Yvan Torrente

Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.

Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.

Human circulating AC133 + stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. Here we report that a subpopulation of circulating cells expressing AC133, a well-characterized marker of hematopoietic stem cells, also expresses early myogenic markers. Freshly isolated, circulating AC133(+) cells were induced to undergo myogenesis when cocultured with myogenic cells or exposed to Wnt-producing cells in vitro and when delivered in vivo through the arterial circulation or directly into the muscles of transgenic scid/mdx mice (which allow survival of human cells). Injected cells also localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and MYF5. Furthermore, functional tests of injected muscles revealed a substantial recovery of force after treatment. As these cells can be isolated from the blood, manipulated in vitro, and delivered through the circulation, they represent a possible tool for future cell therapy applications in DMD disease or other muscular dystrophies.

Transplantation of Genetically Corrected Human iPSC-Derived Progenitors in Mice with Limb-Girdle Muscular Dystrophy

Genetically corrected mesoangioblasts from human iPSCs derived from limb-girdle muscular dystrophy patients produce muscle fibers expressing the therapeutic gene in a mouse model of the disease. Muscle Progenitors Find Their Way Home Muscular dystrophies are genetic disorders primarily affecting skeletal muscle that result in greatly impaired mobility and, in severe cases, respiratory and cardiac dysfunction. There is no effective treatment, although several new approaches are entering clinical testing including cell therapy. Cell therapy aims to replace lost muscle fibers by transplanting healthy donor muscle progenitor cells or cells from dystrophic patients that have been genetically corrected in vitro. Mesoangioblasts are progenitor cells from blood vessel walls that have shown potential as a cell therapy in animal models of muscular dystrophy. In a new study, Tedesco et al. explore whether genetically corrected mesoangioblasts from patients with limb-girdle muscular dystrophy 2D (LGMD2D) have potential as an autologous cell therapy to treat this disease. The authors quickly found that they could not derive a sufficient number of mesoangioblasts from LGMD2D patients because the muscles of the patients were depleted of these progenitor cells. To overcome this problem, the authors reprogrammed fibroblasts or myoblasts from the LGMD2D patients to obtain human induced pluripotent stem cells (iPSCs) and induced them to differentiate into mesoangioblast-like cells that were then genetically corrected in vitro using a viral vector expressing the defective gene SGCA, which encodes α-sarcoglycan. After intramuscular or intra-arterial injection of these genetically corrected, iPSC-derived mesoangioblasts into mice with LGMD2D (immune-deficient Sgca-null mice), the cells homed to damaged mouse skeletal muscle, engrafted, and formed muscle fibers expressing α-sarcoglycan. Using mouse iPSC-derived mesoangioblasts, the researchers showed that the transplanted engrafted cells imbued muscle with greater strength and enabled the dystrophic mice to run for longer on a treadmill than dystrophic mice that did not receive the cells. This strategy offers the advantage of being able to produce unlimited numbers of genetically corrected progenitor cells, which perhaps could be used in the future as cell therapy for treating LGMD2D and other forms of muscular dystrophy. Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne’s muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan–null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan–null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

Restoration of Human Dystrophin Following Transplantation of Exon-Skipping-Engineered DMD Patient Stem Cells into Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.

Autologous transplantation of muscle-derived CD133(+) stem cells in Duchenne muscle patients

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration

Cell therapy for degenerative muscle diseases such as the muscular dystrophies requires a source of cells with the capacity to participate in the formation of new muscle fibers. We investigated the myogenic potential of human fetal mesenchymal stem cells (hfMSCs) using a variety of stimuli. The use of 5‐azacytidine or steroids did not produce skeletal muscle differentiation, whereas myoblast‐conditioned medium resulted in only 1%–2% of hfMSCs undergoing muscle differentiation. However, in the presence of galectin‐1, 66.1% ± 5.7% of hfMSCs, but not adult bone marrow‐derived mesenchymal stem cells, assumed a muscle phenotype, forming long, multinucleated fibers expressing both desmin and sarcomeric myosin via activation of muscle regulatory factors. Continuous exposure to galectin‐1 resulted in more efficient muscle differentiation than pulsed exposure (62.3% vs. 39.1%; p < .001). When transplanted into regenerating murine muscle, galectin‐1‐exposed hfMSCs formed fourfold more human muscle fibers than nonstimulated hfMSCs (p = .008), with similar results obtained in a scid/mdx dystrophic mouse model. These data suggest that hfMSCs readily undergo muscle differentiation in response to galectin‐1 through a stepwise progression similar to that which occurs during embryonic myogenesis. The high degree of myogenic conversion achieved by this method has relevance for the development of therapies for muscular dystrophies.

