Transplantation of hepatocytes in nonhuman primates: a preclinical model for the treatment of hepatic metabolic diseases

BACKGROUND: The transplantation of isolated hepatocytes in large animals, including nonhuman primates, must be evaluated before clinical trials are performed. However, in the absence of large transgenic animals and large-animal (as opposed to small-animal) models of genetic deficiencies, it is difficult to evaluate the fate of transplanted hepatocytes, their localization, survival, and function within the parenchyma of the host liver. In this work, we aimed to develop a technique for delivering hepatocytes to the liver of a nonhuman primate and to evaluate their localization and functionality in the short term. METHODS: A 20% hepatectomy was performed in 34 cynomolgus monkeys (Macaca fascicularis) and hepatocytes were isolated. Hepatocytes were labeled in vitro with a recombinant retrovirus expressing the beta-galactosidase gene and returned to the liver by infusion through a portal catheter left in place. Liver biopsies were performed 4 and 7 d after transplantation. RESULTS: Twenty-four monkeys underwent surgery to define the necessary technical adjustments and to optimize conditions. Six monkeys died. The whole protocol, including the transplantation of genetically marked hepatocytes and procurement of liver biopsies, was performed in the remaining 10 monkeys. In eight monkeys, transplanted hepatocytes expressing the beta-galactosidase gene were widely distributed in the portal tracts, sinusoids, and hepatocyte plates of the host liver 4 and 7 d after transplantation. CONCLUSIONS: We have developed an experimental nonhuman primate model for the evaluation of hepatocyte transplantation. We demonstrated the engraftment and functioning of transplanted hepatocytes in the host liver 4 and 7 d after transplantation.     

Hepatocyte transplantation using biodegradable matrices in ascorbic acid-deficient rats: comparison with heterotopically transplanted liver grafts

BACKGROUND: Hepatocyte transplantation using polymeric matrices is under investigation as an alternative therapy for metabolic liver diseases. Long-term engraftment of hepatocytes in polymers has been demonstrated. However, the metabolic activity of hepatocytes in such devices has never been assessed in direct comparison with liver grafts. METHODS: Hepatocyte and partial liver transplantation were evaluated in the scurvy-prone osteogenic disorder Shionogi rat model. Biodegradable poly glycolic acid matrices seeded with hepatocytes equivalent to 20% of the recipient's liver mass, or 20% liver grafts were heterotopically transplanted into ascorbic acid- (AsA) deficient recipients. Recipients of cell-free matrices or AsA-deficient liver grafts served as controls. Recipients were set on AsA-free diet after transplantation. Plasma AsA levels, AsA concentrations in liver and adrenal gland tissue, and body weight ratios were assessed and H&E histology was performed. RESULTS: Recipients from the control groups showed symptoms of scurvy at 1 month after cessation of AsA supply. Hepatocyte transplantation and auxiliary liver transplantation prevented symptoms of scurvy and increased plasma and tissue AsA levels and body weight ratios. AsA levels in recipients of 20% liver grafts were comparable to normal control animals. CONCLUSIONS: Hepatocytes transplanted in polymeric matrices are able to compensate for liver-based metabolic deficiencies. Hepatocyte transplantation improves plasma AsA levels in AsA-deficient recipients. However, auxiliary liver grafts are superior to hepatocyte grafts in improving metabolic parameters. Further research work is needed to increase the efficiency of liver cell transplantation with regard to a clinical application.     

Engraftment measurement in human liver tissue after liver cell transplantation by short tandem repeats analysis

Hepatocyte transplantation has been proposed as a technique for bridging patients to whole-organ transplantation, for providing metabolic support during liver failure, and for replacing whole-organ transplantation in certain metabolic liver diseases. Assessment of hepatocyte engraftment has been difficult to measure, and the degree of engraftment needed to correct various liver disorders is still unknown. A sensitive, simple, and specific method of monitoring engraftment of transplanted hepatocytes for the purpose of bridging human liver failure to native regeneration using short tandem repeats (STRs) was evaluated. The analytical sensitivity of the test was evaluated using DNA mixing curves and established as 0.5% (percentage of donor DNA/ recipient DNA). Sex-matched and mismatched cases were included during the validation. The clinical evaluation of the assay was performed using liver samples from two patients who underwent hepatocyte transplantation. We concluded from this study that the AmpFLSTR Profiler Plus PCR Amplification Kit, a well-established technique in forensic medicine, is specific, sensitive, and a reproducible assay for measurement of engraftment after hepatocyte transplantation in both sex-matched and sex-mismatched cases.     

HVEGF165 increases survival of transplanted hepatocytes within portal radicles: suggested mechanism for early cell engraftment

VEGF is a potent angiogenic factor that promotes hepatocyte growth, increases permeability of blood vessels, and induces vasodilatation, and may accelerate engraftment and function of transplanted hepatocytes. The aim was to study the effect of VEGF on early hepatocyte engraftment. Thirty-two Lewis syngeneic female rats underwent 70% partial hepatectomy. Eighteen received 240 ng VEGF165 and 14 received saline for control. Thereafter, intrasplenic transplantation of 10(7) male hepatocytes was done. Semiquantitative analysis of PCR product of the SRY region of the Y-chromosome was performed. Paraffin-embedded sections were stained for H&E and for PCNA immunostaining. By PCR, male hepatocytes were identified in 8 livers out of 14 VEGF-treated rats at 24-48 h, compared with only 1 liver out of 8 controls. Transplanted cells were seen within portal vessels radicles in 7 out of 14 VEGF-treated rats for as long as 48 h posttransplantation, compared with only one control liver at 24 h. There was no histological sign of cell injury to transplanted or adjacent cells. Two weeks after transplantation male transplanted cells were identified in two out of four rats treated with hVEGF165 and in one out of six rats treated with saline. No transplanted cells were detected within portal tracts 14 days after transplantation. hVEGF165 enhances the presence of transplanted hepatocytes within portal vessels after transplantation. We suggest an additional mechanism for cell engraftment, whereby transplanted hepatocytes first stick to each other in the portal radicles. Later they become included in the liver parenchyma as groups of organized cells in a process stimulated by VEGF.     

Construction of liver tissue in vivo with preparative partial hepatic irradiation and growth stimulus: investigations of less invasive techniques and progenitor cells

Background

The selective proliferation of transplanted hepatocytes with a growth stimulus, such as partial hepatectomy or hepatocyte growth factor, concomitant with hepatic irradiation (HIR), which can suppress proliferation of host hepatocytes, has been reported. We have conducted experiments that focused on less invasive and clinically applicable techniques and progenitor cells.

Materials and methods

First, dipeptidyl-peptidase IV-F344 or jaundiced Gunn rats underwent partial HIR (only 30% of whole liver) and portal vein branch ligation (PVBL) of one lobe, followed by intrasplenic hepatocyte transplantation at 1 × 10⁷. Second, after partial HIR and PVBL, two types of progenitor cells were transplanted (i.e., small hepatocytes (SHs) or adipose-derived mesenchymal stem cells.

Results

Sixteen weeks after transplantation, the donor cells constituted > 70% of the hepatocytes of the irradiated lobe, showing connexin 32, phosphoenolpyruvate carboxykinase-1, and glycogen storage. Moreover, the serum bilirubin level had decreased significantly in the jaundiced Gunn rats and remained at this level throughout the 24 wk experimental period. The SHs grew more quickly than the hepatocytes. After 8 wk, around 40% of the host hepatocytes had been replaced by transplanted SHs. Although the donor adipose-derived mesenchymal cells were engrafted after 8 wk, their proliferation was not observed.

Conclusions

HIR, combined with PVBL, can be given to a selective liver lobe and is a less-invasive but effective method for proliferation of transplanted hepatocytes. Even a smaller number of SHs can construct liver tissue with their prevailing proliferative ability.     

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.     

Amplification of engrafted hepatocytes by preparative manipulation of the host liver

Scarcity of donor livers is a major obstacle to the general application of hepatocytes for the development of bioartificial liver assist devices as well as intracorporeal engraftment of hepatocytes for the treatment of inherited metabolic diseases. The number of hepatocytes that can be transplanted into the liver safely in a single sitting also limits the utility of this procedure. These limitations could be addressed by providing preferential proliferative advantage to the transplanted cells. Studies using transgenic mouse recipients or donors have indicated that massive repopulation of the host liver by engrafted hepatocytes requires that the transplanted cells are subjected to a proliferative stimulus to which the host hepatocytes cannot respond. Prevention of host hepatocyte proliferation has been achieved by treatment with a plant alkaloid, retrorsine. Because retrorsine is carcinogenic, we have evaluated preparative irradiation for this purpose. The proliferative stimulus may consist of the loss of hepatic mass (e.g., partial hepatectomy, reperfusion injury or induction of Fas-mediated apoptosis by gene transfer) or administration of stimulants of hepatocellular mitosis (e.g., growth factors or thyroid hormone). Potential applications of these preparative manipulations of the host liver include the treatment of inherited metabolic disorders by transplantation of allogeneic hepatocytes, hepatocyte-mediated ex vivo gene therapy, rescuing liver cancer patients from radiation-induced liver damage, and expansion of human hepatocytes in animal livers.     

Histological identification of purified and cryopreserved allogeneic hepatocytes following transplantation in a murine model without host immunosuppression

Hepatocyte transplantation is a conceptually attractive alternative to whole organ grafting for some inborn metabolic errors and for fulminant liver failure. However, studies of the immunogenicity of transplanted allogeneic hepatocytes have yielded contradictory results. In these experiments, the effect of purification and cryopreservation of the hepatocytes on the ability of these cells to engraft in the mouse allogeneic recipients without immunosuppression was studied. BALB/cByJ mouse crude (unpurified), modified (purified or cryopreserved), or dead (irradiated) hepatocyte preparations labeled with fluorescein dye CFSE were infused either into the portal vein or into the spleen parenchyma of the recipient CBA mice. A histological examination revealed normal appearance of engrafted modified hepatocytes with no signs of acute rejection up to 21 days posttransplant. Many of the intrasplenically implanted hepatocytes migrated into the hepatic sinusoids. The modified hepatocytes showed intact ultrastructural appearance 7 days after transplantation. The numbers of inoculated crude hepatocytes rapidly declined with signs of dense infiltration of mononuclear cells in the graft indicating destructive response. The fluorescence of dead hepatocytes was undetectable. These results suggest that reduced immunogenicity may be responsible for the longer survival time of inoculated, purified or cryopreserved hepatocytes with no adverse morphological effects. Received: 10 August 1998 Received after revision: 2 March 1999 Accepted: 26 March 1999     

Rat hepatocyte engraftment in severe combined immunodeficient x beige mice using mouse-specific anti-fas antibody

BACKGROUND: Hepatocyte transplantation holds promise as a treatment for acute and chronic liver failure; however, robust model systems needed to study xenogeneic hepatocyte transfer are lacking. Severe combined immunodeficient x beige (SCID/bg) hybrid mice readily accept foreign tissue. Repopulation of C.B-17 SCID/bg mouse liver with rat hepatocytes was studied following induction of mouse hepatocyte apoptosis using an anti-mouse agonistic fas monoclonal antibody (Jo2 mAb) that does not engage xenogeneic fas. METHODS: SCID/bg mice were transplanted with 1 x 10(6) fresh adult rat hepatocytes intrasplenically and treated with various doses, routes and frequencies of Jo2 mAb. Rat cell repopulation was characterized by quantitative immunofluorescent antibody (q-IFA) staining specific for rat dipeptidyl peptidase type IV (DPP-IV) and leucine amino peptidase, amplification of rat genomic DNA using polymerase chain reaction and histopathological and serum biochemistry analyses. RESULTS: Analysis of liver sections from mice treated twice weekly for 12 weeks with 0.4 mg/kg Jo2 mAb intraperitoneally consistently demonstrated >50% rat hepatocytes in the parenchymal mass by q-IFA. Rat hepatocyte engraftment protected mice from Jo2 mAb-mediated liver hemorrhage and hepatocyte apoptosis. Serum liver enzyme levels did not increase in Jo2 mAb-treated mice that were highly engrafted with rat hepatocytes, in contrast to matched non-engrafted mice. At 12 weeks post-engraftment, minimal fibrosis and inflammation were apparent and liver architecture had returned to near normal. Jo2 mAb did not induce histopathological abnormalities in other tissues known to express fas antigen (i.e. heart, lung). CONCLUSIONS: This novel model represents a simple and robust system of xenogeneic hepatocyte transplantation that could be applied to studies of liver biology, regeneration and hepatocyte transplantation.     

Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique,safety, and metabolic follow-up

Hepatocyte transplantation is an investigational alternative to orthotopic liver transplantation to treat liver based inborn errors of metabolism. We report successful hepatocyte transplantation in a 4-year-old girl with infantile Refsum disease. Hepatocytes were isolated from the left liver segment of two male donors using a classic two-step perfusion method. Fresh cells were transplanted first and then cryopreserved cells, for a total of 2 billion cells. Total bile acids and abnormal dihydroxycoprostano?c acid markedly decreased in the patient's serum, indicating resolution of cholestasis and re-population of liver cells. Pipecholic acid decreased by 40% and c26:c22 fatty acid ratio by 36% after 18 months. Donor chromosomes sequences were detected on biopsy posttransplant, indicating engraftment. Hepatocyte transplantation is a safe and promising technique in the treatment of rare inborn errors of metabolism. Future improvements of cell viability and prevention of apoptosis may increase engraftment and subsequent re-population.     

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.     

Regulation of hepatocyte engraftment and proliferation after cytotoxic drug-induced perturbation of the rat liver

BACKGROUND: Perturbations in specific liver cell compartments benefit transplanted cell engraftment and/or proliferation. We analyzed whether cytotoxic drugs interfering with the integrity of genomic DNA or cell division could be useful for liver cell transplantation. METHODS: We used dipeptidyl peptidase IV deficient (DPPIV-) rats as recipients of syngeneic F344 rat hepatocytes. Rats were pretreated with doxorubicin, irinotecan, or vincristine prior to cell transplantation and synergistic liver perturbations were induced by drug administration followed by partial hepatectomy or carbon tetrachloride treatments. Transplanted cells were identified by DPPIV histochemistry and cell engraftment and proliferation were analyzed morphometrically. Perturbations in endothelial, Kupffer cell, and hepatocyte compartments were analyzed by electron microscopy, carbon incorporation, and blood tests, respectively. RESULTS: Cell engraftment was improved in rats treated with doxorubicin but not with irinotecan or vincristine. Doxorubicin disrupted endothelial cells for up to seven days without causing Kupffer cell or hepatocellular toxicity. Neither doxorubicin nor vincristine induced liver repopulation in animals up to three months, including after partial hepatectomy or carbon tetrachloride-induced additional liver injury. CONCLUSIONS: Doxorubicin-induced hepatic endothelial damage enhanced cell engraftment, which should be useful in cell therapy strategies.     

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.     

Evaluation of methods of hepatotrophic stimulation in rat heterotopic hepatocyte transplantation using polymers.

BACKGROUND: Hepatocyte transplantation has been studied as an alternative to organ transplantation. Hepatocyte transplant models should provide sufficient cell mass for replacement function and hepatotrophic stimulation of the transplanted cells in heterotopic locations. METHOD: The authors used three-dimensional porous polyvinyl-alcohol matrices as cell carriers, which were implanted between mesenteric leaves of the intestine. In this study, different methods were evaluated for hepatotrophic stimulation. Fifty million transplanted hepatocytes (approximately 10% liver mass) were implanted in Lewis rats. We compared 70% partial hepatectomy, portacaval shunt, cotransplantation of enterocytes, cotransplantation of islets of Langerhans, and methylprednisolone injection to a control group with only hepatocyte transplantation. Portacaval shunt and islet cotransplantation also were used in combination. Specimens were harvested 2 weeks after transplantation, and area per histological cross section compromised by hepatocytes was measured. RESULTS: Seventy percent partial hepatectomy, enterocyte cotransplantation, and methylprednisolone injection resulted in hepatocyte maintenance similar to control group (3,100 +/- 7,592 microm2). Portacaval shunt (96,866 +/- 55,039 microm2) and islet cotransplantation (173,020 +/- 75,977 microm2) yielded a highly significant increase in hepatocyte area. The combination of portacaval shunt and islet cotransplantation resulted in a significant increase compared with using these methods individually (288,930 +/- 86,726 microm2). Additional immunohistochemical stains for active DNA synthesis, insulin, and glucagon demonstrated the proliferative abilities of the hepatocytes and the synthesis of insulin and glucagon in the cotransplanted islets. CONCLUSION: Hepatocyte transplantation can be performed using polymer carriers and that hepatocyte survival and maintenance can be improved with portacaval shunt and islet cotransplantation.     

Enhanced proliferation of hepatic progenitor cells in rats after portal branch occlusion.

It is known that hepatic progenitor cells increase in number after liver injury caused by carcinogens, but this injury cannot be reproduced in humans. In order to create a practical source of hepatic progenitor cells, changes in the number of liver epithelial cells (LECs), a type of hepatic progenitor cell, were examined following partial interruption of the portal flow. Efficiency in this isolation procedure was investigated, and isolated LECs were transplanted into livers to demonstrate their differentiation into hepatocytes in vivo.A volume of 70% of Sprague-Dawley rat's livers was exposed to portal vein ligation. LECs, identified as alpha-fetoprotein (AFP)-positive and albumin-negative cells, were counted and LECs isolated from the portal vein ligated-lobe were characterized by immunostaining and Western blotting. Isolated cells were subjected to a 1-week-culture, and the number of colonies formed on dishes was counted. The cells were then transplanted to the livers of genetic analbuminemic rats and identified by immunohistochemistry. The number of LECs in the portal ligated-lobes on day 7 was 14.7 +/- 6.5 cells/1,000 hepatocytes: 18 times higher than numbers in a normal liver. A significant increase was noted from day 3 until day 28. Isolated LECs were AFP-positive, albumin-negative, and cytokeratin-19-positive cells. The number of colonies on the 7th day following portal vein ligation was 42 times higher than in a normal liver. After transplantation of the LECs to the analbuminemic rat, a cluster of albumin-producing cells was present until day 56, suggesting that they differentiate into hepatocytes. We conclude that after portal vein occlusion, the liver can be a good source of hepatic progenitor cell. These results open up the possibility of cellular transplantation for liver functional support in clinical settings.     

