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Short- versus long-term observations following MT through the polyethylene microtube. PKH-67 labeled monkey myoblasts (1 × 10⁶) were suspended in HBSS containing 50 ng of bFGF or IGF-1 and transplanted in SCID mice using the microtube technique. (a) Systemic injection of Evans blue revealed that only rare damaged myofibers (red florescence following Evans blue incorporation) could be observed next to microtube an hour after its insertion and after cells injection (green). (b) Sixty hours posttransplantation, green labeled myogenic cells (PKH-67) could be visualized at more than 700 μm of the microtube. (c) Dystrophin staining revealed that hardly any hybrid myofibers could be observed 14 days posttransplantation. Only occasional dystrophin expressing fibers were found close to the microtube insertion site. Scale bars: 165 μm.
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Duchenne muscular dystrophy (DMD) is an inherited disease and a main target of myogenic cell transplantation (MT). After the failure of the first clinical trials with DMD patients, the poor migration of transplanted cells has been suspected to be a major problem for a more effective clinical application of MT. Previous investigations suggested that...
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Citations
... The growth factor, vascular endothelial growth factor (VEGF) reduced hypoxia-induced death of human myoblasts in vitro and in a mouse model [151,160]. Other growth factors such as insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) were coinjected and promoted the overall migration of human myoblasts in various mouse models and stimulated cell migration and engraftment of monkey myoblasts in a nonhuman primate model [161]. Treatments with these factors stimulated components of proteolytic systems and thereby enhanced cellular migration. ...
The intrinsic regenerative capacity of skeletal muscle makes it an excellent target for cell therapy. However, the potential of muscle tissue to renew is typically exhausted and insufficient in muscular dystrophies (MDs), a large group of heterogeneous genetic disorders showing progressive loss of skeletal muscle fibers. Cell therapy for MDs has to rely on suppletion with donor cells with high myogenic regenerative capacity. Here, we provide an overview on stem cell lineages employed for strategies in MDs, with a focus on adult stem cells and progenitor cells resident in skeletal muscle. In the early days, the potential of myoblasts and satellite cells was explored, but after disappointing clinical results the field moved to other muscle progenitor cells, each with its own advantages and disadvantages. Most recently, mesoangioblasts and pericytes have been pursued for muscle cell therapy, leading to a handful of preclinical studies and a clinical trial. The current status of (pre)clinical work for the most common forms of MD illustrates the existing challenges and bottlenecks. Besides the intrinsic properties of transplantable cells, we discuss issues relating to cell expansion and cell viability after transplantation, optimal dosage, and route and timing of administration. Since MDs are genetic conditions, autologous cell therapy and gene therapy will need to go hand-in-hand, bringing in additional complications. Finally, we discuss determinants for optimization of future clinical trials for muscle cell therapy. Joined research efforts bring hope that effective therapies for MDs are on the horizon to fulfil the unmet clinical need in patients.
Graphical abstract
... (h) Co-engraftment of muscle cells with other cell types involved in muscle repair could enhance muscle regeneration and remedy dystrophic symptoms Kinoshita et al. (1996) Immune system Intramuscular injection of allogeneic myoblasts (LacZ) into NHPs treated with tacrolimus or without immunosuppressant Lymphocyte infiltration and increase in cytokines 7 days post MT in the absence of tacrolimus. ß-Galpositive fibers were not detected 4 weeks post MT Detection of ß-Gal-positive fibers 1, 4, and 12 weeks post MT with significant reduced lymphocyte infiltration and cytokines with tacrolimus compared to no tacrolimus administration Skuk et al. (1999) Notexin Allogeneic myoblasts (LacZ) were injected with or without notexin and varying cell number (4 Â 10 6 , 8 Â 10 6 , 24 Â 10 6 ) with 35-40 injections (separated by 1 mm) 8 Â 10 6 led to most ß-Gal-positive fibers 4 weeks post MT Injection of 8 Â 10 6 myoblasts suspended in notexin led to a 50% increase of ß-Gal fibers 4 weeks post MT Skuk et al. (2002) Injection, immune system Injection of allogeneic myoblasts (LacZ) with varying inter-injection distances (1 mm, 1-1.5 mm, 2 mm) and different concentrations of tacrolimus alone or combined with mycophenolate mofetil Detection of 25-67% ß-Gal-positive fibers 1 month post MT (injection distance 1 mm) Combination of tacrolimus with mycophenolate mofetil enabled a decreased dosage of tacrolimus Lafreniere et al. (2009) ...
... Attempts have been made to modulate key molecular determinants controlling these parameters ( Figure 3). The amount of donor muscle formed reportedly increased after exposure of donor myogenic cells, before or during their implantation, to factors altering the activity of signaling pathways initiated by FGF, IGF, IL-4, Wnt7a, and TGF-β superfamily members (149)(150)(151)(152)(153). Other approaches attempted to increase the survival of grafted cells by preconditioning cells with stressful stimuli or interfering with signaling pathways controlling cell death (154,155). ...
