Adipose stem cells isolated from diabetic mice improve cutaneous wound healing in streptozotocin-induced diabetic mice

Abstract

Background: Adipose-derived mesenchymal stem cells (ASCs) therapy is emerging as a novel therapeutic option for the treatment of a variety of diseases including diabetes and diabetic wound healing. Multiple studies indicate that ASCs could promote wound healing and reverse diabetes. However, whether ASCs from diabetic donors retain their therapeutic functions and the mechanisms of how ASCs contribute to wound healing remain largely unknown. In this study, we explored the cutaneous wound healing ability of ASCs collected from C57BL/6 mice that had been rendered diabetic with streptozotocin (STZ).

Methods: ASCs were harvested from adipose tissues of type 1 diabetic (T1D) or normal C57BL/6 mice. Cell phenotypes were evaluated by flow cytometry analysis, and cell differentiation into adipocytes, chondrocytes, and osteocytes was compared. Secretions of transforming growth factor β (TGF-β1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) by ASCs were assessed by ELISA. Migration and proliferation of fibroblasts co-cultured with T1D ASCs or control ASCs were also compared. The therapeutic effects of T1D and control ASCs in promoting wound closure were measured in vivo in a T1D wound mouse model. Granulation tissues were collected and stained with H&E at 14th day. CD34 and collagen I were detected by immunohistochemistry. Expressions of IL-6, α-SMA, CD31, collagen I, and collagen III were quantified by real-time PCR. GFP-expressing ASCs were used to trace in vivo cell differentiation.

Results: T1D ASCs and control ASCs showed similar expression of cell surface markers (CD29, CD34, CD105) and proliferation pattern. They can both differentiate into different cell types. T1D ASCs secreted similar amounts of VEGF and bFGF, but less TGF-β compared with control ASCs. Like control ASCs, T1D ASCs promoted the proliferation and migration of skin fibroblast cells. When injected in cutaneous wound of T1D mice, T1D ASCs increased wound closure and hair follicle regeneration at a comparable extent as ASCs. Mice receiving T1D ASCs or ASCs exhibited significantly higher expressions of collagen I, collagen III, and CD31 and reduced expression of IL-6 in wound tissues. Immunohistochemistry staining showed increased angiogenesis in mice receiving ASCs as was evident by increased CD34⁺ cells and collagen I staining. GFP⁺ ASCs injection showed that ASCs differentiated into fibroblasts and endothelial cells in vivo.

Conclusions: Our results suggest that T1D ASCs could accelerate cutaneous wound healing. Mechanisms may include increasing fibroblast growth and migration, skin angiogenesis, and differentiation into fibroblasts and endothelial cells. This study provides evidence that diabetic ASCs may be used as a therapeutic option in cutaneous wound healing in diabetic recipients.

Keywords: Adipose stem cells; Cutaneous wound healing; Diabetes.

Fig. 1

Characterization of T1D ASCs. Blood glucose levels (A) and body weights (B) of control and STZ-treated mice at different days after treatments. C Representative cell morphology of T1D ASCs (a) and ASCs (b) at passage 3 (bar = 200 μm). D Growth curves of T1D ASCs and ASCs at passage 3. E Cell surface antigen expression in T1D ASCs and ASCs analyzed by flow cytometry. F Concentration of bFGF (a), VEGF (b), and TGF-β1 (c) secreted by T1D ASCs and ASCs. *p < 0.05, Student’s t test

Fig. 2

Multiple differentiation potential of ASCs harvested from control of T1D donors. A Representative micrographs of ASC-derived adipocytes identified by oil red staining, osteocytes by alizarin red staining, and chondrocytes by toluidine blue staining. Scale bar = 100 μm. B Expression of Lpl, Glut-4, PPARγ, FABP-4, and C/EBPα mRNA level in adipocytes derived from control or T1D ASCs. C Expression of Runx2, Alp, and Opn mRNA level in osteocytes from control or T1D ASCs. D Expression of Sox9, Has2, and Acan mRNA level when control and T1D ASCs were differentiated into chondrocytes. Data are from at least three individual experiments.*p < 0.05, **p < 0.01, Student’s t test

Fig. 3

T1D ASCs promote wound healing in T1D mouse wound. A Representative images of excisional wound model using a donut-shaped splint to keep the wounds open. B Photomicrographs of wounds treated with PBS, ASCs, or T1D ASCs at 0, 3, 7, and 14 days. Control normal mice wounds were treated with PBS; T1D mice wounds were treated with PBS, T1D ASCs, or ASCs respectively. C The wound closure rates of T1D mice treated with PBS, ASCs, or T1D ASCs. PBS, control normal mice were treated with PBS. Date expressed as the mean ± SD; n = 8–10 per group; *p < 0.05, **p < 0.01 vs. PBS group, ANOVA test

Fig. 4

T1D ASCs promote wound vascularity and collagen I expression. H&E staining of wound sections of normal control group treated with PBS (A), T1D mice wounds treated with PBS (B), ASCs (C), or T1D ASCs (D) 14 days post-wounding. CD34 (red arrows) immunohistochemistry staining of wound sections of normal control group treated with PBS (E); T1D mice wounds treated with PBS (F), ASCs (G), or T1D ASCs (H) 14 days post-wounding. Collagen I protein immunohistochemistry staining of wound sections of normal control group treated with PBS (I); T1D mice wounds treated with PBS (J), ASCs (K), or T1D ASCs (L) 14 days post-wounding. Bars = 100 μm. M Gene expression analysis by qRT-PCR using actin as the endogenous control. *p < 0.05, **p < 0.01, ***p < 0.001 vs. PBS controls. Triplicate results were averaged and mean ± SD is shown, ANOVA test

Fig. 5

T1D ASCs enhance the migration, proliferation, and α-SMA and collagen I/III expression of fibroblasts in vitro. A Representative images of “wounds” in the scratch assay before and 24 h after co-culture with T1D or control ASCs. B Migration rates of fibroblasts. C Representative images of mice fibroblasts before and 5 days after co-cultured with T1D or control ASCs. D Cell numbers of fibroblasts co-cultured with T1D ASCs or ASCs for 5 days. E Gene expression of α-SMA, collagen I, and collagen III. Bars = 200 μm. Each bar represents the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, Student’s t test

Fig. 6

Differentiation of ASCs in mouse wound area. A–C Representative images show injected GFP-positive ASCs in mice wound. D Positive identification of injected ASCs in wound dermal tissue is indicated by GFP staining (green). E Red stain indicates CD34. F Overlay demonstrates co-localization of GFP and CD34 signal (white arrows). G Positive identification of injected ASCs in wound dermal tissue indicated by the GFP staining (green). H Red stain indicates vimentin. I Overlay demonstrates co-localization of GFP and vimentin signal (white arrows). Bar = 100 μm