Briggs, D; (2013) An investigation into sub-populations of satellite cells and myoblasts. Doctoral thesis , UCL (University College London).

Abstract

One potential therapy for muscular dystrophy is myoblast transplantation. This will require cell expansion before transplantation and donor cells must therefore retain their stem cell capacity after propagation. I used a mouse model with the aim of sub-populating freshly isolated satellite cells and cultured satellite cell-derived myoblasts to obtain those that retain their ability to contribute to muscle regeneration upon grafting into dystrophin-deficient mdx-nude mouse muscles. Using flow cytometric cell sorting, satellite cells or myoblasts were separated on the basis of proliferative state and the level of expression of reactive oxygen species (ROS). DNA dyes that separated cells in G0 from those proliferating in G1 were useful for analysis but were toxic, limiting further comparisons. A donor transgenic mouse, which expresses GFP only in activated satellite cells, was used to separate quiescent GFP– from activated GFP+ satellite cells. In culture, the sorted GFP– satellite cells became activated, turned on GFP and showed enhanced proliferation over GFP+ satellite cells. When engrafted into mdx-nude mice, GFP– satellite cells regenerated more muscle than GFP+ satellite cells. Freshly isolated satellite cells showed low levels of ROS, but upon expansion in culture, ROS levels increased. Myoblasts were sorted into ROSlow and ROShigh populations and compared. ROSlow cells had a higher proliferative capacity in vitro than ROShigh cells and this corresponded with an increased ability to regenerate muscle in vivo. Expansion in low oxygen improved myoblast growth. Furthermore, myoblasts that had been grown in 5% O2 contributed to more muscle fibres of donor origin than the same number of myoblasts that had been grown in 20% O2. Transgenic mice expressing GFP or β-gal, under ubiquitous promoters, were compared, to find the most reliable marker for donor-cell transplantation, with the bactinGFP mouse selected for use. In summary, selecting for cell populations that have a greater proliferative ability increases the capacity of transplanted myoblasts to regenerate muscle fibres in vivo.

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