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
| dc.contributor.advisor | Van de Vyver, Mari | en_ZA |
| dc.contributor.author | Mehrbani Azar, Yashar | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: General Internal Medicine. | en_ZA |
| dc.date.accessioned | 2019-11-05T14:16:57Z | |
| dc.date.accessioned | 2019-12-11T06:39:44Z | |
| dc.date.available | 2020-07-21T03:00:15Z | |
| dc.date.issued | 2019-12 | |
| dc.description | Thesis (PhD)--Stellenbosch University, 2019. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Obesiteitsverwante tipe 2-diabetes mellitus (T2DM) is ’n komplekse siekte wat verskeie komorbiditeite veroorsaak, waaronder wonde wat sukkel om te genees. Sowat 15-25% van alle diabetespasiënte het las van sulke wonde, en bykans 50% van alle diabetesverwante hospitaalopnames kan hieraan toegeskryf word. Mesenchimale stamsel- (MSS-)terapie is ’n belowende behandelingsmoontlikheid omdat MSS’e die kliniese status van ’n wond kan ‘aanvoel’ en die mikro-omgewing deur parakriene seine kan herstel om regenerasie te bevorder. Die patologiese aard van die nis-mikro-omgewing beperk egter die gebruik van outoloë MSS-terapie by diabetespasiënte omdat verlengde blootstelling van endogene MSS’e aan die diabetiese omgewing in vivo die selle se vermoë aantas om op omgewingseine te reageer. Daarom berus die verdere ontwikkeling van outoloë selterapie op (a) ’n beter begrip van hoe die patogenese van T2DM die multifunksionele eienskappe van MSS’e beïnvloed, en (b) die ontwikkeling van nuwe strategieë om die funksie van hierdie aangetaste MSS’e te herstel voordat dit vir oorplanting gebruik word. Hierdie studie ondersoek of ex vivo-antioksidant- [N-asetielsisteïen (7.5 mM NAC) en askorbiensuur2-fosfaat-behandeling (0.6 mM AAP)] die parakriene responsiwiteit, groeitempo, migrasievermoë en lewensvatbaarheid van aangetaste diabetiese MSS’e kan herstel, en, indien wel, of hierdie herstelde toestand in die teenwoordigheid van diabetiese wondvog (DWV) gehandhaaf kan word. Beenmurgafkomstige MSS’e is op die ouderdom van agt weke geïsoleer by wildetipe-C57BL/6Jmuise (gesonde kontrole: MSCWT) (n = 24) en vetsugtige diabetiese B6.Cg-Lepob/J-muise (aangetas/disfunksioneel: MSCob) (n = 24). Die ex vivo-behandelingsgroepe (MSCWT teenoor MSCo b) het bestaan uit (a) geen behandeling (basislynfenotipe), (b) DWV-gestimuleer (basislynreaksie), (c) vooraf met antioksidante gekondisioneer (voorafgekondisioneerde fenotipe), en (d) vooraf met antioksidante gekondisioneer, met daaropvolgende DWV-stimulering (voorafgekondisioneerde reaksie). DWV vir hierdie ex vivo-proefnemings is oor ’n tydperk van 28 dae bekom uit bilaterale, dorsale eksisiewonde van volle dikte wat op diabetiese muise (B6.Cg-Lepob/J) (n = 7) aangebring is. Die optimale konsentrasie antioksidante is met behulp van ’n dosisreaksieproefneming met geïmmortaliseerde C3H10/T1/2-selle bepaal. Hierdie studie toon dat die uitbreiding van primêre MSS’e (MSCWT en MSCob) in die teenwoordigheid van antioksidante die ex vivo-lewensvatbaarheid van selle verbeter en ’n beskermende uitwerking teen die toksisiteit van DWV het. Daarbenewens is die parakriene responsiwiteit van MSCWT en MSCob (met en sonder voorafkondisionering met antioksidante) op molekulêre vlak (mRNAuitdrukking van 84 sitokiene en reseptors, qPCR-mikrorangskikking) sowel as proteïenvlak bereken (Luminex-essai, 23-pleks, kraalrangskikking). Op die basislyn is ooruitdrukking van 31 gene (meer as tweevoudig) en onderuitdrukking van 39 gene (meer as tweevoudig) by MSCob teenoor MSCWT opgemerk. In gekondisioneerde media is beduidende basislynverskille (p < 0.05) opgemerk vir twee pro-inflammatoriese sitokiene (TNFα en IFNγ), vier chemokiene (KC, G-CSF, Eotaxin en MCP1) en een anti-inflammatoriese sitokien (IL10). Ná DWV-stimulering is beduidende verskille (p < 0.05) opgemerk in die uitskeiding van twee chemokiene (GM-CSF en Eotaxin), drie pro-inflammatoriese sitokiene (TNFα, IFNγ en IL9) en vier anti-inflammatoriese sitokiene (IL10, IL4, IL13 en IL3). Voorafkondisionering met antioksidante het die waargenome TNFα-oorreaksie by MSCob aansienlik gedemp en die uitskeiding van IL10 verbeter. Dít dui daarop dat gekombineerde ex vivo-behandeling van outoloë MSS’e met NAC en AAP moontlik ’n doeltreffende strategie kan wees om die parakriene funksie van aangetaste MSS’e voor oorplanting te herstel. Ondanks beter lewensvatbaarheid en ’n herstelde parakriene reaksie, kon voorafkondisionering met antioksidante egter nie die proliferasie- en migrasievermoë van ernstig aangetaste diabetiese MSS’e red nie. | af_ZA |
| dc.description.version | Doctorate | |
| dc.embargo.terms | 2020-05-06 | |
| dc.format.extent | 149 pages | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/106940 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch -- Stellenbosch University. | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Regenerative Medicine | en_ZA |
| dc.subject | Stem cells | en_ZA |
| dc.subject | Diabetics -- Care | en_ZA |
| dc.subject | Wound healing | en_ZA |
| dc.title | 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 | en_ZA |
| dc.type | Thesis | en_ZA |