Stem cell impairment associated with type 2 diabetes mellitus : investigating the effects of obesity-associated inflammation on mesenchymal stem cell function

Seboko, Ascentia Mathapelo (2017-12)

Thesis (MSc)--Stellenbosch University, 2017.

AFRIKAANSE OPSOMMING : Agtergrond: Suid Afrika het die hoogste voorkoms van vetsug (obesiteit), veral in swart vrouens, van al die Afrika-lande suid van die Sahara. Hierdie populasie het dus ʼn hoë risiko om tipe II (insulien-weerstandige) diabetes mellitus (T2DM) en verwante sekondêre toestande soos kroniese wonde te ontwikkel. Vetweefsel afkomstige mesenchiem stam selle, word dikwels gebruik vir die behandeling van kroniese wonde, maar stamsel-terapie wat gebruik maak van diabetiese pasiënte se eie stam selle (liggaamseie sel terapie), is onsuksesvol. Daar word vermoed dat metaboliese siektes soos T2DM die funksionering van vetweefsel-stamselle (ADSCs) aantas, alhoewel dit nog nie duidelik is op watter stadium van die siekte dit gebeur nie. In hierdie navorsingstudie het ons die hipotese ondersoek dat die kroniese inflammasie wat gepaardgaan met vetsug en T2DM bydra tot die verswakte funksionering van ADSCs. Metodes: Altesaam sewe-en-veertig (n=47) vrouens tussen 18 en 45 jaar oud, woonagtig in die voorstedelike gebiede naby Tygerberg hospitaal, het aan die studie deelgeneem. Die deelnemers is in vier groepe verdeel: a) gesond en skraal (n=10) (liggaamsmassa-indeks (LMI) ≤ 25 kg/m2); b) gesond, nie-diabeties maar vetsugtig (n=11) (LMI ≥ 30 kg/m2); c) vetsugtig met metaboliese sindroom (n=19) en d) reeds gediagnoseerde T2DM (n=7). Pasiënte het gesondheid-, leefstyl- en voedingsvraelyste voltooi. Antropometrie en ‘n “dual energy x-ray absorptiometry” (DXA) skandering is uitgevoer om die liggaamsamestelling te assesseer. Bloedmonsters is versamel om die metaboliese (vastende bloedsuiker, totale cholesterol, HDL, LDL, triglycerides) en inflammatoriese profiele (CRP, SDF-1α, IL-6, IL-8, IL-10, TNF-α en IFN-γ) van pasiënte te analiseer. Om die vas te stel of daar ʼn verwantskap bestaan tussen sistemiese inflammasie tydens die verskillende stadiums van siekte-progressie en ADSC beskadiging, is in vitro selkultuur eksperimente uitgevoer waarin ADSCs (Poetics sellyn) behandel is met die bloedsera van individuele deelnemers. Veranderinge in sel-lewensvatbaarheid (MTT toets), selverdeling (BrdU toets) en sel migrasie (as ʼn aanduiding van wondheling) is gemeet onder standaard selkultuur-kondisies. Resultate: Sistemiese inflammasie was duidelik waarneembaar in die gesond vetsugtige (CRP 29± 8 pg/mL) en metaboliese sindroom vetsugtige (CRP 50.8 ± 24 pg/mL) groepe. Serumvlakke van die anti-inflammatoriese sitokien IL-10 was beduidend laer in T2DM deelnemers (0.42 ± 0.63 pg/mL) (p < 0.05). Serumvlakke van IL-6, IL-8, TNF-α en IFN-γ het gevarieer, as gevolg van individuele variasie in die deelnemers van die verskillende groepe. Betekenisvolle verband tussen IL-6 vlakke en sel-verdeling is waargeneem, veral in die gesond skraal (p < 0.01) en metaboliese sindroom (p < 0.01) groepe. ʼn Veband tussen serumvlakke van IL-8 en sel-migrasie, was ook duidelik. Die stimulerende effek van serum op selverdeling was afwesig in serum afkomstig van vetsugtige deelnemers. Gevolgtrekking: Hierdie studie is die eerste om te bewys dat die funksionering van vetweefsel-stam selle negatief beïnvloed kan word deur vetsug-gedrewe versteurings in die delikate sistemiese inflammatoriese balans, onafhanklik van die metaboliese sindroom.

Thesis

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.

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