Analysis of Allogenicity of Mesenchymal Stem Cells in Engraftment and Wound Healing in Mice

Studies have shown that allogeneic (allo-) bone marrow derived mesenchymal stem cells (BM-MSCs) may enhance tissue repair/regeneration. However, recent studies suggest that immune rejection may occur to allo-MSCs leading to reduced engraftment. In this study, we compared allo-BM-MSCs with syngeneic BM-MSCs or allo-fibroblasts in engraftment and effect in wound healing. Equal numbers of GFP-expressing allo-BM-MSCs, syngeneic BM-MSCs or allo-fibroblasts were implanted into excisional wounds in GFP-negative mice. Quantification of GFP-expressing cells in wounds at 7, 14 and 28 days indicated similar amounts of allogeneic or syngeneic BM-MSCs but significantly reduced amounts of allo-fibroblasts. With healing progression, decreasing amounts of allogeneic and syngeneic BM-MSCs were found in the wound; however, the reduction was more evident (2 fold) in allo-fibroblasts. Similar effects in enhancing wound closure were found in allogeneic and syngeneic BM-MSCs but not in allo-fibroblasts. Histological analysis showed that allo-fibroblasts were largely confined to the injection sites while allo-BM-MSCs had migrated into the entire wound. Quantification of inflammatory cells in wounds showed that allo-fibroblast- but not allo-BM-MSC-treated wounds had significantly increased CD45+ leukocytes, CD3+ lymphocytes and CD8+ T cells. Our study suggests that allogeneic BM-MSCs exhibit ignorable immunogenicity and are equally efficient as syngeneic BM-MSCs in engraftment and in enhancing wound healing

Epigenetic Dysregulation in Mesenchymal Stem Cell Aging and Spontaneous Differentiation

BACKGROUND: Mesenchymal stem cells (MSCs) hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation

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