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© The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Myoblast preparation for transplantation into injured myocardium

Monika Seidel1, Natalia Rozwadowska1, Kinga Tomczak1,2 and Maciej Kurpisz1,*

1 Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-479 Poznan, Poland
2 Children's Hospital Boston, Harvard Medical School, MA, USA

* Corresponding author. Tel: +48 6579 202; fax: +48 8233 235.E-mail address: kurpimac{at}man.poznan.pl


   Abstract
Top
 Abstract
Introduction
 Myoblast subsets
 Myoblast--application and...
 Future perspectives
 Acknowledgement
 References
 
Skeletal muscle stem cells are defined as the quiescent mononucleated cells localized outside the sarcolemma but within the basal lamina of muscle fibre. Extensive research on myoblasts revealed considerable diversity within this population: side population (SP), muscle-derived stem cells (MDSCs), and satellite cells (myoblasts). These cells possess different phenotypes and unique plasticity. In order to isolate single population, numerous techniques were developed; this includes fluorescence activated cell sorter (FACS) sorting and pre-plating. Because all clinical trials based on skeletal muscle-derived cells involved myoblast population, more detailed attention has been focused on their expression profiling. These studies have provided the list of gene clusters which express remarkable changes upon differentiation.

Owing to the myoblasts accessibility, their autologous origin, ease of their isolation, and in vitro expansion, these cells have gradually become one of the major tools for ex vivo therapy. The major limitation of the myoblast therapy is poor survival of cells. In this context, the isolation and identification of MDSCs in animals triggered a new hope in the field of myopathies treatment.

Although MDSCs give new possibilities for cell therapy, it is clear that taking MDSCs into the clinics requires more detailed understanding of the signals that govern their behaviour and fate.

Key Words: Satellite cells • Myoblasts • Heart regeneration


   Introduction
Top
 Abstract
 Introduction
Myoblast subsets
 Myoblast--application and...
 Future perspectives
 Acknowledgement
 References
 
Myoblast transplantation has been applied as a therapy for skeletal muscle-related diseases, as a gene delivery vehicle, and as a way to learn whether the implanted muscle cell precursors can improve functions of the infarcted myocardium. The population of cells obtained from digested skeletal muscle oligobiopsy contains a variety of cell types, i.e. fibroblasts, endothelial cells, macrophages, and circulating bone marrow cells. Myogenic cells isolated from skeletal muscle have also shown to be highly heterogeneous. Using various techniques, populations of cells with different phenotypes as well as distinct stem cell-like properties were identified.

Historically, skeletal muscle stem cells were defined as the quiescent mononucleated cells localized outside the sarcolemma but within the basal lamina of muscle fibre. Since then, the prospective application of these cells led to numerous studies focused on the muscle stem cell biology, their markers, and cell isolation and propagation in vitro.

The research on functional muscle stem cells is mainly based on implanted cell survival in post-infarction heart and, in the case of Duchenne muscular dystrophy (DMD), evaluation of dystrophin expression (after transplantation into mdx mice, which is an animal model of this muscle disorder). These models constitute the basis for the comparison of myoblast-like population properties obtained from skeletal muscles.


   Myoblast subsets
Top
 Abstract
 Introduction
 Myoblast subsets
Myoblast--application and...
 Future perspectives
 Acknowledgement
 References
 
Side population
Mouse bone marrow was the first source of the rare cell population identified by Hochest 33342 dye pump and FACS sorting. The obtained population was defined as side population (SP) cells.1 On the basis of the same methodology, numerous tissues have been studied and tissue-specific SP cells were isolated afterwards.2 SP cells isolated from the skeletal muscle are multipotential progenitors that do not express muscle markers. Immunohistological techniques revealed that Abcg2 protein (Abcg2 transporter expression is the main determinant of SP population because of its crucial role in Hochest 33342 efflux) is present on cells located in the muscle interstitium, i.e. outside the basal lamina.3 Muscle SP cells are multipotential progenitors, which were initially found to reconstitute a haematopoietic system after lethal irradiation of mice. The intravenously administered SP cells were localized in muscle and bone marrow, which supports the hypothesis that these cells are able to target skeletal muscle from circulation. The SP cells fused into myofibres in vivo,2 but were unable to differentiate into myotubes in vitro unless they were co-cultured with the main population of myoblasts.4

