Hesham Basma

Differentiation and Transplantation of Human Embryonic Stem Cell–Derived Hepatocytes

BACKGROUND & AIMS The ability to obtain unlimited numbers of human hepatocytes would improve the development of cell-based therapies for liver diseases, facilitate the study of liver biology, and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, potentially can differentiate into any cell type, and therefore could be developed as a source of human hepatocytes. METHODS To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein-receptor expression. Characterization was performed by real-time polymerase chain reaction, immunohistochemistry, immunoblot, functional assays, and transplantation. RESULTS Embryonic stem cell-derived hepatocytes expressed liver-specific genes, but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes, and showed human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. CONCLUSIONS Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein-receptor expression and potentially could be used in drug discovery research and developed as therapeutics.

Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell–derived hepatocytes

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter–based cell sorting. ES cell–derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell–derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell–derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

Reduced miR-146a Increases Prostaglandin E2 in Chronic Obstructive Pulmonary Disease Fibroblasts

RATIONALE Persistent inflammation plays a major role in chronic obstructive pulmonary disease (COPD) pathogenesis, but its mechanisms are incompletely defined. Overproduction of the inflammatory mediator prostaglandin (PG) E₂ by COPD fibroblasts contributes to reduced repair function. OBJECTIVES The present study determined if fibroblasts from subjects with COPD overproduce PGE₂ after stimulation with the inflammatory cytokines IL-1β and tumor necrosis factor-α, and further defined the mechanism for overproduction. METHODS Fibroblasts were isolated from parenchymal tissue obtained from smokers with and without COPD undergoing lung surgery. PGE₂, cyclooxygenases (COX), and miR-146a in these cells were evaluated by in vitro studies. MEASUREMENTS AND MAIN RESULTS After stimulation with inflammatory cytokines, COPD fibroblasts produced 2.7-fold more PGE₂ compared with controls with similar smoking history. The increase in PGE₂ depended on induction of COX-2, which increased to a greater degree in fibroblasts from subjects with COPD. Cytokines also induced microRNA miR-146a expression in both fibroblasts, but significantly less in COPD fibroblasts. miR-146a caused degradation of COX-2 mRNA; reduced expression prolonged COX-2 mRNA half-life in fibroblasts from subjects with COPD. Cytokine-stimulated PGE₂ production and miR-146a expression in cultured fibroblasts correlated with clinical severity assessed by expiratory airflow and diffusion capacity. CONCLUSIONS miR-146a seems to play a pathogenetic role in the abnormal inflammatory response in COPD. Increased half-life of inflammatory mRNAs is a mechanism of abnormal inflammation in this disease.

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

Cytomegalovirus (CMV) immediate early promoter is a powerful promoter frequently used for driving the expression of transgenes in mammalian cells. However, this promoter gradually becomes silenced in stably transfected cells. We employed Chinese Hamster Ovary (CHO) and human pancreatic cancer (Panc 1) cells stably tansfected with three glycogenes driven by a CMV promoter to study the activation of silenced glycogenes. We found that butyrate, tricostatin A (TSA), and 5-aza-2′-deoxycytidine (5-Aza-dC) can activate these CMV-driven glycogenes. The increase in mRNA and protein of a glycogene occurred 8–10 h after butyrate treatment, suggesting an indirect effect of butyrate in the activation of the transgene. The enhanced expression of the trangenes by butyrate and TSA, known inhibitors of histone deacetylase, was independent of the transgene or cell type. However, the transgene can be activated by these two agents in only a fraction of the cells derived from a single clone, suggesting that inactivation of histone deacetylase can only partially explain silencing of the transgenes. Combination treatment of one or both agents with 5-Aza-dC, a known inhibitor of DNA methylase, resulted in a synergistic activation of the transgene, suggesting a cross-talk between histone acetylation and DNA demethylation. Understanding the mechanisms of the inactivation and reactivation of CMV promoter-controlled transgenes should help develop an effective strategy to fully activate the CMV promoter-controlled therapeutic genes silenced by the host cells. Published in 2005.

MicroRNA-146a modulates human bronchial epithelial cell survival in response to the cytokine-induced apoptosis.

MicroRNA plays an important role in cell differentiation, proliferation and cell death. The current study found that miRNA-146a was up-regulated in human bronchial epithelial cells (HBECs) in response to stimulation by TGF-beta1 plus cytomix (a mixture of IL-1beta, IFN-gamma and TNF-alpha). TGF-beta1 plus cytomix (TCM) induced apoptosis in HBECs (3.4+/-0.6% of control vs 83.1+/-4.0% of TCM treated cells, p<0.01), and this was significantly blocked by the miRNA-146a mimic (8.8+/-1.5%, p<0.01). In contrast, a miRNA-146a inhibitor had only a modest effect on cell survival but appeared to augment the induction of epithelial-mesenchymal transition (EMT) in response to the cytokines. The MicroRNA-146a mimic appears to modulate HBEC survival through a mechanism of up-regulating Bcl-XL and STAT3 phosphorylation, and by this mechanism it could contribute to tissue repair and remodeling.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis.

