In vitro mesenchymal stem cell differentiation after mechanical stimulation.

OBJECTIVES: Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into adipocytic, chondrocytic and osteocytic lineages on suitable stimulation. We have hypothesized that mechanical loading may influence MSC differentiation and alter their phenotype accordingly. MATERIALS AND METHODS: Mouse bone marrow-derived MSC were established in vitro by differential adherence to plastic culture plates and grown in low glucose medium with 10% foetal calf serum and growth factors. Cells grew out and were subcultured up to 20 times. Differentiation protocols were followed for several cell lineages. Clones with trilineage potential were seeded in type I collagen gels and incubated in a tensioning force bioreactor and real-time cell-derived forces were recorded. Gels were fixed and sectioned for light and electron microscopy. RESULTS: Cell monolayers of parent and cloned mouse bone marrow-derived MSC differentiated into adipocytes, osteocytes and chondrocytes, but not into cardiomyocytes, myotubes or neuronal cells. When cast into type I collagen gels and placed in tensioning bioreactors, MSC differentiated into fibroblast-like cells typical of tissue stroma, and upregulated α-smooth muscle actin, but rarely upregulated desmin. Electron microscopy showed collagen and elastin fibre synthesis into the matrix. CONCLUSIONS: These experiments confirmed that MSC cell fate choice depends on minute, cell-derived forces. Applied force could assist in commercial manufacture of cultured bio-engineered prostheses for regenerative medicine as it mimics tissue stresses and constitutes a good model for development of tissue substitutes.

Intestinal metaplasia in liver of rats after partial hepatectomy and treatment with acetylaminofluorene.

OBJECTIVES: The liver is widely recognized for its ability to self-regenerate after damage. Hepatocyte replication is the primary source of liver restoration, although hepatic stem cells (of one kind or another) may be a secondary font, only brought into effect when primary regeneration is severely compromised. MATERIALS AND METHODS: In experiments using small rodents, such an injury can be inflicted by surgically removing a large portion of the liver followed by treatment with hepatotoxin 2-acetylaminofluorene. Regeneration by hepatocyte replication is blocked and thus, stem cell involvement is promoted. However, other responses may be stimulated and this study describes the presence of mucinous glandular structures in the healing liver after two-thirds of its volume was removed via hepatectomy followed by treatment with 2-acetylaminofluorene. RESULTS: Unique observation of intestinal metaplastic cells was seen under alcian blue/periodic acid-Schiff staining. CONCLUSION: The existence of this phenotype (along with oval cells and small hepatocyte-like cells) is evidence of multipotency of progenitors involved in the hepatic healing response.

Cell proliferation rates in an artificial tissue-engineered environment.

Worldwide, and particularly in Europe, Japan and the USA, cardiovascular disease is a major killer. It can be treated using tissue or organ transplant surgery, but donor organs may be scarce. Tissue engineering is the integration of engineering principles and biology to produce satisfactory synthetic replacement body parts, using viable cells in a suitable matrix, for regenerative medicine. The aim of this study was to measure and compare cell proliferation kinetics after different time intervals of myofibroblasts in a synthetic matrix, thus to be able to deduce the period that a transplanted-cell population can be expected to survive in a tissue-engineered environment. Porcine aortic wall cells were grown in a porous sponge scaffold, that later could be fashioned into aortic or heart valve substitutes. Freshly acquired cells were seeded on identical sponges and were grown under normal culture conditions for a period of 4 weeks. Seeding concentration was a million cells per sponge. Cells progressively populated the sponges, both covering the surface and infiltrating the depth of the matrix, via sponge pores. Samples were taken at 1 week and at 4 weeks, and the rate of cell proliferation was determined by the metaphase arrest technique. Specimens were also taken for light and electron microscopy to determine whether these transplanted cells were capable of synthesizing their own extracellular matrix.

Heart valve and arterial tissue engineering.

In the industrialized world, cardiovascular disease alone is responsible for almost half of all deaths. Many of the conditions can be treated successfully with surgery, often using transplantation techniques; however, autologous vessels or human-donated organs are in short supply. Tissue engineering aims to create specific, matching grafts by growing cells on appropriate matrices, but there are many steps between the research laboratory and the operating theatre. Neo-tissues must be effective, durable, non-thrombogenic and non-immunogenic. Scaffolds should be bio-compatible, porous (to allow cell/cell communication) and amenable to surgery. In the early days of cardiovascular tissue engineering, autologous or allogenic cells were grown on inert matrices, but patency and thrombogenicity of grafts were disappointing. The current ethos is toward appropriate cell types grown in (most often) a polymeric matrix that degrades at a rate compatible with the cells' production of their own extracellular matrical proteins, thus gradually replacing the graft with a living counterpart. The geometry is crucial. Computer models have been made of valves, and these are used as three-dimensional patterns for mass-production of implant scaffolds. Vessel walls have integral connective tissue architecture, and application of physiological level mechanical forces conditions bio-engineered components to align in precise orientation. This article reviews the concepts involved and successes achieved to date.

