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Skeletal myocytes demonstrate robust expression of components of the contractile apparatus. They form striated, multinucleated myocytes that contract in response to acetylcholine by day 10, post-revival. Unprecedented batch-to-batch consistency allows for their use in screening applications.
Video capturing the rapid reprogramming from iPSCs to skeletal myocytes upon induction of MYOD1 expression. 10-day time course.
By day three post-revival, ioSkeletal Myocytes demonstrate classical myocyte morphology and express the myocyte genes Desmin, Myogenin, and Myosin Heavy Chain, as assessed by qRT-PCR. By day 10 post-revival, skeletal myocytes express the major proteins of myofilaments, including Myosin Heavy Chain, Desmin, Dystrophin, Troponin, and form striated, multinucleated, myocytes.
Immunofluorescence staining at day 10 post-revival demonstrates robust expression of components of the contractile apparatus; including Desmin (A), Dystrophin (B), and Myosin Heavy Chain (C), along with the muscle transcription factor Myogenin (C). Cells also demonstrate expression of Troponin with visible striated fibers and multinucleation (D).
Cells express the insulin-regulated glucose transporter GLUT4, critical for metabolic studies.
ioSkeletal Myocytes express the insulin-regulated glucose transporter GLUT4 by day seven, providing a unique human muscle cell model for metabolic research.
(A) RT-qPCR, at day 10 post-revival, demonstrating expression of GLUT4 in the ioSkeletal Myocytes, compared to undifferentiated hiPSCs and ioGlutamatergic Neurons. (B) Immunocytochemistry, at day seven post-revival, demonstrates expression of GLUT4 in peri-nuclear regions and striations in the ioSkeletal Myocytes*. (C) Western blotting of differentiated 3T3-L1 adipocytes and maturing ioSkeletal Myocytes demonstrates GLUT4 expression in a time-dependent manner*.
*Douggal, Norris & Daniel Fazakerley, Wellcome-MRC Institute of Metabolic Science.
By day 10 post-revival, skeletal myocytes demonstrate strong contactility in response to treatment with acetylcholine. Spontaneous contraction is also observed during continuous culture (data not shown).
Day 10 post-revival skeletal myocytes; 50µM acetylcholine.
1. Pawlowski, M. et al. Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons, Skeletal Myocytes, and Oligodendrocytes. Stem Cell Reports. 8(4), 803-812 (2017).