Engraftment Of Hepatocytes Research Articles

Regulation of Hepatocyte Engraftment and Proliferation after Cytotoxic Drug-Induced Perturbation of the Rat Liver

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. 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. 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. Doxorubicin-induced hepatic endothelial damage enhanced cell engraftment, which should be useful in cell therapy strategies.

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.

Vascular Endothelial Growth Factor-Releasing Scaffolds Enhance Vascularization and Engraftment of Hepatocytes Transplanted on Liver Lobes

Hepatocyte transplantation within porous scaffolds (HT) is being explored as a treatment strategy for end-stage liver diseases and enzyme deficiencies. One of the main issues in this approach is the limited viability of transplanted cells because vascularization of the scaffold site is either too slow or insufficient. We now address this by enhancing scaffold vascularization before cell transplantation via sustained delivery of vascular endothelial growth factor (VEGF), and by examining the liver lobes as a platform for transplanting donor hepatocytes in close proximity to the host liver. The vascularization kinetics of unseeded VEGF-releasing scaffolds on rat liver lobes were evaluated by analyzing the microvascular density and tissue ingrowth in implants harvested on days 3, 7, and 14 postimplantation. Capillary density was greater at all times in VEGF-releasing scaffolds than in the control scaffold without VEGF supplementation; on day 14, it was 220 +/- 33 versus 139 +/- 23 capillaries/mm2 (p < 0.05). Furthermore, 35% of the newly formed capillaries in VEGF-releasing scaffolds were larger than 16 microm in diameter, whereas in control scaffolds only 10% exceeded this size. VEGF had no effect on tissue ingrowth into the scaffolds. HT onto the implanted VEGF-releasing or control scaffolds was performed after 1 week of prevascularization on the liver lobe in Lewis rats. Fifty implants were harvested on days 1, 3, 7, and 12 and the area of viable hepatocytes was evaluated. The enhanced vascularization improved hepatocyte engraftment; 12 days after HT, the intact hepatocyte area (136,910 microm2/cross-section) in VEGF-releasing scaffolds was 4.6 higher than in the control group. This study shows that sustained local delivery of VEGF induced vascularization of porous scaffolds implanted on liver lobes and improved hepatocyte engraftment.

HVEGF165Increases 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.

Hepatocyte transplantation

Hepatocyte transplantation

Stable hepatocyte transplantation using fibrin matrix.

Fibrin matrix, a naturally derived biodegradable polymer matrix, was evaluated as a scaffold for hepatocyte transplantation in an athymic mouse model. One week after transplantation, opaque conglomerates of the transplanted hepatocytes and fibrin matrix were found on the intestinal mesentery, whereas no transplanted hepatocytes were observed in control groups (transplantation of hepatocytes suspended in culture medium). The hepatocytes in the conglomerates retained hepatocyte-specific functions, as examined with histochemical and immunohistochemical stainings. Stable hepatocyte engraftment may thus be achieved by hepatocyte transplantation using fibrin matrix.

Serum from patients with fulminant hepatic failure causes hepatocyte detachment and apoptosis by a beta(1)-integrin pathway.

Hepatocyte transplantation is restricted by the impaired ability of hepatocytes to engraft and survive in the damaged liver. Understanding the mechanisms that control this process will permit the development of strategies to improve engraftment. We studied changes in liver matrix during acute injury and delineated the mechanisms that perturb the successful adhesion and engraftment of hepatocytes. Collagen IV expression was increased in sinusoidal endothelium and portal tracts of fulminant hepatic failure explants, whereas there were minimal changes in the expression of fibronectin, tenascin, and laminin. Using an in vitro model of cellular adhesion, hepatocytes were cultured on collagen-coated plates and exposed to serum from patients with liver injury to ascertain their subsequent adhesion and survival. There was a rapid, temporally progressive decrease in the adhesive properties of hepatocytes exposed to such serum that occurred within 4 hours of exposure. Loss of activity of the beta1-integrin receptor, which controls adhesion to collagen, was seen to precede this loss of adhesive ability. Addition of the beta1-integrin activating antibody (TS2/16) to cells cultured with liver injury serum significantly increased their adhesion to collagen, and prevented significant apoptosis. In conclusion, we have identified an important mechanism that underpins the failure of infused hepatocytes to engraft and survive in liver injury. Pretreating cells with an activating antibody can improve their engraftment and survival, indicating that serum from patients with liver injury exerts a defined nontoxic biological effect. This finding has important implications in the future of cellular transplantation for liver and other organ diseases.

