Necdin enhances muscle reconstitution of dystrophic muscle by vessel-associated progenitors, by promoting cell survival and myogenic differentiation.

Improving stem cell therapy is a major goal for the treatment of muscle diseases, where physiological muscle regeneration is progressively exhausted. Vessel-associated stem cells, such as mesoangioblasts (MABs), appear to be the most promising cell type for the cell therapy for muscular dystrophies and have been shown to significantly contribute to restoration of muscle structure and function in different muscular dystrophy models. Here, we report that melanoma antigen-encoding gene (MAGE) protein necdin enhances muscle differentiation and regeneration by MABs. When necdin is constitutively overexpressed, it accelerates their differentiation and fusion in vitro and it increases their efficacy in reconstituting regenerating myofibres in the α-sarcoglycan dystrophic mouse. Moreover, necdin enhances survival when MABs are exposed to cytotoxic stimuli that mimic the inflammatory dystrophic environment. Taken together, these data demonstrate that overexpression of necdin may be a crucial tool to boost therapeutic applications of MABs in dystrophic muscle.

Partial dysferlin reconstitution by adult murine mesoangioblasts is sufficient for full functional recovery in a murine model of dysferlinopathy.

Dysferlin deficiency leads to a peculiar form of muscular dystrophy due to a defect in sarcolemma repair and currently lacks a therapy. We developed a cell therapy protocol with wild-type adult murine mesoangioblasts. These cells differentiate with high efficiency into skeletal muscle in vitro but differ from satellite cells because they do not express Pax7. After intramuscular or intra-arterial administration to SCID/BlAJ mice, a novel model of dysferlinopathy, wild-type mesoangioblasts efficiently colonized dystrophic muscles and partially restored dysferlin expression. Nevertheless, functional assays performed on isolated single fibers from transplanted muscles showed a normal repairing ability of the membrane after laser-induced lesions; this result, which reflects gene correction of an enzymatic rather than a structural deficit, suggests that this myopathy may be easier to treat with cell or gene therapy than other forms of muscular dystrophies.

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.

Cell therapy of primary myopathies.

Mesoangioblasts are multipotent progenitors of mesodermal tissues. In vitro mesoangioblasts differentiate into many mesoderm cell types, such as smooth, cardiac and striated muscle, bone and endothelium. After transplantation mesoangioblasts colonize mostly mesoderm tissues and differentiate into many cell types of the mesoderm. When delivered through the arterial circulation, mesoangioblasts significantly restore skeletal muscle structure and function in a mouse model of muscular dystrophy. Their ability to extensively self-renew in vitro, while retaining multipotency, qualifies mesoangioblasts as a novel class of stem cells. Phenotype, properties and possible origin of mesoangioblasts are addressed in the first part of this paper. In the second part we will focus on the cell therapy approach for the treatment of Muscular Dystrophy and we will describe why mesangioblasts appear to be promising candidates for this strategy.

Spectrum of mutations in fucosidosis.

Fucosidosis is a lysosomal storage disorder characterised by progressive psychomotor deterioration, angiokeratoma and growth retardation. It is due to deficient alpha-l-fucosidase activity leading to accumulation of fucose-containing glycolipids and glycoproteins in various tissues. Fucosidosis is extremely rare with less than 100 patients reported worldwide, although the disease occurs at a higher rate in Italy, in the Hispanic-American population of New Mexico and Colorado, and in Cuba. We present here a review study of the mutational spectrum of fucosidosis. Exon by exon mutation analysis of FUCA1, the structural gene of alpha-l-fucosidase, has identified the mutation(s) in nearly all fucosidosis patients investigated. The spectrum of the 22 mutations detected to date includes four missense mutations, 17 nonsense mutations consisting of seven stop codon mutations, six small deletions, two large deletions, one duplication, one small insertion and one splice site mutation. All these mutations lead to nearly absent enzymatic activity and severely reduced cross-reacting immunomaterial. The observed clinical variability is, therefore, not due to the nature of the fucosidosis mutation, but to secondary unknown factors.

Rox, a novel bHLHZip protein expressed in quiescent cells that heterodimerizes with Max, binds a non-canonical E box and acts as a transcriptional repressor.

Proteins of the Myc and Mad family are involved in transcriptional regulation and mediate cell differentiation and proliferation. These molecules share a basic-helix-loop-helix leucine zipper domain (bHLHZip) and bind DNA at the E box (CANNTG) consensus by forming heterodimers with Max. We report the isolation, characterization and mapping of a human gene and its mouse homolog encoding a new member of this family of proteins, named Rox. Through interaction mating and immunoprecipitation techniques, we demonstrate that Rox heterodimerizes with Max and weakly homodimerizes. Interestingly, bandshift assays demonstrate that the Rox-Max heterodimer shows a novel DNA binding specificity, having a higher affinity for the CACGCG site compared with the canonical E box CACGTG site. Transcriptional studies indicate that Rox represses transcription in both human HEK293 cells and yeast. We demonstrate that repression in yeast is through interaction between the N-terminus of the protein and the Sin3 co-repressor, as previously shown for the other Mad family members. ROX is highly expressed in quiescent fibroblasts and expression markedly decreases when cells enter the cell cycle. Moreover, ROX expression appears to be induced in U937 myeloid leukemia cells stimulated to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The identification of a novel Max-interacting protein adds an important piece to the puzzle of Myc/Max/Mad coordinated action and function in normal and pathological situations. Furthermore, mapping of the human gene to chromosome 17p13.3 in a region that frequently undergoes loss of heterozygosity in a number of malignancies, together with the biochemical and expression features, suggest involvement of ROX in human neoplasia.

Kallmann syndrome gene on the X and Y chromosomes: implications for evolutionary divergence of human sex chromosomes.

The recently identified gene for X-linked Kallmann syndrome (hypogonadotropic hypogonadism and anosmia) has a closely related homologue on the Y chromosome. The X and Y copies of this gene are located in a large region of X/Y homology, on Xp22.3 and Yq11.2, respectively. Comparison of the structure of the X-linked Kallmann syndrome gene and its Y homologue shed light on the evolutionary history of this region of the human sex chromosomes. Our data show that the Y homologue is not functional. Comparative analysis of X/Y sequence identity at several loci on Xp22.3 and Yq11.2 suggests that the homology between these two regions is the result of a complex series of events which occurred in the recent evolution of sex chromosomes.

A gene deleted in Kallmann's syndrome shares homology with neural cell adhesion and axonal path-finding molecules.

Kallmann's syndrome (clinically characterized by hypogonadotropic hypogonadism and inability to smell) is caused by a defect in the migration of olfactory neurons, and neurons producing hypothalamic gonadotropin-releasing hormone. A gene has now been isolated from the critical region on Xp22.3 to which the syndrome locus has been assigned: this gene escapes X inactivation, has a homologue on the Y chromosome, and shows an unusual pattern of conservation across species. The predicted protein has significant similarities with proteins involved in neural cell adhesion and axonal pathfinding, as well as with protein kinases and phosphatases, which suggests that this gene could have a specific role in neuronal migration.

Characterization of a murine gene expressed from the inactive X chromosome.

In mammals, equal dosage of gene products encoded by the X chromosome in male and female cells is achieved by X inactivation. Although X-chromosome inactivation represents the most extensive example known of long range cis gene regulation, the mechanism by which thousands of genes on only one of a pair of identical chromosomes are turned off is poorly understood. We have recently identified a human gene (XIST) exclusively expressed from the inactive X chromosome. Here we report the isolation and characterization of its murine homologue (Xist) which localizes to the mouse X inactivation centre region and is the first murine gene found to be expressed from the inactive X chromosome. Nucleotide sequence analysis indicates that Xist may be associated with a protein product. The similar map positions and expression patterns for Xist in mouse and man suggest that this gene may have a role in X inactivation.

A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome.

X-chromosome inactivation results in the cis-limited dosage compensation of genes on one of the pair of X chromosomes in mammalian females. Although most X-linked genes are believed to be subject to inactivation, several are known to be expressed from both active and inactive X chromosomes. Here we describe an X-linked gene with a novel expression pattern--transcripts are detected only from the inactive X chromosome (Xi) and not from the active X chromosome (Xa). This gene, called XIST (for Xi-specific transcripts), is a candidate for a gene either involved in or uniquely influenced by the process of X inactivation.