miRNA signature of healthy vs impaired mesenchymal stem cells as a biomarker for autologous stem cell therapy
| dc.contributor.advisor | Van de Vyver, Mari | en_ZA |
| dc.contributor.advisor | Scholefield, Janine | en_ZA |
| dc.contributor.author | Seboko, Ascentia Mathapelo | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. | en_ZA |
| dc.date.accessioned | 2021-09-29T09:40:43Z | en_ZA |
| dc.date.accessioned | 2022-02-22T10:17:04Z | en_ZA |
| dc.date.available | 2021-09-29T09:40:43Z | en_ZA |
| dc.date.issued | 2021-12 | en_ZA |
| dc.description | Thesis (PhD)--Stellenbosch University, 2021. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: Background: Globally, approximately 463 million adults are living with diabetes. This growing prevalence of diabetes places an increased burden on the health systems of low-to-middle income countries such as South Africa. The pathological microenvironment associated with diabetes leads to severe co-morbidities that include chronic foot ulcers. Non-healing ulcers affect approximately 15% of diabetic patients. Novel approaches in the treatment of chronic ulcers make use of mesenchymal stem cell (MSC) therapies. However, autologous MSCs from diabetic patients are less successful than allogeneic donor MSCs. This is due to the impairment of MSCs as result of the pathological diabetic environment. MicroRNAs (miRNAs) have gained immense popularity as biomarkers of disease and have been associated with the development of metabolic diseases. This study hypothesised that MSCs with compromised therapeutic potential, have a unique miRNA signature that could potentially be used as a biomarker to predict if a specific patient would be a good candidate for autologous MSC therapy. Methods: Three models were utilised: 1) Healthy control and impaired diabetic bone marrow MSCs (BMSC) isolated from wildtype (C57BL6/J) and obese diabetic B6.Cg-Lepob/J (ob/ob) mice. BMSC function was assessed by comparing the a) ex vivo growth rate, b) secreted and intracellular IL6 (ELISA), and c) osteogenic (Alizarin Red S staining) and adipogenic (Oil Red O staining) differentiation capacity. Broad miRNA profiling (> 600 miRNAs) was performed using the NanoString mouse v1.5 miRNA Expression Assay (BMSCs) and RT-qPCR (whole blood). 2) Synthetic miRNA mimics were transfected into C3H10T1/2 cells with subsequent assessment of cellular proliferation and migration capacity. 3) The expression of miRNAs of interest was assessed in the serum and PBMCs of overweight/obese (OW/OB) (n=14) and diabetic (n=16) patients who were subdivided into: a) predicted good healers and b) predicted poor healers based on serum MMP9 levels. Health, lifestyle, and dietary questionnaires were completed by participants and blood samples collected to determine their metabolic (blood glucose, HbA1c) profile. Results: The miRNA profile of BMSCs was affected by both sex (male vs female) and metabolic phenotype (healthy vs impaired). MSC impairment was prominent in male ob/ob mice and coincided with the upregulation of 19 miRs and downregulation of 3 miRs compared to their healthy control counterparts (p < 0.05). Five miRNAs of interest (miR-142-5p, miR-200b, miR-202-5p, miR-384-3p and miR-466g) emerged as having a potential role in the functional capacity of BMSCs. C3H10T1/2 cells transfected with miR-202-5p had an increased proliferation rate (p < 0.01) compared to non-transfected cells. All 5 miRNAs of interest reduced the rate of wound closure in vitro following transfection (p < 0.05) compared to non-transfected cells. This was due to more random migration patterns and a reduction in directionality. No association could be observed between the metabolic profile, predicted healing capacity and miRNA expression in the PBMCs of participants. Conclusion: The data confirmed that a unique miRNA signature exists between healthy vs impaired BMSCs. Numerous confounding factors, including sex-specific differences, make the use of these miRNAs as biomarkers unlikely. This study did, however, confirm a role of miRNAs in BMSCs function. | en_ZA |
