Glucose tolerance improvement in diabetic mice after MSC transplantation.

<p>At day 130 after diabetes induction, glucose tolerance was assessed in <i>normal</i>, <i>diabetic</i>, <i>diabetic + 1xMSCs</i>, <i>diabetic/insulin</i>, <i>diabetic/insulin + 1xMSCs</i> and <i>diabetic/insulin + 2xMSCs</i>. Data correspond to mean ± s.e.m. for 9 animals per experimental group. <i>p<0.05, diabetic vs. diabetic/insulin + 1xMSC</i>.</p

Hyperglycemia reversion in diabetic mice after MSC transplantation.

<p>Diabetic mice received or not MSCs (A). Diabetic mice received or not insulin prophylaxis and/or MSCs (B). Diabetic mice received or not insulin prophylaxis and one or two doses of MSCs (C). Blood glucose levels were determined every three days in alert nonfasted animals. Data correspond to mean ± s.e.m. for 9 animals per experimental group. Only statistical significant values among diabetic mice are shown.</p

Experimental groups.

<p>Vehicle A: 0.1 M citrate buffer, pH 4.5. STZ: intraperitoneal 40 mg/kg/day, for five consecutive days. Vehicle B: 5% mice plasma. MSCs: intravenous 0.5×10⁶ healthy donor bone marrow-derived MSCs resuspended in 0.2 mL vehicle B. Insulin pellets: subcutaneous implantation of two timed-release porcine insulin pellets per animal. Triangle represents exogenous insulin levels.</p

Additional file 1: of Systemically administered allogeneic mesenchymal stem cells do not aggravate the progression of precancerous lesions: a new biosafety insight

Figure S1. Characterization of MSCs isolated from bone marrow of Syrian golden hamster. Immunophenotype (a) and differentiation potential (b). Dashed line, mean fluorescence intensity of isotype control. Bar = 100 μm (n = 4). (TIF 1623 kb

Effects of allo-mBM-MSCs or allo-acd-mMSCs on wound closure in murine excisional wounds.

<p>(<b>A)</b> Representative images of the wounds in NOD mice under vehicle, allo-mBM-MSCs or allo-acd-mMSCs treatments at day 0 (before treatment), 4, 8, 10, 12 and 16 for NOD wounds. (<b>B)</b> Wound closure analysis. All wounds were measured using digital calipers at day 0, 2, 4, 6, 8, 10, 12 and 16 post-operation. The wound closure rate is plotted as the reduction percentage of original wound area over time (n = 25, 25 and 27 for vehicle, allo-mBM-MSCs and allo-acd-mMSCs groups, respectively). Analysis of variance (ANOVA) was used, * indicates a significant difference (p < 0.05). Abbreviations: NOD, Non-Obese Diabetic; allo-mBM-MSCs, mouse bone marrow-derived allogeneic MSCs and allo-acd-mMSCs, mouse bone marrow acelullar derivatives allogeneic MSCs.</p

Histological analysis of leucocyte infiltration, granulation tissue and collagen fibers of skin wounds 16 days after treatment with allo-mBM-MSCs and allo-acd-mMSCs in NOD mice.

<p>Frequency of animals with the histological scores for: <b>(A)</b> leukocyte infiltration in the dermis, (<b>B</b>) granulation tissue formation stage and (<b>C</b>) quantitative analysis of the intensity of dermal collagen fibers. (n = 25, 25 and 27 for vehicle, allo-mBM-MSCs and allo-acd-mMSCs groups, respectively). Pearson’s chi-square test and variance (ANOVA) analysis were used, * indicates a significant difference p < 0.0001 and ** p < 0.05. Abbreviations: NOD, Non-Obese Diabetic; allo-mBM-MSCs, mouse bone marrow-derived allogeneic MSCs and allo-acd-mMSCs, mouse bone marrow acelullar derivatives allogeneic MSCs.</p

The role of bone marrow mesenchymal stromal cell derivatives in skin wound healing in diabetic mice