In advanced cirrhosis, impaired function is caused by intrinsic damage to the native liver cells and from the abnormal microenvironment in which the cells reside. The extent to which each plays a role in liver failure and regeneration is unknown. To examine this issue, hepatocytes from cirrhotic and age‐matched control rats were isolated, characterized, and transplanted into the livers of noncirrhotic hosts whose livers permit extensive repopulation with donor cells. Primary hepatocytes derived from livers with advanced cirrhosis and compensated function maintained metabolic activity and the ability to secrete liver‐specific proteins, whereas hepatocytes derived from cirrhotic livers with decompensated function failed to maintain metabolic or secretory activity. Telomere studies and transcriptomic analysis of hepatocytes recovered from progressively worsening cirrhotic livers suggest that hepatocytes from irreversibly failing livers show signs of replicative senescence and express genes that simultaneously drive both proliferation and apoptosis, with a later effect on metabolism, all under the control of a central cluster of regulatory genes, including nuclear factor κB and hepatocyte nuclear factor 4α. Cells from cirrhotic and control livers engrafted equally well, but those from animals with cirrhosis and failing livers showed little initial evidence of proliferative capacity or function. Both, however, recovered more than 2 months after transplantation, indicating that either mature hepatocytes or a subpopulation of adult stem cells are capable of full recovery in severe cirrhosis. Conclusion: Transplantation studies indicate that the state of the host microenvironment is critical to the regenerative potential of hepatocytes, and that a change in the extracellular matrix can lead to regeneration and restoration of function by cells derived from livers with end‐stage organ failure. (HEPATOLOGY 2011)

Prostaglandin E2 Inhibits Human Lung Fibroblast Chemotaxis through Disparate Actions on Different E-Prostanoid Receptors

The migration of fibroblasts is believed to play a key role in both normal wound repair and abnormal tissue remodeling. Prostaglandin E (PGE)(2), a mediator that can inhibit many fibroblast functions including chemotaxis, was reported to be mediated by the E-prostanoid (EP) receptor EP2. PGE(2), however, can act on four receptors. This study was designed to determine if EP receptors, in addition to EP2, can modulate fibroblast chemotaxis. Using human fetal lung fibroblasts, the expression of all four EP receptors was demonstrated by Western blotting. EP2-selective and EP4-selective agonists inhibited both chemotaxis toward fibronectin in the blindwell assay and migration in a wound-closure assay. In contrast, EP1-selective and EP3-selective agonists stimulated cell migration in both assay systems. These results were confirmed using EP-selective antagonists. The role of both EP2 and EP4 receptors in mediating the PGE(2) inhibition of chemotaxis was also confirmed by small interfering RNA suppression. Furthermore, the role of EP receptors was confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE(2) can act on multiple EP receptors in human lung fibroblasts, to exert disparate effects. Alterations in EP receptor expression may have the potential to alter PGE(2) action. Targeting specific EP receptors may offer therapeutic opportunities in conditions characterized by abnormal tissue repair and remodeling.

Vitamin D modulates prostaglandin E2 synthesis and degradation in human lung fibroblasts.

Vitamin D insufficiency has been increasingly recognized in the general population worldwide and has been associated with several lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and respiratory tract infections. Fibroblasts play a critical role in tissue repair and remodeling, which is a key feature of COPD and asthma. Fibroblasts modulate tissue repair by producing and modifying extracellular matrix components and by releasing mediators that act as autocrine or paracrine modulators of tissue remodeling. The current study was designed to investigate if vitamin D alters fibroblast release of key autocrine/paracrine repair factors. First, we demonstrated that human fetal lung (HFL)-1 cells express the vitamin D receptor (VDR) and that vitamin D, 25-hydroxyvitamin D [25(OH)D], or 1,25-dihydroxyvitamin D [1,25(OH)2D] induce VDR nuclear translocation and increase VDR-DNA binding activity. We next demonstrated that vitamin D, 25(OH)D, and 1,25(OH)2D significantly reduced prostaglandin (PG)E2 production by human lung fibroblasts (HFL-1) but had no effect on transforming growth factor β1, vascular endothelial growth factor, or fibronectin production. Vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited IL-1β-induced microsomal PGE synthase (mPGES)-1 expression; in contrast, all three forms of vitamin D stimulated 15-hydroxy PG dehydrogenase, an enzyme that degrades PGE2. Cyclooxygenase-1 and -2 and the other two PGE2 synthases (mPGES-2 and cytosolic PGE synthase) were not altered by vitamin D, 25(OH)D, or 1,25(OH)2D. Finally, the effect of PGE2 inhibition by 25(OH)D was observed in adult lung fibroblasts. These findings suggest that vitamin D can regulate PGE2 synthesis and degradation and by this mechanism can modulate fibroblast-mediated tissue repair function.

Prostaglandin E2 Stimulates the Production of Vascular Endothelial Growth Factor through the E-Prostanoid–2 Receptor in Cultured Human Lung Fibroblasts

Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E(2) (PGE(2)) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE(2) of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbeccos modified Eagles medium, with or without PGE(2), and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE(2) and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE(2) was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase-A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE(2). The increased release of VEGF induced by PGE(2) was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE(2) can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall.