Saturday, March 19, 2011

Cell therapy strategies and improvements for muscular dystrophy



Edited by R De Maria

M Quattrocelli1, M Cassano1, S Crippa1, I Perini1 and M Sampaolesi1,2

1. 1Translational Cardiomyology, SCIL Katholieke Universiteit Leuven, Herestraat 49 bus 814, Leuven 3000, Belgium
2. 2Human Anatomy, University of Pavia, Via Forlanini 8, Pavia 27100, Italy

Correspondence: M Sampaolesi, Translational Cardiomyology, Stem Cell Research Institute, University Hospital Gasthuisberg, Herestraat 49, Leuven B-3000, Belgium. Tel: +32 0163 30295; Fax: +32 0163 30294; E-mail: maurilio.sampaolesi@med.kuleuven.be

Received 9 July 2009; Revised 8 September 2009; Accepted 21 September 2009; Published online 30 October 2009.
Top of page
Abstract

Understanding stem cell commitment and differentiation is a critical step towards clinical translation of cell therapies. In past few years, several cell types have been characterized and transplanted in animal models for different diseased tissues, eligible for a cell-mediated regeneration. Skeletal muscle damage is a challenge for cell- and gene-based therapeutical approaches, given the unique architecture of the tissue and the clinical relevance of acute damages or dystrophies. In this review, we will consider the regenerative potential of embryonic and somatic stem cells and the outcomes achieved on their transplantation into animal models for muscular dystrophy or acute muscle impairment.
Keywords:

muscular dystrophy; animal models; cell therapy; stem cells
Abbreviations:

ABCG2, ATP-binding cassette, sub-family G (WHITE), member 2; ALP, alkaline phosphatase; αsg, α-sarcoglycan; bg, natural killer (NK) cell-deficient mice (beige); BM, bone marrow; BMD, Becker muscular dystrophy; BMSCs, bone marrow-derived stem cells; BMT, bone marrow transplantation; βsg, β-sarcoglycan; c-MYC, cellular myelocytomatosis oncogene; cDNA, complementary DNA; Cxcr4, chemokine (C-X-C motif) receptor 4; DMD, Duchenne muscular dystrophy; ESCs, embryonic stem cells; FACS, fluorescence-activated cell sorting; Flk1, fetal liver kinase 1; GFP, green fluorescent protein; GLP–GMP, good laboratory practice–good manufacturing practice; GRMD, Golden Retriever muscular dystrophy; Gy, Gray (unit); HCT, haemopoietic cell transplantation; hMADS, human multi-potent adipose-derived stem cells; HLA, human leukocyte antigen; Hmgb1, high mobility group box 1; IGF1, Insulin-like growth factor 1; iPS, induced pluripotent stem cells; KLF4, Kruppel-like factor 4; KO, knockout; KSN, mice strain with high natural killer activity; LGMD, limb-girdle muscular dystrophy; MABs, mesoangioblasts; Mac1, integrin alpha M; MagicF1, cMet-activating genetically improved factor1; MAPCs, multipotent adult progenitor cells; mdx, muscular dystrophy X-linked (?); μDYS, micro-dystrophin; MGF, mechano growth factor; MMP9, matrix metallopeptidase 9; mRNA, messenger ribonucleic acid; MyoD, myoblast determination protein; Myf5, myogenic factor 5; NCAM, neural cell adhesion molecule; NG2, chondroitin sulfate proteoglycan 4; NICD, Notch1 intracellular domain; NOD, non-obese diabetic; OCT4, octamer-binding transcription factor 4; Pax3, paired box gene 3; PDGFRα, platelet derived growth factor receptor alpha; PIGF, phosphatidylinositol glycan anchor biosynthesis, class F; PTC124, 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid (C15H9FN2O3); RT-PCR, retrotranscription-based polymerase chain reaction; Sca1, stem cell antigen 1; scid, severe combined immuno-deficiency; SDF1, stromal-derived factor 1; SMPs, skeletal muscle precursors; SOX2, SRY (sex determining region Y)-box 2; SP, side population; TNF-α, tumor necrosis factor-α; VEGFR2, vascular endothelial growth factor receptor 2; Wnt3a, wingless-type MMTV integration site family, member 3A

Muscular dystrophies are a heterogeneous group of inherited diseases, primarily characterized by severe and chronic skeletal muscle degeneration. Duchenne muscular dystrophy (DMD) is the most severe disease among similar dystrophic diseases and is caused by frame-shift deletions, duplications, or point mutations in dystrophin gene. Patient mobility is highly affected, usually resulting in wheelchair dependency, and death occurs due to respiratory or cardiac failure.1, 2

New strategies for the treatment of this disease are currently being investigated and are categorized by two approaches: endogenous activation and exogenous delivery (Figure 1). The first strategy consists of re-activating endogenous cells to achieve muscle hypertrophy, counteracting the mass/force loss in inflamed fibres. To reach this goal, a growing range of small molecules or recombinant proteins has been tested, including insulin-like growth factor 1 (IGF1),3, 4 MagicF15 or valproic acid.6 The second strategy, on the contrary, relies on exogenous tools (gene and/or cell therapies) to improve muscle regeneration, thus providing new, functional fibres to the dystrophic muscle. Gene therapy targets the genetic defects, attempting to overcome pathological mutations by providing the muscle with the correct form of the gene7 or by correcting the splicing through the exon-skipping vectors8, 9 or drugs, such as PTC124.10 Cell therapy, however, is based on stem cell-driven muscle regeneration, by systemic or local injections.
Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Combining endogenous activation and exogenous delivery to enhance muscle regeneration. Currently, two therapeutical approaches are investigated for the regeneration of the skeletal muscle: the endogeneous activation of physiological repair potential, such as targeting of satellite, circulating or perycite cells through small molecules, or the exogenous delivery of stem cells or genetic tools, such as antisense oligonucleotides (ODNs), drugs (PTC124) or viral vectors. M-SP, muscle side population; BM-SP, bone marrow side population; BMSCs, bone marrow-derived stem cells; MABs: mesoangioblasts; MADS, multipotent adipose-derived stem cells; iPS, induced pluripotent stem cells, not yet injected in animal models for muscular dystrophy; ESCs, embryonic stem cells
Full figure and legend (192K)

