Acute conversion of patient-derived Duchenne muscular dystrophy iPSC into myotubes reveals constitutive and inducible over-activation of TGFß-dependent pro-fibrotic signaling.

BACKGROUND: In Duchenne muscular dystrophy (DMD), DYSTROPHIN deficiency exposes myofibers to repeated cycles of contraction/degeneration, ultimately leading to muscle loss and replacement by fibrotic tissue. DMD pathology is typically exacerbated by excessive secretion of TGFß and consequent accumulation of pro-fibrotic components of the extra-cellular matrix (ECM), which in turn impairs compensatory regeneration and complicates the efficacy of therapeutic strategies. It is currently unclear whether DMD skeletal muscle fibers directly contribute to excessive activation of TGFß. Development of skeletal myofibers from DMD patient-derived induced pluripotent stem cells (iPSC), as an "in dish" model of disease, can be exploited to determine the myofiber contribution to pathogenic TGFß signaling in DMD and might provide a screening platform for the identification of anti-fibrotic interventions in DMD. METHODS: We describe a rapid and efficient method for the generation of contractile human skeletal muscle cells from DMD patient-derived hiPSC, based on the inducible expression of MyoD and BAF60C (encoded by SMARCD3 gene), using an enhanced version of piggyBac (epB) transposone vectors. DMD iPSC-derived myotubes were tested as an "in dish" disease model and exposed to environmental and mechanical cues that recapitulate salient pathological features of DMD. RESULTS: We show that DMD iPSC-derived myotubes exhibit a constitutive activation of TGFß-SMAD2/3 signaling. High-content screening (HCS)-based quantification of nuclear phosphorylated SMAD2/3 signal revealed that DMD iPSC-derived myotubes also exhibit increased activation of the TGFß-SMAD2/3 signaling following exposure to either recombinant TGFß or electrical pacing-induced contraction. CONCLUSIONS: Acute conversion of DMD patient-derived iPSC into skeletal muscles, by the ectopic expression of MyoD and BAF60C, provides a rapid and reliable protocol for an "in dish" DMD model that recapitulates key pathogenic features of disease pathology, such as the constitutive activation of the TGFß/SMAD signaling as well as the deregulated response to pathogenic stimuli, e.g., ECM-derived signals or mechanical cues. Thus, this model is suitable for the identification of new therapeutic targets in DMD patient-specific muscles.

Differentiation of control and ALS mutant human iPSCs into functional skeletal muscle cells, a tool for the study of neuromuscolar diseases.

Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disease characterized by progressive loss of motoneurons, muscle atrophy and paralysis. Recent evidence suggests that ALS should be considered as a multi-systemic disease, in which several cell types contribute to motoneuron degeneration. In this view, mutations in ALS linked genes in other neural and non-neural cell types may exert non-cell autonomous effects on motoneuron survival and function. Induced Pluripotent Stem Cells (iPSCs) have been recently derived from several patients with ALS mutations and it has been shown that they can generate motoneurons in vitro, providing a valuable tool to study ALS. However, the potential of iPSCs could be further valorized by generating other cell types that may be relevant to the pathology. In this paper, by taking advantage of a novel inducible system for MyoD expression, we show that both control iPSCs and iPSCs carrying mutations in ALS genes can generate skeletal muscle cells. We provide evidence that both control and mutant iPSC-derived myotubes are functionally active. This in vitro system will be instrumental to dissect the molecular and cellular pathways impairing the complex motoneuron microenvironment in ALS.

ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons.

Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patient fibroblasts, whereas in the case of the severe FUS(P525L) mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.

Survey of Mymarommatidae and their occurrence in agricultural systems in Brazil.

Mymarommatidae surveys were carried out through the use of yellow pan traps in crops of green dwarf coconut, Cocos nucifera L. (Arecales: Arecaceae), papaya, Carica papaya L. (Brassicales: Caricaceae), citrus, Citrus spp. L. (Sapindales: Rutaceae), and guava, Psidium guajava L. (Myrtales: Myrtaceae), in the northern Espirito Santo State, Brazil. 146 specimens of mymarommatids were collected, of which 71, 55, 16, and 4 exemplars were obtained in the area cultivated with guava, papaya, citrus, and coconut, respectively. The mean numbers of mymarommatids collected in the period from April to June 2011 were significantly higher than those obtained in the other nine months. Two genera, Mymaromma and Mymaromella, were identified The most abundant genus was Mymaromma, comprising 93.8% of the total collection; however, the genus Mymaromella was encountered in all crops. This is the first record of the presence of mymarommatids in these agricultural systems.