Hepatocyte Isolation From Unused/Rejected Livers for Transplantation: Initial Step Toward Hepatocyte Transplantation,the First Experience From Iran

Background

Hepatocyte transplantation is being used in patients with liver-based metabolic disorders and acute liver failure. Hepatocytes can be isolated from unused/rejected livers under sterile conditions.

Objectives

The quality of the hepatocytes is very important and the main and initial step in hepatocyte transplantation is hepatocyte isolation. In this study we tried to set up the methods of hepatocyte isolation in order to use the high quality cells in acute liver failure or congenital metabolic disorders.

Materials and Methods

In this study, during a year, hepatocytes were isolated from 7 unused/rejected livers among more than 300 harvested livers in Shiraz University of Medical Sciences. The two step collagenase perfusion method was used under GMP (Good manufacturing practice) for hepatocyte isolation.

Results

Highly quality hepatocytes with high viability and low contamination were isolated. The mean viability was 71.8% ± 21.7. In the first 4 cases microbial contamination by Staphylococci, Diphtheroid and Klebsiella was detected, however the last 3 cases were free of any micro organisms. After 5 weeks of cryopreservation in -140°C, the cell viability was still acceptable.

Conclusions

Hepatocyte isolation can be performed as the main and initial step for cell transplantation from unused/rejected liver. It is the first experience in Iran.     

Pluripotent Stem Cell-derived Strategies to Treat Acute Liver Failure: Current Status and Future Directions

Liver disease has long been a heavy health and economic burden worldwide. Once the disease is out of control and progresses to end-stage or acute organ failure, orthotopic liver transplantation (OLT) is the only therapeutic alternative, and it requires appropriate donors and aggressive administration of immunosuppressive drugs. Therefore, hepatocyte transplantation (HT) and bioartificial livers (BALs) have been proposed as effective treatments for acute liver failure (ALF) in clinics. Although human primary hepatocytes (PHs) are an ideal cell source to support these methods, the large demand and superior viability of PH is needed, which restrains its wide usage. Thus, a finding alternative to meet the quantity and quality of hepatocytes is urgent. In this context, human pluripotent stem cells (PSC), which have unlimited proliferative and differential potential, derived hepatocytes are a promising renewable cell source. Recent studies of the differentiation of PSC into hepatocytes has provided evidence that supports their clinical application. In this review, we discuss the recent status and future directions of the potential use of PSC-derived hepatocytes in treating ALF. We also discuss opportunities and challenges of how to promote such strategies in the common applications in clinical treatments.     

Hepatocyte transplantation: State of the art

Advances in biotechnology have allowed hepatocyte transplantation as a relevant proposition to treat liver disease. This procedure may change the crescent mortality in liver transplantation waiting lists due global organ shortage. Recent clinical trials have described promising results of hepatocyte transplantation for acute, acute-on-chronic and metabolic liver disease. In this report, we discuss progresses regarding hepatocyte culture, cryopreservation systems, hepatocyte immortalization, suitable recipient site for hepatocyte engraftment, cell differentiation and fusion into hepatocytes, current clinical trials, and summarize the bioartificial liver systems. These progressions motivate expectation concerning hepatocyte transplantation as a consistent therapy for liver disease.     

Cryopreserved mouse hepatocytes retain regenerative capacity in vivo

BACKGROUND & AIMS: Substitution of hepatocyte transplantation for whole liver transplants in selected individuals with liver disease could significantly expand the number of patients to benefit from use of scarce donor livers. However, successful hepatocyte transplantation may require that donor cells retain normal functional and proliferative capabilities and that they be readily available. Banking of cryopreserved hepatocytes would fulfill the latter requirement. Cryopreservation protocols have been developed that minimize hepatocyte injury and allow preservation of metabolic activity. The aim of this study was to assess cryopreserved hepatocyte proliferative capacity in vivo after thawing. METHODS: Fresh and frozen/thawed mouse hepatocytes were transferred separately into the livers of recipient mice with transgene-induced liver disease, an environment that is permissive for clonal expansion of donor cell populations. Fresh and cryopreserved donor cells were compared for their ability to proliferate and replace damaged parenchyma. RESULTS: Although cryopreservation decreased hepatocyte viability, individual viable frozen/thawed hepatocytes demonstrated clonal replicative potential identical to that of fresh hepatocytes. Even after storage for 32 months in liquid nitrogen, transplanted hepatocytes constituting 0.1% of total adult hepatocyte number could repopulate a mean of 32% of recipient liver parenchyma. CONCLUSIONS: These findings suggest that cryopreserved hepatocytes represent an appropriate source of cells for therapeutic hepatocyte transplantation.     

