Self-talk vs crosstalk: extracellular particles derived from myoblasts and fibroblasts and their effects on myoblast function and related myogenic regulatory factors

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dc.contributor.advisorMyburgh, Kathryn Helenen_ZA
dc.contributor.advisorMcColl, Rhysen_ZA
dc.contributor.authorHagemann, Kyle Nicholasen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Physiological Sciences.en_ZA
dc.date.accessioned2024-02-29T09:35:21Z
dc.date.accessioned2024-04-26T10:57:11Z
dc.date.available2024-02-29T09:35:21Z
dc.date.available2024-04-26T10:57:11Z
dc.date.issued2024-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2024. en_ZA
dc.description.abstractENGLISH ABSTRACT: In the intricate environment of intercellular communication, small extracellular vesicles (EVs) have emerged as mediators, facilitating the transfer of bioactive molecules and influencing recipient cell behaviour. Myoblasts, the precursors of muscle cells, and fibroblasts are both found in the muscle niche. They release small EVs that play a crucial yet incompletely understood role in orchestrating various aspects of muscle biology. This study delves into the interactions between myoblast- and fibroblast-derived extracellular particles (myo-EPs and fibro-EPs) and myoblast function, with a specific focus on uptake, proliferation, the SMAD pathway, and cellular migration. Myoblast (C2C12) and fibroblast (L929) EPs were harvested from conditioned media using differential ultracentrifugation and characterised according to guidelines (MISEV 2018). Their effects on myoblasts were analysed using a combination of light and confocal microscopy, and western blotting. Four conditions were compared: Normal growth media (NGM), EP-depleted media (EP-DM) control, Myo-EP and Fibro-EP. Preferential uptake of myo-EPs into myoblasts after 48 hours was shown (EP-DM control: 0.9396 spots per cell ± 0.6313 spots per cell; Myo-EP: 16.55 spots per cell ± 12.60 spots per cell; Fibro-EP: 9.668 spots per cell ± 4.882 spots per cell). No effect of EPs on proliferation was observed over 48 hours for either Myo-EPs or Fibro-EPs. Both EP types were shown to contain TGF-β, an activator of the SMAD pathway. Follistatin was also found in the myo-EPs. There was no change in the SMAD pathway associated proteins p-SMAD2/3 (NGM: 1; EP-DM: 1.328 ± 0.5896; Myo-EP: 1.149 ± 0.5299; Fibro- EP: 0.8192 ± 0.3035) or SMAD6/7 (NGM: 1; EP-DM control: 1.033 ± 0.2187; Myo- EP: 1.059 ± 0.4896; Fibro-EP: 1.000 ± 0.4976) expression after 48 hours. There was no change in myoD expression (NGM: 1; EP-DM: 1.489 ± 0.5930; Myo-EP: 1.561 ± 0.7696; Fibro-EP: 1.561 ± 0.1693), however myogenin expression was significantly increased in the three conditions compared to the NGM (NGM: 1; EP-DM: 7.397 ± 1.913; Myo-EP: 7.578 ± 2.073; Fibro-EP: 8.024 ± 1.987). Myoblast migration was altered by fibro-EPs, with these myoblasts having a more directionless migration pattern (Rayleigh p-values: NGM: p < 6.90244E-11; EP-DM control: p < 4.58659E-08; Myo-EP: p < 2.1185E-12; Fibro-EP: p < 4.76142E-07). Cellular communication is crucial for normal cell function, and small EVs have come to be known as an integral mediator of this communication. In this study, we indicated preferential uptake of myoblast EPs into myoblasts over fibroblast EPs. Although uptake was shown, these EPs had little effect on myoblast proliferation, as well as SMAD pathway associated proteins within the time course assessed. Cell migration characteristics were also observed, and it was shown that fibroblast EPs decreased the directionality of C2C12 myoblasts.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: In die komplekse omgewing van intersellulêre kommunikasie, het klein ekstrasellulêre vesikels (EVs) na vore gekom as belangrike rolspelers, wat die oordrag van bioaktiewe molekules fasiliteer en die gedrag van die ontvangende sel beïnvloed. Mioblaste, die voorlopers van spierselle, en fibroblaste word beide gevind in die spiernis. Hulle stel klein EVs vry wat se rol in verskeie aspekte van spier biologie nog nie ten volle beskryf is nie. Hierdie studie ondersoek die interaksies tussen ekstrasellulêre partikels afkomend van mioblaste (mio-EPs) en fibroblaste (fibro-EPs) en mioblast funksie, met ‘n spesifieke fokus op opname, sel toename, the SMAD-weg en migrasie. Mioblast (C2C12) en fibroblast (L929) EPs was versamel uit gekondisioneerde media deur middel van differensiële ultrasentrifugering en gekarakteriseer volgens die MISEV 2018 riglyne. Hul effekte op mioblaste was geanaliseer deur ‘n kombinasie van lig en konfokale mikroskopie, asook western blot. Vier kondisies is vergelyk: normale groeimedia (NGM), kontrole, Mio-EP en Fibro-EP. Voorkeur opname van Mio-EPs deur mioblaste is na 48 uur getoon (Kontrole: 0.9396 ± 0.6313 spikkels per sel; Mio-EP: 16.55 ± 12.60 spikkels per sel; Fibro-EP: 9.668 ± 4.882 spikkels per sel). Geen effek van EPs op sel toename was geobserveer na 48 uur vir Mio-EPs of fibro-EPs nie. Beide EP-tipes is getoon om TGF-β te bevat., ‘n aktiveerder van die SMAD-weg. Follistatin is ook in die myo-EP's gevind. Daar was geen verandering in SMAD-weg verwante proteïene p-SMAD2/3 (NGM: 1; kontrole: 1.328 ± 0.5896; Mio-EP: 1.149 ± 0.5299; Fibro-EP: 0.8192 ± 0.3035) of SMAD6/7 (NGM: 1; kontrole: 1.033 ± 0.2187; Mio- EP: 1.059 ± 0.4896; Fibro-EP: 1.000 ± 0.4976) se uitdrukking na 48 uur nie. Daar was geen verandering in myoD uitdrukking nie (NGM: 1; kontrole: 1.489 ± 0.5930; Mio-EP: 1.561 ± 0.7696; Fibro-EP: 1.561 ± 0.1693), maar myogenin uitdrukking was aansienlik verhoog in die drie kondisies in vergelyking met die NGM (NGM: 1; kontrole: 7.397 ± 1.913; Mio-EP: 7.578 ± 2.073; Fibro-EP: 8.024 ± 1.987). Mioblast migrasie was verander deur fibro- EPs, met hierdie mioblaste wat ‘n meer rigtinglose migrasie patroon getoon het (Raleigh p- waardes: NGM: p < 6.90244E-11; Kontrole: p < 4.58659E-08; Mio-EP: p < 2.1185E-12; Fibro- EP: p < 4.76142E-07). Sellulêre kommunikasie is noodsaaklik vir normale selfunksie, en klein EVs het bekend geword as integrale bemiddelaars van hierdie kommunikasie. In hierdie studie, het ons die voorkeur opname van mioblast-EPs deur mioblaste bo fibroblast-EPs getoon. Alhoewel opname getoon is, het hierdie EPs minimale effek op mioblast toename, sowel as die SMAD-weg verwante proteïene gehad binne die tydsverloop waarin dit geasseseer is.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxiii, 78 pages : illustrations (some color)en_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130255
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMyoblasts -- Growth -- Regulationen_ZA
dc.subject.lcshFibroblasts -- Regenerationen_ZA
dc.subject.lcshCell interactionen_ZA
dc.subject.lcshFollistatinen_ZA
dc.subject.lcshGrowth factors -- Inhibitorsen_ZA
dc.subject.lcshExtracellular vesiclesen_ZA
dc.subject.nameUCTDen_ZA
dc.titleSelf-talk vs crosstalk: extracellular particles derived from myoblasts and fibroblasts and their effects on myoblast function and related myogenic regulatory factorsen_ZA
dc.typeThesisen_ZA
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