View source for Myoblast to myotube (wt and DMD)

{{TimeCourse
|TCOverview= Differentiation of human primary skeletal myoblasts derived from healthy donors and patients affected by Duchenne Muscular Dystrophy. <br>Skeletal muscle regenerates thanks to the proliferation of mononuclear myogenic precursor cells called myoblasts, which have the ability to fuse and become part of multinucleated myotubes, which at later mature into myofibers. The fusion of myoblasts is specific to skeletal muscle. Myoblasts express FGF receptor and IGF expression is increased during myoblast differentiation in culture. The human skeletal muscle myoblast culture is a convenient in vitro model for the study of cellular development and differentiation process. Duchenne muscular dystrophy (DMD) is a genetically well-defined disorder being associated with mutations in the dystrophin gene (1). Increasing evidence indicates that disruption of the dystrophin-associated protein complex (DAPC) at the sarcolemma affects not only the structure of muscle fibers, but impact global genome expression (coding and non coding transcripts) through deregulation of the nNOS-HDAC2 pathway (2,3)<br><br>References:<br>[1] Davies, K. E. & Nowak, K. J. Molecular mechanisms of muscular dystrophies: old and new players. Nat Rev Mol Cell Biol 7, 762-773, (2006).<br>[2] Cacchiarelli, D. et al. MicroRNAs involved in molecular circuitries relevant for the Duchenne muscular dystrophy pathogenesis are controlled by the dystrophin/nNOS pathway. Cell Metab 12, 341-351, (2010). <br>[3] Colussi, C. et al. Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy. FASEB J 23, 2131-2141, (2009).<br>
|TCQuality_control=MyoD and Myogenin are transcription factors. MyoD is slightly upregulated (2-4 times), Myogenin is an early marker of differentiation, in general it is highly expressed in myocytes (day 1 to 4) before the fusion of these cells in polynucleated myotubes (day 4-5 to 12) when Myogenin expression is reduced. The class of Myosins genes are progressively upregulated for the entire process. Among genes that show the opposite trend you should find ID1, ID2 and ID3, known to inhibit differentiation, which are progressively repressed during differentiation.<br><html><img src='/resource_browser/images/TC_qc/1000px-Human_Myoblast_differentiation_to_myotubes.png' onclick='javascript:window.open("/resource_browser/images/TC_qc/1000px-Human_Myoblast_differentiation_to_myotubes.png", "imgwindow", "width=1000,height=375");' style='width:700px;cursor:pointer'/></html>Figure 1: CAGE expression of marker genes in TPM.<br>
|TCSample_description=Human primary cultures are derived directly from excised human tissue and cultured either as explants or after dissociation into a single cell suspension by enzyme digestion. Human primary myoblasts from healthy donors and DMD patients were obtained from Telethon BioBank, C. Besta Institute, Milan, Italy. All of the patients satisfied the accepted clinical criteria for DMD. They had undergone DNA diagnosis and were identified as carriers of specific exons deletions in the dystrophin gene. Details for individuals recruited for the study are listed in Table I. Muscle cells were cultured in DMEM supplemented with 20% FBS, human recombinant insulin (Sigma) 10 mg/ml, bFGF (Tebu-Bio) 25 ng/ml, EGF (Tebu-Bio) 10 ng/ml (proliferating medium), and induced to differentiate in DMEM supplemented with 2% horse serum (differentiating medium). For time course experiments, cells were harvested in proliferating condition (Myoblasts, Day 0) and at different days during the differentiation process (Myotubes, Day 1,2,3,4,6,8,10,12).<br><html><img src='/resource_browser/images/TC_qc/500px-Myoblasts_Table1.png'></html>
|Time_Course=
|category_treatment=Differentiation
|collaborators=Valerio Orlando
|description=human_Myoblast_differentiation_to_myotubes
|germ_layer=mesoderm
|libraryids=CNhs13847,CNhs13848,CNhs13849,CNhs13850,CNhs13851,CNhs13852,CNhs13853,CNhs13854,CNhs14566,CNhs14567,CNhs14570,CNhs14571,CNhs14572,CNhs14573,CNhs14574,CNhs14575,CNhs14576,CNhs14577,CNhs14578,CNhs14579,CNhs14580,CNhs14581,CNhs14582,CNhs14583,CNhs14585,CNhs14586,CNhs14587,CNhs14588,CNhs14589,CNhs14590,CNhs14591,CNhs14592,CNhs14594,CNhs14595,CNhs14596,CNhs14597,CNhs14598,CNhs14599,CNhs14600,CNhs14602,CNhs14603,CNhs14604,CNhs14605,CNhs14606,CNhs14607,CNhs14609,CNhs14610,CNhs14611,CNhs14612,CNhs14613
|number_time_points=9
|page_name=human_Myoblast_differentiation_to_myotubes
|primary_cells=primary cells
|series=IN_VITRO DIFFERENTIATION SERIES
|species=Human (Homo sapiens)
|tet_config=https://fantom.gsc.riken.jp/5/suppl/tet/Myoblast_differentiation_to_myotubes.tsv.gz
|tet_file=https://fantom.gsc.riken.jp/5/tet#!/search/?filename=hg19.cage_peak_phase1and2combined_tpm_ann_decoded.osc.txt.gz&file=1&c=1&c=932&c=933&c=934&c=929&c=930&c=931&c=938&c=940&c=935&c=936&c=937&c=944&c=945&c=946&c=941&c=942&c=943&c=949&c=950&c=951&c=947&c=948&c=955&c=956&c=957&c=952&c=953&c=954&c=961&c=962&c=963&c=958&c=960&c=967&c=968&c=969&c=964&c=965&c=966&c=973&c=974&c=970&c=971&c=972&c=979&c=980&c=981&c=976&c=977&c=978
|time_points=day00
|time_span=12 days
|timepoint_design=Staged in-vitro diff
|tissue_cell_type=Myoblast>>myotube
|zenbu_config=https://fantom.gsc.riken.jp/zenbu/gLyphs/#config=BpiY0BEbLZpjpmfh4nGpyB
}}