Hepatocyte transplantation for metabolic disorders, experience at King's College hospital and review of literature

Hepatocyte transplantation is emerging as a potential treatment for liver based metabolic disorders and acute liver failure. To date, clinical studies have shown that hepatocyte transplantation could be used as a "bridge" to liver transplantation in a few patients with acute liver failure and has changed the phenotype of metabolic patients in terms of reducing the severity of illness. For many years, research studies have been carried out to optimise conditions for a) hepatocyte isolation in order to obtain the highest possible number of viable hepatocytes isolated from various sources of unused donor liver tissues, and b) cryopreservation and storage of hepatocytes for immediate cell availability in emergency cases. In this review, we summarise the worldwide clinical experience in hepatocyte transplantation for liver based metabolic disorders including the experience of our centre at King's College Hospital, London (United Kingdom). We briefly comment on the possible future developments and improvements needed in this field.     

成人原代肝细胞的分离、培养及冻存

目的:建立一种稳定的成人原代肝细胞分离、培养、冻存方法,为肝细胞移植、生物人工肝支持系统治疗急慢性肝病以及肝细胞体外应用模型提供潜在的肝细胞资源.方法:20例供肝采用离体两步胶原酶灌注技术分离成人原代肝细胞.选择7个不同的预培养时间点(2、6、12、24、36、48和72h),分离所获肝细胞按上述不同预培养时间在4℃人无血清培养基(HepatoZYME-SFM)中培养,然后收集预培养肝细胞转移到含100mL/L胎牛血清和DMSO的HepatoZYME-SFM中,再立即放入-80℃异丙醇冷冻盒过夜,次日投入液氮.分析比较各肝细胞在解冻后细胞活力率、贴壁率、白蛋白分泌及尿素合成.结果:在部分肝叶切除后使用离体两步胶原酶灌流技术分离所得肝细胞活率和贴壁率分别是75.0%±4.6%和72.0%±6.0%.4℃预培养12或24h被证明是最适预培养时间,这两个时间点的白蛋白分泌高于其他时间点(P<0.05).与立即冷冻组相比较,预培养12或24h肝细胞解冻后活力(61.4%±4.8%,62.0%±5.6%vs53.4%±4.2%)、贴壁率(63.2%±5.8%,62.6%±3.6%vs55.2%±4.6%)、白蛋白分泌及...     

Animal model for liver cell banking from non-heart beating donors after prolonged ischaemia time

BACKGROUND AND AIM: Although there is a growing interest on the use of non-heart beating donors to enlarge the liver donor pool, livers with prolonged warm ischaemia time are not currently considered for organ transplantation. We hypothesised that these organs may represent a source of hepatocytes for cell transplantation and/or use in bioartificial liver devices. Thus, we investigated if prolonged ischaemia could influence the recovery and viability of functional hepatocytes dissociated from rat livers. METHODS: Hepatocytes were isolated from the liver within 15 min after death (t=15 min) and after 4, 8 and 12h of ischaemia. Cells were either maintained in culture or cryopreserved. In all products, we evaluated cell recovery and viability, hepatocyte markers and cellular functions, including albumin and urea production. RESULTS: The number of cells per gram of tissue was similar at 15 min, 4 and 8h, while it was significantly decreased at 12h. About 0.2 x 10(6) viable cells expressing hepatocyte markers and producing albumin and urea were isolated up to 8h of ischaemia per gram of tissue. CONCLUSIONS: Recovery of viable and functional hepatocytes seems possible after prolonged ischaemia time. These data warrant the evaluation of hepatocyte isolation from human livers of non-heart beating donors.     