This review will focus on in vivo cell therapy strategies and improvements in the treatment of sarcoglycan/dystrophin complex-related dystrophies, such as Duchenne or limb-girdle muscular dystrophies.
Top of page
Animal Models for Sarcoglycan/Dystrophin Complex-Related Muscular Dystrophies

Sarcoglycan/dystrophin complex-related muscular dystrophies are caused by disruption of the sarcoglycan–dystrophin complex that normally anchors the actin fibres to the sarcolemma, generally resulting in chronic muscle wastage, progressive fibrotic infiltrations and force decrease. Moreover, cardiac involvement is often described, in terms of chronic dilatative cardiomyopathy, scar infiltrations, aneurisms and repeated microinfarctions.

DMD is the most severe form and it is caused by frame-shift mutations or huge deletions in the dystrophin gene. It is one of the largest gene in the human genome and encodes a 14-kb mRNA. In this type of dystrophy, the protein is completely or partially lost. A less severe phenotype is observed in Becker dystrophy, in which mutations still affect the dystrophin gene, but myofibres retain a truncated and low-active isoform of the protein. Some forms of limb-girdle muscular dystrophy (LGMD2) are also caused by mutations in sarcoglycan complex proteins, for example, α- or β-sarcoglycan (αsg or βsg) depletion, and can result in severe pathological phenotypes.

Several animal models have been developed to study muscular dystrophies, particularly for DMD and LGMD2. The most widely used model for dystrophy is the mdx mouse that carries an X-linked mutation in the dystrophin gene, thus mimicking, at least in principle, the DMD genotype in humans. In mdx mice, the effects of degeneration are less severe, mainly due to the presence of relatively high numbers of revertant fibres (1–3%)11 and an upregulation of utrophin. Utrophin is a smaller analogue of the dystrophin and may account for the partial compensatory effect on muscle wastage.12, 13

Recently, it has been demonstrated that mdx satellite cells undergo telomere erosion, which may also contribute to the inability of these cells to continuously repair DMD muscle. It is possible that muscle stem cells or myogenic progenitors that maintain telomerase activity until late passages, may contribute in part to the muscular regeneration, that provides mdx mice with a normal life span.14

Feasible models of LGMD2 are α-sarcoglycan- and β-sarcoglycan-knockout mice. These mice are very close to the human phenotype of LGMD2D and LGMD2E, respectively, as they show chronic skeletal muscle degeneration and, in the case of βsg-KO mice, dilatative cardiomyopathy.15, 16 In α-sarcoglycan-KO mice, αsg gene is disrupted through a neomycin cassette insertion between exons 1 and 9, through homologous recombination of the flanking regions. Similarly, in the β-sarcoglycan-KO mice, the region encompassing exons 3–6 of βsg gene is disrupted. LGMD mice are considered a better animal model than mdx mice because of their lack of revertant fibres, which often render mdx mice-related results controversial.17

Canine models of DMD are being also extensively studied.18, 19 The larger fibres in canine muscles mimic the human dystrophy effects better than that of the mouse. At present, there are two major colonies of dystrophic dogs all over the world, bearing the same mutation in different genetic backgrounds; a colony of Golden Retrievers and one of Beagle dogs. These animals are both derived through cross-breeding of a naturally born, affected founder because transgenic creation would be unethical in these animal models. The mutation lies in intron 6 of dystrophin gene and results in aberrant splicing that causes a premature transcription stop codon. Dystrophic dogs, from both varieties, show extremely affected motility, posing, salivation, severe chronic scar infiltrations and skeletal muscle degeneration. In these animals, revertant fibres are also almost undetectable, thus providing a good model to analyze regeneration effects of cell and genetic therapies.
Top of page
Cell Models for In Vivo Skeletal Muscle Regeneration

To date, the only clinical treatments for muscular dystrophy are steroid administration and non-invasive intermittent positive pressure ventilation. These treatments result in amelioration of symptoms and improved quality of life.20 Despite the benefits of slightly longer lifespan, alleviation of pain and surgical management of scoliosis, this therapy shows many side effects, such as weight increase, and has no real beneficial effects on skeletal muscle architecture and force.21 Thus, cell therapy represents a theoretical valuable alternative. The main goal of cell therapy is to directly regenerate wasted, adult muscle fibres through systemic or targeted injection of stem cells, which function to block muscle loss and restore, at least partially, the normal muscular activity. It is currently a difficult task to conjugate high efficiencies in cell motility, homing, engraftment and differentiation into the complex environment of a severely inflammed and degenerated muscle. Several cell models have been tested in vivo, with diverse results (Table 1). Three main classes of stem cells with a myogenic differentiation potential have been considered for cell therapy protocols in preclinical studies for the treatment of muscular dystrophy: (i) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSs); (ii) bone marrow-derived stem cells (BMSCs) and circulating progenitors; and (iii) local myogenic-committed progenitors.
Table 1 - Stem cell types for the treatment of chronic or acute skeletal muscle damage.
Table 1 - Stem cell types for the treatment of chronic or acute skeletal muscle damage - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorFull table

ESCs and iPS cells

Embryonic stem cells (ESCs) are generally considered the most promising natural source of pluripotent cells for cell therapy, but few attempts have been reported in muscle regeneration and efficiencies are still quite low. Importantly, ethical issues around their procurement and use, such as blastocyst disgregation and oocyte requirement, have raised a lot of concerns and limited the use of human ESCs (hESCs) for research purposes in several countries.

The critical step in using ESCs resides in cell conditioning before engraftment, a necessary step that increases the differentiation rate towards myogenesis and avoids teratoma formation. A heterogeneous suspension, derived from a co-culture of male embryoid bodies and female freshly isolated dystrophic satellite cells, has been demonstrated to produce few chimeric, dystrophin-positive myofibres in injured muscles of female mdx mice.22 Promising results have been obtained with hESCs, cultivated to enrich in mesenchymal precursors. The CD73+ NCAM+ sub-fraction was able to differentiate into myotubes in vitro and, remarkably, to regenerate up to 7% of injured skeletal muscle in immunodeficient mice.23 Recently, another strategy has been developed using paraxial mesoderm progenitors that are isolated from differentiating ESCs. The PDGFRα+/Flk1− fraction of Pax3-induced, embryoid body-derived cells shows activation of myogenic transcription factors in vitro and good differentiation in dystrophin+ fibres on transplantation into both cardiotoxin-injured and dystrophic muscle. Injected mice also show an amelioration of the contractility force. In this case, intra-arterial administration of cells resulted in higher engraftment than intravenous injection.24 In another study, paraxial mesoderm progenitors were isolated as PDGFRα+/VEGFR2+ cells, which also have been successfully tested in cardiotoxin-injured quadriceps of KSN nude mice.25

A new source of pluripotent cells comes from the reprogramming of adult murine or human somatic cells, by means of pluripotency transcription factor expression. Human iPS (induced pluripotent stem, hiPS) cells are reprogrammed from differentiated, adult cells, such as fibroblasts, to an ES-like status, by retroviral-mediated transduction of OCT4, SOX2, KLF4 and, even if dispensable, c-MYC.26, 27, 28 As hiPS cells are created by reprogramming adult cells into a flexible, embryonic-like state, they have been claimed as alternative pluripotent cells to overcome the ethical issues regarding the use of ESCs. However, some reservations exist regarding the in vivo safety of hiPS cells, which must be addressed in the future. Recently, a wide range of disease-specific hiPS cells has been generated from patients with various Mendelian or complex diseases, including Duchenne and Becker muscular dystrophy.29 These cells could represent a greater advancement in terms of plasticity and life span, in comparison with other cell lines tested to date. Moreover, these DMD- or BMD-specific hiPS cells show the same genetic background of the donor, thus representing a good model for in vitro drug testing and, if genetically corrected, a suitable cell line for extensive skeletal muscle repair.
Bone marrow-derived and circulating progenitors

Mesenchymal stem cells have been tested in acute and chronic muscle wastage, but results are still controversial. After bone marrow transplantation (BMT) in dystrophic mice, BMSCs are able to migrate and contribute to the formation of new Myf5+ fibres through repeated rounds of inflammation and regeneration, which is typical of the mdx mouse muscles.30 In humans, the clinical case of a young DMD patient (deletion of the exon 45 in the dystrophin gene) has been reported, which 12 years after BMT, showed donor nuclei fused to 0.5% of dystrophic myofibres.31 Real-time RT-PCR analysis detected a small amount of a truncated, in-frame isoform of dystrophin, lacking exons 44 and 45, and trace amounts of the wild-type gene (0.0005%), although a direct correlation between the BMT-derived nuclei and the dystrophin isoform expression was missing. In addition, human mesenchymal stem cells isolated from synovial membrane of adult donors, on intramuscular delivery into tibialis anterior of mdx mice, efficiently produce new, functional myofibres, without any sign of fusion. These cells also contribute to the long-term satellite cell population and restore mechano growth factor (MGF) expression in treated muscles.32 The myogenic potential of these cells seems to be strongly related to the microenvironment surrounding the delivered cells because when injected systemically in dystrophic mice, they are observed in almost all tissues.

Given the broad availability of the source, human multipotent adipose-derived stem cells (hMADS) have been investigated as a possible alternative for muscle regeneration. hMADS are CD44+, CD90+ and CD105+, confirming their mesenchymal lineage. Once injected intramuscularly into tibialis anterior of immunocompetent and immunosuppressed mdx mice, they fuse with host fibres, resulting, 6 months later, in a large number of chimaeric myofibres expressing human dystrophin. Interestingly, no differences are reported between immuno-competent and immuno-suppressed hosts and it also seems that hMADS significantly reduce necrosis in the dystrophic muscle.33 These promising results have since been enhanced by priming the adipose-derived mesenchymal cells by co-culturing with myoblasts34 or forced MyoD expression.35

A sub-population of circulating, haematopoietic stem cells expressing CD133, which constitutes another interesting and easily isolatable cell pool, has been reported to express early myogenic markers.36 Intramuscular or intra-arterial injection of genetically corrected CD133+ progenitors, isolated from both peripheral blood and muscles of DMD patients, results in a significant recovery of muscle morphology, function and re-expression of human dystrophin in scid/mdx mice.37