A model to quantitate cell transplant function after liver-specific injury

Introduction: As the study of hepatic cytotherapeutics requires 1) selective pressure to improve engraftment of transplanted cells, and 2) a method to assess the hepatocyte-specific function of transplanted cells, we created a model to quantitate the functional engraftment of transplanted cells following liver-specific injury. Methods: Transgenic mice expressing human alpha-1 antitrypsin (hAAT) linked to the albumin promoter were used for donor cells. Liver injury was mediated through adenovirus transfection of urokinase-type tissue plasminogen activator (uPA). After uPA administration, 10 x 6 hAAT hepatocytes were injected into juvenile SCID mice. Control animals were uninjured. Recipients were bled serially after transplantation and the serum samples assessed for hAAT via ELISA. Results: Injection of uPA increased transaminase levels transiently, confirming hepatocyte injury. Uninjured SCID mice expressed 250 nanograms/mL of hAAT after injection of 10 x 6 hepatocytes. Mice receiving uPA prior to hepatocyte administration expressed 25 micrograms/mL of hAAT. Conclusions: uPA administration results in a 100-fold increase in transplanted hepatocyte engraftment and function. This model offers a method to confer positive selection for liver engraftment and yields a quantifiable increase in the hepatocyte-specific function of transplanted cells. This data establishes a basis to compare the kinetics of engraftment and function of cellular transplants from various sources after acute liver injury.

Route of hepatocyte delivery affects hepatocyte engraftment in the spleen.

In laboratory animals, intrasplenic hepatocyte transplantation corrects the physiologic abnormalities associated with decompensated liver disease. The clinical experience with hepatocyte transplantation for cirrhosis has been disappointing when compared with laboratory experience. The route of hepatocyte delivery may influence hepatocyte engraftment and function. Outbred pigs were recipients of allogeneic pig hepatocytes. Donor hepatocytes were isolated by collagenase perfusion and labeled using 5(6)-carboxyfluorescein diacetate succinimidyl-ester (CMFSE). Cells were introduced into pig spleens by infusion through the splenic artery or by direct splenic puncture. Direct intrasplenic injection produced engraftment that was far superior to that obtained using splenic artery infusion. Splenic artery infusion produced a gastric erosion and large areas of splenic necrosis secondary to vascular occlusion with hepatocytes, whereas direct splenic injection was associated with clinically insignificant intraabdominal hemorrhage. The route of hepatocyte delivery may influence hepatocyte engraftment and explain the disparity in efficacy of hepatocyte transplantation between the laboratory and clinic.

Transplanted human cord blood cells give rise to hepatocytes in engrafted mice.

Recent studies suggest that rodent hepatocytes may be derived from hematopoietic stem cells. In the current study, the potential hematopoietic origin of hepatocytes was addressed using xenogeneic transplantation of human cord blood cells. CD34(+) or CD45(+) human cord blood cells were transplanted into "conditioned" newborn NOD/SCID/beta2-microglobulin(null) mice. At 4 to 5 months post-transplantation, livers of the recipient mice were cryosectioned and examined for evidence of human hepatocyte engraftment using RT-PCR to detect human albumin mRNA, immunohistochemistry to detect human hepatocytic proteins, and fluorescence in situ hybridization (FISH) to detect the presence of human centromeric DNA. Analysis of the bone marrow of transplanted mice revealed that 21.0-45.9% of the cells were human CD45(+) cells. FISH analysis of frozen sections of transplanted mouse liver revealed the presence of engrafted cells positive for human centromeric DNA. That engrafted human cells functioned as hepatocytes was indicated by the expression of human albumin mRNA, as judged by RT-PCR. FISH analysis with human and mouse centromeric DNA probes excluded spontaneous cell fusion as the cause for the generation of human hepatocytes. Human cord blood cells can give rise to hepatocytes in a xenogeneic transplantation model. This model will be useful to further characterize the cord blood progenitors of hepatocytes.

The effect of heparanase on hepatocyte engraftment after intrasplenic cell transplantation in the rats

The effect of heparanase on hepatocyte engraftment after intrasplenic cell transplantation in the rats

The effects of different growth factors on human bone marrow stromal cells differentiating into hepatocyte-like cells.

Stem cells are self-renewable and are thought to be the pluripotent cells that can differentiate into a variety of cells or tissue types. For many years, adult stem cells harvested from bone marrow have been used therapeutically. In 1999, Pittenger et al. published the methods to induce differentiation of human mesenchymal stem cells into different cell types of mesodermal lineages. Theise et al. have demonstrated that hepatocytes, from endodermal lineage, can be derived from bone marrow in human and mice. Hepatocyte engraftment from bone marrow cells have been confirmed by double FISH (fluorescence in situ hybridization) after bone marrow transplantation. Lagasse et al. further demonstrated that purified hematopoietic stem cells from adult mouse bone marrow rescued fumarylacetoacetate hydrolase (FAH) deficient mice and restored the biochemical function of their liver. Alison et al.5showed that the human adult hematopoietic stem cell population is capable of yielding an epithelial lineage in chronically damaged liver. ß2m-/Thy-1+cells from rat and human bone marrow were integrated with hepatic cell plates and were differentiated into mature hepatocytes after intraportal infusion into rat livers studied by Avital et al.6ßzm7Thy-1+cells also differentiated into mature hepatocytes in a culture system simulating liver regeneration and containing cholestatic serum. Oh et al.’ and Schwartz et al. have succeeded in differentiating rat bone marrow cells and multipotent adult progenitor cells from bone marrow into hepatocyte-like cells by addition of growth factors in the culture medium. Hamazaki et al. induced hepatic maturation in mouse embryonic stem cellsin vitro.Other studies suggest that the microenvironment of embryonic liver, including growth factors and cell-cell interactions, is critical for regulating liver development. Acidic FGF and HGF were known to play important roles in the early-and mid-developmental stages of liver development. So far, none have succeeded to induce human bone marrow stromal cells (BMSC) differentiation into hepatocytes in culture.KeywordsHepatocyte Growth FactorHuman Mesenchymal Stem CellHuman Bone Marrow Stromal CellMature HepatocyteHepatic DifferentiationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

263 Heparanase increases hepatocyte engraftment and early endothelial cell proliferation after intrasplenic cell transplantation in hepatectomized rats

263 Heparanase increases hepatocyte engraftment and early endothelial cell proliferation after intrasplenic cell transplantation in hepatectomized rats

124 Hepatic integrin-dependent extracellular matrix (ECM) component interactions regulate hepatocyte engraftment in the liver

124 Hepatic integrin-dependent extracellular matrix (ECM) component interactions regulate hepatocyte engraftment in the liver

Bone marrow-derived liver stem cell and mature hepatocyte engraftment in livers undergoing rejection

Background. The definitive therapy for end-stage liver disease is orthotopic liver transplantation (OLT). However, rejection is still a major cause of mortality and morbidity following OLT. Hepatocyte transplantation has been used experimentally to treat liver diseases. The aim of this study was to investigate whether bone marrow-derived liver stem cells (BDLSC) and mature hepatocytes could repopulate transplanted livers undergoing rejection. Methods. OLT was carried out from D'Agouti (C3-positive female) into Lewis (C3-negative female) rats. BDLSC were transplanted from Lewis (male) into livers of D'Agouti (female) rats. Group A (n = 9) received intraportal normal saline. Groups B (n = 9) and C (n = 9) underwent intraportal transplantation of mature hepatocytes (Lewis female, 0.75 × 107) and DBLSC (Lewis male, 5 × 104) respectively. All groups received subtherapeutic immunosuppression (Cyclosporin 0.25 mg/kg/d) for 13 days. Liver repopulation was assessed using immunohistochemistry (C3 antigen-negative cells), in-situ hybridization, (Y-chromosome-positive BDLSC) and histologic assessment (hematoxylin and eosin) for rejection. Results. BDLSC and mature hepatocytes repopulated 62 ± 12.3% and 2.5 ± 1.7% of rejecting livers, respectively. BDLSC demonstrated formation of hepatic lobules and portal triads with little evidence of rejection 36 days after discontinuation of immunosuppression. Conclusions. BDLSC can repopulate livers undergoing severe rejection. Moreover, BDLSC can differentiate into hepatocytes and cholangiocytes. This finding may have important clinical implications. (Surgery 2002:132:384-90.)