成人脂肪肝整肝肝细胞分离及大量冻存的实验研究

目的建立成人脂肪肝整肝肝细胞的分离以及人肝细胞大量冻存技术,为生物人工肝提供稳定的人肝细胞来源。方法采用胶原酶经肝静脉逆行灌注的方法分离成人重度脂肪肝的整肝肝细胞,并比较常规冻存和程序冻存后肝细胞在细胞活性、贴壁率、LDH漏出量及白蛋白合成能力的差异。结果采用添加N-乙酰半胱氨酸(NAC)的胶原酶灌注液分离肝细胞的产量为(7.4±0.5)×10~6 cells/g肝组织,活性为(81.4±3.4)%,而未添加NAC组的肝细胞产量为(5.6±0.8)×10~6 cells/g肝组织和活性为(67.3±5.0)%,差异均有统计学意义(P0.05)。分离的人肝细胞采用程序冻存后在细胞活性、贴壁率及白蛋白合成能力方面均相应高于常规冻存组(P0.05),LDH漏出量低于常规冻存组(P0.05)。结论应用添加NAC的胶原酶灌注液经肝静脉逆行灌注可提高脂肪肝整肝肝细胞分离的活性及产量,采用程序冻存的方法可以提高冻存人肝细胞的活性,满足生物人工肝对肝细胞的需要。     

Hepatocyte isolation from resected benign tissues: Results of a 5-year experience

AIM To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease.METHODS We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi(n = 7) and liver hemangioma(n = 17). Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation.RESULTS Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 10~6 hepatocytes/g liver, with 76.4% ± 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells(5.4 ± 1.71 × 10~6 cells/g vs 3.49 ± 2.31 × 10~6 cells/g, P 0.05) than the samples of intrahepatic duct calculi. However, there seems to be no clear difference in cell viability(80.3% ± 9.67% vs 76.4% ± 10.7%, P 0.05). We obtained a cell yield of 5.31 ± 1.87 × 10~6 hepatocytes/g liver when the samples weighed 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found(3.08 ± 1.86 × 10~6 cells/g vs 5.31 ± 1.87 × 10~6 cells/g, P 0.05).CONCLUSION Benign diseased livers are valuable sources for largenumber hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation.     

Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.     

Hepatocyte cell therapy in liver disease

Liver disease is a leading cause of morbidity and mortality. Liver transplantation remains the only proven treatment for end-stage liver failure but is limited by the availability of donor organs. Hepatocyte cell therapy, either with bioartificial liver devices or hepatocyte transplantation, may help address this by delaying or preventing liver transplantation. Early clinical studies have shown promising results, however in most cases, the benefit has been short lived and so further research into these therapies is required. Alternative sources of hepatocytes, including stem cell-derived hepatocytes, are being investigated as the isolation of primary human hepatocytes is limited by the same shortage of donor organs. This review summarises the current clinical experience of hepatocyte cell therapy together with an overview of possible alternative sources of hepatocytes. Current and future areas for research that might lead towards the realisation of the full potential of hepatocyte cell therapy are discussed.     

N-acetylcysteine and liberase improve success of hepatocyte isolation and viability of hepatocytes isolated from normal and diseased liver

