Not an Inside Job: How Can Transplantation of Relatively Few Exogenous Satellite Cells Do What Thousands of Endogenous Cells Cannot?

When muscle fibers are damaged, they release growth factors that stimulate the proliferation of muscle stem cells termed satellite cells (Figure 1). The progeny of these newly proliferating satellite cells then fuse with the damaged muscle fibers so as to repair them. This process is generally able to maintain muscle strength and size but can tend to fail during the aging process and in certain disease states, such as in dystrophic muscle. This has spurred efforts to develop molecular and cellular therapies to stimulate the repair process. It is against this backdrop that Hall et al.1Hall JK Banks GB Chamberlain JS Olwin BB Prevention of muscle aging by myofiber-associated satellite cell transplantation.Sci Transl Med. 2010; 2: 57-83Crossref Scopus (91) Google Scholar recently reported the startling finding that the transplantation of a small number of exogenous satellite cells—still localized within their cellular niche in close proximity to the muscle fiber—into damaged mouse muscle produced significant repair and growth of the muscle that persisted for nearly 2 years. Equally surprisingly, the transplanted cells were retained during this period in the absence of any immunosuppression, even though they were immunologically incompatible with those of the host. A key question arising from these remarkable findings is how fewer than 10 transplanted satellite cells can give rise to significant muscle hypertrophy, whereas the literally thousands of endogenous satellite cells already present in the muscle are unable to do so. It is also unclear why the transplanted cells were not rejected. Answering these key questions may have significant impact on the clinical development of cell transplantation for muscle repair and may open up new strategies for enhancing the inherent regenerative capacity of muscle. Several groups have tried over many years to develop a therapy for muscular dystrophies based on the transplantation of myoblasts—the downstream progeny of satellite cells—which have been shown to stimulate muscle repair.2Skuk D Tremblay JP Myoblast transplantation: the current status of a potential therapeutic tool for myopathies.J Muscle Res Cell Motil. 2003; 24: 285-300Crossref PubMed Google Scholar In mouse experiments, the success of such attempts has required the transplantation of hundreds of thousands of myoblasts per muscle following pretreatment of the muscle by myotoxin injection, which mediates injury signals and induces muscle regeneration, and irradiation, which reduces the competition from the host satellite cells.3Kinoshita I Vilquin JT Guérette B Asselin I Roy R Tremblay JP Very efficient myoblast allotransplantation in mice under FK506 immunosuppression.Muscle Nerve. 1994; 17: 1407-1415Crossref PubMed Scopus (163) Google Scholar In recent years, several studies have reported that transplantation of a few freshly isolated satellite cells4Montarras D Morgan J Collins C Relaix F Zaffran S Cumano A et al.Direct isolation of satellite cells for skeletal muscle regeneration.Science. 2005; 309: 2064-2067Crossref PubMed Scopus (833) Google Scholar,5Cerletti M Jurga S Witczak CA Hirshman MF Shadrach JL Goodyear LJ et al.Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.Cell. 2008; 134: 37-47Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar,6Sacco A Doyonnas R Kraft P Vitorovic S Blau HM Self-renewal and expansion of single transplanted muscle stem cells.Nature. 2008; 456: 502-506Crossref PubMed Scopus (642) Google Scholar or of a few myofibers still associated with their satellite cells within their niche position7Collins CA Olsen I Zammit PS Heslop L Petrie A Partridge TA et al.Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche.Cell. 2005; 122: 289-301Abstract Full Text Full Text PDF PubMed Scopus (1042) Google Scholar produced better transplantation results (i.e., more hybrid muscle fibers formed in part by the transplanted cells) than delivery of these large numbers of myoblasts. However, such dramatic muscle hypertrophy as that reported by Hall et al. has not been previously observed following the transplantation of more than 1 million myoblasts, the transplantation of intact muscle fibers, or the transplantation of freshly isolated satellite cells. In the new study, the workers incubated freshly isolated muscle fibers for 4 to 5 hours with their associated satellite cells in a relatively standard growth medium supplemented with fibroblast growth factor 2 and horse serum. Fibroblast growth factor 2 is known to stimulate the proliferation of myoblasts. One to five of these myofibers were then injected into the muscle of a normal mouse that had been treated with BaCl2 or cardiotoxin to mediate the muscle fiber–injury signals necessary to initiate repair. The authors reported that this treatment protocol led to increased muscle strength and 35% muscle hypertrophy. Importantly, approximately 80% of the newly regenerated muscle fibers expressed the green fluorescent protein (GFP) carried by the transplanted donor satellite cells, indicating their exogenous origin. It is important to note that only that a tiny fraction (as little as one in a thousand) of the nuclei within the myofiber must carry the GFP transgene in order to be GFP-positive. Moreover, this transplantation prevented for as long as 24 months the age-related reductions in muscle mass, function, and strength observed in control animals. Neither the injection of the BaCl2 alone nor the transplantation of muscle fibers in the absence of muscle injury produced these extraordinary effects. Thus, the transplantation of a few donor satellite cells in a muscle in which regeneration was induced seems to have prevented the onset of age-associated muscle atrophy and weakness, suggesting new therapeutic avenues if these phenomena can be confirmed by further research and are reproduced in large-animal models and humans. It is intriguing that transplantation of 10, at most, satellite cells attached to muscle fibers produced a greater degree of muscle hypertrophy than the thousands of endogenous host satellite cells, which are already present in the host muscle and were thus also exposed to the same altered environment produced by myofiber transplantation and the muscle damage regimen. Indeed, other than their distinct genetic background, the only difference between the donor-derived satellite cells and the recipients' endogenous satellite cells was the short ex vivo incubation of the donor cells. The only significant difference here was that the ex vivo incubation of the cells was performed with the satellite cells remaining within their niche position attached to the myofibers. The authors conclude that this brief treatment might have altered the cells permanently in some fashion so as to lead to the astonishing ability to proliferate and form new myofibers during the subsequent 21 months. Another possible interpretation is that the ex vivo culture protocol has led not to any permanent changes to the cells but, rather, to an intense proliferation of the cells during the first few days following transplantation into the muscle and that this increased density of satellite cells in the muscles was retained during the subsequent 21 months. Muscle fiber nuclei are normally located at the periphery of the cell. Following regeneration, the nuclei move to the center of the fiber—a phenomenon referred to as centronucleation. Hall et al. attributed the presence of centronucleation observed at 21 months after transplantation to recent regeneration of the fibers. However, centronucleation has also been observed several months after cardiotoxin damage in rats.8Couteaux R Mira JC d'Albis A Regeneration of muscles after cardiotoxin injury. I. Cytological aspects.Biol Cell. 1988; 62: 171-182Crossref PubMed Scopus (114) Google Scholar The burst of proliferation of the transplanted cells relative to the host cells resulted not only in the formation of a high percentage of hybrid cells expressing the donor reporter gene but, more importantly, in the formation of abundant satellite cells that could subsequently be transplanted to other mice. Neither the ability to form abundant daughter satellite cells nor their ability to be retransplanted is unique to myofiber-associated satellite cells. Several groups have reported the same phenomenon following intramuscular transplantation of both mouse and human myoblasts.9Skuk D Paradis M Goulet M Chapdelaine P Rothstein DM Tremblay JP Intramuscular transplantation of human postnatal myoblasts generates functional donor-derived satellite cells.Mol Ther. 2010; 18: 1689-1697Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar The important difference between the report by Hall et al. and these previous studies is the very high density of the satellite cells of donor origin. Their protocol thus generated an important proliferation burst of the 10 transplanted cells but not of the thousands of host cells, resulting in the formation not only of a high percentage of hybrid cells expressing the donor reporter gene but also, more importantly, of a population of donor-derived satellite cells that ultimately exceeded the original satellite cell population of the host. The impressive muscle hypertrophy in Hall and colleagues' study required destruction of the host muscle with myotoxic substances such as BaCl2, but cardiotoxin8Couteaux R Mira JC d'Albis A Regeneration of muscles after cardiotoxin injury. I. Cytological aspects.Biol Cell. 1988; 62: 171-182Crossref PubMed Scopus (114) Google Scholar can produce similar results. It is important to note that both agents leave satellite cells unscathed. The damage to the host myofibers releases growth factors, which favors the proliferation of satellite cells, the formation of new myofibers, and the repopulation of the satellite cell population by the donor cells. Muscle growth is normally limited by signaling through the myostatin pathway. Indeed, Benabdallah et al.10Benabdallah BF Bouchentouf M Rousseau J Bigey P Michaud A Chapdelaine P et al.Inhibiting myostatin with follistatin improves the success of myoblast transplantation in dystrophic mice.Cell Transplant. 2008; 17: 337-350Crossref PubMed Scopus (47) Google Scholar,11Benabdallah BF Bouchentouf M Tremblay JP Improved success of myoblast transplantation in mdx mice by blocking the myostatin signal.Transplantation. 2005; 79: 1696-1702Crossref PubMed Scopus (42) Google Scholar showed that blocking myostatin improves myoblast transplantation, leading to the formation of larger and more abundant myofibers. These findings raise the intriguing possibility that the satellite cells transplanted by Hall et al. might have been rendered less susceptible to myostatin inhibition, thus permitting their strong proliferation burst. A major challenge for therapeutic application of satellite cell transplantation is the development of a clinically acceptable protocol to deliver the satellite cells into human muscles that maintains their capacity for renewal and proliferation. Hall et al. transplanted fully intact mouse myofibers, and the transplantation of satellite cells that remained attached to these myofibers probably maintained the stem cell niche functionally intact, which may underlie their important proliferative capacity. One question that arises is whether it is necessary that the muscle fibers to which the satellite cells are attached remain intact or whether fragments of myofibers, which are more easily obtained for use in humans because of the longer length of the fibers, would yield similar success. Another key question is whether the niche environment is important or whether it is necessary only to transplant freshly isolated satellite cells as reported by other researchers.6Sacco A Doyonnas R Kraft P Vitorovic S Blau HM Self-renewal and expansion of single transplanted muscle stem cells.Nature. 2008; 456: 502-506Crossref PubMed Scopus (642) Google Scholar Indeed, could a simple 4-hour ex vivo culture of these isolated satellite cells produce the same beneficial effects? An important question raised by this new study is whether these results could find clinical application for the treatment of muscle aging in human patients. The use of myotoxins to stimulate muscle repair would not be acceptable in patients because of potential risks of systemic toxicity. In a clinical trial recently conducted by our group,12Skuk D Goulet M Roy B Piette V Côté CH Chapdelaine P et al.First test of a “high-density injection” protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen months follow-up.Neuromuscul Disord. 2007; 17: 38-46Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar,13Skuk D Goulet M Roy B Chapdelaine P Bouchard JP Roy R et al.Dystrophin expression in muscles of Duchenne muscular dystrophy patients after high-density injections of normal myogenic cells.J Neuropathol Exp Neurol. 2006; 65: 371-386Crossref PubMed Scopus (179) Google Scholar muscle damage was produced in the host following multiple intramuscular injections at 1-mm intervals. As is the case when using myotoxins, this procedure favors fusion of the implanted cells with host myofibers. However, this transplantation protocol, involving hundreds of injections, is not acceptable to ethics committees overseeing clinical application. Thus, new options to induce muscle injury are sorely needed and are not obviated by these new findings. Future work should aim to produce beneficial growth-stimulatory effects in the absence of muscle damage, perhaps by identifying the optimal cocktail of growth factors. As already noted, the myofiber-associated satellite cells transplanted into the immunocompetent mice expressed GFP, which would be expected to be recognized as a foreign protein by the immune system, and were obtained from mice of a genetic background different from that of the recipient animals. Such conditions would generally be expected to result in acute rejection of the implant. Despite the absence of immunosuppression, abundant myofibers and satellite cells were GFP-positive 21 months later. This very surprising observation merits further investigation because it implies that neither the transplanted cells nor the myoblasts, myotubes, or myofibers derived from them were rejected by the recipient's immune system. A similar observation was made by Cerletti et al.,5Cerletti M Jurga S Witczak CA Hirshman MF Shadrach JL Goodyear LJ et al.Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.Cell. 2008; 134: 37-47Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar who reported the absence of rejection following the transplantation of allogenic satellite cells for up to 4 months. However, the mechanism responsible for this lack of rejection was not investigated in the studies by Cerlutti et al. and Hall et al. Is this effect due to central tolerance (i.e., deletion in the thymus of lymphocytes that may react with donor antigens, including GFP) or to some sort of peripheral tolerance mechanism (i.e., the induction of regulatory T cells)? Another, more likely, possibility is that despite ongoing rejection the muscle fibers are continuously repaired by the large pool of satellite cells produced by the transplant protocol. It is possible that the donor satellite cells are not rejected because they do not express the major histocompatibility complex class II necessary for the presentation of antigens to the cytotoxic lymphocytes. The survival of the donor satellite cells would ensure the formation of new muscle fibers expressing GFP despite their repeated rejection, because only a minority of the nuclei in these fibers may be of donor origin. If a tolerogenic phenomenon is indeed responsible, it must be verified in large animals such as nonhuman primates, given that tolerance is much more easily obtained in mice than in humans. In summary, the findings of Hall et al. reinforce the remarkable potential of satellite cells as transplantable stem cells to repair muscles. At the same time, however, the study has raised several important unanswered questions, especially if one considers the translation of these intriguing but preliminary findings from the specific experimental conditions performed in mice to larger animals and humans. However, if a clinically acceptable method to stimulate the proliferation of the endogenous satellite cells within a patient's own muscles could be developed, this would represent a powerful strategy to prevent muscle aging and perhaps delay the progression of some muscular dystrophies. If this could be combined with the transplantation of healthy donor satellite cells or genetically corrected satellite cells generated from a patient's own cells, this approach could also lead to a treatment for several muscular dystrophies. On the other hand, the report of such dramatic effects on muscle structure and function as the consequence of the transplantation of so few cells will probably generate much excitement and expectation. However, given that some important aspects of the study are vague (rationale and method of myofiber isolation and transplantation) or were omitted (analysis of the absence of acute rejection), a cautious and critical approach to these results would be prudent. Indeed, this is particularly important in the field of muscular dystrophies, given the severity of some of these diseases and the daunting expectations that this study might raise among patients.

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.