HEPATOCYTE TRANSPLANTATION FOR LIVER BASED METABOLIC DISORDER
Anil Dhawan*, Ragai R Mitry PhD**, Robin D Hughes PhD***
Institute of Liver Studies, King's College London School of Medicine, King's College Hospital, London.*, Institute of Liver Studies, King's College London School of Medicine, King's College Hospital, London.**, Institute of Liver Studies, King's College London School of Medicine, King's College Hospital, London. ***
Introduction: Liver transplantation is the accepted method of treatment for liver based metabolic disorders. Advances in surgical techniques, now allow the use of auxiliary liver transplantation in the management of patients with liver-based metabolic defects such as Crigler-Najjar syndrome type I, urea cycle defects, and familial hypercholesterolemia. The success of auxiliary liver transplantation in humans (1) has supported the observation in animal experiments that relatively small amounts of liver tissue can provide sufficient function to correct the underlying metabolic defects. This has further increased the interest in using human hepatocytes for cell transplantation in the management of liver-based metabolic conditions.

There are a number of potential advantages of hepatocyte transplantation if the technique can be proved successful. It avoids the risks and undertaking of major surgery and as the native liver is still in place can help bridge a patient to whole organ transplant or leave the option of gene therapy if and when it becomes available in future. Hepatocyte transplantation has been used as a treatment for liver based metabolic diseases such as Crigler-Najjar syndrome type I (4), glycogen storage disease type 1a (5), urea cycle defects (6, 7) & congenital deficiency of coagulation factor VII. (8).

Isolation of Hepatocytes: There are well-established protocols for isolation of human hepatocytes from unused segments of donor livers (7, 8), based on collagenase digestion of cannulated liver tissue at 37C. After isolation the hepatocytes are purified by centrifugation and assessed for cell viability and yield. Hepatocytes are better used fresh for cell transplantation as function deteriorates even when kept at 4C for more than 24 hours. For longer-term storage hepatocytes are cryopreserved, but often-insufficient viable hepatocytes are recovered on thawing. The best results are currently obtained by cryopreservation in a mixture of the organ preservation media University of Wisconsin solution and 10% dimethylsulfoxide using a controlled-rate cell freezer (9). The frozen hepatocytes can then be stored at -140 o C until required for clinical use.

Sources of Liver Tissue: The main source of human hepatocytes for transplantation is from unused donor livers and liver segments from which hepatocytes of high viability can be isolated. The quality of cells obtained from livers rejected for clinical transplantation is often not sufficient for clinical hepatocyte transplantation (1). Over the 5-year period since the start of the project at King's, a total of 120 livers or liver segments have been processed to prepare hepatocytes. Overall the mean viability of the hepatocytes obtained was 65%. Newer sources being livers obtained form non heart beating donors, marginal graft (Steatotic, liver trauma) and segment four with or without caudate lobe from split liver technique where one liver is used for two recipients. (one liver there).

GMP Laboratory: An aseptic environment is required to prepare cells on a large scale in conditions of Good Manufacturing Practices (GMP), so that the isolated cells are safe to be administered to humans. The Cell Isolation Unit is a purpose-built facility consisting of four interconnected rooms. Air entering the laboratory passes through HEPA filters to remove any particles and an air-handling unit maintains a temperature-controlled environment inside the Unit. There is a gradient of air pressures between the rooms, which maintains a positive air pressure differential, with the highest pressure in the Aseptic Room, where the tissue processing is performed. Operators have to wear sterile clean-room suits. Standard operating procedures (SOP) are followed for all aspects of work in the Cell Isolation Unit. A comprehensive quality control system, which monitors all aspects of laboratory performance, is in operation.

The Cell Isolation Unit at King's is accredited by the Medicines and Healthcare products Regulatory Agency (MHRA) as a Tissue Bank, being the only one in the UK for isolation of human hepatocytes. Cryopreserved hepatocytes for clinical use are stored in cell freezer bags in the vapour phase of liquid nitrogen inside an automated storage container. This hepatocyte bank means that hepatocytes are available for immediate use in urgent cases of pediatric liver disease.

All donated organs/tissues are screened for viral infection, including hepatitis and HIV according to the UK Transplant Service criteria for whole organ transplantation. The final cell products are screened for microbiological contamination. The hepatocytes can only be only released for cell transplantation if the quality is suitable for hepatocyte transplantation (viability> 60%) and no microorganisms are present (Figure 1). To date 74 isolations have been performed under sterile conditions in the Unit.

