Deciphering the role of mesenchymal stem cells (MSCs) in cutaneous wound healing

Abstract

MSCs (N=9) were isolated from the bone marrow of patients who had received hip replacement therapy. MSC-serum free conditioned media (MSC-CM) were collected at different time points during MSC passage and analysed for protein content using enzyme linked immunosorbent assay (ELISA). The effect of MSC-CM was tested on migration and proliferation of human skin cells (HaCat cell line, primary fibroblast and primary keratinocytes) (N=4) using a 2D scratch assay and tetrazolium salt proliferation assay respectively. For primary keratinocyte experiments, MSC-CM were collected in keratinocyte growth media containing low calcium levels (0.04 mM/L) (LC-CM). The effect of LC-CM was tested on migration and proliferation of primary keratinocytes during different testing conditions; normoxia (N=4), hypoxia (N=4), blocking of stromal derived factor-1α (SDF-1α) (N=4) and inhibition of proliferation (N=4). MSCs were tested for their ability to differentiate into epidermal like cells (ELCs) using both 2D and 3D cultures. A human 3D skin model was developed for wound healing and micro RNA profiling studies. All MSCs met the criteria stipulated by the International Society for Cellular Therapy (ISCT). MSC-CM contained growth factors e.g. keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet derived growth factor-AB (PDGF-AB), transforming growth factor-β1 (TGF-β), macrophage stimulating protein1 (MSP-1) and SDF-1α and RT qPCR analysis demonstrated receptors of these growth factors e.g. FGFR2, c-MET, PDGFRA, TGFβ-R1, RON and CXCR4 respectively on both scratched and non-scratched primary keratinocytes. The main findings from this study showed that MSCs could differentiate into ELCs and MSC-CM were shown to have a positive effect on migration and proliferation of skin cells in 2D and 3D culture. MSC secretions collected at early time points were more effective on cell migration than those collected at later time points during MSC expansion. 2D and 3D studies also showed that cell migration was the first and the major mechanism evoked by MSCCM followed by proliferation and differentiation. Additionally, the 3D skin model developed in this study could be used as a skin replica for wound healing studies at the cellular and molecular level including the use of microRNA profiling. These microRNAs were regulated at different time points during the wound repair suggesting their participation in the different phases of the healing process. In conclusion, MSCs play a multifunctional role in the cellular and molecular mechanisms of the healing process and enhance the healing process via two mechanisms; cell mediated repair by differentiation into ELCs and secretory mediated repair by cytokines.