Mark A. Zern

Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

Hepatic stellate cells: a target for the treatment of liver fibrosis.

Abstract: Hepatic fibrosis is a wound-healing process that occurs when the liver is injured chronically. Hepatic stellate cells (HSC) are responsible for the excess production of extracellular matrix (ECM) components. The activation of HSC, a key issue in the pathogenesis of hepatic fibrosis, is mediated by various cytokines and reactive oxygen species released from the damaged hepatocytes and activated Kupffer cells. Therefore, inhibition of HSC activation and its related subsequent events, such as increased production of ECM components and enhanced proliferation, are crucial goals for intervention in the hepatic fibrogenesis cascade. This is especially true when the etiology is unknown or there is no established therapy for the cause of the chronic injury. This review explores the rationale for choosing HSC as a target for the pharmacological, molecular, and other novel therapeutics for hepatic fibrosis. One focus of this review is the inhibition of two cytokines, transforming growth factor-β and platelet-derived growth factor, which are important in hepatic fibrogenesis. A number of new agents, such as Chinese herbal recipes and herbal extracts, silymarin, S-adenosyl-l-methionine, polyenylphosphatidylcholine, and pentoxifylline are also discussed.

Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis

BACKGROUND & AIMS The accelerated course of hepatic fibrosis that occurs in some patients after liver transplantation is a major clinical problem. This response may be caused by the antirejection therapeutics, and in an earlier report we showed that FK-506 enhanced the fibrogenic process in in vivo and in vitro models of liver fibrosis. In the present study, the aim was to determine whether a new immunosuppressive agent, rapamycin, enhances or inhibits liver fibrosis. METHODS Effects of rapamycin were investigated in a carbon tetrachloride model of hepatic fibrosis in rats and on hepatic stellate proliferation in vitro. RESULTS Rapamycin inhibited extracellular matrix deposition in the rat model of fibrogenesis as determined by histological analysis, collagen content, messenger RNA levels of procollagen and transforming growth factor beta1, and tissue transglutaminase activity. Moreover, rapamycin decreased platelet growth factor-induced proliferation of hepatic stellate cells. CONCLUSIONS These findings indicate that the new antirejection agent rapamycin inhibits hepatic fibrosis and thus may become a valuable addition to the immunosuppression armamentarium.

Targeting hepatocytes for drug and gene delivery: emerging novel approaches and applications.

The asialoglycoprotein receptor (ASGP-R) on mammalian hepatocytes provides a unique means for the development of liver-specific carriers, such as liposomes, recombinant lipoproteins, and polymers for drug or gene delivery to the liver, especially to hepatocytes. The abundant receptors on the cells specifically recognize ligands with terminal galactose or N-acetylgalactosamine residues, and endocytose the ligands for an intracellular degradation process. The use of its natural ligand, i.e. asialofetuin, or synthetic ligands with galactosylated or lactosylated residues, such as galactosylated cholesterol, glycolipids, or galactosylated polymers has achieved significant targeting efficacy to the liver. There are several examples of successful targeted therapy for acute liver injury with asialofetuin-labeled and vitamin E-associated liposomes or with a caspase inhibitor loaded in sugar-carrying polymer particles, as well as for the delivery of a new antiviral agent, 9-(2-phosphonylmethoxyethyl)adenine. Liposome-mediated gene delivery to the liver is more difficult than to other organs, such as to lungs. It is still in its infancy due to difficulties in solving general issues, such as the circulatory stability of liposome-DNA complexes, and lysosomal or endosomal degradation of plasmid DNA. In spite of these existing concerns, several new approaches offer some reason for optimism, for example; intravenous injection of asialofetuin- or galactosylated cholesterol-labeled cationic liposomes has led to high transgene expression in the liver. In addition, specific antisense oligonucleotides against woodchuck hepatitis viruses incorporated into sialoorosomucoid-poly-L-lysine significantly inhibited viral replication in the liver. Finally, galactosylated polymers are promising for gene delivery, but require further studies to verify their potential applications.

Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo.

