In Duchenne muscular dystrophy (DMD) the absence of dystrophin at the sarcolemma delocalizes and downregulates nitric oxide synthase (nNOS); this alters S-nitrosylation of HDAC2 and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes. Several circuitries controlled by the identified microRNAs, such as the one linking miR-1 to the G6PD enzyme and the redox state of cell, or miR-29 to extracellular proteins and the fibrotic process, explain some of the DMD pathogenetic traits. We also show that, at variance with other myomiRs, miR-206 escapes from the dystrophin-nNOS control being produced in activated satellite cells before dystrophin expression; in these cells, it contributes to muscle regeneration through repression of the satellite specific factor, Pax7. We conclude that the pathway activated by dystrophin/nNOS controls several important circuitries increasing the robustness of the muscle differentiation program.

Duchenne muscular dystrophy (DMD)-which is caused by mutations in the dystrophin gene-is one of the most severe myopathies. Among therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional messenger RNA. Here, we report the identification of a microRNA-miR-31-that represses dystrophin expression by targeting its 3 0 untranslated region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR-31 inhibition increases dystrophin rescue. These results indicate that interfering with miR-31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.

Pluripotent stem (PS) cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous therefore clinical translation requires development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin α9β1 and Syndecan2 (SDC2) as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and cGMP-compatible magnetic-based sorting technologies, and that CD54+α9β1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step towards enabling translation of PS cell-based therapies for muscle diseases.

Three-dimensional (3D) porous scaffolds combined with therapeutic stem cells play vital roles in tissue engineering. The adult brain has very limited regeneration ability after injuries such as trauma and stroke. In this study, injectable 3D silk fibroin-based hydrogel scaffolds with encapsulated neural stem cells were developed, aiming at supporting brain regeneration. To improve the function of the hydrogel towards neural stem cells, silk fibroin was modified by an IKVAV peptide through covalent binding. Both unmodified and modified silk fibroin hydrogels were obtained, through sonication, with mechanical stiffness comparable to that of brain tissue. Human neural stem cells were encapsulated in both hydrogels and the effects of IKVAV peptide conjugation on cell viability and neural differentiation were assessed. The silk fibroin hydrogel modified by IKVAV peptide showed increased cell viability and an enhanced neuronal differentiation capability, which contributed to understanding the effects of IKVAV peptide on the behaviour of neural stem cells. For these reasons, IKVAV-modified silk fibroin is a promising material for brain tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

No abstract

Different hydrogel materials have been prepared to investigate the effects of culture substrate on the behaviour of pluripotent cells. In particular, genipin-crosslinked gelatin-silk fibroin hydrogels of different compositions have been prepared, physically characterized and used as substrates for the culture of pluripotent cells. Pluripotent cells cultured on hydrogels remained viable and proliferated. Gelatin and silk fibroin promoted the proliferation of cells in the short and long term, respectively. Moreover, cells cultured on genipin-crosslinked gelatin-silk fibroin blended hydrogels were induced to an epithelial ectodermal differentiation fate, instead of the neural ectodermal fate obtained by culturing on tissue culture plates. This work confirms that specific culture substrates can be used to modulate the behaviour of pluripotent cells and that our genipin-crosslinked gelatin-silk fibroin blended hydrogels can induce pluripotent cells differentiation to an epithelial ectodermal fate. Copyright © 2014 John Wiley & Sons, Ltd.

Many mutations and deletions in the dystrophin gene, responsible for Duchenne muscular dystrophy (DMD), can be corrected at the posttranscriptional level by skipping specific exons. Here we show that long-term benefit can be obtained in the dystrophic mouse model through the use of adeno-associated viral vectors expressing antisense sequences: persistent exon skipping, dystrophin rescue, and functional benefit were observed 74 weeks after a single systemic administration. The therapeutic benefit was sufficient to preserve the muscle integrity of mice up to old age. These results indicate a possible long-term gene therapy treatment of the DMD pathology.

Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSCs) allows the generation of myogenic progenitors endowed with enhanced regenerative capacity. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro-generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared with fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple reinjuries, and contribute to long-term regeneration. Upon engraftment, the transcriptome of reisolated Pax3/Pax7-induced PSC-derived myogenic progenitors changes toward a postnatal molecular signature, particularly in genes involved in extracellular matrix remodeling. These findings demonstrate that Pax3/Pax7-induced myogenic progenitors remodel their molecular signature and functionally mature upon in vivo exposure to the adult muscle environment. pluripotent stem cell | skeletal myogenesis | Pax3 | Pax7 | transcriptome analysis