Clarifying Misconceptions About Myoblast Transplantation in Myology
Authors: Daniel Skuk, Jacques P Tremblay
Journal: Molecular Therapy (2014)

Various misconceptions exist with regard to myoblast transplantation as a potential treatment for Duchenne muscular dystrophy (DMD). It is widely believed that myoblast transplantation is inefficient at inducing the expression of dystrophin in the myofibers of DMD patients, based on the conclusions drawn from several clinical trials conducted between 1991 and 1997. It is also widely believed that this inefficiency derives from both the poor survival and insufficient migration of the transplanted myoblasts as well as their rejection by the recipients' immune system. The source of these misconceptions is an oversimplified interpretation of the existing data along with a failure to recognize more recent evidence to the contrary. We provide here an updated and more nuanced view of the current state of the field of myoblast transplantation. Even today, when referring to clinical trials of myoblast transplantation as a potential treatment for DMD, authors tend to cite early trials published between 1991 and 1997 (reviewed in ref. 1Skuk D Myoblast transplantation for inherited myopathies: a clinical approach.Expert Opin Biol Ther. 2004; 4: 1871-1885Crossref PubMed Scopus (43) Google Scholar) instead of more recent trials published between 2004 and 2007 (refs. 2Skuk D Roy B Goulet M Chapdelaine P Bouchard JP Roy R et al.Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells.Mol Ther. 2004; 9: 475-482Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar,3Skuk 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 (177) Google Scholar,4Skuk D Goulet M Roy B Piette V Cote 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 (155) Google Scholar). In this light it is important to note that the early trials were performed using transplantation parameters (such as the distribution of injections, the number of grafted cells, and the protocol employed to control acute rejection) that lacked empirical support. For example, it was unknown at that time whether implantation of myoblasts by matrices of parallel injections separated by 5 mm (as done in most of the early clinical trials) would lead to efficient engraftment of cells. Subsequent studies in nonhuman primates (NHPs) showed that this inter-injection distance was too large for a significant engraftment.1Skuk D Myoblast transplantation for inherited myopathies: a clinical approach.Expert Opin Biol Ther. 2004; 4: 1871-1885Crossref PubMed Scopus (43) Google Scholar The more recent clinical trials were the fruit of nearly a decade of technical and conceptual improvements in study design and were performed using transplantation parameters previously defined in preclinical experiments on NHPs, the animal model that most closely reflects human physiology in preclinical research.5Herodin F Thullier P Garin D Drouet M Nonhuman primates are relevant models for research in hematology, immunology and virology.Eur Cytokine Netw. 2005; 16: 104-116PubMed Google Scholar These improvements included immunosuppression based on the use of tacrolimus and a better cell implantation technique. This technique—called "high-density injections"1Skuk D Myoblast transplantation for inherited myopathies: a clinical approach.Expert Opin Biol Ther. 2004; 4: 1871-1885Crossref PubMed Scopus (43) Google Scholar—consisted of matrices of closely spaced (1–2 mm or less) parallel and equidistant injections delivering small volumes of cell suspension (5–10 μl per cm of injection trajectory) homogeneously during the withdrawal of the needle in its intramuscular course. As a consequence, the latest trials2Skuk D Roy B Goulet M Chapdelaine P Bouchard JP Roy R et al.Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells.Mol Ther. 2004; 9: 475-482Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar,3Skuk 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 (177) Google Scholar,4Skuk D Goulet M Roy B Piette V Cote 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 (155) Google Scholar have generated better results with regard to expression of donor-derived dystrophin in patients' myofibers. Although there remains much room for improvement in engraftment efficiency in the transplantation protocols, the latest trials demonstrate the importance of employing transplantation protocols that have been developed in appropriate preclinical models. Another misconception is that most grafted myoblasts die shortly after transplantation, leaving so few surviving cells that it is impossible to obtain a satisfactory graft. In reality, the posttransplantation death does not annihilate the population of grafted cells and does not prevent optimal engraftment of the surviving cells. The experimental evidence in mice indicates that a significant fraction of grafted myoblasts die after transplantation but that nevertheless some survive and proliferate so as to replace those that are lost.6Beauchamp 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 (443) Google Scholar,7Skuk 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 (85) Google Scholar On the other hand, myoblast transplantation is dose-dependent: the number of hybrid myofibers increases with the number of grafted myoblasts in both mice8Rando TA Blau HM Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.J Cell Biol. 1994; 125: 1275-1287Crossref PubMed Scopus (799) Google Scholar,9Praud C Montarras D Pinset C Sebille A Dose effect relationship between the number of normal progenitor muscle cells grafted in mdx mouse skeletal striated muscle and the number of dystrophin-positive fibres.Neurosci Lett. 2003; 352: 70-72Crossref PubMed Scopus (11) Google Scholar and NHPs.10Skuk D Goulet M Tremblay JP Intramuscular transplantation of myogenic cells in primates: importance of needle size, cell number and injection volume.Cell Transplant. 2014; 23: 13-25Crossref PubMed Scopus (30) Google Scholar We demonstrated in NHPs that the number of hybrid myofibers increases until it plateaus, with the ceiling of graft efficiency dependent on the number of injections per volume of muscle.10Skuk D Goulet M Tremblay JP Intramuscular transplantation of myogenic cells in primates: importance of needle size, cell number and injection volume.Cell Transplant. 