Earlier Pre-clinical and Clinical Studies
The reader is directed towards excellent recent reviews, which fully describe the background to the field of hepatocyte transplantation. (15, 16, 17). A large number of studies carried out in animal models of human liver disease established the feasibility and efficacy of hepatocyte transplantation into various sites such as liver, spleen, pancreas, peritoneal cavity, and sub-renal capsule. The majority of these studies showed improvement of the biochemical abnormalities, there were concerns whether sufficient number of hepatocytes could be transplanted to normalize function.

The animal studies encouraged human clinical studies of hepatocyte transplantation initially in treatment of patients with acute liver failure. Thirty patients from 6 centres were reviewed by Strom et al (18). Hepatocytes were infused either fresh or after cryopreservation into the splenic artery or portal vein of patients with liver failure. The number of hepatocytes administered was in the range of 107 to 1010 cells. Up to a maximum of 5% of normal liver mass was infused and it is questionable whether this is a sufficient quality to replace the massive lost function in acute liver failure. In these studies a reduction in ammonia and bilirubin levels and improvements in hepatic encephalopathy were reported.
The cell requirement for transplantation may be lower in inherited metabolic liver diseases where the aim is to replace a single deficient enzyme. One of the key early reports was from Fox and colleagues in 1998 (4), who reported treatment of a 10 year old girl with Crigler-Najjar syndrome type I, which showed there was sustained stable expression of bilirubin-UDP-glucuronosyl transferase activity in the liver up to 9 months post-hepatocyte transplantation. The overall experience of hepatocyte transplantation in liver-based genetic liver disease mainly in children including that at King's College Hospital is shown in Table 1.

Clinical Hepatocyte Transplantation at KCH: The focus has been on children with liver-based metabolic disorders. Firstly ABO blood group compatible hepatocytes which meet our criteria for clinical use are needed. It is best to use fresh cells, but these can be supplemented with thawed cryopreserved cells. For transplantation into the liver up to 109 cells per treatment are infused via the portal vein either through an in dwelling catheter in to a branch of inferior mesenteric vein or one placed transhepatically under X-ray screening. It is essential to monitor effects on the hepatic circulation by measuring the hepatic portal venous pressure to avoid the risk of portal hypertension. The aim is to perform repeat cell infusions to provide approximately 10% of total liver mass. It is currently not possible to determine the proportion of administered cells with accuracy, which survive and engraft into the liver and this is an important area for research. The immunosuppression regimen is similar to that given to whole organ transplantation recipients, currently based on tacrolimus and prednisolone. It is likely that this needs to be further optimized for cell transplantation and methods to determine cell rejection developed.

Once the cell isolation methods in the Cell Isolation Unit were established and validated, the first patient was treated in late 2002. A child with an antenatal diagnosis of omithine transcarbamylase deficiency received infusion of hepatocytes through an umbilical vein catheter. The clinical course after transplantation of a total of 1.9 billion hepatocytes showed a significant improvement in terms of maintenance of blood ammonia levels, and an increase in serum urea, while on normal protein diet. Long-term uncertainties about the efficacy of hepatocytes transplantation led to successful auxiliary left lobe orthotopic liver transplantation at 7 months of age. The patient remains well with normal growth and development at 2.5 years of age at the last follow-up.

The next patients were two brothers will severe inherited coagulation factor VII deficiency who received the first use of hepatocyte transplantation in such a condition. Both children received hepatocytes (a total of 1.1 & 2.2 billion) through a Hickman line inserted in the inferior mesenteric vein. Infusion of isolated human hepatocytes improved the coagulation defect ad markedly decreased the requirement for exogenous factor VII (rfVIIa) to ~20% of the before cell transplantation (18). Importantly, one patient received exclusively cryopreserved hepatocytes, indicating that function, at least related to clotting factors, is maintained after cryopreservation. Six months post-hepatocyte transplantation in both cases higher rFVIIa doses were required suggesting loss of transplanted hepatocyte function, possibly associated with sepsis. With hepatocyte replacement in FVII deficiency there is no selective advantage for the transplanted hepatocytes to repopulate the otherwise healthy host liver. Due to increasing problems with venous access and long-term uncertainly of the efficacy of hepatocyte transplantation and has shown similar improvement in coagulation parameters with reduction in factor VII requirement at follow up of 3 months after the procedure.