<div><p>Mesenchymal stromal cells (MSCs) have shown to be a promising tool in cell therapies to treat different conditions. Several pre-clinical and clinical studies have proved that the transplantation of MSCs improves wound healing. Here, we compare the beneficial effects of mouse bone marrow-derived allogeneic MSCs (allo-mBM-MSCs) and their acelullar derivatives (allo-acd-mMSCs) on skin wound healing in Non-Obese Diabetic (NOD) mice. One dose of allo-mBM-MSCs (1×10⁶ cells) or one dose of allo-acd-mMSCs (1X) were intradermally injected around wounds in 8–10 week old female NOD mice. Wound healing was evaluated macroscopically (wound closure) every two days, and microscopically (reepithelialization, dermoepidermal junction, skin appendage regeneration, leukocyte infiltration, vascularization, granulation tissue formation, and density of collagen fibers in the dermis) after 16 days of MSC injection. In addition, we measured growth factors and specific proteins that were present in the allo-acd-mMSCs. Results showed significant differences in the wound healing kinetics of lesions that received allo-acd-mMSCs compared to lesions that received vehicle or allo-mBM-MSCs. In particular, mice treated with allo-acd-mMSCs reached significantly higher percentages of wound closure at day 4, 6 and 8, relative to the allo-mBM-MSCs and vehicle groups (p < 0.05), while wound closure percentages could not be statistically distinguished between the allo-mBM-MSCs and vehicle groups. Also, allo-acd-mMSCs had a greater influence in the skin would healing process. Specifically, they caused a less pronounced inflammatory severe response (p < 0.0001), more granulation tissue formation at an advanced stage (p < 0.0001), and higher density of collagen fibers (p < 0.05) compared to the other groups. Nevertheless, at day 16, both allo-mBM-MSCs and allo-acd-mMSCs revealed a higher effect on the recovery of the quality skin (continuous epidermis; regular dermoepidermal junction and skin appendages) relative to untreated lesions (p < 0.0001), but not between them. On the other hand, ELISA analyses indicated that the allo-acd-mMSCs contained growth factors and proteins relevant to wound healing such as IGF-1, KGF, HGF, VEGF, ANG-2, MMP-1, CoL-1 and PGE2. Compared to allo-acd-mMSCs, the administration of allo-mBM-MSCs is insufficient for wound healing in diabetic mice and delays the therapeutic effect, which maybe explained by the fact that trophic factors secreted by MSCs are critical for skin regeneration, and not the cells per se, suggesting that MSCs may require some time to secrete these factors after their administration.</p></div

Growth factors and protein profile relevant to wound healing found in allo-acd-mMSCs.

<p>ELISA measurement of secretion levels of KGF, CoL-1, MPP-1, VEGF, IGF-1, Ang-2, HGF, and PGE₂ were measured after 24 h allo-mBM-MSCs culture. (n = 24). Abbreviations: allo-mBM-MSCs, mouse bone marrow-derived allogeneic MSCs; allo-acd-mMSCs, mouse bone marrow acelullar derivatives allogeneic MSCs, KGF, keratinocyte growth factor; CoL-1, collagen type 1; MPP-1, matrix metalloproteinase 1; VEGF, vascular endothelial growth factor; Ang-1, angiopoietin 1; IGF-1, human insulin-like growth factor 1; Ang-2, angiopoietin 2; HGF, hepatocyte growth factor and PGE₂, prostaglandin E2.</p

Histological analysis of dermo-epidermal junction and appendage-like structure formation of skin wounds 16 days after treatment with allo-mBM-MSCs and allo-acd-mMSCs in NOD mice.

<p>(A) Wound histological representative images of haematoxylin-eosin staining showing dermo-epidermal junction, which are indicated by arrows. Scale bar = 100 <i>μ</i>m. (B) Frequency of animals with the histological scores for dermo-epidermal junction. The score was assigned based on the junction level and was quantified using ImageJ (scores: IV: 0.1–400 pixels (complete junction), III: 401–750 pixels, II: 751–1,200 pixels, I: 1,201–2,800 pixels and 0: > 2,801 (incomplete junction). (C) Representative images of Masson’s trichrome staining illustrating appendage-like structure in the dermis indicated by arrows. Scale bar = 50 <i>μ</i>m. (D) Frequency of animals with the score for appendage-like structure. (n = 25, 25 and 27 for vehicle, allo-mBM-MSCs and allo-acd-mMSCs groups, respectively). Analysis of Pearson’s chi-square test was used, * indicates a significant difference (p < 0.0001). Abbreviations: NOD, Non-Obese Diabetic; allo-mBM-MSCs, mouse bone marrow-derived allogeneic MSCs; allo-acd-mMSCs and mouse bone marrow acelullar derivatives allogeneic MSCs.</p

Bovine Lactoferricin-derived peptides used in this study.

<p>Bovine Lactoferricin-derived peptides used in this study.</p