Etanercept blocks inflammatory responses orchestrated by TNF-α to promote transplanted cell engraftment and proliferation in rat liver.

UNLABELLED: Engraftment of transplanted cells is critical for liver-directed cell therapy, but most transplanted cells are rapidly cleared from liver sinusoids by proinflammatory cytokines/chemokines/receptors after activation of neutrophils or Kupffer cells (KCs). To define whether tumor necrosis factor alpha (TNF-α) served roles in cell-transplantation-induced hepatic inflammation, we used the TNF-α antagonist, etanercept (ETN), for studies in syngeneic rat hepatocyte transplantation systems. After cell transplantation, multiple cytokines/chemokines/receptors were overexpressed, whereas ETN before cell transplantation essentially normalized these responses. Moreover, ETN down-regulated cell-transplantation-induced intrahepatic release of secretory cytokines, such as high-mobility group box 1. These effects of ETN decreased cell-transplantation-induced activation of neutrophils, but not of KCs. Transplanted cell engraftment improved by several-fold in ETN-treated animals. These gains in cell engraftment were repeatedly realized after pretreatment of animals with ETN before multiple cell transplantation sessions. Transplanted cell numbers did not change over time, indicating absence of cell proliferation after ETN alone. By contrast, in animals preconditioned with retrorsine and partial hepatectomy, cell transplantation after ETN pretreatment significantly accelerated liver repopulation, compared to control rats. CONCLUSION: TNF-α plays a major role in orchestrating cell-transplantation-induced inflammation through regulation of multiple cytokines/chemokines/receptor expression. Because TNF-α antagonism by ETN decreased transplanted cell clearance, improved cell engraftment, and accelerated liver repopulation, this pharmacological approach to control hepatic inflammation will help optimize clinical strategies for liver cell therapy.

Spontaneous origin from human embryonic stem cells of liver cells displaying conjoint meso-endodermal phenotype with hepatic functions.

Understanding the identity of lineage-specific cells arising during manipulations of stem cells is necessary for developing their potential applications. For instance, replacement of crucial functions in organ failure by transplantation of suitable stem-cell-derived cells will be applicable to numerous disorders, but requires insights into the origin, function and fate of specific cell populations. We studied mechanisms by which the identity of differentiated cells arising from stem cells could be verified in the context of natural liver-specific stem cells and whether such differentiated cells could be effective for supporting the liver following cell therapy in a mouse model of drug-induced acute liver failure. By comparing the identity of naturally occurring fetal human liver stem cells, we found that cells arising in cultures of human embryonic stem cells (hESCs) recapitulated an early fetal stage of liver cells, which was characterized by conjoint meso-endoderm properties. Despite this fetal stage, hESC-derived cells could provide liver support with appropriate metabolic and ammonia-fixation functions, as well as cytoprotection, such that mice were rescued from acute liver failure. Therefore, spontaneous or induced differentiation of human embryonic stem cells along the hepatic endoderm will require transition through fetal-like stages. This offers opportunities to prospectively identify whether suitable cells have been generated through manipulation of stem cells for cell therapy and other applications.

Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non-heart-beating donor rats.

UNLABELLED: Organs from non-heart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non-heart-beating donor cells. We studied non-heart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In non-heart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart-beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non-heart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart-beating donors (P < 0.05). Similarly, although hepatocytes from non-heart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non-heart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades. CONCLUSION: Liver tissue remained intact over prolonged periods after death in non-heart-beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from non-heart-beating donors offer opportunities for improving donor cell viability, which will advance the utility of non-heart-beating donor organs for cell therapy or other applications.

Perturbations in ataxia telangiectasia mutant signaling pathways after drug-induced acute liver failure and their reversal during rescue of animals by cell therapy.

