Muscular Dystrophy
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                                   Stem Cell Therapy



Stem cells give rise to every tissue and organ in the body. They are defined by their capability of self-renewal and multipotent long-term proliferation, differentiating into multiple cell lineages. Stem-cell based therapies for the repair and regeneration of various tissues and organs offers an alternative therapeutic solution for a number of diseases, including musculoskeletal, hematopoietic, neurological and cardiovascular diseases.  Cells used for such strategies include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells (ASCs) which include bone marrow-derived stem cells, blood- and muscle-derived CD133+ cells, muscle-derived stem cells (MDSC), side population (SP) cells and mesoangioblasts. (1,2)
ESCs are derived from mammalian embryos in the blastocyst stage and are capable of differentiating into any of the tissues of the body while, iPSCs are cells which are reprogrammed from differentiated, adult somatic cells to an ESC-like status, by pluripotency transcription factor expression. Although the therapeutic potential of ESCs and iPSCs is enormous because of their auto-reproducibility and pluripotency, various limitations to their use are imposed by cell regulations, ethical considerations, and genetic manipulation. In contrast, ASCs are easily available, immunocompatible and no ethical issues are involved related to their use as long as they are of autologous tissue origin. They are intrinsic to various tissues of the body and are capable of maintaining, generating and replacing terminally differentiated cells within their own specific tissue following cell turnover or tissue injury. In addition, a growing number of studies have demonstrated that ASCs, under certain micro environmental conditions, give rise to cell types other than the cell type in the tissue of origin. Based on these advantages, ASCs are gradually becoming a candidate for regenerative medicine. (3)

Cell therapy has evolved as one of the promising treatments for muscular dystrophy. 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. (4) The mechanisms by which stem cells function and reverse the effects of cell death include differentiation, cell fusion, and secretion of cytokines or paracrine effects. (5-7)

Stem cells show high plasticity, i.e. the complex ability to cross lineage barriers and adopt the expression profile and functional phenotypes of the cells unique to other tissues. The plasticity can be explained by transdifferentiation (direct or indirect) and fusion. Transdifferentiation is the acquisition of the identity of a different phenotype through the expression of the gene pattern of other tissue (direct) or through the achievement of a more primitive state and the successive differentiation to another cell type (indirect or de-differentiation). By fusion with a cell of another tissue, a cell can express a gene and acquire a phenotypic element of another parenchyma. (8)During cell fusion, the cells connect and exchange vital cell components. (9)
So far, bone marrow-derived stem cells and their paracrine factors have shown all the necessary attributes for tissue regeneration, namely, angiogenesis, inhibition of apoptosis, anti-inflammation, immunosuppression, homing stimulation of endogenous cells, and possible regulation of specific metabolic pathways.  Thus, research into paracrine factors and mechanisms has shown that stem cell therapy is much more complicated and greatly enhances the potential and variety of therapeutic applications. (10-12)

Angiogenesis consists of several distinct processes which include sprouting and proliferation of pre-existing capillaries to form new networks. This process is tightly regulated by hypoxia through a number of proangiogenic factors, including VEGF, FGFs, P1GF, and hepatocyte growth factor (HGF). (13-15) Furthermore, these cytokines act not only in a coordinated time and concentration-dependent manner, but one cytokine may inhibit or stimulate the effect of another.  

Homing signals are extremely important for the efficacy of cell therapy because it is via these paracrine effects that the precise localization of transplanted cells is possible and can be improved. Cell homing, transmigration, adhesion, and tissue invasion are the result of many complex steps. Naturally, the migration, differentiation and growth are mediated by the tissue, degree of injury and SCs involved. Damaged tissue releases factors that induce homing. The tissue, intended as stromal cells, extracellular matrix, circulating growth and differentiating factors, determines gene activation and a functional reaction on stem cells, such as moving in a specific district, differentiating in a particular cell type or resting in specific niches. These factors can alter the gene expression pattern in SCs when they reside in a new tissue. (16)

