Another New “Super Muscle Stem Cell” Leaves Unaddressed the Real Problems of Cell Therapy for Duchenne Muscular Dystrophy
Authors: Jacques P Tremblay, Daniel Skuk
Journal: Molecular Therapy (2008)

The history of preclinical efforts toward developing cell transplantation therapy to treat Duchenne muscular dystrophy (DMD) has been marked by a series of promising studies in mice, with sometimes surprising results, that generated large expectations that in some cases remain valid and in others have been refuted or slowly forgotten. Therefore, after many years working in the field of cell transplantation in neuromuscular diseases, we respond a priori to any announcement of a proposed "super-cell" for clinical cell regeneration therapy with a healthy dose of skepticism. This skepticism has been reinforced in many cases after a critical reading of the articles in question and has often been further validated by the inability of others to reproduce the reported results. Indeed, with few exceptions, the results published by different teams describing this or that new cell with greater proliferative or restorative capacities have not been reproduced by independent groups. It is against this backdrop that we read the recent report by Cerletti et al.1Cerletti 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 (347) Google Scholar describing the results of transplantation of a new type of muscle stem cell—a subpopulation of satellite cells referred to as "skeletal muscle precursors" (SMPs)—into dystrophic mdx mice. As has been shown with other cell populations previously isolated from muscle, often referred to as "stem cells," the transplantation into mdx mice of this new subpopulation was reported to produce new myofibers that were both hybrid and purely donor-derived, and that expressed the dystrophin protein that is lacking in mdx muscle, thereby leading to partial restoration of muscle function. The article by Cerletti et al.1Cerletti 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 (347) Google Scholar is one of a series of recent studies reporting the efficient engraftment of muscle precursor cells without previous expansion in culture in much smaller numbers than those of cells used for transplantation of myoblasts that had been expanded in vitro before intramuscular injection.2Sacco A Doyonnas R Kraft P Vitorovic S Blau HM Self-renewal and expansion of single transplanted muscle stem cells.Nature. 2008; (published online 17 September 2008 (doi 10.1038/ nature07384))PubMed Google Scholar,3Montarras 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 (829) Google Scholar The successful use of fewer cells in these new studies seems to be consistent with the current view that only a small percentage of myoblasts proliferated in vitro survive after intramuscular transplantation and that the number of molecularly corrected myofibers may therefore be dependent on a small subpopulation of the implanted myoblasts.4Beauchamp JR Morgan JE Pagel CN Partridge TA Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.J Cell Biol. 1999; 144: 1113-1122Crossref PubMed Scopus (445) Google Scholar,5Skuk D Caron NJ Goulet M Roy B Tremblay JP Resetting the problem of cell death following muscle-derived cell transplantation: detection, dynamics and mechanisms.J Neuropathol Exp Neurol. 2003; 62: 951-967Crossref PubMed Scopus (86) Google Scholar Cerletti et al.1Cerletti 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 (347) Google Scholar demonstrated that the transplantation of SMPs improved the contractile force of the mdx muscle by 5.5-fold when 94% engraftment was obtained. There was also a good correlation between the level of cell engraftment and increase in strength. This is good news for cell therapy. However, the exact technique used by Cerletti et al. to detect a 5.5-fold improvement will need to be further analyzed, because the difference in strength between the muscle of the untreated mdx mice and that of normal mice has never been reported to be so high. Indeed, it is surprising that restoration of dystrophin alone (strength was normalized to muscle cross-section) increases strength to such an extent. In addition, some of the properties described for these new SMPs are not exclusive to these cells but are shared with myoblasts. Formation of new myofibers by the fusion of donor cells with one another has already been reported following transplantation of myoblasts in mdx mice in 1995 (ref. 6Kinoshita I Vilquin JT Tremblay JP Mechanism of increasing dystrophin-positive myofibers by myoblast transplantation: study using mdx/beta-galactosidase transgenic mice.Acta Neuropathol. 1996; 91: 489-493Crossref PubMed Scopus (23) Google Scholar). The same applies to the re-engraftment of SMPs into the satellite cell niche. Cerletti et al.1Cerletti 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 (347) Google Scholar suggested that this was a property of the SMPs but not of their more differentiated progeny (see Figure 4E and F in their article). However, the control cells that the authors transplanted in these latter experiments were not muscle precursor cells (i.e., they were neither myoblasts nor daughters of SMPs) but the progeny of a completely different cell that was CD45– Sca-1– Mac-1– CXCR4–β1-integrin– and thus not muscle stem cell. It is not surprising, then, that the transplantation results were negative. In contrast, conversion of transplanted myoblasts into satellite cells and demonstration of their enduring regenerative function have been demonstrated by several groups7Ehrhardt J Brimah K Adkin C Partridge T Morgan J Human muscle precursor cells give rise to functional satellite cells in vivo.Neuromuscul Disord. 