BackgroundSuccessful hepatocyte isolation is crucial for development of cellular transplantation and biochemical research. Most researchers isolate hepatocytes from surplus donor tissue or normal tissue removed during resection of liver tumours. However, most tissue available for research is from explanted diseased liver and donor tissue rejected for transplant. We previously described our experience of hepatocyte isolation using liberase from such livers with a success rate of 51% and median viability of 40%. Liberase is a highly purified collagenase that has been shown to improve the viability of isolated porcine hepatocytes. N-acetylcysteine (NAC) has been shown to improve the viability of human hepatocytes isolated from steatotic donor tissue. The aim of this study was to determine the effect of both reagents in combination on the outcome of hepatocyte isolation from normal and diseased liver.MethodsHepatocytes were isolated from 30 consecutive liver specimens as previously described (old protocol). A further 30 consecutive liver specimens were perfused with buffer containing NAC and standard collagenase substituted by liberase (new protocol). Success was defined as maintenance of cell adhesion and morphology for 48 h and/or their successful use in laboratory studies. Mann-Whitney tests were used to compare results. Fisher's exact test was used for categorical data.FindingsBaseline factors were similar for both groups. The delay to tissue processing was slightly less in the new protocol group (median 2 h vs 4 h, p=0·007). The success rate improved from 40% (12/30) with the old protocol to 70% (21/30) with the new protocol (p=0·037), and the median viable cell yield increased from 7·3 × 10⁴ to 28·3 × 10⁴ cells per g tissue (p=0·003). After multivariable analysis adjusting for the difference in time delay, the success rate (p=0·014) and viable cell yield per g tissue (p=0·001) remained significantly improved.InterpretationNAC and liberase greatly improve the success of hepatocyte isolation and result in a significantly higher viable cell yield. Use of these agents may improve the availability of hepatocytes for transplantation as well as laboratory research.FundingUK Medical Research Council.     

Caspase inhibition reduces apoptotic death of cryopreserved porcine hepatocytes

Cryopreserved porcine hepatocytes are a ready source of metabolic function for use in a bioartificial liver (BAL). However, cryopreservation is associated with a loss of hepatocyte viability. The mechanism of cell death during cryopreservation is incompletely understood, but may involve apoptosis through caspase activation. This study evaluates the cytoprotective effect of a global caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (ZVAD-fmk) during cryopreservation of porcine hepatocytes. Freshly isolated porcine hepatocytes (viability, 97.4% +/- 0.9%) were cryopreserved in 60 micromol/L ZVAD-fmk (+ZVAD group) or without ZVAD-fmk (-ZVAD group) for 24 to 72 hours. Apoptotic and necrotic death were both observed after thawing and after 24 hours of culture. Caspase 3-like activity was significantly reduced by ZVAD-fmk, and was associated with improved viability and reduced apoptotic death of porcine hepatocytes after cryopreservation. Mitochondrial membrane potential (MMP) was increased in cultures of porcine hepatocytes that were cryopreserved in ZVAD-fmk. These results demonstrate the following: 1) Caspase 3-like protease activation and apoptosis occurs in porcine hepatocytes during cryopreservation; and 2) mitochondrial injury in this process is reduced by caspase inhibition.     

Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers

AIMS: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo. METHODS: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration. CONCLUSION: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.     

A highly efficient,stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector

Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture nor efficiently genetically modified and are very sensitive to dissociation before their reimplantation into the recipient. These difficulties have greatly limited the use of an ex vivo approach in clinical trials. In the present study, we have shown that primary human and rat hepatocytes can be efficiently transduced with a FLAP lentiviral vector without the need for plating and culture. Efficient transduction of nonadherent primary hepatocytes was achieved with a short period of contact with vector particles, without modifying hepatocyte viability, and using reduced amounts of vector. We also showed that the presence of the DNA FLAP in the vector construct was essential to reach high levels of transduction. Moreover, transplanted into uPA/SCID mouse liver, lentivirally transduced primary human hepatocytes extensively repopulated their liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, the use of FLAP lentiviral vectors allows, in a short period of time, a high transduction efficiency of human functional and reimplantable hepatocytes. This work therefore opens new perspectives for the development of human clinical trials based on liver-directed ex vivo gene therapy.     

Analysis of the functional integrity of cryopreserved human liver cells including xenografting in immunodeficient mice to address suitability for clinical applications.

