Dystrophin deficiency impairs cell junction formation during embryonic myogenesis from pluripotent stem cells.

Mutations in the gene lead to Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2-4 years of age, but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients, which poses a serious challenge in current standards of care. Here, we investigated the consequences of dystrophin deficiency during skeletal muscle development.

Dystrophin deficiency impairs cell junction formation during embryonic myogenesis.

Mutations in the gene lead to Duchenne muscular dystrophy, a severe X-linked neuromuscular disorder that manifests itself as young boys acquire motor functions. DMD is typically diagnosed at 2 to 4 years of age, but the absence of dystrophin negatively impacts muscle structure and function before overt symptoms appear in patients, which poses a serious challenge in the optimization of standards of care. In this report, we investigated the early consequences of dystrophin deficiency during skeletal muscle development.

Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts.

Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblasts-the effector cells of muscle growth and regeneration-are affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts.

CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis.

Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease.

Myogenesis modelled by human pluripotent stem cells: a multi-omic study of Duchenne myopathy early onset.

Background: Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5000 male births. Symptoms appear in early childhood, with a diagnosis made mostly around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise-even asymptomatically-is still unknown.

miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients.

Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins.

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells.

The paracrine effects of human induced pluripotent stem cells promote bone-like structures via the upregulation of BMP expression in a mouse ectopic model.

Use of human induced pluripotent stem cells (h-iPSCs) for bone tissue engineering is most appealing, because h-iPSCs are an inexhaustible source of osteocompetent cells. The present study investigated the contribution of undifferentiated h-iPSCs and elucidated aspects of the underlying mechanism(s) of the involvement of these cells to new bone formation. Implantation of undifferentiated h-iPSCs seeded on coral particles in ectopic sites of mice resulted in expression of osteocalcin and DMP-1, and in mineral content similar to that of the murine bone.

Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.

Quality control of cell cultures used in new in vitro toxicology assays is crucial to the provision of reliable, reproducible and accurate toxicity data on new drugs or constituents of new consumer products. This chapter explores the key scientific and ethical criteria that must be addressed at the earliest stages of developing toxicology assays based on human pluripotent stem cell (hPSC) lines. It also identifies key considerations for such assays to be acceptable for regulatory, laboratory safety and commercial purposes.

Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

Background: Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address.

Methods: The bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment.

Cytostatic Effect of Repeated Exposure to Simvastatin: A Mechanism for Chronic Myotoxicity Revealed by the Use of Mesodermal Progenitors Derived from Human Pluripotent Stem Cells.

Statin treatment of hypercholesterolemia can lead to chronic myotoxicity which is, in most cases, alleviated by drug withdrawal. Cellular and molecular mechanisms of this adverse effect have been elusive, in particular because of the lack of in vitro models suitable for long-term exposures. We have taken advantage of the properties of human pluripotent stem cell-derived mesodermal precursors, that can be maintained unaltered in vitro for a long period of time, to develop a model of repeated exposures to simvastatin during more than 2 weeks.

The ENKI-2 water-jet system versus Dual Knife for endoscopic submucosal dissection of colorectal lesions: a randomized comparative animal study.

Background And Study Aims: The ENKI-2 water-jet system for endoscopic submucosal dissection (ESD) combines submucosal saline pressure injection with dissection. The aim of this study was to compare ENKI-2 with a standard device in terms of procedure time and perforation rate during colorectal ESD.

Methods: In this randomized comparative study, 10 30-mm-diameter lesions were created in the colon and rectum of 10 healthy adult pigs.

H19 antisense RNA can up-regulate Igf2 transcription by activation of a novel promoter in mouse myoblasts.

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome.

[Prospective evaluation of intrasphincteric injections of autologous muscular cells in patients with stress urinary incontinence following radical prostatectomy].

Purpose: Cell therapy for urinary incontinence management has been experienced in animals with encouraging results, but studies in human beings are lacking. Our primary objective was to assess the safety of intrasphincteric injections of autologous muscular cells in patients with postprostatectomy incontinence (PPI). Secondary objectives focused on complications efficacy.

Intrasphincteric injections of autologous muscular cells in women with refractory stress urinary incontinence: a prospective study.

Introduction And Hypothesis: Cell therapy for stress urinary incontinence (SUI) management has been experienced with encouraging results.

Methods: We conducted an open prospective study on 12 women presenting severe SUI with fixed urethra, after previous failed surgical management. Patients underwent intrasphincteric injections of autologous progenitor muscular cells isolated from a biopsy of deltoid muscle.

Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene.

Transplantation of muscle precursor cells (MPCs) is a promising approach for the treatment of muscular dystrophies. However, preclinical and clinical results have shown that the technology is not yet efficient enough for most therapeutic applications. Among the problems that remain unsolved are low cellular survival, poor proliferation and lack of migration of the transplanted cells.

An automatic image analysis approach to quantify stained cell cultures.

Counting cells in culture is a common task in biotechnology research and production. This process should be automated to provide fast and objective quantification. Flow cytometry is adapted to count cells in suspension.

Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5.

Background: The two myogenic regulatory factors Myf5 and MyoD are basic helix-loop-helix muscle transcription factors undergoing differential cell cycle dependent proteolysis in proliferating myoblasts. This regulated degradation results in the striking expression of these two factors at distinct phases of the cell cycle, and suggests that their precise and alternated disappearance is an important feature of myoblasts, maybe connected to the maintenance of the proliferative status and/or commitment to the myogenic lineage of these cells. One way to understand the biological function(s) of the cyclic expression of these proteins is to specifically alter their degradation, and to analyze the effects of their stabilization on cells.

Fate of implanted syngenic muscle precursor cells in striated urethral sphincter of female rats: perspectives for treatment of urinary incontinence.

Objectives: To analyze the outcome of syngenic skeletal muscle precursor cells (MPCs) after implantation in the striated urethral sphincter of the female rat.

Methods: MPCs were isolated from the striated muscles of the lower limbs and infected with a retrovirus carrying the gene for green fluorescent protein. Approximatively 10(5) cells were injected longitudinally in the striated urethral sphincter of 24 animals using a 10-muL Hamilton syringe.

Dose effect relationship between the number of normal progenitor muscle cells grafted in mdx mouse skeletal striated muscle and the number of dystrophin-positive fibres.

The transplantation of progenitor muscle cells in striated skeletal muscle of mdx mice, a model of dystrophin deficiency, is well known to induce the formation of mosaic fibres expressing dystrophin near the site of injection. We tried to determine if the number of injected cells is related to the number of dystrophin-positive fibres. The grafted cells provided by 5 day-old C57Bl10 mice are syngenic to mdx mice and were cultured to select undifferentiated progenitors.

Noncultured, autologous, skeletal muscle cells can successfully engraft into ovine myocardium.

Background: There is compelling evidence showing that cellular cardiomyoplasty can improve cardiac function. Considering the potential benefit of using noncultured muscle cells (little time, lower cost, reduced risk of contamination), we investigated the feasibility of grafting cells obtained directly after enzymatic dissociation of skeletal muscle biopsies into ovine myocardium. We hypothesized that those noncultured muscle cells would engraft massively.

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells.

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed.