Project description:Investigation of content of microvesicles exocytosed by cardiomyocytes. The aims of the study were to identify DNA and RNA content in microvesicles from cardiomyocytes. DNA and RNA were purified from microveicles from cultured cardiomyocytes. Illumina microarrays were used to detect DNA and mRNA molecules and identify them.
Project description:Introduction: Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in target cells. We have recently shown that cultured cardiomyocytes release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the released exosome characteristics. Material and Methods: Exosomes were isolated from media collected from cultured cardiomyocyte (HL-1) cells with or without growth factor treatment (TGF-beta2 and PDGF-BB), with a series of differential centrifugations. The exosomes were characterized with dynamic light scattering (DLS) and Western blot and analysed with Illumina whole genome microarray gene expression. Results: An average size of 50-80 nm in diameter with no difference between treatment groups was found. Analysis of the mRNA content revealed 623 transcripts in the control group, 691 in the TGF-beta2-treated group and 362 in the PDGF-BB-treated group. 235 transcripts were common for all three groups. Conclusion: We conclude that there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system. To study if the transcriptional content in exosomes derived from untreated and growth factor-treated cultured cardiomyocytes (HL-1) differ, and if so, can this difference be explained, 4 control (untreated) exosome samples, 4 TFG-beta2-treated cardiomyocyte-derived exosome samples and 4 PDGF-BB-treated cardiomyocyte-derived exosomes were studied.
Project description:To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)–derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless, analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed a miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together, these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification. Comparison of mRNA expression profiling of differentiating human-induced pluripotent stem cell (hiPSC)–derived cardiomyocytes, biopsies from fetal, adult and hypertensive human hearts and primary cardiomyocytes
Project description:Here we provide the gene expression patterns of human iPS cell-derived cardiomyocytes used in the motion field imaging assay, adult human heart tissues, fetal human heart tissue and iPS cells. These data provided the causal relationship between phenotype and function in the human iPS cell-derived cardiomyocytes.
Project description:Expression profiling of cultured HL-1 cardiomyocytes subjected to hypoxia for 8 hours. Three samples from each condition were analyzed
Project description:Recent establishment of induced pluripotent stem (iPS) cells opened new avenues for generating human patient-specific stem cell derivatives that can be used for in vitro modeling of human disease, drug development and cell replacement therapy. In this study we analyzed the molecular and functional properties of cardiomyocytes (CM) differentiated from human iPS cells. Clusters of synchronously beating cells were first observed at day 11 of iPS cell differentiation. Beating clusters that were microdissected at day 18 of differentiation expressed high levels of cardiospecific transcripts NKx2.5, alpha-MHC, MLC2v, alpha-actinin and troponin T. Immunocytochemical stainings for alpha-actinin and troponin T revealed cross-striations typical of CM. Functional assessment of iPS cell-derived CM showed that these cells possess intact calcium transients and respond to beta-adrenergic and muscarinic stimulation. Molecular, structural and electrophysiological properties of iPS cell-derived CMs were highly comparable to their human ES cell-derived counterparts at the same differentiation stage. Comparison of global gene expression profiles of human ES and iPS cells and the corresponding microdissected beating areas further confirmed their similarity. We conclude that human iPS cells can differentiate into functional CM, which are indistinguishable from human ES cell-derived CMs and may fulfill the basic requirement for their use in disease modeling, drug screening and future therapeutic applications. Six different experimental groups were included into analysis: undifferentiated human ES cells (1) and undifferentiated human iPS cells (2), human ES cell-derived cardiomyocytes (3) and human iPS cell-derived cardiomyocytes (4) enriched by microdissection of spontaneously contracting embryoid body outgrowths, and human fetal (5) and adult (6) heart tissue. Total RNA samples were prepared from three independent biological replicates in groups 1-4. In groups 5 and 6, single RNA probes were analyzed as three technical replicates.
Project description:Introduction: Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in target cells. We have recently shown that cultured cardiomyocytes release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the released exosome characteristics. Material and Methods: Exosomes were isolated from media collected from cultured cardiomyocyte (HL-1) cells with or without growth factor treatment (TGF-beta2 and PDGF-BB), with a series of differential centrifugations. The exosomes were characterized with dynamic light scattering (DLS) and Western blot and analysed with Illumina whole genome microarray gene expression. Results: An average size of 50-80 nm in diameter with no difference between treatment groups was found. Analysis of the mRNA content revealed 623 transcripts in the control group, 691 in the TGF-beta2-treated group and 362 in the PDGF-BB-treated group. 235 transcripts were common for all three groups. Conclusion: We conclude that there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system.