma albumin levels. Similarly, normal Wistar hepatocytes were infused intraportally into the posterior lobes of Gunn rats (n = 4), which lack the ability to conjugate bilirubin. Pre- and posttransplantation bile was collected following bile duct cannulation. Bile analysis by HPLC, demonstrated a significant (P = 0.04) increase in the level of bilirubin conjugates following transplantation and a corresponding decrease in total serum bilirubin (P = 0.04). Our experimental data demonstrate that direct selective intraportal infusion of hepatocytes is an effective technique of hepatocyte transplantation in the rat.

We have shown that elevated plasma D-glucose levels in experimentally-induced diabetic nude athymic rats can be reduced by intraperitoneal transplantation of microcarrier-attached insulin producing beta cells from the mouse pancreatic beta cell line, beta TC-1. The reduction in the level of hyperglycemia was observed as early as two days following cell transplantation and was associated with a concomitant increase in plasma insulin levels. beta TC-1 cell transplanted diabetic rats had plasma D-glucose levels similar to those found in non-diabetic control animals and remained normoglycemic throughout the 39 day experimental period. The beta TC-1 cell transplanted diabetic rats also had near normalization of body weight, food and water intake and of urine output when compared to control diabetic and non-diabetic rats. Similarly, they exhibited improved blood glucose clearance following intravenous D-glucose administration. These results suggest that beta TC-1 cells regulate D-glucose homeostasis following transplantation into diabetic rat recipients in a manner similar to that of endogenous pancreatic beta cells.

The purpose of this report is to summarize data collected over the past several years in the field of hepatocyte transplantation. Herein, a novel method of intraperitoneal transplantation of hepatocytes attached to collagen-coated microcarriers (or a totally biodegradable collagen sponge) is described, resulting in correction of specific genetic liver defects in rats and improved survival in rats undergoing 90% partial hepatectomy. Transplanted cells/matrix form well-vascularized aggregates in the peritoneal cavity; transplanted hepatocytes can be identified by light microscopy using immunohistochemical methods. Prolongation of transplanted hepatocyte survival and function was demonstrated by pretreatment of cells with ultraviolet irradiation. The ability of transplanted cells to proliferate was examined using both biochemical and morphologic criteria. Finally, a method of harvesting, cryopreserving, and in vivo functional testing of human hepatocytes is described.

Background Distinguishing those patients with fulminant hepatic failure (FHF) and who require transplantation from those FHF patients who will survive with receiving only intensive medical care remains problematic, and this distinction is important because of the chronic shortage of donor livers. Methods To assess the applicability of two prognostic scoring systems, referred to as the London and Clichy criteria, we compared using both systems, at the time of admission, for 43 FHF patients (15 M/28 F; age: 3716 yrs). Acetaminophen (ACM) was the etiology for 16 patients, while the remaining 27 had other etiologies. All the patients received intensive care, and 18 (8 ACM/10 non-ACM) had investigational BAL support. Results For the ACM toxicity, neither the London nor the Clichy criteria exhibited acceptable sensitivity (71 vs 86%, respectively), specificity (78 vs 56%, respectively), a positive predictive value (71 vs 60%, respectively), a negative predictive value (78 vs 83%, respectively) or predictive accuracy (75 vs 69%, respectively) to predict patient survival without transplantation. In contrast, applying the London and Clichy criteria to the FHF patients with non-ACM etiologies showed a sensitivity of 96 vs 80%, respectively, a specificity of 100 vs 100%, respectively, a positive predictive value of 100 vs 100%,, respectively a negative predictive value of 67 vs 29%, respectively and a predictive accuracy of 96% vs 82%, respectively. Conclusions Overall, the London criteria more accurately predicted the need for transplantation, and neither the London criteria nor the Clichy prognostic criteria accurately predicted the outcome of those patients who suffered with FHF due to ACM. BAL support may have contributed to the survival of the patients with ACM toxicity and who didn't undergo transplantation, and this survival exceeded the predictions of both prognostic systems. Additional multicenter studies should be conducted to refine these prognostic scoring systems, and this will help physicians rapidly identify those FHF patients who can survive without undergoing liver transplantation.