2014; 23: 13-25Crossref PubMed Scopus (30) Google Scholar Therefore, posttransplant cell death does not prevent optimal engraftment under a given set of transplantation conditions. That being said, the development of strategies to reduce early cell death should be encouraged so as to limit the number of cells required for successful engraftment. We and others examined very carefully the early posttransplantation survival of myoblasts using various methods to analyze cell death, survival, and proliferation, leading to various hypotheses to explain post transplantation myoblast death and the testing of corresponding strategies to enhance their survival.11Bouchentouf M Skuk D Tremblay JP Early and massive death of myoblasts transplanted into skeletal muscle: responsible factors and potential solutions.Curr Opin Organ Transplant. 2007; 12: 664-667Crossref Scopus (7) Google Scholar By contrast, similar rigorous analyses were lacking for other types of candidate myogenic cells, leading to the impression that myoblasts were particularly sensitive to early cell death following transplantation. We also note that most studies of early posttransplantation survival of myoblasts were performed in mice (in some cases involving transplantation of human myoblasts in mice, thus introducing additional complicating factors). However, there are no analyses of the survival of human myoblasts transplanted into humans. Clearly, this topic deserves more detailed study in a model, such as NHPs, whose physiology resembles more closely that of humans with respect to both the characteristics of the cultured myoblasts and immunological responses to transplantation. It has also been thought that grafted myoblasts exhibit poor capacity to migrate, and that this contributes to the inefficiency of their transplantation. In fact, grafted myoblasts do migrate,12Skuk D Goulet M Tremblay JP Transplanted myoblasts can migrate several millimeters to fuse with damaged myofibers in nonhuman primate skeletal muscle.J Neuropathol Exp Neurol. 2011; 70: 770-778Crossref PubMed Scopus (29) Google Scholar,13Skuk D Goulet M Tremblay JP Electroporation as a method to induce myofiber regeneration and increase the engraftment of myogenic cells in skeletal muscles of primates.J Neuropathol Exp Neurol. 2013; 72: 723-734Crossref PubMed Scopus (15) Google Scholar but in a manner most closely associated with their physiological function to support myofiber regeneration rather than in one ideally suited to facilitate their transplantation as a potential therapy. It is well known that myoblasts migrate in response to chemoattractant factors released at sites of damaged myofibers.14Goetsch KP Myburgh KH Niesler CU In vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair.J Muscle Res Cell Motil. 2013; 34: 333-347Crossref PubMed Scopus (28) Google Scholar Therefore, if the only regenerating myofibers following myoblast transplantation are those that were damaged by the injection needle, the grafted cells will migrate toward and fuse predominantly with such myofibers.12Skuk D Goulet M Tremblay JP Transplanted myoblasts can migrate several millimeters to fuse with damaged myofibers in nonhuman primate skeletal muscle.J Neuropathol Exp Neurol. 2011; 70: 770-778Crossref PubMed Scopus (29) Google Scholar If the myofiber damage is more extensive, the grafted myoblasts will migrate and fuse more diffusely throughout the injected muscle, a phenomenon that was observed not only in mice but also in NHPs.13Skuk D Goulet M Tremblay JP Electroporation as a method to induce myofiber regeneration and increase the engraftment of myogenic cells in skeletal muscles of primates.J Neuropathol Exp Neurol. 2013; 72: 723-734Crossref PubMed Scopus (15) Google Scholar Certainly, myoblasts do not migrate so as to fuse indiscriminately with undamaged myofibers (and this applies probably to other putative myogenic cells). As such, cell engraftment requires the induction of myofiber regeneration. Possible strategies to overcome this limitation include (i) inducing chemoattractant gradients to drive the spread of myogenic cells throughout the muscle, (ii) conferring on these cells the ability to penetrate the basal lamina without the help of inflammatory cells, (iii) inhibiting potential repulsive factors in intact myofibers, and (iv) inducing in quiescent myofibers the molecular signals required for cell fusion.13Skuk D Goulet M Tremblay JP Electroporation as a method to induce myofiber regeneration and increase the engraftment of myogenic cells in skeletal muscles of primates.J Neuropathol Exp Neurol. 2013; 72: 723-734Crossref PubMed Scopus (15) Google Scholar A final misconception is that rejection of grafted myoblasts limits their transplantation. In fact this issue might be irrelevant given the existence of effective immunosuppressive regimens designed to control acute rejection that are commonly used in clinical transplantation. One can debate the suitability of subjecting a myopathic patient to a regimen of immunosuppression depending on the degree of benefit expected from the transplantation of myoblasts (or other myogenic cells), but it is incorrect to say that myoblast transplantation is inefficient because of immune rejection. In some cases, rejection does not occur, such as following transplantation of autologous myoblasts, as was performed in a recent phase I/IIa clinical trial in patients with oculopharyngeal muscular dystrophy.15Perie S Trollet C Mouly V Vanneaux V Mamchaoui K Bouazza B et al.Autologous myoblast transplantation for oculopharyngeal muscular dystrophy: a phase I/IIa clinical study.Mol Ther. 2014; 22: 219-225Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Collectively, these common misconceptions have been used as a rationale to support the search for other myogenic cells for use in experimental transplantation. To date, however, myoblasts remain the only myogenic cells that have been shown unequivocally to engraft in humans2Skuk D Roy B Goulet M Chapdelaine P Bouchard JP Roy R et al.Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells.Mol Ther. 2004; 9: 475-482Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar,3Skuk 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 (177) Google Scholar,4Skuk D Goulet M Roy B Piette V Cote 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 (155) Google Scholar (Figure 1). They are the only cells that have been shown to induce the production of normal donor-derived dystrophin in significant numbers of myofibers of DMD patients, and there is also evidence that grafted myoblasts have the capacity to form new, small myofibers in DMD patients and probably to give rise to satellite cells (the skeletal muscle stem cell).3Skuk 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 (177) Google Scholar It remains possible that in the future another type of cell will outperform myoblasts for transplantation in DMD patients, but meanwhile it is important to recognize what has been achieved in clinical studies of myoblast transplantation.