Can supernatant from immortalized adipose tissue MSC replace cell therapy? An in vitro study in chronic wounds model

Mesenchymal stem cells (MSCs) secrete a cocktail of growth factors and cytokines, which could promote tissue regeneration and wound healing. Therefore, in clinical practice, post-culture MSC supernatant treatment could be a more attractive alternative to autologous stem cell transplantation. In this study, we compared the regenerative properties of supernatants harvested from four newly established human adipose tissue mesenchymal stem cell lines (HATMSCs) derived from chronic wound patients or healthy donors.

HATMSC supernatants were produced in a serum-free medium under hypoxia and their content was analyzed by a human angiogenesis antibody array. The regenerative effect of HATMSCs supernatants was investigated in an in vitro model of chronic wound, where cells originating from human skin, such as microvascular endothelial cells (HSkMEC.2), keratinocytes (HaCaT), and fibroblasts (MSU-1.1), were cultured in serum-free and oxygen-reduced conditions. The effect of supernatant treatment was evaluated using an MTT assay and light microscopy. In addition, fibroblasts and HATMSCs were labeled with PKH67 and PKH26 dye, respectively, and the effect of supernatant treatment was compared to that obtained when fibroblasts and HATMSCs were co-cultured, using flow cytometry and fluorescent microscopy.

A wide panel of angiogenesis-associated cytokines such as angiogenin, growth-regulated oncogene (GRO), interleukin-6 and 8 (IL-6, IL-8), vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF-1), and matrix metalloproteinase (MMP) were found in all tested HATMSCs supernatants. Moreover, supernatant treatment significantly enhanced the survival of fibroblasts, endothelial cells, and keratinocytes in our chronic wound model in vitro. Importantly, we have shown that in in vitro settings, HATMSC supernatant treatment results in superior fibroblast proliferation than in the case of co-culture with HATMSCs.

Our results suggest that therapy based on bioactive factors released by the immortalized atMSC into supernatant has important effect on skin-derived cell proliferation and might preclude the need for a more expensive and difficult cell therapy approach to improve chronic wound healing.