2007; 17: 631-638Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar,8Gross JG Morgan JE Muscle precursor cells injected into irradiated mdx mouse muscle persist after serial injury.Muscle Nerve. 1999; 22: 174-185Crossref PubMed Scopus (95) Google Scholar,9Heslop L Beauchamp JR Tajbakhsh S Buckingham ME Partridge TA Zammit PS Transplanted primary neonatal myoblasts can give rise to functional satellite cells as identified using the Myf5(nlacZl+) mouse.Gene Ther. 2001; 8: 778-783Crossref PubMed Scopus (90) Google Scholar,10Yao SN Kurachi K Implanted myoblasts not only fuse with myofibers but also survive as muscle precursor cells.J Cell Sci. 1993; 105: 957-963PubMed Google Scholar and are thus not new phenomena with SMPs. In fact, our group recently demonstrated in a clinical trial of myoblast transplantation in DMD patients that some transplanted cells were found 1 month after transplantation in a satellite cell position.11Skuk 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 (178) Google Scholar Importantly, certain practical limitations of cell therapy for DMD are not addressed in the new study. One of the main problems with cell therapy for DMD is that the transplanted cells fuse only with myofibers that are undergoing repair or hypertrophy. Thus, myoblasts injected intramuscularly fuse mainly with the myofibers damaged by the injection needle. Injection at every 2 mm in a muscle results in clear rows of myofibers expressing a reporter gene present in the transplanted cells (β-Gal for transplantation in monkeys) or dystrophin (transplantation in a DMD patient). Because muscle precursor cells fuse only with damaged myofibers, Cerletti et al.1Cerletti 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 (347) Google Scholar injected the myofibers along with cardiotoxin so as to induce muscle damage with the aim of obtaining better engraftment of the donor cells. Thus, SMPs do not resolve the problem that muscle precursor cells fuse only with damaged myofibers. Nevertheless, Cerletti and colleagues suggest that their SMPs have a better regeneration index (RI) than other muscle precursors. RI is calculated as the number of myofibers expressing a gene present in the donor cells (GFP or dystrophin) following the transplantation of 100,000 cells. The index can be used to compare different muscle precursor cells only if exactly the same transplantation procedure is used. The key factors include the number of injected cells, the particular muscle-damaging drug (e.g., cardiotoxin, notexin, or bupivicaine), the type of needle, and the number of injections—all factors that vary the number of damaged myofibers and thus the number of myofibers with which the donor cells can fuse. The number of donor cells needed to treat most of the muscles in a DMD patient is not a limiting factor when myoblasts are transplanted, because roughly 105 satellite cells can be extracted from 1 g of muscle from a human donor.12Webster C Pavlath GK Parks DR Walsh FS Blau HM Isolation of human myoblasts with the fluorescence-activated cell sorter.Exp Cell Res. 1988; 174: 252-265Crossref PubMed Scopus (110) Google Scholar Each of these satellite cells can be expanded in culture to obtain 17 million myoblasts (i.e., about 25 cell divisions) without reaching senescence and without losing myogenic capacity. This generates 17 × 1011 myoblasts from 1 g of muscle, enough to treat 6 × 104 cm3 of muscle if 30 million cells are injected per cubic centimeter as done in our recent clinical trial.11Skuk 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 (178) Google Scholar This is not the case for the SMPs isolated by Cerletti et al. Indeed, only 50,000 SMPs were isolated per gram of muscle. They transplanted 11,000 SMPs (i.e., the cells derived from 0.22 g of muscle) in the tibialis anterior (TA) muscle to generate about 400 myofibers positive for enhanced green fluorescent protein (eGFP)—or about 13% of the myofibers of an adult TA. Given that a mouse TA weighs less than 0.05 g, this means that the SMPs from at least four donor TAs must be isolated to treat one TA. The authors also transplanted 20,000 SMPs in the soleus, which weighs about 0.015 g. These 20,000 SMPs were obtained from 0.4 g of muscle, or the equivalent of 26 soleus muscles. So far, the SMPs cannot be expanded without differentiating into myoblasts and would thus no longer be stem cells with special biological properties. This means that to treat all the muscles of a DMD patient (weighing up to 80 kg in adults) and considering the muscle mass to be 40–50% of the total weight,13Landon DN Skeletal muscle: normal morphology, development and innervation.in: Mastaglia FL Walton J Skeletal Muscle Pathology. Churchill Livingstone, Edinburgh, UK1982: 1-87Google Scholar SMPs would need to be extracted from the total muscles of 4 to 26 cadaveric adult donors, depending on the number of SMPs injected per gram of muscle and the age of the DMD patient. Without effective expansion of the SMPs (while preserving their stem cell characteristics), treatment of a person with DMD in this fashion would seem to be impossible, because any realistic approach to cell therapy must be based on a type of cell that can be extensively expanded ex vivo before transplantation. Moreover, it is difficult to believe that the purification of stem cells by fluorescence-activated