Muscular dystrophies are a heterogeneous group of genetic diseases, characterized by progressive degeneration of skeletal and cardiac muscle. Despite the intense investigation of different therapeutic options, a definitive treatment has not been yet developed for this debilitating class of pathologies. Cell-based therapies in muscular dystrophies have been pursued experimentally for the last three decades. Several cell types with different characteristics and tissues of origin, including myogenic stem and progenitor cells, stromal cells, and pluripotent stem cells, have been investigated over the years and have recently entered in the clinical arena with mixed results. In this review, we will do a roundup of the past attempts and describe the updated status of cell-based therapies aimed at counteracting the skeletal and cardiac myopathy present in dystrophic patients. We will present current challenges, summarize recent progresses, and make recommendations for future research and clinical trials.
... (h) Co-engraftment of muscle cells with other cell types involved in muscle repair could enhance muscle regeneration and remedy dystrophic symptoms Kinoshita et al. (1996) Immune system Intramuscular injection of allogeneic myoblasts (LacZ) into NHPs treated with tacrolimus or without immunosuppressant Lymphocyte infiltration and increase in cytokines 7 days post MT in the absence of tacrolimus. ß-Galpositive fibers were not detected 4 weeks post MT Detection of ß-Gal-positive fibers 1, 4, and 12 weeks post MT with significant reduced lymphocyte infiltration and cytokines with tacrolimus compared to no tacrolimus administration Skuk et al. (1999) Notexin Allogeneic myoblasts (LacZ) were injected with or without notexin and varying cell number (4 Â 10 6 , 8 Â 10 6 , 24 Â 10 6 ) with 35-40 injections (separated by 1 mm) 8 Â 10 6 led to most ß-Gal-positive fibers 4 weeks post MT Injection of 8 Â 10 6 myoblasts suspended in notexin led to a 50% increase of ß-Gal fibers 4 weeks post MT Skuk et al. (2002) Injection, immune system Injection of allogeneic myoblasts (LacZ) with varying inter-injection distances (1 mm, 1-1.5 mm, 2 mm) and different concentrations of tacrolimus alone or combined with mycophenolate mofetil Detection of 25-67% ß-Gal-positive fibers 1 month post MT (injection distance 1 mm) Combination of tacrolimus with mycophenolate mofetil enabled a decreased dosage of tacrolimus Lafreniere et al. (2009) ...
... Moreover, treatments with these factors also stimulated components of proteolytic systems and enhanced cellular migration in vitro. As observed with human myoblasts, monkey myoblasts co-injected with these growth factors also showed increased intramuscular migration in SCID mice [130]. A short term ex vivo treatment using Wnt7a, a member of the Wingless-INT (WNT) family, on satellite stem cells markedly increased cell dispersion and engraftment, which ultimately resulted in improved muscle function of dystrophic muscles [131] (Figure 3). ...
Muscular dystrophies (MD) are a group of genetic diseases that lead to skeletal muscle wasting and may affect many organs (multisystem). Unfortunately, no curative therapies are available at present for MD patients, and current treatments mainly address the symptoms. Thus, stem-cell-based therapies may present hope for improvement of life quality and expectancy. Different stem cell types lead to skeletal muscle regeneration and they have potential to be used for cellular therapies, although with several limitations. In this review, we propose a combination of genetic, biochemical, and cell culture treatments to correct pathogenic genetic alterations and to increase proliferation, dispersion, fusion, and differentiation into new or hybrid myotubes. These boosted stem cells can also be injected into pretreate recipient muscles to improve engraftment. We believe that this combination of treatments targeting the limitations of stem-cell-based therapies may result in safer and more efficient therapies for MD patients. Matricryptins have also discussed.
... There were experiments in mice to achieve the first objective by enhancing the migrative capacity of myoblasts [109][110][111][112][113][114]. However, this seems not beneficial for myoblast transplantation in monkeys, at least if there is not a context of myofiber regeneration to recruit the grafted cells [109]. ...
... Moreover, analyzed myoblasts were not able to fuse with undamaged muscle fibers, regardless of the growth factors used. 22 In our own studies we showed that the Sdf-1 could improve migration of satellite cell derived myoblasts and C2C12 myoblasts in vitro in metalloproteinase (MMP) dependent manner. 23 We also documented that Sdf-1 treatment enhanced embryonic stem cells (ESCs) and bone marrow derived mesenchymal stem cells (BM-MSC) migration and fusion with myoblasts in vitro, what was connected with the increase in tetraspanin CD9 expression. ...