Cyclophosphamide disrupts hepatic sinusoidal endothelium and improves transplanted cell engraftment in rat liver

To determine whether disruption of the hepatic sinusoidal endothelium will facilitate engraftment of transplanted cells, we treated Fischer 344 (F344) rats lacking dipeptidyl peptidase IV (DPPIV) activity with cyclophosphamide (CP). Electron microscopy showed endothelial injury within 6 hours following CP, and, after 24 and 48 hours, the endothelium was disrupted in most hepatic sinusoids. CP did not affect Kupffer cell function. Similarly, CP had no obvious effects on hepatocytes. Intrasplenic transplantation of F344 rat hepatocytes followed by their localization with DPPIV histochemistry showed 3- to 5-fold increases in the number of transplanted cells in CP-treated animals. Transplanted cells integrated in the liver parenchyma more rapidly in CP-treated animals, and hybrid bile canaliculi developed even 1 day after cell transplantation, which was not observed in control animals. To demonstrate whether improved cell engraftment translated into superior liver repopulation, recipient animals were conditioned with retrorsine and two-thirds partial hepatectomy (PH), which induces transplanted cell proliferation. CP treatment of these animals before cell transplantation significantly increased the number and size of transplanted cell foci. In conclusion, disruption of the hepatic sinusoidal endothelium was associated with accelerated entry and integration of transplanted cells in the liver parenchyma. These results provide insights into hepatocyte engraftment in the liver and will help in optimizing liver-directed cell therapy. (H EPATOLOGY 2002;36:112-121.)

Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices.

We investigated heterotopic hepatocyte transplantation on biodegradable polymers as a potential treatment for end-stage liver disease. The primary problem has been insufficient engraftment of transplanted cells partly because of insufficient vascularization. Increasing vascularization through locally delivered angiogenic factors may increase angiogenesis and hepatocyte engraftment. We studied the effect of local delivery of basic fibroblast growth factor (bFGF) on angiogenesis and hepatocyte engraftment within tissue-engineered liver constructs. Poly-l-lactic acid discs were fabricated and coated with either a mixture of saline, sucralfate, and Hydron (control group) or bFGF, sucralfate, and Hydron (bFGF group). bFGF release from polymers in vitro was tested using an ELISA. Hepatocytes were isolated from Lewis rats, seeded on control (n=9) or bFGF (n=11) polymers, and implanted into the small bowel mesentery of syngeneic animals. Specimens were harvested after 2 weeks and analyzed for hepatocyte engraftment. Microvascular density was compared between control (n=6) and bFGF groups (n=5). Three hundred twenty-three thousandths of a microgram of bFGF were incorporated per polymer. Greater than 99% of the bFGF was released into solution by 72 hr in vitro. Two weeks after implantation, microvascular density, as measured by capillaries per high-powered field (c/hpf), was significantly greater in the bFGF group (43.8 c/hpf), compared with the control group (30.5 c/hpf; P<0.005). Specimens from the bFGF group (mean engraftment, 61,355 microm2) showed a 2.5-fold increase in hepatocyte engraftment as compared with control (24,197 microm2; P<0.002). The angiogenic growth factor bFGF can be incorporated into degradable polymers used as delivery devices for hepatocyte transplantation. Implantation of these devices increases angiogenesis into the device and increases hepatocyte engraftment.

VEGF effect on hepatocyte engraftment after intrasplenic cell transplantation in rats

VEGF effect on hepatocyte engraftment after intrasplenic cell transplantation in rats

Hepatitis C virus replication in mice with chimeric human livers.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

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.