Superior insights into molecular mechanisms of liver failure, which are not fully understood, will help strategies for inducing liver regeneration. We examined hepatotoxic mechanisms in mice homozygous for the severe combined immune deficiency mutation in the protein kinase, DNA-activated, catalytic polypeptide. Mice were treated with rifampicin, phenytoin, and monocrotaline. The ensuing acute liver failure was characterized by serological, histological, and mRNA studies. Subsequently, we studied whether transplantation of hepatocytes could rescue animals with liver failure. We found extensive liver damage in these animals, with mortality over several days. The expression of multiple hepatic genes was rapidly altered, including those representing pathways in oxidative/metabolic stress, inflammation, DNA damage-repair, and ataxia telangiectasia mutant (Atm) signaling pathways. This led to liver cell growth arrest involving cyclin-dependent kinase inhibitor 1A. Transplantation of hepatocytes with microcarriers in the peritoneal cavity efficiently rescued animals with liver failure. Molecular abnormalities rapidly reversed, including in hepatic Atm and downstream signaling pathways; and residual hepatocytes overcame cyclin-dependent kinase inhibitor 1A-induced cell growth arrest. Reseeding of the liver with transplanted hepatocytes was not required for rescue because native hepatocytes overcame cell growth-arrest to regenerate the liver. This likely resulted from paracrine signaling from hepatocytes in the peritoneal cavity. We concluded that Atm signaling played critical roles in the pathological features of liver failure. These studies should help redirect examination of pathophysiologic and therapeutic mechanisms in liver failure.

Isolated small intestinal segments support auxiliary livers with maintenance of hepatic functions.

We determine here the functional integrity of auxiliary livers in containers fashioned from the small intestine. Liver microfragments from dipeptidyl peptidase 4 (DPP4)-deficient rats were transplanted into syngeneic normal animals with isolated intestinal segments characterized by mucosal denudation but intact vascular supply. Transplanted liver fragments were restored to confluent tissue with normal hepatic architecture and development of DPP4-positive vessels, indicating angiogenesis and revascularization. Auxiliary liver units expressed multiple hepatotrophic and angiogenic genes, and transplanted tissues remained intact for up to the 6-week duration of the studies with neither ischemic injury nor significant hepatocellular proliferation. Hepatic metabolic, transport and synthetic functions were preserved in auxiliary livers, including uptake and biliary excretion of (99m)Tc-mebrofenin in syngeneic recipients of liver from F344 rats, as well as secretion of albumin in allografted Nagase analbuminemic rats. This ability to produce functionally competent auxiliary livers in vascularized intestinal segments offers therapeutic potential for liver disease and genetic deficiency.

Enzymatically labeled chromosomal probes for in situ identification of human cells in xenogeneic transplant models.

Analysis of the viability, differentiation, clonogenicity and function of human stem/progenitor cells requires suitable xenograft models. However, the identification of transplanted cells has been generally difficult. Fluorescence in situ hybridization is a tedious method for analyzing tissues, and localization of transplanted cells with X or Y chromosome probes is limited by the sparse signals produced. Therefore, we examined the possibility of generating either pan-nuclear signals with a total human DNA probe or multiple nuclear signals with a pan-centromeric human DNA probe. The probes were labeled with digoxigenin to make reaction products visible by light microscopy and to allow the use of immunohistochemistry methods incorporating various color schemes to demonstrate specific properties of transplanted cells. The ability to localize all types of nucleated human cells with such probes will facilitate studies of stem cell biology and cell and gene therapy, as well as the development of new animal models.

Hepatic stellate cells promote hepatocyte engraftment in rat liver after prostaglandin-endoperoxide synthase inhibition.

