The effect of the TGF-β isoforms on progenitor cell recruitment and differentiation into cardiac and skeletal muscle
Date
2007-12
Authors
Schabort, Elske Jeanne
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and
the ability to differentiate into multiple cell-lineages.
The potential for the application of stem cells in clinical settings has had a profound effect on
the future of regenerative medicine. However, to be of greater therapeutic use, selection of
the most appropriate cell type, as well as optimisation of stem cell incorporation into the
damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell
population which mediate postnatal muscle growth. Following injury or in diseased
conditions, these cells are activated and recruited for new muscle formation. In contrast, the
potential of resident adult stem cell incorporation into the myocardium has been challenged
and the response of cardiac tissue, especially to ischaemic injury, is scar formation.
Following muscle damage, various growth factors and cytokines are released in the afflicted
area 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 and
has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation
of cell growth and regeneration following activation and stimulation of receptor-signalling
pathways. By improving the understanding of how TGF-β affects these processes, it is
possible to gain insight into how the intercellular environment can be manipulated to improve
stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis
were 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 to
possible improved mechanisms for muscle repair.
The effect of short- and long-term treatment with all three TGF-β isoforms were investigated
on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle
satellite 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 C2C12
cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and
together with total nuclear counts, used to assess the effect of TGF-β on myoblast
proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural
protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal
differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms
regulate differentiation, further analysis included determination of MyoD localisation and the
rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of
connexin-43 and MHC were analysed, together with the determination of embryoid body
numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to
analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as
well 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-β
treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with
TGF-β1 could potentially enhance differentiation. Further research is however required in this
area, especially since migration was greatly reduced in these cells.
Taken together, results demonstrated variable effects following TGF-β treatment depending
on the cell type and the duration of TGF-β application. Circulating and/or treatment
concentrations of this growth factor could therefore be manipulated depending on the area of
injury to improve regenerative processes. Alternatively, when selecting appropriate stem or
progenitor cells for therapeutic application, the effect of the immediate environment and
subsequent interaction between the two should be taken into consideration for optimal
beneficial results.
Description
Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007.
Keywords
TGF-B isoforms, Progenitor cells, Differentiation, Myogenesis, Transforming growth factors-beta, Stem cells -- Research, Stem cells -- Transplantation, Myocardium -- Regeneration, Cell differentiation, Musculoskeletal system -- Wounds and injuries -- Treatment, Dissertations -- Physiology (Human and animal), Theses -- Physiology (Human and animal)