Hepatic stellate cell activation is a main feature of liver fibrogenesis. We have previously shown that phagocytosis of apoptotic bodies by stellate cells induces procollagen α1 (I) and transforming growth factor beta (TGF‐β) expression in vitro. Here we have further investigated the downstream effects of phagocytosis by studying NADPH oxidase activation and its link to procollagen α1 (I) and TGF‐β1 expression in an immortalized human stellate cell line and in several models of liver fibrosis. Phagocytosis of apoptotic bodies in LX‐1 cells significantly increased superoxide production both in the extracellular and intracellular milieus. By confocal microscopy of LX‐1 cells, increased intracellular reactive oxygen species (ROS) were detected in the cells with intracellular apoptotic bodies, and immunohistochemistry documented translocation of the NADPH oxidase p47phox subunit to the membrane. NADPH oxidase activation resulted in upregulation of procollagen α1 (I); in contrast, TGF‐β1 expression was independent of NADPH oxidase activation. This was also confirmed by using siRNA to inhibit TGF‐β1 production. In addition, with EM studies we showed that phagocytosis of apoptotic bodies by stellate cells occurs in vivo. In conclusion, these data provide a mechanistic link between phagocytosis of apoptotic bodies, production of oxidative radicals, and the activation of hepatic stellate cells. (HEPATOLOGY 2006;43:435–443.)

Differentiation of Human and Mouse Embryonic Stem Cells Along a Hepatocyte Lineage

Embryonic stem (ES) cells may differentiate along a hepatocyte lineage; however, currently there are no reports of culture conditions yielding high levels of hepatocyte-specific gene expression in these cells. We investigated culture conditions for differentiating ES cells into hepatocyte-like cells in vitro. Various combinations of culture media, growth and differentiation factors, and substratum precoatings were evaluated, and it was determined that a combination of Iscoves modified Dulbeccos medium with 20% fetal bovine serum, human insulin, dexamethasone, and collagen type I precoating was optimal for directing mouse ES cells along a hepatocyte lineage. Treatment of mouse ES cell with the optimal condition led to prealbumin gene expression 20% as high, and albumin synthesis 7% as high, as in mouse liver. The optimal culture condition also induced albumin gene expression in differentiated human ES cells 1% as high as in normal human hepatocytes as shown by Western blot analysis, and cells were positive for human albumin by immunocyto-chemistry. In addition, our optimal condition led to high levels of albumin gene expression in primary mouse hepatocytes after 35 days of culture, levels 10-fold higher than with other hepatocyte differentiation media. In conclusion, our optimal condition directed both mouse and human ES cells along a hepatocyte lineage. This represents the initial step in establishing cell lines that can be employed in cell-based therapeutics in humans and for toxicology and pharmacology studies.

Liver fibrosis causes downregulation of miRNA-150 and miRNA-194 in hepatic stellate cells, and their overexpression causes decreased stellate cell activation

Activation of hepatic stellate cells (HSC) results in their proliferation and in the secretion of extracellular matrix (ECM) proteins, which leads to hepatic fibrosis. microRNAs (miRNAs) have been shown to regulate various cell functions, such as proliferation, differentiation, and apoptosis. Hence, we have analyzed the miRNAs that were differentially expressed in HSC isolated from sham-operated and bile duct-ligated rats. Expression of two miRNAs, miRNA-150 and miRNA-194, was reduced in HSC isolated from fibrotic rats compared with sham-operated animals. These two miRNAs were overexpressed in LX-2 cells, and their ability to inhibit cell proliferation, the expression of smooth muscle alpha-actin (SMA), a marker for activation, and collagen type I, a marker for ECM secretion, was determined. Overexpression of these two miRNAs resulted in a significant inhibition of proliferation (P < 0.05) and reduced SMA and collagen I levels compared with either untreated cells or nonspecific miRNA-expressing cells. Next, the protein targets of these two miRNAs were found using bioinformatics approaches. C-myb was found to be a target for miRNA-150, and rac 1 was found to be one of the targets for miRNA-194. Therefore, we studied the expression of these two proteins by overexpressing these two miRNAs in LX-2 cells and found that overexpression of miRNA-150 and miRNA-194 resulted in a significant inhibition of c-myb and rac 1 expression, respectively. We conclude that both miRNA-150 and miRNA-194 inhibit HSC activation and ECM production, at least in part, via inhibition of c-myb and rac 1 expression.

Differentiation and Characterization of Metabolically Functioning Hepatocytes from Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) may provide a cell source for functional hepatocytes for clinical applications and drug development. Initially, the hESC population was enriched to be more than 85% definitive endoderm (DE) as assessed by the expression of CXCR4, SOX17, and FOXA2. We then successfully converted DE into hepatic progenitors with 93% of the cells being positive for α‐feto protein within 9 days. The percentage of albumin positive cells gradually increased to 90% at days 20‐22 after differentiation. Moreover, our hESC‐derived hepatocytes (hEH) developed a complete biotransformation system including phase I and II metabolizing enyzmes and phase III transporters. Nuclear receptors, which are critical in regulating the expression of metabolizing enzymes, were also expressed by our hEH. Using ultraperformance liquid chromatography‐tandem mass spectrometry technology, we identified seven metabolic pathways of the drug bufuralol including four newly‐reported ones in our hEH, which are the same as those in freshly isolated human primary hepatocytes (hPH). In addition, the results of the metabolism of four drugs indicate that our hEH have the capacity to metabolize these drugs at levels that are comparable to hPH. In conclusion, we have generated a relatively homogenous population of hepatocytes from hESCs, which appear to have complete metabolic function that is comparable to primary liver cells. These results represent a significant step towards the efficient differentiation of mature hepatocytes for cell‐based therapeutics as well as for pharmacology and toxicology studies. STEM CELLS 2010;28:674–686

Epithelial mesenchymal transition and hedgehog signaling activation are associated with chemoresistance and invasion of hepatoma subpopulations

BACKGROUND & AIMS Our previous studies showed that CD133, EpCAM, and aldehyde dehydrogenase (ALDH) are useful markers to identify cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) tissues. The present study aims to evaluate chemosensitivity and invasion capability of HCC based on CSC marker profiles, and to explore the underlying molecular mechanisms. METHODS Hepatoma cell lines were separated into subpopulations according to CD133, EpCAM, and ALDH expression profiles. Epithelial mesenchymal transition (EMT) and hedgehog (Hh) signaling were examined to identify their links with chemoresistance and aggressive invasion. RESULTS Well-differentiated cell lines were positive for CD133(+)/ALDH(high) and CD133(+)/EpCAM(+) at 1.5-15% and 2.3-8.3%; whereas, poorly-differentiated cells were almost all negative for these markers. FACS-enriched CD133(+)/ALDH(high) and CD133(+)/EpCAM(+) Hep3B and Huh-7 cells formed more spheroids in vitro. CD133(-)/ALDH(low) HLE cells were more resistant to cisplatin, doxorubicin or sorafenib than their positive counterparts. CD133(-)/EpCAM(-) Huh-7 cells or CD133(-)/ALDH(-) HLE cells exhibited a higher invasion rate than their positive counterparts. HLE and HLF cells acquired EMT in double negative subpopulations. Hh activity in Huh-7 CD133(-)/EpCAM(-) cells was higher than in their positive counterparts, and the inhibition of Hh activity by cyclopamine resulted in reduced cell proliferation. CONCLUSIONS Well-differentiated CD133(+)/ALDH(high) or CD133(+)/EpCAM(+) cells appear to be a CSC/initiating subpopulation; whereas, in poorly-differentiated hepatoma cells, EMT and enhanced hedgehog signaling activity may be responsible for their chemoresistance and invasion. These findings underscore the significance of EMT and enhanced Hh signaling in liver cancer stem or initiating cells.

De novo nonalcoholic fatty liver disease after liver transplantation

Hepatic steatosis is a recognized problem in patients after orthotopic liver transplant (OLT). However, de novo development of nonalcoholic fatty liver disease (NAFLD) has not been well described. The aim of this study was to determine the prevalence and predictors of de novo NAFLD after OLT. A retrospective analysis was performed on 68 OLT patients with donor liver biopsies and posttransplantation liver biopsies. Individual medical charts were reviewed for demographics, indication for OLT, serial histology reports, genotypes for hepatitis C, comorbid conditions, and medications. Liver biopsies were reviewed blindly and graded according to the Brunt Scoring System. Multivariate logistic regression analysis was used to study the risk factors for developing NAFLD. The interval time from OLT to subsequent follow‐up liver biopsy was 28 ± 18 months. A total of 12 patients (18%) developed de novo NAFLD, and 6 (9%) developed de novo NASH. The regression model indicated that the use of angiotensin‐converting enzyme inhibitors (ACE‐I) was associated with a reduced risk of developing NAFLD after OLT (odds ratio, 0.09; 95% confidence interval, 0.010‐0.92; P = 0.042). Increase in body mass index (BMI) of greater than 10% after OLT was associated with a higher risk of developing NAFLD (odds ratio, 19.38; 95% confidence interval, 3.50‐107.40; P = 0.001). In conclusion, de novo NAFLD is common in the post‐OLT setting, with a significant association with weight gain after transplant. The use of an ACE‐I may reduce the risk of developing post‐OLT NAFLD. Liver Transpl, 2006.