BACKGROUND: The availability of well-characterized human liver cell populations that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after cryopreservation and engraft in immunodeficient mice. METHODS: We isolated cells from discarded human livers and studied the properties of cryopreserved cells. The viability of thawed cells was established with multiple in vitro assays, including analysis of liver gene expression, ureagenesis, cytochrome P450 activity, and growth factor-induced cell proliferation. The fate of transplanted cells was analysed in immunodeficient NOD-SCID mice. RESULTS: After thawing, the viability of human hepatocytes exceeded 60%. Cells attached to culture dishes, proliferated following growth factor stimulation and exhibited liver-specific functions. After transplantation in NOD-SCID mice, cells engrafted in the peritoneal cavity, a heterologous site, as well as the liver itself, retained hepatic function and proliferated in response to liver injury. Transplanted hepatocytes were integrated in the liver parenchyma. Occasionally, transplanted cells were integrated in bile ducts. CONCLUSIONS: Cryopreserved human liver cell showed the ability to retain functional integrity and to reconstitute both hepatic and biliary lineages in mice. These studies offer suitable paradigms aimed at characterizing liver cells prior to transplantation in people.     

Kinetics and functional assay of liver repopulation after human cord blood transplantation

BACKGROUND AND AIMS: To evaluate donor cell engraftment and the kinetics of cell repopulation in the injured mouse liver following human umbilical cord blood cell transplantation. METHODS: Nonobese diabetic/severe immunodeficient mice were treated with allyl alcohol to induce liver injury. Twenty-four hours later, umbilical cord blood derived mononuclear cells were transplanted by intra-splenic injection. Mice were sacrificed from 1 to 180 days after transplantation. Temporal changes in the ratio of human cells and fluorescence counts of human sex-determining region Y alleles in mouse liver were determined to evaluate the kinetics of cell repopulation. Mouse liver and sera were examined for the presence of human albumin. RESULTS: Human cell repopulation was extremely rapid in the first week following transplantation, with a doubling time of 1.16-1.39 days apparent. Thereafter cell doubling rate slowed significantly. Cells displaying characteristics of human hepatocytes were still evident at 180 days. Human albumin was detected in mouse liver and sera. CONCLUSION: These findings confirm those from previous studies demonstrating that cells derived from human umbilical cord blood have the capacity to differentiate into cells with human hepatocyte characteristics in mouse liver following injury. Moreover, the detailed information collected regarding the kinetics of human cell repopulation in mouse liver will be of relevance to future studies examining the use of umbilical cord blood cells in liver transplantation therapy.     

The capability of isolated hepatocytes and liver slices of donor livers to predict graft function after liver transplantation

AIMS/BACKGROUND: In liver transplantation, adequate function tests for donor livers and transplanted livers are of utmost importance to provide an objective basis for decision-making. Isolated hepatocyte and/or slice preparations from human donor liver tissue may be suitable to test the quality of the organ to be transplanted. METHODS: Surgical waste material remaining after reduced size or split liver transplantation in children was used to prepare slices and isolated hepatocytes. The viability of these preparations as well as drug transport and metabolism functions were determined and related to graft function in 32 liver recipients. RESULTS: The in vitro tests used in the present study apparently did not select non-viable livers. In vitro preparations of the primary non-function grafts which occurred in the investigated group showed normal viability, metabolic and uptake function. CONCLUSION: These results indicate that either the presently used viability tests are not sensitive enough to detect potential organ failure or that other factors besides the hepatocyte viability at the time of transplantation are of paramount importance to the graft function of the recipient, such as complications during and after transplantation or the viability of the non-parenchymal cells.     

Hepatocyte transplantation: new horizons and challenges

Hepatocyte transplantation represents an alternative strategy for treating liver disease. Liver repopulation following acute liver failure could, potentially, eliminate the requirement for orthotopic liver transplantation. Similarly, the ability to repopulate the liver with disease-resistant hepatocytes offers new opportunities for correcting genetic disorders and treating patients with chronic liver disease. Recent advances concerning the fate of transplanted cells in the recipient liver, the efficacy of cell therapy in outstanding animal models of human disease, and the isolation of progenitor liver cells capable of differentiating into mature hepatocytes have renewed optimism in regard to treating people with hepatocyte transplantation. Recruitment of an increasing number of investigators to the field and the success of recent pilot studies indicate that hepatocyte transplantation will become routine clinical practice in the near future.     

Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells

The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT. CONCLUSION: The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure.