Establishing reliable criteria for isolating myogenic cell fractions with stem cell properties and enhanced regenerative capacity
Introduction
Transplantation of myogenic progenitor or precursor cells, commonly referred to in the literature as myoblast transplantation, has been investigated as a treatment for many muscle-related disorders and also as a gene delivery system for therapeutic recombinant proteins. This approach has most recently received attention for its therapeutic potential in the repair of cardiac muscle (cellular cardiomyoplasty) [2], [3]. However, much of what is known regarding myogenic cell transplantation has evolved from studies investigating its therapeutic potential for the treatment of the most severe form of muscular dystrophy, Duchenne muscular dystrophy (DMD). DMD is a lethal X chromosome-linked recessive disorder characterized by an absence or marked deficiency of the cytoskeletal protein dystrophin [4].
The necessity of restoring dystrophin expression to alleviate the progression of DMD and its associated debilitating symptoms has led to proposed experimental therapies involving both viral and cell-based gene delivery. Cell-based transplantation approaches have been investigated extensively in both animals and humans. Several clinical trials have been performed and, unfortunately, have met with a very limited degree of success [5], [6], [7], [8]. As a whole, little evidence of significant contribution to myofiber regeneration and dystrophin expression by the donor cells is typically observed.
In spite of these apparent clinical setbacks, ongoing investigations in animal models continue. Such studies have since revealed several inherent limitations that are thought to impede the use of the direct myogenic cell-injection approach to treat DMD. These include the low migratory capacity of the cells from the injection site and potential specific and nonspecific immune responses to both donor cells and the dystrophin protein itself [9]. Further, significant evidence for a large and rapid donor cell demise following injection has also been implicated as one of the primary reasons for the lack of clinical success [6], [10], [11], [12], [13]. This donor cell demise cannot only be attributed to specific cellular immune responses (poor restoration is encountered despite matching of donor–host major histocompatibility loci [14], [15]), has yet to be clarified by animal model experimentation, and has since been observed within immunocompromised and immunosuppressed, normal and dystrophic animal models, under conditions considered optimal for muscle cell transplantation. Such conditions include pretreatment of the host muscle with myonecrotic agents and irradiation to prime the muscle for a regeneration response and reduce host cell competition with donor cells [10], [16], [17], [18], [19], [20].
It is apparent that the initial transplantation environment, which includes nonspecific inflammatory reactions along with necrotic and possibly other undefined factors, influences initial cell survival [21], [22], [23]. Improvement in transplantation efficiency within animal models has been achieved through delivery of anti-inflammatory or anti-complement agents [20], [24]. However, experimental observations regarding cell survival suggest that specific populations of myogenic progenitor cells may be more suited to survive and flourish in the initial posttransplantation environment and contribute to the regeneration process [18], [25]. Still, in combination with current delivery techniques, the estimated yield of new muscle and its associated nuclei formed by transplanted cells falls well short of parity and is currently not amenable to treating the major muscle groups of the body [26]. The need to define and isolate efficient progenitor populations in terms of regenerative ability and dissemination quality has recently led to a focus on muscle-derived cells with stem cell-like properties. A review of currently described populations, as well as theories pertaining to their potential developmental origin, is presented within numerous reviews [21], [27], [28], [29], [30]. Some of these muscle-derived stem cell (MDSC) populations have demonstrated a remarkable ability to regenerate myofibers following transplantation [31]. However, the characteristics or qualities possessed by such cells that permit them to display this desired behavior are currently unknown.
The goal of the investigations described here was to further establish cellular traits that may be used to more easily identify efficient progenitor cells within culture, before their use in regenerative therapies, and also to generate potential mechanisms that may be used to explain why discrepancies in efficiency are observed. One possible explanation for the variation in regenerative efficiency stems from heterogeneity in the proliferative and/or fusion behavior of the donor populations. Such behavior has been previously described by numerous investigators [32], [33], [34], [35], [36], [37]. Thus, it is both of scientific and therapeutic interest to examine the potential association between inherent population expansion and regenerative capability within the myogenic progenitor compartment. Specifically, from previous observations made in the mouse model [1] it is proposed that the ability of a myogenic progenitor population, and the ability of its subsequent progeny's, to expand in number while consequently maintaining an undifferentiated state is reflected in their capacity to regenerate new myofibers following transplantation. With any isolation strategy, eventual clinical utilization requires that such observations not be limited in a species-dependent manner. Thus, within a rat animal model evaluation of both regeneration and fusion parameters, as previously established for the mouse model, are presented.
Section snippets
Cell isolation
A modified version of the preplate technique [31], [38], [39] was utilized to obtain various myogenic cultures from dissociated skeletal muscle. Both hindlimb gastrocnemius muscles from a single female rat (Sprague–Dawley, 6–10 weeks old) were combined for enzymatic digestion and cell isolation. The preplating protocol, including adhesion times and supplemented culture medium, was performed using the same materials and methodology described previously [1]. Enzymatic dissociation was performed
Desmin and CD34 expression
The average percentage of myogenic cells, as determined through desmin expression, within each preplate population from PP1 to PP6 was 14 ± 2, 34 ± 13, 58 ± 4, 86 ± 4, 86 ± 2, and 81, respectively. MDSC cultures contained an average percentage of 66 ± 1 desmin-expressing cells.
Flow cytometric evaluation of nonhematopoietic cells contained within the rat cell cultures revealed minimal CD34 expression. Less than 2% of the cells within each preplate (PP1–PP6) expressed CD34, regardless of donor
Discussion
We have sought to determine cellular traits that may be used to identify efficient myogenic progenitor cells within culture, with regard to their regenerative capacity following isolation from muscle biopsy and transplantation into skeletal muscle. Utilizing the same previously described procedures and parameters to define their behavior within culture we have observed, within a second animal model, that a given myogenic cell populations proliferation and fusion behavior in vitro correlates
Acknowledgements
The authors would like to acknowledge funding support from the National Institutes of Health (NIH, 1R01 AR49684-01, HL 069368), the Muscular Dystrophy Association (MDA), the Parent Project (USA), and the Orris C. Hirtzel and Beatrice Dewey Hirtzel Memorial Foundation. This work was also supported by the William F. Donaldson Endowed Chair at the Children's Hospital of Pittsburgh and the Henry J. Mankin Endowed Chair at the University of Pittsburgh. This paper is based on a presentation at a
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