The skin regeneration potential of a pro-angiogenic secretome from human skin-derived multipotent stromal cells

Abstract

Multipotent stromal cells stimulate skin regeneration after acute or chronic injuries. However, many stem cell therapy protocols are limited by the elevated number of cells required and poor cell survival after transplantation. Considering that the beneficial effects of multipotent stromal cells on wound healing are typically mediated by paracrine mechanisms, we examined whether the conditioned medium from skin-derived multipotent stromal cells would be beneficial for restoring the skin structure of mice after wounding. A proteomic characterization of skin-derived multipotent stromal cell-conditioned medium was performed, and the angiogenic function of this secretome was investigated in vitro using an endothelial cell tube formation assay. We then applied the skin-derived multipotent stromal cell-conditioned medium directly to full-thickness excisional wounds or embedded it into carrageenan or poly(vinyl alcohol) hydrogels to monitor tissue regeneration in mice. Biological processes related to wound healing and angiogenesis were highlighted by the analysis of the skin-derived multipotent stromal cell secretome, and a pro-angiogenic capacity for promoting tubule-like structures was first confirmed in vitro. Skin wounds treated with skin-derived multipotent stromal cell-conditioned medium also displayed increased angiogenesis, independently of the association of the conditioned medium with hydrogels. However, improvements in wound closure and epidermis or decreased inflammatory cell presence were not observed. Hence, the use of the secretome obtained from human skin-derived multipotent stromal cells may be a potential strategy to aid the natural skin repair of full-thickness lesions mainly based on its pro-angiogenic properties.

Keywords: Secretome; angiogenesis; skin-derived multipotent stromal cell; wound healing.

Figure 1.

Gene ontology (GO) analysis of (a) cellular components and (b) biological processes based on the proteins identified in the SD-MSC secretome. A REVIGO scatterplot shows representative clusters of the GO analysis performed with g:Profiler. The log10 p-value of each GO term after REVIGO analysis is plotted on the x-axis, while the frequency of GO term in the GO Annotation Database is displayed on the y-axis (log size). Bubble colors indicate log10 p-value (from smaller p-values on blue color to highest p-values on red color).

Figure 2.

CM derived from SD-MSCs induces tube formation in HUVEC cultures. (a) Representative images of the tubule-like structures that appeared in each condition (200 µm scale bar). Analysis of the number of (b) meshes and (c) nodes formed after 12 h for the CM treatment (CM), positive controls (Pos; EBM-2 fully supplemented, as described in M&M) or negative controls (Neg; EBM-2 without any supplementation). ***p < 0.001; ****p < 0.0001.

Figure 3.

Histomorphological analysis of reepidermalization during skin healing in mice: untreated control (CT) and effects of PVA, carrageenan (CG), conditioned medium (CM), or associations between hydrogels and CM. Wound closure was evaluated by measuring the wound area on the (a) 3rd and (b) 14th day post-treatment. Inflammatory infiltrates were estimated by evaluating the leukocyte density in the lesions on the (c) 3rd day post-treatment (n = 5 animals for each condition). The (d) thickness of granulation tissue and the (e) thickness and (f) uniformity of the epidermis were assessed on the 14th day post-treatment. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4.

In vivo pro-angiogenic effect of the CM derived from SD-MSCs. Representative H&E-stained images of the granulation tissue showing blood vessels (black arrows—capillaries containing a lumen with red blood cells) in the (a) untreated control group (CT) and treated groups: (b) CG, (c) CG + CM, (d) PVA, (e) PVA + CM, and (f) CM. Scale bars = 100 µm. (g) Graph representing the mean values obtained by counting the blood vessels/wound. *p < 0.05; **p < 0.01; ***p < 0.001.