Several studies investigated the molecular phenotype of murine muscle SP cells. It was proved that most of these cells have been positive for Sca-1 antigen, but the reports concerning other surface antigens such as CD34, CD31 (endothelial cell marker), and CD45 (haematopoietic cell marker) were inconsistent. The reproducible method for muscle SP cell isolation was proposed by Kunkel and co-workers,5 furthermore showing that isolation parameters, such as dye concentration and tissue dissociation, can affect the homogeneity of isolated cells. The pure fraction of cells positive for Sca-1 and negative for CD45 antigens was obtained by standard cell isolation. The transcriptome analysis of this population showed the lack of CD31 mRNA, whereas FACS sorting revealed the presence of CD34 antigen on almost 50% of cells.3

On the contrary, recent studies showed that the majority of isolated SP muscle cells (Hochest staining) were positive for Sca-1 (90%) and CD31 (>85%), but almost all of them were negative for CD45. More than a half of these cells were also positive for CD34 antigen. After detailed FACS sorting, SP cells were divided into three subpopulations on the basis of the CD31 and CD45 markers (CD31– CD45––muSP-DN; CD31+CD45–muSP-31; and CD31– CD45+–muSP-45).6 These cell populations were shown to be distinct in myogenic and haematopoietic potentials for both in vitro and in vivo conditions.

muSP-31 was the main population of SP cells obtained from healthy muscles. During the regeneration process, these cells showed neither any proliferation activity nor myo-, osteo-, or adipogenic potential. Gene expression screening showed that these cells were positive for endothelial markers (such as Tie2, Flk1, vWF, and others), suggesting their close relationship to committed endothelial cells.

muSP-45 population was increased during regeneration, but surprisingly, these cells were negative for Ki67 antigen, suggesting that their proliferation activity was rather low. Transplantation experiments confirmed the bone marrow origin of mSP CD45+ population. These cells did not express any of the investigated myogenic or endothelial markers, but in the case of the regenerating muscle, the muSP-45 cells showed a weak expression of {alpha}SMA, PDGFRß, and follistatin. The CD45+ cells from SP population were shown to exhibit both myogenic and haematopoietic potentials, the latter was found to be restricted only to this population.7

The novel population of SP cells (CD31– CD45–), called muSP-DN, exhibited the capacity for mesenchymal differentiation (osteo-, adipo-, and myogenic characteristics). Their differentiation potential was restricted to mesenchymal lineage; in fact, the lack of haematopoietic colonies was soon revealed and these cells failed to rescue the lethally irradiated mice. During muscle regeneration, the number of muSP-DN cells was increased. These cells actively proliferated after injury as indicated by Ki67 immunostaining. Under non-regenerating conditions, muSP-DN population was negative for mesenchymal cell markers, whereas upon muscle regeneration, it became positive for both mesenchymal and developmental regulators. Bcrp1 (Abcg2) expression analysis showed that these cells lack the Brcp1 transporter responsible for SP phenotype of muSP-CD31–. As muSP-DN cells were shown to actively exclude Hochest dye, this finding brought into question the exact localization of muSP-DN cells.6

The Pax7, a paired box transcription factor responsible for myogenic specification, was absent in SP cells. The Pax–/– knockout mice did not have any alteration in both number and content of muscle side cell population. The Pax–/– SP cells were shown to possess 10-fold higher ability to form haematopoietic colonies in comparison with wild SP.8

The analysis of the transcriptional profile of the crude SP fraction obtained from skeletal muscle revealed that SP cells shared some transcripts with embryonic stem cells. The skeletal muscle and bone marrow SP overlapped almost half of mRNA, although 483 transcripts were specific to skeletal muscle SP.3

The haematopoietic potential of the cells obtained (without Hochest staining) from human muscle was measured by Tsuboi et al.9 The CD45– cell population was found to be deprived of haematopoietic activity. On the contrary, the CD45 + cells from human muscle gave rise to haematopoietic colonies. Retrospective study of the individuals who had undergone bone marrow (BM) transplantation showed that all CD45+ cells were of donor origin.

