Electromechanical integration of cardiomyocytes derived from human embryonic stem cells

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.     

Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes

Assessment of early ultrastructural development and cell-cycle regulation in human cardiac tissue is significantly hampered by the lack of a suitable in vitro model. Here we describe the possible utilization of human embryonic stem cell (ES) lines for investigation of these processes. With the use of the embryoid body (EB) differentiation system, human ES cell-derived cardiomyocytes at different developmental stages were isolated and their histomorphometric, ultrastructural, and proliferative properties were characterized. Histomorphometric analysis revealed an increase in cell length, area, and length-to-width ratio in late-stage EBs (>35 days) compared with early (10-21 days) and intermediate (21-35 days) stages. This was coupled with a progressive ultrastructural development from an irregular myofibrillar distribution to an organized sarcomeric pattern. Cardiomyocyte proliferation, assessed by double labeling with cardiac-specific antibodies and either [3H]thymidine incorporation or Ki-67 immunolabeling, demonstrated a gradual withdrawal from cell cycle. Hence, the percentage of positively stained nuclei in early-stage cardiomyocytes ([3H]thymidine: 60 +/- 10%, Ki-67: 54 +/- 23%) decreased to 36 +/- 7% and 9 +/- 16% in intermediate-stage EBs and to <1% in late-stage cardiomyocytes. In conclusion, a reproducible temporal pattern of early cardiomyocyte proliferation, cell-cycle withdrawal, and ultrastructural maturation was noted in this model. Establishment of this unique in vitro surrogate system may allow to examine the molecular mechanisms underlying these processes and to assess interventions aiming to modify these properties. Moreover, the detailed characterization of the ES cell-derived cardiomyocyte may be crucial for the development of future cell replacement strategies aiming to regenerate functional myocardium.     

Meta-analysis of gene expression microarrays with missing replicates

Background  

Many different microarray experiments are publicly available today. It is natural to ask whether different experiments for the same phenotypic conditions can be combined using meta-analysis, in order to increase the overall sample size. However, some genes are not measured in all experiments, hence they cannot be included or their statistical significance cannot be appropriately estimated in traditional meta-analysis. Nonetheless, these genes, which we refer to as incomplete genes, may also be informative and useful.     

Epigenetic regulation of cell type-specific expression patterns in the human mammary epithelium

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type-specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type-specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation.     

Active anthocyanin degradation in <Emphasis Type="Italic">Brunfelsia calycina</Emphasis> (yesterday–today–tomorrow) flowers

Anthocyanins are the largest group of plant pigments responsible for colors ranging from red to violet and blue. The biosynthesis of anthocyanins, as part of the larger phenylpropanoid pathway, has been characterized in great detail. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, very little is known about the stability and catabolism of anthocyanins in plants. In this study we present a preliminary characterization of active in planta degradation of anthocyanins, requiring novel mRNA and protein synthesis, in Brunfelsia calycina flowers. Brunfelsia is a unique system for this study, since the decrease in pigment concentration in its flowers (from dark purple to white) is extreme and rapid, and occurs at a specific and well-defined stage of flower development. Treatment of detached flowers with protein and mRNA synthesis inhibitors, at specific stages of flower development, prevented degradation. In addition, treatment of detached flowers with cytokinins delayed senescence without changing the rate of anthocyanin degradation, suggesting that degradation of anthocyanins is not part of the general senescence process of the flowers but rather a distinctive and specific pathway. Based on studies on anthocyanin degradation in wine and juices, peroxidases are reasonable candidates for the in vivo degradation. A significant increase in peroxidase activity was shown to correlate in time with the rate of anthocyanin degradation. An additional indication that oxidative enzymes are involved in the process is the fact that treatment of flowers with reducing agents, such as DTT and glutathione, caused inhibition of degradation. This study represents the first step in the elucidation of the molecular mechanism behind in vivo anthocyanin degradation in plants.     

Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.     

Release of euchromatic segments by MgCa-dependent autodigestion of chromatin

Mouse TLT hepatoma chromatin was incubated in the presence of added MgCl₂ and CaCl₂ but in the absence of added exogenous nuclease. Fraction-ation of such autodigested chromatin by glycerol density gradient centrifugation resulted in a heterochromatin-enriched pellet and euchromatin-enriched non-pelletable fractions. This was determined by satellite DNA content analyses and analysis of nascent RNA distribution. Electron microscope examination of the chromatin revealed it to consist of a series of connected spherical particles (nucleosomes) having a diameter of 370 ± 70Å. This native structure of chromatin was not damaged by the autodigestion process.     

THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO₄ with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO₄. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.     

Vimentin binding to phosphorylated Erk sterically hinders enzymatic dephosphorylation of the kinase

Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.