Analysis of gene expression in hTERT/cdk4 immortalized human myoblasts compared to their primary populations in both undifferentiatied (myoblast) and differentiated (myotube) states
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE79263 | GEO | 2016/03/16
SECONDARY ACCESSION(S): PRJNA315301
REPOSITORIES: GEO
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Project description:hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research, being used as therapeutically-pertinent models of various neuromuscular disorders and in numerous fundamental studies of muscle cell function. However, the cell cycle is linked to other cellular processes such as integrin regulation, the PI3K/Akt pathway, and microtubule stability, raising the question as to whether transgenic modification of the cell cycle results in secondary effects that could undermine the validity of these cell models. Here we subjected healthy and disease lines to intensive transcriptomic analysis, comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states, and testing their myogenic character by comparison with non-myogenic (CD56-negative) cells. We found that immortalization has no measurable effect on the myogenic cascade or on any other cellular processes, and that it was protective against the systems level effects of senescence that are observed at higher division counts of primary cells. This dataset includes gene expression profiles for 94 samples comprising primary myoblasts and their corresponding immortalized clones in both differentiated and undifferentiated states (average of 4 cell culture replicates each) from 5 human subjects (2 healthy and 3 Duchenne muscular dystropy - DMD), together with primary populations of non-myogenic (CD56-ve) cells from the muscles of 8 other human subjects. Total RNA was extracted from, myoblasts, myotubes (after 9 days of differentiation), or CD56-ve cells by dissolving cell pellets in TRIzol then using PureLink RNA Mini Kit.
2016-03-16 | E-GEOD-79263 | biostudies-arrayexpress
Project description:hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research, being used as therapeutically-pertinent models of various neuromuscular disorders and in numerous fundamental studies of muscle cell function. However, the cell cycle is linked to other cellular processes such as integrin regulation, the PI3K/Akt pathway, and microtubule stability, raising the question as to whether transgenic modification of the cell cycle results in secondary effects that could undermine the validity of these cell models. Here we subjected healthy and disease myoblasts to intensive transcriptomic analysis, comparing immortalized cell populations, and clonal lines derived from them, with their parent primary populations. We found that immortalization has no measurable effect on the myogenic cascade or on any other cellular processes, and that it was protective against the systems level effects of senescence that are observed at higher division counts of primary cells.
2019-09-16 | GSE79303 | GEO
Project description:Enhancing the proliferation and myogenic commitment of progenitor cells during fetal development enhances muscle growth and lean production in offspring. During the early development, a pool of skeletal stem cells (SSCs) proliferates and then diverge into either myoblast. Myoblast further fusion and develop into myotube. However, the landscape from muscle stem cells to myotubes in goats is not yet clear. This study aims to characterize the changes in the expression profile of skeletal muscle stem cells that differentiate into myoblasts, then migrate and fuse to form myotubes.
2021-11-11 | PXD029693 |
Project description:Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.
| S-EPMC3879278 | biostudies-literature
Project description:STAC3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon STAC3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, STAC3 was initially expressed in myotomal adaxial cells and in fast muscle fibres post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibres, and decreased levels of the hedgehog receptor patched. The function of STAC3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. STAC3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of STAC3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton and the pro-myogenic factors IGFBP-5 and IGF2 were down regulated. RNAi-treated cells had suppressed AKT signalling and exogenous IGF2 was unable to rescue the phenotype, however, IGF/AKT signalling was not blocked. Overexpression of STAC3, which results in increased levels of IGFBP-5 mRNA, did not lead to increased differentiation. In synchronized cells, STAC3 mRNA was most abundant during the G1 phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and decreased proportion of cells in G1 phase when compared to controls, suggesting a role in the G1 phase checkpoint. These results identify STAC3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates. We identified STAC3 as being essential for myogenic differentiation. The goal of the microarray analysis was to identify genes that were up- or down-regulated in C2C12 cells when STAC3 expression was inhibited by interfering RNA (RNAi). The microarray compares the C2C12 mouse myogenic cell line between control samples to samples treated with an interfering RNA directed at STAC3. The mouse C2C12 cell line was used at two time points: day 0, which was 24 hours after adding the RNAi, with cells growing in growth media (non-differentiating), and day 1, which was 48 hours after the interfering RNAi was added, and 24 hours after switching to differentiation media. RNAi-treated cells were transfected with an RNAi directed against STAC3, and control cells were transfected with a high-GC content control RNAi. Total RNA was extracted from control and RNAi-treated samples at two time points, at day 0 (24 h after treating with RNAi, while samples were still in growth media), and at day 1 (cells grown in differentiation media for 24h, 48 h after RNAi treatment). At each time point, STAC3 RNAi-treated cells were compared to controls. For the array comparison, at day 0, triplicate biological samples + one technical replicate were used for the controls, and four biological samples were used for the RNAi-treated cells. At day 1, four biological samples and one technical replicate were used for controls and RNAi-treated cells. Technical replicate identifiers are appended by _r.
2012-12-01 | E-GEOD-34474 | biostudies-arrayexpress
Project description:STAC3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon STAC3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, STAC3 was initially expressed in myotomal adaxial cells and in fast muscle fibres post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibres, and decreased levels of the hedgehog receptor patched. The function of STAC3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. STAC3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of STAC3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton and the pro-myogenic factors IGFBP-5 and IGF2 were down regulated. RNAi-treated cells had suppressed AKT signalling and exogenous IGF2 was unable to rescue the phenotype, however, IGF/AKT signalling was not blocked. Overexpression of STAC3, which results in increased levels of IGFBP-5 mRNA, did not lead to increased differentiation. In synchronized cells, STAC3 mRNA was most abundant during the G1 phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and decreased proportion of cells in G1 phase when compared to controls, suggesting a role in the G1 phase checkpoint. These results identify STAC3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates. We identified STAC3 as being essential for myogenic differentiation. The goal of the microarray analysis was to identify genes that were up- or down-regulated in C2C12 cells when STAC3 expression was inhibited by interfering RNA (RNAi). The microarray compares the C2C12 mouse myogenic cell line between control samples to samples treated with an interfering RNA directed at STAC3. The mouse C2C12 cell line was used at two time points: day 0, which was 24 hours after adding the RNAi, with cells growing in growth media (non-differentiating), and day 1, which was 48 hours after the interfering RNAi was added, and 24 hours after switching to differentiation media.
2012-12-01 | GSE34474 | GEO
Project description:Comparison of undifferentiated C2C12 myoblast to 4 day differentiated myotubes. Keywords: other
2005-09-07 | GSE3243 | GEO
Project description:Wnt/M-NM-2-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and M-NM-2-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However, both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of M-NM-2-catenin signaling during myogenic differentiation remain unknown. Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of M-NM-2-catenin/Tcf complex formation, reduced basal M-NM-2-catenin in cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased membrane-bound M-NM-2-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated M-NM-2-catenin (Tyr654) during myogenic differentiation. These results suggest that various Wnt ligands control subcellular M-NM-2-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via M-NM-2-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation. Control cells (day 0) prior to differentiation induction with n=4; differentiated for two days with n=3; differentiated for four days with n=3.
2011-07-15 | E-GEOD-30077 | biostudies-arrayexpress
Project description:Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However, both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown. Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation. These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.
2011-07-15 | GSE30077 | GEO
Project description:The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes that they regulate remain poorly defined. Here we find that activity of p38α/β is important not only for early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treating C2 myoblasts with the pro-myogenic growth factor, IGF-I, we were able to overcome the early block in differentiation imposed by co-incubation with the p38 chemical inhibitor, SB202190. Yet, IGF-I could not overcome the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts showed that the fusion block was reversible, as multinucleated myofibers were detected several hours later, and reached ~90% of the culture within 30 hours. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of up-regulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion, which should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis.
2018-10-24 | PXD002413 | Pride