Browsing by Author "Seboko, Ascentia Mathapelo"

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    miRNA signature of healthy vs impaired mesenchymal stem cells as a biomarker for autologous stem cell therapy
    (Stellenbosch : Stellenbosch University, 2021-12) Seboko, Ascentia Mathapelo; Van de Vyver, Mari; Scholefield, Janine; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
    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.
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    Stem cell impairment associated with type 2 diabetes mellitus : investigating the effects of obesity-associated inflammation on mesenchymal stem cell function
    (Stellenbosch : Stellenbosch University, 2017-12) Seboko, Ascentia Mathapelo; Van de Vyver, Mari; Ferris, William; Lopes, John; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division Medical Physiology
    ENGLISH ABSTRACT : Background: South Africa has the highest prevalence of obesity in sub-Saharan Africa, particularly in Black women. This population is thus at a higher risk of developing obesity-associated type 2 diabetes mellitus (T2DM) and its associated co-morbidities such as non-healing wounds. Adipose tissue-derived mesenchymal stem cells (ADSCs) have been widely utilized in the treatment of chronic wounds, however, autologous stem cell therapies using endogenous ADSCs from T2DM patients have proven unsuccessful. Metabolic disorders such as T2DM are thus thought to compromise the functional capacity of mesenchymal stem cells. The underlying molecular mechanisms that contribute to the functional decline of mesenchymal stem cells is still unclear and it is not yet known at which stage of disease progression ADSCs become compromised. In this research study, it was hypothesised that the progressive worsening of chronic systemic inflammation during disease progression from obesity towards T2DM, contributes to the decline of ADSCs’ multifunctional properties. Methods: A total of forty-seven (n=47) reproductive aged (18-45 years) Black Xhosa women from peri-urban areas surrounding the Tygerberg hospital, were included in this study. Participants were subdivided into: a) healthy lean (n=10) (BMI ≤ 25 kg/m2); b) healthy obese (n=11) (BMI ≥ 30 kg/m2); c) obese metabolic syndrome (n=19) and d) previously diagnosed T2DM (n=7) groups. Health, lifestyle and dietary questionnaires were completed by participants. Anthropometric measurements and a dual energy x-ray absorptiometry (DXA) scan were performed in order to assess body composition. Blood samples were collected in order to assess each participant’s metabolic- (fasting blood glucose, total cholesterol, HDL, LDL, triglycerides) and inflammatory (CRP, SDF-1α, IL-6, IL-8, IL-10, TNF-α, IFN-γ) profiles. To establish whether a relationship exists between systemic inflammation at different stages of disease progression and stem cell impairment, in vitro experiments were performed in which ADSCs (Poietics cell line) were exposed to participant-derived serum for a period of 48h. Changes in cellular viability (MTT-based assay), proliferation (BrdU) and migration (wound healing assay) were assessed using standard tissue culture techniques. Results: Systemic inflammation was evident in the healthy obese (CRP 29.8 ± 8 pg/mL) and obese metabolic syndrome (CRP 50.8 ± 24 pg/mL) participants. Additionally, circulating levels of the anti-inflammatory cytokine IL-10, were significantly reduced in T2DM participants (0.42 ± 0.63 pg/mL) (p < 0.05) compared to the healthy lean and obese groups. Due to individual variability within the different groups, there were no significant differences observed in circulating levels of IL-6, IL-8, TNF-α and IFN-γ.However, there was a significant correlation between circulating levels of IL-6 and the proliferation of ADSCs, particularly in the healthy lean (p < 0.01) and metabolic syndrome (p < 0.01) groups. Furthermore, serum levels of IL-8 significantly correlated with the migration of ADSCs (p < 0.01). Healthy lean participant serum had a mitogenic effect on ADSCs, which was not observed in the obese groups. Conclusion: This study demonstrated for the first time, that the disruption in the delicate systemic inflammatory balance as a result of obesity, regardless of metabolic syndrome, may have an adverse effect on the functional capacity of ADSCs.