Experimental scheme.

<p>Experimental scheme.</p

Antibody-based protein array analysis of fibroblast- or MSC-conditioned medium

<p>Human or murine dermal fibroblast- or BM-MSC-conditioned medium under hypoxic conditions for 24 h was analyzed with antibody-based protein array. The intensity of each dot was measured. “−”, not detected; +/−, weakly detected; + ∼ ++++, intensity of positive detection; N/A, not tested; protein levels in medium under hypoxic conditions are indicated as up, down or NC (no change) compared to normoxic conditions.</p

mRNA levels of cytokines and extracellular matrix molecules in BM-MSCs and fibroblasts.

<p>Total RNA extracted from BM-MSCs (MSC) or dermal fibroblasts (FB) treated under hypoxic conditions was analyzed by Real-Time PCR for mRNA expression of genes as indicated in the figure. Fold changes vs dermal fibroblasts are shown. Data are mean±SD; n = 3; *<i>P</i><0.05 vs FB. KGF, keratinocyte growth factor; HB-EGF, heparin-binding EGF-like growth factor; TGFb1, Transforming growth factor-β1; Ang, angiopoietin; SDF1, stromal cell-derived factor-1; MIP, macrophage inflammatory protein; SCF, stem cell factor; EPO, erythropoietin; TPO, thrombopoietin; G-CSF, granulocyte colony stimulating factor; MCP1, monocyte chemotactic protein-1; MIG, monokine induced by gama interferon.</p

Murine primers for Real-Time PCR

<p>Murine primers for Real-Time PCR</p

Engraftment of BM-MSCs into the wounded skin.

<p>(A) Allo-fibroblasts or allo-MSCs in wounds. Representative fluorescence microscopic images of day 7 wound sections showing that the injected allogeneic GFP⁺fibroblasts (allo-FB) were confined to the injection site and surrounded by a layer of inflammatory and fibroblast-like cells (arrow heads, left panel). Weak GFP signals were detected in some of allo-fibroblasts. After immunostaining for GFP, topically applied allo-fibroblasts (green) were shown to be poorly incorporated into the tissue (middle and right panels of upper row) and in many of them nuclei were not shown (arrow heads, middle panel of upper row), indicating cell death, while similarly applied allo-MSCs (green) were closely integrated into the wound (lower row, representative images from three mice). Wound beds are indicated by arrows. Nuclei were stained blue with Hoechst. scale bar, 50 µm. (B) Wounds treated with allogeneic or syngeneic BM-MSCs or vehicle medium (sham) in Balb/C or C57BL/6 mice at 1 or 2 weeks were enzymatically dissociated as discribed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007119#s2" target="_blank">Materials and Methods</a>” and single-cell suspensions were analyzed by flow cytometry to detect percentages of GFP-positive cells. One representative result is shown. Cells from sham wounds were used for negative controls and gate setting. (C) Cell engraftment. Taking the initially implanted one million cells per wound as 100%, proportions of engrafted BM-MSCs or fibroblasts at different times after transplantation are shown. *<i>P</i><0.001 (allo-fibroblast vs MSC, n = 6 or 7).</p

Analysis of cells in wounds.

<p>(A) FACS analysis of cells derived from each wound indicated that wounds treated with concentrated BM-MSC-conditioned medium (MSC-M) at 7 or 14 days had increased percentages of CD4/80 positive monocytes/macrophages compared to wounds treated with vehicle control medium (vehicle-M) or concentrated fibroblast-conditioned medium (FB-M). (B&C) Percentages of cells in wound after FACS analysis (n = 5∼6, *<i>P</i><0.05). (D) Representative images of confocal microscopy of day 7 wounds treated with vehicle medium, concentrated fibroblast- or BM-MSC-conditioned medium after immunostaining for CD68 (red). Nuclei were stained blue with Hoechst. scale bar, 20 µm.</p

Effects of BM-MSC-conditioned medium on cell migration and proliferation.

<p>(A) migration of CD14⁺ monocytes. CD14⁺ monocytes were isolated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001886#s2" target="_blank">Methods</a>” and equal numbers of the cells were loaded to the top chambers. Control (CTL) vehicle medium, fibroblast (FB-M)- or BM-MSC (MSC-M)-conditioned medium at different concentrations were added to the bottom chambers. Cells migrated into the bottom chambers were counted. Triple wells were used. Data shown represent mean±SD of 3 independent experiments (<i>P</i><0.01). (B) Keratinocyte migration. Equal numbers of murine dermal keratinocytes were added to the top chambers. Media in the bottom chambers were as indicated. Cells migrated to the down-side of the filter were stained, photographed (6 fields per well) and counted. Triple wells were used for each treatment and data shown represent mean±SD of 3 independent experiments (<i>P</i><0.001). (C) Keratinocyte proliferation. 0.5×10⁵ murine dermal keratinocytes per well were incubated with vehicle-M, FB-M, MSC-M or keratinocyte SFM supplemented with EGF (5 ng/ml, EGF) for different times and cell numbers were counted. Triple wells were used for each treatment. Values shown represent mean±SD of 4 independent experiments (* <i>P</i><0.01). (D) HUVEC migration. The bottom chambers contained vehicle-M, FB-M or MSC-M at various dilutions. Cells migrated to the down-side of the filter were stained, photographed (6 fields per well) and counted. Triple wells were used for each treatment and data shown represent mean±SD of three independent experiments (*<i>P</i><0.01 vs vehicle-M; #<i>P</i><0.01 vs FB-M) (E) HUVEC proliferation. Equal numbers of HUVECs were grown in vehicle-, FB- or MSC-conditioned basal endothelial growth medium (EGM-2) supplemented with 2% FBS or complete EGM-2 and incubated for 3 days. Cell numbers were counted. Experiments were performed in triplicate wells (n = 3, * <i>P</i><0.001).</p

Surface marker profile of dermal fibroblasts and BM-MSCs

<p>Representative results of three FACS analyses of murine dermal fibroblasts and BM-MSCs. − ∼ ++++ represent percentages of cells expressing surface markers as indicated: “−” ≤ 2%; “+” 3∼10%; “++” 11∼50%; “+++” 51∼90%; “++++” 91∼100%.</p

Protein levels of cytokines in BM-MSC-conditioned medium.

<p>(A) Antibody-based protein array analysis human dermal fibroblast (FB)- or BM-MSC-conditioned medium under hypoxic conditions. Similar results were obtained from three independent experiments and results from one of them are shown. The abbreviations are donated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001886#pone-0001886-t002" target="_blank">Table 2</a>. (B) ELISA measurement of cytokines in murine fibroblast- or BM-MSC- conditioned medium under hypoxic conditions. Data are expressed as means±SD (n = 3, *<i>P</i><0.01). PDGF-BB, platelet-derived growth factor-BB. Other abbreviations can be found in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001886#pone-0001886-g001" target="_blank">Figure 1</a>.</p

Changes of MSCs in culture.

<p>Cultured in growth medium, MSCs in passage (P) 10 were larger and fatter compared to cells in P1 (A & B). Real-Time PCR analysis showed expressional changes of genes involved in stem cell pluripotency and osteogenic differentiation (C). The experiment was repeated three time with similar results, and representative results from one experiment were shown (*<i>P</i><0.01). ALP, asalkaline phosphatase; OPN, osteopontin.</p