3 research outputs found
miRNA signature of healthy vs impaired mesenchymal stem cells as a biomarker for autologous stem cell therapy
No full textThesis (PhD)--Stellenbosch University, 2021.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.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.Doctorat
Stem cell impairment associated with type 2 diabetes mellitus : investigating the effects of obesity-associated inflammation on mesenchymal stem cell function
Get PDFThesis (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.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