In contrast, several studies have reported of absent or incomplete muscle repair by mesenchymal or haematopoietic stem cells. On being intravenously injected into a mouse model for LGMD2F (mice lacking δ-sarcoglycan), bone marrow side population cells engraft and fuse into skeletal fibres, but do not restore δ-sarcoglycan expression.38 Green fluorescent protein-positive bone marrow (GFP+ BM) cells, delivered through retro-orbital injection, fuse with ~3% of fibres in the tibialis anterior of treated mdx mice, but almost no dystrophin expression is detected in GFP+ myofibres. However, where dystrophin is detected, its expression is spatially more limited than in revertant fibres.39 These data have been confirmed in another study, in which it has been demonstrated that greater than or equal to80% of BM-derived muscle-incorporated nuclei in the transplanted dystrophic mouse are ‘silent’. Incorporated nuclei fail to express myogenic factors, including dystrophin, and this ‘silencing’ is still retained even in the presence of strong chromatin-remodelling agents, such as 5′-azacytidine.40 It has also been demonstrated that haematopoietic cell transplantation (HCT) alone result neither in any skeletal fibre regeneration nor in expression of dystrophin or other muscle genes.41 Nevertheless, an interesting role for HCT in muscle regeneration may come from immunotolerance effects towards allogeneic myoblast engraftment. DMD dogs have been successfully treated using both peripheral HCT and freshly isolated myoblasts from the same healthy donor. Donor myoblast-related dystrophin expression increased up to ~7% of wild-type levels and was maintained for at least 24 weeks, without any pharmacological immunosuppression.42

Finally, bone marrow-derived multipotent adult progenitor cells (MAPCs) were observed to durably repair muscles in ischaemic limbs, by efficient revascularization of necrotic tissues.43
Local myogenic-committed progenitors

Satellite cells are quiescent unipotent myoprecursors, located between the fibre and the basal lamina; during embryogenesis, they form during the second wave of myogenesis and, after contributing massively to the first post-natal muscle growth, they stop proliferating and reach their niche.44 They can also be re-activated on muscle damage, re-entering cell cycle and contributing to the formation of new muscle fibres.45 Given their natural commitment, it's easy to imagine satellite cells as a major candidate for muscle regeneration in muscular dystrophies. On single fibre transplantation into radiation-ablated mdx tibialis anterior, donor satellite cells multiply and expand, re-populating the satellite cell pool and differentiate into functional myofibres.46 Pax7+ CD34+ GFP+ satellite cells, isolated from Pax3GFP/+ mice diaphragms, have also been demonstrated to be a good cell model for mdx irradiated muscle treatment, resulting in the restoration of dystrophin expression in many skeletal fibres and contributing to the resident satellite compartment.47 Injections were administered intramuscularly and, notably, two major problems arose; satellite cells have a very low migration capability and, furthermore, cells showed impaired engraftment capability when expanded in vitro, even if for few days.47

To test the regeneration capability of satellite cells at a clonal level, single-cell dilutions of CD34+ integrinα+ luciferase-expressing satellite cells were injected into the skeletal muscle of a NOD/SCID mouse depleted of resident satellite cells by 18 Gy irradiation. It was shown by in vivo imaging that a single satellite cell can reconstitute the satellite compartment and, on further damage, can rapidly re-enter a new proliferation wave, generating new myofibres.48

Recently, interesting findings resulted from a prospective isolation of skeletal muscle precursors (SMPs), consisting of a CD45− Sca1− Mac1− Cxcr4+ β1-integrin+ subset within the endogenous satellite compartment. When injected into cardiotoxin-injured muscles in immunodeficient mdx mice, SMPs robustly contributed to muscle regeneration (up to 94%) by fusing with pre-existing fibres or stimulating de novo myogenesis. Muscle histology and contractile force in treated mice were significantly better than those in untreated mice. Furthermore, SMPs contributed greatly to the endogenous satellite population, undergoing new cycles of re-activation on subsequent induced damage.49 However, as freshly isolated SMPs were injected, without in vitro expansion, the migration capability remains restricted in the areas surrounding the intramuscular injection site.

One of the major concerns about satellite cells is that, probably, they are not a homogeneous population. As argued by Collins et al.,46 the variable engraftment rate of satellite cells from a single myofibre transplantation could be due to the functional heterogeneity of the satellite cell pool and of their niche of origin. Satellite cell heterogeneity is still a contentious issue and has been extensively reviewed in several studies50, 51 and is still open for debate.52

Muscle side population (SP) cells are defined as Sca1+ CD45+ cells, able to rapidly efflux the Hoechst dye 33342, and are being investigated as potential myogenic progenitors. They are associated with the muscle vasculature and are spontaneously committed towards the haematopoietic lineage. On co-culture with myoblasts, they can form myotubes in vitro and, if injected intramuscularly into crushed tibialis of a scid/bg immunodeficient mouse, can give rise to up to 1% of regenerating fibres.53 The efficiency of SP-mediated muscle regeneration has been increased to 5–8% by injection into the femoral artery of mdx5cv DMD mice, resulting in Pax7 and desmin expression by donor cells, after extravasation and recruitment to inflammation sites.54 Impressive results have been obtained with the identification of a rare subset (0.25%) of SP cells, characterized by both satellite- and SP-related markers, such as Sca1+/ABCG2+/Syndecan4+/Pax7+, and found in the typical satellite compartment, under the basal lamina. Once sorted from the mononuclear fraction of the hind limb, they can grow in association with single muscle fibres and can robustly undergo myogenic differentiation in vitro. On intramuscular injection in the presence of 1.2% BaCl2, these satellite-SP cells have been shown to efficiently compete with endogenous satellite cells in regenerating the wild-type muscle. Injected cells resulted in 30% fibre regeneration and, strikingly, in up to 75% reconstitution of the endogenous satellite cell pool. The newly generated satellite cells were able to undergo new rounds of proliferation and muscle repair on subsequent injuries. Furthermore, the same long-term effects have been proven through injections into dystrophic mdx4cv tibialis anterior muscles, producing up to 70% regenerating fibres.55 However, BaCl2-induced muscle damage is not a widely used and accepted regeneration model, and this must be taken into consideration when interpreting these findings.

Recently, a new type of vessel-associated muscle-derived stem cells has been investigated as a suitable potential model for chronic muscle therapy, namely mesoangioblasts (MABs). They can be isolated from the dorsal aorta of E9.5 embryo56 or from adult skeletal muscle of mice, dogs and humans.57, 58, 59 MABs are CD34+, Sca1+, PDGFRα+, PDFGRβ+, NG2+ and ALP+, thus supporting the idea that they are a sub-group of the pericytic population.60 They show high proliferation rates in vitro, without transformation potential, and display, in in vitro and embryonic chimaera systems, multipotent differentiation capability towards myogenic, osteogenic, chondrogenic and adipogenic lineages. On intra-arterial injection into inflammated muscles of αsg-KO mice or DMD Golden Retriever dogs, they are capable of consistently regenerating (up to 50%) the muscle architecture, sarcoglycan/dystrophin expression and electrophysiological properties of wasted dystrophic muscle.57, 58 Similarly, good results have been achieved through human MAB transplantation into scid–mdx immunodeficient dystrophic mice.59

Treatment of dogs with MABs resulted in very good and long-term results in some animals, in terms of general motility restoration and whole muscle regeneration, whereas other animals did not show a good engraftment and any clinical improvement.58 This confirms the general idea that background variability in dystrophies in larger organisms, such as dogs or humans, has to be evaluated for effective cell therapies.

Furthermore, another class of myogenic precursors has been isolated from endothelial population of adult human muscle, through FACS-mediated prospective isolation of CD56+ CD34+ CD144+ cells. These myoendothelial progenitors, on injection into injured muscle of scid mice can achieve muscle engraftment and fibre neo-formation at an higher degree than CD56+ canonical myoprecursors.61
Top of page
Cell Conditioning and Priming

To increase engraftment specificity and differentiation potential, cell transplantation can be combined with pre-injection cell conditioning or with genetic manipulation.

Cell migration limitations are often a major cause of low engraftment efficiencies in skeletal muscle, which is a very complex tissue with severely limited cell motility, particularly in the presence of large fibrotic or necrotic areas. Thus, some attempts have been made to assist migration by conditioning cells with migration-enhancing soluble factors or by overexpressing commiting/mobilizing proteins before applying them to the degenerated muscle.

A lot of interest has been shown in Notch signalling, as a potential enhancer of myogenic commitment. Rat BMSCs, transfected with a plasmid for the intracellular domain of Notch1 (NICD) and injected locally or intravenously into injured muscles of rats or nude mice, account for a very high level of regenerating fibres (up to 89%).62 Given that NICD is the active signalling form of the receptor, it could be possible that finer genetic tools to activate Notch signalling could enhance myogenesis by donor cell injection, although recently it has been demonstrated that a temporal switch from Notch to Wnt3a signalling activation is necessary during normal adult myogenesis.63

Encouraging in vivo results have come from cell therapy experiments involving soluble factor-dependent cell conditioning. Hmgb1, a cytokine secreted by activated macrophages and monocytes, is able to increase the recruitment of MABs out of the vessels into the muscle. Heparin–Sepharose beads, loaded with Hmgb1 and injected into the femoral artery, were shown to promote the trans-endothelial migration of intra-arterial-injected embryonic MABs into non-injured tibialis anterior of wild-type mice.64 Pre-treatment of MABs with mobilization cytokines, such as SDF1 and TNFα, highly increase MAB homing into dystrophic muscle of αsg-null mice, thus reducing approximately 50% the aspecific homing into filter organs. Their regenerative effect was magnified by TNFα priming and α4-integrin overexpression.65 Furthermore, improving the angiogenic potential in necrotic areas could help cell therapy. For example, tendon fibroblasts expressing placenta growth factor (PIGF, an angiogenic factor) and matrix metalloproteinase 9 (MMP9), injected intramuscularly into aged αsg-KO mice, result in a dramatic increase in the extension of regenerated dystrophin+ muscle areas after intra-arterial delivery of wild-type MABs.66 Such results corroborate the idea that a deeper knowledge of cytokines and angiogenic factors regulating the inflammation-dependent recruitment of myoprecursors will serve to improve the benefits mediated by cell therapy.
Top of page
Genetic Manipulation for Autologous Cell Therapy

Correction of the dystrophic genotype in the transplanted cells could allow the usage of autologous cells, instead of heterologous wild-type cells, thus avoiding immunosuppressive drugs.

A widely used strategy relies on lentiviral transduction of muscle-regenerating cells to allow integration and expression of the disrupted gene. To correct DMD, several alternatives of the dystrophin gene (which is too long to be introduced in a lentiviral vector) exist, such as micro- or mini-dystrophin. These are shorter isoforms of the native protein, which retain a partial functionality, thus providing, in principle, a possible molecular rescue on differentiation towards newly formed myofibres. mdx5cv mice, injected intravenously with autologous muscle-SP cells that were previously transduced with human micro-dystrophin (hμDYS)-expressing lentiviruses, show some skeletal fibres positive for the human version of the lacking protein.67 Similarly, very good results came from transplantation of hμDYS-transduced human pericytes into mdx–scid mice.59 In GRMD dog treatment, autologous MABs, transduced with human micro-dystrophin, induced a quite widespread expression of dystrophin and other proteins of the sarcoglycan complex in analyzed muscles and a partial recovery of the histological architecture. However, treated dogs had poorly restored general motility and force.58 These results may support the idea that, especially in higher organisms, mini- or micro-genes are not so feasible, or, at least, show highly variable and only short-term effects. Genetic correction can be addressed also for treatment of other genetic defects resulting in dystrophy, as when αsg-KO MABs, carrying a lentiviral αsg cDNA construct under a constitutive promoter, are injected into LGMD2D mice, there is extensive fibre regeneration, mobility and muscle force recovery.57
Top of page
Conclusions and Perspectives

Stem cell-based therapy is the most attractive approach for the treatment of DMD and other muscular dystrophies, and research in this direction has moved rapidly in past few years. Experiments in small and large animal models are paving the way for clinical experimentation, but it would be imprudent to predict a ‘cure’ from these first attempts. Nevertheless, several clinical trials have been started or planned, involving myoblast or perycyte injection from HLA-matched donors and, given the growing variety of possible myogenic progenitors in literature, the number will increase during the near future. Other clinical trials are focusing on gene- or antibody-based strategies, such as adeno-associated viruses carrying γ-sarcoglycan or μ-dystrophin and antibody-triggered myostatin blockade.21

Furthermore, encouraging results have come from clinical trials related to exon-skipping technology. Specific exons carrying mutations can be skipped by antisense oligonucleotides administration to restore the reading frame and result in the production of internally deleted, but functional dystrophin. Recently, two clinical trials involving two different drugs, AVI-4658 (developed by the MDEX Consortium, United Kingdom and manufactured by AVI BioPharma, Bothell, WA, USA) and PRO051 (developed by University of Leiden, The Nederlands in collaboration with Prosensa B.V.), were performed on Duchenne patients. In both trials, biopsy data showed that injections of antisense oligonucleotides, to skip exon 51, into dystrophic muscles, successfully induced new dystrophin production, with no adverse events. These pioneering studies are now followed by randomized controlled trials of systemic therapies both in The Netherlands and the United Kingdom.

Therefore, it is reasonable to expect encouraging results that may also drive a combination of the stem cell- and gene-based therapies. It is critical to better understand the biological properties of stem cells and their paracrine role in the treatment of muscular diseases, to realize the potential positive effects of these new cures.

The scientific community largely accepts the presence of adult stem cells in all tissues but their origin is still controversial. We and other authors suggest pericytes as a source of stem cells present in skeletal and cardiac muscles.59, 60 They are influenced by their surrounding when maintaining a specific cell commitment, although this has to be clarified in pathological tissues.

Interestingly, other research groups have identified mesenchymal cells as stem cells for all tissues,68 raising questions regarding the possibility that the primary source of cell plasticity is confined to the bone marrow. It could be possible that cells move from bone marrow towards pericyte compartment, to adopt a specific cell fate, influenced by local niche. To further elucidate their origin, it is necessary to generate transgenic animals to track endogenous stem cells during muscle development and regeneration.

Despite the fact that current regulatory restrictions will defer the clinical translation of new approaches, that are successful in animal models, several trials have been started. Moreover, the actual huge costs of GLP–GMP (good laboratory and manufacture practice) stem cell technology limit the feasibility of cell therapy treatment for patients affected by muscular dystrophy. However, strategies to lower costs are being investigated to develop treatments that are available for large numbers of patients. In conclusion, it is critical to better understand the biological properties of stem cells and their paracrine role in the treatment of muscular diseases, to realize the potential positive effects of these new cures. Therefore, it is reasonable to expect encouraging results from the on-going trials that may also drive a combination of stem cell- and gene-based therapies for the treatment of muscular dystrophies.
Top of page
References

