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.     

Hepatocyte survival depends on beta1-integrin-mediated attachment of hepatocytes to hepatic extracellular matrix.

BACKGROUND: A major drawback of allogeneic hepatocyte transplantation is the lack of sustained survival of the transplanted cells in the recipient liver parenchyma. The purpose of this study was to determine the effect of the presence or absence of hepatic extracellular matrix (ECM) molecules on hepatocyte survival and function following hepatocyte isolation for transplantation purposes, and the role of beta1-integrin molecules therein. METHODS: Hepatocytes, either untreated or treated with anti-beta1 integrin antibodies or RGD peptides, were seeded on wells precoated with collagen type I, type IV, laminin, fibronectin or polyhydroxyethylmehacrylate. The extent of attachment and apoptosis was evaluated. RESULTS: When hepatocytes were added into wells precoated with either fibronectin, or collagen type IV, rapid spreading and prolonged survival occurred, in contrast to hepatocytes that were seeded in wells precoated with collagen type I or polyhydroxyethylmehacrylate. Pretreatment of the cells with anti-beta1-integrin antibodies resulted in reduction of cell attachment to laminin, fibronectin, collagen I, and collagen IV. Synthetic RGD (arginine-glycine-aspartate)-peptides and anti-beta1 antibodies inhibited apoptosis of cultured hepatocytes. CONCLUSIONS: Our findings indicate that embedding of hepatocytes within their normal liver ECM surroundings maintains their survival. When detached from their natural surrounding hepatocytes enter into apoptosis, unless treated with anti-beta1-integrin antibodies or RGD peptides. This knowledge will allow improvement of hepatocyte transplantation efficiency.     

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.     

Hepatocyte transplantation for total liver repopulation

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

Immunosuppression using the mTOR inhibition mechanism affects replacement of rat liver with transplanted cells

Successful grafting of tissues or cells from mismatched donors requires systemic immunosuppression. It is yet to be determined whether immunosuppressive manipulations perturb transplanted cell engraftment or proliferation. We used syngeneic and allogeneic cell transplantation assays based on F344 recipient rats lacking dipeptidyl peptidase IV enzyme activity to identify transplanted hepatocytes. Immunosuppressive drugs used were tacrolimus (a calcineurin inhibitor) and its synergistic partners, rapamycin (a regulator of the mammalian target of rapamycin [mTOR]) and mycophenolate mofetil (an inosine monophosphate dehydrogenase inhibitor). First, suitable drug doses capable of inducing long-term survival of allografted hepatocytes were identified. In pharmacologically effective doses, rapamycin enhanced cell engraftment by downregulating hepatic expression of selected inflammatory cytokines but profoundly impaired proliferation of transplanted cells, which was necessary for liver repopulation. In contrast, tacrolimus and/or mycophenolate mofetil perturbed neither transplanted cell engraftment nor their proliferation. Therefore, mTOR-dependent extracellular and intracellular mechanisms affected liver replacement with transplanted cells. In conclusion, insights into the biological effects of specific drugs on transplanted cells are critical in identifying suitable immunosuppressive strategies for cell therapy.     

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.     

Therapeutic liver repopulation for metabolic liver diseases: Advances from bench to bedside

Metabolic liver diseases are characterized by inherited defects in hepatic enzymes or other proteins with metabolic functions. Therapeutic liver repopulation (TLR), an approach of massive liver replacement by transplanted normal hepatocytes, could be used to provide the missing metabolic function elegantly. However, partial and transient correction of the underlying metabolic defects due to very few integrated donor cell mass remains the major obstacle for the effective and widespread use of this approach. Little engraftment and proliferation insufficiency lead to the poor outcome. This article reviews the advances in the mechanisms of initial engraftment and selective proliferation and suggests some effective treatment strategies, from pharmacological preconditioning to stem cell transplantation, to optimize liver repopulation with liver cell transplantation. Enhancing cell viability and plating efficiency, increasing sinusoidal spaces, regulation of sinusoidal endothelial cell barrier and controlling inflammatory reaction may promote initial cell engraftment. Liver‐directed irradiation, reversible portal vein embolization and fetal liver stem/progenitor cell transplantation induce preferential proliferation of donor cells substantially without severe side‐effects. Furthermore, it seems better to use combined approaches to achieve a high level of liver repopulation for the management of metabolic liver diseases.     

