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.     

Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion

BACKGROUND & AIMS: Studies have indicated that stem cells have unexpected plasticity and can differentiate down a multitude of nonhematopoietic cell lineages in rodents. Our aim was to identify whether human cord blood cells, which are a rich source of stem cells, would be able to differentiate into hepatocytes when infused into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. We also wanted to test whether such differentiated cells were the result of cellular fusion or true stem cell transdifferentiation. METHODS: Unsorted mononuclear cell preparations of human cord blood were infused into sublethally irradiated NOD-SCID mice. After death, immunohistologic analysis of murine livers was performed using human specific hepatocyte, biliary, and endothelial markers. Fluorescent in situ hybridization (FISH) for mouse and human DNA was also performed. RESULTS: We show that human cord blood cells have the ability to engraft into NOD-SCID liver and become mature hepatocytes. We were unable to identify any biliary or endothelial differentiation. Furthermore, we do not detect any evidence of cell fusion in any of the human cells found in the mouse liver, suggesting that human cord blood cells are capable of true transdifferentiation into hepatocytes in vivo. CONCLUSIONS: We conclude that hepatocytes can derive from human cord blood cells when infused into NOD-SCID mice in the absence of fusion. The demonstration that human stem cell differentiation can occur in this murine model permits comprehensive study of human stem cell plasticity in vivo.     

Efficient ex vivo gene transfer into non-human primate hepatocytes using HIV-1 derived lentiviral vectors

BACKGROUND/AIMS: Lentivirus-mediated ex vivo gene therapy is becoming a promising approach for the treatment of liver metabolic disorders. However, the feasibility of this approach needs to be studied in large animal models. The purpose of this study was to evaluate the efficacy of ex vivo gene transfer into Macaca hepatocytes with two different HIV-1 derived lentiviral vectors. METHODS: A self-inactivating lentivector was constructed to express GFP under the control of the hepatic apolipoprotein A-II promoter. Freshly isolated and thawed hepatocytes were transduced in suspension with lentiviral vectors expressing the GFP gene under the control of a ubiquitous promoter (EF1-alpha) and the apolipoprotein A-II promoter. Transduced thawed hepatocytes were transplanted into the spleen of newborn mice, and livers analyzed 4 and 12 weeks after transplantation. RESULTS: We show that lentivectors are efficient in transducing hepatocytes in suspension either freshly isolated or cryopreserved. We also show that thawed and transduced hepatocytes engrafted and participated in liver growth after transplantation into newborn mice and that the apolipoprotein A-II promoter is functional. CONCLUSIONS: Our data show that transplantation of transduced hepatocytes into monkeys should allow to evaluate the fate of transplanted cells and transgene expression in a pre-clinical model of ex vivo gene therapy.     

Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus

Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and hepatotropic viruses, for which no permissive cell lines exist. Here, we report partial repopulation of the liver of immunodeficient urokinase-type plasminogen activator (uPA)/recombinant activation gene-2 (RAG-2) mice with normal human hepatocytes isolated from the adult liver. In the transplanted mice, the production of human albumin was demonstrated, indicating that human hepatocytes remained functional in the mouse liver for at least 2 months after transplantation. Inoculation of transplanted mice with human hepatitis B virus (HBV) led to the establishment of productive HBV infection. According to human-specific genomic DNA analysis and immunostaining of cryostat liver sections, human hepatocytes were estimated to constitute up to 15% of the uPA/RAG-2 mouse liver. This is proof that normal human hepatocytes can integrate into the mouse hepatic parenchyma, undergo multiple cell divisions, and remain permissive for a human hepatotropic virus in a xenogenic liver. This system will provide new opportunities for studies on etiology and therapy of viral and nonviral human liver diseases, as well as on hepatocyte biology and hepatocellular transplantation.     

Ectopic expression of murine CD47 minimizes macrophage rejection of human hepatocyte xenografts in immunodeficient mice

Macrophages play an important role in the rejection of xenogeneic cells and therefore represent a major obstacle to generating chimeric mice with human xenografts that are useful tools for basic and preclinical medical research. The signal inhibitory regulatory protein α (SIRPα) receptor is a negative regulator of macrophage phagocytic activity and interacts in a species-specific fashion with its ligand CD47. Furthermore, SIRPα polymorphism in laboratory mouse strains significantly affects the extent of human CD47-mediated toleration of human xenotransplants. Aiming to minimize macrophage activity and thus optimize human cell engraftment in immunodeficient mice, we lentivirally transduced murine CD47 (Cd47) into human liver cells. Human HepG2 liver cells expressing Cd47 were less frequently contacted and phagocytosed by murine RAW264.7 macrophages in vitro than their Cd47-negative counterparts. For the generation of human-mouse chimeric livers in immunodeficient BALB-ΔRAG/γ(c) -uPA (urokinase-type plasminogen activator) mice, freshly thawed cryopreserved human hepatocytes were transduced with a lentiviral expression vector for Cd47 using a refined in vitro transduction protocol immediately before transplantation. In vivo, Cd47-positive human primary hepatocytes were selectively retained following engraftment in immunodeficient mice, leading to at least a doubling of liver repopulation efficiencies. Conclusion: We conclude that ectopic expression of murine Cd47 in human hepatocytes selectively favors engraftment upon transplantation into mice, a finding that should have a profound impact on the generation of robust humanized small animal models. Moreover, dominance of ectopically expressed murine Cd47 over endogenous human CD47 should also widen the spectrum of immunodeficient mouse strains suitable for humanization. (HEPATOLOGY 2012).     

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.     

Endothelial progenitor cell transplantation improves the survival following liver injury in mice

BACKGROUND & AIMS: Neovascularization, which is vital to the healing of injured tissues, recently has been found to include both angiogenesis, which involves in mature endothelial cells, and vasculogenesis, involving endothelial progenitor cells. The aim of this study was to clarify the possible roles of endothelial progenitor cells during postnatal liver regeneration. METHODS: To determine how endothelial progenitor cells participate in liver regeneration, human or mouse endothelial progenitor cells were transplanted into the mice with carbon tetrachloride-induced acute liver injury. Survival rate of the mice in endothelial progenitor cell-transplanted and control groups was calculated. Separately, livers removed temporally from both groups were examined. RESULTS: At an early stage, transplanted human endothelial progenitor cells were seen mainly surrounding hepatic central veins where hepatocytes showed extensive necrosis; later, the transplanted cells formed tubular structures. More of these cells were observed along hepatic sinusoids. Transplantation of human or mouse endothelial progenitor cells improved survival of the mice following liver injury (from 28.6% to 85.7%, P < .0005 and from 33.3% to 80.0%, P < .001, respectively), accompanied by greater proliferation of hepatocytes. Human endothelial progenitor cells produced several growth factors, such as hepatocyte growth factor, transforming growth factor-alpha, heparin-binding epidermal growth factor-like growth factor, and vascular endothelial growth factor, and also elicited endogenous growth factors. CONCLUSIONS: Endogenous and exogenous growth factors and direct neovascularization after endothelial progenitor cell transplantation promoted liver regeneration, thus improving survival after liver injury. Transplantation of endothelial progenitor cells could represent a new therapeutic strategy for promoting liver regeneration.     

Proliferation of rat small hepatocytes after long-term cryopreservation

BACKGROUND/AIMS: The demand for clinical use of hepatocytes is escalating because cell transplantation will be an alternative to orthotopic liver transplantation and the shortage of liver donors is a serious problem throughout the world. However, the supply of fresh differentiated hepatocytes is limited and methods for cryopreservation of hepatocytes that can proliferate with hepatic functions are not satisfactorily established. METHODS: Colonies of small hepatocytes were collected and then maintained at -80 degrees C for more than 6 months. Albumin secretion and mRNA expression of thawed cells were measured by enzyme linked immunosorbent assay and Northern blotting, respectively, and the expression of hepatic functions was examined by immunoblotting. The ultrastructure of cryopreserved cells was also examined. RESULTS: About 60% of the cryopreserved colonies attached on dishes and then proliferated. The average area of small hepatocyte colonies was about 7.5 times larger at day 15 than at day 1. Albumin production increased with time in culture. In addition, the cells produced other serum proteins such as transferrin and fibrinogen, and expressed carbamoyl phosphate synthetase I and tryptophan 2,3-dioxygenase. CONCLUSIONS: Small hepatocytes maintain growth ability and hepatic differentiated functions even after long-term cryopreservation.     

Hepatocyte transplantation activates hepatic stellate cells with beneficial modulation of cell engraftment in the rat

We investigated whether transplanted hepatocytes interact with hepatic stellate cells, as cell-cell interactions could modulate their engraftment in the liver. We transplanted Fischer 344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. Activation of hepatic stellate cells was analyzed by changes in gene expression, including desmin and alpha-smooth muscle actin, matrix proteases and their inhibitors, growth factors, and other stellate cell-associated genes with histological methods or polymerase chain reaction. Furthermore, the potential role of hepatic ischemia, Kupffer cells, and cytokine release in hepatic stellate cell activation was investigated. Hepatocyte transplantation activated desmin-positive hepatic stellate cells, as well as Kupffer cells, including in proximity with transplanted cells. Inhibition of Kupffer cells by gadolinium chloride, blockade of tumor necrosis factor alpha (TNF-alpha) activity with etanercept or attenuation of liver ischemia with nitroglycerin did not decrease this hepatic stellate cell perturbation. After cell transplantation, soluble signals capable of activating hepatic stellate cells were rapidly induced, along with early upregulated expression of matrix metalloproteinases-2, -3, -9, -13, -14, and their inhibitors. Moreover, prior depletion of activated hepatic stellate cells with gliotoxin decreased transplanted cell engraftment. In conclusion, cell transplantation activated hepatic stellate cells, which, in turn, contributed to transplanted cell engraftment in the liver. Manipulation of hepatic stellate cells might provide new strategies to improve liver repopulation after enhanced transplanted cell engraftment.     

Directed differentiation of human embryonic stem cells into functional hepatic cells

The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next, the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation, these cells expressed the adult liver cell markers tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp7A1, Cyp3A4 and Cyp2B6. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion, glycogen storage, indocyanine green, and low-density lipoprotein uptake, and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice, these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition, we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. CONCLUSION: We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development, and form the basis for hepatocyte transplantation and drug tests.     

Hepatic differentiation of amniotic epithelial cells

Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell-derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)-treated SCID/beige mice, na?ve hAECs differentiated into hepatocyte-like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. CONCLUSION: Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation.     

Hepatocyte transplantation: Studies in preclinical models

Summary Transplantation of allogeneic or genetically modified autologous hepatocytes may be an alternative to whole-liver transplantation for the treatment of hereditary metabolic liver diseases. Human hepatocytes have already been transplanted in patients, demonstrating the safety and feasibility of both approaches. Although a few cases of allogeneic transplantation have resulted in long-term engraftment and function, only a partial and transient correction of the disease was achieved. This may partly result from a lack of proliferation of transplanted cells. In rodents, transplanted hepatocytes do not proliferate in adult quiescent livers and repopulate recipient livers only when they display a proliferative advantage over resident hepatocytes. Most of these models are not transposable to humans, however. Our aim is to develop preclinical approaches to hepatocyte transplantation in nonhuman primates. We have defined a strategy that increases the engraftment efficiency of transplanted hepatocytes by inducing their proliferation together with that of resident hepatocytes. We have also immortalized simian fetal hepatic progenitor cells and shown that these cells do not proliferate in situ after transplantation into the livers of immunodeficient mice. By contrast early human hepatoblasts repopulate mouse livers more efficiently. However, if we consider the number of cells to be transplanted (one to several billion), the means of expanding and differentiating stem or progenitor cells other than hepatocytes will have to be determined prior to envisaging treating patients. Presented at the 42nd Annual Meeting of the SSIEM, Paris, 6–9 September, 2005. Competing interests: None declared     

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.     

