Supplementary document for Single-active-element demultiplexed multi-photon source - 6552945.pdf

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Differences in Gene Expression and Cytokine Release Profiles Highlight the Heterogeneity of Distinct Subsets of Adipose Tissue-Derived Stem Cells in the Subcutaneous and Visceral Adipose Tissue in Humans

<div><p>Differences in the inherent properties of adipose tissue-derived stem cells (ASC) may contribute to the biological specificity of the subcutaneous (Sc) and visceral (V) adipose tissue depots. In this study, three distinct subpopulations of ASC, i.e. ASC_SVF, ASC_Bottom, and ASC_Ceiling, were isolated from Sc and V fat biopsies of non-obese subjects, and their gene expression and functional characteristics were investigated. Genome-wide mRNA expression profiles of ASC_SVF, ASC_Bottom and ASC_Ceiling from Sc fat were significantly different as compared to their homologous subsets of V-ASCs. Furthermore, ASC_SVF, ASC_Ceiling and ASC_Bottom from the same fat depot were also distinct from each other. In this respect, both principal component analysis and hierarchical clusters analysis showed that ASC_Ceiling and ASC_SVF shared a similar pattern of closely related genes, which was highly different when compared to that of ASC_Bottom. However, larger variations in gene expression were found in inter-depot than in intra-depot comparisons. The analysis of connectivity of genes differently expressed in each ASC subset demonstrated that, although there was some overlap, there was also a clear distinction between each Sc-ASC and their corresponding V-ASC subsets, and among ASC_SVF, ASC_Bottom, and ASC_Ceiling of Sc or V fat depots in regard to networks associated with regulation of cell cycle, cell organization and development, inflammation and metabolic responses. Finally, the release of several cytokines and growth factors in the ASC cultured medium also showed both inter- and intra-depot differences. Thus, ASC_Ceiling and ASC_Bottom can be identified as two genetically and functionally heterogeneous ASC populations in addition to the ASC_SVF, with ASC_Bottom showing the highest degree of unmatched gene expression. On the other hand, inter-depot seem to prevail over intra-depot differences in the ASC gene expression assets and network functions, contributing to the high degree of specificity of Sc and V adipose tissue in humans.</p> </div

Quantitative analysis of specific genes previously identified in the microarray analysis and found to be differentially expressed in the distinct ASC subsets.

<p>mRNA levels were analyzed by <i>q</i>RT-PCR, as described under Methods. Open bars, Sc-ASC; filled bars, V-ASC. All data represent mean ± SE from 9 independent adipose tissue donors. *<i>p</i><0.05 vs. V-ASC; #<i>p</i><0.05 vs. other ASC populations from the same adipose tissue depot (ANOVA test followed by Fisher’s post-hoc test). SVF, stromo-vascular; B, bottom; C, ceiling.</p

Significant top list of associated network functions from IPA in Sc and V ASCs.

<p>Significant top list of associated network functions from IPA in Sc and V ASCs.</p

Isolation and characterization of human ASC.

<p><i>A</i>. Morphology of human ASC populations, as detected under bright field. <i>B</i>. Expression of specific markers measured by <i>q</i>RT-PCR. Cells were harvested at passage 0, and RNA was analyzed for markers of adipose-derived stem cells (CD105, CD44, CD49d), human leukocytes and macrophages (CD45, CD11b), and mature endothelial cells (CD31).+or – signs indicate the relative levels of marker expression. Results are from 5 independent adipose tissue donors.</p

Release of cytokines from Sc-ASC and V-ASC populations.

<p>Culture medium from Sc-ASC (open bars) and V-ASC (filled bars) subsets (10⁶ cells) was collected after a 16-h period, and levels of specific cytokines were determined using the multiplex technique, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057892#s2" target="_blank">Materials and Methods</a>. Data represent the mean ± SE of results from 9 independent adipose tissue donors. *<i>p</i><0.05 vs. V; #<i>p</i><0.05 vs. other ASC subsets from the same adipose tissue depot (ANOVA test followed by Fisher’s post-hoc test).</p

Significant top list of canonical pathways from IPA in Sc and V ASCs.

<p>Significant top list of canonical pathways from IPA in Sc and V ASCs.</p

Venn diagrams summarizing the number of differentially expressed genes in Sc and V ASC populations.

