The effect of the TGF-β isoforms on progenitor cell recruitment and differentiation into cardiac and skeletal muscle

[摘要] Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal andthe ability to differentiate into multiple cell-lineages.The potential for the application of stem cells in clinical settings has had a profound effect onthe future of regenerative medicine. However, to be of greater therapeutic use, selection ofthe most appropriate cell type, as well as optimisation of stem cell incorporation into thedamaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cellpopulation which mediate postnatal muscle growth. Following injury or in diseasedconditions, these cells are activated and recruited for new muscle formation. In contrast, thepotential of resident adult stem cell incorporation into the myocardium has been challengedand the response of cardiac tissue, especially to ischaemic injury, is scar formation.Following muscle damage, various growth factors and cytokines are released in the afflictedarea which influences the recruitment and incorporation of stem cells into the injured tissue.Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines andhas at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulationof cell growth and regeneration following activation and stimulation of receptor-signallingpathways. By improving the understanding of how TGF-β affects these processes, it ispossible to gain insight into how the intercellular environment can be manipulated to improvestem cell-mediated repair following muscle injury. Therefore, the main aims of this thesiswere to determine the effect of the three TGF-β isoforms on proliferation, differentiation,migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this topossible improved mechanisms for muscle repair.The effect of short- and long-term treatment with all three TGF-β isoforms were investigatedon muscle progenitor cell proliferation and differentiation using the C2C12 skeletal musclesatellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems.Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, andtogether with total nuclear counts, used to assess the effect of TGF-β on myoblastproliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structuralprotein myosin heavy chain (MHC) were used as protein markers to assess early and terminaldifferentiation, respectively. To establish possible mechanisms by which TGF-β isoformsregulate differentiation, further analysis included determination of MyoD localisation and therate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels ofconnexin-43 and MHC were analysed, together with the determination of embryoid bodynumbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed toanalyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), aswell as fusion of C2C12 cells (Chapter 8).Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells,differentiation results, however, indicated that especially following long-term incubation,TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes.Similarly, myocyte migration and fusion were also negatively regulated following TGF-βtreatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment withTGF-β1 could potentially enhance differentiation. Further research is however required in thisarea, especially since migration was greatly reduced in these cells.Taken together, results demonstrated variable effects following TGF-β treatment dependingon the cell type and the duration of TGF-β application. Circulating and/or treatmentconcentrations of this growth factor could therefore be manipulated depending on the area ofinjury to improve regenerative processes. Alternatively, when selecting appropriate stem orprogenitor cells for therapeutic application, the effect of the immediate environment andsubsequent interaction between the two should be taken into consideration for optimalbeneficial results.

[发布机构] Stellenbosch University