Specific features of SP cells identified in a variety of tissues by optimal isolation procedures remain to be determined. The unambiguous recognition of muscle SP cells heterogeneity and phenotype will provide the tools for careful selection of the donor cells for stem cell therapy not only for skeletal muscle disorder but also for heart failure (HF) or urinary incontinence.

Muscle-derived stem cells
The pre-plating method, based on the differential adherence abilities of freshly isolated muscle cells, has been used to obtain muscle-derived stem cells (MDSCs). The late adherent cells (named pp6) showed the myogenic (desmin and c-met) and stem cell (Sca-1, Flk-1, and CD34) markers.10

Within the late pre-plated myogenic cells, nearly 95% expressed desmin in contrast to early pre-plated cells (5%) and were positive for Sca-1 but negative for CD34 and c-kit markers.11 However, MDSCs obtained by other investigators showed a distinct phenotype: low desmin (<40% positive), Sca-1+, CD34+, and Bcl2+. FACS analysis showed the presence of CD34 and Sca-1 antigens, and RT–PCR revealed transcripts for MyoD, Bcl2, and MNF nuclear factors.12 In comparison with the early pre-plated cells, MDSCs gave rise to a significantly higher number of dystrophin positive myofibres when transplanted into muscles of mdx mice. After in vitro stimulation, MDSCs expressed both neural and endothelial cell markers. Moreover, transplantation experiments suggested that injected (not stimulated) MDSC differentiated towards other lineages (nerves and blood vessels). However, one cannot exclude fusion as the mechanism of new phenotype acquisition.12 Additionally, MDSCs obtained by the pre-plating technique reconstituted the haematopoietic system in the lethally irradiated mice.13 The transdifferentiation of highly purified rat myogenic cells (pp6) into osteogenic lineage (pp6) was induced by bone morphogenic protein (BMP-2) in in vitro culture.14

MDSCs could retain their phenotype for more than 30 passages without any sign of tumour transformation and karyotype imbalance.12 The recent work from Huard's group was focused on the self-renewal and long-term culture of muscle stem cells (pp6). MDSCs expanded for 300 population doublings (PDs) (theoretical yield of 10100 cells in 6 months). During the long-term in vitro culture, the stem cell marker expression (Sca-1 and CD34) remained stable until 200 PDs and was subsequently diminished afterwards. Both fusion and differentiation potentials seemed to be reduced in contrast to the anchorage-independent growth on soft agar that appeared to be increased. Furthermore, one out of eight mice injected with considerably expanded MDSC developed neoplastic tumour.15

Described features of the MDSCs were studied in a rodent model (mainly in mouse). There are very limited data concerning the cell surface antigens and molecular markers of human MDSCs. The same term, i.e. human MDSCs, was used in the case of cells isolated without serial pre-plating from brachioradialis muscle. These cells were cultured in non-standard conditions, i.e. in serum-free medium. They did not express CD34, CD45, CD31, FLK-1, BCL2, or V-cadherins, but showed a weak signal for CD105 and CD133 and were strongly positive for desmin. Although human MDSCs were able to differentiate in vitro into neural lineages and expressed some of the neuronal markers after in vivo engraftment, they failed to transdifferentiate.16 MDSCs from human muscle remain to be fully identified with regard to their phenotype and localization as the precise isolation of these cells and culture procedures constitutes the groundwork for subsequent therapeutic success.

Satellite cells and myoblasts
Satellite cells (Scs) were identified over 40 years ago and, at this time, were thought to be committed to muscle stem cells with a major role in skeletal muscle repair and growth. Their location beneath the basal lamina of mature myofibres constitutes the second part of the definition.17 The muscle Scs developed from Pax3 positive cells in the hypaxial somite.18 The Sc number in adult muscle stabilized between 1 and 5% of subluminar nuclei in mice, whereas during a neonatal period, it could reach as much as 32%.19 Because of intensive research, this population is relatively well known with respect to the cell origin, molecular markers, and the process of quiescence and activation.