| dc.description.abstract | AFRIKAANS OPSOMMING: Agtergrond: Wêreldwyd leef ongeveer 463 miljoen volwassenes met diabetes. Dié groeiende voorkoms van diabetes plaas groot druk op gesondheidsorg stelsels in lae-tot-middel inkomste lande soos Suid Afrika. Diabetes skep ‘n patologiese mikro-omgewing in die liggaam wat lei tot die ontwikkeling van ernstige komorbiditeite. ‘n Voorbeeld hiervan is diabetiese voetsere. Ongeveer 15% van diabetes lyers ontwikkel voetsere. Nuwe benaderings vir die behandeling van kroniese sere maak gebruik van mesenchimale stam sel (MSS) terapie. Maar, outologiese MSS verkry van diabetiese pasiënte is minder suksesvol as geskenkte allogeniese MSS, as gevolg van blootstelling aan die diabetiese patologiese mikro-omgewing. Mikro-RNS (miRNS) het grootliks bekendheid verwerf om te dien as biomerker vir siektetoestande en was al geassosieer met die ontwikkeling van metaboliese siektes. Hierdie studie se hipotese lei, MSS met benadeelde terapeutiese potensiaal het ‘n unieke miRNS kenmerk wat moontlik as biomerker gebruik kan word om te voorspel of ‘n spesifieke pasiënt ‘n goeie kandidaat sal wees vir outologiese MSS behandeling. Metodes: Drie modelle was gebruik om die hipotese te ondersoek: 1) Gesonde kontrole en benadeelde diabetiese beenmurg MSS (BMSS) was geïsoleer van wilde tipe (C57BL6/J) en vetsugtige diabetiese B6.Cg-Lepob/J (ob/ob) muise. BMSS funksie was geassesseer deur a) ex vivo groeitempo, b) afgeskeide en intrasellulêre IL6 (ELISA) vlakke, en c) osteogenese (Alizarin Red S kleurstof) en adipogenese (Oil Red O kleurstof) differensiasie kapasiteit, te vergelyk. Breë miRNS profilering (> 600 miRNS) was uitgevoer deur NanoString muis v1.5 miRNS uitdrukking toets (BMSS) en RT-qPCR (heel bloed) te gebruik. 2) Sintetiese miRNS nabootsers was getransfekteer binne-in C3H10T1/2 selle waarop assessering van sellulêre proliferasie en migrasie kapasiteit gevolg het. 3) Die uitdrukking van die miRNS van belang in die serum en PBMS van oorgewig/vetsugtige (OW/OB) (n=14) en diabetiese (n=16) pasiënte was ondersoek. Hierdie pasiënte was nog verder onderverdeel in kategorieë: a) voorspelde goeie genesers en b) voorspelde swak genesers, gebaseer op serum MMP9 vlakke. Vraelyste oor gesondheid, leefstyl en dieet was deur deelnemers voltooi en bloed monsters was geneem om hul metaboliese (bloedglukosevlakke, HbA1c) profiel te bepaal. Resultate: Die miRNS profiel van BMSS was deur beide geslag (manlik vs. vroulik) en metaboliese fenotipe (gesond vs. benadeeld) geaffekteer. Benadeling van MSS was prominent in manlike ob/ob muise en het gesamentlik met 19 op gereguleerde miRs en 3 af gereguleerde miRs gepaard/ geval, in vergelyking met die/hul gesonde kontrole eweknieë (p < 0.05). Vyf miRNS van belang (miR-142-5p, miR-200b, miR-202-5p, miR-384-3p and miR-466g) het ‘n potensiële rol in die funksionele kapasiteit van BMSS getoon. Verhoogde proliferasie tempo (p < 0.01) was waargeneem in miR-202-5p getransfekteerde C3H10T1/2 selle in vergelyking met selle wat nie getransfekteer was nie. In vergelyking met nie-getransfekteerde selle, het al vyf miRNS van belang die tempo van wond sluiting in vitro verlaag na transfektering (p < 0.05). Lukrake migrasie patrone en die vermindering in rigtinggewendheid was die redes daarvoor. Geen assosiasie was tussen die metaboliese profiel, voorspelde genesing kapasiteit en miRNS uitdrukkings in PBMS van deelnemers waargeneem nie. Gevolgtrekking: Die data bevestig dat ‘n unieke miRNS kenmerk tussen gesonde vs. benadeelde BMSS bestaan. Verskeie faktore veroorsaak egter verwarring in die gebruik van miRNS as biomerker. Geslag spesifieke verskille maak die gebruik van miRNS as biomerker onwaarskynlik. Hierdie studie het egter bevestig dat miRNS ‘n rol speel in BMSS funksie. | en_ZA |
| dc.description.version | Doctorate | en_ZA |
| dc.embargo.terms | 2022-03-29 | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/124217 | en_ZA |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | MicroRNA | en_ZA |
| dc.subject | Mesenchymal stem cells | en_ZA |
| dc.subject | Biochemical markers | en_ZA |
| dc.subject | Stem cell therapy | en_ZA |
| dc.subject | Diabetes | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | miRNA signature of healthy vs impaired mesenchymal stem cells as a biomarker for autologous stem cell therapy | en_ZA |
| dc.type | Thesis | en_ZA |