Stem cells are present in many adult tissues, where they play an important role in postnatal growth and tissue regeneration. Our data in mice demonstrate that direct cell sorting using a distinct combination of cell surface markers allows the prospective identification of a novel subset of skeletal muscle satellite cells that exhibits properties of adult muscle stem cells. SMPs are committed myogenic precursor cells, isolated by negative selection for the cell surface markers CD45, Sca1, and Mac1 and positive selection for β1-integrin and CXCR4. Unlike previous approaches (Dezawa et al., 2005
; Kuang et al., 2007
; Montarras et al., 2005
; Sampaolesi et al., 2003
), isolation of SMPs is based on normally expressed cell surface markers, and therefore is broadly applicable to accomplish direct muscle stem cell isolation and analysis from different mouse strains and ages without the need for specialized transgenic lines or extended periods of cell culture.
To examine the therapeutic potential of SMPs, we conducted transplant studies into mdx
mice, a model of Duchenne Muscular Dystrophy. Wild-type GFP+
SMPs contributed robustly to the regeneration of mature muscle fibers in recipient mice at a frequency that was directly proportional to the number of precursor cells injected and that in some recipients approached complete donor chimerism for myofibers (). Engraftment of wild-type SMPs into mdx
muscle thus provides a highly effective mechanism for the introduction of the dystrophin gene into dystrophin-deficient muscle, consequently restoring dystrophin protein expression on the majority of myofibers (), reducing muscle inflammation and fibrosis (Figure S7B
), and significantly improving physiological muscle function ().
In addition to participating in the repair of differentiated muscle fibers, transplanted SMPs also re-enter the satellite cell niche, contributing the majority of Pax7+
cells found on donor-derived myofibers (). These engrafted precursor cells could be recruited to participate again in the repair of future muscle injury (). The high level of contribution of donor GFP+
SMPs to mature GFP+
myofibers and to the pool of satellite cells associated with GFP+
fibers likely reflects both unique, cell autonomous properties of the SMP population, as well as cell non-autonomous aspects of the transplant model. Specifically, the lower endogenous frequency of SMPs in mdx
muscle () and the cytotoxic effects of cardiotoxin pre-treatment on SMP numbers (Figure S8
), may lessen potential competition from endogenous cells during transplant and facilitate high level contribution of the introduced GFP+
cells. Alternatively, cardiotoxin pre-treatment may induce or enhance production of SMP support factors, and provide a more conducive environment for myogenic engraftment. However, it is important to note that muscle engraftment by SMPs was not dependent on highly ablative pre-injury, as these cells also contributed at high efficiency (up to 94% of total myofibers) after transfer into uninjured muscle ( and S7
). In addition, recipient immunosuppression (Montarras et al., 2005
) was not necessary for high level SMP engraftment or for maintenance of grafted fibers for up to 4 months (data not shown). Thus, robust donor cell engraftment can be achieved in fully immunocompetent animals and can be maintained for extended periods of time following SMP transplant in mice, which should encourage investigation of analogous strategies for cell replacement therapy in humans.
Taken together, these data demonstrate that, like hematopoietic stem cells in the bone marrow (Kondo et al., 2003
), SMPs represent a transplantable population of tissue-specific stem cells that can undergo self-renewing proliferation as well as lineage-selective differentiation in response to injury. Notably, this capacity for enduring regenerative function is a unique activity of this distinct subset of primitive muscle stem cells, and is not provided by their more differentiated daughters (), a finding that helps to explain the modest clinical benefit observed in earlier studies employing transplantation of differentiated myoblasts for the treatment of muscle degenerative disease (Miller et al., 1997
; Peault et al., 2007
). Thus, even in tissues composed of typically very long-lived progeny – like the skeletal muscle – stem cells appear to exhibit specialized activities, uniquely providing robust engraftment and long-term contribution to tissue regeneration. While the singular properties of muscle stem cells that are critical for these important activities remain largely undiscovered (as is the case for most stem cell populations identified to date), stem cell-specific attributes, including their ability to generate large numbers of progeny, their extensive self-renewal potential, or other more poorly understood characteristics (such as their ability to home to appropriate niches, or to sense environmental signals) likely promote their robust regenerative activity. The ability to prospectively identify and directly purify muscle stem cells (SMPs) from both normal and diseased skeletal myofibers will be a tremendous benefit for the future elucidation of both the normal processes that control muscle specification and the ways in which these processes are deregulated by disease. Such studies ultimately will build a molecular portrait of the positive and negative regulatory pathways controlling adult myogenesis, and will enable more refined interventions to enhance muscle function by restoring appropriate numbers and endogenous activity of these cells, which can be significantly perturbed by injury, disease and aging ( and (Blau et al., 1983
; Conboy et al., 2003
; Conboy et al., 2005
; Luz et al., 2002
; Wright, 1985
)). Finally, as demonstrated here, direct transplantation of highly purified muscle stem cells represents a promising therapeutic option for muscle degenerative disorders, as these cells provide an effective source of immediately available muscle regenerative cells as well as a reserve pool that can maintain muscle regenerative activity in response to future challenges.