A variety of protein markers were found to be associated with Scs, among them, CD34, M-cadherin, syndecan-3 and syndecan-4, and c-met and Pax7 were the most broadly studied. FACS separation of Sc isolated from Pax3GFP/+mice revealed the phenotype of quiescent cells: Pax7+, CD34+, CD45–, and Sca1–. This allowed to isolate the wild-type Scs.20 It is notable that the markers of quiescent, activated Scs and their progeny are distinct; moreover, propagation in in vitro condition may rapidly change the phenotype of the studied cells. Quiescent cells appeared to be negative for the myogenic regulatory factors (MyoD, Myf5, myogenin, and MRF4), but during activation, their expression was upregulated.21 Moreover, some transcripts were flip-flopped to alternative splice variants when silent Sc started to proliferate (CD34+ from truncated to a full-length form, MNF ß-form to MNF {alpha}-form).22,23

The transcription factor, Pax7, was found to be expressed in both Scs and proliferating myoblasts but during differentiation, it was downregulated. Furthermore, a proportion of Sc retained Pax7 expression and remained undifferentiated, possibly reconstituting a quiescent pool of the Sc. The Pax7–/– knockout mice displayed severe functional impairment of Sc without affecting overall organization of muscle fibre and a significant reduction in fibre diameter was also observed. It has been recently shown that the lack of Sc in Pax7 mutant mice occurred as the result of cell cycle defect accompanied by apoptosis.24

Unfortunately, the immunohistochemical labelling of Pax7 in biopsied samples of normal or pathological human skeletal muscles as well as in vitro culture argued strongly against the use of Pax7 as a marker for human Sc. The expression of Pax7 was not restricted to Scs, but it was also found in the nuclei of myofibres. Furthermore, some Pax7 negative Scs were also found.25

Scs have been considered as unipotent myogenic stem cell precursors for a long time, which upon appropriate stimulation, expressed muscle regulatory factor (MRF) and then proliferated to undergo terminal differentiation. However, in vitro culture of primary myoblasts with BMP or adipogenic inducers resulted in their transdifferentiation into osteocytes and adipocytes, respectively.26 In another study, this phenomenon was found to occur without any stimulation in in vitro Sc cultures after cell isolation from a single muscle fibre. Thus, it was shown once again that Sc possessed mesenchymal plasticity and could give rise to non-myogenic descendants. However, the mechanism of choosing the differentiation pathway by a particular cell remains to be determined.27

Almost all data concerning phenotype, culture behaviour, differentiation, and molecular markers of heterogeneous cells (isolated from skeletal muscle) originated in rodents. There are hardly any data with regard to human and other primate muscle cells; thus, direct interspecies comparisons should be avoided. The large-scale gene expression study of human myoblast differentiation or its ageing showed that there is a significant distinction between rodent and human myogenesis.28,29

Myoblast transcriptome analysis
In the recent years, several studies have analysed the whole transcriptome of differentiating myoblasts using various genome-wide examination techniques such as high-density oligonucleotide microarrays.30–35 Myogenic differentiation programme can be selectively triggered in cultured C2C12 myoblasts upon withdrawal of media-derived growth factors and mitogens. After switching to differentiation medium (day 0), cells withdraw from the cell cycle, start fusing (around day 2), and subsequently form elongated myotubes that twitch spontaneously (day 6). Moran et al.32 presented analysis of approximately 11 000 murine transcripts of four time points of differentiating C2C12 mouse skeletal muscle cells, whereas Shen et al.33 conducted a more focused analysis of events during C2C12 myoblast cell cycle withdrawal using cDNA microarrays containing approximately 10 000 non-redundant murine IMAGE clones. Delgado et al.31 have studied the transcriptional events occurring within the first 24 h of serum withdrawal and initiation of differentiation over the 1-day time course. Tomczak et al.35 used approximately 24 000 probe sets for murine genes on Affymetrix chips over eight time points spanning 12 days of muscle differentiation. Moreover, the microarray and chromatin immunoprecipitation assays in mouse embryo fibroblasts were used to examine discrete subprogrammes of gene expression regulated by MyoD. These studies were complemented by large-scale gene expression profiling of five time points (days 0, 1, 4, 6, and 14) in primary human myoblast cultures by Sterrenburg et al.34

All these reports provide lists of genes whose expression changes over time in a highly organized manner. There is roughly a 30% overlap between the mouse and human differentially expressed genes. Furthermore, clusters of genes shown in these reports have shapes, suggesting similar expression dynamics of genes induced during the process of myogenesis. It is well known that skeletal muscle differentiation is controlled by a complex series of transcriptional factors including the MRFs and MEF2 family of MADS-box (abbreviation for MCM1, AGAMOUS, DEFICIENS, and SRF) transcription factors that need to be expressed to define the cells to a skeletal muscle fate. In a study of Tomczak et al.,35 the primary MRFs, Myf5 and MyoD, were induced early in the time course, but Myf5 expression decreased gradually and MyoD transcripts peaked on day 0. In contrast, the secondary MRFs, myogenin and Myf6, were turned on later in the time course. Myogenin expression was induced at day 0 and had the highest expression values on day 2. Myf6 was not expressed until day 4. Four probe sets for Mef2c were upregulated by day 2 as well. Thus, as expected from the previous studies, each myogenic transcription factor exhibited unique expression kinetics. Describing in much detail the genes involved in the process of myogenesis is beyond the scope of current review. However, expression profiling studies have also provided useful insights into the skeletal muscle regeneration process that was analysed after muscle damage in mice.36,37

Zhao et al.37 looked at the temporal expression profiling of specific time points during regeneration after cardiotoxin (CTX) injury to discover novel downstream targets of MyoD. Slug, a gene coding for zinc finger protein belonging to the Snail family, has been identified and in vivo experiments confirmed that MyoD binds to the promoter of this gene. This data suggest that the Slug protein is important for muscle regeneration.

A very comprehensive analysis of transcriptional changes in the mouse model of regeneration using microarrays and semi-quantitative RT–PCR was described by Yun and Wold.36 After the analysis of genes important for cell cycle control and DNA replication during the early stages of muscle regeneration, these researchers looked at the group of imprinted genes and also genes with functions to inhibit cell cycle progression induced upon activation of Scs to differentiate. Re-entry of Sc into the cell cycle from quiescence requires activation of the cyclin-dependent kinase(s)/retinoblastoma/E2f transcription factor signalling pathway. The expression of eight genes related to cell cycle or myogenesis known to be induced during regeneration (cyclins A2, B, p107, Mcmd2, Mcmd3, MyoD, E2f1, and E2f2) was abolished in muscles lacking Sc activity after gamma irradiation. Interestingly, regeneration was severely compromised in E2F1 null mice but not affected in E2f2 null mice, suggesting that these two factors play distinct roles in adult skeletal muscle regeneration.

In addition, the results of gene expression studies were carried out in muscle disease processes in which regeneration takes place.38–40 In DMD patients, there is an upregulation of many structural genes (Myl and Tnnt isoforms),38,40 whereas some other structural genes were found to be downregulated in both DMD and alpha-sarcoglycan-deficient patients (alpha-1 syntrophin and alpha-tubulin).38 These global gene expression analyses add critical new information to existing pathophysiological models of dystrophin and alpha-sarcoglycan deficiency. Despite the fact that the microarrays detect only changes in transcriptional levels, mRNA expression profiling remains a powerful technique, particularly in temporal experiments in vitro. Besides confirming the changing expression patterns of known skeletal muscle genes over time, it allows the identification of novel candidate genes involved in myogenesis. It seems especially compelling to analyse, in more detail, genes that are induced shortly before and at the time of muscle fusion. The switch from a proliferative state to a differentiative state in the classical C2C12 time course occurs primarily between days –1 and +2. The expression profiling papers presented earlier agree that genes clustering at this important time are primarily involved in the signalling pathways, transcription, cell adhesion, cell cycle regulation, cell cycle arrest, or apoptosis. As expected, these studies detected the decrease of cyclins Ccna2, Ccnb2, and Ccnd1 at days –1 and 032–34 and the increase of Ccnd3, Rb, and cyclin-dependent kinase inhibitors that need to be upregulated for permanent withdrawal of myoblasts from cell cycle. Conversely, the negative regulators of cell division, Cdkn1a (P21) and Cdkn1c (P57), were required for myogenic differentiation, and in these experiments, were induced between days –1 and 2.32,33,35

Interestingly, together with the genes that are known to be upregulated in the process of cell fusion, such as Vcam1, or adhesion, such as Itga5 (fibronectin receptor alpha), several genes were not previously associated with a role in skeletal muscle differentiation. For example, Bersteins' group identified 17 differentially expressed genes, not previously associated with muscle development, that are likely to play important regulatory roles in early myoblast differentiation and cell cycle withdrawal. Two of these, dimethylarginine dimethylaminohydrolase (DDAH2) and glycosyl phosphatidylinositol-anchored protein (Ly-6A) were chosen as the best candidates for further studies.33 Beggs' group identified prostaglandin E receptor 4 (Ptger4) as a good candidate for a novel gene involved in the process of muscle fusion as this gene was specifically upregulated on day 0 (fold change 2.1).35

Moreover, not all transcripts whose expression was remarkable in these experiments (~30%) were annotated at the moment and many remain unknown for their potential role(s) in muscle differentiation. Interestingly, multiple probe sets for unknown genes were classified together with known genes involved in every step of muscle differentiation. Further characterization and more detailed studies of each will lead to a better understanding of muscle fusion and differentiation.


   Myoblast—application and function
Top
 Abstract
 Introduction
 Myoblast subsets
 Myoblast--application and...
Future perspectives
 Acknowledgement
 References
 
The principal role of Scs is skeletal muscle regeneration. These cells become activated upon muscle injury, and they start to proliferate, differentiate, and fuse together to form multinucleated myotubes that mature upon innervation to become functional muscle fibres.41 In the beginning, Scs attracted much attention because of their prospective application in sport medicine where increased efficiency of muscle regeneration is highly desirable. Almost immediately, myoblasts were also engaged in muscular dystrophy therapy: first as a source of correct genes (e.g. dystrophin in DMD) in allogenic transplantation and later as vehicles for ex vivo therapy. So far, transplantation of cultured myoblasts has been used as a sole or combined therapy to treat HF, myocardial infarction, non-ischaemic cardiomyopathy, and urinary incontinence, to name a few.42–46 Owing to the myoblasts accessibility, their autologous origin, ease of their isolation, and in vitro expansion, these cells have gradually become one of the major tools for ex vivo therapy aiming to deliver relevant genes into the blood stream. The improvement in the transfection methods and refinement of transgenic expression regulation47,48 considerably accelerated research in this area. The potential myoblast application for ex vivo therapy has been demonstrated for insulin,47 erythropoietin,49 interleukin-10,50 and many others.

Novel ways of treatment—muscle-related diseases
Most myopathies possess a molecular mutation that affects the structural or cytoskeletal proteins in skeletal muscle. DMD, the most common and the most devastating form, was the first disease entity in which the potential benefit of skeletal myoblast transplantation was noticed. DMD is characterized by the absence of a cytoskeletal protein called dystrophin, resulting in muscle fragility and widespread degeneration. This process leads to the continuous degeneration–regeneration cycles that ultimately exhaust the Sc pool.51 In 1989, Partridge et al.52 showed that intramuscular injection of C2C12 cells (an immortal mouse myoblast cell line) could efficiently reconstitute dystrophin-positive fibres in mdx mice. The first clinical trial using skeletal myoblasts to treat DMD was conducted by Hooper53 in 1990. Law et al.54,55 reported that multiple injections of skeletal myoblasts obtained from healthy donors into the muscles of DMD patients resulted in a dystrophin expression in myofibres up to 6 years after transplantation. However, the small size and the oval shape of some of these fibres implied that they were not functionally competent.54 The results of other clinical trials were much less encouraging and failed to prove any beneficial effect of this strategy in humans. The reasons for the failure of such approaches in humans were numerous: first, C2C12 mouse cells have an unlimited life span and are syngeneic with mdx mice (lack of such equivalent in humans); secondly, the massive death of grafted cells (due to the inflammation and then due to cell-mediated immunity directed against donor antigens and against wild-type of dystrophin); and thirdly, the limited distribution of injected cells because of their low migration capacities.46 In order to circumvent at least some of these hurdles, the procedures involving pharmacological partial immunosuppression, the application of neutralizing antibodies directed against the surface molecules of infiltrating cells and modifications of the muscle connective tissue, were undertaken.46 Because these attempts improved myoblast survival only to some extent, new experimental approaches, aiming to eliminate immunological problems, were designed. The idea of ex vivo gene therapy was to isolate autologous skeletal myoblasts, expand them in vitro, transfect with dystrophin or utrophin (a related protein that compensates for the loss of dystrophin, its presence at neuromuscular junctions in DMD patients eliminates the immune response problem56), and finally to re-inject into DMD patient's muscles. It turned out, however, that this approach faced at least two serious problems: first, finding of the appropriate vector able to accommodate extremely large dystrophin gene and secondly, a considerably limited life span of myogenic cells isolated from DMD patients.

The length of the dystrophin coding sequence, which is ~11 kb, considerably reduces the efficacy of the transfection of primary cultured myogenic cells. In this context, the idea of using truncated versions of dystrophin cDNA had seemed attractive, but, unfortunately, turned out to be of a questionable functional efficacy. Viral vectors (adenoviruses and herpes viruses) capable of delivering the complete coding sequence of dystrophin are mainly episomal. The lack of genomic integration leads to transgenic loss during proliferation and excludes their application in DMD therapy.57 Additionally, viral vectors are considered less safe than non-viral vectors. In regard to the clinical application, this issue seems to be a serious problem and needs to be carefully addressed. Quenneville et al.58 developed a non-viral approach to efficiently and stably transfecting human myoblasts. This method combines nucleofection (electroporation of cells maintained in special solution) with {Phi}C31 integrase system responsible for site-specific integration of the transgene. Co-nucleofection of the {Phi}C31 integrase plasmid and a large plasmid containing enhanced green fluorescent protein (eGFP)-full-length dystrophin fusion gene, bearing attB sequence, produced fluorescent human myoblasts able to form fluorescent myotubes after 1 month of in vitro culture. Unfortunately, the majority of cells with a high expression of the dystrophin gene underwent morphological changes and did not survive.58 As normal myoblasts do not express the dystrophin and because the protein expression appears only after the myotube formation, these results strongly suggest that the rapid death of the transfected cells was due to dystrophin toxicity. In order to eliminate this serious obstacle, a new approach involving the use of a specific promoter, active only in differentiated myotubes (e.g. under the muscle creatine kinase promoter), must be elucidated.

The poor myoblast survival rate upon transplantation remains the major limitation of the DMD therapy and accounts for the failure of human clinical trials. The regenerative capacity of human Scs strictly depends on the number of available cells and their proliferative potential.29 It is known that the quantity and quality of Scs decrease with age, especially during childhood and adolescence, but remain relatively stable during maturity and senescence.29,59 This trend reflects the influence of the degeneration–regeneration cycles on the limited stem cell pool and explains the extremely low division capacity of Scs isolated from DMD subjects. The decreased proliferative capacity correlates with a decline in the length of telomeres observed both in old donors and in children suffering from different types of muscular dystrophies.29 Unfortunately, the transfection of human myoblasts with the telomerase gene did not affect their programme of differentiation in vitro and failed to immortalize these cells.29 Because muscle age-related atrophy was shown to be at least partly correlated with a general decrease in the circulating level of insulin-like growth factor (IGF-1),29 a new approach aiming to use this factor has emerged. IGF-1 exhibits dual effects on both myoblast proliferation and differentiation and is able to provoke muscle hypertrophy in rodent muscles.29 Barton-Davis et al.60 reported that viral-mediated expression of IGF-1 blocked the ageing-related loss of skeletal muscle function, which seemed to be a consequence of activation of Scs. Musaro et al.61 generated a model of persistent, functional myocyte hypertrophy using a tissue-restricted transgene encoding a locally acting isoform of IGF-1 that is expressed in skeletal muscles. The hypertrophic myocytes retained the proliferative response to muscle injury characteristic of younger animals.61 These results suggest that the possible application of this factor might increase the Sc numbers and extend their proliferation abilities; two conditions that have to be met for the effective treatment of DMD. Benabdallah et al.62 reported that improved outcomes of myoblast transplantation might be achieved by blocking the myostatin signal. Myostatin is known to reduce skeletal muscle regeneration. Transplantation of myoblasts obtained from the transgenic mice carrying the dominant-negative myostatin receptor into mdx recipients resulted in the formation of abundant and large dystrophin-positive fibres.62

The isolation and identification of MDSCs in animals12,42,63 triggered a new hope in the field of myopathies treatment. The unique features of MDSCs, such as long-time proliferative ability (over 30 passages in in vitro culture), a strong capacity for self-renewal, and multilineage differentiation potential,12,64 constitute a basis for the improvement of cell transplantation. Additionally, these cells exhibit the immune-privileged behaviour, which might play a role in the long-term persistence of dystrophin restoration within dystrophic muscles.64 As expected, MDSC transplantation turned out to be much more efficient than myoblast therapy.65 Qu-Petersen et al.12 reported that MDSC injection into the muscle of mdx mice resulted in 10 times more dystrophin-positive myofibres (30 and 90 days after transplantation) when compared with myoblast treatment. Jankowski et al.66 claim that the decreased fusion potential of MDSC observed in vitro is associated with increased dystrophin restoration and regeneration capacity in vivo. According to this hypothesis, the rapid and intense fusion considerably limits the number of cells capable of proliferating and participating in the regeneration process. In this regard, cells that remain in an undifferentiated and unfused state retain the potential to multiply and thus increase the yield of myogenic nuclei derived from grafted cells. A negative side of delayed fusion between donor cells and host myofibres is the potential initiation of immune response, as the fusion seems to protect grafted cells from the host's immune reactions.66 An important consideration for cell therapy of DMD is the possibility of systemic transplantation of MDSCs as the intramuscular injections of these cells failed to restore target muscles (diaphragm and intercostal muscles) of DMD patients.64 The ability of MDSCs to regenerate dystrophic muscles upon systemic delivery, however to a lesser extent than direct intramuscular injection, has been demonstrated in mdx mice. A homing process was shown to be enhanced by a needle injury of target muscles.63

These preliminary results strongly support the advantageous position of MDSCs over myoblasts. There is, however, a long way to go before these cells become clinically applicable. First of all, MDSCs require further characterization, which is absolutely essential for the successful isolation of these cells. There is also a great need for the understanding of homing, differentiation, and signalling processes that enable systemic delivery of these cells and thus improve the final clinical outcome.


   Future perspectives
Top
 Abstract
 Introduction
 Myoblast subsets
 Myoblast--application and...
 Future perspectives
Acknowledgement
 References
 
MDSCs seem to display abilities that broaden their potential clinical use far beyond muscle regeneration in dystrophies. The new studies comparing the efficacy of skeletal myoblasts with MDSC transplantation in HF suggest that MDSCs possess enhanced regeneration capacity when compared with myoblasts and are capable of improving cardiac function.64 Their ability to differentiate into endothelial cells may eliminate the necessity to trigger angiogenesis and thus omit the myoblast transfection procedures. Human MDSC transduced with recombinant human bone morphogenetic protein-2 have been shown to stimulate osteogenesis which may place these cells among the strong candidates for bone disorder treatments. Additionally, a recent study has shown that MDSCs transplanted into the bladders of subjects with urinary incontinence not only differentiated towards smooth muscle cells but also improved bladder contractility.64 MDSCs give new possibilities for cell therapy but still, and most of all, constitute a great area of intensive investigation. It is clear that taking MDSCs into the clinics requires more detailed understanding of the signals that govern their behaviour and fate.


   Acknowledgement
Top
 Abstract
 Introduction
 Myoblast subsets
 Myoblast--application and...
 Future perspectives
 Acknowledgement
References
 
This study was supported by the Ministry of Science, PBZ/KBN/099/P05/10.

Conflict of interest: none declared.


   References
Top
 Abstract
 Introduction
 Myoblast subsets
 Myoblast--application and...
 Future perspectives
 Acknowledgement
 References
 

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