Antioxidant Preconditioning Improves the Paracrine Responsiveness of Mouse Bone Marrow Mesenchymal Stem Cells to Diabetic Wound Fluid

Abstract

Mesenchymal stem cells (MSCs) are a promising therapeutic tool for the treatment of nonhealing diabetic wounds. The pathological nature of the niche microenvironment limits the use of autologous cell therapy in diabetic patients. Prolonged exposure of endogenous MSCs to a pathological microenvironment in vivo reduces their ability to respond to environmental cues. This study investigated the effectiveness of ex vivo antioxidant treatment [N-acetylcysteine (7.5 mM NAC) and Ascorbic acid 2-phosphate (0.6 mM AAP)] to restore the paracrine function of diabetic MSCs. Healthy control [bone marrow stem cells derived from wild-type mice (SC^WT)] (source: wild-type C57BL/6J mice) (n = 12) and impaired/dysfunctional [bone marrow stem cells derived from ob/ob mice (SC^ob)] (source: obese diabetic, B6.Cg-Lep^ob/J mice) (n = 12) MSCs were isolated. Ex vivo treatment groups (SC^WT vs. SC^ob) were as follows: (1) no treatment (baseline phenotype), (2) stimulated with diabetic wound fluid (DWF) (baseline response), (3) antioxidant preconditioning (preconditioned phenotype), and (4) antioxidant preconditioned with subsequent stimulation with DWF (preconditioned response). The paracrine responsiveness on both the molecular (mRNA expression of 80 cytokines and receptors, quantitative polymerase chain reaction microarray) and protein (23-plex bead-array Luminex assay) level was assessed. At baseline, 31 genes were overexpressed (> × 2-fold) and 39 genes were underexpressed (> × 2-fold) in SC^ob versus SC^WT. In conditioned media, significant differences (P < 0.05) were detected at baseline for two proinflammatory cytokines [tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ)], four chemokines [keratinocyte chemoattractant (KC), granulocyte colony-stimulating factor (GCSF), Eotaxin, and macrophage chemoattractant protein (MCP1)], and one anti-inflammatory cytokine [interleukin 10 (IL10)]. Following stimulation with DWF, significant differences (P < 0.05) were detected in the secretion of two chemokines [granulocyte macrophage colony-stimulating factor (GMCSF) and Eotaxin], three proinflammatory cytokines (TNFα, IFNγ, and IL9), and four anti-inflammatory cytokines (IL10, IL4, IL13, and IL3). Antioxidant preconditioning significantly dampened the excessive TNFα response observed in SC^ob and improved the secretion of IL10. Taken together these data suggest that the combined ex vivo treatment of autologous stem cells with NAC and AAP could potentially be an effective strategy to restore the paracrine function of impaired diabetic MSCs before transplantation.