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, 8 (3), e57892

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

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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

Sebastio Perrini et al. PLoS One.

Abstract

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. ASCSVF, ASCBottom, and ASCCeiling, 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 ASCSVF, ASCBottom and ASCCeiling from Sc fat were significantly different as compared to their homologous subsets of V-ASCs. Furthermore, ASCSVF, ASCCeiling and ASCBottom from the same fat depot were also distinct from each other. In this respect, both principal component analysis and hierarchical clusters analysis showed that ASCCeiling and ASCSVF shared a similar pattern of closely related genes, which was highly different when compared to that of ASCBottom. 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 ASCSVF, ASCBottom, and ASCCeiling 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, ASCCeiling and ASCBottom can be identified as two genetically and functionally heterogeneous ASC populations in addition to the ASCSVF, with ASCBottom 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.

Conflict of interest statement

Competing Interests: This study was partly funded by NovoNordisk. Francesco Giorgino is a PLOS ONE Editorial Board member. However, there are no patents, products in development or marketed products to declare. This does not alter the adherence of the authors to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Isolation and characterization of human ASC.
A. Morphology of human ASC populations, as detected under bright field. B. Expression of specific markers measured by qRT-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.
Figure 2
Figure 2. mRNA expression levels of contaminating mesenchymal lineage markers (CD45, CD31, CD11b) (A) and adipose-derived stem cell markers (CD105, CD44, CD49d) (B), measured by qRT-PCR.
ASCSVF were analyzed at passage 0 (P0) and at passage 4 (P4). Open bars, Sc-ASC; filled bars, V-ASC. *p<0.001 vs. P0. Results are from 5 independent adipose tissue donors.
Figure 3
Figure 3. Venn diagrams summarizing the number of differentially expressed genes in Sc and V ASC populations.
A. Genes found to be differentially expressed by comparing Sc-ASC and V-ASC subsets (inter-depot analysis). B. Genes found to be differentially expressed by comparing ASCSVF, ASCBottom, and ASCCeiling from the Sc adipose tissue (Sc intra-depot analysis). C. Genes found to be differentially expressed by comparing ASCSVF, ASCBottom, and ASCCeiling from the V adipose tissue (V intra-depot analysis). In each panel, figures for conjoint (and non-conjoint) differentially expressed genes are also indicated.
Figure 4
Figure 4. Global views of gene expression utilizing the Principal Component Analysis (PCA).
A. PCA comparing gene expression of Sc-ASC (blue) and V-ASC (red) subsets. B. PCA comparing gene expression of ASCSVF (red), ASCBottom (blue), and ASCCeiling (green) from Sc and V fat depots, respectively. PCA analyses were performed by Partek GS software using default settings that include a threshold to remove low background level intensities. PCA percent mapping on the top of each plot indicates the explained variability on the first coordinate.
Figure 5
Figure 5. Quantitative analysis of specific genes previously identified in the microarray analysis and found to be differentially expressed in the distinct ASC subsets.
mRNA levels were analyzed by qRT-PCR, as described under Methods. Open bars, Sc-ASC; filled bars, V-ASC. All data represent mean ± SE from 9 independent adipose tissue donors. *p<0.05 vs. V-ASC; #p<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.
Figure 6
Figure 6. Release of cytokines from Sc-ASC and V-ASC populations.
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 Materials and Methods. Data represent the mean ± SE of results from 9 independent adipose tissue donors. *p<0.05 vs. V; #p<0.05 vs. other ASC subsets from the same adipose tissue depot (ANOVA test followed by Fisher’s post-hoc test).

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Grant support

This work was supported in part by grants from the Ministero dell’Università e Ricerca (Italy), the COST Action BM0602, the Fondazione della Società Italiana di Diabetologia per la Ricerca in Diabetologia e Malattie Metaboliche (Fo.Ri.SID), and NovoNordisk to F. Giorgino. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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