Masatoshi Suzuki, DVM, PhD – Slide of the Week

Suzuki Slide of the Week

Title: Derivation of Mature Skeletal Myotubes Directly Prepared from Human induced Pluripotent Stem Cells

Legend: Human induced pluripotent stem cells (iPSCs) can be sufficiently differentiated into skeletal muscle stem cells (myogenic progenitors) and mature skeletal myotubes in a defined culture without genetic modification. Top: Skeletal muscle differentiation of iPSC-derived myogenic progenitors. Human iPSCs were maintained as spherical aggregates (EZ spheres) in suspension medium containing high concentration of growth factors (FGF-2 and EGF) for 6 weeks. Myogenic progenitors in EZ spheres were plated on coverslips and terminally differentiated in culture for 2-12 weeks. The stage of muscle cells can be determined by the expression of Pax7 (myogenic progenitors), MyoD (myoblasts), myogenin (MyoG; committed myocytes), and myosin heavy chain (MHC). Bottom left: Sarcomere formation in iPSC-derived myotubes. Sarcomere formation is critical for morphologically modeling the functional units of muscle contraction. Titin staining revealed that striated patterns were clearly visible in the myotubes at 12 weeks. Lower image shows MHC staining in the same cell preparations used for titin labeling. Bottom right: Ultrastructures of iPSC-derived myotubes. After 12 weeks of terminal differentiation, mature sarcomeres were observed assembled into myofibrils. Morphological hallmarks, including I-band of actin filaments and A-band with distinct M-line across myosin filaments, were clearly visible. Sarcomere Z lines appeared to be reasonably aligned and gave rise to a striated pattern.

Citation: Jiwlawat S, Lynch E, Glaser J, Smit-Oistad I, Jeffrey J, Van Dyke J, Suzuki M (2017). Differentiation and sarcomere formation in skeletal myocytes directly prepared from human induced pluripotent stem cells using a sphere-based culture. Differentiation, 96:70-81.

Abstract: Human induced-pluripotent stem cells are a promising resource for propagation of myogenic progenitors. Our group recently reported a unique protocol for the derivation of myogenic progenitors directly (without genetic modification) from human pluripotent cells using free-floating spherical culture. Here we expand our previous efforts and attempt to determine how differentiation duration, culture surface coatings, and nutrient supplements in the medium influence progenitor differentiation and formation of skeletal myotubes containing sarcomeric structures. A long differentiation period (over 6 weeks) promoted the differentiation of iPSC-derived myogenic progenitors and subsequent myotube formation. These iPSC-derived myotubes contained representative sarcomeric structures, consisting of organized myosin and actin filaments and could spontaneously contract. We also found that a bioengineering approach using three-dimensional artificial muscle constructs could facilitate the formation of elongated myotubes. Lastly, we determined how culture surface coating matrices and different supplements would influence terminal differentiation. While both Matrigel and laminin coatings showed comparable effects on muscle differentiation, B27 serum-free supplement in the differentiation medium significantly enhanced myogenesis compared to horse serum. Our findings support the possibility of creating an in vitro model of contractile sarcomeric myofibrils for disease modeling and drug screening to study neuromuscular diseases.

About the Lab: The Suzuki group has demonstrated the therapeutic benefits of ex vivo gene therapy (stem cell-based growth/trophic factor delivery) targeting the skeletal muscle to prevent degeneration of motor neurons and associated neuromuscular junctions during ALS. Although most ALS research has focused on mechanisms of motor neuron cell death, degeneration is also observed in skeletal muscle, particularly at the neuromuscular connection. Glial cell line-derived neurotrophic factor and vascular endothelial growth factor (VEGF) promote survival of motor neurons and their neuromuscular junctions in neuromuscular disorders, such as ALS. Most recently, the lab delivered a combination of GDNF and/or VEGF to muscles using hMSCs; the hMSCs survive and synthesize and release growth factors, which slow disease progression in familial ALS model rats.

Slide of the Week Archives