The skeletal muscle regeneration occurs due to the presence of tissue specific stem cells - satellite cells. These cells, localized between sarcolemma and basal lamina, are bound to muscle fibers and remain quiescent until their activation upon muscle injury. Due to pathological conditions, such as extensive injury or dystrophy, skeletal muscle regeneration is diminished. Among the therapies aiming to ameliorate skeletal muscle diseases are transplantations of the stem cells. In our previous studies we showed that Sdf-1 (stromal derived factor −1) increased migration of stem cells and their fusion with myoblasts in vitro. Importantly, we identified that Sdf-1 caused an increase in the expression of tetraspanin CD9 - adhesion protein involved in myoblasts fusion. In the current study we aimed to uncover the details of molecular mechanism of Sdf-1 action. We focused at the Sdf-1 receptors - Cxcr4 and Cxcr7, as well as signaling pathways induced by these molecules in primary myoblasts, as well as various stem cells - mesenchymal stem cells and embryonic stem cells, i.e. the cells of different migration and myogenic potential. We showed that Sdf-1 altered actin organization via FAK (focal adhesion kinase), Cdc42 (cell division control protein 42), and Rac-1 (Ras-Related C3 Botulinum Toxin Substrate 1). Moreover, we showed that Sdf-1 modified the transcription profile of genes encoding factors engaged in cells adhesion and migration. As the result, cells such as primary myoblasts or embryonic stem cells, became characterized by more effective migration when transplanted into regenerating muscle.
... Using fewer cells per injection improves survival, and using multiple injections can be used to increase transplanted cell numbers and circumvent migration issues [9,46]. Alternatively, promigratory factors can aid distribution of cells and thereby potentiate improvements in muscle function [47,48]. ...
In Duchenne muscular dystrophy (DMD) and other muscle wasting disorders, cell therapies are a promising route for promoting muscle regeneration by supplying a functional copy of the missing dystrophin gene and contributing new muscle fibers. The clinical application of cell-based therapies is resource intensive, and it will therefore be necessary to address key limitations that reduce cell engraftment into muscle tissue. A pressing issue is poor donor cell survival following transplantation, which in preclinical studies limits the ability to effectively test the impact of cell-based therapy on whole muscle function. We therefore sought to improve engraftment and the functional impact of in vivo myogenically converted dermal fibroblasts (dFbs) using a pro-survival cocktail (PSC) that includes heat shock followed by treatment with insulin-like growth factor-1, a caspase inhibitor, a Bcl-XL peptide, a K<sub>ATP</sub> channel opener, basic fibroblast growth factor, Matrigel and cyclosporine A. Advantages of dFbs include compatibility with the autologous setting, ease of isolation, and greater proliferative potential than DMD satellite cells. dFbs expressed tamoxifen-inducible MyoD and carried a mini-dystrophin gene driven by a muscle-specific promoter. After transplantation into muscles of mdx mice, a 70% reduction in donor cells was observed by day 5, and a 94% reduction by day 28. However, treatment with PSC gave a nearly 3-fold increase in donor cells in early engraftment, and greatly increased the number of donor-contributed muscle fibers and total engrafted area in transplanted muscles. Furthermore, dystrophic muscles that received dFbs with PSC displayed reduced injury with eccentric contractions and an increase in maximum isometric force. Thus, enhancing survival of myogenic cells increases engraftment and improves structure and function of dystrophic muscle.
... There were experiments in mice to achieve the first objective by enhancing the migrative capacity of myoblasts [109][110][111][112][113][114]. However, this seems not beneficial for myoblast transplantation in monkeys, at least if there is not a context of myofiber regeneration to recruit the grafted cells [109]. ...
Duchenne muscular dystrophy is a genetic degenerative myopathy that causes progressive and irreversible loss of skeletal muscles. The transplantation of myogenic cells is an experimental strategy for the potential treatment of this disease. Although the repertoire of myogenic cells appears to have expanded in recent years, myoblasts are the only cells that have demonstrated to significantly engraft in clinical trials. In this chapter, we present the most relevant of the clinical experience made so far, in which our laboratory has had a greater involvement in the development of better transplantation protocols.
... Myogenic cells have also been coinjected with growth factors or with other cell types that improve cell survival, migration, and engraftment (20,38,164,173). For instance, short ex vivo treatment of satellite cells with Wnt7a considerably increases dispersion and engraftment that ultimately results in improved function of dystrophic muscles (23). ...
Skeletal muscles are essential for vital functions such as movement, postural support, breathing, and thermogenesis. Muscle tissue is largely composed of long, postmitotic multinucleated fibers. The life-long maintenance of muscle tissue is mediated by satellite cells, lying in close proximity to the muscle fibers. Muscle satellite cells are a heterogeneous population with a small subset of muscle stem cells, termed satellite stem cells. Under homeostatic conditions all satellite cells are poised for activation by stimuli such as physical trauma or growth signals. After activation, satellite stem cells undergo symmetric divisions to expand their number or asymmetric divisions to give rise to cohorts of committed satellite cells and thus progenitors. Myogenic progenitors proliferate, and eventually differentiate through fusion with each other or to damaged fibers to reconstitute fiber integrity and function. In the recent years, research has begun to unravel the intrinsic and extrinsic mechanisms controlling satellite cell behavior. Nonetheless, an understanding of the complex cellular and molecular interactions of satellite cells with their dynamic microenvironment remains a major challenge, especially in pathological conditions. The goal of this review is to comprehensively summarize the current knowledge on satellite cell characteristics, functions, and behavior in muscle regeneration and in pathological conditions. © 2015 American Physiological Society. Compr Physiol 5:1027-1059, 2015.