Human skin-derived stem cells migrate throughout forebrain and differentiate into astrocytes after injection into adult mouse brain.

Recent evidence indicates that neural stem cell properties can be found among a mammalian skin‐derived multipotent population. A major barrier in the further characterization of the human skin‐derived neural progenitors is the inability to isolate this population based on expression of cell surface markers. Our work has been devoted to purified human skin‐derived stem cells that are capable of neural differentiation, based on the presence or absence of the AC133 cell surface marker. The enriched skin‐derived AC133+ cells express the CD34 and Thy‐1 antigens. These cells cultured in a growth medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) proliferate, forming spheres, and differentiate in vitro into neurons, astrocytes, and rarely into oligodendrocytes. Single cells from sphere cultures initiated from human purified AC133+ cells were replated as single cells and were able to generate new spheres, demonstrating the self‐renewing ability of these stem cell populations. Brain engraftment of cells obtained from human purified AC133+‐derived spheres generated different neural phenotypes: immature neurons and a most abundant population of well differentiated astrocytes. The AC133‐derived astrocytes assumed perivascular locations in the frontal cortex. No donor‐derived oligodendrocytes were found in the transplanted mouse brains. Several donor small, rounded cells that expressed endothelial markers were found close to the host vessel and near the subventricular zone. Thus, mammalian skin AC133‐derived cells behave as a multipotent population with the capacity to differentiate into neural lineages in vitro and, prevalently, endothelium and astrocytes in vivo, demonstrating the great plasticity of these cells and suggesting potential clinical application.

The involvement of microRNAs in neurodegenerative diseases.

Neurodegenerative diseases (NDDs) originate from a loss of neurons in the central nervous system and are severely debilitating. The incidence of NDDs increases with age, and they are expected to become more common due to extended life expectancy. Because no cure is available, these diseases have become a major challenge in neurobiology. The increasing relevance of microRNAs (miRNAs) in biology has prompted investigation into their possible involvement in neurodegeneration in order to identify new therapeutic targets. The idea of using miRNAs as therapeutic targets is not far from realization, but important issues need to be addressed before moving into the clinics. Here, we review what is known about the involvement of miRNAs in the pathogenesis of NDDs. We also report the miRNA expression levels in peripheral tissues of patients affected by NDDs in order to evaluate their application as biomarkers of disease. Finally, discrepancies, innovations, and the effectiveness of collected data will be elucidated and discussed.

Functional changes in Duchenne muscular dystrophy A 12-month longitudinal cohort study

Objective: The aim of the study was to assess different outcome measures in a cohort of ambulant boys with Duchenne muscular dystrophy (DMD) over 12 months in order to establish the spectrum of possible changes in relation to age and steroid treatment. Methods: The study is a longitudinal multicentric cohort study. A total of 106 ambulant patients with DMD were assessed using the 6-minute walk test (6MWT) and North Star Ambulatory Assessment (NSAA) at baseline and 12 months. Clinical data including age and steroid treatment were collected. Results: During the 12 months of the study, we observed a mean decline of 25.8 meters in the 6MWT with a SD of 74.3 meters. On NSAA, the mean decline was 2.2 points with a SD of 3.7. Not all the boys with DMD in our cohort showed a decline over the 12 months, with young boys showing some improvement in their 6MWT and NSAA scores up to the age of 7. NSAA and the 6MWT had the highest correlation (r = 0.52, p < 0.001). Conclusions: This study provides longitudinal data of NSAA and 6MWT over a 12-month period. These data can be useful when designing a clinical trial.

Mesenchymal stem cell transplantation for neurodegenerative diseases.

Neurodegenerative diseases are characterized by a progressive degeneration of selective neural populations. The lack of effective treatment and the characteristic of their pathology make these diseases appropriate candidates for cell therapy. Mesenchymal stem cells (MSCs) are multipotent stem-like cells that are capable of differentiating into mesenchymal and nonmesenchymal lineages. Their regenerative capacity after in vivo transplantation into animal models of neurodegenerative diseases has suggested that they could be useful against human diseases. Human bone marrow-derived MSCs (hMSCs) can be easily amplified in vitro and their transdifferentiation has been claimed in vitro and in vivo in neural cells. There are some doubts concerning the exact mechanisms responsible for the beneficial outcome observed after MSC transplantation into neurodegenerating tissues. Possible interpretations include cell replacement, trophic factor delivery, and immunomodulation. This review mainly concerns hMSCs transplantation in neurodegenerative diseases, because it has proven to be feasible, safe, and potentially effective. Although they have been used in hundreds of clinical trials, mixed results and no functional and long-lasting integration have so far been observed. hMSCs transplantations therefore still have their “dark side.” However, the challenge in well-planned clinical trials merits discussion.