Two other children treated in 2003 were suffering from progressive familial intrahepatic cholestasis (PFIC2) a genetic disease (23) where the liver is lacking the bile salt export pump (BSEP). As a result of this defect, bile flow is severely impaired and patients rapidly develop liver cirrhosis and need liver transplantation. Both children with PFIC2 received infusion of fresh hepatocyte through a portal vein catheter. Each child received a single infusion of one third of a billion fresh hepatocytes. The rationale was that the injected hepatocytes would have a selective advantage over their own defective hepatocytes to repopulate the native liver, as had been shown in a mouse model of progressive familial intrahepatic cholestasis (24), where up to 70% of host hepatocytes were replaced with donor cells. However, both patients had a whole liver transplant 5 and 14 months later respectively as their liver had continued to deteriorate. Existing fibrosis in the hepatic sinusoids is likely to have impaired engraftment of transplanted hepatocytes into the liver structure. Earlier treatment, if feasible, may be the best approach in this situation.

     Two children age 18 months and 3 years have received hepatocyte transplantation for Crigler-Najjar syndrome. First patient showed reduction of bilirubin by more than 50% with reduction in phototherapy but decrease in function of transplanted hepatocytes was observed after 7 months. His immunosuppression was stopped and received liver transplant 4 months later. Interestingly the explant liver showed evidence of engraftment on short tandem repeat assay for donor HLA and the bile specimen obtained at the operation showed bile conjugates indicating presence and function of transplanted cells up to 8 months. A three year old girl who underwent hepatocyte transplantation for Crigler-Najjar syndrome 2 months back has also shown decrease in bilirubin by 30% and is currently under follow up on immunosuppression.

The Future : Considerable progress has been made in bringing hepatocyte transplantation to the bedside. However, the promise of hepatocyte transplantation from animal experiments performed in ideal experimental conditions usually without the need for immunosuppression has not yet been fully borne out. There are a number of areas for improvement and development.

In terms of the limited supply of livers currently available to isolate hepatocytes, fatty livers are those most commonly rejected for clinical transplantation and represent an important potential source of hepatocytes. Thus improvement of the outcome of isolation and purification of hepatocytes from fatty livers is an important goal, so that these cells could be used for transplantation. There is a need to improve the storage of hepatocytes, both for longer periods in the cold so they can be used fresh after a number of days and also better cryopreservation protocols for longer-term storage. Some progress has been made in our laboratories in understanding the mechanisms of hepatocyte freezing damage and preventing the loss of hepatocyte function after cryopreservation by development of protocols incorporating cryo/cytoprotectant agents (25). It is clear that many injected cells do not engraft into the recipient liver and are either cleared by the reticuloendothelial system or lose viability during this early phase. The outcome of hepatocyte transplantation would benefit from methods to enhance engraftment and subsequent repopulation of the liver, although the options for this in man will be limited.

There is no doubt that stem cells/stem cell-derived hepatocytes should offer the potential to overcome the current limitations of both supply of hepatocytes and the extent of repopulation of the liver after transplantation (26). Sources of stem cells for therapy could be fetal livers, cord blood, embryos, and possibly bone marrow. There is a focus of research worldwide on liver stem cell biology and there is no doubt that there are many hurdles to cross before clinical application will be possible. If these are overcome, then stem cells could be differentiated into all types of liver cells, be easier to cryopreserve and thaw with good function, have proliferative capacity in vitro and in vivo, and may be less immunogenic. As another approach, hepatocytes could be genetically manipulated in vitro to upregulate gene expression to enhance enzymatic activity and function eg. Ornithine transcarbamylase, bilirubin glucuronyl transferase or possibly render them immune-tolerant. Methods to transfect hepatocytes are available and those using non-viral rectors are of particular interest as they avoid risks due to viral vectors.

     In summary considerable experience has been gained so far in the handling of hepatocytes and techniques for hepatocyte transplantation allowing clinical hepatocyte transplantation. This will give a good basis for the future application of new technologies particularly those based on stem cells which it is hoped will increase the utilization of cell transplantation.

Acknowledgements: We would like to thank the Children's Liver Disease Foundation and King's College Hospital Charitable Trust for financial support. This work would not have been possible without the contributions of Mr. Nigel Heaton and Mr. Mohamed Rela transplant surgeons, Dr. John Korani, Consultant Radiologist, together with the other member of the liver transplant surgical team and transplant coordinators at KCH. s
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