Transcriptome sequencing of human Duchenne muscular dystrophy and CRISPR-corrected myogenic cultures during secondary myogenic differentiation.
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ABSTRACT: We sought to investigate whether the reduced differentiation and fusion competence in DMD-R3381X cultures was due to aberrant myogenic gene expression. We performed transcriptome sequencing and analysis of DMD-R3381X and CORR-R3381X myogenic cultures during secondary differentiation at 0, 24 and 120 hours timepoints.
Project description:To investigate transcriptomes of skeletal muscle cells in health and Duchene muscular dystrophy (DMD), we performed RNA sequencing of DMD-K2957fs and CRISPR-corrected (CORR-K2957fs ) myogenic cultures during secondary differentiation at 5 time points.
Project description:Transcriptome sequencing of human Duchenne muscular dystrophy and CRISPR-corrected myogenic cultures during secondary myogenic differentiation.
Project description:In Duchenne Muscular Dystrophy (DMD), the absence of the subsarcolemmal dystrophin protein leads to repeated myofiber damages inducing cycles of muscle regeneration that is driven by muscle stem cells (MuSCs). With time, MuSC regenerative capacities are overwhelmed, leading to fibrosis and muscle atrophy. Whether MuSCs from DMD muscle have intrinsic alterations or are primed by their degenerative/regenerative environment is still debated. We investigated cell behavior and gene expression in human using MuSCs derived from DMD or healthy muscles. We found that proliferation, differentiation and fusion were not altered in DMD-MuSCs, but with time, they lost the expression of the myogenic marker CD56 twice as fast as healthy MuSCs. The rapid drift towards a fibroblast-like cell identity was observed at the clonal level, and resulted from the altered expression of epigenetic enzymes required to maintain the myogenic cell fate. Indeed, the reexpression of CBX3, SMC3, H2AFV and H3F3B prevented the MuSC identity drift. Amongst the epigenetic changes, a closing of chromatin at the gene encoding the transcription factor MEF2B caused a down-regulation of its expression and a loss of the myogenic fate. Thus, MEF2B is a key mediator of the myogenic identity in human MuSCs, that is altered in DMD pathology.
Project description:Duchenne Muscular Dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced Pluripotent Stem Cells (iPSC) to a late myogenic stage. This allows to recapitulate classical DMD phenotypes (mislocalization of proteins of the Dystrophin glycoprotein associated complex (DGC), increased fusion, myofiber branching, force contraction defects and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are due to anti-inflammatory properties. Our work introduces a new human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.
Project description:Genome-wide gene expression analysis of MyoD-infected DMD-specific iPSCs (GM05112-M5.1) on days 0 (untreated), day 3 and day 8 post Dox treatment, human primary myoblasts (undifferentiated and as differentiated myotubes), and undifferentiated iPSCs from healthy donors (iPSCs-1 and iPSCs-2). DMD-specific iPSCs were infected with lentivirus expressing MyoD under the control of Tet-inducible promoter and another lentivirus expressing the transactivator. To initiate myogenic differentiation, iPSCs were treated with 1µg/ml Dox. RNA was isolated 0, 3 and 8 days later and gene expression analysis was performed.
Project description:Transcriptome analysis of human DMD-K2957fs and CRISPR-corrected (CORR-K2957fs ) myogenic cultures during secondary myogenic differentiation.
Project description:Skeletal muscle is highly vascularized. Beyond oxygen and nutriment supply, new functions for vessels have been recently identified, via the interactions that vessel cells establish with muscle progenitor cells. These latter cells closely interact with endothelial cells for their expansion and their differentiation, while periendothelial cells are involved in muscle cell self-renewal and return to quiescence. Thus, vessels play a central role in the tissue remodeling after an injury, while the mechanisms are poorly understood. We investigated myogenic/endothelial cell (MPCs/ECs) interactions in two paradigmatic contexts of regenerating muscle in the child: Juvenile Dermatomyositis (JDM), which is characterized by a transient loss of capillaries, and Duchenne Muscular Dystrophy (DMD), which is associated with an increase in vessel density. We showed in vitro specific interactions between myoblasts isolated from muscle of JDM and DMD patients and endothelial cells. In vitro myogenesis/angiogenesis studies demonstrated that MPCs exhibited various angiogenic properties depending on the pathological environment. In DMD, MPCs promoted the development of an anarchic, although strong, ECs stimulation, leading to the formation of weakly functional vessels. DMD cells presented an unbalanced homeostasis with nonspecific deregulation of several processes involved in muscle and vessel development. On the contrary, in JDM, MPCs enhanced the vessel reconstruction to efficiently restore the vessel function via the expression of a set of specific angiogenic effectors. MPCs exhibit a strong specific type I IFNs signature and ECs a dysregulation of their angiogenic capacities, suggesting a drastic reprogrammation of these cells in response to an inflammatory environment during JDM.
Project description:gene expression data is from RNA extracted from muscle biopsy samples taken from boys with Duchenne muscular dystrophy (DMD) or pathologically normal controls (CTRL). Each muscle biospy was examined in detail histologically by Dr. Eric P. Hoffman at Children's National Medical Center to determine stage of disease. In addition, the absence or presence of dystrophin was determined via western blot analyses. We utilized Human U133 2.0 arrays to examine the transcriptome of each muscle, and then we compared differential gene expression between DMD patient muscles and CTRL muscules. We set the FDR p value for significance at 0.05 and at least a 1.5 fold difference in DMD/CTRL compared differential gene exrpression between DMD versus Control
Project description:In Duchenne Muscular Dystrophy (DMD), the absence of the subsarcolemmal dystrophin protein leads to repeated myofiber damages inducing cycles of muscle regeneration that is driven by muscle stem cells (MuSCs). With time, MuSC regenerative capacities are overwhelmed, leading to fibrosis and muscle atrophy. Whether MuSCs from DMD muscle have intrinsic alterations or are primed by their degenerative/regenerative environment is still debated. We investigated gene expression in human using primary MuSCs derived from DMD or healthy muscles. Cells were isolated from the muscle and bulk expanded then purified as CD56positive cells (CD56 is a myogenic marker that characterizes the myogenic nature of the cells). Human MuSCs loose the expression of the CD56 with time. We analyzed gene expression by CD56positive and CD56negative cells originating from the same initial CD56positive MuSC population.
Project description:Large animal models for Duchenne muscular dystrophy (DMD) are indispensible for preclinical evaluation of novel diagnostic procedures and treatment strategies. To evaluate functional consequences of Duchenne muscular dystrophy (DMD) in skeletal muscle and myocardium, we used a new genetically engineered dystrophin KO pig model displaying hallmarks of human DMD. Heart and skeletal muscle tissue samples of DMD pigs and wild-type (WT) controls at three different ages were analyzed by label-free proteomics.