The third international hackathon for applying insights into large-scale genomic composition to use cases in a wide range of organisms

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Money's Role in Keynes' General Theory Considered

MicroRNAs are key modulators at molecular level in different biological processes, including determination of cell fate and differentiation. Herein, microRNA expression profiling experiments were performed on syngeneic cardiac (CStC) and bone marrow (BMStC) mesenchymal stromal cells cultured in standard growth medium and then in vitro exposed to adipogenic, osteogenic, cardiomyogenic and endothelial differentiation media. Analysis identified a tissue-specific microRNA signature composed of 16 microRNAs that univocally discriminated cell type of origin and that were completely unaffected by in vitro differentiation media: 4 microRNAs were over-expressed in cardiac stromal cells, and 12 were overexpressed or present only in bone marrow stromal cells. Further, results revealed microRNA subsets specifically modulated by each differentiation medium, irrespective of the cell type of origin, and a subset of 7 microRNAs that were down-regulated by all media with respect to growth medium. Finally, we identified 16 microRNAs that were differentially modulated by the media when comparing the two tissues of origin. The existence of a tissue-specific microRNA signature surviving to any differentiation stimuli, strongly support the role if microRNAs determining cell identity related to tissue origin. Moreover, we identified microRNA subsets modulated by different culture conditions in a tissue-specific manner, pointing out their importance during differentiation processes

miRs specifically influenced by differentiation media.

<p>(<b><i>A</i></b>) Heatmap representing the expression of 80 miRs significantly modulated by differentiation stimuli (FDR≤0.01) independently from the tissue of origin. Unsupervised hierarchical analysis groups in five distinct clusters both Cardiac (CStC) and Bone Marrow (BMStC) Stromal Cells exposed to the same medium, as highlighted by translucent purple wedges drawn from the five main nodes. Clustering was done using Pearson’s correlation (centered) and average linkage method. The relative expression level of each miR is represented with a green, black, and red color scale (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107269#pone-0107269-g003" target="_blank">Figure 3</a>). Gene-annotation enrichment analysis showed relevant GO biological processes and KEGG pathways potentially targeted by miRs modulated by (<b><i>B</i></b>) Adipogenic Medium (AM), (<b><i>C</i></b>) Osteogenic Medium (OM), (<b><i>D</i></b>) Cardiomyogenic Medium (CM), and (<b><i>E</i></b>) Endothelial Medium (EM). EASE score <i>P</i>-values are reported for every term.</p

Gene set enrichment analysis of 4 miRs differentially modulated by differentiation media in CStC <i>vs.</i> BMStC.

<p>*Number of genes belonging to a given GO or KEGG gene set with respect to the total validated targets for a given miR.</p>†<p>Percentage of genes belonging to a given GO or KEGG gene set with respect to the total validated targets for a given miR.</p>‡<p>EASE score <i>P</i>-values.</p><p>Gene set enrichment analysis of 4 miRs differentially modulated by differentiation media in CStC <i>vs.</i> BMStC.</p

Unsupervised hierarchical clustering of miRs influenced by tissue of origin and/or differentiation media and/or interaction.

<p>Two-way ANOVA identified 115 miRs significantly modulated (FDR≤0.01). Samples and miRs were clustered using Pearson’s correlation (centered) and average linkage method. Each combination of cell type and differentiation medium was grouped in distinct clusters. The relative expression level of each miR is represented with a blue, black, and orange color scale, ranging from samples with −2 to +2 standard deviations from the mean (blue indicates below median; black, equal to, and orange, above median). CStC, Cardiac and BMStC, Bone Marrow Stromal Cells; GM, growth medium; AM, Adipogenic Medium; OM, Osteogenic Medium; CM, Cardiomyogenic Medium; EM, Endothelial Medium.</p

miRs for which the effect of the media differed between CStC and BMStC.

<p>Mean expression levels ± SEM are plotted for 16 miRs showing a significant interaction effect between tissue origin and media, at 2-way ANOVA, for both Cardiac (CStC) and Bone Marrow (BMStC) Stromal Cells (open circle and filled squares, respectively). Post-hoc comparison between CStC and BMStC indentified miRs differentially modulated by differentiation media. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. GM, growth medium; AM, Adipogenic Medium; OM, Osteogenic Medium; CM, Cardiomyogenic Medium; EM, Endothelial Medium.</p

Morphology and response to <i>in vitro</i> differentiation.

<p>(<b><i>A</i></b>) Cardiac (CStC) and Bone Marrow (BMStC) Stromal Cells cultured in standard growth medium (GM). (<b><i>B</i></b>) CStC and BMStC exposed to adipogenic media (AM) show intracellular lipid accumulation as evidenced by Oil-red O staining. (<b><i>C</i></b>) Von Kossa staining of CStC and BMStC after osteogenic treatment: mineralized matrix is visualized by black dots. (<b><i>D</i></b>) Immunostaining for α-sarcomeric actin, a marker of cardiomyogenic differentiation. (<b><i>E</i></b>) Ac-LDL uptake assay: red in cytoplasm represents DiI-labeled acetylated LDL. Original magnifications: 10× for panels A, B, and C, and 40× for panels D and E. (<b><i>F</i></b>) Percentage of CStC and BMStC positive to Oil-red O staining in GM and after exposure to AM. (<b><i>G</i></b>) Accumulation of alkaline phosphatase was evaluated in CStC and BMStC cultured in GM and exposed for 21 days to osteogenic medium (OM). (<b><i>H</i></b>) RT-qPCR analysis for α-sarcomeric actin expression in CStC and BMStC after 3 weeks culture in GM and cardiomyogenic medium (CM). (<b><i>I</i></b>) Bar graph showing quantitative results for the Ac-LDL-DiI uptake in CStC and BMStC cultured in GM and exposed to endothelial medium (EM). (<b><i>L</i></b>) Representative flow cytofluorograms and bar graph indicating the percentage of VEGFR2 positive cells evaluated by FACS expression in CStC and BMStC before and after 3 weeks of EM culture. All the bar graphs show the mean values of 3 independent experiments ± SD (unpaired Student <i>t</i>-test: *<i>P</i><0.001 <i>vs.</i> corresponding GM, §<i>P</i><0.001 <i>vs.</i> BMStC).</p

Differentiation cluster network based on functionally enriched GO terms and KEGG pathways.

<p>Functional differences of the over-represented GO Biological Processes (BP) and KEGG pathways for the adipogenic, osteogenic, and cardiomyogenic differentiation stimuli in CStC are shown. GO BP and pathway terms are represented as nodes and functional groups are linked to their biological function. Node labels show the most significant or relevant group gene set. Node size represents the term enrichment significance. Node color represents specific cluster membership, <i>i.e.</i> adipogenic (blue), osteogenic (green) or cardiomyogenic (red) differentiation clusters; grey nodes represent unspecific cluster related terms. Node color gradient (from lighter to darker) refers to the proportion of genes (ascending) of a specific differentiation cluster.</p