Background and aims: Reversible Ischemia/Reperfusion liver injury was used to induce engraftment and hepatic parenchymal differentiation of exogenous β2-microglubulin -/ Thy1+ bone marrow-derived cells. This method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, was further tested to correct the metabolic disorder in a rat model of congenital hyperbilirubinemia. Methods and results: Analysis by confocal laser microscopy of fluorescence-labelled cells and by immunohistochemistry for β2-microglubulin, 72 hours after intraportal delivery, demonstrated engraftment of infused cells in liver parenchyma of rats with Ischemia/Reperfusion, but not in control animals with non-injured liver. Transplantation of bone marrow-derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP-positive hepatocytes in Ischemia/Reperfusion liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP-negative bone marrow-derived cells into GFP-positive transgenic rats resulted in the appearance of GFP-negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow-derived cells into hyperbilirubinemic Gunn rats with deficient bilirubin conjugation after Ischemia/Reperfusion damage resulted in 30% decrease of serum bilirubin, in the appearance of bilirubin conjugates in bile and in the expression of normal UDP-glucuronyltransferase enzyme evaluated by PCR. Conclusions: Ischemia/Reperfusion injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow-derived cells. Transplantation of bone marrow-derived cells from non-affected animals resulted in the partial correction of hyperbilirubinemia in the Gunn rat.

There is a need to develop artificial means of liver replacement and/or assistance with the aim of either supporting patients with borderline functional liver cell mass until their liver regenerates, or until a donor liver becomes available for transplantation. Selective plasma filtration is a novel approach to blood purification therapy designed to reduce the level of circulating toxins of hepatic and renal failure, mediators of inflammation and inhibitors of hepatic regeneration. The results of preclinical studies indicate that treatment of pigs with experimentally-induced fulminant hepatic failure is safe and effective in extending survival time and arresting brain swelling. In addition, the amount of ammonia, aromatic amino acids, IL6, TNFalpha and C3a removed during the 6-h treatment in the present study was higher by 34% to 175% than the total plasma content of those substances at the start of therapy.

Pancreatic islet transplantation is limited by shortage of donor organs. Although β-cell lines could be used, their secretion of insulin is characteristically glucose independent and immunoisolation is required. Here we show that intrasplenic transplantation of encapsulated glucose-responsive mouse insulinoma cells reversed streptozotocin (STZ)-induced diabetes in rats. MIN-6 cells derived from a transgenic mouse expressing SV 40 large T antigen in pancreatic β-cells were transfected with minigene encoding for human glucagon-like-peptide-1 under the control of rat insulin promoter. The cells were encapsulated in alginate/poly-L-lysine and used for cell transplantation in STZ-diabetic rats. Rats with nonfasting blood glucose (n-FBG) greater than 350 mg/dl were used. In group I rats (n = 6) 20 million encapsulated cells were injected into the spleen. Group II rats (n = 6) received empty capsules. n-FBG was measured biweekly. After 4 and 8 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed in group I; normal rats served as controls. Plasma insulin level was measured every other week (RIA). After 8 weeks, spleens were removed 1 day before sacrifice. In rats transplanted with cells the n-FBG was 100—150 mg/dl until the end of the study. After splenectomy, all cell recipients became diabetic (glucose 400 ± 20 mg/dl). Transplanted rats showed increase in body weight and insulin production (3.3 ± 1.0 ng/ml versus 0.92 ± 0.3 ng/ml; p < 0.01) and had normal IPGTT. Spleens contained capsules with insulin-positive cells. Overall, data from this work indicate that intrasplenic transplantation of xenogeneic encapsulated insulin-producing cells without immunosuppression reversed diabetes in rats. Excellent survival and function of the transplanted cells was due to the fact that the cells were separated from the bloodstream by the immunoisolatory membrane only and insulin was delivered directly to the liver (i.e., in a physiological manner).

Background. Encapsulated cell therapy might be a promising approach to enable cell transplantation without immunosuppression. This study investigates the viability and hepatic function of hepatocytes encapsulated with alginate/poly-L-lysine in vitro and the effect of the intrasplenic transplantation of cultured encapsulated hepatocytes on survival in 90% hepatectomized rats as a preliminary step toward allogeneic hepatocyte transplantation without immunosuppression. Materials and Methods. Rat hepatocytes were isolated and encapsulated using alginate/poly-L-lysine. Encapsulated hepatocytes were cultured for 28 days to measure cell viability, liver function, and morphology. Rats were treated with a 90% partial hepatectomy and then immediately underwent the intrasplenic transplantation of the cultured encapsulated hepatocytes, the capsule alone, or the allogeneic hepatocytes without the capsule. The survival rate, liver function, and cell morphology were assessed after transplantation. Results. The cultured encapsulated hepatocytes maintained their viability and showed better metabolic activity than day 0 cultured encapsulated hepatocytes. The encapsulated cells strongly expressed albumin and were positive for periodic acid-Schiff staining. Electron microscopy demonstrated that the microencapsulated hepatocytes retained the structural elements of hepatic cytoplasm and nuclei. Intrasplenic transplantation of the encapsulated hepatocytes increased the survival rate and improved the hepatic function. Encapsulated hepatocytes transplanted into rat spleen survived well and retained their hepatic function. Moreover, dramatic liver regeneration was observed 48 hr after transplantation in the group that received intrasplenic transplantations of encapsulated hepatocytes. Conclusions. The intrasplenic transplantation of cultured encapsulated hepatocytes improved the survival rate of an acute liver failure rat model induced by a 90% partial hepatectomy.

Background Several studies have identified beta2-microglobulin-negative (β2M − ) cells as a potential stem cell fraction in the bone marrow of rats and humans. We studied the ability of bone marrow–derived β2M − cells to differentiate into cardiomyocytes and reconstitute the myocardium in a model of myocardial infarction. Methods β2M − cells were purified from bone marrow of Lewis rats using a magnetic activated cell-sorting technique. β2M − cells, 2.5 × 10 6 cells in 100 μl of phosphate-buffered saline (PBS), were transplanted 7 days after infarction into a transmural myocardial scar induced by cryoinjury in Lewis rats ( n = 9). Control Group 1( n = 10) received a 100-μl injection of PBS, and Control Group 2 ( n = 15) received no injection. The β2M − cells were labeled before transplantation, using the membrane fluorescent intercalated dye, PKH26. Repopulation was examined at 6 and 8 weeks after transplantation. Differentiation of β2M − cells into cardiac myocytes was determined by the colocalization of troponin and PKH26 to the same cell, utilizing immunohistochemistry, ultraviolet photomicroscopy and fluorescence microscopy on 6-μm serial sections. Area of engraftment within the scar was calculated by planimetry. Results The treatment group had multiple islands of de novo–formed myocardium within the fibrous matrix of the transmural scar (mean area 35 ± 4.2% of scar area at 6 and 8 weeks). These cells colocalized cardiac-specific troponin and PKH26. Using these techniques, no myocardial islands were seen in the control groups. Before transplantation, β2M − cells were troponin-negative. Conclusions This study demonstrates that β2M − cells represent a novel sub-population of bone marrow–derived stem cells capable of successful and substantial engraftment in areas of transmural myocardial scar, with de novo formation of cardiac myocytes. The functional significance of this observation is being studied.

Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against immune rejection. We hypothesized that this could be achieved with intrasplenic transplantation of encapsulated cells because the cells would have instant access to oxygen and nutrients while being separated from the host immune system. In order to provide proof of the concept, primary rat hepatocytes and human hepatoblastoma-derived HepG2 cells were used as model cells. Rat hepatocytes were encapsulated in 100-kDa hollow fibers and cultured for up to 28 days. Rat spleens were implanted with hollow fibers that were either empty or contained 1 × 107 rat hepatocytes. Human HepG2 cells were encapsulated using alginate/poly-l-lysine (ALP) and also transplanted into the spleen; control rats were transplanted with free HepG2 cells. Blood human albumin levels were measured using Western blotting and spleen sections were immunostained for albumin. Hepatocytes in monolayer cultures remained viable for only 6–10 days, whereas the cells cultured in hollow fibers remained viable and produced albumin throughout the study period. Allogeneic hepatocytes transplanted in hollow fibers remained viable for 4 weeks (end of study). Free HepG2 transplants lost viability and function after 7 days, whereas encapsulated HepG2 cells remained viable and secreted human albumin at all time points studied. ALP capsules, with or without xenogeneic HepG2 cells, produced no local fibrotic response. These data indicate that intrasplenic transplantation of encapsulated cells results in excellent survival and function of the transplanted cells and that the proposed technique has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.

Background. The definitive therapy for end-stage liver disease is orthotopic liver transplantation (OLT). However, rejection is still a major cause of mortality and morbidity following OLT. Hepatocyte transplantation has been used experimentally to treat liver diseases. The aim of this study was to investigate whether bone marrow-derived liver stem cells (BDLSC) and mature hepatocytes could repopulate transplanted livers undergoing rejection. Methods. OLT was carried out from D'Agouti (C3-positive female) into Lewis (C3-negative female) rats. BDLSC were transplanted from Lewis (male) into livers of D'Agouti (female) rats. Group A (n = 9) received intraportal normal saline. Groups B (n = 9) and C (n = 9) underwent intraportal transplantation of mature hepatocytes (Lewis female, 0.75 × 107) and DBLSC (Lewis male, 5 × 104) respectively. All groups received subtherapeutic immunosuppression (Cyclosporin 0.25 mg/kg/d) for 13 days. Liver repopulation was assessed using immunohistochemistry (C3 antigen-negative cells), in-situ hybridization, (Y-chromosome-positive BDLSC) and histologic assessment (hematoxylin and eosin) for rejection. Results. BDLSC and mature hepatocytes repopulated 62 ± 12.3% and 2.5 ± 1.7% of rejecting livers, respectively. BDLSC demonstrated formation of hepatic lobules and portal triads with little evidence of rejection 36 days after discontinuation of immunosuppression. Conclusions. BDLSC can repopulate livers undergoing severe rejection. Moreover, BDLSC can differentiate into hepatocytes and cholangiocytes. This finding may have important clinical implications. (Surgery 2002:132:384-90.)

Liver transplantation is the only clinically effective method of treating acute liver failure. However, wider application of this therapeutic modality is restricted primarily by shortage of donor organs. In the search for alternative methods of liver replacement therapy, investigators have focused on transplantation of normal allogeneic hepatocytes and on the development of liver support systems utilizing isolated hepatocytes. Since all human livers suitable for cell harvest are being used for transplantation, hepatocyte therapy using human tissue would require growing of cells in vitro. Unfortunately, although hepatocytes have tremendous capacity to proliferate in vivo, their ability to grow in culture is severely limited. Stromal cells from bone marrow and other blood-forming organs have been found to support hematopoiesis. In this paper, we show that bone marrow-derived stromal cells (BMSCs) enhance proliferation and support differentiation of rat hepatocytes in culture. Further, we demonstrate that in hepatocyte/BMSC co-cultures, clonal expansion of small hepatocytes (SH) is increased. Using semipermeable membrane cultures, we established that direct cell–cell contact is necessary for stimulation of cell proliferation. We also show that BMSCs which are in direct contact with hepatocytes and SH colonies express Jagged1. This suggests a potential role for Notch signaling in the observed effects. Finally, we present evidence that the expression and activity of liver specific transcirption factors, CCAAT/enhancer binding proteins and liver specific key enzymes such as tryptophan 2,3-dioxygenase, are improved in hepatocyte/BMSC co-cultures. In conclusion, results of this study indicate that BMSCs could facilitate proliferation and differentiation of primary rat hepatocytes and their progenitors (SH) in vitro. © 2001 Wiley-Liss, Inc.

Orthotopic liver transplantation is the only definitive therapy for patients with fulminant hepatic failure (FHF). However, due to shortage of organs, a large number of patients die before a liver can be procured for transplantation. In FHF the need for a liver is particularly urgent because of rapid deterioration in the patients' condition with the onset of cerebral edema and intracranial hypertension leading to irreversible brain damage. It is thus necessary to develop an extracorporeal liver support system to help maintain patients alive and neurologically intact until an organ becomes available for transplantation. Multiple attempts have been made, ranging from the use of plasma exchange to utilization of charcoal columns and extracorporeal devices loaded with liver tissue to develop liver support systems for treating patients with acute severe liver failure. None of these systems has achieved wide clinical use, and FHF due to multiple causes continues to be associated with significant morbidity and mortality. In this paper, the authors review the history of extracorporeal liver support for acute liver failure and discuss their experience with a hollow fiber bioartificial liver support system utilizing porcine hepatocytes in the treatment of patients with acute liver failure.