Intracellular expression of the truncated extracellular domain of c-erbB-3/HER3.

The ERBB3 gene is expressed as a 6.2- and a 1.4-kb transcript. The former encodes the full-length transmembrane protein and the latter a truncated extracellular fragment consisting of 140 amino acids of the c-erbB-3 protein followed by 43 unique residues. We have examined the expression of the two ERBB3 transcripts by Northern blotting in cancer cell lines and normal human fetal and adult tissues. We expressed the truncated receptor fragment and showed that it was glycosylated, probably with a single N-linked complex sugar chain, and that the protein was a 58-kDa disulphide-linked dimer. We were able to crosslink iodinated neuregulin (NRG)-1beta to the full-length solubilised receptor but not to the truncated dimeric protein. Using Western blot analysis, the truncated protein was shown to be present in cell lysates and, using immunoelectron microscopy, in vesicular structures within cells and associated with the plasma cell membrane.

E1B-deleted adenovirus (dl1520) gene therapy for patients with primary and secondary liver tumors.

Clinical studies were performed with a recombinant mutant adenovirus with an E1B 55-kDa deletion, dl1520, to assess its toxicity and efficacy in patients with irresectable primary and secondary liver tumors. A phase I study showed that dl1520 was well tolerated when administered directly intratumorally, intraarterially, or intravenously up to a dose of 3 x 10(11) PFU. Ultrastructural examination of tissue showed the presence of adenovirus in cell cytoplasm around the nucleus and revealed two dissimilar end points of cell death after virus infection: a preapoptotic sequence and necrosis. A phase II study showed that the combination of dl1520 and 5-fluorouracil (5-FU), when infused into the hepatic artery, was well tolerated. Further improvement in the recombinant vector design will be needed in order to achieve better clinical response.

Branched chain amino acids induce apoptosis in neural cells without mitochondrial membrane depolarization or cytochrome c release: implications for neurological impairment associated with maple syrup urine disease.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain alpha-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular alpha-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, alpha-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their alpha-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway.

Detection of adenovirus and initiation of apoptosis in hepatocellular carcinoma cells after Ad-p53 treatment.

Transcription of the p53 gene can regulate progression of apoptosis in a wide variety of tissues. Three categories of human hepatocyte culture have been used to show the initiation of apoptosis after treatment with p53-bearing adenovirus. Chang liver cells are derived from normal liver tissue and express native p53, whereas hepatocellular carcinoma (HCC)-derived cell lines were Hep3B (p53-deleted) and PLC/PRF/5 (p53-mutant). Cultures were infected with Ad-p53 (15 particles per cell; 36 hours), and after treatment, morphological changes in all cell categories were observed by electron microscopy. Infection was evident in the cytoplasm of all treated cell types: after entry across the plasma membrane viruses translocated and came to rest surrounding and adjacent to nuclei, cytoplasm proximal to nuclear membranes became dense with virus- and membrane-derived debris, but intact viruses did not enter nuclei. Apoptosis, recognized morphologically by characteristic chromatin and cytoplasmic condensation, occurred more frequently in HCC-derived cells, and the ultimate fate of apoptotic bodies was phagocytosis and degradation by neighboring cells.

Immunolabeling for electron microscopy.

The protocols in this chapter concern postembedding immunolabeling for transmission electron microscopy; other schedules, such as pre-embedding methods, frozen tissue processes, and procedures for scanning electron microscopy, can be found elsewhere (1). In principle, immunolabeling at the electron microscope (EM) level follows the same precepts as immunolabeling at the light microscope level; in tissues or cells, the location of an antigen of interest is identified by a specific antibody, and must be visualized appropriately for investigation. Electron microscopy permits us to distinguish subcellu-lar organelles, and therefore ultrastructural localization of antigen position. At the EM level, however, the "visualizing step" needs to be provided by an electron-dense entity, most often a heavy metal, which reflects incident electrons; this is in contrast to the final step of light microscope level techniques, in which the final reaction product is sought to be colored (and where there is an element of choice of which color to use). Tissue processing for EM is considerably more severe than that for light microscopy, and thus maintenance of antigenicity in tissue is more taxing. Before immunolabeling for electron microscopy can be fruitful, the first step is to ensure that the antigen of interest is present (or is still present) in the tissue; this is done by performing a thorough procedure at the light microscope level on wax-embedded sections. Once a positive result has been obtained, studies can progress to the ultrastructural level. If the presence of an antigen cannot be demonstrated in a wax-embedded block, it will not be demonstrable in a resin-embedded EM block of the same tissue. In such a case, pre-embedding and frozen tissue techniques can be of use at both light and electron microscope levels.

