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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

Novel hepatic progenitor cell surface markers in the adult rat liver.

Unlabelled: Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2-acetylaminofluorene treatment followed by partial hepatectomy (2-AAF/PH) by using rat genome 230 2.

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).

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.

Integrin and extracellular matrix interactions regulate engraftment of transplanted hepatocytes in the rat liver.

Background & Aims: Recognition and circumvention of the hepatic endothelial barrier is critical in the engraftment of transplanted cells. We examined whether interactions between integrin and extracellular matrix component receptors could be manipulated for improving transplanted cell engraftment and liver repopulation.

Methods: Fischer 344 rat hepatocytes were transplanted into syngeneic dipeptidyl peptidase IV-deficient rats.

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.

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.

Embryonic mouse STO cell-derived xenografts express hepatocytic functions in the livers of nonimmunosuppressed adult rats.

Cells derived from embryonic mouse STO cell lines differentiate into hepatocytes when transplanted into the livers of nonimmunosuppressed dipeptidylpeptidase IV (DPPIV)-negative F344 rats. Within 1 day after intrasplenic injection, donor cells moved rapidly into the liver and were found in intravascular and perivascular sites; by 1 month, they were intrasinusoidal and also integrated into hepatic plates with approximately 2% efficiency and formed conjoint bile canaliculi. Neither donor cell proliferation nor host inflammatory responses were observed during this time.

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.

Analysis of the functional integrity of cryopreserved human liver cells including xenografting in immunodeficient mice to address suitability for clinical applications.

Background: The availability of well-characterized human liver cell populations that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after cryopreservation and engraft in immunodeficient mice.

Methods: We isolated cells from discarded human livers and studied the properties of cryopreserved cells.

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.