Experimental scheme.

<p>Experimental scheme.</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

Gene expression, histone acetylation and DNA methylation in early and late passage MSCs.

<p>(A) Real-Time PCR analysis of the expression of Nanog, REX1 and CD133 in culture passage (P) 1, passage 6 MSCs cultured in growth medium or passage 6 MSCs growth medium supplemented with bFGF (P6-bFGF). bFGF treatment started from passage 1 cells. (B) TERT histone H3 acetylation (** <i>P</i><0.01 versus P1 and P6 in bFGF-supplemented culture). (C) TERT gene expression (Real-Time PCR analysis) and, (D) DNA methylation in CpG islands in the promoter region of TERT in passage 1 and 6 MSCs cultured in growth medium versus passage 6 MSCs cultured in growth medium supplemented with bFGF (P6-bFGF).</p

Fluorescence-activated cell sorting (FACS) analysis of MSCs.

<p>Passage 3 MSCs were analyzed by FACS after staining with FITC- or PE-conjugated control isotype IgG (gray peaks) or antibodies against indicated cell surface proteins (filled red or green peaks).</p

Osteogenic gene expression, histone acetylation and DNA methylation in early and late passage MSCs.

<p>(A) Runx2 and (D) ALP histone H3 acetylation (** <i>P</i><0.01), (B) Runx2 and (E) ALP gene expression (Real-Time PCR analysis), and (C) DNA methylation in CpG islands in the promoter region of Runx2 and (F) in the in the promoter region and exon 1 region of ALP in passage (P) 1 and 6 MSCs cultured in growth medium versus passage 6 MSCs cultured in growth medium supplemented with bFGF (P6-bFGF).</p

Multipotent gene expression, histone acetylation and DNA methylation in early and late passage MSCs.

<p>Gene expression, histone acetylation and DNA methylation. (A) Sox2 and (D) Oct4 histone H3 acetylation (** <i>P</i><0.01); (B) Sox2 and (E) Oct4 gene expression (Real-Time PCR analysis), and (C) DNA methylation in CpG islands in the promoter region and exon 1 region of Sox2 and (F) Oct in passage (P) 1 and 6 MSCs cultured in growth medium versus passage 6 MSCs cultured in growth medium supplemented with bFGF (P6-bFGF).</p

Differentiation of MSCs.

<p>Cultured in appropriate induction media, (A & B) MSCs differentiated into adipocytes (after oil red staining, A represents non-induced and B represents induced), (C & D) osteoblasts (after Alizarin Red S staining, C represents non-induced and D represents induced), and (E & F) chondrocytes (after Alcian Blue staining, E represents non-induced and F represents induced).</p

Effects of bFGF on MSC proliferation and spontaneous differentiation.

<p>(A) Growth curves of MSCs cultured in growth medium (control) and in growth medium supplemented with bFGF (bFGF). (B) MSCs cultured in growth medium (control) or in growth medium supplemented with bFGF (bFGF) for 3 days after Alizarin Red S staining. (C) Mean proportion of Alizarin Red S positive areas per field after quantification of 4 randomly selected fields (** <i>P</i><0.01).</p

Morphological changes and spontaneous estrogenic differentiation of MSCs.

<p>Cultured in growth medium, (A & B) MSCs became larger and fatter upon expansion, (C) A few cells became extremely larger and flatter and were positive (in red) for alkaline phosphatase (ALP) stain. (D) Real-Time PCR analysis showed that the expression of genes associated with osteogenesis such as collagen type I (Col I), alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN) and osteopontin (OPN) increased progressively with cell passages. P1 to P10 represents MSC passage number. Induction indicates MSCs after incubation with osteogenic induction medium for 14 days.</p