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.     

Intrasplenic hepatocyte transplantation prolonged the survival in Nagase analbuminemic rats with liver failure induced by common bile duct ligation

It has already been established that hepatocyte transplantation (HTx) in animal models, such as both chemically and surgically induced acute liver failure, liver-based metabolic disease, and cirrhosis, resulted in significant improvement of liver function and survival. However, the efficacy of hepatocyte transplantation in secondary cholestatic liver disease is not well known. In this study, we transplanted hepatocytes into the spleen of Nagase analbuminemic rats (NARs) with common bile duct ligation (CBDL) to evaluate the function of transplanted hepatocytes by both of serum albumin levels and total bilirubin levels. CBDL was carried out on NARs to induce liver failure. Lewis rat hepatocytes were transplanted in NARs 7 days after CBDL. Animals, in groups of four, underwent the following interventions: group 1--intrasplenic transplantation of 30 x 106 primary Lewis rat hepatocytes in NARs with CBDL (n=4), group 2--intrasplenic injection of 0.5 ml DMEM in NARs with CBDL (n=4); group 3--CBDL only (n=4); group 4--intrasplenic transplantation of 30 x 106 primary Lewis rat hepatocytes in NARs (n=4). Both bilirubin levels and albumin levels in NARs with CBDL were significantly improved post-HTx. Animals receiving hepatocyte transplantation survived longer than animals in nontransplant control groups. This study indicates that hepatocytes can be transplanted to temporarily provide life-supporting liver-specific metabolic function and prolong the survival in recipient rats with liver failure induced by CBDL.     

Transplantation of hepatocytes: temporary elimination of scavenger cells prevents early loss of grafted cells

Transplants of isolated syngeneic and allogeneic hepatocytes are rapidly disintegrated, irrespective of the site of engraftment; be it spleen, liver, portal vein, peritoneum, or subcutaneous tissue. Host scavenger cells are responsible for this reaction. We designed a method overcoming early disintegration of the grafted hepatocytes. It consisted of administration of anti-asialoGM1 antiserum eliminating natural killer cells; sublethal whole body irradiation; and reconstitution with syngeneic bone marrow cells, ligation of host bile duct, intrasplenic hepatocyte transplantation, and three consecutive partial hepatectomies. Six months after transplantation a glycogen-rich, trabeculae-forming, dividing hepatocytes, situated along strands of newly-formed fibrous tissue and numerous dilated blind bile cannaliculae were observed. There was evidently more bile canaliculae in hosts with ligated bile duct than nonligated controls. This is the first study showing fibrous tissue formed at the site of hepatocyte implantation, and stellate cells are presumably involved in this process.     

Monocrotaline, an alternative to retrorsine-based hepatocyte transplantation in rodents

Retrorsine has been used extensively to inhibit proliferation of resident hepatocytes in various transplantation models. Here we report a successful alternative to currently unavailable retrorsine that can be used in cellular transplantation models. Based on structural and molecular similarities, we investigate the use of monocrotaline (MCT) in cell transplantation studies in rodents. In this study, MCT was given to rats intraperitoneally in two injections 2 weeks apart. Two weeks after the final injection, a partial hepatectomy followed by splenic hepatocyte transplantation was performed. The results indicate that MCT, at two doses of 30 mg/kg, highly enhances liver repopulation by donor hepatocytes following partial hepatectomy and produces 15.3 +/- 4.9% liver repopulation within the first 6 weeks following transplantation. Additionally, we tested the effectiveness of MCT in a murine model. Using two injections of 50 mg/kg each, given 2 weeks apart, hepatocyte proliferation in the native liver was inhibited and subsequent oval cell transplants engrafted at 18 +/- 21.3% after 16 weeks posttransplantation. In conclusion, MCT can be used as an effective selective pressure for donor hepatocytes in cell transplantation to the liver in rodents.