MicroRNA Profiling during Cardiomyocyte-Specific Differentiation of Murine Embryonic Stem Cells Based on Two Different miRNA Array Platforms

MicroRNA (miRNA) plays a critical role in a wide variety of biological processes. Profiling miRNA expression during differentiation of embryonic stem cells will help to understand the regulation pathway of differentiation, which in turn may elucidate disease mechanisms. The identified miRNAs could then serve as a new group of possible therapeutic targets. In the present paper, miRNA expression profiles were determined during cardiomyocyte-specific differentiation and maturation of murine embryonic stem (ES) cells. For this purpose a homogeneous cardiomyocyte population was generated from a transgenic murine ES cell line. Two high throughput array platforms (Affymetrix and Febit) were used for miRNA profiling in order to compare the effect of the platforms on miRNA profiling as well as to increase the validity of target miRNA identification. Four time points (i.e. day 0, day 12, day 19 and day 26) were chosen for the miRNA profiling study, which corresponded to different stages during cardiomyocyte-specific differentiation and maturation. Fifty platform and pre-processing method-independent miRNAs were identified as being regulated during the differentiation and maturation processes. The identification of these miRNAs is an important step for characterizing and understanding the events involved in cardiomyocyte-specific differentiation of ES cells and may also highlight candidate target molecules for therapeutic purposes

Evidence That Gene Activation and Silencing during Stem Cell Differentiation Requires a Transcriptionally Paused Intermediate State

A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3′ transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98–99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification

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

c-kitpos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

Resident c-kit positive (c-kitpos) cardiac stem cells (CSCs) could be considered the most appropriate cell type for myocardial regeneration therapies. However, much is still unknown regarding their biological properties and potential.We produced clones of high and low expressing GATA-4 CSCs from long-term bulk-cultured c-kitpos CSCs isolated from adult rat hearts. When c-kitpos GATA-4 high expressing clonal CSCs (cCSCs) were co-cultured with adult rat ventricular cardiomyocytes, we observed increased survival and contractility of the cardiomyocytes, compared to cardiomyocytes cultured alone, co-cultured with fibroblasts or c-kitpos GATA-4 low expressing cCSCs. When analysed by ELISA, the concentration of IGF-1 was significantly increased in the c-kitpos GATA-4 high cCSC/cardiomyocyte co-cultures and there was a significant correlation between IGF-1 concentration and cardiomyocyte survival. We showed the activation of the IGF-1 receptor and its downstream molecular targets in cardiomyocytes co-cultured with c-kitpos GATA-4 high cCSCs but not in cardiomyocytes that were cultured alone, co-cultured with fibroblasts or c-kitpos GATA-4 low cCSCs. Addition of a blocking antibody specific to the IGF-1 receptor inhibited the survival of cardiomyocytes and prevented the activation of its signalling in cardiomyocytes in the c-kitpos GATA-4 high cCSC/cardiomyocyte co-culture system. IGF-1 supplementation or IGF-1 high conditioned medium taken from the co-culture of c-kitpos GATA-4 high cCSCs plus cardiomyocytes did extend the survival and contractility of cardiomyocytes cultured alone and cardiomyocytes co-cultured with c-kitpos GATA-4 low cCSCs.c-kitpos GATA-4 high cCSCs exert a paracrine survival effect on cardiomyocytes through induction of the IGF-1R and signalling pathway

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

In cell-based drug development, researchers attempt to create drugs that kill a pathogen without necessarily understanding the details of how the drugs work. In contrast, target-based drug development entails the search for compounds that act on a specific intracellular target—often a protein known or suspected to be required for survival of the pathogen. The latter approach to drug development has been facilitated greatly by the sequencing of many pathogen genomes and the incorporation of genome data into user-friendly databases. The present paper shows how the database TDRtargets.org can identify proteins that might be considered good drug targets for diseases such as African sleeping sickness, Chagas disease, parasitic worm infections, tuberculosis, and malaria. These proteins may score highly in searches of the database because they are dissimilar to human proteins, are structurally similar to other “druggable” proteins, have functions that are easy to measure, and/or fulfill other criteria. Researchers can use the lists of high-scoring proteins as a basis for deciding which potential drug targets to pursue experimentally

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity

What do parents perceive are the barriers and facilitators to accessing psychological treatment for mental health problems in children and adolescents? A systematic review of qualitative and quantitative studies