Expanding hepatocytes in vitro before cell transplantation: donor age-dependent proliferative capacity of cultured human hepatocytes

BACKGROUND: For hepatocyte transplantation as well as experimental purposes, it would be advantageous to be able to expand human hepatocytes in vitro. However, under serum-free conditions, even with supplements of HGF (hepatic growth factor) and EGF (epidermal growth factor), proliferation of human hepatocytes is hampered. The aim of this study was to identify differences in the proliferative capacity of cultured primary human hepatocytes related to the age of the liver donors. METHODS: Proliferation was determined by BrdU-uptake, ploidy was measured using propidium iodide staining and flow cytometry, and the expression of cell cycle related proteins was determined by Western blotting. RESULTS: During the initial culture, juvenile hepatocytes proliferated better than adult hepatocytes. The proliferation rate declined to barely detectable levels after 8 days in culture in both juvenile and adult hepatocytes. The higher proliferative capacity of juvenile hepatocytes was associated with a larger fraction of diploid cells and a higher viability. The expression of regulatory cell cycle related proteins was higher in juvenile than in adult hepatocytes. CONCLUSIONS: The proliferation of human hepatocytes in vitro is critically related to a large fraction of diploid hepatocytes. The expression of regulatory cell cycle proteins reflects the proliferative capacity of cultured human hepatocytes. Juvenile as compared to adult human hepatocytes may be better suited for expansion in culture and could have a stronger repopulation capacity in vivo.     

Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera

A small animal model harboring a functional human liver cell xenograft would be a useful tool to study human liver cell biology, drug metabolism, and infections with hepatotropic viruses. Here we describe the repopulation, organization, and function of human hepatocytes in a mouse recipient and the infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) of the transplanted cells. Homozygous urokinase plasminogen activator (uPA)-SCID mice underwent transplantation with primary human hepatocytes, and at different times animals were bled and sacrificed to analyze plasma and liver tissue, respectively. The plasma of mice that were successfully transplanted contained albumin and an additional 21 human proteins. Liver histology showed progressive and massive replacement of diseased mouse tissue by human hepatocytes. These cells were accumulating glycogen but appeared otherwise normal and showed no signs of damage or death. They formed functional bile canaliculi that connected to mouse canaliculi. Besides mature hepatocytes, human hepatic progenitor cells that were differentiating into mature hepatocytes could be identified within liver parenchyma. Infection of chimeric mice with HBV or HCV resulted in an active infection that did not alter the liver function and architecture. Electron microscopy showed the presence of viral and subviral structures in HBV infected hepatocytes. In conclusion, human hepatocytes repopulate the uPA(+/+)-SCID mouse liver in a very organized fashion with preservation of normal cell function. The presence of human hepatic progenitor cells in these chimeric animals necessitates a critical review of the observations and conclusions made in experiments with isolated "mature" hepatocytes. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).     

Hepatic sinusoidal vasodilators improve transplanted cell engraftment and ameliorate microcirculatory perturbations in the liver