1. Emery AE. The muscular dystrophies. Lancet 2002; 359: 687–695. | Article | PubMed | ISI | ChemPort |
2. Cossu G, Sampaolesi M. New therapies for Duchenne muscular dystrophy: challenges, prospects and clinical trials. Trends Mol Med 2007; 13: 520–526. | Article | PubMed | ChemPort |
3. Abmayr S, Gregorevic P, Allen JM, Chamberlain JS. Phenotypic improvement of dystrophic muscles by rAAV/microdystrophin vectors is augmented by Igf1 codelivery. Mol Ther 2005; 12: 441–450. | Article | PubMed | ChemPort |
4. Pelosi L, Giacinti C, Nardis C, Borsellino G, Rizzuto E, Nicoletti C et al. Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines. FASEB J 2007; 21: 1393–1402. | Article | PubMed | ChemPort |
5. Cassano M, Biressi S, Finan A, Benedetti L, Omes C, Boratto R et al. Magic-factor 1, a partial agonist of Met, induces muscle hypertrophy by protecting myogenic progenitors from apoptosis. PLoS ONE 2008; 3: e3223. | Article | PubMed | ChemPort |
6. Minetti GC, Colussi C, Adami R, Serra C, Mozzetta C, Parente V et al. Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors. Nat Med 2006; 12: 1147–1150. | Article | PubMed | ChemPort |
7. Hoshiya H, Kazuki Y, Abe S, Takiguchi M, Kajitani N, Watanabe Y et al. A highly stable and nonintegrated human artificial chromosome (HAC) containing the 2.4 Mb entire human dystrophin gene. Mol Ther 2008; 17: 309–317. | Article | PubMed | ChemPort |
8. Lu QL, Mann CJ, Lou F, Bou-Gharios G, Morris GE, Xue SA et al. Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 2003; 9: 1009–1014. | Article | PubMed | ISI | ChemPort |
9. Benchaouir R, Meregalli M, Farini A, D'Antona G, Belicchi M, Goyenvalle A et al. Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice. Cell Stem Cell 2007; 1: 646–657. | Article | PubMed | ChemPort |
10. Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007; 447: 87–91. | Article | PubMed | ISI | ChemPort |
11. Hoffman EP, Morgan JE, Watkins SC, Partridge TA. Somatic reversion/suppression of the mouse mdx phenotype in vivo. J Neurol Sci 1990; 99: 9–25. | Article | PubMed | ISI | ChemPort |
12. Weir AP, Burton EA, Harrod G, Davies KE. A- and B-utrophin have different expression patterns and are differentially upregulated in mdx muscle. J Biol Chem 2002; 277: 45285–45290. | Article | PubMed | ISI | ChemPort |
13. Hirst RC, McCullagh KJ, Davies KE. Utrophin upregulation in Duchenne muscular dystrophy. Acta Myol 2005; 24: 209–216. | PubMed | ChemPort |
14. Lund TC, Grange RW, Lowe DA. Telomere shortening in diaphragm and tibialis anterior muscles of aged mdx mice. Muscle Nerve 2007; 36: 387–390. | Article | PubMed | ChemPort |
15. Duclos F, Straub V, Moore SA, Venzke DP, Hrstka RF, Crosbie RH et al. Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice. J Cell Biol 1998; 142: 1461–1471. | Article | PubMed | ISI | ChemPort |
16. Durbeej M, Cohn RD, Hrstka RF, Moore SA, Allamand V, Davidson BL et al. Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E. Mol Cell 2000; 5: 141–151. | Article | PubMed | ISI | ChemPort |
17. Cossu G. Fusion of bone marrow-derived stem cells with striated muscle may not be sufficient to activate muscle genes. J Clin Invest 2004; 114: 1540–1543. | PubMed | ChemPort |
18. Kornegay JN, Tuler SM, Miller DM, Levesque DC. Muscular dystrophy in a litter of golden retriever dogs. Muscle Nerve 1988; 11: 1056–1064. | Article | PubMed | ISI | ChemPort |
19. Sharp NJ, Kornegay JN, Van Camp SD, Herbstreith MH, Secore SL, Kettle S et al. An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics 1992; 13: 115–121. | Article | PubMed | ISI | ChemPort |
20. Daftary AS, Crisanti M, Kalra M, Wong B, Amin R. Effect of long-term steroids on cough efficiency and respiratory muscle strength in patients with Duchenne muscular dystrophy. Pediatrics 2007; 119: e320–e324. | Article | PubMed
21. Cossu G, Sampaolesi M. New therapies for muscular dystrophy: cautious optimism. Trends Mol Med 2004; 10: 516–520. | Article | PubMed | ChemPort |
22. Bhagavati S, Xu W. Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice. Biochem Biophys Res Commun 2005; 333: 644–649. | Article | PubMed | ChemPort |
23. Barberi T, Bradbury M, Dincer Z, Panagiotakos G, Socci ND, Studer L. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat Med 2007; 13: 642–648. | Article | PubMed | ChemPort |
24. Darabi R, Gehlbach K, Bachoo RM, Kamath S, Osawa M, Kamm KE et al. Functional skeletal muscle regeneration from differentiating embryonic stem cells. Nat Med 2008; 14: 134–143. | Article | PubMed | ChemPort |
25. Sakurai H, Okawa Y, Inami Y, Nishio N, Isobe K. Paraxial mesodermal progenitors derived from mouse embryonic stem cells contribute to muscle regeneration via differentiation into muscle satellite cells. Stem Cells 2008; 26: 1865–1873. | Article | PubMed | ChemPort |
26. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131: 861–872. | Article | PubMed | ChemPort |
27. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008; 451: 141–146. | Article | PubMed | ChemPort |
28. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008; 26: 101–106. | Article | PubMed | ChemPort |
29. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A et al. Disease-specific induced pluripotent stem cells. Cell 2008; 134: 877–886. | Article | PubMed | ChemPort |
30. Bittner RE, Schöfer C, Weipoltshammer K, Ivanova S, Streubel B, Hauser E et al. Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anat Embryol (Berl) 1999; 199: 391–396. | Article | PubMed | ChemPort |
31. Gussoni E, Bennett RR, Muskiewicz KR, Meyerrose T, Nolta JA, Gilgoff I et al. Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation. J Clin Invest 2002; 110: 807–814. | Article | PubMed | ISI | ChemPort |
32. De Bari C, Dell'Accio F, Vandenabeele F, Vermeesch JR, Raymackers JM, Luyten FP. Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane. J Cell Biol 2003; 160: 909–918. | Article | PubMed | ChemPort |
33. Rodriguez AM, Pisani D, Dechesne CA, Turc-Carel C, Kurzenne JY, Wdziekonski B et al. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med 2005; 201: 1397–1405. | Article | PubMed | ISI | ChemPort |
34. Di Rocco G, Iachininoto MG, Tritarelli A, Straino S, Zacheo A, Germani A et al. Myogenic potential of adipose-tissue-derived cells. J Cell Sci 2006; 119: 2945–2952. | Article | PubMed | ChemPort |
35. Goudenege S, Pisani DF, Wdziekonski B, Di Santo JP, Bagnis C, Dani C et al. Enhancement of myogenic and muscle repair capacities of human adipose-derived stem cells with forced expression of MyoD. Mol Ther 2009; 17: 1064–1072. | Article | PubMed | ChemPort |