A minority of children and adolescents with mental health problems access treatment. The reasons for poor rates of treatment access are not well understood. As parents are a key gatekeeper to treatment access, it is important to establish parents’ views of barriers/facilitators to accessing treatment. The aims of this study are to synthesise findings from qualitative and quantitative studies that report parents’ perceptions of barriers/facilitators to accessing treatment for mental health problems in children/adolescents. A systematic review and narrative synthesis were conducted. Forty-four studies were included in the review and were assessed in detail. Parental perceived barriers/facilitators relating to (1) systemic/structural issues; (2) views and attitudes towards services and treatment; (3) knowledge and understanding of mental health problems and the help-seeking process; and (4) family circumstances were identified. Findings highlight avenues for improving access to child mental health services, including increased provision that is free to service users and flexible to their needs, with opportunities to develop trusting, supportive relationships with professionals. Furthermore, interventions are required to improve parents’ identification of mental health problems, reduce stigma for parents, and increase awareness of how to access services

Cardiogenic Induction of Pluripotent Stem Cells Streamlined Through a Conserved SDF-1/VEGF/BMP2 Integrated Network

BACKGROUND: Pluripotent stem cells produce tissue-specific lineages through programmed acquisition of sequential gene expression patterns that function as a blueprint for organ formation. As embryonic stem cells respond concomitantly to diverse signaling pathways during differentiation, extraction of a pro-cardiogenic network would offer a roadmap to streamline cardiac progenitor output. METHODS AND RESULTS: To resolve gene ontology priorities within precursor transcriptomes, cardiogenic subpopulations were here generated according to either growth factor guidance or stage-specific biomarker sorting. Innate expression profiles were independently delineated through unbiased systems biology mapping, and cross-referenced to filter transcriptional noise unmasking a conserved progenitor motif (55 up- and 233 down-regulated genes). The streamlined pool of 288 genes organized into a core biological network that prioritized the "Cardiovascular Development" function. Recursive in silico deconvolution of the cardiogenic neighborhood and associated canonical signaling pathways identified a combination of integrated axes, CXCR4/SDF-1, Flk-1/VEGF and BMP2r/BMP2, predicted to synchronize cardiac specification. In vitro targeting of the resolved triad in embryoid bodies accelerated expression of Nkx2.5, Mef2C and cardiac-MHC, enhanced beating activity, and augmented cardiogenic yield. CONCLUSIONS: Transcriptome-wide dissection of a conserved progenitor profile thus revealed functional highways that coordinate cardiogenic maturation from a pluripotent ground state. Validating the bioinformatics algorithm established a strategy to rationally modulate cell fate, and optimize stem cell-derived cardiogenesis

Loss of Caveolin-1 Accelerates Neurodegeneration and Aging

The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease.We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression.Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease

Remote ischemic conditioning: from experimental observation to clinical application: report from the 8th Biennial Hatter Cardiovascular Institute Workshop

In 1993, Przyklenk and colleagues made the intriguing experimental observation that 'brief ischemia in one vascular bed also protects remote, virgin myocardium from subsequent sustained coronary artery occlusion' and that this effect '.... may be mediated by factor(s) activated, produced, or transported throughout the heart during brief ischemia/reperfusion'. This seminal study laid the foundation for the discovery of 'remote ischemic conditioning' (RIC), a phenomenon in which the heart is protected from the detrimental effects of acute ischemia/reperfusion injury (IRI), by applying cycles of brief ischemia and reperfusion to an organ or tissue remote from the heart. The concept of RIC quickly evolved to extend beyond the heart, encompassing inter-organ protection against acute IRI. The crucial discovery that the protective RIC stimulus could be applied non-invasively, by simply inflating and deflating a blood pressure cuff placed on the upper arm to induce cycles of brief ischemia and reperfusion, has facilitated the translation of RIC into the clinical setting. Despite intensive investigation over the last 20 years, the underlying mechanisms continue to elude researchers. In the 8th Biennial Hatter Cardiovascular Institute Workshop, recent developments in the field of RIC were discussed with a focus on new insights into the underlying mechanisms, the diversity of non-cardiac protection, new clinical applications, and large outcome studies. The scientific advances made in this field of research highlight the journey that RIC has made from being an intriguing experimental observation to a clinical application with patient benefit