Electron microscopic assessment of adenovirus-mediated transfer.

This chapter describes the method of preparing and observing hepatocellular carcinoma (HCC) and surrounding normal liver cells infected with therapeutically p (53)-transfected adenovirus (Ad-p (53)), so that morphology of the cells and viruses, and crucially their relationships to each other, are revealed. In standard practice, ultrastructural analysis of viruses carried in body fluids (e.g., stool or mucus) is sufficient for diagnosis, using the technique of phosphotungstate - dark field staining-of the aqueous extract. That method, however, is not suitable when one needs to examine precise subcellular location of viruses in situ, with tissue and cells intact, for complete pathological assessment; here, we describe our method (1) for transmission electron microscopy of the ultrastructure of virus-infected tumors. Tissue fixation, osmication, embedding, section cutting, and observation of Ad-p (53) infection will be included.

Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin.

We have aimed at selective gene delivery to vascular endothelial cells (EC) at sites of inflammation, by targeting E-selectin, a surface adhesion molecule that is only expressed by activated EC. An anti-E-selectin mAb, 1.2B6, was complexed with the adenovirus vector AdZ.FLAG (expressing the FLAG peptide) by conjugating it to an anti-FLAG mAb. Gene transduction of cultured EC was increased 20-fold compared with AdZ.FLAG complexed with a control bsAb providing EC were activated by cytokines. The anti-E-selectin-complexed vector transduced 29 +/- 9% of intimal EC in segments of pig aorta cultured with cytokines ex vivo, compared with less than 0.1% transduced with the control construct (P < 0.05). This strategy could be developed to target endothelium in inflammation with genes capable of modifying the inflammatory response.

Wound healing in the liver with particular reference to stem cells.

The efficiency of liver regeneration in response to the loss of hepatocytes is widely acknowledged, and this is usually accomplished by the triggering of normally proliferatively quiescent hepatocytes into the cell cycle. However, when regeneration is defective, tortuous ductular structures, initially continuous with the biliary tree, proliferate and migrate into the surrounding hepatocyte parenchyma. In humans, these biliary cells have variously been referred to as ductular structures, neoductules and neocholangioles, and have been observed in many forms of chronic liver disease, including cancer. In experimental animals, similar ductal cells are usually called oval cells, and their association with impaired regeneration has led to the conclusion that they are the progeny of facultative stem cells. Oval cells are of considerable biological interest as they may represent a target population for hepatic carcinogens, and they may also be useful vehicles for ex vivo gene therapy for the correction of inborn errors of metabolism. This review proposes that the liver harbours stem cells that are located in the biliary epithelium, that oval cells are the progeny of these stem cells, and that these cells can undergo massive expansion in their numbers before differentiating into hepatocytes. This is a conditional process that only occurs when the regenerative capacity of hepatocytes is overwhelmed, and thus, unlike the intestinal epithelium, the liver is not behaving as a classical, continually renewing, stem cell-fed lineage. We focus on the biliary network, not merely as a conduit for bile, but also as a cell compartment with the ability to proliferate under appropriate conditions and give rise to fully differentiated hepatocytes and other cell types.

Pericardial substitution after cardiopulmonary bypass surgery: a trial of an absorbable patch.

Primary closure of the pericardium affords some protection against adhesion formation and the consequent hazards of resternotomy. However, its completion may be impractical and hazardous, and therefore the pursuit of an ideal pericardial substitute has prompted much research. Twenty calves were divided into 3 groups for the study. All animals underwent right posterolateral thoracotomy. The test group (group X), consisting of 6 animals, received a poly-beta-hydroxybutyrate patch (PHB) to close the pericardium following cardiopulmonary bypass (CPB). In group Y (9 animals) the pericardium was left open following CPB. Group Z (5 animals) also had their pericardium left open but did not undergo CPB (non-CPB). The plasminogen activating activity (PAA) of homogenates of pericardial tissue samples were measured in 5 animals in group X, and 5 in group Z. Samples were taken at three time points from the time of pericardiotomy, and at reoperation 4 weeks later. In group X (CPB) there was a significant reduction in the PAA during the operation with some recovery at reoperation. The reduction in the pericardial PAA of group Z (non-CPB) animals did not reach significance. For both group X and group Z the progress of mesothelial damage, compared with that at zero time, showed a significant increase. In addition, their pericardial inflammatory features became more apparent in the later samples but more significantly in group Z. This study demonstrated no significant short-term differences in adhesion formation or postoperative coronary anatomy visibility between any of the groups. At reoperation the patch material contained pronounced macrophage activity but no regenerative mesothelium. There were no infective episodes in any of the animals studied. Furthermore, this study suggests that CPB in comparison to non-CPB has a significant affect on pericardial PAA.

Hepatic extramedullary granulopoiesis after treatment with liver-stimulatory xenobiotics, in lpr mice.

This study demonstrates that extramedullary hematopoiesis occurs in livers of adult lpr mice and, after treatment with each of three xenobiotic compounds--phenobarbital, cyproterone acetate, and nafenopin--it includes granulopoiesis. lpr mice are used as a model of the human disease systemic lupus erythematosus (SLE). The develop a syndrome very similar to that of human sufferers. In untreated lpr mice, mononuclear white blood cells were discernible in hepatic sinusoidal foci; T and B lymphocytes were distinguished from each other by immunocytochemistry at light microscope level. After treatment with any of the xenobiotic compounds, immunolabeling demonstrated the additional presence of granulocytes in foci, and, at electron microscope level neutrophils, eosinophils and their precursors were clearly recognizable.

Relation of impaired energy metabolism to apoptosis and necrosis following transient cerebral hypoxia-ischaemia.

This study investigated whether both mild and severe hypoxia-ischaemia (HI) caused significant numbers of cells to die by apoptosis in the developing brain in vivo. Newborn piglets were subjected to transient global HI and the fraction of all cells in the cingulate gyrus that were apoptotic or necrotic counted 48 h after resuscitation. The mean (S.D.) proportion of apoptotic cells was 11.9% (6.7%) (sham operated controls 4.1% (2.7%)), while 11.4% (8.4%) were necrotic (controls 0.7% (1.3%)) (P<0.05). Apoptotic and necrotic cell counts were both linearly related to the severity of impaired cerebral energy metabolism measured by magnetic resonance spectroscopy (P<0.05), as shown by: (1) the decline in the ratio of nucleotide triphosphates to the exchangeable phosphate pool during HI; (2) the fall in the ratio of phosphocreatine to inorganic phosphate 8 - 48 h after HI; and (3) an increased ratio of lactate to total creatine at both these times. Thus both apoptosis and necrosis occurred in the cingulate gyrus after both severe and mild HI in vivo in proportion to the severity of the insult.

Wild-type p53 induces apoptosis in Hep3B through up-regulation of bax expression.

We demonstrated that introduction and expression of wild-type p53 gene in the human hepatocellular carcinoma cell line, Hep3B, resulted in up-regulation of both p21WAF1/CIP1 and bax gene expression and apoptosis. This cell line contains integrated hepatitis B virus sequences and lacks the expression of both p53 and retinoblastoma tumor suppressor genes because of deletions. Our results suggest that whereas an increased level of bax expression mediates apoptosis, an increased level of p21WAF1/CIP1 expression does not induce arrest of cell growth, presumably because of the deletion of the retinoblastoma gene. This study also confirms reported observations that p53 is a tumor suppressor gene, which induces apoptosis in malignant cells that lack normal p53 activity because of mutation, deletion, or inactivation of the gene by the presence of oncogenic viral proteins.

Differential induction of apoptosis in Swiss 3T3 cells by nitric oxide and the nitrosonium cation.

We have investigated the effect of nitric oxide (NO) on apoptosis in Swiss 3T3 fibroblasts and compared it to the effect of the nitrosonium cation (NO+). Both species induced apoptosis, confirmed by electron microscopy, propidium iodide staining, DNA laddering and activation of caspases. The kinetics of triggering apoptosis were different for the two redox species: NO+ required only a 2 hour exposure, whereas NO required 24 hours. Three sources of NO were used: aqueous solutions of NO and two NO donors, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine. The time course of apoptosis induced by these two S-nitrosothiols correlated with their rate of decomposition to NO. The apoptotic effect of NO was reduced in the presence of the NO scavenger oxyhaemoglobin, or the antioxidants N-acetylcysteine and ascorbic acid, whereas in the case of NO+ these antioxidants potentiated apoptosis. Glutathione also had a potentiating effect on the cytotoxicity of NO+. This suggests that cellular antioxidants may play a role in protecting the cell from NO-induced apoptosis while NO+ may trigger apoptosis independently of oxidative stress mechanisms.