Robin D. Hughes

Hepatocyte transplantation for inherited factor VII deficiency.

Hepatocyte transplantation has been investigated in patients with liver-based metabolic disorders and acute liver failure. We report the first use of hepatocyte transplantation in two brothers with severe inherited coagulation factor VII deficiency. Patient 1 received a total of 1.09x10(9) cryopreserved hepatocytes, and patient received 2.18x10(9) fresh and cryopreserved hepatocytes through a Hickman line inserted in the inferior mesenteric vein. Infusion of isolated human hepatocytes improved the coagulation defect and markedly decreased the requirement for exogenous recombinant factor VII (rFVIIa) to approximately 20% of that before cell transplantation. In both patients, episodes of line sepsis were associated with an increase in rFVIIa requirement. Six months posthepatocyte transplantation, higher rFVIIa doses were required, suggesting loss of transplanted hepatocyte function. Because of increasing problems with venous access and long-term uncertainty of the efficacy of hepatocyte transplantation, orthotopic liver transplantation was performed successfully in both cases.

Human hepatocyte transplantation: current experience and future challenges.

Hepatocyte transplantation has shown potential as an additional treatment modality for certain diseases of the liver. To date, patients with liver-based metabolic disorders or acute liver failure have undergone hepatocyte transplantation in several centers around the world. Results from individual patients are promising, especially for the treatment of liver-based metabolic disorders, but the lack of controlled trials makes the interpretation of the findings difficult. The current source of isolated hepatocytes is donor organs that are unused or deemed unsuitable for liver transplantation. Hence the major challenge that this field is facing is the limited supply of donor organs that can provide good quality cells. Alternative sources of cells, including stem cells, are under investigation. This Review discusses the current bench-to-bedside issues and future challenges that need to be faced to allow the wider application of hepatocyte transplantation.

Hepatocyte transplantation for liver-based metabolic disorders.

SummaryHepatocyte transplantation is being investigated as an alternative to orthotopic liver transplantation in patients with liver-based metabolic disorders. The progress made in this field to date is reviewed. Protocols have been developed using collagenase perfusion to isolate human hepatocytes from unused donor liver tissue. Hepatocytes with a high viability can often be obtained and can be cryopreserved for later use, though with loss of function on thawing. For clinical use, hepatocytes must be prepared in clean GMP conditions with cells meeting criteria of function and lack of microbial contamination before patient use. Hepatocytes are infused intraportally into the patients liver, where a proportion of cells will engraft and replace the deficient metabolic function without the need for major surgery. Twenty patients have now received hepatocyte transplantation, including eight children at Kings College Hospital. There was a range of aetiologies of liver disease: familial hypercholesterolaemia, Crigler–Najjar syndrome type 1, urea cycle defects, infantile Refsum disease, glycogen storage disease type Ia, inherited factor VII deficiency and progressive familial intrahepatic cholestasis type 2. Clinical improvement and partial correction of the metabolic abnormality was observed in most cases. Considerable progress has been made in developing the technique, but hepatocyte transplantation is limited by the available supply of liver tissue. Hepatocytes derived from stem cells could provide alternative sources of cells in the future.

Current status of hepatocyte transplantation.

Hepatocyte transplantation (HT) has been performed in patients with liver-based metabolic disease and acute liver failure as a potential alternative to liver transplantation. The results are encouraging in genetic liver conditions where HT can replace the missing enzyme or protein. However, there are limitations to the technique, which need to be overcome. Unused donor livers to isolate hepatocytes are in short supply and are often steatotic, although addition of N-acetylcysteine improves the quality of the cells obtained. Hepatocytes are cryopreserved for later use and this is detrimental to metabolic function on thawing. There are improved cryopreservation protocols, but these need further refinement. Hepatocytes are usually infused into the hepatic portal vein with many cells rapidly cleared by the innate immune system, which needs to be prevented. It is difficult to detect engraftment of donor cells in the liver, and methods to track cells labeled with iron oxide magnetic resonance imaging contrast agents are being developed. Methods to increase cell engraftment based on portal embolization or irradiation of the liver are being assessed for clinical application. Encapsulation of hepatocytes allows cells to be transplanted intraperitoneally in acute liver failure with the advantage of avoiding immunosuppression. Alternative sources of hepatocytes, which could be derived from stem cells, are needed. Mesenchymal stem cells are currently being investigated particularly for their hepatotropic effects. Other sources of cells may be better if the potential for tumor formation can be avoided. With a greater supply of hepatocytes, wider use of HT and evaluation in different liver conditions should be possible.

Improving the techniques for human hepatocyte transplantation: Report from a consensus meeting in London

On September 6 and 7, 2009 a meeting was held in London to identify and discuss what are perceived to be current roadblocks to effective hepatocyte transplantation as it is currently practiced in the clinics and, where possible, to offer suggestions to overcome the blocks and improve the outcomes for this cellular therapy. Present were representatives of most of the active clinical hepatocyte transplant programs along with other scientists who have contributed substantial basic research to this field. Over the 2-day sessions based on the experience of the participants, numerous roadblocks or challenges were identified, including the source of cells for the transplants and problems with tracking cells following transplantation. Much of the discussion was focused on methods to improve engraftment and proliferation of donor cells posttransplantation. The group concluded that, for now, parenchymal hepatocytes isolated from donor livers remain the best cell source for transplantation. It was reported that investigations with other cell sources, including stem cells, were at the preclinical and early clinical stages. Numerous methods to modulate the immune reaction and vascular changes that accompany hepatocyte transplantation were proposed. It was agreed that, to obtain sufficient levels of repopulation of liver with donor cells in patients with metabolic liver disease, some form of liver preconditioning would likely be required to enhance the engraftment and/or proliferation of donor cells. It was reported that clinical protocols for preconditioning by hepatic irradiation, portal vein embolization, and surgical resection had been developed and that clinical studies using these protocols would be initiated in the near future. Participants concluded that sharing information between the groups, including standard information concerning the quality and function of the transplanted cells prior to transplantation, clinical information on outcomes, and standard preconditioning protocols, would help move the field forward and was encouraged.

Ammonia and the neutrophil in the pathogenesis of hepatic encephalopathy in cirrhosis

Hepatic encephalopathy (HE) constitutes a neuropsychiatric syndrome which remains a major clinical problem in patients with cirrhosis. In the severest form of HE, cirrhotic patients may develop varying degrees of confusion and coma. Ammonia has been regarded as the key precipitating factor in HE, and astrocytes have been the most commonly affected cells neuropathologically. Although the evidence base supporting a pivotal role of ammonia is robust, in everyday clinical practice a consistent correlation between the concentration of ammonia in the blood and the manifest symptoms of HE is not observed. More recently the synergistic role of inflammation and infection in modulating the cerebral effects of ammonia has been shown to be important. Furthermore, it has been recognized that infection impairs brain function both in the presence and absence of liver disease. Thus it could be postulated that in the presence of ammonia, the brain is sensitized to a systemic inflammatory stimulus and is able to elicit an inflammatory response involving both proinflammatory and neurotransmitter pathways. Ammonia is not only directly toxic to astrocytes but induces neutrophil dysfunction with the release of reactive oxygen species, which contribute to oxidative stress and systemic inflammation. This may further exacerbate the cerebral effects of ammonia and potentially reduce the capacity of the neutrophil to fight microbial attack, thus inducing a vicious circle. This evidence supports the neutrophil in addition to ammonia as being culpable in the pathogenesis of HE, making the neutrophil a target for future anti‐inflammatory therapeutic strategies in addition to ammonia lowering therapies. (HEPATOLOGY 2010.)

Screening of Hepatoprotective Plant Components using a HepG2 Cell Cytotoxicity Assay

Identification of the active components of plants with hepatoprotective properties requires screening of large numbers of samples during fractionation and purification. A screening assay has been developed based on protection of human liver‐derived HepG2 cells against toxic damage.

One liver, three recipients: segment IV from split-liver procedures as a source of hepatocytes for cell transplantation.

Hepatocyte transplantation is emerging as a possible treatment for patients with acute liver failure and liver-based metabolic disorders. With the limited availability of donor tissue, it is important to find new sources of liver tissue for isolation of high-quality hepatocytes. Segment IV with or without the caudate lobe was removed during three split-liver procedures. Hepatocytes were isolated from the tissues using a collagenase perfusion technique under strict sterile conditions. The mean number of hepatocytes that were isolated was 5.14×108 cells with a mean cell viability of 89%. Two of the hepatocyte preparations were used for cell transplantation in a 1-day-old boy with an antenatal diagnosis of a severe urea cycle defect caused by ornithine transcarbamylase deficiency. The six recipients of split-liver grafts demonstrated no complications related to the removal of segment IV. Segment IV with or without the caudate lobe obtained from split-liver procedures is potentially a good source of high-quality hepatocytes for cell transplantation.

Human hepatocyte isolation and relationship of cell viability to early graft function.

Hepatocyte transplantation is emerging as an additional modality of treatment for patients with acute liver failure or liver-based metabolic disorders. The procedure requires isolation of high-quality hepatocytes from unused donor livers. Hepatocytes were isolated from 20 donor livers (11 right lobes, 3 left lateral segments, 6 whole livers) using a collagenase perfusion technique. Cell viability (median 56%, range 13–95%) and yield (median 1.4 × 109 cells, range 2.0 × 106–1.8 × 1010 cells) varied according to the tissue available. Fatty livers rejected for transplantation gave lower cell viability (median 45%, range 25–59%). There was a significant correlation between age of donor (median 21 years, range 7–66 years) and viability of isolated hepatocytes in vitro (r = −0.683, p = 0.001). The 13 segments of livers were from reduced/split grafts used for clinical transplantation in 9 children and 4 adults. There was no significant correlation between in vitro cell viability and clinical parameters including intensive care stay, serum aspartate aminotransferase, and international normalized ratio (in the first 7 days), and allograft rejection or other early posttransplant complications, in patients transplanted with the corresponding tissue.

Predicting survival in fulminant hepatic failure using serum Gc protein concentrations.

Plasma Gc protein sequesters actin released into the circulation after massive hepatocyte necrosis, but is greatly depleted in the process. In fulminant hepatic failure (FHF), Gc is present in serum both as a complex with actin and as unbound protein, the latter becoming completely exhausted in those patients with the most severe FHF. In the present study, 47 consecutive patients with FHF, 39 of whom were the result of acetaminophen (AC) overdose, were evaluated to determine whether measurement of Gc protein levels could be used to predict survival. Using serum samples obtained shortly after admission as well as later samples, levels for total Gc protein, percentage of Gc complexed with actin, and calculated unbound Gc remaining in serum were compared for survivors and those who died of their illness. The most marked changes were present in unbound Gc levels in nonsurvivors, the mean of which for follow‐up samples was 10% of normal mean values, as compared with 23% of normal mean values in those who survived (P<.01). Using a cutoff value for unbound Gc protein of ±34 μg/mL to predict survival, outcome was correctly predicted in 32 of 47 (68%) patients using early samples, and in 24 of 27 (89%) patients using later sera. No differences were observed between values and/or outcome in AC and non‐AC cases. Measurement of Gc protein level correctly predicted all patients dying of hepatic failure. This single measurement compares favorably with multifactorial predictive models, such as the Kings College model, and might be a useful test for patients being considered for transplantation. (Hepathology 1995;21:101‐105).