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.     

Allogeneic hepatocyte transplantation using fk 506

Hepatocyte transplantation is an intriguing alternative to orthotopic liver transplantation. While engraftment of syngeneic hepatocytes can be achieved with relative ease, engraftment of allogeneic hepatocytes has been far more complicated. We used FK 506 (Tacrolimus), a novel and highly efficient immunosuppressant, which has been reported to augment liver regeneration in rats. Recipients of isolated syngeneic (LEW) and allogeneic (Wistar F.) rat hepatocytes (major histocompatibility barrier) recieved different immunosuppressive regiments with FK 506 or Cyclosporine A (CsA). Mature syngeneic hepatocytes could be retrieved up to post op day 300 with the lowest number of hepatocytes on post op day 20. Following allogeneic transplantation, no mature hepatocytes could be identified after post op day 10, though ductular like structures within the spleen were found in FK 506 but not CsA-treated animals. The epithelial cells of ductular like structures exhibit cytological features of CK-19 positive cells. Our results suggest that under CsA or FK 506 immunosuppression long-term survival of mature allogeneic hepatocytes within the spleen cannot be achieved across a major histocompatibility barrier though FK 506 allows engraftment of allogeneic donor type ductular cells.     

Hepatocyte transplantation for total liver repopulation

Hepatocyte transplantation (HT) is an attractive therapeutic alternative to liver transplantation. A number of experiments have shown the feasibility of total liver parenchymal cell replacement by transplanted hepatocytes. In this review, we would like to highlight researches and clinical reports of HT for liver repopulation. Cellular source of clinical HT should be safety. Immortalized cells, hepatic stem cells, and other stem cells have been used for an experimental model for HT. The exact mechanism of the cell engraftment after HT has not been completely understood, although there were some markers to detect and investigate transplanted cells. In order to achieve liver repopulation following HT, a mild hepatic damage may need to facilitate cell engraftment and replace the host liver by transplanted cells. Hormonal factor may use for the same purpose. Despite the results of preclinical studies promising clinical benefits for cell therapy, the clinical experience of HT has been disappointing, except in a few cases. HT may become an alternative for liver transplantation in the future; however, many efforts should made before establishing an effective method for HT and liver replacement therapy.     

Hepatocyte transplantation through the hepatic vein: a new route of cell transplantation to the liver

The efficiency of hepatocyte transplantation into the liver varies with the method of administration. This study investigated whether retrograde infusion via the hepatic vein provides a sufficient number of donor cells for the liver. Donor hepatocytes were isolated from dipeptidyl peptidase IV (DPPIV(+)) rats and transplanted into DPPIV(-) rat livers either by antegrade portal vein infusion or retrograde hepatic vein infusion. Hepatocyte engraftment ratios and localization were evaluated by histological DPPIV enzymatic staining at 1 week and 8 weeks after the transplantation. No significant differences in engraftment efficiency were observed at either 1 week or 8 weeks after transplantation by either route. However, the localization of the transplanted hepatocytes differed with the administration route. Portal vein infusion resulted in predominantly periportal engraftment, whereas hepatic vein infusion led to pericentral zone engraftment. Immunohistochemical analysis showed that the transplanted hepatocytes engrafted in the pericentral zone after retrograde infusion displayed intense CYP2E1 staining similar to the surrounding native hepatocytes. CYP2E1 staining was further enhanced by administration of isosafrole, an inducing agent for various cytochrome P450 enzymes, including CYP2E1. This study demonstrates a novel approach of transplanting hepatocytes into the liver through retrograde hepatic vein infusion as the means to target cell implantation to the pericentral zone.     

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.     

A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.     

Replacement of liver parenchyma in analbuminemic rats with allogenic hepatocytes is facilitated by intrabone marrow-bone marrow transplantation

