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Myoblast to myotube (wt and DMD): Difference between revisions

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Revision as of 21:44, 3 February 2015

Series:IN_VITRO DIFFERENTIATION SERIES
Species:Human (Homo sapiens)
Genomic View:Zenbu
Expression table:FILE
Link to TET:[{{{tet_file}}} TET]
Sample providers :Valerio Orlando
Germ layer:mesoderm
Primary cells or cell line:primary cells
Time span:12 days
Number of time points:9


Overview

Differentiation of human primary skeletal myoblasts derived from healthy donors and patients affected by Duchenne Muscular Dystrophy
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)

References:
[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).
[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).
[3] Colussi, C. et al. Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy. FASEB J 23, 2131-2141, (2009).

Sample 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).

Quality 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.
Figure 1: CAGE expression of marker genes in TPM.

Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples


13469-144I4Myoblast differentiation to myotubesday00control donor1
13470-144I5Myoblast differentiation to myotubesday01control donor1
13471-144I6Myoblast differentiation to myotubesday02control donor1
13472-144I7Myoblast differentiation to myotubesday03control donor1
13473-144I8Myoblast differentiation to myotubesday04control donor1
13474-144I9Myoblast differentiation to myotubesday06control donor1
13475-145A1Myoblast differentiation to myotubesday08control donor1
13476-145A2Myoblast differentiation to myotubesday10control donor1
13477-145A3Myoblast differentiation to myotubesday12control donor1
13478-145A4Myoblast differentiation to myotubesday00control donor2
13480-145A6Myoblast differentiation to myotubesday02control donor2
13481-145A7Myoblast differentiation to myotubesday03control donor2
13482-145A8Myoblast differentiation to myotubesday04control donor2
13483-145A9Myoblast differentiation to myotubesday06control donor2
13484-145B1Myoblast differentiation to myotubesday08control donor2
13485-145B2Myoblast differentiation to myotubesday10control donor2
13486-145B3Myoblast differentiation to myotubesday12control donor2
13487-145B4Myoblast differentiation to myotubesday00control donor3
13488-145B5Myoblast differentiation to myotubesday01control donor3
13489-145B6Myoblast differentiation to myotubesday02control donor3
13490-145B7Myoblast differentiation to myotubesday03control donor3
13491-145B8Myoblast differentiation to myotubesday04control donor3
13492-145B9Myoblast differentiation to myotubesday06control donor3
13493-145C1Myoblast differentiation to myotubesday08control donor3
13495-145C3Myoblast differentiation to myotubesday12control donor3
13496-145C4Myoblast differentiation to myotubesday00Duchenne Muscular Dystrophy donor1
13497-145C5Myoblast differentiation to myotubesday01Duchenne Muscular Dystrophy donor1
13498-145C6Myoblast differentiation to myotubesday02Duchenne Muscular Dystrophy donor1
13499-145C7Myoblast differentiation to myotubesday03Duchenne Muscular Dystrophy donor1
13500-145C8Myoblast differentiation to myotubesday04Duchenne Muscular Dystrophy donor1
13501-145C9Myoblast differentiation to myotubesday06Duchenne Muscular Dystrophy donor1
13502-145D1Myoblast differentiation to myotubesday08Duchenne Muscular Dystrophy donor1
13503-145D2Myoblast differentiation to myotubesday10Duchenne Muscular Dystrophy donor1
13504-145D3Myoblast differentiation to myotubesday12Duchenne Muscular Dystrophy donor1
13505-145D4Myoblast differentiation to myotubesday00Duchenne Muscular Dystrophy donor2
13506-145D5Myoblast differentiation to myotubesday01Duchenne Muscular Dystrophy donor2
13507-145D6Myoblast differentiation to myotubesday02Duchenne Muscular Dystrophy donor2
13508-145D7Myoblast differentiation to myotubesday03Duchenne Muscular Dystrophy donor2
13509-145D8Myoblast differentiation to myotubesday04Duchenne Muscular Dystrophy donor2
13511-145E1Myoblast differentiation to myotubesday08Duchenne Muscular Dystrophy donor2
13512-145E2Myoblast differentiation to myotubesday10Duchenne Muscular Dystrophy donor2
13513-145E3Myoblast differentiation to myotubesday12Duchenne Muscular Dystrophy donor2
13514-145E4Myoblast differentiation to myotubesday00Duchenne Muscular Dystrophy donor3
13515-145E5Myoblast differentiation to myotubesday01Duchenne Muscular Dystrophy donor3
13516-145E6Myoblast differentiation to myotubesday02Duchenne Muscular Dystrophy donor3
13518-145E8Myoblast differentiation to myotubesday04Duchenne Muscular Dystrophy donor3
13519-145E9Myoblast differentiation to myotubesday06Duchenne Muscular Dystrophy donor3
13520-145F1Myoblast differentiation to myotubesday08Duchenne Muscular Dystrophy donor3
13521-145F2Myoblast differentiation to myotubesday10Duchenne Muscular Dystrophy donor3
13522-145F3Myoblast differentiation to myotubesday12Duchenne Muscular Dystrophy donor3
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