BACKGROUND & AIMS: Hepatic inflammation occurs immediately after cells are transplanted to the liver, but the mechanisms that underlie this process are not fully defined. We examined cyclooxygenase pathways that mediate hepatic inflammation through synthesis of prostaglandins, prostacyclins, thromboxanes, and other prostanoids following transplantation of hepatocytes. METHODS: We transplanted F344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient F344 rats. Changes in cyclooxygenase pathways were analyzed, and specific pathways were blocked pharmacologically; the effects on cell engraftment and native liver cells were determined. RESULTS: Transplantation of hepatocytes induced hepatic expression of prostaglandin-endoperoxide synthases 1 and 2, which catalyze production of prostaglandin H2, as well as the downstream factor thromboxane synthase, which produces thromboxane A2 (a regulator of vascular and platelet responses in inflammation). Transplanted hepatocytes were in proximity with liver cells that expressed prostaglandin-endoperoxide synthases. The number of engrafted hepatocytes increased in rats given naproxen or celecoxib before transplantation but not in rats given furegrelate (an inhibitor of thromboxane synthase) or clopodigrel (an antiplatelet drug). Naproxen and celecoxib did not prevent hepatic ischemia or activation of neutrophils, Kupffer cells, or inflammatory cytokines, but they did induce hepatic stellate cells to express cytoprotective genes, vascular endothelial growth factor and hepatocyte growth factor, and matrix-type metalloproteinases and tissue inhibitor of metalloproteinase-1, which regulate hepatic remodeling. CONCLUSIONS: Activation of cyclooxygenase pathways interferes with engraftment of transplanted hepatocytes in the liver. Pharmacologic blockade of prostaglandin-endoperoxide synthases stimulated hepatic stellate cells and improved cell engraftment.

Hepatocyte transplantation-induced liver inflammation is driven by cytokines-chemokines associated with neutrophils and Kupffer cells.

BACKGROUND & AIMS: Hepatocyte transplantation-induced liver inflammation impairs cell engraftment. We defined whether proinflammatory cytokines and chemokines played roles in regulation of hepatocyte engraftment in the liver. METHODS: We performed studies over up to 3 weeks in rat hepatocyte transplantation systems. Expression of 84 cytokine-chemokine genes was studied by quantitative real-time polymerase chain reactions. Expression of selected up-regulated genes was verified by immunohistochemistry. Hepatic recruitment of neutrophils was demonstrated by myeloperoxidase activity assays, and Kupffer cell activation was established by carbon phagocytosis assays. The role of neutrophils and Kupffer cells in regulating expression of cytokine-chemokine genes as well as cell engraftment was determined by cell depletion studies. RESULTS: Within 6 hours after syngeneic cell transplantation, expression of 25 cytokine-chemokine genes increased by 2- to 123-fold, P < .05. These genes were largely associated with activated neutrophils and macrophages, including chemokine ligands, CXCL1, CXCL2, CCL3, CCL4; chemokine receptors, CXCR1 or CXCR2, CCR1, CCR2; and regulatory cytokines tumor necrosis factor alpha and interleukin-6. Inflammatory cells in the liver immunostained for CCR1, CCR2, CXCR1, and CXCR2, which indicated that up-regulated messenger RNA was appropriately translated. When neutrophils and Kupffer cells were depleted with neutrophil antiserum and gadolinium chloride, respectively, before transplanting cells, cell transplantation-induced cytokine-chemokine responses were attenuated. Virtually all abnormalities subsided in animals treated with neutrophil antiserum plus gadolinium chloride. Moreover, depletion of neutrophils or Kupffer cells improved engraftment of transplanted cells. CONCLUSIONS: Cell transplantation-induced liver inflammation involves proinflammatory cytokine-chemokine systems capable of modulation by neutrophils and Kupffer cells. This offers new directions for optimizing cell therapy strategies.

Bile salt-induced pro-oxidant liver damage promotes transplanted cell proliferation for correcting Wilson disease in the Long-Evans Cinnamon rat model.

Insights into disease-specific mechanisms for liver repopulation are needed for cell therapy. To understand the efficacy of pro-oxidant hepatic perturbations in Wilson disease, we studied Long-Evans Cinnamon (LEC) rats with copper toxicosis under several conditions. Hepatocytes from healthy Long-Evans Agouti (LEA) rats were transplanted intrasplenically into the liver. A cure was defined as lowering of copper to below 250 microg/g liver, presence of ATPase, Cu++ transporting, beta polypeptide (atp7b) messenger RNA (mRNA) in the liver and improvement in liver histology. Treatment of animals with the hydrophobic bile salt, cholic acid, or liver radiation before cell transplantation produced cure rates of 14% and 33%, respectively; whereas liver radiation plus partial hepatectomy followed by cell transplantation proved more effective, with cure in 55%, P < 0.01; and liver radiation plus cholic acid followed by cell transplantation was most effective, with cure in 75%, P < 0.001. As a group, cell therapy cures in rats preconditioned with liver radiation plus cholic acid resulted in less hepatic copper, indicating greater extent of liver repopulation. We observed increased hepatic catalase and superoxide dismutase activities in LEC rats, suggesting chronic oxidative stress. After liver radiation or cholic acid, hepatic lipid peroxidation levels increased, indicating further oxidative injury, although we did not observe overt additional cytotoxicity. This contrasted with healthy animals in which liver radiation and cholic acid produced hepatic steatosis and loss of injured hepatocytes. We concluded that pro-oxidant perturbations were uniquely effective for cell therapy in Wilson disease because of the nature of preexisting hepatic damage.

Hepatic targeting and biodistribution of human fetal liver stem/progenitor cells and adult hepatocytes in mice.

UNLABELLED: Tracking stem/progenitor cells through noninvasive imaging is a helpful means of assessing the targeting of transplanted cells to specific organs. We performed in vitro and in vivo studies wherein adult human hepatocytes and human fetal liver stem/progenitor cells were labeled with indium-111 ((111)In)-oxine and technetium-99m ((99m)Tc)-Ultratag or (99m)Tc-Ceretec. The labeling efficiency and viability of cells was analyzed in vitro, and organ biodistribution of cells was analyzed in vivo after transplantation in xenotolerant nonobese diabetic/severe combined immunodeficiency mice through intrasplenic or intraportal routes. We found that adult hepatocytes and fetal liver stem/progenitor cells incorporated (111)In but not (99m)Tc labels. After radiolabeling, cell viability was unchanged. Transplanted adult hepatocytes or fetal liver stem/progenitor cells were targeted to the liver more effectively by the intraportal rather than the intrasplenic route. Transplanted cells were retained in the liver after intraportal injection and in the liver and spleen after intrasplenic injection, without translocations into pulmonary or systemic circulations. Compared with fetal liver stem/progenitor cells, fewer adult hepatocytes were retained in the spleen after intrasplenic transplantation. The distribution of transplanted cells in organs was substantiated by genetic assays, including polymerase chain reaction amplification of DNA sequences from a primate-specific Charcot-Marie-Tooth element, and in situ hybridization for primate alphoid satellite sequences ubiquitous in all centromeres. CONCLUSION: (111)In labeling of human fetal liver stem/progenitor cells and adult hepatocytes was effective for noninvasive localization of transplanted cells. This should facilitate continued development of cell therapies through further animal and clinical studies.

Adenosine triphosphate-binding cassette subfamily C member 2 is the major transporter of the hepatobiliary imaging agent (99m)Tc-mebrofenin.

UNLABELLED: The organic anion (99m)Tc-N-[2-[(3-bromo-2,4,6-trimethylphenyl)-amino]-2-oxoethyl]-N-(carboxymethyl)-glycine ((99m)Tc-mebrofenin) and its analogs are widely used for hepatobiliary imaging. Identification of the mechanisms directing bile canalicular transport of these agents will provide insights into the basis of their hepatic handling for assessing perturbations. METHODS: We performed studies in animals, including healthy Fischer 344 rats or rats treated with carbon tetrachloride or intrasplenic cell transplantation and healthy Wistar rats or HsdAMC:TR-Abcc2 mutant rats in Wistar background. Onset of hepatic inflammation was verified by analysis of carbon uptake in Kupffer cells. Hepatic clearance of (99m)Tc-mebrofenin was studied with dynamic imaging, and fractional retention of peak hepatic mebrofenin activity after 60 min was determined. Changes in the expression of bile canalicular transporters were analyzed by real-time polymerase chain reaction and Western blots. RESULTS: Carbon tetrachloride and cell transplantation produced hepatic inflammation with activation of Kupffer cells, resulting in a rapid decline in the expression of the bile canalicular transporters Abcb4, Abcb11, and Abcc2. Among these transporters, decreased expression of Abcc2 was most prominent, and this decline persisted for 4 wk. Next, we examined (99m)Tc-mebrofenin excretion in HsdAMC:TR-Abcc2 mutant rats (in which Abcc2 expression is naturally inactivated), compared with their healthy counterparts. In healthy HsdRccHan:WIST rats, only 23% +/- 3% of the peak (99m)Tc-mebrofenin activity was retained after 60 min. By contrast, in HsdAMC:TR-Abcc2 mutant rats, 73% +/- 5% of the peak (99m)Tc-mebrofenin activity was retained (P < 0.001). Moreover, the administration of cyclosporin A markedly inhibited (99m)Tc-mebrofenin excretion in healthy rats, with no further effect on already impaired (99m)Tc-mebrofenin excretion in HsdAMC:TR-Abcc2 mutant rats. Hepatic excretion of (99m)Tc-mebrofenin was largely dependent on Abcc2. This molecular basis of (99m)Tc-mebrofenin excretion will advance studies of pathophysiologic mechanisms in hepatic Abcc2 pathways.

Hepatocyte transplantation and drug-induced perturbations in liver cell compartments.

UNLABELLED: 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.

Molecular pathway-specific 99mTc-N-(3-bromo-2,4,6-trimethyacetanilide) iminodiacetic acid liver imaging to assess innate immune responses induced by cell transplantation.

OBJECTIVES: Inflammatory responses after cell transplantation impair engraftment of transplanted cells. We studied whether perturbations in specific molecular pathways after inflammation in a syngeneic cell transplantation model could be identified by noninvasive imaging. METHODS: After transplanting hepatocytes into the liver of dipeptidyl peptidase IV-deficient Fischer 344 rats, we imaged hepatobiliary excretion of ppmTc-N-(3-bromo-2,4,6-trimethyacetanilide) iminodiacetic acid (99mTc-mebrofenin). Fractional retention of peak hepatic mebrofenin activity over 60-min periods was correlated with parameters of hepatic inflammation. RESULTS: In healthy animals, 28+/-6% 99mTc-mebrofenin activity was in the liver after 60 min, whereas cell transplantation dose-dependently inhibited excretion of 99mTc-mebrofenin, P value of less than 0.001. Resolution of this abnormality in 99mTc-mebrofenin transport required 2 weeks in the setting of prolonged activation of Kupffer cells with increased TNF-alpha and IL-6 expression. Hepatic transport of 00mTc-mebrofenin was promptly restored by anti-inflammatory treatments, including inhibition of cyclooxygenase activity, depletion of neutrophils, or blocking of inflammatory cytokines before cell transplantation. Moreover, these treatments improved transplanted cell engraftment. CONCLUSION: Molecular pathway-based imaging offers appropriate noninvasive means to address activation of innate immune responses. This will help in developing suitable strategies for characterizing and overcoming immune responses for cell and gene therapy.

Systemic and local release of inflammatory cytokines regulates hepatobiliary excretion of 99mTc-mebrofenin.

OBJECTIVES: Imaging agents capable of providing cell compartment-specific information will facilitate studies of pathophysiological mechanisms, natural history of diseases, and therapeutic development. To demonstrate the effects of liver injury on the disposal of the organic anion mebrofenin, we performed animal studies. METHODS: Acute liver injury was induced in Fischer 344 rats with 0.25-1 ml/kg single doses of carbon tetrachloride followed by studies of animals over 4 weeks. The liver injury was analyzed by blood tests and histological grading. Additional rats were treated with lipopolysaccharide, interleukin-6 or tumor necrosis factor-alpha to activate inflammatory events. Hepatic clearance of Tc-mebrofenin was studied with dynamic imaging and fractional retention after 60 min of peak hepatic mebrofenin activity was determined. RESULTS: In healthy rats, only 24+/-2% of peak mebrofenin activity was retained in the liver after 60 min. By contrast, 24 h after carbon tetrachloride, virtually all mebrofenin activity was retained in the liver (P<0.001). Three weeks were required for mebrofenin excretion to become normal after carbon tetrachloride administration. In this situation, we found that Kupffer cell activity was increased. In addition, the abnormality in mebrofenin excretion was reproduced by lipopolysaccharide, which activates Kupffer cells. Moreover, mebrofenin excretion was highly sensitive to interleukin-6 and/or tumor necrosis factor-alpha, which help mediate the Kupffer cell response. CONCLUSION: Hepatobiliary excretion of mebrofenin was affected rapidly and over an extended period by inflammatory cytokines released after liver injury. The remarkable sensitivity of mebrofenin excretion to cytokines suggests that Tc-mebrofenin imaging will be helpful for assessing cytokine-mediated liver inflammation.