After transplantation, hepatocytes entering liver sinusoids are engrafted, whereas cells entrapped in portal spaces are cleared. We studied whether hepatic sinusoidal dilatation will increase the entry of transplanted cells in the liver lobule, improve cell engraftment, and decrease microcirculatory perturbations. F344 rat hepatocytes were transplanted intrasplenically into syngeneic dipeptidyl peptidase IV (DPPIV)-deficient rats. Animals were treated with adrenergic receptor blockers (phentolamine, labetalol), a calcium channel blocker (nifedipine), and splanchnic vasodilators (nitroglycerine, calcitonin gene-related peptide [CGRP], glucagon). Transplanted cells were localized by histochemistry. The hepatic microcirculation was studied with in vivo videomicroscopy. Changes in cell translocations were analyzed by injection of (99m)Tc-labeled hepatocytes. Pretreatment with phentolamine and nitroglycerine increased transplanted cell entry in liver sinusoids, whereas labetalol, nifedipine, CGRP, and glucagon were ineffective. Increased deposition of transplanted cells in sinusoids resulted in greater cell engraftment. In vivo microscopy showed disruption of sinusoidal blood flow immediately after cell transplantation with circulatory restoration requiring more than 12 to 24 hours after cell transplantation. However, in nitroglycerine-treated animals, sinusoidal blood flow was perturbed less. Nitroglycerine did not meaningfully increase intrapulmonary cell translocations. In conclusion, these findings indicate that hepatic sinusoidal capacitance is regulated by alpha-adrenergic- and nitroglycerine-responsive elements. Sinusoidal vasodilatation benefited intrahepatic distribution of transplanted cells and restored hepatic microcirculation after cell transplantation. This shall facilitate optimization of clinical cell transplantation and offers novel ways to investigate vascular mechanisms regulating hepatic sinusoidal reactivity.     

Susceptibility of chimeric mice with livers repopulated by serially subcultured human hepatocytes to hepatitis B virus

We previously identified a small population of replicative hepatocytes in long-term cultures of human adult parenchymal hepatocytes (PHs) at a frequency of 0.01%-0.09%. These hepatocytes were able to grow continuously through serial subcultures as colony-forming parenchymal hepatocytes (CFPHs). In the present study, we generated gene expression profiles for cultured CFPHs and found that they expressed cytokeratin 19, CD90 (Thy-1), and CD44, but not mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) and glucose-6-phosphatase (G6P), confirming that these cells are hepatic progenitor-like cells. The cultured CFPHs were resistant to infection with human hepatitis B virus (HBV). To examine the growth and differentiation capacity of the cells in vivo, serially subcultured CFPHs were transplanted into the progeny of a cross between albumin promoter/enhancer-driven urokinase plasminogen activator-transgenic mice and severe combined immunodeficient (SCID) mice. The cells were engrafted into the liver and were able to grow for at least 10 weeks, ultimately reaching a maximum occupancy rate of 27%. The CFPHs in the host liver expressed differentiation markers such as TO, G6P, and cytochrome P450 subtypes and could be infected with HBV. CFPH-chimeric mice with a relatively high replacement rate exhibited viremia and had high serum levels of hepatitis B surface antigen. CONCLUSION: Serially subcultured human hepatic progenitor-like cells from postnatal livers successfully repopulated injured livers and exhibited several phenotypes of mature hepatocytes, including susceptibility to HBV. In vitro-expanded CFPHs can be used to characterize the differentiation state of human hepatic progenitor-like cells.     

Hepatocyte transplantation and drug-induced perturbations in liver cell compartments

The potential for organ damage after using drugs or chemicals is a critical issue in medicine. To delineate mechanisms of drug-induced hepatic injury, we used transplanted cells as reporters in dipeptidyl peptidase IV-deficient mice. These mice were given phenytoin and rifampicin for 3 days, after which monocrotaline was given followed 1 day later by intrasplenic transplantation of healthy C57BL/6 mouse hepatocytes. We examined endothelial and hepatic damage by serologic or tissue studies and assessed changes in transplanted cell engraftment and liver repopulation by histochemical staining for dipeptidyl peptidase IV. Monocrotaline caused denudation of the hepatic sinusoidal endothelium and increased serum hyaluronic acid levels, along with superior transplanted cell engraftment. Together, phenytoin, rifampicin, and monocrotaline caused further endothelial damage, reflected by greater improvement in cell engraftment. Phenytoin, rifampicin, and monocrotaline produced injury in hepatocytes that was not apparent after conventional tissue studies. This led to transplanted cell proliferation and extensive liver repopulation over several weeks, which was more efficient in males compared with females, including greater induction by phenytoin and rifampicin of cytochrome P450 3A4 isoform that converts monocrotaline to toxic intermediates. Through this and other possible mechanisms, monocrotaline-induced injury in the endothelial compartment was retargeted to simultaneously involve hepatocytes over the long term. Moreover, after this hepatic injury, native liver cells were more susceptible to additional pro-oxidant injury through thyroid hormone, which accelerated the kinetics of liver repopulation. Conclusion: Transplanted reporter cells will be useful for obtaining insights into homeostatic mechanisms involving liver cell compartments, whereas targeted injury in hepatic endothelial and parenchymal cells with suitable drugs will also help advance liver cell therapy.     

Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen

BACKGROUND/AIMS: Hepatocytes immortalized with a temperature-sensitive SV40 large T antigen (SV40Tag) function as well as primary hepatocytes following transplantation to reverse hepatic encephalopathy and improve survival in rodents with liver failure. The continued presence of SV40Tag in the conditionally immortalized hepatocytes may increase the risk of malignant tumor growth in transplant recipients. METHODS: We immortalized hepatocytes using a recombinant retrovirus containing the gene encoding SV40Tag flanked by loxP recombination target sites. Excision of SV40Tag from immortalized cells could then be accomplished by site-specific recombination with Cre-recombinase. RESULTS: Cells immortalized with this recombinant virus expressed SV40Tag and doubled in number every 48 h. After excision of the gene encoding SV40Tag with Cre-recombinase, cells stopped growing, DNA synthesis fell by 90%, and production of liver-specific mRNAs was either increased or became newly detectable. In addition, the morphology and epithelial cell polarity of the cells became more characteristic of differentiated hepatocytes. To determine their malignant potential, immortalized hepatocytes were transfected to express a second oncogene, activated H-ras. SV40Tag+/H-ras+-immortalized cells were capable of anchorage-independent growth and developed into tumors when injected in severe combined immunodeficiency mice. While Cre-recombinase delivery by recombinant adenovirus infection was not 100% efficient, when SV40Tag excision occurred anchorage-independent growth stopped and tumor formation in immunodeficient mice was abolished. Immortalized hepatocytes also contained the gene encoding herpes simplex virus thymidine kinase and treatment with ganciclovir produced complete regression of established tumors in mice. CONCLUSIONS: These studies extend previous work that indicates that a transplantable hepatocyte cell line could be developed for clinical use.     

Identification of expandable human hepatic progenitors which differentiate into mature hepatic cells in vivo

BACKGROUND: Liver diseases include a wide spectrum of both acute and chronic conditions which are associated with significant morbidity and mortality worldwide. Hepatocyte transplantation has therapeutic potential in the treatment of liver diseases, but its clinical use is hampered by the lack of donor tissue. Generation of hepatocytes in vitro from adult or fetal liver cell progenitors or, alternatively, identification of a progenitor population which in vivo can generate mature liver cells could solve this problem. METHODS: CD117+/CD34+/Lin- human fetal liver cells were isolated by magnetic cell sorting and expanded in culture. Both freshly isolated and in vitro expanded cells in various passages were studied for their ability to be functional in hepatic parenchyma following d-galactosamine (GalN) induced injury in nude C57 black mice. RESULTS: Freshly isolated and in vitro expanded CD117+/CD34+/Lin- cells, when transplanted intrasplenically into GalN treated mice, morphologically and functionally differentiated into hepatocytes and cholangiocytes. Human specific albumin, alpha fetoprotein, cytokeratin 19, and antitrypsin mRNA were expressed in mouse liver. In addition, the human progenitor cells expressed glucose-6-phosphatase, glycogen, albumin, gamma glutamyl transpeptidase, and dipeptidyl peptidase IV after transplantation. Expanded cells in various passages maintained their capacity to differentiate into functional liver cells. CONCLUSIONS: Fetal liver CD117+/CD34+/Lin- progenitors and their progeny proliferated in vitro and also functionally differentiated into mature hepatic cells in an acute liver injury model. Successful in vitro expansion of liver progenitor cells provides a basis for developing cell therapy strategies, metabolic and toxicity testing systems, and may serve as a vehicle for gene therapy.