Although hepatocyte transplantation (HCTx) is expected to become a useful therapy for human liver diseases, allogenic hepatocytes still tend to be rejected within a short period due to host immunosurveillance. In the present study, we investigated the effect of prior bone marrow transplantation (BMTx) for the engraftment of allogenic hepatocytes using the analbuminemic rat transplantation model. The hepatocytes of Lewis (LEW) rats were not accepted in the liver of retrorsine (RS)/partial hepatectomy (PH)-treated analbuminemic F344 (F344-alb) rats, which express the disparate major histocompatibility complex (MHC) against that of LEW rats. Prior BMTx with the LEW bone marrow cells (BMCs) after sublethal irradiation achieved acceptance and repopulation of LEW hepatocytes in the liver of the RS/PH-treated F344-alb rats, associated with elevation of serum albumin. The replacement of hepatic parenchyma with albumin positive (Alb(+)) donor hepatocytes and elevation of serum albumin levels were dependent on the bone marrow reconstitution by donor BMCs, which was more efficiently achieved by intrabone marrow (IBM)-BMTx than by intravenous (IV)-BMTx. Our results demonstrate that efficient bone marrow reconstitution by IBM-BMTx enables the replacement of the hepatic parenchyma with allogenic hepatocytes in RS/PH-treated analbuminemic rats without immunosuppressants.     

Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model

BACKGROUND: The use of cell transplantation as an alternative therapy for orthotopic liver transplantation has been widely anticipated due to a chronic donor shortage. We previously reported the method used to enrich hepatic progenitor cells (HPCs) forming cell aggregations. In this study, we transplanted HPCs into the liver injury model mice to determine whether HPC transplantation may improve the liver dysfunction. METHODS: We obtained donor cells from E13.5 fetal livers of green fluorescent protein (GFP) transgenic mice. We transplanted GFP-positive fetal liver cells into the transgenic mice which express diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Subsequently, we induced selective liver injury to recipient mice by DT administration. We then evaluated the engraftment of the transplanted cells and their effect on survivorship. RESULTS: The low dose of DT induced sublethal liver injury and the high dose of DT was lethal to the liver injury model mice. The transplanted GFP-positive cells were engrafted into the recipient livers and expressed albumin, resembling mature hepatocytes. They continued to proliferate, forming clusters. The survival rate at 25 days after transplantation of the cell-transplanted group (8 of 20; 40.0%) was improved significantly (P=0.0047) in comparison to that of the sham-operated group (0 of 20; 0%). CONCLUSIONS: The transplanted cells were engrafted and repopulated the liver of recipient mice, resulting in the improvement of the survival rate of the liver injury model mice. We therefore propose that HPCs are a desirable cell source for cell transplantation.     

Intrasplenic transplantation of encapsulated hepatocytes decreases mortality and improves liver functions in fulminant hepatic failure from 90% partial hepatectomy in rats

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.     

Hepatic support by hepatocyte transplantation in congenitally metabolic diseased rats

Nagase analbuminemic rats (NAR), which lack albumin synthesis in the liver, underwent intrasplenic hepatocyte transplantation (HCTx), and the long-term effects were studied using functional and morphological examinations. Hepatocytes were isolated from congenic F344 rats with collagenase infusion, and 1 × 10⁷ cells were Injected into the spleen of 3-month-old NAR (n = 10). Serum albumin increased with time, reaching 53.9 mg/dl 14 months after HCTx, which was equivalent to 2.1% (maximum 4%) of serum albumin in normal rats. On the other hand, untreated NAR showed persistently low serum albumin levels (0.99 ± 0.23 mg/dl at 10 months). According to immunostaining with anti-rat albumin antibody at 16 months after HCTx, hepatocyte grafts occupied 27–41% of the spleen area and weighed 120–420 mg, Which was equivalent to 0.8–2.9% of a whole liver. Our study demonstrated that grafted hepatocytes can grow in the spleen with the ability to synthesize albumin. HCTx in NAR is a new experimental system to monitor the function and survival of grafted heptocytes without sacrificing the animals by measuring serum albumin levels. Certain manipulations to facilitate the growth of grafted hepatocytes are necessary to achieve sufficient hepatic support in HCTx.     

Characteristics of hepatocytes derived from early ES cells and treatment of surgically induced liver failure rats by transplantation

BACKGROUND: Although liver transplantation has become a standard therapy for diseases such as fulminant hepatitis and cirrhosis, the lack of donor organs remains a major problem. One solution is the development of transplantable hepatocytes. The metabolic characteristics as well as function and adaptation of hepatocytes (R-EES-hep cell) derived from rat early embryonic stem cells were examined after transplantation into rats with surgically induced liver failure. METHODS: Rat hepatocyte cell lines were established from early embryonic stem cells cultured in the presence of embryotrophic factors by colony cloning methods. The cell lines were established from two cell embryos taken from spontaneous dwarf rats using the novel method of Ishiwata et al. Morphologic differentiation as well as albumin and bilirubin production were observed by immunostaining. R-EES-hep cells were transplanted into the spleens of 90% hepatectomized, surgically induced liver failure rats to analyze survival rates. RESULTS: When cultured in type I collagen gel the cells formed cordlike structures resembling the liver. Both albumin and bilirubin production were observed when transplanted; the spleen was converted into a liver-like structure with prolonged survival of the 90% hepatectomized rats for up to 3 months up to the time of killing. CONCLUSIONS: R-EES-hep cells showed many of the distinctive metabolic characteristics of the liver. These cells may be efficient for further research and application for hepatic cell transplantation to treat liver insufficiency patients and as biologic artificial organs.     