cell sorting (FACS) followed by their immediate transplantation into a donor would be acceptable in a clinical situation, because the fluorochrome-coupled antibodies used for the FACS purification would remain bound to the cells. In addition to the problem that any such antibodies would need to be approved for human use, they would trigger an immune response if they were of animal origin and/or coupled to a fluorochrome, and they could lead to complement fixation and destruction of the donor cells by the host macrophages if they were of human origin. In addition, given the number of cells to be purified, the cost of the antibodies and of the FACS procedure would be prohibitive. Another key factor limiting cell therapy for muscular dystrophies is that it currently requires immunosuppression when using allogeneic cells obtained from a normal healthy donor. This is certainly a problem, because immunosuppressive drugs produce several adverse effects. Cerletti et al.1Cerletti 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 (347) Google Scholar suggested that their SMPs could be transplanted without immunosuppression because the myofibers expressing eGFP were still present after 4 months even without immunosuppression. The frequent claim that stem cells have immunological privilege cannot be extrapolated to a graft of stem cells that differentiate after transplantation. Although some stem cells may not express the major histocompatibility complex (MHC) before differentiation, the differentiated cells that they produce will express MHC, and this will lead to rejection in an allogeneic context. The apparent absence of rejection observed by Cerletti et al. at 4 months is probably due to the isolation of their donor cells from C57BL/Ka or C57BL/Ka-β-actin-eGFP mice, whereas the recipients were mdx mice (C57BL/10ScSn-Dmdmdx). The donor and host cells thus differed only for minor antigens. We have shown that transplantation of myoblasts obtained from an MHC-compatible donor can lead to a slower rejection than when they are obtained from an MHC-incompatible donor, if they have differences in minor antigens.14Boulanger A Asselin I Roy R Tremblay JP Role of non-major histocompatibility complex antigens in the rejection of transplanted myoblasts.Transplantation. 1997; 63: 893-899Crossref PubMed Scopus (23) Google Scholar In fact, depending on the histocompatible strains used, rejection of the cells due to the presence of minor antigens can be more or less rapid, and in some cases dystrophin-positive myofibers of donor origin could still be detected 16 weeks after the transplantation of normal myoblasts in the absence of immunosuppression.15Vilquin JT Wagner E Kinoshita I Roy R Tremblay JP Successful histocompatible myoblast transplantation in dystrophin-deficient mdx mouse despite the production of antibodies against dystrophin.J Cell Biol. 1995; 131: 975-988Crossref PubMed Scopus (76) Google Scholar This was due not to any special immune-privileged status of the transplanted myoblasts but simply to the fact that rejection occurs more slowly when it is caused by only minor antigens. Cell therapy is a therapeutic approach that should be pursued despite the promising results obtained by exon skipping in the treatment of DMD.16van Deutekom JC Janson AA Ginjaar IB Frankhuizen WS Aartsma-Rus A Bremmer-Bout M et al.Local dystrophin restoration with antisense oligonucleotide PRO051.N Engl J Med. 2007; 357: 2677-2686Crossref PubMed Scopus (699) Google Scholar,17Yokota T Duddy W Partridge T Optimizing exon skipping therapies for DMD.Acta Myol. 2007; 26: 179-184PubMed Google Scholar Indeed, exon skipping is not a potential solution for several types of dystrophies because in many cases the absence of one or several exons will lead to a nonfunctional protein, whereas the transplantation of normal muscle precursor cells will always introduce a normal version of the defective gene in all recessive dystrophies. In addition, only the direct intramuscular implantation of cells able to form new skeletal muscle is a realistic approach to treat patients who are unlikely to benefit from various forms of gene therapy, such as those whose skeletal muscles have already been largely destroyed by the degenerative process of DMD.18Skuk D Myoblast transplantation for inherited myopathies: a clinical approach.Expert Opin Biol Ther. 2004; 4: 1871-1885Crossref PubMed Scopus (44) Google Scholar However, it appears unlikely that cell therapy for the muscular dystrophies will be revolutionized by reporting new types of "stem" cells suggested to possess particular biological advantages following evaluation under particular experimental conditions in mice. Although they certainly contribute to a better understanding of the properties of various muscle precursor cell populations, such studies ignore the complex challenges of clinical application of cell therapy in general and do not address the critical problems faced by therapeutic strategies involving the intramuscular transplantation of myogenic cells. We believe that the advancement of cell therapy for muscular dystrophies will necessitate sustained work to develop pertinent solutions applicable in the clinical context to (i) reduce the number of intramuscular injections required for efficient donor cell engraftment and (ii) develop strategies to avoid the need for sustained immunosuppression either by transplanting autologous genetically corrected cells or by developing a sustained immunological tolerance to normal allogeneic cells.