<p><i>A</i>. Genes found to be differentially expressed by comparing Sc-ASC and V-ASC subsets (inter-depot analysis). <i>B</i>. Genes found to be differentially expressed by comparing ASC_SVF, ASC_Bottom, and ASC_Ceiling from the Sc adipose tissue (Sc intra-depot analysis). <i>C</i>. Genes found to be differentially expressed by comparing ASC_SVF, ASC_Bottom, and ASC_Ceiling from the V adipose tissue (V intra-depot analysis). In each panel, figures for conjoint (and non-conjoint) differentially expressed genes are also indicated.</p

Effects of TNFα on endothelial activation in HUVEC.

<p>Cells were incubated with 50 ng/ml TNFα for the indicated times or left untreated. <i>A</i>. Leukocyte transmigration test. HL-60 cells were added to HUVEC/wt, stimulated with 50 ng/ml TNFα for 4 h, and left to migrate for 18 h at 37°C. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates from two independent experiments, and are normalized to control. *<i>P</i><0.05 vs. no TNFα. <i>B</i>. TNFα regulation of E-Selectin mRNA levels. E-Selectin mRNA levels were evaluated by qRT-PCR, and normalized using β-actin as internal control. <i>C</i>. TNFα regulation of E-Selectin protein levels. E-Selectin protein levels were evaluated by immunoblotting, using GAPDH as internal control. <i>D</i>. Leukocyte transmigration test. HL-60 cells were added to control HUVEC (treated with negative siRNA) and HUVEC treated with E-Selectin siRNA#1 (50 nM) or siRNA#2 (100 nM) for 48 h, and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates of one experiment and are normalized to control. *<i>P</i><0.05 vs. negative siRNA. <i>E</i>. Effects of SP600125 (left) and PD98059 (right) on TNFα-induced E-Selectin mRNA expression. Cells were pre-treated with 30 mM JNK or MEK inhibitor, respectively, for 2 h and then exposed 50 ng/ml TNFα for the indicated times (untreated cells, black; inhibitor-treated cells, grey; DMSO-treated cells, light grey). E-Selectin mRNA levels were evaluated by qRT-PCR, using β-actin as internal control. *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. TNFα-stimulated cells.</p

Effects of p66^Shc silencing on endothelial activation in HUVEC.

<p><i>A</i>. (top left) Representative immunoblot of p66^Shc Ser³⁶ phosphorylation in HUVEC transfected with 50 nM or 100 nM of siRNA#1 or siRNA#2, and (right) quantification of multiple experiments. <i>Hatched </i><i>bar</i>, cells transfected with a negative control siRNA; black bars, cells treated with siRNA#1 and siRNA#2. (bottom left) Representative immunoblot of p66^Shc protein content in HUVEC transfected with 50 nM or 100 nM of siRNA#1 or siRNA#2, respectively, and (right) quantification of multiple experiments. <i>Hatched </i><i>bar</i>, cells transfected with a negative control siRNA; grey bars, cells treated with siRNA#1; light grey bars, cells treated with siRNA#2. *<i>P</i><0.05 vs. cells transfected with negative siRNA. Neg., negative siRNA. <i>B</i>. Leukocyte transmigration test. HL-60 cells were added to control HUVEC (w.t., Lipofectamine™, and negative siRNA) and HUVEC treated with p66^Shc siRNA#1 (100 nM) or p66^Shc siRNA#2 (50 nM), and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions or following stimulation with 50 ng/ml TNFα for 4 h. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates from two independent experiments, and are normalized to basal HUVEC/wt. *<i>P</i><0.05 vs. no TNFα; ^§P<0.05 vs. HUVEC/wt. <i>C</i>. Effects of siRNA-mediated knockdown of p66^Shc on E-Selectin mRNA levels under basal conditions (left) and following TNFα stimulation (right). HUVEC were transfected with 100 nM siRNA#1 (grey bars) or 50 nM siRNA#2 (light grey bars), and then left untreated or incubated with 50 ng/ml TNFα for 1 h. *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. TNFα-stimulated controls (wild-type, Lipofectamine™, and negative siRNA). Lysates were analyzed by qPCR 48 h following transfection, using β-actin as internal control. Lipo, Lipofectamine™.</p