33 research outputs found
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
miR tissue signature and its potential functional implications.
<p>(<b><i>A</i></b>) A Venn diagram of the 115 significantly modulated miRs helps indentifying those exclusively affected by the tissue of origin (upper left segment) or by one or more differentiation media (upper right), and those influenced by both factors (upper mid intersection) or by the interaction between the two (lower intersection segments). (<b><i>B</i></b>) Unsupervised hierarchical clustering defining the miR tissue signature composed by 16 miRs, distinctive of the tissue of origin and with at least a 2-fold difference. Log<sub>2</sub> transformed miR expression signals were centered by median values and samples and miRs were clustered using Pearson’s correlation (centered) and average linkage method, with leaf order optimization. The dendrogram above shows that this signature is able to divide Cardiac (CStC) and Bone Marrow (BMStC) Stromal Cells in two distinct clusters, irrespective of the culture media exposure. The relative expression level of each miR is represented with a green, black, and red color scale (green indicates below, black equal to, and red above median). (<b><i>C</i></b>) Gene-annotation enrichment analysis revealed GO biological processes (blue bars), molecular functions (green), and KEGG pathways (red) potentially and exclusively targeted by CStC tissue-specific miRs. The x-axis represents the percentage of genes belonging to a given GO or KEGG term with respect to the total predicted and validated targets. EASE score <i>P</i>-values are reported for every term. (<b><i>D</i></b>) Gene categories potentially targeted by BMStC tissue-specific miRs, as revealed by gene-annotation enrichment analysis. GM, growth medium; AM, Adipogenic Medium; OM, Osteogenic Medium; CM, Cardiomyogenic Medium; EM, Endothelial Medium.</p
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
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
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
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
MiR subsets specifically modulated by differentiation media <i>vs.</i> growth medium in both CStC and BMStC.
†<p>Bonferroni corrected <i>P</i>-values.</p><p><sup>*</sup><i>P</i><0.05;</p><p><sup>**</sup><i>P</i><0.01;</p><p><sup>***</sup><i>P</i><0.001. FC: fold-change.</p><p>CStC, Cardiac and BMStC, Bone Marrow Stromal Cells; AM, Adipogenic Medium; OM, Osteogenic Medium; CM, Cardiomyogenic Medium; EM, Endothelial Medium.</p><p>MiR subsets specifically modulated by differentiation media <i>vs.</i> growth medium in both CStC and BMStC.</p
A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids
<div><p>A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary <i>in vitro</i> biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.</p></div
Flow field within the bioreactor.
<p>Flow field visualization of the mutual interaction between the medium (primary phase) and the cells/constructs (dispersed phase) within the culture chamber for ultralow (A and A1) and low-to-moderate (B and B1) shear stress conditions. Flow field is depicted using both linear integral convolution lines (A and B), and a classical streamline representation (A1 and B1). Yellow arrows indicate the flow inlet and outlet. Blue arrows indicate the primary buoyant vortices. Red arrows indicate the secondary vortices.</p