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

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

Regulation of hepatobiliary transport activity and noninvasive identification of cytokine-dependent liver inflammation.

UNLABELLED: Many diseases are associated with cytokine release after inflammatory infiltration, which perturbs organ function. Radioligands capable of noninvasive tracking to assess the integrity of specific biochemical pathways offer potent ways to establish such perturbing mechanisms. METHODS: To demonstrate regulation of hepatobiliary transport in disease, we used (99m)Tc-mebrofenin in a carbon tetrachloride-induced liver injury model in Fischer 344 rats. Healthy rats served as control animals. Image analysis was used to determine (99m)Tc-mebrofenin handling. Liver tests and histologic analysis were used for grading liver injury and hepatic fibrosis. To address the role of inflammatory cytokines, we used in vitro assays with (99m)Tc-mebrofenin-loaded primary rat hepatocytes. RESULTS: In healthy rats, (99m)Tc-mebrofenin was promptly excreted, and after 1 h only 20% +/- 5% (mean +/- SD) of peak (99m)Tc-mebrofenin activity remained in the liver. In contrast, rats treated with carbon tetrachloride for 1 or 3 mo showed 84% +/- 5% and 80% +/- 7% (mean +/- SD), respectively, of peak (99m)Tc-mebrofenin activity in the liver after 1 h (P < 0.001). Abnormal (99m)Tc-mebrofenin transport was associated with necroinflammatory activity and not hepatic fibrosis. This was examined directly in animals, where withdrawal of carbon tetrachloride for 2 wk after significant liver injury produced loss of inflammatory activity without affecting hepatic fibrosis. In this situation, (99m)Tc-mebrofenin transport returned to normal, indicating a central role of inflammatory activity in this process. In vitro assays showed impairment in (99m)Tc-mebrofenin excretion after incubation of cultured hepatocytes with interleukin-6 and further impairment with interleukin-6 plus tumor necrosis factor-alpha. CONCLUSION: The findings indicate that inflammatory cytokines regulate (99m)Tc-mebrofenin transport. This cytokine-mediated process establishes a paradigm for identifying and monitoring organ inflammation, including in viral or alcoholic hepatitis, fatty liver disease, allograft rejection, and responses to gene therapy vectors.

Emerging insights into liver-directed cell therapy for genetic and acquired disorders.

Treatment of acute or chronic liver diseases by cell transplantation is an attractive prospect because organ shortages greatly restrict liver transplantation. Moreover, a variety of genetic deficiency states affecting extrahepatic organs are amenable to liver-directed cell therapy. While the initial clinical experience with liver cell transplantation has been encouraging, further advances in several areas are necessary to improve these results. Insights into how engraftment and proliferation of transplanted cells may be modulated to obtain therapeutically effective masses of transplanted cells will be important in this pursuit. Studies of cell therapy in animal models of specific diseases have provided insights into the development of clinically relevant strategies for various disorders. Also, identification of suitable cell types, including stem/progenitor cells that could be expanded and manipulated in cell culture conditions, has begun to provide important new information for cell therapy. Similarly, advances in cryopreservation of cells and prevention of allograft rejection offer ways to accomplish cell therapy in an effective manner. Taken together, these advances indicate that liver-directed cell therapy will be well positioned in the near future to play significant roles in transplantation medicine.

Development of cell therapy strategies to overcome copper toxicity in the LEC rat model of Wilson disease.