Encouraging and pioneering experiments are being carried out in animal models for various muscular dystrophies. Diseased tissue may be regenerated in vivo by transplantation of healthy cells that can extensively replicate. Progenitor and stem cells have this intrinsic ability, and are used in certain clinical settings to enhance or restore damaged tissue. In attempts to regenerate muscle cells replete with dystrophin in the muscles of patients with dystrophin deficiency, several types of muscle-derived cell transplantation strategies have been tested in animals and in a few DMD patients. A muscle precursor cell, known as the myoblast, was one of the first cell types explored in DMD studies. In 1990, the first muscle stem cell transplantation was carried out in a 9 year old DMD patient, showing dystrophin production. (17) Soon after, many clinical trials in DMD patients were conducted. (18-25) Although, it was found to be a safe procedure, clinical benefits were recorded in none.
Since, myoblast transplantation has not shown effective results mainly due to rapid death of most injected myoblasts and the failure of injected myoblasts to migrate more than ~0.5 mm away from the injection site, (26) satellite stem cells have been explored as an alternative.  These cells which are specialized muscle stem cells, have the ability to both replicate and differentiate into various types of muscle cells. They are positioned between the plasma membrane and the surrounding basal membrane of adult skeletal muscle fibers, and expresses CD34, Pax 3 and Pax 7. (27) These cells upon activation may also express MyoD, Myf-5 and M-Cadherin. CD34 and M-Cadherin are not so consistent markers of human satellite cells. Among the more reliable markers of satellite cells in human muscle is neural cell adhesion molecule (CD56), which, however, also marks lymphocytes that may enter degenerating muscle in large numbers.(28) Dr. Rudnicki's team found that the Wnt7a protein, when introduced into mouse muscle tissue, significantly increased the population of these satellite stem cells and fueled the regeneration process, creating bigger and stronger muscles. Muscle tissue mass was increased by nearly 20 per cent in the study. (29)
A study was carried out to track the pathway of bone marrow derived stem cells (BMSC) to satellite cell to myofiber, unfractionated green fluorescent protein-positive (GFP+), BMSCs were transplanted into irradiated recipients. Irradiation served to both ablate the bone marrow compartment and decrease satellite stem cell numbers in muscle tissue. GFP+, BMSC-derived satellite cells were identified in muscle tissue of bone marrow transplanted (BMT) recipients by morphology and also by their ability to self-renew and differentiate into myotubes in vitro. The cells were karyotyped, and the “re-programmed” cells were diploid. The level of BMSC derived multinucleate muscle fibers in BMT-recipient mice was greatly increased when the animals underwent physical activity for 6 months. (30) This study is important because it provides evidence that BMSC to muscle differentiation occurs via repopulation of the muscle stem cell compartment. In 2008, Wallace et al carried out a study wherein they transplanted adult muscle mononuclear cells (AMMCs) in d-sarcoglycan-null dystrophic mice. They found that AMMCs were 35 times more efficient at restoring sarcoglycan compared to cultured myoblasts. The single injections of AMMCs provided long term benefit for muscular dystrophy and found persistent regeneration after 6 months. (31)
A few other myogenic precursor cell types distinct from satellite cells have also been explored. One of them is the muscle-resident side population (SP) cells. A study has shown that these cells could serve as a vehicle for delivering the dystrophin gene contained in a viral vector into mdx mice. (32) Muscle derived stem cells (MDSCs) or multipotent adult progenitor cells (MAPCs) have also reported to have a high capacity for muscle regeneration. However, compared to conventional satellite cells, many of these SP cells, do not display a promising myogenic potential.(33)

Little progress has also been made towards the use of embryonic stem cells (ESC) to study its potential in muscle regeneration. (34-35) It has been observed that injection of wild type ESCs into the mdx blastocysts produce mice with improved pathology and function. (36)

However, due to high rate of rejection and ethical issues related to ESCs, not many studies have been carried out on humans, which are necessary to demonstrate the therapeutic benefits of ESCs in muscular dystrophy patients.

Blood and muscle derived CD 133+ cells have shown to give rise to dystrophin-positive fibers when transplanted into mdx mice. (37) These cells have been demonstrated as safe, following their intramuscular transplantation. In accordance to which, Torrente et al in 2004 2007, carried out a phase I double blind trial with autologous muscle derived CD 133+ cells in 8 boys affected with DMD and was found safe with no adverse events reports. (38,39)

Experimental studies have shown that sub-populations of human umbilical cord blood (HUCB) cells have myogenic potential and can differentiate into muscle cells. (40,41) A single blind study in mice showing dysferlin deletions was conducted, wherin HUCB cells were transplanted and the expression of dysferlin positive muscle cells was recorded providing support for the hypothesis that a subpopulation of HUCB has myogenic potential. (42)
In 2009, Jazedje et al analyzed the potential of CD34+ cells from umbilical cord blood to do the same. It was observed that, these cells differentiated into mature myotubes after 15 days and dystrophin positive regions were also detected through immunofluorescence analysis. (43)A study was carried out on 82 progressive muscular dystrophy(PMD)  cases, who were treated by using double transplantations of BMSC and CB-MSC. No adverse reactions were reported. It was found that 37.8% obtained a remarkable efficacy, 45.1% were effective and 17.1% had no change. Hence, it was safe, convenient and effective therapy for PMD. (44)

Mesenchymal stem cells (MSC) are also attractive candidates for the treatment of muscular dystrophy.  Bone marrow derived mesenchymal stem cell transplantation in mouse models have shown to result in ultrastructural changes in muscular tissue due to migration of the cells to lesioned muscular tissue via blood circulation and further resulting in repair and regeneration. (45-51)
In immunosupressed mdx mice, 3H-thymidine labeled human bone marrow derived MSCs were transplanted. One month later, the mice showed greater radioactivity in most of the tissues and organs especially in the bone marrow and skeletal muscle compared to the control mice. Dystrophin positive cells were also detected at 1 month. The percentage of dystrophin positive fibres was 6.6% at 1 month and 8.9% at the end of 4 months. (52)
In 2007, a fetal to fetal transplantation strategy was carried out in mice embryo, human fetal MSCs via intramuscular, intravascular and intraperitoneal delivery. Wherein, it was found that intravascular and intraperitoneal delivery led to systemic spread of the cells. (53)

In mdx mice, MSCs derived from adult adipose tissue have homed to, differentiated into skeletal muscles and repaired injured muscle tissue. The repair is correlated to with reconstitution of dystrophin expression on the damaged fibres. (54-55)
At the NeuroGen Brain & Spine Institute, Mumbai, a study was carried out on 72 muscular dystrophy patients who underwent intrathecal autologous bone marrow derived mononuclear cell transplantation, 41 were suffering from Duchene Muscular Dystrophy type, 17 had Limb Girdle Muscular Dystrophy, 11 had Congenital Muscular Dystrophy, 2 had Becker’s Muscular Dystrophy and 1 had Fascioscapulohumeral Dystrophy. The mean follow up of 6 months showed that out of 72, 32 showed improved trunk strength, 30 improved in their lower extremity strength with 11 of them showing gait improvement and 20 improved in upper extremity strength. (56) Induced pluripotent stem (iPS) cells are a recent development which has brought a promise of great therapeutic values. Studies conducted on animal models, have demonstrated iPS cells to have myogenic regenerative potential. (57-58)
Further research is ongoing, and is clearly necessary to make this therapy a viable treatment option for patients with muscular dystrophy.



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