Monocrotaline promotes transplanted cell engraftment and advances liver repopulation in rats via liver conditioning

Disruption of the hepatic endothelial barrier or Kupffer cell function facilitates transplanted cell engraftment in the liver. To determine whether these mechanisms could be activated simultaneously, we studied the effects of monocrotaline, a pyrollizidine alkaloid, with reported toxicity in liver sinusoidal endothelial cells and Kupffer cells. The effects of monocrotaline in Fischer 344 rats were examined by tissue morphology, serum hyaluronic acid levels, and liver tests (endothelial and hepatocyte injury) or incorporation of carbon and (99m)Tc-sulfur colloid (Kupffer cell damage). To study changes in cell engraftment and liver repopulation, Fischer 344 rat hepatocytes were transplanted into syngeneic dipeptidyl peptidase IV-deficient rats followed by histological assays. We observed extensive endothelial injury without Kupffer cell or hepatocyte damage in monocrotaline-treated rats. Monocrotaline enhanced transplanted cell engraftment without changes in transplanted cell numbers or induction of proliferation in native hepatocytes over 3 months. In monocrotaline-treated rats, transplanted cells integrated into the liver parenchyma and survived in vascular spaces. To determine whether native hepatocytes suffered inapparent damage after monocrotaline, we introduced further liver injury with carbon tetrachloride subsequent to cell transplantation. Monocrotaline sensitized the liver to carbon tetrachloride-induced necrosis, which advanced transplanted cell proliferation, leading to significant liver repopulation. During this process, we observed proliferation of bile duct cells and small epithelial cells, although transplanted hepatocytes did not appear to reconstitute bile ducts. The studies showed that perturbation of multiple liver cell compartments by monocrotaline promoted transplanted cell engraftment and proliferation. In conclusion, development of drugs with monocrotaline-like effects will help advance liver cell therapy.     

A human umbilical cord stem cell rescue therapy in a murine model of toxic liver injury

BACKGROUND: Several studies have demonstrated that bone marrow contains a subpopulation of stem cells capable of participating in the hepatic regenerative process, even if some reports indicate quite a low level of liver repopulation by human stem cells in the normal and transiently injured liver. AIMS: In order to overcome the low engraftment levels seen in previous models, we tried the direct intraperitoneal administration of human cord blood stem cells, using a model of hepatic damage induced by allyl alcohol in NOD/SCID mice. METHODS: We designed a protocol based on stem cell infusion following liver damage in the absence of irradiation. Flow cytometry, histology, immunohistochemistry and RT-PCR for human hepatic markers were performed to monitor human cell engraftment. RESULTS: Human stem cells were able to transdifferentiate into hepatocytes, to improve liver regeneration after damage and to reduce the mortality rate both in both protocols, even if with qualitative and quantitative differences in the transdifferentiation process. CONCLUSIONS: We demonstrated for the first time that the intraperitoneal administration of stem cells can guarantee a rapid liver engraftment. Moreover, the new protocol based on stem cell infusion following liver damage in the absence of irradiation may represent a step forward for the clinical application of stem cell transplantation.     

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat

Background

Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous β2‐microglubulin⁻/Thy1⁺ bone marrow derived cells.

Aim

To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia.

Methods and results

Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for β2‐microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non‐injured liver. Transplantation of bone marrow derived cells obtained from GFP‐transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP‐glucuronyltransferase enzyme evaluated by polymerase chain reaction.

Conclusions

I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte‐like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non‐affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.     

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.     

Noninvasive evaluation of liver repopulation by transplanted hepatocytes using 31P MRS imaging in mice

Hepatocyte transplantation (HT) is being explored as a substitute for liver transplantation for the treatment of liver diseases. For the clinical application of HT, a preparative regimen that allows preferential proliferation of transplanted cells in the host liver and a noninvasive method to monitor donor cell engraftment, proliferation, and immune rejection would be useful. We describe an imaging method that employs the creatine kinase (CK) gene as a marker of donor hepatocytes. Creatine kinase is unique among marker genes, because it is normally expressed in brain and muscle tissues and is therefore not immunogenic. Preferential proliferation of transplanted CK-expressing hepatocytes was induced by preparative hepatic irradiation and expression of hepatocyte growth factor using a recombinant adenoviral vector. CK is normally not expressed in mouse liver and its expression by the donor cells led to the production of phosphocreatine in the host liver, permitting (31)P magnetic resonance spectroscopic imaging of liver repopulation by engrafted hepatocytes. In conclusion, this study combined a noninvasive imaging technique to assess donor hepatocyte proliferation with a preparative regimen of partial liver irradiation that allowed regional repopulation of the host liver. Our results provide groundwork for future development of clinical protocols for HT.     

