B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death

Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma

Background: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are.98 % pure, and at least 85 % of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint an

Anthropogenic intensification of short-duration rainfall extremes

Short- duration (1-3 h) rainfall extremes can cause serious damage to societies through rapidly developing (flash) flooding and are determined by complex, multifaceted processes that are altering as Earth's climate warms. In this Review, we examine evidence from observational, theoretical and modelling studies for the intensification of these rainfall extremes, the drivers and the impact on flash flooding. Both short- duration and long- duration (\textgreater1 day) rainfall extremes are intensifying with warming at a rate consistent with the increase in atmospheric moisture (~7% K-1), while in some regions, increases in short- duration extreme rainfall intensities are stronger than expected from moisture increases alone. These stronger local increases are related to feedbacks in convective clouds, but their exact role is uncertain because of the very small scales involved. Future extreme rainfall intensification is also modulated by changes to temperature stratification and large- scale atmospheric circulation. The latter remains a major source of uncertainty. Intensification of short- duration extremes has likely increased the incidence of flash flooding at local scales and this can further compound with an increase in storm spatial footprint to considerably increase total event rainfall. These findings call for urgent climate change adaptation measures to manage increasing flood risks

Serial Analysis of Gene Expression (SAGE): A useful tool to analyze the cardiac transcriptome

Serial analysis of gene expression (SAGE), a functional genomics technique, can be used for global profiling of gene transcripts. It relies on the preparation and sequencing of cDNA concatemers, but it does not require prior knowledge of the genes to be assayed (as with microarrays). Once analyzed, SAGE data provide both a qualitative and quantitative assessment of potentially every transcript present in a particular cell or tissue type. In this chapter, we describe the fundamental principles of SAGE, describe a complete protocol for the generation of SAGE libraries, and show how it has been employed to generate the first SAGE reference data set of the mouse myocardium. Following the protocols described here, investigators should be able to generate unique mouse heart SAGE libraries, which can be directly compared with our reference library. This permits the identification of transcripts that are differentially expressed as a function of time, age, genetic background or transgenic state, among other factors. SAGE is thus a powerful technique that permits a comprehensive analysis of changes in mRNA abundance. The results provide a snapshot of altered patterns of gene expression in response to any genetic or environmental stimulus that can be used to generate new biological hypotheses or test existing paradigms. © Humana Press Inc.link_to_subscribed_fulltex

Human embryonic stem cell-derived cardiomyocytes: Therapeutic potentials and limitations

Embryonic stem cell (ESC) lines, derived from the inner cell mass (ICM) of blastocyst-stage embryos, are pluripotent and have a virtually unlimited capacity for self-renewal and differentiation into derivatives of all three germ layers. Human ESCs (hESCs), in particular, are the subject of intensive investigation for potential applications in developmental biology and medicine. A promising aspect of hESCs is their ability to differentiate into cardiomyocytes (CMs), which generally lack the capacity to regenerate, and therefore their potential for cell-replacement heart therapies. Molecular, cellular and physiological analyses demonstrate that hESC-derived CMs are functionally viable and that they exhibit characteristics typical of heart cells in the early stages of cardiac development. This article reviews the current state of hESC-CM research, their therapeutic potentials and limitations. © 2006 Nova Science Publishers, Inc.link_to_subscribed_fulltex

Embryonic stem cells and cardiomyocyte differentiation: Phenotypic and molecular analyses

Embryonic stem (ES) cell lines, derived from the inner cell mass (ICM) of blastocyst-stage embryos, are pluripotent and have a virtually unlimited capacity for self-renewal and differentiation into all cell types of an embryo-proper. Both human and mouse ES cell lines are the subject of intensive investigation for potential applications in developmental biology and medicine. ES cells from both sources differentiate in vitro into cells of ecto-, endo-and meso-dermal lineages, and robust cardiomyogenic differentiation is readily observed in spontaneously differentiating ES cells when cultured under appropriate conditions. Molecular, cellular and physiologic analyses demonstrate that ES cell-derived cardiomyocytes are functionally viable and that these cell derivatives exhibit characteristics typical of heart cells in early stages of cardiac development. Because terminal heart failure is characterized by a significant loss of cardiomyocytes, the use of human ES cell-derived progeny represents one possible source for cell transplantation therapies. With these issues in mind, this review will focus on the differentiation of pluripotent embryonic stem cells into cardiomyocytes as a developmental model, and the possible use of ES cell-derived cardiomyocytes as source of donor cells.link_to_subscribed_fulltex

Galanin and galanin receptors in embryonic stem cells: Accidental or essential?

Galanin is a peptide consisting of 29 (mouse) or 30 (human) amino acids that was recently identified in undifferentiated mouse embryonic stem (ES) cells through transcriptome analyses. Galanin is known to have important modulatory roles in neuronal cells, but it is currently unclear what biological role, if any, galanin has in stem cells. Here we show that galanin transcripts represent a distinguishing molecular feature of embryonic stem cell lines and that all three galanin receptors subtypes are expressed in mouse ES cells (Gal-R2 > Gal-R3 ≫ Gal-R1). Based on cell culture data, galanin in a dose-dependent manner appears to regulate growth characteristics of ES cells, at least partially, through interactions with leukemia inhibitory factor (LIF), a cytokine implicated in the self-renewal process of ES cells. The regulation of ES cell growth can therefore be added to the list of biological processes regulated by this peptide.link_to_subscribed_fulltex

Linkage of pluripotent stem cell-associated transcripts to regulatory gene networks

These journal issues entitled: Stem Cells, Tissue Regeneration and Repair (free suppl.)Knowledge of the transcriptional circuitry responsible for pluripotentiality and self-renewal in embryonic stem cells is tantamount to understanding early mammalian development and a prerequisite to determining their therapeutic potential. Various techniques have employed genomics to identify transcripts that were abundant in stem cells, in an attempt to define the molecular basis of 'stemness'. In this study, we have extended traditional genomic analyses to identify cis-elements that might be implicated in the control of embryonic stem cell-restricted gene promoters. The strategy relied on the generation of a problem-specific list from serial analysis of gene expression profiles and subsequent promoter analyses to identify frameworks of multiple cis-elements conserved in space and orientation among genes from the problem-specific list. Subsequent experimental data suggest that 2 novel transcription factors, B-Myb and Maz, predicted from these models, are implicated either in the maintenance of the undifferentiated stem cell state or in early steps of differentiation. Copyright © 2008 S. Karger AG.link_to_OA_fulltex

Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling

There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca 2+ imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca 2+ Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca 2+current (I CaL) induces a global Ca 2+ release (CICR), depleting the Ca 2+ content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca 2+release of multiple LCRs throughout the cell. The LCRs generate an inward Na +/Ca 2+exchanger (NCX) current (absent in Na +-free solution) that ignites the next AP. Interfering with SR Ca 2+ cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na +-free solution), or I CaL (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca 2+ loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca 2+ loading and LCR activity could be also increased with a selective activation of SR Ca 2+ pumping by a phospholamban antibody. We conclude that SR Ca 2+ loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I CaL synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca 2+ release and NCX current. Rhythmic ESC automaticity can be achieved by boosting "coupling" factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma. © 2010.link_to_subscribed_fulltex