Construction and transplantation of an engineered hepatic tissue using a polyaminourethane-coated nonwoven polytetrafluoroethylene fabric

BACKGROUND: Acute liver failure (ALF) is a serious condition that has a high mortality rate. Construction of an efficient culture and transplantation engineering system of hepatic tissue is an important approach to treat patients suffering from ALF to provide short-term hepatic support until the damaged liver spontaneously recovers or a donor liver becomes available for transplantation. Here, we evaluate the construction and transplantation of an engineered hepatic tissue (EHT) using primary isolated hepatocytes cultured onto polyaminourethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric. METHODS: The isolated hepatocytes cultured onto PAU-coated PTFE fabric were able to adhere and spread over the individual fibers of the net and formed hepatic clusters after 3 days, such clusters revealed Gap junctions and well-developed bile canaliculi. RESULTS: When PAU-coated PTFE was utilized, ammonia-, and diazepam- metabolizing capacities and albumin production ability were significantly increased compared with collagen control. To test the function of this hepatic tissue in vivo, we transplanted a nonwoven PAU-coated PTFE fabric inoculated with one million hepatocytes on the surface of the spleen of Balb/c mice suffering from ALF induced by 90% hepatectomy, and found that this EHT prolonged the survival of liver failure-induced mice without adverse effects. Ultrastructure analyses showed good attachment of the cells on the surface of PTFE fabric and strong albumin expression seven days after the newly formed hepatic tissue was transplanted. CONCLUSION: We have here demonstrated the efficient construction and transplantation of hepatic tissue using primary hepatocytes and PAU-coated PTFE fabric.     

A novel system for transplantation of isolated hepatocytes utilizing HBsAg-producing transgenic donor cells

Hepatocytes from donor transgenic mice that produce an easily assayable circulating marker have been used to develop a novel hepatocyte transplantation system. Isolated G7 HBV transgenic donor hepatocytes secreting HBsAg were transplanted into congeneic or allogeneic mouse recipients. Serum HBsAg was present three days after hepatocyte transplantation in congeneic animals and persisted indefinitely when hepatocytes were transplanted into the spleen. Transplanted hepatocytes within the splenic pulp were identified by morphologic and histochemical analysis. Migration of hepatocytes injected into the spleen to the liver was demonstrated by in situ hybridization using an RNA probe for HBsAg. Transplantation into nonimmunosuppressed allogeneic recipients resulted in disappearance of detectable hepatocytes in the spleen within two weeks. This novel transplantation system should facilitate studies of hepatocyte engraftment and survival, modulation of allograft rejection, and development of hepatocyte-directed gene therapy.     

Selective repopulation of mice liver after Fas-resistant hepatocyte transplantation

Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopulation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 x 10(6) cells in female MRL-+/+ (wild-type mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intravenously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The pathologic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver.     

DiI荧光示踪剂在大鼠脾内肝细胞移植中的研究

目的 探讨大鼠肝细胞在脾内移植后的生存、迁移及其并发症情况。方法 先用ＤｉＩ荧光示踪剂标记肝细胞再行脾内肝细胞移植。在不同时间段取肝、脾、肺、心、肾 、胸腺组织冷冻切片,在荧光显微镜下用荧光和普通光示踪观察。结果 ＤｉＩ荧光示踪剂能很好地标记肝细胞,６０ｄ实验期内,移植细胞能在所取各器官中生存并保持良好形态,未发现有区域梗塞灶、同时还反映了其在肝实质中的分布。结论 ＤｉＩ是一种新颖、使用简便的荧光     

Intraperitoneal hepatocyte transplantation: morphologic findings and clinical relevance

In order to evaluate if intraperitoneal hepatocyte transplantation could include a therapeutic approach in acute hepatic failure, isolated hepatocytes as well as microcarrier attached hepatocytes were transplanted intraperitoneally and the survival rates of transplanted cells were evaluated morphologically. The histological investigations showed, that the transplanted hepatocytes undergo complete cell necrosis within 3 days due to a lack of substrate, which also cannot be avoided by an attachment to microcarriers. Liver cell necrosis is followed by a peritoneal reaction resulting in granuloma formation. Because liver cell necrosis occurred early after i.p. hepatocyte transplantation a functional support in acute hepatic failure cannot be expected.     

Three different hepatocyte transplantation techniques for enzyme deficiency disease and acute hepatic failure

The effects of three different techniques of hepatocyte transplantation were investigated: transplantation of free hepatocytes into the spleen and intraperitoneal transplantation of microcarrier-attached hepatocytes or of microencapsulated hepatocytes. The liver-supportive functions of these transplanted hepatocytes were analyzed using either the Gunn rat (hyperbilirubinemia) or rats with acute liver failure. In the Gunn rat intraperitoneal transplantation of microcarrier-attached hepatocytes resulted in a significant reduction of plasma bilirubin for 28 days whereas intraperitoneal transplantation of microencapsulated hepatocytes was ineffective notwithstanding immunosuppression by cyclosporin A. Intrasplenic hepatocyte transplantation was only effective in reducing plasma bilirubin for 14 days. During acute liver failure, liver support was achieved temporarily by hepatocyte transplantation in the spleen, by intraperitoneally transplanted microcarrier-attached hepatocytes, and by microencapsulated hepatocytes to equal extents, the microencapsulated hepatocytes being the least effective after 8 h of liver ischemia.     

The role of progenitor cells in repair of liver injury and in liver transplantation

There are three levels of cells in the hepatic lineage that respond to injury or carcinogenesis: the mature hepatocyte, the ductular "bipolar" progenitor cell, and a putative periductular stem cell. Hepatocytes are numerous, and respond rapidly to liver cell loss by one or two cell cycles but can only produce other hepatocytes. The ductular progenitor cells are less numerous, may proliferate for more cycles than hepatocytes, and are generally considered "bipolar," i.e., they can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential, and may be multipotent. Extrahepatic (bone marrow) origin of the periductular stem cells is supported by recent data showing that hepatocytes may express genetic markers of donor hematopoietic cells after bone marrow transplantation. These different regenerative cells with variations in potential for proliferation and differentiation may provide different sources of cells for liver transplantation: hepatocytes for treatment of acute liver damage, liver progenitor cell lines for liver-directed gene therapy, and bone marrow-derived cells for chronic long-term liver replacement. A limiting factor in the success of liver cell transplantation is the condition of the hepatic microenvironment in which the cells must proliferate and set up housekeeping.     

Experimental models of acute and chronic liver failure in nude mice to study hepatocyte transplantation

Although hepatocyte transplantation is a promising therapy for acute liver failure in human, there is still a lack of animal models suffering from hepatic injury in which the benefits of hepatocyte transplantation could be evaluated solely, without the bias caused by immunosuppression. As a consequence, the aim of the study was first to develop reproducible models of partial hepatectomy and of thioacetamide (TA)- or Jo2-induced acute liver failure in nude mice. Chronic liver disease was also investigated by repeated injections of sublethal doses of thioacetamide. Survival rates, routine histologic observations, alanin aminotransferase sera content, Ki67, and caspase 3 immunodetection were investigated both after 40% partial hepatectomy and after toxic-induced damages. Liver injuries were more severe and/or precocious in nude mice than in Balb/c mice for a given treatment with a maximum of acute injury obtained 24 h after single toxic injection, and were found to be transitory and reversible within 10 days. Toxics induced apoptosis followed by necrosis, confirming recent published data. Onset of fibrosis leading to reproducible chronic cirrhosis in nude mice correlated with increasing number of Ki67-positive cells, indicating that high levels of cell proliferation occurred. Chronic cirrhosis progressively reversed to fibrosis when the treatment ceased. Preliminary results demonstrated that engrafted xenogeneic hepatocytes could be detected in the host liver by anti-MHC class I immunohistochemistry. Fractions enriched in 2n or 4n hepatocytes by cell sorting using a flow cytometer were equivalent to the unpurified fraction in terms of engraftment in control nude mice or in nude mice subjected to PH. However, in mice suffering from liver injury 24 h after Jo2 or TA treatment, the engraftment of 2n hepatocytes was about twice that of an unpurified hepatocyte population or of a population enriched in 4n hepatocytes.