Myogenic Differentiation of ES Cells for Therapies in Neuromuscular Diseases: Progress to Date

Coordinated Vascular Endothelial Growth Factor Expression and Signaling During Skeletal Myogenic Differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e07-09-0856 ◽

2008 ◽

Vol 19 (3) ◽

pp. 994-1006 ◽

Cited By ~ 76

Author(s):

Brad A. Bryan ◽

Tony E. Walshe ◽

Dianne C. Mitchell ◽

Josh S. Havumaki ◽

Magali Saint-Geniez ◽

...

Keyword(s):

Skeletal Muscle ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Myogenic Differentiation ◽

Es Cells ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Vascular Endothelial ◽

Skeletal Muscle Differentiation ◽

Endothelial Growth Factor

Angiogenesis is largely controlled by hypoxia-driven transcriptional up-regulation and secretion of vascular endothelial growth factor (VEGF) and its binding to the endothelial cell tyrosine receptor kinases, VEGFR1 and VEGFR2. Recent expression analysis suggests that VEGF is expressed in a cell-specific manner in normoxic adult tissue; however, the transcriptional regulation and role of VEGF in these tissues remains fundamentally unknown. In this report we demonstrate that VEGF is coordinately up-regulated during terminal skeletal muscle differentiation. We reveal that this regulation is mediated in part by MyoD homo- and hetero-dimeric transcriptional mechanisms. Serial deletions of the VEGF promoter elucidated a region containing three tandem CANNTG consensus MyoD sites serving as essential sites of direct interaction for MyoD-mediated up-regulation of VEGF transcription. VEGF-null embryonic stem (ES) cells exhibited reduced myogenic differentiation compared with wild-type ES cells, suggesting that VEGF may serve a role in skeletal muscle differentiation. We demonstrate that VEGFR1 and VEGFR2 are expressed at low levels in myogenic precursor cells and are robustly activated upon VEGF stimulation and that their expression is coordinately regulated during skeletal muscle differentiation. VEGF stimulation of differentiating C2C12 cells promoted myotube hypertrophy and increased myogenic differentiation, whereas addition of sFlt1, a VEGF inhibitor, resulted in myotube hypotrophy and inhibited myogenic differentiation. We further provide evidence indicating VEGF-mediated myogenic marker expression, mitogenic activity, migration, and prosurvival functions may contribute to increased myogenesis. These data suggest a novel mechanism whereby VEGF is coordinately regulated as part of the myogenic differentiation program and serves an autocrine function regulating skeletal myogenesis.

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Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-dependent Stimulation of Myogenic Differentiation

International Journal of Molecular Sciences ◽

10.3390/ijms20174188 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4188

Author(s):

Silvia Sorrentino ◽

Alessandra Barbiera ◽

Gabriella Proietti ◽

Gigliola Sica ◽

Sergio Adamo ◽

...

Keyword(s):

Protein Kinase ◽

Nuclear Export ◽

Nuclear Translocation ◽

Protein Kinase B ◽

Myogenic Differentiation ◽

Neuromuscular Disorders ◽

Neuromuscular Diseases ◽

Molecular Techniques ◽

Physiological Factor ◽

Pi3k Inhibitor Ly294002

Arginine-vasopressin (AVP) promotes muscle differentiation, hypertrophy, and regeneration through the combined activation of the calcineurin and Calcium/Calmodulin-dependent Protein Kinase (CaMK) pathways. The AVP system is impaired in several neuromuscular diseases, suggesting that AVP may act as a physiological factor in skeletal muscle. Since the Phosphoinositide 3-kinases/Protein Kinase B/mammalian Target Of Rapamycin (PI3K/Akt/mTOR) signaling plays a significant role in regulating muscle mass, we evaluated its role in the AVP myogenic effect. In L6 cells AKT1 expression was knocked down, and the AVP-dependent expression of mTOR and Forkhead box O3 (FoxO) was analyzed by Western blotting. The effect of the PI3K inhibitor LY294002 was evaluated by cellular and molecular techniques. Akt knockdown hampered the AVP-dependent mTOR expression while increased the levels of FoxO transcription factor. LY294002 treatment inhibited the AVP-dependent expression of Myocyte Enhancer Factor-2 (MEF2) and myogenin and prevented the nuclear translocation of MEF2. LY294002 also repressed the AVP-dependent nuclear export of histone deacetylase 4 (HDAC4) interfering with the formation of multifactorial complexes on the myogenin promoter. We demonstrate that the PI3K/Akt pathway is essential for the full myogenic effect of AVP and that, by targeting this pathway, one may highlight novel strategies to counteract muscle wasting in aging or neuromuscular disorders.

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hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures

Cells ◽

10.3390/cells10123292 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3292

Author(s):

Sarah Janice Hörner ◽

Nathalie Couturier ◽

Roman Bruch ◽

Philipp Koch ◽

Mathias Hafner ◽

...

Keyword(s):

Schwann Cells ◽

Neuromuscular Junctions ◽

Myogenic Differentiation ◽

Body Movement ◽

Functional Integration ◽

Neuromuscular Diseases ◽

In Vitro Models ◽

Bmp Signaling ◽

Induced Pluripotent

Motoneurons, skeletal muscle fibers, and Schwann cells form synapses, termed neuromuscular junctions (NMJs). These control voluntary body movement and are affected in numerous neuromuscular diseases. Therefore, a variety of NMJ in vitro models have been explored to enable mechanistic and pharmacological studies. So far, selective integration of Schwann cells in these models has been hampered, due to technical limitations. Here we present robust protocols for derivation of Schwann cells from human induced pluripotent stem cells (hiPSC) and their coculture with hiPSC-derived motoneurons and C2C12 muscle cells. Upon differentiation with tuned BMP signaling, Schwann cells expressed marker proteins, S100b, Gap43, vimentin, and myelin protein zero. Furthermore, they displayed typical spindle-shaped morphologies with long processes, which often aligned with motoneuron axons. Inclusion of Schwann cells in coculture experiments with hiPSC-derived motoneurons and C2C12 myoblasts enhanced myotube growth and affected size and number of acetylcholine receptor plaques on myotubes. Altogether, these data argue for the availability of a consistent differentiation protocol for Schwann cells and their amenability for functional integration into neuromuscular in vitro models, fostering future studies of neuromuscular mechanisms and disease.

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