Sinusoidal endothelial cell repopulation following ischemia/reperfusion injury in rat liver transplantation

We evaluated the kinetics by which rat liver sinusoidal endothelial cells (LSECs) are repopulated in the reperfused transplanted liver after 18 hours of cold ischemic storage. We found that the majority of LSECs in livers cold-stored for 18 hours in University of Wisconsin solution are seriously compromised and often are retracted before transplantation. Sinusoids rapidly re-endothelialize within 48 hours of transplantation, and repopulation is coincident with up-regulation of hepatocyte vascular endothelial growth factor expression and vascular endothelial growth factor receptor-2 expression on large vessel endothelial cells and repopulating LSECs. Although re-endothelialization occurs rapidly, we show here, using several high-resolution imaging techniques and 2 different rat liver transplantation models, that engraftment of bone marrow-derived cells into functioning LSECs is routinely between 1% and 5%. CONCLUSION: Bone marrow plays a measurable but surprisingly limited role in the rapid repopulation and functional engraftment of bone marrow-derived LSECs after cold ischemia and warm reperfusion.     

Immediate intraportal transplantation of human bone marrow mesenchymal stem cells prevents death from fulminant hepatic failure in pigs

The effectiveness of human bone marrow mesenchymal stem cell (hBMSC) transplantation to treat acute and chronic liver injury has been demonstrated in animal models and in a few nonrandomized clinical trials. However, no studies have investigated hBMSC transplantation in the treatment of fulminant hepatic failure (FHF), especially in large animal (pig) models. The aim of this study was to demonstrate the safety, effectiveness, and underlying mechanism of hBMSC transplantation for treating FHF in pigs through the intraportal route. Human BMSCs (3 × 10(7) ) were transplanted into pigs with FHF via the intraportal route or peripheral vein immediately after D-galactosamine injection, and a sham group underwent intraportal transplantation (IPT) without cells (IPT, peripheral vein transplantation [PVT], and control groups, respectively, n = 15 per group). All of the animals in the PVT and control groups died of FHF within 96 hours. In contrast, 13 of 15 animals in the IPT group achieved long-term survival (>6 months). Immunohistochemistry demonstrated that transplanted hBMSC-derived hepatocytes in surviving animals were widely distributed in the hepatic lobules and the liver parenchyma from weeks 2 to 10. Thirty percent of the hepatocytes were hBMSC-derived. However, the number of transplanted cells decreased significantly at week 15. Only a few single cells were scattered in the regenerated liver lobules at week 20, and the liver tissues exhibited a nearly normal structure. Conclusion: Immediate IPT of hBMSCs is a safe and effective treatment for FHF. The transplanted hBMSCs may quickly participate in liver regeneration via proliferation and transdifferentiation into hepatocytes during the initial stage of FHF. This method can possibly be used in future clinical therapy. (HEPATOLOGY 2012;56:1044-1052).     

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.     

Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling

BACKGROUND AND AIMS: Few circulating tumor cells implant or cause metastasis. We hypothesized that venous or lymphatic pressure or iatrogenic pressure during resection activates signals governing malignant colonocyte adhesion. METHODS: We studied the effect of 15 mm Hg increased pressure for 30 minutes on adhesion of primary human colon cancer cells and SW620 colonocytes to collagen and endothelial cells. We modulated integrin affinity with extracellular cations. We assessed binding affinity by detachment assay; integrin surface expression by flow cytometry; and focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinase (ERK) activation by Western analysis and Src in vitro kinase assay. We inhibited Src (PP2), FAK (small RNA interference, SiRNA, or FRNK transfection), MEK (PD98059), PKC (calphostin C), and actin destabilization (phalloidin). RESULTS: Pressure and manganese stimulated primary and SW620 colonocyte adhesion to collagen. Pressure also stimulated SW620 adhesion to endothelial monolayers. Pressure strengthened SW620 binding force to matrix without changing integrin surface expression. Pressure activated SW620 FAK and Src, but not ERK. Manganese did not. Calcium-inhibited adhesion but stimulated FAK (but not Src). PP2 prevented pressure activation of Src, Src phosphorylation of FAK576, and pressure-stimulated adhesion but not FAK397 autophosphorylation. FRNK transfection or FAK SiRNA also prevented pressure-stimulated adhesion. FAK SiRNA ablated pressure-activated FAK397, Src, and FAK576 phosphorylation. Neither Src nor FAK inhibition blocked cation effects. Phalloidin prevented pressure-stimulated adhesion. PD98059 or calphostin C did not. CONCLUSIONS: In contrast to divalent cations, extracellular pressure may increase integrin affinity and promote colon cancer adhesion via actin dependent inside-out FAK and Src signals. This mechanotransduced pathway may regulate metastasizing tumor cell adhesion.     

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.     

Kupffer cells participate in early clearance of syngeneic hepatocytes transplanted in the rat liver

BACKGROUND & AIMS: Kupffer cells are activated shortly after deposition of hepatocytes in liver sinusoids, with clearance of a significant fraction of transplanted cells, especially when cells are entrapped in portal spaces. We determined whether perturbation of Kupffer cells would improve transplanted cell engraftment. METHODS: Dipeptidyl peptidase IV-deficient rats were used as recipients of syngeneic Fischer 344 rat hepatocytes. Kupffer cell function was analyzed by measuring phagocytic activity with carbon particle or (99m)Tc-sulfur colloid incorporation. Transplanted cell survival and integration in the liver parenchyma was determined by histochemical analysis of tissues. Transplanted cell proliferation was analyzed in rats conditioned with retrorsine and partial hepatectomy. RESULTS: Gadolinium chloride significantly impaired Kupffer cell function, especially in periportal areas, where transplanted cells were localized. Transplanted cell survival increased by approximately 2-fold in animals treated with gadolinium chloride 24 hours before cell transplantation. In gadolinium-treated rats, more transplanted cells were observed in portal vein radicles, as well as in liver sinusoids, albeit integration of cells in the liver parenchyma was slower in gadolinium-treated rats and cells separated from other hepatocytes in portal vein radicles that failed to exhibit bile canalicular reconstitution. Finally, hepatocyte transplantation in rats primed with retrorsine and partial hepatectomy showed accelerated kinetics of liver repopulation in animals pretreated with gadolinium chloride. CONCLUSIONS: Perturbation of Kupffer cell activity will benefit liver repopulation with cells and further analysis of clinically suitable approaches to exploit this mechanism will be appropriate.     

Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency

BACKGROUND & AIMS: Donor cell engraftment with expression of enzyme activity is the goal of liver cell transplantation for inborn errors of liver metabolism with a view to achieving sustained metabolic control. METHODS: Sequential hepatic cell transplantations using male and female cells were performed in a 3.5-year-old girl with argininosuccinate lyase deficiency over a period of 5 months. Beside clinical, psychomotor, and metabolic follow-up, engraftment was analyzed in repeated liver biopsies (2.5, 5, 8, and 12 months after first infusion) by fluorescence in situ hybridization for the Y-chromosome and by measurement of tissue enzyme activity. RESULTS: Metabolic control was achieved together with psychomotor catch-up, changing the clinical phenotype from a severe neonatal one to a moderate late-onset type. The child was no longer hospitalized and was able to attend normal school. Sustained engraftment of male donor liver cells was shown in repeated biopsies, reaching 19% at 8 months and 12.5% at the 12-month follow-up. XXYY tetraploid donor cells were mainly detected during the infusion period (2.5- and 5-month biopsies), whereas in the follow-up 8-month and 1-year biopsies, diploid donor cell subpopulations had become dominant. Moreover, argininosuccinate lyase activity, originally absent, became measurable in 2 different biopsy samples at 8 months, reaching 3% of control activity, indicating in situ metabolic effect and supporting the clinical evolution to a moderate form of the disease. CONCLUSIONS: Liver cell transplantation can achieve donor cell engraftment in humans in a significant proportion, leading to sustained metabolic and clinical control with psychomotor catch-up.