36. Torrente Y, Belicchi M, Marchesi C, Dantona G, Cogiamanian F, Pisati F et al. Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients. Cell Transplant 2007; 16: 563–577. | PubMed | ChemPort |
37. Benchaouir R, Meregalli M, Farini A, D'Antona G, Belicchi M, Goyenvalle A et al. Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice. Cell Stem Cell 2007; 1: 646–657. | Article | PubMed | ChemPort |
38. Lapidos KA, Chen YE, Earley JU, Heydemann A, Huber JM, Chien M et al. Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle. J Clin Invest 2004; 114: 1577–1585. | Article | PubMed | ChemPort |
39. Chretien F, Dreyfus PA, Christov C, Caramelle P, Lagrange JL, Chazaud B et al. In vivo fusion of circulating fluorescent cells with dystrophin-deficient myofibers results in extensive sarcoplasmic fluorescence expression but limited dystrophin sarcolemmal expression. Am J Pathol 2005; 166: 1741–1748. | PubMed | ChemPort |
40. Wernig G, Janzen V, Schäfer R, Zweyer M, Knauf U, Hoegemeier O et al. The vast majority of bone-marrow-derived cells integrated into mdx muscle fibers are silent despite long-term engraftment. Proc Natl Acad Sci USA 2005; 102: 11852–11857. | Article | PubMed | ChemPort |
41. Dell'Agnola C, Wang Z, Storb R, Tapscott SJ, Kuhr CS, Hauschka SD et al. Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs. Blood 2004; 104: 4311–4318. | Article | PubMed | ISI | ChemPort |
42. Parker MH, Kuhr C, Tapscott SJ, Storb R. Hematopoietic cell transplantation provides an immune-tolerant platform for myoblast transplantation in dystrophic dogs. Mol Ther 2008; 16: 1340–1346. | Article | PubMed | ChemPort |
43. Aranguren XL, McCue JD, Hendrickx B, Zhu XH, Du F, Chen E et al. Multipotent adult progenitor cells sustain function of ischemic limbs in mice. J Clin Invest 2008; 118: 505–514. | PubMed | ChemPort |
44. Biressi S, Molinaro M, Cossu G. Cellular heterogeneity during vertebrate skeletal muscle development. Dev Biol 2007; 308: 281–293. | Article | PubMed | ChemPort |
45. Cossu G, Biressi S. Satellite cells, myoblasts and other occasional myogenic progenitors: possible origin, phenotypic features and role in muscle regeneration. Semin Cell Dev Biol 2005; 16: 623–631. | Article | PubMed | ISI | ChemPort |
46. Collins 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: 289–301. | Article | PubMed | ISI | ChemPort |
47. Montarras 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: 2064–2067. | Article | PubMed | ISI | ChemPort |
48. Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single transplanted muscle stem cells. Nature 2008; 456: 502–506. | Article | PubMed | ChemPort |
49. Cerletti 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: 37–47. | Article | PubMed | ChemPort |
50. Zammit PS. All muscle satellite cells are equal, but are some more equal than others? J Cell Sci 2008; 121: 2975–2982. | Article | PubMed | ChemPort |
51. Kuang S, Rudnicki MA. The emerging biology of satellite cells and their therapeutic potential. Trends Mol Med 2008; 14: 82–91. | Article | PubMed | ChemPort |
52. Partridge T. Denominator problems in a muscle stem cell study? Cell 2008; 135: 997–998; author reply 998–999. | Article | PubMed | ChemPort |
53. Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002; 159: 123–134. | Article | PubMed | ISI | ChemPort |
54. Bachrach E, Perez AL, Choi YH, Illigens BM, Jun SJ, del Nido P et al. Muscle engraftment of myogenic progenitor cells following intraarterial transplantation. Muscle Nerve 2006; 34: 44–52. | Article | PubMed
55. Tanaka KK, Hall JK, Troy AA, Cornelison DD, Majka SM, Olwin BB. Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration. Cell Stem Cell 2009; 4: 217–225. | Article | PubMed | ChemPort |
56. Minasi MG, Riminucci M, De Angelis L, Borello U, Berarducci B, Innocenzi A et al. The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues. Development 2002; 129: 2773–2783. | PubMed | ISI | ChemPort |
57. Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D'Antona G, Pellegrino MA et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 2003; 301: 487–492. | Article | PubMed | ISI | ChemPort |
58. Sampaolesi M, Blot S, D'Antona G, Granger N, Tonlorenzi R, Innocenzi A et al. Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 2006; 444: 574–579. | Article | PubMed | ISI | ChemPort |
59. Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol 2007; 9: 255–267. | Article | PubMed | ChemPort |
60. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008; 3: 301–313. | Article | PubMed | ChemPort |
61. Zheng B, Cao B, Crisan M, Sun B, Li G, Logar A et al. Prospective identification of myogenic endothelial cells in human skeletal muscle. Nat Biotechnol 2007; 25: 1025–1034. | Article | PubMed | ChemPort |
62. Dezawa M, Ishikawa H, Itokazu Y, Yoshihara T, Hoshino M, Takeda S et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005; 309: 314–317. | Article | PubMed | ChemPort |
63. Brack AS, Conboy IM, Conboy MJ, Shen J, Rando TA. A temporal switch from notch to Wnt signaling in muscle stem cells is necessary for normal adult myogenesis. Cell Stem Cell 2008; 2: 50–59. | Article | PubMed | ChemPort |
64. Palumbo R, Sampaolesi M, De Marchis F, Tonlorenzi R, Colombetti S, Mondino A et al. Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation. J Cell Biol 2004; 164: 441–449. | Article | PubMed | ISI | ChemPort |
65. Galvez BG, Sampaolesi M, Brunelli S, Covarello D, Gavina M, Rossi B et al. Complete repair of dystrophic skeletal muscle by mesoangioblasts with enhanced migration ability. J Cell Biol 2006; 174: 231–243. | Article | PubMed | ChemPort |
66. Gargioli C, Coletta M, De Grandis F, Cannata SM, Cossu G. PlGF-MMP-9-expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle. Nat Med 2008; 14: 973–978. | Article | PubMed | ChemPort |
67. Bachrach E, Li S, Perez AL, Schienda J, Liadaki K, Volinski J et al. Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells. Proc Natl Acad Sci USA 2004; 101: 3581–3586. | Article | PubMed | ChemPort |
68. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2008; 2: 313–319. | Article | PubMed | ChemPort |

Top of page
Acknowledgements

We thank Paolo Luban for his support. We are particularly grateful to Guido Tettamanti and Giulio Cossu for critical reading of the paper and for helpful comments, and Shea Carter for the ms proofreading service. This work was supported by FWO Odysseus Program n. G.0907.08; Wicka Funds n. zkb8720, University of Minnesota US; the Italian Ministry of University and Scientific Research (grant n. 2005067555_003, COFIN 2006–08), the Muscular Dystrophy Association, Association Francoise contre les Myopathies, CARIPLO Funds 2007-5639 and 2008-2005.

No comments:

Post a Comment

Recent Comments