In the past three decades, a large number of in vitro and in vivo small and large animal studies of all aspects of hepatocyte transplantation have been carried out to improve the understanding of the process and thus make possible future clinical application of this important tool. Various isolation techniques, including mechanical dissolution by homogenization, pipetting, shaking, and chemical dispersion by citrate, EDTA, EGTA, and sodium tetraphenyl boron have been used in the early stages of hepatocyte harvest development. None of these techniques produced a high yield of viable hepatocytes. For a hepatocyte transplantation method to be useful, the technique must be simple, it should allow early engraftment, it should allow transplantation of an adequate number of hepatocytes, and transplanted hepatocytes should be able to express differentiated functions in vivo for prolonged periods of time. Although significant progress has been achieved in isolated hepatocyte transplantation, the optimal site of implantation has not been determined. In searching for the best transplantation technique and location, hepatocytes have been introduced virtually into every body site, including liver, spleen, thymus, testes, brain, pancreas, lungs, kidneys, peritoneal cavity, subcutaneous tissues, fat pads, and other locations.

Fulminant hepatic failure is a devastating disease that, despite recent therapeutic advances, continues to be associated with high morbidity and mortality. Orthotopic liver transplantation has emerged as the sole modality of treatment that significantly improves survival. However, the critical shortage of donors precludes timely transplantation for all patients. Consequently, almost half of all patients with fulminant hepatic failure die before a graft becomes available. This has generated interest in developing a system that would support patients until either native liver regeneration occurs or an optimal donor liver can be found. Investigators have used biological, artificial and bioartificial techniques in an attempt to improve survival in liver failure. This article reviews the history, the current state of the art and future directions of artificial liver support.

The development of an artificial liver is a major challenge because the liver controls, or has an effect on, almost every aspect of metabolism, blood coagulation and fibrinolysis, the immune system, endocrine responses and detoxification. It is believed that devices utilising intact viable hepatocytes have the greatest potential to provide liver support at this level of complexity. In recent years, advances in hepatocyte isolation and culture techniques, better understanding of hepatocyte-matrix interactions, availability of new biomaterials, and improved hollow-fiber technology have resulted in the development of such hepatocyte-based systems. Some of these bioartificial livers (BAL), including the one developed by our group, are currently being tested in the clinical setting [1–5]. In patients with severe acute hepatic failure, their role is to provide physiologic support and to “buy time” until a new liver is procured and to render patients stable and more likely to survive transplantation. There will also be patients who suffer from severe, but potentially reversible liver injury, in whom BAL therapy can potentially serve as the definitive treatment.

To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor β1 (TGF-β1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 × 107 cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 ± 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-β1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 ± 8.5 vs. 15.5 ± 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-β1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-β1 levels in rats rendered anhepatic. (Hepatology 1998;28:1365-1370.)

For centuries, many cultures have described mythical creatures with bodies that combined human and animal features, often the result of violating taboos. This study attempted to investigate the beliefs of transplant patients about xenografting. A survey was given to 100 patients ranging in age from 17 to 74 years old, with 65 men and 35 women, including 72 whites, 18 Hispanics, 5 African Americans, and 4 Asian Americans. The subjects included liver, heart, kidney, lung, and multi-organ transplant patients. The patients were not aware of plans for xenografting at the center under study. Eighty patients agreed with xenografting in an emergency situation. Ten subjects replied, "under no circumstances." Ninety percent believed animal research has advanced medical science. In descending order, the patients preferred human (96%), monkey (44%), mechanical (43%), pig (42%), or dog (34%) organs. Twenty-four patients thought a xenograft would change their appearance, personality, or eating or sexual habits. Twenty patients believed animals have souls. The patients also documented any ethical concerns about xenografting.

Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation in experimental animal models with genetic disorders of liver metabolism and liver failure. 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, expansion of transplanted cell population, and gene transfer requiring active DNA synthesis. The present study was undertaken to evaluate the proliferative capacity of fetal and adult rat hepatocytes under standardized culture conditions. Fetal (20 days of gestation) and adult hepatocytes were cultured in serum-free media at low densities and treated with growth factors. Proliferation was assessed by [3H]-thymidine incorporation and cell cycle analysis by flow cytometry. In nonstimulated cells, DNA synthesis at 4 h was about × 100 higher and after 10 days in culture ×20 higher in fetal compared to adult hepatocytes. When epidermal growth factor (EGF) was added, maximal DNA synthesis in fetal hepatocytes was seen at 48 h, whereas in adult hepatocytes at 72 h. For adult hepatocytes, the average increase compared to untreated cells was × 13.8 with EGF, ×18.5 with transforming growth factor alpha (TGF-α), and ×7.6 with hepatocyte growth factor (HGF). For fetal hepatocytes, the increase was twofold with either EGF, TGF-α or HGF. EGF-, TGF-α- and HGF-dependent DNA synthesis was inhibited by transfroming growth factor beta-1 (TGF-β1) in both fetal and adult hepatocyte cultures; this antiproliferative effect was significantly stronger in adult hepatocyte cultures. With cyclosporine, EGF-, TGF-α- and HGF-dependent DNA synthesis in fetal hepatocyte cultures decreased by 36–46%, whereas in adult hepatocytes by 19–27%. These results show that in contrast to adult hepatocytes, fetal hepatocytes have high spontaneous proliferative activity independently of growth factors and are relatively resistant to the inhibitory effect of TGF-β1. It was also found that cyclosporine suppresses proliferation of cultured fetal hepatocytes.

Background: Limitation of β-cell growth after intraportal islet transplantation plays an important role in graft failure. To induce transplanted β-cell proliferation, we studied the effect of compensatory liver growth in diabetic rats that had a subtherapeutic islet mass previously injected into the liver. Methods: Syngeneic rats were used as islet donors or recipients; diabetes was induced by streptozocin. Three groups of streptozocin-treated rats were studied. In group 1, 250 islets were selectively transplanted into the posterior liver lobes and 10 days later anterior portal branch ligation (PBL) was performed (n = 18); in group 2, 250 islets were transplanted into the posterior lobes and 10 days later sham PBL was performed (n = 13); in group 3, rats underwent a sham transplantation and PBL (n = 6). Nonfasting blood glucose levels and body weight were monitored. Six rats in groups 1 and 2 were killed 48 hours after PBL, liver sections were stained for proliferating cell nuclear antigen, and islet cell labeling index was calculated. The remaining rats were killed 30 days later. Liver compensatory growth or atrophy was calculated and morphometric determination of β-cell area was assessed on insulin-immunostained sections of the liver. Results: In group 1 rats killed 48 hours after PBL, islet cell labeling index was significantly higher than in group 2 (p < 0.0001). After PBL, we observed normalization of nonfasting blood glucose levels in 10 of 12 rats. At 30 days, posterior liver lobes showed compensatory growth (218.5% ± 18.6%) accompanied by atrophy of the anterior lobes; morphometric study of liver-engrafted islets showed a significant increase of individual β-cell area, compared with group 2 (p < 0.0001). In groups 2 and 3, normoglycemia was not achieved. Conclusions: In streptozocin-diabetic rats, normoglycemia was restored after transplantation of a subtherapeutic islet mass, followed by PBL-induced liver regeneration. Histologic and morphometric results indicating islet cell proliferation suggest that compensatory liver growth might have induced a hypertrophic/hyperplastic response in the intraportally transplanted β-cells. (Surgery 1998;123:398-406.)

OBJECTIVE: The purpose of this study was to develop a bioartificial liver (BAL) to treat patients with severe liver failure until they can be either transplanted or recover spontaneously. SUMMARY BACKGROUND DATA: Severe acute liver failure is associated with high mortality. Liver transplantation has emerged as an effective therapy for patients who did not respond to standard management. However, because of the donor organ shortage and urgent need for transplantation, many patients die before they can be transplanted and others do not survive after transplantation, primarily because of intracranial hypertension. METHODS: Three groups of patients with severe acute liver failure were treated with the BAL. In group 1 (n = 18) were patients with fulminant hepatic failure (FHF), in group 2 (n = 3) were patients with primary nonfunction (PNF) of a transplanted liver, and in group 3 (n = 10) were patients with acute exacerbation of chronic liver disease. Patients in groups 1 and 2 were candidates for transplantation at the time they entered the study, whereas patients in group 3 were not. RESULTS: In group 1, 16 patients were "bridged" successfully to transplantation, 1 patient was bridged to recovery without a transplant, and 1 patient died because of concomitant severe pancreatitis. In group 2, all patients were bridged successfully to retransplantation. In group 3, two patients were supported to recovery and successful transplants at later dates; the other eight patients, although supported temporarily with the BAL, later died because they were not candidates for transplantation. CONCLUSIONS: The authors' clinical experience with the BAL has yielded encouraging results. A randomized, controlled, prospective trial (phase II-III) is being initiated to determine the efficacy of the system.

Whole organ transplantation is the only clinically effective method of treating fulminant hepatic failure and chronic liver failure due to specific genetic, hepatocellular, and anatomic defects of liver function. However, wider application of liver transplantation is limited by shortage of organ donors, high cost, a relatively high morbidity rate, and need for life long immunotherapy. As a result, investigators have attempted to develop alternative methods to treat liver insufficiency. These ranged from use of plasma exchange to use of detoxification columns and extracorporeal devices loaded with various liver tissue preparations. Several liver support systems were developed in the 1950s and 1960s, but it was not until recently that advances in hepatocyte isolation and culture, improved understanding of hepatocyte-matrix interactions, availability of new biomaterials, improved hollow fiber technology, and better understanding of flow and mass transport across semipermeable membranes resulted in the development of a new generation of liver assist devices. Some of these devices are being tested in the clinical setting. In this article, the authors review past experience with liver support systems, critically examine the current status of the field by drawing primarily on their own experience, and attempt to speculate on the future direction of liver assist system development.

The optimal site for implantation of isolated hepatocytes has not been established. We have developed a novel technique which allows repeated infusion of hepatocytes into the portal system via an indwelling catheter. Seven Nagase Analbuminemic rats (NAR) underwent single intra-portal infusion of 2 × 107 isolated normal albumin-producing rat hepatocytes. Another seven NAR rats underwent placement of indwelling catheters into the portal venous system via the gastroduodenal vein. Each of them received six batches of 5 × 106 normal albumin producing hepatocytes. Seven control NAR rats were infused repeatedly (intraportally) with saline only. Plasma albumin (ELISA) showed significant increase in experimental animals and was more pronounced (p < 0.05) in rats transplanted repeatedly than in those given a single dose of cells. Immunohistochemical staining of the liver sections confirmed the presence of transplanted albumin producing hepatocytes. Rats transplanted with a single large batch of isolated hepatocytes showed liver tissue damage, whereas those subjected to repeated cell infusions had normal liver histology. We have developed a novel intraportal transplantation method which allows successful engraftment of a large number of isolated hepatocytes.

Renewed interest in the transplantation of isolated hepatocytes into the liver as a potential therapy for liver disease has stimulated the development of methods for the identification of donor cells within the recipient organ. We describe a method for cellular tagging and in vivo identification of intraportally transplanted hepatocytes using an intracellular fluorescent dye, 5(6)-carboxyfluorescein diacetate, succinimidyl-ester (CFSE). Rat and porcine hepatocytes were isolated and labelled with CFSE. The optimal conditions for labelling consisted of a buffered saline suspension of hepatocytes (5 × 106 cells/mL) in 20.0 μM CFSE incubated for 15 min at 37°C. In vitro, labelled hepatocytes were cultured either on fibronectin-coated chamber slides or in culture flasks. Cultures were evaluated in situ by fluorescence photomicrography or by fluorescence-activated cell sorting (FACS) after cell detachment. Cell viability was assessed serially and cultured, labelled hepatocytes retained the dye for up to 3 wk (last day of study). CFSE did not effect hepatocyte viability and there was no evidence of intercellular diffusion of the dye. In vivo, syngeneic Lewis rats underwent selective portal vein infusion of freshly isolated, labelled hepatocytes (2.0 × 107 cells/2.0 mL saline/animal) into the posterior liver lobes. All recipients were sacrificed 48 h and 96 h later and their livers examined. Transplanted hepatocytes were identified by fluorescence microscopy in tissue sections and by FACS following collagenase digestion of the liver tissue. CFSE persisted in a population of viable, engrafted hepatocytes. FACS analysis demonstrated that 9 ± 3% of the hepatocytes in the posterior liver lobes were labelled 48 and 96 h after transplantation. At 96 h following transplantation, multiple engrafted hepatocytes could be observed by fluorescence microscopy around the central veins. CFSE labelling allows for both in vitro identification and in vivo localization of donor hepatocytes. Furthermore, it appears to be more stable and specific for labelling hepatocytes than other tested dyes (especially DiI).