AIMS: Therapeutic replacement of organs with healthy cells requires disease-specific strategies. As copper toxicosis due to ATP7B deficiency in Wilson disease produces significant liver injury, disease-specific study of transplanted cell proliferation will offer insights into cell and gene therapy mechanisms. MATERIALS & METHODS: We used Long-Evans Cinnamon (LEC) rats to demonstrate the effects of liver preconditioning with radiation and ischemia reperfusion, followed by transplantation of healthy Long-Evans Agouti rat hepatocytes and analysis of hepatic atp7b mRNA, bile copper, liver copper and liver histology. RESULTS: LEC rats without cell therapy or after transplantation of healthy cells without liver conditioning accumulated copper and showed liver disease during the study period. Liver conditioning incorporating hepatic radiation promoted transplanted cell proliferation and reversed Wilson disease parameters, although with interindividual variations and time lags for improvement, which were different from previous results of liver repopulation in healthy animals. CONCLUSION: Cell therapy will correct genetic disorders characterized by organ damage. However, suitable mechanisms for inducing transplanted cell proliferation will be critical for therapeutic success.

Stage-specific regulation of adhesion molecule expression segregates epithelial stem/progenitor cells in fetal and adult human livers.

PURPOSE: Regulated expression of cell adhesion molecules could be critical in the proliferation, sequestration, and maintenance of stem/progenitor cells. Therefore, we determined fetal and adult stage-specific roles of cell adhesion in liver cell compartments. METHODS: We performed immunostaining for the adhesion molecules, E-cadherin and Ep-CAM, associated proteins, beta-catenin and alpha-actinin, hepatobiliary markers, albumin, alpha-fetoprotein, and cytokeratin-19, and the proliferation marker, Ki-67. Expression of albumin was verified by in situ mRNA hybridization. RESULTS: In the fetal liver, hepatoblasts showed extensive proliferation with wide expression of E-cadherin, beta-catenin, and alpha-actinin, although Ep-CAM was expressed in these cells less intensely and focally in the cell membrane to indicate weak cell adhesion. Hepatoblasts in ductal plate and bile ducts showed less proliferation and Ep-CAM was intensely expressed in these cells throughout the cell membrane, indicating strong adhesion. In some ductal plate cells, beta-catenin was additionally in the cytoplasm and nucleus, suggesting active cell signaling by adhesion molecules. In adult livers, cells were no longer proliferating and E-cadherin, beta-catenin, and alpha-actinin were expressed in hepatocytes throughout, whereas Ep-CAM was expressed in only bile duct cells. Some cells in ductal structures of the adult liver with Ep-CAM coexpressed albumin and cytokeratin-19, indicating persistence of fetal-like stem/progenitor cells. CONCLUSIONS: Regulated expression of Ep-CAM supported proliferation in fetal hepatoblasts through weak adhesion and helped in biliary morphogenesis by promoting stronger adhesion in hepatoblasts during this process. Restriction of Ep-CAM expression to bile ducts in the adult liver presumably facilitated sequestration of stem/progenitor cells. This stage-specific and cell compartment-related regulation of adhesion molecules should be relevant for defining how liver stem/progenitor cells enter, exit, and remain in hepatic niches during both health and disease.

Phenotype reversion in fetal human liver epithelial cells identifies the role of an intermediate meso-endodermal stage before hepatic maturation.

Understanding the biological potential of fetal stem/progenitor cells will help define mechanisms in liver development and homeostasis. We isolated epithelial fetal human liver cells and established phenotype-specific changes in gene expression during continuous culture conditions. Fetal human liver epithelial cells displayed stem cell properties with multilineage gene expression, extensive proliferation and generation of mesenchymal lineage cells, although the initial epithelial phenotype was rapidly supplanted by meso-endodermal phenotype in culture. This meso-endodermal phenotype was genetically regulated through cytokine signaling, including transforming growth factor beta, bone morphogenetic protein, fibroblast growth factor and other signaling pathways. Reactivation of HNF3alpha (FOXA1) transcription factor, a driver of hepatic specification in the primitive endoderm, indicated that the meso-endodermal phenotype represented an earlier developmental stage of cells. We found that fetal liver epithelial cells formed mature hepatocytes in vivo, including after genetic manipulation using lentiviral vectors, offering convenient assays for analysis of further cell differentiation and fate. Taken together, these studies demonstrate plasticity in fetal liver epithelial stem cells, offer paradigms for defining mechanisms regulating lineage switching in stem cells, and provide potential avenues for regulating cell phenotypes for applications of stem cells, such as for cell therapy.