Browsing by Author "Mehrbani Azar, Yashar"

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    An investigation into the effects of ex vivo antioxidant treatment on the regenerative potential of mesenchymal stem cells following prolonged exposure to a pathological microenvironment associated with diabetes mellitus in vivo
    (Stellenbosch -- Stellenbosch University., 2019-12) Mehrbani Azar, Yashar; Van de Vyver, Mari; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: General Internal Medicine.
    ENGLISH ABSTRACT: Obesity-associated type-2 diabetes mellitus (T2DM) is a multifactorial disease that causes severe comorbidities such as non-healing wounds. Non-healing diabetic wounds affect 15-25% of all diabetic patients and are responsible for nearly 50% of all diabetes-related hospital admissions. Mesenchymal stem cell (MSC) therapy is a promising therapeutic option, as MSCs can ‘sense’ the clinical status of the wound and restore the micro-environment through paracrine signalling to promote regeneration. However, the pathological nature of the niche micro-environment does limit the use of autologous MSC therapy in diabetic patients, since prolonged exposure of endogenous MSCs to the diabetic environment in vivo reduces their ability to respond to environmental cues. Thus, the advancement of autologous cell therapy depends on (a) a better understanding of how the pathogenesis of T2DM affects the multifunctional properties of MSCs, and (b) the development of new strategies to restore the function of these impaired MSCs before they are used for transplantation. This study investigated whether ex vivo antioxidant [N-acetylcysteine (7.5 mM NAC) and ascorbic acid 2-phosphate (0.6 mM AAP)] treatment could restore the paracrine responsiveness, growth rate, migration ability and viability of impaired diabetic MSCs, and, if so, whether this restored state could be maintained in the presence of diabetic wound fluid (DWF). Bone marrow-derived MSCs were isolated from eight-week-old wild-type C57BL/6J mice (healthy control: MSCWT) (n = 24) and obese diabetic B6.Cg-Lepob/J mice (impaired/dysfunctional: MSCob) (n = 24). The ex vivo treatment groups (MSCWT vs MSCob) were (a) no treatment (baseline phenotype), (b) DWF-stimulated (baseline response), (c) antioxidant-preconditioned (preconditioned phenotype), and (d) antioxidant-preconditioned with subsequent DWF stimulation (preconditioned response). For these ex vivo experiments, DWF was harvested over a period of 28 days from bilateral, dorsal, full-thickness excisional wounds created on obese diabetic mice (B6.Cg-Lepob/J) (n = 7). The optimum concentration of antioxidants was determined using a dose-response experiment in immortalised C3H10/T1/2 cells. This study demonstrated that the expansion of primary MSCs (MSCWT and MSCob) in the presence of antioxidants improved the ex vivo viability of cells and had a protective effect against the toxicity of DWF. The paracrine responsiveness of MSCWT and MSCob (with and without antioxidant preconditioning) was furthermore determined at both the molecular level (mRNA expression of 84 cytokines and receptors, qPCR microarray) and protein level (23-plex bead-array Luminex assay). At baseline, 31 genes were overexpressed (more than twofold) and 39 genes were under-expressed (more than twofold) in MSCob versus MSCWT. In conditioned medium, significant baseline differences (p < 0.05) were detected for two pro-inflammatory cytokines (TNFα and IFNγ), four chemokines (KC, G-CSF, Eotaxin and MCP1) and one anti-inflammatory cytokine (IL10). Following DWF stimulation, significant differences (p < 0.05) were detected in the secretion of two chemokines (GM-CSF and Eotaxin), three pro-inflammatory 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 MSCob and improved the secretion of IL10. This suggests that combined ex vivo treatment of autologous MSCs with NAC and AAP could potentially be an effective strategy to restore the paracrine function of impaired diabetic MSCs before transplantation. However, despite improved viability and a restored paracrine response, antioxidant preconditioning could not rescue the proliferation and migration capacity of severely impaired diabetic MSCs.