OBJECTIVE: The authors developed an extracorporeal liver support system and tested its efficacy in experimental animals with liver failure. The first clinical use of this system to treat a patient with liver failure is reported. SUMMARY BACKGROUND DATA: Multiple attempts have been made, ranging from plasma exchange to use of charcoal columns, to develop liver support systems for treating patients with acute severe liver failure. None of these systems has achieved wide clinical use. There is a need for providing liver support as a "bridge" to transplantation and for treating patients with potentially reversible liver dysfunction. METHODS: A hybrid liver support system has been developed consisting of plasma perfusion through a charcoal column and a porous hollow fiber module inoculated with 5 x 10(9) matrix-attached hepatocytes. The system was tested in dogs with ischemic liver failure (n = 7) who underwent plasmapheresis; a control group (n = 6) underwent charcoal perfusion alone. A patient with liver failure was treated with this hybrid system. RESULTS: After 6 hours of hybrid liver support treatment, animals had significantly decreased serum ammonia and lactate levels, increased glucose level, normal prothrombin time, and increased systolic blood pressure compared with controls treated with charcoal perfusion alone. Use of the system to treat a patient was well tolerated with evidence of clinical improvement. CONCLUSIONS: Plasma perfusion through a system consisting of a charcoal column and matrix-attached porcine hepatocytes had significant beneficial effects in animals with liver failure and was well tolerated by a patient with liver failure.

Introduction: The effects of cold ischemic preservation and degree of donor-recipient histoincompatibility on graft coronary arteriosclerosis(GCA)were examined using a rat heterotopic cardiac transplant model. Methods: Three donor-recipient rat strain combinations were utilized. Lewis-F344 rats which vary slightly in class I Major Histocompatibility (MHC) Loci genes, ACI-Lewis which vary significantly in MHC class I and II genes, and Lewis-Lewis (control), donor-recipient strain combinations were subjected to varying lengths of cold ischemic preservation prior to transplantation(0, 4, and 24h in University-of-Wisconsin solution at 4’C).There were 9 rats per group.Grafts were harvested at 90 days post-op. and sections of the epicardial arteries were examined (H&E and van Gieson elastin stains).Intimal area ratio (IAR = intimal area/area within internal elastic lamina), intimal thickness score (ITS-based on % luminal compromise, perimeter of involvement), and rejection score (RS) were determined for each specimen using computerized image morphometry. Results: IAR, ITS and RS were significantly higher at 24hrs vs. 0hrs (p ∗ . IAR (hr) LEW-LEW (%) LEW-F344 ∗ (%) LEW-ACI ∗ (%) 0 0.38 ± 0.15 0.33 ± 0.16 0.57 ± 0.16 4 0.34 ± 0.11 0.42 ± 0.21 0.72 ± 0.29 24 0.41 ± 0.18 0.63 ± 0.14 ITS (hr) 0 0.36 ± 1.00 0.67 ± 0.84 2.00 ± 1.40 4 0.14 ± 0.29 0.82 ± 0.94 2.80 ± 2.10 24 0.91 ± 0.91 1.90 ± 0.87 RS (hr) 0 0.11 ± .33 3.30 ± 0.89 3.10 ± 1.4 4 0.56 ± .88 3.80 ± 0.71 5.00 ± 0.0 24 1.10 ± 1.4 4.50 ± 0.53 4.40 ± .71 ∗ p Conclusions: GCA is significantly increased by greater MHC disparity and longer cold ischemic times. GCA may be caused by an Immune-mediated antigen dependent vasculopathy secondary to histoincompatability. In the control group, longer cold ischemic times did not have a significant effect on GCA, suggesting that better histocompatibility may mitigate the adverse effects of longer ischemic times.