Kraskiewicz, Honorata (Laboratory of Biology of Stem and Neoplastic Cells, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences)


"Introduction: Mesenchymal stem cells (MSCs) can improve chronic wound healing, and recently it was suggested that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells. However, MSCs still are not the standard of care in wound healing due to several limitations such as patient-specific difference in MSCs, poor survival of transplanted cells, and technical considerations such as standardization of isolation, characterization, expansion and delivery. To overcome difficulties related to the translation of cell therapy into clinic we propose an innovative, standardized skin treatment option, a conditioned medium (CM) from recently established by our research group Human Adipose Tissue Mesenchymal Stem Cell (HATMSC) line (1). In this study we evaluate the biological activity of HATMSCs-produced factors following incorporation into collagen hydrogel as a potential treatment for chronic wounds (2).
Methodology: Biocompatibility and biological activity of hydrogel-released HATMSC2-origin bioactive factors were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) using ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR.
Results: The results showed significant 3-fold increase in metabolic activity of fibroblast (p < 0.001) and 2-flold of endothelial cells and keratinocytes (p < 0.01) following 3 day culture in the presence of HATMSC2-origin growth factors loaded hydrogels compared to unloaded gels. The supplementation of hydrogel with HATMSCs supernatant improves the tube formation process in angiogenic test in vitro. Moreover, we have confirmed the expression of pro-angiogenic miRNA (miR210, miR126 and miR296) in the HATMSC2 secretome indicating that supernatant can support proangiogenic processes in tissue regeneration. Hydrogel release study showed that there is a substantial difference in the levels of IL-8 and MCP-1 between unloaded hydrogels and supernatant–loaded hydrogels. For example, on day 3 for MSU-1.1 cells the levels of MCP-1 and IL-8 in hydrogel treated groups were 0.4 pg/mL and 156.2 pg/mL, while in cells treated with supernatant-loaded hydrogel the these level were much higher, 45.5 pg/mL and 1723.7 pg/mL, respectively. This suggest that the hydrogel used in this study is an appropriate carrier of HATMSC-originated trophic factors.
Conclusions: This study demonstrated that the therapeutic effect of the HATMSC2-produced bioactive factors (IL-8, MCP-1, proangiogenic miRNAs) is maintained following incorporation into collagen- hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. These results suggest the possible beneficial effect of dressing, composed of hydrogel loaded with HATMSCs bioactive factors, on the wound healing process in the context of restoration of proper angiogenesis.
1. Kraskiewicz, H. et al., Stem Cell Res. Ther. 11, (1), 29 (2020).
2. Kraskiewicz, H. et al., Int. J. Mol. Sci. Nov 12;22(22):12241 (2021)."


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