Genome-Wide Profiling of MicroRNAs in Adipose Mesenchymal Stem Cell Differentiation and Mouse Models of Obesity

In recent years, there has been accumulating evidence that microRNAs are key regulator molecules of gene expression. The cellular processes that are regulated by microRNAs include e.g. cell proliferation, programmed cell death and cell differentiation. Adipocyte differentiation is a highly regulated cellular process for which several important regulating factors have been discovered, but still not all are known to fully understand the underlying mechanisms. In the present study, we analyzed the expression of 597 microRNAs during the differentiation of mouse mesenchymal stem cells into terminally differentiated adipocytes by real-time RT-PCR. In total, 66 miRNAs were differentially expressed in mesenchymal stem cell-derived adipocytes compared to the undifferentiated progenitor cells. To further study the regulation of these 66 miRNAs in white adipose tissue in vivo and their dependence on PPARγ activity, mouse models of genetically or diet induced obesity as well as a mouse line expressing a dominant negative PPARγ mutant were employed

Expression of perivascular and endothelial cell-specific markers in wound repair.

<p>Analysis of PECAM1, desmin, α-SMA and Sca1 expression in the wounded skin. (A) Dorsal view of full thickness wounds on the back skin of mice one (D1), seven (D7) and 14 (D14) days post injury. Representative H&E-stained cryosections of selected wounds (arrowhead) during inflammation (D1), granulation (D7) and remodeling (D14) are shown. (B-D) Immunostaining of (B) PECAM1/desmin, (C) PECAM1/α-SMA or (D) PECAM1/Sca1 expression at the different stages of wound healing. The individual monochrome signals for PECAM1, desmin, α-SMA and Sca1 are shown in overviews. Squares within the images represent closeups of overlays for the PECAM1/desmin, PECAM1/α-SMA PECAM1/Sca1 stainings (B-D). Bars 1 cm (A, top), 1 mm (A, lower panel), 100 µm (B).</p

Distribution of PECAM1 and Sca1 protein on skin- and wound-derived cells.

<p>(A) Flow cytometry analysis of Sca1 and PECAM1 expression in CD45^- non-hematopoietic cells isolated from newborn (nb), three weeks (3 wk) and three months (3 mo) old dermis. Sca1⁺ (1, red box), PECAM1⁺ (2, green ellipse) and PECAM1⁺/Sca1⁺ (3, black ellipse) cell populations are highlighted. (B) Dot plots of Sca1 and PECAM1 expression in cell suspensions isolated from full thickness wounds one (D1), seven (D7) and 14 days (D14) post injury of eight weeks old mice. Percentage of positive cell populations at the different time points of flow cytometry analysis (lower panel) is given with standard deviation and significant changes were determined using the unpaired two-tailed student’s T-test (n≥3, **p≤0.01, n.s = not significant).</p

Myofibroblast-like cell formation and modulation of CD38 receptor activity. (A)

<p>Representative cell cycle analysis of skin- and wound-derived Sca1⁺, PECAM1⁺ and PECAM1⁺/Sca1⁺ cells seven days post injury using propidium iodide (PI) stain in flow cytometry analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053262#pone.0053262.s001" target="_blank">figure S1</a>). The relative percentage of cells in G1 (green), S (ochre) and G2 (blue) are highlighted. (B) Flow cytometric detection of α-SMA in wound-derived Sca1⁺, PECAM1⁺/Sca1⁺ and PECAM1⁺ cells seven days post injury (n = 7 mice). (C) Immunofluorescence analysis of α-SMA expression in cultured wound-derived Sca1⁺, PECAM1⁺ and PECAM1⁺/Sca1⁺ cells. Nucleoli were detected using DAPI stain. (D) Morphometric analysis of wounds in immunodeficient mice stimulated with rat anti-CD38 or isotype matched antibodies (n = 4). Distances between edges of the panniculus carnosus (δ pc), hair follicles (δ A) and the area of the granulation tissue (g) were determined. Statistics: unpaired two-tailed student’s T-test (*p≤0.05, **p≤0.01). Bars 100 µm (C), 500 µm (D).</p

Expression profile of isolated Sca1⁺, PECAM1⁺ and PECAM1⁺/Sca1⁺

<p><b>cells.</b> Marker expression in the sorted cell populations isolated from dermis or wounds seven days post injury. (A) Relative mRNA expression levels of perivascular (<i>Desmin</i>, <i>Pdgfrb</i>, <i>Angpt1</i>), endothelial (<i>Angpt2</i>, <i>Tie2</i>, <i>Pecam1</i>), progenitor cell-specific markers (<i>Sca1</i>, <i>Cd34</i>) and of the CD38 receptor (<i>Cd38</i>) were determined by semiquantitative RT-PCR in non-hematopoietic Sca1⁺, PECAM1⁺ and PECAM1⁺/Sca1⁺ cell populations. The band intensities of the electrophoretic gels were processed and quantified using Image J software. The relative expression intensity of the individual genes compared to <i>Gapdh</i> is given. The lack of Sca1 or Pecam1 expression in sorted PECAM1⁺ or Sca1⁺ cells demonstrated the purity of the cell fractions. (B) Flow cytometric detection of TIE2, CD34, CD38 expression at the cell surface of Sca1⁺ (red), PECAM1⁺ (green) and PECAM1⁺/Sca1⁺ (black) cells isolated from dermis or full thickness wounds seven days post injury.</p